Specific Mutation Patterns Research Articles

O086 Genomic analysis of response to neoadjuvant chemotherapy in oesophageal adenocarcinoma

Abstract Introduction Oesophageal adenocarcinoma (OAC) is the ninth most common cancer worldwide with a mortality of over 500,000 deaths yearly. Neoadjuvant chemotherapy (NAC) followed by surgery is the standard of care (SOC) for locally advanced OAC. Although almost all patients receive chemotherapy as SOC, fewer than 20% obtain a clinically meaningful response and benefit before surgery. The OAC genome is complex and heterogeneous between patients, and it is not yet understood whether specific mutational patterns result in chemotherapy sensitivity or resistance. Methods To identify associations between genomic events and response to NAC in OAC, a comparative genomic analysis was performed in 65 patients using whole-genome sequencing. We defined response to NAC using Mandard Tumour Regression Grade TRG), with responders classified as TRG1-2 (n=27) and non-responders classified as TRG4- 5 (n=38). Results We report a higher non-synonymous mutation burden in responders (median 2.08/Mb vs 1.70/Mb, P=0.036) and elevated copy number variation (CNV) in non-responders (282 vs 136/patient, P<0.001). We identified CNVs unique to each group, with cell cycle (CDKN2A, CCND1), c-Myc (MYC), RTK/PIK3 (KRAS, EGFR) and gastrointestinal differentiation (GATA6) pathway genes being specifically altered in non-responders. Of particular interest was the identification of the Neuron Navigator-3 (NAV3), a known tumour suppressor downstream of EGFR, which was mutated exclusively in 22% of non-responders. Conclusion We characterise genetic features and mutations that are uniquely associated with response to NAC. We envision a treatment pipeline that incorporates driver mutation profiling in OAC, combining response prediction with targeted therapies enhancing response to NAC and improving survival outcomes. Take-home message Developing a method of determining an OAC patient's response to neoadjuvant chemotherapy before treatment is administered is desperately needed and will improve patient outcome and quality of life. We identified a number of aberrations in the genome that were unique to non-responders to chemotherapy compared to responders, particularly a known tumour suppressor gene namely Neuron Navigator-3, suggesting that these events may contribute to chemoresistance in these patients. Our work characterises pre-existing genomic alterations that have potential as biomarkers for resistance or sensitivity to NAC.

Whole-genome analysis and mutation pattern of SARS-CoV-2 during first and second wave outbreak in Gwangju, Republic of Korea

To investigate the specific genomic features and mutation pattern, whole and near-complete SARS-CoV-2 genome sequences were analyzed. Clinical samples were collected from 18 COVID-19–positive patients and subjected to nucleic acid purification. Cell culture was performed to extract various SARS-CoV-2 isolates. Whole-genome analysis was performed using next-generation sequencing, and phylogenetic analyses were conducted to determine genetic diversity of the various SARS-CoV-2 isolates. The next-generation sequencing data identified 8 protein-coding regions with 17 mutated proteins. We identified 51 missense point mutations and deletions in 5′ and 3′ untranslated regions. The phylogenetic analysis revealed that V and GH are the dominant clades of SARS-CoV-2 circulating in the Gwangju region of South Korea. Moreover, statistical analysis confirmed a significant difference between viral load (P < 0.001) and number of mutations (P < 0.0001) in 2 mutually exclusive SARS-CoV-2 clades which indicates frequent genomic alterations in SARS-CoV-2 in patients with high viral load. Our results provide an in-depth analysis of SARS-COV-2 whole genome which we believe, can shed light in the understanding of SARS-COV-2 pathogenesis and mutation pattern which can aid in the development of prevention methods as well as future research into the pathogenesis of SARS-CoV-2 and therapeutic development.

Uncovering Beta-Lactam Susceptibility Patterns in Clinical Isolates of Mycobacterium tuberculosis through Whole-Genome Sequencing.

