**The Role of Haptoglobin and Its Genetic Polymorphism in Cancer: A Review**

Maria Clara Bicho, Alda Pereira da Silva, Rui Medeiros and Manuel Bicho

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56695

## **1. Introduction**

thrombopoiesis through thrombopoietin: role in inflammatory thrombocytosis.

[70] Klinger MH, Jelkmann W. Role of blood platelets in infection and inflammation. J In‐

[71] Müller F, Mutch NJ, Schenk WA, Smith SA, Esterl L, Spronk HM, Schmidbauer S, Gahl WA, Morrissey JH, Renné T. Platelet polyphosphates are proinflammatory and

[72] Morrissey JH, Choi SH, Smith SA. Polyphosphate: an ancient molecule that links pla‐

[73] Caudrillier A, Kessenbrock K, Gilliss BM, Nguyen JX, Marques MB, Monestier M, Toy P, Werb Z, Looney MR. Platelets induce neutrophil extracellular traps in transfu‐

[74] Oikonomopoulou K, Ricklin D, Ward PA, Lambris JD. Interactions between coagula‐ tion and complement--their role in inflammation.Semin Immunopathol.

[75] Markiewski MM, Nilsson B, Ekdahl KN, Mollnes TE, Lambris JD. Complement and coagulation: strangers or partners in crime?Trends Immunol. 2007;28:184-192.

[76] Krisinger MJ, Goebeler V, Lu Z, Meixner SC, Myles T, Pryzdial EL, Conway EM.Thrombin generates previously unidentified C5 products that support the termi‐

Blood. 2001;98:2720-2725.

54 Acute Phase Proteins

2012;34:151-165.

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telets, coagulation, and inflammation. Blood. 2012;119:5972-5979

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nal complement activation pathway. Blood. 2012;120:1717-1725.

#### **1.1. The acute phase response (APR) and haptoglobin (Hp)**

Acute phase response is a stereotyped innate nonspecific reaction of the body proceeding specific immune reactions. It´s a systemic homeostatic reaction of the organism to local and or systemic disturbances caused by infections, tissue injury, trauma, immunologic disorders and neoplasias (Ron D *et al* 1990, Trautwein C *et al* 1994, Gruys E *et al* 2005). Proinflammatory cytokines are released at the place of tissue injury, diffuses locally and systemically to the vascular system and activates receptors on different target cells resulting in the activation of hypothalamic-pituitary-adrenal axis (HPAA), results in the production of growth hormone secretion and induces changes in the concentration of several plasma proteins (Ron D *et al* 1990, Trautwein C *et al* 1994, Gruys E *et al* 2005).

These acute phase proteins (APPs) can be positive (higher levels in plasma) or negative (lower levels in plasma). The alteration on mRNA in hepatocytes is due to simultaneous influence of systemic cytokines (IL1, IL6 and TNFα), glucocorticoids and catecholamines (Bowman BH 1993, Ron D *et al* 1990, Trautwein C *et al* 1994).

Haptoglobin together with fibrinogen, α-globulins with antiprotease-activity and lipopoly‐ saccharide binding protein belong to the group of positive APPs that increase 3-fold in mammals (Trautwein C *et al* 1994, Gruys E *et al* 2005).

Haptoglobin (Hp) is an acute phase α2 plasma glycoprotein that is a component of innate immunity, which also may influence acquired immunity. Through both types of immunity,

Hp is involved in the pathogenesis of tumours and infections (Langlois MR and Delanghe JR 1996, Van Vlierberghe H. *et al* 2004, Levy AP *et al* 2010).

ethnic groups) and Hp1 allele is more predominant in populations subjected to malaria burden (Giblett ER 1968, Langlois MR and Delanghe JR 1996, Wobeto VP et al 2008, Levy AP et al

The Role of Haptoglobin and Its Genetic Polymorphism in Cancer: A Review

http://dx.doi.org/10.5772/56695

57

In close linkage with haptoglobin gene there is another one, 2-2 Kb downstream from Hp locus, coding for Hp related (Hpr) plasma protein with 91% sequence identity to Hp1. The α-chain of Hpr contains a hydrophobic signal peptide that may explain its association to lipoprotein

The Hp gene is expressed primarily in hepatocytes and more recently has been described in other locations, such as keratinocytes, airway epithelial cells of lung, leucocytes, fibrocytes, adipocytes and endometrial cells, particularly during the blastocyst implantation (Friederichs WE *et al* 1995, Olson EG *et al* 1997 Wang *et al* 2005, Shaw JL *et al* 2007, Yang F 2000 *et al*, Larsen

Haptoglobin synthesis is induced by cytokines such as interleukin-6 (IL-6), interleukin-1 (IL-1) and tumour necrosis factor (TNFα) released by the macrophages, after activation of the innate immunity cells by PAMPs (pathogen associated molecular patterns) such as lipopolysacchar‐ ide, a TLR4 (Toll Like Receptor) activator (Raynes JG et al 1991, Kaisho T and Alkira S 2002). Glucocorticoids and catecholamines activate haptoglobin synthesis previously induced by interleukins (increased), whereas insulin exerts an opposite action, despite the presence of these interleukins (Ron D et al 1990, Campos SP and Baumann H 1992 Nascimento CO et al 2004, Gruys E et al 2005 and XiaLi-xin et al 2008). Hypoxia also induces indirectly its synthesis

**4. Haptoglobin metabolism, actions and respective mechanisms**

Haptoglobin has a pronounced anti-inflammatory action, which is explained by its ability to bind to heme of haemoglobin, forming a Hp-Hb complex. This is characterized by stability and high affinity to its specific type scavenger receptor (CD163) located in the hepatocyte and the phagocytic-type cells such as circulating monocytes, resident macrophages (M2) and liver Kupffer cells. The CD163 is a membrane protein 130-kDa, whose long extracellular region has nine cysteine-rich domains of scavenger-type receptor (Graversen JH *et al* 2002, Nielsen MJ *et al* 2010, Akila P *et al* 2012). The expression of receptors (CD 163), Hp and hemoxygenase (HO-1), is strongly activated by antinflammatory cytokines, such as interleukins (IL6, IL10), growth factors (M-CSF) and glucocorticoids (Moestrup SK and Møller H 2004). In contrast CD 163 is down regulated by IL4 and GM-CSF, Interferon γ and TNF (Nielsen MJ *et al* 2010, Vallelian F

After binding to its receptor the Hp-Hb complex is internalized in the form of endosome, followed by fusion with lysosomes, proteolysis of globin and intracellular release of heme to

particles (HDL) or membranes (Kuhajda FP *et al* 1989 a, b).

K *et al* 2006, and Theilgaard-Mönch K *et al* 2006).

(Wenger RH et al 1995, Oh Mi-Kyung et al 2011).

*et al* 2011 and Akila P *et al* 2012).

**3. Haptoglobin locals of its synthesis and regulation**

2010).

## **2. Haptoglobin (Hp) synthesis, gene structure, variants and its geographic distribution**

Haptoglobin locus is on chromosome 16q22 and its gene is transcribed and translated into a single peptide which undergoes post-translational processing resulting in a smaller α-chain and a longer β-chain linked by disulphide bridge (Giblett ER 1968, Langlois MR and Delanghe JR 1996, Wicher KB and Fries E 2007).

In 1955, Smithies, using thin layer starch gel electrophoresis identified the three phenotypes of Hp (1-1, 2-1, 2-2), corresponding to the α-chain length interindividual genetic variation.

The three genotypes are shown in electrophoresis in polyacrylamide gel electrophoresis (PAGE) (fig 1).

This genetic variation results from an internal duplication of a gene segment (exons 3 and 4), correspondent to α-chain of Hp1 giving rise to a larger one, characteristic of Hp2 (Maeda *et al* 1984, Wicher KB and Fries E 2007).

**Figure 1.** Typical pattern of haptoglobin bands in a polyacrylamide gel electrophoresis (PAGE). Shown are the pheno‐ types: Hp 1-1, is characterized by a fast migration band; Hp 2-2 is characterized by slower multiple bands; Hp 2-1, characterized by a mixed pattern of two allelic forms. The ultrafast bands are no haptoglobin bound haemoglobin chains (Linke RP 1984, Guerra J *et al* 1997).

This inter-individual variation is found only in humans and aroused about 100,000 years ago in Southeast Asia. The great majority of other mammals have only one band corresponding to the human Hp1-1, except the sheep, deer and cows (Ruminantia), which have only slow bands corresponding to Hp2-2 (Bowman BH and Kurosky A 1982, Wicher KB and Fries 2007).

The appearance of Hp2 can represent an important evolutionary genetic contribution for interpopulational diversity in human pathology (ER Giblett 1968, Maeda et al 1984, Wicher KB and Fries 2007). This allele is predominant in the human population (about 80% in some

4

ethnic groups) and Hp1 allele is more predominant in populations subjected to malaria burden (Giblett ER 1968, Langlois MR and Delanghe JR 1996, Wobeto VP et al 2008, Levy AP et al 2010).

