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Molecular subclusters of follicular lymphoma: a report from the United Kingdom’s Haematological Malignancy Research Network

Visual Abstract

• Subtypes of follicular lymphoma are identified by modelbased clustering of genetic mutations in a large (n = 548) population-based cohort.
• These subtypes imply distinct mechanistic subsets of follicular lymphoma with implications for patient risk and treatment opportunities.

Introduction
The molecular pathogenesis of follicular lymphoma (FL) is closely linked to that of diffuse large B-cell lymphoma (DLBCL) and the physiological germinal center (GC) reaction. 1 Key factors connecting these malignancies to GC biology include BCL2 and BCL6 gene translocations, deregulating apoptotic and transcriptional control, along with aberrant somatic hypermutation (aSHM). [2][3][4][5][6][7][8] The latter provides a mechanistic link between aberrant targeting of enhanced mutagenesis that underpins antibody affinity maturation and the increased rate of B-cell neoplasia associated with the GC reaction. Targets of aSHM have been identified in observational and experimental contexts. 8,9 Mutation profiling has detected additional pathways deregulated in FL; recurrent mutations in epigenetic regulators KMT2D (MLL2) and CREBBP have identified these as candidate early initiating FL pathogenic events. [10][11][12] EZH2 has emerged as a promising therapeutic target following the identification of mutations targeting this histone methylase in FL and studies establishing the importance of this epigenetic regulator in GC biology. 13 Beyond epigenetic regulators, recurrent mutations have been detected in genes on various pathways, including cytokine/STAT activation and the B-cell receptor. 12,[14][15][16] In DLBCL, mutational heterogeneity resolves into recurrent patterns, which have been independently identified by several groups. [17][18][19][20][21][22] While existing data suggest less mutational heterogeneity in FL than DLBCL, FL is not a homogenous entity; whether recurrent patterns of cooccurring mutations allow distinct subtypes of FL to be resolved remains an open question.
In contrast to the aggressive but potentially curable DLBCL, FL is by nature indolent and generally incurable. Seminal studies have addressed the question of FL progression. 12,15 Aggressive disease may remain morphologically consistent with FL or undergo transformation to a pattern that meets the diagnostic criteria for DLBCL. Morphological transformation to DLBCL is linked to branching clonal evolution and emergence of mutations and subclones that are rare or undetectable at diagnosis, while progression without transformation may be linked to the persistence of subclones that are detectably present from diagnosis. 12,23 Predicting patients with FL at high risk of early disease progression and/or transformation is clinically important. For DLBCL, recurrent patterns of cooccurring mutations can be used to identify risk groups and suggest novel treatment strategies. [18][19][20][21] For FL, a molecular risk predictor, the m7-FLIPI, 24 which integrates mutational with conventional risk factors, has been derived from clinical trials; however, the extent to which patterns of mutation may inform outcome in a wider patient population remains uncertain. 25 Using targeted sequencing to classify DLBCL diagnostic samples from our large "real-world" population-based cohort, we recently reported mutational profiling findings that both confirmed and extended those of others, identifying 5 robust DLBCL subclusters. 20 Here, we extend this approach to 548 patients with FL with the aim of assessing whether comparable subclusters can be defined in FL using the same panel, sequencing and assessing surplus material archived at the time of diagnosis for somatic mutations against a pan-hematological malignancy panel of 293 genes. Our analysis, which identifies 3 subsets of FL based on somatic mutational patterns, has implications for pathogenesis, risk stratification, and future treatment strategies.

Mutation signature analysis
All variant calls for each sample were analyzed with Signal (https:// signal.mutationalsignatures.com/) 29 using default parameters. The nucleotide exchange patterns in the triplet context, along with contributing mutational signature estimates, were generated with default parameters (Bootstraps: 100; Threshold: 5; P value = .05) for either all mutations across defined sets of genes linked to mutational clusters or for individual cases.

