Infectious Aetiology of Cancer: Developing World Perspective

Infection attributable cancers contribute over 1/4th of all cancers in the developing countries (26.3%) compared to the developed countries (7.7%), (Parkin, 2006). Overwhelming majority are related to viral infections. In contrast to other carcinogens where it is usually a ‘hit and run’ kind of situation, with infectious agents particularly viruses one may precisely demonstrate and prove its presence and integration within host neoplastic cells. Oncogenic DNA viral genome incorporates itself directly into host cells DNA while oncogenic RNA viral genome is transcribed into host cell DNA by reverse transcriptase. Neoplastic transformation usually follows. Oncogenic mechanisms include acting as promoter, transforming protooncogenes into oncogenes. Credit goes to Dr Peyton Rous, a noble laureate pathologist who demonstrated that it was possible to transmit tumours from one animal to other like transmission of an infection. Human tumours with proven or proposed viral aetiology include ‘Human papillomavirus (HPV)’, Epstein-Barr Virus (EBV), Hepatitis B and C viruses, RNA retroviruses like ‘Human T-lymphotropic virus (HTLV1)’, ‘Human Herpes Virus-8 (HHV-8). Bacteria with proven carcinogenic potential include ‘Helicobacter pylori’. Among fungi aflatoxins produced by ‘Aspergillus flavus’ are potent carcinogens. Among parasites ‘Schistosoma’ and ‘Clonorchis sinensis’ are implicated in the causation of cancer.


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countries. In Karachi it ranked 2 nd with an identical risk in both genders (Bhurgri et al, 2003). However if combined with pharynx and larynx cancers which have the same histologic type (squamous cell carcinoma) & risk factors it ranks number 1. Major risk factors in the developed world include 'smoking' and 'alcohol; however in the developing world though smoking is a common major risk factor, role of alcohol drinking is possibly a minor risk factor particularly in developing muslim countries. In subcontinent (Pakistan, India & Bangladesh) alternate chewing habits like betel quid and areca nut are major risk factors. Areca nut is now declared by WHO as a bonafide carcinogen. People using paan (betel leaf) are about 8-9 times more likely to develop oral cancers as compared to non-users (Merchant et al, 2000). Smokeless tobacco, including 'gudka' and 'niswar' is an extremely addictive substance with a high rate of use in younger age groups, as well is contributing toward endemic rise of oral cancers in Pakistan (Ali et al, 2009& Nair et al, 2004. (Figure 1) This habit commonly leads to a pre-malignant condition 'Submucosal fibrosis' which commonly transforms into OSCC. Poor oral hygiene is another contributory factor in this population. A significant proportion of OSCC patients however deny exposure to conventional and well known risk factors. This has led to search of other risk factors and associations including microbes (Scully et al, 1985). The striking commonality between oral cavity and cervical cavity paved the way to look for epitheliotropic viruses like HPV. Although these two areas are anatomically different, the squamous epithelium found in both areas has several similarities. For instance the squamous epithelium of ecto-cervix and oral cavity including pharynx and larynx are composed of squamous epithelium with a thin layer of keratin or no keratin. In both areas the epithelium is subject to microtrauma of various types as well as to bacteria and varying chemical irritants. Most common malignancy at both anatomic sites is also SCC with varied differentiation. (Figure 2) These factors may directly expose to HPV infections of cells resulting in malignant transformation. Furthermore the HPV subtypes isolated from lesions of squamous epithelium of cervix are similar to the type found in both normal epithelium and various lesions of the oral cavity, pharynx and larynx. These include HPV subtypes16, 18, 31 & 33. The reported prevalence of HPV in OSCC varies widely in various studies depending on the population and ethnicity studied and/or sensitivity of the methods used and viral DNA sequence targeted. HPV in particular HPV-16, like in cervix is implicated in the aetiology of OSCC (Gillison, 2004;Miller & Johnstone, 2001) About 40 -60% of patients with tumours of oropharynx are reported to be positive for HPV infection (Gillison, 2004;Kreimer et al, 2005). HPV-positive tumours are distinct from HPV-negative tumours in their biological characteristics and clinical behaviour. Data from retrospective analyses as well as a prospective clinical trial demonstrated that HPV-positive oropharyngeal tumours are more sensitive to chemotherapy and radiation treatment and have a markedly improved prognosis and favourable clinical outcome compared with HPV-negative tumours (Fakhry et al, 2008; Recently, another significant observation has emerged in terms of HPV status of oropharyngeal tumours and racial disparities. Black Americans are known to have a higher incidence of and mortality from head and neck squamous cell carcinoma (HNSCC) than the whites and present with more advanced disease at a younger age (Goodwin et al, 2008;Morse & Kerr 2006;Ryerson et al, 2008;Shiboski et al, 2007). The greatest survival difference between blacks and whites was detected specifically in oropharyngeal cancers, but there was no racial difference between the overall survival rates of patients with nonoropharyngeal tumours . Most importantly, the recently published prospective analysis demonstrated that a marked difference exists between black and white Americans in terms of HPV infection. HPV positivity was about 9-fold higher in white (34%) than in black (4%) patients, directly correlating HPV infection with significant survival disparities between the two populations . Clearly, the HPV status of patients with OSCC would be an important determinant for prognosis and treatment options in the future. Recently, in a retrospective study of 140 patients with primary OSCC and a long-term follow up, Ali et al reported from Karachi, Pakistan, 68% of cases to be positive for HPV (Ali et al, 2008). Approximately 90% of these cases were infected with HPV16, (Figure 3 & 4) the predominant subtype in the US population as well. HPV infection was detected entirely in tumours of the cheek and tongue in the oral cavity; this was consistent with the occurrence frequency in the Karachi population for oral cancers which is as follows: 55.9% for cheek, 28.4% for tongue, 6.8% for palate, 4.4% for gum, 3.1% for lip and 1.4% for floor of the mouth (Bhurgri et al, 2003). Furthermore, though HPV positive patients had comparatively prolonged overall survival when compared with HPV negative patients but the difference was not statistically significant (P=0.97) ( Figure 5). Betel quid chewers were comparatively more prone to HPV positivity (OR=2.34; 95 CI= 1.1-4.31). These findings are in contrast with the results from US studies where the ratio of oropharyngeal tumours with respect to other sites was 2:1 and the HPV-positive tumours were consistently associated with a better clinical outcome in terms of both overall and disease-free survival (Fakhry et al, 2008;). The reason(s) for these different findings are not clear.