In close linkage with haptoglobin gene there is another one, 2-2 Kb downstream from Hp locus, coding for Hp related (Hpr) plasma protein with 91% sequence identity to Hp1. The α-chain of Hpr contains a hydrophobic signal peptide that may explain its association to lipoprotein particles (HDL) or membranes (Kuhajda FP *et al* 1989 a, b).

## **3. Haptoglobin locals of its synthesis and regulation**

Hp is involved in the pathogenesis of tumours and infections (Langlois MR and Delanghe JR

**2. Haptoglobin (Hp) synthesis, gene structure, variants and its geographic**

Haptoglobin locus is on chromosome 16q22 and its gene is transcribed and translated into a single peptide which undergoes post-translational processing resulting in a smaller α-chain and a longer β-chain linked by disulphide bridge (Giblett ER 1968, Langlois MR and Delanghe

In 1955, Smithies, using thin layer starch gel electrophoresis identified the three phenotypes of Hp (1-1, 2-1, 2-2), corresponding to the α-chain length interindividual genetic variation. The three genotypes are shown in electrophoresis in polyacrylamide gel electrophoresis

This genetic variation results from an internal duplication of a gene segment (exons 3 and 4), correspondent to α-chain of Hp1 giving rise to a larger one, characteristic of Hp2 (Maeda *et*

Free Haemoglobin (Hb)

Hp α 2 –Hb polymers

Hp α 1 -Hb

**Figure 1.** Typical pattern of haptoglobin bands in a polyacrylamide gel electrophoresis (PAGE). Shown are the pheno‐ types: Hp 1-1, is characterized by a fast migration band; Hp 2-2 is characterized by slower multiple bands; Hp 2-1, characterized by a mixed pattern of two allelic forms. The ultrafast bands are no haptoglobin bound haemoglobin

This inter-individual variation is found only in humans and aroused about 100,000 years ago in Southeast Asia. The great majority of other mammals have only one band corresponding to the human Hp1-1, except the sheep, deer and cows (Ruminantia), which have only slow bands corresponding to Hp2-2 (Bowman BH and Kurosky A 1982, Wicher KB and Fries 2007).

The appearance of Hp2 can represent an important evolutionary genetic contribution for interpopulational diversity in human pathology (ER Giblett 1968, Maeda et al 1984, Wicher KB and Fries 2007). This allele is predominant in the human population (about 80% in some

4

1996, Van Vlierberghe H. *et al* 2004, Levy AP *et al* 2010).

**distribution**

56 Acute Phase Proteins

(PAGE) (fig 1).

JR 1996, Wicher KB and Fries E 2007).

*al* 1984, Wicher KB and Fries E 2007).

chains (Linke RP 1984, Guerra J *et al* 1997).

The Hp gene is expressed primarily in hepatocytes and more recently has been described in other locations, such as keratinocytes, airway epithelial cells of lung, leucocytes, fibrocytes, adipocytes and endometrial cells, particularly during the blastocyst implantation (Friederichs WE *et al* 1995, Olson EG *et al* 1997 Wang *et al* 2005, Shaw JL *et al* 2007, Yang F 2000 *et al*, Larsen K *et al* 2006, and Theilgaard-Mönch K *et al* 2006).

Haptoglobin synthesis is induced by cytokines such as interleukin-6 (IL-6), interleukin-1 (IL-1) and tumour necrosis factor (TNFα) released by the macrophages, after activation of the innate immunity cells by PAMPs (pathogen associated molecular patterns) such as lipopolysacchar‐ ide, a TLR4 (Toll Like Receptor) activator (Raynes JG et al 1991, Kaisho T and Alkira S 2002).

Glucocorticoids and catecholamines activate haptoglobin synthesis previously induced by interleukins (increased), whereas insulin exerts an opposite action, despite the presence of these interleukins (Ron D et al 1990, Campos SP and Baumann H 1992 Nascimento CO et al 2004, Gruys E et al 2005 and XiaLi-xin et al 2008). Hypoxia also induces indirectly its synthesis (Wenger RH et al 1995, Oh Mi-Kyung et al 2011).

## **4. Haptoglobin metabolism, actions and respective mechanisms**

Haptoglobin has a pronounced anti-inflammatory action, which is explained by its ability to bind to heme of haemoglobin, forming a Hp-Hb complex. This is characterized by stability and high affinity to its specific type scavenger receptor (CD163) located in the hepatocyte and the phagocytic-type cells such as circulating monocytes, resident macrophages (M2) and liver Kupffer cells. The CD163 is a membrane protein 130-kDa, whose long extracellular region has nine cysteine-rich domains of scavenger-type receptor (Graversen JH *et al* 2002, Nielsen MJ *et al* 2010, Akila P *et al* 2012). The expression of receptors (CD 163), Hp and hemoxygenase (HO-1), is strongly activated by antinflammatory cytokines, such as interleukins (IL6, IL10), growth factors (M-CSF) and glucocorticoids (Moestrup SK and Møller H 2004). In contrast CD 163 is down regulated by IL4 and GM-CSF, Interferon γ and TNF (Nielsen MJ *et al* 2010, Vallelian F *et al* 2011 and Akila P *et al* 2012).

After binding to its receptor the Hp-Hb complex is internalized in the form of endosome, followed by fusion with lysosomes, proteolysis of globin and intracellular release of heme to hemoxygenase (HO-1) with concomitant formation of biliverdin that is converted in bilirrubin, CO (carbon monoxide) and release of iron to ferritin where is compartmentalised (Graversen JH *et al* 2002, Nielsen MJ *et al* 2010, Vallelian F *et al* 2011, Akila P *et al* 2012).

The Hp-Hb complex also binds nitric oxide or nitrogen monoxide (NO), produced by cytokine activated macrophages, thus preventing their physiological and pathological actions (Langlois MR and Delanghe JR 1996, Azarov I *et al* 2008, Alayash A 2011). Also this action is phenotype dependent, because Hp2-2/Hb complex scavenge more NO than Hp1-1/Hb due to its longer

The Role of Haptoglobin and Its Genetic Polymorphism in Cancer: A Review

http://dx.doi.org/10.5772/56695

59

The Hp is also a potent endogenous inhibitor of prostaglandin synthesis, resulting in antiinflammatory action. The inhibitory effects of Hp2-2 and Hp2-1 are less pronounced than those of Hp1-1 (Kendall PA *et al* 1979, Langlois MR and Delanghe JR 1996, Saeed SA *et al* 2007). Haptoglobin has also bacteriostatic effects, because the capture and compartmentalization of the iron of Hb made it no longer available for bacterial growth. The Hp 2-2 is more efficient than the other phenotypes in this action against S*treptococcus*. There are also microorganisms that can remove iron from the Hp-Hb complex (Langlois MR and Delanghe JR 1996, Weinberg

The role of Hp in angiogenesis has been identified as one of the factors for modulation of differentiation and proliferation of endothelial cells during the formation of new vessels (Cid MC *et al* 1993, Park SJ 2009). Free Hb can promote indirectly carcinogenesis through the iron that is necessary for cell growth. The withholding of iron inhibits cell growth and depresses

The local increased concentration of Hp in chronic inflammatory processes is important for the ischemic tissue reparation, promoting collateral vessel formation. Of the three genetic

In resident tissues macrophages (M2 type), carbon monoxide (CO) resulting from the intra‐ cellular degradation Hp-Hb complex appears to be involved in anti-inflammatory effects of interleukin 10 (IL-10). The suppression of these immune and inflammatory responses results from its ability to decrease the antigen presentation and cytokine synthesis. This mechanism of regulation is more active in patients with the Hp1-1 phenotype that has a greater clearance of their complexes with their CD163 receptors present on monocytes, than for those carrying the phenotype Hp2-2 (Nielsen MJ *et al* 2010, Vallelian F *et al* 2011 and Akila P *et al* 2012).

In macrophages, after the endocytosis of the Hp-Hb complex and CD163, increased levels of cytoplasmic iron ocurr, inducing the synthesis of ferritin, a primary iron storage, which can subtract it from inflammation site (Cozzi *et al* 2004). The activation of the CD 163 also induces a signal mediated by protein tyrosine kinase, leading to the secretion of anti-inflammatory cytokines and giving rise to a connection between the clearance function of the Hp and their immunomodulatory functions (Van Vlierberghe *et al* 2004, Guetta *et al* 2007, Nielsen MJ *et al*

Haptoglobin can also modulate the immune response by binding to receptors on immune cells, such as CD22 on B lymphocytes and β2 integrin (CD11b/CD18) in neutrophils or LFA-1 (lymphocyte function associated antigen-1) in T lymphocytes (EL Ghmati SM *et al* 1996, Giannoni E *et al* 2003, Bottini N *et al* 2005). The Hp may bind to neutrophils, inhibiting NADPH oxidase activation and the production of reactive forms of oxygen associated with inflamma‐

the immune system (Langlois MR and Delanghe JR 1996, Weinberg ED 1996).

forms Hp2.2 is the most angiogenic (Cid MC *et al* 1993).

