BACKGROUND/OBJECTIVES:Circular RNAs (circRNAs) are emerging players in human diseases, with functions as part of competing endogenous networks. Given the importance of messenger RNA (mRNA) regulation in human diseases and the potential of circRNAs in this regulation, we studied the circRNA-mRNA couple in blood within a cohort of 712 patients suspected of having hereditary colorectal cancer (CRC) and 249 matched controls. METHODS:The circRNA-mRNA couple was studied by SEALigHTS (Splice and Expression Analyses by exon Ligation and High-Throughput Sequencing) with a panel of 23 genes involved in CRC predisposition, comprising 788 probes designed at exon ends, enabling the exploration of all exon-exon junctions. Following reverse transcription and probe hybridization on cDNA, nearby probes were ligated, and the number of ligations was quantified using unique molecular identifiers and sequencing. RESULTS:We identified 220 circular junctions, including 47 novel ones. The circRNA/mRNA ratio was 2.42-fold higher in patients compared to controls (p < 10-16), irrespective of age at cancer onset. This increase was mainly driven by POLD1 (fold change 3.84) and a single circPOLD1(3,2) with oncogenic potential Conclusions: This study supports the existence of a physiological balance between circRNA and mRNA that can be disrupted under pathological conditions. It rules out a competitive mechanism between circular and linear transcripts in CRC predisposition and raises questions about the role of specific circRNAs in the development of CRC, either as a cause or a consequence.

Diffuse Large B-cell lymphoma (DLBCL) is the most common aggressive lymphoma in the Western world. First-line immunochemotherapy fails in approximately 30-40% of patients, with refractory and relapse patients presenting a dismal prognosis. Currently, these high-risk patients cannot be accurately identified at diagnosis. Using statistical modeling and machine learning approaches applied to large public DLBCL datasets, we identified a novel predictive signature based on the reactivation of eight normally silent tissue-dependent genes associated with survival. We then developed a multiplex RT-MLPseq based assay, compatible with formalin-fixed paraffin-embedded (FFPE) samples and transferable into routine clinical practice, enabling analysis of expression of these eight genes and validated their prognosis impact in an independent real-life cohort. This signature could be integrated with current prognostic indices and molecular classifications to improve patient stratification and guide treatment selection toward a personalized theragnostic approach, thereby enhancing management of non-responder patients.

ABSTRACT:Primary mediastinal large B-cell lymphoma (PMBL) achieves excellent outcomes with dose-dense immunochemotherapy, yet response assessment by positron emission tomography (PET) remains limited. In this prospective multicenter observational study, we evaluated the clinical relevance of circulating tumor DNA (ctDNA) minimal residual disease (MRD) in patients with newly diagnosed PMBL and assessed whether MRD enhances outcome discrimination beyond PET. Plasma and PET images were collected at baseline and after 2 and 4 cycles. ctDNA and tumor biopsy were analyzed by high-depth, error-corrected sequencing (limit of detection ∼10-3). Associations between MRD, PET response, and progression-free survival (PFS) were evaluated. Among 84 patients, baseline ctDNA was detected in 98%. After 4 cycles of treatment with R-CHOP14 (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone, administered every 14 days) or R-ACVBP (rituximab, doxorubicin, cyclophosphamide, vindesine, bleomycin, prednisone), 87.7% had undetectable MRD. Persistence of minimal residual disease after 4 cycles of therapy (MRD4) was associated with shorter PFS (hazard ratio [HR], 78.1; 95% confidence interval [CI], 9.5-641.8). The 1-year PFS was 98.4% (95% CI, 95.4%-100%) for patients with undetectable MRD4 vs 33.3% (95% CI, 13.2%-84.0%) for patients with detectable MRD4 (P < 10^-4). MRD4 showed a higher positive predictive value (89% vs 50%) for disease progression than PET4, (PET after 4 cycles of chemotherapy) maintaining a similar negative predictive value (100% vs 93%). In multivariate analysis, only PET4-/MRD4- remained associated with PFS (adjusted HR, 0.07; 95% CI, 0.01-0.90). Plasma ctDNA represents a highly abundant source of genetic markers for tumor fingerprinting and disease monitoring. MRD detection after frontline therapy strongly complements PET response criteria in predicting outcome. These findings support the use of ctDNA monitoring as a valuable tool for future risk-adapted strategies in PMBL. This trial was registered at www.clinicaltrials.gov as NCT04980222.

