Abstract Digital PCR (dPCR) is a powerful technology for absolute quantification of nucleic acids, valued for its accuracy, sensitivity, and repeatability. Yet, the commercialization of different instruments with proprietary software has introduced challenges to data analysis, interoperability, and comparability. Therefore, we present the Digital PCR Data Essentials Standard (DDES) – a lightweight, human- and machine-readable, and cross-platform data standard developed in collaboration with the dPCR community. The standard consists of three file types designed to enable both manual inspection and automated analysis: (i) a main file summarizing experiment and reaction-level (meta-)data; (ii) an assay file describing targets and detection chemistry, and (iii) intensity files capturing partition-level raw fluorescence data per reaction. DDES supports a wide range of current dPCR applications, including singleplex and multiplex assays, endpoint and real-time readouts, and will be curated to implement future dPCR developments. By harmonizing the data structure, DDES lays out the foundation for FAIR dPCR data practices and supports improved software compatibility, collaborative and reproducible research, and future dPCR data repositories.

ABSTRACT Background The level of measurable residual disease (MRD) is one of the most important features correlating depths of response and long‐term outcomes in multiple myeloma (MM) and MRD evaluation is currently the gold standard tool for assessing treatment response. Nevertheless, reproducibility across laboratories is a major concern, as discrepancies among results make comparability impractical. Aims: herein, we report preliminary results from the “Italian MM‐MRD network” project. Patients & Methods MRD in bone marrow (BM) samples have been measured from newly diagnosed MM patients using next‐generation flow‐cytometry (NGF) or next‐generation sequencing (NGS) approaches in different laboratories. Results The NGF workgroup (7 laboratories) implemented the Euro‐Flow Standard‐Operating‐Protocol to reach minimum 1 × 10−5 sensitivity. The inter‐operator retrospective study (Stage 1) showed high inter‐center concordance in monoclonal plasma cells detection (ICC = 0.90, p < 0.001), whereas moderate concordance was observed in the inter‐laboratory correlation (Stage 2) in in‐vivo samples (ICC = 0.63, p < 0.001), reaching a median limit‐of‐detection (LOD) and limit‐of‐quantification (LOQ) of 8 × 10−6 and 2 × 10−5, respectively. Greater variability was also observed in the analysis of other BM cell populations. The NGS workgroup (4 laboratories) employed a targeted amplicon‐based approach to detect clonotypic IGH/IGK gene rearrangements in diagnostic samples, subsequently used to track MRD in mock samples. The experimental design was divided into three quality‐control (QC) rounds, focused on finding a shared strategy for clonotype identification (QC1: 100% concordance among centers), or quantifying MRD in mock samples (concordance: 81% [QC2]; 91% [QC3]). The 10−5‐sensitivity level was successfully reached in most of tested dilutions (QC2: 19/20 = 95%; QC3: 19/23 = 83%). Conclusion Overall, this pilot study provided preliminary data for MRD harmonization across Italian centers, paving the way for an expanded network, aiming at reducing variability, improving comparability, and enabling broader use of MRD‐monitoring in clinical practice.

The present study comprehensively dissects the molecular landscape of elderly mantle cell lymphoma (MCL) patients enrolled in the phase II V-RBAC trial of the Fondazione Italiana Linfomi. Of the 140 patients enrolled in the trial, 132 had available gDNA extracted from lymph node biopsies or bone marrow aspirates and were included in the analysis. A CAPP-Seq assay targeting 146 genes relevant to MCL pathogenesis was employed to identify gene mutations and copy number variations. ATM was the most frequently mutated gene, detected in 55 patients (41.7%), followed by TP53 and KMT2D in 31 patients (23.5%). ATM deletion was observed in 32 patients (24%), while CDKN2A loss in 29 (22%). Beyond TP53 mutations, three other molecular lesions, including CDKN2A loss, CD36 mutations and single-hit ATM abnormalities (either mutation or deletion) were independently associated with progression-free survival after adjustment for high-risk trial-defining features, namely Ki-67 >30% and blastoid variant. Notably, patients harboring single-hit ATM alterations without any additional risk factors achieved durable long-term remission, while CD36 mutations were associated with adverse survival. Both findings represent previously unrecognized aberrations that in this cohort independently and inversely associated with survival. The four variables were integrated into a 4-factor molecular prognostic model internally validated using a bootstrapping approach, which identified four distinct patient subgroups with significantly different outcomes. These findings support the importance of i) molecular profiling in MCL, ii) risk-adapted trials like V-RBAC, and iii) the integration of other biological markers with TP53 mutations for a more precise risk assessment in MCL. (NCT03567876)

Abstract Waldenström macroglobulinemia (WM) is a rare, indolent B-cell lymphoproliferative disorder, often preceded by a history of immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance (IgM-MGUS). In this retrospective, multicenter study, we collected real-life data from 577 patients with IgM gammopathy (221 symptomatic WM [sWM], 245 asymptomatic WM [aWM], 111 IgM-MGUS) from 22 Spanish centers with a validation cohort of 166 patients (73 sWM, 71 aWM, 22 IgM-MGUS) from the University Hospital of Torino, Italy. The median overall survival (OS) was 126.7 months for the Spanish cohort and 202.8 for the Torino cohort. A multivariate analysis identified age >65 years, male gender, diagnosis of sWM, and β-2-microglobulin >3 as significant predictors of a shorter OS. In addition, age >65 years, bone marrow (BM) biopsy infiltration, hemoglobin <11.5 g/dL, and platelets <100 000/μL were associated with a shorter time to first treatment (TTFT). Pooling data from both cohorts revealed that a baseline BM quantitative MYD88 L265P to wild-type MYD88 ratio of >0.162 (either by droplet digital polymerase chain reaction [PCR] or quantitative PCR), together with multiparameter flow cytometry (MFC) infiltration >4.39%, had a significant impact on OS and TTFT. The combination of MYD88 and MFC levels enabled stratification of patients into high-, intermediate-, and low-risk groups with patients with high-risk IgM gammopathy showing increased disease-related death in a competing risk analysis.

