The interaction between lymphoma cells and immune microenvironment cells and the impact of this functional interplay on therapeutic responses remain largely unexplored. Here, we utilized murine models with oncogenically active MYD88 and additional genetic lesions co-triggered at selected B cell stages to generate human-like lymphomas harboring the MYD88L265P mutation. Lymphomas exhibited behaviors ranging from clinically indolent small-cell tumors to aggressive diffuse large B-cell lymphoma (DLBCL). Genetically diverse lymphoma cells employ distinct immune evasion mechanisms that shape unique lymphoma microenvironment (LME) states. In this setting, clonally expanded T-cells function as a double-edged sword, either sustaining indolent lymphoma cell survival or promoting antitumor responses in DLBCL. Consequently, the efficacy of standard-of-care and novel immunotherapies was determined using individual T-cell features. Furthermore, the experimental targeting of newly identified immune mechanisms has improved therapeutic responses in vivo. Our results elucidate that genetically driven LME landscapes influence therapeutic outcomes across distinct lymphoma subtypes, providing proof-of-concept for personalized treatment based on immune LME information.

The role of autologous stem cell transplantation (ASCT) in older patients (pts) with multiple myeloma (MM) is being re-evaluated in light of effective anti-CD38-based regimens now available for non-transplant eligible (NTE) pts. In this context, we retrospectively compared outcomes of transplant-eligible (TE) elderly pts receiving ASCT after Dara-VTD induction versus NTE pts treated with anti-CD38-based regimens, evaluating the impact of frailty status on outcomes. We analyzed 122 unselected pts aged 65–75 years (median age 72) with newly diagnosed MM at our institution between April 2016 and December 2024. ASCT eligibility was based on clinical judgment. TE pts received Dara-VTD induction, ASCT, Dara-VTD consolidation and lenalidomide maintenance (ASCT cohort), while NTE pts received anti-CD38-based regimens (no-ASCT cohort). Response rate, progression-free survival (PFS), overall survival (OS), and treatment-related toxicity were assessed. The IMWG frailty score was retrospectively applied to evaluate outcomes according to frailty status. Out of 122 pts recorded, 22 (18%) were ISS stage 3 and 40 (32%) had high risk (HR) cytogenetics. Median age was 68 vs 73 years in ASCT vs no-ASCT cohorts (p<0.0001), while HR cytogenetics and ISS-3 distribution were well balanced between cohorts. Thirty-six (29%) were assigned to the ASCT cohort (5 receiving double ASCT); 86 (71%) constituted the no-ASCT cohort: DRd 54%, D-VMP 16%, Isa-VRd 1%. According to the IMWG frailty score, 27 pts (22%) were classified as fit, 91 (75%) as intermediate-fit (unfit), and 4 (3%) as frail. In the ASCT cohort, 47% were fit and 53% were intermediate-fit. In the no-ASCT cohort, 11% were fit, 84% intermediate-fit, and 5% frail. ORR was 94% in the ASCT cohort vs 91% in the no-ASCT cohort (p=0.7), and ≥VGPR rates were 75% vs 62%, respectively (p=0.5). sCR+CR rates were higher in the ASCT cohort (53% vs 24%, p=0.032). MRD assessed by multiparametric flow cytometry (sensitivity 10⁻⁵) was available in 17 ASCT pts before maintenance, of which 13 (76%) were negative. After a median follow-up of 24 months, PFS was similar between ASCT and no-ASCT (median NR vs 75.5 months; 3-year PFS 83% vs 76%, p=0.55), as was OS (median NR in both cohorts; 3-year OS 93% vs 86%; p=0.41). In the ASCT cohort, PFS was longer in fit pts compared to unfit, although not statistically significant (3-year PFS 94% vs 73%; median NR in both groups; p=0.191). OS showed a similar trend (3-year OS 100% vs 87%; median NR; p=0.208). Among ASCT pts, the subgroup aged >70 years (n=4) showed outcomes comparable to those aged 65–70, with no PFS and OS events reported at the time of analysis. In the no-ASCT cohort, PFS differed significantly according to frailty status (p=0.04): 3-year PFS was 100% in fit pts, 70% in unfit, and 50% in frail; median PFS was 80, 75, and 29 months, respectively. OS was not significantly different between groups (p=0.3), with 3-year OS of 100%, 83%, and 100%, and median OS NR, NR, and 45 months, respectively. No significant differences in PFS or OS were observed when comparing pts aged 65–70 and 71–75 years within each treatment cohort. Notably, PFS in unfit pts receiving ASCT was comparable to that observed in the no-ASCT cohort (3-year PFS 73% vs 70%; p=0.891). Grade ≥3 hematologic toxicities were significantly more frequent in the ASCT cohort, with neutropenia in 92% vs 51% (p<0.0001) and thrombocytopenia in 89% vs 16% (p<0.0001). Infections of any grade occurred in 92% of ASCT pts vs 67% in the no-ASCT cohort (p=0.0017), with grade ≥3 infections reported in 64% vs 34% (p=0.0027), respectively. Temporary treatment discontinuation was comparable between groups. However, when considering the temporary discontinuation of at least one drug within the treatment regimen, the rate was significantly higher in the ASCT cohort (67% vs 43%; p=0.028), mainly due to thalidomide-related complications. These findings support the feasibility and effectiveness of ASCT in carefully selected pts aged >70 years. However, modern anti-CD38-based regimens represent a valid alternative in the 65–75 age group, offering comparable outcomes with a more favorable safety profile. The IMWG frailty score emerges as a valuable tool to guide therapeutic decisions regarding treatment intensity and transplant eligibility, particularly in unfit pts, whose outcomes were comparable to those treated with non-intensive regimens.

