Germline assessment for alloHSCT candidates over 50 years: A 'Fast-Track' screening in myeloid neoplasms.

Patients aged 50 or above diagnosed with myeloid neoplasms (MNs) are typically not candidates for germline testing. However, approximately 8% carry pathogenic germline variants. Allogeneic haematopoietic stem cell transplantation (alloHSCT) remains an option for those aged over 50; neglecting germline testing could mask the risk for relative donor cell-derived MN. We propose a germline-augmented somatic panel (GASP), combining MN predisposition genes with a myeloid somatic panel for timely germline variant identification when initial testing is not indicated. Out of our 133 whole-exome-sequenced MN cases aged over 50 years, 9% had pathogenic/likely variants. GASP detected 92%, compared to 50% with somatic-only panel. Our study highlights the relevance of germline screening in MN, particularly for alloHSCT candidates without established germline-testing recommendations.

NLRP3 inflammasome activation and symptom burden in KRAS-mutated CMML patients is reverted by IL-1 blocking therapy.

Chronic myelomonocytic leukemia (CMML) is frequently associated with mutations in the rat sarcoma gene (RAS), leading to worse prognosis. RAS mutations result in active RAS-GTP proteins, favoring myeloid cell proliferation and survival and inducing the NLRP3 inflammasome together with the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which promote caspase-1 activation and interleukin (IL)-1ß release. Here, we report, in a cohort of CMML patients with mutations in KRAS, a constitutive activation of the NLRP3 inflammasome in monocytes, evidenced by ASC oligomerization and IL-1ß release, as well as a specific inflammatory cytokine signature. Treatment of a CMML patient with a KRASG12D mutation using the IL-1 receptor blocker anakinra inhibits NLRP3 inflammasome activation, reduces monocyte count, and improves the patient's clinical status, enabling a stem cell transplant. This reveals a basal inflammasome activation in RAS-mutated CMML patients and suggests potential therapeutic applications of NLRP3 and IL-1 blockers.

NOTCH1 mutation in chronic lymphocytic leukaemia is associated with an enhanced cell cycle G1/S transition and specific cyclin overexpression: Preclinical ground for targeted inhibition.

Studies prior to next-generation sequencing (NGS) showed that the frequent indolent course of chronic lymphocytic leukaemia (CLL) is related to most cells remaining quiescent in the G0 -G1 cell cycle phase, due to the expression of dysregulated cyclin genes. Of note, the activating nature of the NOTCH1 mutation in T lymphoblastic leukaemia also drives the dysregulation of cell cycle genes. Our goal was to comprehensively revisit the cell cycle in NOTCH1-mutated CLL (NOTCH1MUT ) to test for potential therapeutic targets. Among 378 NGS-annotated CLL cases, NOTCH1MUT cells displayed a unique transcriptome profile of G0 -G1 cell cycle components, with an overexpression of early-phase effectors, reaching a 38-, 27- and ninefold change increase for the complex elements CCND3, CDK4 and CDK6, respectively. This NOTCH1MUT cells' profile was related to more cells traversing through the cell cycle. In-vitro targeted inhibition of NOTCH1 gamma-secretase and CDK4/6 reversed the distribution of cells through the cycle phases and enhanced the killing of NOTCH1MUT CLL cells, suggesting new therapeutic approaches.

Prognosis in Myelodysplastic Syndromes: The Clinical Challenge of Genomic Integration.

Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic neoplasms characterized by ineffective hematopoiesis and myelodysplasia with a variable spectrum of clinical-biological features that can be used to build a prognostic estimation. This review summarizes the current most widely used prognostic scoring systems and gives a general view of the prognostic impact of somatic mutations in MDS patients.

Cancer testis antigens in myelodysplastic syndromes revisited: a targeted RNA-seq approach.

Cancer-Testis antigens (CTA) are named after the tissues where they are mainly expressed: in germinal and in cancer cells, a process that mimics many gametogenesis features. Mapping accurately the CTA gene expression signature in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) is a prerequisite for downstream immune target-discovery projects. In this study, we take advantage of the use of azacitidine to treat high-risk MDS and CMML to draw the CTAs landscape, before and after treatment, using an ad hoc targeted RNA sequencing (RNA-seq) design for this group of low transcript genes. In 19 patients, 196 CTAs were detected at baseline. Azacitidine did not change the number of CTAs expressed, but it significantly increased or decreased expression in nine and five CTAs, respectively. TFDP3 and DDX53, emerged as the main candidates for immunotherapeutic targeting, as they showed three main features: i) a significant derepression on day +28 of cycle one in those patients who achieved complete remission with hypomethylating treatment (FC = 6, p = .008; FC = 2.1, p = .008, respectively), ii) similar dynamics at the protein level to what was observed at the RNA layer, and iii) to elicit significant specific cytotoxic immune responses detected by TFDP3 and DDX53 HLA-A*0201 tetramers. Our study addresses the unmet landscape of CTAs expression in MDS and CMML and revealed a previously unrecognized TFDP3 and DDX53 reactivation, detectable in plasma and able to elicit a specific immune response after one cycle of azacitidine.

Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease.