2010, Vallelian F *et al* 2011 and Akila P *et al* 2012).

tion (Moestrup SK and Møller H, 2004, Guetta *et al* 2007).

half-life (Azarov I *et al* 2008, Levy AP *et al* 2010, Alayash A 2011).

ED 1996, Van Vlierberghe *et al* 2004).

The small protein Hp1-1 is excreted in the urine when occurs kidney damage, however, the Hp2-1 and Hp2-2 are always retained (Fagoonee S 2005). The clearance of free haemoglobin (Hb) after intravascular haemolysis by the haptoglobin is higher in individuals carrying the Hp1 allele (Giblett ER 1968, Langlois MR and Delanghe JR 1996, Moestrup SK and Møller H 2004, Nielsen MJ *et al* 2010, Vallelian F *et al* 2011, Akila P *et al* 2012).

The free Hb has the ability to catalyse the formation of hydroxyl radicals (OH·), from the hydrogen peroxide, with highly damaging effects to the cellular constituents and extracellular macromolecules (Sadrzadeh SMH 1984, Gutteridge JMC 1987).

The Hp-Hb complex, reduces the loss of Hb in urine and concomitant loss of iron and its transport is done mainly to the liver. As a result, the removal of free Hb has much important consequences for the organisms, preventing renal injury that may occur when the free Hb passes through the glomerular filter (Fagoonee S *et al* 2005). Also Hp prevents the promotion of free radicals and its accumulation in endothelial cells, catalysed by heme, where it causes vessel injury (Nielsen MJ *et al* 2010, Vallelian F *et al* 2011 and Akila P *et al* 2012). However, there is a great variability in these responses, which is dependent of Hp polymorphism having individuals with the Hp2-2 a lower antioxidant capacity than those with other phenotypes. Furthermore at the extra-vascular interstitial level, the antioxidant capacity of carriers of Hp2-2 is lower, because of its higher molecular mass that restricts its extravascular diffusion (Langlois MR and Delanghe JR 1996, Van Vierberghe *et al* 2004, Fagoonee S. *et al* 2005, Levy AP *et al* 2010).

Levels of haptoglobin in plasma or serum are lower in healthy infants than adults whose concentrations are between 0.38 and 2.08g/l (Langlois MR and Delanghe JR 1996). These steady state levels are consequence of haptoglobin half-life of 3.5 days and Hp-Hb complex of ten minutes (Sadrzadeh SMH and Bozorgmehr J 2004). The Hp can also be detected in urine and other organic fluids (Langlois MR and Delanghe JR 1996, Sadrzadeh SMH and Bozorgmehr J 2004). The half-life of Hp-Hb complex is phenotype dependent being Hp1-1 shorter than Hp2-2 (Levy AP *et al* 2010). Plasma concentrations are also phenotype dependent, people with Hp1-1 having the highest, Hp2-1 intermediate and Hp2-2 lesser concentrations in plasma (Langlois MR and Delanghe JR 1996).

Haptoglobin levels are quantified by chemical and immunochemical methods, from these the most utilised are the immunonephelometric and immunoturbidimetric methods that are automated (Langlois MR and Delanghe JR 1996, Sadrzadeh SMH and Bozorgmehr J 2004).

The haptoglobin polymorphism is most commonly determined by starch or polyacrylamide electrophoresis (Fig 1). When plasma levels are lower than 0.10g/l PCR based assays are utilised (Linke RP 1984, Langlois MR and Delanghe JR 1996, Guerra A *et at* 1997, Levy AP *et al* 2010). More recently in both, levels measurement and phenotype, are utilised new proteomic methods based on two dimensional gel electrophoresis and quantitative determination by mass spectrometry (MALDI-TOF-MS and SELDI-TOF-MS) methods (Gast M-C *et al* 2008, Chen C-B *et al* 2008).

The Hp-Hb complex also binds nitric oxide or nitrogen monoxide (NO), produced by cytokine activated macrophages, thus preventing their physiological and pathological actions (Langlois MR and Delanghe JR 1996, Azarov I *et al* 2008, Alayash A 2011). Also this action is phenotype dependent, because Hp2-2/Hb complex scavenge more NO than Hp1-1/Hb due to its longer half-life (Azarov I *et al* 2008, Levy AP *et al* 2010, Alayash A 2011).

hemoxygenase (HO-1) with concomitant formation of biliverdin that is converted in bilirrubin, CO (carbon monoxide) and release of iron to ferritin where is compartmentalised (Graversen

The small protein Hp1-1 is excreted in the urine when occurs kidney damage, however, the Hp2-1 and Hp2-2 are always retained (Fagoonee S 2005). The clearance of free haemoglobin (Hb) after intravascular haemolysis by the haptoglobin is higher in individuals carrying the Hp1 allele (Giblett ER 1968, Langlois MR and Delanghe JR 1996, Moestrup SK and Møller H

The free Hb has the ability to catalyse the formation of hydroxyl radicals (OH·), from the hydrogen peroxide, with highly damaging effects to the cellular constituents and extracellular

The Hp-Hb complex, reduces the loss of Hb in urine and concomitant loss of iron and its transport is done mainly to the liver. As a result, the removal of free Hb has much important consequences for the organisms, preventing renal injury that may occur when the free Hb passes through the glomerular filter (Fagoonee S *et al* 2005). Also Hp prevents the promotion of free radicals and its accumulation in endothelial cells, catalysed by heme, where it causes vessel injury (Nielsen MJ *et al* 2010, Vallelian F *et al* 2011 and Akila P *et al* 2012). However, there is a great variability in these responses, which is dependent of Hp polymorphism having individuals with the Hp2-2 a lower antioxidant capacity than those with other phenotypes. Furthermore at the extra-vascular interstitial level, the antioxidant capacity of carriers of Hp2-2 is lower, because of its higher molecular mass that restricts its extravascular diffusion (Langlois MR and Delanghe JR 1996, Van Vierberghe *et al* 2004, Fagoonee S. *et al* 2005, Levy AP *et al* 2010). Levels of haptoglobin in plasma or serum are lower in healthy infants than adults whose concentrations are between 0.38 and 2.08g/l (Langlois MR and Delanghe JR 1996). These steady state levels are consequence of haptoglobin half-life of 3.5 days and Hp-Hb complex of ten minutes (Sadrzadeh SMH and Bozorgmehr J 2004). The Hp can also be detected in urine and other organic fluids (Langlois MR and Delanghe JR 1996, Sadrzadeh SMH and Bozorgmehr J 2004). The half-life of Hp-Hb complex is phenotype dependent being Hp1-1 shorter than Hp2-2 (Levy AP *et al* 2010). Plasma concentrations are also phenotype dependent, people with Hp1-1 having the highest, Hp2-1 intermediate and Hp2-2 lesser concentrations in plasma (Langlois

Haptoglobin levels are quantified by chemical and immunochemical methods, from these the most utilised are the immunonephelometric and immunoturbidimetric methods that are automated (Langlois MR and Delanghe JR 1996, Sadrzadeh SMH and Bozorgmehr J 2004). The haptoglobin polymorphism is most commonly determined by starch or polyacrylamide electrophoresis (Fig 1). When plasma levels are lower than 0.10g/l PCR based assays are utilised (Linke RP 1984, Langlois MR and Delanghe JR 1996, Guerra A *et at* 1997, Levy AP *et al* 2010). More recently in both, levels measurement and phenotype, are utilised new proteomic methods based on two dimensional gel electrophoresis and quantitative determination by mass spectrometry (MALDI-TOF-MS and SELDI-TOF-MS) methods (Gast M-C *et al* 2008, Chen

JH *et al* 2002, Nielsen MJ *et al* 2010, Vallelian F *et al* 2011, Akila P *et al* 2012).

2004, Nielsen MJ *et al* 2010, Vallelian F *et al* 2011, Akila P *et al* 2012).

macromolecules (Sadrzadeh SMH 1984, Gutteridge JMC 1987).

MR and Delanghe JR 1996).

58 Acute Phase Proteins

C-B *et al* 2008).

The Hp is also a potent endogenous inhibitor of prostaglandin synthesis, resulting in antiinflammatory action. The inhibitory effects of Hp2-2 and Hp2-1 are less pronounced than those of Hp1-1 (Kendall PA *et al* 1979, Langlois MR and Delanghe JR 1996, Saeed SA *et al* 2007).

Haptoglobin has also bacteriostatic effects, because the capture and compartmentalization of the iron of Hb made it no longer available for bacterial growth. The Hp 2-2 is more efficient than the other phenotypes in this action against S*treptococcus*. There are also microorganisms that can remove iron from the Hp-Hb complex (Langlois MR and Delanghe JR 1996, Weinberg ED 1996, Van Vlierberghe *et al* 2004).