Approximately 95% of lymphoplasmacytic lymphomas (LPL) are IgM secreting and are characterized as Waldenstrom Macroglobulinemia (WM). Conversely, non-IgM secreting LPL are rare. As part of the 12th International Workshop on WM (IWWM-12), a consensus panel of experts was tasked to develop recommendations for the management and response assessment of non-IgM LPL. The panel considered that in view of available molecular, pathological and clinical data, non-IgM LPL should be considered as a separate sub-entity of LPL. The panel further recommended that the IWWM-2 consensus criteria used for IgM LPL (WM) treatment initiation, should also be used for non-IgM LPL and be independent of IgG or IgA paraprotein level unless symptomatic hyperviscosity is present. The panel agreed that based on current evidence, there is insufficient data to support a different clinical management for non-IgM vs IgM (WM) LPL. Moreover, the panel advised that patients with non-IgM LPL should be treated in a similar manner to patients with IgM LPL independent of MYD88 mutation status until more is known about its impact on treatment outcomes for non-IgM LPL patients. The panel therefore recommends the use of the IWWM-11 IgM LPL (WM) response criteria for cases of non-IgM LPL with a monoclonal IgA or IgG paraprotein component, but creating a specific panel to develop formal response criteria for this LPL subset was also recommended.

Background: A single risk assessment using the IMWG 20/2/20 model may be insufficient for individualized management of SMM pts. Serial assessments could improve risk-stratification through the identification of stable vs evolving patterns, but bone marrow (BM) aspirates cannot be performed repeatedly for quantification and characterization of tumor cells. Aim: Investigate the role of CTCs as an alternative to BM tumor cells for dynamic risk assessment of SMM. Methods: The iMMunocell study enrolled 324 untreated SMM pts from 2017 to 2024. A total of 1,250 blood samples were collected every 6 months during 3 years to monitor CTCs and immune profiles using next-generation flow cytometry. CTCs were isolated by FACS for longitudinal exome sequencing. Clinical data were updated every 6 months, resulting in a total of 13,659 longitudinal datapoints that included serological biomarkers, BM assessment of tumor burden with conventional microscopy and flow cytometry, cytogenetics, and imaging with MRI and/or PET/CT. Dynamic risk assessment using single or combined features measured in ≥3 timepoints was performed with CoNNector. Baseline and longitudinal immune monitoring data will be presented at the meeting. Results: Median age of the 324 SMM pts was 67 years. Patient distribution into low, intermediate and high-risk disease according the IMWG 20/2/20 model was 40%, 36% and 24%. With a median follow-up of 46 months, 108 of the 324 (33%) pts progressed to active MM. At baseline, CTCs were detected in 235 of the 324 (73%) SMM pts (median 0.002%, range 0.0002% - 0.42%). The optimal CTC cutoff to stratify pts in two risk groups was 0.008% (HR: 3.5, p<.001). Pts having >0.008% CTCs at baseline showed median time-to-progression (TTP) of 26 months vs not reached in those with ≤0.008% CTCs. Higher CTC cutoffs such as ≥0.25% defined a subgroup of pts who all progressed to active MM in 2y. In a multivariate analysis of TTP including the risk factors >2g/dL M-component, >20 sFLC ratio, >20% BM tumor cells and >0.008% CTCs, all except BM tumor cells showed independent prognostic value. The C-indexes of the 20/2/0.008 and the 20/2/20 models were 0.85 and 0.81. Being minimally-invasive, the 20/2/0.008 model could be reassessed every 6 months. It was noteworthy that 37% of pts had their risk of transformation modified over time. Of particular interest were the 26% of pts who converted from low/intermediate into high-risk SMM according to the 20/2/0.008 model. These showed a median TTP similar to pts who were high-risk at baseline (23 and 26 months). In 206 pts with ≥3 CTC assessments (1,055 in