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CDK4/6 inhibitors combined with endocrine therapy have demonstrated higher antitumor activity than endocrine therapy alone for the treatment of advanced estrogen receptor-positive breast cancer. Some of these tumors are de novo resistant to CDK4/6 inhibitors and others develop acquired resistance. Here, we show that p16 overexpression is associated with reduced antitumor activity of CDK4/6 inhibitors in patient-derived xenografts (n = 37) and estrogen receptor-positive breast cancer cell lines, as well as reduced response of early and advanced breast cancer patients to CDK4/6 inhibitors (n = 89). We also identified heterozygous RB1 loss as biomarker of acquired resistance and poor clinical outcome. Combination of the CDK4/6 inhibitor ribociclib with the PI3K inhibitor alpelisib showed antitumor activity in estrogen receptor-positive non-basal-like breast cancer patient-derived xenografts, independently of PIK3CA, ESR1 or RB1 mutation, also in drug de-escalation experiments or omitting endocrine therapy. Our results offer insights into predicting primary/acquired resistance to CDK4/6 inhibitors and post-progression therapeutic strategies. This study has been supported by the Susan G. Komen Foundation (CCR15330331) and by the Catalan Agency AGAUR (2017 SGR 540) [to V.S.]. V.S. received funds from the Instituto de Salud Carlos III: grants PI13/01714, CP14/00228, MV15/00041, CPII19/00033 and PI20/00892. M.P. received a Juan de la Cierva Grant from the Ministerio de Economía y Competitividad (FJCI-2015-25412), L.Mo. a grant from FI-AGAUR (2019 FI_B 01199), F.B-M. a grant from the Fundación Científica Asociación Española Contra el Cáncer (AECC_Postdoctoral17-1062) and M.S-G, a Marie Slodowska-Curie Innovative Training Networks PhD fellowship (H2020-MSCA-ITN-2015_675392). This work was supported by Breast Cancer Research Foundation (BCRF-19-08), Instituto de Salud Carlos III Project Reference number AC15/00062 and the EC under the framework of the ERA-NET TRANSCAN-2 initiative co-financed by FEDER, Instituto de Salud Carlos III (CB16/12/00449 and PI19/01181) and Asociación Española Contra el Cáncer (to J.A.). R.B.C. laboratory is supported by Breast Cancer Now (grant numbers: MAN-Q1 and MAN-Q2), NIHR Manchester Biomedical Research Centre (IS-BRC-1215-20007) and EdiREX Horizon 2020 grant No.731105. The xenograft program in the C.C. laboratory is supported by Cancer Research UK and also received funding from an EU H2020 Network of Excellence (EuroCAN). This work has been supported by NIH grants P30 CA008748 and RO1CA190642-01, the CDMRP grant BC171535P1, and the Breast Cancer Research Foundation [to M.S.]. A.P. received funds from Instituto de Salud Carlos III—PI16/00904 and PI19/01846, Breast Cancer Now—2018NOVPCC1294, Breast Cancer Research Foundation-AACR Career Development Awards for Translational Breast Cancer Research 19-20-26-PRAT, Fundació La Marató TV3 201935-30, the European Union’s Horizon 2020 research and innovation program H2020-SC1-BHC-2018-2020. IRB Barcelona is a recipient of a Severo Ochoa Centre of Excellence Award from the Spanish Ministry of Economy and Competitiveness (MINECO; Government of Spain) and is supported by CERCA (Generalitat de Catalunya). C. S. Verma reports grants from MSD International and grants from Ipsen outside the submitted work.

ObjectiveLynch Syndrome (LS) carriers have a significantly increased risk of developing colorectal cancer (CRC) during their lifetimes. Further stratification of this patient population may help in identifying additional risk factors that predispose to colorectal carcinogenesis. In most LS patients CRC may arise from adenomas, although an alternative non-polypoid carcinogenesis pathway has been proposed for PMS2 carriers. Using data from our institutional LS cohort, our aim was to describe our current colorectal screening outcomes with a focus on the incidence of adenomas in the context of different MMR genotypes and patient demographics such as gender, race, and ethnicity.DesignWe collected demographics, genetic, colonoscopy, and pathology results from a total of 163 LS carriers who obtained regular screening care at MD Anderson Cancer Center. Data were extracted from the electronic health records into a REDCap database for analysis. Logistic regressions were performed to measure the association between MMR variants and the likelihood of adenomas, advanced adenomas, and CRC. Then, we analyzed the cumulative incidences of these outcomes for the first 36 months following enrollment using Kaplan-Meier incidence curves, and Cox proportional hazard regressions.ResultsOn multivariate analysis, age (≥45 years old) was associated with an increased risk of developing adenomas (P=0.034). Patients with a prior or active cancer status were less likely to develop adenomas (P=0.015), despite of the lack of association between surgical history with this outcome (P=0.868). We found no statistically significant difference in likelihood of adenoma development between MLH1 and MSH2/EPCAM, MSH6, and PMS2 carriers. Moreover, we observed no statistically significant difference in the likelihood of advanced adenomas or CRC for any measured covariates. On Cox proportional hazard, compared to MLH1 carriers, the incidence of adenomas was highest among MSH2/EPCAM carriers during for the first 36-months of follow-up (PP=0.073). No MMR carrier was more likely to develop advanced adenomas. No difference in the incidence of CRC by MMR gene (P=0.198).ConclusionScreening recommendations for CRC in LS patients should be based on specific MMR variants and should also be tailored to consider patient demographics.

T Cells comprise many subtypes of specified lymphocytes, and their differentiation and function take place in different tissues. This cellular diversity is also observed in the multiple ways T-cell transformation gives rise to a variety of T-cell neoplasms. This review covers the main types of T-cell malignancies and their specific characteristics, emphasizing recent advances at the cellular and molecular levels as well as differences and commonalities among them.

Background Although intensity-modulated radiation therapy (IMRT) is considered the standard of care for the treatment of squamous cell carcinoma of the anus (SCCA), few large series have reported oncologic outcomes and toxicities. In this retrospective report, we aim to describe outcomes and toxicities after IMRT-based chemoradiation (CRT) for the treatment of SCCA, evaluate the impact of dose escalation (>54 Gy), and compare concurrent fluoropyrimidine in combination with either mitomycin or with cisplatin as chemosensitizers. Methods Patients treated at The University of Texas MD Anderson Cancer Center between January 1, 2003 and December 31, 2018 with IMRT-based CRT were included. Median time to locoregional recurrence, time to colostomy, and overall survival were estimated using the Kaplan–Meier method. Results A total of 428 patients were included; median follow-up was 4.4 years. Three hundred and thirty-four patients (78.0%) were treated with concurrent cisplatin and fluoropyrimidine, and 160 (37.4%) with >54 Gy. Two- and 5-year freedom from locoregional failure, freedom from colostomy failure, and overall survival were 86.5% and 81.2%, respectively, 90.0% and 88.3%, respectively, and 93.6% and 85.8%, respectively. Neither dose escalation nor mitomycin-based concurrent chemotherapy resulted in improved outcomes. Mitomycin-based concurrent chemotherapy was associated with in approximately 2.5 times increased grade 3 or greater acute toxicity. Radiation dose >54 Gy was associated with approximately 2.6 times increased Grade 3 or greater chronic toxicity. Conclusions Our results suggest IMRT-based CRT with concurrent fluoropyrimidine and cisplatin is a safe and feasible option for patient with SCCA and may cause less acute toxicity. The role for radiation dose escalation is unclear and requires further study.

Lung cancer in never smokers (LCINS) is a common cause of cancer mortality but its genomic landscape is poorly characterized. Here high-coverage whole-genome sequencing of 232 LCINS showed 3 subtypes defined by copy number aberrations. The dominant subtype (piano), which is rare in lung cancer in smokers, features somatic UBA1 mutations, germline AR variants and stem cell-like properties, including low mutational burden, high intratumor heterogeneity, long telomeres, frequent KRAS mutations and slow growth, as suggested by the occurrence of cancer drivers' progenitor cells many years before tumor diagnosis. The other subtypes are characterized by specific amplifications and EGFR mutations (mezzo-forte) and whole-genome doubling (forte). No strong tobacco smoking signatures were detected, even in cases with exposure to secondhand tobacco smoke. Genes within the receptor tyrosine kinase-Ras pathway had distinct impacts on survival; five genomic alterations independently doubled mortality. These findings create avenues for personalized treatment in LCINS.