The role of Hp in angiogenesis has been identified as one of the factors for modulation of differentiation and proliferation of endothelial cells during the formation of new vessels (Cid MC *et al* 1993, Park SJ 2009). Free Hb can promote indirectly carcinogenesis through the iron that is necessary for cell growth. The withholding of iron inhibits cell growth and depresses the immune system (Langlois MR and Delanghe JR 1996, Weinberg ED 1996).

The local increased concentration of Hp in chronic inflammatory processes is important for the ischemic tissue reparation, promoting collateral vessel formation. Of the three genetic forms Hp2.2 is the most angiogenic (Cid MC *et al* 1993).

In resident tissues macrophages (M2 type), carbon monoxide (CO) resulting from the intra‐ cellular degradation Hp-Hb complex appears to be involved in anti-inflammatory effects of interleukin 10 (IL-10). The suppression of these immune and inflammatory responses results from its ability to decrease the antigen presentation and cytokine synthesis. This mechanism of regulation is more active in patients with the Hp1-1 phenotype that has a greater clearance of their complexes with their CD163 receptors present on monocytes, than for those carrying the phenotype Hp2-2 (Nielsen MJ *et al* 2010, Vallelian F *et al* 2011 and Akila P *et al* 2012).

In macrophages, after the endocytosis of the Hp-Hb complex and CD163, increased levels of cytoplasmic iron ocurr, inducing the synthesis of ferritin, a primary iron storage, which can subtract it from inflammation site (Cozzi *et al* 2004). The activation of the CD 163 also induces a signal mediated by protein tyrosine kinase, leading to the secretion of anti-inflammatory cytokines and giving rise to a connection between the clearance function of the Hp and their immunomodulatory functions (Van Vlierberghe *et al* 2004, Guetta *et al* 2007, Nielsen MJ *et al* 2010, Vallelian F *et al* 2011 and Akila P *et al* 2012).

Haptoglobin can also modulate the immune response by binding to receptors on immune cells, such as CD22 on B lymphocytes and β2 integrin (CD11b/CD18) in neutrophils or LFA-1 (lymphocyte function associated antigen-1) in T lymphocytes (EL Ghmati SM *et al* 1996, Giannoni E *et al* 2003, Bottini N *et al* 2005). The Hp may bind to neutrophils, inhibiting NADPH oxidase activation and the production of reactive forms of oxygen associated with inflamma‐ tion (Moestrup SK and Møller H, 2004, Guetta *et al* 2007).

Changes of the ratio of lymphocytes Th1 and Th2 are important for the determination of susceptibility to viral and parasitic infections, for allergies, for antitumor responses and autoimmunity (Gleeson ME 2006, Clerici M *et al* 1998). It was shown that Hp plays a modu‐ lating role of the Th1/Th2 ratio, promoting a Th2 dominant response, which is more pro‐ nounced in patients with the Hp1-1 and 2-1 phenotypes (Bottini N *et al* 2005, Guetta *et al* 2007).

**The objective** of this chapter is to review the scientific evidence of haptoglobin role, as an immune innate protein in the several aspects of cancer biology and its possible clinical importance as a genetic and a circulating biomarker for that pathology.

**The methodology** for this review is based in the search in the literature of relevant studies in cancer concerning both the circulating levels of haptoglobin (including the recent described fucosylated glicans), haptoglobin related (Hpr) and the genetic variation studies, in the Medline Data Bases and the related papers, since the first reports in the sixties of the last century until actuality. A special attention will be a consideration of cancer associated with human papillomavirus (HPV). The keywords used in the search will be haptoglobin, cancer genetics, circulating levels and clinics.

## **5. Haptoglobin (Hp) and its related pathway as biomarkers in cancer**

Genetic polymorphism of haptoglobin leads to its functional differences resulting in interin‐ dividual variation of the related intermediate phenotypes at the different biological levels that can constitute circulating biological markers of clinical importance not only for the suscepti‐ bility but also for the prognostic and response to treatment at the diverse levels of natural history of the neoplasia disease (Bicho MC 2011). We will review by organs and systems the studies that evidence those aspects.

In table 1, we describe the association of Hp polymorphism in several populations with CNS head and neck, lung, blood and skin malignancies.

For the central nervous system it was demonstrated that haptoglobin is transcribed and expressed (proteomic methods) in human glioblastome cells and it is significantly associated with greater plasmatic levels in the higher grades compared with lower ones and those of control subjects (Sanchez DJ *et al* 2001, Kumar DM *et al* 2010). Also it was demonstrated that Hp increases in vitro glioblastome cell migration (Kumar DM *et al* 2010), table 1.

Hp1 allele is more frequent in women with adenocarcinoma (Beckman G *et al* 1986). More recently other group confirmed the association of local higher levels of the Hp l expression in pulmonary adenocarcinomas in opposition to squamous cell carcinomas (SCC) and small cell

Abbreviations: ALL-Acute lymphatic leukaemia; AML- Acute myeloid leukaemia; CML- Chronic myeloid leukaemia; CLL-

Kaposi´s sarcoma Hp1.1 phenotype more prone Speeckaert R *et al* 2011

**Conclusions References**

The Role of Haptoglobin and Its Genetic Polymorphism in Cancer: A Review

N=135/163 Hp2 allele tumour volume dependency Chen C-B *et al* 2008

serology Speeckaert R *et al*<sup>2009</sup>

Israel Associated Hp1-1 with ALL, AML, CML Nevo S and Tatarsky I 1986

Brazil Campregher PV *et al*<sup>2004</sup>

in kidney transplanted patients Speeckaert R *et al*<sup>2012</sup>

Hp1-1 and Hp 2-1 less prone to positive EBV

Sanchez DJ *et al* 2001; Kumar DM *et al* 2010

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61

Lee CC *et al* 2009

Beckman G *et al* 1986

Mitchell RJ *et al* 1988 Germinis A et al 1983

U 1988

Fröhlander N and Stendahl

There are five references for blood malignancies such as acute and chronic lymphoid leukemia and acute myeloid leukemia from three different ethnic groups Sweden, Israel and Brazil (Caucasians and Afro-descendants) and only one sample of Ashkenazy Jews (Germinis A *et al* 1983, Nevo S and Tatarsky I 1986, Mitchell RJ *et al* 1988, Fröhlander N and Stendahl U 1988,

Cutaneous malignancies and in particular squamous cell carcinoma (SCC)/Bowen´s disease are more frequent in kidney transplanted patients, that are more prone to disease when are

carcinomas (Abdullah M 2009).

Chronic lymphatic leukaemia; ML- Myeloma.

**Table 1.** Haptoglobin and Cancer: Various Tumours.

**Neoplasia Population (N)**

Human glioblastome

Head/neck squamous

cell cancer

Nasopharynx carcinoma

Acute lymphoid leukemia

Leukemias

Acute myeloid leukemia

Squamous cell

**(Control/Neo)**

N=26/96

N=134/49 Taiwan

N=918/208 Belgium

N=2331/110 Sweden

N= 211

N=197/188

ALL, AML, CML, IgA ML N=134 Australia Higher Hp 1-1 association

India Hp2 allele higher grades

Lung cancer N=309 Sweden Hp1 allele more frequent in adenocarcinoma in females

carcinoma (SCC) N=300 Belgium Hp phenotype 1.1 more prone to develop SCC

No association

Campregher PV *et al* 2004).

Head and neck squamous cell cancer (HNSCC) is a term that collectively refers to cancer of oral cavity, salivary glands, larynx and pharynx. After a first study the authors whose objective is discovery of circulating biomarkers associated with those tumours, demonstrate in HNSCC in general and nasopharynx in particular, the haptoglobin overexpression, in a stage and tumour volume dependency (Chen C-B *et al* 2008, Lee CC *et al* 2009). Another group confirmed the involvement of the Hp phenotype in infection with Epstein-Barr virus (EBV) that is associated with nasopharynx carcinoma (Speeckaert R *et al* 2009).

In the eighties several studies of association with cancer of acute phase proteins in particular haptoglobin were done, that is the case for the lung cancer in 309 Swedish patients where the


Abbreviations: ALL-Acute lymphatic leukaemia; AML- Acute myeloid leukaemia; CML- Chronic myeloid leukaemia; CLL-Chronic lymphatic leukaemia; ML- Myeloma.