total), CoNNector identified two subgroups with undetectable/low vs high CTC levels over time. The latter pts showed a 4.8-fold increased risk of progression (p=.008). Besides CTCs, only serial hemoglobin levels were associated with transformation into active MM (HR: 12.5, p<.001). Serial levels of the M-component, sFLC ratio, β2m and creatinine were not significantly associated with risk of progression. Other features such as BM tumor burden, cytogenetics and imaging were rarely available in ≥3 timepoints and could not be analyzed over time. A dynamic model using serial CTC and hemoglobin levels stratified pts having 0, 1 and 2 risk factors with progression rates of 0%, 5.5% and 54% at 2y (p<.001). Longitudinal characterization of CTC exomes in a total of 40 samples from 14 pts enabled genomic profiling with unprecedented periodicity. For example, a patient with stable SMM and CTC exomes in six consecutive samples showed relatively stable mutational and copy number burden, while the cancer cell fraction of mutated NRAS and +1q21 changed over time. Surprisingly, SMM pts who progressed to active MM did not show a notable accumulation of mutations and copy number alterations in longitudinal CTC exomes. Conclusions: iMMunocell is one of the largest, comprehensive and dynamic studies performed in SMM. Our results support the complementary evaluation of the 20/2/20 and 20/2/0.008 risk models at baseline, and the longitudinal evaluation of the minimally-invasive 20/2/0.008 model for improved prediction of outcomes. Of note, the combined assessment of serial CTC and hemoglobin levels resulted in the most effective dynamic model and enabled the identification of SMM pts with virtually no risk of progression at 2y. Furthermore, CTCs can be used for periodic evaluation of genetic alterations towards a better understanding of its impact in SMM evolution.

Background: Evidence suggests that CTCs are responsible for MM spreading and therefore prognostic. However, some pts unexpectedly have undetectable CTCs and we hypothesized that this feature defines a distinct MM subtype. Aim: Investigate the clinical and biological features of MM pts with undetectable CTCs. Methods: This study included 3,146 pts. Disease characteristics and clinical outcomes associated with undetectable CTCs defined by next-generation flow (NGF) with a limit of detection (LOD) of 2x10-6 were investigated in 1,093 transplant-eligible and ineligible newly diagnosed MM (NDMM) pts enrolled in the GEM2012MENOS65, CLARIDEX and GEM2017FIT clinical trials. Validation was performed in a European pooled-analysis of 1,601 NDMM pts having CTC assessments by flow cytometry with LOD of ≤1x10-5. We further investigated the prognostic value of undetectable CTCs after 2 cycles of induction in the GEM2017FIT trial (n=251), and prior salvage therapy in 242 relapsed MM (RRMM) pts enrolled in the GEM KyCyDex and SeliBorDara trials. Risk of transformation in the absence of CTCs was investigated in 1,487 MGUS and 324 smoldering MM (SMM) pts. Molecular alterations associated with CTC egress were evaluated by exome and RNA sequencing. Mechanistic validation of selected genes was performed in MM5080 cells derived from Trp53-BIcγ1 mice, a model that recapitulates tumor dissemination from early to late stages of MM progression. Results: Among NDMM pts, 10% showed undetectable CTCs before treatment. Compared to those with detectable CTCs, they displayed significantly less anemia and tumor burden, only one had R-ISS 3 and MGUS-like profiles were more frequent. In contrast, the incidence of plasmacytomas was higher (33% vs 15%, p<.001). Altogether, pts with undetectable CTCs showed clinical features linked to macrofocal disease. When compared to NDMM pts with detectable CTCs, those with undetectable CTCs showed significantly higher 5y rates of PFS (80% vs 50%) and OS (92% vs 72%). In multivariate analyses of PFS and OS including transplant-eligibility and the R-ISS, undetectable CTCs showed independent prognostic value for PFS (HR: 0.5; p=.005) and OS (HR: 