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Contents

Chapter 1 **The Association of Chronic** 

Chapter 3 **Circulating Markers in** 

**Inflammation and Gastroenteropancreatic Neuroendocrine Tumors (GEP-NETs) 1** 

Chapter 2 **Chromogranin A and Neuroendocrine Tumors 11** 

**Neuroendocrine Tumors (GEP NETs) 19**  Sara Massironi, Matilde Pia Spampatti,

Federica Cavalcoli and Maddalena Peracchi

Chapter 5 **Gastrointestinal Neuroendocrine Tumors 47**  Ozcan Yildiz and Suheyla Serdengecti

Roberta Elisa Rossi, Dario Conte, Clorinda Ciafardini,

Jennifer Keiser, Emily Bergsland and Eric Nakakura

**of Neuroendocrine Carcinoma of Unknown Primary 37** 

Angela Prestifilippo, Giusi Blanco, Maria Paola Vitale and Dario Giuffrida

**Gastroenteropancreatic** 

Chapter 4 **The Diagnosis and Management** 

Maja Cigrovski Berković, Davorka Herman Mahečić, Vedran Tomašić, Davor Hrabar and Vanja Zjačić-Rotkvić

### Contents


Ozcan Yildiz and Suheyla Serdengecti

**1** 

**The Association of Chronic** 

Davor Hrabar2 and Vanja Zjačić-Rotkvić<sup>1</sup>

*"Sestre milosrdnice", Zagreb,* 

*"Sestre milosrdnice", Zagreb,* 

*Croatia* 

**Inflammation and Gastroenteropancreatic** 

Maja Cigrovski Berković1, Davorka Herman Mahečić1, Vedran Tomašić2,

*1Department of Endocrinology, Diabetes and Metabolism University Hospital Centre* 

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare and heterogeneous neoplasms with overall increasing incidence, but not an associated increase in survival rate over the past few decades. Tumors originate from at least 16 different cells of diffuse endocrine system (DES), scattered through mucosa of gastrointestinal tract. They are mainly sporadic, but sometimes exhibit familial inheritance. Tumors often preserve the ability to synthesize, store and secrete numerous hormones and biogenic amines which sometimes lead to distinct hypersecretory and clinically recognizable syndromes (such as carcinoid, Zollinger-Ellison, WDHA etc.).1 The resulting clinical symptoms are generally well

More often, GEP-NETs remain clinically silent until late, when they present with mass effect, and have unfortunately already locally or distantly spread. In the later case tumor growth and spread are not always well controlled by either biotherapy or chemotherapy. Although many biochemical and tissue markers for GEP-NETs already exist, sensitive and

According to our unpublished data chromogranin A (CgA) and 5-hydroxyindolacetic acid (5-HIAA), currently used as standard biochemical markers of neuroendocrine tumors were only positive in 76.84% and 20.79% of GEP-NET cases respectively. Tumor markers were analyzed in 101 patients (61.2% with localized and 38.8% with metastatic disease) diagnosed with GEP-NETs. According to same investigation, CgA levels were much higher when tumors were part of MEN1 syndrome, while 5-HIAA levels were higher in case of metastatic disease, especially when hepatic metastases were present. When 5-HIAA values were compared among patients with different tumor localizations, the highest values were detected in patients with functional midgut tumors. This is consistent with data of other

controlled by somatostatin analogs and/or interferon-α.2

specific markers that predict tumor growth and behavior are lacking.3

authors on biochemical diagnostics of gastrointestinal neuroendocrine tumors.4

**1. Introduction** 

*2Department of Gastroenterology and Hepathology University Hospital Centre* 

**Neuroendocrine Tumors (GEP-NETs)** 

## **The Association of Chronic Inflammation and Gastroenteropancreatic Neuroendocrine Tumors (GEP-NETs)**

Maja Cigrovski Berković1, Davorka Herman Mahečić1, Vedran Tomašić2, Davor Hrabar2 and Vanja Zjačić-Rotkvić<sup>1</sup> *1Department of Endocrinology, Diabetes and Metabolism University Hospital Centre "Sestre milosrdnice", Zagreb, 2Department of Gastroenterology and Hepathology University Hospital Centre "Sestre milosrdnice", Zagreb,* 

*Croatia* 

#### **1. Introduction**

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare and heterogeneous neoplasms with overall increasing incidence, but not an associated increase in survival rate over the past few decades. Tumors originate from at least 16 different cells of diffuse endocrine system (DES), scattered through mucosa of gastrointestinal tract. They are mainly sporadic, but sometimes exhibit familial inheritance. Tumors often preserve the ability to synthesize, store and secrete numerous hormones and biogenic amines which sometimes lead to distinct hypersecretory and clinically recognizable syndromes (such as carcinoid, Zollinger-Ellison, WDHA etc.).1 The resulting clinical symptoms are generally well controlled by somatostatin analogs and/or interferon-α.2

More often, GEP-NETs remain clinically silent until late, when they present with mass effect, and have unfortunately already locally or distantly spread. In the later case tumor growth and spread are not always well controlled by either biotherapy or chemotherapy. Although many biochemical and tissue markers for GEP-NETs already exist, sensitive and specific markers that predict tumor growth and behavior are lacking.3

According to our unpublished data chromogranin A (CgA) and 5-hydroxyindolacetic acid (5-HIAA), currently used as standard biochemical markers of neuroendocrine tumors were only positive in 76.84% and 20.79% of GEP-NET cases respectively. Tumor markers were analyzed in 101 patients (61.2% with localized and 38.8% with metastatic disease) diagnosed with GEP-NETs. According to same investigation, CgA levels were much higher when tumors were part of MEN1 syndrome, while 5-HIAA levels were higher in case of metastatic disease, especially when hepatic metastases were present. When 5-HIAA values were compared among patients with different tumor localizations, the highest values were detected in patients with functional midgut tumors. This is consistent with data of other authors on biochemical diagnostics of gastrointestinal neuroendocrine tumors.4

The Association of Chronic Inflammation and

Fig. 1. Development of GEP-NETs.

Loss of mismatch repair

Methylation Acethylation TGFα

TRANSFORMATION PROLIFERATION

**MALIGNANT ALTERATION** 

**INITIATION** 

**METASTASING**

Gastroenteropancreatic Neuroendocrine Tumors (GEP-NETs) 3

Normal neuroendocrine cells

 Hyperplastic cells

 Dysplastic cells

Well differentiated tumor

Well differentiated carcinoma

Poorly differentiated carcinoma

Comparative studies of pancreatic adenocarcinoma and pancreatic neuroendocrine tumors (pNETs) have helped in giving insight into cellular biology of those specific tumors. Unlike pancreatic adenocarcinomas, pNETs do not exhibit mutations in K-Ras oncogene or p53 tumor suppressor gene, which are often mutated in the former. Also, the pattern of genomic alterations of pNETs differs from that of gastrointestinal NETs, where losses on

Metastasis

**2. HIT** 

LOH Loss of TSG

Growth factors TGF, VEGF, bFGF

**1. HIT** Inactivation

*MEN1, VHL, NF1, TSC*

Inactivation *MEN1*

Chromosome instability

Loss of adhesion Induction of VEGF

It seems that specifics of pNET development are gains and losses of chromosomes, which also appear to influence disease stage. Specifically, genomic gains are common on chromosomes 4pq, 5pq, 7pq, 9q, 12q, 14q, 17pq, 18q and 20q, while losses occur on

chromosome 18q are almost a rule (occur in 38-88% of tumors).13

Unfortunately, the correct diagnose of GEP-NETs is delayed for 7-10 years, additionally adding burden to anyhow complex and challenging tumor management.3 So, in clinical practice, more reliable serum markers as well as precise tumor localization of small, initial lesions together with incorporation of a histological grading system with implemented prognostic implications would help in optimal treatment of patients. The mentioned needs to be supported by better understanding of tumor cell biology and mechanistic regulation of underlying growth processes.5

In general, majority of GEP-NETs are represented by well-differentiated cells, and one would expect low proliferating rate, but unfortunately, tumors often present metastatic at the time of diagnosis. This is one of the most intriguing characteristics, and has triggered scientific research aiming to demonstrate specific molecular features that could explain mechanisms underneath the ability of tumor cells to detach from primary malignancy and gain excess to the surrounding structures.6

Although development of GEP-NETs is still unclear, significant breakthrough has been made in elucidating molecular genetics of neuroendocrine tumors exhibiting a hereditary background. Those rare tumor types (5-10% of all GEP-NETs) are often caused by mutations in tumor suppressor genes MEN1, VHL, NF-1, TSC1, and TSC2 which in turn lead to development of NETs as a part of multiple endocrine neoplasia type 1, von Hippel Lindau disease, neurofibromatosis type 1 and tuberous sclerosis complex respectively.7 Besides tumor suppression genes, studies have also demonstrated involvement of oncogenes, each of which may be associated with several different abnormalities that include point mutations, gene deletions, DNA methylation, chromosomal losses and chromosomal gains (Figure 1).3,8,9

Perhaps the best characterized is the genetic background of the MEN1 syndrome, which in addition to neuroendocrine tumors of duodenum and pancreas includes adenomas/ hyperplasia of other endocrine glands (parathyroid hyperplasia/hyperparathyroidism, pituitary adenomas and adrenal cortical adenomas). It involves mutations of the MEN-1 tumor suppressor gene. This chromosome 11q13 gene encodes protein menin which interacts with a number of proteins involved in the transcriptional regulation and genome stability, so it has been proposed to be a key player in regulating NET cell proliferation.8

The *MEN-1* gene, although conferring a high disease risk in MEN-1 patients where it represents a putative tumor suppressor gene accounts for less than 40 percent of sporadic GEP-NET cases.10 Thus, the genes involved in neuroendocrine tumorigenesis and the cellular roles of their proteins on proliferation and/or apoptotic pathways remain largely unknown. Studies of comparative genomic hybridization and allelic loss analysis have detected a large number of genomic regions with loss or gain of genetic material, further elucidating genetic differences between GEP-NETs of various primary localizations, and proving the heterogeneity of the tumors.11

In general, foregut GEP-NETs often show loss of 11q, while tumors of midgut origin frequently show losses on chromosome 18q. The genetic abnormalities in hindgut NETs have not been well characterized, but it was noticed that larger tumors tend to express transforming growth factor-alpha (TGF-α) more frequently, while epidermal growth factor receptor (EGFR) was expressed in all lesions.12

Unfortunately, the correct diagnose of GEP-NETs is delayed for 7-10 years, additionally adding burden to anyhow complex and challenging tumor management.3 So, in clinical practice, more reliable serum markers as well as precise tumor localization of small, initial lesions together with incorporation of a histological grading system with implemented prognostic implications would help in optimal treatment of patients. The mentioned needs to be supported by better understanding of tumor cell biology and mechanistic regulation of

In general, majority of GEP-NETs are represented by well-differentiated cells, and one would expect low proliferating rate, but unfortunately, tumors often present metastatic at the time of diagnosis. This is one of the most intriguing characteristics, and has triggered scientific research aiming to demonstrate specific molecular features that could explain mechanisms underneath the ability of tumor cells to detach from primary malignancy and

Although development of GEP-NETs is still unclear, significant breakthrough has been made in elucidating molecular genetics of neuroendocrine tumors exhibiting a hereditary background. Those rare tumor types (5-10% of all GEP-NETs) are often caused by mutations in tumor suppressor genes MEN1, VHL, NF-1, TSC1, and TSC2 which in turn lead to development of NETs as a part of multiple endocrine neoplasia type 1, von Hippel Lindau disease, neurofibromatosis type 1 and tuberous sclerosis complex respectively.7 Besides tumor suppression genes, studies have also demonstrated involvement of oncogenes, each of which may be associated with several different abnormalities that include point mutations, gene deletions, DNA methylation, chromosomal losses and chromosomal gains

Perhaps the best characterized is the genetic background of the MEN1 syndrome, which in addition to neuroendocrine tumors of duodenum and pancreas includes adenomas/ hyperplasia of other endocrine glands (parathyroid hyperplasia/hyperparathyroidism, pituitary adenomas and adrenal cortical adenomas). It involves mutations of the MEN-1 tumor suppressor gene. This chromosome 11q13 gene encodes protein menin which interacts with a number of proteins involved in the transcriptional regulation and genome stability, so it has been proposed to be a key player in regulating NET cell

The *MEN-1* gene, although conferring a high disease risk in MEN-1 patients where it represents a putative tumor suppressor gene accounts for less than 40 percent of sporadic GEP-NET cases.10 Thus, the genes involved in neuroendocrine tumorigenesis and the cellular roles of their proteins on proliferation and/or apoptotic pathways remain largely unknown. Studies of comparative genomic hybridization and allelic loss analysis have detected a large number of genomic regions with loss or gain of genetic material, further elucidating genetic differences between GEP-NETs of various primary localizations, and

In general, foregut GEP-NETs often show loss of 11q, while tumors of midgut origin frequently show losses on chromosome 18q. The genetic abnormalities in hindgut NETs have not been well characterized, but it was noticed that larger tumors tend to express transforming growth factor-alpha (TGF-α) more frequently, while epidermal growth factor

underlying growth processes.5

(Figure 1).3,8,9

proliferation.8

gain excess to the surrounding structures.6

proving the heterogeneity of the tumors.11

receptor (EGFR) was expressed in all lesions.12

Fig. 1. Development of GEP-NETs.

Comparative studies of pancreatic adenocarcinoma and pancreatic neuroendocrine tumors (pNETs) have helped in giving insight into cellular biology of those specific tumors. Unlike pancreatic adenocarcinomas, pNETs do not exhibit mutations in K-Ras oncogene or p53 tumor suppressor gene, which are often mutated in the former. Also, the pattern of genomic alterations of pNETs differs from that of gastrointestinal NETs, where losses on chromosome 18q are almost a rule (occur in 38-88% of tumors).13

It seems that specifics of pNET development are gains and losses of chromosomes, which also appear to influence disease stage. Specifically, genomic gains are common on chromosomes 4pq, 5pq, 7pq, 9q, 12q, 14q, 17pq, 18q and 20q, while losses occur on

The Association of Chronic Inflammation and

 **INFECTION** 

Fig. 2. Connection between the endocrine system and cytokines.

clearly elucidated.28

Cytokines and growth factors seem to largely contribute to the development and progression of GEP-NETs13,17,26,27, but their involvement in the autocrine stimulation of tumor cells, either in genesis and/or in the progression of GEP-NETs has not yet been

GEP-NETs represent a tumor entity with an extraordinary high vascularization along with an abundant production and secretion of growth factors, especially vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin like growth factor (IGF), fibroblast growth factor (FGF) and transforming

survival.25

Gastroenteropancreatic Neuroendocrine Tumors (GEP-NETs) 5

important association is still not understood completely. The development of a tumor is associated with the growth and expansion of not only tumor cells but also stroma, vessels and infiltrating inflammatory cells, and it is the interaction between these different cell types that propagates tumor growth. Cytokines found in tumors, acting on paracrine and autocrine loops, are most likely the key players in the mentioned communication24, and for some of them link has been found between the serum and/or tumor tissue level and cancer

**STRESS** CRF

 **MIF** 

Hypothalamus

ACTH

Glucocorticoids

Pituitary

chromosomes 1p, 3p, 6q, 10p, 11pq, X and Yq. It is interesting that nonfunctioning pNETs harbor more genetic changes than those functional; in particular they exhibit more losses of 3p and mutations in MEN1 gene. The locus 3p is especially interesting while it harbors several tumor suppressor genes like VHL and retinoic receptor-beta (RAR-β). The later, involved in induction of apoptosis, has been found hypermethylated in 25% of pNETS.14

In addition to tumor suppressor genes, some oncogenes have also been found altered in pNETs. Those specifically include over expression of growth factor-related genes such as insulin like growth factor binding protein 3 (IGFBP3), cell adhesion and migration molecules as well as endothelial elements, suggesting an important role of tumor microenvironment.15

Dysregulation of DNA methylation patterns is a central feature of colon carcinogenesis, and was also found to be present in development of gastrointestinal neuroendocrine tumors (especially carcinoids). This finding is interesting from the nutrigenomic point of view, and it raises the possibility of tumor prevention with folate and vitamin B12 supplementation.16,17

Positive immunohistochemistry staining for different cytokines and growth factors in the GEP-NETs as well as occurrence of GEP-NETs in the setting of inflammatory bowel disease led to the belief that chronic inflammation may play a crucial role in their development and that a number of more prevalent, low penetrance genes contribute to GEP-NET susceptibility in a larger population of patients.18

With respect to the role of inflammatory signals in promoting the development of cancer, there is now emerging evidence for an important relationship between macrophage migration inhibitory (MIF) factor expression, oncogenesis and tumor progression. It seems that in different tumors MIF directly promotes tumorigenesis by inhibiting p53 accumulation, promotes cellular proliferation through activation of members of the MAPK family and through induction of COX-2/PGE-2 influences tumor growth and viability. MIF was found to be co-secreted with adrenocorticotrophic hormone (ACTH) by the anterior pituitary, and it has the ability to override its antiinflammatory effects, thus promoting the inflammation and favouring protumor microinvironment.19

It seems that immune system through the network of different cytokines and growth factors may also play permissive role in GEP-NET development (Figure 2).20

It is now widely acknowledged that chronic inflammatory conditions can both pave the way for and sustain conditions favorable for carcinogenesis and tumor progression. Although the molecular mechanisms of this causal relationship remain to be elucidated, there is strong evidence of association between chronic inflammation and aproximately 1/5 of human cancers confirmed by numerous epidemiologic, gene association and molecular studies.21

Overall, it appears that chronic inflammation more often stimulates then inhibits tumor development. The persistence of chronic inflammation plays a critical role in initiating, sustaining and advancing tumor growth, and thus modulating the immune response may still be an alluring goal for therapeutic intervention.22,23

Although a pathogenic role for chronic inflammation has been suggested in multiple tumor systems in tumor initiation, progression and metastatic potential, the mechanism of this

chromosomes 1p, 3p, 6q, 10p, 11pq, X and Yq. It is interesting that nonfunctioning pNETs harbor more genetic changes than those functional; in particular they exhibit more losses of 3p and mutations in MEN1 gene. The locus 3p is especially interesting while it harbors several tumor suppressor genes like VHL and retinoic receptor-beta (RAR-β). The later, involved in induction of apoptosis, has been found hypermethylated in 25% of pNETS.14

In addition to tumor suppressor genes, some oncogenes have also been found altered in pNETs. Those specifically include over expression of growth factor-related genes such as insulin like growth factor binding protein 3 (IGFBP3), cell adhesion and migration molecules as well as endothelial elements, suggesting an important role of tumor

Dysregulation of DNA methylation patterns is a central feature of colon carcinogenesis, and was also found to be present in development of gastrointestinal neuroendocrine tumors (especially carcinoids). This finding is interesting from the nutrigenomic point of view, and it raises the possibility of tumor prevention with folate and vitamin B12

Positive immunohistochemistry staining for different cytokines and growth factors in the GEP-NETs as well as occurrence of GEP-NETs in the setting of inflammatory bowel disease led to the belief that chronic inflammation may play a crucial role in their development and that a number of more prevalent, low penetrance genes contribute to GEP-NET

With respect to the role of inflammatory signals in promoting the development of cancer, there is now emerging evidence for an important relationship between macrophage migration inhibitory (MIF) factor expression, oncogenesis and tumor progression. It seems that in different tumors MIF directly promotes tumorigenesis by inhibiting p53 accumulation, promotes cellular proliferation through activation of members of the MAPK family and through induction of COX-2/PGE-2 influences tumor growth and viability. MIF was found to be co-secreted with adrenocorticotrophic hormone (ACTH) by the anterior pituitary, and it has the ability to override its antiinflammatory effects, thus promoting the

It seems that immune system through the network of different cytokines and growth factors

It is now widely acknowledged that chronic inflammatory conditions can both pave the way for and sustain conditions favorable for carcinogenesis and tumor progression. Although the molecular mechanisms of this causal relationship remain to be elucidated, there is strong evidence of association between chronic inflammation and aproximately 1/5 of human cancers confirmed by numerous epidemiologic, gene association and molecular studies.21

Overall, it appears that chronic inflammation more often stimulates then inhibits tumor development. The persistence of chronic inflammation plays a critical role in initiating, sustaining and advancing tumor growth, and thus modulating the immune response may

Although a pathogenic role for chronic inflammation has been suggested in multiple tumor systems in tumor initiation, progression and metastatic potential, the mechanism of this

microenvironment.15

supplementation.16,17

susceptibility in a larger population of patients.18

inflammation and favouring protumor microinvironment.19

still be an alluring goal for therapeutic intervention.22,23

may also play permissive role in GEP-NET development (Figure 2).20

important association is still not understood completely. The development of a tumor is associated with the growth and expansion of not only tumor cells but also stroma, vessels and infiltrating inflammatory cells, and it is the interaction between these different cell types that propagates tumor growth. Cytokines found in tumors, acting on paracrine and autocrine loops, are most likely the key players in the mentioned communication24, and for some of them link has been found between the serum and/or tumor tissue level and cancer survival.25

Fig. 2. Connection between the endocrine system and cytokines.

Cytokines and growth factors seem to largely contribute to the development and progression of GEP-NETs13,17,26,27, but their involvement in the autocrine stimulation of tumor cells, either in genesis and/or in the progression of GEP-NETs has not yet been clearly elucidated.28

GEP-NETs represent a tumor entity with an extraordinary high vascularization along with an abundant production and secretion of growth factors, especially vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin like growth factor (IGF), fibroblast growth factor (FGF) and transforming

The Association of Chronic Inflammation and

development.33,34

nonfunctioning pNETs.36

treatment*.*<sup>38</sup>

**2. References** 

Gastroenteropancreatic Neuroendocrine Tumors (GEP-NETs) 7

Cytokine genes are highly polymorphic, with polymorphisms frequently located in regions of DNA that regulate transcription, or posttranscriptional events, thus influencing functional activity. Recently published studies connected proinflammatory cytokine genes SNPs with cancer susceptibility and severity, putting them in the spot light as cancermodifier genes.31 This is particularly true for cytokine gene polymorphisms and gastrointestinal malignancy, where many authors suggest the role of inflammationmediated oncogenesis.16,18,32 It seems likely that they also contribute to GEP-NET

Genetic polymorphisms directly influence interindividual variation in the cytokine response, and this clearly contributes to an individual's ultimate clinical outcome. Many single nucleotide polymorphisms (SNPs) have been detected within the cytokine gene sequences, particularly within the promoter regions. Several of these SNPs may be associated with differential level of gene transcription, thus influencing levels of cytokines and growth factors in sera and tumor tissue and ultimately altering the disease prognosis by

However, for the ultimate outcome, not only cytokines or growth factors but also (tumor) cell type and stimulus may also be important.35 In our investigation of the role of IL-6 in GEP-NETs we have found the significantly higher proportion of high expression genotypes (*-174 C/G* and *G/G*) in the nonfunctioning pNETs, discriminating them from functional pNETs and gastrointestinal NETs (mainly of midgut origin). Mentioned patients had also higher concentrations of IL-6 in their sera (it was overall elevated in 36.8% of patients), suggesting the potential role of IL-6 as a novel diagnostic and prognostic marker of

A number of studies have reported associations between TNF-α promoter SNPs with high expression alleles (-*238A, -308A, -1031C*) and susceptibility to cancer.20,37 Our ongoing studies have strongly confirmed the role of TNF-α *-1031C* (high expression) allele as a potential risk factor for developing GEP-NET. Also, we have found the higher level of the -*308* high expression genotypes (*AG, AA*) as well as high expression -*308A* allele among the patients contracting foregut GEP-NETs than in those with midgut tumors. This finding may provide better insight in the role of cytokines in the development of different GEP-NET types and differentiation, and possibly open new prospective in GEP-NET

[1] Plӧckinger U, Rindi G, Arnold A, Eriksson B, Krenning EP, DeHerder WW, Goede A,

[2] Cigrovski Berković M, Altabas V, Herman D, Hrabar D, Goldoni V, Vizner B, Zjačić-

Gastrointestinal Tumors. Neuroendocrinology 2004;80:394-424.

Endocrine-Related Cancer 2003;10:427-436.

Caplin Guidelines for the Diagnosis and Treatment of Neuroendocrine

Rotkvić V. A Single-Centre Experience with Octreotide in the Treatment of Different Hypersecretory Syndromes in Patients with Functional Gastroenteropancreatic Neuroendocrine Tumors. Coll Antropol. 2007;31:531-534. [3] Rindi G, Bordi C. Highlights of the biology of endocrine tumors of the gut and pancreas.

influencing anti-tumor immunologic response or pathways of (neo)angiogenesis.

growth factor-α (TGF-α), which according to both observational and mechanistic data connect chronic inflammation with gastrointestinal carcinogenesis.20,23

MEN-1 patients have a higher serum level of fibroblast growth factor (FGF), which correlates with the amount of tumor-associated fibroblastic response. Furthermore, insulinlike growth factor-I (IGF-I) receptors found on GEP-NET cells suggest an autocrine trophic function for the mentioned growth factor in these tumors.27 Patients with carcinoid syndrome were found to have positive immunohistochemistry for TGF -β on the right sided heart valves, as a consequence of NET progression and metastasis.29

For further cancer evolution angiogenesis plays an important role. Proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), IL1 and IL6 once again participate in this process by inducing the production of angiogenic factors, mainly VEGF. The role of vascular endothelial growth factor (VEGF) in the new vessel formation of these highly vascularized tumors is increasingly studied, and it appears to be involved in the metastasing process of the mentioned tumors. Higher levels of cytokines and growth factors detected in GEP-NETs are responsible for neurotrophic effects, smooth muscle cell hypertrophy and proliferation of both intimal and adventitial elastic tissue of the mesenteric blood vessels leading to vascular elastosis sometimes associated with ischemic changes of the near-by tissue (Figure 3).6,30

Fig. 3. Tumor cell markers of neuroendocrine cell.

growth factor-α (TGF-α), which according to both observational and mechanistic data

MEN-1 patients have a higher serum level of fibroblast growth factor (FGF), which correlates with the amount of tumor-associated fibroblastic response. Furthermore, insulinlike growth factor-I (IGF-I) receptors found on GEP-NET cells suggest an autocrine trophic function for the mentioned growth factor in these tumors.27 Patients with carcinoid syndrome were found to have positive immunohistochemistry for TGF -β on the right sided

For further cancer evolution angiogenesis plays an important role. Proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), IL1 and IL6 once again participate in this process by inducing the production of angiogenic factors, mainly VEGF. The role of vascular endothelial growth factor (VEGF) in the new vessel formation of these highly vascularized tumors is increasingly studied, and it appears to be involved in the metastasing process of the mentioned tumors. Higher levels of cytokines and growth factors detected in GEP-NETs are responsible for neurotrophic effects, smooth muscle cell hypertrophy and proliferation of both intimal and adventitial elastic tissue of the mesenteric blood vessels leading to vascular elastosis sometimes associated with ischemic

**VEGFR**

**TGFβR**

**MENIN**

connect chronic inflammation with gastrointestinal carcinogenesis.20,23

heart valves, as a consequence of NET progression and metastasis.29

changes of the near-by tissue (Figure 3).6,30

**COX2**

**IGFR**

 **CgA** 

Fig. 3. Tumor cell markers of neuroendocrine cell.

Cytokine genes are highly polymorphic, with polymorphisms frequently located in regions of DNA that regulate transcription, or posttranscriptional events, thus influencing functional activity. Recently published studies connected proinflammatory cytokine genes SNPs with cancer susceptibility and severity, putting them in the spot light as cancermodifier genes.31 This is particularly true for cytokine gene polymorphisms and gastrointestinal malignancy, where many authors suggest the role of inflammationmediated oncogenesis.16,18,32 It seems likely that they also contribute to GEP-NET development.33,34

Genetic polymorphisms directly influence interindividual variation in the cytokine response, and this clearly contributes to an individual's ultimate clinical outcome. Many single nucleotide polymorphisms (SNPs) have been detected within the cytokine gene sequences, particularly within the promoter regions. Several of these SNPs may be associated with differential level of gene transcription, thus influencing levels of cytokines and growth factors in sera and tumor tissue and ultimately altering the disease prognosis by influencing anti-tumor immunologic response or pathways of (neo)angiogenesis.

However, for the ultimate outcome, not only cytokines or growth factors but also (tumor) cell type and stimulus may also be important.35 In our investigation of the role of IL-6 in GEP-NETs we have found the significantly higher proportion of high expression genotypes (*-174 C/G* and *G/G*) in the nonfunctioning pNETs, discriminating them from functional pNETs and gastrointestinal NETs (mainly of midgut origin). Mentioned patients had also higher concentrations of IL-6 in their sera (it was overall elevated in 36.8% of patients), suggesting the potential role of IL-6 as a novel diagnostic and prognostic marker of nonfunctioning pNETs.36

A number of studies have reported associations between TNF-α promoter SNPs with high expression alleles (-*238A, -308A, -1031C*) and susceptibility to cancer.20,37 Our ongoing studies have strongly confirmed the role of TNF-α *-1031C* (high expression) allele as a potential risk factor for developing GEP-NET. Also, we have found the higher level of the -*308* high expression genotypes (*AG, AA*) as well as high expression -*308A* allele among the patients contracting foregut GEP-NETs than in those with midgut tumors. This finding may provide better insight in the role of cytokines in the development of different GEP-NET types and differentiation, and possibly open new prospective in GEP-NET treatment*.*<sup>38</sup>

#### **2. References**


The Association of Chronic Inflammation and

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2003; 88:1367-1373.

Cancer 2003;10:459-462.

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cancers. Cancer Treat Res 2006;130:39-65.

carcinogenesis. Cell cycle 2009;8 (13):2005-13.

Neuroendocrinology 80(suppl 1):94-98.

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[22] Jackson L, Evers BM. Chronic inflammation and pathogenesis of GI and pancreatic

[23] Höpfner M, Schuppan D, Scherübl H. Treatment of gastrointestinal neuroendocrine

[24] Gonda TA, Tu S, Wang TC. Chronic inflammation, the tumor microenvironment and

[25] Westbrook AM, Szakmary A, Schiestl RH. Mechanisms of intestinal inflammation and

[26] Wiedenmann B, Pape UF 2004 From basic to clinical research in gastroenteropancreatic

[27] Wild A et al. Frequent methylation-associated silencing of the tissue inhibitor of

[28] Barakat MT, Meeren K, Bloom SR. Neuroendocrine tumors. Endocrine-Related Cancer

[29] Lester WM, Gotlieb AI 1991 The cardiovascular system. In *Functional Endocrine Pathology*, vol. 2, pp 724-747. Eds k Kovacs and SL Asa, Boston: Blackwell [30] Ardill JES, Erikkson B. The importance of the measurment of circulating markers in

[31] Seike M et al. Use of a cytokine gene expression signature in lung adenocarcinoma and

[32] Bidwell J et al. Cytokine gene polymorphism in human disease: on-line databases.

[33] Wilkening S et al. Interleukin promoter polymorphisms and prognosis in colorectal

[34] Cigrovski Berkovic M. The role of cytokines and growth factors in development and

[35] MacArthur M, Hold GL, El-Omar EM. Inflammation and Cancer II. Role of chronic

[36] Cigrovski Berković M, Jokić M, Marout J, Radošević S, Zjačić-Rotkvić V, Kapitanović, S.

(GEP-NETs). Experimental and Molecular Pathology 2007;83:474-479. [37] Anderson GM, Nakada MT, Dewitte M. Tumor necrosis factor-α in the pathogenesis

and treatment of cancer. Curr Opin Pharmacol 2004; 4314-4320.

tumors with inhibitors of growth factor receptors and their signaling pathways: Recent advances and future perspectives. World J Gastroenterol 2008;14(16):2461-

development of associated cancers: Lessons learned from mouse models. Mutat Res

neuroendocrine tumor disease-the clinician-scientist perspective.

metalloproteinase-3 gene in pancreatic endocrine tumors. J Clin Endocrinol Metab

patients with neuroendocrine tumors of the pancreas and gut. Endocrine-related

the surrounding tissue as a prognostic classifier. J Natl Cancer Inst 2007;99:1257-

progression of gastroenteropancreatic neuroendocrine tumors (GEP-NETs).

inflammation and cytokine gene polymorphisms in the pathogenesis of gastrointestinal malignancy. Am J Physiol Gastrointest Liver Physiol

IL-6-174 C/G polymorphism in the gastroenteropancreatic neuroendocrine tumors


[4] Ardill JE. Circulating markers for endocrine tumors of the gastroenteropancreatic tract.

[5] Cigrovski Berkovic M, Jokic M, Zjacic-Rotkvic V, Kapitanovic S. The role of cytokines

[7] Zikusoka MN, Kidd M, Eick G, Latich I, Modlin IM. The molecular genetics of gastroenteropancreatic neuroendocrine tumors. Cancer 2005;04:2292-2309. [8] Perren A, Komminoth P, Heitz PU. Molecular genetics of gastroenteropancreatic

[9] Chan AO, Kim SG, Bedeir A, Issa JP, Hamilton SR, Rashid A 2003 CpG island methylation in carcinoid and pancreatic endocrine tumors. Oncogene 22:924-934. [10] Pannett AA, Thakker RV 2001 Somatic mutations in MEN type 1 tumors, consistent with the Knudson "two-hit" hypothesis. J Clin Endocrinol Metab 86:4371-4374. [11] Duerr E-M, Chung DC. Molecular Genetics of pancreatic neuroendocrine tumors. In: A

[12] Leotlela PD, Jauch A, Holtgrave-Grez H, Thakker RV. Genetics of neuroendocrine tumors and carcinoid tumors. Endocrine Related Cancer 2003;10:437-450. [13] Öberg K. Carcinoid tumors-current considerations. In: A century of advances in

[14] Speel EJ et al. Genetic evidence for early divergence of small functioning and

[15] Östman A. Tumor stroma-a perspective of therapeutic and prognostic opportunities. In:

[16] House MG et al. Aberrant hypermethylation of tumor suppresor genes in pancreatic

[17] Shimizu T et al. Growth characteristics of rectal carcinoid tumors. Oncology

[18] Terris B et al. Expression of vascular endothelial growth factor in digestive

[19] Conroy H, Mawhinney L, S. C. Donnelly SC. Inflammation and cancer: macrophage

[20] Wulbrand U, Wied M, Zofel P, Goke B, Arnold R, Fehmann HC. Growth factor receptor

[21] De Marzo AM et al. Inflammation in prostate carcinogenesis. Nat Rev Cancer

migration inhibitory factor (MIF)—the potential missing link. Q J Med 2010;

expression in human gastroenteropancreatic neuroendocrine tumors. European J of

(GEP-NETs): mini review. Periodicum Biologorum. 2007;109:111-114. [6] Delle Fave G, Corleto VD. Oncogenes, growth factors, receptor expression and

and their polymorphisms in the gastroenteropancreatic neuroendocrine tumors

proliferation markers in digestive neuroendocrine tumors. A critical reappraisal.

century of advances in neuroendocrine tumor biology and treatment. (Ed. Modlin

neuroendocrine tumor biology and treatment. (Ed. Modlin IM, Oberg K.),

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2007;7:256–269.

