These authors contribute equally to this work.

#### **References**


[6] Wolf GT. Surgical margins in the genomic era: The Hayes Martin Lecture, 2012. *Arch Otolaryngol Head Neck Surg*. 2012;138(11):1001–13.

from various upper aerodigestive sites with distinctly diverse embryological and biological characteristics [47]. As a result, there is little definitive data with regard to clinical implications of CSCs within HNSCC, the primary exception being prognostic value. Furthermore, no single biomarker for CSC cells in HNSCC has proven absolute in distinguishing this vital subpopulation. The continued study of current prospective CSC markers in HNSCC, combined with the investigation of putative CSC biomarkers from other malignancies, will undoubtedly augment our knowledge and improve our understanding of the pathogenesis of HNSCC. In addition, further knowledge regarding the biomarkers and regulation of normal, native stem cells in the head and neck region will serve as a strong foundation for oncological research. Ultimately, CSCs may prove to be useful diagnostic and prognostic markers for HNSCC,

guiding therapy and treatment through personalized approaches and interventions.

, Reigh-Yi Lin\*

Department of Otolaryngology – Head and Neck Surgery, Saint Louis University School of

[1] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. *CA*: A cancer journal for

[2] Anderson RT, Keysar SB, Bowles DW, Glogowska MJ, Astling DP, Morton JJ, et al. The dual pathway inhibitor rigosertib is effective in direct patient tumor xenografts of head and neck squamous cell carcinomas. *Mol Canc Therapeut*. 2013;12(10):1994–

[3] Chen Z. The cancer stem cell concept in progression of head and neck cancer. *J Oncol*.

[4] Bhaijee F, Pepper DJ, Pitman KT, Bell D. Cancer stem cells in head and neck squa‐ mous cell carcinoma: a review of current knowledge and future applications. *Head*

[5] Shah A, Patel S, Pathak J, Swain N, Kumar S. The evolving concepts of cancer stem cells in head and neck squamous cell carcinoma. *Scientific World J*. 2014;2014:8.

, Nathan Lindquist#

108 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

\*Address all correspondence to: rlin7@slu.edu

These authors contribute equally to this work.

clinicians. 2013;63(1):11–30.

Medicine, Saint Louis, Missouri, USA

**Author details**

Kaveh Karimnejad#

#

**References**

2005.

2009;2009:8.

*Neck*. 2012;34(6):894–9.


cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage. *J Cell Sci*. 1996;109 (Pt 5):1017–28.

[36] Jones PH, Watt FM. Separation of human epidermal stem cells from transit amplify‐ ing cells on the basis of differences in integrin function and expression. *Cell*. 1993;73 (4):713–24.

[23] Calenic B, Ishkitiev N, Yaegaki K, Imai T, Costache M, Tovaru M, et al. Characteriza‐ tion of oral keratinocyte stem cells and prospects of its differentiation to oral epithe‐ lial equiv- alents. Romanian journal of morphology and embryology = *Rev Roumaine*

[24] Okubo T, Clark C, Hogan BL. Cell lineage mapping of taste bud cells and keratino‐ cytes in the mouse tongue and soft palate. *Stem Cells* (Dayton, Ohio). 2009;27(2):442–

[25] Nakamura T, Endo K, Kinoshita S. Identification of human oral keratinocyte stem/ pro- genitor cells by neurotrophin receptor p75 and the role of neurotrophin/p75 sig‐

[26] Li X, Shen Y, Di B, Li J, Geng J, Lu X, et al. Biological and clinical significance of p75NTR expression in laryngeal squamous epithelia and laryngocarcinoma. *Acta*

[27] Ishii A, Muramatsu T, Lee JM, Higa K, Shinozaki N, Jung HS, et al. Expression of p75 (NGFR), a proliferative and basal cell Marker, in the buccal mucosa epithelium dur‐

[28] Huang SD, Yuan Y, Liu XH, Gong DJ, Bai CG, Wang F, et al. Self-renewal and che‐ mother- apy resistance of p75NTR positive cells in esophageal squamous cell carcino‐

[29] Tanaka T, Komai Y, Tokuyama Y, Yanai H, Ohe S, Okazaki K, et al. Identification of stem cells that maintain and regenerate lingual keratinized epithelial cells. *Natur Cell*

[30] Allegra E, Trapasso S, Pisani D, Puzzo L. The role of BMI1 as a biomarker of cancer stem cells in head and neck cancer: a review. *Oncology*. 2014;86(4):199–205.

[31] Luo X, Okubo T, Randell S, Hogan BL. Culture of endodermal stem/progenitor cells of the mouse tongue. In vitro cellular & developmental biology. *Animal*. 2009;45(1–2):

[32] Raimondi AR, Molinolo A, Gutkind JS. Rapamycin prevents early onset of tumori‐ genesis in an oral-specific K-ras and p53 two-hit carcinogenesis model. *Canc Res*.

[33] Mackenzie IC. Stem cells in oral mucosal epithelia. *Oral Biosci Med*. 2005;2(2):95–103.

[34] Calenic B, Ishkitiev N, Yaegaki K, Imai T, Kumazawa Y, Nasu M, et al. Magnetic sep‐ ara- tion and characterization of keratinocyte stem cells from human gingiva. *J Perio‐*

[35] Michel M, Torok N, Godbout MJ, Lussier M, Gaudreau P, Royal A, et al. Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing

ing re- epithelialization. *Acta Histochem Cytochem*. 2014;47(4):145–53.

*Morphol Embryolog*. 2010;51(4):641–5.

110 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

*Oto-laryngol*. 2012;132(3):314–24.

mas. *BMC Canc*. 2009;9:9.

*Biol*. 2013;15(5):511–8.

2009;69 (10):4159–66.

*dontal Res*. 2010;45(6):703–8.

44–54.

naling. *Stem Cells* (Dayton, Ohio). 2007;25(3):628–38.

50.


publica- tion of the Federation of American Societies for Experimental Biology. 1998;12 (12):1241–51.

[62] Han J, Fujisawa T, Husain SR, Puri RK. Identification and characterization of cancer stem cells in human head and neck squamous cell carcinoma. *BMC Canc*. 2014;14:173.

[49] Lessard J, Sauvageau G. Bmi-1 determines the proliferative capacity of normal and

[50] Park IK, Morrison SJ, Clarke MF. Bmi1, stem cells, and senescence regulation. *J Clin*

[51] Yin AH, Miraglia S, Zanjani ED, Almeida-Porada G, Ogawa M, Leary AG, et al. AC133, a novel marker for human hematopoietic stem and progenitor cells. *Blood*.

[52] Corbeil D, Marzesco AM, Wilsch-Brauninger M, Huttner WB. The intriguing links between prominin-1 (CD133), cholesterol-based membrane microdomains, remodel‐ ing of apical plasma membrane protrusions, extracellular membrane particles, and

[53] Zhou L, Wei X, Cheng L, Tian J, Jiang JJ. CD133, one of the markers of cancer stem

[54] Canis M, Lechner A, Mack B, Zengel P, Laubender RP, Koehler U, et al. CD133 is a predictor of poor survival in head and neck squamous cell carcinomas. *Canc Biomark‐*

[55] Yu CC, Hu FW, Ph DC, Chou MY. Targeting CD133 in the enhancement of chemo‐ sen- sitivity in oral squamous cell carcinomas-derived side population cancer stem

[56] Clay MR, Tabor M, Owen JH, Carey TE, Bradford CR, Wolf GT, et al. Single-marker identification of head and neck squamous cell carcinoma cancer stem cells with alde‐

[57] Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a pre‐

[58] Charafe-Jauffret E, Ginestier C, Iovino F, Tarpin C, Diebel M, Esterni B, et al. Alde‐ hyde dehydrogenase 1-positive cancer stem cells mediate metastasis and poor clini‐ cal outcome in inflammatory breast cancer. Clin Canc Res: an official journal of the

[59] Krishnamurthy S, Dong Z, Vodopyanov D, Imai A, Helman JI, Prince ME, et al. En‐ dothe- lial cell-initiated signaling promotes the survival and self-renewal of cancer

[60] Baumann P, Cremers N, Kroese F, Orend G, Chiquet-Ehrismann R, Uede T, et al. CD24 expression causes the acquisition of multiple cellular properties associated

[61] Aigner S, Ramos CL, Hafezi-Moghadam A, Lawrence MB, Friederichs J, Altevogt P, et al. CD24 mediates rolling of breast carcinoma cells on P-selectin. *FASEB J*: official

(neuro) epithelial cell differentiation. *FEBS Lett*. 2010;584(9):1659–64.

cells in Hep-2 cell line. *Laryngoscope*. 2007;117(3):455–60.

*ers*: section A of Disease Markers. 2012;12(2):97–105.

hyde dehydrogenase. *Head Neck*. 2010;32(9):1195–201.

dictor of poor clinical outcome. *Cell Stem Cell*. 2007;1(5):555–67.

American Associ- ation for Cancer Research. 2010;16(1):45–55.

with tumor growth and metastasis. *Canc Res*. 2005;65(23):10783–93.

stem cells. *Canc Res*. 2010;70(23):9969–78.

leukaemic stem cells. *Nature*. 2003;423(6937):255–60.

112 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

*Invest*. 2004;113(2):175–9.

1997;90 (12):5002–12.

cells. *Head Neck*. 2014.


## **Adult Acute Myeloid Leukemia – A Possible Relation to Disease Invasion and the Impact of Independent Prognostic Markers Associated with Survival Outcome**

Mohamed El-Refaei and Fahd Al Qahtani

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61179

#### **Abstract**

Over the past decade, leukemia exists and frequently occurs in adults. Radiation ex‐ posure, hereditary syndromes, smoking, age, and many other unknown factors are generally the major risk factors for leukemia. Acute myeloid leukemia (AML) is a hematological malignancy that is dispersed from its beginning and may be perceived as a prototype of metastatic cancer, yet leukemia is considered a highly malignant ne‐ oplasms responsible for a large number of cancer-related deaths. In addition, to unin‐ hibited proliferation, leukemic cells dispense early from the bone marrow into the peripheral blood, followed by an infiltration of various organs such as lymph nodes, liver, spleen, lungs, intestinal tract, skin, or mucous membranes. Several studies are concerned with the critical role of angiogenesis in the development and growth of sol‐ id tumors and hematological malignancies. Moreover, angiogenic mediators created by AML cells act through external or internal autocrine loops, thereby directly indors‐ ing cell survival, spread, and disease development. In recent years, many researchers focus on angiopioetins (Ang), an innovative family of angiogenic mediators, which have shown to be vital regulators of angiogenesis and vascular stability. Ang-1 and its antagonist Ang-2 act via the receptor tyrosine kinase sTie 2, which is expressed in en‐ dothelial cells (ECs) of the vasculature and in subset of hematopoietic stem cells. Binding of Ang-1 causes phosphorylation of sTie2 and ensures the integrity of the vasculature by stimulating interactions between ECs and endothelial support cells. This chapter reviews the incidence, mortality, pathogenesis, and diagnostic proce‐ dures of AML. As well as aims at evaluating serum levels of endostatin, MMP- 9, and uPAR in acute myeloid leukemia patients before chemotherapy and after achieving complete remission. At the same time, the chapter also assesses the pretreatment lev‐ els of plasma Ang-1, Ang-2, and sTie2, and the calculated ratio of Ang2/sTie2 receptor in a cohort of AML patients also studies their impact on the AML patients' overall sur‐ vival.

**Keywords:** AML, endostatin, MMP- 9, uPAR, Angi-1, Angi-2, sTie2

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **1. Introduction**

Cancer is considered as one of the major causes of mortality in the world. Despite the recent advances in science, cancer has not been cured yet [1]. Healthy cells are different from cancer cells. Cancer cells can do what others cells cannot such as: become resistant to growth inhibi‐ tion; evade apoptosis; invade, metastasize, duplicate without limits; and support angiogenesis [2]. Cancer death rates remained approximately the same in the United States from 1975 through 2002, unlike heart disease. It is predicted that there will be about 15 million new cancer cases worldwide diagnosed and roughly 12 million cancer patients could die by 2020 [3].

Leukemia is a cancer of the body's blood-forming tissues. It's caused by the rapid production of abnormal white blood cells. The high number of abnormal white blood cells are not able to fight infection. The white blood cells can debilitate the ability of the bone marrow to produce red blood cells and platelets, figure 1 [4]. Leukemia develops when blood stem cells in the bone marrow change. The bone marrow will no longer grow and behave abnormally. The cells that are abnormal are called leukemia cells, figure 2. Leukemia cells will then start to crowd out the normal blood cells, causing the normal blood cells to not do their normal jobs over time [5].

**Figure 1.** Stem cells and blood cell production.

There are many types of leukemia that exist. There are forms of leukemia that are more common in children, while other forms of leukemia only occur in adults. There are many types of risk factors for leukemia and they include, among others, age, hereditary syndromes, radiation exposure, smoking, and other unknown factors [6]. Furthermore, depending on the type of leukemia the signs and symptoms will vary. The type of blood that the stem cell leukemia develops determines the type of leukemia. Abnormal lymphoid stem cells develop lymphocytic leukemias, also known as lymphoblastic leukemias. Abnormal myeloid stem cells develop myelogenous leukemias [7, 8].

A genetically heterogeneous clonal disorder called acute myeloid leukemia (AML) is charac‐ terized by the accumulation of somatic genetic alterations in hematopoietic progenitor cells that transform mechanisms of self-renewal, differentiation, and proliferation [9]. Approxi‐ mately 55% of adults with AML are detected by non-random clonal chromosome aberrations (i.e., balanced translocations, deletions, inversions, monosomies, and trisomies). These chromosome changes have been recognized as the most important prognostic factor of complete remission, risk of relapses, and long-term survival and have contributed to disease designation [10, 11]. A number of gene mutations and deregulated expression of genes have been identified in recent years, clarifying the immense heterogeneity of cytogeneticallydefined AML subsets, especially the larger subsets of AML showing normal karyotype [12, 13].

**Figure 2.** The normal and leukemic cells in the bone marrow.

**1. Introduction**

116 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

**Figure 1.** Stem cells and blood cell production.

develop myelogenous leukemias [7, 8].

Cancer is considered as one of the major causes of mortality in the world. Despite the recent advances in science, cancer has not been cured yet [1]. Healthy cells are different from cancer cells. Cancer cells can do what others cells cannot such as: become resistant to growth inhibi‐ tion; evade apoptosis; invade, metastasize, duplicate without limits; and support angiogenesis [2]. Cancer death rates remained approximately the same in the United States from 1975 through 2002, unlike heart disease. It is predicted that there will be about 15 million new cancer cases worldwide diagnosed and roughly 12 million cancer patients could die by 2020 [3].

Leukemia is a cancer of the body's blood-forming tissues. It's caused by the rapid production of abnormal white blood cells. The high number of abnormal white blood cells are not able to fight infection. The white blood cells can debilitate the ability of the bone marrow to produce red blood cells and platelets, figure 1 [4]. Leukemia develops when blood stem cells in the bone marrow change. The bone marrow will no longer grow and behave abnormally. The cells that are abnormal are called leukemia cells, figure 2. Leukemia cells will then start to crowd out the normal blood cells, causing the normal blood cells to not do their normal jobs over time [5].

There are many types of leukemia that exist. There are forms of leukemia that are more common in children, while other forms of leukemia only occur in adults. There are many types of risk factors for leukemia and they include, among others, age, hereditary syndromes, radiation exposure, smoking, and other unknown factors [6]. Furthermore, depending on the type of leukemia the signs and symptoms will vary. The type of blood that the stem cell leukemia develops determines the type of leukemia. Abnormal lymphoid stem cells develop lymphocytic leukemias, also known as lymphoblastic leukemias. Abnormal myeloid stem cells Numerous studies have shown that angiogenesis is a crucial part in the growth and develop‐ ment of hematological malignancies and solid tumors [14]. Furthermore, there are several angiogenic mediators produced by AML cells that act through internal and external loops; thus promoting cell survival, spread of the disease, and proliferation [15]. It has been proven that basic fibroblast growth factor (b FGF) and vascular endothelial growth factor (VEGF) are major regulators of tumors with angiogenesis in AML; cellular VEGF represents an adverse prognostic factor [16].

In addition, many studies have focused on angiopoietins (Ang), which are a family of novel angiogenic mediators. Ang have been shown to be important regulators of angiogenesis and vascular stability [17]. Ang-1 and its antagonist, Ang-2, act through the tyrosine kinase sTie 2 receptor. These are expressed in endothelial cells (ECs) of the vasculature and in a subset of hematopoietic stem cells [18].

Acute leukemias are known to be a rare disease, but have a disproportionately large effect on cancer survival statistics [19]. They are the most common type of leukemia in adults, yet they continue to have the least survival rate of all other leukemias. Although, the rates have improved remarkably in the younger age groups, the prognosis in older patients continues to be very poor [20, 21].

### **2. Incidence and mortality**

Incidence of acute leukemia accounts for <3% of all cancers. Leukemia constitutes as the leading cause of death due to cancer in children and persons<39 years old [22]. The most frequent form of leukemia is AML. AML accounts for about 25% of all leukemias in the Western world in adults [23]. U.S., Australia, and western Europe have the highest AML incidence worldwide [24]. AML in the U.S. during the years 1975–2003 was approximately 3.4 occurrences per 100,000 persons (= 2.5 per 100,000 persons when age-adjusted to the world standard popula‐ tion) [25].

The mortality associated with AML, just like its incidence, varies with factors such as age, gender, and race. The age-adjusted mortality rate in the U.S. seem to increase with age and peaks at 17.6 per 100,000 persons in people aging between 80 to 84. The age adjusted mortality rate for females in the years between 2000–2003 was 2.2 per 100,000, where it appears in 3.5 per 100,000 in males in the same period. Estimates show that approximately 7,800 adults will die annually of AML in the U.S. [26, 27]. However, an estimated 54,270 new cases of leukemia are expected in 2015 [28].

#### **3. Etiology**

Several risk factors have been associated with AML. Known risk factors only account for a small number of cases that were observed [29]. This will include age, antecedent hematolog‐ ic disease, and genetic disorders, as well as exposures to chemical or other occupational hazards, radiation, viruses, chemical, and previous chemotherapy [30, 31]. There are several congenital conditions that may increase the risk of leukemia; the most common is most likely Down syndrome, which is associated with a 10- to 18-fold increase in the risk of AML [32].

#### **4. Pathogenesis**

A repercussion of specific chromosome translocations of the pathogenesis of AML are associated with the appearance of oncogenic fusion proteins. One of the fusion proteins is generally a transcription factor where the other partner is a variable in function and often involved in the control of apoptosis and cell survival. AML-associated fusion proteins function as aberrant transcriptional regulators that interfere with the process of myeloid differentiation, which determines the stage-specific arrest of maturation and enhance cell survival in a celltype specific manner as a consequence [33].

The French-American-British (FAB) system (Table 1) described AML subtypes as M0 through M7. However, AML has been reclassified by the World Health Organization (WHO) into four categories in an attempt to predict the prognosis and biologic properties of AML subcategories more accurately and to enhance the clinical relevance of the system [34].


**Table 1.** AML classification.

continue to have the least survival rate of all other leukemias. Although, the rates have improved remarkably in the younger age groups, the prognosis in older patients continues to

Incidence of acute leukemia accounts for <3% of all cancers. Leukemia constitutes as the leading cause of death due to cancer in children and persons<39 years old [22]. The most frequent form of leukemia is AML. AML accounts for about 25% of all leukemias in the Western world in adults [23]. U.S., Australia, and western Europe have the highest AML incidence worldwide [24]. AML in the U.S. during the years 1975–2003 was approximately 3.4 occurrences per 100,000 persons (= 2.5 per 100,000 persons when age-adjusted to the world standard popula‐

The mortality associated with AML, just like its incidence, varies with factors such as age, gender, and race. The age-adjusted mortality rate in the U.S. seem to increase with age and peaks at 17.6 per 100,000 persons in people aging between 80 to 84. The age adjusted mortality rate for females in the years between 2000–2003 was 2.2 per 100,000, where it appears in 3.5 per 100,000 in males in the same period. Estimates show that approximately 7,800 adults will die annually of AML in the U.S. [26, 27]. However, an estimated 54,270 new cases of leukemia

Several risk factors have been associated with AML. Known risk factors only account for a small number of cases that were observed [29]. This will include age, antecedent hematolog‐ ic disease, and genetic disorders, as well as exposures to chemical or other occupational hazards, radiation, viruses, chemical, and previous chemotherapy [30, 31]. There are several congenital conditions that may increase the risk of leukemia; the most common is most likely Down syndrome, which is associated with a 10- to 18-fold increase in the risk of AML [32].

A repercussion of specific chromosome translocations of the pathogenesis of AML are associated with the appearance of oncogenic fusion proteins. One of the fusion proteins is generally a transcription factor where the other partner is a variable in function and often involved in the control of apoptosis and cell survival. AML-associated fusion proteins function as aberrant transcriptional regulators that interfere with the process of myeloid differentiation, which determines the stage-specific arrest of maturation and enhance cell survival in a cell-

be very poor [20, 21].

tion) [25].

are expected in 2015 [28].

**3. Etiology**

**4. Pathogenesis**

type specific manner as a consequence [33].

**2. Incidence and mortality**

118 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

#### **5. WHO classification**

According to WHO, AML classifies depending on morphology, immunophenotype, genetics, and the combination of clinical features [35]. In the hopes that future work will elucidate molecular pathways that may be amenable to targeted therapies, the classification tries to identify biologic entities (Table 2) [36, 37]. The subgroup "AML with recurrent genetic abnormalities" amounts to some primary AML entities. "AML with t (8;21) (q22;q22); RUNX1- RUNX1T1" and "AML with inv (16) (p13. 1q22) or t (16;16) (p13. 1;q22); CBFB-MYH11" are considered as AML regardless of bone marrow blast counts. In "APL with t (15;17) (q22;q12); PML-RARA", RARA translocations with other partner genes are recognized separately. The former category "AML with 11q23 (MLL) abnormalities" was redefined as "AML with t (9;11) (p22;q23); MLLT3-MLL" and is now a unique entity; balanced translocations other than that involving MLLT3 should be specified in the diagnosis. Three new cytogenetically defined entities were incorporated: "AML with t (6;9) (p23;q34); DEK-NUP214"; "AML with inv (3) (q21q26. 2) or at (3;3) (q21; q26. 2); RPN1-EVI1"; and "AML (megakaryoblastic) with t (1; 22) (p13;q13); RBM15-MKL1", a rare leukemia most common in infants. Two new provisional entities defined by the presence of gene mutations were added, "AML with mutated NPM1 [nucleophosmin (nucleolarphosphoproteinB23, Numatrin)]" and "AML with mutated CEBPA[CCAAT/enhancer binding protein (C/EBP), alpha]". There is growing evidence that these two gene mutations represent primary genetic lesions (so-called class II mutations) that impair hematopoietic differentiation.