ABSTRACTThe increasing threat of drug resistance and a stagnated pipeline of novel therapeutics endanger the eradication of tuberculosis. Beta-lactams constitute promising additions to the current therapeutic arsenal and two carbapenems are included in group C of medicines recommended by the WHO for use in longer multidrug-resistant tuberculosis regimens. However, the determinants underlining diverse Mycobacterium tuberculosis phenotypes to beta-lactams remain largely undefined. To decipher these, we present a proof-of-concept study based on a large-scale beta-lactam susceptibility screening for 172 M. tuberculosis clinical isolates from Portugal, including 72 antimycobacterial drug-resistant strains. MICs were determined for multiple beta-lactams and strains were subjected to whole-genome sequencing to identify core-genome single-nucleotide variant-based profiles. Global and cell wall-targeted approaches were then followed to detect putative drivers of beta-lactam response. We found that drug-resistant strains were more susceptible to beta-lactams, but significant differences were not observed between distinct drug-resistance profiles. Sublineage 4.3.4.2 strains were significantly more susceptible to beta-lactams, while the contrary was observed for Beijing and 4.1.2.1 sublineages. While mutations in beta-lactamase or cell wall biosynthesis genes were uncommon, a rise in beta-lactam MICs was detected in parallel with the accumulation of mutations in peptidoglycan cross-linking or cell division genes. Finally, we exposed that putative beta-lactam resistance markers occurred in genes for which relevant roles in cell wall processes have been ascribed, such as rpfC or pknA. Genetic studies to validate the relevance of the identified mutations for beta-lactam susceptibility and further improvement of the phenotype-genotype associations are needed in the future.IMPORTANCE Associations between differential M. tuberculosis beta-lactam phenotypes and preexisting antimycobacterial drug resistance, strain sublineage, or specific mutational patterns were established. Importantly, we reveal that highly drug-resistant isolates of sublineage 4.3.4.2 have an increased susceptibility to beta-lactams compared with other strains. Thus, directing beta-lactams to treat infections by specific M. tuberculosis strains and refraining its use from others emerges as a potentially important strategy to avoid resistance development. Individual mutations in blaC or genes encoding canonical beta-lactam targets, such as peptidoglycan transpeptidases, are infrequent and do not greatly impact the MICs of potent carbapenem plus clavulanic acid combinations. An improved understanding of the global effect of cumulative mutations in relevant gene sets for peptidoglycan and cell division processes on beta-lactam susceptibility is also provided.

Characterization of Vaccine Breakthrough Cases during Measles Outbreaks in Milan and Surrounding Areas, Italy, 2017-2021.

Despite the existence of an effective live-attenuated vaccine, measles can appear in vaccinated individuals. We investigated breakthrough measles cases identified during our surveillance activities within the measles/rubella surveillance network (MoRoNet) in Milan and surrounding areas (Northern Italy). Between 2017 and 2021, we confirmed measles virus (genotypes B3 or D8) infections in 653 patients and 51 of these (7.8%) were vaccinees. Among vaccinated individuals whose serum was available, a secondary failure was evidenced in 69.4% (25/36) of cases while 11 patients (30.6%) were non-responders. Non-responders were more frequently hospitalized and had significantly lower Ct values in both respiratory and urine samples. Median age and time since the last immunization were similar in the two groups. Importantly, we identified onward transmissions from vaccine failure cases. Vaccinees were involved in 20 outbreaks, in 10 of them they were able to transmit the virus, and in 8 of them, they were the index case. Comparing viral hemagglutinin sequences from vaccinated and non-vaccinated subjects did not show a specific mutation pattern. These results suggest that vaccination failure was likely due to the poor immune response of single individuals and highlights the importance of identifying breakthrough cases and characterizing their clinical and virologic profiles.

Multiagent Chemotherapy and Stereotactic Body Radiation Therapy in Patients with Unresectable Pancreatic Adenocarcinoma: A Prospective Nonrandomized Controlled Trial

PurposeIn a prospective multicenter study, gemcitabine monotherapy followed by stereotactic body radiation therapy (SBRT) was well tolerated with outcomes comparable to chemoradiation for locally advanced pancreatic cancer (LAPC). Recent trials have reported improved survival with multiagent chemotherapy (MA-CTX) alone. This prospective trial explored whether SBRT could be safely delivered after MA-CTX. Herein, we report the long-term outcomes of adding SBRT after MA-CTX in LAPC patients and evaluate whether genetic profiles of specimens obtained before SBRT influence outcomes. Methods and MaterialsThis prospective nonrandomized controlled phase 2 trial enrolled 44 LAPC and 4 locally recurrent patients after multidisciplinary evaluation between 2012 and 2015 at a high-volume pancreatic cancer center. For induction CTX, most received modified FOLFIRINOX (mFFX), or gemcitabine and nab-paclitaxel (GnP) followed by 5-fraction SBRT for all. During fiducial placement, biopsies were obtained with DNA extracted for targeted sequencing using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets platform. ResultsMedian induction CTX duration was ≥4 months, and 31 patients received mFFX (65%). Among 44 LAPC patients, 17 (39%) were surgically explored, and 12 of 16 (75%) achieved a R0 resection. Median overall survival (mOS) was 20.2 and 14.6 months from diagnosis and SBRT, respectively. One- and 2-year OS from SBRT was 58% and 28%. The mOS after resection was 28.6 and 22.4 months from diagnosis and SBRT, respectively. Median local progression-free survival was 23.9 and 15.8 months from diagnosis and SBRT, respectively. The mOS for pre-SBRT CA 19-9 ≤180 U/mL versus >180 was 23.1 and 11.3 months, respectively (hazard ratio, 0.53; P = .04). Only 1 patient (2.1%) had late grade ≥2 gastrointestinal toxic effects attributable to SBRT. Despite significant pretreatment with chemotherapy, 88% of tumor specimens were effectively sequenced; survival outcomes were not significantly associated with specific mutational patterns. Quality of life was prospectively collected pre- and post-SBRT with the EORTC QLQ-C30 and PAN26 questionnaires showing no significant change. ConclusionsSBRT was safely administered with MA-CTX with minimal toxicity. A high proportion of LAPC patients underwent R0 resection with favorable survival outcomes.