Clin Invest 1998;28:1038-1049.

endocrine tumors. Ann NY Acad Sci 2004;1014:199-208.


**2** 

*Italy* 

Angela Prestifilippo, Giusi Blanco, Maria Paola Vitale and Dario Giuffrida

*Istituto Oncologico del Mediterraneo, Viagrande - CT* 

**Chromogranin A and Neuroendocrine Tumors** 

Neuroendocrine tumors (NETs) are neoplasms that arise from cells of the endocrine and nervous systems. Many are benign, while some are cancers. They most commonly occur in

A neuroendocrine tumor is suspected when classical clinical symptoms occur but the large majority of NETs does not show any specific symptomatology (Oberg et al., 1999). Accordingly, the biochemical diagnosis is of great value, with the validation of radioimmunoenzymatic assays for various circulating peptide hormones in the last decade, clinical awareness and ability to diagnose NET is increased . However, due to the relative low incidence of NETs and the very large number of measurable hormones, clinicians need to know which measurable variables have an established clinical value and are cost effective

Flushing of the face and neck (appearance of deep red color, usually with sudden onset)

the intestine, but are also found in the lung and in the rest of the body.

Neuroendocrine tumors can be functional and nonfunctional.

Fig. 1. Neuroendocrine System GEP: Gastroenteropancreatic In the case of funtional NETs signs and symptoms include:

Diarrhea, nausea, vomiting, rapid heart rate

**1. Introduction** 

(Giuffrida et al., 2006)

[38] Berkovic M, Cacev T, Zjacic-Rotkvic V, Kapitanovic S. TNF-α promoter SNPs in gastroenteropancreatic neuroendocrine tumors (GEP-NET). Neuroendocrinology 2006;84:346-352.

## **Chromogranin A and Neuroendocrine Tumors**

Angela Prestifilippo, Giusi Blanco, Maria Paola Vitale and Dario Giuffrida *Istituto Oncologico del Mediterraneo, Viagrande - CT Italy* 

#### **1. Introduction**

10 Neuroendocrine Tumor

[38] Berkovic M, Cacev T, Zjacic-Rotkvic V, Kapitanovic S. TNF-α promoter SNPs in

2006;84:346-352.

gastroenteropancreatic neuroendocrine tumors (GEP-NET). Neuroendocrinology

Neuroendocrine tumors (NETs) are neoplasms that arise from cells of the endocrine and nervous systems. Many are benign, while some are cancers. They most commonly occur in the intestine, but are also found in the lung and in the rest of the body.

A neuroendocrine tumor is suspected when classical clinical symptoms occur but the large majority of NETs does not show any specific symptomatology (Oberg et al., 1999). Accordingly, the biochemical diagnosis is of great value, with the validation of radioimmunoenzymatic assays for various circulating peptide hormones in the last decade, clinical awareness and ability to diagnose NET is increased . However, due to the relative low incidence of NETs and the very large number of measurable hormones, clinicians need to know which measurable variables have an established clinical value and are cost effective (Giuffrida et al., 2006)

Neuroendocrine tumors can be functional and nonfunctional.

Fig. 1. Neuroendocrine System GEP: Gastroenteropancreatic

In the case of funtional NETs signs and symptoms include:


Chromogranin A and Neuroendocrine Tumors 13

Chromogranin A might promote the generation of secretory granules. Chromogranin A is the precursor to several functional peptides including vasostatin, pancreastatin, catestatin and parastatin. These peptides negatively modulate the neuroendocrine function of the releasing cell (autocrine) or nearby cells (paracrine). Other peptides derived from

Chromogranin A concentrations are normally low. An increased level in a symptomatic person may indicate the presence of a tumor but not what type it is or where it is. The quantity of CgA is not associated with the severity of the symptoms but with the mass and

The possibility to measure Chromogranin A (CgA) plasma levels by means of radio- or immunoenzymatic assay represents a tremendous step forward in the management of

Chromogranin can be dosed. The IRMA method is based on two monoclonal antibodies raised against the unprocessed central domain of the human CgA, allowing sensitive detection of total human CgA. Recombinant human CgA was used as calibrator and the standard curve concentration ranged from 22 to 1200 ng/ml, with a minimal detectable level of 10ng/ml. Inter-assay coefficients of variation were 3.4 and 4.5% at 124.7and 355.2 ng/ml, respectively. Intra-assay coefficients of variation were 5.1, 3.0, and 7.8% for the following

The ELISA assay is based on two polyclonal rabbit antibodies directed toward a 23 kDa carboxyl-terminal fragment of human CgA, therefore measuring more human CgA fragments . The calibrators were extracted from urine of patients with carcinoids and the standard curve concentraction ranged from 5 to 650 U/l, with a minimal detectable level of 5U/l. Inter-assay coefficients of variation were 3.4, 3.9 and 6.8 at 11.5, 52.7, and 358U/l, respectively. Intra-assay coefficients of variation were 4.5, 3.8 and 8.5% for the following ranges 5-10, 15-25 and 250-450 U/l, respectively (Zatelli et al., 2007). The three most commonly available employed assays for CgA measurement, has been compared in a group of NET patients and has been found that sensitivities vary between 67 and 93%, while specificities were 1 to 85% for all three (Stridsberg et al., 2003). A recent multicenter prospective comparison between two methods, immunoradiometric and ELISA, found a 36% clinical discordance rate. These results were mirrored with a difference of 5-fold interlaboratory variation rate in a recent Italian study aimed at assessing CgA detection performance as applied to immunoradiometric and ELISA assays (Janson et al., 1997). A further prospective analysis underlined CgA to be a practical marker in patients with NET, however, with limited diagnostic power. A cut-off of 53 ng/ml for IRMA and 16 U/l for ELISA for discriminating between healthy controls and NET patients yielded only moderate

sensitivities (71.3 and 83%, respectively) and specificities (71 and 85%, respectively).

The Chromogranin A test is used often as a tumor marker. It may be ordered in combination with or in place of 5-HIAA to help diagnose carcinoid tumors. It is also used to help monitor the effectiveness of treatment and detect recurrence of this tumor. Sometimes it may be ordered with specific hormones, such as catecholamines, to help diagnose and monitor a

chromogranin A with uncertain function include chromostatin, WE-14 and GE-25.

the functional activity of the tumor (Wu et al., 2000)

ranges 15-25, 90-110, and 500-700ng/ml, respectively.

patients with NETs.

**3.1 Chromogranin: Laboratory test** 

**3.2 Chromogranin related to net** 

Wheezing, coughing, difficulty breathing

### **2. Tumor markers**

Symptoms that are exhibited in the functional NETs is related to the release of circulating hormones and peptides such as catecholamines, insulin, 5-hydroxyindoleacetic acid (5- HIAA), gastrin, calcitonin and others. Although there are many kinds of NETs, they are treated as a group because the cells of these neoplasms share common features, such as looking similar, having special secretory granules, and often producing biogenic amines and polypeptide hormones 5-hydroxytryptamine (5-HT) or serotonin is product by functional neuroendocrine tumors (NETs) originating from the midgut. Serotonin is a tryptophanderived biogenic amine involved in smooth muscle contraction, blood pressure regulation and both peripheral and central nervous system neurotransmission. Approximately 2% of dietary tryptophan is converted into serotonin. Serotonin is synthesized and stored in enterochromaffin cells of the gastrointestinal tract (80% of total body serotonin content), in dense granules of platelets (storage only) and in the serotoninergic neurons of the central nervous system. The urinary breakdown metabolite of serotonin is 5-hydroxyindole acetic acid (5 - HIAA) which is particularly useful in the diagnosis and follow-up of NETs with carcinoid syndrome. Serum measurements of serotonin are possible in these patients; however, large individual variation makes them unreliable for diagnosis and in follow-up. Universally, 5-HIAA is the most frequently performed assay in the clinical setting of the carcinoid syndrome (O'Toole et al., 2009).

The generic markers of NETs are Neurone Specific Enolase (NSE) and Cromogranine A. Neurone-Specific Enolase is an useful immunohistochemical marker of NETs. Neverthelles ,its serum mesurament has not, except for patients with small cell lung cancer and neuroblastoma, because of relatively low sensitivity and specificity of the marker (Giovannella et al., 1999).

### **3. Chromogranin**

Chromogranin A is an acidic glycoprotein expressed in the secretory granules of most normal and neoplastic neuroendocrine cell types, where it is released togheter with peptide hormones and biogenic amines. In humans, chromogranin A protein is encoded by the CHGA gene (Helman et al., 1988)

Fig. 2. CHGA structure

The chromogranin family consists of at least three different watersoluble acidic glycoproteins (CgA, CgB, and secretogranin II, sometimes called chromogranin C). Upon stimulation, CgA and other peptide hormones and neuropeptides are released. CgA is also secreted from neuroendocrine derived tumors including foregut, midgut and hindgut gastrointestinal NETs, pheochromocytomas, neuroblastomas, medullary thyroid carcinomas, some pituitary tumors, functioning and non-functioning pancreatic NETs and other amine precursor uptake and decarboxylation tumors.

Symptoms that are exhibited in the functional NETs is related to the release of circulating hormones and peptides such as catecholamines, insulin, 5-hydroxyindoleacetic acid (5- HIAA), gastrin, calcitonin and others. Although there are many kinds of NETs, they are treated as a group because the cells of these neoplasms share common features, such as looking similar, having special secretory granules, and often producing biogenic amines and polypeptide hormones 5-hydroxytryptamine (5-HT) or serotonin is product by functional neuroendocrine tumors (NETs) originating from the midgut. Serotonin is a tryptophanderived biogenic amine involved in smooth muscle contraction, blood pressure regulation and both peripheral and central nervous system neurotransmission. Approximately 2% of dietary tryptophan is converted into serotonin. Serotonin is synthesized and stored in enterochromaffin cells of the gastrointestinal tract (80% of total body serotonin content), in dense granules of platelets (storage only) and in the serotoninergic neurons of the central nervous system. The urinary breakdown metabolite of serotonin is 5-hydroxyindole acetic acid (5 - HIAA) which is particularly useful in the diagnosis and follow-up of NETs with carcinoid syndrome. Serum measurements of serotonin are possible in these patients; however, large individual variation makes them unreliable for diagnosis and in follow-up. Universally, 5-HIAA is the most frequently performed assay in the clinical setting of the

The generic markers of NETs are Neurone Specific Enolase (NSE) and Cromogranine A. Neurone-Specific Enolase is an useful immunohistochemical marker of NETs. Neverthelles ,its serum mesurament has not, except for patients with small cell lung cancer and neuroblastoma, because of relatively low sensitivity and specificity of the marker

Chromogranin A is an acidic glycoprotein expressed in the secretory granules of most normal and neoplastic neuroendocrine cell types, where it is released togheter with peptide hormones and biogenic amines. In humans, chromogranin A protein is encoded by the

The chromogranin family consists of at least three different watersoluble acidic glycoproteins (CgA, CgB, and secretogranin II, sometimes called chromogranin C). Upon stimulation, CgA and other peptide hormones and neuropeptides are released. CgA is also secreted from neuroendocrine derived tumors including foregut, midgut and hindgut gastrointestinal NETs, pheochromocytomas, neuroblastomas, medullary thyroid carcinomas, some pituitary tumors, functioning and non-functioning pancreatic NETs and

other amine precursor uptake and decarboxylation tumors.

Wheezing, coughing, difficulty breathing

carcinoid syndrome (O'Toole et al., 2009).

(Giovannella et al., 1999).

CHGA gene (Helman et al., 1988)

**3. Chromogranin** 

Fig. 2. CHGA structure

**2. Tumor markers** 

Chromogranin A might promote the generation of secretory granules. Chromogranin A is the precursor to several functional peptides including vasostatin, pancreastatin, catestatin and parastatin. These peptides negatively modulate the neuroendocrine function of the releasing cell (autocrine) or nearby cells (paracrine). Other peptides derived from chromogranin A with uncertain function include chromostatin, WE-14 and GE-25.

Chromogranin A concentrations are normally low. An increased level in a symptomatic person may indicate the presence of a tumor but not what type it is or where it is. The quantity of CgA is not associated with the severity of the symptoms but with the mass and the functional activity of the tumor (Wu et al., 2000)

The possibility to measure Chromogranin A (CgA) plasma levels by means of radio- or immunoenzymatic assay represents a tremendous step forward in the management of patients with NETs.

#### **3.1 Chromogranin: Laboratory test**

Chromogranin can be dosed. The IRMA method is based on two monoclonal antibodies raised against the unprocessed central domain of the human CgA, allowing sensitive detection of total human CgA. Recombinant human CgA was used as calibrator and the standard curve concentration ranged from 22 to 1200 ng/ml, with a minimal detectable level of 10ng/ml. Inter-assay coefficients of variation were 3.4 and 4.5% at 124.7and 355.2 ng/ml, respectively. Intra-assay coefficients of variation were 5.1, 3.0, and 7.8% for the following ranges 15-25, 90-110, and 500-700ng/ml, respectively.

The ELISA assay is based on two polyclonal rabbit antibodies directed toward a 23 kDa carboxyl-terminal fragment of human CgA, therefore measuring more human CgA fragments . The calibrators were extracted from urine of patients with carcinoids and the standard curve concentraction ranged from 5 to 650 U/l, with a minimal detectable level of 5U/l. Inter-assay coefficients of variation were 3.4, 3.9 and 6.8 at 11.5, 52.7, and 358U/l, respectively. Intra-assay coefficients of variation were 4.5, 3.8 and 8.5% for the following ranges 5-10, 15-25 and 250-450 U/l, respectively (Zatelli et al., 2007). The three most commonly available employed assays for CgA measurement, has been compared in a group of NET patients and has been found that sensitivities vary between 67 and 93%, while specificities were 1 to 85% for all three (Stridsberg et al., 2003). A recent multicenter prospective comparison between two methods, immunoradiometric and ELISA, found a 36% clinical discordance rate. These results were mirrored with a difference of 5-fold interlaboratory variation rate in a recent Italian study aimed at assessing CgA detection performance as applied to immunoradiometric and ELISA assays (Janson et al., 1997). A further prospective analysis underlined CgA to be a practical marker in patients with NET, however, with limited diagnostic power. A cut-off of 53 ng/ml for IRMA and 16 U/l for ELISA for discriminating between healthy controls and NET patients yielded only moderate sensitivities (71.3 and 83%, respectively) and specificities (71 and 85%, respectively).

#### **3.2 Chromogranin related to net**

The Chromogranin A test is used often as a tumor marker. It may be ordered in combination with or in place of 5-HIAA to help diagnose carcinoid tumors. It is also used to help monitor the effectiveness of treatment and detect recurrence of this tumor. Sometimes it may be ordered with specific hormones, such as catecholamines, to help diagnose and monitor a

Chromogranin A and Neuroendocrine Tumors 15

metastatic disease, respectively. Nonetheless, the prognostic value of CgA in patients with

Chronic elevation of gastrin levels provokes hyperplasia of the neuroendocrine cells of the stomach, and these cells are able to secrete CgA (D'Adda et al., 1990) . In patients with chronically elevated CgA and Zollinger Ellison Syndrome (ZES), has been demonstrated that the CgA concentrations can be normalized by gastrectomy alone, without resection of the gastrin producing tumor. A more recently described case report of false-positive CgA was due to the presence of heterophile antibodies (HAb), which can bind to animal antigens and may be present in up to 40% of the normal population (Levinson et al., 2007); in the CgA immunometric assays, HAb interferences may be circumnavigated by using a

CgA laboratory tests that have been developed and validated will all be slightly different, and their results will not be interchangeable. For this reason, if someone is having more than one CgA test performed (such as for monitoring), all test are sent to the same laboratory. The very frequent elevation of CgA in patients with pheochromocytomas/paragangliomas confirms that it may be the marker of choice for these diseases, being more convenient than

The highest CgA levels were noted in patients with metastatic carcinoid tumors and neuroendocrine carcinomas of gastrointestinal origin. Conversely, the lowest values were found in patients with advanced SCLC. Some data support the notion that CgA is less useful

It is noteworthy that elevated plasma CgA levels cannot differentiate between neuroendocrine and non neuroendocrine neoplasms. Slightly elevated CgA levels, in fact, were identified in more than 40% of patients with advanced non-endocrine tumors, a proportion that was not so different from that of patients with SCLC (Nobels, 1997, Stivanello, 2001). The detection of elevated plasma CgA in non-endocrine tumors mainly indicates that there is a neuroendocrine differentiation and a proliferation of neuroendocrine

Drugs that stimulate secretion of neuroendocrine cells can lead to artifactual chromogranin A elevations. In particular, proton pump inhibitors (e.g., omeprazole), which are used in the treatment of esophageal and gastroduodenal ulcer disease and dyspepsia, will result in significant elevations of serum chromogranin A levels, often to many times above the normal range. If medically feasible, proton pump inhibitor therapy should be discontinued

Chromogranin A and its peptide fragments are cleared by a combination of hepatic metabolism and renal excretion. In patients with significant impairment of liver or kidney function, serum chromogranin levels are often substantially elevated and single

in undifferentiated neuroendocrine neoplasms (Blanco, 2007; Stivanello, 2011).

False-positive elevation of CgA may occur in the following circumstances:


catecholamines either measured in plasma or in urine.

cells at advanced stage of many carcinomas.

drug week of serum chromogranin A levels.

NET has not been confirmed to date.




Habblocking tube.

pheochromocytoma. It may also be used to detect the presence of other neuroendocrine tumors, even those that do not secrete hormones . Plasma CgA levels (2-18 u/l) were found elevated in a variety of NETs, including pheocromocytoma, carcinoid tumors, pancreatic islet cell tumors, medullary carcinoma of the thyroid, small-cell lung cancer and so forth (Verderio et al., 2007).

#### **Positive Cromogranin A related to Neuroendocrine Tumors:**


Table 1. CgA and Neuroendocrine Tumors

The sensitivity and specificity of circulating CgA in any NETs vary between 70% and 95%. The highest accuracy has been observed in tumors characterized by an intense secretory activity, but its specificity and sensitivity remain very high also in non-functioning tumors.

Although CgA specificity cannot compete with that of the specific hormonal products secreted by many NETs, this molecule has very useful clinical applications in subjects with NETs for whom either no marker is available or the marker is inconvenient for routine clinical use generally, if concentrations of CgA are elevated prior the treatment and then fall, the treatment is likely to have been effective. CgA concentrations may be elevated but not monitored with conditions, such as liver disease, inflammatory bowel disease, renal insufficiency, and with stress. These possible causes for elevated CgA levels should be considered when interpreting test results, as false positive.

Overall CgA has been found to be clinically informative and moderately sensitive in the majority of studies devoted to this topic. CgA was found of a large mixed NET patient cohort, CgA was more sensitive than neurone-specific enolase (Baudin et al., 1998) . While performances have been limited in low-level cut-offs due to the overlap with control populations, very high levels of serum CgA are rarely found outside the setting of NETs with the exception of patients on gastric acid secretory blockers, especially PPIs (Sanduleanu et al., 2001) or those with hypergastrinaemia. Specificity of CgA in the diagnosis of NETs depends on the tumor type and burden (100% specificities have been reported in patients with metastatic disease ), the quality of the control populations used and the cut-off values employed. Elevated CgA was found to be more sensitive than high urinary 5- HIAA levels in patients with metastatic midgut lesions (87 vs. 76%, respectively). A significant positive relation between the serum levels of CgA and the tumor mass in NETs, has been demonstrated; however, the distinction between high and low tumor volume may be open to question, infact, high CgA concentrations were found in all patients with gastrinoma, although tumor was small in volume (Nobels et al., 1997). In a mixed series of 128 patients with NET, increased CgA levels were found in 29% and 67% of patients with locoregional or metastatic disease, respectively. Nonetheless, the prognostic value of CgA in patients with NET has not been confirmed to date.

False-positive elevation of CgA may occur in the following circumstances:


14 Neuroendocrine Tumor

pheochromocytoma. It may also be used to detect the presence of other neuroendocrine tumors, even those that do not secrete hormones . Plasma CgA levels (2-18 u/l) were found elevated in a variety of NETs, including pheocromocytoma, carcinoid tumors, pancreatic islet cell tumors, medullary carcinoma of the thyroid, small-cell lung cancer and so forth

The sensitivity and specificity of circulating CgA in any NETs vary between 70% and 95%. The highest accuracy has been observed in tumors characterized by an intense secretory activity, but its specificity and sensitivity remain very high also in non-functioning tumors. Although CgA specificity cannot compete with that of the specific hormonal products secreted by many NETs, this molecule has very useful clinical applications in subjects with NETs for whom either no marker is available or the marker is inconvenient for routine clinical use generally, if concentrations of CgA are elevated prior the treatment and then fall, the treatment is likely to have been effective. CgA concentrations may be elevated but not monitored with conditions, such as liver disease, inflammatory bowel disease, renal insufficiency, and with stress. These possible causes for elevated CgA levels should be

Overall CgA has been found to be clinically informative and moderately sensitive in the majority of studies devoted to this topic. CgA was found of a large mixed NET patient cohort, CgA was more sensitive than neurone-specific enolase (Baudin et al., 1998) . While performances have been limited in low-level cut-offs due to the overlap with control populations, very high levels of serum CgA are rarely found outside the setting of NETs with the exception of patients on gastric acid secretory blockers, especially PPIs (Sanduleanu et al., 2001) or those with hypergastrinaemia. Specificity of CgA in the diagnosis of NETs depends on the tumor type and burden (100% specificities have been reported in patients with metastatic disease ), the quality of the control populations used and the cut-off values employed. Elevated CgA was found to be more sensitive than high urinary 5- HIAA levels in patients with metastatic midgut lesions (87 vs. 76%, respectively). A significant positive relation between the serum levels of CgA and the tumor mass in NETs, has been demonstrated; however, the distinction between high and low tumor volume may be open to question, infact, high CgA concentrations were found in all patients with gastrinoma, although tumor was small in volume (Nobels et al., 1997). In a mixed series of 128 patients with NET, increased CgA levels were found in 29% and 67% of patients with locoregional or

**Positive Cromogranin A related to Neuroendocrine Tumors:** 

Ectopic Adrenocorticotropic Hormone Producing Tumors

considered when interpreting test results, as false positive.

(Verderio et al., 2007).

Merkel Cell Tumor

 Pheocromocytoma Small Cell Lung Cancer

Prostate Cancer

 Gastroenteropancreatic NETs Anterior Pituitary tumors Parathyroid tumors

Medullary Thyroid Carcinoma

Ganglioneuroma / Neuroblastoma

Table 1. CgA and Neuroendocrine Tumors


Chronic elevation of gastrin levels provokes hyperplasia of the neuroendocrine cells of the stomach, and these cells are able to secrete CgA (D'Adda et al., 1990) . In patients with chronically elevated CgA and Zollinger Ellison Syndrome (ZES), has been demonstrated that the CgA concentrations can be normalized by gastrectomy alone, without resection of the gastrin producing tumor. A more recently described case report of false-positive CgA was due to the presence of heterophile antibodies (HAb), which can bind to animal antigens and may be present in up to 40% of the normal population (Levinson et al., 2007); in the CgA immunometric assays, HAb interferences may be circumnavigated by using a Habblocking tube.

CgA laboratory tests that have been developed and validated will all be slightly different, and their results will not be interchangeable. For this reason, if someone is having more than one CgA test performed (such as for monitoring), all test are sent to the same laboratory.

The very frequent elevation of CgA in patients with pheochromocytomas/paragangliomas confirms that it may be the marker of choice for these diseases, being more convenient than catecholamines either measured in plasma or in urine.

The highest CgA levels were noted in patients with metastatic carcinoid tumors and neuroendocrine carcinomas of gastrointestinal origin. Conversely, the lowest values were found in patients with advanced SCLC. Some data support the notion that CgA is less useful in undifferentiated neuroendocrine neoplasms (Blanco, 2007; Stivanello, 2011).

It is noteworthy that elevated plasma CgA levels cannot differentiate between neuroendocrine and non neuroendocrine neoplasms. Slightly elevated CgA levels, in fact, were identified in more than 40% of patients with advanced non-endocrine tumors, a proportion that was not so different from that of patients with SCLC (Nobels, 1997, Stivanello, 2001). The detection of elevated plasma CgA in non-endocrine tumors mainly indicates that there is a neuroendocrine differentiation and a proliferation of neuroendocrine cells at advanced stage of many carcinomas.

Drugs that stimulate secretion of neuroendocrine cells can lead to artifactual chromogranin A elevations. In particular, proton pump inhibitors (e.g., omeprazole), which are used in the treatment of esophageal and gastroduodenal ulcer disease and dyspepsia, will result in significant elevations of serum chromogranin A levels, often to many times above the normal range. If medically feasible, proton pump inhibitor therapy should be discontinued drug week of serum chromogranin A levels.

Chromogranin A and its peptide fragments are cleared by a combination of hepatic metabolism and renal excretion. In patients with significant impairment of liver or kidney function, serum chromogranin levels are often substantially elevated and single

Chromogranin A and Neuroendocrine Tumors 17

tumor location and functional status. In gastrointestinal neuroendocrine tumours the measurement of general and specific markers offers important information for the clinician treating patients. This information is useful for the initial diagnosis and during the followup for monitoring patients with non functional disease and under medical treatment. Several of the markers are good prognostic markers for both carcinoid and pancreatic

This marker seems to be less useful in undifferentiated tumors such as Small Cell Lung Cancer. Elevated CgA plasma levels allow the identification of the coexistence of neuroendocrine differentiation in the context of non-endocrine malignancies and this could have diagnostic, prognostic, and possibly therapeutic implications. A dynamic evaluation of this marker in the follow-up of NETs provides useful information on the disease recurrence in NED cases or on the treatment efficacy in advanced cases submitted to cytotoxic or

Elevated CgA can occur in normal individuals and in patients with non-NET tumors

CgA is the most practical and useful general serum tumor marker in patients with NET

Reference intervals and individual patient results differ significantly between different

 False-positive results are possible in patients with hypergastrinaemia (especially on anti- secretory medications or chronic atrophic gastritis type A) and in the presence of heterophile antibodies (care in patients autoimmune disease or those sensitized to

Where possible, proton pump inhibitors should be interrupted, leaving a clearance of at

Ardill, J.E.S. & Erikkson,B (2003). The importance of the measurement of circulating markers

Arnaldi, G., Cardinaletti, M. & Polenta, B. (2007). Biological markers of neuroendocrine tumors: false positives and negatives , Rivista Medica, Vol .13, No.2, pp.15-21 Baudin, E., Gigliotti, A. & Ducreux, M. (1998). Neuron-specific enolase and chromogranin A asmarkers of neuroendocrine tumours, *British Journal of cancer*, Vol .78, pp.1102–1107 Blanco,G., Martino, M., & Giuffrida, D. (2007). Clinical and therapeutical approach in poorly differentiated neuroendocrine tumors, *Rivista Medica,* Vol.13, No. 2,pp. 51-54 Campana, D., Nori, F. ,& Tomassetti, P. (2007). Chromogranin A: Is It a Useful Marker of Neuroendocrine Tumors, *Journal of Clinical Oncology,* Vol.25, No. 15, pp.1967-1973 D'Adda, T. Corleto,V. & Pilato, FP. (1990). Quantitative ultrastructure of endocrine cells of

in patients with neuroendocrine tumours of the pancreas and gut, *Endocrine-Related* 

oxyntic mucosa in Zollinger-Ellison syndrome. Correspondence with light

microscopic findings, *Gastroenterology,* Vol.99, pp. 17–26

although the levels are usually lower than in patients with NET

chromogranin A assays and cannot be directly compared Serial measurements should be performed using the same assay

rodent proteins (mouse monoclonal antibodies))

least 3 half-lives, prior to CgA plasma sampling.

*Cancer,* Vol. 10, pp.459-642

 Sensitivity of elevated CgA varies according to NET tumor type and volume Reference laboratories should be preferred for clinical samples assays

If assays are changed, patients should undergo a new baseline measurement

disease (Ardill & Erikkson , 2003).

biologic therapy (Zatelli et al., 2007) **CgA: General Remarks and Assays** 

**5. References** 

chromogranin A measurements are uninterpretable. Serial measurements may have some value in selected patients if the disturbance in hepatic or renal function remains stable, but results must be interpreted with extreme caution. There is no universal calibration standard for serum chromogranin A assays. In addition, different chromogranin A assays, which use different antibodies or antibody combinations, will display different cross-reactivity with the various chromogranin A fragments. Therefore, reference intervals and individual patient results differ significantly between different chromogranin A assays and cannot be directly compared. Serial measurements should be performed with the same assay, or, if assays are changed, patients should be rebaselined. As with all immunometric assays, there is a low but definite possibility of false-positive results in patients with heterophile antibodies.

These antibodies are rarely found in the normal population, but are observed at increased rates in persons with autoimmune disease or after prior sensitization to rodent proteins (patients who have received diagnostic or therapeutic mouse monoclonal antibodies). Blocking reagents have been added to this assay to minimize the likelihood of heterophile antibody interference. However, test results that do not fit the clinical picture should always be discussed with the laboratory.

A "hook effect" can occur at extremely high chromogranin A concentrations, resulting in a lower measured chromogranin A concentration than is actually contained in the specimen. This is not expected to impact the utility of the assay for initial diagnosis, as levels will typically remain significantly above the reference range, even in the presence of hooking. However, hooking may complicate the interpretation of serial chromogranin A measurements in rare patients with extremely high levels. Normally it would be useful to dose dilute and remeasure all specimens >625 ng/mL to minimize the risk of this occurring. However, if there is the clinical suspicion of hooking, then retesting after further specimen dilution should be requested.

There are some pitfalls in the interpretation of CgA levels. Among them, renal impairment is one of the most important. All the patients with chronic renal failure presented very high levels of CgA, thus suggesting that serum creatinine should always be measured concomitantly with plasma CgA (Stridsberg et al., 2003)

Circulating CgA was found to be a reliable marker for the follow-up of patients with neuroendocrine tumors. CgA levels were with not evident disease (NED) , CgA levels were within normality. In advanced cases submitted to systemic treatment, a clear relationship was found between changes in CgA levels and disease response. This marker decreased in all patients showing a tumour shrinkage after cytotoxic treatment, increased in the great majority of patients showing progressive disease, and did not change in most cases depicting a disease stabilization. Discrepancies between tumor and biochemical changes in non-responding patients are attributable to the concomitant administration of somatostatin analog (Campana et al., 2007)

The correlation between CgA levels and tumor mass is lost during treatment with somatostatin so that CgA may not be used as a marker of tumor response when a cytotoxic regimen is administered in combination with a somatostatin analog (O'Toole et al., 2009).

#### **4. Conclusion**

Many data confirm the general view that CgA is the best circulating neuroendocrine marker available up to now. Its clinical application involves all differentiated NETs, irrespective of

chromogranin A measurements are uninterpretable. Serial measurements may have some value in selected patients if the disturbance in hepatic or renal function remains stable, but results must be interpreted with extreme caution. There is no universal calibration standard for serum chromogranin A assays. In addition, different chromogranin A assays, which use different antibodies or antibody combinations, will display different cross-reactivity with the various chromogranin A fragments. Therefore, reference intervals and individual patient results differ significantly between different chromogranin A assays and cannot be directly compared. Serial measurements should be performed with the same assay, or, if assays are changed, patients should be rebaselined. As with all immunometric assays, there is a low but definite possibility of false-positive results in patients with heterophile antibodies.

These antibodies are rarely found in the normal population, but are observed at increased rates in persons with autoimmune disease or after prior sensitization to rodent proteins (patients who have received diagnostic or therapeutic mouse monoclonal antibodies). Blocking reagents have been added to this assay to minimize the likelihood of heterophile antibody interference. However, test results that do not fit the clinical picture should always

A "hook effect" can occur at extremely high chromogranin A concentrations, resulting in a lower measured chromogranin A concentration than is actually contained in the specimen. This is not expected to impact the utility of the assay for initial diagnosis, as levels will typically remain significantly above the reference range, even in the presence of hooking. However, hooking may complicate the interpretation of serial chromogranin A measurements in rare patients with extremely high levels. Normally it would be useful to dose dilute and remeasure all specimens >625 ng/mL to minimize the risk of this occurring. However, if there is the clinical suspicion of hooking, then retesting after further specimen

There are some pitfalls in the interpretation of CgA levels. Among them, renal impairment is one of the most important. All the patients with chronic renal failure presented very high levels of CgA, thus suggesting that serum creatinine should always be measured

Circulating CgA was found to be a reliable marker for the follow-up of patients with neuroendocrine tumors. CgA levels were with not evident disease (NED) , CgA levels were within normality. In advanced cases submitted to systemic treatment, a clear relationship was found between changes in CgA levels and disease response. This marker decreased in all patients showing a tumour shrinkage after cytotoxic treatment, increased in the great majority of patients showing progressive disease, and did not change in most cases depicting a disease stabilization. Discrepancies between tumor and biochemical changes in non-responding patients are attributable to the concomitant administration of somatostatin

The correlation between CgA levels and tumor mass is lost during treatment with somatostatin so that CgA may not be used as a marker of tumor response when a cytotoxic regimen is administered in combination with a somatostatin analog (O'Toole et al., 2009).

Many data confirm the general view that CgA is the best circulating neuroendocrine marker available up to now. Its clinical application involves all differentiated NETs, irrespective of

be discussed with the laboratory.

dilution should be requested.

analog (Campana et al., 2007)

**4. Conclusion** 

concomitantly with plasma CgA (Stridsberg et al., 2003)

tumor location and functional status. In gastrointestinal neuroendocrine tumours the measurement of general and specific markers offers important information for the clinician treating patients. This information is useful for the initial diagnosis and during the followup for monitoring patients with non functional disease and under medical treatment. Several of the markers are good prognostic markers for both carcinoid and pancreatic disease (Ardill & Erikkson , 2003).