Oncogenic HPV pathways
The chief oncoproteins of HPV16 are encoded by the genes E6 and E7. The E6 protein targets the tumour suppressor gene p53 for degradation. In fact, degradation of p53 in HPV positive cells is fully dependent on the presence of E6 , Figure 6). The E7 oncoprotein is involved in suppression of retinoblastoma protein (pRb) function. Reduced pRb expression is common in HPV-positive tonsillar cancer.

Mode of transmission
Two questions immediately come to mind, first how HPV gets there and second why patients with HPV association will have better survival. In response to question 1, haematogenous spread from genital tract is proposed besides atypical sexual habits. In response to question 2 one possible explanation is that HPV infection may lead to genome instability, paradoxically making tumour cells more susceptible to radiotherapy.

Epstein-Barr virus (EBV)
EBV was initially discovered from cell cultures of a high grade B-cell lymphoma 'Endemic (African) Burkitt Lymphoma (BL)', which is highly prevalent in paraequatorial Africa and New Guinea. The disease affects children and adolescents and has strong association with malaria. Endemic BL commonly involves extra-nodal sites particularly jaw. In rest of the world 'sporadic form of BL' is seen having a weaker association with EBV and commonly affecting gastro-intestinal tract (GIT) particularly small intestine. EBV associated other lymphomas include 'classic Hodgkin lymphoma (cHL)' particularly 'mixed cellularity' type (60%), 'B-cell lymphoma in immunosuppressed', 'mature T-cell lymphoproliferative disorders' in particular 'Angioimmunoblastic T-cell lymphoma (AILT)', 'Angiocentric (Nasal) T-cell lymphoma'. Non-lymphoid associations include 'Nasopharyngeal carcinoma'.

EBV & mature T-cell non-Hodgkin lymphoma (T-NHL)
EBV association with certain subsets of T-NHL is now well established. In a study conducted by us in Pakistan (Noorali et al, 2003), mature T-NHL comprised 22.2% of total mature NHLs. These cases were characterised on the basis of morphology, immunohistochemistry and T-cell receptor (

EBV & angioimmunoblastic T-cell lymphoma (AILT)
AILT is an uncommon form of mature T-NHL characterised by systemic disease that occurs predominantly in middle-aged and elderly patients. The clinico-pathologic syndrome is characterised by fever, night sweats, weight loss, generalised lymphadenopathy, hepatomegaly and splenomegaly. Histologic examination of lymph nodes typically shows effacement of lymph nodes architecture, a polymorphous infiltrate including immunoblasts, lymphocytes, plasma cells, eosinophils, epithelioid histiocytes and a prominent arborizing postcapillary vasculature (Figure 8). In a study conducted by us a total of 13 well characterised cases of AILT based on morphology, IHC and TcR gene rearrangement studies were analysed for EBV by PCR and ISH (EBER). Association of EBV was seen in 11 out of 13 cases (84.6%) by PCR. By ISH (EBER), EBV was detected in 8 out of 9 cases (88.8%) cases. (Figure 8) So all in all strongest correlation of EBV was seen in this type of T-NHL. (Noorali et al, 2005).