0.4, p=.02). Pts with undetectable CTCs achieving MRD negative CR displayed unprecedented 5y rates of PFS and OS (92% and 98%). In the European pooled analysis, undetectable CTCs was confirmed as an independent prognostic factor of PFS (HR: 0.5; p<.001) and OS (HR: 0.4, p<.001). Of note, a 2x10-6 LOD was required to define this group of pts with unprecedented prognosis since detection of CTCs in between ≥0.0002% and <0.001% was associated with inferior survival in the Spanish and European cohorts. Absence of CTCs was also associated with longer PFS after cycle 2 of induction in transplant-ineligible NDMM (HR: 0.47, p=.003) and before a new line of therapy in RRMM (HR: 0.5, p=.01). Although the absence of CTCs was associated with lower risk of progression in MGUS (HR: 0.1, p<.001) and SMM (HR:0.4, p<.001), 1% of MGUS and 10% of SMM pts with sustained undetectable CTCs progressed to active MM. These results suggest the presence of a subgroup of pts with undetectable CTCs throughout the disease course, which displays less aggressive tumors and favorable clinical outcomes by the time they progress to active MM. We next investigated the biology of bone marrow tumor cells in this subgroup. While the median number of coding mutations was similar, copy number alterations were less frequent in pts with undetectable vs detectable CTCs (29 vs 61, p=.03). The former displayed lower frequency of del(17p) (5% vs 11%, p=.03) and +1q21 (23% vs 48%, p<.001). RNAseq uncovered 66 differentially expressed genes, 9 of which were located on 1q, in line with the genetic findings. To gain more insights into the functional role of candidate genes we selected LGALS1, which was among the genes showing the highest correlation between mRNA expression and CTC levels, for CRISPR/Cas9 knockout experiments in MM5080 cells inoculated in Trp53-BIcγ1 mice. Compared to Lgals1 wild-type, biallelic loss of Lgals1 resulted in longer OS (median not reached vs 46 days). Conclusions: NDMMpts with undetectable CTCs have less genetic alterations and lower expression of genes linked to CTC egress, which results in less aggressive and macrofocal disease. Thus, this study recognizes a new myeloma subtype that accounts for 10% of NDMM pts, who show unprecedented survival and can be defined by undetectable CTCs with NGF.

Functional high-risk (FHR) multiple myeloma (MM) patients, defined as those with early relapse despite optimal initial therapy, represent an unmet clinical need. Diffusion-weighted whole-body MRI (DW-MRI) is increasingly used in MM management due to its high sensitivity in assessing treatment response. The Myeloma Response Assessment and Diagnosis System (MYRADS) established the Response Assessment Category (RAC), a 5-point scale ranging from complete response (RAC 1) to progressive disease (RAC 5), which independently stratifies patients with different outcomes after autologous stem cell transplantation (ASCT). The relative fat fraction (rFF), derived from DW-MRI, provides additional prognostic insights into bone marrow composition. This study aimed to evaluate whether the combined assessment of RAC and rFF could identify FHR MM patients at risk of early relapse, defined as progression within 18 months post-autologous stem cell transplantation (ASCT). Ninety-seven MM patients were retrospectively analyzed after ASCT, before maintenance, with a median follow-up of 47 months. An rFF threshold of 17.2% predicted early relapse with 83% sensitivity and 85% specificity. Patients with rFF >17.2% had significantly improved post-ASCT progression-free survival (PFS, median not reached [NR] vs. 13.7 months, HR 0.18; 95% CI: 0.08-0.43) and overall survival (OS, 3-year rate: 96% vs. 62%, HR 0.12; 95% CI: 0.03-0.45) compared to rFF ≤17.2%. Patients with RAC 1/rFF High had the best outcomes, while RAC ≥2/rFF Low had the worst prognosis (median PFS: NR vs. 12.3 months, HR 0.21; 95% CI: 0.07-0.62). rFF complements RAC for response assessment after ASCT, enabling early identification of FHR patients with poor prognosis.