This marker seems to be less useful in undifferentiated tumors such as Small Cell Lung Cancer. Elevated CgA plasma levels allow the identification of the coexistence of neuroendocrine differentiation in the context of non-endocrine malignancies and this could have diagnostic, prognostic, and possibly therapeutic implications. A dynamic evaluation of this marker in the follow-up of NETs provides useful information on the disease recurrence in NED cases or on the treatment efficacy in advanced cases submitted to cytotoxic or biologic therapy (Zatelli et al., 2007)

#### **CgA: General Remarks and Assays**


#### **5. References**


**3** 

*Italy* 

**Circulating Markers in** 

*1Gastroenterology Unit II,* 

**Gastroenteropancreatic** 

Sara Massironi1, Matilde Pia Spampatti1,2,

Federica Cavalcoli1 and Maddalena Peracchi2

**Neuroendocrine Tumors (GEP NETs)** 

Roberta Elisa Rossi1,2, Dario Conte1,2, Clorinda Ciafardini1,2,

 *Fondazione IRCCS Ca' Granda – Ospedale Maggiore Policlinico, Milano, 2Postgraduate School of Gastroenterology, Università degli Studi di Milano,* 

Neuroendocrine Tumors (NETs) constitute a heterogeneous group of neoplasms which originate from neuroendocrine cells of diffuse endocrine system. They may synthesize, store, and secrete peptides and neuroamines that can cause distinct clinical syndromes. On

Gastro-Entero-Pancreatic (GEP) NETs originate from both pancreatic islet cells or gastroenteric tissue (from diffuse neuroendocrine cells distributed throughout the gut) and are rare neoplasms, representing about 2% of all the gastrointestinal tumors. Due to their rarity, they are difficult to diagnose and the begnning of the diagnostic process is often based on the measurement of circulating markers, before planning expensive and invasive diagnostic tests (2, 3). A critical point is that the frequent late diagnosis of NETs is due to failure to identify symptoms or to establish the biochemical diagnosis; in fact 60-80% of NETs are metastatic at diagnosis. A prompt identification by the use of specific biomarkers

Circulating tumor biomarkers can be divided into general and specific biomarkers. The neuroendocrine cells that give rise to NETs have many common features, including the synthesis of peptides, biologically inactive, that act as general markers, but have also the capacity to secrete a variety of specific biomarkers that characterize a precise biochemical function (4). Individual amines and peptide hormones are indeed specific to certain types of

There are several families of secretory proteins found in high concentrations

in neuroendocrine cells and, in particular, neuroendocrine tumor cells.

the other hand, many are clinically silent until late presentation with mass effects (1).

**1. Introduction** 

NETs (**Table 1**).

**2. General biomarkers** 

is therefore useful to recognize these tumors (1).


## **Circulating Markers in Gastroenteropancreatic Neuroendocrine Tumors (GEP NETs)**

 Sara Massironi1, Matilde Pia Spampatti1,2, Roberta Elisa Rossi1,2, Dario Conte1,2, Clorinda Ciafardini1,2, Federica Cavalcoli1 and Maddalena Peracchi2 *1Gastroenterology Unit II, Fondazione IRCCS Ca' Granda – Ospedale Maggiore Policlinico, Milano, 2Postgraduate School of Gastroenterology, Università degli Studi di Milano, Italy* 

#### **1. Introduction**

18 Neuroendocrine Tumor

Giovanella, L., La Rosa, S. & Garancini, S.(1999). Chromogranin-A as a serum marker for

Giuffrida, D., Blanco,G. & Mare, M. (2006).A Clinical Approach to Neuroendocrine Tumors,

Helman, LJ., Ahn, TG. & Israel, MA. (1988). Molecular cloning and primary structure of

Hsiao, RJ., Mezger, MS. & O'Connor, DT. (1990). Chromogranin A in uremia: progressive retention of immunoreactive fragments, *Kidney International,* Vol.37, pp. 955–964 Janson, ET., Holmberg, L.& Stridsberg, M., (1997). Carcinoid tumors: analysis of prognostic

Levinson, SS. & Miller, JJ. (2002). Towards a better understanding of heterophile (and the

Nobels, FR., Kwekkeboom, DJ. & Coopmans, W. (1997). Chromogranin A as serum marker

Oberg, K., Janson, ET. & Eriksson ,B. (1999) Tumour markers in neuroendocrine tumours, *Italian Journal Gastroenterology and Hepatology,* Vol.*3*1, pp.160-162 (suppl 2) O' Toole, D., Grossman, A. & al other Mallorca Consensus Conference Partecipans (2009)

Sanduleanu, S., De Bruïne,A .& Stockbrügger, RW.(2001). Serum chromogranin A as a

Stivanello, M., Berruti, A. & An & Dogliotti, L. (2001). Circulating chromogranin A in the

Stridsberg, M., Husebye, ES. (1997). Chromogranin A and chromogranin B are sensitive

Stridsberg, M., Eriksson, B. & Janson, ET. (2003). A comparison between three commercial

Verderio, P. Dittadi, R. & Marubini, E. (2007). An Italian program of external quality control

Wu, JT., Erickson, AJ. & Sun, CF. (2000). Elevated serum chromogranin A is detectable in

Zatelli, M.C., Torta, M . & On behalf of the Italian CromoNet Working Group (2007).

*Clinical Chemistry and Laboratory Medicine ,* Vol. 45, pp. 1244–1250

markers, *Neuroendocrinology*, Vol. 90, pp. 194-202

*laboratory Science,* Vol. 30, No.2, pp. 175–8

Study,*Endocrine-Related Cancer,* Vol. 14, pp.473.482

experience, *Annals of Oncology* Vol. 12, supp l.2, pp.73-77

*Supportive and Palliative Cancer Care,* Vol.2, No. 2, pp.17-19

Vol.14, pp. 160–166

pp. 685–690

Vol.263, No. 23, pp. 11559–63

*Elsevier,* Vol. 325, pp. 1–15

Vol.82, pp. 2622–2628

36, pp. 67–73

neuroendocrine tumors: comparison with neuron-specific enolase and correlation with immunohistochemical findings, *The international Journal of Biological Markers*,

human chromogranin A (secretory protein I) cDNA, *Journal of Biological Chemistry*

factors and survival in 301 patients from a referral center, *Annals of Oncology,* Vol. 8,

like) antibody interference with modern immunoassays, *Clinica Chimica Acta* 

for neuroendocrine neoplasia: comparison with neuron-specific enolase and the subunit of glycoprotein hormones, *Journal Clinical Endocrinology and metabolism*,

ENETS Guidelines for the standards of care in neuroendocrine tumors biochemical

screening test for gastric enterochromaffin-like cell hyperplasia during acidsuppressive therapy*,European Journal Clinical of Investigation,* Vol. 31, No.9, pp. 802-11

assessment of patients with neuroendocrine tumours. A single institution

circulating markers for phaeochromocytoma, *European Journal of endocrinology,* Vol.

kits for chromogranin A measurements*,Journal Endocrinology,* Vol. 177, pp. 337–341

for chromogranin A (CgA) assay: performance evaluation of CgA determination,

patients with carcinomas at advanced disease stages*, Annals of Clinical and* 

Chromogranin A as a marker of neuroendocrine neoplasia: an Italian Multicenter

Neuroendocrine Tumors (NETs) constitute a heterogeneous group of neoplasms which originate from neuroendocrine cells of diffuse endocrine system. They may synthesize, store, and secrete peptides and neuroamines that can cause distinct clinical syndromes. On the other hand, many are clinically silent until late presentation with mass effects (1).

Gastro-Entero-Pancreatic (GEP) NETs originate from both pancreatic islet cells or gastroenteric tissue (from diffuse neuroendocrine cells distributed throughout the gut) and are rare neoplasms, representing about 2% of all the gastrointestinal tumors. Due to their rarity, they are difficult to diagnose and the begnning of the diagnostic process is often based on the measurement of circulating markers, before planning expensive and invasive diagnostic tests (2, 3). A critical point is that the frequent late diagnosis of NETs is due to failure to identify symptoms or to establish the biochemical diagnosis; in fact 60-80% of NETs are metastatic at diagnosis. A prompt identification by the use of specific biomarkers is therefore useful to recognize these tumors (1).

Circulating tumor biomarkers can be divided into general and specific biomarkers. The neuroendocrine cells that give rise to NETs have many common features, including the synthesis of peptides, biologically inactive, that act as general markers, but have also the capacity to secrete a variety of specific biomarkers that characterize a precise biochemical function (4). Individual amines and peptide hormones are indeed specific to certain types of NETs (**Table 1**).

#### **2. General biomarkers**

There are several families of secretory proteins found in high concentrations in neuroendocrine cells and, in particular, neuroendocrine tumor cells.

Circulating Markers in Gastroenteropancreatic Endocrine Tumors (GEP NETs) 21

Chromogranin A (CgA) has been claimed to be the best general neuroendocrine marker so far available. CgA is a 49 kDa monomeric, hydrophilic, acidic glycoprotein of 460 amino acid and is widely expressed in neuroendocrine cells, where it constitutes one of the most abundant components of secretory granules, and it is secreted from neuroendocrinederived tumors including functioning and non-functioning GEP NETs, pheochromocytomas, neuroblastomas, medullary thyroid carcinomas and some pituitary tumors. CgA is secreted to the extracellular space, so it's easily detectable in the blood. CgA is co-secreted with the amines and peptides that are present in the neurosecretory granules even if it can be elevated in both functionally active and non-functional NETs. CgA seems to be a "common denominator" peptide in all the components of the diffuse

The precise function of CgA remains unknown, but it is thought to be involved in the packaging and processing of neuropeptide precursor and peptide hormones. It may also play a role in the organization of the secretory granule matrix. Moreover CgA has diverse physiological interactions: CgA (or its derivatives) is an inhibitor of catecholamine, insulin, and leptin, having a role in carbohydrate and lipid metabolism; moreover it inhibits parathormone secretion; on the other hand, CgA increases glucagon and amylase release. In addition to its effects on endocrine organs, CgA also regulates reproductive functions and has a role also in the regulation of cardiovascular function: CgA elevations have been reported in essential hypertension (CgA levels correlates with the severity of hypertension) and in chronic heart failure correlating with grade of cardiac dysfunction and mortality. A role of CgA in the regulation of inflammatory response has also been described. In fact increased CgA levels correlate with serum TNF-α receptor levels in a number of inflammatory diseases including rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, chronic obstructive pulmonary disease and chronic heart failure. Patients with sepsis show the highest increase of CgA; CgA positively correlates with inflammatory markers as C-reactive protein and procalcitonin. It remains to be elucidated the pathophysiological relevance of these correlations. Some authors suggest that CgA participates in a negative feedback that limits the activation of endothelial cells (1).

Circulating CgA concentrations are sensitive even if non specific markers of NETs. CgA has been reported to be more sensitive than urinary 5-hydroxyindoleacetic acid (5-HIAA) as well as than pancreatic polypeptide concentrations. The highest values were noted in ileal NETs (200 times the upper normal limit) and IN GEP-NETs associated with MEN1 (150 times the upper normal limit) while gastric type I, pituitary, and parathyroid tumors had lower values (ranging from 2 to 4 times normal). Both functioning and nonfunctioning pancreatic NETs had intermediate values (60–80 times the upper limit of normal) as did Zollinger- Ellison Syndrome (ZES), multiple endocrine neoplasia (MEN)-1, type II and III gastric entero-chromaffin-like (ECL)omas (80–100 times normal). It has also been proposed that CgA is more frequently elevated in well-differentiated tumors compared to poorly differentiated tumors of the midgut, suggesting that the loss of CgA expression in poorly differentiated neuroendocrine carcinomas indicates their incomplete or partial endocrine differentiation. In fact, poorly differentiated NE carcinomas rarely express CgA because of the rarity of large, dense-core granules. The presence of high plasma levels of CgA at diagnosis is an independent prognostic factor that indicates a reduced overall survival.

**2.1.1 Chromogranin A (CgA)** 

neuroendocrine system (7).

They include the granins, neuron specific enolase (NSE), and pancreatic polypeptide (PP). Both chromogranin A (CgA) and NSE show increased concentration levels in many patients with NETs, but CgA is recognized as the most effective and the only general biomarker that has been extensively investigated (1-5).


CgA=Chromogranin A; 5-HIAA= 5-hydroxyindoleacetic acid; PP= Pancreatic polypeptide; VIP= vasointestinal peptide; NKA= neurokinin A; PYY= peptide YY

Table 1. Syndromes, symptoms and secretory products from GEP NETs

#### **2.1 Granins**

The chromogranin family consists of at least three different water soluble acidic glycoproteins (CgA, CgB, and secretogranin II, sometimes called chromogranin C). These proteins are 27 to 100 kDa in size and contain 10% acidic (glutamic or aspartic acid) residues, as well as multiple single and dibasic amino acid residues. All of the granins are found as major components of the soluble core of dense-core secretory granules in NE cells and are secreted from these cells in a physiologically regulated manner. Granins are major constituents of large dense-core secretory vesicles and are co-secreted with peptide hormones and amines. Electron dense or translucent secretory granules are in fact prototypical features of the neuroendocrine cells (1-6).

#### **2.1.1 Chromogranin A (CgA)**

20 Neuroendocrine Tumor

They include the granins, neuron specific enolase (NSE), and pancreatic polypeptide (PP). Both chromogranin A (CgA) and NSE show increased concentration levels in many patients with NETs, but CgA is recognized as the most effective and the only general biomarker that

diarrhea, GERD

Ileal Carcinoid Diarrhea, flushing, sweating CgA, serotonin, NKA

Appendix Carcinoid Diarrhea, flushing, sweating CgA, serotonin, HIAA,

Rectal None None CgA, PYY(10%)

diarrhea

sweating

diarrhea

erythema

Somatostatinoma None Mild diabetes, gallstones CgA, somatostatin

CgA=Chromogranin A; 5-HIAA= 5-hydroxyindoleacetic acid; PP= Pancreatic polypeptide; VIP=

The chromogranin family consists of at least three different water soluble acidic glycoproteins (CgA, CgB, and secretogranin II, sometimes called chromogranin C). These proteins are 27 to 100 kDa in size and contain 10% acidic (glutamic or aspartic acid) residues, as well as multiple single and dibasic amino acid residues. All of the granins are found as major components of the soluble core of dense-core secretory granules in NE cells and are secreted from these cells in a physiologically regulated manner. Granins are major constituents of large dense-core secretory vesicles and are co-secreted with peptide hormones and amines. Electron dense or translucent secretory granules are in fact

VIP-oma WDHA Watery diarrhea CgA, VIP

 PP-oma None None CgA, PP Non functioning None None CgA, PP

Table 1. Syndromes, symptoms and secretory products from GEP NETs

Epigastric pain, peptic ulcer,

Epigastric pain, peptic ulcer,

Hypoglycemia, dizziness,

Epigastric pain, peptic ulcer,

CgA, gastrin (>50%), PP

Somatostatin (<10%)

CgA, gastrin (>50%)

CgA, insulin, proinsulin, C-peptide

CgA, glucagon, glycentin

CgA, gastrin, PP (35%)

and SP, 5HIAA

(35%),

NKA

**Tumor Site Syndrome Symptoms Biomarkers**  Gastric (type 1 and 2) None Upper GI CgA, gastrin

Gastric (type 3) None Upper GI CgA

Ellison

Ellison

triad

Ellison

Glucagonoma None Necrolytic migratory

vasointestinal peptide; NKA= neurokinin A; PYY= peptide YY

prototypical features of the neuroendocrine cells (1-6).

has been extensively investigated (1-5).

Duodenal Zollinger-

Meckel diverticulum Zollinger-

Insulinoma Whipple's

Gastrinoma Zollinger-

Pancreas

**2.1 Granins** 

Chromogranin A (CgA) has been claimed to be the best general neuroendocrine marker so far available. CgA is a 49 kDa monomeric, hydrophilic, acidic glycoprotein of 460 amino acid and is widely expressed in neuroendocrine cells, where it constitutes one of the most abundant components of secretory granules, and it is secreted from neuroendocrinederived tumors including functioning and non-functioning GEP NETs, pheochromocytomas, neuroblastomas, medullary thyroid carcinomas and some pituitary tumors. CgA is secreted to the extracellular space, so it's easily detectable in the blood. CgA is co-secreted with the amines and peptides that are present in the neurosecretory granules even if it can be elevated in both functionally active and non-functional NETs. CgA seems to be a "common denominator" peptide in all the components of the diffuse neuroendocrine system (7).

The precise function of CgA remains unknown, but it is thought to be involved in the packaging and processing of neuropeptide precursor and peptide hormones. It may also play a role in the organization of the secretory granule matrix. Moreover CgA has diverse physiological interactions: CgA (or its derivatives) is an inhibitor of catecholamine, insulin, and leptin, having a role in carbohydrate and lipid metabolism; moreover it inhibits parathormone secretion; on the other hand, CgA increases glucagon and amylase release. In addition to its effects on endocrine organs, CgA also regulates reproductive functions and has a role also in the regulation of cardiovascular function: CgA elevations have been reported in essential hypertension (CgA levels correlates with the severity of hypertension) and in chronic heart failure correlating with grade of cardiac dysfunction and mortality. A role of CgA in the regulation of inflammatory response has also been described. In fact increased CgA levels correlate with serum TNF-α receptor levels in a number of inflammatory diseases including rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, chronic obstructive pulmonary disease and chronic heart failure. Patients with sepsis show the highest increase of CgA; CgA positively correlates with inflammatory markers as C-reactive protein and procalcitonin. It remains to be elucidated the pathophysiological relevance of these correlations. Some authors suggest that CgA participates in a negative feedback that limits the activation of endothelial cells (1).

Circulating CgA concentrations are sensitive even if non specific markers of NETs. CgA has been reported to be more sensitive than urinary 5-hydroxyindoleacetic acid (5-HIAA) as well as than pancreatic polypeptide concentrations. The highest values were noted in ileal NETs (200 times the upper normal limit) and IN GEP-NETs associated with MEN1 (150 times the upper normal limit) while gastric type I, pituitary, and parathyroid tumors had lower values (ranging from 2 to 4 times normal). Both functioning and nonfunctioning pancreatic NETs had intermediate values (60–80 times the upper limit of normal) as did Zollinger- Ellison Syndrome (ZES), multiple endocrine neoplasia (MEN)-1, type II and III gastric entero-chromaffin-like (ECL)omas (80–100 times normal). It has also been proposed that CgA is more frequently elevated in well-differentiated tumors compared to poorly differentiated tumors of the midgut, suggesting that the loss of CgA expression in poorly differentiated neuroendocrine carcinomas indicates their incomplete or partial endocrine differentiation. In fact, poorly differentiated NE carcinomas rarely express CgA because of the rarity of large, dense-core granules. The presence of high plasma levels of CgA at diagnosis is an independent prognostic factor that indicates a reduced overall survival.

Circulating Markers in Gastroenteropancreatic Endocrine Tumors (GEP NETs) 23

Dako assay results are expressed in U/l, and normal range is within 19 U/l; with the ED kit CgA levels are expressed in nmol/l and normal range is < 41 nmol/l. Concerning plasma and serum measurement, a strong positive linear relationship has been reported between plasma and serum CgA values, indicating that CgA measurement can be undertaken in both sample types. In conclusion, because CgA concentrations are of considerable clinical relevance, substantial characterization and standardization to ensure uniform reporting are

The granin family comprises eight members including CgA and its derivated peptides, CgB, CgC (secretogranin II [SgII]), SgIII, SgIV, SgV, SgVI and VGF, but their value as circulating

Several other CgA-derived peptides, resulting by posttranslational processing have been isolated from extracts of human endocrine tumors. These molecules results in a series of smaller biologically active peptides, such as pancreastatin (corresponding to CgA residues 250–301), catestatin (corresponding to CgA residues 352–372), and vasostatin I and II (corresponding to CgA residues 1–76 and 1–113, respectively). These CgA derived peptides affect secretion of other hormones, play a role in vasoconstriction, and regulate metabolism. Among them, the most clinically interesting is pancreastatin. An endoprotease, the prohormone convertase-1 (PC-1), is involved in the processing of the precursor protein chromogranin A (CGA) to a smaller peptide called pancreastatin (PST), a 49-aminoacid peptide that inhibits insulin secretion, somatostatin release, exocrine pancreatic secretion and gastric acid secretion. PST is found in human stomach- and colon extracts and in a liver metastasis of gastrinoma. Pancreastatin was used before the complete sequence of CgA had been elucidated and before there were any reliable assays that could measure the whole molecule of CgA, as an epitope for antibody production. Pancreastatin antisera were used in immunohistochemistry and RIA to assess the presence of CgA in cells and the concentration of CgA in the circulation. But pancreastatin levels do not equale to CgA concentrations in the circulation. The molecule was found to be significantly increased in patients with NETs metastasized to the liver and concentrations are proportional to the number of hepatic metastases. Monitoring of liver metastases may remain the main advantage of pancreastatin assay (2). It is interesting that pancreastatin is not increased in patients with gastric achlorhydria or hypochlorhydria. Thus, false-positives are less problematic with the pancreastatin assay. It may be a very early biomarker for liver tumor activity, even when

CgB is the second most abundant member of the chromogranin family. Like CgA, it is a strongly acid protein containing approximately 25% acidic amino acid residues. It has 14 dibasic cleavage points but has been less well studied than CgA. Unlike CgA, CgB does not seem to have increased concentrations in patients with renal failure, in patients with atrophic gastritis, or those receiving acid-suppressing therapy. The interest to measure CgB in addition to CgA in patients with GEP NETs is therefore increased. Moreover, in tumors where CgA is not found, CgB may be increased. Such patients include those with MEN 1 and those with tumors in the duodenum or rectum. In addition, CgB is a major granin of the human adrenal medulla and may be a more sensitive marker of

markers for endocrine tumors has not been investigated extensively extensively (1).

needed (1, 7, 8).

CgA is normal (5).

pheochromocytomas (2, 5).

**2.1.2 Other granin family peptides** 

Effective treatment is often associated with decrease in CgA values. CgA correlates with tumor burden and recurrence. Measurement of CgA may help in the effective diagnosis of NET and has a major utility in predicting disease recurrence, outcome, and efficacy of therapy, so delineating the prognosis (5).

Elevated CgA can occur in normal individuals and in patients with non-NET tumors, although the levels are usually lower than in patients with NET. Less than 1% of CgA tests that are more than 20 times greater than the reference range are false positive. Levels of CgA secretion vary on a day-to-day basis in healthy subjects as well as in individuals with NETs. The mean day-to-day variation of CgA is approximately 25%. Food intake may increase CgA levels, therefore, CgA should be measured in fasting patients to ensure standardization of the results. There are conflicting results on the impact of exercise on CgA. Significant increases in CgA concentration have been reported in healthy subjects, but in patients with heart disease, long-term exercise had no impact on CgA. Finally extreme physical stress also causes CgA elevations. High-serum levels of CgA have also been demonstrated in patients with other malignancies including colon, lung, breast, liver and prostate cancer. Overall CgA has been found to be clinically informative and moderately sensitive in the majority of the studies, and more sensitive than NSE. In prostate cancer elevated CgA seems to indicate a poor prognosis; in small-cell lung carcinoma CgA levels were more frequently elevated and were also higher in cases of more extensive disease; NE differentiation occurs in 34% of primary colorectal cancer (1).

False-positive elevation of CgA may also occur in the following non-neoplastic circumstances: impaired renal function, Parkinson disease, untreated hypertension and pregnancy, steroid treatment or glucocorticoid excess, chronic atrophic gastritis (type A), treatment with proton pump inhibitors (PPI), inflammatory bowel disease, liver disease, hyperthyroidism. In renal failure CgA increases due to a decreased plasma clearance, reaching levels found in neuroendocrine neoplasia. In autoimmune chronic atrophic gastritis, elevated circulating CgA levels are caused by chronic hypergastrinemia and stimulation of ECL cell proliferation. Raised circulating CgA levels in addition to raised gastrin in atrophic gastritis, confounds the diagnosis of gastrinoma in many patients who present with dyspeptic symptoms. But the major cause of elevated CgA levels is the widespread use of PPIs and other acid suppressive medications. All PPI users, even with low dosage (10 mg/d) have elevated fasting CgA levels. The normalization of CgA levels occurs by withdrawal of PPI in 1-2 weeks (1, 10).

There is no universal standard calibration for serum or plasma chromogranin A assays. In addition, different chromogranin A assays, which use different antibodies or antibody combinations, will display different cross-reactivity with the various chromogranin A fragments. Therefore, reference intervals and individual patient results differ significantly between different chromogranin A assays and cannot be directly compared. Several commercially available radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays (ELISAs) have been developed for the measurement of circulating CgA concentrations. Moreover many diagnostic laboratories use in-house assays. The three main different commercial kits are CgA-RIA CT (CIS Bio International, Gif-sur-Yvette Cedex, France), Dako CgA ELISA kit (Dako A/S, Glostrup, Denmark), and CgA EuroDiagnostica (ED) (Malmo¨, Sweden). All three assays use different standards. In the CIS kit, CgA concentration is expressed in ng/ml and normal range is < 99g/l, while with the Dako assay results are expressed in U/l, and normal range is within 19 U/l; with the ED kit CgA levels are expressed in nmol/l and normal range is < 41 nmol/l. Concerning plasma and serum measurement, a strong positive linear relationship has been reported between plasma and serum CgA values, indicating that CgA measurement can be undertaken in both sample types. In conclusion, because CgA concentrations are of considerable clinical relevance, substantial characterization and standardization to ensure uniform reporting are needed (1, 7, 8).

#### **2.1.2 Other granin family peptides**

22 Neuroendocrine Tumor

Effective treatment is often associated with decrease in CgA values. CgA correlates with tumor burden and recurrence. Measurement of CgA may help in the effective diagnosis of NET and has a major utility in predicting disease recurrence, outcome, and efficacy of

Elevated CgA can occur in normal individuals and in patients with non-NET tumors, although the levels are usually lower than in patients with NET. Less than 1% of CgA tests that are more than 20 times greater than the reference range are false positive. Levels of CgA secretion vary on a day-to-day basis in healthy subjects as well as in individuals with NETs. The mean day-to-day variation of CgA is approximately 25%. Food intake may increase CgA levels, therefore, CgA should be measured in fasting patients to ensure standardization of the results. There are conflicting results on the impact of exercise on CgA. Significant increases in CgA concentration have been reported in healthy subjects, but in patients with heart disease, long-term exercise had no impact on CgA. Finally extreme physical stress also causes CgA elevations. High-serum levels of CgA have also been demonstrated in patients with other malignancies including colon, lung, breast, liver and prostate cancer. Overall CgA has been found to be clinically informative and moderately sensitive in the majority of the studies, and more sensitive than NSE. In prostate cancer elevated CgA seems to indicate a poor prognosis; in small-cell lung carcinoma CgA levels were more frequently elevated and were also higher in cases of more extensive disease; NE differentiation occurs in 34% of

False-positive elevation of CgA may also occur in the following non-neoplastic circumstances: impaired renal function, Parkinson disease, untreated hypertension and pregnancy, steroid treatment or glucocorticoid excess, chronic atrophic gastritis (type A), treatment with proton pump inhibitors (PPI), inflammatory bowel disease, liver disease, hyperthyroidism. In renal failure CgA increases due to a decreased plasma clearance, reaching levels found in neuroendocrine neoplasia. In autoimmune chronic atrophic gastritis, elevated circulating CgA levels are caused by chronic hypergastrinemia and stimulation of ECL cell proliferation. Raised circulating CgA levels in addition to raised gastrin in atrophic gastritis, confounds the diagnosis of gastrinoma in many patients who present with dyspeptic symptoms. But the major cause of elevated CgA levels is the widespread use of PPIs and other acid suppressive medications. All PPI users, even with low dosage (10 mg/d) have elevated fasting CgA levels. The normalization of CgA levels

There is no universal standard calibration for serum or plasma chromogranin A assays. In addition, different chromogranin A assays, which use different antibodies or antibody combinations, will display different cross-reactivity with the various chromogranin A fragments. Therefore, reference intervals and individual patient results differ significantly between different chromogranin A assays and cannot be directly compared. Several commercially available radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays (ELISAs) have been developed for the measurement of circulating CgA concentrations. Moreover many diagnostic laboratories use in-house assays. The three main different commercial kits are CgA-RIA CT (CIS Bio International, Gif-sur-Yvette Cedex, France), Dako CgA ELISA kit (Dako A/S, Glostrup, Denmark), and CgA EuroDiagnostica (ED) (Malmo¨, Sweden). All three assays use different standards. In the CIS kit, CgA concentration is expressed in ng/ml and normal range is < 99g/l, while with the

therapy, so delineating the prognosis (5).

primary colorectal cancer (1).

occurs by withdrawal of PPI in 1-2 weeks (1, 10).

The granin family comprises eight members including CgA and its derivated peptides, CgB, CgC (secretogranin II [SgII]), SgIII, SgIV, SgV, SgVI and VGF, but their value as circulating markers for endocrine tumors has not been investigated extensively extensively (1).

Several other CgA-derived peptides, resulting by posttranslational processing have been isolated from extracts of human endocrine tumors. These molecules results in a series of smaller biologically active peptides, such as pancreastatin (corresponding to CgA residues 250–301), catestatin (corresponding to CgA residues 352–372), and vasostatin I and II (corresponding to CgA residues 1–76 and 1–113, respectively). These CgA derived peptides affect secretion of other hormones, play a role in vasoconstriction, and regulate metabolism. Among them, the most clinically interesting is pancreastatin. An endoprotease, the prohormone convertase-1 (PC-1), is involved in the processing of the precursor protein chromogranin A (CGA) to a smaller peptide called pancreastatin (PST), a 49-aminoacid peptide that inhibits insulin secretion, somatostatin release, exocrine pancreatic secretion and gastric acid secretion. PST is found in human stomach- and colon extracts and in a liver metastasis of gastrinoma. Pancreastatin was used before the complete sequence of CgA had been elucidated and before there were any reliable assays that could measure the whole molecule of CgA, as an epitope for antibody production. Pancreastatin antisera were used in immunohistochemistry and RIA to assess the presence of CgA in cells and the concentration of CgA in the circulation. But pancreastatin levels do not equale to CgA concentrations in the circulation. The molecule was found to be significantly increased in patients with NETs metastasized to the liver and concentrations are proportional to the number of hepatic metastases. Monitoring of liver metastases may remain the main advantage of pancreastatin assay (2). It is interesting that pancreastatin is not increased in patients with gastric achlorhydria or hypochlorhydria. Thus, false-positives are less problematic with the pancreastatin assay. It may be a very early biomarker for liver tumor activity, even when CgA is normal (5).

CgB is the second most abundant member of the chromogranin family. Like CgA, it is a strongly acid protein containing approximately 25% acidic amino acid residues. It has 14 dibasic cleavage points but has been less well studied than CgA. Unlike CgA, CgB does not seem to have increased concentrations in patients with renal failure, in patients with atrophic gastritis, or those receiving acid-suppressing therapy. The interest to measure CgB in addition to CgA in patients with GEP NETs is therefore increased. Moreover, in tumors where CgA is not found, CgB may be increased. Such patients include those with MEN 1 and those with tumors in the duodenum or rectum. In addition, CgB is a major granin of the human adrenal medulla and may be a more sensitive marker of pheochromocytomas (2, 5).

Circulating Markers in Gastroenteropancreatic Endocrine Tumors (GEP NETs) 25

useful for the diagnosis and the monitoring of patients with neuroendocrine tumors (especially neuroblastoma, small cell lung cancer, less important for GEP NETs); it has other several applications, including the assessment of neuronal damage during stroke. Elevated NSE levels are indicative of poorly differentiated tumors. NSE levels seems not to be related

In addition to general markers, there are biomarkers specific for particular GEP-NET associated syndromes. The most typical is carcinoid syndrome and the specific marker is 5- Hydroxyindole Acetic Acid. Other specific markers including insulin, gastrin, vasoactive intestinal peptide, glucagon, bradykinin, substance P, neurotensin, human chorionic gonadotropin, neuropeptide K, and neuropeptide L are each of some value in precisely

5-HIAA is the urinary breakdown of serotonin, which is synthesized and stored in enterochromaffin cells of the gastrointestinal tract (80% of total body serotonin content), in dense granules of platelets and in the serotoninergic neurons of the central nervous system. Serotonin is a ubiquitous tryptophan-derived biogenic amine, involved in homeostasis,

Carcinoid syndrome is the typical clinical picture of metastatic ileal carcinoid, occurring in about 18% of patients and is characterized by flushing, diarrhea, abdominal pain; less frequent events are lacrymation, profuse sweating, telangiectasias, cardiac fibrosis, and cutaneous manifestations pellagra-like due to lack of niacin. This syndrome is caused by the massive release of serotonin, which is no longer metabolized in the liver, and other

> prolonged fit, red-purple, localized to face and trunk.

Serotonin, histamine,

prostaglandins, gastrin

P substance, serotonin

ischemia, fibrosis

hepatomegaly, intestinal

P substance, prostaglandins

*Midgut* tumors: quick fit, pink-red.

Profuse sweating 15 Serotonin, histamine Telangiectasias 25 Face Unknown cause

Pellagra 5 Dermatitis deficit of niacin

Diarrhea 70 Secretory Serotonin, histamine, VIP,

Valvulopathies (tricuspid valve, pulmonary valve). Right heart failure. Dyspnea

substances, such as tachykinins, prostaglandins, and bradykinins (3) (**Table 2**).

**Clinical features (%) Characteristics Mediators** 

Abdominal pain 40 Long lasting Obstruction,

to any secretory activity of the tumor (11, 12).

**3.1 5-Hydroxyindole acetic acid (5-HIAA)** 

vasoconstriction and neurotransmission (7).

Flushing 90 *Foregut* tumors:

10 (left)

Table 2. Characteristics of carcinoid syndrome

Heart disease 30 (right)

defining the functionality of individual NETs (see above **Table 1**).

**3. Specific biomarkers** 

Fig. 1. The granin family and fragments of CgA. The 8 granin proteins include chromogranin A, CgB, CgC (SgII), SgIII, SgIV, 7B2 (SgV), neuroendocrine secretory protein 55 (NESP55 or SgVI), and VGF nerve growth factor–inducible (VGF).

