Relapses in early‐stage follicular lymphoma frequently develop via a divergent evolution from their clonally related precursor cells

Follicular lymphoma (FL) develops through a stepwise acquisition of cooperative genetic changes with t(14;18)(q32;q21)/IGH::BCL2 occurring early at the pre‐B stage of B‐cell development. Patients with FL typically show an indolent clinical course, remitting and relapsing with the eventual development of resistance to treatments. Interestingly, the majority of transformed FL do not progress directly from FL but originate from their clonally related lymphoma precursor (CLP) cells. To examine whether such divergent tumour evolution also underpins the relapses in patients with early‐stage FL, we investigated by targeted next‐generation sequencing 13 cases (stage I = 9, stage II = 4), who showed complete remission (mean: 5 years; range: 1–11.5 years) following local radiotherapy but subsequently relapsed (≥2 in 5). A clonal relationship between the diagnostic FL and relapses was confirmed in 11 cases. In six cases, common and distinct variants were seen between the paired diagnostic and relapsed lymphomas, indicating their divergent evolution from a CLP. In two cases, different B‐cell clones were involved in the diagnostic and relapsed lymphomas, including one case involving two different BCL2 translocations. In the remaining five cases, the relapsed lymphoma developed via a linear progression (n = 4) or a mixed evolutionary path (n = 1). These findings may bear important implications in the routine diagnosis and management of relapsed FL. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Introduction
Follicular lymphoma (FL) is an indolent B-cell lymphoma characterised by a spectrum of somatic genetic changes, including t(14;18)(q32;q21)/IGH::BCL2, which occurs early at the pre-B stage of B-cell development in the bone marrow as a result of erroneous VDJ recombination.The majority of patients with FL undergo an indolent clinical course, remitting and relapsing, and eventually developing resistance to standard therapies, while 30% of cases may show transformation into a more aggressive lymphoma, often diffuse large B-cell lymphoma (DLBCL), also known as transformed FL (tFL) [1].Remarkably, the majority of tFL do not progress directly from FL but originate from their clonally related lymphoma precursor (CLP) cells [2][3][4].Most cases investigated in the literature are paired FL and transformed FL and do not have detailed information on clinical stage or treatments and their responses, particularly the complete remission (CR) status.In this study, we investigated the evolution of lymphoma relapse in a series of well-documented patients with early-stage FL, who had achieved CR following local radiotherapy.As there is no systemic therapy involved and, thus, no therapeutic effect on CLP cells, these cases provide a unique opportunity to study the natural history of CLP cells, greatly complementing previous studies on this topic.

Case materials
The use of archival tissues for research was approved by the ethics committees of the institutions involved.A total of 13 patients with early-stage FL (nine stage I, four stage II) were successfully investigated, and all achieved CR (mean: 6 years; range: 1-11.5 years) following local radiotherapy but subsequently relapsed (Figure 1).Seven cases had one lymphoma relapse, while the remaining six cases had two to five relapses.In seven cases, the relapsed lymphoma was FL, while in the remaining cases, the relapsed lesions were tFL (n = 2) or both FL and tFL (n = 4), including an EBV-positive LBCL (case 11).Paired formalin-fixed paraffin-embedded diagnostic and relapsed lymphoma biopsies were available in each case.

Interphase fluorescence in situ hybridisation (FISH)
FISH was performed to investigate BCL2 translocation using BCL2 Break Apart probes (Vysis, Abbott Park, IL, USA).

DNA extraction and quality assessment
Histology was reviewed, and areas rich in lymphoma cells (>30%) in each specimen were microdissected and subsequently subjected to DNA extraction and a qualitycontrol PCR [5].

Clonality analysis of IG gene rearrangements
This was performed by combining the BIOMED-2 PCR assays and Illumina MiSeq sequencing (Illumina Inc., San Diego, CA, USA) [6].

Targeted next-generation sequencing (NGS)
A customised panel of 191 genes for FL and DLBCL was used for mutational analyses (supplementary material, Table S1).Targeted NGS was performed with 100 ng DNA using TWIST capture (TWIST Bioscience, San Francisco, CA, USA) and the Illumina NextSeq platform.In seven specimens with suboptimal DNA quality, targeted sequencing was performed in duplicate (supplementary material, Tables S2 and S3).The gene panel and NGS methodology are detailed in Supplementary materials and methods [5][6][7].