BACKGROUND:Bendamustine and rituximab combined with intermediate-dose cytarabine (RBAC) is one of the standard initial treatments for older, fit patients with mantle cell lymphoma. We aimed to investigate whether the addition of venetoclax to RBAC would improve progression-free survival in patients with high-risk mantle cell lymphoma. METHODS:FIL_V-RBAC was a multicentre, single-arm, phase 2 study done in 35 institutions of the Fondazione Italiana Linfomi in Italy. Treatment-naive patients with a histological diagnosis of mantle cell lymphoma, aged 65 years or older and fit according to the Fondazione Italiana Linfomi modified comprehensive geriatric assessment (or younger than 65 years and ineligible for high-dose chemotherapy with Eastern Cooperative Oncology Group performance status of 2 or less), were classified after enrolment as having low-risk or high-risk disease, based on the presence of blastoid morphology, Ki67 30% or higher, TP53, or 17p deletion. Patients with a low-risk profile received RBAC intravenously (rituximab 375 mg/m2 and day 1; bendamustine 70 mg/m2 on days 1 and 2; and cytarabine 500 mg/m2 on days 1, 2, and 3) every 4 weeks for 6 cycles. Patients with a high-risk profile received four cycles of RBAC followed by fixed-duration oral venetoclax consolidation (4 months, 800 mg/day) and maintenance (20 months, 400 mg/day). The primary endpoint was 2-year progression-free survival for patients with a high-risk profile who received at least one dose of RBAC. This trial was registered with ClinicalTrials.gov, NCT03567876, and this is the final report. FINDINGS:Between Sept 10, 2018, and July 26, 2021, 155 patients were screened for inclusion, 140 of whom were enrolled and analysed for study endpoints. Median age was 72 (IQR 69-76), 107 (76%) patients were male, 33 (24%) were female, and all were White. 54 (39%) patients had a high-risk profile (28 [20%] with TP53 mutations, 19 [14%] with 17p deletions, 34 [24%] with Ki67 ≥30%, and 13 [9%] with a blastoid morphology) and 86 (61%) had a low-risk profile. After a median follow-up of 45 months (IQR 40-55), the 2-year progression-free survival in the high-risk group was 60% (95% CI 48-74) and the median progression-free survival was 37 months (95% CI 19-not reached). The most frequent grade 3 or worse adverse events during venetoclax consolidation were neutropenia (12 [28%] of 43 patients), followed by thrombocytopenia (three [7%]) and skin reactions (three [7%]). During venetoclax maintenance, the most frequent grade 3 or worse adverse events were neutropenia (seven [19%] of 37 patients), followed by thrombocytopenia (two [5%]) and anaemia (two [5%]). One (1%) of 140 patients had a treatment-related death (tumour lysis syndrome during first induction with RBAC in a patient with a high-risk profile). INTERPRETATION:To our knowledge, this is the first prospective study to stratify patients with mantle cell lymphoma to different treatments according to their risk profile. Our results suggest that the addition of fixed-duration venetoclax improves the performance of RBAC in patients with a high-risk disease profile. Our findings point to the importance of identifying patients with high-risk disease at initial diagnosis. FUNDING:Fondazione Italiana Linfomi-Ente del Terzo Settore, Leukemia and Lymphoma Society, and Ministry of Health, Italy, and AbbVie. TRANSLATION:For the Italian translation of the abstract see Supplementary Materials section.