Chemotherapies may increase mutagenesis of healthy cells and change the selective pressures in tissues, thus influencing their evolution. However, their contributions to the mutation burden and clonal expansions of healthy somatic tissues are not clear. Here, exploiting the mutational footprint of some chemotherapies, we explore their influence on the evolution of hematopoietic cells. Cells of Acute Myeloid Leukemia (AML) secondary to treatment with platinum-based drugs show the mutational footprint of these drugs, indicating that non-malignant blood cells receive chemotherapy mutations. No trace of the 5-fluorouracil (5FU) mutational signature is found in AMLs secondary to exposure to 5FU, suggesting that cells establishing the leukemia could be quiescent during treatment. Using the platinum-based mutational signature as a barcode, we determine that the clonal expansion originating the secondary AMLs begins after the start of the cytotoxic treatment. Its absence in clonal hematopoiesis cases is consistent with the start of the clonal expansion predating the exposure to platinum-based drugs., The mutational effects of chemotherapies on healthy cells are unclear. Here, the authors show that the mutational signature of platinum-based drugs -but not 5-fluorouracil- is detectable in secondary acute myeloid leukemia, implying that the clonal expansion begins after the start of therapy.

Invasive malignant melanoma (MM) is an aggressive tumor with no curative therapy in advanced stages. Chemotherapy has not demonstrated its efficacy in MM and current treatment for tumors carrying the most frequent BRAFV600E mutation consists of BRAF inhibitors alone or in combination with MAPK pathway inhibitors. We previously found that BRAF inhibition prevents activation of the DNA-damage repair (DDR) pathway in colorectal cancer thus potentiating the effect of chemotherapy. We now show that different chemotherapy agents inflict DNA damage in MM cells, which is efficiently repaired, associated with activation of the ATM-dependent DDR machinery. Pharmacologic inhibition of BRAF impairs ATM and DDR activation in these cells, leading to sustained DNA damage. Combination treatments involving DNA-damaging agents and BRAF inhibitors increase tumor cell death in vitro and in vivo, and impede MM regrowth after treatment cessation. We propose to reconsider the use of chemotherapy in combination with BRAF inhibitors for MM treatment.

Tyrosine kinase inhibitors were found to be clinically effective for treatment of patients with certain subsets of cancers carrying somatic mutations in receptor tyrosine kinases. However, the duration of clinical response is often limited, and patients ultimately develop drug resistance. Here, we use single-cell RNA sequencing to demonstrate the existence of multiple cancer cell subpopulations within cell lines, xenograft tumors and patient tumors. These subpopulations exhibit epigenetic changes and differential therapeutic sensitivity. Recurrently overrepresented ontologies in genes that are differentially expressed between drug tolerant cell populations and drug sensitive cells include epithelial-to-mesenchymal transition, epithelium development, vesicle mediated transport, drug metabolism and cholesterol homeostasis. We show analysis of identified markers using the LINCS database to predict and functionally validate small molecules that target selected drug tolerant cell populations. In combination with EGFR inhibitors, crizotinib inhibits the emergence of a defined subset of EGFR inhibitor-tolerant clones. In this study, we describe the spectrum of changes associated with drug tolerance and inhibition of specific tolerant cell subpopulations with combination agents., It has been proposed that resistance to targeted therapies in non-small cell lung carcinoma (NSCLC) is due to a nonhomogeneous cell population. Here the authors analyse preclinical NSCLC models using single-cell RNA-seq and identify drug tolerant cell states and subpopulations, as well as associated genes.

Mutations in the VAV1 guanine nucleotide exchange factor 1 have been recently found in peripheral T cell lymphoma and nonsmall-cell lung cancer (NSCLC). To understand their pathogenic potential, we generated a gene-edited mouse model that expresses a VAV1 mutant protein that recapitulates the signalling alterations present in the VAV1 mutant subclass most frequently found in tumours. We could not detect any overt tumourigenic process in those mice. However, the concurrent elimination of the Trp53 tumour suppressor gene in them drives T cell lymphomagenesis. This process represents an exacerbation of the normal functions that wild-type VAV1 plays in follicular helper T cells. We also found that, in combination with the Kras oncogene, the VAV1 mutant version favours progression of NSCLC. These data indicate that VAV1 mutations play critical, although highly cell-type-specific, roles in tumourigenesis. They also indicate that such functions are contingent on the mutational landscape of the tumours involved., The XRB’s project leading to these results has received funding from the RTI2018-096481-B-100 grant cofounded by MCIN/AEI/10.13039/501100011033 and the European Research Development Fund “A way of making Europe”, the Spanish Association against Cancer (GC16173472GARC), the CastillaLeon autonomous government (CSI252P18, CSI145P20, CLC-2017-01), and “la Caixa” Banking Foundation (HR20-00164). XRB’s institution is supported by the Programa de Apoyo a Planes Estrategicos de Investigacion de Estructuras de Investigaci on de Excelencia of the Castilla-Leon autonomous government (CLC-2017- 01), JR-V received funding from the Carlos III Health Institute (PI20/01724). LF-N contract has been supported by the Salamanca local section of the Spanish Association against Cancer

Altres ajuts: European Food and Safety Authority, EFSA.PRAS.2018.04-CT1; Consejo Nacional de Ciencia y Tecnología, CB-2012-01-183467; Centro de Investigación Biomédica en Red en Cáncer, PID2019-104695RBI00; Asociación Española Contra el Cáncer, AECC INVES211226MOLI

Recently, distinct mutational footprints observed in metastatic tumors, secondary malignancies and normal human tissues have been demonstrated to be caused by the exposure to several chemotherapeutic drugs. These characteristic mutations originate from specific lesions caused by these chemicals to the DNA of exposed cells. However, it is unknown whether the exposure to these chemotherapies leads to a specific footprint of larger chromosomal aberrations. Here, we address this question exploiting whole genome sequencing data of metastatic tumors obtained from patients exposed to different chemotherapeutic drugs. As a result, we discovered a specific copy number footprint across tumors from patients previously exposed to platinum-based therapies. This footprint is characterized by a significant increase in the number of chromosomal fragments of copy number 1-4 and size smaller than 10 Mb in exposed tumors with respect to their unexposed counterparts (median 14-387% greater across tumor types). The number of chromosomal fragments characteristic of the platinum-associated CN footprint increases significantly with the activity of the well known platinum-related footprint of single nucleotide variants across exposed tumors.

(100-120 words) Alterations in lipid metabolism are being increasingly implicated in the initiation, aggressiveness, and progression to metastasis of cancers. Recent advances have shown that high levels of fat availability and altered lipid metabolism can promote the metastatic process by: i) providing energy for cancer cells, thereby supporting invasion and colonization; ii) promoting their survival by enhancing anti-oxidative, anti-apoptotic mechanisms and therapy resistance; and iii) inhibiting the immune surveillance from detecting/removing tumor cells. These findings are especially disturbing given the alarmingly high fat intake within our modern diet. However, the reprogramming of fat metabolism exhibited by cancer cells and intratumor immune cells promises to generate new pharmacological opportunities to treat metastasis.

Lifestyle factors related to energy balance, such as excess body weight, poor diet, and physical inactivity, are associated with risk of sporadic endometrial cancer (EC) and colorectal cancer (CRC). There are limited data on energy balance-related lifestyle factors and EC or CRC risk among individuals with Lynch syndrome, who are at extraordinarily higher risk of developing EC or CRC. We conducted a systematic review of evidence related to weight status, weight change, dietary habits, and physical activity on EC and CRC risk among individuals with Lynch syndrome. Findings are reported narratively. We searched Medline, EMBASE, CENTRAL, PubMed, and clinicaltrials.gov up to June 14(th), 2018. In total, 1,060 studies were identified and 16 were included. Three studies were related to EC and 13 to CRC. Overall, evidence suggests that weight status/weight change may not be associated with EC risk and multivitamin and folic-acid supplementation may be associated with decreased EC risk. Early-adulthood overweight/obese weight-status and adulthood weight-gain may be associated with increased CRC risk, whereas multivitamin supplementation, tea and high fruit intake, and physical activity may be associated with decreased CRC risk. Current evidence proposes that recommendations related to weight, some dietary habits, and physical activity recommended for the general public are also relevant to individuals with Lynch syndrome. More research is needed, specifically prospective cohorts and randomized controlled trials, to determine if tailored recommendations are needed among individuals with Lynch syndrome.

