A comprehensive biomarker analysis of microsatellite unstable/mismatch repair deficient colorectal cancer cohort treated with immunotherapy

The search for immunotherapy biomarkers in Microsatellite Instability High/Deficient Mismatch Repair system (MSI-H/dMMR) metastatic colorectal cancer (mCRC) is an unmet need. Sixteen patients with mCRC and MSI-H/dMMR (determined by either immunohistochemistry or polymerase chain reaction) treated with PD-1/PD-L1 inhibitors at our institution were included. According to whether the progression-free survival with PD-1/PD-L1 inhibitors was longer than 6 months or shorter, patients were clustered into the IT-responder group (n: 9 patients) or IT-resistant group (n: 7 patients), respectively. In order to evaluate determinants of benefit with PD-1/PD-L1 inhibitors, we performed multimodal analysis including genomics (through NGS panel tumour-only with 431 genes) and the immune microenvironment (using CD3, CD8, FOXP3 and PD-L1 antibodies). The following mutations were more frequent in IT-resistant compared with IT-responder groups: B2M (4/7 versus 2/9), CTNNB1 (2/7 versus 0/9), and biallelic PTEN (3/7 versus 1/9). Biallelic ARID1A mutations were found exclusively in the IT-responder group (4/9 patients). Tumour mutational burden did not correlate with immunotherapy benefit, neither the rate of indels in homopolymeric regions. Of note, biallelic ARID1A mutated tumours had the highest immune infiltration and PD-L1 scores, contrary to tumours with CTNNB1 mutation. Immune microenvironment analysis showed higher densities of different T cell subpopulations and PD-L1 expression in IT-responders. Misdiagnosis of MSI-H/dMMR inferred by discordances between immunohistochemistry and polymerase chain reaction was only found in the IT-resistant population (3/7 patients). Biallelic ARID1A mutations and Wnt signalling activation through CTNNB1 mutation were associated with high and low T cell immune infiltrates, respectively, and deserve special attention as determinants of response to PD-1/PD-L1 inhibitors. The non-MSI-H phenotype in dMMR is associated with poor benefit to immunotherapy. Our results suggest that mechanisms of resistance to immunotherapy are multi-factorial

Early-Stage Breast Cancer Detection in Breast Milk

Breast cancer; Breast milkCáncer de mama; Leche maternaCàncer de mama; Llet maternaBreast cancer occurring during pregnancy (PrBC) and postpartum (PPBC) is usually diagnosed at more advanced stages compared with other breast cancer, worsening its prognosis. PPBC is particularly aggressive, with increased metastatic risk and mortality. Thus, effective screening methods to detect early PrBC and PPBC are needed. We report for the first time that cell-free tumor DNA (ctDNA) is present in breast milk (BM) collected from patients with breast cancer. Analysis of ctDNA from BM detects tumor variants in 87% of the cases by droplet digital PCR, while variants remain undetected in 92% of matched plasma samples. Retrospective next-generation sequencing analysis in BM ctDNA recapitulates tumor variants, with an overall clinical sensitivity of 71.4% and specificity of 100%. In two cases, ctDNA was detectable in BM collected 18 and 6 months prior to standard diagnosis. Our results open up the potential use of BM as a new source for liquid biopsy for PPBC detection. Significance: For the first time, we show that BM obtained from patients with breast cancer carries ctDNA, surpassing plasma-based liquid biopsy for detection and molecular profiling of early-stage breast cancer, even prior to diagnosis by image.We thank the patients who participated in the study and donated samples for analysis for their generous contribution, with particular thanks to the first patient, Maite, and her daughter Àneu, who inspired us to initiate this study (oral consent to name the patient and her daughter was provided by the patient, and her legal partner provided written consent after patient's exitus). We are grateful to Javier Carmona for his valuable contributions and support in the manuscript's conceptualization, preparation, and revision. VHIO would like to acknowledge the Cellex Foundation for providing research facilities and equipment and the CERCA Programme from the Generalitat de Catalunya for their support of this research. The authors from VHIO acknowledge the State Agency for Research (Agencia Estatal de Investigación) for the financial support as a Center of Excellence Severo Ochoa (CEX2020-001024-S/AEI/10.13039/501100011033). This research is financially supported by the “El paseíco de la mama” Foundation. C. Saura was the recipient of a II FERO-GHD grant from the FERO Foundation (FERO/5086), a Junior Clinical award from the Spanish Association Against Cancer Foundation (FAECC; CLJUN212026ORTI), and a SEOM-Daiichi Sankyo grant for its support on the Breast Cancer Research Projects 2021 (SEOM/FECMA2022) and received funding from the Department of Health (Generalitat de Catalunya SLT008/18/00198) and from the Instituto de Salud Carlos III (ISCIII) and Fondo Europeo de Desarrollo Regional (FEDER), cofunded by the European Union (PI21/01020). C. Ortiz was the recipient of a Junior Clinician award from the FAECC (CLJUN212026ORTI) and a SEOM-Daiichi Sankyo grant for its support on the Breast Cancer Research Projects 2021 (SEOM/FECMA2022), and received funding from the Department of Health (Generalitat de Catalunya SLT008/18/00198). N. Bayó-Puxan received funding from the Department of Health (Generalitat de Catalunya SLT008/18/00205), MCIN/AEI/10.13039/501100011033 (GPE2022-001029) and MCIN/AEI/10.130.39/501100011033, and the European Union “Next GenerationEU/PRTR” (ECT2020-000827). J.M. Miquel received funding from the Department of Health (Generalitat de Catalunya SLT008/18/00205), MCIN/AEI/10.130.39/501100011033, and the European Union “Next GenerationEU/PRTR” (ECT2020-000827). J. Arribas is funded by the Breast Cancer Research Foundation (BCRF-23-008), Instituto de Salud Carlos III (project reference numbers AC15/00062, CB16/12/00449, and PI22/00001), and the European Commission under the framework of the ERA-NET TRANSCAN-2 initiative cofinanced by FEDER and Asociación Española Contra el Cáncer. A. Vivancos was the recipient of a project award from the FAECC (AVP/18/AECC/3219) and received funding from the Advanced Molecular Diagnostic (DIAMAV) program from the FERO Foundation (8361) and from ISDIN for supporting the development of liquid biopsy applications at the Cancer Genomics Lab (1848). M. Sansó was the recipient of a II FERO-GHD grant from the FERO Foundation (FERO/5086) and an investigator award from the FAECC (INVES19056SANS), and received funding from the Health Research Institute of the Balearic Islands (IdISBa), the RADIX-Janssen program (RADIX/JANSSEN21/01), and the Miguel Servet Program funded by the ISCIII (CP22/00131)