Abstract P2-12-15: Neoadjuvant liposomal doxorubicin and carboplatin is effective and tolerable for the treatment of early stage triple negative breast cancer

Abstract Background: The addition of neoadjuvant platinums to standard chemotherapy regimens for triple negative breast cancer (TNBC) improves rates of pathologic complete response (pCR). Our prior trial combining carboplatin (CAR) with liposomal doxorubicin (DOX) for metastatic TNBC showed good response rates with minimal side effects and allows for higher platinum dosing. We hypothesized that the doublet of DOX+CAR is effective and tolerable in the neoadjuvant setting for TNBC and tumor genomics may identify biomarkers of complete response and actionable targets. Methods: A phase II single arm trial was conducted for patients (pts) diagnosed with stage II-III TNBC. Patients received 4 cycles of neoadjuvant carboplatin (AUC 5) and liposomal doxorubicin (30mg/m2) administered every 28 days, then underwent definitive breast surgery followed by 12 weeks of adjuvant paclitaxel 80 mg/m2 administered weekly. Primary and secondary clinical endpoints were rate of pCR and two year recurrence free survival (RFS) and overall survival (OS), respectively. Cardiac safety of the combination was assessed. Fresh residual tumor samples were obtained at time of surgery for generation of patient derived xenografts (PDX). Whole genome sequencing (WGS) and RNA sequencing will be performed to evaluate specific patterns of small-variant mutations and patterns of structural variants to identify which patients are likely to have pCR. Results: From 2/2015 to 6/2021, 62 pts were enrolled, 8 pts withdrew consent either prior to treatment or completion of neoadjuvant therapy and/or surgery. Median age of the cohort was 55.4 years. There was high participation by under-represented groups: 17.0% African American, 20.8% Asian, 13.2% Hispanic. Most histologies were invasive ductal carcinoma, but included apocrine, pleomorphic lobular, and metaplastic subtypes. 53 pts completed all 4 cycles of neoadjuvant DOX+CAR, followed by definitive surgery; 1 pt progressed after 1 cycle and proceeded to surgery thereafter. 30.2% (16) pts achieved pCR, and residual cancer burden (RCB) will be reported in patients with residual disease. Of the 53 pts who completed 4 cycles of treatment, 5 pts progressed (recurrence or death) within two years from time of surgery (4 distant and 1 local recurrence), yielding the 2-year recurrence-free-survival rate of 90.3% (95%CI 81.0%, 99.6%). The most common toxicities during DOX+CAR were grade 1 fatigue in 50 pts (92.6%), grade 1 anemia in 44 pts (81.5%), and grade 3/4 neutropenia in 16 pts (29.6%); these pts received GCSF support with subsequent cycles; febrile neutropenia occurred in 1 pt (1.9%) in this group. Only 10 pts (18.5%) had grade 1 alopecia, 5 pts (9.3%) had grade 2 alopecia, 2 pts (3.7%) had grade 3 thrombocytopenia. There were no delays in treatment due to cardiotoxicity or complications from surgical healing. Of the 37 pts who had residual disease, PDX was attempted in 21 pts, and 16 (76%) PDX were established, including those for 4 patients experiencing recurrence. WGS and RNA sequencing data from pre-treatment and residual disease samples will be analyzed and reported.Conclusion: 4 cycles of neoadjuvant DOX+CAR achieved rate of pCR similar to standard regimens and has good tolerability. Post chemotherapy PDX is feasible and may help identify targeted approaches for patients with resistant disease. These results warrant further evaluation of this combination for early stage TNBC. Citation Format: Nancy Chan, Shou-en Lu, Yue Wang, Gregory M Riedlinger, Coral Omene, Mridula George, Jyoti Malhotra, Maria Kowzun, Firas G Eladoumikdachi, Lindsay B Potdevin, Shicha Kumar, Kant Matsuda, Shruti Desai, Nayana Patel, Deborah L Toppmeyer, Shridar Ganesan, Kim Hirshfield. Neoadjuvant liposomal doxorubicin and carboplatin is effective and tolerable for the treatment of early stage triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-12-15.