Genomic profiling of colorectal cancer aims to identify actionable somatic mutations but can also discover incidental germline findings.The purpose of this study was to report the detection of pathogenic germline variants that confer heritable cancer predisposition.This was a retrospective study.The study was conducted at a tertiary-referral institution.Between 2012 and 2015, 1000 patients with advanced cancer underwent targeted exome sequencing of a 202-gene panel. The subgroup of 151 patients with advanced colorectal cancer who underwent matched tumor-normal (blood) sequencing formed our study cohort.Germline variants in 46 genes associated with hereditary cancer predisposition were classified according to a defined algorithm based on in silico predictions of pathogenicity. Patients with presumed pathogenic variants were examined for type of mutation, as well as clinical, pedigree, and clinical genetic testing data.We measured detection of pathogenic germline variants.A total of 1910 distinct germline variants were observed in 151 patients. After filtering, 15 pathogenic germline variants (9.9%) were found in 15 patients, arising from 9 genes of varying penetrance for colorectal cancer (APC (n = 2; 13%), ATM (n = 1; 6%), BRCA1 (n = 2; 13%), CDH1 (n = 2; 13%), CHEK2 (n = 4; 27%), MSH2 (n = 1; 7%), MSH6 (n = 1; 7%), NF2 (n = 1; 7%), and TP53 (n = 1; 7%)). Patients with pathogenic variants were diagnosed at a younger age than those without (median, 45 vs 52 y; p = 0.03). Of the 15 patients, 7 patients (46.7%) with variants in low/moderate- penetrant genes for colorectal cancer would likely have not been tested based on clinical and pedigree criteria, where 2 harbored clinically actionable variants (CDH1 and NF2, 28.5% of 7).This study was limited by its small sample size and advanced-stage patients.Tumor-normal sequencing can incidentally discover clinically unsuspected germline variants that confer cancer predisposition in 9.9% of patients with advanced colorectal cancer. Precision medicine should integrate clinical cancer genetics to inform and interpret the actionability of germline variants and to provide follow-up care to mutation carriers. See Video Abstract at http://links.lww.com/DCR/A906.

MNT, a transcription factor of the MXD family, is an important modulator of the oncoprotein MYC. Both MNT and MYC are basic-helix-loop-helix proteins that heterodimerize with MAX in a mutually exclusive manner, and bind to E-boxes within regulatory regions of their target genes. While MYC generally activates transcription, MNT represses it. However, the molecular interactions involving MNT as a transcriptional regulator beyond the binding to MAX remain unexplored. Here we demonstrate a novel MAX-independent protein interaction between MNT and REL, the oncogenic member of the NF-κB family. REL participates in important biological processes and it is altered in a variety of tumors. REL is a transcription factor that remains inactive in the cytoplasm in an inhibitory complex with IκB and translocates to the nucleus when the NF-κB pathway is activated. In the present manuscript, we show that MNT knockdown triggers REL translocation into the nucleus and thus the activation of the NF-κB pathway. Meanwhile, MNT overexpression results in the repression of IκBα, a bona fide REL target. Both MNT and REL bind to the IκBα gene on the first exon, suggesting its regulation as an MNT-REL complex. Altogether our data indicate that MNT acts as a repressor of the NF-κB pathway by two mechanisms: (1) retention of REL in the cytoplasm by MNT interaction, and (2) MNT-driven repression of REL-target genes through an MNT-REL complex. These results widen our knowledge about MNT biological roles and reveal a novel connection between the MYC/MXD and NF-κB pathways, two of the most prominent pathways in cancer.

Inherited defects in base-excision repair (BER) predispose to adenomatous polyposis and colorectal cancer (CRC), yet our understanding of this important DNA repair pathway remains incomplete. By combining detailed clinical, histological and molecular profiling, we reveal biallelic germline loss-of-function (LOF) variants in the BER gene MBD4 to predispose to adenomatous polyposis and –uniquely amongst CRC predisposition syndromes– to myeloid neoplasms. Neoplasms from MBD4-deficient patients almost exclusively accumulate somatic CpG>TpG mutations, resembling mutational signature SBS1. MBD4-deficient adenomas harbour mutations in known CRC driver genes, although AMER1 mutations were more common and KRAS mutations less frequent. We did not find an increased risk for colorectal tumours in individuals with a monoallelic MBD4 LOF variant. We suggest that this condition should be termed MBD4-associated neoplasia syndrome (MANS) and that MBD4 is included in testing for the genetic diagnosis of polyposis and/or early-onset AML.

Current therapy against colorectal cancer is based on DNA-damaging agents that eradicate highly proliferative malignant cells. Whether sublethal chemotherapy affects tumor cell behavior and impacts on patient outcome is primarily unstudied. We now show that sublethal chemotherapy imposes a quiescent-like state to p53 wildtype human colorectal cancer (CRC) cells that is linked to the acquisition of a fetal phenotype downstream of YAP1, similar to that observed after intestinal damage. CRC cells displaying this fetal phenotype exhibit tumor- initiating activity comparable to untreated cells but superior metastatic capacity. Notably, nuclear YAP1 accumulation, or detection of the fetal signature in tumors predict poor prognosis in CRC patients carrying p53 wildtype tumors. Collectively, our results uncover a potential adverse response of tumor cells to suboptimal chemotherapy, and identify nuclear YAP1 and fetal conversion of colorectal tumors as biomarkers for prognosis and therapy prescription.Statement of significanceChemotherapy induces a quiescent-like phenotype to colorectal cancer cells that is linked to the acquisition of a YAP1-dependent fetal signature. Notably, this signature is predictive of patient outcome in different cohorts of human colorectal cancer.

Introduction: Clonal evolution drives cancer development due to the emergence and/or selection of proliferatively advantageous subclones. Its understanding may facilitate the design of anticipation-based management strategies. Richter transformation (RT) is a paradigmatic tumor evolution in which chronic lymphocytic leukemia (CLL), an indolent neoplasia of mature B-cells, transforms into a high-grade lymphoma, usually diffuse large B-cell lymphoma (DLBCL), conferring a dismal prognosis. The evolutionary trajectories of RT and its driving (epi)genomic mechanisms remain largely unknown. Aims: To reconstruct the evolutionary history of RT and to reveal the molecular processes underlying this transformation. Methods: We characterized the whole genome (WGS), epigenome (DNA methylation, H3K27ac, ATAC-seq), and transcriptome (RNA-seq), combined with single-cell DNA and RNA sequencing analyses, of 19 CLL patients developing RT before (n=3) or after treatment with chemoimmunotherapy (n=6) and targeted therapies (BCR or BCL2 inhibitors, n=10). We analyzed 54 longitudinal samples covering up to 19 years of disease course. Results: Our WGS analyses uncovered that RT is characterized by a remarkable structural complexity. We also identified a novel treatment-independent RT-specific mutational process, which we named SBS-RT. The genetic driver landscape of RT is a compendium of alterations in genes involved in cell cycle, MYC, and NF-κB pathways, frequently targeted in single catastrophic events including chromothripsis and chromoplexy. The WGS-based phylogenic reconstruction and single-cell DNA/RNA-seq analyses identified a very early diversification of CLL leading to emergence of RT-cells carrying specific genetic drivers and transcriptomic profiles of RT already at CLL diagnosis. These small subclones were dormant for 6-19 years until rapid expansion associated with the clinical transformation. While the DNA methylome kept track of the cell of origin and proliferative history of RT cells, their chromatin configuration and transcriptional program converged into the overexpression of cell cycle regulators, Toll-like receptors, MYC, MTORC1, and OXPHOS related transcripts, as well as downregulation of BCR pathway. This phenotypic shift was related to de novo activation of key transcription factors. In vitro experiments confirmed that RT cells have a 4-fold higher oxygen consumption at routine respiration and electron transfer system capacity compared to CLL. The resistance of RT to BCR inhibition is consistent with its high OXPHOS and low BCR signaling, which mimics de novo DLBCL-OXPHOS insensitive to BCR inhibition. This OXPHOShigh-BCRlow transcriptional axis of RT can be exploited therapeutically. Conclusions: These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover therapeutic targets for the, once expanded, lethal Richter transformation. Citation Format: Ferran Nadeu, Romina Royo, Ramon Massoni-Badosa, Beatriz Garcia-Torre, Martí Duran-Ferrer, Kevin J. Dawson, Marta Kulis, Ander Diaz-Navarro, Neus Villamor, Juan L. Melero, Vicente Chapaprieta, Ana Dueso-Barroso, Julio Delgado, Riccardo Moia, Sara Ruiz-Gil, Domenica Marchese, Núria Verdaguer-Dot, Mónica Romo, Maria Rozman, Gerard Frigola, Alfredo Rivas-Delgado, Tycho Baumann, Miguel Alcoceba, Marcos González, Fina Climent, Pau Abrisqueta, Josep Castellví, Francesc Bosch, Marta Aymerich, Anna Enjuanes, Sílvia Ruiz-Gaspà, Armando López-Guillermo, Pedro Jares, Sílvia Beà, Dolors Colomer, Núria López-Bigas, Josep LlGelpí, David Torrents, Peter J. Campbell, Ivo Gut, Pablo M. Garcia-Roves, Davide Rossi, Gianluca Gaidano, Xose S. Puente, Holger Heyn, Francesco Maura, José I. Martín-Subero, Elías Campo. Early seeding of Richter transformation in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3795.