**Table 1.** Haptoglobin and Cancer: Various Tumours.

Changes of the ratio of lymphocytes Th1 and Th2 are important for the determination of susceptibility to viral and parasitic infections, for allergies, for antitumor responses and autoimmunity (Gleeson ME 2006, Clerici M *et al* 1998). It was shown that Hp plays a modu‐ lating role of the Th1/Th2 ratio, promoting a Th2 dominant response, which is more pro‐ nounced in patients with the Hp1-1 and 2-1 phenotypes (Bottini N *et al* 2005, Guetta *et al* 2007). **The objective** of this chapter is to review the scientific evidence of haptoglobin role, as an immune innate protein in the several aspects of cancer biology and its possible clinical

**The methodology** for this review is based in the search in the literature of relevant studies in cancer concerning both the circulating levels of haptoglobin (including the recent described fucosylated glicans), haptoglobin related (Hpr) and the genetic variation studies, in the Medline Data Bases and the related papers, since the first reports in the sixties of the last century until actuality. A special attention will be a consideration of cancer associated with human papillomavirus (HPV). The keywords used in the search will be haptoglobin, cancer genetics,

**5. Haptoglobin (Hp) and its related pathway as biomarkers in cancer**

Genetic polymorphism of haptoglobin leads to its functional differences resulting in interin‐ dividual variation of the related intermediate phenotypes at the different biological levels that can constitute circulating biological markers of clinical importance not only for the suscepti‐ bility but also for the prognostic and response to treatment at the diverse levels of natural history of the neoplasia disease (Bicho MC 2011). We will review by organs and systems the

In table 1, we describe the association of Hp polymorphism in several populations with CNS

For the central nervous system it was demonstrated that haptoglobin is transcribed and expressed (proteomic methods) in human glioblastome cells and it is significantly associated with greater plasmatic levels in the higher grades compared with lower ones and those of control subjects (Sanchez DJ *et al* 2001, Kumar DM *et al* 2010). Also it was demonstrated that

Head and neck squamous cell cancer (HNSCC) is a term that collectively refers to cancer of oral cavity, salivary glands, larynx and pharynx. After a first study the authors whose objective is discovery of circulating biomarkers associated with those tumours, demonstrate in HNSCC in general and nasopharynx in particular, the haptoglobin overexpression, in a stage and tumour volume dependency (Chen C-B *et al* 2008, Lee CC *et al* 2009). Another group confirmed the involvement of the Hp phenotype in infection with Epstein-Barr virus (EBV) that is

In the eighties several studies of association with cancer of acute phase proteins in particular haptoglobin were done, that is the case for the lung cancer in 309 Swedish patients where the

Hp increases in vitro glioblastome cell migration (Kumar DM *et al* 2010), table 1.

associated with nasopharynx carcinoma (Speeckaert R *et al* 2009).

importance as a genetic and a circulating biomarker for that pathology.

circulating levels and clinics.

60 Acute Phase Proteins

studies that evidence those aspects.

head and neck, lung, blood and skin malignancies.

Hp1 allele is more frequent in women with adenocarcinoma (Beckman G *et al* 1986). More recently other group confirmed the association of local higher levels of the Hp l expression in pulmonary adenocarcinomas in opposition to squamous cell carcinomas (SCC) and small cell carcinomas (Abdullah M 2009).

There are five references for blood malignancies such as acute and chronic lymphoid leukemia and acute myeloid leukemia from three different ethnic groups Sweden, Israel and Brazil (Caucasians and Afro-descendants) and only one sample of Ashkenazy Jews (Germinis A *et al* 1983, Nevo S and Tatarsky I 1986, Mitchell RJ *et al* 1988, Fröhlander N and Stendahl U 1988, Campregher PV *et al* 2004).

Cutaneous malignancies and in particular squamous cell carcinoma (SCC)/Bowen´s disease are more frequent in kidney transplanted patients, that are more prone to disease when are carriers of Hp 1.1 phenotypes particularly after ten years of the transplantation (Speeckaert R *et al* 2012). The same happens in the development of Kaposi´s sarcoma in HIV positive patients, even after adjustment for age, gender and AIDS status (Speeckaert R *et al* 2011).

demonstrate any association (Germenis A *et al* 1983, Dimopoulos MA *et al* 1984). These results were consistent with a recent report from an association study realized in an African popula‐ tion where the Authors didn´t also demonstrate any association of the Hp polymorphism with PSA (Prostate Specific Antigen) and prostate cancer patients survival (Mavondo GA *et al* 2012). However there were demonstrated higher circulation levels of monoclonal antibodies against glycosyl epitopes presents in the beta chain of Hp in prostate carcinoma compared with BPH

Serum levels of haptoglobin were elevated in kidney and bladder cancer concomitantly with a metabolite of Prostaglandin F2α, however only in bladder cancer was demonstrated in 264 Germans a statistically significant lower frequency of Hp2-2 genotype (Dunzendorfer U *et al*

Failed to demonstrate any Hp

Any association of the polymorphism with PSA and

For breast cancer despite consistency of overrepresentation of Hp 1 allele in three earlier studies (Tsamantains C *et al* 1980, Kaur H *et al* 1984, Bartel U *et al* 1985) and only one negative study (Hudson BL *et al* 1982) a more recent study demonstrate that Hp phenotype distribution in patients is family history-dependent. For these authors the frequency of Hp 1-1 and Hp 2-1 phenotypes is higher in the familial group and the opposite for the no familial group (Awa‐ dallah S and Atoum MF 2004). Moreover, in the recent study whose objective was to search for circulating proteins, predictive of recurrences and free survival of high risk primary breast cancer, with proteomic techniques (SELDI-TOF-MS) the authors, based on disturbances of iron (low levels of ferritin light chain is associated with good prognosis), identified Hp 2 allele as risk factor, nonetheless validated in an independent, sample and technique group of patients (Gast M-C W *et al* 2008). Also, it may have clinical important value, as a biomarker for recurrence in early breast cancer patients, the haptoglobin related (Hpr) protein in tissues and

The first references from the sixties of the last century about gynaecologic tumours indicate contradictory results between the authors when they were analyzed as a whole in what concerns to the frequency of Hp1 allele (Larkin MF 1967, Milunicova A *et al* 1969). However, a posterior reference of Bartel U et al 1985 confirms a higher Hp1-1 genotype frequency in 246

association

survival

**Conclusions References**

The Role of Haptoglobin and Its Genetic Polymorphism in Cancer: A Review

Lower frequency of Hp 2-2 Benkman HG *et al* 1987

*et al* 1984

Mavondo GA *et al* 2012

Germenis A *et al* 1983; Dimopoulos MA

http://dx.doi.org/10.5772/56695

63

that decreased after radical prostatectomy (Saito S *et al* 2008), table 3.

1981; Bemkman HG *et al* 1987).

Prostate cancer N=155/115

Bladder cancer N=264

**Neoplasia Population (N)**

**(Control/Neo)**

Greek patients

N=122/74 Africa, Botswana Zimbabwe

Germany

**Table 3.** Haptoglobin and Cancer: Urological Tumours.

plasma (Kuhajda FP *et al* 1989 a-b).

Tumours of gastrointestinal tract where also studied and the single reference to haptoglobin polymorphism in colon cancer refers to one association in 184 Greek patients of Hp1-1 phenotype (Archimandritis A *et al* 1993), table 2.

More recently it was shown that Hp is produced in a large molecular complex with the beta chain of urokinase in cancer cells as well as in capillary endothelial cells (Harvey S *et al* 2009). This cancer-associated glycoform of Hp (β-chain) is a ligand for Galectin-3, a beta-galactoside binding protein implicated in tumour progression and metastases of colorectal cancers (Bresalier RS *et al* 2004).


**Table 2.** Haptoglobin and Cancer: Digestive Tumours.

Geographic differences have been reported regarding the influence of the Hp alleles in cancer risk. In India, where the frequency of Hp2 allele is high at the population level (84%) the risk for gastric cancer of the Hp2-2 phenotype carriers is 4.04 and the risk for oesophageal is 3.86 for Hp2-1 phenotype carriers (Jayanthi M *et al* 1989). On the contrary in another geographic localization (Greece) a study of a similar number of gastric carcinoma patients didn't show any difference for the same polymorphism (Theodoropoulos G *et al* 1992).

Deng R *et al* demonstrated in 2007 that, in pancreatic carcinoma patients, the frequency of Hp 2-2 is higher compared with chronic pancreatitis patients and normal controls. Haptoglobin of these patients is not elevated in serum, but it is abnormally fucosylated in β-chain that has four N-glycans sites, the same happens but not so extensively in hepatocellular, gastric and colorectal carcinomas. The fucosylation of Hp seems to be induced by a factor secreted by these tumours itself (Nakano et al 2009, Miyoshi E, Nakano M 2008)

Early references of the distribution of haptoglobin polymorphism in Greek patients with prostate carcinoma compared with prostate benign hypertrophy (BPH) patients failed to demonstrate any association (Germenis A *et al* 1983, Dimopoulos MA *et al* 1984). These results were consistent with a recent report from an association study realized in an African popula‐ tion where the Authors didn´t also demonstrate any association of the Hp polymorphism with PSA (Prostate Specific Antigen) and prostate cancer patients survival (Mavondo GA *et al* 2012). However there were demonstrated higher circulation levels of monoclonal antibodies against glycosyl epitopes presents in the beta chain of Hp in prostate carcinoma compared with BPH that decreased after radical prostatectomy (Saito S *et al* 2008), table 3.

Serum levels of haptoglobin were elevated in kidney and bladder cancer concomitantly with a metabolite of Prostaglandin F2α, however only in bladder cancer was demonstrated in 264 Germans a statistically significant lower frequency of Hp2-2 genotype (Dunzendorfer U *et al* 1981; Bemkman HG *et al* 1987).


