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Background: Tumor mutational burden (TMB) is an FDA-approved biomarker for immune checkpoint inhibitor (ICI) therapy. However, its predictive value varies among tumor types and molecular contexts. We investigated whether a very high TMB identifies a biologically distinct subset and whether a higher cutoff provides additional clinical insights beyond the conventional high TMB threshold. Methods: We analyzed 133 patients with advanced solid tumors and TMB ≥ 10 mutations/Mb (mut/Mb) who underwent tumor genomic profiling using a 523-gene DNA/RNA next-generation sequencing panel. Tumors were stratified into prespecified TMB categories: 10–20 mut/Mb (TMB-H) and >20 mut/Mb (TMB-VH). The clinical characteristics, ICI outcomes (in the treated subset), and pathway-level genomic features were compared between groups. Results: TMB-VH was observed in 42/133 (31.6%) patients and spanned more than 20 tumor types. MSI was markedly more prevalent in TMB-VH than in TMB-H tumors (38.1% vs. 2.2%; Fisher’s exact p = 8.9 × 10−8). Pathway-level comparisons did not identify statistically significant differences after false discovery rate correction (all q > 0.05), and the observed patterns were descriptive in nature. In the ICI-treated subset with complete follow-up, objective response did not differ according to the TMB group. Overall survival (OS) was also similar between groups, whether measured from metastatic diagnosis (log-rank p = 0.937) or from ICI initiation (log-rank p = 0.814), although OS was numerically longer in the TMB-VH group in both analyses without reaching statistical significance. Conclusions: In this cohort study, TMB-VH was strongly associated with MSI but not independently associated with improved ICI outcomes. Larger multicenter cohorts are needed to validate pathway-oriented patterns and clarify the clinical utility of extreme TMB thresholds across various histologies. Integrating the functional context (e.g., MSI status, gene-level context, and pathway-level features) with TMB magnitude may enable more robust, tumor-aware biomarker models for immunotherapy selection.

Genomic characterization of patients with myeloproliferative neoplasms (MPN) may lead to better diagnostic classification, prognostic assessment, and treatment decisions. These goals are particularly important in myelofibrosis (MF). We performed target Next Generation Sequencing for a panel of 255 genes and Chromosome Microarray Analysis (CMA) in 27 patients with MF. Patients were classified according to genomic findings and we compared the performance of a personalized prognostication system with IPSS, MIPSS70 and MIPSS70 + v2. Twenty-six patients presented mutations: 11.1% had single driver mutations in either JAK2, CALR or MPL; 85.2% had mutations in non-restricted genes (median: 2 per patient). CMA was abnormal in 91.7% of the 24 cases with available data. Copy-Number-Neutral Loss-of-Heterozygosity was the most common finding (66.7%). Del13q was the most frequent copy number variation, and we could define a 2.4 Mb minimally affected region encompassing RB1, SUCLA2 and CLLS2 loci. The largest genomic subgroup consisted of patients with mutations in genes involved with chromatin organization and splicing control (40.7%) and the personalized system showed better concordance and accuracy than the other prognostic systems. Comprehensive genomic characterization reveals the striking genetic complexity of MF and, when combined with clinical data, led, in our cohort, to better prognostication performance.

RAS-pathway mutations are recurrent events in myeloid malignancies. However, there is limited data on the significance of RAS-pathway mutations in patients with myelofibrosis (MF). We analyzed next-generation sequencing data of 16 genes, including RAS-pathway genes, from 723 patients with primary and secondary MF across three international centers and evaluated their significance. N/KRAS variants were present in 6% of patients and were typically sub-clonal (median VAF = 20%) relative to other genes variants. RAS variants were associated with advanced MF features including leukocytosis (p = 0.02), high somatic mutation burden (p < 0.01) and the presence of established “molecular high-risk” (MHR) mutations. MF patients with N/KRAS mutations had shorter 3-year overall survival (OS) (34% vs 58%, p < 0.001) and higher incidence of acute myeloid leukemia at 3 years (18% vs 11%, p = 0.03). In a multivariate Cox model, RAS mutations were associated with decreased OS (HR 1.93, p < 0.001). We created a novel score to predict OS incorporating RAS mutations, and it predicted OS across training and validation cohorts. Patients with intermediate risk/high-risk DIPSS with RAS mutations who received ruxolitinib had a nonsignificant longer 2-year OS relative to those who did not receive ruxolitinib. These data demonstrate the importance of identifying RAS mutations in MF patients.

Abstract Comprehensive genomic profiling is highly recommended for treatment decision in nonsquamous, non-small cell lung cancer (NSCLC). However, rare genomic alterations are still being unveiled, with scarce data to guide therapy. Herein, we describe the treatment journey of a 56-year-old, never-smoker Caucasian woman with a metastatic NSCLC harboring a CD47-MET fusion, initially classified as a variant of unknown significance. She had undergone 3 lines of therapy over the course of 3 years, including chemotherapy, immunotherapy, and anti-angiogenic therapy. After reanalysis of her next-generation sequencing data in our service, the fusion was reclassified as likely oncogenic. The patient was started with fourth-line capmatinib, with a good tolerance so far and a complete metabolic response in the active sites of disease, currently ongoing for 18 months. In conclusion, we highlight the sensitivity of a novel MET fusion to capmatinib and emphasize the need for comprehensive panels in NSCLC and molecular tumor board discussions with specialized centers when rare findings arise. This case report highlights the sensitivity of a novel MET fusion to capmatinib and emphasizes the need for comprehensive panels in non-small cell lung cancer and molecular tumor board discussions with specialized centers when rare findings arise.