Background:In the phase I Epi-RCHOP study (NCT02889523), we reported that R-CHOP-tazemetostat was well tolerated with the recommended phase II dose, consistent with monotherapy. Methods:Phase II included newly diagnosed diffuse large B cell lymphoma patients aged 60-80 years who received six cycles of rituximab-CHOP (R-CHOP) with continuous tazemetostat (800 mg BID), plus two cycles of tazemetostat and rituximab (cycles 7 and 8), from July 31, 2020 to July 18, 2022. Primary endpoint was positron emission tomography complete metabolic response (CMR). Sample size was calculated with H0 of 70% and H1 assumption of 80%. Findings:The trial enrolled 122 patients: median age 70 (60-80), 90.2% with stage III-IV, and 73.8% with International Prognostic Index 3-5. Overall, 100 patients (82%) received eight cycles, while 22 had premature treatment discontinuation (PTD), including 12 during the first two cycles. Reasons for PTD were consent withdrawal (N = 10), adverse events (N = 6), death (N = 2), protocol deviation (N = 2), progressive disease (N = 1), and physician decision (N = 1). The median percentage of relative dose intensity of tazemetostat and R-CHOP exceeded 90%, but required a protocol amendment and reduction in vincristine dosage at 1 mg full dose. At the end of treatment or PTD, 92/122 patients (75.4%) achieved CMR, eight (6.6%) partial metabolic response, five (4.1%) progressive disease, two (1.6%) died (septic shock), and 15 (12.3%) were not evaluated. Sensitivity analysis, excluding ten non-evaluated patients who withdrew consent, showed CMR in 82.1%. After a median follow-up of 18.5 months (IQR: 15.4-21), estimated progression-free and overall survival at 18 months were 77.7% (95% CI: 67.5-85.1%) and 88.8% (95% CI: 79.9-93.9%), respectively. Interpretation:R-CHOP plus tazemetostat is feasible with a promising CMR in elderly DLBCL patients. Complementary biomarker studies are needed for a more personalized approach. Funding:This study was sponsored under a grant from Ipsen.

ABSTRACT:The GAINED study was a randomized phase 3 trial comparing obinutuzumab (G) with rituximab (R) plus ACVBP (doxorubicin, cyclophosphamide, and prednisone, combined with either vindesine or bleomycin) or CHOP14 (cyclophosphamide, doxorubicin, vincristine, and prednisone, administered on a 14-day schedule) induction, followed by positron emission tomography (PET)-guided consolidation. This post hoc analysis aimed to detail the outcomes of patients with primary mediastinal B-cell lymphoma (PMBL), verified through expert pathological review and the use of gene expression profiling (GEP) and next-generation sequencing. Of 620 centrally reviewed patients, 138 (22.3%) confirmed PMBL cases were analyzed. Baseline characteristics included a median age of 33.5 years, 63.8% female, 55.1% stage III to IV, 90.6% elevated lactate dehydrogenase, 87.6% Eastern Cooperative Oncology Group performance status score of 0 to 1, 62.3% extranodal involvement, 52.6% age-adjusted International Prognostic Index (aaIPI) of 2% to 3%, and 53.6% bulk (>10 cm). Induction regimens were R/G-CHOP14 (56.9%) and R/G-ACVBP (43.1%). Postinduction treatments, based on interim PET results, included: standard consolidation chemotherapy (59.8%) if change in maximum standardized uptake value (ΔSUVmax) of >66% after cycle 2 and >70% after cycle 4 (PET2-/4-), intensive treatment and autologous transplantation (26.8%) if PET2+/4-, and salvage therapy (13.4%) if PET4+ (ΔSUVmax of ≤70%). Among patients with GEP data (n = 107), 38 (35.5%) were PDL1high/PDL2high. Key somatic mutations data (n = 87) included SOCS1 (70.1%), B2M (56.3%), STAT6 (49.4%), TNFAIP3 (47.1%), GNA13 (39.1%), CIITA (37.9%), CD58 (36.8%), and TP53 (29.9%). After a median follow-up of 39.5 months, 2-year progression-free survival (PFS) and overall survival (OS) rates were 86.2% and 93.2%, respectively. In a multivariate model including bulk, aaIPI, and ΔSUVmax PET2/PET4, only bulk and ΔSUVmax PET4 of ≤70% were associated with shorter PFS (hazard ratio, 4.39 [95% confidence interval (CI), 1.28-15.11] and 4.95 [95% CI, 1.71-14.3], respectively), whereas none were associated with OS. The ΔSUVmax-based interim PET4 response emerged as the strongest predictor of patient outcomes in this selected clinical trial population. This trial was registered at www.ClinicalTrials.gov as #NCT01659099.