**Table 3.** Haptoglobin and Cancer: Urological Tumours.

carriers of Hp 1.1 phenotypes particularly after ten years of the transplantation (Speeckaert R *et al* 2012). The same happens in the development of Kaposi´s sarcoma in HIV positive patients,

Tumours of gastrointestinal tract where also studied and the single reference to haptoglobin polymorphism in colon cancer refers to one association in 184 Greek patients of Hp1-1

More recently it was shown that Hp is produced in a large molecular complex with the beta chain of urokinase in cancer cells as well as in capillary endothelial cells (Harvey S *et al* 2009). This cancer-associated glycoform of Hp (β-chain) is a ligand for Galectin-3, a beta-galactoside binding protein implicated in tumour progression and metastases of colorectal cancers

**Conclusions References**

No association Theodoropoulos G *et al* 1992

Greece Association of Hp1-1 phenotype Archimandritis A *et al*<sup>1993</sup>

India Risk for Hp2-2 carriers Jayanthi M *et al*<sup>1989</sup>

China Frequency of Hp 2-2 is higher Deng R *et al*<sup>2007</sup>

Geographic differences have been reported regarding the influence of the Hp alleles in cancer risk. In India, where the frequency of Hp2 allele is high at the population level (84%) the risk for gastric cancer of the Hp2-2 phenotype carriers is 4.04 and the risk for oesophageal is 3.86 for Hp2-1 phenotype carriers (Jayanthi M *et al* 1989). On the contrary in another geographic localization (Greece) a study of a similar number of gastric carcinoma patients didn't show

Deng R *et al* demonstrated in 2007 that, in pancreatic carcinoma patients, the frequency of Hp 2-2 is higher compared with chronic pancreatitis patients and normal controls. Haptoglobin of these patients is not elevated in serum, but it is abnormally fucosylated in β-chain that has four N-glycans sites, the same happens but not so extensively in hepatocellular, gastric and colorectal carcinomas. The fucosylation of Hp seems to be induced by a factor secreted by these

Early references of the distribution of haptoglobin polymorphism in Greek patients with prostate carcinoma compared with prostate benign hypertrophy (BPH) patients failed to

even after adjustment for age, gender and AIDS status (Speeckaert R *et al* 2011).

Oesophageal cancer Higher risk for Hp2-1 phenotype Jayanthi M *et al* 1989

any difference for the same polymorphism (Theodoropoulos G *et al* 1992).

tumours itself (Nakano et al 2009, Miyoshi E, Nakano M 2008)

phenotype (Archimandritis A *et al* 1993), table 2.

**(Control/Neo)**

N=2026/184

N=104/100

N=114/2026 Greece

N=11/11

**Table 2.** Haptoglobin and Cancer: Digestive Tumours.

(Bresalier RS *et al* 2004).

Colon cancer

62 Acute Phase Proteins

Gastric cancer

Pancreatic cancer

**Neoplasia Population (N)**

For breast cancer despite consistency of overrepresentation of Hp 1 allele in three earlier studies (Tsamantains C *et al* 1980, Kaur H *et al* 1984, Bartel U *et al* 1985) and only one negative study (Hudson BL *et al* 1982) a more recent study demonstrate that Hp phenotype distribution in patients is family history-dependent. For these authors the frequency of Hp 1-1 and Hp 2-1 phenotypes is higher in the familial group and the opposite for the no familial group (Awa‐ dallah S and Atoum MF 2004). Moreover, in the recent study whose objective was to search for circulating proteins, predictive of recurrences and free survival of high risk primary breast cancer, with proteomic techniques (SELDI-TOF-MS) the authors, based on disturbances of iron (low levels of ferritin light chain is associated with good prognosis), identified Hp 2 allele as risk factor, nonetheless validated in an independent, sample and technique group of patients (Gast M-C W *et al* 2008). Also, it may have clinical important value, as a biomarker for recurrence in early breast cancer patients, the haptoglobin related (Hpr) protein in tissues and plasma (Kuhajda FP *et al* 1989 a-b).

The first references from the sixties of the last century about gynaecologic tumours indicate contradictory results between the authors when they were analyzed as a whole in what concerns to the frequency of Hp1 allele (Larkin MF 1967, Milunicova A *et al* 1969). However, a posterior reference of Bartel U et al 1985 confirms a higher Hp1-1 genotype frequency in 246 German patients with gynaecological and breast tumours. When only ovarian cancer samples were considered, two references, one Polish and another Swedish, indicate they are associated respectively with Hp 1 allele and Hp2-1 phenotype in patients with family history (Dobrys‐ zycka W and Wavas M. 1983, Fröhlander N and Stendahl U 1988).

Cervical neoplasia is a good model that illustrates haptoglobin and its polymorphism influence in the several steps of its natural history interacting with oncogenic and non-oncogenic HPV (Human Papillomavirus) and other co-factors such as sexual steroid hormones and smoking habits (Bicho MC 2011).

Preliminary reports on the role of this haptoglobin polymorphism in the development of cervical cancer were conflicting, with two authors (Milunicova and Bartel) indicating that Hp1 allele carriers were at risk of cancer development. In opposition, Larkin et al report the Hp2 allele as the most represented in their cervical cancer cases (Milunicova A *et al* 1969, Bartel U *et al* 1985 U, Larkin M 1967). Those reports were published previous to the, nowadays confirmed, association of oncogenic HPV types as the primary etiologic factor of cervical cancer and the HPV effect was not evaluated in the control populations. However, HPV is a necessary but not a sufficient cause of cervical cancer and it is also important the presence the other cofactors host related. One of these co-factors can be the immune response of the host. It has been proposed a role for haptoglobin a one of such co-factors (Mahmud SM *et al* 2007, Bicho MC *et al* 2006 and 2009).

In the case control study conducted in Canada (307 cases vs 358 control women), Mahmud et al examined the association of Hp phenotype with high grade cervical intraepithelial neoplasia (CIN III), a precursor lesion of invasive carcinoma (ICC). The control group had to present a normal cytology and HPV genotyping was performed to evaluate the HPV oncogenic type status. Accordingly, only when the risk analysis is restricted to the HPV positive women, an association was observed and Hp 1-1 carriers have almost a threefold increased susceptibility to the development of CIN III (OR=2.7, 95% IC: 1.0-7.2) (Mahmud SM et al 2007). In a recent study, we report an increased susceptibility for women that are Hp 1-1 carriers to develop ICC (OR=4.62, 95% IC: 1.86-11.48) (Bicho MC 2011). These results are consistent with another study performed in a different geographic localization (Ghana) and indicating a significant protec‐ tive effect for the Hp2 allele in homozygous women (Quaye IK et al 2009). In another report, we studied the influence of Hp polymorphism on the risk for the development of HSIL and ICC (n=196) under the influence of sex steroid hormones. We found that the risk for an interaction is proportionally higher with the number of Hp 1 allele presents (Bicho MC et al 2009). However, when the interaction between Hp polymorphism with smoking habits was studied the Hp 2 allele in homozygoty increased the risk to develop HSIL and ICC (Bicho MC et al 2006).

#### **6. Discussion**

During the first thirty years (from the sixties to nineties of the last century) of cancer association studies, genetic blood markers, including haptoglobin were concomitantly studied with descriptive studies of allele distribution in the different populations (Giblett ER 1968, Langlois MR and Delanghe JR 1996, Wobeto VP *et al* 2008, Levy AP *et al* 2010). These preliminary reports usually were cross-sectional case control studies, that didn't enter in consideration with the biological plausibility in cancer linked to the genetic variation. Diverse geographic regions, with very different distribution of alleles, various genetic variations backgrounds and above

**Neoplasia Population (N)**

Breast cancer

Ovarian cancer

Cervical cancer

**(Control/Neo)**

N=109 Greece

N=50/50 India

N=246 Germany

N=129/200

Familial (N=42) Non familial (N=86)

> N=371 USA

N=114/132

N=182 Swedish

N=170/85

N=430/526

N=430/526 USA

N= 396/196 Portugal

N=120/60 Ghana

**Table 4.** Haptoglobin and Cancer: Gynaecological Tumours.

**Conclusions References**

The Role of Haptoglobin and Its Genetic Polymorphism in Cancer: A Review

Tsamantains C *et al* 1980

http://dx.doi.org/10.5772/56695

65

Kaur H *et al* 1984

Bartel U *et al* 1985

Atoum MF 2004

Fröhlander N and Stendahl U 1988

Overrepresentation of Hp 1

Overrepresentation of Hp 1

Overrepresentation of Hp 1

Jordania Higher frequency of Hp 1 allele Awadallah S

Higher frequency of Hp 1 and

Associated Hp2-1 phenotype

Checoslovakia Hp1 allele carriers at risk Milunicova A *et al*<sup>1969</sup>

Germany Hp1 allele carriers at risk Bartel U *et al*<sup>1985</sup>

Hp2 allele as the most

Hp 1-1 is higher CIN III

hormone ingestion

In ICC women the risk for Hp 1-1 carriers is greater in steroid

Protective effect of the Hp2

allele in homozygoty Quaye IK *et al*<sup>2009</sup>

represented

N=358/307 Canada In HPV positive women, risk for

with family history

USA No association Hudson BL *et al* 1982

Polland Associated Hp 1 allele Dobryszycka W and Wavas M.