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EBV & Mycosis fungoides (MF)
MF is an indolent T-cell lymphoma of skin. In a study conducted by us a total of 14 well characterised cases of MF were analysed for EBV by PCR and ISH (EBER). EBV was identified in 3 out of 6 cases (50%) by PCR but all these were negative on ISH (EBER). This discrepancy is most likely caused by low copy number of infected cells in tissue sections not amplified as in PCR based studies (Noorali et al, 2002).

EBV & anaplastic large cell lymphoma (ALCL)
ALCL is a peculiar type of T-NHL. In a recent study by us (Syed et al, 2011) ALCL was turned out to be the most common T-NHL in the archives of the largest referral centre of Pakistan. This variant of T-NHL however has the weakest association with EBV (Noorali et al 2004).

HTLV1 is a RNA oncogenic virus which is associated with 'adult T-cell leukemia /lymphoma'
and is endemic in southern Japan and Caribbean basin. Like HIV which causes AIDS, HTLV1 also shows tropism for CD4+ T cells, hence this subset is the main victim for neoplastic transformation. In our local studies HTLV1 association was absent in mature Tlymphoproliferative disorders. This is in line with relatively low burden of HIV-AIDS in Pakistan so far (Noorali et al, 2004). (Figure 9)

Role of EBV detection by PCR, ISH & IHC in diagnostic pathology
The ability to amplify specific regions of DNA from paraffin-embedded tissue by PCR has a profound impact on diagnostic pathology. For routine histopathological diagnosis of various lymphoproliferative disorders EBV-ISH (EBV-encoded nuclear RNA -1(EBER-1) and IHC by using an antibody to 'Latent Membrane Protein-1 (LMP-1) are frequently used in diagnostic dilemmas. For instance in the differential diagnosis of cHL and ALCL, EBER or LMP-1 positivity in neoplastic cells will strongly favour cHL as EBV association with ALCL is very weak. (Figure 10)

EBV & nasopharyngeal carcinoma
Nasopharyngeal carcinomas are particularly common in some parts of Africa and southern China. In former they constitute most frequent childhood cancer while in the later adults are mostly affected. Association of EBV with nasopharyngeal carcinoma is well established. In fact this association is literally 100%. EBV associated protein LMP-1 is expressed in most cases. (Figure 11) Fig. 11. Photomicrograph of a case of Nasopharyngeal carcinoma (H& E, A) stained with an antibody to LMP-1 (B), note cytoplasmic staining of neoplastic cells.

HHV8 & Kaposi sarcoma
Relatively recently in 1994 'Human  was identified in an AIDS patient with cutaneous 'Kaposi sarcoma (KS)'. Later it was found that over 95% KS are associated with HHV-8. This virus is largely transmitted sexually. An antibody against HHV-8 shows positive reactivity in about 100% of cases and is a useful tool to confirm the diagnosis.
Although 'Kaposi sarcoma' is uncommon in our practice in Pakistan, it is highly prevalent in developing world with high AIDS incidence. (Figure 12 (Figure 13). In fact the pathogenesis of gastric 'MALT Lymphoma' is believed to be caused by repeated antigenic stimulation of the immune system in the stomach by HP. The role of HP in the pathogenesis of 'gastric MALTomas' can be illustrated by the fact that 75% of the patients who have gastric MALToma undergo remission if treated with antibiotics to eradicate HP (Ono et al, 2008). About half the people in the world have HP colonized in their gastrointestinal tract. Of these most remain asymptomatic. Despite the fact that, a high prevalence of HP is reported from Pakistan , the prevalence of 'gastric MALTomas' is very low in our experience. Seroprevalence of HP infection in the Pakistani population has been reported as high as 58%. This correlates with the 'Asian enigma' described by various authors where less developed Asian countries like Pakistan, India, Bangladesh and Thailand have lower rates of gastric carcinoma compared to well developed countries like Japan and China, despite a higher prevalence of HP infection in the population. HP has been established to have a role in the aetiology of gastric carcinoma and its paradoxical high prevalence in areas with few cases of gastric carcinoma has long puzzled researchers. Available evidences do not support difference in HP strains as the sole explanation for this enigma.