BackgroundLi-Fraumeni syndrome (LFS) predisposes individuals to a wide range of cancers from childhood onwards, underscoring the crucial need for accurate interpretation of germlineTP53variants for optimal clinical management of patients and families. Several unclassified variants, particularly those potentially affecting splicing, require specialised testing. One such example is the NM_000546.6:c.1101-2A>C (rs587781664) variant, located at the splice acceptor site of the last intron ofTP53, identified in a female patient with breast cancer diagnosed in her 20s.MethodsTo interpret this variant, which has been classified as a variant of uncertain significance (VUS), we developed specific assays including a p53 functional assay, RT-QMPSF, Splice and Expression Analyses by exon Ligation and High-Throughput Sequencing and long RT-droplet digital PCR.ResultsWe demonstrated a loss of p53 transcriptional activity, and a half reduction in TP53 mRNA expression. Additionally, we detected the use of a novel alternative last exon downstream of exon 11, which we have named exon 12. This transcript, typically detectable at low levels in most individuals, was found to be more highly expressed in the c.1101-2A>C carrier, predominantly transcribed from the mutant allele due to the disruption of the splice acceptor site in intron 10.ConclusionBy combining these approaches, we successfully reclassified this intronic VUS as ‘pathogenic’, enabling appropriate genetic counselling for the patient and her family. Additionally, we identified a novel TP53 alternative transcript that is expressed in both physiological and pathological contexts, with heightened expression in the patient with LFS. This discovery provides a basis for further investigation into the role of TP53 isoforms in LFS oncogenesis.

IntroductionCirculating tumor DNA (ctDNA) has become a well-established dynamic biomarker in Large B Cell Lymphoma (LBCL). Previous studies have shown that decrease in ctDNA quantities after standard R-CHOP treatment is significantly correlated with prognosis (Kurtz et al, Blood 2015 and Kurtz et al, J Clin Oncol 2018). However, no studies have described the very early kinetics of ctDNA following immunochemotherapy. Moreover, low-pass whole-genome sequencing (lpWGS) of cfDNA can identify DNA copy number alterations (CNAs) and be used to define focal CNA score (FCS). High FCS, denoting genomic instability, has been shown to be linked to inferior response rates to CAR-T therapy for LBCL patients (Cherng et al, Blood 2022). We developed a prospective clinical trial, LYMPHOCLEAR, which included 24 LBCL patients with planned first-line (L1) R-CHOP treatment, for whom ctDNA samples were taken at multiple timepoints within the first 48 hours post R-CHOP and at C2D1. We sought to describe early ctDNA kinetics and propose a clinically relevant model in order to potentially identify a prognostic early ctDNA analysis timepoint. We also sought to describe variants at each timepoint to see whether distinct natural evolutions could be observed very early post R-CHOP. We sought to evaluate whether the impact of FCS on response could be observed at very early timepoints. Patients and Methods We included 24 LBCL patients (17 DLBCL, 4 PMBL, 2 HGBCL, 1 B Cell lymphoma NOS) who required hospitalization for C1 R-CHOP, according to their physician's decision. Median age was 64.5 years (range 24-85). IPI was 1-2 for 30.4% of patients and 3-5 for 69.6%. Ann Arbor stage was 4 in 70.8% of patients, with bulky mass in 29%. We performed capture-based NGS and lpWGS using DNA extracted from patient plasma samples at H0, H4, H8, H12, H16, H24 and H48 post R-CHOP initiation and at C2D1. All patients had a FDG PET-CT performed at baseline, post C2 and at end of treatment (EOT). Total Metabolic Tumor Values (TMTV) were measured using a fixed SUVmax > 4 threshold. A latent class mixed model (LCMM) was used to modelize ctDNA kinetics and delineate groups of patients with similar trajectories (Proust-Lima et al, J. Stat. Softw. 2017). Results The application of a LCMM segregated four classes of ctDNA kinetics with robust statistical characteristics: Bayesian Information Criterion (BIC) was 330 and the post-hoc probability of a patient belonging to a specific class was close to 0.9. Classes 1 (n=3) and 2 (n=2) had lower baseline ctDNA values (median log 10 1.71 and 0.72 respectively) but class 1 had decreasing values from H0 to H48 while class 2 had increasing values from H0 to H48. Classes 3 (n=5) and 4 (n=14) had higher baseline ctDNA values (median log 10 3.42 and 3.21 respectively) but ctDNA quantities increased from H0 to H48 in class 3 whereas they decreased in class 4. Classes 1 and 2 had lower baseline TMTV and LDH than classes 3 and 4 (TMTV: 370.2cm3 and 61.1cm3 respectively versus 1347.3cm3 