Introduction The TRIANGLE trial (Dreyling et al, Lancet 2024) compared three treatment arms in untreated, younger patients with mantle cell lymphoma (MCL): arm I (IR-CHOP/R-DHAP + ibrutinib maintenance [Im]), arm A+I (IR-CHOP/R-DHAP + ASCT + Im), and arm A (standard-of-care R-CHOP/R-DHAP + ASCT). Ibrutinib-containing regimens demonstrated superior failure-free survival (FFS) and overall survival (OS). Prior studies (Hadzidimitriou et al, Blood 2011) have shown skewed immunoglobulin (IG) repertoires in MCL, with preferential IG heavy chain (IGH) gene usage. However, large, homogeneous cohorts have not clearly linked IGHV usage or IGHV mutational status to clinical outcomes. Moreover, the interaction between biological risk factors such as TP53 aberrations and IGH repertoire is still unknown. Here, we assess the prognostic significance of IGHV repertoire after adjustment for baseline risk factors. Methods IGH clonal rearrangement analysis for minimal residual disease (MRD) was performed on bone marrow or peripheral blood samples centralized in 7 EuroMRD Network laboratories. Samples were analyzed by Sanger or amplicon-based NGS (VH-FR1/JH-3 primers) and were processed via IMGT/V-QUEST or ARResT/Interrogate to assign IGH rearrangements and germline FR1-IGHV identity. Diagnostic lymph node biopsies were assessed for p53 immunohistochemistry expression as a surrogate of TP53 alterations, scored as low (<50%) or high (≥50%). Missing baseline values were imputed by Chained Equations (MICE) model. The prognostic impact of IGHV genes and IGHV mutational status on FFS was analyzed with Kaplan-Meier curves and multivariable Cox regression adjusted for baseline prognostic factors. Results Out of the 870 enrolled patients, 560 had an available IGHV sequence for analysis. Baseline characteristics and outcomes were comparable to the remaining TRIANGLE patients and, in the selected patients, ibrutinib-containing arms had superior outcomes compared with the control arm (3y-FFS: arm A 70% vs arm A+I and I: 85%, P=0.001). The most common IGHV families were IGHV 3-21 (n=123, 22%), 4-34 (n=59, 11%) and 1-8 (n=42, 7.5%) whereas IGHD 3-3 (n=60, 11%) and IGHJ 4 (n=230, 41%) were the most frequent IGHD and IGHJ families, respectively. Univariable Cox regression focused on IGHV family usage identified the VH 3-21, 3-30, 3-48, and 3-74 genes as associated with improved FFS which were therefore grouped together, namely VHcomb patients (N=171). VHcomb patients were younger, had lower MIPI scores, and showed superior 3-year FFS compared to other families (VHother) (87% vs. 77%, P=0.002). Stratified by treatment arm, VHcomb had significantly better FFS vs. VHother in arm A (87% vs. 62%, P<0.001), but no difference was observed in ibrutinib-containing arms (87% vs. 85%, P=0.22). Interestingly, after adjusting for p53, MIPI and Ki67, VHcomb remained associated with improved FFS in arm A (Hazard ratio [HR] 0.46 [0.23-0.92], P=0.029), while no statistically significant impact was observed in arm A+I: HR 1.08 [0.55-2.13], P=0.82; but still a trend in arm I: HR 0.51 [0.23-1.13], P=0.096. Subsequently, to investigate an optimal cut-off point for FR1-IGHV gene identity, a Cox regression with restricted cubic splines was performed, identifying 97% as the best FR1-IGHV gene homology cut-off for prognostic discrimination of FFS after adjustment for MIPI, histologic subtype, Ki67, and treatment arm. Patients with FR1-IGHV gene identity > 97% (FR1-IGHV unmutated, n=448 [80%]) showed a trend towards worse FFS at later follow-up (log rank P=0.056) compared to patients with FR1-IGHV gene identity ≤ 97% (FR1-IGHV mutated, n=110 [20%]). No significant differences in FFS were observed according to FR1-IGHV mutation status after adjusting for p53, MIPI and Ki67 in multivariable analysis both in standard and ibrutinib-containing regimens. Conclusions This is the largest study investigating IGH repertoire in a prospective phase 3 trial in MCL. FR1-IGHV unmutated patients showed inferior FFS. The IGHV 3-21, 3-30, 3-48, and 3-74 rearrangements were associated with improved FFS in the chemo-immunotherapy arm, independently of p53 alterations. On the other hand, the addition of ibrutinib may potentially mitigate the prognostic impact of the IGHV families. Although requiring validation, these findings support a BCR-related prognostic role in MCL, independent from MIPI, Ki67 and p53 alterations and potentially modulated by ibrutinib.