#### **2.2 Pancreatic polypeptide (PP)**

Circulating PP is a single chain, 36-aminoacid peptide arising from the PP cells of the pancreas and is expressed in neuroendocrine cells of the gut and the pancreas. The function of PP is to self regulate pancreatic secretion activities (endocrine and exocrine), it also has effects on hepatic glycogen levels and gastrointestinal secretions.

Elevated PP concentration are found in patients with NETs, both pancreatic (20-50%) and gastrointestinal carcinoids (30-50%). Before methods for the measurements of CgA were available, PP was used as a general marker for endocrine tumors, although it is poorly specific. PP is now useful in the diagnosis and monitoring of NETs where no other general marker is raised and in PP-omas. High levels of circulating PP can also be found in diabetes, renal impairment, chronic inflammation, alcoholism and in elder patients. Its secretion is increased after a protein meal, fasting, exercise, and acute hypoglycemia and is decreased by somatostatin and intravenous glucose (2, 11).

#### **2.3 Neuron Specific Enolase (NSE)**

NSE is the neuron-specific isomer of the glycolytic enzyme 2-phospho-D-glycerate hydroxylase or enolase. This isomer is present in neurons and neuroendocrine cells and can be used as a biomarker for tumors derived from these cells. As well as CgA, NSE is a marker useful for the diagnosis and the monitoring of patients with neuroendocrine tumors (especially neuroblastoma, small cell lung cancer, less important for GEP NETs); it has other several applications, including the assessment of neuronal damage during stroke. Elevated NSE levels are indicative of poorly differentiated tumors. NSE levels seems not to be related to any secretory activity of the tumor (11, 12).

### **3. Specific biomarkers**

24 Neuroendocrine Tumor

Chromogranin A (CgA) and CgAderived peptides

Fig. 1. The granin family and fragments of CgA. The 8 granin proteins include

55 (NESP55 or SgVI), and VGF nerve growth factor–inducible (VGF).

VGF

Secretogranin V (Sg V or 7B2)

Secretogranin VI (SgVI or NESP55)

effects on hepatic glycogen levels and gastrointestinal secretions.

**2.2 Pancreatic polypeptide (PP)** 

somatostatin and intravenous glucose (2, 11).

**2.3 Neuron Specific Enolase (NSE)** 

chromogranin A, CgB, CgC (SgII), SgIII, SgIV, 7B2 (SgV), neuroendocrine secretory protein

Secretogranin IV (Sg IV)

Granin family

> Secretogranin III (SgIII)

Chromogranin B (CgB)

> Chromogranin C (CgC or secretogranin II)

Circulating PP is a single chain, 36-aminoacid peptide arising from the PP cells of the pancreas and is expressed in neuroendocrine cells of the gut and the pancreas. The function of PP is to self regulate pancreatic secretion activities (endocrine and exocrine), it also has

Elevated PP concentration are found in patients with NETs, both pancreatic (20-50%) and gastrointestinal carcinoids (30-50%). Before methods for the measurements of CgA were available, PP was used as a general marker for endocrine tumors, although it is poorly specific. PP is now useful in the diagnosis and monitoring of NETs where no other general marker is raised and in PP-omas. High levels of circulating PP can also be found in diabetes, renal impairment, chronic inflammation, alcoholism and in elder patients. Its secretion is increased after a protein meal, fasting, exercise, and acute hypoglycemia and is decreased by

NSE is the neuron-specific isomer of the glycolytic enzyme 2-phospho-D-glycerate hydroxylase or enolase. This isomer is present in neurons and neuroendocrine cells and can be used as a biomarker for tumors derived from these cells. As well as CgA, NSE is a marker In addition to general markers, there are biomarkers specific for particular GEP-NET associated syndromes. The most typical is carcinoid syndrome and the specific marker is 5- Hydroxyindole Acetic Acid. Other specific markers including insulin, gastrin, vasoactive intestinal peptide, glucagon, bradykinin, substance P, neurotensin, human chorionic gonadotropin, neuropeptide K, and neuropeptide L are each of some value in precisely defining the functionality of individual NETs (see above **Table 1**).

#### **3.1 5-Hydroxyindole acetic acid (5-HIAA)**

5-HIAA is the urinary breakdown of serotonin, which is synthesized and stored in enterochromaffin cells of the gastrointestinal tract (80% of total body serotonin content), in dense granules of platelets and in the serotoninergic neurons of the central nervous system. Serotonin is a ubiquitous tryptophan-derived biogenic amine, involved in homeostasis, vasoconstriction and neurotransmission (7).

Carcinoid syndrome is the typical clinical picture of metastatic ileal carcinoid, occurring in about 18% of patients and is characterized by flushing, diarrhea, abdominal pain; less frequent events are lacrymation, profuse sweating, telangiectasias, cardiac fibrosis, and cutaneous manifestations pellagra-like due to lack of niacin. This syndrome is caused by the massive release of serotonin, which is no longer metabolized in the liver, and other substances, such as tachykinins, prostaglandins, and bradykinins (3) (**Table 2**).


Table 2. Characteristics of carcinoid syndrome

Circulating Markers in Gastroenteropancreatic Endocrine Tumors (GEP NETs) 27

HIAA. Untreated patients with malabsorption (celiac disease, tropical sprue, Whipple disease, intestinal stasis and cystic fibrosis), may have increased tryptophan metabolites. Also certain medications may interfere with the assay: paracetamol, fluorouracil, methysergide, naproxen and caffeine may cause false positive results. On the contrary levodopa, aspirin, adrenocorticotrophic hormone (ACTH), methyldopa, and phenothiazines may give a false negative results (13). Somatostatin analogs are known to decrease levels of 5-HIAA. Moreover, patients with renal impairment and those with hemodyalisis may have

5-HIAA does not seem to be a useful prognostic factor in patients with carcinoid syndrome, because of the fluctuating release of serotonin in NETs of the midgut. On the other hand, several studies found high 5-HIAA levels to be an independent survival factor. Overall, in these studies, higher concentrations of urinary 5-HIAA are associated with a worse prognosis, and persistently low 5-HIAA excretion predicts more favorable survival in patients with disseminated disease. The intra-individual variation of 5-HIAA may be high. When the collection is required for the diagnosis it is useful to have two consecutive 24-

Serotonin plays a key role in development of peritoneal and cardiac fibrosis via activation of the 5HT2B receptor and a cascade of connective tissue growth factors. Reductions in plasma serotonin levels correlate with a decreased incidence of carcinoid heart disease (CHD). Moreover, urinary 5-HIAA excretion also correlates with the severity of CHD and prognosis

Insulin is a peptide hormone composed of 51 amino acids and has a molecular weight of 5808 Da. It is produced in the islets of Langerhans in the pancreas, within the β-cells. In βcells, insulin is synthesized from the proinsulin precursor molecule. Insulin is a hormone central in the regulation of carbohydrate and fat metabolism in the body. Since the main action of insulin is reducing blood glucose levels, by increasing glycogen synthesis and promoting storage of glucose in liver (and muscle) cells, insulin excess (such as an insulinoma) induces hypoglycemia. In patients with suspected insulinoma, the insulin and its precursors or breakdown products should be tested, even if further biochemical tests include the 72-hour fast, which is the gold standard for establishing the diagnosis of insulinoma. Insulinomas secrete proinsulin, insulin and C-peptide intermittently, and, although insulin concentrations in the circulation may often be within reference range,

insulin is at most times inappropriately high for the blood glucose concentration (14).

Insulinoma is the most common secretory NET of the pancreas that produces a symptomatic clinical syndrome. More than 80% of insulinomas are benign. Insulinoma is uncommon, although it is the second most common pancreatic NET to occur in patients with MEN1 (5).

Gastrin is a hormone that stimulates secretion of gastric acid (HCl) by the parietal cells of the stomach and aids in gastric motility. It is released by G cells in the stomach, duodenum, and the pancreas. Gastrinoma (gastrin-producing tumor) is the second most common

falsely low 5-HIAA levels.

hours collections and to take the mean value (7).

in patients with carcinoid syndrome.

**3.2 Insulin** 

**3.3 Gastrin** 


This syndrome is typical of metastatic well-differentiated midgut NETs, even if other clinical conditions may mimic symptoms and signs (**Table 3**).

CgA=Chromogranin A; 5-HIAA= 5-hydroxyindoleacetic acid; PP= Pancreatic polypeptide; VIP= vasointestinal peptide; CEA= carcino-embryonic antigen; VMA= vanillylmandelic acid; Epi= epinephrine; Norepi= norepinephrine; MIBG= metaiodobenzylguanidine; HRV= heart rate variability; 2hs PP= 2-hour postprandial blood sugar; FSH= follicle-stimulating hormone; EEG= electroencephalography; ACTH= adrenocorticotropic hormone.

Table 3. Differential diagnosis of flushing and diagnostic tests

The 24-h measurement of 5-HIAA is a useful specific marker for serotonin-producing NETs. The overall sensitivity and specificity of urinary 5-HIAA in the presence of the carcinoid syndrome is 70 and 90%, respectively. Therefore this marker is the most frequently performed assay in the clinical setting of the carcinoid syndrome. Midgut carcinoids are most liable to produce the carcinoid syndrome with 5-HIAA elevation, thus attesting to a high specificity in this setting (approximately 75% of midgut NETs are associated with a positive urinary 5-HIAA test). Functional symptoms in NETs originating from the midgut are in fact mostly due to the secretion of 5-hydroxytryptophan (5-HTP) or serotonin. The sensitivity is lower in patients with midgut carcinoid tumors without the carcinoid syndrome and in patients with fore- and hindgut NETs due to less serotonin production from these tumors than midgut ones. Elevated 5-HIAA levels in the urine are highly suggestive of an ileal NET, although some NETs found in the lung and pancreas also secrete serotonin (7, 11).

High-performance-liquid-chromatography (HPLC) with electrochemical detection is currently recommended to measure 5-HIAA. In some laboratory automated assays or those using mass spectrometry are available.

There are false positive 5-HIAA urinary levels as well as false negative ones. Some foods contain high levels of serotonin which may increase the levels of urinary 5-HIAA and their consumption should be avoided 3 days prior to urine collection (i.e. plums, pineapples, bananas, eggplants, tomatoes, avocados, and walnuts). For this reason patients need to be on a diet free of tryptophan/serotonin-rich foods to avoid false elevations in urinary 5HIAA. Untreated patients with malabsorption (celiac disease, tropical sprue, Whipple disease, intestinal stasis and cystic fibrosis), may have increased tryptophan metabolites. Also certain medications may interfere with the assay: paracetamol, fluorouracil, methysergide, naproxen and caffeine may cause false positive results. On the contrary levodopa, aspirin, adrenocorticotrophic hormone (ACTH), methyldopa, and phenothiazines may give a false negative results (13). Somatostatin analogs are known to decrease levels of 5-HIAA. Moreover, patients with renal impairment and those with hemodyalisis may have falsely low 5-HIAA levels.

5-HIAA does not seem to be a useful prognostic factor in patients with carcinoid syndrome, because of the fluctuating release of serotonin in NETs of the midgut. On the other hand, several studies found high 5-HIAA levels to be an independent survival factor. Overall, in these studies, higher concentrations of urinary 5-HIAA are associated with a worse prognosis, and persistently low 5-HIAA excretion predicts more favorable survival in patients with disseminated disease. The intra-individual variation of 5-HIAA may be high. When the collection is required for the diagnosis it is useful to have two consecutive 24 hours collections and to take the mean value (7).

Serotonin plays a key role in development of peritoneal and cardiac fibrosis via activation of the 5HT2B receptor and a cascade of connective tissue growth factors. Reductions in plasma serotonin levels correlate with a decreased incidence of carcinoid heart disease (CHD). Moreover, urinary 5-HIAA excretion also correlates with the severity of CHD and prognosis in patients with carcinoid syndrome.

#### **3.2 Insulin**

26 Neuroendocrine Tumor

This syndrome is typical of metastatic well-differentiated midgut NETs, even if other clinical

Medullary carcinoma of the thyroid CgA, CEA, Calcitonin, Ca++ infusion, RET

Pheocromocytoma CgA, Plasma free metanephrines, urine

proto-oncogene

stimulation, MIBG

metanephrines, VMA, Epi, Norepi, glucagon

Carcinoid CgA, Serotonin, 5-HIAA, PP, VIP

Mastocytosis Plasma histamine, urine tryptase

2hs PP= 2-hour postprandial blood sugar; FSH= follicle-stimulating hormone; EEG=

CgA=Chromogranin A; 5-HIAA= 5-hydroxyindoleacetic acid; PP= Pancreatic polypeptide; VIP= vasointestinal peptide; CEA= carcino-embryonic antigen; VMA= vanillylmandelic acid; Epi=

epinephrine; Norepi= norepinephrine; MIBG= metaiodobenzylguanidine; HRV= heart rate variability;

The 24-h measurement of 5-HIAA is a useful specific marker for serotonin-producing NETs. The overall sensitivity and specificity of urinary 5-HIAA in the presence of the carcinoid syndrome is 70 and 90%, respectively. Therefore this marker is the most frequently performed assay in the clinical setting of the carcinoid syndrome. Midgut carcinoids are most liable to produce the carcinoid syndrome with 5-HIAA elevation, thus attesting to a high specificity in this setting (approximately 75% of midgut NETs are associated with a positive urinary 5-HIAA test). Functional symptoms in NETs originating from the midgut are in fact mostly due to the secretion of 5-hydroxytryptophan (5-HTP) or serotonin. The sensitivity is lower in patients with midgut carcinoid tumors without the carcinoid syndrome and in patients with fore- and hindgut NETs due to less serotonin production from these tumors than midgut ones. Elevated 5-HIAA levels in the urine are highly suggestive of an ileal NET, although some NETs found in the lung and pancreas also secrete

High-performance-liquid-chromatography (HPLC) with electrochemical detection is currently recommended to measure 5-HIAA. In some laboratory automated assays or those

There are false positive 5-HIAA urinary levels as well as false negative ones. Some foods contain high levels of serotonin which may increase the levels of urinary 5-HIAA and their consumption should be avoided 3 days prior to urine collection (i.e. plums, pineapples, bananas, eggplants, tomatoes, avocados, and walnuts). For this reason patients need to be on a diet free of tryptophan/serotonin-rich foods to avoid false elevations in urinary 5-

conditions may mimic symptoms and signs (**Table 3**). **Clinical Condition Tests** 

Menopause FSH Epilepsy EEG

VIP-oma CgA, PP, VIP

Diabetic autonomic neuropathy HRV, 2hs PP glucose

Panic attack Pentagastrin, ACTH

Mitral valve prolapse Cardiac echo

electroencephalography; ACTH= adrenocorticotropic hormone. Table 3. Differential diagnosis of flushing and diagnostic tests

serotonin (7, 11).

using mass spectrometry are available.

Insulin is a peptide hormone composed of 51 amino acids and has a molecular weight of 5808 Da. It is produced in the islets of Langerhans in the pancreas, within the β-cells. In βcells, insulin is synthesized from the proinsulin precursor molecule. Insulin is a hormone central in the regulation of carbohydrate and fat metabolism in the body. Since the main action of insulin is reducing blood glucose levels, by increasing glycogen synthesis and promoting storage of glucose in liver (and muscle) cells, insulin excess (such as an insulinoma) induces hypoglycemia. In patients with suspected insulinoma, the insulin and its precursors or breakdown products should be tested, even if further biochemical tests include the 72-hour fast, which is the gold standard for establishing the diagnosis of insulinoma. Insulinomas secrete proinsulin, insulin and C-peptide intermittently, and, although insulin concentrations in the circulation may often be within reference range, insulin is at most times inappropriately high for the blood glucose concentration (14).

Insulinoma is the most common secretory NET of the pancreas that produces a symptomatic clinical syndrome. More than 80% of insulinomas are benign. Insulinoma is uncommon, although it is the second most common pancreatic NET to occur in patients with MEN1 (5).

#### **3.3 Gastrin**

Gastrin is a hormone that stimulates secretion of gastric acid (HCl) by the parietal cells of the stomach and aids in gastric motility. It is released by G cells in the stomach, duodenum, and the pancreas. Gastrinoma (gastrin-producing tumor) is the second most common

Circulating Markers in Gastroenteropancreatic Endocrine Tumors (GEP NETs) 29

it relaxes the lower esophageal sphincter ,the fundic smooth muscle and suppress gastric acid secretion. These effects work together to increase motility. Like secretin, it stimulates secretion of water and bicarbonate and stimulates secretion of chloride and water from large intestine; in small intestine inhibits absorption and the contractile effect of CCK. Moreover it enhances the release of insulin and glucagon. VIP has also significant effects on the cardiovascular system. It causes coronary vasodilation as well as it has a positive inotropic

VIPoma is much less common that insulinoma and gastrinoma with an incidence of approximately 0.02 per 100,000 per year. VIPoma is characterized by watery diarrhea, hypokalemia and achlorhydria (WDHA syndrome or pancreatic cholera syndrome, or also called Verner Morrison syndrome). Due to VIP effects as a potent stimulator of intestinal secretion and inhibitor of gastric acid secretion, the massive amounts of secreted VIP cause profound and chronic **w**atery **d**iarrhea (fasting stool volume > 750 to 1000 mL/day) and resultant dehydration, **h**ypokalemia, **a**chlorhydria (hence WDHA-syndrome), acidosis, vasodilation (flushing and hypotension), hypercalcemia and hyperglycemia. The watery diarrhea may be intermittent at the onset , but it may rapidly escalate and reach a volume of 15-20L per day, causing profound alteration in fluids and electrolytes control. Hypokalemic acidosis is due to bicarbonate and potassium loss across the bowel mucosa; it may provoke asthenia and tetanic contraction. Gastric achlorhydria occurs only in 50% of patients, while hypochlorhydria is usually present. Abdominal pain and weight loss are also common features. Other signs are hypercalcemia, related to VIP direct action on bone metabolism, and flushes that may cause some confusion with classical midgut carcinoid

The majority of VIPomas occurs in the pancreas, while about 10-15% arises in the ganglionic chain and most common in the adrenal medulla. In children, besides, VIP-producing tumors may occur in ganglioneuroma and neuroblastoma. About 50-60% of VIP-secreting tumors are malignant and present hepatic involvement. It may arise in contest of MEN1 syndrome

Glucagon is a hormone secreted by alpha cells (α-cells) of the islets of Langerhans of the pancreas and from the L cells in the intestinal mucosa. Glucagon is a 29-amino acid polypeptide and its main action is to raise blood glucose levels. From these two sites, proglucagon is processed differently. In the pancreas, proglucagon is processed to produce glucagon, glycentin-related peptide, intervening peptide, and the major glucagon fragment. Intestinal proglucagon undergoes alternative posttranslational processing that generates glycentin, sometimes referred to as gut glucagon, glucagon-like peptide 1 (GLP1), and

Plasma glucagon is a specific marker for Glucagonoma. Glucagonoma occurs at approximately the same frequency as VIPoma. Circulating glucagon concentrations are typically more than 5-fold higher than the reference range. Both pancreatic glucagon and glycentin are measured in high concentrations. Considering the importance of glucagon in the control of blood glucose, one would expect a glucagon-secreting tumor to produce a profound syndrome. However this is not the case and glucagonoma usually presents late

and chronotropic effect. VIP helps to regulate prolactin secretion (2).

syndrome (5) (see above, **Table 3**).

glucagon-like peptide 2 (GLP2) (2).

(2).

**3.5 Glucagon** 

secretory pancreatic NET. It can rise from both cells in the duodenum and in the pancreas, with just more than half malignant at presentation. Approximately 25% to 35% of gastrinomas are associated with MEN1. Gastrinoma is the most common GEP NET associated with MEN1. Gastrinomas secrete gastrin but gastrin can circulate in numerous forms. Progastrin, gastrin 34, gastrin 17, and C-terminally extended gastrins may all circulate in high concentrations in patients with gastrinoma. In the Zollinger-Ellison syndrome, gastrin is produced at excessive levels. Normal values are generally less than 100 pg/mL (2, 5).

Gastrinoma is not the only cause of hypergastrinemia, since there are several causes for hypergastrinemia that often require numerous and expensive diagnostic investigations. Hypergastrinemia is most frequently due to hypochlorhydria and only seldom the underlying cause is gastrinoma. The most frequent condition that causes hypochlorhydria is the use of antacids or medicines that suppress stomach acid. Also autoimmune gastritis, where the immune system attacks the parietal cells leading to hypochlorhydria (low stomach acidity) is a possible cause. In this condition, hypochlorhydria results in an elevated gastrin level in an attempt to compensate for increased pH in the stomach. Eventually, all the parietal cells are lost and achlorhydria results to a loss of negative feedback on gastrin secretion. Other causes of hypergastrinemia are G-cell hyperplasia (overactivity of gastrinproducing cells in the stomach), Helicobacter pylori infection of the stomach, mucolipidosis type IV (5, 15) (**Table 4**).


Table 4. Conditions associated with hypergastrinemia

#### **3.4 VIP**

Vasoactive intestinal peptide (VIP) is a peptide hormone containing 28-amino acid residues. It is produced in many areas of the human body including the gut, pancreas and suprachiasmatic nuclei of the hypothalamus in the brain.

In normal physiology VIP acts as a neuromodulator and not as an hormone, since it circulates in low quantity even an increase of about 20-50% of normal reference range is significant. VIP is released from neurons, peripheral ganglia, throughout the GI tract, in the urogenital system, respiratory tract and blood vessels. VIP has several effects on the digestive system:

it relaxes the lower esophageal sphincter ,the fundic smooth muscle and suppress gastric acid secretion. These effects work together to increase motility. Like secretin, it stimulates secretion of water and bicarbonate and stimulates secretion of chloride and water from large intestine; in small intestine inhibits absorption and the contractile effect of CCK. Moreover it enhances the release of insulin and glucagon. VIP has also significant effects on the cardiovascular system. It causes coronary vasodilation as well as it has a positive inotropic and chronotropic effect. VIP helps to regulate prolactin secretion (2).

VIPoma is much less common that insulinoma and gastrinoma with an incidence of approximately 0.02 per 100,000 per year. VIPoma is characterized by watery diarrhea, hypokalemia and achlorhydria (WDHA syndrome or pancreatic cholera syndrome, or also called Verner Morrison syndrome). Due to VIP effects as a potent stimulator of intestinal secretion and inhibitor of gastric acid secretion, the massive amounts of secreted VIP cause profound and chronic **w**atery **d**iarrhea (fasting stool volume > 750 to 1000 mL/day) and resultant dehydration, **h**ypokalemia, **a**chlorhydria (hence WDHA-syndrome), acidosis, vasodilation (flushing and hypotension), hypercalcemia and hyperglycemia. The watery diarrhea may be intermittent at the onset , but it may rapidly escalate and reach a volume of 15-20L per day, causing profound alteration in fluids and electrolytes control. Hypokalemic acidosis is due to bicarbonate and potassium loss across the bowel mucosa; it may provoke asthenia and tetanic contraction. Gastric achlorhydria occurs only in 50% of patients, while hypochlorhydria is usually present. Abdominal pain and weight loss are also common features. Other signs are hypercalcemia, related to VIP direct action on bone metabolism, and flushes that may cause some confusion with classical midgut carcinoid syndrome (5) (see above, **Table 3**).

The majority of VIPomas occurs in the pancreas, while about 10-15% arises in the ganglionic chain and most common in the adrenal medulla. In children, besides, VIP-producing tumors may occur in ganglioneuroma and neuroblastoma. About 50-60% of VIP-secreting tumors are malignant and present hepatic involvement. It may arise in contest of MEN1 syndrome (2).

#### **3.5 Glucagon**

28 Neuroendocrine Tumor

secretory pancreatic NET. It can rise from both cells in the duodenum and in the pancreas, with just more than half malignant at presentation. Approximately 25% to 35% of gastrinomas are associated with MEN1. Gastrinoma is the most common GEP NET associated with MEN1. Gastrinomas secrete gastrin but gastrin can circulate in numerous forms. Progastrin, gastrin 34, gastrin 17, and C-terminally extended gastrins may all circulate in high concentrations in patients with gastrinoma. In the Zollinger-Ellison syndrome, gastrin is produced at excessive levels. Normal values are generally less than 100

Gastrinoma is not the only cause of hypergastrinemia, since there are several causes for hypergastrinemia that often require numerous and expensive diagnostic investigations. Hypergastrinemia is most frequently due to hypochlorhydria and only seldom the underlying cause is gastrinoma. The most frequent condition that causes hypochlorhydria is the use of antacids or medicines that suppress stomach acid. Also autoimmune gastritis, where the immune system attacks the parietal cells leading to hypochlorhydria (low stomach acidity) is a possible cause. In this condition, hypochlorhydria results in an elevated gastrin level in an attempt to compensate for increased pH in the stomach. Eventually, all the parietal cells are lost and achlorhydria results to a loss of negative feedback on gastrin secretion. Other causes of hypergastrinemia are G-cell hyperplasia (overactivity of gastrinproducing cells in the stomach), Helicobacter pylori infection of the stomach, mucolipidosis

Vasoactive intestinal peptide (VIP) is a peptide hormone containing 28-amino acid residues. It is produced in many areas of the human body including the gut, pancreas and

In normal physiology VIP acts as a neuromodulator and not as an hormone, since it circulates in low quantity even an increase of about 20-50% of normal reference range is significant. VIP is released from neurons, peripheral ganglia, throughout the GI tract, in the urogenital system, respiratory tract and blood vessels. VIP has several effects on the

HYPERGASTRINEMIA WITHOUT GASTRIC ACID HYPERSECRETION:

HYPERGASTRINEMIA WITH GASTRIC ACID HYPERSECRETION:

Atrophic gastritis (with or without pernicious anemia) Gastric cancer without involvement of the gastric antrum Therapy with H-2 blockers or proton pump inhibitors (PPIs)

Table 4. Conditions associated with hypergastrinemia

suprachiasmatic nuclei of the hypothalamus in the brain.

pg/mL (2, 5).

type IV (5, 15) (**Table 4**).

Antral G cell hyperplasia

Massive bowel resection Chronic renal impairment

Gastrojejunostomy or Billroth II

Gastrinoma

Duodenal ulcer

Pyloric stenosis Hypercalcemia

digestive system:

**3.4 VIP** 

Glucagon is a hormone secreted by alpha cells (α-cells) of the islets of Langerhans of the pancreas and from the L cells in the intestinal mucosa. Glucagon is a 29-amino acid polypeptide and its main action is to raise blood glucose levels. From these two sites, proglucagon is processed differently. In the pancreas, proglucagon is processed to produce glucagon, glycentin-related peptide, intervening peptide, and the major glucagon fragment. Intestinal proglucagon undergoes alternative posttranslational processing that generates glycentin, sometimes referred to as gut glucagon, glucagon-like peptide 1 (GLP1), and glucagon-like peptide 2 (GLP2) (2).

Plasma glucagon is a specific marker for Glucagonoma. Glucagonoma occurs at approximately the same frequency as VIPoma. Circulating glucagon concentrations are typically more than 5-fold higher than the reference range. Both pancreatic glucagon and glycentin are measured in high concentrations. Considering the importance of glucagon in the control of blood glucose, one would expect a glucagon-secreting tumor to produce a profound syndrome. However this is not the case and glucagonoma usually presents late

Circulating Markers in Gastroenteropancreatic Endocrine Tumors (GEP NETs) 31

Growth-hormone-releasing-hormone (GHRH) is a 44-amino acid hormone released from neurosecretory nerve terminals of the arcuate neurons, and is carried by the hypothalamichypophyseal portal system to anterior pituitary gland, where stimulates growth hormone (GH) secretion. Hypothalamic tumors, including hamartomas, choristomas, gliomas, and gangliocytomas, may produce excessive GHRH with subsequent GH hypersecretion and resultant acromegaly. Peripheral GHRH levels are not elevated in patients with hypothalamic GHRH-secreting tumors, supporting the notion that excess eutopic hypothalamic GHRH secretion into the hypophyseal portal system does not appreciably enter the systemic circulation. Excessive ectopic peripheral production of GHRH is present in several tumors, including carcinoid tumors, pancreatic cell tumors, small-cell lung cancers, adrenal adenomas, and pheochromocytomas, which have been reported to secrete GHRH. In these cases, peripheral GHRH levels are elevated even if acromegaly in these patients, is uncommon. For these reasons measuring GHRH plasma levels provides a precise and cost-effective test for the diagnosis of ectopic acromegaly. Elevated circulating GHRH levels, a normal or small-size pituitary gland, or clinical and biochemical features of other tumors known to be associated with extra-pituitary acromegaly, are all indications for

Calcitonin is a 32-amino acid peptide released, in normal physiology, only in non-follicular C-cells of the thyroid . It is produced as a 136-amino acid precursor (pro-calcitonin) and processed in secretory granules to the active form. The synthesis and release of calcitonin are closely related to calcium serum levels. Calcitonin is raised in medullary thyroid cancer, where concentration may be thousands-fold the reference range. Medullary thyroid cancers frequently arise as part of multiple endocrine neoplasia type 2 (MEN2) syndrome. Calcitonin may also be raised in some pancreatic neuroendocrine tumor, especially those that are multi-hormone producing and bronchial carcinoid. Usually ectopic-produced

NETs secreting insulin are termed insulinomas and are almost exclusively intrapancreatic in nature*.* Insulinomas secrete proinsulin, insulin and C-peptide intermittently, and, although insulin concentrations in the circulation may often be within reference range, insulin is at

The diagnosis is suggested in the presence of the Whipple's triad: symptoms of hypoglycemia, glucose < 2.5 mmol/l (45 mg/dl) and relief of symptoms with administration of glucose. Hypoglycemia-induced clinical signs are classically present in the early morning pre-prandial phase or maybe exercise-induced. The typical signs are due to activation of adrenergic nervous system (palpations, sweating pallor, anxiety) and neuroglycopenia (personality changes, and loss of consciousness). The latter symptom reflects both the severity and duration of hypoglycemia. Although these symptoms are profound, they may

**3.7.2 Growth-hormone-releasing-hormone (GHRH)** 

extra-pituitary production of GHRH (18).

calcitonin is a large molecule without biochemical activity (2).

most times inappropriately high for the blood glucose concentration.

**3.7.3 Calcitonin** 

**4. Provocative tests** 

**4.1 Insulinoma: 72-hour fast** 

with extensive metastatic spread, mild diabetes, and a characteristic rash (necrolytic migratory erythema) (5).

Increased secretion of glucagon is also caused by other causes, like decreased plasma glucose (indirectly), increased catecholamines - norepinephrine and epinephrine, increased plasma amino acids (to protect from hypoglycemia if an all-protein meal is consumed), sympathetic nervous system, acetylcholine, cholecystokinin. Decreased secretion of glucagon is caused by somatostatin, insulin, increased free fatty acids and keto acids into the blood, increased urea production (16).

#### **3.6 Somatostatin**

Somatostatin is a peptide hormone that regulates the endocrine system and affects neurotransmission and cell proliferation. Somatostatin is classified as an inhibitory hormone, that exerts its effects mainly on anterior pituitary, by inhibiting the release of GH, opposing the effects of Growth Hormone-Releasing Hormone (GHRH), and inhibiting the release of thyroid-stimulating hormone (TSH) and on gastrointestinal system, by suppressing the release of other gastrointestinal and pancreatic hormones, decreasing gastric emptying, and reducing smooth muscle contractions and blood flow within the intestine (2).

Pancreatic somatostatinoma is a tumor of the delta cells of the endocrine pancreas that produces somatostatin. It is uncommon and symptoms are vague, thus diagnosis is frequently delayed. Clinical syndrome is characterized by a triad of : mild diabetes mellitus, steatorrhea and gallstones. Also hypochlorhydria can be associated. Patients may present with clinical endocrine syndrome due to elevated somatostatin that can cause diabetes mellitus, by inhibiting insulin secretion, steatorrhea by inhibiting cholecystokinin and secretin, gallstones by inhibiting cholecystokinin which normally induce gallbladder myocytes contraction, and hypochlorhydria caused by inhibiting gastrin, which normally stimulates acid secretion. However, more frequently patients present with symptoms related with tumor bulk. In presence of a somatostatinoma circulating somatostatin concentrations may be more than 100 times the reference range (2, 5).

#### **3.7 Other circulating markers**

#### **3.7.1 Corticotropin-releasing hormone (CRH)**

Corticotropin-releasing hormone, a 41-amino acid peptide derived from a 191-amino acid preprohormone, acts as hormone and neurotransmitter in the stimulation of pituitary synthesis of ACTH in stress response. In normal physiology CRH is produced by parvocellular neuroendocrine cells (contained within the paraventricular nucleus of hypothalamus).

Ectopic CRH production is rare, it may occur in patients with medullary thyroid carcinoma (about 33%) and pheochromocytoma (19%), carcinoid (5%) and small cell lung carcinoma (about 10 %) and prostate cancer (17). The main clinical feature is Cushing 's syndrome; levels of cortisol are elevated (>900 nmol/l) as ACTH, DHEA-S. Overnight administration of dexamethasone doesn't suppress cortisol secretion.

#### **3.7.2 Growth-hormone-releasing-hormone (GHRH)**

Growth-hormone-releasing-hormone (GHRH) is a 44-amino acid hormone released from neurosecretory nerve terminals of the arcuate neurons, and is carried by the hypothalamichypophyseal portal system to anterior pituitary gland, where stimulates growth hormone (GH) secretion. Hypothalamic tumors, including hamartomas, choristomas, gliomas, and gangliocytomas, may produce excessive GHRH with subsequent GH hypersecretion and resultant acromegaly. Peripheral GHRH levels are not elevated in patients with hypothalamic GHRH-secreting tumors, supporting the notion that excess eutopic hypothalamic GHRH secretion into the hypophyseal portal system does not appreciably enter the systemic circulation. Excessive ectopic peripheral production of GHRH is present in several tumors, including carcinoid tumors, pancreatic cell tumors, small-cell lung cancers, adrenal adenomas, and pheochromocytomas, which have been reported to secrete GHRH. In these cases, peripheral GHRH levels are elevated even if acromegaly in these patients, is uncommon. For these reasons measuring GHRH plasma levels provides a precise and cost-effective test for the diagnosis of ectopic acromegaly. Elevated circulating GHRH levels, a normal or small-size pituitary gland, or clinical and biochemical features of other tumors known to be associated with extra-pituitary acromegaly, are all indications for extra-pituitary production of GHRH (18).

