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High-dose melphalan (HDM) improves progression-free survival in multiple myeloma (MM), yet melphalan is a DNA-damaging alkylating agent; therefore, we assessed its mutational effect on surviving myeloma cells by analyzing paired MM samples collected at diagnosis and relapse in the IFM 2009 study. We performed deep whole-genome sequencing on samples from 68 patients, 43 of whom were treated with RVD (lenalidomide, bortezomib, and dexamethasone) and 25 with RVD + HDM. Although the number of mutations was similar at diagnosis in both groups (7137 vs 7230; P = .67), the HDM group had significantly more mutations at relapse (9242 vs 13 383, P = .005). No change in the frequency of copy number alterations or structural variants was observed. The newly acquired mutations were typically associated with DNA damage and double-stranded breaks and were predominantly on the transcribed strand. A machine learning model, using this unique pattern, predicted patients who would receive HDM with high sensitivity, specificity, and positive prediction value. Clonal evolution analysis showed that all patients treated with HDM had clonal selection, whereas a static progression was observed with RVD. A significantly higher percentage of mutations were subclonal in the HDM cohort. Intriguingly, patients treated with HDM who achieved complete remission (CR) had significantly more mutations at relapse yet had similar survival rates as those treated with RVD who achieved CR. This similarity could have been due to HDM relapse samples having significantly more neoantigens. Overall, our study identifies increased genomic changes associated with HDM and provides rationale to further understand clonal complexity.

The ribonuclease DIS3 is one of the most frequently mutated genes in the hematological cancer multiple myeloma, yet the basis of its tumor suppressor function in this disease remains unclear. Herein, exploiting the TCGA dataset, we found that DIS3 plays a prominent role in the DNA damage response. DIS3 inactivation causes genomic instability by increasing mutational load, and a pervasive accumulation of DNA:RNA hybrids that induces genomic DNA double-strand breaks (DSBs). DNA:RNA hybrid accumulation also prevents binding of the homologous recombination (HR) machinery to double-strand breaks, hampering DSB repair. DIS3-inactivated cells become sensitive to PARP inhibitors, suggestive of a defect in homologous recombination repair. Accordingly, multiple myeloma patient cells mutated for DIS3 harbor an increased mutational burden and a pervasive overexpression of pro-inflammatory interferon, correlating with the accumulation of DNA:RNA hybrids. We propose DIS3 loss in myeloma to be a driving force for tumorigenesis via DNA:RNA hybrid-dependent enhanced genome instability and increased mutational rate. At the same time, DIS3 loss represents a liability that might be therapeutically exploited in patients whose cancer cells harbor DIS3 mutations.

Somatic mutations in splicing factor genes frequently occur in myeloid neoplasms. While SF3B1 mutations are associated with myelodysplastic syndromes (MDS) with ring sideroblasts, SRSF2P95 mutations are found in different disease categories, including MDS, myeloproliferative neoplasms (MPN), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), and acute myeloid leukemia (AML). To identify molecular determinants of this phenotypic heterogeneity, we explored molecular and clinical features of a prospective cohort of 279 SRSF2P95-mutated cases selected from a population of 2663 patients with myeloid neoplasms. Median number of somatic mutations per subject was 3. Multivariate regression analysis showed associations between co-mutated genes and clinical phenotype, including JAK2 or MPL with myelofibrosis (OR = 26.9); TET2 with monocytosis (OR = 5.2); RAS-pathway genes with leukocytosis (OR = 5.1); and STAG2, RUNX1, or IDH1/2 with blast phenotype (MDS or AML) (OR = 3.4, 1.9, and 2.1, respectively). Within patients with SRSF2-JAK2 co-mutation, JAK2 dominance was invariably associated with clinical feature of MPN, whereas SRSF2 mutation was dominant in MDS/MPN. Within patients with SRSF2-TET2 co-mutation, clinical expressivity of monocytosis was positively associated with co-mutated clone size. This study provides evidence that co-mutation pattern, clone size, and hierarchy concur to determine clinical phenotype, tracing relevant genotype-phenotype associations across disease entities and giving insight on unaccountable clinical heterogeneity within current WHO classification categories.