Gcn5 facilitates Pol II progression, rather than recruitment to nucleosome-depleted stress promoters, in Schizosaccharomyces pombe

In the fission yeast, the MAP kinase Sty1 and the transcription factor Atf1 regulate up to 400 genes in response to environmental signals, and both proteins have been shown to bind to their promoters in a stress-dependent manner. In a genetic search, we have isolated the histone H3 acetyltransferase Gcn5, a component of the SAGA complex, as being essential for oxidative stress survival and activation of those genes. Upon stress, Gcn5 is recruited to promoters and coding sequences of stress genes in a Sty1- and Atf1-dependent manner, causing both an enhanced acetylation of histone H3 and nucleosome eviction. Unexpectedly, recruitment of RNA polymerase II (Pol II) is not impaired in Δgcn5 cells. We show here that stress genes display a 400-bp long nucleosome depleted region upstream of the transcription start site even prior to activation. Stress treatment does not alter promoter nucleosome architecture, but induces eviction of the downstream nucleosomes at stress genes, which is not observed in Δgcn5 cells. We conclude that, while Pol II is recruited to nucleosome-free stress promoters in a transcription factor dependent manner, Gcn5 mediates eviction of nucleosomes positioned downstream of promoters, allowing efficient Pol II progression along the genes

A Comprehensive Biomarker Analysis of Microsatellite Unstable/Mismatch Repair Deficient Colorectal Cancer Cohort Treated with Immunotherapy

Biomarkers; Colorectal cancer; ImmunotherapyBiomarcadors; Càncer colorectal; ImmunoteràpiaBiomarcadores; Cáncer colorrectal; InmunoterapiaThe search for immunotherapy biomarkers in Microsatellite Instability High/Deficient Mismatch Repair system (MSI-H/dMMR) metastatic colorectal cancer (mCRC) is an unmet need. Sixteen patients with mCRC and MSI-H/dMMR (determined by either immunohistochemistry or polymerase chain reaction) treated with PD-1/PD-L1 inhibitors at our institution were included. According to whether the progression-free survival with PD-1/PD-L1 inhibitors was longer than 6 months or shorter, patients were clustered into the IT-responder group (n: 9 patients) or IT-resistant group (n: 7 patients), respectively. In order to evaluate determinants of benefit with PD-1/PD-L1 inhibitors, we performed multimodal analysis including genomics (through NGS panel tumour-only with 431 genes) and the immune microenvironment (using CD3, CD8, FOXP3 and PD-L1 antibodies). The following mutations were more frequent in IT-resistant compared with IT-responder groups: B2M (4/7 versus 2/9), CTNNB1 (2/7 versus 0/9), and biallelic PTEN (3/7 versus 1/9). Biallelic ARID1A mutations were found exclusively in the IT-responder group (4/9 patients). Tumour mutational burden did not correlate with immunotherapy benefit, neither the rate of indels in homopolymeric regions. Of note, biallelic ARID1A mutated tumours had the highest immune infiltration and PD-L1 scores, contrary to tumours with CTNNB1 mutation. Immune microenvironment analysis showed higher densities of different T cell subpopulations and PD-L1 expression in IT-responders. Misdiagnosis of MSI-H/dMMR inferred by discordances between immunohistochemistry and polymerase chain reaction was only found in the IT-resistant population (3/7 patients). Biallelic ARID1A mutations and Wnt signalling activation through CTNNB1 mutation were associated with high and low T cell immune infiltrates, respectively, and deserve special attention as determinants of response to PD-1/PD-L1 inhibitors. The non-MSI-H phenotype in dMMR is associated with poor benefit to immunotherapy. Our results suggest that mechanisms of resistance to immunotherapy are multi-factorial.This research was funded by Merck Research Grants (Call 2018) in the Area of Colorectal Cancer Clinical Investigation