Statistical analysis
To identify subclusters defined by genetic features, binary mutation data were modeled as a finite mixture of Bernoulli distributions, with the number of clusters selected using the Bayesian Information Criterion (BIC), while the Akaike Information Criterion (AIC) was applied to identify the finer structure. 20 As the signal-to-noise ratio using all available genetic features was low, varying thresholds were applied, selecting only genetic features for the cluster analysis occurring in specified proportions of the study cohort (supplemental Methods section 2.4). The definitive analysis was chosen by finding clear BIC/AIC signals for cluster identification. Thirty-one genetic features were used in the cluster analysis; of these, all 22 targets of somatic hypermutations were included, as well as 9 nonsomatic hypermutation features that occurred in 60 or more patients. These features are listed in supplemental Table 1

Mutation distribution in FL
Demographic and clinical characteristics of the source cohort (n = 852), the full study cohort (n = 548), and those for whom detailed clinical and treatment data were available (source cohort 830 of 852; study cohort 538 of 548) are summarized in Table 1, with the data flow outline in supplemental Figure 1.
Using criteria previously applied to DLBCL, 22 genes were identified as potential targets of aSHM, 20 many of which belonged to the most frequently mutated FL genes ( Figure 1B,C). Of these, BCL2 was the commonest target. Aberrant targeting of SHM to the BCL2 gene in the context of t(14;18) is thought to reflect the recruitment of the SHM machinery by the translocated IGH regulatory elements. 7 Different mechanisms of aSHM targeting are implicated in genes that are not linked by translocation to the IGH locus; these include both recruitment of AID to transcribing polymerase in strongly expressed genes and the impact of differential repair. 34 Setting BCL2 aside for this reason, the most commonly mutated aSHM targets were: BCL7A, HIST1H1E, SOCS1, PIM1, HISTH1C1, BTG1, and SGK1 ( Figure 1C). Considered either with or without BCL2 mutations (supplemental Figure 2A,B, respectively), FL can broadly be divided into those with or without evidence of mutation at 1 or more potential aSHM target genes. Indeed, a significant tail of cases showed evidence of mutation in multiple aSHM target genes, with a maximum of 13 mutated genes per individual patient. Rituximab, cyclophosphamide, vincristine, prednisolone (R-CVP; n = 215); R-CHOP (n = 81); chlorambucil only (n = 42); FCR (n = 10); other (n = 7).