Background Critically ill COVID-19 patients are burdened by high mortality. Evaluation to improve patients’ management was performed using strict patient criteria for Intensive Care Unit (ICU) admission, a low- positive end-expiratory pressure (PEEP) setting and standard thromboembolism treatment. Objectives of this study was to report the effects of this strategy on P/F-ratio evolution during mechanical ventilation (MV), ICU length of stay (LOS) and MV length.Methods A retrospective analysis was conducted on all consecutive patients with acute respiratory distress due to COVID-19 pneumonia admitted into ICU from March 2nd to January 15th, 2021. Patients were treated with a low-PEEP strategy (PEEP 10 cmH2O if BMI < 30 Kg m-2, PEEP 12 cmH2O if BMI 30-50 Kg m-2, PEEP 15 cmH2O if BMI > 50 Kg m-2) and therapeutic anticoagulation in case of thrombosis or D-dimer greater than 1’500 ng ml-1.Results 79 patients were on invasive MV. Average applied PEEP was 11 ± 2.9 cmH2O for BMI < 30 Kg m-2, 16 ± 3.18 cmH2O for BMI > 30 Kg m-2. After low-PEEP application, patients’ P/F ratio presented daily improvement from admission during next 72 hours (pConclusions A combination of low-PEEP treatment resulted in P/F persistent daily ratio improvement during first 72 hours after OTI. A low-PEEP strategy could be beneficial in hemodynamic than respiratory terms.

Esophageal adenocarcinoma (EAC) is associated with a marked genomic instability, which underlies disease progression and development of resistance to treatment. In this study, we used an integrated genomics approach to identify a genomic instability signature. Here we show that elevated expression of this signature correlates with poor survival in EAC as well as three other cancers. Knockout and overexpression screens establish the relevance of these genes to genomic instability. Indepth evaluation of three genes (TTK, TPX2 and RAD54B) confirms their role in genomic instability and tumor growth. Mutational signatures identified by whole genome sequencing and functional studies demonstrate that DNA damage and homologous recombination are common mechanisms of genomic instability induced by these genes. Our data suggest that the inhibitors of TTK and possibly other genes identified in this study have potential to inhibit/reduce growth and spontaneous as well as chemotherapy-induced genomic instability in EAC and possibly other cancers., Subodh Kumar et al. identify a gene signature correlated with genomic instability and poor survival in esophageal adenocarcinoma (EAC), using a combination of integrative genomic analysis of patient data and laboratory validation in cell line models and mice. They find that inhibitors of some of the identified proteins, including TTK, could be used to reduce genomic evolution as well as inhibit growth of EAC cells.

The DNA damage response (DDR) is the signaling cascade that recognizes DNA double-strand breaks (DSBs) and promotes their resolution via the DNA repair pathways of non-homologous end joining (NHEJ) or homologous recombination (HR). We and others have shown that DDR activation requires DROSHA; however, whether DROSHA exerts its functions by associating with damage sites, what controls its recruitment, and how DROSHA influences DNA repair remains poorly understood. Here, we show that DROSHA associates with DSBs independently of transcription. Neither H2AX, nor ATM or DNA-PK kinase activities are required for recruitment of DROSHA to break sites. Rather, DROSHA interacts with RAD50, and inhibition of the MRN complex by mirin treatment abolishes this interaction. MRN complex inactivation by RAD50 knockdown or mirin treatment prevents DROSHA recruitment to DSBs and, as a consequence, also prevents 53BP1 (also known as TP53BP1) recruitment. During DNA repair, DROSHA inactivation reduces NHEJ and boosts HR frequency. Indeed, DROSHA knockdown also increases the association of downstream HR factors such as RAD51 to DNA ends. Overall, our results demonstrate that DROSHA is recruited at DSBs by the MRN complex and directs DNA repair towards NHEJ., Summary: DROSHA is part of the primary response to DNA damage and is recruited at double-strand breaks in an MRN-dependent manner. DROSHA fosters NHEJ repair and inhibits HR by controlling 53BP1 recruitment.