AbstractAmong blood cancers, multiple myeloma (MM) represents the second most common neoplasm and is characterized by the accumulation and proliferation of monoclonal plasma cells within the bone marrow. Despite the last few decades being characterized by the development of different therapeutic strategies against MM, at present such disease is still considered incurable. Although MM is highly heterogeneous in terms of genetic and molecular subtypes, about 67% of MM cases are associated with abnormal activity of the transcription factor c-Myc, which has so far revealed a protein extremely difficult to target. We have recently demonstrated that activation of fibroblast growth factor (FGF) signaling protects MM cells from oxidative stress-induced apoptosis by stabilizing the oncoprotein c-Myc. Accordingly, secretion of FGF ligands and autocrine activation of FGF receptors (FGFR) is observed in MM cells and FGFR3 genomic alterations represent some 15–20% MM cases and are associated with poor outcome. Thus, FGF/FGFR blockade may represent a promising strategy to indirectly target c-Myc in MM. On this basis, the present review aims at providing an overview of recently explored connections between the FGF/FGFR system and c-Myc oncoprotein, sustaining the therapeutic potential of targeting the FGF/FGFR/c-Myc axis in MM by using inhibitors targeting FGF ligands or FGF receptors. Importantly, the provided findings may represent the rationale for using FDA approved FGFR TK inhibitors (i.e. Pemigatinib, Futibatinib, Erdafitinib) for the treatment of MM patients presenting with an aberrant activation of this axis.

Somatic SETBP1 mutations are found in various myeloid disorders covering both myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS). To characterize the early steps of SETBP1-mediated leukemogenesis, we generated a conditional mouse model expressing SETBP1 G870S mutant in the entire hematopoietic tissue through Cre-mediated recombination driven by the Vav1 promoter. In all mice signs of a hematological disease appeared between 30 and 90 days: longitudinal analysis revealed accumulation of white blood cells (WBC) in virtually all heterozygous SETBP1 G870S mice, with a marked imbalance between the lymphoid and myeloid lineages in favor of the latter, and an increase in mature myeloid cells in absence of circulating blasts or non-segmented myeloid precursors. Kaplan-Meier analysis revealed a dramatic decrease in event-free survival in SETBP1 G870S mice. BM histology showed overt myeloid hyperplasia with fibrosis and no evidence of dysplasia except for the megakaryocytic lineage. Exploration of visceral organs highlighted the presence of severe hepatosplenomegaly with massive infiltration by myeloid elements, disruption of normal tissue architecture, and signs of extramedullary hematopoiesis. Single-cell RNA-sequencing (scRNA) on BM Lin - cells identified Mecom, Setbp1 and Hoxa9 among the top upregulated genes in SETBP1 G870S precursors. Pseudotime analysis of scRNA data revealed the presence of 261 spatially autocorrelated genes. Of them 158 were also differentially expressed in SETBP1 G870Svs control mice. Spi1, encoding for the master regulator of hematopoietic differentiation PU.1, was significantly upregulated in SETBP1 G870S precursors. PU.1 promotes the maturation of bone marrow early precursors towards the granulocytic/monocytic lineages by directly impairing the transcription of Gata2 and Gata1. In line with these data, Gata2 and Gata1 expression was profoundly suppressed in the early myeloid precursors of SETBP1 G870S mice, which associated with the down-modulation of markers of the erythroid lineage, such as the Carbonic Anhydrase 1, in the MEP differentiation branch. Ourmouse model recapitulates many clinical features of primary myelofibrosis (PMF). As up to 10% of PMF are triple-negative for the classical JAK2, CALR, and MPL mutations (TN-PMF), we set out to assess SETBP1 mutations in this context. We analyzed 36 TN-PMF patients by exome sequencing. In 29 we did not find any evidence of somatic mutations; in the remaining 7 (7/36; 19.4%) high VAF SETBP1 degron mutations were identified. In two patients SETBP1 was found as a single somatic variant, while in the others it coexisted with ASXL1, NRAS TET2 SRSF2, RIT1 CBL or CSF3R mutations. Notably, SETBP1 mutations were the only shared alterations among all seven patients. A markedly reduced overall survival was observed for SETBP1 positive patients, with a median survival time of 24 months (median survival not reached at 60 months for SETBP1 negative patients). To dissect the clonal architecture of SETBP1 positive TN-PMF at single-cell resolution, we applied single-cell targeted DNA sequencing on 3 SETBP1-mutated TN-PMF samples using the Tapestri technology, showing that, as opposite to MDS/MPN, in all TN-PMF cases SETBP1 is a very early clonal event. Therefore, our study suggests a clear partition of TN-PMF into two groups, the first one characterized by oncogenic, high VAF SETBP1 mutations accompanied by other oncogenic variants and poor prognosis and the other one characterized by no evidence of an active clonal process or of driver oncogenic events and much lower aggressiveness. SETBP1 positive TN-PMF disorders lie in a gray zone comprised between the MPN and MPN/MDS boundary. In MDS/MPN, SETBP1 mutations are often found as mid or late events. In contrast, we show here that in TN-PMF SETBP1 appears to be one of the earliest hits, therefore highlighting a potentially relevant biological difference occurring in the two subsets. In this context the presence of early SETBP1 mutations seems to promote the occurrence of a myeloid disorder characterized by the triad: leukocytosis without differentiation block or dysplasia, BM fibrosis and progressive splenomegaly, hence recapitulating many clinical features of overt or prefibrotic/early PMF and resembling the SETBP1 mouse model.