FL genetic subclusters
We found that clustering FL using all available mutation data provided very weak evidence of structure. However, this could be attributable to a degree of noise in an entity showing relative homogeneity of mutational profile in comparison, for example, to DLBCL. Accordingly, we undertook a process of filtering for signalto-noise ratio (supplemental Methods 2.4), resolving a set of 31 primary genetic features (including BCL2) that were the most informative for cluster analysis.
Supplemental Figure 3 shows the distribution of gene mutations that occurred in ≥10 patients (differentially mutated between clusters or not). Several frequently mutated genes were not significantly differentially associated with BIC or AIC clusters; notably, these include EZH2 as well as IRF8 in the context of AIC clusters.
Comparing clusters obtained using the 2 criteria, 55 of 58 cases belonging to AIC FL_aSHM also belonged to the smaller BIC FL_A cluster (Table 2). Thus, both BIC and AIC converged on a subset of FL with multiple aSHM target gene mutations. By contrast, FL_B, the larger BIC cluster, was mostly split between the FL_STAT6 and the FL_Com AIC clusters (115-355), with only 3 subjects assigned to FL_aSHM AIC. Segregation of an additional AIC cluster was reflected in the separation of a subgroup of cases principally characterized by STAT6 mutation (FL_STAT6). Of note, in an independent data set, 35 clustering with AIC rediscovered the separation of FL cases with high aSHM burden from those without (supplemental Methods section 2.3). In this external dataset, cases with STAT6 mutation were not present at sufficient frequency to allow their separation. This may be due to the inclusion criteria and skewing of STAT6 mutation in relation to clinical stage and associated treatment choice.
Translocation of BCL2 and, to a lesser extent, BCL6, are key pathogenetic events in FL. Our approach focused specifically on the question of clustering based on gene mutation rather than structural genomic features. Among cases with translocation data accrued during routine diagnosis, we found no evidence of bias between the mutation-defined subgroups for the absence/presence of a detected BCL2 translocation (BCL2 translocation positive/tested: FL_aSHM 21/25; FL_STAT6 25/32; FL_Com 101/114). We note that this does not exclude the potential for selective enrichment of STAT6 mutation among BCL2 translocation-negative FL, as has been previously reported, but argues that STAT6 mutation may occur as a feature of both BCL2 translocation-positive and -negative FL cases. [36][37][38][39] Mutational profiles in FL subgroups BIC clustering only on aSHM targets (excluding BCL2) confirmed the segregation based on these features alone, supporting the conclusion that a significant subset of FL is characterized by a mutational pattern consistent with higher aSHM activity. Seeking further evidence of differential mutational mechanisms between FL subsets, we carried out a mutational signature analysis. Although mutational signatures are conventionally analyzed at the whole genome sequencing level, we reasoned that mutational mechanisms acting at relevant oncogenes/tumor suppressors are of particular interest, and our data, while biased in terms of genomic coverage, allowed meaningful comparison between cases and sets of genes in the panel. Therefore, all single base substitutions across the tiled regions in the target panel were considered across the set of FL cases and analyzed using the Signal platform, 29 providing a case-by-case representation of substitutions in their triplet context.
A wide range of substitutions per case (8-167) was observed, and the absolute substitution burden was skewed regardless of mutation signature between the FL subgroups ( Figure 4A). Aggregating all the substitutions observed across groups of genes increased the depth of data across tiled regions and demonstrated that the pattern of substitution differed between the genes defined as targets of aSHM relative to the substitution pattern across the remainder of the genes used in our clustering analysis, or the extended group of all lymphoma genes not defined as aSHM targets (supplemental Figure 5). The pattern of substitutions at aSHM targets was similar to that previously described as aSHM targets genome-wide in the K1 signature by Ye and colleagues 40 and also related to the RCH signature 41 with a preference for G(C>T)T and G(C>T)C conforming to the RCY context (R = A/G, Y = C/T) that characterizes the core of SHM hotspots ( Figure 4B).
The aSHM K1 signature demonstrated a cosine similarity of 0.947 relative to the observed substitution pattern at aSHM target genes in our FL cases, contrasting with a cosine similarity of 0.398 for the substitution pattern at non-aSHM targets ( Figure 4B). Among predicted contributions of reference pan-cancer mutation signatures provided by Signal, the aSHM targets differed from other genes in the panel, with marked skewing to the RefSig30 mutation signature (predicted contribution: 69% at aSHM; 17% at non-aSHM clustering genes; 25% at all non-aSHM panel genes) (supplemental Figure 5). While the number of mutations observed in the targeted panel was limiting for many cases, an estimated contribution of reference pan-cancer mutation signatures could be predicted for a substantial subset of individual cases. We, therefore, used these predictions to assess whether the observed skewing between FL clusters was maintained at the level of individual cases. RefSig30 was the dominant mutational signature assigned to cases in the FL_aSHM subgroup, while FL_Com cases showed a mixed pattern predominantly reflecting RefSig_1 as well as the admixture of RefSig30 in some cases ( Figure 4C). By contrast, FL_STAT6 was enriched for cases with a predicted contribution of RefSig1 but not RefSig30. We acknowledge the limitation inherent in assessing mutational signatures using targeted sequencing panels relative to whole genome or exome studies. However, we reason that individual cases are treated equally in this analysis, allowing conclusions to be drawn from and colleagues. 40 This pattern is also closely related to the cAID pattern identified by Chapuy and colleagues in DLBCL, 18 and the SHM-like mutational pattern at IG-VDJ regions described by Hubschmann and colleagues. 42 In contrast, FL_STAT6 cases have a lower overall substitution burden across our lymphoma oncogene panel, showing little evidence of aSHM-related mutation patterns in these key oncogenic regions. Together these data support the conclusion that the FL_aSHM subgroup identifies a distinct etiological subgroup at the level of mutation signature.