**Table 2.** Acute myeloid leukemia and related precursor neoplasms and acute leukemias of ambiguous lineage (WHO).

### **6. Diagnostic procedures**

#### **6.1. Morphology**

**Categories**

**Acute myeloid leukemia with recurrent genetic abnormalities**

AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); *RPN1*-*EVI1* AML (megakaryoblastic) with t (1;22) (p13;q13); *RBM15*-*MKL1*

**Acute myeloid leukemia withmyelodysplasia-related changes**‡

Acute panmyelosis with myelofibrosis (syn.: acute myelofibrosis; acute myelosclerosis) **Myeloid sarcoma (syn.: extramedullary myeloid tumor; granulocytic sarcoma;chloroma)**

Transient abnormal myelopoiesis (syn.: transient myeloproliferative disorder)

Mixed phenotype acute leukemia with t(9;22)(q34;q11.2); *BCR*-*ABL1* Mixed phenotype acute leukemia with t(v;11q23); *MLL* rearranged

*Provisional entity: Natural killer(NK)–celllymphoblastic leukemia/lymphoma*

**Table 2.** Acute myeloid leukemia and related precursor neoplasms and acute leukemias of ambiguous lineage (WHO).

**Acute myeloid leukemia, not otherwise specified (NOS)** Acute myeloid leukemia with minimal differentiation

AML with inv (16) (p13. 1q22) or t (16;16) (p13. 1;q22); *CBFB*-*MYH11*

120 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

AML with t (8;21) (q22;q22); *RUNX1-RUNX1T1*

APL with t (15;17) (q22;q12); *PML*-*RARA*\* AML with t (9;11) (p22;q23); *MLLT3*-*MLL*† AML with t (6;9) (p23;q34); *DEK*-*NUP214*

*Provisional entity: AML with mutated NPM1 Provisional entity: AML with mutated CEBPA*

**Therapy-relatedmyeloid neoplasms**§

Acute myelomonocytic leukemia Acute monoblastic/monocytic leukemia

Erythroleukemia, erythroid/myeloid Acute megakaryoblastic leukemia Acute basophilic leukemia

**Myeloid proliferations related to Down syndrome**

Myeloid leukemia associated with Down syndrome **Blastic plasmacytoid dendritic cell neoplasm Acute leukemias of ambiguous lineage** Acute undifferentiated leukemia

Mixed phenotype acute leukemia, B/myeloid, NOS Mixed phenotype acute leukemia, T/myeloid, NOS

Acute erythroid leukemia Pure erythroid leukemia

Acute myeloid leukemia without maturation Acute myeloid leukemia with maturation

AML is first described by its morphology figure 3, or what the cancer cells look like when put under a microscope. The type of normal, immature white blood cell most closely resembles classified AML. The cancer that is in the cells that normally produce neutrophils is a subtype called myeloid leukemia in most patients with AML.

Using a May-Grunwald-Giemsa or a Wright-Giemsa stain, blood and marrow smears are morphologically examined.Using this method, they can give information about blood diseases (e.g., anemia, leukemia) that will change the aspect, leukocytes, number, size, or shape of erythrocytes and platelets [38]. 200 leukocytes on blood smears and 500 nucleated cells on marrow smears, with the latter containing spicules, is recommended to be count‐ ed. A marrow or blood blast count of 20% or more is required to be a diagnosis of AML, except for AML with t (8;21), t(15;17), t (16;16), or inv (16) and in a few cases of erythroleuke‐ mia. Included in the blast count are myeloblasts, monoblasts, and megakaryoblasts. AML with monoblasts, monocytic, or myelomonocytic differentiation and promonocytes, howev‐ er, not abnormal monocytes are counted as blast equivalents. Only rare instances of pure erythroid leukemia can erythroblasts be not counted as blasts. Occasionally, the cytoplasm of the immature cells may contain abundant basophilic cytoplasm, containing variable numbers of indistinct coalescent granules. The diagnosis is M1 in case the immature cells are <10%, and the diagnosis will be AML-M2 if the immature cells are >10%. AML M2 baso showed a higher number of basophils along with the typical M2 morphology and was sometimes correlated with the t(6;9) [39].

**Figure 3.** AML-M2 morphology. Note the presence of myeloid maturation.

#### **6.2. Immunophenotyping**

Immunophenotyping was performed by using a direct immunofluorescent technique and flow cytometry on peripheral blood specimens anti-coagulated by heparin of a newly diagnosed acute leukemia. For most markers, a commonly used criterion is 20% or more of leukemic cells expressing the marker [40], whereas for selected markers (e.g., cytoplasmic CD3, MPO, TdT, CD34, and CD117) a lower cutoff has been applied (10%). Phenotype acute leukemia (MPAL) diagnoses lineage assignment, and Measurement of Minimal Residual Disease is used to detect aberrant immunophenotypes (MRD), which is necessary for quantification of expression patterns of several surfaces and cytoplasmic antigens [41]. For morphological evaluation flow cytometry, determination of blast count should not be used as a substitute.

Immunophenotyping establishes the diagnosis of AML with minimal differentiation, acute megakaryoblastic leukemia, and acute leukemias of ambiguous lineage [42]. An AML with minimal differentiation is without morphologic and cytochemical evidence of myeloid differentiation [43].

Acute megakaryoblastic leukemia has 20% or more blasts, of which 50% or more are of megakaryocytic lineage; megakaryoblasts express less commonly CD42 one or more platelet glycoproteins CD41 and/or CD61. Acute Undifferentiated Leukemia (AUL) or those with blasts that express markers of more than one lineage (i.e., MPAL), are acute leukemias of ambiguous lineage, are rare leukemias, and comprise cases that show no evidence of lineage differing. AULs often express CD34 and/or CD38 and/or HLA-DR but lack lineage associated markers. Distinct blast populations or one blast population with markers of different lineages on the same cell or a combination of both can be contained in MPAL. WHO defines MPAL and encompasses subsets that are with or without genetic abnormalities [44].

#### **6.3. Cytogenetic and molecular genetics**

Chromosome preparation from peripheral blood and/or bone marrow was done according to standard techniques after culturing for 24 or 48 hours [45]. Colcemid treatment, hypotonic shock, and 3:1 methanol: acetic acid fixation and chromosome analysis were carried out on Gbanded metaphase and were included in routine methods for metaphase spread preparations. According to the 1995 International System for Human Cytogenetic Nomenclature at least four metaphase were karyotyped and described [46]. In order to confirm or exclude the presence of leukemia-associated gene rearrangements, molecular genetic studies were performed. Thus, either fluorescence with LSI Dual Color Break Apart Rearrangement probe for Mixed Lineage Leukemia (MLL) or Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for detecting AML rearrangements were used [47].

#### **6.4. Genome-wide studies**

The major ways to distinguish AML subtypes involve hematopathologic diagnosis with flow cytometry and cytogenetic/molecular analyzes. Essential in the distinction among the sub‐ classes of AML is the notion that different subtypes of AML express different proteins, either at the cell surface, defining AML types distinguishable based on flow cytometry, or as a result of chromosomal rearrangements or gene mutations. Therefore, it is logical that a technique that can define a transcriptional gene expression globally should be able to distinguish among AML subtypes [48]. Identifiying novel genetic abnormalities and the promise of making the systematic characterization of cancer genomes feasible has resulted in progress in genomics technology. For example, gene- and micro RNA-expression profiling have proven valuable for the discovery of novel leukemia subgroups and of prognostic signatures [49]. Uniparental disomy (UPD) is when a genome-wide single nucleotide polymorphism (SNP)–based map‐ ping arrays, giving both copy number and allele-specific information, led to the identification of a novel mechanism involved in the pathogenesis of AML [50]. UPD is due to a mitotic recombination event and may render a cell homozygous for a pre-existing mutation positioned in the affected genomic region. The power of SNP genotyping as a tool for gene discovery is shown by several recent studies [51]. Hopefully, high-throughput DNA sequence analysis will become possible at an affordable cost, while analyses of genomic copy number will continue to be informative with regard to the selection of candidate leukemia genes. This may ultimately result in the development of comprehensive, disease- and allele-specific oncogene mutation profiling strategies [52].

#### **6.5. Additional diagnostic tests**

acute leukemia. For most markers, a commonly used criterion is 20% or more of leukemic cells expressing the marker [40], whereas for selected markers (e.g., cytoplasmic CD3, MPO, TdT, CD34, and CD117) a lower cutoff has been applied (10%). Phenotype acute leukemia (MPAL) diagnoses lineage assignment, and Measurement of Minimal Residual Disease is used to detect aberrant immunophenotypes (MRD), which is necessary for quantification of expression patterns of several surfaces and cytoplasmic antigens [41]. For morphological evaluation flow

Immunophenotyping establishes the diagnosis of AML with minimal differentiation, acute megakaryoblastic leukemia, and acute leukemias of ambiguous lineage [42]. An AML with minimal differentiation is without morphologic and cytochemical evidence of myeloid

Acute megakaryoblastic leukemia has 20% or more blasts, of which 50% or more are of megakaryocytic lineage; megakaryoblasts express less commonly CD42 one or more platelet glycoproteins CD41 and/or CD61. Acute Undifferentiated Leukemia (AUL) or those with blasts that express markers of more than one lineage (i.e., MPAL), are acute leukemias of ambiguous lineage, are rare leukemias, and comprise cases that show no evidence of lineage differing. AULs often express CD34 and/or CD38 and/or HLA-DR but lack lineage associated markers. Distinct blast populations or one blast population with markers of different lineages on the same cell or a combination of both can be contained in MPAL. WHO defines MPAL and

Chromosome preparation from peripheral blood and/or bone marrow was done according to standard techniques after culturing for 24 or 48 hours [45]. Colcemid treatment, hypotonic shock, and 3:1 methanol: acetic acid fixation and chromosome analysis were carried out on Gbanded metaphase and were included in routine methods for metaphase spread preparations. According to the 1995 International System for Human Cytogenetic Nomenclature at least four metaphase were karyotyped and described [46]. In order to confirm or exclude the presence of leukemia-associated gene rearrangements, molecular genetic studies were performed. Thus, either fluorescence with LSI Dual Color Break Apart Rearrangement probe for Mixed Lineage Leukemia (MLL) or Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for detecting

The major ways to distinguish AML subtypes involve hematopathologic diagnosis with flow cytometry and cytogenetic/molecular analyzes. Essential in the distinction among the sub‐ classes of AML is the notion that different subtypes of AML express different proteins, either at the cell surface, defining AML types distinguishable based on flow cytometry, or as a result of chromosomal rearrangements or gene mutations. Therefore, it is logical that a technique that can define a transcriptional gene expression globally should be able to distinguish among AML subtypes [48]. Identifiying novel genetic abnormalities and the promise of making the

cytometry, determination of blast count should not be used as a substitute.

122 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

encompasses subsets that are with or without genetic abnormalities [44].

**6.3. Cytogenetic and molecular genetics**

AML rearrangements were used [47].

**6.4. Genome-wide studies**

differentiation [43].

A patient with AML may need additional diagnostic tests and procedures that can confirm the diagnosis. Leukemia usually does not form tumors, so imaging tests are not used for diagnosis and are often used to check infections or other problems that may occur. X-rays routinely looks for suspected lung infection. Moreover, imaging in a few cases may be done to determine disease extent if it is thought to be spread beyond the bone marrow and blood.

#### *6.5.1. Computed tomography (CT)*

The computed tomography (CT) scan is usually needed if it is suspected that leukemia is growing in an organ, such as the spleen. Unlike a regular x-ray, CT scans can illustrate in detail soft tissues. In some cases, a CT scan can be used to guide the biopsy needle into areas of abnormality, such as an abscess [53].

#### *6.5.2. Magnetic resonance imaging (MRI) scan*

Asymptomatic patients are diagnosed with acute leukemia after the identification of abnormal peripheral blood counts. However, magnetic resonance imaging (MRI) has found that abnormal bone marrow signals appear in patients who have not been previously diagnosed with leukemia.The differentiation between benign marrow edema and tumorous involvement of the bone marrow is the measurement of the tissue microstructure the diffusion-weighted imaging reflects the random motion of water protons. MRI enables precise assessment of bone marrow infiltration early and before osteolytic changes become visible by conventional radiology imaging or CT scans [54].

#### *6.5.3. Ultrasound*

Ultrasound uses sound waves and ultrasound echoes and then produces a picture of internal organs or masses. Usually for this test, a small, microphone-like instrument called a transducer is placed on the skin. These transducers emit sound waves and pick up echoes as they bounce off organs in the body. The echos are then converted into an image that is displayed on a computer. Ultrasounds are used to look at the lymph nodes near the surface of the body or look at the enlarged organs inside the abdomen such as the kidneys, liver, and spleen [55].

#### **7. Prognostic factors**

The patient characteristics and general health condition of those related to characteristics particular to the AML clone are prognostic factors that may be subdivided. Patient character‐ istics and general health condition usually predict treatment-related mortality (TRM) and becomes important as patient age increases. Characteristics particular to the AML clone predicts resistance to at least conventional therapy. The following studies are aimed at assessing the pretreatment levels of plasma Ang-1, Ang-2, and sTie2, and the calculated ratio of Ang-2/sTie2 receptor in AML patients. Moreover, it aims to evaluate serum levels of endostatin, MMP- 9, and uPAR in AML patients before chemotherapy and after achieving complete remission. At the same time, it also studies the impact in the lives AML patients and their overall survival.

#### **7.1. Circulating angiopoietin-2 is a strong prognostic factor in AML**

The prognostic significance and over expression of cellular angiopoietin in the isolated peripheral AML blast and AML bone marrow is not demonstrable. Loges (2005) [56] showed that patients with high cellular Ang-2 had extended overall survival compared to those with low Ang-2 expression. Previously, we assessed the pretreatment levels of plasma Ang-1, Ang-2, and sTie2, and the calculated ratio of Ang-2/sTie2 receptor in a cohort of 71 AML patients in order to evaluate the impact in the lives of AML patients and their overall survival.

#### *7.1.1. Materials*

Seventy-one newly-diagnosed AML patients were tested in this study. Table 3 shows the patients' characteristics. All 71 patients were tested and followed up to 24 months (Table 3) or up until their deaths. Patients were followed in the oncology department at Mansoura Cancer Institute in Egypt where they were treated with approved protocols. 3+7 protocols (Daunor‐ ubicin 45 mg/m2 iv days 1–3; Cytarabine 100 mg/m2 /day continuous infusion for 7 days) were used to treat the AML patients. Upon post remission, there was a high dose of cytarabine. Salvage therapy (HAM protocol) was applied (Cytarabine 3 gm/m2 bid iv 3 hours infusion days 1–3; Mitoxantrone 10 mg/m2 iv days 3–5) for patients who did not respond to the induction therapy. Complete hematological remission patients (bone marrow blast cells <5% in bone marrow) were submitted to consolidation therapy that contained a high dose cytrabine containint regimine (2 g/m2 /2hours/day × 4 days) + Daunorubicin 45 mg/m2 /day ×3 days. The M3 patients received all trans retinoic acid and went through chemotherapy. Nineteen normal, healthy subjects made up the normal control group.


**Table 3.** Patients' characteristics.

#### *7.1.2. Methods*

off organs in the body. The echos are then converted into an image that is displayed on a computer. Ultrasounds are used to look at the lymph nodes near the surface of the body or look at the enlarged organs inside the abdomen such as the kidneys, liver, and spleen [55].

124 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

The patient characteristics and general health condition of those related to characteristics particular to the AML clone are prognostic factors that may be subdivided. Patient character‐ istics and general health condition usually predict treatment-related mortality (TRM) and becomes important as patient age increases. Characteristics particular to the AML clone predicts resistance to at least conventional therapy. The following studies are aimed at assessing the pretreatment levels of plasma Ang-1, Ang-2, and sTie2, and the calculated ratio of Ang-2/sTie2 receptor in AML patients. Moreover, it aims to evaluate serum levels of endostatin, MMP- 9, and uPAR in AML patients before chemotherapy and after achieving complete remission. At the same time, it also studies the impact in the lives AML patients and

The prognostic significance and over expression of cellular angiopoietin in the isolated peripheral AML blast and AML bone marrow is not demonstrable. Loges (2005) [56] showed that patients with high cellular Ang-2 had extended overall survival compared to those with low Ang-2 expression. Previously, we assessed the pretreatment levels of plasma Ang-1, Ang-2, and sTie2, and the calculated ratio of Ang-2/sTie2 receptor in a cohort of 71 AML patients in order to evaluate the impact in the lives of AML patients and their overall survival.

Seventy-one newly-diagnosed AML patients were tested in this study. Table 3 shows the patients' characteristics. All 71 patients were tested and followed up to 24 months (Table 3) or up until their deaths. Patients were followed in the oncology department at Mansoura Cancer Institute in Egypt where they were treated with approved protocols. 3+7 protocols (Daunor‐

used to treat the AML patients. Upon post remission, there was a high dose of cytarabine.

therapy. Complete hematological remission patients (bone marrow blast cells <5% in bone marrow) were submitted to consolidation therapy that contained a high dose cytrabine

M3 patients received all trans retinoic acid and went through chemotherapy. Nineteen normal,

/2hours/day × 4 days) + Daunorubicin 45 mg/m2

/day continuous infusion for 7 days) were

iv days 3–5) for patients who did not respond to the induction

bid iv 3 hours infusion

/day ×3 days. The

**7.1. Circulating angiopoietin-2 is a strong prognostic factor in AML**

ubicin 45 mg/m2 iv days 1–3; Cytarabine 100 mg/m2

healthy subjects made up the normal control group.

days 1–3; Mitoxantrone 10 mg/m2

containint regimine (2 g/m2

Salvage therapy (HAM protocol) was applied (Cytarabine 3 gm/m2

**7. Prognostic factors**

their overall survival.

*7.1.1. Materials*

In a sterile tube with Ethylenediaminetetraacetic acid (EDTA), 6 ml of peripheral blood were collected from each AML patient at presentation and before the start of induction chemother‐ apy. For ten minutes in a refrigerated centrifuge, the plasma was separated by centrifugation at 1500 Xg. The separated plasma was reserved at -70ºC, thawed in use. Using commercially available kits from R&D systems (Minneapolis, MN, USA) and according to the manufacturer's instructions, Enzyme-linked immunosorbent assay (ELISA) were performed. Briefly, patient samples using the anticoagulant were collected with Ethylenediaminetetraacetic acid (EDTA) and stored at -80o C. Plasma samples were transferred to separate microplates, each containing a specific antibody for Ang-1, Ang-2, or sTie2. At room temperature, the mixtures were incubated for 2 hours. Plates were washed 4 times to remove unbound antigen. Enzyme-linked polyclonal antibodies specific for each angiogenic factor were then added, then incubated for 2 hours, followed by another washing step. At color development, adding of the substrate was stopped and a standard curve was used to compare the intensity of the color measure. Optical density of each well was determined at 570 nm.

#### *7.1.3. Statistics*

The software package SPSS version 10 has been used in this study. Mann-Whitney rank sum test for independent groups were used to analyze the differences in angiogenic factor level between AML and control groups. Furthermore, the Spearman rank correlation coefficient (Rs) was applied to assess the correlations between continuous variables. The Kaplan-Meier method was used for survival curves estimation. Overall, survival was the primary outcome of the studies and was calculated from the date of the first diagnosis to the death of the patient from any cause. In order to evaluate the predictive effect of each angiogenic factor, the univariate and multivariate Cox regression analysis was performed (figure 4, 5). Optimal cutoff points depend on 50 percentile of each angiogenic factor.

**Figure 4.** Kaplan-Meier survival analysis of AML patients according to Ang-1 levels. AML patients with high Ang-1 levels (≥260) displayed significantly poor survival rates than those with low Ang-1 levels (≤260) (P=0.018).

#### *7.1.4. Results*

#### *7.1.4.1. Comparison of plasma levels of Ang-1, Ang-2, sTie2, and Ang-2/sTie2ratio in AML patients versus healthy controls*

The plasma levels (median and range) of Ang-1, Ang-2, and sTie2 in pre-therapeutic AML patients and healthy volunteers are illustrated in Table 3. Circulating levels of Ang-2 and the calculated Ang-2/sTie2 ratio are decidedly higher in AML patients compared with controls

**Figure 5.** Kaplan-Meier survival analysis of AML patients according to Ang-2/sTie ratio. AML patients with a high Ang-2/sTie ratio (>279) displayed significantly poor survival rates than those with low Ang-2/sTie ratio (≤279) (P=0.004).

(P=0.002, P=0.015 respectively). In AML patients, the Ang-1 and sTie2 levels were not signifi‐ cantly compared with the controls (Table 4).


**Table 4.** Plasma angiopoietins levels in AML patients as compared to controls.

stopped and a standard curve was used to compare the intensity of the color measure. Optical

The software package SPSS version 10 has been used in this study. Mann-Whitney rank sum test for independent groups were used to analyze the differences in angiogenic factor level between AML and control groups. Furthermore, the Spearman rank correlation coefficient (Rs) was applied to assess the correlations between continuous variables. The Kaplan-Meier method was used for survival curves estimation. Overall, survival was the primary outcome of the studies and was calculated from the date of the first diagnosis to the death of the patient from any cause. In order to evaluate the predictive effect of each angiogenic factor, the univariate and multivariate Cox regression analysis was performed (figure 4, 5). Optimal cut-

**Figure 4.** Kaplan-Meier survival analysis of AML patients according to Ang-1 levels. AML patients with high Ang-1

*7.1.4.1. Comparison of plasma levels of Ang-1, Ang-2, sTie2, and Ang-2/sTie2ratio in AML patients*

The plasma levels (median and range) of Ang-1, Ang-2, and sTie2 in pre-therapeutic AML patients and healthy volunteers are illustrated in Table 3. Circulating levels of Ang-2 and the calculated Ang-2/sTie2 ratio are decidedly higher in AML patients compared with controls

levels (≥260) displayed significantly poor survival rates than those with low Ang-1 levels (≤260) (P=0.018).

density of each well was determined at 570 nm.

126 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

off points depend on 50 percentile of each angiogenic factor.

*7.1.3. Statistics*

*7.1.4. Results*

*versus healthy controls*

*7.1.4.2. The association between plasma levels of Ang-1, Ang-2, sTie2, calculated Ang-2/sTie2 ratio, and clinico-pathological features*

A significant correlation was obtained between Ang-2, determined Ang-2/sTie2 ratio, as well as patients' age with P value <0.05. On the other hand, no significant correlation was seen with sTie2 receptor that gives a P value of 0.786. Moreover, significant correlation was observed with Ang-2, sTie2, and WBCs with r=0.338, P=0.004, r=0.263, P=0.027. At the same time, no significant correlation has been seen in Ang-1 and calculated Ang-2/sTie2 ratio. The cell blast percentage in peripheral smears were significantly correlated to Ang-2 and sTie2 receptors with the following data obtained (r=0.365 P=0.002, r=0.387 P=0.001); no significant correlation with Ang-1 or Ang-2/sTie2 ratio (Table 5). Significant correlation appears clearly between LDH and Ang-1, Ang-2, and sTie2, contrary with Ang-2/sTie2 ratio. A positive correlation between


Ang-2 and Ang-2/sTie2 ratio with cytogenetic grades was found. On the other hand, a negative correlation was obtained with sTie2 receptor levels (P>0.1).

**Table 5.** Correlation angiopoietins and other prognostic markers.

*7.1.4.3. Association between plasma levels of Ang-1, Ang-2, sTie2, calculated Ang-2/sTie2 ratio, and overall survival*

Cytogenetic 0. 258 0.030\* 0.426 0.000\* 0.170 0.157 0.333 0.005\*

In the study, univariate Cox proportional hazard analysis was dependent on 50% as a cutoff. This was done to assess the effect of circulating Ang-1, Ang-2, sTie2 levels, and Ang-2/sTie2 ratio on AML overall survival. The clinic-pathological variable as cytogenetic settled that a significant effect on the overall survival (p<0.005) variable as cytogenetic (intermediate vs. good vs. poor) and LDH (≤450 vs. >450).

AML survival was significantly associated with angiogenic factors Ang-1 (≤260 vs. >260), Ang-2 (≤1400 vs. >1400), Ang-2/sTie ratio (≤279 vs. >297) but sTie2 showed no effect. The death of Ang-2 was higher for the relative risk (RR) when the base line >1400 pg/ml (RR 5.7, with 95% confidence interval (CI))(0.061–0.50, p=0.001) had high significance with this ratio. CIs were 0.084–0.653, P=0.004 and the RR was 4.1 95%. No significant role of sTie2 >3.9 ng/ml (RR 1.24 95%, CI 0.481–3.220, p=0.652) table 6. Additionally, we performed multivariate Cox regression analysis incorporating all variables that were in significant effect on univariate analysis. The calculated Ang-2/sTie2 ratio was identified to be as the most prognostic factor with significant independent impact on survival (p = 0.000) (Table 7).

