Molecular mechanisms promoting long-term cytopenia after BCMA CAR-T therapy in Multiple Myeloma.

Hematological toxicity is a common side effect of CAR-T therapies, particularly severe in relapsed/refractory multiple myeloma (MM) patients. In this study, we analyzed a cohort of 48 patients treated with BCMA CAR-T cells to characterize the kinetics of cytopenia, identify predictive factors and determine potential mechanism underlying these toxicities. The overall incidence of cytopenia was 95.7%, and grade>3 thrombocytopenia and neutropenia, one month after infusion, was observed in 57% and 53% of the patients, being still present after one year in 4 and 3 patients respectively. Presence of cytopenia at baseline and high peak inflammatory markers highly correlated with cytopenia persisting up to three months. To determine potential mechanisms underpinning cytopenias, we evaluated the paracrine effect of BCMA CAR-T cells on HSPCs differentiation using an ex-vivo myeloid differentiation model. Phenotypic analysis showed that supernatants from activated CAR-T cells (spCAR) halted HSPCs differentiation, promoting more immature phenotypes, with reduced expression of granulocytic, monocytic and erythroid markers, which could be prevented with a combination of IFNγ, TNFα/ß, TGFß, IL-6 and IL-17 inhibitors. Single-cell RNA-seq demonstrated upregulation of transcription factors associated with early stages of hematopoietic differentiation in the presence of spCAR (GATA2, RUNX1, CEBPA) and decreased activity of key regulons involved in neutrophil and monocytic maturation (ID2, MAFB). Our results suggest that CAR-T cell activation negatively influences hematopoietic differentiation through paracrine effects inducing HSPCs maturation arrest. Moreover, our study contributes to the understanding of severe cytopenia observed after CAR-T therapy in MM and provides potential treatments to prevent or decrease its severity.

Current Therapy of the Patients with MDS: Walking towards Personalized Therapy.

Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis, dysplasia and peripheral cytopenias. Nowadays, MDS therapy is selected based on risk. The goals of therapy are different in low-risk and high-risk patients. In low-risk MDS, the goal is to decrease transfusion needs and to increase the quality of life. Currently, available drugs for newly diagnosed low-risk MDS include growth factor support, lenalidomide and immunosuppressive therapy. Additionally, luspatercept has recently been added to treat patients with MDS with ring sideroblasts, who are not candidates or have lost the response to erythropoiesis-stimulating agents. Treatment of high-risk patients is aimed to improve survival. To date, the only currently approved treatments are hypomethylating agents and allogeneic stem cell transplantation. However, the future for MDS patients is promising. In recent years, we are witnessing the emergence of multiple treatment combinations based on hypomethylating agents (pevonedistat, magrolimab, eprenetapopt, venetoclax) that have proven to be effective in MDS, even those with high-risk factors. Furthermore, the approval in the US of an oral hypomethylating agent opens the door to exclusively oral combinations for these patients and their consequent impact on the quality of life of these patients. Relapsed and refractory patients remain an unmet clinical need. We need more drugs and clinical trials for this profile of patients who have a dismal prognosis.

Molecular Heterogeneity of High Grade Colorectal Adenocarcinoma.

High grade colorectal carcinomas (HG-CRCs), which comprise 15% of colorectal carcinomas, are underrepresented in reported molecular studies. Clinicopathological, immunohistochemical, and molecular features of 40 HG-CRCs are described. Moreover, glandular and solid areas of 25 tumors were separately analyzed. The expression of MLH1, PMS2, MSH2, MSH6, p53, E-cadherin, CDX2, CK20, CD8, PDL1, PAN-TRK, c-MET, SMARCB1, ARID1A, SMARCA2, and SMARCA4 was analyzed by immunohistochemistry. Promoter MLH1 methylation was analyzed in tumors with MLH1/PMS2 loss. Next-generation sequencing was used to screen 161 genes for hotspot mutations, copy number variations and gene fusions. In this series, 72.5% of HG-CRCs showed mismatch repair deficiency (MMRd). MMR deficient tumor and MMR proficient (MMRp) tumors showed striking molecular differences. Thus, whereas BRAF mutations were only observed in MMRd tumors, mutations in KRAS and TP53 were more frequent in MMR proficient tumors. Moreover, gene fusions (NTRK1 and MET) were detected only in MMRd tumors, whereas gene amplification (MYC, CCND1 and EGFR) predominated in MMRp/TP53-mutated tumors. Loss of expression of proteins involved in chromatin remodeling, such as ARID1A, was observed only in MMRd HG-CRCs, which also showed more frequently PD-L1 expression and a higher number of tumor infiltrating lymphocytes. The separate analysis of glandular and solid areas indicated that the clonal or subclonal nature of the molecular alterations also depended on MMR status. Mutations in genes such as TP53 and KRAS were always clonal in MMRp-CRCs but occurred as subclonal events in MMRd-CRCs. Gene amplification was implicated in the progression of MMRp tumors, but not in MMRd tumors, in which clonal diversity was due to accumulation of mutations in genes of different pathways such as NOTCH, MMR, or PIK3CA. In summary, intertumor and intratumor molecular heterogeneity in HG-CRCs is mainly due to MMR status.

Molecular Features of Metaplastic Breast Carcinoma: An Infrequent Subtype of Triple Negative Breast Carcinoma.

Metaplastic breast carcinoma (MBC) is a heterogeneous group of infrequent invasive carcinomas that display differentiation of the neoplastic epithelium towards squamous cells and/or mesenchymal-type elements. Most MBC have a triple negative phenotype and poor prognosis. Thus, MBC have worse survival rates than other invasive breast carcinomas, including other triple negative breast carcinomas (TNBC). In this study, we reviewed the molecular features of MBC, pointing out the differences among subtypes. The most frequently mutated genes in MBC were TP53 and PIK3CA. Additionally, mutations in the other genes of the PI3K/AKT pathway indicated its importance in the pathogenesis of MBC. Regarding copy number variations (CNVs), MYC was the most frequently amplified gene, and the most frequent gene loss affected the CDKN2A/CDKN2B locus. Furthermore, the pattern of mutations and CNVs of MBC differed from those reported in other TNBC. However, the molecular profile of MBC was not homogeneous among histological subtypes, being the alterations in the PI3K pathway most frequent in spindle cell carcinomas. Transcriptomic studies have demonstrated an epithelial to mesenchymal program activation and the enrichment of stemness genes in most MBC. In addition, current studies are attempting to define the immune microenvironment of these tumors. In conclusion, due to specific molecular features, MBC have a different clinical behavior from other types of TNBC, being more resistant to standard chemotherapy. For this reason, new therapeutic approaches based on tumor molecular characteristics are needed to treat MBC.