Stem cell-like breast cancer cells with acquired resistance to metformin are sensitive to inhibitors of NADH-dependent CtBP dimerization.

Altered flux through major metabolic pathways is a hallmark of cancer cells and provides opportunities for therapy. Stem cell-like cancer (SCLC) cells can cause metastasis and therapy resistance. They possess metabolic plasticity, theoretically enabling resistance to therapies targeting a specific metabolic state. The C-terminal binding protein (CtBP) transcriptional regulators are potential therapeutic targets in highly glycolytic cancer cells, as they are activated by the glycolytic coenzyme nicotinamide adenine dinucleotide (NADH). However, SCLC cells commonly exist in an oxidative state with low rates of glycolysis. Metformin inhibits complex I of the mitochondrial electron transport chain; it can kill oxidative SCLC cells and has anti-cancer activity in patients. SCLC cells can acquire resistance to metformin through increased glycolysis. Given the potential for long-term metformin therapy, we have studied acquired metformin resistance in cells from the claudin-low subtype of breast cancer. Cells cultured for 8 weeks in sub-IC50 metformin concentration proliferated comparably to untreated cells and exhibited higher rates of glucose uptake. SCLC cells were enriched in metformin-adapted cultures. These SCLC cells acquired sensitivity to multiple methods of inhibition of CtBP function, including a cyclic peptide inhibitor of NADH-induced CtBP dimerization. Single-cell mRNA sequencing identified a reprogramming of epithelial-mesenchymal and stem cell gene expression in the metformin-adapted SCLC cells. These SCLC cells demonstrated an acquired dependency on one of these genes, Tenascin C. Thus, in addition to acquisition of sensitivity to glycolysis-targeting therapeutic strategies, the reprograming of gene expression in the metformin-adapted SCLC cells renders them sensitive to potential therapeutic approaches not directly linked to cell metabolism.

Fcγ receptors: genetic variation, function, and disease.

Fcγ receptors (FcγRs) are key immune receptors responsible for the effective control of both humoral and innate immunity and are central to maintaining the balance between generating appropriate responses to infection and preventing autoimmunity. When this balance is lost, pathology results in increased susceptibility to cancer, autoimmunity, and infection. In contrast, optimal FcγR engagement facilitates effective disease resolution and response to monoclonal antibody immunotherapy. The underlying genetics of the FcγR gene family are a central component of this careful balance. Complex in humans and generated through ancestral duplication events, here we review the evolution of the gene family in mammals, the potential importance of copy number, and functionally relevant single nucleotide polymorphisms, as well as discussing current approaches and limitations when exploring genetic variation in this region.

Surface IgM expression and function are associated with clinical behavior, genetic abnormalities, and DNA methylation in CLL.

Chronic lymphocytic leukemia (CLL) with unmutated (U-CLL) or mutated (M-CLL) immunoglobulin gene heavy-chain variable region (IGHV) displays different states of anergy, indicated by reduced surface immunoglobulin M (sIgM) levels and signaling, consequent to chronic (super)antigen exposure. The subsets also differ in the incidence of high-risk genetic aberrations and in DNA methylation profile, preserved from the maturational status of the original cell. We focused on sIgM expression and function, measured as intracellular Ca(2+) mobilization following stimulation, and probed correlations with clinical outcome. The relationship with genetic features and maturation status defined by DNA methylation of an 18-gene panel signature was then investigated. sIgM levels/signaling were higher and less variable in U-CLL than in M-CLL and correlated with disease progression between and within U-CLL and M-CLL. In U-CLL, increased levels/signaling associated with +12, del(17p) or NOTCH1 mutations. In M-CLL, there were fewer genetic lesions, although the methylation maturation status, generally higher than in U-CLL, varied and was increased in cases with lower sIgM levels/signaling. These features revealed heterogeneity in M-CLL and U-CLL with clear clinical correlations. Multivariate analyses with phenotype, genetic lesions, or DNA methylation maturation status identified high sIgM levels as a new potential independent factor for disease progression. Multiple influences on sIgM include the cell of origin, the clonal history of antigen encounter in vivo, and genetic damage. This simple marker compiles these different factors into an indicator worthy of further investigations for prediction of clinical behavior, particularly within the heterogeneous M-CLL subset.

Exome sequence read depth methods for identifying copy number changes.

