**4. Pathogenesis of lymphoma in IBD**

Lymphoma is a clonal expansion of B- and T- lymphocytes caused by the accumulation of a series of genetic mutations affecting proto-oncogenes and tumour suppressor genes. This results in dysregulated proliferation, evasion of immune surveillance mechanisms and inhibition of apoptosis (Jaffe et al., 2001). Significant progress has been made in to the understanding of these mechanisms at a molecular level. The activation of oncogenes by aberrant chromosomal translocations as well as the inactivation of tumour suppressor genes by chromosomal deletion or mutation are both important mechanisms of lymphomagenesis (Kuppers et al., 1999). Oncogenic viruses such as EBV and HTLV1 can also introduce foreign genetic sequences into the lymphocyte genome causing disruption of normal function (Neri et al., 1991).

There are a number of genetic, environmental, infectious and iatrogenic factors amongst patients with inflammatory bowel disease which can predispose to increased susceptibility to these mechanisms for the development of lymphoma:


Evaluating Lymphoma Risk in Inflammatory Bowel Disease 321

2001).

Wong et al describe a case of synchronous colonic adenocarcinoma and lymphoma and demonstrated that EBV was present in the lymphomatous tissue but not in the invasive adenomatous tissue (Wong et al., 2003). A series of IBD patients from the Mayo clinic identified 12 patients diagnosed with lymphoma between 1993 and 2000, half of whom were on azathioprine therapy. The lymphomas of five out of these six patients on azathioprine were EBV positive whereas only one out of the six azathioprine-naïve patients was EBV positive (Dayharsh et al., 2002). This study suggests a link between azathioprine therapy and EBV driven lymphoma in IBD though the numbers were too small to reach statistical significance. In the CESAME prospective study of over 21,000 French IBD patients, 9 of the 13 cases of lymphoma in patients on azathioprine were EBV positive with up to 16 years exposure to the drug (Beaugerie et al., 2009a). Reijasse et al measured EBV viral loads in patients with Crohn's disease and EBV sero-positive controls. There was no difference in viral loads between the two groups irrespective of immunomodulator or biological therapy but a minority of patients did have transient, very high EBV viral loads (Reijasse et al., 2004). It is not clear, whether these peaks in EBV viral load are associated with lymphoma risk but this does appear to be the case in post-transplant patients where EBV viral load can predict this outcome (Stevens et al.,

The pathobiology of EBV and its role in lymphomagenesis is complex. The hostincorporated EBV genome encodes a number of proteins with similarities to a variety of cytokines, anti-apoptotic molecules and signal transducers that can immortalise and

The risk of other oncogenic viruses such as HLTV1 is not well described in the IBD literature. A recent meta-analysis suggested a lower prevalence of *Helicobacter Pylori* infection in IBD patients compared to control groups but its association with gastric

In order to evaluate any causality between IBD and the risk of lymphoma, it is extremely important to appreciate the quality of safety data available. Frequently, this information is

Randomised controlled drug trials collate information regarding adverse events but they are powered to elucidate differences in efficacy and not safety. They also tend to have relatively small numbers and a short follow up period which may not reflect the true incidence of late or delayed adverse events. Some useful safety information is available from observational studies of large populations. These have large numbers and long follow up but are susceptible to indication bias and often have other confounding factors. Case controlled series have an efficient methodology but may be hampered by the shortcomings of control selection. The most common form of safety data comes from case reports and case series which are able to identify rare risks. However, the inherent positive bias with this form of evidence, does not allow it to be utilised for risk quantification or for providing proof of causality. Post marketing surveillance, a form of *pharmaco-vigilence*, is another important source of safety data. This information may be made available through institutional

mutate infected cells (Sokol and Beaugerie, 2009).

MALT-oma is well documented (Luther et al., 2010).

**5. Lymphoma risk in the literature** 

flawed and difficult to interpret.

**5.1 Quality of data** 

NOD1 which in its wild-type activates nuclear factor kappa B (NF-κB) (Ogura et al., 2001). NF-κB is a tightly regulated mediator of T- and B-lymphocytes and alterations in its signalling pathway have been implicated in a number of malignancies including lymphoma (Jost and Ruland, 2007). Although some plausibility exists, this link remains to be established.