Graft versus host disease (GvHD) is the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). Here we report studies of a patient with chronic GvHD (cGvHD) carrying persistent CD4+ T cell clonal expansion harboring somatic mTOR, NFKB2, and TLR2 mutations. In the screening cohort (n = 134), we detect the mTOR P2229R kinase domain mutation in two additional cGvHD patients, but not in healthy or HSCT patients without cGvHD. Functional analyses of the mTOR mutation indicate a gain-of-function alteration and activation of both mTORC1 and mTORC2 signaling pathways, leading to increased cell proliferation and decreased apoptosis. Single-cell RNA sequencing and real-time impedance measurements support increased cytotoxicity of mutated CD4+ T cells. High throughput drug-sensitivity testing suggests that mutations induce resistance to mTOR inhibitors, but increase sensitivity for HSP90 inhibitors. Our findings imply that somatic mutations may contribute to aberrant T cell proliferations and persistent immune activation in cGvHD, thereby paving the way for targeted therapies.

An early increase of CD56bright natural killer subset as dominant effect and predictor of response to extracorporeal photopheresis for graft-versus-host disease.

BACKGROUND: CD56bright natural killer (NK) regulatory cells were recently shown to display a differential impact on the risk of developing extensive chronic graft-versus-host disease (GVHD). To date no study has definitively established which immune populations are most responsible for the immunomodulatory effects or response to extracorporeal photopheresis (ECP) for GVHD. STUDY DESIGN AND METHODS: To test the role of CD56bright NK cells in ECP, a prospective enhanced flow cytometry follow-up of immune subsets (CD19+, CD3+, CD3+/CD4+, CD3+/CD8+, CD3-/CD56+, CD3-/CD56bright , and CD3-/CD56dim ) was performed in 32 patients with GVHD who underwent 552 procedures. RESULTS: An early increase of CD56bright NK cells was found as a hallmark effect to ECP, particularly during the first 3 months of treatment. This was also supported by the ability to predict for complete responses when this increase was expressed as a higher CD56bright versus CD56dim NK cells ratio. Among the immune subsets tested, the only variable that had direct influence on response to ECP was a CD56bright/dim ratio more than 0.16 (hazard ratio [HR] 4.32, p = 0.014; HR 5.8, p = 0.007, at 2 and 3 months of ECP treatment, respectively). CONCLUSION: These findings argue for exploring strategies for priming a CD56bright NK cell expansion during ECP and providing additional and potentially relevant data for revisiting the underpinning cellular mechanisms of ECP that could generate that expansion.

Bone marrow biopsy superiority over PET/CT in predicting progression-free survival in a homogeneously-treated cohort of diffuse large B-cell lymphoma.

Several studies have reported uneven results when evaluating the prognostic value of bone marrow biopsy (BMB) and PET/CT as part of the staging of diffuse large B-cell lymphoma (DLBCL). The heterogeneity of the inclusion criteria and not taking into account selection and collinearity biases in the analysis models might explain part of these discrepancies. To address this issue we have carried a retrospective multicenter study including 268 DLBCL patients with a BMB and a PET/CT available at diagnosis where we estimated both the prognosis impact and the diagnostic accuracy of each technique. Only patients treated with R-CHOP/21 as first line (n = 203) were included in the survival analysis. With a median follow-up of 25 months the estimated 3-year progression-free survival (PFS) and overall survival (OS) were 76.3% and 82.7% respectively. In a multivariate analysis designed to avoid a collinearity bias with IPI categories, BMB-BMI [bone marrow involvement](+) (HR: 3.6) and ECOG PS > 1 (HR: 2.9) were independently associated with a shorter PFS and three factors, age >60 years old (HR: 2.4), ECOG PS >1 (HR: 2.4), and abnormally elevated B2-microglobulin levels (HR: 2.2) were independently associated with a shorter OS. In our DLBCL cohort, treated with a uniform first-line chemotherapy regimen, BMI by BMB complemented performance status in predicting those patients with a higher risk for relapse or progression. In this cohort BMI by PET/CT could not independently predict a shorter PFS and/or OS.

The role of bone marrow biopsy and FDG-PET/CT in identifying bone marrow infiltration in the initial diagnosis of high grade non-Hodgkin B-cell lymphoma and Hodgkin lymphoma. Accuracy in a multicenter series of 372 patients.

Bone marrow infiltration (BMI), categorized as an extra-nodal site, affects stage and is associated with poor prognosis in newly diagnosed lymphoma patients. We have evaluated the accuracy of PET/CT and bone marrow biopsy (BMB) to assess BMI in 372 lymphoma patients [140 Hodgkin Lymphoma (HL) and 232 High Grade B-cell non-Hodgkin Lymphoma (HG B-NHL), among them 155 Diffuse Large B-Cell Lymphoma (DLCL)]. For HL cases, and taking into account PET/CT, sensitivity, negative predictive value (NPV) and accuracy were 96.7, 99.3, and 99.3% while those of BMB were 32.3, 83.8, and 85%, respectively. For HG B-NHL and considering PET/CT, sensitivity, NPV, and accuracy were 52.7, 81.7, and 84.1%, while those of BMB were 77.6, 90.2, and 90.7%, respectively. In the HG B-NHL group, 25 patients would have been under-staged without BMB. These results lead us to recommend PET/CT and the avoidance of BMB to assess BMI in HL. In the case of HG B-NHL, bone marrow status should be assessed firstly by means of PET/CT; only in either focal or diffuse PET/CT with low borderline SUV max values or in negative cases, should BMB be carried out afterwards. In the HG B-NHL setting and at the present moment, both techniques are complementary.