Oncogene deregulation triggers replication stress, which can be detected in precancerous lesions and is a source of subsequent genomic instability in cancer. Studies on the mechanisms underlying DNA replication stress caused by frequently deregulated oncogenes, like MYC or RAS, can identify novel mechanisms of oncogenesis and point to actionable pathways with potential for anticancer therapy. To understand the molecular mechanisms underlying this cellular response, we used high-content microscopy to monitor the dynamic behavior of >60 GFP-tagged proteins linked to replication stress or DNA damage in primary human cells. Iterations of this screen identified a proteomic signature specific to MYC- and RAS-driven replication stress that was significantly different from responses activated by other sources of replication stress (i.e. DNA damaging drugs). These findings indicated that the replication stress response to oncogenes is somewhat unique. MYC-dependent replication stress responses typically activated the INO80 chromatin-remodeling complex — a highly conserved complex necessary for maintenance of genome stability in yeast. Core components of the INO80 complex assembled onto chromatin at MYC-bound sites and were quantitatively enriched at active replication forks. These molecular events were independent of transcription, and their disruption by pharmacological or genetic means specifically altered replication dynamics and cellular growth in cells overexpressing MYC. Notably, we identified recurrent somatic mutations in several genes encoding for INO80 complex components across multiple cancer types, with an abundance of nonsense and splice-site mutations consistent with loss of function. Most of these mutant variants behaved as hypomorphs when tested in cell-based assays: they were insensitive to MYC deregulation and failed to rescue cell proliferation in knock-out cells with MYC overexpression. Computational predictions also suggested that many of these mutations had been selected as potential cancer drivers. Consistent with this notion, we found that in vivo, INO80 complex mutations shortened tumor latency in a mouse model of MYC-dependent lymphomagenesis. Collectively, these findings indicate that the replication stress response to oncogene deregulation is unique in its molecular architecture, and modification of this response by partial loss-of-function mutations in cancer facilitates tumor progression. Citation Format: Rosa Vinas-Castells, Alja Kozulic-Pirher, Tomás Aparicio, Sandra Casas Recasens, Mark P. Roberto, Rachel Sue, Francisco Martinez, Nuria López-Bigas, Jean Gautier, David Dominguez-Sola. Molecular architecture of replication stress response to oncogene deregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1517.

Dysregulation of the c-Myc oncogene occurs in a wide variety of hematologic malignancies, and its overexpression has been linked with aggressive tumor progression. Here, we show that poly (ADP-ribose) polymerase 1 (PARP-1) and PARP-2 exert opposing influences on progression of c-Myc-driven B-cell lymphoma. PARP-1 and PARP-2 catalyze the synthesis and transfer of ADP-ribose units onto amino acid residues of acceptor proteins in response to DNA strand breaks, playing a central role in the response to DNA damage. Accordingly, PARP inhibitors have emerged as promising new cancer therapeutics. However, the inhibitors currently available for clinical use are not able to discriminate between individual PARP proteins. We found that genetic deletion of PARP-2 prevents c-Myc-driven B-cell lymphoma, whereas PARP-1 deficiency accelerates lymphomagenesis in the E¿-Myc mouse model of aggressive B-cell lymphoma. Loss of PARP-2 aggravates replication stress in preleukemic E¿-Myc B cells, resulting in accumulation of DNA damage and concomitant cell death that restricts the c-Myc-driven expansion of B cells, thereby providing protection against B-cell lymphoma. In contrast, PARP-1 deficiency induces a proinflammatory response and an increase in regulatory T cells, likely contributing to immune escape of B-cell lymphoma, resulting in an acceleration of lymphomagenesis. These findings pinpoint specific functions for PARP-1 and PARP-2 in c-Myc-driven lymphomagenesis with antagonistic consequences that may help inform the design of new PARP-centered therapeutic strategies, with selective PARP-2 inhibition potentially representing a new therapeutic approach for the treatment of c-Myc-driven tumors., The J.Y. laboratory is funded by the Spanish Ministerio de Economía, Industria y Competitividad (grant SAF2017-83565-R), Spanish Ministerio de Ciencia e Innovación (grant PID2020-112526RB-I00), and Fundación Científica de la Asociación Española Contra el Cáncer (grant PROYEI6018YÉLA). Work in the J.E.S. laboratory is supported by a core grant to the Laboratory of Molecular Biology from the Medical Research Council (U105178808). The F.D. laboratory is supported by a Laboratory of Excellence grant (ANR-10-LABX-0034_Medalis) to Strasbourg University, Centre National de la Recherche Scientifique. The P.N. laboratory is supported by grants from the Spanish Ministry of Economy and Competitiveness/Instituto de Salud Carlos III–Fondo Europeo de Desarrollo Regional (FEDER; PI17/00199 and PI20/00625) and the Generalitat de Catalunya (2017-SGR-225). The P.M. laboratory acknowledges support from Centres de Recerca de Catalunya/Generalitat de Catalunya and Fundació Josep Carreras-Obra Social la Caixa for core support, the Spanish Ministry of Economy and Competitiveness (grant SAF-2019-108160-R), the Fundación Uno entre Cienmil, the Obra Social La Caixa (grant LCF/PR/HR19/52160011), and the German Josep Carreras Leukamie Stiftung. Work at the G.R. and P.M. laboratories are cofinanced by the European Regional Development Fund through the Interreg V-A Spain-France-Andorra Program (project PROTEOblood; grant EFA360/19). The O.F.-C. laboratory is funded by grants from the Spanish Ministry of Science, Innovation and Universities (RTI2018-102204-B-I00; cofinanced with European FEDER funds) and the European Research Council (ERC-617840). T.V.-H. was supported by a Marie Sklodowska Curie fellowship (GA792923). The A.B. laboratory is supported by the Spanish Ministry of Economy and Competitiveness (grant PID2019-104695RB-I00).

Anti-EGFR antibodies such as cetuximab are active against KRAS/NRAS wild-type colorectal cancers (CRCs), but acquired resistance invariably evolves. It is unknown which mutational mechanisms enable resistance evolution and whether adaptive mutagenesis (a transient cetuximab-induced increase in mutation generation) contributes in patients. Here, we investigate these questions in exome sequencing data from 42 baseline and progression biopsies from cetuximab-treated CRCs. Mutation loads did not increase from baseline to progression, and evidence for a contribution of adaptive mutagenesis was limited. However, the chemotherapy-induced mutational signature SBS17b was the main contributor of specific KRAS/NRAS and EGFR driver mutations that are enriched at acquired resistance. Detectable SBS17b activity before treatment predicted shorter progression-free survival and the evolution of these specific mutations during subsequent cetuximab treatment. This result suggests that chemotherapy mutagenesis can accelerate resistance evolution. Mutational signatures may be a new class of cancer evolution predictor.