Results and discussion
Interphase FISH showed BCL2 translocation in six cases, BCL2 amplification (up to eight copies) in one case and BCL2 copy number gain (three to four copies) in three cases.In three cases, both diagnostic and relapsed lymphomas were investigated by interphase FISH, with identical or similar results between the paired samples.
Adequate sequencing coverage (>98% with a minimum of 100 reads after deduplication) for all the targeted genes was achieved in each specimen investigated.The variants detected were typical of those seen in FL and DLBCL.There was no significant difference in the number of potentially pathogenic mutations detected between the diagnostic (mean: 7.8/sample; range: 1-13) and relapsed FL (mean: 7.9/sample; range: 2-13) excluding tFL.Both diagnostic and relapsed FL (excluding tFL) showed similar mutation frequencies in BCL2, CREBBP, EP300, KMT2D, TNFRSF14 and STAT6 (supplementary material, Figure S1).There was no association between STAT6 mutation and BCL2 translocation status or CD23 expression by immunohistochemistry.
To investigate the clonal evolution of the diagnostic and relapsed lymphomas, we compared all somatic variants in the paired samples, including pathogenic, benign and synonymous variants and variants in BCL2 5'-and 3'-untranslated region (UTR).The clonal relationship between the diagnostic FL and relapses was confirmed in 11 cases by their shared somatic variants.
In six cases, both common and distinct variants were seen between the paired diagnostic and relapsed lymphomas, indicating divergent evolution of these lesions from a CLP (Figure 2).The malignant status of the inferred CLP cells is uncertain.In situ follicular B-cell neoplasia (ISFN), a premalignant lesion, may harbour variable numbers (zero to four) and combinations of pathogenic mutations [6,8,9].In four of these cases (nos. 1, 3, 5 and 6), the inferred CLP cells only harboured one to three potentially pathogenic mutations (excluding benign changes) and are thus probably premalignant.In the remaining two cases (nos. 2 and 4), the inferred CLP cells harboured ≥7 potentially pathogenic mutations in addition to IGH::BCL2, and they are likely malignant or in the phase of malignant transformation.
In two cases, there was evidence of two B-cell clones that gave rise to multiple lymphomas independently.In one case (no.7), different IGH rearrangements were seen between FL-D and tFL-R2 (Figure 3).There were also no shared somatic variants between the diagnostic FL and the two 'relapsed' lesions, in keeping with their derivation from different B-cell clones.In the other case (no.8), interphase FISH with the BCL2 breakapart probe demonstrated a BCL2 translocation in each of these lymphomas but displayed different constellation patterns.FL-D and FL-R1 showed evidence of an unbalanced BCL2 translocation (one or two co-localised red/green and one red signal), while FL-R2 and FL-R3 displayed a classic breakage pattern (one co-localised red/green, one red and one green signal) (Figure 3).Immunohistochemistry showed strong and uniform BCL2 expression in the lymphoma cells of these specimens.PCR for IGH::BCL2 genomic fusion did not yield any products within the expected size range in each of these specimens.Nonetheless, clonality analyses displayed distinctly sized FR2-JH products between FL-R1 and FL-R2, indicating different IGH rearrangements, and this was further confirmed by sequencing these PCR products (Figure 3).In line with these findings, FL-D and FL-R1 shared one CREBBP mutation, while FL-R2 and FL-R3 had four common mutations.However, there were no common variants between FL-D/FL-R1 and FL-R2/ FL-R3.Taken together, these findings indicate that FL-D/ FL-R1 and FL-R2/FL-R3 originated from two different B-cell clones, which also harboured different BCL2 translocations.
In four cases (nos.9-12), mutations were largely cumulative in consecutive lesions, suggesting linear progression of the relapsed lymphoma (Figure 4).In each of these cases, there were variable numbers (one to 15) of shared somatic variants between the diagnostic and relapsed lymphomas.Although more genetic changes might be demonstrated by more extensive sequencing beyond the 191 FL/DLBCL-associated genes investigated, this is unlikely to alter the conclusion of linear progression in these relapsed lesions.In the remaining case (no.13), the relapsed lymphoma appeared first to follow a linear progression, then a divergent evolutionary path (Figure 4).
As many of the genes mutated in ISFN are affected by the off-target activities of the somatic hypermutation machinery and may confer no or moderate oncogenic activities, there exists a considerable challenge in stratifying the risk posed by these mutations and their combinations in lymphoma development [10,11].Nonetheless, CREBBP mutations within the KAT domain have been found preferentially in the lymphoma precursor cells that eventually develop into overt FL [12].In line with this, the majority of CREBBP mutations found in the present study are within the KAT domain, seen in nine cases, including in the inferred CLP cells in two of the eight cases where divergent evolution enables CLP prediction (supplementary material, Figure S2).
The preceding findings raise critical clinical implications in the routine diagnosis and management of recurrent lymphoma in patients with FL.In routine clinical follow-up of patients with FL after therapy, re-biopsy is usually not considered unless a high-grade transformation is suspected.Even when a re-biopsy is performed, the routine diagnostic workup only includes IG gene clonality and BCL2 translocation analyses, not mutation profiling.Importantly, neither IG gene clonality nor BCL2 translocation analyses can address the evolutionary path of the relapsed lymphoma, despite providing evidence about clonal relationship to the original FL.
As the majority of relapsed lymphomas (both FL and DLBCL) in patients with FL originate from their clonally related CLP cells via a divergent evolution or a different B-cell clone, these relapsed lymphomas are in fact a new tumour, rather than a true relapse.As new lymphomas, they responded to the same or similar treatments without the need for escalating the therapy, unless they were high-grade tumours.In this context, it is important to ascertain whether a recurrent lymphoma is a new tumour or a true relapse.
The aforementioned findings, along with the evidence of divergent evolution in the majority of tFL, challenge the current therapeutic strategy of FL because the IGH::BCL2-positive premalignant cells largely underpin the lymphoma recurrence and their low proliferation and indolent nature most likely render them poorly responsive to immunochemotherapy designed for FL.Studies of ISFN have shown that IGH::BCL2-positive premalignant cells can actively transit in peripheral lymphoid tissues undergo relentless clonal expansion through germinal Evolution of recurrent follicular lymphoma  centre reactions while at risk of acquiring genetic changes due to exposure to somatic hypermutation and switch recombination activities, as well as apoptosis evasion by BCL2 overexpression [6,8,9,13].This insidious clonal evolution is formidable, inevitably propelling independent malignant transformation and, hence, the development of a new lymphoma.This likely explains why FL is incurable based on the current therapeutic strategies despite being a low-grade tumour and highlights the need to control the IGH::BCL2-positive premalignant cell population in order to achieve a cure.
In summary, recurrent lymphomas in patients with early-stage FL frequently develop via a divergent evolution from their clonally related CLP cells or a different B-cell clone, further underscoring the potential of these premalignant cells in multi-lymphoma development.