The findings from this study did not coordinate with Loges et al. (2005), wherein cellular Ang-2 was identified as the predictor of AML patients with favorable prognosis [56]. On the basis that the source of circulating Ang-2 from not only leukemic blasts, but also from other cell types such as endothelial cells [57,58], show the differences between soluble Ang-2 and cellular expression of Ang-2. Other hematological malignancies or solid tumors show limited studies considering prognostic relevance. Moreover, angiosarcoma [59], breast cancer [60], multiple myeloma, chronic myeloid leukemia [61], and recently in acute myeloid leukemia [58], higher levels of Ang-2 have been detected. An attractive therapeutic target when introducing antiangiogenic strategies in the treatment of AML could be strategies in the treatment of AML for Ang-2.

Adult Acute Myeloid Leukemia – A Possible Relation to Disease Invasion and the Impact of Independent... http://dx.doi.org/10.5772/61179 129


**Table 6.** Univarate analysis of overall survival in AML patients.

Ang-2 and Ang-2/sTie2 ratio with cytogenetic grades was found. On the other hand, a negative

Age (years) 0.254 0.030\* 0.336 0.004\* -0.033 0.786 0.287 0.015\*

Peripheral blood 0.183 0.127 0.365 0.002\* 0.387 0.001\* 0.169 0.158 Blast in BM% 0.137 0.254 0.305 0.010\* 0.317 0.007\* 0.187 0.119 LDH 0.513 0.000\* 0.362 0.002\* 0.262 0.027 0.146 0.225 Cytogenetic 0. 258 0.030\* 0.426 0.000\* 0.170 0.157 0.333 0.005\*

*7.1.4.3. Association between plasma levels of Ang-1, Ang-2, sTie2, calculated Ang-2/sTie2 ratio, and*

In the study, univariate Cox proportional hazard analysis was dependent on 50% as a cutoff. This was done to assess the effect of circulating Ang-1, Ang-2, sTie2 levels, and Ang-2/sTie2 ratio on AML overall survival. The clinic-pathological variable as cytogenetic settled that a significant effect on the overall survival (p<0.005) variable as cytogenetic (intermediate vs.

AML survival was significantly associated with angiogenic factors Ang-1 (≤260 vs. >260), Ang-2 (≤1400 vs. >1400), Ang-2/sTie ratio (≤279 vs. >297) but sTie2 showed no effect. The death of Ang-2 was higher for the relative risk (RR) when the base line >1400 pg/ml (RR 5.7, with 95% confidence interval (CI))(0.061–0.50, p=0.001) had high significance with this ratio. CIs were 0.084–0.653, P=0.004 and the RR was 4.1 95%. No significant role of sTie2 >3.9 ng/ml (RR 1.24 95%, CI 0.481–3.220, p=0.652) table 6. Additionally, we performed multivariate Cox regression analysis incorporating all variables that were in significant effect on univariate analysis. The calculated Ang-2/sTie2 ratio was identified to be as the most prognostic factor

The findings from this study did not coordinate with Loges et al. (2005), wherein cellular Ang-2 was identified as the predictor of AML patients with favorable prognosis [56]. On the basis that the source of circulating Ang-2 from not only leukemic blasts, but also from other cell types such as endothelial cells [57,58], show the differences between soluble Ang-2 and cellular expression of Ang-2. Other hematological malignancies or solid tumors show limited studies considering prognostic relevance. Moreover, angiosarcoma [59], breast cancer [60], multiple myeloma, chronic myeloid leukemia [61], and recently in acute myeloid leukemia [58], higher levels of Ang-2 have been detected. An attractive therapeutic target when introducing antiangiogenic strategies in the treatment of AML could be strategies in the treatment of AML for

with significant independent impact on survival (p = 0.000) (Table 7).

) 0.170 0.155 0.338 0.004\* 0.263 0.027\* 0.205 0.086

**Ang-1 Ang-2 sTie2 receptors Ang-2/sTie2 ratio**

correlation was obtained with sTie2 receptor levels (P>0.1).

128 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

**Table 5.** Correlation angiopoietins and other prognostic markers.

good vs. poor) and LDH (≤450 vs. >450).

WBCs (x103

*overall survival*

Ang-2.


\*\*Multivariate regression analysis of angiogenesis markers with other variants clarified that the ratio is the most foretelling predictor (p=0.000).

**Table 7.** Multivariate regression analysis of all variants.

Ang-2 blocks the therapeutic efficacy that has been manifested in solid tumors [62] and a recombinant Fc fusion protein against the action of angiopoietins is now being studied in a phase I study with patients that have advanced solid tumors. This study has concluded that the calculated ratio between Ang-2/sTie is a factor that should be a part of the decision-making process when choosing to use anti-angiogenic therapy.

#### **7.2. Endostatin levels associated with favorable outcome**

Inhibiting angiogenesis and tumor growth have been highly effective using endostatin [63, 64]. By inhibiting proliferation-inducing apoptosis in endothelial cells, it may mediate these biological effects [65]. Endostatin has been shown to induce regression of metastatic tumor in an animal model. The relation between endostatin levels and patients' outcome is scarce and the data surrounding endostatin levels in AML patients are controversial [66]. Tumor derived proteases generate the extracellular matrix protein collagen XVIII and is the C terminal antiangiogenic fragment. It is not clear in AML patients the levels of prognostic relevance of serum endostatin. The study took serum levels of endostatin before chemotherapy and after complete remission in acute leukemia patients. The study also took the patients' outcome and correlated the endostatin levels.

#### *7.2.1. Patients serum sample*

Samples from 8 females and 22 males, with a median age of 37 within a range of 19 to 66 years old, with AML had been taken before chemotherapy. Also, 20 out of 30 patients were tested again once they were in complete remission (CR). From the healthy normal person group, ten samples were taken and matched with the same age and sex and were evaluated as the control group for reference. Enzyme linked immunosorbent assay (ELISA) were determined using serum endostatin (sE) levels.

#### **7.3. Study results**

**95% confidence interval for B**

**Lower**

130 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

Ang-1 0.000 0.000 -0.068 0.946 Ang-2 0.000 0.000 1.829 0.072 Ang-2/sTie ratio -0.002 -0.001 -3.939 0.000\*\* WBCs -0.007 0.002 -1.092 0.279 Peripheral Blast -0.016 0.004 -1.266 0.210 Cell % -0.010 0.008 -0.299 0.766 Blast in BM % 0.000 0.000 -0.732 0.467

\*\*Multivariate regression analysis of angiogenesis markers with other variants clarified that the ratio is the most foretelling

Ang-2 blocks the therapeutic efficacy that has been manifested in solid tumors [62] and a recombinant Fc fusion protein against the action of angiopoietins is now being studied in a phase I study with patients that have advanced solid tumors. This study has concluded that the calculated ratio between Ang-2/sTie is a factor that should be a part of the decision-making

Inhibiting angiogenesis and tumor growth have been highly effective using endostatin [63, 64]. By inhibiting proliferation-inducing apoptosis in endothelial cells, it may mediate these biological effects [65]. Endostatin has been shown to induce regression of metastatic tumor in an animal model. The relation between endostatin levels and patients' outcome is scarce and the data surrounding endostatin levels in AML patients are controversial [66]. Tumor derived proteases generate the extracellular matrix protein collagen XVIII and is the C terminal antiangiogenic fragment. It is not clear in AML patients the levels of prognostic relevance of serum endostatin. The study took serum levels of endostatin before chemotherapy and after complete remission in acute leukemia patients. The study also took the patients' outcome and correlated

Samples from 8 females and 22 males, with a median age of 37 within a range of 19 to 66 years old, with AML had been taken before chemotherapy. Also, 20 out of 30 patients were tested again once they were in complete remission (CR). From the healthy normal person group, ten

**bound Upper bound <sup>T</sup> <sup>P</sup>**

**Model**

LDH

predictor (p=0.000).

the endostatin levels.

*7.2.1. Patients serum sample*

**Table 7.** Multivariate regression analysis of all variants.

process when choosing to use anti-angiogenic therapy.

**7.2. Endostatin levels associated with favorable outcome**

#### *7.3.1. Eendostatin levels in AML*

In the control group, endostatin levels ranged from 5–20 ng/ml and have a median of 11.8 ng/ ml. Pre-treatment serum endostatin (sE) levels ranged from 3–70 ng/ml and have a median of 14.8. The post-treatment sE levels ranged from 15.8–78 gm/ml with a median of 35 ng/ml figure 6. There was no statistically significant differences that were found between pre-treatment sE levels and normal controls. The post-treatment levels were statistically higher in pre-treatment controls. CR and sE and the relationship between them were then evaluated. Twenty out of the 30 AML patients reached CR. The patients who achieved CR had higher sE levels shown from the Wilcoxon tests.

**Figure 6.** The Box-plot diagram showing the serum endostatin range levels in AML patients pre- and post-treatment compared with controls. Both 25th and 75th percentiles are illustrated with the upper and lower lines of each box. The median indicated with the line appears in each box. Pre-treatment compared to control (P>0.05); post treatment com‐ pared to control (P=0.000); and pre-treatment compared to post treatment (P= 0.001).

At the time of diagnosis as illustrated in Table 8, the sE levels increased markedly in the survived group when compared to those who died; the survived AML patients and control groups had a range of 9.6–70 ng/ml and median 38.15 compared with the levels of the died group that had a range of 3–25.5 ng/ml and median 14.8 (P= 0.04) and control group with a range 5–20 ng/ml and median 11.8 (P=0.026).


**Table 8.** Baseline serum endostatin levels in died AML patients as compared to the survived group as well as normal controls.

The baseline sElevel was not significantly correlated to age, hemoglobin level, peripheral WBCs counts, platelet counts, blast cells percentage in bone marrow, and BCDR (P>0.05) (Table 9).


**Table 9.** Correlation between baseline endostatin levels and some clinical and laboratory parameters.

The prognostic value of sE was then evaluated by dividing AML patients into low and high sE groups using the 75 percentile level of AML group (i.e., 20.5 ng/ml) as the cut off. As illustrated in Table 10 and Figure 7, high sE patients survived for a significantly longer time than low sE patients (P=0.02).


**Table 10.** Forty-eight weeks disease-free survival of the studied cases in relation to the pre-treatment serum endostatin level.

**Group Mean ± SD Median Range P value**

Survived (n=6) 56.58± 60.39 38.15 (9.6-70) 0.026

**Table 8.** Baseline serum endostatin levels in died AML patients as compared to the survived group as well as normal

The baseline sElevel was not significantly correlated to age, hemoglobin level, peripheral WBCs counts, platelet counts, blast cells percentage in bone marrow, and BCDR (P>0.05) (Table

**r P**

Died (n=19) 12.87± 6.07 14.5 (3-25.5)

132 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

Control (n=10) 11.73± 4.28 11.8 (5-20)

**Features Endostatin**

Age 0.166 0.444 Hemoglobin g/dl 0.068 0.745

Bone Marrow Blast cell % 0.029 0.889 BCDR - 0.01 0.98

**Table 9.** Correlation between baseline endostatin levels and some clinical and laboratory parameters.

BCDR (blast cell distribution ratio) = peripheral blast cell % / Bone marrow blast cell %

Above 75% 6 8 66.67% 36.33

/cmm 0.112 0.593

/cmm 0.101 0.630

The prognostic value of sE was then evaluated by dividing AML patients into low and high sE groups using the 75 percentile level of AML group (i.e., 20.5 ng/ml) as the cut off. As illustrated in Table 10 and Figure 7, high sE patients survived for a significantly longer time

Endostatin level Total Died % Censored Mean Survival time P value

Below 75% 19 17 10.53% 18.84 0.02

**Table 10.** Forty-eight weeks disease-free survival of the studied cases in relation to the pre-treatment serum endostatin

controls.

9).

WBCs× 103

level.

Platelet count × 103

than low sE patients (P=0.02).

**Figure 7.** The overall survival of AML patients, according to pre-treatment concentrations of serum endostatin. The 75% concentration level of serum endostatin was used as cut off value.

The study showed that there was not much difference in the endostatin serum levels of the pre-treatment AML patients compared to the normal control group. The baseline for AML patients was lower than at CR. There was no correlation between pre-treatment endostatin levels, age, platelet counts, bone marrow blast cell counts, peripheral white blood cell counts, and blast cell distribution ratio. AML patients were divided into high and low sE groups using the 75 percentile of sE levels of the patient group and showed the prognostic value of sE. The study showed that the group with the high sE levels survived a longer time than the patients that had a low sE levels. The results conclude that the elevated endostatin levels in AML diagnosis is a good prognostic marker for patients' outcome. Wide-scale study is recommend‐ ed in order to establish the clinical value of this study.

#### **7.4. Role of metalloproteinase and urokinase in acute myeloid leukemia**

An important role of the matrix metalloproteinase (MMPs) and urokinase may be important when trying to find tumor invasion and metastasis. An anchored membrane protein that promotes generation of plasmin on the surface of cell types is the Urokinase-type plasminogen activator receptor (uPAR-CD87), which is a glycosyl phosphatidyl inositol (GIP) and facilitates cellular tissue invasion and extravasation. Inflammatory cells or inaction of neoplastic promotes invasion from UPAR by proteolysis of urokinase [67, 68].

The collagens elastin and gelatin are a family of enzymes with the common ability to degrade various components of ECM [69]; they are also called MMPs. This family of enzymes all help the physiological processes that occur when the tissue is remodeling and repairing. The determination of these parameters in the blood are recommended as non-invasive tools in cancer diagnosis and monitoring may be reflected in body fluids called Cellular Concentration of MMPs [70].

Components of ECM, cytokines, hormones, growth factors, or a variety of biochemical stimuli all modulate the transcription of MMPs [71]. Various extracellular proteinases such as plasmin and urokinase are catalyzed by the activation of pro-MMP. Crucial roles of MMPs and soluble urokinase-type plasminogen activator receptor (suPAR) in the invasiveness of many malig‐ nant disorders have been accumulated in evidences from recent studies. The studies assessed the levels of MMP-9, uPAR in AML patients and compared them with previous clinicopatho‐ logical status [72].

#### *7.4.1. Methods and Patients*

Twenty-five males and 18 females who were recently diagnosed with AML were used in the study. The ages ranged from 26 to 73 years old, with a mean of 46.8. The AML patients were studied at the time they were diagnosed, after starting chemotherapy, and during any relapses. During the induction of chemotherapy, seven patients died. The French-American-British (FAB) study group and immunophenotypic studies performed the diagnosis [73]. There were no patients that had a history of chemotherapy or radiotherapy that were diagnosed with hematological disorders. The criteria proposed by Cheson et al.[74] is the Morphologic CR. Peripheral blood counting was obtained through WBC and a percentage of blast cells. Blast cell distribution ratio (BCDR) was equal to the ratio between peripheral absolute blast cell counts to absolute bone marrow blast cell count.

The following FAB subtypes 2 M7, 4 M3, 4 M4, 4 M6, 6 M1, 7 M5, and 16 M2 were included in the AML patients. The normal subject group was made up of 10 normal subjects that matched the age and sex of the patients were used as the control group. A high-dose combination chemotherapy containing indarubicin, cytarabine, mitoxantrone, and etopside were treated in the AML patients. Remission induction therapy included one or two courses of cytarabine, etoposide, and idarubicin. The patients that achieved morphological CR received one course of intensive therapy with cytarabine and mitoxantrone. And lastly, all patients got consolida‐ tion treatments. All AML patients were then followed around for up to a year. EDTA tubes were used to collect blood samples and kept in ice before the plasma was separated.

The plasma was then removed within 2 hours by centrifugation for 30 minutes at 4°Celsius at 1800 g and stored frozen at -70°Celsius until the examination. Ficoll-Hypaque centrifugation was used to separate the mononuclear cell fraction. Most of the mononuclear cells in AML patients were blast cells that ranged from 0–96%. 1% Triton X-100 and protease inhibitors that were contained in cells were lysed in PBS. Lysates were centrifuged at 14,000 g for 10 minutes and 4°Celsius and the supernatants were stored at -70°Celsius until it was examined. The colorimetric assay kit determined the total protein. UPAR and ELISA were used to analyze the same amount of protein from each sample.

#### *7.4.2. Immunophenotypic by flow cytometry*

#### *7.4.2.1. Preparation samples*

Erythrocyte-lysed BM samples were used to diagnose all immunophenotyping study cases. A mircroscope adjusted to 1 x 106 in each tube quantified the number of cells. The following monoclonal antibodies were used to analyze antigen expression with fluorochrome-conjugat‐ ed:

CD34 CD33 CD14 CD11b CD45 CD10 CD19 CD22 CD3 CD5

Components of ECM, cytokines, hormones, growth factors, or a variety of biochemical stimuli all modulate the transcription of MMPs [71]. Various extracellular proteinases such as plasmin and urokinase are catalyzed by the activation of pro-MMP. Crucial roles of MMPs and soluble urokinase-type plasminogen activator receptor (suPAR) in the invasiveness of many malig‐ nant disorders have been accumulated in evidences from recent studies. The studies assessed the levels of MMP-9, uPAR in AML patients and compared them with previous clinicopatho‐

Twenty-five males and 18 females who were recently diagnosed with AML were used in the study. The ages ranged from 26 to 73 years old, with a mean of 46.8. The AML patients were studied at the time they were diagnosed, after starting chemotherapy, and during any relapses. During the induction of chemotherapy, seven patients died. The French-American-British (FAB) study group and immunophenotypic studies performed the diagnosis [73]. There were no patients that had a history of chemotherapy or radiotherapy that were diagnosed with hematological disorders. The criteria proposed by Cheson et al.[74] is the Morphologic CR. Peripheral blood counting was obtained through WBC and a percentage of blast cells. Blast cell distribution ratio (BCDR) was equal to the ratio between peripheral absolute blast cell

The following FAB subtypes 2 M7, 4 M3, 4 M4, 4 M6, 6 M1, 7 M5, and 16 M2 were included in the AML patients. The normal subject group was made up of 10 normal subjects that matched the age and sex of the patients were used as the control group. A high-dose combination chemotherapy containing indarubicin, cytarabine, mitoxantrone, and etopside were treated in the AML patients. Remission induction therapy included one or two courses of cytarabine, etoposide, and idarubicin. The patients that achieved morphological CR received one course of intensive therapy with cytarabine and mitoxantrone. And lastly, all patients got consolida‐ tion treatments. All AML patients were then followed around for up to a year. EDTA tubes

were used to collect blood samples and kept in ice before the plasma was separated.

The plasma was then removed within 2 hours by centrifugation for 30 minutes at 4°Celsius at 1800 g and stored frozen at -70°Celsius until the examination. Ficoll-Hypaque centrifugation was used to separate the mononuclear cell fraction. Most of the mononuclear cells in AML patients were blast cells that ranged from 0–96%. 1% Triton X-100 and protease inhibitors that were contained in cells were lysed in PBS. Lysates were centrifuged at 14,000 g for 10 minutes and 4°Celsius and the supernatants were stored at -70°Celsius until it was examined. The colorimetric assay kit determined the total protein. UPAR and ELISA were used to analyze the

Erythrocyte-lysed BM samples were used to diagnose all immunophenotyping study cases. A

in each tube quantified the number of cells. The following

logical status [72].

*7.4.1. Methods and Patients*

counts to absolute bone marrow blast cell count.

134 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

same amount of protein from each sample.

*7.4.2. Immunophenotypic by flow cytometry*

*7.4.2.1. Preparation samples*

mircroscope adjusted to 1 x 106

CD7

Direct immunofluorescence was performed by first incubating 1 × 106 cells with the specific monoclonal antibody for 15 minutes in the dark at room temperature. To assess background fluorescence intensity, an isotype-matched negative control was used. Cells were lysed (FACS lysis solution) for 5 minutes and centrifuged at 250 g for 5 minutes. Before being re-suspended in PBS and examined, the cells were washed twice with phosphate-buffered saline (PBS). The EGIL recommendations were followed for immunologic criteria for lineage assignment [75].

#### *7.4.2.2. Data analysis*

EPICS flow cytometry was measured. For data acquisitions, the Cell quest software program was used. Over 10,000 events/tubes were measured..

Thresholds for positivity were based on isotype negative controls. Analytical gates were set on desired viable cells based on forward light scatter and side light scatter combined with exclusion of normal cells using a CD45 tube. The positivity threshold was 20% for all markers except for cytoplasmic or intra-nuclear antigens for which a 10% threshold was used [76].

#### *7.4.2.3. Assay of suPAR and uPAR in cell lysates*

As previously described [77], ELISA and suPAR were used. Immuno-plates were coated overnight with polyclonal antihuman uPAR antibodies. The wells were incubated with standard dilutions of purified recombinant suPAR or with 1:10 dilutions of plasma after blocking and washing. The volume equal to 20 ug of protein was put into each well for the cell lysates to determine protein concentrations. A mixture of monoclonal antihuman upper antibodies were rinsed and then incubated after antigen binding and then followed by alkaline phosphatase-conjugated antibodies. P-nitrophenyl phosphatase substrate was allowed to develop at room temperature to get a color reaction. Absorbance was read at 405 nm. The assay is 0.03 ng/ml lower detection limit. To estimate the total uPAR load in the cells in the circulation, the exact amount of uPAR in lysates was multiplied by the mononuclear cell count in the peripheral blood. This was tested without specific monoclonal antibodies.

#### *7.4.2.4. Total MMP-9 enzyme-linked immunosorbent assay of plasma*

Using a linked immunosorbent assay kit for human total MMP 9 according to the manual was used to determine total MMP-9 activities in the plasma. The diluted plasma samples or MMPs standards were mixed with 100 ul of 50 ul anti-MMP immunoglobulin G labeled with horseradish peroxidase in 10 mmol/l ethylenediaminetetra acetic acid. A 100-μl aliquot of the mixture was transferred to each well that was previously coated with an anti-MMP immuno‐ globulin G. The horseradish peroxidase-bound activity was detected by adding 100 μl volume of 0.15 mol/l citric acid sodium phosphate buffer at pH 4.9, containing 2.0 g/l of o-phenylene‐ dianmine and 0.02% (v/v) hydrogen peroxide, followed by incubation at room temperature for 20 minutes. Adding 100 μl sulphuric acid 1 mol/l will stop the action. The micro-plate reader was adjusted to 492 nm for absorbance measurement. The human MMPs values were obtained from the standard curve.

#### *7.4.3. Results*

#### *7.4.3.1. AML patients suPAR, cellular uPAR and MMP-9 levels versus control*

At diagnosis the AML patients' suPAR and cellular PAR levels that were considered in mean were highly significant compared to the healthy controls with P=0.001 for both. However, during AML remission, the suPAR levels declined, achieving levels close to the control levels. On the contrary, during relapse, the levels increased again and came close to diagnosis levels (Table 11, Figure 8).


**Table 11.** suPAR, cellular uPAR and MMP-9 in AML patients vs. controls.

Adult Acute Myeloid Leukemia – A Possible Relation to Disease Invasion and the Impact of Independent... http://dx.doi.org/10.5772/61179 137

**Figure 8.** Soluble MMP-9 concentration levels at different AML status.

develop at room temperature to get a color reaction. Absorbance was read at 405 nm. The assay is 0.03 ng/ml lower detection limit. To estimate the total uPAR load in the cells in the circulation, the exact amount of uPAR in lysates was multiplied by the mononuclear cell count in the

Using a linked immunosorbent assay kit for human total MMP 9 according to the manual was used to determine total MMP-9 activities in the plasma. The diluted plasma samples or MMPs standards were mixed with 100 ul of 50 ul anti-MMP immunoglobulin G labeled with horseradish peroxidase in 10 mmol/l ethylenediaminetetra acetic acid. A 100-μl aliquot of the mixture was transferred to each well that was previously coated with an anti-MMP immuno‐ globulin G. The horseradish peroxidase-bound activity was detected by adding 100 μl volume of 0.15 mol/l citric acid sodium phosphate buffer at pH 4.9, containing 2.0 g/l of o-phenylene‐ dianmine and 0.02% (v/v) hydrogen peroxide, followed by incubation at room temperature for 20 minutes. Adding 100 μl sulphuric acid 1 mol/l will stop the action. The micro-plate reader was adjusted to 492 nm for absorbance measurement. The human MMPs values were obtained

At diagnosis the AML patients' suPAR and cellular PAR levels that were considered in mean were highly significant compared to the healthy controls with P=0.001 for both. However, during AML remission, the suPAR levels declined, achieving levels close to the control levels. On the contrary, during relapse, the levels increased again and came close to diagnosis levels

> (ng/ mg protein/ 109 cells/l)

At diagnosis Remission Replace At diagnosis Remission Replace

Controls 1.02±0.14 0.26±0.02 42.05±2.18

P value 0.001 0.02 0.003 0.001 0.001 >0.05 0.001

3.01±0.38 0.7±0.05 2.1±0.17 2.1± 0.17 11.21±1.71 44.5±0.76 13.56±1.07

MMP-9 (ng/ml)

peripheral blood. This was tested without specific monoclonal antibodies.