and 703.1cm3 and LDH: 212 and 220 respectively versus 775 and 340), also highlighting their lower tumor burden. We confirmed that patients with Deauville score (DS) 4-5 at PET2 had significantly higher ctDNA quantities at C2D1 than patients with DS 1-3 (p=0.0048). Interestingly, we also showed that ctDNA values at H4 tend to be higher in patients with positive PET2 and that median ctDNA variant allele frequency at H4 was higher in patients with DS 4-5 at EOT PET.As expected, FCS was positively and strongly correlated to ctDNA quantities and tended to be higher in patients with DS 4-5 at EOT PET. Additional data regarding cfDNA fragmentome evolution at these early timepoints will be reported. As compared to baseline profiles, we observed that additional mutations over time mainly targeted intronic and µ Switch regions, suggesting very early enrichment of ctDNA by additional subclones following immuno-chemotherapy. ConclusionsWe have shown that it is possible to describe the kinetics of ctDNA quantities and variant types in the first 48 hours post R-CHOP treatment initiation, leading to the identification of 4 patient classes, as well as the observation of very early ctDNA enrichment by additional subclones. We confirmed known impact of C2D1 ctDNA quantity but also highlighted potential impact of H4 ctDNA quantity and VAF on patient response. If confirmed in a larger cohort, this would allow for exceedingly early detection of poor responders to L1 R-CHOP.

Few data exist regarding the tumor B-cell receptor (BCR) repertoire and lymphoid microenvironment in primary mediastinal B-cell lymphoma (PMBL). We applied 5' rapid amplification of cDNA ends (5'RACE) to tumor RNA samples from 137 PMBL patients with available gene expression profiling and next-generation sequencing data. We obtained 5'RACE results for 75/137 (54.7%) patients, with clinical characteristics as follows: median [min-max] age, 33 [18-64] years; female, 53.3%; ECOG score 0-1, 86.7%; stage I-II, 57.3%; 1st-line treatment with anti-CD20 plus ACVBP, 72%; CHOP14, 14.7%; CHOP21, 13.3%. Among the 60 biopsies that expressed a productive BCR, we highlighted a strong somatic hypermutation profile with 58 (96.7%) patients carrying mutated IgVH, defined as <98% identity to the germline sequence. We then identified a subgroup of 12/75 patients (16%) with a worse prognosis (progression-free survival (PFS): HR [95% CI]=17 [3.2-88]; overall survival (OS): HR=21 [2.1-210]) associated with the highest clonal dominance status (HCD), defined by the dominant clonotype representing >81.1% and >78.6% of all CDR3 sequences for IgVH and IgVL, respectively. Compared to other patients, this subgroup had similar clinical characteristics but a greater median allele frequency for all somatic variants, decreased BCR diversity, and greater expression of PDL1/PDL2 and MS4A1 genes, suggesting a greater tumoral infiltration. According to a multivariate model integrating AID expression and BCR diversity, only HCD status was associated with outcome (PFS: HR=14.6 [2.46-86.8]; OS: HR=11.4 [1-128.8]). We confirmed this poorer prognosis in an independent cohort, in which 6/37 (16%) patients exhibited HCD (PFS: HR=12 [3-46]; OS: HR=17 [1.8-170]).

Diagnostic analysis of mRNA is essential because altered splicing is a frequent cause of genetic diseases. High-throughput splicing studies remain difficult to implement in routine diagnostics. This is why SEALigHTS (splice and expression analyses by ligation and high throughput sequencing), a cost-effective and easy-to-implement technique designed for simultaneous analysis of RNA from multiple patients on a panel of genes, was developed using probes designed at exon extremities. After reverse transcription and probing on cDNA, neighboring probes are ligated and the number of ligations quantified by using unique molecular identifiers and sequencing. A panel covering 42 genes (ie, 2195 probes) involved in breast/ovarian and colorectal cancer predispositions was designed. After a training phase on 40 samples, SEALigHTS was validated in another laboratory on 56 samples carrying various splicing variations previously characterized by RNA sequencing, with a sensitivity of 96% and specificity of 94%. Subsequently, in a series of 37 selected patients and 114 consecutive patients from the genetics clinic with a concomitant DNA panel, five new diagnoses were made, revealing the impact on splicing of a cryptic genomic variant (deep intronic, retrotransposon insertion), and the unexpected impact on splicing of six genomic variations was unmasked. Beyond increased diagnostic yield and classification of variants of uncertain significance, this comprehensive DNA and RNA combined approach highlights unexpected splicing defects and addresses genotype-phenotype correlation issues.