Background:Covalent BTK inhibitors are the backbone of treatment for patients with Waldenström's Macroglobulinemia (WM). Addition of rituximab to ibrutinib has shown a remarkable efficacy in WM in the iNNOVATE trial, including patients carrying CXCR4 mutations or MYD88 wildtype (Dimopoulos et al, NEJM 2018, Buske et al. JCO 2022). In addition, the proteasome inhibitor Bortezomib (B) has shown significant activity in WM as single agent or combined with Rituximab, Dexamethasone and Cyclophoshamide (DRC) (Buske et al., JCO 2023). The ECWM-2 trial of the European Consortium for Waldenström's Macroglobulinemia (NCT03620903) aimed at evaluating the efficacy and toxicity of Bortezomib-Ibrutinib/Rituximab (B-IR) as first line treatment in WM. Methods:In this multicenter European single-arm phase II trial, treatment naïve patients with WM requiring therapy received 6 cycles (C) (d=28) of Bortezomib (1.6 mg/ m2 s.c. d1,8,15), Rituximab (375 mg/m2 i.v (C1d1), 1400 mg absolute s.c (C2-6 d1) and Ibrutinib (420 mg p.o. daily) followed by maintenance with Rituximab (1400 mg absolute s.c; D1 every 2nd month) combined with Ibrutinib for 24 months and subsequent ibrutinib treatment until progression or non-tolerated toxicity. Primary endpoint was the 1-year progression free survival (PFS) rate (1YPFS). Secondary endpoints included response rates, PFS, overall survival (OS), and toxicity. Plasma cell-free DNA (cfDNA) and digital droplet PCR (ddPCR) was used for identifying and quantifying MYD88L265P mutational burden in patients at different time points before and during treatment. Results: 53 patients were included and started trial treatment. Median age was 63 years (range 36-84), 62% were male and 70% of patients had intermediate/high risk according to the ISSWM prognostic score. Median baseline hemoglobin was 10.1 g/dl (7.1-14.5) and median baseline IgM 33.9 g/l (3.05-102.87). Mutational status was available for 51 patients with 31 pts (60.8%) showing mutated MYD88 (MYD88MT) and CXCR4 wildtype (CXCR4WT), 18 patients (35.3%) MYD88MT/CXCR4MT and 2 pts (3.9%) MYD88 wildtype (MYD88WT). Of note, no patients showed progression after a median follow-up of 37 months. The primary endpoint 1YPFS was 93% (38/41, p<0.001 in a one-sided exact binomial test to reject 1YPFS ≤ 60%), with 3 deaths. 2YPFS was 0.88 (95% CI: 0.79-0.97). OS probabilities were identical to PFS probabilities due to the fact of lacking progressions. B-IR reduced rapidly deep responses with an overall response rate (ORR) and a major response rate (MRR) of 98% and 70%, respectively, after 3 cycles, with a median reduction of IgM serum levels by 74%. At best response 98% of all patients achieved a MRR with 100% ORR. The proportion of patients with VGPR/CR increased over time with 19% versus 28% versus 38% after 6 cycles, 12 cycles and at best response, respectively. Median time to major response was 2.8 months. Responses were largely independent of CXCR4 mutations with an MRR of 76 and 70 % in the CXCR4WT vs CXCR4MT patients, respectively, at end of induction. In total 104 cfDNA plasma samples from 32/53 patients were analyzed by ddPCR for MYD88L265P mutation: 93% of patients (30/32) showed MYD88L265P at baseline (median AF: 3.8%; range 50%-0.24%) with 41% of samples reaching MRD negativity and an almost 1 log reduction among MRD positive cases (median residual AF: 0.63%; range 5.6%-0.1%) after 3 cycles and a MRD negativity rate of 65% (median residual AF of 0.16% (range 2.34%-0.035%) at end of induction. Grade ≥3 AEs related to treatment occurred in 45% of all patients. Most common grade ≥3 AEs included COVID-19 pneumonia (13.0 %), lower respiratory tract infection (11.1 %), and anemia (7.4%). Overall, 12 pts (22.6 %) developed infections grade ≥3. Peripheral sensory neuropathy occurred in 8 patients (all grade 1 and 2). There have been 8 deaths in the course of the study in total, 5 caused by COVID-19 and three caused by respiratory tract infection. Conclusion: With a 1YPFS of 93%, a major response rate of 98%, 65% MRD negativity after 6 cycles of treatment and no observed progression of disease after a median follow-up of 37 months, B-IR shows impressive efficacy in WM. All deaths were caused by respiratory infections, for which COVID-19 was confirmed as the cause in the majority of cases, reflecting patient recruitment of this trial in the COVID-19 pandemic. These data characterize B-IR as a novel and powerful treatment option for patients with WM.