SummaryJAK2V617F can mimic growth factor signaling, leading to PI3K/AKT/mTOR activation and inhibition of autophagy. We hypothesized that selective inhibition of JAK1/2 by ruxolitinib could induce autophagy and limit drug efficacy in myeloproliferative neoplasms (MPN). Therefore, we investigated the effects of ruxolitinib treatment on autophagy-related genes and cellular processes, to determine the potential benefit of autophagy inhibitors plus ruxolitinib in JAK2V617F cells, and to verify the frequency and clinical impact of autophagy-related gene mutations in patients with MPNs. In SET2 JAK2V617F cells, ruxolitinib treatment induced autophagy and modulated 26 out of 79 autophagy-related genes. Ruxolitinib treatment reduced the expressions of important autophagy regulators, including mTOR/p70S6K/4EBP1 and the STAT/BCL2 axis, in a dose- and time-dependent manner. Pharmacological inhibition of autophagy was able to significantly suppress ruxolitinib-induced autophagy and increased ruxolitinib-induced apoptosis. Mutations in autophagy-related genes were found in 15.5% of MPN patients and were associated with increased age and a trend towards worse survival. In conclusion, ruxolitinib induces autophagy in JAK2V617F cells, potentially by modulation of mTOR-, STAT- and BCL2-mediated signaling. This may lead to inhibition of apoptosis. Our results suggest that the combination of ruxolitinib with pharmacological inhibitors of autophagy, such as chloroquine, may be a promising strategy to treat patients with JAK2V617F-mutated MPNs.

Prognostic stratification in acute myeloid leukemia (AML) relies, mostly, on cytogenetics and molecular features of leukemic blasts. The LeukemiaNet prognostic scoring system has been proposed as a standardized way of evaluating prognosis in AML. We have analysed outcomes in 65 AML cases (median age of 54 years, range 18–82) treated at five hematology centers in Brazil stritified according to the European Leukemia Net (ELN) recommendations for cytogenetic and molecular analysis. We classified patients as favorable (N = 13), intermediate-1 (N = 25), intermediate-2 (N = 15), or adverse risk (N = 9). Bone marrow transplantation (BMT) was performed in 13 patients (21%). Median follow-up was 12 months. The median overall survival (OS) for all patients was 12.4 months. Median OS was 19.8, 12.4, 10.1, and 10.4 months (p = 0.24) for patients in the favorable, intermediate-1, intermediate-2, and adverse groups, respectively. Among patients receiving BMT, median OS was 26.8 months. The ELN is a valuable tool for prognostic stratification of AML patients treated in Brazil. Nevertheless, its usefulness is limited when compared to data from developed countries.

The ETV6 gene encodes an ETS family transcription factor that is involved in a myriad of chromosomal rearrangements found in hematological malignancies and other neoplasms. A recurrent ETV6 translocation, previously described in patients with acute myeloid leukemia (AML) (Genes Chromosomes Cancer 51:328–337,2012, Leuk Res 35:e212-214, 2011), whose partner has not been identified is t(7;12)(p15;p13). We herein report that the t(7;12)(p15;p13) fuses ETV6 to ANLN , a gene not previously implicated in the pathogenesis of hematological malignancies, and we demonstrate that this translocation leads to high expression of the fusion transcript in the myeloid and lymphoid lineages.

Aims. To develop a fast and robust DNA-based assay to detect insertions and deletions mutations in exon 34 that encodes the PEST domain of NOTCH1 in order to evaluate patients with chronic lymphocytic leukemia (CLL). Methods. We designed a multiplexed allele-specific polymerase chain reaction (PCR) combined with a fragment analysis assay to detect specifically the mutation c.7544_7545delCT and possibly other insertions and deletions in exon 34 of NOTCH1. Results. We evaluated our assay in peripheral blood samples from two cohorts of patients with CLL. The frequency of NOTCH1 mutations was 8.4% in the first cohort of 71 unselected CLL patients. We then evaluated a second cohort of 26 CLL patients with known cytogenetic abnormalities that were enriched for patients with trisomy 12. NOTCH1 mutations were detected in 43.7% of the patients with trisomy 12. Conclusions. We have developed a fast and robust assay combining allele-specific PCR and fragment analysis able to detect NOTCH1 PEST domain insertions and deletions.

Background Acute myeloid leukemia (AML) is a myeloid neoplasm associated with a high morbidity and mortality. The diagnosis, risk stratification and therapy selection in AML have changed substantially in the last decade with the progressive incorporation of clinically relevant molecular markers. Methods In this work, our aim was to describe a real-world genomic profiling experience in AML and to demonstrate the impact of the European Leukemia Net 2022 update on risk stratification in AML. Results and Discussion One hundred and forty-one patients were evaluated with an amplicon-based multi-gene next-generation sequencing (NGS) panel. The most commonly mutated genes were FLT3, DNMT3A, RUNX1, IDH2, NPM1, ASXL1, SRSF2, NRAS, TP53 and TET2. Detection of FLT3 ITD with NGS had a sensitivity of 96.3% when compared to capillary electrophoresis. According to ELN 2017, 26.6%, 20.1%, and 53.3% of patients were classified as having a good, moderate, or unfavorable risk. When ELN 2022 was used, 15.6%, 27.8%, and 56.6% of patients were classified as favorable, moderate, or unfavorable risk, respectively. When ELN 2022 was compared to ELN 2017, thirteen patients (14.4%) exhibited a different risk classification, with a significant decrease in the number of favorable risk patients, what has immediate clinical impact. Conclusions In conclusion, we have described a real-world genomic profiling experience in AML and the impact of the 2022 ELN update on risk stratification.