STAT6 mutant FL
Recurrent STAT6 mutations have been previously characterized in FL. 12,14 Here, the separation of FL clusters characterized by STAT6 mutation using the AIC and the difference in mutation pattern observed in such cases provides strong evidence for this as a distinct entity. Of the 100 patients with STAT6 mutation, 99 had a single mutation, with 1 patient having 2. Fifty-six of 101 mutations fell at the 419D hotspot, with 24 of 101 being D/G mutations, followed by 13 of 101 D/N and 9 of 101 D/H (supplemental Figure 6). The second most frequent (14 of 101) was at position 377 E/K, with 7 of 101 at 372 E/K. These patterns are consistent with those previously described, demonstrating the enrichment for mutations affecting residues associated with the STAT6 DNA-binding domain. 14 This is a feature of both FL and other B-cell lymphomas with STAT6 mutations and is associated with STAT6 activation. 14,43 Hence, the FL_STAT6 cluster defined by STAT6 mutation in our data is consistent with a subgroup characterized by deregulated activation of this pathway and low levels of aSHM.
It has been suggested that STAT6 and CREBBP mutations may represent a cooperative mutational pairing in FL, 14 a proposal that our AIC clustering supports. Examining patterns of cooccurrence of mutations in STAT6 and the other 5 commonest targets (KMT2D, CREBBP, BCL2, TNRFS14, and EZH2_646) independently of clustering confirmed that STAT6 mutation occurs more frequently than expected with CREBBP mutation. This was evident both for the STAT6/CREBBP pairing alone (adjusted P = .018), the combination of STAT6/CREBBP with TNFRSF14 (adjusted P = .0043), and to a lesser extent TNFRSF14 and EZH2_646 (adjusted P = .031). Furthermore, STAT6 and BCL2 comutation (adjusted P = .022) and STAT6, BCL2, and KMT2D comutation (adjusted P = .013) were significantly less common than expected if mutations were independent. In the absence of STAT6 mutation, the fourfold combination of EZH2_646, BCL2, KMT2D, and TNFRSF14 mutation occurred more commonly (adjusted P = .031), as did the fivefold combination including CREBBP (adjusted P = .027). However, these combinations were less frequent and enrichments less significant than the 2 commonest STAT6 patterns. The combinations of STAT6/CREBBP and STAT6/ CREBBP/TNFRSF14 provide the strongest evidence for significant coenrichment among dominant mutational targets, further supporting the validity of the STAT6-, CREBBP-, and TNFRSF14-enriched FL_STAT6 clusters.

Demographics and clinical features of AIC subclusters
No statistically significant differences in sex or FLIPI were evident (Table 3). However, patients in FL_aSHM were, on average, 4.8 years older than those in the other 2 clusters (P = .007). Stage distributions also differ: FL_aSHM has a comparative excess of stage III, FL_STAT6 of stage I and II, and FL_Com of stage IV. As might be expected given this, the underpinning frequency of documented bone marrow involvement at diagnosis in FL_Com was also higher: 204 of 375 (54%) and 63 of 173 (36%), respectively (P < .001). This was not attributable to differences in bone marrow investigation, which were, by contrast, skewed in favor of FL_aSHM (91.23%) compared with FL_Com (85.29%) (supplemental Methods section 2.8).
Overall survival (OS) was lower for FL_aSHM relative to FL_Com ( Figure 5A) (P = .03), but this did not persist after adjustment for age at diagnosis, and no differences were evident for BIC clusters ( Figure 5B). Although no survival differences were detected for patients initially managed by "Watch and Wait" (Figure 5C,D), AIC cluster differences were detected in those initially managed in other ways (listed in Table 1) (P = .04), with weak evidence for BIC clusters (P = .08) ( Figure 5E,F). As before, neither of these differences persisted following age adjustment. 20 BIC clusters were predictive of POD24 status 44