The Consensus Panel 3 (CP3) of the 12th International Workshop on Waldenström macroglobulinemia (IWWM-12) has reviewed and incorporated current data to make recommendations for the management of patients with high-risk WM (HR-WM). Recognizing the considerable heterogeneity in survival outcomes and identifying a subgroup of patients with a very poor prognosis, the key recommendations from CP3 include: (1) Risk stratifying patients with smoldering WM (SWM) and active (symptomatic) WM at diagnosis (2) Using the degree of i) bone marrow lymphoplasmacytosis, ii) serum beta-2 microglobulin (β2M) elevation, iii) IgM increase, iv) serum albumin decrease and the presence of wild-type MYD88 status markers that adversely dictate the time-to-progression from smoldering to active WM to the define HR-SWM. (3) Among patients with active WM, the presenting parameters: advanced chronological age, low serum albumin, elevated serum lactate dehydrogenase, elevated β2M and the presence of TP53 alterations (TP53 mutation or deletion 17p) unfavorably impact the prognosis and should be utilized to risk-stratify patients into the HR category. (4) The panel encourages screening for genetic alterations at diagnosis, prior to initiating therapy and also with rapidly advancing disease or refractoriness to ongoing therapy, which might result from clonal evolution. Although limited data directing the selection and sequencing of therapies exist, a risk-adapted approach and clinical trial participation for patients with HR-WM are highly encouraged.

Background. TP53 mutations are emerging as a relevant prognostic and predictive biomarker in Waldenström Macroglobulinemia (WM). In two retrospective studies, TP53 mutations were associated with shorter progression-free survival (PFS) and overall survival (OS) with chemoimmunotherapy (CIT) (Poulain S et al, Clin Cancer Res 2017; Gustine JN et al, Br J Haematol 2019), while in a post-hoc analysis of the ASPEN study, TP53 mutations correlated with shorter PFS in patients treated with BTK inhibitors (Tam CS et al, Blood Adv 2024). Aim of the study. In the multicenter, observational prospective BIOWM trial (NCT03521596) sponsored by Fondazione Italiana Linfomi (FIL),we assessed TP53 mutations in paired bone marrow (BM) and peripheral blood (PB) samples of patients with untreated WM or IgM-Monoclonal Gammopathy of Undetermined Significance (IgM-MGUS), in order to assess the rate and prognostic impact of TP53 mutations and to evaluate whether these mutations can be detected with adequate accuracy in cell-free DNA (cfDNA) extracted from plasma. Methods. We analyzed paired BM and PB samples collected at the time of diagnosis (T0) or before treatment (T1). In patients with stable disease not requiring treatment, an additional PB sample was collected during follow-up at 12 months (FT1) and at 24 months (FT2). Targeted next-generation sequencing (NGS) of a 15-gene panel including MYD88, CXCR4, TP53, ARID-1A, NOTCH2, KMT2D, CD79b, CARD11, KLF2, TRAF3, HIST1H1E, PRDM1, MYBBP1A, BTK and PLCγ2 was performed on DNA extracted from BM and from plasma. OS and PFS were analyzed using the Kaplan-Meier method. The prognostic impact of TP53 mutations - either at diagnosis or acquired later - was estimated using a Cox regression model with TP53 mutation as time-varying covariate (TVC). Results. TP53 mutation status was assessed in 279 patients, including 210 with WM and 69 with IgM-MGUS. The median follow-up of the study population was 66 months. Overall, 31/279 patients (11%), including 21/210 patients with WM (10%) and 10/69 with IgM-MGUS (14%), harbored a TP53 mutation. The median variant allele frequency (VAF) was 30% (Inter Quartile Range, IQR 12-40) in WM and 6.8% (IQR 2.2-21) in IgM-MGUS patients (P=0.036). Of 31TP53 mutated patients, 20 were mutated at T0 or T1, while 11 acquired the mutation later during the watch-and-wait phase (FT1 or FT2). The total number of oncogenic mutations detected was 35, including 28 missense mutations and 7 nonsense mutations, and the majority of them (32/35, 91%) occurred in the central DNA binding domain encompassing exons 4-8. The rate of TP53 mutations was higher in cfDNA as compared with BM samples (10% versus 4%, P=0.001). Twenty-one of 31 TP53mutated patients (68%) harbored also the MYD88 (L265P) mutation by NGS, with a median VAF of 30% (IQR: 19-40%). By droplet digital polymerase chain reaction (ddPCR) the MYD88 (L265P) mutation was detected in 30/31TP53 mutated patients. As compared with TP53 wild-type patients, the subgroup of TP53 mutated patients was borderline enriched in CXCR4 mutations (35% versus 22%, P=0.113), and significantly enriched in KMT2D mutations (26% versus 9.7%, P=0.015) and NOTCH2 mutations (23% versus 6.5%, P=0.007). Among TP53 mutated patients, 10 have been treated as they met IWWM treatment criteria, 9 with chemo-immunotherapy (7 with Rituximab+Bendamustine, 2 with Dexamethasone+Rituximab+Cyclophosphamide) and 1 with single agent Cyclophosphamide. WM patients with a TP53 mutation had a trend towards a shorter time to first treatment (TTFT) (hazard ratio, HR 2.24, 95% CI 0.67-7.57, P=0.192) and towards an inferior OS as compared with wild-type ones (HR 1.99, 95% CI 0.97-4.09, P=0.055). In WM patients who were treated so far, we did not find a significant difference in PFS between TP53-mutated and wild-type patients (HR 0.65, 95% CI 0.28-1.49, P=0.310). Conclusions. To the best of our knowledge, this is the first prospective study demonstrating an impact of TP53 mutation status on OS in WM patients. Due to the low number of patients treated so far, an impact of TP53 mutations on PFS could not be confirmed at this time. Cell-free DNA extracted from plasma demonstrated to be at least accurate as BM DNA for the assessment of TP53 mutation status, offering an alternative non-invasive, patient-friendly approach for the detection and monitoring of these mutations. The impact of low VAF (<10%) TP53 mutations in patients with IgM-MGUS deserves further investigation.