Broad spectrum of regorafenib activity on mutant KIT and absence of clonal selection in gastrointestinal stromal tumor (GIST): correlative analysis from the GRID trial

BackgroundIn the phase 3 GRID trial, regorafenib improved progression-free survival (PFS) independent of KIT mutations in exons 9 and 11. In this retrospective, exploratory analysis of the GRID trial, we investigated whether a more comprehensive KIT mutation analysis could identify mutations that impact treatment outcome with regorafenib and a regorafenib-induced mutation pattern.MethodsArchived tumor samples, collected at any time prior to enrollment in GRID, were analyzed by Sanger sequencing (n = 102) and next-generation sequencing (FoundationONE; n = 47). Plasma samples collected at baseline were analyzed by BEAMing (n = 163) and SafeSEQ (n = 96).ResultsIn archived tumor samples, 67% (68/102) had a KIT mutation; 61% (62/102) had primary KIT mutations (exons 9 and 11) and 12% (12/102) had secondary mutations (exons 13, 14, 17, and 18). At baseline, 81% of samples (78/96) had KIT mutations by SafeSEQ, including the M541L polymorphism (sole event in 6 patients). Coexisting mutations in other oncogenes were rare, as were mutations in PDGFR, KRAS, and BRAF. Regorafenib showed PFS benefit across all primary and secondary KIT mutational subgroups examined. Available patient-matched samples taken at baseline and end of treatment (n = 41; SafeSEQ), revealed heterogeneous KIT mutational changes with no specific mutation pattern emerging upon regorafenib treatment.ConclusionThese data support the results of the GRID trial, and suggest that patients may benefit from regorafenib in the presence of KIT mutations and without the selection of particular mutation patterns that confer resistance. The study was not powered to address biomarker-related questions, and the results are exploratory and hypothesis-generating.

BRCA1 deficiency specific base substitution mutagenesis is dependent on translesion synthesis and regulated by 53BP1

Defects in BRCA1, BRCA2 and other genes of the homology-dependent DNA repair (HR) pathway cause an elevated rate of mutagenesis, eliciting specific mutation patterns including COSMIC signature SBS3. Using genome sequencing of knock-out cell lines we show that Y family translesion synthesis (TLS) polymerases contribute to the spontaneous generation of base substitution and short insertion/deletion mutations in BRCA1 deficient cells, and that TLS on DNA adducts is increased in BRCA1 and BRCA2 mutants. The inactivation of 53BP1 in BRCA1 mutant cells markedly reduces TLS-specific mutagenesis, and rescues the deficiency of template switch–mediated gene conversions in the immunoglobulin V locus of BRCA1 mutant chicken DT40 cells. 53BP1 also promotes TLS in human cellular extracts in vitro. Our results show that HR deficiency–specific mutagenesis is largely caused by TLS, and suggest a function for 53BP1 in regulating the choice between TLS and error-free template switching in replicative DNA damage bypass.

O-OGC06 Genomic analysis of response to neoadjuvant chemotherapy in oesophageal adenocarcinoma

Abstract Background Oesophageal adenocarcinoma (OAC) is the ninth most common cancer worldwide, with an estimated mortality of over 500,000 deaths yearly. Neoadjuvant chemotherapy (NAC) followed by surgery is the standard of care (SOC) for locally advanced OAC. Although almost all patients receive chemotherapy as SOC, fewer than 20% obtain a clinically meaningful response and benefit before surgery. The OAC genome is complex and heterogeneous between patients, and it is not yet understood whether specific mutational patterns may result in chemotherapy sensitivity or resistance. Methods To identify associations between genomic events and response to NAC in OAC, a comparative genomic analysis was performed in 65 patients using whole-genome sequencing. We defined response to NAC using Mandard Tumour Regression Grade (TRG), with responders classified as TRG1-2 (n = 27) and non-responders classified as TRG4-5 (n = 38). Results We report a higher non-synonymous mutation burden in responders (median 2.08/Mb vs 1.70/Mb, P = 0.036) and elevated copy number variation (CNV) in non-responders (282 vs 136/patient, P&amp;lt;0.001). We identified CNVs unique to each group, with cell cycle (CDKN2A, CCND1), c-Myc (MYC), RTK/PIK3 (KRAS, EGFR) and gastrointestinal differentiation (GATA6) pathway genes being specifically altered in non-responders. Of particular interest was the identification of the Neuron Navigator-3 (NAV3), a known tumour suppressor downstream of EGFR, which was mutated exclusively in non-responders with a frequency of 22%. Conclusions Our work characterises genetic features and mutations that are uniquely associated with response to NAC. We envision a treatment pipeline that incorporates driver mutation profiling in OAC, combining response prediction with targeted therapies to enhance response to NAC and improve survival outcomes.