Figure 1 .
Figure 1.Summary of cases with early-stage FL and clinical follow-up data.Complete remission (CR) was achieved following each treatment unless indicated (nCR: non-complete remission).CT, chemotherapy; RT, radiotherapy; tFL, transformed FL; WW, watch and wait.

Figure 2 .
Figure 2. Cases showing divergent evolution in FL relapse.CLP, predicted clonally related lymphoma precursor cells, and their mutations common to both diagnostic and recurrent lymphoma are indicated; CR, complete remission; D, diagnostic biopsy; FL, follicular lymphoma with grade indicated; R, relapsed lesions; SHM: somatic hypermutation; variants in blue are potentially pathogenic, while variants in green are synonymous or benign changes or present in untranslated region (UTR); tFL: transformed FL.

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Makker et al © 2023 The Authors.The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.www.pathsoc.orgJ Pathol 2024; 262: 289-295 www.thejournalofpathology.com

Figure 3 .
Figure 3. Cases showing involvement of different B-cell clones in FL relapse.In case 7, the FL at diagnosis and recurrent FL (FL-R1 and tFL-R2) do not harbour common mutations and also bear different IGH gene rearrangement, so they are derived from different B-cell clones.In case 8, FL-D/FL-R1 and FL-R2/FL-R3 originate from two different B-cell clones as shown by their different mutation patterns, BCL2 breakapart FISH signal constellation and IGH rearrangements.Arrows in FISH panel indicate breakage of green and red probe signals.CLP, predicted clonally related lymphoma precursor cells and their mutations common to both diagnostic and recurrent lymphoma are indicated; CR, complete remission; D, diagnostic biopsy; R, relapsed lesions; SHM, somatic hypermutation; variants in blue are potentially pathogenic, while variants in green are synonymous or benign changes or present in untranslated region (UTR).