Introduction IgM gammopathies encompass a heterogeneous group of hematological conditions, ranging from IgM monoclonal gammopathies of uncertain significance (IgM-MGUS) to asymptomatic Waldenström's Macroglobulinemia (aWM) and symptomatic WM (WM). This study aimed to investigate the potential of cell-free RNA (cfRNA) as a biomarker source in IgM gammopathies. Methods Diagnostic blood plasma samples were collected from a retrospective and prospective series of IgM gammopathy patients enrolled in the Fondazione Italiana Linfomi (FIL) “BIO-WM” trial (NCT03521516). The blood plasma was analyzed of 60 patients with IgM-MGUS (n=15), aWM (n=32), and WM (n=13), along with healthy controls (HC) (n=28), collected using EDTA or Cell-Free DNA BCT (Streck) tubes. RNA was extracted from 200 µL of plasma using the miRNeasy serum/plasma kit and sequenced on a NovaSeq 6000 instrument with the SMARTer Stranded Total RNA-seq pico v3 library preparation kit. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathways enrichment analyses were performed using the clusterProfiler and ReactomePA packages, respectively. Bulk deconvolution was performed using the Tabula Sapiens v1 basis matrix. Flow cytometry and MYD88L265P mutational dPCR data were also available. Results Due to superior quality of cfRNA in EDTA tubes (median number of genes with a count >10: n=4338 for EDTA versus n=35 for Streck) only data from 49/60 patients (12 IgM-MGUS, 30 aWM, 7 WM) and 14 HC were reported. The Streck tube's poor performance was observed in all the samples of both patients and HC, so it was not influenced by other preanalytics before cfRNA extraction. Compared to IgM-MGUS and HC, higher cfRNA concentrations were found both in aWM (p=0.037, 95% CI [3.5, 250 pg/mL]; p=0.039; 95% CI [2.3, 240 pg/mL]) and WM patients (p=0.036, 95% CI [30, 470 pg/mL]; p=0.031, 95% CI [13, 450 pg/mL]). CfRNA concentrations significantly correlated with IgM serum levels (p=0.0047, R=0.40) and bone marrow infiltration (p=0.0019, R=0.43). Numerous differentially abundant coding and non-coding genes (DAGs) were identified in the subgroups. A comparison between aWM and WM to IgM-MGUS patients revealed 259 and 510 DAGs, respectively, including RASSF6 and G0S2, which have been previously reported in WM. DAG are often lymphoma related genes and seem to capture disease related biological differences between the groups. Of note, aWM and WM patients showed enrichment in neurodegeneration, platelet activation, signaling, and aggregation, as well as neutrophil degranulation pathways. Interestingly, MYD88L265P plasma levels showed a weak negative correlation with normalized cfRNA counts of MYD88 (p=0.015, R=-0.36). TREML1, ITGA2B and PF4V1 are cfRNA transcripts that show high potential for classifying between HC, IgM-MGUS and (a)WM. However, more stringent analysis identified PF4V1 as a potential single marker to distinguish between HC, IgM-MGUS, and aWM/WM, with One-vs-Rest multiclass ROC AUCs of 0.98, 0.82, and 0.88, respectively. Survival analysis of aWM/WM patients showed a higher blood plasma abundance ofa 6-gene unfavorable signature (ECH1, EIF3B, DHX9, EPRS1, ATP5PF, HIST1H1C) linked to all-cause mortality (p<0.0001). Finally, a 3-gene unfavorable signature (PAFAH1B1, ARAF, SMG7) was associated with time to relapse or progression (p<0.0001). Conclusion This is the first reported evidence that a cell-free transcriptome signature observed in EDTA blood plasma can differentiate between distinct subgroups of IgM gammopathies. Moreover, cfRNA might assist in prognostication and therapy response prediction and elucidate underlying biological pathways. Further studies are warranted to confirm its role as a less invasive alternative to bone marrow examination.

