Abstract: Mantle cell lymphoma (MCL), a rare and aggressive B-cell non-Hodgkin lymphoma, mainly develops in the lymph node (LN) and creates a protective and immunosuppressive niche that facilitates tumor survival, proliferation and chemoresistance. To capture disease heterogeneity and tumor microenvironment (TME) cues, we have developed the first patient-derived MCL spheroids (MCL-PDLS) that recapitulate tumor oncogenic pathways and immune microenvironment in a multiplexed system that allows easy drug screening, including immunotherapies. MCL spheroids, integrated by tumor B cells, monocytes and autologous T-cells self-organize in disc-shaped structures, where B and T-cells maintain viability and proliferate, and monocytes differentiate into M2-like macrophages. RNA-seq analysis demonstrated that tumor cells recapitulate hallmarks of MCL-LN (proliferation, NF-kB and BCR), with T cells exhibiting an exhaustion profile (PD1, TIM-3 and TIGIT). MCL-PDLS reproduces in vivo responses to ibrutinib and demonstrates that combination of ibrutinib with nivolumab (anti-PD1) may be effective in ibrutinib-resistant cases by engaging an immune response with increased interferon gamma and granzyme B release. In conclusion, MCL-PDLS recapitulates specific MCL-LN features and in vivo responses to ibrutinib, representing a robust tool to study MCL interaction with the immune TME and to perform drug screening in a patient-derived system, advancing toward personalized therapeutic approaches.

Abstract: Optimal selection of high‐risk patients with stage II colon cancer is crucial to ensure clinical benefit of adjuvant chemotherapy. Here, we investigated the prognostic value of genomic intratumor heterogeneity and aneuploidy for disease recurrence. We combined targeted sequencing, SNP arrays, fluorescence in situ hybridization, and immunohistochemistry on a retrospective cohort of 84 untreated stage II colon cancer patients. We assessed the clonality of copy‐number alterations (CNAs) and mutations, CD8 + lymphocyte infiltration, and their association with time to recurrence. Prognostic factors were included in machine learning analysis to evaluate their ability to predict individual relapse risk. Tumors from recurrent patients displayed a greater proportion of CNAs compared with non‐recurrent (mean 31.3% versus 23%, respectively; p  = 0.014). Furthermore, patients with elevated tumor CNA load exhibited a higher risk of recurrence compared with those with low levels [ p  = 0.038; hazard ratio (HR) 2.46], which was confirmed in an independent cohort ( p  = 0.004; HR 3.82). Candidate chromosome‐specific aberrations frequently observed in recurrent cases included gain of the chromosome arm 13q ( p  = 0.02; HR 2.67) and loss of heterozygosity at 17q22–q24.3 ( p  = 0.05; HR 2.69). CNA load positively correlated with intratumor heterogeneity ( R  = 0.52; p   〈  0.0001). Consistently, incremental subclonal CNAs were associated with an elevated risk of relapse ( p  = 0.028; HR 2.20), which we did not observe for subclonal single‐nucleotide variants and small insertions and deletions. The clinico‐genomic model rated an area under the curve of 0.83, achieving a 10% incremental gain compared with clinicopathological markers ( p  = 0.047). In conclusion, tumor aneuploidy and copy‐number intratumor heterogeneity were predictive of poor outcome and improved discriminative performance in early‐stage colon cancer. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract: Next-generation sequencing (NGS) has transitioned from research to clinical routine, yet the comparability of different technologies for mutation profiling remains an open question. We performed a European multicenter (n=6) evaluation of three amplicon-based NGS assays targeting 11 genes recurrently mutated in chronic lymphocytic leukemia. Each assay was assessed by two centers using 48 pre-characterized chronic lymphocytic leukemia samples; libraries were sequenced on the Illumina MiSeq instrument and bioinformatics analyses were centralized. Across all centers the median percentage of target reads ≥100x ranged from 94.2-99.8%. To rule out assay-specific technical variability, we first assessed variant calling at the individual assay level i.e. pairwise analysis of variants detected amongst partner centers. After filtering for variants present in the paired normal sample and removal of PCR/sequencing artefacts, the panels achieved 96.2% (Multiplicom), 97.7% (TruSeq) and 90% (HaloPlex) concordance at a VAF 〉 0.5%. Reproducibility was assessed by looking at the inter-laboratory variation in detecting mutations and 107/115 (93% concordance) of mutations were detected by all 6 centers, while the remaining 8/115 (7%) variants were undetected by a single center and 6/8 of these variants concerned minor subclonal mutations (VAF 〈 5%). We sought to investigate low-frequency mutations further by using a high-sensitivity assay containing unique molecular identifiers, which confirmed the presence of several minor subclonal mutations. Thus, while amplicon-based approaches can be adopted for somatic mutation detection with VAFs 〉 5%, after rigorous validation, the use of unique molecular identifiers may be necessary to reach a higher sensitivity and ensure consistent and accurate detection of low-frequency variants.