Whole genome global insight of antibiotic resistance gene repertoire and virulome of high - risk multidrug-resistant Uropathogenic Escherichiacoli

Elucidation of genetic determinants via whole genome sequence (WGS) analyses can help understand the high risk multidrug-resistant (MDR) Uropathogenic Escherichia coli (UPEC) associated with urinary tract infections (UTI) and its evasion strategies from treatment. We investigated the WGS of 30 UPEC strains from UTI samples across the world (2016–2019) and found 25 UPEC strains carrying 2–23 antibiotic resistance genes (ARGs) scattered across 1–3 plasmids per strain. Different ARGs (blaTEM, blaCTXM, blaNDM, blaOXA, blaCMY) encoding extended-spectrum beta-lactamases (TEM, CTXM, CMY) and carbapenemases (NDM, OXA) were found in 24/30, ARGs encoding aminoglycoside modifying enzymes (AAC, APH, AAD) variants in 23/30, trimethoprim ARGs (dfrA17, dfrA12, dfrA5, dfrB4 variants) encoding dihydrofolate reductase in 19/30 and sulfonamide ARGs (sul1, sul2, sul3) encoding dihydropteroate synthase and macrolide ARGs (mph1) encoding macrolide 2’ phosphotransferase in 15/30 UPEC strains. Collectively the ARGs were distributed in different combinations in 40 plasmids across UPEC strains with 20 plasmids displaying co-occurrence of multiple ARGs conferring resistance to beta lactam, aminoglycoside, sulfonamide, trimethoprim and macrolide antibiotics. These resistance plasmids belonged to seven incompatibility groups (IncF, IncI, IncC, IncH, IncN, IncB and Col), with IncFI and IncFII being the predominant resistance plasmids. Additionally, we observed co-occurrence of specific mutation pattern in quinolone resistance determining region (QRDR) viz., DNA gyrase (gyrA: S83L, D87N), and topoisomerase IV (parC: S80I, E84V; parE: I529L) in 18/30 strains. The strains also harbored diverse virulence genes, such as fimH, gad, iss, iha, ireA, iroN, cnf1 and san. Multilocus sequence typing (MLST) reconfirmed ST131(n = 10) as the predominant global high-risk clonal strain causing UTI. In summary, our findings contribute to better understand the plasmid mediated ARGs and its encoded enzymes that may contribute in antibiotic inactivation/modification or alteration in the antibiotic target site in high risk MDR hypervirulent UPEC strains causing UTI. The study reinforces the need to characterize and design appropriate inhibitors to counterattack different enzymes and devise strategies to curtail resistance plasmid.

1265P Combined detection of tumor markers and ctDNA in cerebrospinal fluid: A good strategy in diagnosis of leptomeningeal metastases

The main purpose is to select a more effective method for the diagnosis of leptomeningeal metastases (LM) by comparing various detection methods. The secondary purpose is to identify specific mutation patterns of genes in cerebrospinal fluid (CSF) with LM and to explore the value of CSF tumor markers and circulating tumor DNA (ctDNA) testing in guiding clinical treatment.

Hepatic IFNL4 Gene Activation in Hepatocellular Carcinoma Patients with Regard to Etiology.

Hepatocellular carcinoma (HCC) is a malignancy with a leading lethality. The etiology is quite diverse, ranging from viral infections to metabolic disorders or intoxications, and associates with specific somatic mutational patterns and specific host immunological phenotypes. Particularly, hepatitis C virus (HCV)-infected liver is featured by an activation of interferon (IFN)-stimulated genes (ISGs; IFN signature), which we suppose is driven by type III IFNL4. Taking advantage of the TCGA collection of HCC patients of various different etiologies, this study aimed at validating our previous findings on hepatic IFNL4 gene activation in HCV infection in an independent and larger cohort of patients with advanced liver disease. In a cohort of n = 377 cases, the entirety of the sequencing data was used to assess the IFNL genotypes, and the cases were stratified for etiology. The number of IFNL4 transcripts within nonmalignant and malignant tissues was found to be more abundant in patients with HCV or HCV/HBV infections when compared to other risk factors. Moreover, in patients with HCV infection as a risk factor, a close, positive relationship was found between ISG activation and the number of functional IFNL4 transcripts. Data on this independent TCGA sample support the concept of an IFNL4-dependent HCV-driven activation of hepatic ISGs. In addition to that, they add to the understanding of etiology-related host immunological phenotypes in HCC.