© The Author(s) 2021., MNT, a transcription factor of the MXD family, is an important modulator of the oncoprotein MYC. Both MNT and MYC are basic-helix–loop–helix proteins that heterodimerize with MAX in a mutually exclusive manner, and bind to E-boxes within regulatory regions of their target genes. While MYC generally activates transcription, MNT represses it. However, the molecular interactions involving MNT as a transcriptional regulator beyond the binding to MAX remain unexplored. Here we demonstrate a novel MAX-independent protein interaction between MNT and REL, the oncogenic member of the NF-κB family. REL participates in important biological processes and it is altered in a variety of tumors. REL is a transcription factor that remains inactive in the cytoplasm in an inhibitory complex with IκB and translocates to the nucleus when the NF-κB pathway is activated. In the present manuscript, we show that MNT knockdown triggers REL translocation into the nucleus and thus the activation of the NF-κB pathway. Meanwhile, MNT overexpression results in the repression of IκBα, a bona fide REL target. Both MNT and REL bind to the IκBα gene on the first exon, suggesting its regulation as an MNT–REL complex. Altogether our data indicate that MNT acts as a repressor of the NF-κB pathway by two mechanisms: (1) retention of REL in the cytoplasm by MNT interaction, and (2) MNT-driven repression of REL-target genes through an MNT–REL complex. These results widen our knowledge about MNT biological roles and reveal a novel connection between the MYC/MXD and NF-κB pathways, two of the most prominent pathways in cancer., The work was supported by grant SAF2017-88026-R from Agencia Estatal de Investigación, Spanish Government, to J.L. and M.D.D. J.L.-P. and M.C.L.-N. were recipients of F.P.U. fellowships and L.G.-G. of a F.P.I. fellowship from the Spanish Government.

Background: Previous studies in mice indicated that Paneth cells and c-Kit-positive goblet cells represent the stem cell niche of the small intestine and colon, respectively, partly by supporting Wnt and Notch activation. Whether these cell populations play a similar role in human intestinal cancer remains unexplored. Methods: We performed histopathological evaluation and immunohistochemical analysis of early colorectal adenomas and carcinoma adenoma from patients at the Hospital del Mar in Barcelona. We then determined the possible correlation between the different parameters analyzed and with patient outcomes. Results: Paneth cells accumulate in a subset of human colorectal adenomas directly associated with Notch and Wnt/β-catenin activation. Adenoma areas containing Paneth cells display increased vessel density in the lamina propria and higher levels of the stem cell marker EphB2. In an in-house cohort of 200 colorectal adenoma samples, we also observed a significant correlation between the presence of Paneth cells and Wnt activation. Kaplan–Meier analysis indicated that early adenoma patients carrying Paneth cell-positive tumors display reduced disease-free survival compared with patients with Paneth cell-free lesions. Conclusions: Our results indicate that Paneth cells contribute to the initial steps of cancer progression by providing the stem cell niche to adenoma cells, which could be therapeutically exploited.

Increasing evidence supports a potential role for STAT3 as a tumor driver in cutaneous T-cell lymphomas (CTCL). The mechanisms leading to STAT3 activation are not fully understood; however, we recently found that miR-124, a known STAT3 regulator, is robustly silenced in MF tumor-stage and CTCL cells. Objective: We studied here whether deregulation of miR-124 contributes to STAT3 pathway activation in CTCL. Methods: We measured the effect of ectopic mir-124 expression in active phosphorylated STAT3 (p-STAT3) levels and evaluated the transcriptional impact of miR-124-dependent STAT3 pathway regulation by expression microarray analysis. Results: We found that ectopic expression of miR-124 results in massive downregulation of activated STAT3 in different CTCL lines, which resulted in a significant alteration of genetic signatures related with gene transcription and proliferation such as MYC and E2F. Conclusions: Our study highlights the importance of the miR-124/STAT3 axis in CTCL and demonstrates that the STAT3 pathway is regulated through epigenetic mechanisms in these cells. Since deregulated STAT3 signaling has a major impact on CTCL initiation and progression, a better understanding of the molecular basis of the miR-124/STAT3 axis may provide useful information for future personalized therapies.

The prevalence of Lynch syndrome (LS) varies significantly in different populations, suggesting that ethnic features might play an important role. We enrolled 3330 consecutive Chinese patients who had surgical resection for newly diagnosed colorectal cancer. Universal screening for LS was implemented, including immunohistochemistry for mismatch repair (MMR) proteins, BRAFV600E mutation test and germline sequencing. Among the 3250 eligible patients, MMR protein deficiency (dMMR) was detected in 330 (10.2%) patients. Ninety-three patients (2.9%) were diagnosed with LS. Nine (9.7%) patients with LS fulfilled Amsterdam criteria II and 76 (81.7%) met the revised Bethesda guidelines. Only 15 (9.7%) patients with absence of MLH1 on IHC had BRAFV600E mutation. One third (33/99) of the MMR gene mutations have not been reported previously. The age of onset indicates risk of LS in patients with dMMR tumors. For patients older than 65 years, only 2 patients (5.7%) fulfilling revised Bethesda guidelines were diagnosed with LS. Selective sequencing of all cases with dMMR diagnosed at or below age 65 years and only of those dMMR cases older than 65 years who fulfill revised Bethesda guidelines results in 8.2% fewer cases requiring germline testing without missing any LS diagnoses. While the prevalence of LS in Chinese patients is similar to that of Western populations, the spectrum of constitutional mutations and frequency of BRAFV600E mutation is different. Patients older than 65 years who do not meet the revised Bethesda guidelines have a low risk of LS, suggesting germline sequencing might not be necessary in this population.

Rearrangements involving the mixed lineage leukemia gene (MLL) are found in the majority of leukemias that develop within the first year of age, known as infant leukemias, and likely originate during prenatal life. MLL rearrangements are also present in about 10% of other pediatric and adult acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). These translocations and others occurring in early life are associated with a dismal prognosis compared with adult leukemias carrying the same translocations. This observation suggests that infant and adult leukemias are biologically distinct but the underlying molecular mechanisms for these differences are not understood. In this work, we induced the same MLL chromosomal translocation in the embryo at the time of fetal liver hematopoiesis and in the adult hematopoietic tissues to develop disease models in mice that recapitulate human infant and adult leukemias, respectively. We successfully obtained myeloid leukemia in adult mice after MLL-ENL recombination induction using the interferon inducible Mx1-Cre line. Using this same Cre line, we generated embryonic MLL-ENL leukemias, which were more aggressive than the corresponding adult leukemias. In conclusion, we have developed a novel MLL-ENL embryonic leukemia model in mice that can be used to study some aspects of infant leukemia ontogeny.

Lynch syndrome (LS) is a genetic condition secondary to germline alterations in the DNA mismatch repair (MMR) genes with 30% of changes being variants of uncertain significance (VUS). Our aim was to perform an in silico reclassification of VUS from a large single institutional cohort that will help prioritizing functional validation. A total of 54 VUS were detected with 33 (61%) novel variants. We integrated family history, pathology, and genetic information along with supporting evidence from eight different in silico tools at the RNA and protein level. Our assessment allowed us to reclassify 54% (29/54) of the VUS as probably damaging, 13% (7/54) as possibly damaging, and 28% (15/54) as probably neutral. There are more than 1,000 VUS reported in MMR genes and our approach facilitates the prioritization of further functional efforts to assess the pathogenicity to those classified as probably damaging. Cancer Prev Res; 10(10); 580–7. ©2017 AACR.