Abstract: INTRODUCTION: Cancer pathogenesis is usually characterized by a long evolutionary process where genomic driver events accumulate over time, conferring advantage to distinct subclones, allowing their expansion and progression. METHODS: To investigate the multiple myeloma (MM) evolutionary history, we characterized the mutational processes' landscape and activity over time utilizing a large cohort of 89 whole genomes and 973 exomes. To improve the accuracy of mutational signatures analysis, we analyzed both the 3' and 5' nucleotide context of each mutation and we developed the novel fitting algorithm mmSig, which fits the entire mutational catalogue of each patient with the mutational signatures involved in MM pathogenesis. The contribution of each mutational signature was then corrected based on the cosine similarity between the original 96-mutational profile and the reconstructed profile generated without that signature. To reconstruct the genetic evolutionary history of each patient's cancer, we integrated two approaches. First dividing all mutations into clonal (early) or subclonal (late), then subdivided the clonal mutations into duplicated mutations (present on two alleles and therefore acquired before the duplication) or non-duplicated mutations (detected on a single allele), reflecting either pre-gain and post-gain mutations on the minor allele, or post-gain mutations acquired on one of the duplicated alleles. RESULTS Eight mutational signatures were identified, seven of which showed significant similarity with the most recent mutational signature catalogue (i.e SBS1, SBS2, SBS5, SBS8, SBS9, SBS13 and SBS18). The new mutational signature (named SBS-MM1) was observed only among relapsed patients exposed to alkylating agents (i.e melphalan). The etiology of this specific signature was further confirmed by analyzing recent whole genomes public data from human-induced pluripotent stem cells exposed to melphalan (Kucab et al, Cell 2019). Reconstructing the chronological activity of each mutational signature, we identified four different routes to acquire the full mutational spectrum in MM based on the differential temporal activity of AID (SBS9) and APOBEC (SBS2 and SBS13). Our data indicate that AID activity is not limited to the first contact with the GC, but persists in the majority of patients, behaving similarly to a B-memory cells, capable of re-entering the germinal center upon antigen stimulation to undergo clonal expansion several times before MM diagnosis. Next, we confirmed the clock-like nature (i.e constant mutation rate) of SBS5 in MM and other post-germinal center disorders such as chronic lymphocytic leukemia and B-cell lymphomas. Based on the SBS5 mutation rates and the corrected ratio between duplicated and non-duplicated mutations within large chromosomal gains, we could time the acquisition of the first copy number gain during the life history of each MM patient. Intriguingly, the first MM chromosomal duplication was acquired on average 38 years (ranges 11-64) before sample collection. In 23/27 (85%) cases the first multi gain event occurred before 30 years of age, and in 13/27 (48%) before 20 years reflecting a long and slow process potentially influenced and accelerated by extrinsic and intrinsic factors. DISCUSSION Our analysis provides a glimpse into the early stages of myelomagenesis, where acquisition of the first key drivers precedes cancer diagnosis by decades. Defining the time window when transformation occurs opens up for new avenues of research: to identify causal mechanisms of disease initiation and evolution, to better define the optimal time to start therapy, and ultimately develop early prevention strategies. Disclosures Bolli: CELGENE: Honoraria; JANSSEN: Honoraria; GILEAD: Other: Travel expenses. Corradini:Janssen: Honoraria, Other: Travel Costs; Jazz Pharmaceutics: Honoraria; KiowaKirin: Honoraria; Servier: Honoraria; Takeda: Honoraria, Other: Travel Costs; Kite: Honoraria; Novartis: Honoraria, Other: Travel Costs; Gilead: Honoraria, Other: Travel Costs; Roche: Honoraria; Sanofi: Honoraria; BMS: Other: Travel Costs. Anderson:Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board. Moreau:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Papaemmanuil:Celgene: Research Funding. Avet-Loiseau:takeda: Consultancy, Other: travel fees, lecture fees, Research Funding; celgene: Consultancy, Other: travel fees, lecture fees, Research Funding. Munshi:Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Abbvie: Consultancy. Landgren:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Theradex: Other: IDMC; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Other: IDMC; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract: Introduction: B-cell receptor (BCR) signaling is crucial for chronic lymphocytic leukemia (CLL) biology. IGLV3-21-expressing B-cells may acquire a single point mutation (R110) that triggers autonomous BCR signaling conferring aggressive behavior. Epigenetic studies have defined three CLL subtypes based on methylation signatures reminiscent of pre- and post-germinal center B-cells named naïve-like (n-CLL), intermediate (i-CLL) and memory-like CLL (m-CLL) with different biological features. i-CLL carry a borderline IGHV mutational load and a significant higher usage of IGHV3-21/IGLV3-21. The integration of these factors might translate into novel insights in CLL pathogenesis with implications on the proposed stratification of the patients. Aim: To determine the clinical and biological features of the IGLV3-21R110 in CLL in the light of the epigenetic subtypes and immunogenetic, genomic and transcriptomic landscapes of the tumors. Methods: We characterized the immunoglobulin (IG) gene of 584 CLL cases from whole-genome/exome and RNA sequencing using our recently developed algorithm IgCaller (Nadeu et al., Nat. Commun. 2020) and MiXCR, respectively. The genomic makeup of the tumors was obtained from whole-genome/exome sequencing while RNA sequencing data for 369 cases was used for gene expression analyses. Expression levels of WNT5A and WNT5B were verified by quantitative PCR with reverse transcriptase. Primary end points were time to first treatment (TTFT) and overall survival (OS) calculated from the date of diagnosis. All patients gave written informed consent. The study was approved by the Ethics Committee of the Hospital Clínic of Barcelona. Results: The IGLV3-21R110 was detected in 6.5% of cases being similarly distributed between mutated (6.5%) and unmutated (6.6%) IGHV cases (P=0.56). In contrast, the IGLV3-21R110 was found in 30/79 (38%) i-CLL compared to only 5/291 (1.7%) m-CLL and 1/189 (0.5%) n-CLL (P & lt;0.001). All stereotyped subset #2 cases carried IGLV3-21R110 while 62% of IGLV3-21R110 i-CLL had non-stereotyped IG genes. IGLV3-21R110 i-CLL had a borderline IGHV mutational status (median 97.7%) that was higher than i-CLL lacking the IGLV3-21R110 (median 96.2%, P=0.005). IGLV3-21R110 i-CLL had significantly higher number of SF3B1 and ATM mutations, and total number of driver alterations. Nonetheless, the R110 mutation was the sole alteration in one i-CLL case and accompanied only by del(13q) in three. Although composite regarding IGHV mutational status, IGLV3-21R110 i-CLL transcriptomically resembled naïve-like/unmutated IGHV CLL and had a specific expression signature of 64 genes with overexpression of WNT5A and WNT5B as hallmarks. No differences were observed in the expression profile of subset #2 and non-subset #2 IGLV3-21R110 i-CLL tumors. On the other hand, i-CLL lacking the IGLV3-21R110 phenotypically mirrored memory-like/mutated IGHV cases. In relation to prognosis, IGLV3-21R110 i-CLL had a short TTFT and OS similar to n-CLL/unmutated IGHV cases whereas non-IGLV3-21R110 i-CLL had a good prognosis similar to memory-like/mutated IGHV. Therefore, i-CLL cases, which have been associated with an intermediate prognosis between m-CLL and n-CLL in previous studies, can be divided in two subgroups of cases with opposed clinical evolutions based on the IGLV3-21R110. Indeed, the IGLV3-21R110 and n-CLL subtype retained independent prognostic value in multivariate analyses while the i-CLL lost its prognostic prediction both for TTFT and OS. The prognostic value of the IGLV3-21R110 was also independent of the IGHV mutational status. In terms of applicability in the clinics, all n-CLL cases were classified as unmutated IGHV and 98% of m-CLL were mutated IGHV. Thus, either a complete IG characterization (IGHV mutational status and IGLV3-21R110) or the integration of the n-CLL subtype and IGLV3-21R110 identified virtually the same subset of patients with aggressive disease. Conclusions: The IGLV3-21R110 defines a CLL subset with borderline IGHV mutations, specific driver alterations, a gene expression signature including WNT5A/B overexpression, and an unfavorable prognosis independent of the IGHV mutational status and epigenetic subtypes. Our findings support the identification of IGLV3-21R110 CLL as a particular subgroup of the disease with relevance in the risk stratification of the patients. Disclosures Nadeu: Janssen: Honoraria. Campo:NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America..