Mutational profile of hereditary breast and ovarian cancer – Establishing genetic testing guidelines in a developing country

PurposeBecause many countries lack the capacity to follow the international guidelines for genetic testing, we suggest the specific approach for establishing local genetic testing guidelines that could be applied in developing countries. We focus on hereditary breast (BC) and ovarian cancer (OC) in Serbia. MethodsFrom the cohort of 550 persons who were referred for genetic counseling at the Institute for Oncology and Radiology of Serbia, 392 were selected. Personal and family histories were collected and germline DNA was sequenced with NGS in a panel of 20 genes. ResultsPathogenic (PV) and likely-pathogenic variants (LPV) were detected in 8 genes with the frequency of 23.7%. The most frequent were in BRCA1(7.6%), BRCA2(4.8%), PALB2(4.1%) and CHEK2(3.8%). They were also detected in ATM(1.8%), NBN(0.8%), TP53(0.5%) and RAD51C(0.3%). Whereas high carrier probability (CP), bilateral BC, BC and OC in the same patient and family history (FH) of BC/OC, were the strongest predictors for BRCA1/2 PV/LPV, lower CP values and early age of BC onset without FH were associated with higher frequency of PALB2 and CHEK2 PV/LPV. ConclusionsPopulation specific studies to identify specific mutational patterns and predictors of PV/LPV should be conducted in order to make scientifically sound and cost-effective guidelines for genetic testing in developing countries.

Genomic Analysis of Response to Neoadjuvant Chemotherapy in Esophageal Adenocarcinoma.

Simple SummaryEsophageal adenocarcinoma (EAC) is a cancer with very poor survival outcomes. Patients are treated with pre-operative chemotherapy or chemoradiotherapy before surgery. However, four out of every five patients do not respond to pre-operative therapy and these patients (non-responders) have significantly worse outcomes. Identifying non-responders prior to therapy would allow alternative treatment pathways to be offered to these patients. In this study, we analyze whole genome sequences of pre-treatment biopsies from 65 patients and find that non-responders display chromosomal instability and increased gene copy number alterations. We report a distinct profile of copy number alterations in non-responders compared to responders, predominantly in genes involved in cell cycle control and RTK/Ras signaling. Mutations in the tumor suppressor NAV3 are also found exclusively in non-responders. These genetic profiles present potential drug targets for investigation in EAC patients who would not respond to pre-operative chemotherapy.Neoadjuvant therapy followed by surgery is the standard of care for locally advanced esophageal adenocarcinoma (EAC). Unfortunately, response to neoadjuvant chemotherapy (NAC) is poor (20–37%), as is the overall survival benefit at five years (9%). The EAC genome is complex and heterogeneous between patients, and it is not yet understood whether specific mutational patterns may result in chemotherapy sensitivity or resistance. To identify associations between genomic events and response to NAC in EAC, a comparative genomic analysis was performed in 65 patients with extensive clinical and pathological annotation using whole-genome sequencing (WGS). We defined response using Mandard Tumor Regression Grade (TRG), with responders classified as TRG1–2 (n = 27) and non-responders classified as TRG4–5 (n =38). We report a higher non-synonymous mutation burden in responders (median 2.08/Mb vs. 1.70/Mb, p = 0.036) and elevated copy number variation in non-responders (282 vs. 136/patient, p < 0.001). We identified copy number variants unique to each group in our cohort, with cell cycle (CDKN2A, CCND1), c-Myc (MYC), RTK/PIK3 (KRAS, EGFR) and gastrointestinal differentiation (GATA6) pathway genes being specifically altered in non-responders. Of note, NAV3 mutations were exclusively present in the non-responder group with a frequency of 22%. Thus, lower mutation burden, higher chromosomal instability and specific copy number alterations are associated with resistance to NAC.

Acute Myeloid Leukemia Classification System That Associates Normal Myeloid-Cell Subset Gene Signatures with Prognosis