Purpose: Determination of microsatellite instability (MSI) by PCR is the gold standard; however, IHC of mismatch repair (MMR) proteins is frequently performed instead. The reliability of these methods on postneoadjuvant therapy specimens is unknown. We examined the effect of neoadjuvant therapy on MSI results by PCR and IHC. Experimental design: A total of 239 colorectal cancers resected after neoadjuvant therapy were assessed for MSI with PCR and IHC. PCR and IHC results for matched paired pre- and posttreatment specimens were compared. In parallel, 2 isogenic cell lines conditioned for MMR functioning and 2 different patient-derived xenografts (PDXs) were exposed to chemotherapy, radiation, or both. We also examined whether establishment of PDXs induced MSI changes in 5 tumors. IHC and MSI were tested after treatment to assess for changes. Results: We identified paired pre- and posttreatment specimens for 37 patients: 2 with PCR only, 34 with IHC only, and 1 with both. All 3 patients with PCR had microsatellite stable pre- and posttreatment specimens. Of the 35 patients with IHC, 30 had intact MMR proteins in pre- and posttreatment specimens, 1 had equivocal MLH1 staining in the pretreatment and loss in the posttreatment specimen, and 4 had intact pretreatment MSH6 but variable posttreatment staining. In the experimental setting, no changes in MSI status were detected after treatment or tumor implantation in animals. Conclusions: Our findings show that the expression of MMR proteins, commonly MSH6, can change after neoadjuvant therapy and confirm PCR as the gold-standard test for MSI after neoadjuvant therapy. Clin Cancer Res; 23(17); 5246–54. ©2017 AACR.

Melanoma of the skin is a common cancer only in Europeans, whereas it arises in internal body surfaces (mucosal sites) and on the hands and feet (acral sites) in people throughout the world. Here we report analysis of whole-genome sequences from cutaneous, acral and mucosal subtypes of melanoma. The heavily mutated landscape of coding and non-coding mutations in cutaneous melanoma resolved novel signatures of mutagenesis attributable to ultraviolet radiation. However, acral and mucosal melanomas were dominated by structural changes and mutation signatures of unknown aetiology, not previously identified in melanoma. The number of genes affected by recurrent mutations disrupting non-coding sequences was similar to that affected by recurrent mutations to coding sequences. Significantly mutated genes included BRAF, CDKN2A, NRAS and TP53 in cutaneous melanoma, BRAF, NRAS and NF1 in acral melanoma and SF3B1 in mucosal melanoma. Mutations affecting the TERT promoter were the most frequent of all; however, neither they nor ATRX mutations, which correlate with alternative telomere lengthening, were associated with greater telomere length. Most melanomas had potentially actionable mutations, most in components of the mitogen-activated protein kinase and phosphoinositol kinase pathways. The whole-genome mutation landscape of melanoma reveals diverse carcinogenic processes across its subtypes, some unrelated to sun exposure, and extends potential involvement of the non-coding genome in its pathogenesis.

Squamous cell carcinoma (SCC) of the rectum is a rare disease with

In a classical view of carcinogenesis, carcinogens directly cause mutations. In this issue, Riva et al. explore the effects of 20 known or suspected human carcinogens and find discernible mutational signatures for only three of them.

Cancer therapy has improved considerably in the last years; however, therapeutic resistance is still a major problem that impedes full response to the treatment and the main cause of patient relapse and death. Numerous kinases have been reported to be overactivated in cancer and induce resistance to current therapies. Targeting kinases has proven to be useful for overcoming chemotherapy resistance and thus improving patient outcomes. Inhibitor of kappaB kinase alpha (IKKα) is a serine/threonine kinase that was first described as part of the IKK complex in the nuclear factor-κB (NF-κB) pathway, which regulates several physiological and physiopathological processes such as immunity, inflammation, and cancer. However, the IKKα subunit has been shown to be dispensable for NF-κB activation and responsible of multiple pro-tumorigenic functions. Furthermore, we identified a nuclear active form of IKKα kinase IKKα(p45) that promotes tumor growth and therapy resistance, independent of canonical NF-κB. Improved understanding of resistance mechanisms will facilitate drug discovery and provide new effective therapies. Here, we review the recent publications on the implications of IKKα in cancer initiation, development, and resistance.

β-Catenin/CTNNB1 is critical for leukemia initiation or the stem cell capacity of several hematological malignancies. This review focuses on a general evaluation of β-catenin function in normal T-cell development and T-cell acute lymphoblastic leukemia (T-ALL). The integration of the existing literature offers a state-of-the-art dissection of the complexity of β-catenin function in leukemia initiation and maintenance in both Notch-dependent and independent contexts. In addition, β-catenin mutations are screened for in T-ALL primary samples, and it is found that they are rare and with little clinical relevance. Transcriptional analysis of Wnt family members (Ctnnb1, Axin2, Tcf7, and Lef1) and Myc in different publicly available T-ALL cohorts indicates that the expression of these genes may correlate with T-ALL subtypes and/or therapy outcomes.

Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A*01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote "off-the-shelf" precision immunotherapies, alleviating limitations of personalized treatments.

e15601 Background: We evaluated demographic, treatment, and survival outcomes of adults age 18 to 49 years treated at our institution with long course chemoradiotherapy (CRT) followed by total mesorectal excision (TME) for locally advanced rectal cancer. Additionally, we compared outcomes between those age < 45 vs. > 45 years. Methods: The records of 219 patients diagnosed with non-metastatic, clinical T3, T4, or node positive rectal adenocarcinoma and treated between April 2000 and November 2017 were reviewed for age, sex, and presenting symptoms; clinical stage and microsatellite stable (MSS)/DNA mismatch repair (MMR) proficiency status; treatments delivered and sequence; pathologic response to pre-operative therapies; and the development of locoregional recurrence (LRR), distant metastasis (DM), and secondary pelvic malignancy. The Kaplan-Meier method and Log-Rank test were used to calculate and compare disease-free survival (DFS) and overall survival (OS) rates from the date of TME. Results: The median age at diagnosis was 44 years (range 19-49) and there was no sex predominance. Rectal bleeding was the most common presenting symptom (91%), with a median time to diagnosis of 5 months. Clinical tumor/nodal categories were T1-2 in 4%, T3 in 87%, T4 in 7%, N0 in 17%, and N1–2 in 80% of patients. MSS/MMR proficient disease was identified in 95% of tumors with status reported (n = 170). CRT followed by TME and post-operative chemotherapy was the most frequent treatment sequence (n = 196), with capecitabine (n = 176) and FOLFOX (n = 115) as the predominant concurrent and post-operative chemotherapies, respectively. Pathologic complete response at both primary and nodal sites occurred in 15% of all cases and 16% of MSS/MMR proficient cases. There was no difference in sex, tumor category, nodal category, MSS/MMR proficiency status, or pathologic complete response, by age ( < 45 years [n = 111] vs. > 45 years [n = 108]). At a median DFS follow-up time of 5.0 years, there were 11 LRR, 40 DM (including 11 DM detected prior to/at time of TME), and 1 synchronous presentation of LRR and DM. The 5-year rate of DFS was 70.4% for age < 45 years and 85.3% for age > 45 years ( P = 0.02). At an OS median follow-up time of 7.5 years, there were 38 deaths. The 5-year rate of OS was 87.7% for age < 45 years and 94.4% for age > 45 years ( P = 0.126). Two patients developed non-rectal pelvic malignancies. Conclusions: The outcomes reported here from one of the largest single-institution series for young-onset, locally advanced rectal cancer could serve as a benchmark to evaluate newer treatment approaches. Rectal bleeding was the leading presenting symptom, with approximately half-year delay from development of symptoms to diagnosis. Most tumors were MSS/MMR proficient. At 5 years’ follow-up time, the DFS rate was lower for patients age < 45 years when compared to those > 45 years. Secondary pelvic malignancies were a rare occurrence.