FL heterogeneity and transformation
Ninety-six of 548 FL cases transformed to DLBCL during follow-up (Table 1). Over a quarter of these (26 of 96) had samples taken at both timepoints, but no evidence of transformation imbalance was found (FL_aSHM = 13.8%; FL_STAT6 = 17.4%; FL_Com = 18.1% transformed; P = .72), and cluster was not predictive of time-to-transformation (P = .9). However, univariate modeling of time-to-transformation to DLBCL identified 2 genes with adverse HRs: TP53 (HR, 3.13; 95% CI, 1.71-5.76) and CDKN2A   showed weak evidence of an association. One gene, IRF8 (HR, 0.50; 95% CI, 0.23-1.07; P = .074), showed weak evidence of an association with a favorable risk. Time-to-transformation was not associated with the burden of aSHM, but there was weak evidence of association with non-aSHM (P = .052).
Genetic abnormality frequencies were assessed for the 26 subjects with paired FL and DLBCL samples ( Figure 6; supplement Figure 7): 17 of 26 had ≥2 unique to the DLBCL sample, with a maximum of 8 additional genetic abnormalities detected. We considered that there was evidence of branching evolution where mutations unique to both FL and DLBCL were identified, along with a set of shared mutations in both samples, and in only 9 of 26 was this evidence lacking. Analysis of variant allele fraction (vAF) (Figure 7; supplemental Figure 8) for shared and disparate mutations between samples further supports the maintenance of high vAFs for common clonal mutations alongside the emergence of new mutations that were not identified at first diagnosis, consistent with prior publications. 12,15,23,45,46 Examining mutations across individual FL and DLBCL pairs (Figure 7) demonstrated common retention of core oncogenic FL events, including mutation of BCL2, KMT2D, CREBBP, and STAT6 on transformation.