Background: Acute Myeloid Leukemia (AML) represents a group of heterogeneous clonal disorders of hematopoietic progenitor cells that are driven by multiple genetic events that impair cell differentiation and apoptosis and invoke uncontrolled cell proliferation. Consequently, immature myeloid blasts rapidly accumulate in the bone marrow and block the generation of mature end stage cells, which eventually causes potentially fatal infections and hemorrhages. AML is characterized by extensive genetic and phenotypic heterogeneity, which currently is diagnostically classified based on blast morphology defined by the French-American-British (FAB) Cooperative Group, cytogenetic abnormalities, and improved knowledge of genomic and epigenomic alterations. However, this has not yet resulted in treatment progress. AML patients are still routinely treated with a standard first-line combinatorial chemotherapy regimen with an overall survival rate remaining below 20%. Therefore, there is an unmet need for novel methods to stratify patients into therapy-relevant subtypes.Concept and Aims: We hypothesize that phenotypic heterogeneity of AML is a consequence of dysregulated differentiation and that classification based on transcriptional reminiscence of cell of origin (COO) during AML transformation is of pathogenetic impact and therefore provide diagnostic and prognostic value. It is the aim of this project to generate and validate a COO classification system based on normal myeloid-cell-subset-associated gene signatures (MAGS) to stratify AML patients into therapy-relevant and prognostic subtypes.MAGS Generation: Based on gene expression profiles of publicly available sorted normal myeloid subpopulations, we generated a COO classifier including the CD34+/CD38- hematopoietic stem cells (HSC), CD34+/CD38+/CD45RA- megakaryocyte-erythroid progenitors (MEP), and CD34+/CD38+/CD45RA+ granulocytic-monocytic progenitors (GMP) using regularized multinomial regression with 3 discrete outcomes and the elastic net penalty. The regularization parameters were chosen by cross validation.Results: MAGS prediction accuracy was validated in an independent dataset (n = 38; accuracy = 0.79) of sorted normal myeloid subpopulations. The prognostic value of MAGS assignment was studied by survival analyses using the Kaplan Meier method, log-rank test statistics, and simple and multiple Cox proportional hazards regression analysis in 2 clinical cohorts (TCGA: n = 115; GSE6891: n = 431). MAGS assignment had significant prognostic associations with overall survival (Figure 1; Table 1A) revealing that the linage committed MAGS subtypes GMP and MEP showed superior prognosis compared to the undifferentiated AMLs captured by the MAGS HSC subtype. Furthermore, multiple Cox regression analysis with MAGS assignment (HSC, GMP, MEP), FAB score (M0-M6), cytogenetics (good, intermediate, poor), and age (dichotomized into <55 and ≥55) as independent explanatory variables showed that MAGS assignment provides independent prognostic information (Table 1B), indicating that the MAGS classification captures distinct pathogenetic and prognostic knowledge not already explained by the FAB- or cytogenetic classifications. Molecular characterization of MAGS subtypes by differential gene expression analysis, gene set enrichment analysis, and identification of subtype specific mutation patterns of well-documented AML driver mutations indicated a reduced proliferation rate as well as overrepresentation of EVI1 mutations associated with the poor responding HSC subtype.Conclusion: In accordance with our previous reports in malignant B-cell disorders, we have documented a novel classification system that associates normal myeloid progenitor subsets with AML subtypes of distinct pathogenetic and prognostic importance not already explained by the FAB- or cytogenetic classifications. Further pathogenetic studies are needed to develop a diagnostic platform using MAGS-assigned subtypes to improve disease management. [Display omitted] DisclosuresSeverinsen:ARIAD: Research Funding; Celgene: Research Funding; Novartis: Research Funding. El-Galaly:Roche: Other: Travel funding; Takeda: Other: Travel funding.

Clinical relevance of clonal hematopoiesis in persons aged ≥80 years

AbstractClonal hematopoiesis of indeterminate potential (CHIP) is associated with increased risk of cancers and inflammation-related diseases. This phenomenon becomes common in persons aged ≥80 years, in whom the implications of CHIP are not well defined. We performed a mutational screening in 1794 persons aged ≥80 years and investigated the relationships between CHIP and associated pathologies. Mutations were observed in one-third of persons aged ≥80 years and were associated with reduced survival. Mutations in JAK2 and splicing genes, multiple mutations (DNMT3A, TET2, and ASXL1 with additional genetic lesions), and variant allele frequency ≥0.096 had positive predictive value for myeloid neoplasms. Combining mutation profiles with abnormalities in red blood cell indices improved the ability of myeloid neoplasm prediction. On this basis, we defined a predictive model that identifies 3 risk groups with different probabilities of developing myeloid neoplasms. Mutations in DNMT3A, TET2, ASXL1, or JAK2 were associated with coronary heart disease and rheumatoid arthritis. Cytopenia was common in persons aged ≥80 years, with the underlying cause remaining unexplained in 30% of cases. Among individuals with unexplained cytopenia, the presence of highly specific mutation patterns was associated with myelodysplastic-like phenotype and a probability of survival comparable to that of myeloid neoplasms. Accordingly, 7.5% of subjects aged ≥80 years with cytopenia had presumptive evidence of myeloid neoplasm. In summary, specific mutational patterns define different risk of developing myeloid neoplasms vs inflammatory-associated diseases in persons aged ≥80 years. In individuals with unexplained cytopenia, mutational status may identify those subjects with presumptive evidence of myeloid neoplasms.