3549 Background: Strategies to detect and prevent young-onset colorectal cancer (YOCRC, diagnosed under age 50) are critical. Established high-risk screening guidelines (SGs) aim to detect/prevent YOCRCs arising from hereditary syndromes. For non-hereditary YOCRCs, average-risk screening is being considered at an earlier age, but family history (FH)-based increased-risk screening has been poorly studied. We aimed to define the proportion of non-hereditary YOCRC with a FH, and to determine whether existing SGs could have detected/prevented these cases. Methods: 394 consecutive YOCRC patients presenting for surgical resection were reviewed for tumor MMR status, pedigree and genetic testing. Those with known/suspected hereditary syndrome (by phenotype, MMR status, and/or germline mutation) were excluded (N = 65). Pedigrees (N = 329) were analyzed for first- or second-degree relatives (FDR, SDR) with CRC and the ages of diagnosis. The gap between the recommended age for FH-based CRC screening and the age of YOCRC diagnosis was calculated. Results: 89 (27%) non-hereditary YOCRC patients had a FH of CRC. The median age of diagnosis was 45; the tumors were mostly from the distal colon (22%) and rectum (60%), and stage III (48%) and IV (27%). Twenty-one (24%) patients had 22 FDRs with CRCs diagnosed at age 64 (median); and 71 (80%) patients had 92 SDRs with CRCs diagnosed at age 65 (median). Thirteen (15%) had a FH of YOCRC. The existing SGs consider 39 patients (44%) at increased-risk, and the remaining, average-risk (Table). Screening would have begun prior to the YOCRC diagnoses in 28 (31% [or 46, 52%]) patients. But YOCRC diagnosis preceded the recommended screening age in the remaining 61(69% [or 43, 48%]) patients by a median of 5.3 [or 3.9] years (Table). Conclusions: FH is found in 27% of the non-hereditary YOCRC patients; 15% has a FH of YOCRC. In nearly half of the patients, YOCRC was diagnosed several years earlier than the recommended age for FH-based screening, even assuming perfect SG adoption and starting average risk screening at age 45. Refining existing FH-based SGs can potentially be impactful.[Table: see text]

Traditional germline sequencing and deletion/duplication analysis does not detect Lynch syndrome-causing mutations in all individuals whose colorectal or endometrial tumors demonstrate mismatch repair (MMR) deficiency. Unique inversions and other rearrangements of the MMR genes have been reported in families with Lynch syndrome. In 2014, a recurrent inversion of MSH2 exons 1-7 was identified in five families suspected to have Lynch syndrome. We aimed to describe our clinical experience in identifying families with this specific inversion. Four probands whose Lynch syndrome-associated tumors demonstrated absence of MSH2/MSH6 staining and who had negative MMR germline testing were evaluated for the MSH2 inversion of exons 1-7, offered during initial genetic workup or upon routine clinical follow-up. All four probands tested positive for the MSH2 inversion. Proband cancer diagnoses included colon and endometrial adenocarcinoma and sebaceous adenoma. A variety of Lynch syndrome-associated cancers were reported in the family histories, although only one family met Amsterdam II criteria. Thirteen at-risk relatives underwent predictive testing. MSH2 inversion of exons 1-7 was found in four probands previously suspected to have Lynch syndrome based on family history and tumor testing. This testing should be offered routinely to patients with tumors demonstrating loss of MSH2/MSH6 staining.

Background Although the safety of combination chemotherapy without primary tumor resection (PTR) in patients with stage IV colon cancer has been established, questions remain regarding a potential survival benefit with PTR. The objective of this study was to compare mortality rates in patients who had colon cancer with unresectable metastases who did and did not undergo PTR. Methods An observational cohort study was conducted among patients with unresectable metastatic colon cancer identified from the National Cancer Data Base (2003-2005). Multivariate Cox regression analyses with and without propensity score weighting (PSW) were performed to compare survival outcomes. Instrumental variable analysis, using the annual hospital-level PTR rate as the instrument, was used to account for treatment selection bias. To account for survivor treatment bias, in situations in which patients might die soon after diagnosis from different reasons, a landmark method was used. Results In the total cohort, 8641 of 15,154 patients (57%) underwent PTR, and 73.8% of those procedures (4972 of 6735) were at landmark. PTR was associated with a significant reduction in mortality using Cox regression (hazard ratio [HR], 0.45; 95% confidence interval [CI], 0.44-0.47) or PSW (HR, 0.46; 95% CI, 0. 44-0.49). However, instrumental variable analysis revealed a much smaller effect (relative mortality rate, 0.91; 95% CI, 0.87-0.96). Although a smaller benefit was observed with the landmark method using Cox regression (HR, 0.6; 95% CI, 0.55-0.64) and PSW (HR, 0.59; 95% CI, 0.54-0.64), instrumental variable analysis revealed no survival benefit (relative mortality rate, 0.97; 95% CI, 0.87-1.06). Conclusions Among patients with unresectable metastatic colon cancer, after adjustment for confounder effects, PTR was not associated with improved survival compared with systemic chemotherapy; therefore, routine noncurative PTR is not recommended. Cancer 2017;123:1124-1133. © 2016 American Cancer Society.

Notch activation is instrumental in the development of most T-cell acute lymphoblastic leukemia (T-ALL) cases, yet Notch mutations alone are not sufficient to recapitulate the full human disease in animal models. We here found that Notch1 activation at the fetal liver (FL) stage expanded the hematopoietic progenitor population and conferred it transplantable leukemic-initiating capacity. However, leukemogenesis and leukemic-initiating cell capacity induced by Notch1 was critically dependent on the levels of β-Catenin in both FL and adult bone marrow contexts. In addition, inhibition of β-Catenin compromised survival and proliferation of human T-ALL cell lines carrying activated Notch1. By transcriptome analyses, we identified the MYC pathway as a crucial element downstream of β-Catenin in these T-ALL cells and demonstrate that the MYC 3' enhancer required β-Catenin and Notch1 recruitment to induce transcription. Finally, PKF115-584 treatment prevented and partially reverted leukemogenesis induced by active Notch1. This research was funded by the Worldwide Cancer Research (formerly AICR, 13-0064), Fundación AECC (Cancer infantil), Ministerio de Economía y Competitividad (SAF2013-40922-R), Red Temática de Investigación Cooperativa en Cáncer (RD12/0036/0054), Agència de Gestió d’Ajuds Universitaris i de Recerca (AGAUR) (2014SGR-124) to AB

Constitutional mismatch repair deficiency syndrome (CMMRD) is a rare autosomal recessive predisposition to colorectal polyposis and other malignancies, often childhood-onset, that is caused by biallelic inheritance of mutations in the same mismatch repair gene. Here, we describe a patient with a clinical diagnosis of CMMRD based on colorectal polyposis and young-onset endometrial cancer who was identified to have two alterations in trans in PMS2: one known pathogenic mutation (c.1831insA; p.Ile611Asnfs*2) and one novel variant of uncertain significance (c.505C>G; p.Arg169Glu), a missense alteration. We describe the clinical and molecular features in the patient harboring this novel alteration c.505C>G, who meets clinical criteria for CMMRD and exhibits molecular evidence supporting a diagnosis of CMMRD. Although experimental validation is needed to confirm its pathogenicity, PMS2 c.505C>G likely has functional consequences that contributes to our patient's phenotype based on the patient's clinical presentation, tumor studies, and bioinformatics analysis.

WOS: 000379156300012, PubMed ID: 26819058, The European Hematology Association (EHA) Roadmap for European Hematology Research highlights major achievements in diagnosis and treatment of blood disorders and identifies the greatest unmet clinical and scientific needs in those areas to enable better funded, more focused European hematology research. Initiated by the EHA, around 300 experts contributed to the consensus document, which will help European policy makers, research funders, research organizations, researchers, and patient groups make better informed decisions on hematology research. It also aims to raise public awareness of the burden of blood disorders on European society, which purely in economic terms is estimated at (sic)23 billion per year, a level of cost that is not matched in current European hematology research funding. In recent decades, hematology research has improved our fundamental understanding of the biology of blood disorders, and has improved diagnostics and treatments, sometimes in revolutionary ways. This progress highlights the potential of focused basic research programs such as this EHA Roadmap. The EHA Roadmap identifies nine 'sections' in hematology: normal hematopoiesis, malignant lymphoid and myeloid diseases, anemias and related diseases, platelet disorders, blood coagulation and hemostatic disorders, transfusion medicine, infections in hematology, and hematopoietic stem cell transplantation. These sections span 60 smaller groups of diseases or disorders. The EHA Roadmap identifies priorities and needs across the field of hematology, including those to develop targeted therapies based on genomic profiling and chemical biology, to eradicate minimal residual malignant disease, and to develop cellular immunotherapies, combination treatments, gene therapies, hematopoietic stem cell treatments, and treatments that are better tolerated by elderly patients., Biotechnology and Biological Sciences Research CouncilBiotechnology and Biological Sciences Research Council (BBSRC) [BB/L023776/1, BB/I00050X/1, BB/K021168/1]; Cancer Research UKCancer Research UK [11831]; Medical Research CouncilMedical Research Council UK (MRC) [G1000801a]; Novo Nordisk FondenNovo Nordisk [NNF12OC1015986]; British Heart FoundationBritish Heart Foundation [FS/09/039/27788]; Cancer Research UKCancer Research UK [12765]; Medical Research CouncilMedical Research Council UK (MRC) [MR/L022982/1, MC_UU_12009/8, MC_U137981013, MC_PC_12009]