*7.4.3.1. AML patients suPAR, cellular uPAR and MMP-9 levels versus control*

suPAR (ng/ml) cellular uPAR

**Table 11.** suPAR, cellular uPAR and MMP-9 in AML patients vs. controls.

*7.4.2.4. Total MMP-9 enzyme-linked immunosorbent assay of plasma*

136 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

from the standard curve.

*7.4.3. Results*

(Table 11, Figure 8).

AML patients On the one hand, soluble MMP-9 levels were significantly lower in AML patients at diagnosis as compared to normal control, and elevated during AML remission and declined again during relapse (Table 11, Figure 9).

**Figure 9.** suPAR levels in different AML status.

On the other hand, AML patients who achieved complete induction remission have lower levels for both MMP-9 and suPAR as compared to patients who resist remission (P=0.001 for both, Table 12).


**Table 12.** suPAR and MMP-9 levels at diagnosis in AML patients who achieved complete remission vs. who did not.

The levels of sMMP-9 and suPAR were significantly different according to AML status (Table 13). The MMP-9, cellular uPAR and suPAR diagnosis levels were uneven when classified according to FAB subtypes being highest among M5 (P<0.05 for all) (Tables 13 and 14).


**Table 13.** suPAR and MMP-9 levels in different AML states.


**Table 14.** suPAR, cellular uPAR, MMP-9 levels in different FAB subtypes.

#### *7.4.3.2. suPAR, MMP-9, and cellular uPAR levels and extramedullary involvement*

In order to study whether MMP-9 and uPAR have significant effects in the AML blast cell invasion, the AML patient's serum levels of MMP-9, suPAR, and cellular uPAR were compared in the presence and absence of extramedullary involvement (Table 15). The AML subgroup with extramedullary involvement exhibit a significant elevation in MMP-9, cellular uPAR, and suPAR with a value P<0.05; P=0.001; 0.001, respectively.

#### *7.4.3.3. Peripheral blast cells count, BCDR, cellular uPAR, suPAR, and MMP-9 correlation*

A significant correlation was noticed between suPAR and MMP-9 and the peripheral blast cells counts (r=0.88; P=0.001; r=0.65; P=0.001), as well as BCDR (r=0.84; P=0.001; r=0.65; P=0.001). At the same time, there is a significant correlation between suPAR and cellular uPAR (r=0.88; P=0.001). Moreover, MMP-9 is correlated significantly with suPAR (r=0.84; p=0.001) as shown in Table 16.


**AML achieved remission suPAR (ng/ml) MMP-9(ng/ml)**

Yes (27) 1.72±0.19 14.47±1.15 No (13) 3.27±0.20 34.02±5.2 P 0.001 0.001

**Table 12.** suPAR and MMP-9 levels at diagnosis in AML patients who achieved complete remission vs. who did not.

The levels of sMMP-9 and suPAR were significantly different according to AML status (Table 13). The MMP-9, cellular uPAR and suPAR diagnosis levels were uneven when classified according to FAB subtypes being highest among M5 (P<0.05 for all) (Tables 13 and 14).

**suPAR (ng/ml) MMP-9(ng/ml)** Diagnosis Remission Relapse Diagnosis Remission Relapse (n=43) (n= 36) (n=10) (n=43) (n= 36) (n=10) 3.01± 0.38 0.07±0.05 2.1±0.17 11.12±1.7 44.5±0.76 13.56±1.07

M1(n-6) M2(n=6) M3(n=4) M4(n=4) M5(n=7) M6(n=4) M7(n=2) P

0.33±0.1 0.8±0.15 0.4±0.11 0.96±0.14 1.26±0.21 0.53±0.1 0.11±0.11 <0.05

suPAR 1.9±0.01 3.31±0.57 2.1±0.43 3.6±0.41 5.3±0.4 0.85±0.4 0.80±0.1 0.03

MMP-9 16.2±1.8 18.2±2.5 18.7±4.0 16.1±0.3 28.1±7.1 9.1±2.3 7.7±o.95 <0.05

In order to study whether MMP-9 and uPAR have significant effects in the AML blast cell invasion, the AML patient's serum levels of MMP-9, suPAR, and cellular uPAR were compared in the presence and absence of extramedullary involvement (Table 15). The AML subgroup with extramedullary involvement exhibit a significant elevation in MMP-9, cellular uPAR, and

A significant correlation was noticed between suPAR and MMP-9 and the peripheral blast cells counts (r=0.88; P=0.001; r=0.65; P=0.001), as well as BCDR (r=0.84; P=0.001; r=0.65; P=0.001). At the same time, there is a significant correlation between suPAR and cellular uPAR (r=0.88; P=0.001). Moreover, MMP-9 is correlated significantly with suPAR (r=0.84; p=0.001) as shown

*7.4.3.3. Peripheral blast cells count, BCDR, cellular uPAR, suPAR, and MMP-9 correlation*

*7.4.3.2. suPAR, MMP-9, and cellular uPAR levels and extramedullary involvement*

**Table 13.** suPAR and MMP-9 levels in different AML states.

138 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

**Table 14.** suPAR, cellular uPAR, MMP-9 levels in different FAB subtypes.

suPAR with a value P<0.05; P=0.001; 0.001, respectively.

cellular uPAR

in Table 16.

**Table 15.** suPAR, cellular uPAR, and MMP-9 in AML patients with extramedullary infiltration vs. those without.


**Table 16.** Correlation between suPAR, MMP-9, and peripheral blast cells count, BCDR, and cellular uPAR.

The levels of suPAR, cellular uPAR, and MMP-9 at diagnosis were significantly higher in died AML patients group as compared to the survived group (Table 17).


**Table 17.** suPAR, cellular uPAR, and MMP-9 in AML survivors vs. AML non-survivors.

Elevated WBC count and suPAR levels had no correlation in a study in patients with chronic myeloid leukemia in chronic phase; it was also found that all suPAR levels in patient popula‐ tions fell within the normal range. Moreover, it shows evidence that serum suPAR and MMP-9 are strong prognostic variables in AML patients. Levels were significantly higher in AML nonsurvivors when compared to AML survivors in super and mMP-9 diagnostic levels. suPAR and MMP-9 derived from AML blast cells could be a possible explanation for the prognostic value. suPAR and MMP-9 levels are higher in patients with extra-medullary infiltration according to the hypothesis. In conclusion, MMP-9 and suPAR levels might be used as a marker for disease activity and might contribute to blast cell dissemination. MMP-9 and suPAR may be target molecules in the strategy of treatmenting AML.

#### **8. Treatment**

AML has traditionally been considered a medical emergency, delaying induction chemother‐ apy until molecular testing results return; it may benefit some patients but harm others. Advanced age and adverse cytogenetics would show a lower CR rate and that risks associated with giving immediate intensive therapy to patients in whom poor prognostic characteris‐ tics with the risk of waiting to initiate treatment for additional test results to return [78]. Therapy for AML includes remission induction followed by post-remission chemotherapy for most patients. The aim of induction therapy in new patients diagnosed with AML is to lay down complete remission (CR), which in turn could optimize the cure and disease-free survival rate [79].

#### **8.1. Remission induction therapy**

The backbone of remission induction therapy consists of an anthracenedione or anthracycline combined with cytosine arabinoside (cytarabine, Ara-C), a regimen that has not changed since it was first introduced 30 years ago [80]. Daunorubicin is given at a dose of 45 mg/m2 /d×3 days, or mitoxantrone or idarubicin are given at doses of 12 mg/m2 /d×3 days in combination with cytarabine, which is administered as a continuous infusion at 100 or 200 mg/m2 /d×7 days (7+3 chemotherapy). Increasing the doses of cytarabine or the anthracycline, compares different anthracycliness or anthracenedione adding more drugs or using growth factors that prime agents or support care [81]; improved CR rates and disease-free survival commonly come at the price of increased treatment-related mortality than offsetting potential survival advantag‐ es. The median survival for older AML patients that follow these intensive approaches are typically 10 to 12 months, with higher median of survival for those entering CR, compared to non-responders or those achieving CR with incomplete platelet recovery (CRi).

#### **8.2. Post-remission therapy**

Usually, approaches to therapy for older AML patients who are in post-remission involve cytarabine and it is administered for a few days and then introduced in the remission setting. However, this is done alone or in combination with auntracene dione or antracycline for 1 to 2 cycles. Severe neurological toxicity in one-third of patients is associated with high doses of postremission cytrarbine. There is no added benefit to survival from more intense postremission therapy or adding other agents. There is no added benefit from maintenance therapy through a more protracted course or post-remission therapy [82]. No randomized study shows that despite recommendations, no post-remission therapy or over post-remission therapy have any survival advanatages. Post-remission therapy that is becoming more common is Stem Cell Transplantation (SCT) is being considered more. SCT is done at the cost of high treatment mortality and offers the chance of a cure. SCTs have limited uses to the population and the limited matched donors who are related to patients have limited applicability. Ongoing nonmyeloablative approaches studies have demonstrated the feasibility of more survival rates. Ablative approaches may not provide advantages over non-myeloabaltive regimens for older AML patients [83].

#### **8.3. Role of transplantation**

**8. Treatment**

survival rate [79].

**8.1. Remission induction therapy**

**8.2. Post-remission therapy**

AML patients [83].

AML has traditionally been considered a medical emergency, delaying induction chemother‐ apy until molecular testing results return; it may benefit some patients but harm others. Advanced age and adverse cytogenetics would show a lower CR rate and that risks associated with giving immediate intensive therapy to patients in whom poor prognostic characteris‐ tics with the risk of waiting to initiate treatment for additional test results to return [78]. Therapy for AML includes remission induction followed by post-remission chemotherapy for most patients. The aim of induction therapy in new patients diagnosed with AML is to lay down complete remission (CR), which in turn could optimize the cure and disease-free

The backbone of remission induction therapy consists of an anthracenedione or anthracycline combined with cytosine arabinoside (cytarabine, Ara-C), a regimen that has not changed since

chemotherapy). Increasing the doses of cytarabine or the anthracycline, compares different anthracycliness or anthracenedione adding more drugs or using growth factors that prime agents or support care [81]; improved CR rates and disease-free survival commonly come at the price of increased treatment-related mortality than offsetting potential survival advantag‐ es. The median survival for older AML patients that follow these intensive approaches are typically 10 to 12 months, with higher median of survival for those entering CR, compared to

Usually, approaches to therapy for older AML patients who are in post-remission involve cytarabine and it is administered for a few days and then introduced in the remission setting. However, this is done alone or in combination with auntracene dione or antracycline for 1 to 2 cycles. Severe neurological toxicity in one-third of patients is associated with high doses of postremission cytrarbine. There is no added benefit to survival from more intense postremission therapy or adding other agents. There is no added benefit from maintenance therapy through a more protracted course or post-remission therapy [82]. No randomized study shows that despite recommendations, no post-remission therapy or over post-remission therapy have any survival advanatages. Post-remission therapy that is becoming more common is Stem Cell Transplantation (SCT) is being considered more. SCT is done at the cost of high treatment mortality and offers the chance of a cure. SCTs have limited uses to the population and the limited matched donors who are related to patients have limited applicability. Ongoing nonmyeloablative approaches studies have demonstrated the feasibility of more survival rates. Ablative approaches may not provide advantages over non-myeloabaltive regimens for older

/d×3 days,

/d×7 days (7+3

/d×3 days in combination with

it was first introduced 30 years ago [80]. Daunorubicin is given at a dose of 45 mg/m2

cytarabine, which is administered as a continuous infusion at 100 or 200 mg/m2

non-responders or those achieving CR with incomplete platelet recovery (CRi).

or mitoxantrone or idarubicin are given at doses of 12 mg/m2

140 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

Although associated with an improved anti-leukemic effect compared with chemotherapy, the oral solution (OS) did not express consistently improve as shown from several prospective trials of evaluated autologous SCT. There remains a debate about what patients benefit from OS and in what type of patient an aggressive treatment should be reserved for and how SCT from a matched sibling donor has been part of standard care for 25 years [84]. Balancing the relapse risk that the patient faces with chemotherapy compared to the risk of the procedure itself is a decision that the patient faces. If a patient has a low relapse risk, they have a higher chance of responding if they relapse, so holding off on SCT to second CR is more practical. The risk factors that affect SCT could be influenced by many items such as the cytomegalovirus status of the donor and host, the age of the recipient and the donor, and the parity of a female donor. The degree of matching and the comorbidities present in the patient can be evaluated in a risk score [85]. A further complication in assessing the data for SCT in first CR is the method of assessment.

#### **8.4. Molecular genetic implications for diagnosis and therapeutics**

Molecular revelations have recently defined further prognostic cohorts. FMS-like tyrosine kinase 3 (FLT3) is important in the development of myeloid and lymphoid lineages may occur in 25% to 30% [86] for internal tandem duplications and domain or mutations of kinase activating loop. FLT3 ITD has proliferative advantage and anti-apoptotic signals and predicts shorter CR duration. FLT3-ITD to FLT3 wild-type has a high allele ratio and long length of duplication and location of insertion [87]. This information can help with the decision to go through allogeneic SCT even though the benefits may not be unanimous. FLT3 inhibitory activity has had little effect as monotherapy and at least three randomized trials are going in combination with chemo, which should also take account for molecular subsets. Fifty percent of cytogenetically normal acute myeloid leukemia (CN-AML) of the other intermediate group has nucleophosmin 1 (NPM1) mutations that result in delocalizing in the cytoplasm [88]. NPM1 mutations in the absence of FLT3-ITD are favorable outcomes that are similar to CBF leukemia in younger AML patients [89]. These types of patients can benefit from consolidated chemo and do not require CR after SCT. There are suggestions that NPM1 patients should receive additional all-trans-retinoic acid therapy, but this has not been proven. The prognostic value of the molecular biomarkers (e.g., NPM1) could be useful in predicting the outcome in older patients [90].

#### **9. Conclusion and outcomes**

AML in older adults is considered a difficult disease to be cured, representing one of the most challenging groups to be treated in oncology. However, advances in AML treatment have resulted in improved remission (an absence of signs and symptoms) and cure rates. This improvement may benefit treatment with intensive or alternative chemotherapy that is appropriate for the patient, enhanced by supportive performance status and cytogenetics. Remission rates in adult AMLs are universally related to age, with an expected remission rate of more than 65% for those younger than 60 years. Earlier, the median survival of adult patients with AML at the beginning of treatment was 40 days. Recently, AML patients younger than 60 years have complete response rates of 70% to 80% after induction chemotherapy. Overall, survival is only about 50% for those who go into complete remission. Given the desperate nature of survival outcomes, clinical trials should be considered at diagnosis, along with considering the aggressiveness of therapy and patient-oriented treatment goals.

#### **Acknowledgements**

The authors wish to express their deepest gratitude to Professor Salah Aref, Hematology Department, Faculty of Medicine, Mansoura University, Egypt.

#### **Author details**

Mohamed El-Refaei1,2\* and Fahd Al Qahtani2

\*Address all correspondence to: melrefaei2000@yahoo.com

1 Molecular Biology Department, Genetic Engineering and Biotechnology Institute, Sadat City University, Egypt

2 Faculty of Medicine, AlBaha University, Kingdom of Saudi Arabia

#### **References**


Remission rates in adult AMLs are universally related to age, with an expected remission rate of more than 65% for those younger than 60 years. Earlier, the median survival of adult patients with AML at the beginning of treatment was 40 days. Recently, AML patients younger than 60 years have complete response rates of 70% to 80% after induction chemotherapy. Overall, survival is only about 50% for those who go into complete remission. Given the desperate nature of survival outcomes, clinical trials should be considered at diagnosis, along with

The authors wish to express their deepest gratitude to Professor Salah Aref, Hematology

1 Molecular Biology Department, Genetic Engineering and Biotechnology Institute, Sadat

[1] El-Refaei MF, El-Naa MM (2010) Inhibitory effect of caffeic acid phenethyl ester on mice bearing tumor involving angiostatic and apoptotic activities. Chemico-Biologi‐

[3] Bray F, Moller B (2006) Predicting the future burden of cancer. Nat Rev Can J. 6:63–

[4] Wade JC (2006) Viral infections in patients with hematological malignancies. Hema‐

[5] Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ et al. (2009) The 2008 revision of the World Health Organization (WHO) classification of myeloid neo‐ plasms and acute leukemia: Rationale and important changes. Blood. 114:937–951.

[2] Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell J. 100(1):57–70.

considering the aggressiveness of therapy and patient-oriented treatment goals.

Department, Faculty of Medicine, Mansoura University, Egypt.

142 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

\*Address all correspondence to: melrefaei2000@yahoo.com

2 Faculty of Medicine, AlBaha University, Kingdom of Saudi Arabia

tology Am SocHematolEduc Program. 368-374.

Mohamed El-Refaei1,2\* and Fahd Al Qahtani2

cal Interactions. 186:152–156.

**Acknowledgements**

**Author details**

City University, Egypt

**References**

74.


[35] Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ et al. (2009) The 2008 revision of the World Health Organization (WHO) classification of myeloid neo‐ plasms and acute leukemia: Rationale and important changes. Blood. 114:937.

[20] Redaelli A, Lee JM, Stephens JM, Pashos CL (2003) Epidemiology and clinical burden

[21] Sandler DP, Ross JA (1997) Epidemiology of acute leukemia in children and adults.

[22] Wingo PA, Cardinez CJ, Landis SH, Greenlee RT, Ries LA et al. (2003) Long-term trends in cancer mortality in the United States, 1930–1998. Cancer. 97(12 Suppl):3133–

[23] RiesLAG HD, Krapcho M, Mariotto A et al. (2003) Cancer Statistics Review, 1975– 2003. Bethesda, MD: National Cancer Institute. Based on November 2005 SEER data

[24] Jemal A, Thomas A, Murray T, Thun M (2002) Cancer statistics. CA Cancer J Clin.

[25] Ries LAG EM, Kosary CL, Hankey BF et al. (2003) Cancer Statistics Review, 1975– 2000. Bethesda, MD: National Cancer Institute. Available at URL: http://seer.cancer.

[26] American Cancer Society (2005) Cancer Facts and Figures Atlanta. American Cancer

[27] Sekeres MA, Stone RM, Zahrieh D, Neuberg D, Morrison V et al. (2004) Decisionmaking and quality of life in older adults with acute myeloid leukemia or advanced

[28] American Cancer Society (2015) Cancer Facts & Figures Atlanta: American Cancer

[29] Sandler DP, Collman GW (1987) Cytogenetic and environmental factors in the etiolo‐

[30] Pogoda JM, Preston-Martin S, Nichols PW, Ross RK (2002) Smoking and risk of acute myeloid leukemia: results from a Los Angeles County case-control study. Am J Epi‐

[32] Evans DI, Steward JK (1972) Down's syndrome and leukaemia. Lancet. 2(7790):1322.

[33] Alcalay M, Orleth A, Sebastiani C Meani N, Chiaradonna F et al. (2011) Common themes in the pathogenesis of acute myeloid leukemia. Oncogene. 20:5680–5694.

[34] Harris N, Jaffe E, Diebold J, Flandrin G, Muller-Hermelink HK et al. (1997) World Health Organization classification of neoplastic diseases of the hematopoietic and lymphoid tissues: Report of the Clinical Advisory. Committee meeting—Airlie

gy of the acute leukemias in adults. Am J Epidemiol. 126:1017–1032.

[31] Pui CH (1995) Childhood leukemias. N Engl J Med. 332:1618–1630.

House Virginia. J ClinOncol. 17:3835–3849.

of acute myeloid leukemia. Expert Rev Anticancer Ther. 3:695–710.

Seminar Oncol. 24(1):3–16.

submission, posted to the SEER website, 2006.

144 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

Gov/csr/1975\_2000 [accessed June 2004].

myelodysplastic syndrome. Leukemia. 18:809–816.

3275.

52:23–47.

Society.

Society.

demiol.155:546–553.


[61] Quartarone E, Alonci A, Allegra A, Bellomo G, Calabro L, D'Angelo A et al. (2006) Differential levels of soluble angiopoietin-2 and Tie-2 in patients with haematological malignancies. Eur J Haematol. 77:480–485.

[49] Wouters BJ, Lo°wenberg B, Delwel R (2009) A decade of genome-wide gene expres‐ sion profiling in acute myeloid leukemia: Flashback and prospects. Blood. 113(2):

[50] Marcucci G, Radmacher MD, Maharry K, Maharry K, Mrózek K et al. (2008). Micro RNA expression in cytogenetically normal acute myeloid leukemia. N Engl J Med.

[51] Raghavan M, Lillington DM, Skoulakis S, Debernardi S, Chaplin T et al. (2005) Ge‐ nome-wide single nucleotide polymorphism analysis reveals frequent partial unipar‐ ental disomy due to somatic recombination in acute myeloid leukemias. Cancer Res.

[52] Mullighan CG, Miller CB, Radtke I, Phillips LA, Dalton J et al. (2008) BCR- ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros. Nature.

[53] Owonikoko T, Agha M, Balassanian R, Smith R, Raptis A (2007) Gemtuzumab thera‐ py for isolated extramedullary AML relapse following allogeneic stem-cell trans‐

[54] El Rassi F, Little BP, Holloway S, Roberts D, Khoury HJ (2012) Early diagnosis of acute myeloid leukemia by computed tomography scan. American Society of Clinical

[55] Xu ZF, Xu HX, Xie XY, Liu GJ, Zheng YL, Lu MD (2010) Renal cell carcinoma and renal angiomyolipoma: Differential diagnosis with real-time contrast-enhanced ultra‐

[56] Loges S, Heil G, Bruweleit M, Schoder V, Butzal M, Fischer U et al. (2005) Analysis of concerted expression of angiogenic growth factors in acute myeloid leukemia: Ex‐ pression of angiopoietin-2 represents an independent prognostic factor for overall

[57] Schliemann C, Bieker R, Thoennissen N, Gerss J, Liersch R et al. (2007) Circulating angiopoietin-2 is a strong prognostic factor in acute myeloid leukemia. Leukemia.

[58] Schliemann C, Bieker R, Padro T, Kessler T, Hintelmann H et al. (2007) Expression of angiopoietins and their receptor Tie2 in the bone marrow of patients with acute mye‐

[59] Amo Y, Masuzawa M, Hamada Y, Katsuoka K (2004) Observations on angiopoietin 2

[60] Cine GJ, Blann AD, Stonelake PS, Ryan P, Lip GY (2003) Plasma angiopoietin-1, an‐ giopoietin-2 and Tie-2 in breast and prostate cancer: A comparison with VEGF and

291-298.

358(18):1919–1928.

65(2):375–378.

Oncology.

21:1901–1906.

453(7191):110–114.

plant. Nat ClinPractOncol. 4(8):491-5.

146 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

sonography. J Ultrasound Med. 29(5):709–17.

loid leukemia. Haematologica. 91:1203–1211.

in patients with angiosarcoma. Sry Dermatol. 150:1028–1029.

survival. J ClinOncol. 23:1109–1117.