Genome-Wide Screen of Genes Required for Caffeine Tolerance in Fission Yeast

Isabel A. Calvo et al...Background An excess of caffeine is cytotoxic to all eukaryotic cell types. We aim to study how cells become tolerant to a toxic dose of this drug, and the relationship between caffeine and oxidative stress pathways. Methodology/Principal Findings We searched for Schizosaccharomyces pombe mutants with inhibited growth on caffeine-containing plates. We screened a collection of 2,700 haploid mutant cells, of which 98 were sensitive to caffeine. The genes mutated in these sensitive clones were involved in a number of cellular roles including the H2O2-induced Pap1 and Sty1 stress pathways, the integrity and calcineurin pathways, cell morphology and chromatin remodeling. We have investigated the role of the oxidative stress pathways in sensing and promoting survival to caffeine. The Pap1 and the Sty1 pathways are both required for normal tolerance to caffeine, but only the Sty1 pathway is activated by the drug. Cells lacking Pap1 are sensitive to caffeine due to the decreased expression of the efflux pump Hba2. Indeed, ?hba2 cells are sensitive to caffeine, and constitutive activation of the Pap1 pathway enhances resistance to caffeine in an Hba2-dependent manner. Conclusions/Significance With our caffeine-sensitive, genome-wide screen of an S. pombe deletion collection, we have demonstrated the importance of some oxidative stress pathway components on wild-type tolerance to the drug.This work was supported by Direccion General de Investigacion of Spain Grant BFU2006-02610, and by the Spanish program Consolider-Ingenio 2010 Grant CSD 2007-0020, to E.H.Peer reviewe

Clinical value of next generation sequencing of plasma cell-free DNA in gastrointestinal stromal tumors

ADN tumoral circulant; Tumor de l'estroma gastrointestinal; ImatinibADN tumoral circulante; Tumor del estroma gastrointestinal; ImatinibCirculating tumor DNA; Gastrointestinal stromal tumor; ImatinibBackground Gastrointestinal stromal tumor (GIST) initiation and evolution is commonly framed by KIT/PDGFRA oncogenic activation, and in later stages by the polyclonal expansion of resistant subpopulations harboring KIT secondary mutations after the onset of imatinib resistance. Thus, circulating tumor (ct)DNA determination is expected to be an informative non-invasive dynamic biomarker in GIST patients. Methods We performed amplicon-based next-generation sequencing (NGS) across 60 clinically relevant genes in 37 plasma samples from 18 GIST patients collected prospectively. ctDNA alterations were compared with NGS of matched tumor tissue samples (obtained either simultaneously or at the time of diagnosis) and cross-validated with droplet digital PCR (ddPCR). Results We were able to identify cfDNA mutations in five out of 18 patients had detectable in at least one timepoint. Overall, NGS sensitivity for detection of cell-free (cf)DNA mutations in plasma was 28.6%, showing high concordance with ddPCR confirmation. We found that GIST had relatively low ctDNA shedding, and mutations were at low allele frequencies. ctDNA was detected only in GIST patients with advanced disease after imatinib failure, predicting tumor dynamics in serial monitoring. KIT secondary mutations were the only mechanism of resistance found across 10 imatinib-resistant GIST patients progressing to sunitinib or regorafenib. Conclusions ctDNA evaluation with amplicon-based NGS detects KIT primary and secondary mutations in metastatic GIST patients, particularly after imatinib progression. GIST exhibits low ctDNA shedding, but ctDNA monitoring, when positive, reflects tumor dynamics.This research is supported by a Fero Fellowship Award (C.S.), Asociación Española Contra el Cáncer (J.P. Barcelona) (C.S.), and ISCIII PI16/01371 (C.S.). C.S. and A.V. acknowledge to the Cellex Foundation for providing facilities and equipment. None of the funding bodies had access nor influence in the design, collection, analysis, interpretation and writing the manuscript

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P < 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)