Introduction. R-BAC (rituximan-bendamustine-cytarabine) is an highly effective first-line immunochemotherapy regimen for elderly fit patients with mantle cell lymphoma (MCL), allowing a considerable progression-free survival (PFS), particularly in low-risk (LR) patients, even without maintenance. On the other hand, outcomes in high-risk (HR) patients are still unsatisfactory, even with this therapy. To address this issue, the Fondazione Italiana Linfomi (FIL) designed the risk-tailored “V-RBAC” trial (NCT03567876), that showed how the addition of venetoclax to R-BAC in HR cases improved the performance of the induction strategy. Methods. This study is a multicenter, phase 2 trial for elderly (>64 years) fit MCL patients, offering consolidation (800 mg/day) and maintenance (400 mg/day up to 18 months) with venetoclax monotherapy after abbreviated R-BACx4 for HR patients; HR was defined as the presence of any risk factor among blastoid morphology, Ki67 ≥30%, or TP53 mutation/deletion. To investigate the efficacy of this novel regimen, HR patients were studied for minimal residual disease (MRD) both by standardized allele-specific oligonucleotide-droplet digital PCR (ddPCR), targeting either IGHV or IGH::BCL1 rearrangements, and by multiparametric flow cytometry (MFC). Bone marrow (BM) and peripheral blood (PB) samples were centralized in the four EuroMRD labs of the FIL MRD Network at baseline and at predetermined time points (before consolidation, after consolidation and at the end of treatment). Clinical data and primary endpoint of the trial were already reported[Visco, ASH 2023], we here describe in detail the MRD results. Results. Between September 2018 and July 2021, 54 HR MCL patients were prospectively enrolled in the trial. A reliable molecular marker for MRD by ddPCR was retrieved in 49 cases (91%), namely 29 IGHV only, 4 IGH::BCL1 only and 16 both markers. Overall, only 35 patients were evaluable for MRD in at least one timepoint, as 6 patients progressed during the induction and 8 interrupted the treatment due to toxicity or investigators decision. Nonetheless, the RBAC regimen resulted highly effective in clearing the MRD even in this HR population, with 25/33 (75%) patients achieving MRD negativity in BM and 28/35 (80%) in PB after four cycles of induction therapy. Moreover, after four cycles of venetoclax consolidation the rate of MRD negativity further raised to 86% (26/30) in BM and 90% (28/31) in PB, highlighting the activity of this drug in clearing the MRD at least in a subset of cases. Finally, after 18 months of venetoclax maintenance, the MRD negativity rate was 93% (13/14) in BM and 88% (15/17) in PB. Based on these data, we investigated the capacity of MRD to be a prognostic marker for PFS. Overall, MRD positivity at any timepoint was associated with an higher risk of relapse (HR 5.75, 95%CI 1.93-17.18, p=0.002 in BM and HR 7.89, 95%CI 2.65-23.53, p<0.001 in PB, respectively). In particular, the three patients still MRD positive after venetoclax consolidation experienced a dismal outcome with a relapse event after 7, 9 and 25 months, respectively. Finally, a direct comparison between ddPCR and MFC approach to detect MRD in PB was performed: detailed data are still under evaluation and will be presented during the meeting. Conclusion. We here described the MRD monitoring results in the risk-tailored phase II trial FIL V-RBAC. Four cycles of R-BAC regimen provided high rates of molecular MRD negativity in HR elderly fit MCL patients, further improved by a venetoclax consolidation. Patients MRD positive after induction or consolidation still experience dismal outcome and should be considered for alternative, experimental approaches.

This multicentre phase II study Fondazione Italiana Linfomi (FIL)-bortezomib plus rituximab plus bendamustine (BRB) tested a combination of bendamustine (90 mg/m2 on days 1-2), rituximab (375 mg/m2 intravenously on day 1) and bortezomib (1.3 mg/m2 sc on days 1, 8, 15, 22) every 28 days for six cycles in 38 symptomatic patients with relapsed/refractory Waldenstrom macroglobulinaemia (RR-WM). Moreover, MYD88L265P and CXCR4S338X mutations were tested by droplet digital polymerase chain reaction (ddPCR) both at baseline and at the end of treatment in 21 patients. Overall response rate at the end of therapy was 84.6%, including 4 (11%) complete remission, 15 (39%) very good partial response, 12 (32%) partial responses according to IWWM response criteria. At 18, 24 and 30 months, progression-free survival was 84.2% (95% CI 68.2%-92.6%), 81.5% (95%CI 65.1-90.7) and 78.8% (95%CI 62.0-88.8) respectively. At 18 months, the Overall survival was 92.1% (95%CI 77.5%-97.4%). Overall, 19 patients (50%) experienced grade 3-4 haematological toxicity, mainly thrombocytopenia, and grade 1-3 neuropathy rate was about 10% and required bortezomib dose reduction but did not result in treatment interruption. Moreover, BRB treatment induced the high rates of undetectable molecular minimal residual disease (MRD) at the end of the therapy. BRB regimen used as second line is an effective and well-tolerated salvage treatment for relapsed refractory Waldenstrom macroglobulinaemia patients. MRD monitoring showed promising efficacy in clearing the residual disease.