Dimethyl fumarate induces ferroptosis and impairs NF-κB/STAT3 signaling in DLBCL

AbstractDespite the development of novel targeted drugs, the molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) still poses a substantial therapeutic challenge. DLBCL can be classified into at least 2 major subtypes (germinal center B cell [GCB]-like and activated B cell [ABC]-like DLBCL), each characterized by specific gene expression profiles and mutation patterns. Here we demonstrate a broad antitumor effect of dimethyl fumarate (DMF) on both DLBCL subtypes, which is mediated by the induction of ferroptosis, a form of cell death driven by the peroxidation of phospholipids. As a result of the high expression of arachidonate 5-lipoxygenase in concert with low glutathione and glutathione peroxidase 4 levels, DMF induces lipid peroxidation and thus ferroptosis, particularly in GCB DLBCL. In ABC DLBCL cells, which are addicted to NF-κB and STAT3 survival signaling, DMF treatment efficiently inhibits the activity of the IKK complex and Janus kinases. Interestingly, the BCL-2–specific BH3 mimetic ABT-199 and an inhibitor of ferroptosis suppressor protein 1 synergize with DMF in inducing cell death in DLBCL. Collectively, our findings identify the clinically approved drug DMF as a promising novel therapeutic option in the treatment of both GCB and ABC DLBCLs.

Shaping the BRCAness mutational landscape by alternative double-strand break repair, replication stress and mitotic aberrancies.

Tumours with mutations in the BRCA1/BRCA2 genes have impaired double-stranded DNA break repair, compromised replication fork protection and increased sensitivity to replication blocking agents, a phenotype collectively known as ‘BRCAness’. Tumours with a BRCAness phenotype become dependent on alternative repair pathways that are error-prone and introduce specific patterns of somatic mutations across the genome. The increasing availability of next-generation sequencing data of tumour samples has enabled identification of distinct mutational signatures associated with BRCAness. These signatures reveal that alternative repair pathways, including Polymerase θ-mediated alternative end-joining and RAD52-mediated single strand annealing are active in BRCA1/2-deficient tumours, pointing towards potential therapeutic targets in these tumours. Additionally, insight into the mutations and consequences of unrepaired DNA lesions may also aid in the identification of BRCA-like tumours lacking BRCA1/BRCA2 gene inactivation. This is clinically relevant, as these tumours respond favourably to treatment with DNA-damaging agents, including PARP inhibitors or cisplatin, which have been successfully used to treat patients with BRCA1/2-defective tumours. In this review, we aim to provide insight in the origins of the mutational landscape associated with BRCAness by exploring the molecular biology of alternative DNA repair pathways, which may represent actionable therapeutic targets in in these cells.

A bacterial mutational footprint in colorectal cancer genomes.

Changes in the microbiome are associated with the development of colorectal cancer, but causal explanations have been lacking. We recently demonstrated that pks+ Escherichia coli induce a specific mutational pattern using intestinal organoids and these mutations are present in the genomes of colorectal cancer. This finding warrants further studies on the microbial role in oncogenic mutation induction, cancer development and future preventive strategies.

Whole-Genome Sequencing of Retinoblastoma Reveals the Diversity of Rearrangements Disrupting RB1 and Uncovers a Treatment-Related Mutational Signature.

Simple SummaryRetinoblastoma, a childhood cancer of the eye, is thought to be caused by inactivating mutations of both copies of the RB1 gene. The majority of RB1 mutations can be detected by clinical screening. However, retinoblastoma cases exist where mutations in RB1 have not been detected. We used whole-genome sequencing to investigate the landscape of mutations in a cohort of sporadic retinoblastomas, including cases where mutations in both copies of RB1 had not been previously identified. We looked for mutations in cancer driver genes and revealed a wide variety of structural rearrangements disrupting RB1. In addition, we investigated mutation burden and specific mutation patterns (mutational signatures), uncovering a treatment-related mutational signature in a tumour exposed to chemotherapy. The power of whole-genome sequencing to identify RB1 mutations of all mutation types can have significant relevance to the clinical management of retinoblastoma patients and genetic counselling of their families.The development of retinoblastoma is thought to require pathological genetic changes in both alleles of the RB1 gene. However, cases exist where RB1 mutations are undetectable, suggesting alternative pathways to malignancy. We used whole-genome sequencing (WGS) and transcriptomics to investigate the landscape of sporadic retinoblastomas derived from twenty patients, sought RB1 and other driver mutations and investigated mutational signatures. At least one RB1 mutation was identified in all retinoblastomas, including new mutations in addition to those previously identified by clinical screening. Ten tumours carried structural rearrangements involving RB1 ranging from relatively simple to extremely complex rearrangement patterns, including a chromothripsis-like pattern in one tumour. Bilateral tumours obtained from one patient harboured conserved germline but divergent somatic RB1 mutations, indicating independent evolution. Mutational signature analysis showed predominance of signatures associated with cell division, an absence of ultraviolet-related DNA damage and a profound platinum-related mutational signature in a chemotherapy-exposed tumour. Most RB1 mutations are identifiable by clinical screening. However, the increased resolution and ability to detect otherwise elusive rearrangements by WGS have important repercussions on clinical management and advice on recurrence risks.