Discussion
Our analysis of 548 patients with FL from a United Kingdom population-based cohort supports the existence of significant nonrandom patterns of cooccurring mutations. These findings are consistent with previous studies examining the most common targets of mutation in FL, underlining the importance of CREBBP, KMT2D, and TNFRSF14. Similarly, hotspot mutation of EZH2-646 was the single most common mutated residue in FL, followed quite closely by hotspot mutation in STAT6-419, highlighting the previously suggested importance of this site. 14 Our clustering approach demonstrated that FL could be resolved into mutational subgroups with specific mechanistic differences. FL_aSHM links to high levels of aSHM; FL_STAT6 identifies STAT6 mutated cases as a distinct cluster, which was significantly associated with CREBBP alone, and in combination with TNFRSF14. FL_Com was enriched for KMT2D mutation but was principally defined by the absence of the enriched mutations in FL_STAT6 and low mutation frequencies of aSHM targets. Because we focused on a preselected target panel that was matched to our previous DLBCL study, 20 we cannot exclude the possibility that FL_Com may be further subdivided by mutation at targets that are either missing from our panel or are in noncoding regions. In particular, RRAGC, ATP6V1B2, VMA21, as well as CTSS and EEF1A1 have emerged as important mutations in FL, with frequency estimates varying from <10% to <20%, 47,48 and in the case of EEF1A1, for example, being enriched in the duodenal subtype of FL which is underrepresented in our study. 15,46 We note that ATP6AP1, another gene linked to RRAGC, was included in the extended regions of our panel but was only identified as mutated in a single FL case. STAT6 mutation has been found to be enriched in selected series of FL with diffuse growth pattern, CD23 expression, and/or absence of t(14;18). [36][37][38][39] Our analysis identifies the FL_STAT6 cluster independent of such criteria but sharing associated molecular features, providing further evidence for this as a distinct molecular subtype of FL. In these prior studies of FL enrichment of STAT6 with CREBBP and TNFRSF14 mutation has been a consistent finding which is shared with our analysis. None of these prior studies appear to have found coenrichment of mutations in RRAGC, ATP6V1B2, VMA21, CTSS, or EEF1A1, arguing that the mutation status of this group of genes though interesting is unlikely to impact FL_STAT6 membership. In terms of the FL_aSHM subgroup, we have shown that this is identifiable in an additional external independent data set, which includes mutational information on EEF1A1 and ATP6V1B2, neither of which showed significant effects on clustering in this data. It is unlikely that the relatively rare mutation of genes outside our targeted panel would impact the dominant effect of the aSHM mutation pattern, but these may have importance in the context of subdivision within the FL_Com group.
That high aSHM load is the most robust feature subdividing a significant subset of FL is notable, implying that either targeting or resolution of the aSHM process is deregulated in these cases. 34 Analysis of mutational signatures provides independent evidence of the mutagenic process, separate from the assumption of aSHM targeting based on prior evidence. The mutational pattern observed both at the specific subsets of presumptive aSHM targets across our FL case series and within the individual FL_aSHM cases across all substitutions detected is closely related to that identified for the aSHM process in previous genome-wide studies. 42 The observed substitution preference for G(C>T)T and G(C>T)C conforms to the RCY context (R = A/G, Y = C/T) that characterizes the core of SHM hotspots. An interesting feature is that this mutation pattern and mutation of target genes that contribute to the FL_aSHM subgroup are more frequently observed in DLBCL. 20,40 However, we were unable to show a strong association between FL_aSHM and risk of transformation. While aSHM presents a common etiological mechanism, it is likely that the consequent mutations in individual cases are of significance in determining clinical behavior; indeed, in the context of DLBCL, mutations in SOCS1 and SGK1 contribute to the identification of a specific subset, and separate from the alternate aSHM target BTG1, while all of these genes contribute to the identification of the FL_aSHM subgroup. It may be of particular interest to note mutations in SGK1 that lead to the expression of stable active truncated isoforms. 49 Additionally, several histone H1 encoding genes, including HIST1H1E, are among the most frequent targets associated with FL_aSHM, supporting a common mechanism of downstream epigenetic deregulation. Recent work has illustrated the profound effect of histone H1 mutation on chromatin structure and oncogenesis. 50 Mutation of HIST1H2AC may act in a related fashion since alterations  in histone H2A usage have also been implicated as common oncogenic events in cancer. 51 Further evaluation will be needed to assess the potential association between aSHM and histonemediated epigenetic perturbation, but this may represent a frequently shared characteristic of the FL_aSHM subset.
The association between mutations targeting DNA-damage responses (TP53 and CDKN2A) and risk of transformation makes intuitive sense since these pathways are well characterized in aggressive lymphoma and cancer progression more generally. Furthermore, copy number alterations affecting TP53 and CDKN2A loci have been linked to adverse outcomes in FL. 52 The weak associations of GNA13 and BTK mutations with shorter time-totransformation, and IRF8 with longer time-to-transformation, are suggestive of an association with potential pathways linked to GC biology. [53][54][55] Of these, loss of GNA13 has been identified as strongly cooperative with BCL2 and BCL6 oncogene expression in the transformation of human in vitro lymphoma models, 56 and has been implicated as a gene with an enhanced mutation in transformed FL. 15 However, even in our large cohort, the low rate of progression limited statistical power, and cases lacking these features also transformed. This could also explain the lack of evidence supporting an association between mutational clusters and transformation.
An important difference between our analyses of FL and DLBCL is that while recurrent patterns of mutation, in keeping with differences in disease biology, can be identified in both, these show limited prognostic significance in our FL data. In part, this reflects a diversity of management strategies and the intrinsically indolent nature of the FL disease process. However, overall our analyses suggest that mutational stratification has limited prognostic value in FL and that in terms of clinical response, these mutational combinations generate phenocopies in reaction to conventional treatment strategies. The underlying mechanistic difference may, however, offer opportunities for the development of future treatment strategies related to molecular subtypes.  We acknowledged that the sequencing was based on tumor-only FFPE samples. Even with very stringent filtering, some germline mutations and sequencing artifacts cannot be excluded from such mutational analyses. However, we note that these are not likely to significantly impact the identification of driver mutation calls or the identification of mutational signatures linked to specific patterns of base substitution.
In conclusion, our population-based analysis of mutation patterns in FL provides evidence in support of distinct mechanistic entities, with aSHM burden and STAT6 mutation status providing key determinants of this molecular heterogeneity. In contrast to DLBCL, we did not find evidence that tumor genetics is a major determinant of patient outcome in FL. Instead, we speculate that tumor-extrinsic factors related to the microenvironment or host immune system may have a greater influence on patient survival.