Copy number variants (CNVs) play important roles in a number of human diseases and in pharmacogenetics. Powerful methods exist for CNV detection in whole genome sequencing (WGS) data, but such data are costly to obtain. Many disease causal CNVs span or are found in genome coding regions (exons), which makes CNV detection using whole exome sequencing (WES) data attractive. If reliably validated against WGS-based CNVs, exome-derived CNVs have potential applications in a clinical setting. Several algorithms have been developed to exploit exome data for CNV detection and comparisons made to find the most suitable methods for particular data samples. The results are not consistent across studies. Here, we review some of the exome CNV detection methods based on depth of coverage profiles and examine their performance to identify problems contributing to discrepancies in published results. We also present a streamlined strategy that uses a single metric, the likelihood ratio, to compare exome methods, and we demonstrated its utility using the VarScan 2 and eXome Hidden Markov Model (XHMM) programs using paired normal and tumour exome data from chronic lymphocytic leukaemia patients. We use array-based somatic CNV (SCNV) calls as a reference standard to compute prevalence-independent statistics, such as sensitivity, specificity and likelihood ratio, for validation of the exome-derived SCNVs. We also account for factors known to influence the performance of exome read depth methods, such as CNV size and frequency, while comparing our findings with published results.

The outcome of Chronic lymphocytic leukaemia patients with 97% IGHV gene identity to germline is distinct from cases with <97% identity and similar to those with 98% identity.

IGHV gene mutational status has prognostic significance in chronic lymphocytic leukaemia (CLL) but the percentage of mutations that correlates best with clinical outcome remains controversial. We initially studied 558 patients from diagnosis and found significant differences in median time to first treatment (TTFT) among Stage A patients and in overall survival (OS) for the whole cohort, between cases with <97% and 97-98·99% identity and between cases with 97-98·99% and ≥99% identity, when cases from the IGHV3-21 Stereotype Subset #2 were excluded. A significant difference in progression-free survival (PFS) and OS between those with <97% and 97-98·99% identity, but not between those with 97-98·99% and ≥99% identity was also observed in a validation cohort comprising 460 patients in the UK CLL4 trial. Cox Regression analyses in the Stage A cohort revealed that a model which incorporated <97%, 97-98·99% and ≥99% identity as subgroups, was a better predictor of TTFT in CLL than using the 98% cut-off. Multivariate analysis selected the three mutational subgroups as independent predictors of TTFT in Stage A patients, and of OS in the diagnostic cohort. This study highlights that cases with 97% identity should not be considered to have the same prognosis as other cases with mutated IGHV genes defined as <98% identity to germline.

The clinical significance of NOTCH1 and SF3B1 mutations in the UK LRF CLL4 trial.

NOTCH1 and SF3B1 mutations have been previously reported to have prognostic significance in chronic lymphocytic leukemia but to date they have not been validated in a prospective, controlled clinical trial. We have assessed the impact of these mutations in a cohort of 494 patients treated within the randomized phase 3 United Kingdom Leukaemia Research Fund Chronic Lymphocytic Leukemia 4 (UK LRF CCL4) trial that compared chlorambucil and fludarabine with and without cyclophosphamide in previously untreated patients. We investigated the relationship of mutations in NOTCH1 (exon 34) and SF3B1 (exon 14-16) to treatment response, survival and a panel of established biologic variables. NOTCH1 and SF3B1 mutations were found in 10% and17% of patients, respectively. NOTCH1 mutations correlated with unmutated IGHV genes, trisomy 12, high CD38/ ZAP-70 expression and were associated with reduced overall (median 54.8 vs 74.6 months, P = .02) and progression-free (median 22.0 vs 26.4 months, P = .02) survival. SF3B1 mutations were significantly associated with high CD38 expression and with shorter overall survival (median 54.3 vs 79.0 months, P < .001). Furthermore, multivariate analysis, including baseline clinical variables, treatment, and adverse prognostic factors demonstrated that although TP53 alterations remained the most informative marker of dismal survival in this cohort, NOTCH1 (HR 1.58, P = .03) and SF3B1 (HR 1.52, P = .01) mutations have added independent prognostic value.

ATM mutation rather than BIRC3 deletion and/or mutation predicts reduced survival in 11q-deleted chronic lymphocytic leukemia: data from the UK LRF CLL4 trial.