Antibody–drug conjugates (ADC) improved survival in patients with HER2-positive MBC. To date, there is no prognostic biomarker in routine practice for these patients. HER2-ECD is associated with poor prognosis but has not yet been studied in patients receiving ADC. This monocentric retrospective study assessed in HER2-positive MBC patients shows the prognostic value on OS and PFS of (1) baseline HER2-ECD and CA15-3 and (2) HER2-ECD and CA15-3 after 3 months of treatment with TDM-1. At baseline, patients were divided according to the median value of HER2-ECD and CA15-3 from study population. For kinetic assessments, we compared survival outcomes according to the evolution of HER2-ECD and CA15-3 (stable/decrease vs increase). 40 patients were included. For both biomarkers, baseline values were not prognostic for OS neither PFS. Patients with stable or decrease HER2-ECD at 3 months had a significantly longer OS (median 43 versus 15.3 months, p < 0.0001) and PFS (median 9.4 versus 2.9 months, p = 0.0018) than patients with decrease, confirmed in multivariate analysis (p = 0.004 for OS and < 0.0001 for PFS). In contrast, CA15-3 kinetic was only prognostic for PFS (median 9.6 versus 4.9 months, p = 0.019), confirmed in multivariate analysis (p = 0.008). In this retrospective cohort, HER2-ECD kinetic was a prognostic biomarker for OS and PFS in patients with an HER2-positive MBC treated with TDM-1.

Ligation-mediated RT-PCR is a rapid and highly sensitive solution to detect the presence of gene fusions in cancers. Here, we provide a detailed protocol which allows the detection of hundreds of these tumor markers in a single assay, in a rapid turnaround time.

Introduction: The GAINED study (NCT01659099) was a randomized phase 3 trial comparing obinutuzumab (G) to rituximab (R) plus ACVBP or CHOP14 induction, followed by PET-guided consolidation. No significant survival differences were found between the arms (Le Gouill et al. 2021). This post-hoc analysis aimed to detail the outcomes of primary mediastinal B-cell lymphoma (PMBL) patients included in the trial, verified through expert pathological review and the use of a molecular gene-expression profiling-based (GEP) classifier. Methods: Conducted by LYSA across 99 centers in Belgium and France from 2012 to 2015, the GAINED study had the following key inclusion criteria : patients aged 18-60 years, CD20+ diffuse large B-cell lymphoma (DLBCL) according to the 2008 WHO classification, aaIPI ≥1, and eligibility for autologous stem cell transplant (ASCT). Patients with a ΔSUVmax >66% after cycle 2 and >70% after cycle 4 (PET2−/4−) received standard consolidation chemotherapy, while those with PET2+/4− received intensive treatment with ASCT. PET4+ patients (ΔSUVmax ≤70%) were given salvage therapy. We here focused on clinical characteristics, treatment responses, and outcomes, incorporating metabolic tumor volume (MTV, fixed SUV4 method) and biological data including cell-free DNA (cfDNA) and soluble PDL1 (sPDL1) levels, mutational landscape (custom targeted NGS panel), and GEP (LymphoSign test). Primary endpoints included were progression-free survival (PFS) and overall survival (OS). False discovery rate correction was applied for multiple testing. Results: Of the initial 670 patients, 138 (20.6%) confirmed PMBL cases were analyzed. Baseline characteristics included a median age of 33.5 years, 63.8% female, 55.1% stage III-IV, 90.6% elevated LDH, 87% ECOG 0-1, 62.3% extranodal involvement, 52.2% aaIPI 2-3, 50.7% MTV ≥360 cm³, and 53.6% with bulky (>10cm) mediastinal masses. Induction regimens were R/G-CHOP14 (56.5%) and R/G-ACVBP (42.8%). Post-induction treatments, based on interim PET (iPET) results, included: consolidation chemotherapy (55.1%), ASCT (24.6%), and salvage therapy (12.3%). After a median follow-up of 39.5 months, 2-year PFS and OS rates were 86.2%, and 93.2%. PET2−/4− had superior PFS (p<0.001) but similar OS (p=0.123) compared to PET2+/4− and PET4+ patients; the 2-year PFS and OS were: 93.5% vs 82.4% vs 58.8% and 96% vs 90.9% vs 85.6% respectively. ACVBP plus ASCT (n=38) and CHOP14 plus ASCT (n=38) had similar 2-year PFS and OS: 94.6% vs 92.1% and 97.1% vs 