FIt-1. Eur J Clin Invest. 33:883–890.


mutations in a large cohort of young adult patients with acute myeloid leukemia. Blood. 111:2776–2784.

[87] Falini B, Bolli N, Liso A, Martelli MP, Mannucci R et al. (2009) Altered nucleophos‐ min transport in acute myeloid leukaemia with mutated NPM1: Molecular basis and clinical implications. Leukemia. 23:1731–1743.

[75] Munoz L, Aveentin A, Villamor N, Junca J, Acebedo G et al. (2003) Immunopheno‐ typic findings in acute leukemia with Flt3 internal Tandem duplication. Hematologi‐

[76] Stephens R, Pedersen W, Nielsen A, Hamers HJ, Hoyer-Hansen et al. (1997) ELISA determination of soluble urokinase receptor in blood from healthy donors and cancer

[77] Sekeres MA (2008) Treatment of older adults with acute myeloid leukemia: State of

[78] Burnett AK, Hills RK, Milligan D, Goldstone AH, Prentice AG et al. (2010) Attempts to optimize induction and consolidation treatment in acute myeloid leukemia: Re‐

[79] Rai KR, Holland JF, Glidewell OJ, Weinberg V, Brunner K et al. (1981) Treatment of acute myelocytic leukemia: A study by cancer and leukemia group B. Blood. 58:1203–

[80] Rowe JM, Neuberg D, Friedenberg W, Bennett JM, Paietta E et al. (2004) A phase 3 study of three induction regimens and of priming with GM-CSF in older adults with acute myeloid leukemia: A trial by the Eastern Cooperative Oncology Group. Blood.

[81] Stone RM, Berg DT, George SL, Dodge RK, Paciucci PA et al. (2001) Postremission therapy in older patients with de novo acute myeloid leukemia: A randomized trial comparing mitoxantrone and intermediate-dose cytarabine with standard-dose cy‐

[82] Alyea EP, Kim HT, Ho V, Cutler C, Gribben J et al. (2005) Comparative outcome of nonmyeloablative and myeloablative allogeneic hematopoietic cell transplantation

[83] Koreth J, Schlenk R, Kopecky KJ, Honda S, Sierra J et al. (2009) Allogeneic stem cell transplantation for acute myeloid leukemia in first complete remission: Systematic

[84] Gratwohl A, Stern M, Brand R, Apperley J, Baldomero H et al. (2009) European Group for Blood and Marrow Transplantation and the European Leukemia Net: Risk score for outcome after allogeneic hematopoietic stem cell transplantation: A retro‐

[85] Dohner H, Estey EH, Amadori S, Mead AJ, Burnett AK et al. (2010) Diagnosis and management of acute myeloid leukemia in adults: Recommendations from an inter‐ national expert panel, on behalf of the European Leukemia Net. Blood. 115:453–474.

[86] Gale RE, Green C, Allen C, Mead AJ, Burnett AK et al. (2008) The impact of FLT3 in‐ ternal tandem duplication mutant level, number, size, and interaction with NPM1

review and meta-analysis of prospective clinical trials. JAMA. 301:2349-2361.

the art and current perspectives. Haematologica. 93:1769–1772.

sults of the MRCAML12 trial. J Clin Oncol. 28(4):586–595.

for patients older than 50 years of age. Blood 105:1810–4.

spective analysis. Cancer. 115:4715–4726.

ca. 88(6):637–645.

12.

103:479–85.

tarabine. Blood. 98:548–53.

patients. Clin Chem. 43(10):1868–1876.

148 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis


### **Pharmacological Inhibition of Intracellular Signaling Pathways in Radioresistant Anaplastic Thyroid Cancer**

Dmitry Bulgin and Alexey Podcheko

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62541

#### **Abstract**

Anaplastic thyroid cancer (ATC) is highly aggressive and has a poor therapeutic response and leads to high mortality. It has been shown that activation of intracellular c-Jun N-ter‐ minal kinase (JNK) and c-ABL signaling pathways is one of the manifestations of the highly resistant response to radiotherapy in ATC. Pharmacological inhibition of these pathways in combination with radiotherapy is a potential treatment modality of ATC.

**Keywords:** Anaplastic thyroid cancer, JNK signaling pathway, c-ABL signaling pathway, ionizing radiation, radioresistance, anthrapyrazolone, imatinib

#### **1. Introduction**

Thyroid malignant neoplasms are the most frequent endocrine tumor. They are classified into two categories, differentiated carcinoma and undifferentiated carcinoma (anaplastic carcino‐ ma), based on the histological differentiation (Figure 1).

In the clinical course, differentiated thyroid carcinomas such as follicular cancer and papillary cancer have relatively good prognosis. ATC is among the most aggressive solid malignancies in human with a bad prognosis. In spite of active therapeutic and surgical treatment, ATC provides mean survival time less than 8 months after diagnosis [1]. ATC is widely metastatic, and it is highly resistant to regular therapeutic approaches such as surgical treatment, chemotherapy, or radiotherapy. It was confirmed that thyroid cells are relatively resistant to ionizing radiation (IR)-induced apoptosis [2, 3].

Currently, it is mainly approved that external beam radiotherapy of ATC should be combined with different anti-tumor pharmacological agents to have better local control of the tumor [4]. The main goal of this combination is to reduce the clonogenic capacity and radioresistance of

classified into two categories, differentiated carcinoma and undifferentiated carcinoma

(anaplastic carcinoma), based on the histological differentiation (Figure 1).

Figure 1. Thyroid malignant neoplasms histology: (a) follicular carcinoma; (b) papillary carcinoma; and **Figure 1.** Thyroid malignant neoplasms histology: (a) follicular carcinoma; (b) papillary carcinoma; and (c) anaplastic carcinoma (courtesy of professor Masahiro Nakashima, Nagasaki University, Japan). Hematoxylin and eosin stain. Original magnification ×100.

ATC cells with the aim of further improving the radiotherapy effect. It is conceivably that molecular-targeted pharmacological agents can decrease cancer resistance to radiotherapy through modulation of DNA repair, cell death pathway, intracellular signal transduction [5, 6], or senescence-like terminal growth arrest [7]. (c) anaplastic carcinoma. Hematoxylin and eosin stain. Original magnification ×100. In the clinical course, differentiated thyroid carcinomas such as follicular cancer and

papillary cancer have relatively good prognosis. Anaplastic thyroid cancer (ATC) is

among the most aggressive solid malignancies in human with a bad prognosis. In spite

#### **2. Radiation therapy**

Radiation therapy, like most anti-tumor treatments, achieves its therapeutic effect by inducing different types of cell death in tumors [8]. Over the past decade, our knowledge is rapidly increasing regarding the discovery of various molecular pathways involved in determining cell death after IR exposure [9]. The biological target of IR in the cell is DNA (Figure 2). of active therapeutic and surgical treatment, ATC provides mean survival time less than

Double-strand breaks (DSBs) are the most destructive DNA alterations, which, if left unre‐ paired, may have serious consequences for cell survival, as they lead to genomic instability, chromosome aberrations, or cell death. DSBs are irreparable and more responsible than the single-strand DNA breaks for most of cell death in tumor as well as surrounding normal cells. Cells respond to DNA damage by activating complex processes at the level of molecules and genes to detect and repair DNA alterations. The formation of DSBs activated phosphorylation of H2AX (the subtype of histone H2A). The phosphorylated form of H2AX is called γ-H2AX [10]. Phosphorylation of H2AX plays a key role in DNA reparation, and it is necessary for the assembly of DNA repair molecules at the sites containing damaged chromatin as well as for activation of checkpoint proteins, which arrest the cell cycle progression [11]. The evaluation of γ-H2AX levels may allow not only to control the efficiency of anti-tumor therapy but also to predict cancer cell sensitivity to DNA-damaging anti-tumor agents and toxicity of antitumor treatment toward normal cells. It is possible to detect H2AX phosphorylation by specific γ-H2AX antibody and thus to detect DNA damage and repair *in situ* in individual cells. The presence of γ-H2AX in chromatin can be exposed shortly after induction of DSBs in the form of discrete nuclear foci (Figure 3) [12]. The presence of γ-H2AX-containing nuclear foci can be measured by microscopy, flow cytometry, and Western blotting of tissue/cell lysates [13]. 8 months after diagnosis [1]. ATC is widely metastatic, and it is highly resistant to regular therapeutic approaches such as surgical treatment, chemotherapy, or radiotherapy. It was confirmed that thyroid cells are relatively resistant to ionizing radiation (IR)-induced apoptosis [2, 3]. Currently, it is mainly approved that external beam radiotherapy of ATC should be combined with different anti-tumor pharmacological agents to have better local control of the tumor [4]. The main goal of this combination is to reduce the clonogenic capacity

2

and radioresistance of ATC cells with the aim of further improving the radiotherapy

Pharmacological Inhibition of Intracellular Signaling Pathways in Radioresistant Anaplastic Thyroid Cancer http://dx.doi.org/10.5772/62541 153

ATC cells with the aim of further improving the radiotherapy effect. It is conceivably that molecular-targeted pharmacological agents can decrease cancer resistance to radiotherapy through modulation of DNA repair, cell death pathway, intracellular signal transduction [5,

In the clinical course, differentiated thyroid carcinomas such as follicular cancer and

papillary cancer have relatively good prognosis. Anaplastic thyroid cancer (ATC) is

(c) anaplastic carcinoma. Hematoxylin and eosin stain. Original magnification ×100.

(a) (b) (c)

Figure 1. Thyroid malignant neoplasms histology: (a) follicular carcinoma; (b) papillary carcinoma; and

**Figure 1.** Thyroid malignant neoplasms histology: (a) follicular carcinoma; (b) papillary carcinoma; and (c) anaplastic carcinoma (courtesy of professor Masahiro Nakashima, Nagasaki University, Japan). Hematoxylin and eosin stain.

classified into two categories, differentiated carcinoma and undifferentiated carcinoma

(anaplastic carcinoma), based on the histological differentiation (Figure 1).

152 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

Radiation therapy, like most anti-tumor treatments, achieves its therapeutic effect by inducing different types of cell death in tumors [8]. Over the past decade, our knowledge is rapidly increasing regarding the discovery of various molecular pathways involved in determining cell death after IR exposure [9]. The biological target of IR in the cell is DNA (Figure 2).

of active therapeutic and surgical treatment, ATC provides mean survival time less than

among the most aggressive solid malignancies in human with a bad prognosis. In spite

Double-strand breaks (DSBs) are the most destructive DNA alterations, which, if left unre‐ paired, may have serious consequences for cell survival, as they lead to genomic instability, chromosome aberrations, or cell death. DSBs are irreparable and more responsible than the single-strand DNA breaks for most of cell death in tumor as well as surrounding normal cells. Cells respond to DNA damage by activating complex processes at the level of molecules and genes to detect and repair DNA alterations. The formation of DSBs activated phosphorylation of H2AX (the subtype of histone H2A). The phosphorylated form of H2AX is called γ-H2AX [10]. Phosphorylation of H2AX plays a key role in DNA reparation, and it is necessary for the assembly of DNA repair molecules at the sites containing damaged chromatin as well as for activation of checkpoint proteins, which arrest the cell cycle progression [11]. The evaluation of γ-H2AX levels may allow not only to control the efficiency of anti-tumor therapy but also to predict cancer cell sensitivity to DNA-damaging anti-tumor agents and toxicity of antitumor treatment toward normal cells. It is possible to detect H2AX phosphorylation by specific γ-H2AX antibody and thus to detect DNA damage and repair *in situ* in individual cells. The presence of γ-H2AX in chromatin can be exposed shortly after induction of DSBs in the form of discrete nuclear foci (Figure 3) [12]. The presence of γ-H2AX-containing nuclear foci can be measured by microscopy, flow cytometry, and Western blotting of tissue/cell lysates [13].

regular therapeutic approaches such as surgical treatment, chemotherapy, or

radiotherapy. It was confirmed that thyroid cells are relatively resistant to ionizing

Currently, it is mainly approved that external beam radiotherapy of ATC should be

combined with different anti-tumor pharmacological agents to have better local control

of the tumor [4]. The main goal of this combination is to reduce the clonogenic capacity

and radioresistance of ATC cells with the aim of further improving the radiotherapy

8 months after diagnosis [1]. ATC is widely metastatic, and it is highly resistant to

2

6], or senescence-like terminal growth arrest [7].

radiation (IR)-induced apoptosis [2, 3].

**2. Radiation therapy**

Original magnification ×100.

**Figure 2.** The biological target of IR in the cell is DNA. IR-induced DNA damage of cancer cells can lead to cell death.

**Figure 3.** IR induces γ-H2AX nuclear foci formation in ATC cell lines: (a) non-radiated; (b) in 24 hours after 10 Gy IR treatment (EXS-300 X-irradiator, Toshiba, Tokyo, Japan; 200 kV, 15 mA, 0.83 Gy/min). Compared to alternative meth‐ ods of DNA damage assessment, the immunocytochemical approach is less cumbersome and offers much greater sen‐ sitivity. Fluorescent immunocytochemistry. Confocal fluorescent microscopy. Original magnification ×400.

The main goal of radiotherapy is to deprive tumor cells of their multiplication potential and finally to destroy the cancer cells. After IR exposure, cell death may occur by one or more of the following mechanisms: immediate or delayed apoptosis, mitotic-linked death (mitotic catastrophe), autophagy, and terminal growth arrest (senescence) associated with necrosis (Figure 4).

**Figure 4.** Mechanisms of cancer cell death triggered by IR.

Radiotherapy does not destroy cancer cells right away. It takes hours, days, or weeks of antitumor therapy before cancer cells start to die after which cancer cells continue dying for weeks to months after ending of radiotherapy. The efficiency of radiotherapy has much to gain by understanding the cell death mechanisms that are induced in tumor cells following irradiation (Table 1). Strategies to use specific pharmacological agents that can inhibit the activity of key molecules in intracellular signaling pathways combined with IR might potentiate therapy and enhance tumor cell death [14].



**Table 1.** Anti-proliferative response and cell death pathways observed upon radiotherapy.

#### **3. JNK signaling pathway**

the following mechanisms: immediate or delayed apoptosis, mitotic-linked death (mitotic catastrophe), autophagy, and terminal growth arrest (senescence) associated with necrosis

Radiotherapy does not destroy cancer cells right away. It takes hours, days, or weeks of antitumor therapy before cancer cells start to die after which cancer cells continue dying for weeks to months after ending of radiotherapy. The efficiency of radiotherapy has much to gain by understanding the cell death mechanisms that are induced in tumor cells following irradiation (Table 1). Strategies to use specific pharmacological agents that can inhibit the activity of key molecules in intracellular signaling pathways combined with IR might potentiate therapy and

**Types of cell death Definition and characteristics Associated changes Detection methods**

Sometimes not considered genetically determined. Drop of ATP levels. ROS over-generation.

Stimulated by cyclin D1 activation and by Myc. Can be inhibited by loss of

wild-type p53. Caspase activation. Early permeability to vital dyes. Staining with propidium iodide.

Sub-G1 peak in flow cytometry.

Electron microscopy.

Annexin-V staining. Quantification of ∆Ψm. Internucleosomal laddering.

(Figure 4).

**Figure 4.** Mechanisms of cancer cell death triggered by IR.

154 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

enhance tumor cell death [14].

Necrosis Cells visibly swell with

Apoptosis Programmed cell death.

breakdown of cell membrane.

disintegrated cell organelles.

Cells shrink with blebbing of

Condensed chromatin and DNA fragmentation.

Typical nuclei with vacuolization and

cell membranes.

c-Jun N-terminal kinases (JNKs) are multifunctional kinases, also known as stress-activated protein kinases be a part of superfamily of mitogen-activated protein kinases (MAPKs) that are involved in many physiological and pathological processes (Figure 5). At first, the JNKs were originally identified as ultraviolet-responsive protein kinases by their capacity to phosphorylate the N-terminal of the transcription factor c-Jun and by their activation in response to various stresses [15]. Initial research works have shown that JNKs can be triggered by various stimuli including growth factors [16, 17], cytokines [18], and stress factors [19]. It was demonstrated that IR with level of 10 Gy induced JNK activation with a maximum at 30 minutes and return to baseline at 12 hours after exposure in ATC cell lines [7].

Other observations have demonstrated the crucial role of JNK pathway in mediating apoptotic signaling in many cell death paradigms [20]. JNK signal transduction pathway regulates the cellular reaction to IR and activating radiation-induced apoptosis [21]. However, it was shown

**Figure 5.** Various extracellular and intracellular stimuli can activate JNKs. Constant JNK activation influences tumouri‐ genesis by both transcription-independent and transcription-dependent mechanisms involved in cell transformation, proliferation, survival, migration, suppression of cell death, and inflammatory processes in tumor.

that JNK cascade, via the stimulation of c-Jun and ATF-2 transcription factors, may provide DNA repair and cell survival (Figure 6) [22].

**Figure 6.** Role of JNK kinase in DNA repair after IR-induced extensive damage.

For that reason, it was proposed that JNK pathway inhibition could result in sensitization of distinct types of tumor cells to DNA damage.

#### **4. c-ABL tyrosine kinase signaling pathway** signaling processes. The c-ABL is highly expressed in normal and cancer cells [23, 24].

c-ABL is an ubiquitously expressed tyrosine kinase that involves in various cellular signaling processes. The c-ABL is highly expressed in normal and cancer cells [23, 24]. c-ABL is frequently overexpressed in ATC cell lines (Figure 7) and thyroid cancer tissue samples (Figure 8). c-ABL is frequently overexpressed in ATC cell lines (Figure 7) and thyroid cancer tissue samples (Figure 8). c-ABL is an ubiquitously expressed tyrosine kinase that involves in various cellular signaling processes. The c-ABL is highly expressed in normal and cancer cells [23, 24].

(a) (b)

anti-c-ABL. Original magnification ×200.

that JNK cascade, via the stimulation of c-Jun and ATF-2 transcription factors, may provide

**Figure 5.** Various extracellular and intracellular stimuli can activate JNKs. Constant JNK activation influences tumouri‐ genesis by both transcription-independent and transcription-dependent mechanisms involved in cell transformation,

proliferation, survival, migration, suppression of cell death, and inflammatory processes in tumor.

For that reason, it was proposed that JNK pathway inhibition could result in sensitization of

DNA repair and cell survival (Figure 6) [22].

156 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

**Figure 6.** Role of JNK kinase in DNA repair after IR-induced extensive damage.

distinct types of tumor cells to DNA damage.

Figure 7. c-ABL expression in ATC cell lines: (a) FRO cell line and (b) ARO cell line. Fluorescent **Figure 7.** c-ABL expression in ATC cell lines: (a) FRO cell line and (b) ARO cell line. Fluorescent immunocytochemis‐ try. Confocal fluorescent microscopy. Original magnification ×400. immunocytochemistry. Confocal fluorescent microscopy. Original magnification ×400.

Figure 8. The different expression pattern of c-ABL: (a) high level of expression of c-ABL in anaplastic **Figure 8.** The different expression pattern of c-ABL: (a) high level of expression of c-ABL in anaplastic thyroid carcino‐ ma; (b) follicular carcinoma; and (c) goiter. Immunohistochemistry: antibodies used were anti-c-ABL. Original magnifi‐ cation ×200 (a,b) and ×100 (c).

(a) (b) (c) Figure 8. The different expression pattern of c-ABL: (a) high level of expression of c-ABL in anaplastic thyroid carcinoma; (b) follicular carcinoma; and (c) goiter. Immunohistochemistry: antibodies used were thyroid carcinoma; (b) follicular carcinoma; and (c) goiter. Immunohistochemistry: antibodies used were anti-c-ABL. Original magnification ×200. Previous experiments indicated that c-ABL is involved in regulation of the cell cycle Previous experiments indicated that c-ABL is involved in regulation of the cell cycle and the cellular genotoxic stress response pathways. It was demonstrated that the growth arrest was accompanied by the down-regulation of c-ABL phosphorylation and of cyclins A and B1 levels and by the up-regulation of the cell cycle inhibitor p21cip1. Also, it was presented that p21cip1 expression is associated with improved survival in patients after adjuvant radiotherapy [25]. The cellular reaction elicited by c-ABL depends upon its location in cells. Accumulation of c-ABL in the cytoplasm results in cell survival and proliferation. By contrast, nuclear c-ABL

Previous experiments indicated that c-ABL is involved in regulation of the cell cycle

growth arrest was accompanied by the down-regulation of c-ABL phosphorylation and

and the cellular genotoxic stress response pathways. It was demonstrated that the

and the cellular genotoxic stress response pathways. It was demonstrated that the

of cyclins A and B1 levels and by the up-regulation of the cell cycle inhibitor p21cip1.

Also, it was presented that p21cip1 expression is associated with improved survival in

9

becomes activated and induces apoptosis following genotoxic stress [26]. DNA damage caused by IR and other DNA-damaging agents has been shown to result in activation of the c-ABL (Figure 9) [27]. [27].

induces apoptosis following genotoxic stress [26]. DNA damage caused by IR and other

**Figure 9.** Nuclear targeting of c-ABL in response to DNA damage. It was demonstrated that IR induces redistribution of c-ABL between nucleus and

It was demonstrated that IR induces redistribution of c-ABL between nucleus and cytoplasm in ATC cells (Figure 10) [28]. cytoplasm in ATC cells (Figure 9) [28].

Figure 10. IR induces redistribution of c-ABL between nucleus and cytoplasm in ATC cells. Translocation **Figure 10.** IR induces redistribution of c-ABL between nucleus and cytoplasm in ATC cells. Translocation of c-ABL in FRO cells after IR treatment (EXS-300 X-irradiator, Toshiba, Tokyo, Japan; 200 kV, 15 mA, 0.83 Gy/min): (a) non-radiat‐ ed; (b) in 6 hours after 10 Gy IR treatment. Fluorescent immunocytochemistry. Confocal fluorescent microscopy. Origi‐ nal magnification ×400.

of c-ABL in FRO cells after IR treatment (EXS-300 X-irradiator, Toshiba, Tokyo, Japan; 200 kV, 15 mA, 0.83 Gy/min): (a) non-radiated; (b) in 6 hours after 10 Gy IR treatment. Fluorescent Notably, nuclear targeting of c-ABL is required for the induction of apoptosis in response to DNA damage. Overexpression of c-ABL activates cell cycle arrest in G1, which requires kinase activity and nuclear localizing signals and depends on the wild-type p53 tumor suppressor (Figure 10). In addition, c-ABL binds p53 and enhances the DNA binding and transcriptional activity of p53 [29, 30].

Notably, nuclear targeting of c-ABL is required for the induction of apoptosis in

10

immunocytochemistry. Confocal fluorescent microscopy. Original magnification ×400.

Previous studies have revealed that loss of wild-type p53 function by mutation of the gene can lead differentiated thyroid cancer to anaplastic change [31, 32]. ATC is harbor mutations of p53 in 80–90% of cases and characterized by aggressive course of disease. It was previously demonstrated that thyroid cancer cells with p53 mutation are relatively resistant to IR-induced apoptosis [33]. Relationships between c-ABL and p53 revealed dependence of p53-deficient cells from c-ABL for enhanced proliferation, suggesting that pharmacologic inhibition of c-ABL may have therapeutic value in the p53-deficient cancer cells [34]. Hence, pharmacological inhibition of c-ABL kinase activity can modify the response of ATC cells to IR and could be a promising treatment modality.