ATM mutation and BIRC3 deletion and/or mutation have independently been shown to have prognostic significance in chronic lymphocytic leukemia. However, the relative clinical importance of these abnormalities in patients with a deletion of 11q encompassing the ATM gene has not been established. We screened a cohort of 166 patients enriched for 11q-deletions for ATM mutations and BIRC3 deletion and mutation and determined the overall and progression-free survival among the 133 of these cases treated within the UK LRF CLL4 trial. SNP6.0 profiling demonstrated that BIRC3 deletion occurred in 83% of 11q-deleted cases and always co-existed with ATM deletion. For the first time we have demonstrated that 40% of BIRC3-deleted cases have concomitant deletion and mutation of ATM. While BIRC3 mutations were rare, they exclusively occurred with BIRC3 deletion and a wild-type residual ATM allele. In 11q-deleted cases, we confirmed that ATM mutation was associated with a reduced overall and progression-free survival comparable to that seen with TP53 abnormalities, whereas BIRC3 deletion and/or mutation had no impact on overall and progression-free survival. In conclusion, in 11q-deleted patients treated with first-line chemotherapy, ATM mutation rather than BIRC3 deletion and/or mutation identifies a subgroup with a poorer outcome.

Molecular landscape of proliferative verrucous leukoplakia: a systematic review.

Proliferative verrucous leukoplakia (PVL) is a rare oral potentially malignant disorder characterised by multifocal origin and unpredictable long-term evolution to oral squamous cell carcinoma (OSCC) or oral verrucous carcinoma (OVC). Currently no predictive biomarkers are in clinical use. We aimed to explore the genomic profile of PVL. A total of 685 cases in 26 studies were included in this review. Genomic data were presented in 15% of studies and biomarker analysis was reported in 85% of studies. At first clinical presentation, PVL is characterised by a high loss of heterozygosity (LOH), similar to OSCC, and low copy number alterations (CNA). As these progress, more CNAs and mutations in CDKN2A and alterations to ELAVL1 expression are noted, but no TP53 mutations are identified. There is significantly lower LOH at 17p in early PVL compared with OSCC (p = 0.037). Deletions in chromosomal loci 17q12, 5q31.1 and amplifications in 7q11.2, 7q22 are shared between early lesions and OVC. PVL shows CNAs at 11q31. WNT signalling pathway genes (SUZ12, CTTN and FOLR3) are enriched in CN-altered regions. PVL stroma shows significantly lower α-SMA and higher CD34 expression than OVC and OSCC. The exact genomic landscape is currently unclear, and further studies are necessary to unravel this mystery.

Single-cell analysis reveals prognostic fibroblast subpopulations linked to molecular and immunological subtypes of lung cancer.

Fibroblasts are poorly characterised cells that variably impact tumour progression. Here, we use single cell RNA-sequencing, multiplexed immunohistochemistry and digital cytometry (CIBERSORTx) to identify and characterise three major fibroblast subpopulations in human non-small cell lung cancer: adventitial, alveolar and myofibroblasts. Alveolar and adventitial fibroblasts (enriched in control tissue samples) localise to discrete spatial niches in histologically normal lung tissue and indicate improved overall survival rates when present in lung adenocarcinomas (LUAD). Trajectory inference identifies three phases of control tissue fibroblast activation, leading to myofibroblast enrichment in tumour samples: initial upregulation of inflammatory cytokines, followed by stress-response signalling and ultimately increased expression of fibrillar collagens. Myofibroblasts correlate with poor overall survival rates in LUAD, associated with loss of epithelial differentiation, TP53 mutations, proximal molecular subtypes and myeloid cell recruitment. In squamous carcinomas myofibroblasts were not prognostic despite being transcriptomically equivalent. These findings have important implications for developing fibroblast-targeting strategies for cancer therapy.

Identification of ATPAF1 as a novel candidate gene for asthma in children.