Background and purpose. Anti-myelin-associated glycoprotein (MAG) demyelinating polyneuropathy (PNP) is a rare and underdiagnosed condition, associated with monoclonal Immunoglobulin M (IgM) gammopathy of undeterminate significance (MGUS) or Waldenstroem macroglobulinemia (WM), that might deeply compromise patients' (pts) quality of life (QoL). Rituximab (RTX) has been largely used, with good efficacy if timely infused. We reported safety and efficacy of RTX in a small, retrospective, homogeneous series of anti-MAG PNP pts treated in a single Institution [Parisi, EJN 2022]. We here present updated, long-term clinical, hematological and neurophysiological results of that original series, along with a novel series of pts. Methods. Anti-MAG PNP pts with relevant clinical impairment were evaluated by a multidisciplinary team prior to, during and after treatment with RTX. Neurological evaluation included electromyography (EMG), INCATds, mISS, PGIC scale, serum anti-MAG titer, and physical examination. Hematological assessment included bone marrow biopsy (BMB) and BM aspirate, complete blood count, paraprotein quantification, serum immunofixation and IgM level; multiparameter flow cytometry (MFC) and droplet digital PCR for MYD88L265P, CXCR4 gene mutations were performed in unsorted BM samples. IgM MGUS or WM diagnoses were defined according to IWWM criteria [Semin, Oncol 2003]. RTX (375 mg/m2 weekly) was administered in “cohort 1” for 4 courses and in “cohort 2” for 8 courses. Hematological and neurological evaluations were performed before (T0), 1 year (T1) and 2 years (T2) after RTX, including CD19+ cells count in peripheral blood (PB) and evaluation of hematological response. PNP relapse (REL) was defined as increase ≥1 point at INCATds score or ≥2 points at mISS. Results. Between 2017 and 2023, 41 pts with confirmed anti-MAG PNP and IgM gammopathy were treated with RTX at the Hematology Division of Torino University Hospital; 7 pts had already received prior RTX while 35 were treatment naïve. Median age at the time of BMB was 71 years (range 50-82), 28 pts (68%) were male, 26 pts (63%) had WM vs 15 pts (37%) IgM MGUS. Median IgM baseline level was 507 mg/dL (range 121-1479 mg/dL), and median anti-MAG titer 1:60000 (range 1:5000-1:200000). Median BMB invasion by lymphoplasmacytic lymphoma (LPL) was 20% (range 5-60%) in those with confirmed WM; median clonal B lymphocyte and plasma cells (PCs) levels in BM aspirate by MFC were respectively 0.17% (range 0-35%) and 0% (range 0-2.34%). 35/38 evaluable pts (92%) carried MYD88L265P mutation in BM with a median level of 5x10-3 (range 4.7x10-4 - 1x10-1) while 6 out of the 19 evaluable pts (13 WM and 6 MGUS) were CXCR4MUT in BM with a median level of 1.75x10-3 (range 6x10-4-3.3x10-2). Interestingly, the median time between clinical PNP onset to RTX was 22 months (range 4-174). 22 pts were in cohort 1 (4 RTX), 19 in cohort 2 (8 RTX). Major response rate (MRR) was 47% at T1 and 58% at T2. Median baseline level of CD19+ B cells in PB in 7 evaluable pts was 67/µL (range 5-637/µL), while after RTX 7 out of 12 pts had no detectable CD19+ cells. Neurological responses (EMG, clinical scales and QoL) were initially reported elsewhere [Parisi, EJN 2022] and will be updated after the follow-up (FU) completion. Median FU was 79.5 months. 9 pts (22%, 7 WM and 2 MGUS) were retreated with RTX for clinical PNP relapse with a median time to next treatment of 27 months (range 11-65). Median CD19+ in PB was 41 c/µL (range 0-129 c/µL) prior to retreatment. 7 out of 9 REL pts came from cohort 1 (p=0.0535), but we should acknowledge that the median FU was longer in cohort 1 than in cohort 2 (89 vs 57 months, respectively, p=0.34). Finally, one REL patient required additional treatment achieving clinical response with zanubrutinib due to PNP progression, and another patient died for unrelated cardiovascular disease. Conclusions. This study describes a homogeneous, consistent and well characterized series of pts affected by IgM gammopathies and anti-MAG PNP effectively treated with RTX in a single center; likelihood of PNP recurrence after RTX seems to be correlated to the number of cycles given, but additional FU is needed to confirm this hypothesis. This work highlights how the cooperation between hematologists and neurologists might improve diagnostics, monitoring and outcome in this rare condition.