#### **5. Senescence-like terminal growth arrest**

becomes activated and induces apoptosis following genotoxic stress [26]. DNA damage caused by IR and other DNA-damaging agents has been shown to result in activation of the c-ABL

induces apoptosis following genotoxic stress [26]. DNA damage caused by IR and other

DNA-damaging agents has been shown to result in activation of the c-ABL (Figure 9)

It was demonstrated that IR induces redistribution of c-ABL between nucleus and cytoplasm

Figure 10. IR induces redistribution of c-ABL between nucleus and cytoplasm in ATC cells. Translocation

**Figure 10.** IR induces redistribution of c-ABL between nucleus and cytoplasm in ATC cells. Translocation of c-ABL in FRO cells after IR treatment (EXS-300 X-irradiator, Toshiba, Tokyo, Japan; 200 kV, 15 mA, 0.83 Gy/min): (a) non-radiat‐ ed; (b) in 6 hours after 10 Gy IR treatment. Fluorescent immunocytochemistry. Confocal fluorescent microscopy. Origi‐

of c-ABL in FRO cells after IR treatment (EXS-300 X-irradiator, Toshiba, Tokyo, Japan; 200 kV, 15 mA,

Notably, nuclear targeting of c-ABL is required for the induction of apoptosis in response to DNA damage. Overexpression of c-ABL activates cell cycle arrest in G1, which requires kinase activity and nuclear localizing signals and depends on the wild-type p53 tumor suppressor (Figure 10). In addition, c-ABL binds p53 and enhances the DNA binding and transcriptional

0.83 Gy/min): (a) non-radiated; (b) in 6 hours after 10 Gy IR treatment. Fluorescent

Notably, nuclear targeting of c-ABL is required for the induction of apoptosis in

immunocytochemistry. Confocal fluorescent microscopy. Original magnification ×400.

It was demonstrated that IR induces redistribution of c-ABL between nucleus and

10

(Figure 9) [27].

[27].

**Figure 9.** Nuclear targeting of c-ABL in response to DNA damage.

Figure 9. Nuclear targeting of c-ABL in response to DNA damage.

158 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

(a) (b)

in ATC cells (Figure 10) [28].

nal magnification ×400.

activity of p53 [29, 30].

cytoplasm in ATC cells (Figure 9) [28].

Senescence is a physiological process of changes in cell metabolism associated with a series of inductive, permissive, and restrictive communications that limit the cell proliferative capacity. Senescent cells are viable but non-dividing, stop to synthesize DNA, and become enlarged and flattened with an increased granularity. Recent data show that senescence may act as an acute, drug- or IR-induced growth arrest program in numerous stromal and epithelial tumors [35]. It was found that IR induces senescence-like phenotype (SLP) associated with terminal growth arrest in ATC cell lines and also in primary thyrocyte line in time- and dose-dependent manner [36].

The induction of SLP in thyroid cells can be identified by the following:


#### **6. Anthrapyrazolone as a specific inhibitor of JNK signaling pathway**

Anthrapyrazolone is a synthetic polyaromatic small molecule–specific inhibitor of c-JNK signaling (Figure 13). Anthrapyrazolone acts as a reversible ATP-competitive inhibitor with an identical capability toward JNK1, JNK2, and JNK3 with >20-fold selectivity versus various tested kinases other than JNKs [37, 38].

In cell cultures, anthrapyrazolone shows dose-dependent inhibition of c-Jun phosphorylation in the range of 5–50 μM [38]. It was demonstrated that combination of anthrapyrazolone and IR treatment inhibited ATC cell growth [7]. Numerous SA-β-Gal–positive cells were markedly increased when anthrapyrazolone was combined with IR (Figure 14).

in primary thyrocyte line in time- and dose-dependent manner [36].

The induction of SLP in thyroid cells can be identified by the following:

like phenotype (SLP) associated with terminal growth arrest in ATC cell lines and also



**Figure 11.** IR induces SLP associated with terminal growth arrest in human ATC cell lines: (a and c) non-irradiated; (b and d) in 120 hours after 10 Gy IR treatment; ATC cells exhibited typical features of SLP. Induction of SA-β-Gal activity (green) was mostly observed in large cells with increased granularity and flattened shape. SA-β-Gal staining method. (a) and (b) Bright-field microscopy, original magnification ×200. (c) and (d) Confocal fluorescent microscopy; original magnification ×400. ×200. (c) and (d) Confocal fluorescent microscopy; original magnification ×400.

12 **Figure 12.** IR-induced SA-β-Gal activity in the primary culture of human thyroid follicular cells: (a) thyroglobulin-posi‐ tive (brown) cells in primary culture, anti-thyroglobulin immunocytochemistry; (b) non-irradiated cells, double stain‐ ing for SA-β-Gal and thyroglobulin; and (c) in 120 hours after 10 Gy IR treatment, double staining for SA-β-Gal and thyroglobulin. Original magnification ×200.

Pharmacological Inhibition of Intracellular Signaling Pathways in Radioresistant Anaplastic Thyroid Cancer http://dx.doi.org/10.5772/62541 161

**Figure 13.** Chemical structure of anthrapyrazolone.

signaling pathway retarded DNA repair [7].

anthrapyrazolone. UV-fluorescent microscope.

anthrapyrazolone. UV-fluorescent microscope.

**Figure 11.** IR induces SLP associated with terminal growth arrest in human ATC cell lines: (a and c) non-irradiated; (b and d) in 120 hours after 10 Gy IR treatment; ATC cells exhibited typical features of SLP. Induction of SA-β-Gal activity (green) was mostly observed in large cells with increased granularity and flattened shape. SA-β-Gal staining method. (a) and (b) Bright-field microscopy, original magnification ×200. (c) and (d) Confocal fluorescent microscopy; original

×200. (c) and (d) Confocal fluorescent microscopy; original magnification ×400.

Figure 11. IR induces SLP associated with terminal growth arrest in human ATC cell lines: (a and c) non-

irradiated; (b and d) in 120 hours after 10 Gy IR treatment; ATC cells exhibited typical features of SLP.

Induction of SA--Gal activity (green) was mostly observed in large cells with increased granularity and

flattened shape. SA--Gal staining method. (a) and (b) Bright-field microscopy, original magnification

like phenotype (SLP) associated with terminal growth arrest in ATC cell lines and also





in primary thyrocyte line in time- and dose-dependent manner [36].

vital staining with PKH-2 dye

160 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

immunocytochemistry (Figure 12)

The induction of SLP in thyroid cells can be identified by the following:

12

(a) (b) (c)

**Figure 12.** IR-induced SA-β-Gal activity in the primary culture of human thyroid follicular cells: (a) thyroglobulin-posi‐ tive (brown) cells in primary culture, anti-thyroglobulin immunocytochemistry; (b) non-irradiated cells, double stain‐ ing for SA-β-Gal and thyroglobulin; and (c) in 120 hours after 10 Gy IR treatment, double staining for SA-β-Gal and

magnification ×400.

thyroglobulin. Original magnification ×200.

anthrapyrazolone; and (c) in 120 hours after 10 Gy IR treatment combined with anthrapyrazolone. SA--

Figure 14. Anthrapyrazolone potentiates SLP in irradiated ATC cells: (a) non-radiated; (b) treatment with **Figure 14.** Anthrapyrazolone potentiates SLP in irradiated ATC cells: (a) non-radiated; (b) treatment with anthrapyra‐ zolone; and (c) in 120 hours after 10 Gy IR treatment combined with anthrapyrazolone. SA-β-Gal staining method. Bright-field microscopy. Original magnification ×100. damage in alkaline single cell gel electrophoresis (Comet assay), in ATC cells treated

anthrapyrazolone; and (c) in 120 hours after 10 Gy IR treatment combined with anthrapyrazolone. SA-- Gal staining method. Bright-field microscopy. Original magnification ×100. It was observed a robust increase of tail moment (Figure 15), which represents DNA damage in alkaline single cell gel electrophoresis (Comet assay), in ATC cells treated with anthrapyr‐ azolone plus IR compared to IR alone, suggesting that inhibition of JNK signaling pathway retarded DNA repair [7]. with anthrapyrazolone plus IR compared to IR alone, suggesting that inhibition of JNK signaling pathway retarded DNA repair [7].

with anthrapyrazolone plus IR compared to IR alone, suggesting that inhibition of JNK Figure 15. Anthrapyrazolone potentiates DNA damage in irradiated ATC cells: (a) non-radiated; (b) in 30 **Figure 15.** Anthrapyrazolone potentiates DNA damage in irradiated ATC cells: (a) non-radiated; (b) in 30 minutes after 10 Gy IR treatment; and (c) in 5 minutes after 10 Gy IR treatment combined with anthrapyrazolone. UV-fluorescent microscope.

minutes after 10 Gy IR treatment; and (c) in 5 minutes after 10 Gy IR treatment combined with

(a) (b) (c)

explore whether SLP induced by combination of anthrapyrazolone plus IR was

In this study, DNA repair rates after 10 Gy exposure were analyzed by Comet assay to

Figure 15. Anthrapyrazolone potentiates DNA damage in irradiated ATC cells: (a) non-radiated; (b) in 30

damaged DNA embedded in agarose gels are subjected to an electric field and generate

a characteristic pattern of DNA distribution forming a tail that, after staining with

associated with accumulation of DNA damage. In this method, individual cells with

In this study, DNA repair rates after 10 Gy exposure were analyzed by Comet assay to

explore whether SLP induced by combination of anthrapyrazolone plus IR was

minutes after 10 Gy IR treatment; and (c) in 5 minutes after 10 Gy IR treatment combined with

14

In this study, DNA repair rates after 10 Gy exposure were analyzed by Comet assay to explore whether SLP induced by combination of anthrapyrazolone plus IR was associated with accumulation of DNA damage. In this method, individual cells with damaged DNA embedded in agarose gels are subjected to an electric field and generate a characteristic pattern of DNA distribution forming a tail that, after staining with fluorescence dye, can be analyzed by fluorescence microscopy. The extent and length of the comet's tail correlates with the severity of DNA damage [39].

Thus, one of the mechanisms that may contribute to the combination treatment effects is likely the delay of DNA repair evoked by JNK pathway inhibition. Treatment with anthrapyrazolone significantly delayed DNA rejoining after 10 Gy IR and increased the radiosensitivity of ATC cells.

#### **7. Imatinib as a selective inhibitor of c-ABL tyrosine kinase activity**

Imatinib (also known as STI571 or Gleevec®) is a tyrosine kinase inhibitor with selectivity toward BCR/ABL, c-ABL, platelet-derived growth factor receptor (PDGFR), and c-KIT (Figure 16) [40].

**Figure 16.** Chemical structure of imatinib.

p21cip1. Original magnification ×200.

growth inhibition (Figure 18) [41].

with single dose of 5 Gy IR treatment.

Figure 17. (a) ATC xenograft model (ATC cells were implanted s.c. into male athymic mice); (b) cell **Figure 17.** (a) ATC xenograft model (ATC cells were implanted s.c. into male athymic mice); (b) cell cycle inhibitor p21cip1 expression in non-radiated mouse ATC cells xenograft; (c) imatinib 10 μM combined with single dose of 10 Gy IR in 120 hours after treatment. Imatinib suppressed *in vivo* growth of ATC cells implanted into nude mice. (b and c) immunohistochemistry, antibodies used were anti-p21cip1. Original magnification ×200.

cycle inhibitor p21cip1 expression in non-radiated mouse ATC cells xenograft; (c) imatinib 10 µM

combined with single dose of 10 Gy IR in 120 hours after treatment. Imatinib suppressed *in vivo* growth

of ATC cells implanted into nude mice. (b and c) immunohistochemistry, antibodies used were anti-

It was demonstrated that imatinib-induced S- and G2–M cell cycle arrest, leading to cell

(a) (b)

Figure 18. To evaluate G2 arrest in ATC cells, changes in the percent mitotic cells should be determined:

(a) in 120 hours after imatinib (10 µM) treatment and (b) in 120 hours after imatinib (10 µM) combined

The anti-tumor effect of imatinib is potentiated in adjunctive therapy with IR, not only

due to inhibition of proliferative cell growth with transient cell cycle arrest and

Imatinib impedes the growth of ATC cell lines *in vitro* through selective inhibition of c-ABL tyrosine kinase activity [41]. *In vivo*, imatinib combined with IR promotes p21cip1 expression in mice bearing ATC xenograft model (Figure 17). p21cip1. Original magnification ×200. p21cip1. Original magnification ×200.

of ATC cells implanted into nude mice. (b and c) immunohistochemistry, antibodies used were anti-

cycle inhibitor p21cip1 expression in non-radiated mouse ATC cells xenograft; (c) imatinib 10 µM

cycle inhibitor p21cip1 expression in non-radiated mouse ATC cells xenograft; (c) imatinib 10 µM

combined with single dose of 10 Gy IR in 120 hours after treatment. Imatinib suppressed *in vivo* growth

combined with single dose of 10 Gy IR in 120 hours after treatment. Imatinib suppressed *in vivo* growth

It was demonstrated that imatinib-induced S- and G2–M cell cycle arrest, leading to cell growth inhibition (Figure 18) [41]. It was demonstrated that imatinib-induced S- and G2–M cell cycle arrest, leading to cell It was demonstrated that imatinib-induced S- and G2–M cell cycle arrest, leading to cell

with single dose of 5 Gy IR treatment.

growth inhibition (Figure 18) [41].

[41].

growth inhibition (Figure 18) [41].

In this study, DNA repair rates after 10 Gy exposure were analyzed by Comet assay to explore whether SLP induced by combination of anthrapyrazolone plus IR was associated with accumulation of DNA damage. In this method, individual cells with damaged DNA embedded in agarose gels are subjected to an electric field and generate a characteristic pattern of DNA distribution forming a tail that, after staining with fluorescence dye, can be analyzed by fluorescence microscopy. The extent and length of the comet's tail correlates with the severity

162 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

Thus, one of the mechanisms that may contribute to the combination treatment effects is likely the delay of DNA repair evoked by JNK pathway inhibition. Treatment with anthrapyrazolone significantly delayed DNA rejoining after 10 Gy IR and increased the radiosensitivity of ATC

**7. Imatinib as a selective inhibitor of c-ABL tyrosine kinase activity**

Imatinib (also known as STI571 or Gleevec®) is a tyrosine kinase inhibitor with selectivity toward BCR/ABL, c-ABL, platelet-derived growth factor receptor (PDGFR), and c-KIT (Figure

(a) (b) (c)

Figure 17. (a) ATC xenograft model (ATC cells were implanted s.c. into male athymic mice); (b) cell

**Figure 17.** (a) ATC xenograft model (ATC cells were implanted s.c. into male athymic mice); (b) cell cycle inhibitor p21cip1 expression in non-radiated mouse ATC cells xenograft; (c) imatinib 10 μM combined with single dose of 10 Gy IR in 120 hours after treatment. Imatinib suppressed *in vivo* growth of ATC cells implanted into nude mice. (b and c)

cycle inhibitor p21cip1 expression in non-radiated mouse ATC cells xenograft; (c) imatinib 10 µM

immunohistochemistry, antibodies used were anti-p21cip1. Original magnification ×200.

combined with single dose of 10 Gy IR in 120 hours after treatment. Imatinib suppressed *in vivo* growth

of ATC cells implanted into nude mice. (b and c) immunohistochemistry, antibodies used were anti-

It was demonstrated that imatinib-induced S- and G2–M cell cycle arrest, leading to cell

(a) (b)

Figure 18. To evaluate G2 arrest in ATC cells, changes in the percent mitotic cells should be determined:

(a) in 120 hours after imatinib (10 µM) treatment and (b) in 120 hours after imatinib (10 µM) combined

The anti-tumor effect of imatinib is potentiated in adjunctive therapy with IR, not only

due to inhibition of proliferative cell growth with transient cell cycle arrest and

16

of DNA damage [39].

cells.

16) [40].

**Figure 16.** Chemical structure of imatinib.

p21cip1. Original magnification ×200.

growth inhibition (Figure 18) [41].

with single dose of 5 Gy IR treatment.

(a) (b)

Figure 18. To evaluate G2 arrest in ATC cells, changes in the percent mitotic cells should be determined: **Figure 18.** To evaluate G2 arrest in ATC cells, changes in the percent mitotic cells should be determined: (a) in 120 hours after imatinib (10 μM) treatment and (b) in 120 hours after imatinib (10 μM) combined with single dose of 5 Gy IR treatment. The anti-tumor effect of imatinib is potentiated in adjunctive therapy with IR, not only due to inhibition of proliferative cell growth with transient cell cycle arrest and

(a) in 120 hours after imatinib (10 µM) treatment and (b) in 120 hours after imatinib (10 µM) combined with single dose of 5 Gy IR treatment. The anti-tumor effect of imatinib is potentiated in adjunctive therapy with IR, not only due to inhibition of proliferative cell growth with transient cell cycle arrest and apoptosis but also due to the terminal growth arrest associated with SLP (Figure 19) [41]. apoptosis but also due to the terminal growth arrest associated with SLP (Figure 19)

Figure 19. Phenotypic changes associated with SLP in ATC cells: (a) ATC cell line without treatment; (b) in 72 hours after imatinib (10 µM) treatment; (c) in 72 hours after imatinib (10 µM) treatment combined **Figure 19.** Phenotypic changes associated with SLP in ATC cells: (a) ATC cell line without treatment; (b) in 72 hours after imatinib (10 μM) treatment; (c) in 72 hours after imatinib (10 μM) treatment combined with single dose of 5 Gy IR. Enlarged and flattened morphology and increased granularity of ATC cells. Original magnification ×200.

16

#### **8. Conclusion**

Intracellular JNK and c-ABL signaling pathways are essential components of ATC cell proliferation and survival after radiation therapy. Hence, pharmacological inhibition of these pathways in combination with radiotherapy may be a potential treatment modality of ATC.

#### **Author details**

Dmitry Bulgin1\* and Alexey Podcheko2

\*Address all correspondence to: molmed1999@yahoo.com

1 Center for Regenerative Medicine "ME-DENT", Rovinj, Croatia

2 American University of Integrative Sciences, St. Maarten School of Medicine, Sint Maarten, Caribbean Netherlands

#### **References**


[7] Bulgin D, Podtcheko A, Takakura S, *et al*. Selective pharmacologic inhibition of c-Jun NH2-terminal kinase radiosensitizes thyroid anaplastic cancer cell lines via induction of terminal growth arrest. Thyroid 2006;16:217–224. DOI:10.1089/thy.2006.16.217

**8. Conclusion**

**Author details**

Caribbean Netherlands

1995;55:2075–2080.

**References**

Dmitry Bulgin1\* and Alexey Podcheko2

\*Address all correspondence to: molmed1999@yahoo.com

164 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

groups. Am J Clin Oncol. 2002;25:442–446.

Cancer Res. 2000;6:323–325.

Clin Cancer Res. 2000;6:4343–4350.

1 Center for Regenerative Medicine "ME-DENT", Rovinj, Croatia

Intracellular JNK and c-ABL signaling pathways are essential components of ATC cell proliferation and survival after radiation therapy. Hence, pharmacological inhibition of these pathways in combination with radiotherapy may be a potential treatment modality of ATC.

2 American University of Integrative Sciences, St. Maarten School of Medicine, Sint Maarten,

[1] Ain KB. Anaplastic thyroid carcinoma: behavior, biology, and therapeutic ap‐

[2] Namba H, Hara T, Tukazaki T, *et al.* Radiation-induced G1 arrest is selectively medi‐ ated by the p53-WAF1/Cip1 pathway in human thyroid cells. Cancer Res.

[3] Yang T, Namba H, Hara T, *et al.* p53 induced by ionizing radiation mediates DNA end-jointing activity, but not apoptosis of thyroid cells. Oncogene 1997;14:1511–1519.

[4] Heron DE, Karimpour S, Grigsby PW. Anaplastic thyroid carcinoma: comparison of conventional radiotherapy and hyperfractionation chemoradiotherapy in two

[5] Harari PM, Huang SM. Modulation of molecular targets to enhance radiation. Clin

[6] Bianco C, Bianco R, Tortora G, *et al*. Antitumor activity of combined treatment of hu‐ man cancer cells with ionizing radiation and anti-epidermal growth factor receptor monoclonal antibody C225 plus type I protein kinase A antisense oligonucleotide.

proaches. Thyroid 1998;8:715–726. DOI:10.1089/thy.1998.8.715


[34] Whang YE, Tran C, Henderson C, *et al*. c-ABL is required for development and opti‐ mal cell proliferation in the context of p53 deficiency. Proc Natl Acad Sci USA. 2000;97:5486–5491.

[20] Watters D. Molecular mechanisms of ionizing radiation-induced apoptosis. Immunol

[21] Kuwabara M, Takahashi K, Inanami O. Induction of apoptosis through the activation of SAPK/JNK followed by the expression of death receptor Fas in X-irradiated cells. J

[22] Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell 2000;103:239–

[23] Van Etten R.A. Cycling, stressed-out and nervous: cellular functions of c-ABL.

[24] Greuber EK, Smith-Pearson P, Wang J, Pendergast AM. Role of ABL family kinases in cancer: from leukaemia to solid tumours. Nat Rev Cancer. 2013;13:559–571. DOI:

[25] Cheng L, Lloyd RV, Weaver AL, *et al.* The cell cycle inhibitors p21WAF1 and p27KIP1 are associated with survival in patients treated by salvage prostatectomy af‐

[26] Yoshida K, Miki Y. Enabling death by the ABL tyrosine kinase: mechanisms for nu‐ clear shuttling of c-ABL in response to DNA damage. Cell Cycle 2005;4:777–779. [27] Shafman T, Khanna KK, Kedar P, *et al.* Interaction between ATM protein and c-ABL in response to DNA damage. Nature 1997;387:520–523. DOI:10.1038/387520a0

[28] Podtcheko A, Ohtsuru A, Namba H, *et al.* Inhibition of ABL tyrosine kinase potenti‐ ates radiation-induced terminal growth arrest in anaplastic thyroid cancer cells. Ra‐

[29] Yuan ZM, Huang Y, Whang Y, *et al.* Role for c-ABL tyrosine kinase in growth arrest response to DNA damage. Nature 1996;382:272–274. DOI:10.1038/382272a0

[30] Sionov RV, Moallem E, Berger M, *et al*. c-ABL neutralizes the inhibitory effect of Mdm2 on p53. J Biol Chem. 1999;274:8371–8374. DOI:10.1074/jbc.274.13.8371

[31] Ito T, Seyama T, Mizuno T, *et al.* Unique association of p53 mutations with undiffer‐ entiated but not with differentiated carcinomas of the thyroid gland. Cancer Res.

[32] Fagin JA, Matsuo K, Karmakar A, *et al.* High prevalence of mutations of the p53 gene in poorly differentiated human thyroid carcinomas. J Clin Invest. 1993;91:179–184.

[33] Namba H, Hara T, Tukazaki T, *et al.* Radiation-induced G1 arrest is selectively medi‐ ated by the p53-WAF1/Cip1 pathway in human thyroid cells. Cancer Res.

Trends Cell Biol. 1999;9:179–186. DOI:10.1016/S0962-8924(99)01549-4

ter radiation therapy. Clin Cancer Res. 2000;6:1896–1899.

Cell Biol. 1999;77:263–271. DOI:10.1046/J.1440-1711.1999.00824.X

Radiat Res. 2003;44:203–209.

10.1038/nrc3563

diat Res. 2006;165:35–42.

1992;52:1369–1371.

DOI:10.1172/JCI116168

1995;55:2075–2080.

252. DOI:10.1016/S0092-8674(00)00116-1

166 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis


**Chapter 7**

### **Modern Technologies for Timely Detection and Differential Diagnosis of Gastric Cancer**

Vladimir Levkin, Nina Gagarina, Sergey Kharnas, Gaziyav Musaev, Artem Shiryaev and Dmitry Bulgin

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62540

#### **Abstract**

The diagnostic potentialities of laser spectro- and videofluorescence endoscopy, complex transabdominal US examination, dynamic multihelical computed tomography (MHCT) with the possibility of constructing multiplanar reformations, and virtual gastroscopy were studied with a view to diagnosing gastric cancer (GC). It was established that laser spectral fluorescence with the drug Alasens (5-aminolevulinic acid) is a highly revealing method for diagnosis and differential diagnosis of GC. The sensitivity of the method is 96%, and its specificity is 78%. Well-defined videofluorescence was noted in 91.3% of pa‐ tients with GC. The possibility of detecting cancer with complex trans-ultrasonography in the pyloroantral division and in the lower third of the body of the stomach constitutes 95.6% attaining absolute values in T3 and T4. Dynamic MHCT allows 97% detection of GC attaining absolute values, beginning with T2 invasion depth; tumor localization is ir‐ relevant. Comparative visual assessment of the quality of a virtual image and conven‐ tional video esophagogastroduodenoscopy (EGDS) was made. The study demonstrated a sufficiently high level of virtual images whose quality was not inferior to that of conven‐ tional images in intraluminal tumor growth. The indications for the application of this technique require further specification.