Background: Monoclonal gammopathies are defined as plasma cell (PC) neoplasms. However, it is postulated that the cell of origin is a B lymphocyte. A better understanding of this potential discrepancy is paramount for accurate diagnosis and monitoring. Aim: Identify the cell of origin and tumor reservoirs containing driver genetic alterations within the B lineage of patients with Waldenström's macroglobulinemia (WM), light-chain amyloidosis (AL) and multiple myeloma (MM). Methods: 71 individuals were studied: 18 healthy adults older than 58 (range, 58 - 85), 13 WM, 17 AL and 23 MM patients. Single cell RNA and B cell receptor sequencing (scRNA/BCRseq) was performed using 10X in B cell precursors, mature B cells and PC isolated by FACS (n=32). Exome sequencing was performed in sorted CD34 progenitors, B-cell precursors, mature B cells, normal and clonal PC, and T cells as control (n=28). To avoid contamination, normal cell types were isolated before and after transplant in MM patients with negative MRD at 10-6 using NGF. Results: scRNA/BCRseq yielded paired transcriptomes and immunoglobulin gene rearrangements in 129,794, which were classified into B cell precursors (n=7,810), mature B cells (n=84,231) and PC (n=37,753). In healthy adults, clonal expansions were rare and mainly observed in PC. Interestingly, non-tumor clones were significantly increased in mature B cells and PC from WM patients, and in B cell precursors from MM patients. These findings suggest a higher frequency of oligoclonality in the B lineage of patients with monoclonal gammopathies. Next, we investigated the presence of patient-specific tumor BCR in the normal cell compartments of each monoclonal gammopathy. Surprisingly, tumor BCR were detected in 1.7% and 0.3% B-cell precursors from WM and MM patients, as well as in 0.1% and 0.4% mature B cells from AL and MM patients. Phenotypic hallmarks of the B cell differentiation (eg, CD10, CD19, CD20, CD34, PRDM1, or CD138) were similarly expressed in non-tumor vs tumor-related B cell precursors, mature B cells and PC. However, tumor-related cell types from patients showed on average 640 differentially expressed genes when compared to the normal counterpart in healthy adults. These results confirm the immature phenotype of clonotypic cells and uncover altered transcriptomes possibly due to tumor microenvironments. The median numbers of somatic mutations in CD34 progenitors, B-cell precursors, mature B cells and normal PC from healthy adults (>58y) vs patients with monoclonal gammopathies were 97 vs 431 (P = .002), 177 vs 503 (P = .02), 108 vs 564 (P = .004) and 189 vs 573 (P = .009). An average of 31% and 34% of somatic mutations were shared between CD34 progenitors and normal PC from healthy adults and patients, respectively. These data indicates a continuum of mutated cells throughout the B lymphopoiesis, which is increased in monoclonal gammopathies. In AL patients, there were 30%, 34%, 31% and 28% shared mutations between clonal PC and CD34 progenitors, B cell precursors, mature B cells and normal PC. In MM patients, the respective percentages were 20%, 17%, 11% and 17% shared mutations between clonal PC and CD34 progenitors, B cell precursors, mature B cells and normal PC. Similar results were observed before and after transplant. In contrast to WM patients in whom MYD88 mutations were observed in normal cell types (Rodriguez S, Science Ad 2022), driver mutations (eg, in RAS genes) as well as copy number alterations were private in clonal PC, and absent in normal cell types from AL and MM patients. We next hypothesized that clonotypic B cells with a normal phenotype could result in sporadic cases of positive MRD by NGS while negative by NGF. Such cases should have PFS similar to patients with double negative MRD because clonotypic B cells lacked key genetic alterations and therefore cannot drive relapse. Thus, we analyzed PFS in 103 MM patients enrolled in the GEM2012MENOS65 trial who had simultaneous MRD assessment using NGF and NGS. Of the 103 MM patients, 7 were NGF-/NGS+ despite MRD levels above the limit of detection of NGF. After a median follow-up of 80 months, these patients had a median PFS not reached, similarly to those who were NGF-/NGS-. Conclusion: Monoclonal gammopathies stem from a clonal B lymphopoiesis but tumor reservoirs are contained within phenotypically aberrant cells. These results shed light on the cell of origin and have implications on monitoring of patients with WM, AL and MM.