#### **3.7.3 Calcitonin**

30 Neuroendocrine Tumor

with extensive metastatic spread, mild diabetes, and a characteristic rash (necrolytic

Increased secretion of glucagon is also caused by other causes, like decreased plasma glucose (indirectly), increased catecholamines - norepinephrine and epinephrine, increased plasma amino acids (to protect from hypoglycemia if an all-protein meal is consumed), sympathetic nervous system, acetylcholine, cholecystokinin. Decreased secretion of glucagon is caused by somatostatin, insulin, increased free fatty acids and keto acids into the

Somatostatin is a peptide hormone that regulates the endocrine system and affects neurotransmission and cell proliferation. Somatostatin is classified as an inhibitory hormone, that exerts its effects mainly on anterior pituitary, by inhibiting the release of GH, opposing the effects of Growth Hormone-Releasing Hormone (GHRH), and inhibiting the release of thyroid-stimulating hormone (TSH) and on gastrointestinal system, by suppressing the release of other gastrointestinal and pancreatic hormones, decreasing gastric emptying, and reducing smooth muscle contractions and blood flow within the

Pancreatic somatostatinoma is a tumor of the delta cells of the endocrine pancreas that produces somatostatin. It is uncommon and symptoms are vague, thus diagnosis is frequently delayed. Clinical syndrome is characterized by a triad of : mild diabetes mellitus, steatorrhea and gallstones. Also hypochlorhydria can be associated. Patients may present with clinical endocrine syndrome due to elevated somatostatin that can cause diabetes mellitus, by inhibiting insulin secretion, steatorrhea by inhibiting cholecystokinin and secretin, gallstones by inhibiting cholecystokinin which normally induce gallbladder myocytes contraction, and hypochlorhydria caused by inhibiting gastrin, which normally stimulates acid secretion. However, more frequently patients present with symptoms related with tumor bulk. In presence of a somatostatinoma circulating somatostatin

Corticotropin-releasing hormone, a 41-amino acid peptide derived from a 191-amino acid preprohormone, acts as hormone and neurotransmitter in the stimulation of pituitary synthesis of ACTH in stress response. In normal physiology CRH is produced by parvocellular neuroendocrine cells (contained within the paraventricular nucleus of

Ectopic CRH production is rare, it may occur in patients with medullary thyroid carcinoma (about 33%) and pheochromocytoma (19%), carcinoid (5%) and small cell lung carcinoma (about 10 %) and prostate cancer (17). The main clinical feature is Cushing 's syndrome; levels of cortisol are elevated (>900 nmol/l) as ACTH, DHEA-S. Overnight administration of

concentrations may be more than 100 times the reference range (2, 5).

migratory erythema) (5).

**3.6 Somatostatin** 

intestine (2).

**3.7 Other circulating markers** 

hypothalamus).

**3.7.1 Corticotropin-releasing hormone (CRH)** 

dexamethasone doesn't suppress cortisol secretion.

blood, increased urea production (16).

Calcitonin is a 32-amino acid peptide released, in normal physiology, only in non-follicular C-cells of the thyroid . It is produced as a 136-amino acid precursor (pro-calcitonin) and processed in secretory granules to the active form. The synthesis and release of calcitonin are closely related to calcium serum levels. Calcitonin is raised in medullary thyroid cancer, where concentration may be thousands-fold the reference range. Medullary thyroid cancers frequently arise as part of multiple endocrine neoplasia type 2 (MEN2) syndrome. Calcitonin may also be raised in some pancreatic neuroendocrine tumor, especially those that are multi-hormone producing and bronchial carcinoid. Usually ectopic-produced calcitonin is a large molecule without biochemical activity (2).

#### **4. Provocative tests**

#### **4.1 Insulinoma: 72-hour fast**

NETs secreting insulin are termed insulinomas and are almost exclusively intrapancreatic in nature*.* Insulinomas secrete proinsulin, insulin and C-peptide intermittently, and, although insulin concentrations in the circulation may often be within reference range, insulin is at most times inappropriately high for the blood glucose concentration.

The diagnosis is suggested in the presence of the Whipple's triad: symptoms of hypoglycemia, glucose < 2.5 mmol/l (45 mg/dl) and relief of symptoms with administration of glucose. Hypoglycemia-induced clinical signs are classically present in the early morning pre-prandial phase or maybe exercise-induced. The typical signs are due to activation of adrenergic nervous system (palpations, sweating pallor, anxiety) and neuroglycopenia (personality changes, and loss of consciousness). The latter symptom reflects both the severity and duration of hypoglycemia. Although these symptoms are profound, they may

Circulating Markers in Gastroenteropancreatic Endocrine Tumors (GEP NETs) 33

hyperchlorhydria (e.g., Helicobacter pylori infection, gastric outlet obstruction, renal failure, antral G cell syndromes, short bowel syndrome, retained antrum) (see above

The secretin stimulation test can differentiate patients with gastrinomas from those with hypergastrinemia of different etiologies and identify those patients with gastrinoma and only mild hypergastrinemia (**Figure 2**). Secretin is normally secreted by duodenal S-cells in response to a low luminal pH following food-stimulated acid secretion. Its primary action is to cause the release of bicarbonate rich pancreatic juice from pancreatic acinar cells, thus neutralizing the acidic juice delivered from the stomach. Under physiological conditions, secretin decreases antral gastrin secretion. However in presence of a gastrinoma, secretin stimulates the release of stored gastrin by gastrinoma cells, and therefore most patients with these tumors have a dramatic rise in serum gastrin in response to a secretin infusion. In contrast, normal gastric G cells are inhibited by secretin, and therefore serum gastrin concentrations do not rise in patients with other causes of hypergastrinemia. The secretin stimulation test is performed by administering 2 U of secretin/kg body weight intravenously over one minute; a baseline serum gastrin is measured twice before the secretin is administered and 2, 5, 10, 15, and 20 minutes later. Several criteria have been proposed to define a positive test; the most commonly accepted one is a rise in serum gastrin by 200 pg/mL (95 pmol/L) or more, which is diagnostic for the presence of gastrinoma in more than 90% of cases. The use of this test has recently been re-evaluated, suggesting a lower cut-off (a rise >120 pg/mL in serum gastrin concentration) to define a positive test, thus obtaining an increase in the sensitivity (94%) without loss of specificity

Elevated fasting serum gastrin

Gastric juice pH

Gastrin < 1000 pg/mL

Provocation test (secretin)

MEN1

pH > 5

Stop Antacid drugs

Upper endoscopy with multiple biopsies

> Helicobacter Pylori ?

Atrophic gastritis

Autoimmune? (APCA)

Fig. 2. Suggested algorithm for the investigation of hypergastrinemia.

pH < 5

ZES

**Table 4**) (2).

(100%) (7, 15).

Gastrin >1000 pg/mL

Tumor localization and staging

be intermittent and diagnosis is not always straightforward. The 72-hour fast is the gold standard for diagnosing insulinoma and it attests autonomous insulin secretion and the failure of appropriate insulin suppression in the presence of hypoglycemia. In fact, a carefully supervised 72-hour starvation usually precipitates hypoglycemia within the first 36 to 48 hours. A 72-hour period is universally recognized as the most appropriate duration although some groups have proposed a shorter fast of 48 h. Symptoms appear within 12 h for one third of patients, 80% within 24 h, 90% with 48 h and approaching 100% within 72 h. Absolute values of glucose and insulin are the most important variables and any measurable insulin is abnormal when blood glucose drops to 2.5 mol/l (45 mg/dl). The patient should be monitored in a supervised environment and fasting should be accompanied by an intravenous line. Absolute blood (venous) determinations should be performed at least 2–4 times per day and bedside measurements can be used in the presence of clinical symptoms to determine if more definitive measurements should be made. Blood should also be drawn for insulin measurement concurrently with glucose estimations, and assay for insulin and Cpeptide when the hypoglycemia is confirmed. The differential diagnosis is insulin abuse (known insulin-requiring diabetes or factitious hypoglycemia): it can be detected when insulin is increased but not pro-insulin or C-peptide, so the measurement of increased proinsulin or C-peptide secures the diagnosis of insulinoma. On rare occasions the abuse of sulphonylureas and related insulin secretagogues result in a clinical picture similar to patients with insulinoma and may be diagnosed only by a positive drug screen. Even patients on regular therapy with oral hypoglycemic medications in the setting of renal impairment may show symptoms that mimic insulinoma (5, 7).

#### **4.2 Gastrinoma: Secretin test**

Gastrinoma is the second most common secretory pancreatic NET with just more than half malignant at presentation. Gastrinoma is the most common GEP NET associated with MEN1, with approximately 25-35%of gastrinomas being associated with MEN1. Gastrinomas may be found in the duodenum (50-70%) and less commonly in the pancreas (20-40%) and secrete excess of gastrin, leading to ulceration in the stomach, duodenum and the small intestine. Hydrochloric acid (HCl) also causes hyperperistalsis and inhibits the activity of lipase causing severe diarrhea. The clinical syndrome is therefore characterized by the classical triad of gastric acid hypersecretion, severe peptic ulceration, and non-beta cell islet tumor of pancreas (gastrinoma) and this is called Zollinger Ellison syndrome (ZES). Despite a clear clinical syndrome, the primary gastrinoma may be often smaller than can be visualized by any current radiological method. Therefore, circulating gastrin remains a useful tool for diagnosis. The diagnosis of ZES can be established by the demonstration of elevated fasting serum gastrin (FSG) in the presence of low gastric pH (<5.0). In fact, in the presence of gastric acid (pH<5.0), a serum gastrin value greater than 1000 pg/mL is virtually diagnostic of the disorder. However, about two-thirds of patients with the Zollinger-Ellison syndrome have serum gastrin concentrations less than 10 times the upper limit of normal (generally between 150 and 1000 pg/mL), so FSG alone is not adequate for a conclusive diagnosis of ZES. Moreover hypergastrinemia can be seen in patients with achlorhydria associated with chronic atrophic fundus gastritis (e.g., pernicious anemia), proton pump inhibitor drugs and in other conditions with

be intermittent and diagnosis is not always straightforward. The 72-hour fast is the gold standard for diagnosing insulinoma and it attests autonomous insulin secretion and the failure of appropriate insulin suppression in the presence of hypoglycemia. In fact, a carefully supervised 72-hour starvation usually precipitates hypoglycemia within the first 36 to 48 hours. A 72-hour period is universally recognized as the most appropriate duration although some groups have proposed a shorter fast of 48 h. Symptoms appear within 12 h for one third of patients, 80% within 24 h, 90% with 48 h and approaching 100% within 72 h. Absolute values of glucose and insulin are the most important variables and any measurable insulin is abnormal when blood glucose drops to 2.5 mol/l (45 mg/dl). The patient should be monitored in a supervised environment and fasting should be accompanied by an intravenous line. Absolute blood (venous) determinations should be performed at least 2–4 times per day and bedside measurements can be used in the presence of clinical symptoms to determine if more definitive measurements should be made. Blood should also be drawn for insulin measurement concurrently with glucose estimations, and assay for insulin and Cpeptide when the hypoglycemia is confirmed. The differential diagnosis is insulin abuse (known insulin-requiring diabetes or factitious hypoglycemia): it can be detected when insulin is increased but not pro-insulin or C-peptide, so the measurement of increased proinsulin or C-peptide secures the diagnosis of insulinoma. On rare occasions the abuse of sulphonylureas and related insulin secretagogues result in a clinical picture similar to patients with insulinoma and may be diagnosed only by a positive drug screen. Even patients on regular therapy with oral hypoglycemic medications in the setting of renal

Gastrinoma is the second most common secretory pancreatic NET with just more than half malignant at presentation. Gastrinoma is the most common GEP NET associated with MEN1, with approximately 25-35%of gastrinomas being associated with MEN1. Gastrinomas may be found in the duodenum (50-70%) and less commonly in the pancreas (20-40%) and secrete excess of gastrin, leading to ulceration in the stomach, duodenum and the small intestine. Hydrochloric acid (HCl) also causes hyperperistalsis and inhibits the activity of lipase causing severe diarrhea. The clinical syndrome is therefore characterized by the classical triad of gastric acid hypersecretion, severe peptic ulceration, and non-beta cell islet tumor of pancreas (gastrinoma) and this is called Zollinger Ellison syndrome (ZES). Despite a clear clinical syndrome, the primary gastrinoma may be often smaller than can be visualized by any current radiological method. Therefore, circulating gastrin remains a useful tool for diagnosis. The diagnosis of ZES can be established by the demonstration of elevated fasting serum gastrin (FSG) in the presence of low gastric pH (<5.0). In fact, in the presence of gastric acid (pH<5.0), a serum gastrin value greater than 1000 pg/mL is virtually diagnostic of the disorder. However, about two-thirds of patients with the Zollinger-Ellison syndrome have serum gastrin concentrations less than 10 times the upper limit of normal (generally between 150 and 1000 pg/mL), so FSG alone is not adequate for a conclusive diagnosis of ZES. Moreover hypergastrinemia can be seen in patients with achlorhydria associated with chronic atrophic fundus gastritis (e.g., pernicious anemia), proton pump inhibitor drugs and in other conditions with

impairment may show symptoms that mimic insulinoma (5, 7).

**4.2 Gastrinoma: Secretin test** 

hyperchlorhydria (e.g., Helicobacter pylori infection, gastric outlet obstruction, renal failure, antral G cell syndromes, short bowel syndrome, retained antrum) (see above **Table 4**) (2).

The secretin stimulation test can differentiate patients with gastrinomas from those with hypergastrinemia of different etiologies and identify those patients with gastrinoma and only mild hypergastrinemia (**Figure 2**). Secretin is normally secreted by duodenal S-cells in response to a low luminal pH following food-stimulated acid secretion. Its primary action is to cause the release of bicarbonate rich pancreatic juice from pancreatic acinar cells, thus neutralizing the acidic juice delivered from the stomach. Under physiological conditions, secretin decreases antral gastrin secretion. However in presence of a gastrinoma, secretin stimulates the release of stored gastrin by gastrinoma cells, and therefore most patients with these tumors have a dramatic rise in serum gastrin in response to a secretin infusion. In contrast, normal gastric G cells are inhibited by secretin, and therefore serum gastrin concentrations do not rise in patients with other causes of hypergastrinemia. The secretin stimulation test is performed by administering 2 U of secretin/kg body weight intravenously over one minute; a baseline serum gastrin is measured twice before the secretin is administered and 2, 5, 10, 15, and 20 minutes later. Several criteria have been proposed to define a positive test; the most commonly accepted one is a rise in serum gastrin by 200 pg/mL (95 pmol/L) or more, which is diagnostic for the presence of gastrinoma in more than 90% of cases. The use of this test has recently been re-evaluated, suggesting a lower cut-off (a rise >120 pg/mL in serum gastrin concentration) to define a positive test, thus obtaining an increase in the sensitivity (94%) without loss of specificity (100%) (7, 15).

Fig. 2. Suggested algorithm for the investigation of hypergastrinemia.

Circulating Markers in Gastroenteropancreatic Endocrine Tumors (GEP NETs) 35

concentrations in patients with pernicious anemia. This test has not been widely evaluated; however, further studies are required to determine the overall accuracy of the glucagon

Numerous biochemical markers have been identified in association with GEP-NETs, but few have the specificity or predictive value of CgA or urinary 5-HIAA, and their measurement is complex. Recent studies have proposed that alkaline phosphatase and neurokinin A are better predictors of survival in metastatic NETs than CgA but additional rigorous data to

Circulating biomarkers offer a useful diagnostic tool in conjunction with radiology and tissue pathology for NETs. However, these biomarkers are more reliable when used to monitor disease progression, response to treatment, and for early detection of recurrence

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prognostic indicators. Endocrinol Metab Clin North Am. 2010;39:777-90. [6] Conlon JM. Granin-derived peptides as diagnostic and prognostic markers for endocrine

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[8] Lawrence B, Gustafsson BI, Kidd M, Pavel M, Svejda B, Modlin IM. The clinical

neuroendocrine tumors. Endocrinol Metab Clin North Am. 2011;40:111-34. [9] Donica H, Malecha-Jędraszek A, Strosławska E, Burska A, Szubstarski F. Significance of

[10] Vezzosi D, Walter T, Laplanche A, Raoul JL, Dromain C, Ruszniewski P, d'Herbomez

biological function and clinical utility in neuro endocrine tumor disease. Ann Surg

Neuroendocrine tumors of the gastro-entero-pancreatic system. World J

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Plöckinger U; Mallorca Consensus Conference participants; European Neuroendocrine Tumor Society. ENETS Consensus Guidelines for the Standards of Care in Neuroendocrine Tumors: biochemical markers. Neuroendocrinology.

relevance of chromogranin A as a biomarker for gastroenteropancreatic

plasma chromogranin A determination in neuroendocrine tumour (NET) diagnosis.

M, Guigay J, Mitry E, Cadiot G, Leboulleux S, Lombard-Bohas C, Borson-Chazot F,

stimulation test (15).

**5. Conclusions** 

after treatment.

**6. References** 

support this assertion are required.

Oncol. 2010;17:2427-43.

Ann Clin Biochem. 2008;45:539-59.

Gastroenterol. 2008:21;14:5377-84.

tumors. Regul Pept. 2010;165:5-11.

Folia Histochem Cytobiol. 2010;48:603-10.

2009;90:194-202.

Another critical point regarding secretin test is to establish whether the the same diagnostic operative characteristics of the test remains unchanged even during treatment with inhibitors of acid secretion. In fact the standard secretin test was performed with patients off antacids and anticholinergics for at least 12 hours and H2 antagonists and proton pump inhibitors were not available at the time the test was described. However, patients with severe ZES can develop complications (such as acute bleeding and perforation) if acid suppression is discontinued. Thus, discontinuation of acid suppression should be performed cautiously. While the accuracy of the test in patients taking proton pump inhibitors (PPIs) has not been well established, some authors suggest that PPIs do not interfere with the interpretation of the secretin stimulation test and that PPIs do not need to be discontinued. However, the leading experts suggest PPIs should be discontinued for one-two weeks (1, 7, 10).

The secretin stimulation test has also been shown to be a valuable predictor of recurrence of gastrinoma following surgery (15).

#### **4.3 Other provocative tests for gastrinoma**

In addition to the secretin test, several other stimulation tests have been developed to attempt to differentiate between neoplastic and non-neoplastic causes of hypergastrinemia. These include calcium, meal and glucagon stimulation tests.

#### **4.3.1 Calcium stimulation test**

The calcium provocation test is based on the principle that calcium administration stimulates the release of stored gastrin from gastrinoma cells, as well as for secretin test. Serum gastrin concentrations are measured at 0, 30, 60, 90, 120, 150, 180 minutes intervals during an intravenous infusion of 10% calcium gluconate (5 mg/kg body weight over 3 h). More than 80% of gastrinoma patients show an increase in serum gastrin of >200 pg/mL within the third hour of calcium infusion, usually with a positive response at 120 to 180 min. The sensitivity of this test is low (about 43%), thus it can't replace the secretin test, but it could be used in patients with an high clinical suspicion of ZES and a negative secretin test (15).

#### **4.3.2 Meal stimulation test**

The physiological response to a standard test meal (of two eggs and toast) is an increase in plasma gastrin of up to two to threefold. Patients with ZES demonstrate no or only a very minimal increase in serum gastrin concentration after ingestion of such a protein rich test meal, whereas patients with other causes of hypergastrinemia show a more pronounced increase in serum gastrin following the same protein test meal. However, data on these results seems to be conflicting and a significant overlap in patients with ZES and with other antral syndromes is observed, thus the meal stimulation test for investigation of the cause of hypergastrinemia is not currently recommended (15).

#### **4.3.3 Glucagon stimulation test**

Glucagon stimulation test consists in a rise in serum gastrin concentration following the administration of glucagon to patients with ZES, but a paradoxical fall in serum gastrin concentrations in patients with pernicious anemia. This test has not been widely evaluated; however, further studies are required to determine the overall accuracy of the glucagon stimulation test (15).

### **5. Conclusions**

34 Neuroendocrine Tumor

Another critical point regarding secretin test is to establish whether the the same diagnostic operative characteristics of the test remains unchanged even during treatment with inhibitors of acid secretion. In fact the standard secretin test was performed with patients off antacids and anticholinergics for at least 12 hours and H2 antagonists and proton pump inhibitors were not available at the time the test was described. However, patients with severe ZES can develop complications (such as acute bleeding and perforation) if acid suppression is discontinued. Thus, discontinuation of acid suppression should be performed cautiously. While the accuracy of the test in patients taking proton pump inhibitors (PPIs) has not been well established, some authors suggest that PPIs do not interfere with the interpretation of the secretin stimulation test and that PPIs do not need to be discontinued. However, the leading

The secretin stimulation test has also been shown to be a valuable predictor of recurrence of

In addition to the secretin test, several other stimulation tests have been developed to attempt to differentiate between neoplastic and non-neoplastic causes of hypergastrinemia.

The calcium provocation test is based on the principle that calcium administration stimulates the release of stored gastrin from gastrinoma cells, as well as for secretin test. Serum gastrin concentrations are measured at 0, 30, 60, 90, 120, 150, 180 minutes intervals during an intravenous infusion of 10% calcium gluconate (5 mg/kg body weight over 3 h). More than 80% of gastrinoma patients show an increase in serum gastrin of >200 pg/mL within the third hour of calcium infusion, usually with a positive response at 120 to 180 min. The sensitivity of this test is low (about 43%), thus it can't replace the secretin test, but it could be used in patients with an high clinical suspicion of ZES and a negative secretin test

The physiological response to a standard test meal (of two eggs and toast) is an increase in plasma gastrin of up to two to threefold. Patients with ZES demonstrate no or only a very minimal increase in serum gastrin concentration after ingestion of such a protein rich test meal, whereas patients with other causes of hypergastrinemia show a more pronounced increase in serum gastrin following the same protein test meal. However, data on these results seems to be conflicting and a significant overlap in patients with ZES and with other antral syndromes is observed, thus the meal stimulation test for investigation of the cause of

Glucagon stimulation test consists in a rise in serum gastrin concentration following the administration of glucagon to patients with ZES, but a paradoxical fall in serum gastrin

experts suggest PPIs should be discontinued for one-two weeks (1, 7, 10).

gastrinoma following surgery (15).

**4.3.1 Calcium stimulation test** 

**4.3.2 Meal stimulation test** 

**4.3.3 Glucagon stimulation test** 

(15).

**4.3 Other provocative tests for gastrinoma** 

These include calcium, meal and glucagon stimulation tests.

hypergastrinemia is not currently recommended (15).

Numerous biochemical markers have been identified in association with GEP-NETs, but few have the specificity or predictive value of CgA or urinary 5-HIAA, and their measurement is complex. Recent studies have proposed that alkaline phosphatase and neurokinin A are better predictors of survival in metastatic NETs than CgA but additional rigorous data to support this assertion are required.

Circulating biomarkers offer a useful diagnostic tool in conjunction with radiology and tissue pathology for NETs. However, these biomarkers are more reliable when used to monitor disease progression, response to treatment, and for early detection of recurrence after treatment.

#### **6. References**


**4** 

**The Diagnosis and Management of** 

Jennifer Keiser, Emily Bergsland and Eric Nakakura

In 1907, the pathologist Siegfried Oberndorfer first coined the term "carcinoid" to describe neoplasms located in the submucosa of the ileum (Oberndorfer 1907). These small intestinal neoplasms were typically small and often multifocal. Their borders were well circumscribed. Although he initially erroneously asserted that carcinoid tumors did not

One hundred years after Oberndorfer's initial description of carcinoid tumors, the preferred term for these neoplasms is neuroendocrine tumors (NETs). Compared with other neoplasms, NETs are rare. However, the incidence of NETs is increasing, occurring in 5.25 individuals per 100,000 persons per year in the United States (Yao, Hassan et al. 2008). Neuroendocrine tumors most commonly arise from the gastrointestinal (GI) tract and bronchopulmonary (BP) tree. Histologically, NETs vary from well differentiated to poorly differentiated and are characterized by expression of neuroendocrine markers like chromogranin A and synaptophysin. Uptake of tracer using somatostatin receptor

Hormone production occurs in a minority of patients, but can cause a range of clinical syndromes, including: hypoglycemia (insulin), recurrent ulcers/diarrhea (gastrin), glucose intolerance (glucagon), watery diarrhea (vasointestinal peptide), and diarrhea, flushing, palpitations, right-sided heart valve dysfunction (serotonin). Poorly differentiated NETs are rare but can arise in nearly any location. They are associated with a poor prognosis and have a high predilection for metastases. As such, systemic chemotherapy (with a small cell lung cancer regimen) is the mainstay of therapy. Treatment for localized well differentiated tumors is surgical. Patients with advanced disease may benefit from treatment to control hormone-mediated symptoms and/or disease progression. Treatment options are evolving and include somatostatin analogs, liver-directed approaches, systemic chemotherapy, peptide receptor radionuclide therapy, interferon, and newer targeted agents (e.g. sunitinib and everolimus). The recent approval of everolimus and sunitinib in pancreatic NET specifically highlights the importance of identifying the primary site. Despite extensive

metastasize, he later recognized their malignant potential.

scintigraphy is also common in well differentiated tumors.

**1. Introduction** 

**Neuroendocrine Carcinoma** 

**of Unknown Primary** 

*University of California San Francisco* 

*Family Comprehensive Center* 

*UCSF Helen Diller* 

*USA* 

Ducreux M, Baudin E. Chromogranin A measurement in metastatic welldifferentiated gastroenteropancreatic neuroendocrine carcinoma: screening for false positives and a prospective follow-up study. Int J Biol Markers. 2011;26:94- 101.


## **The Diagnosis and Management of Neuroendocrine Carcinoma of Unknown Primary**

Jennifer Keiser, Emily Bergsland and Eric Nakakura *University of California San Francisco UCSF Helen Diller Family Comprehensive Center USA* 

#### **1. Introduction**

36 Neuroendocrine Tumor

[11] Vinik AI, Gonzales MR. New and emerging syndromes due to neuroendocrine tumors.

[12] Yao JC, Pavel M, Phan AT, Kulke MH, Hoosen S, St Peter J, Cherfi A, Oberg KE.

[13] Ramage JK, Ahmed A, Ardill J, Bax N, Breen DJ, Caplin ME, Corrie P, Davar J, Davies

[14] Guettier JM, Gorden P. Insulin secretion and insulin-producing tumors. Expert Rev

[15] Murugesan SV, Varro A, Pritchard DM. Review article: Strategies to determine whether

[16] Van Beek AP, de Haas ER, van Vloten WA, Lips CJ, Roijers JF, Canninga-van Dijk MR.

[17] Shahani S, Nudelman R. Ectopic corticotropin-releasing-hormone (CRH) syndrome

[18] Doga M, Bonadonna S, Burattin A, Giustina A. Ectopic secretion of growth hormone-

common benign cause. Aliment Pharmacol Ther. 2009;29:1055-68.

Endocrinol Metab Clin North Am. 2011;40:19-63.

tumours (NETs). Gut. 2011 Nov 3. [Epub ahead of print]

101.

12. [Epub ahead of print]

Endocrinol Metab. 2010;5:217-227.

Eur J Endocrinol 2004; 151:531-7.

Ann Oncol 2001;12 Suppl 2:S89-94.

Path 2010;5:56

Ducreux M, Baudin E. Chromogranin A measurement in metastatic welldifferentiated gastroenteropancreatic neuroendocrine carcinoma: screening for false positives and a prospective follow-up study. Int J Biol Markers. 2011;26:94-

Chromogranin A and Neuron-Specific Enolase as Prognostic Markers in Patients with Advanced pNET Treated with Everolimus. J Clin Endocrinol Metab. 2011 Oct

AH, Lewington V, Meyer T, Newell-Price J, Poston G, Reed N, Rockall A, Steward W, Thakker RV, Toubanakis C, Valle J, Verbeke C, Grossman AB. Guidelines for the management of gastroenteropancreatic neuroendocrine (including carcinoid)

hypergastrinaemia is due to Zollinger-Ellison syndrome rather than a more

The glucagonoma syndrome and necrolytic migratory erythema: a clinical review.

from metastatic small cell carcinoma: a case report and review of literature. Diagn

releasing hormone (GHRH) in neuroendocrine tumors: relevant clinical aspects.

In 1907, the pathologist Siegfried Oberndorfer first coined the term "carcinoid" to describe neoplasms located in the submucosa of the ileum (Oberndorfer 1907). These small intestinal neoplasms were typically small and often multifocal. Their borders were well circumscribed. Although he initially erroneously asserted that carcinoid tumors did not metastasize, he later recognized their malignant potential.

One hundred years after Oberndorfer's initial description of carcinoid tumors, the preferred term for these neoplasms is neuroendocrine tumors (NETs). Compared with other neoplasms, NETs are rare. However, the incidence of NETs is increasing, occurring in 5.25 individuals per 100,000 persons per year in the United States (Yao, Hassan et al. 2008). Neuroendocrine tumors most commonly arise from the gastrointestinal (GI) tract and bronchopulmonary (BP) tree. Histologically, NETs vary from well differentiated to poorly differentiated and are characterized by expression of neuroendocrine markers like chromogranin A and synaptophysin. Uptake of tracer using somatostatin receptor scintigraphy is also common in well differentiated tumors.

Hormone production occurs in a minority of patients, but can cause a range of clinical syndromes, including: hypoglycemia (insulin), recurrent ulcers/diarrhea (gastrin), glucose intolerance (glucagon), watery diarrhea (vasointestinal peptide), and diarrhea, flushing, palpitations, right-sided heart valve dysfunction (serotonin). Poorly differentiated NETs are rare but can arise in nearly any location. They are associated with a poor prognosis and have a high predilection for metastases. As such, systemic chemotherapy (with a small cell lung cancer regimen) is the mainstay of therapy. Treatment for localized well differentiated tumors is surgical. Patients with advanced disease may benefit from treatment to control hormone-mediated symptoms and/or disease progression. Treatment options are evolving and include somatostatin analogs, liver-directed approaches, systemic chemotherapy, peptide receptor radionuclide therapy, interferon, and newer targeted agents (e.g. sunitinib and everolimus). The recent approval of everolimus and sunitinib in pancreatic NET specifically highlights the importance of identifying the primary site. Despite extensive

The Diagnosis and Management of Neuroendocrine Carcinoma of Unknown Primary 39

pancreas is unlikely to account for a large fraction of NETs of unknown primary once the

Various diagnostic methods have been explored for detecting the primary site in the context of nonpancreatic, well differentiated NETs , which typically arise in the foregut, midgut or hindgut (i.e., carcinoid tumors). Published studies focusing on the diagnostic work-up are almost always limited by a small sample size. In particular, CT, somatostatin receptor scintigraphy , enteroclysis, capsule endoscopy, and magnetic resonance imaging enteroclysis have all been found to have some utility for identifying the primary tumor (Picus, Glazer et al. 1984; Sugimoto, Lorelius et al. 1995; Bader, Semelka et al. 2001; van Tuyl, Kuipers et al. 2004; Johanssen, Boivin et al. 2006). Recently, the sensitivity of diagnostic methods for locating primary tumors in a larger number of patients with well differentiated NET liver metastases was reported by Wang et al. (Wang, Parekh et al. 2010). Computed tomography (35% [n=78]) and somatostatin receptor scintigraphy (26% [n=42]) were not sensitive in detecting the primary sites. For patients with colonic NETs, colonoscopy

In the study by Wang et al., 15 patients with NET liver metastases and unknown primary tumor underwent surgical exploration, 7 of which were laparoscopic. The primary tumor was located in most (87%) patients (Wang, Parekh et al. 2010). All identified tumors were in the small intestine. They were small in diameter (1.4 cm), and more than half (54%) were mutifocal. Another study also found that surgical exploration successfully localized and resected occult primary NETs in 17 of 22 (77%) patients (Boudreaux, Putty et al. 2005). Based on these data, for patients with well differentiated (low and intermediate grade) unknown primary tumors and NET liver metastases, a multidisciplinary team assessment for possible surgical exploration and resection of occult primary tumors should be considered. Before surgery, a CT scan, somatostatin receptor scintigraphy, and upper/lower endoscopy should be done (Wang, Parekh et al. 2010). EUS may be of value in identifying the location of an

Immunohistochemical analyses using various markers are also emerging as potentially powerful tools for identification of the primary site for NETs. Expression of PAX8 or ISL1 is suggestive of a pancreatic primary tumor (Schmitt, Riniker et al. 2008; Haynes, Sangoi et al. 2011). Although CK7 and CK20 have been reported to be specific for BP- and GI-NETs, respectively (Cai, Banner et al. 2001), these findings were not corroborated by a larger study (Chu, Wu et al. 2000). TTF expression appears to be specific for BP-NETs (Agoff, Lamps et al. 2000; Oliveira, Tazelaar et al. 2001; Du, Goldstraw et al. 2004; Saqi, Alexis et al. 2005; Lin, Saad et al. 2007; Srivastava and Hornick 2009); however, its sensitivity is low and variable (Matoso, Singh et al. 2009). CDX2 is specific for GI-NETs, but it also has low and variable sensitivity and is not a good marker for pancreatic NETs (Saqi, Alexis et al. 2005; Lin, Saad et al. 2007; Srivastava and Hornick 2009). NKX2.2 is a highly sensitive and specific marker for GI-NETs, including NETs of the stomach, duodenum, ampulla of Vater, pancreas, ileum, and colon (Wang, Gallego-Arteche et al. 2009; Wang, Iezza et al. 2010). In the future, use of a

panel of markers may be particularly informative (Srivastava and Hornick 2009).

Chromosomal abnormality and molecular gene profiling may have an increasing role in identifying tumor type. Jiao et al. recently reported that mutations of DAXX/ATRX, MEN1, and mTOR pathway genes are frequent in pancreatic NETs (Jiao, Shi et al. 2011).

appropriate work-up has been done.

detected most primary tumors (87% [n=15]).

insulinoma (Khashab, Yong et al. 2011).

evaluation, some patients are diagnosed with neuroendocrine carcinoma of unknown primary, presenting a major therapeutic challenge in the face of therapies that are increasingly disease- and site-specific.

#### **2. Epidemiology**

Patients with neuroendocrine carcinoma of unknown primary present unique clinical challenges. In a recent epidemiological study, a primary tumor site was not identified in up to 4,752 (13%) of 35,618 patients with NETs (Yao, Hassan et al. 2008).