94.7%. ACVBP plus sequential consolidation chemotherapy and CHOP14 8 cycles had also comparable 2-year PFS and OS: 81.8% vs 82.6% and 90.9% vs 90.9%. Among patients with GEP data (n=107), 38 (35.5%) were PDL1high/PDL2high. Key somatic mutations data (n=87) included SOCS1 (70.1%), B2M (56.3%), STAT6 (49.4%), TNFAIP3 (47.1%), GNA13 (39.1%), CIITA (37.9%), CD58 (36.8%), and TP53 (29.9%). Univariate analysis highlighted that bulky mass (HR 4.37 [95%CI: 1.48-12.92]), stage III-IV (HR 4.06 [1.38-12.01]), and PET4+ (HR 3.18 [1.07-9.48]) were associated with shorter PFS, whereas PET2- and ΔSUVmax PET4 (continuous variable) where associated with longer PFS (HR=0.48 [0.16-1.44] and 0.26 [0.15-0.43], respectively). Additionally, stage III-IV (HR 9.9 [1.28-76.57]) was linked to shorter OS, whereas ΔSUVmax PET4 was associated with longer OS (HR=0.36 [0.21-0.62]). None of the other factors assessed (aaIPI, induction chemotherapy regimen, R or G, cfDNA and sPDL1 level, PDL1high/PDL2high status, B2M,CD58, TP53 alterations, MTV) were significantly associated with outcomes. Finally, in multivariate models, bulky mass adjusted on aaIPI remained significantly associated with inferior PFS (HR 3.84 [1.11-13.27], p=0.034). When MTV and ΔSUVmax PET2 and PET4 were also included, only ΔSUVmax PET4 was associated with longer PFS (HR=0.21 [0.08-0.55], p=0.001) and OS (HR=0.46 [0.23-0.93], p=0.032). Conclusion: This analysis highlights the strong representation, distinct characteristics and excellent outcomes of PMBL patients in the GAINED trial, emphasizing the importance of expert histopathological and molecular characterization for accurate diagnosis. The iPET response, especially ΔSUVmax PET4, emerged as the primary predictor of outcomes in this selected clinical trial population. Tailoring consolidation treatments based on iPET results may have mitigated the impact of baseline adverse characteristics.

Introduction Follicular lymphoma (FL) exhibits a highly variable course, sometimes resulting in histological transformation (HT) to higher-grade lymphoma, complicating clinical management and the prediction of individual outcomes. Each patient's tumor expresses a unique cell surface immunoglobulin (Ig) that may recognize antigens and/or transduce signals. Methods We performed high-throughput RNA sequencing (5' RACE) to simultaneously target the B- and T-cell Ig repertoires (BCR and TCR) in order to assess clonal dominance from bulk RNA samples of diagnostic FFPE biopsies. The data were correlated retrospectively with clinical data, gene expression profiling (RT-MLPseq, Lymphosign® signature), and next-generation sequencing data (custom targeted panel) from two independent monocentric FL patient cohorts treated with standard first-line immunochemotherapy. All patients provided written consent, and diagnoses were confirmed by Lymphopath network pathologists. Our aims were to predict progression-free survival (PFS), progression within 24 months (POD24), overall survival (OS), and HT risk using the preeminence of dominant clonotypes and the Simpson diversity index to measure intraclonal heterogeneity. The Simpson index, ranging from 0 to 1, measures the diversity of CDR3 sequences, with a value approaching 1 indicating uniformity. Log-rank tests were used to assess OS, PFS, and HT via Kaplan-Meier estimates. Univariate Cox regressions were conducted on relevant variables. Results The training cohort included 89 FL patients (grade 1-2: 88.8%; grade 3A: 11.2%, diagnosis between 2006 and 2019) with median age 62 [35-83] years; female sex, 49.4%; ECOG 0-1, 96.6%; stage III-IV, 89.9%; high tumor burden (GELF), 86.5%; R-CHOP-like treatment, 87.6%; FLIPI score 3-5, 48.3%; BCL2 t(14;18), 82.4% (missing data: n=4); HT, n=10 (4.5%); POD24, n=18 (20.2 We correlated B-cell clonal dominance with survival data and identified a subgroup of 10 out of 89 patients (11.2%) where the dominant clones represented more than 76.2% of all CDR3 sequences for IgVH, with a Simpson index greater than 0.89. Both criteria must be met to define high clonal dominance (HCD), with thresholds determined by receiver operating characteristic (ROC) analysis for OS events. These HCD