BackgroundIntensive Care Unit (ICU) management of COVID-19 patients with severe hypoxemia is associated with high mortality. We implemented a ‘care map’, as a standardized multidisciplinary approach to improve patients monitoring using: uniform patient selection for ICU admission, a low-PEEP strategy and a pharmacologic strategic thromboembolism management.MethodsA standardized protocol for managing COVID-19 patients and ICU admissions was implemented through accurate Early Warning Score (EWS) monitoring and thromboembolism prophylaxis at hospital admission. Dyspnea, mental confusion or SpO2 less than 85% were criteria for ICU admission. Ventilation approach employed low PEEP values (about 10 cmH2O in presence of lung compliance > 40 mL/cmH2O) and FiO2 as needed. In presence of lower lung compliance (< 40 mL/cmH2O) PEEP value was increased to about 14 cmH2O.FindingsFrom March 16th to April 12nd 2020, 41 COVID-19 patients were admitted to our ICU from a total of 310 patients. 83% (34) of them needed mechanical ventilation. The ventilation approach chosen employed low PEEP value based on BMI (PEEP 11± 3.8 (10-12) cmH2O if BMI < 30 Kg/m2; PEEP 15± 3.26 (12-18) cmH2O if BMI >30 Kg/m2). To date, ten patients (24%) died, four (9.7%) received mechanical ventilation, two were transferred to another hospital and 25 (60.9%) were discharged from ICU after a median of nine days.InterpretationA multimodal approach for COVID-19 patients is mandatory. The knowledge of this multi-organ disease is growing rapidly, requiring improvements in the standard of care. Our approach implements an accurate pre-ICU monitoring and strict selection for ICU admission, and allows to reduce mechanical ventilation, ICU stay and mortality.FundingNo funding has been required.

Identification of genes driving genomic evolution can provide novel targets for cancer treatment and prevention. Here we show identification of a genomic instability gene signature, using an integrated genomics approach. Elevated expression of this signature correlated with poor survival in esophageal adenocarcinoma (EAC) as well as three other human cancers. Knockout and overexpression screens confirmed the relevance of this signature to genomic instability. Indepth evaluation of TTK (a kinase), TPX2 (spindle assembly factor) and RAD54B (recombination protein) further confirmed their role in genomic instability and tumor growth. Mutational signatures identified by whole genome sequencing and functional studies demonstrated that DNA damage and homologous recombination were common mechanisms of genomic instability induced by these genes. Consistently, a TTK inhibitor impaired EAC cell growth in vivo, and increased chemotherapy-induced cytotoxicity while inhibiting genomic instability in surviving cells. Thus inhibitors of TTK and other genes identified in this study have potential to inhibit/delay genomic evolution and tumor growth. Such inhibitors also have potential to increase chemotherapy-induced cytotoxicity while reducing its harmful genomic impact in EAC and possibly other cancers.

We recently shown that high-dose melphalan (HDM) followed by autologous stem cell transplant (ASCT) as first line therapy in young ( A significant change in frequency of driver mutations including RAS/RAF, FAM46C, TP53, and DIS3 was not observed at the time of relapse. Clonality level was increased only for KRAS (p=0.054), while all other specific driver genes had similar clonality level at diagnosis and relapse. Interestingly, a significant increase in mutations involving MYO16 and SLC7A8 genes was observed at relapse in both arms, implicating components of the induction regimen (RVD). Investigating the mutational signature utilization in only newly acquired mutations identified 4 signatures: APOBEC, HR Double Strand Repair, clock-like signature, and unknown. k-means clustering analysis of samples based on signature utilization showed four distinct clusters. All patients clustering with high DNA repair signature utilization were in the HDM arm (65% HDM patients), the majority of whom achieved CR or sCR (74%); these patients acquired 8308 (range 3302-19107) new mutations between diagnosis and relapse. None of the RVD only treated patients were in this cluster. The remaining 35% HDM group patients were clustered with RVD samples and showed unknown signature utilization. Furthermore, motif enrichment analysis identified CYWR and ATGAGATV (p < 1e-130) as enriched motifs around the new mutations in HDM compared to RVD cohort. Importantly and as expected, DNA damage repair pathway genes were frequently targeted in the HDM group: 72% HDM samples accumulated DDR gene mutations vs. only 17% in the RVD alone arm (p < 0.001). At the time of relapse, 100% HDM arm patients had at least one DDR gene mutation and 80% had two or more, while only 37% RVD only group had one or more such mutation. Finally, we have reconstructed phylogenetic and evolutionary trajectories based on mutation and copy-number data from samples at diagnosis and relapse. The clonal composition in both arms was similar at diagnosis; however, HDM caused a significant shift to more subclonal mutations at relapse. chromothripsis and chromoplexy events were detected in 30% patients at diagnosis, which remained constant at relapse regardless of treatment. In summary, we describe significant accumulation of mutations following high dose melphalan. This fundamental molecular change in the disease at relapse, suggests the need for reappraisal of the optimal use and sequencing of high dose melphalan in the era of novel agents. Disclosures Fulciniti: NIH: Research Funding. Richardson:Celgene/BMS, Oncopeptides, Takeda, Karyopharm: Research Funding. Thakurta:Oxford University: Other: visiting professor; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Perrot:Amgen, BMS/Celgene, Janssen, Sanofi, Takeda: Consultancy, Honoraria, Research Funding. Moreau:Sanofi: Consultancy, Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Honoraria; Novartis: Honoraria; Abbvie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Anderson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Parmigiani:Phaeno Biotehnologies: Current equity holder in publicly-traded company; CRA Health: Current equity holder in publicly-traded company; Foundation Medicine Institute: Consultancy; Delphi Diagnostics: Consultancy; BayesMendel Laboratory: Other: Co-lead. Munshi:Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy; Legend: Consultancy.

