Intermediate steps in FL development

The long latency period to disease presentation seen in allo-HSCT derived FL cases, suggests FL development is protracted, taking years to develop the disease. This ties in with the multi-step pathway to lymphomagenesis, requiring several genetic hits in addition to the t14;18 translocation. Whilst studies have suggested epigenetic alterations to be early events in disease evolution, the exact steps and time points at

46 which they are acquired is still unknown. However, now we have begun to identify potential precursor cells and lesions in which such genetic mutations may arise and accumulate in to develop overt disease. The current model of FL pathogenesis from these presumed precursor lesions is summarised in Figure 1.4.

Although circulating t14;18 positive cells termed follicular lymphoma-like B cells (FLLCs) are found in healthy individuals who never go on to develop the disease, they harbour features which are reminiscent of FL cells including expression of the GC B cell marker CD10, and upregulation of BCL2. Furthermore, FLLCs have intraclonal diversity (Agopian et al., 2009), suggestive of repetitive GC entry where they can undergo AID-mediated SHM and/or CSR. Indeed, FLLCs have been shown to have a GC-experienced memory B cell phenotype (Roulland et al., 2006b, Hirt et al., 2007), supporting their increased exposure to AID. One study also found that some FLLCs have aberrant ongoing AID activity which is normally downregulated following GC exit (Agopian et al., 2009). As AID introduces aberrant mutations in non-Ig genes which are usually proto-oncogenes, such as BCL6, PIM1, MYC and PAX5 (Rossi et al., 2005, Pasqualucci et al., 2001, Liso et al., 2006) increased exposure may increase genomic instability, leading to accumulation of mutations in FLLCs and their eventual transformation to overt FL.

The direct relationship between FLLCs and FL was established in a study which found that the frequency of FLLCs can be used as a predictive marker for FL development. Pre- diagnostic blood samples were available for 100 FL patients (Roulland et al., 2014). Samples were available due to the patients being involved in an earlier study before the occurrence of overt disease. t14;18 positive cells were screened using sensitive PCR assays and clonal relationships between FLLCs and tumour samples were assessed by analysing the t14;18 breakpoints. All FLs and identified t14;18 positive cells in the paired pre-diagnostic blood sample were clonally related, establishing the FLLCs as the disease precursor cell. Furthermore, pre-diagnostic blood samples from these patients had a higher frequency of t14;18 positive cells when compared to samples taken from a control group who didn’t go on to develop the disease (n=218). They concluded that individuals with t(14;18) frequency reaching 1 in every 10,000 blood cells had a 23-fold greater risk of progression to FL. This predictive value did not change depending on

47 whether FLLCs were harvested close to disease onset or many years before. This highlights the long latency period between pre-malignancy to diagnosis, suggesting the overt disease is preceded by a preclinical, asymptomatic phase of clonal growth and establishment of a precursor clone that takes years, if not decades to manifest. This supports the hypothesis of a long-lived and dormant CPC population. This was also seen in cases of donor-derived FL, in which t14;18 positive cells detected at transplantation within DLIs contained the same BCL2:IGH rearrangement as that found in the FL clone of the donor and recipient years later (Weigert et al., 2012).

t14;18 positive cells are also found incidentally in GCs of 2-3% of reactive lymph nodes of healthy adults, forming unconventional foci within a background of BCL2 negative B cell follicles (Cong et al., 2002, Henopp et al., 2011). They are monoclonally derived and colonise several GCs. They are termed in situ follicular neoplasia (ISFN), formerly known as FL in situ. In a study of 21 patients with FLIS, <5% progressed to a clonally related FL (Jegalian et al., 2011). However, the study was limited by the small cohort size and a restricted median follow up 118 months. A study by Mamessier et al utilised high- resolution comparative genomic hybridization microarrays on laser-capture micro- dissected ISFN cases to reveal that there is a degree of genetic instability in these lesions, albeit at a much lower number when compared to overt FL (Mamessier et al., 2014). A study by Schmidt utilised paired FL-ISFN samples to analyse the genetic relationship between FL and its pre-malignant lesion (Schmidt et al., 2014). ISFN samples showed little to no secondary genetic changes. Only one case had an EZH2 mutation shared between biopsies. The low genetic complexity and semblance of ISFN with clonally related FL highlights that several intermediate steps are necessary for disease manifestation from precursor cells.

As FLLCs and ISFN are considered precursors in lymphomagenesis and express t14;18, we can infer that the survival advantage offered by constitutive expression of BCL2 enables the B cell to acquire and accumulate genetic aberrations over the years, if not decades, to enable malignant transformation. The relationship between FLLCs and ISFN remains undetermined, however, Cheung et al found that in one ISFN case, a paired blood sample contained B cells with an identical t14;18 rearrangement to the ISFN cells

48 (Cheung et al., 2009b). This supports the hypothesis that FLLCs represent the circulating counterpart of ISFNs. The selection and commitment of some ISFN to develop into FL whilst others do not suggest a diverse number of unknown factors at play in contributing to lymphomagenesis.

Whilst in ISFN the lymph node architecture is preserved, in partial involvement by FL (PFL), the affected follicles are larger with ill-defined marginal zones and attenuated mantle cuffs. Therefore, histologically, PFL has a greater semblance to FL. This is supported in a follow-up study that found that 53% of untreated PFL patients went on to develop overt FL over a fourteen year period (Jegalian et al., 2011) compared to only 5% of FLIS patients. PFL also has significantly more genetic alterations compared to FLIS as evidenced in array comparative genomic hybridization studies (Mamessier et al., 2014). Some of the amplified genes were also found to be amplified in low-grade FL, indicating a selective pressure for the high malignant transformation of PFL patients. These recurrent mutations included TNFRSF14, EZH2, and MLL2. However it’s important to note that genomic alterations and gains/losses within the PFL cases was still low compared to overt FL and that most cases present with low stage disease, suggesting that PFL does not simply represent a partial colonisation of the lymph node by FL, but is rather a precursor lesion (Adam et al., 2005).

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Figure 1.4: Model of FL pathogenesis based on presumed t14;18 positive precursor cells. In the bone

marrow (BM), naïve B cells acquire the t(14:18) translocation as a result of defective VDJ recombination. Cells exit the BM and enter peripheral circulation in which cells are termed FLLC. Cells colonise secondary lymphoid tissues where they undergo the GC reaction, characterised by somatic hypermutation (SHM) and class switch recombination (CSR). Constitutive expression of BCL2 protein provides a survival advantage, rescuing cells from apoptosis regardless of BCR affinity. ISFN is believed to represent this pre malignant precursor stage. Cells undergo clonal expansion within the GC and can exit into peripheral circulation. They are able to re-enter the GC multiple times as seen by FLLCs presenting a GC-experienced memory B cell phenotype (Roulland et al., 2006b, Hirt et al., 2007), where they have opportunity to increase their genomic instability through repeated exposure to AID. As PFL has a greater genomic complexity than ISFN (Mamessier et al., 2014), they are believed to represent a more differentiated precursor cell. The gain of specific genetic and epigenetic hits ( ) then leads to overt disease and t-FL.