patients, compared to others, had shorter OS (HR=5.1 [1.87-14.17], Cox p=0.002), a higher HT risk (log-rank p=0.042, HR=3.7 [0.9546-14.32], Cox p=0.06) but no significant difference in PFS and POD24 (PFS: HR=1.6 [0.66-3.75], p=0.304; POD24: HR= 0.51 [0.07-3.8], p= 0.51). Causes of deaths (n=18) between HCD and non-HCD patients were lymphoma (50% vs 75%, p=0.34), toxicity (0% vs 8.33%, p=1) or secondary malignancy (50% vs 16.67%, p=0.27). HCD patients had similar clinical characteristics, CD19 and MS4A1 gene expression, and median allele frequency for all somatic variants (26% vs. 25.7%), suggesting similar tumoral infiltration between HCD and non-HCD cases. There was no significant difference in the number of acquired IgVH/IgVL CDR3 N-glycosylation (N-gly) sites between HCD and non-HCD patients (median = 1 site, p=0.23). TP53 variants were more common in the HCD group (n=3, 30%, vs. n=8, 10.13%). HCD patients had only IgM isotype, lacking class switch recombination (CSR), unlike non-HCD patients who had IgG, IgE, or IgA isotypes (45.6%, p=0.005). All patients except one (a non-HCD patient with a BCL2 t(14;18) but no N-gly site) had mutated IgVH (<98% identity to the germline sequence). The validation cohort consisted of 45 patients (grade 1-2: 91.3%; grade 3A: 8.7%, diagnosis between 2006 and 2021) with median age 64 [41-79] years; female sex, 43.5%; ECOG 0-1, 93.5%; stage III-IV, 95.7%; high tumor burden, 100%; R-CHOP-like treatment, 100%; FLIPI score 3-5, 67.4%. Using the same cutoffs as the training cohort, we identified 3/45 patients (6.7%, IgM isotype: n=2, mutated IgVH: n=3) with worse prognosis (PFS: HR [95% CI] = 5.7 [1.19-26.86], p=0.029; OS: HR=17 [2.39-122.8], p=0.005) and higher HT risk (log-rank p<0.001, HR not estimable) associated with HCD status. Conclusions Using the 5' RACE assay, we identified a homogeneous subpopulation of highly proliferative lymphoma cells in two FL patient sets, associated with higher HT risk and death. HCD status may define a new entity of more aggressive FL, characterized by lack of CSR and lower clonal diversification, enhancing our understanding of FL biology and improving patient management.

Introduction: Primary mediastinal B-cell lymphoma (PMBL) is a rare and aggressive B-cell lymphoma. Its diagnosis primarily relies on small core biopsies. Therefore, alternative sources of tumor DNA are needed for genotyping when the tissue biopsy has been exhausted during routine diagnostics. Methods: In this preliminary analysis, we present the baseline mutational profiling results of patients enrolled in the ongoing prospective CAMIL study (NCT04824950). The study aims to evaluate the diagnostic performance of ctDNA minimal residual disease (MRD) in PMBL. Adult patients newly diagnosed with PMBL and treated with combination immunochemotherapy were prospectively included across 30 LYSA centers between 2021 and 2023. Before administering any anti-lymphoma treatment, except for corticosteroids, blood was collected in Streck® Cell-Free DNA BCT for the isolation of cfDNA and in PAXgene® Blood DNA tubes for the isolation of gDNA of PBMC which was used as germline controls. A Cancer Personalized Profiling by Deep Sequencing (CAPP-seq) ctDNA protocol was used to analyze ctDNA (~380 kb selector, 155 genes, including 30 non-coding regions targeted by aberrant somatic hypermutation [aSHM]; coverage >2000x in >80% of the region of interest). A background error-suppressed approach was used for variant calling (sensitivity: 10-3). Non-target reads were utilized for the detection of somatic copy number abnormalities (SCNA) and FACTERA was used for the detection of fusions. Results: The study recruited 87 patients, of which 86 had baseline plasma samples collected before the start of first-line treatment. ctDNA was detected in 84/86 (98%) patients. ctDNA fragmentation patterns showed 53% of patients had predominantly mononucleosomal fragments (median size [range] : 181 [151-208] bp), while 47% had a shift towards submononucleosomal lengths (159 [134-181] bp). The median baseline ctDNA level was 703.7 hGE/mL; the median variant allele frequency was 8.8%; the median number of variants per gene and per sample