Abstract: Introduction: The t(14;19)(q32;q13) is a rare cytogenetic abnormality found in & lt;0.1% of all B-cell neoplasms. The molecular features of this translocation are not well characterized. IGH-BCL3 rearrangement has been found in some tumors identified as "atypical" chronic lymphocytic leukemia (CLL) with aggressive clinical evolution. This translocation has also been observed in other B-cell neoplasms without clear evidence of the target gene. The mechanisms generating this translocation, the genomic profile of alterations of these cases, and whether different molecular features may be associated with specific entities are not known. Aim: To elucidate the genomic features of B-cell neoplasms carrying the t(14;19) and their relationship to pathological characteristics of the tumors. Materials and methods: We sequenced the whole-genome (WGS) of 13 cases in which the t(14;19) had been identified by conventional cytogenetics and/or FISH using a BCL3 break-apart probe. In six of these cases we performed RNA-seq. Pathological and clinical revision was conducted in all cases, 8 of them with tissue biopsies. Results: The breakpoints of the t(14;19) were characterized at base-pair resolution using WGS. All breakpoints in chr14 were found within any of the class switch recombination (CSR) regions suggesting an aberrant CSR as the mechanism causing this alteration. The breakpoints on chr19 were found upstream (13 kb) the 5' untranslated region (UTR) of BCL3 in 8/13 (61.5%) cases. One additional case had the breakpoint further upstream (49 kb) of BCL3 truncating CEACAM16. The four remaining cases had breakpoints downstream of BCL3; two cases within CBLC, one in BCAM, and one after NECTIN2. Of note, the further upstream BCL3 case and the downstream BCL3 cases had mutated IGHV, while all upstream BCL3 cases had unmutated IGHV. Based on RNA-seq data, all upstream BCL3 cases (n=5) showed an upregulation of BCL3, while one downstream case with RNA-seq available showed upregulation of NECTIN2 and low levels of BCL3. The pathology review identified the four downstream BCL3 cases as marginal zone lymphomas whereas the cases with breakpoints upstream BCL3 (n=3 with tissue available) and the case further upstream BCL3 were classified as "atypical" CLL. We next characterized the genomic landscape of these tumors based on the breakpoint on chr19 (upstream and downstream BCL3). The analysis of the WGS showed a lower number of mutations, copy number alterations (CNA), and structural variants (SV) in the upstream BCL3 group compared to the downstream BCL3 cases (mean of 2429.5 vs 6271.7 somatic mutations, 3.1 vs 11.7 CNA, and 4.4 vs 18 SV, respectively). In terms of specific driver mutations, the downstream BCL3 group carried mutations in genes previously described in MZL, such as KMT2D, NOTCH2, or KLF2 found in two cases. All but one case with the breakpoint upstream BCL3 carried trisomy 12 (tri12), which was absent in all cases with a downstream breakpoint. Finally, we performed a differential expression analysis between 5 atypical CLL cases with BCL3 rearrangements vs 4 CLL without t(14;19) [all unmutated IGHV]. This analysis showed 578 genes upregulated and 720 genes downregulated in the BCL3-rearranged cases (q & lt;0.05), including remarkable differences in the expression of previously described CLL hallmark genes, such as upregulation of EBF1 and downregulation of LEF1, FMOD, ADTRP, CLNK, IGSF3, TCF4. An analysis of the RNA-seq data of 294 CLL cases lacking the t(14;19) (Puente et al., Nature 2015) indicated that this transcriptional program was not related to IGHV mutational status nor to the presence of tri12. Nonetheless, we identified a small set of tri12 mutated IGHV CLL lacking the t(14;19) with a similar modulation of the expression of the above hallmark genes. Conclusions: We have characterized the breakpoints of the t(14;19) at base-pair resolution and evidenced marked molecular and pathological differences of the tumors according to the location of the breakpoint. Tumors carrying the breakpoint downstream BCL3 exhibit a higher genomic complexity, driver alterations, and pathological features corresponding to MZL. Contrarily, tumors with the breakpoint upstream of BCL3 upregulate BCL3 and display lower genomic complexity as well as CLL-like features. Nonetheless, these cases have a different gene expression profile compared to conventional CLL characterized by LEF1 downregulation and EBF1 overexpression. Disclosures Navarro: Nocartis: Honoraria; Roche: Honoraria; EUSA: Consultancy, Research Funding; Pharma: Consultancy; GILEAD: Research Funding; Pharma: Research Funding.