**Keywords:** Gastric cancer, 5-aminolevulinic acid (Alasens), laser spectro- and videofluor‐ escence, complex transabdominal US examination, dynamic multihelical-computed to‐ mography, virtual endoscopy

#### **1. Introduction**

Despite the fact that during the previous decades the incidence of gastric cancer (GC) has been tending to decrease in many countries of the world, this disease continues to occupy a leading

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

place in the structure of oncological diseases and is one of the commonest causes of death from malignant neoplasms [1]. It is common knowledge that the success of treatment of patients with GC primarily depends on the timely diagnosis (tumor detection or primary diagnosis).

The main methods that enable us to detect gastric tumor and to verify the diagnosis are esophagogastroduodenoscopy with biopsy and roentgenologic examination of the upper gastrointestinal tract using the double contrast technique. The advantages of these methods are known and indisputable. At the same time, their shortcomings are also well known, namely the difficulty in detecting early forms of cancer as well as tumors with the endophytic submucosal character of growth. A relatively high number of both false-negative and falsepositive results necessitates repeat endoscopic examinations and biopsies [2].

At present, laser fluorescence spectroscopy is one of the promising methods for the early diagnosis of malignant tumors, including GC [3, 4]. This method allows us to enhance the efficacy of the standard endoscopic examination in cancer and proved so informative that it is referred to as 'optic biopsy.' We distinguish autofluorescence, i.e., fluorescence of endogenic porphyrins whose concentration in the tumor cells is higher than in normal cells, and secon‐ dary fluorescence, i.e., the fluorescence of special exogenous photosensitizers, which are tropic to tumor cells. Neither method is free from typical disadvantages: photosensitizers are accumulated not only in the tumor but also in the skin and visible mucous membranes where they are retained for a long time and cause photodermatitis if the light regimen is compro‐ mised. The main disadvantages of autofluorescence monitoring are a low contrast ratio and the necessity of high-precision costly equipment.

Laser fluorescence spectroscopy has offered new possibilities with the development of 5 aminolevulinic acid (5-ALA), which induces the synthesis and accumulation of photoactive protoporphyrin IX in tumor cells. This results in intense fluorescence, which can be registered not only with spectroanalyzers but also with special highly sensitive fluorescent endoscopes, which opens up new horizons for clinical practice.

With the appearance of new radiodiagnostic technologies and the perfection of the already available [such as ultrasonography, endo-ultrasonography, computed tomography (CT), magnetic resonance imaging, and positron emission tomography] techniques, their possibili‐ ties in modern diagnostics of GC are actively being assessed [5–7]. In particular, numerous investigations demonstrated the potentialities of a complex transabdominal US examination with water loading as a method for primary diagnosis of GC [5].

Computed tomography is conventionally considered to be a method for secondary diagnosis of GC and, above all, detection of remote metastases. However, the studies conducted recently give evidence of the fact that the results of multihelical computed tomography (MHCT) are on a par with those of the X-ray and endoscopic methods in detecting GC [8]. Moreover, this method allows, after preliminarily insufflating the stomach, the performance of the so-called virtual gastroscopy. Thus, dynamic MHCT additionally allows a wider field of view of the organ in the modes of constructing multiplanar reformations and virtual endoscopy and, in contrast to conventional videoendoscopy, is characterized by the absence of blind zones [7, 9]. The drawbacks of the method are as follows: the construction of multiplanar and virtual images is time-consuming; the absence of delicate shades of color at the boundary of lesion; the impossibility to perform biopsy; and radiation exposure [7, 9]. Most of the works devoted to primary diagnostics of GC with MHCT are mainly of pilot character.

The aim of this study is to look into the diagnostic possibilities of laser spectro- and video‐ fluorescent endoscopy, complex trans-US examination, MHCT with the possibility of con‐ structing multiplanar reformations, and virtual gastroscopy in the primary diagnosis of GC.

#### **2. Materials and methods**

place in the structure of oncological diseases and is one of the commonest causes of death from malignant neoplasms [1]. It is common knowledge that the success of treatment of patients with GC primarily depends on the timely diagnosis (tumor detection or primary diagnosis).

The main methods that enable us to detect gastric tumor and to verify the diagnosis are esophagogastroduodenoscopy with biopsy and roentgenologic examination of the upper gastrointestinal tract using the double contrast technique. The advantages of these methods are known and indisputable. At the same time, their shortcomings are also well known, namely the difficulty in detecting early forms of cancer as well as tumors with the endophytic submucosal character of growth. A relatively high number of both false-negative and false-

At present, laser fluorescence spectroscopy is one of the promising methods for the early diagnosis of malignant tumors, including GC [3, 4]. This method allows us to enhance the efficacy of the standard endoscopic examination in cancer and proved so informative that it is referred to as 'optic biopsy.' We distinguish autofluorescence, i.e., fluorescence of endogenic porphyrins whose concentration in the tumor cells is higher than in normal cells, and secon‐ dary fluorescence, i.e., the fluorescence of special exogenous photosensitizers, which are tropic to tumor cells. Neither method is free from typical disadvantages: photosensitizers are accumulated not only in the tumor but also in the skin and visible mucous membranes where they are retained for a long time and cause photodermatitis if the light regimen is compro‐ mised. The main disadvantages of autofluorescence monitoring are a low contrast ratio and

Laser fluorescence spectroscopy has offered new possibilities with the development of 5 aminolevulinic acid (5-ALA), which induces the synthesis and accumulation of photoactive protoporphyrin IX in tumor cells. This results in intense fluorescence, which can be registered not only with spectroanalyzers but also with special highly sensitive fluorescent endoscopes,

With the appearance of new radiodiagnostic technologies and the perfection of the already available [such as ultrasonography, endo-ultrasonography, computed tomography (CT), magnetic resonance imaging, and positron emission tomography] techniques, their possibili‐ ties in modern diagnostics of GC are actively being assessed [5–7]. In particular, numerous investigations demonstrated the potentialities of a complex transabdominal US examination

Computed tomography is conventionally considered to be a method for secondary diagnosis of GC and, above all, detection of remote metastases. However, the studies conducted recently give evidence of the fact that the results of multihelical computed tomography (MHCT) are on a par with those of the X-ray and endoscopic methods in detecting GC [8]. Moreover, this method allows, after preliminarily insufflating the stomach, the performance of the so-called virtual gastroscopy. Thus, dynamic MHCT additionally allows a wider field of view of the organ in the modes of constructing multiplanar reformations and virtual endoscopy and, in contrast to conventional videoendoscopy, is characterized by the absence of blind zones [7, 9]. The drawbacks of the method are as follows: the construction of multiplanar and virtual images

positive results necessitates repeat endoscopic examinations and biopsies [2].

the necessity of high-precision costly equipment.

170 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

which opens up new horizons for clinical practice.

with water loading as a method for primary diagnosis of GC [5].

The studies were conducted in the Burdenko Surgical Clinic of the Sechenov First Moscow State Medical University from 2003 to 2010.

The results of laser spectral fluorescence diagnostics (FD) were analyzed in 62 patients with malignant (37, 59.7%) and benign (35, 40.3%) diseases. Videofluorescence was observed in 25 patients (23 with GC and 2 with benign diseases). The study was conducted using 5-ALA– based Alasens (GNTs NIOPIK, Research Institute of Organic Semi-Products and Dyes), which was preliminarily dissolved in 200 mL of water (10–20 mg/kg of body mass) and ingested per os 1.5–2 h before the study. A LESA-01 spectroanalyzer (Biospek and the Institute of Optic Physics, Russian Academy of Sciences, Russia) was used for monitoring fluorescence. To pinpoint videofluorescence (Figure 2), a special attachment for the standard endoscope consisting of a high-sensitivity camera (0.003 lx) and a system of light filters and a photodiode laser with a wavelength of 630 nm and a power of 1.5 W were used.

Eighty-seven patients with GC underwent complex trans-US examination. The study was conducted using SIQUOIA apparatuses (ACUSON, the United States) operating in the realtime mode and furnished with a convex transducer with a frequency of 2.5–3.5 MHz and a linear probe with a frequency of 7.0–11.0 MHz. A two-stage method with the water test proposed by Worlicek et al. (1989) was used when ultrasonography was performed [9].

Ninety patients with verified GC and 10 patients with suspected GC were subjected to dynamic MHCT. The study was conducted using a Toshiba 320-slice tomograph with 16-cm detector that allowed three-dimensional (3D) dynamic scanning with visualization of the whole stomach in the dynamic mode without table feed. The native sequence was then performed, a 16-cm examination zone along the *Z*-axis (embracing the whole stomach) was chosen, and the abdominal cavity and the small pelvis were scanned. The subsequent procedure included the construction of multiplanar reformations and dynamic video files. When virtual gastro‐ scopy was constructed (16 patients), the stomachs were preliminarily insufflated through a probe or by using a special effervescent mixture consisting of citric acid and sodium bicar‐ bonate (4.0–6.0 g).

To objectively evaluate the information content of the methods used for diagnosing GC and assessing its extent of spread, the diagnostic results were compared with the intraoperative and histological findings. The international TNM classification (UICC 2002) was used for staging GC.

The clinical data were analyzed with the standard methods for statistical processing using Microsoft Excel, SPSS 14.0, and MedCalc 5.0.

Only 18 patients were diagnosed as having GC prior to the performance of FD.

#### **3. Results** Later, this diagnosis was verified by both the fluorescence intensity data (PRIX,

#### **3.1. Laser spectral and videofluorescent diagnostics of gastric cancer** the contrast ratio 2.1–18.6) and histological findings.

Only 18 patients were diagnosed as having GC prior to the performance of FD. Later, this diagnosis was verified by both the fluorescence intensity data (PRIX, the contrast ratio 2.1– 18.6) and histological findings. Of 31 patients with the preliminary diagnosis of gastric ulcer, FD performance

Of 31 patients with the preliminary diagnosis of gastric ulcer, FD performance revealed a high-fluorescence intensity (the contrast ratio 2.1–9.6) at the edges of ulceration in 12 patients (Figure 1). revealed a high-fluorescence intensity (the contrast ratio 2.1–9.6) at the edges of ulceration in 12 patients (Figure 1).

Figure 1. (A) Spectrogram and (B) histogram in gastric cancer and (C) the scheme and the principle of operation of LESA-01. **Figure 1.** (A) Spectrogram and (B) histogram in gastric cancer and (C) the scheme and the principle of operation of LESA-01.

Target biopsy of the mucous membrane was performed at these points; histological Target biopsy of the mucous membrane was performed at these points; histological examina‐ tion of the biopsy material confirmed the diagnosis of GC. In the remaining 14 patients, the FD data (the contrast ratio 0.7–0.8) coincided with the histological findings; the diagnosis of gastric ulcer was verified.

histological findings; the diagnosis of gastric ulcer was verified.

examination of the biopsy material confirmed the diagnosis of GC. In the

remaining 14 patients, the FD data (the contrast ratio 0.7–0.8) coincided with the

Esophagogastroduodenoscopy revealed atypical regions of the mucosa suggestive of cancer in five patients. High-intensity fluorescence of ALA-induced PRIX (the contrast ratio 2.1–8.0) was observed in four patients when FD was carried out. Target biopsy of the mucous mem‐ brane of the stomach was performed, and the diagnosis of GC was made. Likewise, a patient with a gastric polyp (the contrast ratio 3.3) was diagnosed as having carcinoma in situ based on the results of histological examination.

In four patients, the preliminary diagnosis of GC was rejected after spectroscopy, which was verified by the data of the subsequent morphological examination.

High-intensity fluorescence of ALA-induced PRIX was detected at 1–2 points of the region studied in five observations. As evidenced by the results of histological examination, a markedly pronounced inflammatory reaction was revealed in four of five of these patients. Numerous authors [3] report that inflammation may be associated with increased generation of ALA-induced PPIX.

Taking into account the foregoing, these patients can be included in the risk group but not assigned to those with false-positive results. These patients require preventive endoscopic examinations at least once a year.

In one observation, during FD the contrast ratio constituted 1.1; however, morphological examination revealed highly differentiated adenocarcinoma. This result can be attributed to false-negative ones.

On the whole, the contrast ratio constituted 4.55 ± 0.2 across the GC group (from 3.83 ± 0.6 in carcinoma in situ to 6.13 ± 0.2 in stage IV). In patients with gastric ulcer, the contrast ratio was 1.08 ± 0.7. The difference between the contrast ratio values in malignant and benign diseases is statistically significant (*p* ≤ 0.05). The sensitivity of the diagnostic method with the study of Alasens-induced fluorescence was 96%, and its specificity was 78%.

#### **3.2. Videofluorescence study**

The clinical data were analyzed with the standard methods for statistical processing using

Only 18 patients were diagnosed as having GC prior to the performance of FD.

Later, this diagnosis was verified by both the fluorescence intensity data (PRIX,

Of 31 patients with the preliminary diagnosis of gastric ulcer, FD performance

revealed a high-fluorescence intensity (the contrast ratio 2.1–9.6) at the edges of

Only 18 patients were diagnosed as having GC prior to the performance of FD. Later, this diagnosis was verified by both the fluorescence intensity data (PRIX, the contrast ratio 2.1–

Of 31 patients with the preliminary diagnosis of gastric ulcer, FD performance revealed a high-fluorescence intensity (the contrast ratio 2.1–9.6) at the edges of ulceration in 12 patients

3

2

1

0

А. В.

**Figure 1.** (A) Spectrogram and (B) histogram in gastric cancer and (C) the scheme and the principle of operation of

Target biopsy of the mucous membrane was performed at these points; histological examina‐ tion of the biopsy material confirmed the diagnosis of GC. In the remaining 14 patients, the FD data (the contrast ratio 0.7–0.8) coincided with the histological findings; the diagnosis of

Figure 1. (A) Spectrogram and (B) histogram in gastric cancer and (C) the scheme

123

Target biopsy of the mucous membrane was performed at these points; histological

examination of the biopsy material confirmed the diagnosis of GC. In the

remaining 14 patients, the FD data (the contrast ratio 0.7–0.8) coincided with the

histological findings; the diagnosis of gastric ulcer was verified.

**3.1. Laser spectral and videofluorescent diagnostics of gastric cancer**

the contrast ratio 2.1–18.6) and histological findings.

Microsoft Excel, SPSS 14.0, and MedCalc 5.0.

172 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

ulceration in 12 patients (Figure 1).

and the principle of operation of LESA-01.

18.6) and histological findings.

**3. Results**

(Figure 1).

С.

LESA-01.

gastric ulcer was verified.

When performing endoscopic examination of the fluorescent images of the stomach, distinct fluorescence of the tumor in 21 (91.3%) of 23 patients with GC was noted (Figure 2).

In two patients, the diagnosis of GC was excluded during complex examination and, accord‐ ingly, videofluorescence was absent (no false-negative results were noted). In addition, the foci of severe dysplasia and malignancy were seen to be fluorescing, which was later confirmed by the results of analysis of both the fluorescence spectra and the biopsy findings.

#### **3.3. Complex trans-ultrasonography in the primary diagnosis of GC**

To assess the possibilities of complex transabdominal US examination as a potential screening method in detecting GC, we examined patients with different stages and different localization of the neoplastic process.

When performing endoscopic examination of the fluorescent images of the

stomach, distinct fluorescence of the tumor in 21 (91.3%) of 23 patients with GC

В.

Figure 2. (A) Prototype of the videofluorescence unit and (B) Malignant gastric **Figure 2.** (A) Prototype of the videofluorescence unit and (B) Malignant gastric polyp (videoendoscopy and video‐ fluorescence).

#### *3.3.1. T1 invasion depth* polyp (videoendoscopy and videofluorescence).

To reveal disease at this stage of development of the pathological process is a difficult task due to a small short local extension and a low depth of the tumor lesion (Figure 3).

According to the postoperative histological findings, 28 patients had T2 invasion depth; the performance of ultrasonography was successful in detecting the tumor in 20 patients (in 8 patients using the standard technique; in 12 patients, US + ingestion of water). In six observa‐ tions, the tumor was localized in the regions inaccessible to US examination: the cardia and the upper third of the stomach body; in two observations, in the pyloroantral zone and the lower third of the body at the lesser curvature. In two patients, the diagnosis of GC was excluded during complex examination and, accordingly, videofluorescence was absent (no false-negative results were noted). In addition, the foci of severe dysplasia and malignancy were seen to be

In two observations, ultrasonography was the first diagnostic procedure that enabled us to suspect early antral cancer in the form of local thickening of the gastric wall with 1.5-cm Modern Technologies for Timely Detection and Differential Diagnosis of Gastric Cancer http://dx.doi.org/10.5772/62540 175

Figure 3. (A) Echogram and (B) the scheme of the infiltrative lesion of the gastric **Figure 3.** (A) Echogram and (B) the scheme of the infiltrative lesion of the gastric wall. T1 invasion depth.

extension, which was subsequently verified by the esophagogastroduodenoscopy (EGDS) + biopsy and X-ray findings. wall. T1 invasion depth.

Thus, complex US examination was instrumental in detecting a tumor at T1 invasion depth in 72% of observations, and when the tumor was localized in the pyloroantral division of the stomach and in the lower third of the organ, this level increased to 90%. The sensitivity and specificity of this technique in measuring the T1 invasion depth constituted 42.9% and 91.8%, respectively. According to the postoperative histological findings, 28 patients had T2 invasion depth; the performance of ultrasonography was successful in detecting the tumor in

#### *3.3.2. T2 invasion depth* 20 patients (in 8 patients using the standard technique; in 12 patients, US +

*3.3.1. T1 invasion depth*

fluorescence).

was noted (Figure 2).

174 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

lower third of the body at the lesser curvature.

polyp (videoendoscopy and videofluorescence).

To reveal disease at this stage of development of the pathological process is a difficult task due

В.

Figure 2. (A) Prototype of the videofluorescence unit and (B) Malignant gastric

**Figure 2.** (A) Prototype of the videofluorescence unit and (B) Malignant gastric polyp (videoendoscopy and video‐

When performing endoscopic examination of the fluorescent images of the

stomach, distinct fluorescence of the tumor in 21 (91.3%) of 23 patients with GC

А.

According to the postoperative histological findings, 28 patients had T2 invasion depth; the performance of ultrasonography was successful in detecting the tumor in 20 patients (in 8 patients using the standard technique; in 12 patients, US + ingestion of water). In six observa‐ tions, the tumor was localized in the regions inaccessible to US examination: the cardia and the upper third of the stomach body; in two observations, in the pyloroantral zone and the

and, accordingly, videofluorescence was absent (no false-negative results were

noted). In addition, the foci of severe dysplasia and malignancy were seen to be

In two patients, the diagnosis of GC was excluded during complex examination

In two observations, ultrasonography was the first diagnostic procedure that enabled us to suspect early antral cancer in the form of local thickening of the gastric wall with 1.5-cm

to a small short local extension and a low depth of the tumor lesion (Figure 3).

According to the histological findings, T2 invasion depth was noted in 19 patients; complex ultrasonography revealed a tumor in 18 patients. In one patient, the tumor was localized in the cardiac region and in the upper third of the stomach body, i.e., the regions inaccessible to US examination. At the first stage of complex US examination, a tumor was detected in 16 patients and in two more patients at the second stage with the water test. However, in numerous cases, it was not simple to reveal a tumor with category T2 invasion depth. This was only possible when the organ was thoroughly and meticulously examined. ingestion of water). In six observations, the tumor was localized in the regions inaccessible to US examination: the cardia and the upper third of the stomach body; in two observations, in the pyloroantral zone and the lower third of the body

Thus, the detection rate for T2 invasion depth lesions was 95% if the tumor was localized in the pyloric and antral divisions of the stomach or involved in its body. The sensitivity and specificity in determining T2 invasion depth constituted 63.2% and 81%, respectively. at the lesser curvature.

#### *3.3.3. T3 invasion depth* In two observations, ultrasonography was the first diagnostic procedure that

According to the histological findings, T3 invasion depth was noted in 16 patients. In all observations, except one (a tumor invading the upper third and the cardiac division of the stomach with a low degree of extension), we succeeded in detecting the tumor easily enough resorting to both techniques: the standard ultrasonography and US supplemented with water enabled us to suspect early antral cancer in the form of local thickening of the gastric wall with 1.5-cm extension, which was subsequently verified by the

Thus, complex US examination was instrumental in detecting a tumor at T1

invasion depth in 72% of observations, and when the tumor was localized in the

pyloroantral division of the stomach and in the lower third of the organ, this level

esophagogastroduodenoscopy (EGDS) + biopsy and X-ray findings.

loading. At this depth of lesion, it was the classical 'hollow organ' symptom that was most commonly observed in the US image. At this pathological stage, we visualized not only considerable thickening of the stomach wall but also a large extent of spread of the tumor along the longitudinal axis of the organ involving several anatomical regions. All this facilitated the task of tumor detection.

Thus, ultrasound examination supplemented with the water test did not increase the infor‐ mation content in terms of tumor detection at this stage of the disease but allowed us to evaluate more thoroughly the extent of infiltration, to determine the depth of involvement by the tumor, and to improve visualization of the organs of the retroperitoneal space.

Complex transabdominal US examination enabled us to reveal the tumor in 94% of cases in T3 invasion depth. If the localization site was the pylorus, antrum, and body of the stomach, the detection rate was 100%. The sensitivity and specificity in relation to measuring the invasion depth were 75% and 83.6%, respectively.

#### *3.3.4. T4 invasion depth*

As judged by the results of the histological examination of biopsy material, T4 invasion depth was noted in 14 patients. In all the observations of patients with category T4 malignant lesions, the tumor was revealed easily as the 'hollow organ' symptom due to a large extent of spread using both techniques: the standard ultrasonography and US with water loading.

In our study, invasion of the adjacent organs, the great vessels, and fatty tissue by the tumor was suspected during ultrasonic examination in four patients, which was later confirmed intraoperatively using the same procedure and by revision of the organs of the abdominal cavity.

Thus, the possibility of detecting tumor lesions of the stomach with T4 invasion depth was absolute. As far as the sensitivity and specificity of the method are concerned, they are 85.7% and 93.7%, respectively.

#### **3.4. Dynamic MHCT in the diagnostics of gastric cancer**

Until recently, CT was regarded as a technique for secondary diagnosis of GC. As dynamic MHCT is a more informative diagnostic method, we studied the possibilities of primary diagnostics of GC, including the so-called virtual endoscopy.

#### *3.4.1. T1 invasion depth*

Based on the data of postoperative histological examination, T1 invasion depth was diagnosed in nine patients. We succeeded in detecting the tumor in seven of nine observations (78%). In all the cases, T1 invasion depth was characterized by the non-uniform contrast of the mucous– submucous layer and peristaltic disorder, even if the wall thickening was insignificant. In two observations of early GC (carcinoma in situ), we failed to reveal the tumor.

Thus, the sensitivity of MHCT in revealing T1 invasion depth constituted 22%; its specificity is 100%; the positive predictive value (PPV) is 100%; and the negative predictive value (NPV) is 93%.