Alternative splicing is one of the multiple post-transcriptional mechanisms that contribute to functional diversification in eukaryotic cells. Cancer-associated alternative splicing events have been reported in different tumor types, establishing new putative targets for diagnostic tools and therapies. Here, we explore the transcriptional program at exon resolution of T-Cell Acute Lymphoblastic Leukemia (T-ALL), an aggressive hematological malignancy characterized by the outgrowth of immature T-cells. To date, we have characterized the alternative splicing configuration of more than 120 RNA-Seq samples from four different T-ALL cohorts, normal T-cell maturation stages, and two cell lines, using vastdb. Exon skipping and intron retention are the most frequent events across all samples, with similar incidence in both cancer driver and non-driver genes. Interestingly, we observe that intron retention is significantly higher in immature and mature T-cells compared to leukemic cells. Conversely, exon skipping is higher in leukemic samples. These results suggest an impairment in the splicing machinery that might be driven by RNA binding proteins (RBPs) deregulation in leukemia. To further investigate that, we aim to discover which are the RBPs associated with isoform changes by constructing co-expression networks. The computational assessment of T-ALL specific isoforms will eventually help to improve the diagnosis, as well as the discovery of putative neoantigens targeted by CAR therapy.

Hematopoietic stem cells (HSCs) constitute the base of the hematopoietic system, giving rise to every blood cell type. Because of their clinical potential, numerous approaches have been tackled trying to generate HSCs in vitro for regenerative medicine purposes. However, due to the complexity HSCs development has, still there is not a robust way to generate bona-fide HSCs, bringing out the necessity to explore alternatives to identify genetic programmes that could lead in a robust generation of this kind of cells. In this way, gene activation by alternative CRISPR (clustered regularly interspaced short palindromic repeats) tools is a novel approach to activate endogenous genes by the simple use of gRNAs targeting promoter regions. This platform can be used in CRISPRa-based screening using library of gRNAs targeting gene promoters in a genome-wide and unbiased fashion, or alternatively can be used to activate the transcription of specific groups of genes. To test the best CRISPRa system to be used in mESCs, we generated mESCs containing the two most widely used CRISPRa systems (SunTag and SAM) that can induce a gene activation constitutively. In mESCs, SunTag induces a more robust gene expression than SAM in all the genes tested. In parallel, we have generated two inducible CRISPRa (iCRISPRa) mESC lines, VPR and iSAM. We will present the comparison of all the different CRISPRa systems in mESC and in hemogenic differentiated cells to determine the most robust iCRISPRa system in mouse embryonic stem cells differentiation system.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease comprising up to 25% of all acute leukemia cases. Despite intensive chemotherapy, up to 50% of T-ALL patients fail to achieve a complete remission, making relapse and resistance to therapy the greatest challenge in the treatment of this disease. Activating mutations in Notch are the most prevalent genetic alterations in T-ALL. However, genomic studies suggest that other signaling pathways are also pivotal. We previously showed that β-catenin, a key element in hematopoietic stem cell regulation, is required for T-cell leukemia initiation through Myc transcription modulation downstream of Notch1. To further explore the molecular mechanisms that underlie β-catenin dependent leukemia initiation and maintenance, here we take a comprehensive genomic strategy integrating: 1) the chromatin occupancy of β-catenin and its associated transcription factors TCF and LEF, in T-ALL cell lines by ChIP-Seq, including external data in different models, 2) the transcriptomic map of cells upon β-catenin gene silencing by RNA-Seq, and 3) a gene co-expression network using transcriptome data from public T-ALL primary samples. We identified almost 400 DNA-binding sites located in regulatory regions for β-catenin, co-occupied with TCF and LEF in T-ALL cell lines and, intriguingly, highly enriched for ZBTB33 DNA motif. Interestingly, our data showed a prominent role of β-catenin in regulating RNA processing functions. Also, we further characterize β-catenin modulated genes in different leukemic mouse models. Surprisingly, we detected a low coincidence of β-catenin chromatin binding as compared to human cell lines. Altogether these results allow for a better comprehension of β-catenin activity and its essentiality in T-ALL leukemogenesis, and eventually the opportunity to design β-catenin target therapies for T-ALL treatment.

A conserved Notch function is crucial for the specification and generation of Hematopoietic Stem Cells (HSC) across evolutionary distinct organisms. By using genetically modified organisms, most of the elements that are functionally relevant have been identified mainly in mouse and zebrafish. However, due to the disruptive nature of these studies, it is still uncertain the physiological sequence of events that lead to and result from this specific Notch function. This is especially important in terms of reproducing this activity in vitro. The Notch system provides cells with a binary decision mechanism that takes place among neighboring cells. In vertebrate systems, several Notch ligands and receptors co-exist in the same cell and cells compete for activating the Notch receptor that will condition the fate of that cell. In the AGM, HSCs develop from hemogenic endothelial cells that reside in the ventral side of the dorsal aorta. Cells in the developing aortic endothelium co-express different types of Notch ligands and receptors. Several groups including ours have contributed to define the elements involved in this signal, but still several gaps prevent to have a complete understanding. In addition to the Notch1 receptor, Jagged1 and hes1, we have recently shown how Dll4 has also an important structural function in the hematopoietic cluster composition of the aorta. We provided data on how blocking Dll4 with a specific antibody impinges on the number of cells recruited into the cluster and the HSC activity. Thanks to current reagents and methodologies, we now have taken a step backwards to analyze the dynamics of Notch ligands and receptors at the protein and RNA level by single cell index sorting. We are characterizing the different hemogenic and hematopoietic clusters by the expression and activity of the Notch pathway. Latest results will be presented in the meeting.

Background: Current treatment for locally advanced rectal cancer (LARC) includes multimodality therapy in the neoadjuvant and adjuvant settings. Response to neoadjuvant therapy (NT) is prognostic of long-term outcomes. While long-course chemoradiation (CRT) had been the traditional NT regimen, novel regimens adding systemic therapy and/or eliminating radiation have been introduced. We examined if circulating tumor cells provide prognostic information independent of the specific treatment regimen utilized. Specifically, we aimed to define the significance of CTC-defined minimal residual disease (MRD) in LARC. Methods: A prospective longitudinal protocol enrolled LARC patients (N=100; clinical stage II=4; stage III=96) undergoing NT. Peripheral blood was collected at baseline (t1, treatment-naive), after NT (t2, intraoperatively before tumor manipulation), after resection (t3, 2-8 weeks postoperatively), and after completion of adjuvant therapy (t4, 2-12 weeks after completion). CTC was enumerated by the CellSearch® platform within 72 hours of collection. Patients were followed for disease-free survival (DFS). Results: At t1, CTCs were detected in 28/90 (31.1%) patients with available samples with a median CTC count of 1.5 [IQR: 1.0, 2.75]. NT consisted of long-course pelvic CRT (65%), extended NT with systemic chemotherapy and long- or short-course pelvic CRT (22%), and a radiation-sparing regimen with systemic chemotherapy only (13%). At t2, CTCs were detected in 34/75 (45.3%) patients with a median CTC count of 1 [IQR: 1, 1]. Type of NAT did not correlate with CTC positivity (p=0.637). Ten (9.4%) patients declined surgery, while cPR occurred in 12/93 (12.9%) surgical patients. Postoperatively (t3), CTC was detected in 12/47 (25.5%) patients, with a median count of 1.5 (IQR: 1.0-3.0). After completing adjuvant chemotherapy (t4), only 3/29 (10.3%) patients had detectable CTC. With a median follow-up of 47.50 months from diagnosis for the entire cohort, CTC positivity at t4 significantly stratified DFS (p=0.035). Conclusions: One third of locally advanced RC patients harbored detectable CTCs at baseline. CTC detection after completion of curative-intent multimodality therapy (i.e., MRD) correlated with long-term DFS. Citation Format: Lucas Lee, Carol Hall, Antony Lucci, Brian Bednarski, Miguel Rodriguez-Bigas, George Chang, Y. Nancy You. Circulating tumor cell-defined minimal residual disease in locally advanced rectal cancer treated with multimodality therapy [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A47.