No association Gast M-C W *et al* 2008

1983

Larkin M 1967

Bicho MC 2011

Mahmud SM *et al* 2007

allele

allele

allele

Hp 2 alleles


**Table 4.** Haptoglobin and Cancer: Gynaecological Tumours.

German patients with gynaecological and breast tumours. When only ovarian cancer samples were considered, two references, one Polish and another Swedish, indicate they are associated respectively with Hp 1 allele and Hp2-1 phenotype in patients with family history (Dobrys‐

Cervical neoplasia is a good model that illustrates haptoglobin and its polymorphism influence in the several steps of its natural history interacting with oncogenic and non-oncogenic HPV (Human Papillomavirus) and other co-factors such as sexual steroid hormones and smoking

Preliminary reports on the role of this haptoglobin polymorphism in the development of cervical cancer were conflicting, with two authors (Milunicova and Bartel) indicating that Hp1 allele carriers were at risk of cancer development. In opposition, Larkin et al report the Hp2 allele as the most represented in their cervical cancer cases (Milunicova A *et al* 1969, Bartel U *et al* 1985 U, Larkin M 1967). Those reports were published previous to the, nowadays confirmed, association of oncogenic HPV types as the primary etiologic factor of cervical cancer and the HPV effect was not evaluated in the control populations. However, HPV is a necessary but not a sufficient cause of cervical cancer and it is also important the presence the other cofactors host related. One of these co-factors can be the immune response of the host. It has been proposed a role for haptoglobin a one of such co-factors (Mahmud SM *et al* 2007, Bicho MC *et*

In the case control study conducted in Canada (307 cases vs 358 control women), Mahmud et al examined the association of Hp phenotype with high grade cervical intraepithelial neoplasia (CIN III), a precursor lesion of invasive carcinoma (ICC). The control group had to present a normal cytology and HPV genotyping was performed to evaluate the HPV oncogenic type status. Accordingly, only when the risk analysis is restricted to the HPV positive women, an association was observed and Hp 1-1 carriers have almost a threefold increased susceptibility to the development of CIN III (OR=2.7, 95% IC: 1.0-7.2) (Mahmud SM et al 2007). In a recent study, we report an increased susceptibility for women that are Hp 1-1 carriers to develop ICC (OR=4.62, 95% IC: 1.86-11.48) (Bicho MC 2011). These results are consistent with another study performed in a different geographic localization (Ghana) and indicating a significant protec‐ tive effect for the Hp2 allele in homozygous women (Quaye IK et al 2009). In another report, we studied the influence of Hp polymorphism on the risk for the development of HSIL and ICC (n=196) under the influence of sex steroid hormones. We found that the risk for an interaction is proportionally higher with the number of Hp 1 allele presents (Bicho MC et al 2009). However, when the interaction between Hp polymorphism with smoking habits was studied the Hp 2 allele in homozygoty increased the risk to develop HSIL and ICC (Bicho MC

During the first thirty years (from the sixties to nineties of the last century) of cancer association studies, genetic blood markers, including haptoglobin were concomitantly studied with

zycka W and Wavas M. 1983, Fröhlander N and Stendahl U 1988).

habits (Bicho MC 2011).

64 Acute Phase Proteins

*al* 2006 and 2009).

et al 2006).

**6. Discussion**

descriptive studies of allele distribution in the different populations (Giblett ER 1968, Langlois MR and Delanghe JR 1996, Wobeto VP *et al* 2008, Levy AP *et al* 2010). These preliminary reports usually were cross-sectional case control studies, that didn't enter in consideration with the biological plausibility in cancer linked to the genetic variation. Diverse geographic regions, with very different distribution of alleles, various genetic variations backgrounds and above all have different environments that interacts with genomes to give highly variable pheno‐ types, may explain controversial results.

integrin and LFA located respectively in B cells neutrophils and T cells (EL Ghmati SM *et al* 1996, Giannoni E *et al* 2003, Bottini N *et al* 1999 and 2005, Arredouani MS *et al* 2005, Lu JY *et al* 2007). A third mechanism can be the interaction of environmental factors such as sex steroid hormones and glucocorticoids that activates predominantly Th2 arm of acquired immunity in synergy with Hp1 allele as happens in cervical cancer (Gleeson M 2006, Bicho MC *et al* 2009,

The Role of Haptoglobin and Its Genetic Polymorphism in Cancer: A Review

http://dx.doi.org/10.5772/56695

67

An increased prevalence of Hp 2-2 genotype is observed in some tumours leading to the hypothesizes of haptoglobin involvement in the mechanisms associated with the carcinogen‐ esis and tumorigenesis of the chronic inflammation (head and neck carcinomas, glioblastome,

The lower antioxidative capacity and inhibition of prostaglandin synthase associated to the genotype Hp 2-2 are the best known explanatory mechanisms (Wen WN et al 2001, Saeed SA *et al* 2007). Indirect effects of prostaglandins on carcinogenesis are mediated through the stabilization of HIF1α and the resultant expression of angiogenic factors like EPO (erythro‐ poietin), VEGF (vascular endothelial growth factor), that have synergic effects with Hp2-2 (Cid MC *et al* 1993, Liu XH *et al* 2002, Acs G *et al* 2003, Ye YN *et al* 2004, Mihailović M *et al* 2005,

Another mechanism involved is the withholding of iron in macrophages that is necessary for the proliferation of immune cell (Touitou Y *et al* 1985, Weinberg ED 1996, Cozzi A *et al* 2004). The effects of smoking habits are modulated by Hp2-2, because the effects in the nicotine down regulation of haptoglobin expression and also the effects of CO producing local hypoxia and

More studies are necessary to complete our understanding about the role of this important acute phase protein, its levels variations, particularly the fucosylated isoforms and its regula‐ tion, the Hpr and its polymorphism and its immunomodulation role in cancer. Finally, future studies may focus in the importance of haptoglobin polymorphism conducting to a pharma‐

, Rui Medeiros3

1 Genetics Laboratory Faculty of Medicine, University of Lisbon, Portugal

3 Portuguese Institute of Oncology (IPOFG) Oporto, Portugal

and Manuel Bicho1,2

Akila P *et al* 2012).

gastric carcinoma).

**7. Perspectives**

**Author details**

Palayoor ST *et al* 2009, Park SJ 2009).

the immune depression (Ye YN *et al* 2005).

cogenetic approach to chemoprevention.

Maria Clara Bicho1,2, Alda Pereira da Silva1

2 Rocha Cabral Institute Lisbon, Portugal

Moreover, the lack of reproducibility of the several studies may also reflect methodological differences in the criteria of case definitions and selection of controls, and in what concerns to the influence of environment of factors such as microbiological (HPV, EBV, M. tuberculosis, H. Pylori, Plasmodia), smoking habits, sun exposition, xenobiotic and sex steroid hormones (Beckman G *et al* 1986, Benkmann HG *et al* 1987, Bicho MC *et al* 2006 and 2009, Mahmud *et al* and 2007, Abdullah M *et al* 2009, Speeckaert R *et al* 2011 and 2012).

The usual cross-sectional approach of these studies didn´t take into account the somewhat different influences of the genotypes on the natural story of the cancer that courses in multistep way (Zur Hausen H 2002). The great majority of the studies were done in patients with distant phenotypes (advanced stage cancer) and take not in consideration the subclinical disease. This isn´t evidenced in those times by lack of knowledge of physiopathology and lack of reliable biomarkers (circulating and imaging) that gives a more dynamic picture of the situation.

It was not common, the realization of measurements of serum and plasma levels of the Hp independently of phenotype in part due too time consuming of the technics (Langlois MR and Delanghe JR 1996). In these cases, not even the local processes are reflected in circulation but also it is demonstrated the existence of a local tissue environment in what Hp functions in paracrine and autocrine way (Yang F *et al* 2000, Xie Y, *et al* 2000, Sharpe-Timms *et al* 2002, Wang H *et al* 2005, Shaw JLV *et al* 2007).

The natural history of cervical cancer seems to be dependent of genetic polymorphism of haptoglobin in its interaction with HPV and cofactors such as sex steroid hormones and smoking habits (Bicho MC *et al* 2006 and 2009, Mahmud *et al* and 2007).

Also there are reports of the different influences of Hp alleles in a context of familiar history for the breast and ovarian cancers (Fröhlander N and Stendahl U 1988, Awadallah S and Atoum MF 2004).

For the clarification of these issues a better knowledge of the physiopathology mechanisms of action of the Hp alleles is necessary.