 **Therapeutic immune modulation** – Immunomodulatory drugs such as the thiopurines, (azathioprine and mercaptopurine), methotrexate, and the anti-TNF drugs (infliximab, adalimumab and certolizumab) have become standard treatment for complicated IBD. These drugs exert their effects through a number of mechanisms which are incompletely understood. It is recognised that AZA and its metabolites suppress intracellular inosinic acid synthesis which interferes with intracellular purine synthesis resulting in a down regulation of B- and T-cell proliferation (Bacon and Salmon, 1987). Thiopurine nucleotides also incorporate into lymphocyte DNA disrupting structure, repair mechanisms and promoting mutagenesis (Ling et al., 1992). There is also evidence that azathioprine renders DNA highly sensitive to damage to ultraviolet (UVA) radiation and this may account for the increased risk of non-melanomatous skin cancer in patients treated with thiopurines (O'Donovan et al., 2005). A recent study showed that IBD patients on thiopurine therapy had significantly more somatic mutations in circulating T-lymphocytes than in a thiopurine-naïve control group (Nguyen et al., 2009).

The impact of anti-TNF drugs on the risk of mutagenesis has not been adequately studied. It is conceivable that interruption of TNF signalling disrupts immune surveillance mechanisms and alters the normal detection and elimination of cells with chromosomal abnormalities.

At higher doses, methotrexate is cytotoxic, whereas the lower doses used in IBD patients are known to alter T-cell derived cytokines in inflammatory states. It inhibits pro-inflammatory cytokines such as interleukin-12, interferon-γ and tumour necrosis factor-α whilst promoting anti-inflammatory cytokines such as interleukin-10 (van Dieren et al., 2006). These cytokines have fundamental effects on lymphocyte proliferation and function but the specific mechanisms which may contribute to potential lymphoma development are not known.

 **Immunosuppression –** The increased risk of lymphoma in patients with immunodeficiency states such as HIV infection (Serraino et al., 1992) and posttransplant immunosuppression (Grulich et al., 2007a) is well recognised and many of these cases are EBV positive. The increased risk of opportunistic infections amongst IBD patients on immunomodulators therapy is also well documented. Toruner et al identified 100 cases of opportunistic infections over an 8 year period on their database of IBD patients from the Mayo Clinic and found that treatment with thiopurines conferred an Odds Ratio of 3.1 (Toruner et al., 2008). The majority of these opportunistic infections were caused by viruses including cytomegalovirus, Herpes simplex virus and Epstein-Barr virus.

EBV is a widely disseminated human Herpes virus which has been associated with a number of different types of B-cell lymphoma, particularly mixed cellularity and lymphocyte depleted classical Hodgkin's lymphoma, Burkitt's lymphoma and posttransplant lymphoproliferative disorder (PTLD). EBV viral load can predict risk of PTLD (Stevens et al., 2001) and cases of infectious mononucleosis with early transformation to lymphoma have been described (Owen et al., 2010). Interestingly,

 **Therapeutic immune modulation** – Immunomodulatory drugs such as the thiopurines, (azathioprine and mercaptopurine), methotrexate, and the anti-TNF drugs (infliximab, adalimumab and certolizumab) have become standard treatment for complicated IBD. These drugs exert their effects through a number of mechanisms which are incompletely understood. It is recognised that AZA and its metabolites suppress intracellular inosinic acid synthesis which interferes with intracellular purine synthesis resulting in a down regulation of B- and T-cell proliferation (Bacon and Salmon, 1987). Thiopurine nucleotides also incorporate into lymphocyte DNA disrupting structure, repair mechanisms and promoting mutagenesis (Ling et al., 1992). There is also evidence that azathioprine renders DNA highly sensitive to damage to ultraviolet (UVA) radiation and this may account for the increased risk of non-melanomatous skin cancer in patients treated with thiopurines (O'Donovan et al., 2005). A recent study showed that IBD patients on thiopurine therapy had significantly more somatic mutations in circulating T-lymphocytes than in a thiopurine-naïve control group