#### **3. Diagnostic methods used to identify the primary site**

Currently, there are no clear recommendations for how best to identify the primary site in patients with advanced neuroendocrine carcinoma and an elusive primary site.

#### **3.1 Rationale for identifying the primary site**

The value of identifying the primary site depends largely on the tumor's differentiation and grade. Recognizing the fact that randomized trials are lacking, patients with advanced extrapulmonary poorly differentiated or high grade (e.g. small or large cell) NETs are typically treated with a small cell lung cancer chemotherapy regimen. In contrast, identification of the primary site in patients with well differentiated (low and intermediate grade) NETs may directly influence treatment decisions and prognosis. Well differentiated NETs of the midgut are often associated with symptoms consistent with traditional carcinoid syndrome and may cause bleeding and obstruction from the primary tumor and regional adenopaty/fibrosis but tend to be relatively indolent. In contrast, rectal tumors do not typically cause hormone-mediated symptoms.

Some systemic treatments have selective use in NETs of known primary sites. Octreotide has been proven to improve outcomes in midgut carcinoids (Rinke, Muller et al. 2009), whereas the molecularly targeted agents sunitinib and everolimus are indicated for treatment of pancreatic NETs specifically (Raymond, Dahan et al. 2011; Yao, Shah et al. 2011). Furthermore, pancreatic NETs tend to be more sensitive to chemotherapy than carcinoids (Nakakura, Venook et al. 2007). Within carcinoid tumors, emerging data suggests that tumors of bronchial and thymic origin may be particular sensitive to temozolomide (Ekeblad, Sundin et al. 2007). Therefore, identification of the primary site may assist in prioritizing systemic treatment options and may shed light on the role of surgical intervention in a given patient (especially in the setting of resectable metastatic disease).

#### **3.2 Methods for identifying the primary site**

Imaging identifies most pancreatic NET primaries. In a recent study, computed tomography (CT) identified the majority of pancreatic NETs (84% [n=231]) (Khashab, Yong et al. 2011). This sensitivity was greater in nonfunctional pancreatic NETs (92% [n=173]). In the same study, 56 patients had combined CT and endoscopic ultrasound (EUS) imaging; EUS detected 91% [n=22] of pancreatic NETs (i.e., insulinomas) not identified on CT. Therefore, as most pancreatic NET primaries are found with CT, EUS, or hormone marker tests, the

evaluation, some patients are diagnosed with neuroendocrine carcinoma of unknown primary, presenting a major therapeutic challenge in the face of therapies that are

Patients with neuroendocrine carcinoma of unknown primary present unique clinical challenges. In a recent epidemiological study, a primary tumor site was not identified in up

Currently, there are no clear recommendations for how best to identify the primary site in

The value of identifying the primary site depends largely on the tumor's differentiation and grade. Recognizing the fact that randomized trials are lacking, patients with advanced extrapulmonary poorly differentiated or high grade (e.g. small or large cell) NETs are typically treated with a small cell lung cancer chemotherapy regimen. In contrast, identification of the primary site in patients with well differentiated (low and intermediate grade) NETs may directly influence treatment decisions and prognosis. Well differentiated NETs of the midgut are often associated with symptoms consistent with traditional carcinoid syndrome and may cause bleeding and obstruction from the primary tumor and regional adenopaty/fibrosis but tend to be relatively indolent. In contrast, rectal tumors do not

Some systemic treatments have selective use in NETs of known primary sites. Octreotide has been proven to improve outcomes in midgut carcinoids (Rinke, Muller et al. 2009), whereas the molecularly targeted agents sunitinib and everolimus are indicated for treatment of pancreatic NETs specifically (Raymond, Dahan et al. 2011; Yao, Shah et al. 2011). Furthermore, pancreatic NETs tend to be more sensitive to chemotherapy than carcinoids (Nakakura, Venook et al. 2007). Within carcinoid tumors, emerging data suggests that tumors of bronchial and thymic origin may be particular sensitive to temozolomide (Ekeblad, Sundin et al. 2007). Therefore, identification of the primary site may assist in prioritizing systemic treatment options and may shed light on the role of surgical intervention in a given patient (especially in the setting of resectable metastatic disease).

Imaging identifies most pancreatic NET primaries. In a recent study, computed tomography (CT) identified the majority of pancreatic NETs (84% [n=231]) (Khashab, Yong et al. 2011). This sensitivity was greater in nonfunctional pancreatic NETs (92% [n=173]). In the same study, 56 patients had combined CT and endoscopic ultrasound (EUS) imaging; EUS detected 91% [n=22] of pancreatic NETs (i.e., insulinomas) not identified on CT. Therefore, as most pancreatic NET primaries are found with CT, EUS, or hormone marker tests, the

patients with advanced neuroendocrine carcinoma and an elusive primary site.

to 4,752 (13%) of 35,618 patients with NETs (Yao, Hassan et al. 2008).

**3. Diagnostic methods used to identify the primary site** 

**3.1 Rationale for identifying the primary site** 

typically cause hormone-mediated symptoms.

**3.2 Methods for identifying the primary site** 

increasingly disease- and site-specific.

**2. Epidemiology** 

pancreas is unlikely to account for a large fraction of NETs of unknown primary once the appropriate work-up has been done.

Various diagnostic methods have been explored for detecting the primary site in the context of nonpancreatic, well differentiated NETs , which typically arise in the foregut, midgut or hindgut (i.e., carcinoid tumors). Published studies focusing on the diagnostic work-up are almost always limited by a small sample size. In particular, CT, somatostatin receptor scintigraphy , enteroclysis, capsule endoscopy, and magnetic resonance imaging enteroclysis have all been found to have some utility for identifying the primary tumor (Picus, Glazer et al. 1984; Sugimoto, Lorelius et al. 1995; Bader, Semelka et al. 2001; van Tuyl, Kuipers et al. 2004; Johanssen, Boivin et al. 2006). Recently, the sensitivity of diagnostic methods for locating primary tumors in a larger number of patients with well differentiated NET liver metastases was reported by Wang et al. (Wang, Parekh et al. 2010). Computed tomography (35% [n=78]) and somatostatin receptor scintigraphy (26% [n=42]) were not sensitive in detecting the primary sites. For patients with colonic NETs, colonoscopy detected most primary tumors (87% [n=15]).

In the study by Wang et al., 15 patients with NET liver metastases and unknown primary tumor underwent surgical exploration, 7 of which were laparoscopic. The primary tumor was located in most (87%) patients (Wang, Parekh et al. 2010). All identified tumors were in the small intestine. They were small in diameter (1.4 cm), and more than half (54%) were mutifocal. Another study also found that surgical exploration successfully localized and resected occult primary NETs in 17 of 22 (77%) patients (Boudreaux, Putty et al. 2005). Based on these data, for patients with well differentiated (low and intermediate grade) unknown primary tumors and NET liver metastases, a multidisciplinary team assessment for possible surgical exploration and resection of occult primary tumors should be considered. Before surgery, a CT scan, somatostatin receptor scintigraphy, and upper/lower endoscopy should be done (Wang, Parekh et al. 2010). EUS may be of value in identifying the location of an insulinoma (Khashab, Yong et al. 2011).

Immunohistochemical analyses using various markers are also emerging as potentially powerful tools for identification of the primary site for NETs. Expression of PAX8 or ISL1 is suggestive of a pancreatic primary tumor (Schmitt, Riniker et al. 2008; Haynes, Sangoi et al. 2011). Although CK7 and CK20 have been reported to be specific for BP- and GI-NETs, respectively (Cai, Banner et al. 2001), these findings were not corroborated by a larger study (Chu, Wu et al. 2000). TTF expression appears to be specific for BP-NETs (Agoff, Lamps et al. 2000; Oliveira, Tazelaar et al. 2001; Du, Goldstraw et al. 2004; Saqi, Alexis et al. 2005; Lin, Saad et al. 2007; Srivastava and Hornick 2009); however, its sensitivity is low and variable (Matoso, Singh et al. 2009). CDX2 is specific for GI-NETs, but it also has low and variable sensitivity and is not a good marker for pancreatic NETs (Saqi, Alexis et al. 2005; Lin, Saad et al. 2007; Srivastava and Hornick 2009). NKX2.2 is a highly sensitive and specific marker for GI-NETs, including NETs of the stomach, duodenum, ampulla of Vater, pancreas, ileum, and colon (Wang, Gallego-Arteche et al. 2009; Wang, Iezza et al. 2010). In the future, use of a panel of markers may be particularly informative (Srivastava and Hornick 2009).

Chromosomal abnormality and molecular gene profiling may have an increasing role in identifying tumor type. Jiao et al. recently reported that mutations of DAXX/ATRX, MEN1, and mTOR pathway genes are frequent in pancreatic NETs (Jiao, Shi et al. 2011).

The Diagnosis and Management of Neuroendocrine Carcinoma of Unknown Primary 41

particular, treatment with somatostatin receptor analog therapy (i.e., octreotide) should be considered, especially for those with carcinoid syndrome. The results from the PROMID study suggest that octreotide has an antitumor effect in midgut carcinoids in addition to controlling hormone-mediated symptoms--the indication for which it has FDA approval (Rinke, Muller et al. 2009). In this placebo-controlled, double-blind, prospective study, patients treated with octreotide had a 14.3 month median time to progression compared to 6 months in the placebo group (Rinke, Muller et al. 2009). Of note, the primary site was unknown in 21 patients, and these patients were considered eligible for the study; however, NETs of the lung, pancreas, and other sites were excluded. Importantly, the optimal timing of treatment in patients with nonfunctional tumors (i.e., at diagnosis or after documented disease progression) and the value specifically in unknown primary tumors remains

Unfortunately, aside from octreotide, systemic treatment options are extremely limited for advanced, nonpancreatic, well differentiated NETs (Chan and Kulke 2011; Strosberg, Cheema et al. 2011). Interferon is associated with stability and biochemical control in some cases, but radiographic responses are rare. There are little data to support cytotoxic chemotherapy in well differentiated NETs, although streptozotocin-based regimens may have limited activity at the expense of toxicity (Nakakura, Venook et al. 2007). Temozolamide-based regimens have emerged as potentially active in pancreatic and poorly differentiated NETs (Strosberg, Fine et al. 2011; Welin, Sorbye et al. 2011). Nevertheless, many believe that cytotoxic chemotherapy should be an option for patients who have failed other therapies, recognizing that no standard regimen exists (Boudreaux, Klimstra et al. 2010). Additional treatments are needed, and enrollment on clinical trials is encouraged.

Emerging data suggest that vascular endothelial growth factor (VEGF) and mTOR are valid targets for therapy in pancreatic NETs, as evidenced by the recent approval of sunitinib and everolimus in this disease (Raymond, Dahan et al. 2011; Yao, Shah et al. 2011). The value of these agents in well differentiated NETs arising outside of the pancreas is uncertain. In the RADIANT-2 study, patients with progressive nonpancreatic well differentiated NETs and a history of carcinoid syndrome were randomized to receive everolimus or placebo. While a trend towards improved progressive free survival was seen with the mTOR inhibitor, the results were not statistically significant, hence the agent is not approved for this indication. Phase III trials with a VEGF inhibitor have not been completed in carcinoid, although SWOG-0518 is ongoing (bevacizumab plus octreotide LAR vs. placebo plus octreotide LAR). For patients with clinically significant progressive disease in the liver, liver directed therapy (e.g., arterial embolization, chemoembolization, radioembolization, or ablative therapy), may be considered although randomized trials are lacking (Nakakura, Venook et al. 2007). Accumulating data with peptide receptor radionuclide therapy (PRRT) suggest that this modality might hold promise for patients with advanced NETs (Bodei, Pepe et al. 2010).

**4.2 Treatment of poorly differentiated (high grade) NETs of unknown primary** 

For patients with poorly differentiated (high grade) neuroendocrine carcinoma of unknown primary, chemotherapy with a small cell lung cancer regimen (i.e., platinum-based therapy, such as etoposide and cisplatin) should be considered (Nakakura, Venook et al. 2007;

somewhat uncertain.

Cunningham et al. suggest that chromosome 18 aberrations are common in both sporadic and familial ileal NETs (Cunningham, Diaz de Stahl et al. 2011). Recently, Erlander et al. recently reported on the use of a 92-gene real-time PCR assay for tumor classification used to compare the gene expression profile of an unknown sample to that of known tumors in a reference database (Erlander, Ma et al. 2011). This reference database includes NETs, such as small intestinal, BP, and pancreatic NETs. While these advances related to chromosome and gene profiling hold promise for the identification of unknown primary NETs, additional work is needed before these techniques can become standard of care.

#### **4. Treatment and outcome**

Tumor grade is of paramount importance in determining the treatment and outcome for patients with neuroendocrine carcinoma of unknown primary (Stoyianni, Pentheroudakis et al. 2011). As such, a precise determination of grade is essential. Additional tissue should be obtained if the initial sample is inadequate. Current data support that measures of proliferation rate are critical in determining grade (i.e., Ki67 index and mitotic counts). Therefore, grade is a standard feature of pathology reports describing NETs and forms the basis of the most recent NET pathology guidelines (Klimstra, Modlin et al. 2010).

#### **4.1 Treatment of well differentiated (low and intermediate grade) unknown primary NETs**

For patients with well differentiated (low and intermediate grade) NET liver metastases and an unknown primary tumor, a multidisciplinary evaluation for possible surgical exploration and resection of occult primary tumors should be considered. Interestingly, Dr. Oberndorfer's initial description of carcinoids included 6 patients with tumors arising from the ileum (Oberndorfer 1907). Over 100 years later, the jejunum/ileum remains one of the most common sites from which GI-NETs arise (Modlin, Shapiro et al. 2004). Despite their small size, NETs of the small intestine cause a characteristic fibrosis of the mesentery, leading to bowel obstruction, ischemia, or perforation in approximately one-third of patients (Makridis, Oberg et al. 1990; Boudreaux, Putty et al. 2005). Thus, the accepted standard of treatment for locoregional disease is resection of the small intestinal primary tumor and regional lymph nodes/fibrosis.

For a limited number of patients with liver metastases, hepatic resection appears to improve survival (Chen, Hardacre et al. 1998; Norton, Fraker et al. 2006). If complete resection of metastases is feasible, this should be considered. However, most patients are not candidates for liver resection because of extensive disease. For these patients, resection of the primary tumor could be beneficial. Two studies reported that resection of the midgut carcinoid primary tumor may be associated with improved outcome, even in the setting of unresectable liver metastases (Hellman, Lundstrom et al. 2002; Givi, Pommier et al. 2006). For carefully selected patients with unresectable NET liver metastases, some recommend that the primary tumor be localized and resected, even in asymptomatic patients (Boudreaux, Putty et al. 2005; Givi, Pommier et al. 2006; Wang, Parekh et al. 2010).

If surgery is not deemed prudent, patients with unknown primaries should be treated similarly as if they have advanced well differentiated NETs, assuming a pancreatic primary has been excluded (Nakakura, Venook et al. 2007; Stoyianni, Pentheroudakis et al. 2011). In

Cunningham et al. suggest that chromosome 18 aberrations are common in both sporadic and familial ileal NETs (Cunningham, Diaz de Stahl et al. 2011). Recently, Erlander et al. recently reported on the use of a 92-gene real-time PCR assay for tumor classification used to compare the gene expression profile of an unknown sample to that of known tumors in a reference database (Erlander, Ma et al. 2011). This reference database includes NETs, such as small intestinal, BP, and pancreatic NETs. While these advances related to chromosome and gene profiling hold promise for the identification of unknown primary NETs, additional

Tumor grade is of paramount importance in determining the treatment and outcome for patients with neuroendocrine carcinoma of unknown primary (Stoyianni, Pentheroudakis et al. 2011). As such, a precise determination of grade is essential. Additional tissue should be obtained if the initial sample is inadequate. Current data support that measures of proliferation rate are critical in determining grade (i.e., Ki67 index and mitotic counts). Therefore, grade is a standard feature of pathology reports describing NETs and forms the

basis of the most recent NET pathology guidelines (Klimstra, Modlin et al. 2010).

**4.1 Treatment of well differentiated (low and intermediate grade) unknown primary** 

For patients with well differentiated (low and intermediate grade) NET liver metastases and an unknown primary tumor, a multidisciplinary evaluation for possible surgical exploration and resection of occult primary tumors should be considered. Interestingly, Dr. Oberndorfer's initial description of carcinoids included 6 patients with tumors arising from the ileum (Oberndorfer 1907). Over 100 years later, the jejunum/ileum remains one of the most common sites from which GI-NETs arise (Modlin, Shapiro et al. 2004). Despite their small size, NETs of the small intestine cause a characteristic fibrosis of the mesentery, leading to bowel obstruction, ischemia, or perforation in approximately one-third of patients (Makridis, Oberg et al. 1990; Boudreaux, Putty et al. 2005). Thus, the accepted standard of treatment for locoregional disease is resection of the small intestinal primary tumor and

For a limited number of patients with liver metastases, hepatic resection appears to improve survival (Chen, Hardacre et al. 1998; Norton, Fraker et al. 2006). If complete resection of metastases is feasible, this should be considered. However, most patients are not candidates for liver resection because of extensive disease. For these patients, resection of the primary tumor could be beneficial. Two studies reported that resection of the midgut carcinoid primary tumor may be associated with improved outcome, even in the setting of unresectable liver metastases (Hellman, Lundstrom et al. 2002; Givi, Pommier et al. 2006). For carefully selected patients with unresectable NET liver metastases, some recommend that the primary tumor be localized and resected, even in asymptomatic patients

If surgery is not deemed prudent, patients with unknown primaries should be treated similarly as if they have advanced well differentiated NETs, assuming a pancreatic primary has been excluded (Nakakura, Venook et al. 2007; Stoyianni, Pentheroudakis et al. 2011). In

(Boudreaux, Putty et al. 2005; Givi, Pommier et al. 2006; Wang, Parekh et al. 2010).

work is needed before these techniques can become standard of care.

**4. Treatment and outcome** 

regional lymph nodes/fibrosis.

**NETs** 

particular, treatment with somatostatin receptor analog therapy (i.e., octreotide) should be considered, especially for those with carcinoid syndrome. The results from the PROMID study suggest that octreotide has an antitumor effect in midgut carcinoids in addition to controlling hormone-mediated symptoms--the indication for which it has FDA approval (Rinke, Muller et al. 2009). In this placebo-controlled, double-blind, prospective study, patients treated with octreotide had a 14.3 month median time to progression compared to 6 months in the placebo group (Rinke, Muller et al. 2009). Of note, the primary site was unknown in 21 patients, and these patients were considered eligible for the study; however, NETs of the lung, pancreas, and other sites were excluded. Importantly, the optimal timing of treatment in patients with nonfunctional tumors (i.e., at diagnosis or after documented disease progression) and the value specifically in unknown primary tumors remains somewhat uncertain.

Unfortunately, aside from octreotide, systemic treatment options are extremely limited for advanced, nonpancreatic, well differentiated NETs (Chan and Kulke 2011; Strosberg, Cheema et al. 2011). Interferon is associated with stability and biochemical control in some cases, but radiographic responses are rare. There are little data to support cytotoxic chemotherapy in well differentiated NETs, although streptozotocin-based regimens may have limited activity at the expense of toxicity (Nakakura, Venook et al. 2007). Temozolamide-based regimens have emerged as potentially active in pancreatic and poorly differentiated NETs (Strosberg, Fine et al. 2011; Welin, Sorbye et al. 2011). Nevertheless, many believe that cytotoxic chemotherapy should be an option for patients who have failed other therapies, recognizing that no standard regimen exists (Boudreaux, Klimstra et al. 2010). Additional treatments are needed, and enrollment on clinical trials is encouraged.

Emerging data suggest that vascular endothelial growth factor (VEGF) and mTOR are valid targets for therapy in pancreatic NETs, as evidenced by the recent approval of sunitinib and everolimus in this disease (Raymond, Dahan et al. 2011; Yao, Shah et al. 2011). The value of these agents in well differentiated NETs arising outside of the pancreas is uncertain. In the RADIANT-2 study, patients with progressive nonpancreatic well differentiated NETs and a history of carcinoid syndrome were randomized to receive everolimus or placebo. While a trend towards improved progressive free survival was seen with the mTOR inhibitor, the results were not statistically significant, hence the agent is not approved for this indication. Phase III trials with a VEGF inhibitor have not been completed in carcinoid, although SWOG-0518 is ongoing (bevacizumab plus octreotide LAR vs. placebo plus octreotide LAR).

For patients with clinically significant progressive disease in the liver, liver directed therapy (e.g., arterial embolization, chemoembolization, radioembolization, or ablative therapy), may be considered although randomized trials are lacking (Nakakura, Venook et al. 2007). Accumulating data with peptide receptor radionuclide therapy (PRRT) suggest that this modality might hold promise for patients with advanced NETs (Bodei, Pepe et al. 2010).

#### **4.2 Treatment of poorly differentiated (high grade) NETs of unknown primary**

For patients with poorly differentiated (high grade) neuroendocrine carcinoma of unknown primary, chemotherapy with a small cell lung cancer regimen (i.e., platinum-based therapy, such as etoposide and cisplatin) should be considered (Nakakura, Venook et al. 2007;

The Diagnosis and Management of Neuroendocrine Carcinoma of Unknown Primary 43

poorly differentiated (high grade) neuroendocrine carcinoma of unknown primary clinically resemble those with aggressive small cell lung cancer: Patients with advanced high grade tumors tend to experience an aggressive course (regardless of primary site). As such, they should be assessed for possible treatment with a small cell lung cancer regimen (i.e.,

Agoff, S. N., L. W. Lamps, et al. (2000). Thyroid transcription factor-1 is expressed in

Bader, T. R., R. C. Semelka, et al. (2001). MRI of carcinoid tumors: spectrum of appearances in the gastrointestinal tract and liver. *J Magn Reson Imaging* 14(3): 261-269. Bodei, L., G. Pepe, et al. (2010). Peptide receptor radionuclide therapy (PRRT) of

Boudreaux, J. P., D. S. Klimstra, et al. (2010). The NANETS consensus guideline for the

Boudreaux, J. P., B. Putty, et al. (2005). Surgical treatment of advanced-stage carcinoid tumors: lessons learned. *Ann Surg* 241(6): 839-845; discussion 845-836. Cai, Y. C., B. Banner, et al. (2001). Cytokeratin 7 and 20 and thyroid transcription factor 1 can

Chan, J. A. and M. H. Kulke (2011). New treatment options for patients with advanced

Chen, H., J. M. Hardacre, et al. (1998). Isolated liver metastases from neuroendocrine

Chu, P., E. Wu, et al. (2000). Cytokeratin 7 and cytokeratin 20 expression in epithelial

Cunningham, J. L., T. Diaz de Stahl, et al. (2011). Common pathogenetic mechanism

Du, E. Z., P. Goldstraw, et al. (2004). TTF-1 expression is specific for lung primary in typical

Ekeblad, S., A. Sundin, et al. (2007). Temozolomide as monotherapy is effective in treatment of advanced malignant neuroendocrine tumors. *Clin Cancer Res* 13(10): 2986-2991. Erlander, M. G., X. J. Ma, et al. (2011). Performance and clinical evaluation of the 92-gene real-time PCR assay for tumor classification. *J Mol Diagn* 13(5): 493-503. Givi, B., S. J. Pommier, et al. (2006). Operative resection of primary carcinoid neoplasms in

neuroendocrine tumors. *Curr Treat Options Oncol* 12(2): 136-148.

neoplasms: a survey of 435 cases. *Mod Pathol* 13(9): 962-972.

extrapulmonary small cell carcinomas but not in other extrapulmonary

neuroendocrine tumors with somatostatin analogues. *Eur Rev Med Pharmacol Sci* 

diagnosis and management of neuroendocrine tumors: well-differentiated neuroendocrine tumors of the Jejunum, Ileum, Appendix, and Cecum. *Pancreas*

help distinguish pulmonary from gastrointestinal carcinoid and pancreatic

tumors: does resection prolong survival? *J Am Coll Surg* 187(1): 88-92; discussion

involving human chromosome 18 in familial and sporadic ileal carcinoid tumors.

and atypical carcinoids: TTF-1-positive carcinoids are predominantly in peripheral

patients with liver metastases yields significantly better survival. *Surgery* 140(6):

platinum- or irinotecan-based chemotherapy regimens).

neuroendocrine tumors. *Mod Pathol* 13(3): 238-242.

endocrine tumors. *Hum Pathol* 32(10): 1087-1093.

*Genes Chromosomes Cancer* 50(2): 82-94.

location. *Hum Pathol* 35(7): 825-831.

891-897; discussion 897-898.

**6. References** 

14(4): 347-351.

39(6): 753-766.

92-83.

Stoyianni, Pentheroudakis et al. 2011). A study of etoposide and cisplatin in patients with poorly differentiated NETs had an objective response rate of 41.5%, an 8.9 month median progression-free survival, and 15 month median overall survival (Mintry, Baudin et al. 1999). Other regimens include carboplatin- and irinotecan- based combinations, which are also small cell lung cancer regimens (Strosberg, Coppola et al. 2010). These tumors carry a poor prognosis.

#### **4.3 Outcome of well differentiated (low and intermediate grade) unknown primary tumors**

Patients with well differentiated neuroendocrine carcinoma of unknown primary may exhibit a relatively indolent course similar to those with advanced well differentiated NETs arising from the GI tract and BP tree (Stoyianni, Pentheroudakis et al. 2011). In a study by Kirshborn et al., patients with advanced midgut NETs (i.e., arising from the jejunum, ileum, and cecum) and those with neuroendocrine carcinoma of unknown primary shared similar serotonin levels as assessed by urine 24 hours hydroxyindoleacetic acid and serotonin levels, as well as by serum and platelet serotonin levels (Kirshbom, Kherani et al. 1998). Moreover, patients with advanced midgut NETs and neuroendocrine carcinoma of unknown primary shared similar 10-year survival rates (28% and 22%, respectively).

In a larger study, Yao et al. analyzed the outcomes of nearly 36,000 patients with NETs and found that primary site was probably the most useful predictor of outcome for patients with well differentiated NETs (Yao, Hassan et al. 2008). Patients with a liver primary and localized disease (i.e., disease confined to the liver) had a 5-year survival rate of 43%; this compared favorably with a 5-year survival rate of 54% for patients with a jejunum/ileum primary and distant disease. Not surprisingly, for patients with a liver primary and distant disease (i.e., widely metastatic disease), the prognosis was poor with a 0% survival rate at 10-years.

#### **4.4 Outcome of poorly differentiated (high grade) NETs of unknown primary**

Patients with poorly differentiated (high grade) neuroendocrine carcinoma of unknown primary clinically resemble those with aggressive small cell lung cancer (Nakakura, Venook et al. 2007; Stoyianni, Pentheroudakis et al. 2011). That is, their disease frequently responds to platinum-based therapy but inevitably becomes refractory. These patients have a poor prognosis with a median survival typically less than 1 year.

#### **5. Conclusion**

The diagnosis and management of patients with neuroendocrine carcinoma of unknown primary is challenging. Tumor grade directs treatment and is prognostic. Patients with well differentiated (low and intermediate grade) NETs may benefit from identification of the primary site because it may influence prognosis, but also because the available treatments we have are increasingly site-specific (e.g. octreotide for midgut tumors, and chemotherapy, everolimus and sunitinib for pancreatic NETs). In some settings, the type and timing of surgical intervention may also be dictated by the primary site. In contrast, patients with poorly differentiated (high grade) neuroendocrine carcinoma of unknown primary clinically resemble those with aggressive small cell lung cancer: Patients with advanced high grade tumors tend to experience an aggressive course (regardless of primary site). As such, they should be assessed for possible treatment with a small cell lung cancer regimen (i.e., platinum- or irinotecan-based chemotherapy regimens).

#### **6. References**

42 Neuroendocrine Tumor

Stoyianni, Pentheroudakis et al. 2011). A study of etoposide and cisplatin in patients with poorly differentiated NETs had an objective response rate of 41.5%, an 8.9 month median progression-free survival, and 15 month median overall survival (Mintry, Baudin et al. 1999). Other regimens include carboplatin- and irinotecan- based combinations, which are also small cell lung cancer regimens (Strosberg, Coppola et al. 2010). These tumors carry a

**4.3 Outcome of well differentiated (low and intermediate grade) unknown primary** 

Patients with well differentiated neuroendocrine carcinoma of unknown primary may exhibit a relatively indolent course similar to those with advanced well differentiated NETs arising from the GI tract and BP tree (Stoyianni, Pentheroudakis et al. 2011). In a study by Kirshborn et al., patients with advanced midgut NETs (i.e., arising from the jejunum, ileum, and cecum) and those with neuroendocrine carcinoma of unknown primary shared similar serotonin levels as assessed by urine 24 hours hydroxyindoleacetic acid and serotonin levels, as well as by serum and platelet serotonin levels (Kirshbom, Kherani et al. 1998). Moreover, patients with advanced midgut NETs and neuroendocrine carcinoma of unknown primary shared similar 10-year survival rates (28% and 22%,

In a larger study, Yao et al. analyzed the outcomes of nearly 36,000 patients with NETs and found that primary site was probably the most useful predictor of outcome for patients with well differentiated NETs (Yao, Hassan et al. 2008). Patients with a liver primary and localized disease (i.e., disease confined to the liver) had a 5-year survival rate of 43%; this compared favorably with a 5-year survival rate of 54% for patients with a jejunum/ileum primary and distant disease. Not surprisingly, for patients with a liver primary and distant disease (i.e., widely metastatic disease), the prognosis was poor with a 0% survival rate at

Patients with poorly differentiated (high grade) neuroendocrine carcinoma of unknown primary clinically resemble those with aggressive small cell lung cancer (Nakakura, Venook et al. 2007; Stoyianni, Pentheroudakis et al. 2011). That is, their disease frequently responds to platinum-based therapy but inevitably becomes refractory. These patients have a poor

The diagnosis and management of patients with neuroendocrine carcinoma of unknown primary is challenging. Tumor grade directs treatment and is prognostic. Patients with well differentiated (low and intermediate grade) NETs may benefit from identification of the primary site because it may influence prognosis, but also because the available treatments we have are increasingly site-specific (e.g. octreotide for midgut tumors, and chemotherapy, everolimus and sunitinib for pancreatic NETs). In some settings, the type and timing of surgical intervention may also be dictated by the primary site. In contrast, patients with

**4.4 Outcome of poorly differentiated (high grade) NETs of unknown primary** 

prognosis with a median survival typically less than 1 year.

poor prognosis.

respectively).

10-years.

**5. Conclusion** 

**tumors** 


The Diagnosis and Management of Neuroendocrine Carcinoma of Unknown Primary 45

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metastatic pulmonary from well-differentiated neuroendocrine tumors of other


*Istanbul University/Cerrahpasa Medical Faculty,Department of Internal Medicine,* 

Gastrointestinal carcinoid tumors date back to 1888 when Otto Lubarsch found multiple tumors in the distal ileum of two patients at autopsy (Lubarsch, 1888). The German pathologist Siegfried Oberndorfer was the first who used the term "Karzinoide Tumoren", the English translation of which is "carcinoid tumor". He coined this term because these tumors behaved less aggressively than true carcinomas (Oberndorfer, 1907). Oberndorfer contributed to the Department of Pathology of Istanbul University where he remained full professor and director until his death in 1944 (Dogan & Hot, 2010; Klöppel et al., 2007). In 1914, Gosset and Masson first mentioned neuroendocrine features of carcinoid tumors. In 1954, Thorson and co-workers described the term "carcinoid syndrome" after they found that the tumors contained serotonin and patients with small intestinal carcinoid tumor and liver metastases presented with the characteristic symptoms of diarrhea, flushing, asthma, and right heart failure (Thorson et al., 1954). It was soon recognized as a distinct entity after they were reported in several organs belonging to bronchopulmonary system and gastrointestinal tract (Yalcin, 2006). Although better named as "neuroendocrine tumors" after the WHO meeting in 2000, these tumors have been traditionally called "carcinoids" especially at some specific locations such as the gastrointestinal tract. Therefore, the terms "carcinoid tumor" and "neuroendocrine tumor" are

Although it might not reflect the true incidence due to their indolent nature, the overall incidence of carcinoid tumors in the United.States has been estimated to be 1 to 2 cases per 100,000 people (Godwin, 1975; Modlin & Sandor, 1997). Yao and co-workers reviewed 35,825 neuroendocrine tumors (NETs) compiled from 1973 to 2004 (Yao et al., 2008). According to this review, 41% was foregut NETs, 26% midgut NETs, and 19% hindgut NETs. In the remaining 13%, the primary tumor site was unknown or could not be classified. These figures were similar to our institution's patient cohort (Yildiz et al., 2009). In Sweden, combined with the autopsy series the incidence has been calculated to be 8.4 cases per 100,000 people. Based on the data from the End Results Group (1950-1969) and the Third National Cancer Survey (1969-1971) the most common site of carcinoid tumors was appendix followed by the rectum, ileum, lung and bronchi, and stomach (Godwin, 1975). Pancreatic endocrine tumors are relatively less frequent and account for 2% of malignant

**1. Introduction** 

used interchangeably throughout the text.

**2. Epidemiology** 

Ozcan Yildiz and Suheyla Serdengecti

*Division of Medical Oncology Istanbul,* 

*Turkey* 

Yao, J. C., M. H. Shah, et al. (2011). Everolimus for advanced pancreatic neuroendocrine tumors. *N Engl J Med* 364(6): 514-523. **5** 

Ozcan Yildiz and Suheyla Serdengecti

*Istanbul University/Cerrahpasa Medical Faculty,Department of Internal Medicine, Division of Medical Oncology Istanbul, Turkey* 

#### **1. Introduction**

46 Neuroendocrine Tumor

Yao, J. C., M. H. Shah, et al. (2011). Everolimus for advanced pancreatic neuroendocrine

Gastrointestinal carcinoid tumors date back to 1888 when Otto Lubarsch found multiple tumors in the distal ileum of two patients at autopsy (Lubarsch, 1888). The German pathologist Siegfried Oberndorfer was the first who used the term "Karzinoide Tumoren", the English translation of which is "carcinoid tumor". He coined this term because these tumors behaved less aggressively than true carcinomas (Oberndorfer, 1907). Oberndorfer contributed to the Department of Pathology of Istanbul University where he remained full professor and director until his death in 1944 (Dogan & Hot, 2010; Klöppel et al., 2007). In 1914, Gosset and Masson first mentioned neuroendocrine features of carcinoid tumors. In 1954, Thorson and co-workers described the term "carcinoid syndrome" after they found that the tumors contained serotonin and patients with small intestinal carcinoid tumor and liver metastases presented with the characteristic symptoms of diarrhea, flushing, asthma, and right heart failure (Thorson et al., 1954). It was soon recognized as a distinct entity after they were reported in several organs belonging to bronchopulmonary system and gastrointestinal tract (Yalcin, 2006). Although better named as "neuroendocrine tumors" after the WHO meeting in 2000, these tumors have been traditionally called "carcinoids" especially at some specific locations such as the gastrointestinal tract. Therefore, the terms "carcinoid tumor" and "neuroendocrine tumor" are used interchangeably throughout the text.