Haptoglobin as a pleiotropic protein has several different functions being the Hp1 allele and correspondent genotypes Hp1-1 and Hp1-2, the more represented in the several cancers reviewed. The innate immune response of the host against the tumour is limited in the subject's carriers of the Hp1 allele through several mechanisms, already reviewed.

It is accepted, in this pathway, the role of Hp-Hb, CD163, HO-1, CO, bilirubin, activation of anti-oxidant intracellular systems (including ferritin), and extrusion of iron through ferropor‐ tin. This pathway is characteristic of immunosuppressive tumor macrophages M2 types that are more active in Hp1 carriers inducing a switch for a Th2 antinflammatory cytokine profile characteristic of lesser Th1 type cytotoxic immune antitumor mechanisms (Van Vlierberghe *et al* 2004, Guetta *et al* 2007, Nielsen MJ *et al* 2010, Vallelian F *et al* 2011 and Akila P *et al* 2012).

This switch can be also dependent of a stronger acute phase response characteristic of Hp1 carriers that can modulate immune cells activity after binding of Hp to its receptors CD22, β2 integrin and LFA located respectively in B cells neutrophils and T cells (EL Ghmati SM *et al* 1996, Giannoni E *et al* 2003, Bottini N *et al* 1999 and 2005, Arredouani MS *et al* 2005, Lu JY *et al* 2007). A third mechanism can be the interaction of environmental factors such as sex steroid hormones and glucocorticoids that activates predominantly Th2 arm of acquired immunity in synergy with Hp1 allele as happens in cervical cancer (Gleeson M 2006, Bicho MC *et al* 2009, Akila P *et al* 2012).

An increased prevalence of Hp 2-2 genotype is observed in some tumours leading to the hypothesizes of haptoglobin involvement in the mechanisms associated with the carcinogen‐ esis and tumorigenesis of the chronic inflammation (head and neck carcinomas, glioblastome, gastric carcinoma).

The lower antioxidative capacity and inhibition of prostaglandin synthase associated to the genotype Hp 2-2 are the best known explanatory mechanisms (Wen WN et al 2001, Saeed SA *et al* 2007). Indirect effects of prostaglandins on carcinogenesis are mediated through the stabilization of HIF1α and the resultant expression of angiogenic factors like EPO (erythro‐ poietin), VEGF (vascular endothelial growth factor), that have synergic effects with Hp2-2 (Cid MC *et al* 1993, Liu XH *et al* 2002, Acs G *et al* 2003, Ye YN *et al* 2004, Mihailović M *et al* 2005, Palayoor ST *et al* 2009, Park SJ 2009).

Another mechanism involved is the withholding of iron in macrophages that is necessary for the proliferation of immune cell (Touitou Y *et al* 1985, Weinberg ED 1996, Cozzi A *et al* 2004).

The effects of smoking habits are modulated by Hp2-2, because the effects in the nicotine down regulation of haptoglobin expression and also the effects of CO producing local hypoxia and the immune depression (Ye YN *et al* 2005).

## **7. Perspectives**

all have different environments that interacts with genomes to give highly variable pheno‐

Moreover, the lack of reproducibility of the several studies may also reflect methodological differences in the criteria of case definitions and selection of controls, and in what concerns to the influence of environment of factors such as microbiological (HPV, EBV, M. tuberculosis, H. Pylori, Plasmodia), smoking habits, sun exposition, xenobiotic and sex steroid hormones (Beckman G *et al* 1986, Benkmann HG *et al* 1987, Bicho MC *et al* 2006 and 2009, Mahmud *et al*

The usual cross-sectional approach of these studies didn´t take into account the somewhat different influences of the genotypes on the natural story of the cancer that courses in multistep way (Zur Hausen H 2002). The great majority of the studies were done in patients with distant phenotypes (advanced stage cancer) and take not in consideration the subclinical disease. This isn´t evidenced in those times by lack of knowledge of physiopathology and lack of reliable biomarkers (circulating and imaging) that gives a more dynamic picture of the situation.

It was not common, the realization of measurements of serum and plasma levels of the Hp independently of phenotype in part due too time consuming of the technics (Langlois MR and Delanghe JR 1996). In these cases, not even the local processes are reflected in circulation but also it is demonstrated the existence of a local tissue environment in what Hp functions in paracrine and autocrine way (Yang F *et al* 2000, Xie Y, *et al* 2000, Sharpe-Timms *et al* 2002, Wang

The natural history of cervical cancer seems to be dependent of genetic polymorphism of haptoglobin in its interaction with HPV and cofactors such as sex steroid hormones and

Also there are reports of the different influences of Hp alleles in a context of familiar history for the breast and ovarian cancers (Fröhlander N and Stendahl U 1988, Awadallah S and Atoum

For the clarification of these issues a better knowledge of the physiopathology mechanisms of

Haptoglobin as a pleiotropic protein has several different functions being the Hp1 allele and correspondent genotypes Hp1-1 and Hp1-2, the more represented in the several cancers reviewed. The innate immune response of the host against the tumour is limited in the subject's

It is accepted, in this pathway, the role of Hp-Hb, CD163, HO-1, CO, bilirubin, activation of anti-oxidant intracellular systems (including ferritin), and extrusion of iron through ferropor‐ tin. This pathway is characteristic of immunosuppressive tumor macrophages M2 types that are more active in Hp1 carriers inducing a switch for a Th2 antinflammatory cytokine profile characteristic of lesser Th1 type cytotoxic immune antitumor mechanisms (Van Vlierberghe *et al* 2004, Guetta *et al* 2007, Nielsen MJ *et al* 2010, Vallelian F *et al* 2011 and Akila P *et al* 2012). This switch can be also dependent of a stronger acute phase response characteristic of Hp1 carriers that can modulate immune cells activity after binding of Hp to its receptors CD22, β2

smoking habits (Bicho MC *et al* 2006 and 2009, Mahmud *et al* and 2007).

carriers of the Hp1 allele through several mechanisms, already reviewed.

and 2007, Abdullah M *et al* 2009, Speeckaert R *et al* 2011 and 2012).

types, may explain controversial results.

66 Acute Phase Proteins

H *et al* 2005, Shaw JLV *et al* 2007).

action of the Hp alleles is necessary.

MF 2004).

More studies are necessary to complete our understanding about the role of this important acute phase protein, its levels variations, particularly the fucosylated isoforms and its regula‐ tion, the Hpr and its polymorphism and its immunomodulation role in cancer. Finally, future studies may focus in the importance of haptoglobin polymorphism conducting to a pharma‐ cogenetic approach to chemoprevention.

## **Author details**

Maria Clara Bicho1,2, Alda Pereira da Silva1 , Rui Medeiros3 and Manuel Bicho1,2

1 Genetics Laboratory Faculty of Medicine, University of Lisbon, Portugal

2 Rocha Cabral Institute Lisbon, Portugal

3 Portuguese Institute of Oncology (IPOFG) Oporto, Portugal

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**Chapter 4**

**Role of SAA in Promoting Endothelial Activation:**

Serum amyloid A (SAA) is a multi-gene family consisting of highly conserved protein sequences that are known to cluster on chromosome 11 in humans [1] and chromosome 7 in mice [2]. The acute-phase proteins SAA1 and SAA2 (mass of ~12 kDa, 104 amino acids) share > 93% identity in primary sequence structure, are secreted predominantly by hepatocytes and are induced by a broad spectrum of inflammatory cytokines [3]. Extra-hepatic production of acute-phase SAA occurs in many organs and tissues of body including vascular smooth muscle cells and endothelial cells (EC) that are also capable of secreting SAA [4, 5]. By contrast, SAA4 is a glycosylated form that is constitutively produced in a wide range of (histologically) normal tissues and cells [6]. The final isoform, SAA3, is a pseudogene that is not transcribed in humans [1]. In rodents, SAA3 is a functional protein that is expressed in extra-hepatic cells, such as macrophages and adipocytes in response to prolactin or lipolysaccaride (LPS) stimuli thereby,

Rapid production of SAA in response to the host inflammatory reaction results in plasma levels increasing up to 1,000-fold under some conditions [9]. This marked increase in circulating SAA is linked to the induction of inflammatory cascades that are characterized by local vascular, systemic and multi-organ responses [10, 11]. A wealth of epidemiological and biological research suggests that SAA is also associated with chronic inflammatory conditions such as cardiovascular diseases (CVD) and atherogenesis [12-14]. For example, significantly elevated levels of SAA are evident at different stages of atherosclerosis [15, 16], which, to some extent, echoes a sustained acute-phase response leading to the chronic production of SAA. In fact, SAA is proposed as a potential regulator of inflammation and endothelial dysfunction, implicating adverse outcomes that complicate CVD [4]. SAA is also synthesized in extra-

> © 2013 Wang et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 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,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Inhibition by High-Density Lipoprotein**

Saul Benedict Freedman and Paul K. Witting

Additional information is available at the end of the chapter

contributing to local inflammation in adipose tissues [7, 8].

Xiaosuo Wang, Xiaoping Cai,

http://dx.doi.org/10.5772/56047

**1. Introduction**