The impact of anti-TNF drugs on the risk of mutagenesis has not been adequately studied. It is conceivable that interruption of TNF signalling disrupts immune surveillance mechanisms and alters the normal detection and elimination of cells with

At higher doses, methotrexate is cytotoxic, whereas the lower doses used in IBD patients are known to alter T-cell derived cytokines in inflammatory states. It inhibits pro-inflammatory cytokines such as interleukin-12, interferon-γ and tumour necrosis factor-α whilst promoting anti-inflammatory cytokines such as interleukin-10 (van Dieren et al., 2006). These cytokines have fundamental effects on lymphocyte proliferation and function but the specific mechanisms which may contribute to

 **Immunosuppression –** The increased risk of lymphoma in patients with immunodeficiency states such as HIV infection (Serraino et al., 1992) and posttransplant immunosuppression (Grulich et al., 2007a) is well recognised and many of these cases are EBV positive. The increased risk of opportunistic infections amongst IBD patients on immunomodulators therapy is also well documented. Toruner et al identified 100 cases of opportunistic infections over an 8 year period on their database of IBD patients from the Mayo Clinic and found that treatment with thiopurines conferred an Odds Ratio of 3.1 (Toruner et al., 2008). The majority of these opportunistic infections were caused by viruses including cytomegalovirus, Herpes simplex virus and

EBV is a widely disseminated human Herpes virus which has been associated with a number of different types of B-cell lymphoma, particularly mixed cellularity and lymphocyte depleted classical Hodgkin's lymphoma, Burkitt's lymphoma and posttransplant lymphoproliferative disorder (PTLD). EBV viral load can predict risk of PTLD (Stevens et al., 2001) and cases of infectious mononucleosis with early transformation to lymphoma have been described (Owen et al., 2010). Interestingly,

to be established.

(Nguyen et al., 2009).

Epstein-Barr virus.

chromosomal abnormalities.

potential lymphoma development are not known.

NOD1 which in its wild-type activates nuclear factor kappa B (NF-κB) (Ogura et al., 2001). NF-κB is a tightly regulated mediator of T- and B-lymphocytes and alterations in its signalling pathway have been implicated in a number of malignancies including lymphoma (Jost and Ruland, 2007). Although some plausibility exists, this link remains Wong et al describe a case of synchronous colonic adenocarcinoma and lymphoma and demonstrated that EBV was present in the lymphomatous tissue but not in the invasive adenomatous tissue (Wong et al., 2003). A series of IBD patients from the Mayo clinic identified 12 patients diagnosed with lymphoma between 1993 and 2000, half of whom were on azathioprine therapy. The lymphomas of five out of these six patients on azathioprine were EBV positive whereas only one out of the six azathioprine-naïve patients was EBV positive (Dayharsh et al., 2002). This study suggests a link between azathioprine therapy and EBV driven lymphoma in IBD though the numbers were too small to reach statistical significance. In the CESAME prospective study of over 21,000 French IBD patients, 9 of the 13 cases of lymphoma in patients on azathioprine were EBV positive with up to 16 years exposure to the drug (Beaugerie et al., 2009a). Reijasse et al measured EBV viral loads in patients with Crohn's disease and EBV sero-positive controls. There was no difference in viral loads between the two groups irrespective of immunomodulator or biological therapy but a minority of patients did have transient, very high EBV viral loads (Reijasse et al., 2004). It is not clear, whether these peaks in EBV viral load are associated with lymphoma risk but this does appear to be the case in post-transplant patients where EBV viral load can predict this outcome (Stevens et al., 2001).

The pathobiology of EBV and its role in lymphomagenesis is complex. The hostincorporated EBV genome encodes a number of proteins with similarities to a variety of cytokines, anti-apoptotic molecules and signal transducers that can immortalise and mutate infected cells (Sokol and Beaugerie, 2009).

The risk of other oncogenic viruses such as HLTV1 is not well described in the IBD literature. A recent meta-analysis suggested a lower prevalence of *Helicobacter Pylori* infection in IBD patients compared to control groups but its association with gastric MALT-oma is well documented (Luther et al., 2010).