In this issue of Blood , Bae et al(1) have elegantly shown that an induced pluripotent stem cell strategy may epigenetically reprogram precursor exhausted B-cell maturation antigen (BCMA)-specific cytotoxic T lymphocytes into hematopoietic progenitor cells, which, in turn, differentiate into functional cognate antigen-specific CD8 alpha beta(+) memory T cells that exert an antitumor effect in multiple myeloma (MM). Overall, these novel studies pave the path to novel strategies for targeting MM cells via an effective antitumor immunity-based approach.

BACKGROUND:Intermittent treatment with tyrosine kinase inhibitors (TKIs) is an option for elderly chronic myeloid leukemia (CML) patients who are often candidates for life-long treatment. MATERIALS AND METHODS:The Italian phase III multicentric randomized Optimize TKIs Multiple Approaches (OPTkIMA) study aimed to evaluate if a progressive de-escalation of TKIs is able to maintain the molecular remission (MR)3.0 and to improve Health-Related Quality of Life (HRQoL) in CML elderly patients. RESULTS:A total of 215 patients in stable MR3.0/MR4.0 were randomized to receive an intermittent TKI schedule 1 month ON-1 month OFF for 3 years (FIXED arm; n = 111) vs. a progressive de-escalation TKI dose up to one-third of the starting dose at the 3rd year (PROGRESSIVE arm; n = 104). Two hundred three patients completed the 3rd year of OPTkIMA study. At the last follow-up, MR3.0 loss was 27% vs. 46% (P = .005) in the FIXED vs PROGRESSIVE arm, respectively. None of these patients experienced disease progression. The 3-year probability of maintaining the MR3.0 was 59% vs. 53%, respectively (P = .13). HRQoL globally improved from the baseline to the 3rd year, without any significant difference between the 2 arms. After the 3rd year, the proportion of patients who was address to TKI discontinuation in the 2 arms was 36% (FIXED) vs. 58% (PROGRESSIVE) (P = .03). CONCLUSIONS:The intensification of intermittent TKI therapy is associated with a higher incidence of MR3.0 loss, but those patients who maintain the MR3.0 molecular response at the end of the study have been frequently considered eligible for TFR. The HRQoL generally improved during the de-escalation therapy in both randomization arms.

Fibroblast growth factors (FGFs) act as proangiogenic and mitogenic cytokines in several cancers, including multiple myeloma (MM). Indeed, corrupted FGF autocrine and paracrine secretion induces an aberrant activation of the FGF receptor (FGFR) signaling sustaining cancer cell spreading and resistance to pharmacological treatments. Thus, FGF traps may represent a promising anti-cancer strategy to hamper the ligand-dependent activation of the FGF/FGFR system. We previously identified NSC12 as the first orally available small molecule FGF trap able to inhibit the growth and progression of several FGF-dependent tumor models. NSC12 is a pregnenolone derivative carrying a 1,1-bis-trifluoromethyl-1,3-propanediol chain in position 17 of the steroid nucleus. Investigation of structure-activity relationships (SARs) provided more potent and specific NSC12 steroid derivatives and highlighted that the C17-side chain is pivotal for the FGF trap activity. Here, a scaffold hopping approach allowed to obtain two FGF trap compounds (22 and 57) devoid of the steroid nucleus and able to efficiently bind FGF2 and to inhibit FGFR activation in MM cells. Accordingly, these compounds exert a potent anti-tumor activity on MM cell lines both in vitro and in vivo and on MM patient-derived primary cells, strongly affecting the survival of both proteasome-inhibitor sensitive and resistant MM cells. These results propose a new therapeutic option for relapsed/refractory MM patients and set the bases for the development of novel FGF traps prone to chemical diversification to be used in the clinic for the treatment of those tumors in which the FGF/FGFR system plays a pivotal role, including MM.