were 7 and 99, respectively. Among the 133 mutated genes, the top 5 encompassed BCL6 (97.6%, almost exclusively non-coding mutations of the intragenic superenhancer [SE]), SOCS1 (88.1%), IGLL5 (84.5%), CD83 (72.6%, almost exclusively non-coding mutations mapping in intron 2), and B2M (71.4%). The five genes with the highest number of different variants were IGLL5 (n=3631), BCL6 (n=2141), SOCS1 (n=876), CD83 (n=351), and RHOH (n=192), all these genes being targets of aSHM mediated by AID. The top 3 SCNA were focal gain of 9p24.1 (60%), gain of chromosome 9 (23.5%), and focal gain of 2p16.1 (22.4%). Within the genomic space covered by our selector, we identified at breakpoint resolution recurrent genomic translocations involving SOCS1 (n=6), IL21R-IL4R (n=3), and MAP3K14-SPATA32 (n=2). We compared the aSHM load of PMBL with that of classical Hodgkin lymphoma (cHL, n=317) and diffuse large B-cell lymphoma (DLBCL, n=235) cases, all assessed using ctDNA with the same CAPP-seq selector, reusing data from our laboratory's database. The median proportion of variants belonging to the AID-signatures was significantly higher in PMBL (58.3%) compared to cHL (31.6%) and DLBCL (42.5%) (p<0.01). The proportion of PMBL harboring mutations within AID-hypermutated SE hotspots was significantly higher (26.7%) compared to cHL (9.8%) and DLBCL (15.1%) (p<0.05). The AID-hypermutated SE hotspots BCL6, SOCS1 and CD83 were more heavily mutated (95%, 86%, 71%) in PMBL than DLBCL (71%, 17%, 23%) and cHL (66%, 49%, 23%). By performing Nonnegative Matrix Factorization-based clustering, we observed that the majority of PMBL cases (n=47, 55.3%) are grouped with cHL cases (n=75, 31.4%) and a small subgroup of DLBCLs (n=4, 1.7%). This cluster is predominantly characterized by 9p24.1 gains (27% of cases), involving JAK2 and PDL1/PDL2, and other significant genetic alterations such as SOCS1 (80.2%), TNFAIP3 (62.7%), B2M (58.7%) and XPO1 (28.6%) mutations. These genetic features collectively argue for pathogenic pathway associated with immune evasion mechanisms and cytokine signaling, particularly involving the JAK/STAT pathway. Conclusion: ctDNA analysis by CAPP-seq in PMBL is informative for 98% of patients at baseline and reflected the strong involvement of AID-associated hypermutation in the lymphomagenesis. Each identified variant at diagnosis can serve as a biomarker for monitoring MRD during treatment.

Chronic lymphocytic leukemia (CLL) is an incurable indolent non-Hodgkin lymphoma characterized by tumor B cells that weakly express a B-cell receptor. The mutational status of the variable region (IGHV) within the immunoglobulin heavy chain (IGH) locus is an important prognosis indicator and raises the question of the CLL cell of origin. Mutated IGHV gene CLL are genetically imprinted by activation-induced cytidine deaminase (AID). AID is also required for IGH rearrangements: class switch recombination and recombination between switch Mu (Sμ) and the 3' regulatory region (3'RR) (Sμ-3'RRrec). The great majority of CLL B cells being unswitched led us to examine IGH rearrangement blockade in CLL. Our results separated CLL into two groups on the basis of Sμ-3'RRrec counts per sample: Sμ-3'RRrecHigh cases (mostly unmutated CLL) and Sμ-3'RRrecLow cases (mostly mutated CLL), but not based on the class switch recombination junction counts. Sμ-3'RRrec appeared to be ongoing in Sμ-3'RRrecHigh CLL cells and comparison of Sμ-3'RRrec junction structural features pointed to different B-cell origins for both groups. In accordance with IGHV mutational status and PIM1 mutation rate, Sμ-3'RRrecHigh CLL harbor a non-germinal center experienced B-cell imprint while Sμ-3'RRrecLow CLL are from AID-experienced B cells from a secondary lymphoid organ. In addition to the proposals already made concerning the CLL cell of origin, our study highlights that analysis of IGH recombinatory activity can identify CLL cases from different origins. Finally, on-going Sμ-3'RRrec in Sμ-3'RRrecHigh cells appeared to presumably be the consequence of high c-MYC expression, as c-MYC overexpression potentiated IGH rearrangements and Sμ-3'RRrec, even in the absence of AID for the latter.