Aim To assess the educational needs, role and perceptions in palliative care issues of radiation oncologists (ROs) and trainees. Background 1/3 of radiotherapy patients are treated with palliative intent. Conversely, education and role that ROs have in the palliative care process are not well established, neither in terms of how they perceive their competence nor whether it is important to improve training, research and attention in palliative care issues at radiotherapy congresses. Material and Methods Literature systematic review in National Library of Medicine and Cochrane databases with 11 relevant issues to be identified. One doctor made first selection of articles, a second one confirmed their eligibility. Results 722 articles reviewed, 19 selected. 100% recognize the importance of palliative care in radiotherapy, 89.4% the need of training in palliative care for ROs, 68.4% the necessity of improving the resident programs, 63.1% the importance of skilled ROs in palliative care, 63.1% the need of better communication skills and pain management (47.3%), 52.6%, the perception of inadequate training in palliative care, 36.8% the lack of research and palliative care topics in radiotherapy meetings, 21% the absence of adequate guidelines regarding palliative care approaches, 42.1% the importance of the ROs in palliative care teams and 26.3% the lack of their involvement. Conclusion Palliative care has an important role in radiotherapy but it seems ROs still need more training. It is necessary to improve training programs, increment palliative care research in radiotherapy, giving more attention to palliative care themes at radiotherapy congresses. This could lead to a better integration of radiotherapists in multidisciplinary palliative care teams in the future.

Multiple myeloma (MM) has a heterogeneous genome, evolving through both pre-clinical and post-diagnosis phases. Here, using sequences from 67 MM genomes serially collected from 30 patients together with public datasets, we establish a hierarchy of driver lesions. Point mutations, structural variants and copy number aberrations define at least 7 genomic subgroups of MM, each with distinct sets of co-operating driver mutations. Complex structural events are major drivers of MM, including chromothripsis, chromoplexy and a replication-based mechanism of templated insertions: these typically occur early. Hyperdiploidy also occurs early, with individual chromosomes often gained in more than one chronological epoch of MM evolution, showing a preferred order of acquisition. Positively selected point mutations frequently occur in later phases of disease development, as do structural variants involving MYC. Thus, initiating driver events of MM, drawn from a limited repertoire of structural and numerical chromosomal changes, shape preferred trajectories of subsequent evolution.

The DNA damage response (DDR) is the signaling cascade through which a cell recognizes DNA lesions, and promotes their resolution via the repair pathways of Non-Homologous End Joining (NHEJ), or Homologous Recombination (HR). We recently demonstrated that DROSHA boosts DDR signaling by processing damage-induced long non-coding RNAs into smaller DNA damage response RNAs (DDRNAs). However, the location at which DROSHA exerts its DDR functions, relative to sites of DNA damage, remains unknown.To investigate DROSHA’s localization during DDR activation, we used the DiVA cellular system, which allows the controlled induction of several DNA double strand breaks (DSBs) in the human genome. Indeed, by genome wide chromatin immunoprecipitation followed by next generation sequencing, we demonstrate that DROSHA associates with DSBs. In support of this, DSB-recruitment of DROSHA is detectable at the single-cell level by Proximity Ligation Assay between DROSHA and known DDR markers, and by DNA damage in situ ligation followed by Proximity Ligation Assay (DI-PLA), which demonstrates proximity of DROSHA to DNA ends. DROSHA recruitment occurs at both genic and inter-genic DSBs, suggesting that its recruitment is independent from ongoing transcription preceding damage generation. DROSHA’s recruitment to DNA lesions occurs throughout the cell cycle, and with a preference for NHEJ-prone DSBs. Consistently, inhibition of the HR pathway increases DROSHA recruitment, and DROSHA knock down strongly impairs NHEJ efficiency in a GFP-reporter cellular system for monitoring NHEJ DNA repair. Overall, these results demonstrate that DROSHA acts locally at sites of DNA damage to promote NHEJ DNA repair.