Introduction: Minimal residual disease (MRD) detection represents a sensitive tool to appropriately measure response in MM. The major concern about MRD detection in real-world setting is the reproducibility of results among different laboratories possibly due to methodological discrepancies. Therefore, a harmonized approach, according to criteria of Next generation Flow (NGF)/ Next generation sequencing (NGS) established by IMWG (Kumar S et al. Lancet Oncol 2016), is warranted. The aim of this project was to create an “Italian MM MRD network” using standardized NGF and NGS-MRD approach; here we report preliminary results of the NGF part. Methods: The “NGF harmonization project” includes 7 Italian Laboratories willing to commit to EuroFlow protocols after an initial MRD survey: Brescia (L1), Catania (L2), Padova (L3), Roma 1 (L4), Roma 2 (L5), S. Giovanni Rotondo (L6) and Torino (L7). Standardization of all flow cytometers settings was performed by implementation of the EuroFlow Standard Operating Protocol (SOP) for instrument setup and compensation for BD FASCCanto II, BD FACSLyric, BC Navios and BC DxFlex flow cytometers (www.euroflow.org). Bone marrow (BM) aspirates were collected from newly diagnosed MM patients treated with 4 cycles of Dara-VTD (Daratumumab, bortezomib, thalidomide, dexamethasone) induction followed by autologous stem cell transplantation (ASCT) in complete-response/very-good-partial-response at day 100 (+/- 15 days) after ASCT. Anonymized samples were split equally and shipped at room temperature to the participati ng laboratories and simultaneously analyzed. Analyses were carried out following NGF methodology (Flores-Montero J et al. Leukemia 2017) by using a two-tube 8-color antibody panel for monoclonal plasma cells identification (Tube 1:CD27, CD138, CD38, CD56, CD45, CD19, CD117, CD81 and Tube 2: CD27, CD138, CD38, CD56, CD45, CD19, cytoplasmic κ and λ light-chain). We aimed to acquire ≥ 3,000,000 events per tube for a sensitivity of at least 1 x 10 -5; a sample was considered MRD positive when ≥ 20 monoclonal plasma cells were detected. Intraclass Correlation Coefficient (ICC) was performed to evaluate degree of correlation and agreement between measurements; standard deviation (SD) and Coefficient of variation (CV) to measure variability of the dataset. Results. In stage 1 of the study, before starting MRD evaluation on freshsamples, 4 laboratories (L1, L2, L3, L7) performed a blinded analysis of the same 5 anonymous flow-cytometry files from a retrospective data repository of MM patients with/without MRD, to evaluate the inter-operator variability: 100% of the participants were concordant that samples #1, #2 and #4 were MRD positive. Sample #3 was considered MRD positive by 75% of participants whereas 75% considered sample #5 as MRD negative (ICC=0.91, 95% CI 0.69-0.98, p<0.001). In stage 2, a total of 7 samples have been distributed and processed after 48 hours from specimen collection, due to different shipping time in all laboratories. The inter-laboratory correlation study showed a concordance of 100 % for the sample #1 and sample #3 considered as MRD positive, 100% of the participants considered sample #5 and sample #6 as MRD negative, sample #2 was considered MRD positive by 71% of participants whereas sample #4 was considered MRD negative by 86 % of participants. Sample #7 was considered as not evaluable from all participants due to a high percentage of dead cells. (ICC=0.61, 95% CI 0.31-0.91, p<0.001) (Fig. 1). The highest difference in term of antigen expression of monoclonal plasma cells was recorded for CD45 and CD27. We obtained a median of 7 x 10 -6 of Limit of detection (LOD, range 6 x 10 -5 - 2 x 10 -6) and 1 x 10 -5 Limit of Quantification (LOQ, range 1 x 10 -4- 5 x 10 -6) for all samples. A centralized revision (in L7 lab) of all FCS files is ongoing to confirm our preliminary results; moreover, the study will be corroborated increasing the number of fresh samples. Conclusion: our preliminary results demonstrate the importance of a harmonized NGF-MRD assessment to improve the accuracy and comparability of MM-MRD testing among different laboratories. Major discrepancies have been found in fresh samples vs the retrospective dataset suggesting an impact of the transportation time and sample processing on the concordance of our results.

Quality control of RNA after human exon enrichement.

Supplementary Data from Src Tyrosine Kinase Regulates Adhesion and Chemotaxis in Waldenstrom Macroglobulinemia