#### **2. Epidemiology**

Although it might not reflect the true incidence due to their indolent nature, the overall incidence of carcinoid tumors in the United.States has been estimated to be 1 to 2 cases per 100,000 people (Godwin, 1975; Modlin & Sandor, 1997). Yao and co-workers reviewed 35,825 neuroendocrine tumors (NETs) compiled from 1973 to 2004 (Yao et al., 2008). According to this review, 41% was foregut NETs, 26% midgut NETs, and 19% hindgut NETs. In the remaining 13%, the primary tumor site was unknown or could not be classified. These figures were similar to our institution's patient cohort (Yildiz et al., 2009). In Sweden, combined with the autopsy series the incidence has been calculated to be 8.4 cases per 100,000 people. Based on the data from the End Results Group (1950-1969) and the Third National Cancer Survey (1969-1971) the most common site of carcinoid tumors was appendix followed by the rectum, ileum, lung and bronchi, and stomach (Godwin, 1975). Pancreatic endocrine tumors are relatively less frequent and account for 2% of malignant

specific TNM classifications and a grading system, which improves prognostic and

**Secretory products of carcinoid tumors Frequency** (%)

5-Hydroxytrypta mine - 5-Hydroxyindoleacetic acid 88 5-Hydroxytryptophan - 5-Hydroxyindoleacetaldehyde - Histamine - Dopamine - *Tachykinins* - Kallikrein - Substance P 32 Neuropeptide K 66

Pancreatic polypeptide 43 Chromogranins 100 Neurotensin 41 Human chorionic gonadotrophin-alpha 28 Human chorionic gonadotropin-beta 12 Motilin 14 Pancreastatin -

Prostaglandins -

Table 1. Secretory products and known frequency of carcinoid tumors

**Foregut Midgut Hindgut**

CgA-positive CgA-positive CgA-positive NSE-positive NSE-positive NSE-positive

CgA: Chromogranin A; SVP-2: Seminal vesicle protein-2; NSE: neuron specific enolase Table 2. Immunestaining of carcinoid tumors regarding their embryonic origin

Synaptophysin-positive Synaptophysin-positive Synaptophysin-positive

Well-differentiated endocrine tumors may exhibit uncertain behavior. They may be angioinvasive. They may also produce serotonin and enteroglucagon. Another subset is well-differentiated endocrine carcinomas once termed "atypical" or "anaplastic". They are subdivided into low-grade and high-grade malignant carcinoids. Low-grade malignant carcinoids are deeply invasive (muscularis propria or beyond) and serotonin-producing

*Argyrophilic Argentaf:*

(O'Dorisio, 2011; Vinik et al., 2008; Norheim et al., 1987; Feldmann & O'Dorisio 1986; Eriksson et al., 1990)

*fin-positive Argyrophilic*

SVP-2-positive

treatment stratification (Schott et al., 2011).

*Amines*

*Peptides*

*Other*

tumors of the gastrointestinal tract. Recent data obtained from the National Cancer Institute Surveillance, Epidemiology, and End Results program (1973 to 1997) showed that, of the 11,427 cases analyzed, the overall incidence rates for carcinoid tumors have increased significantly over the past 25 years, although rates for some sites have decreased (e.g., appendix). The gastrointestinal tract accounted for 54.5% of the tumors. Within the gastrointestinal tract, the small intestine was the most common site (44.7%), followed by the rectum (19.6%), appendix (16.7%), colon (10.6%), and stomach (7.2%). The average age was 60.9 years, and 54.2% were female. The 5-year survival rates for the most common gastrointestinal sites were 75.1% for stomach, 76.1% for small intestine, 76.3% for appendix, and 87.5% for rectum (Maggard et al., 2004).

### **3. Pathology and classification**

Carcinoid tumors are thought to arise primarily from the neuroendocrine cells of the lung and the gastrointestinal tract. In the past, these cells were called "Kulchitsky cells" or "enterochromaffin cells" due to the affinity for soluble silver salts, hence the term argentaffinomas. Although not prognostically useful, historically, carcinoid tumors have been classified according to their embryologic derivation within the primitive gut (Williams & Sandler, 1963): Foregut, midgut, and hindgut carcinoids. Foregut carcinoids include intrathoracic (thymic and bronchial), gastric, esophageal, upper duodenal, and pancreatic carcinoids. They usually produce low levels of serotonin, 5-hydroxytryptophan (5-HTP), histamine, or adrenocorticotropic hormone. Midgut carcinoids include carcinoids of distal duodenum, jejunum, ileum, appendix, proximal colon, liver, ovary, or testes. These tumors have the propensity of producing serotonin at high levels. Finally, hindgut carcinoids include carcinoids of distal colon and rectum. They less likely produce serotonin but may produce somatostatin, peptide YY, 5-HTP, or other hormones (Table 1).

Useful immunohistochemical markers in modern pathology include neuron-specific enolase (NSE), synapthophysin and chromogranin A (CgA) which is also a secretory product of the carcinoid cells used to monitor disease activity (Table 2). On gross examination, carcinoid tumors are small yellow nodules located submucosally. When they invade the serosa, an intense desmoplastic reaction occurs that may lead to intestinal kinking and obstruction.

The WHO 1980 classification had taken into account the histologic features apart from the site of origin (Bosman et al., 2010; table 3). According to this revised system, welldifferentiated neuroendocrine tumors are typical tumors that show characteristic growth pattern and benign behavior. They are indolent tumors, confined to mucosa or submucosa, and less than 1 to 2 cm in diameter.

In the WHO 2000 classification, features such as differentiation and proliferation were retained, but location, tumor size, tumor extent, and angioinvasion were transferred into the TNM (Tumor, Node, Metastasis) staging classification. The WHO 2010 classification states that all GEP-NETs are potentially malignant, but differ in their ability of metastasis (Rindi et al., 2010). Well-differentiated NETs are classified together as neuroendocrine tumors (NETs) G1 or G2. NET G1 is analogous with carcinoid. The term neuroendocrine carcinoma (NEC), unlike NET, refers to all poorly differentiated NETs. NEC is further subdivided into a smallcell and a large-cell variant. In respect of proliferation, all NECs are actively proliferating G3 tumors. Mixed adenoneuroendocrine carcinomas (MANEC) and hyperplastic and preneoplastic lesions are special groups. This classification is complemented by GEP-NET-

tumors of the gastrointestinal tract. Recent data obtained from the National Cancer Institute Surveillance, Epidemiology, and End Results program (1973 to 1997) showed that, of the 11,427 cases analyzed, the overall incidence rates for carcinoid tumors have increased significantly over the past 25 years, although rates for some sites have decreased (e.g., appendix). The gastrointestinal tract accounted for 54.5% of the tumors. Within the gastrointestinal tract, the small intestine was the most common site (44.7%), followed by the rectum (19.6%), appendix (16.7%), colon (10.6%), and stomach (7.2%). The average age was 60.9 years, and 54.2% were female. The 5-year survival rates for the most common gastrointestinal sites were 75.1% for stomach, 76.1% for small intestine, 76.3% for appendix,

Carcinoid tumors are thought to arise primarily from the neuroendocrine cells of the lung and the gastrointestinal tract. In the past, these cells were called "Kulchitsky cells" or "enterochromaffin cells" due to the affinity for soluble silver salts, hence the term argentaffinomas. Although not prognostically useful, historically, carcinoid tumors have been classified according to their embryologic derivation within the primitive gut (Williams & Sandler, 1963): Foregut, midgut, and hindgut carcinoids. Foregut carcinoids include intrathoracic (thymic and bronchial), gastric, esophageal, upper duodenal, and pancreatic carcinoids. They usually produce low levels of serotonin, 5-hydroxytryptophan (5-HTP), histamine, or adrenocorticotropic hormone. Midgut carcinoids include carcinoids of distal duodenum, jejunum, ileum, appendix, proximal colon, liver, ovary, or testes. These tumors have the propensity of producing serotonin at high levels. Finally, hindgut carcinoids include carcinoids of distal colon and rectum. They less likely produce serotonin but may

Useful immunohistochemical markers in modern pathology include neuron-specific enolase (NSE), synapthophysin and chromogranin A (CgA) which is also a secretory product of the carcinoid cells used to monitor disease activity (Table 2). On gross examination, carcinoid tumors are small yellow nodules located submucosally. When they invade the serosa, an intense desmoplastic reaction occurs that may lead to intestinal kinking and obstruction.

The WHO 1980 classification had taken into account the histologic features apart from the site of origin (Bosman et al., 2010; table 3). According to this revised system, welldifferentiated neuroendocrine tumors are typical tumors that show characteristic growth pattern and benign behavior. They are indolent tumors, confined to mucosa or submucosa,

In the WHO 2000 classification, features such as differentiation and proliferation were retained, but location, tumor size, tumor extent, and angioinvasion were transferred into the TNM (Tumor, Node, Metastasis) staging classification. The WHO 2010 classification states that all GEP-NETs are potentially malignant, but differ in their ability of metastasis (Rindi et al., 2010). Well-differentiated NETs are classified together as neuroendocrine tumors (NETs) G1 or G2. NET G1 is analogous with carcinoid. The term neuroendocrine carcinoma (NEC), unlike NET, refers to all poorly differentiated NETs. NEC is further subdivided into a smallcell and a large-cell variant. In respect of proliferation, all NECs are actively proliferating G3 tumors. Mixed adenoneuroendocrine carcinomas (MANEC) and hyperplastic and preneoplastic lesions are special groups. This classification is complemented by GEP-NET-

produce somatostatin, peptide YY, 5-HTP, or other hormones (Table 1).

and 87.5% for rectum (Maggard et al., 2004).

**3. Pathology and classification** 

and less than 1 to 2 cm in diameter.

specific TNM classifications and a grading system, which improves prognostic and treatment stratification (Schott et al., 2011).


(O'Dorisio, 2011; Vinik et al., 2008; Norheim et al., 1987; Feldmann & O'Dorisio 1986; Eriksson et al., 1990)

Table 1. Secretory products and known frequency of carcinoid tumors


CgA: Chromogranin A; SVP-2: Seminal vesicle protein-2; NSE: neuron specific enolase

Table 2. Immunestaining of carcinoid tumors regarding their embryonic origin

Well-differentiated endocrine tumors may exhibit uncertain behavior. They may be angioinvasive. They may also produce serotonin and enteroglucagon. Another subset is well-differentiated endocrine carcinomas once termed "atypical" or "anaplastic". They are subdivided into low-grade and high-grade malignant carcinoids. Low-grade malignant carcinoids are deeply invasive (muscularis propria or beyond) and serotonin-producing

Thymic carcinoids are rare and may be part of multiple endocrine neoplasia type 1 (MEN1). The tumor is usually non-functioning and occurs as an anterior mediastinal mass. The mass may be partly calcified and may cause superior vena cava obstruction. Extension into the pleura, pericardium, great vessels, or regional lymph nodes is commonly seen. Functioning thymic carcinoids usually secrete adrenocorticotrophic hormone (ACTH), which may lead to Cushing's syndrome. In these cases bilateral adrenal hyperplasia may also be seen. Other hormones include corticotrophin-releasing hormone, growth hormone-releasing hormone (GHRH) and 5-HT. Carcinoid syndrome has been described with a multidirectional carcinoma of the thymus with neuroendocrine and sarcomatous components. (Paties et al.,

Pulmonary carcinoid tumors comprise about 2% of primary lung tumors. They are believed to arise from neuroendocrine Kulchitsky's cells of the proximal bronchial mucosa, and bronchopulmonary tract is the second common site of carcinoid tumors after the gastrointestinal system. They have been classified into three groups, according to their malignant potential: Benign or classical bronchial carcinoid, low-grade malignant or atypical carcinoid, and high-grade malignant or poorly differentiated carcinoma of the large cell or

Neuroendocrine tumors of the lung can also be divided into 5 groups: Tumorlet, diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) (Nassar et al., 2011), carcinoid tumor, atypical carcinoid tumor, and small cell carcinoma of the lung. Typical carcinoids of the lung present in the fifth decade of life and are more common in females. These tumors are not related to smoking. Neuroendocrine manifestations are relatively uncommon. Common symptoms are cough, hemoptysis, wheezing, and chest pain (Table 4). Recurrent pneumonia can occur due to obstructing lesions. Cushing's syndrome and acromegaly may be seen due to the secretion of ACTH and GHRH, respectively. The prognosis is excellent for classical bronchial carcinoids. Diagnostic tools for pulmonary carcinoids are generally non-specific. Bronchoscopic biopsy should be used to obtain

Usually found incidentally

Usually found incidentally

Bowel obstruction

Rectal carcinoids Bleeding, pain, constipation, carcinoid syndrome

Cough, hemoptysis, wheezing, chest pain, pneumonia

Nonspecific abdominal pain, carcinoid syndrome

diagnostic tissue, despite the feared complication of hemorrhage.

**Carcinoid tumors Clinical features**

Table 4. Clinical features of neuroendocrine tumors

**5.1.1 Thymic carcinoids** 

**5.1.2 Pulmonary carcinoids** 

1991)

small cell type.

*Foregut*

*Midgut* 

*Hindgut* 

Bronchial carcinoids Gastric carcinoids

Small intestinal carcinoids Appendiceal carcinoids

when metastatic disease develops. High-grade malignant carcinoids, on the other hand, are small to intermediate carcinomas. Appendiceal and rectal carcinoids are rarely malignant, whereas ileal, type III gastric, and colonic ones are frequently malignant. Size is also a predictor of the malignant potential of the carcinoid tumor. More than 66% of carcinoids greater than 2 cm exhibit distant spread when first diagnosed (Capella et al., 1995).


Table 3. Comparison of the WHO classifications of gastroenteropancreatic neuroendocrine tumors. Bosman FT, et al., WHO classification of tumors of the digestive system. Lyon, France: IARC Press; 2010.

#### **4. Clinical chemistry**

Clinically functioning carcinoid tumors produce typical syndromes according to the specific circulating peptide. One of the best-characterized of these peptides is serotonin (5-HT). It is synthesized from 5-hydroxytryptophan (5-HTP) and metabolized to 5-hydroxyindoleacetic acid (5-HIAA) which is excreted in the urine. Measurement of 5-HIAA in the urine is an important tumor marker in the carcinoid syndrome, especially in midgut carcinoids. Recently, measurements of serotonin in platelets have been recommended for the detection of carcinoid tumors that secrete only small amounts of serotonin and/or its precursor 5 hydroxytryptophan (Kema et al., 2000). Tumors originating from foregut carcinoids (lung, pancreas, and stomach) may have relatively low levels of 5-HTP decarboxylase. 5-HTP, 5- HT, and 5-HIAA are excreted in the urine.

#### **5. Clinical features of individual carcinoids**

#### **5.1 Foregut carcinoids**

For the sake of completeness we will discuss thymic and pulmonary carcinoids in the following sections.

#### **5.1.1 Thymic carcinoids**

50 Neuroendocrine Tumor

when metastatic disease develops. High-grade malignant carcinoids, on the other hand, are small to intermediate carcinomas. Appendiceal and rectal carcinoids are rarely malignant, whereas ileal, type III gastric, and colonic ones are frequently malignant. Size is also a predictor of the malignant potential of the carcinoid tumor. More than 66% of carcinoids

> **WHO 2000**  1. Well-differentiated differentiated endocrine tumor

**WHO 2010**  1. Neuroendocrine tumor G1 (Carcinoid)

2. NET G2

5. Mixed

carcinoma

7. Hyperplastic and preneoplastic lesion

3. Small or large-cell neuroendocrine carcinoma G3 (NEC)

adenoneuroendocrine

carcinoma (WDEC)

carcinoma (PDEC)

differentiated (small-cell) endocrine

4. Mixed endocrineexocrine carcinoma

6. Tumor-like lesions

Table 3. Comparison of the WHO classifications of gastroenteropancreatic neuroendocrine tumors. Bosman FT, et al., WHO classification of tumors of the digestive system. Lyon,

Clinically functioning carcinoid tumors produce typical syndromes according to the specific circulating peptide. One of the best-characterized of these peptides is serotonin (5-HT). It is synthesized from 5-hydroxytryptophan (5-HTP) and metabolized to 5-hydroxyindoleacetic acid (5-HIAA) which is excreted in the urine. Measurement of 5-HIAA in the urine is an important tumor marker in the carcinoid syndrome, especially in midgut carcinoids. Recently, measurements of serotonin in platelets have been recommended for the detection of carcinoid tumors that secrete only small amounts of serotonin and/or its precursor 5 hydroxytryptophan (Kema et al., 2000). Tumors originating from foregut carcinoids (lung, pancreas, and stomach) may have relatively low levels of 5-HTP decarboxylase. 5-HTP, 5-

For the sake of completeness we will discuss thymic and pulmonary carcinoids in the

(MEEC)

(TLL)

greater than 2 cm exhibit distant spread when first diagnosed (Capella et al., 1995).

(WDET) 2. Well-differentiated endocrine

3. Poorly

**WHO 1980** 

I. Carcinoid

II. Mucocarcinoid III. Mixed carcinoid-

IV. Pseudotumorous lesion

France: IARC Press; 2010.

**4. Clinical chemistry** 

**5.1 Foregut carcinoids** 

following sections.

adenocarcinoma

HT, and 5-HIAA are excreted in the urine.

**5. Clinical features of individual carcinoids** 

Thymic carcinoids are rare and may be part of multiple endocrine neoplasia type 1 (MEN1). The tumor is usually non-functioning and occurs as an anterior mediastinal mass. The mass may be partly calcified and may cause superior vena cava obstruction. Extension into the pleura, pericardium, great vessels, or regional lymph nodes is commonly seen. Functioning thymic carcinoids usually secrete adrenocorticotrophic hormone (ACTH), which may lead to Cushing's syndrome. In these cases bilateral adrenal hyperplasia may also be seen. Other hormones include corticotrophin-releasing hormone, growth hormone-releasing hormone (GHRH) and 5-HT. Carcinoid syndrome has been described with a multidirectional carcinoma of the thymus with neuroendocrine and sarcomatous components. (Paties et al., 1991)

#### **5.1.2 Pulmonary carcinoids**

Pulmonary carcinoid tumors comprise about 2% of primary lung tumors. They are believed to arise from neuroendocrine Kulchitsky's cells of the proximal bronchial mucosa, and bronchopulmonary tract is the second common site of carcinoid tumors after the gastrointestinal system. They have been classified into three groups, according to their malignant potential: Benign or classical bronchial carcinoid, low-grade malignant or atypical carcinoid, and high-grade malignant or poorly differentiated carcinoma of the large cell or small cell type.

Neuroendocrine tumors of the lung can also be divided into 5 groups: Tumorlet, diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) (Nassar et al., 2011), carcinoid tumor, atypical carcinoid tumor, and small cell carcinoma of the lung. Typical carcinoids of the lung present in the fifth decade of life and are more common in females. These tumors are not related to smoking. Neuroendocrine manifestations are relatively uncommon. Common symptoms are cough, hemoptysis, wheezing, and chest pain (Table 4). Recurrent pneumonia can occur due to obstructing lesions. Cushing's syndrome and acromegaly may be seen due to the secretion of ACTH and GHRH, respectively. The prognosis is excellent for classical bronchial carcinoids. Diagnostic tools for pulmonary carcinoids are generally non-specific. Bronchoscopic biopsy should be used to obtain diagnostic tissue, despite the feared complication of hemorrhage.


Table 4. Clinical features of neuroendocrine tumors

decade of life with mechanical obstruction or vague abdominal pain, sometimes for several years before the diagnosis perhaps due to their submucosal location. These tumors are thought to originate from serotonin-producing intraepithelial endocrine cells and they tend to have high serotonin content. They are multicentric and the most frequent location is the distal ileum. Lymph node and liver metastasis are common but carcinoid syndrome is seen only in 5 to 7% of the patients (Burke et al., 1997; Bilimoria et al., 2009). Metastases tend to occur in liver, bone and lung. Unlike appendiceal carcinoids, smaller tumors have also been found to have metastasized. Nevertheless, tumors measuring 2 cm in diameter have almost

Based on patients, registered in the Swedish Cancer Registry, with small intestinal carcinoids diagnosed from 1960 to 2000 in the duodenum (n= 89) and jejunum/ileum (n=2437), the overall 5-, 10-, and 15 year survivals were, respectively, 60, 46, and 28% for

Carcinoid tumors are the most common cancers of the appendix (Moertel et al., 1968) originating from subepithelial endocrine cells (Lundqvist & Wilander, 1986; Shaw, 1990). They are detected most commonly after appendectomy performed for other reasons and they are found in 0.3-0.9% of patients undergoing appendectomy (Goede et al., 2003). Median age is 4th or 5th decade of life (Modlin & Sandor, 1997). This age range is partly explained by higher appendectomy rate in young adults. Less than 10% of appendiceal carcinoids cause symptoms due to the distal localization of the tumor (Moertel et al., 1987). However, they become symptomatic earlier than the carcinoids of other sites. This may also explain the relatively less frequent metastasis of appendiceal carcinoids regarding tumor size. On the other hand, the size of the tumor is the best predictor of outcome according to the Mayo Clinic series. The critical size has been found to be 2 cm. Therefore, right hemicolectomy is the treatment of choice for those tumors that are more than 2 cm in size as well as those located at the base of the appendix regardless of their size. Local desmoplasia is another indication of right hemicolectomy. Otherwise simple appendectomy suffices as there is no evidence of recurrence after a median follow-up of more than 25 years in the

Less than 1% of colon cancers are carcinoids (Modlin IM, 1997). The usual presentation of colonic carcinoids in patients is bleeding, pain and obstruction (Table 3). In a study of 72 patients, the more common symptoms were pain, anorexia and weight loss (Rosenberg JM, 1985). Patients were usually at their 7th decade of life. The most common location was cecum. Midgut colonic carcinoids, namely proximal colonic ones, are believed to arise from serotonin-secreting epithelial endocrine cells, behave more aggressively and symptomatic tumors need to be approached radically. Fulguration should be avoided in colonic carcinoids due to a risk of perforation. As for prognosis, 5-year survival rate is 70% in patients with local disease; 44% and 20% in patients with regional metastases and distant

duodenal tumors and 56, 36, and 23% for jejunal/ileal tumors (Zar et al., 2004).

always been proved to have distant spreading.

appropriate age group (Moertel et al., 1987).

**5.3 Hindgut carcinoids 5.3.1 Colonic carcinoids** 

spread, respectively.

**5.2.2 Appendiceal carcinoids** 

Treatment should not be radical unless there is lymph node metastasis. Options include lobectomy, segmentectomy, and bronchotomy with tumor enucleation. Curative or palliative metastasectomies are worth trying since they grow slowly. Carcinoid crisis should be prevented with octreotide administration preoperatively. When chemotherapy is indicated in some patients, 5-fluorouracil and streptozotosin or cisplatin and etoposide are most commonly used regimens. Other drugs used in metastatic pulmonary carcinoids are octreotide, 131I-MIBG, and interferon alpha. Stage-by-stage, the outcome is worse for patients with atypical carcinoids than for those with typical carcinoids (Kaplan et al., 2003).

#### **5.1.3 Gastric carcinoids**

Gastric carcinoids are rare neoplasms of the stomach comprising less than 1% of gastric tumors and 8.7% of all gastrointestinal neuroendocrine tumors (Godwin, 1975; Modlin et al., 1997) (Table 4). The incidence is reported to be 1.2 and 1.8/1,000,000 persons/year in white males and females, respectively. Three types have been described: Those associated with chronic atrophic gastritis type A (CAG-A or type I), those associated with Zollinger-Ellison syndrome (type II), and sporadic gastric carcinoid tumors (type III). The first 2 groups of carcinoids are associated with hypergastrinemia. CAG-A associated carcinoids are the most common type (70-80% of cases). The typical patient is a woman in her sixties with pernicious anemia, hypochlorhydria, and hypergastrinemia. High levels of gastrin may mediate the hyperplasia of enterochromaffin-like (ECL) cells in the gastric mucosa and ultimately, carcinoid tumors may develop. They are small indolent, non-functioning tumors and located multifocally in the body or fundus, which are diagnosed incidentally (Rindi et al., 1993; Gough et al., 1994; Moses et al., 1986). Tumors less than 1 cm have been successfully resected and followed endoscopically (Ahlman et al., 1994; Sjoblom et al., 1993). Gastric carcinoids associated with the Zollinger-Ellison syndrome (ZES) almost always occur in patients with multiple endocrine neoplasia type 1 (MEN1 or Wermer's syndrome) suggesting a genetic predisposition. Five percent of gastric carcinoids are of type II. The treatment and prognosis are similar to those of CAG-A associated carcinoids. Within this group, there is also a non-MEN1 associated sporadic ZES which rarely leads to gastric carcinoid tumor development. Interestingly, 99% of them had ECL hyperplasia and abnormal alpha-human chorionic gonadotropin (a-hCG) staining. Sporadic gastric carcinoids (type III), in contrast, are not associated with hypergastrinemia and follow an aggressive clinical course. In addition to being solitary, they are usually more than 1 cm in size and often metastatic at the time of diagnosis. When feasible, radical gastrectomy is the treatment of choice.

#### **5.2 Midgut carcinoids**

#### **5.2.1 Small intestinal carcinoids**

Small bowel is not only the most frequent site of carcinoid tumors (including metastatic carcinoids), but also carcinoids are the commonest primary malignant tumor of the distal small intestine. Their frequency has recently surpassed adenocarcinoma (44% versus 33%) (Bilimoria et al., 2009). More than one third of the small bowel tumors are diagnosed as carcinoids and of all gastrointestinal carcinoids; 42% arise in the small bowel of which the ileum represents the most common localization followed by the duodenum and the jejunum. The annual incidence rate is 0,28 per 100,000. Patients usually present in 6th or 7th

Treatment should not be radical unless there is lymph node metastasis. Options include lobectomy, segmentectomy, and bronchotomy with tumor enucleation. Curative or palliative metastasectomies are worth trying since they grow slowly. Carcinoid crisis should be prevented with octreotide administration preoperatively. When chemotherapy is indicated in some patients, 5-fluorouracil and streptozotosin or cisplatin and etoposide are most commonly used regimens. Other drugs used in metastatic pulmonary carcinoids are octreotide, 131I-MIBG, and interferon alpha. Stage-by-stage, the outcome is worse for patients with atypical carcinoids than for those with typical carcinoids (Kaplan et al., 2003).

Gastric carcinoids are rare neoplasms of the stomach comprising less than 1% of gastric tumors and 8.7% of all gastrointestinal neuroendocrine tumors (Godwin, 1975; Modlin et al., 1997) (Table 4). The incidence is reported to be 1.2 and 1.8/1,000,000 persons/year in white males and females, respectively. Three types have been described: Those associated with chronic atrophic gastritis type A (CAG-A or type I), those associated with Zollinger-Ellison syndrome (type II), and sporadic gastric carcinoid tumors (type III). The first 2 groups of carcinoids are associated with hypergastrinemia. CAG-A associated carcinoids are the most common type (70-80% of cases). The typical patient is a woman in her sixties with pernicious anemia, hypochlorhydria, and hypergastrinemia. High levels of gastrin may mediate the hyperplasia of enterochromaffin-like (ECL) cells in the gastric mucosa and ultimately, carcinoid tumors may develop. They are small indolent, non-functioning tumors and located multifocally in the body or fundus, which are diagnosed incidentally (Rindi et al., 1993; Gough et al., 1994; Moses et al., 1986). Tumors less than 1 cm have been successfully resected and followed endoscopically (Ahlman et al., 1994; Sjoblom et al., 1993). Gastric carcinoids associated with the Zollinger-Ellison syndrome (ZES) almost always occur in patients with multiple endocrine neoplasia type 1 (MEN1 or Wermer's syndrome) suggesting a genetic predisposition. Five percent of gastric carcinoids are of type II. The treatment and prognosis are similar to those of CAG-A associated carcinoids. Within this group, there is also a non-MEN1 associated sporadic ZES which rarely leads to gastric carcinoid tumor development. Interestingly, 99% of them had ECL hyperplasia and abnormal alpha-human chorionic gonadotropin (a-hCG) staining. Sporadic gastric carcinoids (type III), in contrast, are not associated with hypergastrinemia and follow an aggressive clinical course. In addition to being solitary, they are usually more than 1 cm in size and often metastatic at the time of diagnosis. When feasible, radical gastrectomy is the

Small bowel is not only the most frequent site of carcinoid tumors (including metastatic carcinoids), but also carcinoids are the commonest primary malignant tumor of the distal small intestine. Their frequency has recently surpassed adenocarcinoma (44% versus 33%) (Bilimoria et al., 2009). More than one third of the small bowel tumors are diagnosed as carcinoids and of all gastrointestinal carcinoids; 42% arise in the small bowel of which the ileum represents the most common localization followed by the duodenum and the jejunum. The annual incidence rate is 0,28 per 100,000. Patients usually present in 6th or 7th

**5.1.3 Gastric carcinoids** 

treatment of choice.

**5.2 Midgut carcinoids** 

**5.2.1 Small intestinal carcinoids** 

decade of life with mechanical obstruction or vague abdominal pain, sometimes for several years before the diagnosis perhaps due to their submucosal location. These tumors are thought to originate from serotonin-producing intraepithelial endocrine cells and they tend to have high serotonin content. They are multicentric and the most frequent location is the distal ileum. Lymph node and liver metastasis are common but carcinoid syndrome is seen only in 5 to 7% of the patients (Burke et al., 1997; Bilimoria et al., 2009). Metastases tend to occur in liver, bone and lung. Unlike appendiceal carcinoids, smaller tumors have also been found to have metastasized. Nevertheless, tumors measuring 2 cm in diameter have almost always been proved to have distant spreading.

Based on patients, registered in the Swedish Cancer Registry, with small intestinal carcinoids diagnosed from 1960 to 2000 in the duodenum (n= 89) and jejunum/ileum (n=2437), the overall 5-, 10-, and 15 year survivals were, respectively, 60, 46, and 28% for duodenal tumors and 56, 36, and 23% for jejunal/ileal tumors (Zar et al., 2004).

#### **5.2.2 Appendiceal carcinoids**

Carcinoid tumors are the most common cancers of the appendix (Moertel et al., 1968) originating from subepithelial endocrine cells (Lundqvist & Wilander, 1986; Shaw, 1990). They are detected most commonly after appendectomy performed for other reasons and they are found in 0.3-0.9% of patients undergoing appendectomy (Goede et al., 2003). Median age is 4th or 5th decade of life (Modlin & Sandor, 1997). This age range is partly explained by higher appendectomy rate in young adults. Less than 10% of appendiceal carcinoids cause symptoms due to the distal localization of the tumor (Moertel et al., 1987). However, they become symptomatic earlier than the carcinoids of other sites. This may also explain the relatively less frequent metastasis of appendiceal carcinoids regarding tumor size. On the other hand, the size of the tumor is the best predictor of outcome according to the Mayo Clinic series. The critical size has been found to be 2 cm. Therefore, right hemicolectomy is the treatment of choice for those tumors that are more than 2 cm in size as well as those located at the base of the appendix regardless of their size. Local desmoplasia is another indication of right hemicolectomy. Otherwise simple appendectomy suffices as there is no evidence of recurrence after a median follow-up of more than 25 years in the appropriate age group (Moertel et al., 1987).

#### **5.3 Hindgut carcinoids**

#### **5.3.1 Colonic carcinoids**

Less than 1% of colon cancers are carcinoids (Modlin IM, 1997). The usual presentation of colonic carcinoids in patients is bleeding, pain and obstruction (Table 3). In a study of 72 patients, the more common symptoms were pain, anorexia and weight loss (Rosenberg JM, 1985). Patients were usually at their 7th decade of life. The most common location was cecum. Midgut colonic carcinoids, namely proximal colonic ones, are believed to arise from serotonin-secreting epithelial endocrine cells, behave more aggressively and symptomatic tumors need to be approached radically. Fulguration should be avoided in colonic carcinoids due to a risk of perforation. As for prognosis, 5-year survival rate is 70% in patients with local disease; 44% and 20% in patients with regional metastases and distant spread, respectively.

carcinoid syndrome without documented liver metastasis. While patients with small intestinal and proximal colonic carcinoids produce carcinoid syndrome most commonly, it is less commonly seen in bronchial carcinoids and does not occur in rectal carcinoids (Harpole et al., 1992; Smith, 1969; Caldarola et al., 1964). Due to the vagueness of the symptoms or no symptoms at all, diagnosis is delayed approximately 2 to 3 years (Norheim

The diagnosis of a carcinoid tumor depends on the symptomatology of carcinoid syndrome or the presence of other symptoms such as abdominal discomfort. Nevertheless, 60% of the patients with gastrointestinal carcinoids found at surgery were asymptomatic in one study (Thompson et al., 1985). The histopathologic diagnosis is made using immunohistochemical techniques, namely antibodies directed against chromogranin A (CgA), synaptophysin, and NSE. In patients with carcinoid syndrome the diagnosis can be made measuring urinary 5- HIAA levels. Levels of 15 to 60 mg/24 hours are compatible with midgut carcinoid tumors with 60 to 73% of sensitivity and 100% specificity. The most sensitive marker is plasma CgA, albeit not specific, since other neuroendocrine tumors can secrete this substance. Foregut carcinoids, on the other hand, may produce an atypical carcinoid syndrome with minimally elevated or normal urinary 5-HIAA level since they lack the enzyme L-amino-acid decarboxylase. The diagnosis of these tumors rely upon measurement of urinary metabolites of tryptophan such as 5-HTP or 5-HT. Patients should abstain from bananas, avocado, pineapple, walnuts, chocolate, and coffee; and avoid drugs such as chlorpromazine (Bertino & Cole, 1956) salicylates, and L-dopa at least 24 hours before the sample is presented to the

After the diagnosis has been made localization should be determined for the optimal management of the carcinoid tumor. Bronchial carcinoids are located in the airways of the central or middle third of the lung in 80% of cases. Chest x-ray, computed tomography (CT) or bronchoscopy is used to detect these tumors (Nessi et al., 1991). In plain radiography, they appear as a well-demarcated round or ovoid mass, often notched. Small tumors, which are often the case, are best detected by CT scanning. The mass may be visible within the bronchial lumina with an extraluminal component. In peripheral lesions, the mass is typically round or ovoid with a smooth or lobulated border. MRI is reserved for cases in

As midgut carcinoids are small tumors, they are sometimes detected by angiography or somatostatin-receptor scintigraphy (SRS) if not detected by barium enema, CT or MRI. Secondary features such as liver metastases and bowel obstruction are more often visualized than the primary tumor. Liver metastasis can be visualized by CT or MRI. Positron emission tomography (PET) using 11C-5-HTP is another localization modality with a high sensitivity (Orlefors et al., 1998; Sundin et al., 2004). Echocardiography should be performed in all patients with carcinoid syndrome to detect signs of carcinoid heart disease which is

which pulmonary carcinoid is suspected but cannot be visualized on CT.

et al., 1987).

