The Effect of Cytomegalovirus infection on Follicular Lymphoma biology

Follicular lymphoma (FL) is an incurable human B-cell neoplasm that follows a chronic relapsing course requiring multiple treatment episodes and culminating in therapy resistance and/or large-cell transformation. The disease is notable for its clinical variability but the biological basis for this variability is poorly understood. In addition to intrinsic genetic and epigenetic alterations that drive tumour growth, an intricate cross-talk between malignant B cells and the immune microenvironment have been shown to play a significant role in the pathogenesis of FL. This thesis tests the hypothesis that some of the heterogeneity of FL might result from chronic infection with cytomegalovirus (CMV). The latter affects ~50% of the adult population and has been linked to premature immune senescence and expedited age-related alterations in the T cell repertoire. I reasoned that CMV infection might have two distinct effects in FL: (1) by accelerating immune senescence, it might increase the risk of treated-related infection; and (2) by altering the lymphoma microenvironment, it might modulate the effect of chemo-immunotherapy. To address these questions, I was able to access stored PMBCs, serum and tissue samples from patients of known CMV serostatus who were recruited into the PACIFICO trial. The trial compared two different chemo-immunotherapy regimens in older patients with FL and recruited 360 patients between 2009 and 2015. My experimental approach was to compare CMV-positive and CMV-negative patients for blood lymphocyte subsets by flow cytometry (Chapter 3), serum cytokines by Luminex profiling (Chapter 4), accessory cells in the lymphoma microenvironment by immunofluorescence of biopsy material (Chapter 5), and treatment effectiveness and toxicity using follow-up information obtained as part of the trial (Chapter 6). Chapters 3 and 4 employed a cohort of 42 cases (21 of which were CMV- positive) that were carefully selected to be well balanced in other respects. Chapter 5 employed a similar cohort of 42 cases (20 of which were CMV- positive) including 6 that were also in the cohort used for Chapter 4 (it was not possible to use exactly the same cases for Chapters 3, 4 and 5 owing to the limited availability of biopsy material). Chapter 6 related CMV serostatus to clinical outcome in the two cohorts employed for Chapters 3,4 and 5, and also in a third cohort consisting of all 269 patients of known CMV serostatus who were recruited into PACIFICO (109 of which were CMV- positive). Finally, Chapter 7 examined the relationship between CMV-dependent biological variables and treatment efficacy with the aim of shedding light on the mechanisms through which CMV might modulate therapy response in FL. Flow cytometric analysis of PBMCs revealed significant accumulation of end-stage differentiated (TEMRA) T cell subtypes including pE2-CD4 (P=0.046), E-CD4 (P=0.029), EM3-CD4 (P=0.018), E-CD8 (P=0.033) and EM3-CD8 (P=0.005) phenotypes as well as increased NKT cells (P=0.031) in CMV-positive patients. Luminex data showed significantly reduced levels of multiple serum cytokines including IL-9 (P=0.006), IL-17A (P=0.020), FGF-basic (P=0.028), MIP-1α (P=0.039), MIP-1β (P=0.029), IL-6 (P=0.029) and IL-8 (P=0.037) in the CMV-positive group. Taken together, these findings support the hypotheses that CMV infection in FL is associated with accelerated immune senescence with the accumulation of exhausted T cells and reduced cytokine production. Immunofluorescence staining of biopsy material revealed that the CMV-positive group expressed significantly higher levels of CD21 (expressed by follicular dendritic cells; P=0.043) and tended to express higher levels of FOXP3 (expressed by Tregs; P= 0.069) and PD-1 (expressed by exhausted T cells; P=0.097). Analysis of therapy response in the two cohorts used for lymphocyte/cytokine profiling and tissue staining showed an overall trend for higher complete remission (CR) rates in the CMV-positive groups with a significant difference observed in the first cohort after 4 cycles of therapy (P=0.020). This finding could not be confirmed in the overall trial cohort. However, the CMV-positive group in this cohort contained more patients with adverse prognostic features, potentially biasing it towards inferior therapy response. Interestingly, a significant association was observed between the achievement of CR and the number of circulating E-CD4+ T cells but not serum cytokine levels or the tissue accessory cells expressing CD21, FOXP3 or PD-1. Analysis of toxicity data by CMV status showed a significant association between CMV positivity and heterologous infection of any grade. This association was observed in the overall trial cohort (P<0.001) as well as in the cohort used for tissue staining (P=0.017). Taken together, my findings support the hypothesis underlying this thesis, namely that the biology of FL is influenced by chronic CMV infection. Specifically, CMV-positive patients with FL undergo accelerated immune senescence and experience more infections following rituximab-containing chemo-immunotherapy. CMV-positive patients may also achieve better responses, possibly due to the higher numbers of cells expressing Fcγ receptors required for antibody-dependent cell-mediated cytotoxicity (ADCC). Based on my findings, a case could be argued for the routine application of CMV screening in FL prior to treatment with chemo-immunotherapy with the implementation of enhanced infection surveillance in CMV-positive patients. If properly harnessed, these understandings can ultimately improve on the therapeutic strategies in FL management toward a combinatorial approach for direct cytotoxic and indirect immunomodulatory perspectives.