Hierarchy in Somatic Mutations Detected in Circulating and Tissue-Resident Follicular Lymphoma Precursors before Clinical Diagnosis

Background: The most common indolent non-Hodgkin's lymphoma subtype, follicular lymphoma (FL), is characterized by t(14;18) translocation in ~90% of patients. Although such translocations often occur at low levels in healthy older adults, patients developing FL are more likely to have high levels of detectable t(14:18) years before diagnosis. As BCL2 translocations precede but are not sufficient for lymphomagenesis, we analyzed prediagnostic blood and non-malignant lymph node samples to identify other early changes associated with eventual FL, correlated with biopsies of subsequent tumors where available.

Methods: Among 520,000 healthy European participants in the EPIC study, we selected 17 patients who had elevated t(14;18) by qPCR on screening blood samples and subsequently developed FL (Roulland et al 2014 JCO). The median time to lymphoma diagnosis was 2330 days (range 284-4531). Using enhanced CAncer Personalized Profiling by deep Sequencing (CAPP-Seq, Newman et al Nat Biotech 2016), we performed ultra-deep sequencing on prediagnostic blood mononuclear samples (n=17) as well as a subset of paired FL tumor biopsies (n=5). Patients with elevated t(14;18) but no lymphoma diagnosis were sequenced as a control group (n=3). Somatic hypermutation (SHM) was assessed via variant distribution in activation-induced cytidine deaminase associated regions.

We separately identified a second cohort of patients treated at Stanford University who underwent sequential biopsies for lymphadenopathy. For each patient a first biopsy showed histologically non-malignant lymphoid proliferation, but a later biopsy was identified as FL tumor (n=10). The median time between samples was 650 days (range 33-2499). These paired biopsy samples were also deeply sequenced with CAPP-Seq.

Results: Mutations detected on genotyping of EPIC tumor biopsies included CREBBP R1446H (2/5 cases), IRF8 (2/5), TP53, and KMT2D . Ultra-deep sequencing of paired prediagnostic leukocytes (median unique depth 4020-7651x, duplex support 19-31%) recovered CREBBP in one case at an allelic fraction of 0.04%. A concordant SHM pattern was also detected for this case. For FL patients with no available tumor sample (n=12), genotyping of prediagnostic leukocytes detected mutations in CREBBP, TP53, EP300, IRF8, and MYC . No mutation in these genes was detected in the control group. BCL2 translocations were detected in all patients, and fusion sequences obtained by nested PCR and Sanger sequencing were also seen in all cases using CAPP-Seq.

We then genotyped the Stanford paired biopsy cohort, where in 70% of cases at least 1 tumor-confirmed aberration was also seen in the non-lymphoma biopsy. In similar cohorts of other lymphoma histologies, shared mutations were seen in 50% of prediagnostic biopsies (DLBCL n=8, other histologies n=10). Analysis of SHM patterns between biopsies found concordance suggestive of a precursor clonal population in 8 of 10 FL patients (1 low confidence, 1 undetected). Frequently mutated tumor genes, with their rate of recovery in the earlier sample, included CREBBP (71.4% concordance), CARD11 (50%), KMT2D (50%), BCL2 (40%), and HIST1H1E (33%). Despite shared mutations in most patients, the majority of variants were unique to either tumor or prediagnostic sample (median 19% shared SNVs).Mutations frequently detected in tumor but not initial biopsy included EZH2 (3 cases) and EP300 (2 cases).

Conclusions: Collectively, our results definitively confirm prior studies suggesting characteristic mutations including CREBBP as prevailing early lesions of FL. We provide direct evidence via distant paired samples of the presence of such variants in clinically healthy individuals and their persistence in subsequent FL tumors. Although preserved early mutations are frequent in our paired biopsy cohort, when comparing the degree of evolution between precursor lesions and later FL tumors we observed larger clonal shifts than in prior studies comparing established lymphomas in paired biopsies (Scherer et al 2016 Sci Trans Med). This observation was irrespective of histologies of the established pairs, which included FL diagnosis versus progression/relapse, FL diagnosis versus transformation, and DLBCL diagnosis versus progression/relapse. This greater degree of divergence suggests that changes in addition to characteristic FL precursor mutations may be required for lymphomagenesis.