**7. Diagnosis** 

lab (DeVita et al., 2011).

associated with poor survival.

**8. Localization** 

#### **5.3.2 Rectal carcinoids**

Rectal carcinoids constitute 1 to 2% of all rectal cancers and are the most common hindgut carcinoid. They occur most commonly in the 6th decade of life (Modlin & Sandor, 1997). Recent series have shown that they are relatively common (Matsui et al., 1993; Jemore et al., 1992). In one series between 1992 and 1999, they accounted for 27% of all gastrointestinal carcinoid tumors (Modlin et al., 2003). Unlike other carcinoids, they do not usually produce serotonin and therefore do not cause carcinoid syndrome despite extensive metastases. Symptomatic patients have rectal bleeding, pain, and constipation (Table 4) (Harpole et al., 1992; Smith, 1969). They are submucosally located and almost always diagnosed with endoscopic biopsies. Size is the major factor for metastasis. Tumors less than 1 cm in diameter has never been shown to have metastasized during a follow up of up to 25 years according to the Mayo Clinic experience. However, tumors more than 2 cm, which make up about 5%, behave aggressively. For patients with tumors between 1 cm and 2 cm, an individualized approach is appropriate considering age and comorbid conditions.

#### **6. Carcinoid syndrome**

The clinical presentation of the carcinoid tumors varies depending on localization, hormone production, and extent of disease. Midgut carcinoids often present with bowel obstruction or abdominal pain. Patients with rectal carcinoids often seek medical attention with bleeding. Lung carcinoids are found incidentally or they may secrete ACTH or CRH to present with Cushing's syndrome. Growth hormone-releasing hormone secreted by foregut carcinoids may cause acromegaly. Duodenal carcinoids may cause somatostatinoma syndrome. However, the most characteristic clinical feature of carcinoid tumors is carcinoid syndrome.

Carcinoid syndrome, which is almost always seen when the tumor has metastasized to the liver, is the third most common mode of presentation of carcinoid tumors after bowel obstruction and abdominal pain. A small proportion of midgut carcinoids without liver metastasis can still present with carcinoid syndrome suggesting direct liberation of the causative agents into the systemic circulation. Flushing and diarrhea are the most common components of carcinoid syndrome, which are seen up to 89% of the patients during their course of the disease. Flushing is characterized by a sudden onset, deep red or violaceous erythema of the upper body often associated with a feeling of warmth. It is the prominent symptom in midgut carcinoids, which is thought to be due to catecholamine induced kallikrein release. In bronchial carcinoids, the flushing is usually prolonged sometimes hours or days giving rise to permanent dilatation of facial veins and telangiectasia. Gastric carcinoids may cause a characteristic flush that is reddish in color and pachy in distribution reminiscent of an urticarial reaction, which may be inhibited by histamine antagonists (Adamson et al., 1969). Other manifestations include asthma, edema, right heart vulvular lesions, the most common of which is tricuspid regurgitation, a loud sound of the pulmonic component of the 2nd heart sound, pellegra-like skin lesions, peptic ulcer, and arthralgia. Malignant carcinoid syndrome denotes patients with these manifestations combined with liver metastasis and elevated urinary 5-HIAA levels. Some patients may display only a few of the above signs. Usually, a full-blown carcinoid syndrome is seen in patients with extensive hepatic metastases. Some patients may have hepatic metastases with minimal symptoms and they generally look well. Ovarian and bronchial carcinoids may cause carcinoid syndrome without documented liver metastasis. While patients with small intestinal and proximal colonic carcinoids produce carcinoid syndrome most commonly, it is less commonly seen in bronchial carcinoids and does not occur in rectal carcinoids (Harpole et al., 1992; Smith, 1969; Caldarola et al., 1964). Due to the vagueness of the symptoms or no symptoms at all, diagnosis is delayed approximately 2 to 3 years (Norheim et al., 1987).

#### **7. Diagnosis**

54 Neuroendocrine Tumor

Rectal carcinoids constitute 1 to 2% of all rectal cancers and are the most common hindgut carcinoid. They occur most commonly in the 6th decade of life (Modlin & Sandor, 1997). Recent series have shown that they are relatively common (Matsui et al., 1993; Jemore et al., 1992). In one series between 1992 and 1999, they accounted for 27% of all gastrointestinal carcinoid tumors (Modlin et al., 2003). Unlike other carcinoids, they do not usually produce serotonin and therefore do not cause carcinoid syndrome despite extensive metastases. Symptomatic patients have rectal bleeding, pain, and constipation (Table 4) (Harpole et al., 1992; Smith, 1969). They are submucosally located and almost always diagnosed with endoscopic biopsies. Size is the major factor for metastasis. Tumors less than 1 cm in diameter has never been shown to have metastasized during a follow up of up to 25 years according to the Mayo Clinic experience. However, tumors more than 2 cm, which make up about 5%, behave aggressively. For patients with tumors between 1 cm and 2 cm, an

individualized approach is appropriate considering age and comorbid conditions.

The clinical presentation of the carcinoid tumors varies depending on localization, hormone production, and extent of disease. Midgut carcinoids often present with bowel obstruction or abdominal pain. Patients with rectal carcinoids often seek medical attention with bleeding. Lung carcinoids are found incidentally or they may secrete ACTH or CRH to present with Cushing's syndrome. Growth hormone-releasing hormone secreted by foregut carcinoids may cause acromegaly. Duodenal carcinoids may cause somatostatinoma syndrome. However, the most characteristic clinical feature of carcinoid tumors is carcinoid

Carcinoid syndrome, which is almost always seen when the tumor has metastasized to the liver, is the third most common mode of presentation of carcinoid tumors after bowel obstruction and abdominal pain. A small proportion of midgut carcinoids without liver metastasis can still present with carcinoid syndrome suggesting direct liberation of the causative agents into the systemic circulation. Flushing and diarrhea are the most common components of carcinoid syndrome, which are seen up to 89% of the patients during their course of the disease. Flushing is characterized by a sudden onset, deep red or violaceous erythema of the upper body often associated with a feeling of warmth. It is the prominent symptom in midgut carcinoids, which is thought to be due to catecholamine induced kallikrein release. In bronchial carcinoids, the flushing is usually prolonged sometimes hours or days giving rise to permanent dilatation of facial veins and telangiectasia. Gastric carcinoids may cause a characteristic flush that is reddish in color and pachy in distribution reminiscent of an urticarial reaction, which may be inhibited by histamine antagonists (Adamson et al., 1969). Other manifestations include asthma, edema, right heart vulvular lesions, the most common of which is tricuspid regurgitation, a loud sound of the pulmonic component of the 2nd heart sound, pellegra-like skin lesions, peptic ulcer, and arthralgia. Malignant carcinoid syndrome denotes patients with these manifestations combined with liver metastasis and elevated urinary 5-HIAA levels. Some patients may display only a few of the above signs. Usually, a full-blown carcinoid syndrome is seen in patients with extensive hepatic metastases. Some patients may have hepatic metastases with minimal symptoms and they generally look well. Ovarian and bronchial carcinoids may cause

**5.3.2 Rectal carcinoids** 

**6. Carcinoid syndrome** 

syndrome.

The diagnosis of a carcinoid tumor depends on the symptomatology of carcinoid syndrome or the presence of other symptoms such as abdominal discomfort. Nevertheless, 60% of the patients with gastrointestinal carcinoids found at surgery were asymptomatic in one study (Thompson et al., 1985). The histopathologic diagnosis is made using immunohistochemical techniques, namely antibodies directed against chromogranin A (CgA), synaptophysin, and NSE. In patients with carcinoid syndrome the diagnosis can be made measuring urinary 5- HIAA levels. Levels of 15 to 60 mg/24 hours are compatible with midgut carcinoid tumors with 60 to 73% of sensitivity and 100% specificity. The most sensitive marker is plasma CgA, albeit not specific, since other neuroendocrine tumors can secrete this substance. Foregut carcinoids, on the other hand, may produce an atypical carcinoid syndrome with minimally elevated or normal urinary 5-HIAA level since they lack the enzyme L-amino-acid decarboxylase. The diagnosis of these tumors rely upon measurement of urinary metabolites of tryptophan such as 5-HTP or 5-HT. Patients should abstain from bananas, avocado, pineapple, walnuts, chocolate, and coffee; and avoid drugs such as chlorpromazine (Bertino & Cole, 1956) salicylates, and L-dopa at least 24 hours before the sample is presented to the lab (DeVita et al., 2011).

#### **8. Localization**

After the diagnosis has been made localization should be determined for the optimal management of the carcinoid tumor. Bronchial carcinoids are located in the airways of the central or middle third of the lung in 80% of cases. Chest x-ray, computed tomography (CT) or bronchoscopy is used to detect these tumors (Nessi et al., 1991). In plain radiography, they appear as a well-demarcated round or ovoid mass, often notched. Small tumors, which are often the case, are best detected by CT scanning. The mass may be visible within the bronchial lumina with an extraluminal component. In peripheral lesions, the mass is typically round or ovoid with a smooth or lobulated border. MRI is reserved for cases in which pulmonary carcinoid is suspected but cannot be visualized on CT.

As midgut carcinoids are small tumors, they are sometimes detected by angiography or somatostatin-receptor scintigraphy (SRS) if not detected by barium enema, CT or MRI. Secondary features such as liver metastases and bowel obstruction are more often visualized than the primary tumor. Liver metastasis can be visualized by CT or MRI. Positron emission tomography (PET) using 11C-5-HTP is another localization modality with a high sensitivity (Orlefors et al., 1998; Sundin et al., 2004). Echocardiography should be performed in all patients with carcinoid syndrome to detect signs of carcinoid heart disease which is associated with poor survival.

important and somatostatin analogues exert their action primarily by binding to subtype 2 (Kubota et al., 1994). Activation results in inhibition of growth factor production and release as well as antiproliferative effects (Scarpignato & Pelosini, 2001; Buscail et al., 1995; Cordelier et al., 1997 & Cattaneo et al., 1996). Receptor subtype 3 mediates phosphotyrosine phosphatase dependent apoptosis during high dose therapy (3mg daily octreotide or 12 mg daily lanreotide) (Imam et al., 1997). Octreotide is the most widely available drug followed by lanreotide and vapreotide. Subcutaneous administration of these drugs can control the symptoms in most of the patients. The recommended dose of immediate release form of octreotide is 100 mcg 2 or 3 times a day. The dose should be titrated upwards according to the symptoms sometimes up to 3000 mcg a day in 3 to 7 days. Tolerance is a common caveat in long-term use. Long-acting, slow-release formulation of octreotide with 20 and 30 mg of doses (up to 60 mg) every month (every 2 weeks for lanreotide) has enabled patients to enjoy a more comfortable life in terms of controlling symptoms and reducing the number of injections. The immediate release formulation and the long-acting form should overlap at least 2 weeks to achieve maximal symptom control. Patients with breakthrough symptoms may benefit from subcutaneous administration of the immediate release formulation. Treatment should be continued life-long or until troublesome side effects develop. Somatostatin analogues improve symptoms in 88% of patients and in 72%, urinary 5-HIAA

Metastatic carcinoid tumors are indolent tumors and response to chemotherapeutics is generally poor. Classical midgut carcinoids do not show any response to chemotherapy. Since foregut carcinoids are more malignant than others, cytotoxic therapy can be attempted. Combinations include streptozotocin plus 5-fluorouracil, doxorubicin, cisplatin plus etoposide, and dacarbazine plus 5-fluorouracil (Moertel et al., 1991; Di Bartolomeo et al., 1995 & Bajetta et al., 1998). Somatostatin analogues can be combined with the above regimens. A recent study showed that chemotherapy naïve patients with metastatic pancreatic endocrine carcinomas had an exceptionally high and durable response rate with

Oberg and his coworkers have reported a study form Sweden that 42% of patients with carcinoid syndrome had a reduction of their urinary 5-HIAA levels when treated with low dose human leukocyte interferon and recombinant leukocyte interferon-a (Oberg et al., 1986), but there was only 11% tumor regression rate. Mayo Clinic tried higher doses of interferon (Moertel et al., 1991) and objective tumor regression was 20% but it was not durable. Toxicity was the major drawback. Oberg has suggested the dose of interferon be

As neuroendocrine tumors are highly vascular tumors bevacizumab, a monoclonal antibody targeting vascular endothelial growth factor (VEGF), was tested in advanced neuroendocrine tumors. This phase II study resulted in objective responses, reduction of

the combination of temezolamide and capecitabine (Strosberg et al., 2011).

titrated according to the patient's neutrophil count.

excretion decreases (Kvols et al., 1986).

**9.3 Chemotherapy** 

**9.4 Immunotherapy** 

**9.5 Angiogenesis inhibitors** 

Hindgut carcinoids are usually localized at endoscopy. They appear as solitary yellowish submucosal lesions. Endoscopic ultrasound may demonstrate invasion of the full rectal wall (stage T3) or adjacent structures (T4).

In recent years, SRS or octreoscan and iodinated meta-iodobenzylguanidine (123I-MIBG) have been introduced to localize and stage the tumor. Five somatostatin receptors are currently recognized bound to varying degrees by the analogues 111In-octreotide, 111Inlanreotide, and P829, a new technetium-99m (99mTc) analogue (Menda & Khan, 2002). Octreotide receptor imaging is most useful for the prediction of the success of octreotide therapy. If the scan is positive, then therapy will most likely be beneficial. It is also used peroperatively for perioperative tumor detection. Imaging with 123I-MIBG can also be performed for the prediction of therapy success with 131I-MIBG. The frequency of positive imaging in carcinoid tumors is 50-75% for 123I-MIBG and 67-96% for 111ln-octreotide. 123I-MIBG scintigraphy, which is more widely available, appears to be more sensitive for sympatho-adrenomedullary tumors such as pheochromocytomas and paragangliomas, whereas 111ln-octreotide detects more tumors in all other neuroendocrine neoplasms.

#### **9. Management of carcinoid tumors**

Tumor reduction and symptomatic control are the mainstays of treatment of carcinoid tumors. When the tumor is localized, surgical resection the extent of which is determined primarily by the tumor size, is the treatment of choice. Symptomatic control includes lifestyle changes, dietary supplementation, and medical treatments directed to specific symptoms of carcinoid syndrome. Benign cases can respond to avoiding physical and emotional stress, alcohol, spicy food, and certain drugs. Supplemental niacin is recommended in patients whose symptoms are due to serotonin excess to prevent pellegra. Heart failure due to carcinoid heart disease requires drugs such as angiotensin-converting enzyme (ACE) inhibitors and diuretics. Diarrhea may respond to loperamide or diphenoxylate. Side effects of these drugs are the main limiting factor in controlling carcinoid syndrome. Therefore, more specific drugs have been developed, one of which is octreotide, a somatostatin analogue.

#### **9.1 Surgical palliation**

Patients suspected with intestinal obstruction must be relieved of their obstruction even if they have extensive liver metastases. Liver metastases can be resected without jeopardizing survival in select patients (Que et al., 1995; McEntee et al., 1990; & Dousset, 1996). Liver resection has been associated with improved 5 year survival rates in several series and is recommended in appropriate patients to attempt cure or to debulk metastatic disease (Sutton et al., 2003). Embolization of the hepatic artery is another option for liver disease. Liver transplantation for highly selected patients offers a five-year survival rate of 69% according to a French study (Le Treut et al., 1997). Mesenteric ischemia should promptly be recognized and the affected bowel segment resected.

#### **9.2 Somatostatin analogues**

Somatostatin analogues interact with cellular and transmembrane somatostatin receptors coupled with G proteins. Five subtypes have been defined. Subtypes 2, 3 and 5 are most

Hindgut carcinoids are usually localized at endoscopy. They appear as solitary yellowish submucosal lesions. Endoscopic ultrasound may demonstrate invasion of the full rectal wall

In recent years, SRS or octreoscan and iodinated meta-iodobenzylguanidine (123I-MIBG) have been introduced to localize and stage the tumor. Five somatostatin receptors are currently recognized bound to varying degrees by the analogues 111In-octreotide, 111Inlanreotide, and P829, a new technetium-99m (99mTc) analogue (Menda & Khan, 2002). Octreotide receptor imaging is most useful for the prediction of the success of octreotide therapy. If the scan is positive, then therapy will most likely be beneficial. It is also used peroperatively for perioperative tumor detection. Imaging with 123I-MIBG can also be performed for the prediction of therapy success with 131I-MIBG. The frequency of positive imaging in carcinoid tumors is 50-75% for 123I-MIBG and 67-96% for 111ln-octreotide. 123I-MIBG scintigraphy, which is more widely available, appears to be more sensitive for sympatho-adrenomedullary tumors such as pheochromocytomas and paragangliomas, whereas 111ln-octreotide detects more tumors in all other neuroendocrine neoplasms.

Tumor reduction and symptomatic control are the mainstays of treatment of carcinoid tumors. When the tumor is localized, surgical resection the extent of which is determined primarily by the tumor size, is the treatment of choice. Symptomatic control includes lifestyle changes, dietary supplementation, and medical treatments directed to specific symptoms of carcinoid syndrome. Benign cases can respond to avoiding physical and emotional stress, alcohol, spicy food, and certain drugs. Supplemental niacin is recommended in patients whose symptoms are due to serotonin excess to prevent pellegra. Heart failure due to carcinoid heart disease requires drugs such as angiotensin-converting enzyme (ACE) inhibitors and diuretics. Diarrhea may respond to loperamide or diphenoxylate. Side effects of these drugs are the main limiting factor in controlling carcinoid syndrome. Therefore, more specific drugs have been developed, one of which is

Patients suspected with intestinal obstruction must be relieved of their obstruction even if they have extensive liver metastases. Liver metastases can be resected without jeopardizing survival in select patients (Que et al., 1995; McEntee et al., 1990; & Dousset, 1996). Liver resection has been associated with improved 5 year survival rates in several series and is recommended in appropriate patients to attempt cure or to debulk metastatic disease (Sutton et al., 2003). Embolization of the hepatic artery is another option for liver disease. Liver transplantation for highly selected patients offers a five-year survival rate of 69% according to a French study (Le Treut et al., 1997). Mesenteric ischemia should promptly be

Somatostatin analogues interact with cellular and transmembrane somatostatin receptors coupled with G proteins. Five subtypes have been defined. Subtypes 2, 3 and 5 are most

(stage T3) or adjacent structures (T4).

**9. Management of carcinoid tumors** 

octreotide, a somatostatin analogue.

recognized and the affected bowel segment resected.

**9.1 Surgical palliation** 

**9.2 Somatostatin analogues** 

important and somatostatin analogues exert their action primarily by binding to subtype 2 (Kubota et al., 1994). Activation results in inhibition of growth factor production and release as well as antiproliferative effects (Scarpignato & Pelosini, 2001; Buscail et al., 1995; Cordelier et al., 1997 & Cattaneo et al., 1996). Receptor subtype 3 mediates phosphotyrosine phosphatase dependent apoptosis during high dose therapy (3mg daily octreotide or 12 mg daily lanreotide) (Imam et al., 1997). Octreotide is the most widely available drug followed by lanreotide and vapreotide. Subcutaneous administration of these drugs can control the symptoms in most of the patients. The recommended dose of immediate release form of octreotide is 100 mcg 2 or 3 times a day. The dose should be titrated upwards according to the symptoms sometimes up to 3000 mcg a day in 3 to 7 days. Tolerance is a common caveat in long-term use. Long-acting, slow-release formulation of octreotide with 20 and 30 mg of doses (up to 60 mg) every month (every 2 weeks for lanreotide) has enabled patients to enjoy a more comfortable life in terms of controlling symptoms and reducing the number of injections. The immediate release formulation and the long-acting form should overlap at least 2 weeks to achieve maximal symptom control. Patients with breakthrough symptoms may benefit from subcutaneous administration of the immediate release formulation. Treatment should be continued life-long or until troublesome side effects develop. Somatostatin analogues improve symptoms in 88% of patients and in 72%, urinary 5-HIAA excretion decreases (Kvols et al., 1986).

#### **9.3 Chemotherapy**

Metastatic carcinoid tumors are indolent tumors and response to chemotherapeutics is generally poor. Classical midgut carcinoids do not show any response to chemotherapy. Since foregut carcinoids are more malignant than others, cytotoxic therapy can be attempted. Combinations include streptozotocin plus 5-fluorouracil, doxorubicin, cisplatin plus etoposide, and dacarbazine plus 5-fluorouracil (Moertel et al., 1991; Di Bartolomeo et al., 1995 & Bajetta et al., 1998). Somatostatin analogues can be combined with the above regimens. A recent study showed that chemotherapy naïve patients with metastatic pancreatic endocrine carcinomas had an exceptionally high and durable response rate with the combination of temezolamide and capecitabine (Strosberg et al., 2011).

#### **9.4 Immunotherapy**

Oberg and his coworkers have reported a study form Sweden that 42% of patients with carcinoid syndrome had a reduction of their urinary 5-HIAA levels when treated with low dose human leukocyte interferon and recombinant leukocyte interferon-a (Oberg et al., 1986), but there was only 11% tumor regression rate. Mayo Clinic tried higher doses of interferon (Moertel et al., 1991) and objective tumor regression was 20% but it was not durable. Toxicity was the major drawback. Oberg has suggested the dose of interferon be titrated according to the patient's neutrophil count.

#### **9.5 Angiogenesis inhibitors**

As neuroendocrine tumors are highly vascular tumors bevacizumab, a monoclonal antibody targeting vascular endothelial growth factor (VEGF), was tested in advanced neuroendocrine tumors. This phase II study resulted in objective responses, reduction of

Carcinoid syndrome represents an advanced disease and prognosis depends upon the site and extent of the disease. While in patients with localized disease the 5-year survival rate is approximately 65%, patients with advanced disease this figure drops to 36% (Godwin et al., 1975). Female gender and young age at diagnosis are associated with a better prognosis. Presence of metastases, high CgA level, and high proliferation index (Ki-67) are indicators of a poor outcome. Once, carcinoid heart disease was a troublesome complication and a cause of early death. With modern diagnostic and therapeutic technics this is rarely seen today. Development of a secondary malignancy, namely an adenocarcinoma of the large intestine, is another cause of reduced survival in these patients (Godwin et al., 1975). Whether combined adenocarcinoma and carcinoid tumors are a composite tumor or collision tumors

Neuroendocrine tumors continue to evolve with regard to diagnosis, classification epidemiology and treatment possibilities. They also provide a challenging source of testing

Adamson AK, Grahame-Smith DG, Peart WS, & Starr M. (1969). Pharmacological blockade of carcinoid flushing provoked by catecholamines and alcohol. *Lancet* 1969;2:293. Ahlman H, Kolby L, & Lundell L, et al., (1994). Clinical management of gastric carcinoid

Anthony L, Vinik, AI. Pancreas. (2011). Evaluating the Characteristics and the Management

Bajetta E, Rimassa L, & Carnaghi C, et al., (1998). 5-Fluorouracil, dacarbazine, and epirubicin in the treatment of patients with neuroendocrine tumors. *Cancer* 1998;83:372-378. Bilimoria KY, Bentrem DJ, Wayne JD, Ko CY, Bennett CL, & Talamonti MS. (2009). Small

Bosman FT, Carneiro F, Hruban RH, Theise ND (2010). *WHO classification of tumors of the* 

Burke AP, Thomas RM, Elsayed AM, & Sobin LH. (1997). Carcinoids of the jejunum and

Buscail L, Esteve JP, & Saint-Laurent, et al., (1995). Inhibition of cell proliferation by the

Caldarola Vt, Jackman Rj, Moertel Cg, & Dockerty Mb. (1964). Carcinoid tumors of the

survival over the last 20 years. *Ann Surg.* 2009 Jan;249(1):63-71

*digestive system*. Lyon, France: IARC Press; 2010.

of Patients With Neuroendocrine Tumors Receiving Octreotide LAR During a 6- Year Period. *Pancreas*. 40(7):987-994, October 2011. doi:

bowel cancer in the United States: changes in epidemiology, treatment, and

ileum: an immunohistochemical and clinicopathologic study of 167 cases. *Cancer*

somatostatin analogue RC-160 is mediated by somatostatin receptor subtypes SSTR2 and SSTR5 through different mechanisms. *N Proc Natl Acad Sci USA*

novel drugs as their molecular targets have now started to be recognized.

tumors. *Digestion* 1994;55(Suppl 3):77-85.

10.1097/MPA.0b013e31821f66b4)

1997;79:1086-1093.

1995;92:1580-1584.

rectum. *Am J Surg*. 1964;107:844-849.

**10. Prognosis** 

**11. Conclusion** 

**12. References** 

has been debated (Yamashina & Flinner, 1985).

tumor blood flow, and longer PFS in patients with carcinoid than PEG interferon treatment (Yao, 2008). Sunitinib, another molecule inhibiting VEGF receptors, is approved by FDA for the treatment of advanced or unresectable progressive well-differentiated pancreatic neuroendocrine tumors based on the phase III study stating that it improves progressionfree survival, overall survival, and the objective response rate of these tumors as compared with placebo (Raymond et al., 2011).

#### **9.6 Mammalian target of rapamycin (mTOR) inhibitors**

Everolimus inhibits mTOR, an intracellular serine/threonine kinase that regulates multiple signaling pahways. It showed activity in advanced or unresectable low-grade or intermediate-grade pancreatic neuroendocrine tumors (Yao et al., 2011) and approved by FDA for the treatment of advanced, unresctable, or locally advanced pancreatic neuroendocrine tumors.

#### **9.7 Radiation therapy**

External irradiation has been used for the palliation of bone and brain metastases (Schupak & Wallner, 1991 & Kimmig, 1994). Radiolabeled MIBG has been the most widely studied agent. It showed a 30% response rate with 125I-MIBG or 131I-MIBG (Hoefnagel et al., 1987 & Taal et al., 1996). In one study, investigators performed a retrospective review of 98 patients with metastatic carcinoid who were treated at their institution with 131I-MIBG over a 15-year period. The median survival after treatment was 2.3 years. Patients who experienced a symptomatic response had improved survival. For the 56 patients who had 5- HIAA levels monitored, the mean urine 5-HIAA levels decreased significantly after 131I-MIBG treatment. Authors concluded that 131I-MIBG treatment could be recommended in select patients with metastatic carcinoid who progress despite optimal medical management. Improved survival was predicted best by symptomatic response to 131I-MIBG treatment, but not by hormone or radiographic response (Safford et al., 2004). 111lnoctreotide is a somatostatin analogue-based tumor-targeted radioactive agent. It is most often used when imaging with 123I-MIBG fails to detect any tumor. When 111In-octreotide is avidly concentrated within the tumors, then radionuclide labeled octreotide can be administered. Studies with 90Y-labeled octreotide have shown that 83% of patients with carcinoid syndrome had a significant reduction in symptoms (Waldherr et al., 2001).

#### **9.8 Carcinoid heart disease**

In the pre-somatostatin era carcinoid heart disease used to occur in two thirds of patients with the carcinoid syndrome (Lundin et al., 1988). However, in the post-somatostatin era its incidence has dropped dramatically to 5% (Anthony et al., 2011). Right heart is affected most commonly. Tricuspid regurgitation, tricuspid stenosis, pulmonary regurgitation, and pulmonary stenosis may all occur. It is thought that these lesions are due to the secreted factors by the carcinoid cells of the liver metastases into the hepatic vein. Serotonin is blamed to be responsible for the cardiac lesions in carcinoid syndrome. However, its role in the development of cardiac lesions is poorly understood. Left heart is less often affected due to inactivation of serotonin by lung metabolism. Valvular replacement in patients with symptomatic heart disease proved unsuccessful due to the high perioperative mortality (Robiolio et al., 1995 & Connolly et al., 1995).

#### **10. Prognosis**

58 Neuroendocrine Tumor

tumor blood flow, and longer PFS in patients with carcinoid than PEG interferon treatment (Yao, 2008). Sunitinib, another molecule inhibiting VEGF receptors, is approved by FDA for the treatment of advanced or unresectable progressive well-differentiated pancreatic neuroendocrine tumors based on the phase III study stating that it improves progressionfree survival, overall survival, and the objective response rate of these tumors as compared

Everolimus inhibits mTOR, an intracellular serine/threonine kinase that regulates multiple signaling pahways. It showed activity in advanced or unresectable low-grade or intermediate-grade pancreatic neuroendocrine tumors (Yao et al., 2011) and approved by FDA for the treatment of advanced, unresctable, or locally advanced pancreatic

External irradiation has been used for the palliation of bone and brain metastases (Schupak & Wallner, 1991 & Kimmig, 1994). Radiolabeled MIBG has been the most widely studied agent. It showed a 30% response rate with 125I-MIBG or 131I-MIBG (Hoefnagel et al., 1987 & Taal et al., 1996). In one study, investigators performed a retrospective review of 98 patients with metastatic carcinoid who were treated at their institution with 131I-MIBG over a 15-year period. The median survival after treatment was 2.3 years. Patients who experienced a symptomatic response had improved survival. For the 56 patients who had 5- HIAA levels monitored, the mean urine 5-HIAA levels decreased significantly after 131I-MIBG treatment. Authors concluded that 131I-MIBG treatment could be recommended in select patients with metastatic carcinoid who progress despite optimal medical management. Improved survival was predicted best by symptomatic response to 131I-MIBG treatment, but not by hormone or radiographic response (Safford et al., 2004). 111lnoctreotide is a somatostatin analogue-based tumor-targeted radioactive agent. It is most often used when imaging with 123I-MIBG fails to detect any tumor. When 111In-octreotide is avidly concentrated within the tumors, then radionuclide labeled octreotide can be administered. Studies with 90Y-labeled octreotide have shown that 83% of patients with

carcinoid syndrome had a significant reduction in symptoms (Waldherr et al., 2001).

In the pre-somatostatin era carcinoid heart disease used to occur in two thirds of patients with the carcinoid syndrome (Lundin et al., 1988). However, in the post-somatostatin era its incidence has dropped dramatically to 5% (Anthony et al., 2011). Right heart is affected most commonly. Tricuspid regurgitation, tricuspid stenosis, pulmonary regurgitation, and pulmonary stenosis may all occur. It is thought that these lesions are due to the secreted factors by the carcinoid cells of the liver metastases into the hepatic vein. Serotonin is blamed to be responsible for the cardiac lesions in carcinoid syndrome. However, its role in the development of cardiac lesions is poorly understood. Left heart is less often affected due to inactivation of serotonin by lung metabolism. Valvular replacement in patients with symptomatic heart disease proved unsuccessful due to the high perioperative mortality

with placebo (Raymond et al., 2011).

neuroendocrine tumors.

**9.7 Radiation therapy** 

**9.8 Carcinoid heart disease** 

(Robiolio et al., 1995 & Connolly et al., 1995).

**9.6 Mammalian target of rapamycin (mTOR) inhibitors** 

Carcinoid syndrome represents an advanced disease and prognosis depends upon the site and extent of the disease. While in patients with localized disease the 5-year survival rate is approximately 65%, patients with advanced disease this figure drops to 36% (Godwin et al., 1975). Female gender and young age at diagnosis are associated with a better prognosis. Presence of metastases, high CgA level, and high proliferation index (Ki-67) are indicators of a poor outcome. Once, carcinoid heart disease was a troublesome complication and a cause of early death. With modern diagnostic and therapeutic technics this is rarely seen today. Development of a secondary malignancy, namely an adenocarcinoma of the large intestine, is another cause of reduced survival in these patients (Godwin et al., 1975). Whether combined adenocarcinoma and carcinoid tumors are a composite tumor or collision tumors has been debated (Yamashina & Flinner, 1985).

#### **11. Conclusion**

Neuroendocrine tumors continue to evolve with regard to diagnosis, classification epidemiology and treatment possibilities. They also provide a challenging source of testing novel drugs as their molecular targets have now started to be recognized.

#### **12. References**


Kema IP, de Vries EG, & Muskiet FA. (2000). Clinical chemistry of serotonin and

Kaplan B, Stevens CW, Allen P, Liao Z, & Komaki R. (2003). Outcomes and patterns of failure in bronchial carcinoid tumors. *Int J Radiat Oncol Biol Phys* 2003;55:125-131. Kimmig BN. (1994). Radiotherapy for gastroenteropancreatic neuroendocrine tumors. *Ann* 

Klöppel G, Dege K, Remmele W, Kapran Y, Tuzlali S, Modlin IM. (2007). Siegfried

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Gastroenteropancreatic neuroendocrine tumors: 10-year experience in a single

## *Edited by Anthony Lowell*

While the incidence rate of neuroendocrine tumours has been increasing, the survival rate has not. Therefore, there is a great need for state-of-the-art research and new treatment protocols. The chapters in this book contribute to that debate and will be required reading for both researchers and healthcare practitioners, as it offers both overviews of the condition and specialized contributions. Topics covered include the association between chronic inflammation and gastroenteropancreatic neuroendocrine tumours, or GEP-NETs. GEP-NETs are also addressed in a chapter on their circulating markers, which can help in diagnosing this hard to detect condition. One chapter covers the role of Chromogranin A, while another addresses the diagnosis and management of neuroendocrine carcinomas in cases where the primary site is unknown.

Neuroendocrine Tumor

Neuroendocrine Tumor