Multiple Myeloma (MM) is a plasma cells malignancy with number of recent therapeutic options that has improved outcomes with median survival now stretching beyond 8 years. There has been an intense search to identify genomic and laboratory correlates of outcome for high risk patients. However, a subgroup of patients have a long survival but genomic segmentation of this important group which can be potentially cured has not been identified. We here described first such attempt at identifying a subset of patients with long survival. We have analyzed data from 205 newly-diagnosed uniformly-treated MM patients using both deep whole genome (WGS) (80x) and whole transcriptome sequencing (RNAseq). Median number of SNVs, small insertion and deletion per megabase were 2.18 [0.49 - 14.52], 0.077 [0.024 - 0.17] and 0.12 [0.03 - 0.26] respectively. MM subgroups defined by FISH or copy number alterations (CNAs) had significantly different mutational load (Kruskal-Wallis p-value = 0.003). In general, Hyperdiploid MM (HMM) patients had lower SNV load compared to other subgroups and t(14;16) had the highest mutational load per megabase (Dunn test FDR < 0.25). These subgroups also showed significant differences for mutational process utilization. Mutational processes associated spontaneous deamination of 5-methylcytosine to thymine was significantly high in HMM, APOBEC activity was very strong in t(14;16) MM and DNA repair was significantly different in t(6;14), del17p and t(11;14). Importantly, the mutations associated with clonal cell population and thus in early phases of MM cell progression were driven by spontaneous or enzymatic deamination (Signature 1), somatic hypermutation in lymphoid cells (Signature 9) and Signature 17 (ANOVA p value < 1e-16) and surprisingly APOBEC activity was constant among the clones (ANOVA p value > 0.05). However, mutations associated with homologous recombination (HR) and nucleotide excision repair (NER) activity were significantly enriched in the subclonal mutations (ANOVA p value < 1e-16) suggesting their role in later stages of MM progression. Based on this information, we next calculated the homologous recombination repair defects using copy number alterations from WGS data. We identified that HR score generated using WGS copy number information predicted outcome in newly diagnosed myeloma. Further analysis of this data identified that newly diagnosed MM patients with hyperdiploid MM (HMM) and no HR- repair deficiency have superior outcome with 6 year overall survival (OS) probability for this of 100% in IFM/DFCI 2009 cohort (Logrank test p value = 0.0012). We next validated this finding using MMRF CoMMpass dataset and we confirmed 6-yr OS probability of 95% (Logrank test p value = 0.0016) in this independent dataset (Figure 1). We further investigated RNAseq data between these genomically defined groups and identified that chromosome and telomere maintenance pathways (FDR < 0.01) and low bone disease associated genes described by Zhan et al. (FDR = 0.004) were up regulated in low-risk group. Finally, patients who were able to maintain HR, had lower mutation rate for TP53 (0% vs 9%, FDR < 0.05), CSMD1 (0% vs 6%, FDR < 0.05), FAT3/FAT4 mutations (1.7% vs 15%, FDR < 0.05), however surprisingly higher mutation rate for NRAS (42% vs 14%, p value < 0.001). In addition, DNA damage associated processes predicted by SNVs trinucleotides were significantly lower in the low-risk group (12% vs. 20%, p value < 1e-05) providing further support to this data. In conclusion, we report a detailed genomic profile using deep DNA and RNA sequencing and identify a genomically-defined sub group that is predicted to have a long survival. This study also identifies the role of HR in myeloma with potential for its translational application in both prognosis as well as therapy. Figure 1 Disclosures Richardson: Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. Moreau:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Thakurta:Celgene: Employment, Equity Ownership. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Avet-Loiseau:celgene: Consultancy, Other: travel fees, lecture fees, Research Funding; takeda: Consultancy, Other: travel fees, lecture fees, Research Funding. Munshi:Takeda: Consultancy; Oncopep: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Janssen: Consultancy.

FEMS Microbiology Ecology, 84 (3), ISSN:0168-6496, ISSN:1574-6941