Immunoproliferative small intestinal disease (IPSID) & Campylobacter jejuni :
Immunoproliferative small intestinal disease (IPSID) is a special variant of, 'Extranodal marginal zone B cell lymphoma', which affects the small intestine. In early to mid 1960s it was referred to as 'Mediterranean lymphomas', during late 1960s the term 'α-heavy chain disease' was also used for patients with similar clinico-pathological presentations. Later it was realized that both 'Mediterranean lymphomas' and 'α-heavy chain disease' represented a spectrum of the same disease which presents in different stages i.e., benign, intermediate and overtly malignant (stage A, B & C) and the disease was named IPSID (Fine & Stone 2000). IPSID is predominantly found in patients of 'Mediterranean origin'; however a few cases of IPSID are also diagnosed in the subcontinent . IPSID involves the production of truncated alpha heavy chains which may appear in the serum and other body fluids. It can be treated with broad spectrum antibiotics at its early stages. It is postulated that IPSID occurs in patients with repeated intestinal infections. Recent studies suggest association with Campylobacter jejuni (Lecuit et al, 2004). It is postulated that this results in continuous chronic antigenic stimulation of IgA secreting lymphoid tissue common in small intestine with a resultant clonal proliferation of IgA secreting lymphoid cells. Subsequently most cases lose the ability to synthesize light chain. In early stages it may be very difficult to differentiate IPSID, from chronic inflammatory process by the reporting pathologists. In such circumstances it may be impossible to diagnose without the help of clonal studies for IgH chain gene rearrangement ( Figure 14). The other close mimicry includes 'Coeliac disease' as both IPSID and 'Coeliac disease' are characterised by lymphoplasmacytic infiltrate and villous atrophy. In these cases demographics are important; also gluten free diet will lead to improvement of 'Coeliac disease' cases. Intra-intraepithelial lymphocytosis with surface epithelial damage shall also favour Coeliac disease . As some cases of IPSID particularly if untreated may transform into aggressive lymphomas like 'Diffuse large B-cell lymphoma' (DLBCL), recognition of subtle features and follow-up is of paramount importance, particularly in endemic regions.

Hepatitis B virus (HBV), Hepatitis C virus (HCV) & Hepatocellular carcinoma (HCC)
Developing countries bear major burden of 'Hepatitis B & C' for the obvious reasons i.e., insufficient or no screening of transfused blood, multiple use of contaminated needles, drug abuse and overall poor safety standards (Jafri et al, 2006). Pakistan for instance carries a very high burden of hepatitis B & C. There are estimated 7-9 million carriers of hepatitis B with a carrier rate of 3-5% . Genotype D (63.71%) is the most prevalent genotype in Pakistani population . The overall anti-HCV prevalence rate is 14-15% in general population of Pakistan (Idrees et al, 2009). Though hepatitis C is a major culprit for the reasons including increased potential to cause 'chronic liver disease' and 'no vaccination'; hepatitis B is still highly prevalent as well. A large proportion of population is still not vaccinated for hepatitis B, though now it is included in EPI (Extended Program of Immunization) program by the government and all newborns do get it. In a recent study from Pakistan out of 161 subjects with HCC, chronic HCV infection was identified as a major risk factor (63.44% of tested HCC patients) for the development of HCC (Idrees et a, 2009). The time from HCV infection to the clinical appearance of cancer ranged from 10-50 years. In this population with HCC among various genotypes of HCV, genotype 3a was predominant (40.96%), followed by 3b in 15.66%, 1a in 9.63% and 1b in 2.40%. On the face of such a high burden of Hepatitis B & C, hepatocellular carcinoma (HCC) is one of the common malignancies in our practice arising in a background of liver cirrhosis ( Figure 15). Besides several other environmental factors are also playing their role in the causation of HCC. In Karachi, a port city of about 15 million inhabitants with hot and humid climate, it is reported that in wholesale markets selling food commodities without proper packing and preservation, a very high content of 'aspergillus flavus' is isolated which is a known cause of HCC. Unfortunately HCC is a bad cancer and in our experience life expectancy at the time of diagnosis is not more than six months.

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www.intechopen.com Infectious Aetiology of Cancer: Developing World Perspective 331 Fig. 15. Photomicrograph of a liver biopsy in a patient infected with Hepatitis C. Note fibrous band dividing the liver parenchyma into varying size nodules ( A, Trichrome). Figure B shows a well differentiated hepatocellular carcinoma (HCC) arising in this patient (H&E).

Acknowledgement
Dr