#### *3.4.2. T2 invasion depth*

loading. At this depth of lesion, it was the classical 'hollow organ' symptom that was most commonly observed in the US image. At this pathological stage, we visualized not only considerable thickening of the stomach wall but also a large extent of spread of the tumor along the longitudinal axis of the organ involving several anatomical regions. All this facilitated the

Thus, ultrasound examination supplemented with the water test did not increase the infor‐ mation content in terms of tumor detection at this stage of the disease but allowed us to evaluate more thoroughly the extent of infiltration, to determine the depth of involvement by the tumor,

Complex transabdominal US examination enabled us to reveal the tumor in 94% of cases in T3 invasion depth. If the localization site was the pylorus, antrum, and body of the stomach, the detection rate was 100%. The sensitivity and specificity in relation to measuring the

As judged by the results of the histological examination of biopsy material, T4 invasion depth was noted in 14 patients. In all the observations of patients with category T4 malignant lesions, the tumor was revealed easily as the 'hollow organ' symptom due to a large extent of spread

In our study, invasion of the adjacent organs, the great vessels, and fatty tissue by the tumor was suspected during ultrasonic examination in four patients, which was later confirmed intraoperatively using the same procedure and by revision of the organs of the abdominal

Thus, the possibility of detecting tumor lesions of the stomach with T4 invasion depth was absolute. As far as the sensitivity and specificity of the method are concerned, they are 85.7%

Until recently, CT was regarded as a technique for secondary diagnosis of GC. As dynamic MHCT is a more informative diagnostic method, we studied the possibilities of primary

Based on the data of postoperative histological examination, T1 invasion depth was diagnosed in nine patients. We succeeded in detecting the tumor in seven of nine observations (78%). In all the cases, T1 invasion depth was characterized by the non-uniform contrast of the mucous– submucous layer and peristaltic disorder, even if the wall thickening was insignificant. In two

observations of early GC (carcinoma in situ), we failed to reveal the tumor.

using both techniques: the standard ultrasonography and US with water loading.

and to improve visualization of the organs of the retroperitoneal space.

invasion depth were 75% and 83.6%, respectively.

176 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

**3.4. Dynamic MHCT in the diagnostics of gastric cancer**

diagnostics of GC, including the so-called virtual endoscopy.

task of tumor detection.

*3.3.4. T4 invasion depth*

and 93.7%, respectively.

*3.4.1. T1 invasion depth*

cavity.

According to the histological findings, T2 invasion depth was determined in 13 patients. We succeeded in detecting the tumor in all the cases, irrespective of the localization of the neoplastic process in the stomach.

The most characteristic CT symptom at T2 invasion depth, as at T1 invasion depth, is limited thickening of the gastric wall (13.6 ± 5.4 mm) with increased accumulation of the contrast medium and with impaired peristalsis in this division without the involvement of perigastric fatty tissue.

The sensitivity of MHCT in measuring T2 invasion depth constituted 69%; its specificity is 92%; PPV is 57%; and NPV is 95%.

#### *3.4.3. T3 invasion depth*

According to the results of histological examination, the T3 invasion depth group included 24 patients. With T3 invasion depth, marked (19.0 ± 8.4 mm) thickening of the stomach wall, irregularity of the external outline with reticular, and linear thickening of the surrounding fatty tissue are noted.

The accumulation of contrast medium by the tumor was of different character and depended, to a larger degree, on its histological variant. It was not difficult to reveal the tumor lesion at this stage of the process due to the extent of spread when compared to stage T2, not to mention T1. Moreover, at this stage of the disease, not only the stomach wall is significantly thickened, but also the tumor is more extended involving several anatomical regions of the stomach, which was especially well seen when multiplanar images were constructed.

The sensitivity of MHCT in revealing T3 invasion depth constituted 80%; its specificity is 85%; PPV is 71%; and NPV is 91%.

#### *3.4.4. T4 invasion depth*

It should be noted that the T4 invasion depth is associated with the most significant lesion of the organ in terms of depth and the extent of spread. In all our observations (36 patients), it was not difficult to reveal the tumor as with T3 malignant lesions. The CT picture of T4 invasion depth, as visualized with dynamic MHCT, was characterized by marked thickening of the stomach walls (21.3 ± 7.1 mm) with their indistinct outline, infiltration of the adjacent fatty tissue, the absence of the boundary between the gastric wall and the adjacent organs, and invasion of the surrounding organs by the tumor (Figure 4).

The detection of the tumor involving the adjacent organs and tissues was a significant criterion confirming the presence of this stage of disease. Thus, according to the data of our study, when invasion of the surrounding organs by the tumor (Figure 4).

specificity is 85%; PPV is 71%; and NPV is 91%.

**3.4.4. T4 invasion depth**

The sensitivity of MHCT in revealing T3 invasion depth constituted 80%; its

It should be noted that the T4 invasion depth is associated with the most significant

lesion of the organ in terms of depth and the extent of spread. In all our

observations (36 patients), it was not difficult to reveal the tumor as with T3

malignant lesions. The CT picture of T4 invasion depth, as visualized with

dynamic MHCT, was characterized by marked thickening of the stomach walls

(21.3 ± 7.1 mm) with their indistinct outline, infiltration of the adjacent fatty tissue,

the absence of the boundary between the gastric wall and the adjacent organs, and

Figure 4. Dynamic MHCT, gastric cancer, subtotal lesion, T3–T4 invasion depth, **Figure 4.** Dynamic MHCT, gastric cancer, subtotal lesion, T3–T4 invasion depth, multiplanar reformations.

MHCT was performed, we suspected invasion of the adjacent organs and the great vessels by the tumor in 18 patients, which was subsequently verified with intraoperative ultrasono‐ graphic examination and by revision of the organs of the abdominal cavity. multiplanar reformations.

The sensitivity of MHCT in detecting T4 invasion depth was 83%; its specificity is 95%; PPV is 91%; and NPV is 91%. The detection of the tumor involving the adjacent organs and tissues was a

On the whole, the sensitivity of MHCT in detecting GC constituted 97%; its specificity is 100%; PPV is 100%; and NPV is 77%. significant criterion confirming the presence of this stage of disease. Thus,

#### **3.5. Virtual endoscopy** according to the data of our study, when MHCT was performed, we suspected

Some authors consider virtual endoscopy as a potentially screening method of diagnostics and differential diagnostics of GC. In this connection, we evaluated the diagnostic possibilities of the method and the quality of the image obtained in the process of simulation.

#### *3.5.1. Gastric leiomyoma*

Based on the EGDS and X-ray data, two patients had intraluminal tumor growth; in one female patient, the tumor was localized outside the stomach only insignificantly bulging into the lumen of the organ. In all the cases, a virtual image obtained as a result of CT simulation was characterized by a high quality maximally approximating to the image obtained during videoendoscopy. The study enabled us to specify the localization of the tumor, and the planar slices demonstrated the tumor site relative to the stomach wall (intraparietal localization with prolapse into the lumen or extragastral). These data allowed us to preliminarily choose and plan the optimal treatment modality (wedge resection of the stomach was performed in all patients).

When a virtual image is constructed in patients with GC, it is worth pointing out the following. In the cases when exophytic tumor growth (Type 1, according to Bormann's classification) was

noted (three observations; Figure 5), the greatest similarity between a virtual and video image was obtained (irrespective of localization of the tumor in the stomach). similarity between a virtual and video image was obtained (irrespective of

When a virtual image is constructed in patients with GC, it is worth pointing out

the following. In the cases when exophytic tumor growth (Type 1, according to

Figure 5. Video EGDS. The virtual endoscopy. **Figure 5.** Video EGDS. The virtual endoscopy.

localization of the tumor in the stomach).

MHCT was performed, we suspected invasion of the adjacent organs and the great vessels by the tumor in 18 patients, which was subsequently verified with intraoperative ultrasono‐

**Figure 4.** Dynamic MHCT, gastric cancer, subtotal lesion, T3–T4 invasion depth, multiplanar reformations.

Figure 4. Dynamic MHCT, gastric cancer, subtotal lesion, T3–T4 invasion depth,

The detection of the tumor involving the adjacent organs and tissues was a

significant criterion confirming the presence of this stage of disease. Thus,

according to the data of our study, when MHCT was performed, we suspected

The sensitivity of MHCT in revealing T3 invasion depth constituted 80%; its

It should be noted that the T4 invasion depth is associated with the most significant

lesion of the organ in terms of depth and the extent of spread. In all our

observations (36 patients), it was not difficult to reveal the tumor as with T3

malignant lesions. The CT picture of T4 invasion depth, as visualized with

dynamic MHCT, was characterized by marked thickening of the stomach walls

(21.3 ± 7.1 mm) with their indistinct outline, infiltration of the adjacent fatty tissue,

the absence of the boundary between the gastric wall and the adjacent organs, and

specificity is 85%; PPV is 71%; and NPV is 91%.

**3.4.4. T4 invasion depth**

The sensitivity of MHCT in detecting T4 invasion depth was 83%; its specificity is 95%; PPV

On the whole, the sensitivity of MHCT in detecting GC constituted 97%; its specificity is 100%;

Some authors consider virtual endoscopy as a potentially screening method of diagnostics and differential diagnostics of GC. In this connection, we evaluated the diagnostic possibilities of

Based on the EGDS and X-ray data, two patients had intraluminal tumor growth; in one female patient, the tumor was localized outside the stomach only insignificantly bulging into the lumen of the organ. In all the cases, a virtual image obtained as a result of CT simulation was characterized by a high quality maximally approximating to the image obtained during videoendoscopy. The study enabled us to specify the localization of the tumor, and the planar slices demonstrated the tumor site relative to the stomach wall (intraparietal localization with prolapse into the lumen or extragastral). These data allowed us to preliminarily choose and plan the optimal treatment modality (wedge resection of the stomach was performed in all

When a virtual image is constructed in patients with GC, it is worth pointing out the following. In the cases when exophytic tumor growth (Type 1, according to Bormann's classification) was

graphic examination and by revision of the organs of the abdominal cavity.

invasion of the surrounding organs by the tumor (Figure 4).

178 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

the method and the quality of the image obtained in the process of simulation.

is 91%; and NPV is 91%.

**3.5. Virtual endoscopy**

*3.5.1. Gastric leiomyoma*

patients).

PPV is 100%; and NPV is 77%.

multiplanar reformations.

The high quality of virtual images was also noted in the infiltrative and mixed character of growth, when the tumor was localized in the body of the stomach (five patients). In infiltrative tumor growth with the phenomena of antral stenosis, the presence of fluid (primarily, its amount) in the organ lumen affected the quality of the image, which resulted in the appearance of artifacts in the simulation process (in two of five patients). Note that the mucous membrane in the region of lesion and nearby was characterized by excessive serration, although the circular The high quality of virtual images was also noted in the infiltrative and mixed character of growth, when the tumor was localized in the body of the stomach (five patients). In infiltrative tumor growth with the phenomena of antral stenosis, the presence of fluid (primarily, its amount) in the organ lumen affected the quality of the image, which resulted in the appearance of artifacts in the simulation process (in two of five patients). Note that the mucous membrane in the region of lesion and nearby was characterized by excessive serration, although the circular narrowing of the antral division of the stomach characteristic of such a lesion was also well visualized as in videoendoscopy. The planar slices specified the depth of tumor invasion and the status of regional lymphatic vessels and allowed the detection of remote metastases. Unfortunately, the video sequences obtained during the study were characterized by the monotonousness of color images and did not allow us to pinpoint the areas of necrosis and disintegration, hemorrhage of the tumor surface, as well as perineoplastic and inflammatory mucosal changes. The high level (100%) of detection of a tumor lesion of the organ during virtual simulation in our study is determined by the vast extent of the lesion (large-sized leiomyomas and extended stage II–IV GC) and a good level of inflation of the organ.

#### narrowing of the antral division of the stomach characteristic of such a lesion was **4. Discussion**

also well visualized as in videoendoscopy. The planar slices specified the depth of tumor invasion and the status of regional lymphatic vessels and allowed the Despite rapid development of medical technologies, it is difficult to differentiate between benign and malignant tumors of the stomach. In this connection, the development of new and the perfection of available technologies is an important task. One of the most promising methods for increasing the resolving capacity of endoscopic examination is laser spectro- and

videofluorescence. The procedure of endoscopic fluorescent diagnostics only insignificantly lengthens the EGDS performance time (in contrast to chromoendoscopy), and the conclusion as to the character of a pathological process can be formulated immediately in the process of gastroscopy performance. The equipment complex used now for FD is compact (portable) and mobile enough, which allows it to be delivered to the endoscopy room. No additional time for assembling the complex and preparing it for operation under the endoscopy room conditions is required. The rapid elimination of Alasens from the body decreases the likelihood of the development of toxic and photic reactions and makes it a drug of choice for fluorescent diagnostics. All the above mentioned allows us to recommend laser spectral FD combined with the study of Alasens-induced PPIX, and videofluorescence in the future, to use in the clinical setting as an express method of diagnosis, including the early diagnosis, of malignant gastric tumors.

This study showed that complex transabdominal ultrasonography is a highly informative method of diagnosis of GC. The possibility of detecting GC localized in the pyloroantral division and in the lower third of the stomach body during trans-US performance approaches the absolute values (97.4%). At the same time, in isolated lesions of the cardia and the upper third of the stomach body, we failed to reveal the tumor in any of the 10 patients studied. On the whole, the success rate of revealing GC at all tumor sites using complex trans-US exami‐ nation constituted 87.0%. The high level of detection of the neoplastic process in the stomach in our study can be explained by the fact that we knew exactly where the process was localized in most patients (based on the EGDS and X-ray findings) and targeted at the zone concerned. However, this does not decrease the possibilities of the method but, on the contrary, testifies to its high diagnostic potential. In addition to the changes revealed for the first time in the stomach (in two patients with early cancer), the 'hollow organ' symptom was detected in numerous patients at the outpatient step followed by purpose-oriented examination and verification of the diagnosis with the subsequent referral to the clinic for operative treatment. Our experience shows that routine ultrasonography of the organs of the abdominal cavity should not be confined to the examination of the parenchymatous organs only. What is necessary is target visualization in the region of hollow organs (in particular, the stomach), especially in patients with the phenomena of abdominal discomfort. True, ultrasonography is actually the first and sometimes the only diagnostic procedure for most of such patients.

The studies conducted recently emphasized low diagnostic possibilities of conventional CT investigation in detecting GC, especially at stage T1 when the attempts to reveal any changes in the stomach wall were unsuccessful. On the whole, the accuracy of detecting the depth of invasion in more prevalent forms was not satisfactory either (no more than 65%) [10]. The main limiting factors were artifacts from peristaltic movements of the stomach wall, which did not allow us to assess its condition on standard examination. However, at present, the appearance of dynamic 3D MHCT capable of assessing the stomach wall in all the divisions in the fourdimensional mode has significantly increased the diagnostic possibilities of this technique.

In our study, dynamic 3D CT proved to be a highly revealing technique for GC (sensitivity 97%). Such high values were primarily achieved by virtue of the possibility of evaluating peristaltic disorders of the stomach, which was afforded by this CT technique, in contrast to the standard method relying on measuring only the thickness of the stomach wall. Dynamic MHCT enabled us to reveal seven of nine (85%) cases of early GC owing to the characteristic contrast between the mucous and submucous layers and peristaltic disorders without the stomach wall thickening. But it is fair to say that, at present, MHCT is inferior to the conven‐ tional endoscopic examination in revealing early GC.

The progress in computer technology led to the fact that virtual simulation is finding an ever wider application in different fields of surgery and oncology [6]. Our study demonstrated the possibilities of such simulation as applied to the construction of virtual gastroscopy. A highquality virtual image not inferior to that acquired with conventional videoendoscopy was obtained in the case of intraluminal character of neoplastic growth [7, 9]. As it follows from the literature data, virtual endoscopy is not surpassed by conventional endoscopy in terms of its diagnostic possibilities of tumor detection and is more informative than planar slices, especially when the invasion depth is small (T2 and especially T1). Nevertheless, it is fair to say that now virtual endoscopy cannot be considered a real alternative to conventional videoendoscopy as a method of primary diagnostics of GC. At present, the possibilities of the wide application of the complex of CT techniques for differential diagnosis of benign and malignant ulcers with negative results of biopsy are also doubtful, although the first reports are highly encouraging [11]. In our opinion, the use of endo-ultrasonography and laser fluorescence spectroscopy (the so-called optic biopsy) is more justified for these purposes. Virtual simulation extends the possibilities of the method and allows for a more accurate choice of the target zone for a thorough study of the stomach wall with a view to specifying the depth of malignant invasion. However, the rapid progress of computer technology and software will possibly allow us to consider the complex of CT studies, including those in the virtual endoscopy mode, a screening technique in the countries with a high morbidity rate [7, 9]. At present, MHCT has found wide application in various diseases of the organs of the abdominal cavity, retroperitoneal space, and vertebral column. Therefore, we think that the use of such a method for an additional examination of the stomach in the group at higher risk not only for cancer but also for CT study of the organs of the abdominal cavity for other diseases with a view to detecting changes seems to be interesting and promising.

#### **5. Conclusion**

videofluorescence. The procedure of endoscopic fluorescent diagnostics only insignificantly lengthens the EGDS performance time (in contrast to chromoendoscopy), and the conclusion as to the character of a pathological process can be formulated immediately in the process of gastroscopy performance. The equipment complex used now for FD is compact (portable) and mobile enough, which allows it to be delivered to the endoscopy room. No additional time for assembling the complex and preparing it for operation under the endoscopy room conditions is required. The rapid elimination of Alasens from the body decreases the likelihood of the development of toxic and photic reactions and makes it a drug of choice for fluorescent diagnostics. All the above mentioned allows us to recommend laser spectral FD combined with the study of Alasens-induced PPIX, and videofluorescence in the future, to use in the clinical setting as an express method of diagnosis, including the early diagnosis, of malignant gastric

180 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

This study showed that complex transabdominal ultrasonography is a highly informative method of diagnosis of GC. The possibility of detecting GC localized in the pyloroantral division and in the lower third of the stomach body during trans-US performance approaches the absolute values (97.4%). At the same time, in isolated lesions of the cardia and the upper third of the stomach body, we failed to reveal the tumor in any of the 10 patients studied. On the whole, the success rate of revealing GC at all tumor sites using complex trans-US exami‐ nation constituted 87.0%. The high level of detection of the neoplastic process in the stomach in our study can be explained by the fact that we knew exactly where the process was localized in most patients (based on the EGDS and X-ray findings) and targeted at the zone concerned. However, this does not decrease the possibilities of the method but, on the contrary, testifies to its high diagnostic potential. In addition to the changes revealed for the first time in the stomach (in two patients with early cancer), the 'hollow organ' symptom was detected in numerous patients at the outpatient step followed by purpose-oriented examination and verification of the diagnosis with the subsequent referral to the clinic for operative treatment. Our experience shows that routine ultrasonography of the organs of the abdominal cavity should not be confined to the examination of the parenchymatous organs only. What is necessary is target visualization in the region of hollow organs (in particular, the stomach), especially in patients with the phenomena of abdominal discomfort. True, ultrasonography is actually the first and sometimes the only diagnostic procedure for most of such patients.

The studies conducted recently emphasized low diagnostic possibilities of conventional CT investigation in detecting GC, especially at stage T1 when the attempts to reveal any changes in the stomach wall were unsuccessful. On the whole, the accuracy of detecting the depth of invasion in more prevalent forms was not satisfactory either (no more than 65%) [10]. The main limiting factors were artifacts from peristaltic movements of the stomach wall, which did not allow us to assess its condition on standard examination. However, at present, the appearance of dynamic 3D MHCT capable of assessing the stomach wall in all the divisions in the fourdimensional mode has significantly increased the diagnostic possibilities of this technique.

In our study, dynamic 3D CT proved to be a highly revealing technique for GC (sensitivity 97%). Such high values were primarily achieved by virtue of the possibility of evaluating peristaltic disorders of the stomach, which was afforded by this CT technique, in contrast to

tumors.

Fluorescent diagnostics with the study of Alasens-induced protoporphyrin IX is a highly efficacious, simple, and safe method for diagnosing GC. It can be used as a standard step of endoscopic examination in diagnostically complicated cases. The sensitivity of the method is 96%; its specificity is 98%.

The method for detecting videofluorescence with Alasens is a promising screening technique in patients with GC and in precancerous states.

Trans-ultrasonography is a potentially screening method in distal localization of GC. The possibility of detecting GC localized in the pyloroantral division and in the lower third of the body of the stomach using complex trans-ultrasonography was 95.6% attaining the absolute values in the T3 and T4 groups.

Dynamic MHCT is a highly informative method for cancer detection. The possibility of revealing cancer did not depend on the tumor localization and constituted 97% in the group of patients attaining absolute values, beginning with T2 invasion depth.

Indications for use of virtual endoscopy, one of the MHCT modes, as a method for the primary diagnosis of GC and for screening the disease require further study and specification.

Modern ultrasound and CT technologies allow us not only to stage GC but also to carry out primary diagnostics. However, the technical complexity and the high cost of CT, and the impossibility that cancer of the proximal division of the stomach will be detected ultrasono‐ graphically do not allow us to regard them as full-fledged screening techniques. Laser spectroscopy is indicated only in diagnostically difficult cases, and videofluorescent diagnos‐ tics requires higher performance, after which it may rightly be considered a recognized screening method.

#### **Author details**

Vladimir Levkin1\*, Nina Gagarina1 , Sergey Kharnas1 , Gaziyav Musaev1 , Artem Shiryaev1 and Dmitry Bulgin2


#### **References**


[5] Gorovaya NS. Role of complex US examination in the surgical treatment of gastric cancer patients. Cand. Sci. Dissertation (Med.), Moscow, 2009.

body of the stomach using complex trans-ultrasonography was 95.6% attaining the absolute

Dynamic MHCT is a highly informative method for cancer detection. The possibility of revealing cancer did not depend on the tumor localization and constituted 97% in the group

Indications for use of virtual endoscopy, one of the MHCT modes, as a method for the primary

Modern ultrasound and CT technologies allow us not only to stage GC but also to carry out primary diagnostics. However, the technical complexity and the high cost of CT, and the impossibility that cancer of the proximal division of the stomach will be detected ultrasono‐ graphically do not allow us to regard them as full-fledged screening techniques. Laser spectroscopy is indicated only in diagnostically difficult cases, and videofluorescent diagnos‐ tics requires higher performance, after which it may rightly be considered a recognized

diagnosis of GC and for screening the disease require further study and specification.

, Sergey Kharnas1

[1] Davydov MI & Aksel EM. The incidence of malignant tumors and mortality caused by them in Commonwealth of Independent States in 2005. *Vestn Ross Akad Med Nauk*,

[2] Portnoi LM. Modern X-ray diagnostics in gastroenterology and gastroenterooncolo‐

[3] Polsachev VI. Preoperative fluorescent diagnostics of the prevalence of gastric can‐

[4] Polsachev VI. Fluorescent method in the differential diagnosis of benign and malig‐

, Gaziyav Musaev1

, Artem Shiryaev1

and

of patients attaining absolute values, beginning with T2 invasion depth.

182 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

values in the T3 and T4 groups.

screening method.

**Author details**

Dmitry Bulgin2

**References**

2007,11:45–49.

gy. Moscow, Vidar-M, 2003.

cer. *Vestn Khirurg*, 1989,12:17–19.

Vladimir Levkin1\*, Nina Gagarina1

\*Address all correspondence to: doctor-levkin@mail.ru

1 Sechenov First Moscow State Medical University, Moscow, Russia

nant diseases of the stomach. *Vestn Khirurg*, 1992,1:95–97.

2 Center for Regenerative Medicine "ME-DENT", Rovinj, Croatia


### *Edited by Dmitry Bulgin*

Written by an international team of experts in the field of human carcinogenesis, this book discusses recent advances in cancer research, which include the following topics: basic molecular and cellular mechanisms behind cancer growth, new approaches in cancer therapy, and cancer diagnostic. The book serves as a useful source of reference for cancer biologists, medical doctors, and clinical researchers in the fields of cancer diagnosis, prevention, and treatment.

New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

New Aspects in Molecular

and Cellular Mechanisms of

Human Carcinogenesis

*Edited by Dmitry Bulgin*

Photo by Asergieiev / iStock