BACKGROUND: Asthma is a common disease of children with a complex genetic origin. Understanding the genetic basis of asthma susceptibility will allow disease prediction and risk stratification. OBJECTIVE: We sought to identify asthma susceptibility genes in children. METHODS: A nested case-control genetic association study of children of Caucasian European ancestry from a birth cohort was conducted. Single nucleotide polymorphisms (SNPs, n = 116,024) were genotyped in pools of DNA samples from cohort children with physician-diagnosed asthma (n = 112) and normal controls (n = 165). A genomic region containing the ATPAF1 gene was found to be significantly associated with asthma. Additional SNPs within this region were genotyped in individual samples from the same children and in 8 independent study populations of Caucasian, African American, Hispanic, or other ancestries. SNPs were also genotyped or imputed in 2 consortia control populations. ATPAF1 expression was measured in bronchial biopsies from asthmatic patients and controls. RESULTS: Asthma was found to be associated with a cluster of SNPs and SNP haplotypes containing the ATPAF1 gene, with 2 SNPs achieving significance at a genome-wide level (P = 2.26 × 10(-5) to 2.2 × 10(-8)). Asthma severity was also found to be associated with SNPs and SNP haplotypes in the primary population. SNP and/or gene-level associations were confirmed in the 4 non-Hispanic populations. Haplotype associations were also confirmed in the non-Hispanic populations (P = .045-.0009). ATPAF1 total RNA expression was significantly (P < .01) higher in bronchial biopsies from asthmatic patients than from controls. CONCLUSION: Genetic variation in the ATPAF1 gene predisposes children of different ancestries to asthma.

Comparison of optimized methodologies for isolating nuclei from esophageal tissue.

Single-nuclei RNA sequencing allows single cell-based analysis in frozen tissue, ameliorating cell recovery biases associated with enzymatic dissociation methods. The authors present two optimized methods for isolating and sequencing nuclei from esophageal tissue using a commercial EZ and citric acid (CA)-based method. Despite high endogenous RNase activity, these protocols produced libraries of expected fragment length (average length EZ: 745 bp; CA: 1232 bp) with comparable complexity (median Transcript/Gene number, EZ: 496/254; CA: 483/256). CA nuclei showed a higher proportion of ribosomal gene reads, potentially reflecting co-isolation of nuclei and adherent ribosomes. The authors identified 11 cell lineages in the combined datasets, with differences in cell type recovery between the two methods, providing utility dependent on experimental needs.

The spatiotemporal dynamics of microglia across the human lifespan.

Microglia, the brain's resident macrophages, shape neural development and are key neuroimmune hubs in the pathological signatures of neurodevelopmental disorders. Despite the importance of microglia, their development has not been carefully examined in the human brain, and most of our knowledge derives from rodents. We aimed to address this gap in knowledge by establishing an extensive collection of 97 post-mortem tissues in order to enable quantitative, sex-matched, detailed analysis of microglia across the human lifespan. We identify the dynamics of these cells in the human telencephalon, describing waves in microglial density across gestation, infancy, and childhood, controlled by a balance of proliferation and apoptosis, which track key neurodevelopmental milestones. These profound changes in microglia are also observed in bulk RNA-seq and single-cell RNA-seq datasets. This study provides a detailed insight into the spatiotemporal dynamics of microglia across the human lifespan and serves as a foundation for elucidating how microglia contribute to shaping neurodevelopment in humans.

Phosphodiesterase type 5 inhibitors enhance chemotherapy in preclinical models of esophageal adenocarcinoma by targeting cancer-associated fibroblasts.

The chemotherapy resistance of esophageal adenocarcinomas (EACs) is underpinned by cancer cell extrinsic mechanisms of the tumor microenvironment (TME). We demonstrate that, by targeting the tumor-promoting functions of the predominant TME cell type, cancer-associated fibroblasts (CAFs) with phosphodiesterase type 5 inhibitors (PDE5i), we can enhance the efficacy of standard-of-care chemotherapy. In ex vivo conditions, PDE5i prevent the transdifferentiation of normal fibroblasts to CAF and abolish the tumor-promoting function of established EAC CAFs. Using shotgun proteomics and single-cell RNA-seq, we reveal PDE5i-specific regulation of pathways related to fibroblast activation and tumor promotion. Finally, we confirm the efficacy of PDE5i in combination with chemotherapy in close-to-patient and in vivo PDX-based model systems. These findings demonstrate that CAFs drive chemotherapy resistance in EACs and can be targeted by repurposing PDE5i, a safe and well-tolerated class of drug administered to millions of patients world-wide to treat erectile dysfunction.

Prenatal and infant acetaminophen exposure, antioxidant gene polymorphisms, and childhood asthma.

BACKGROUND: Prenatal and infant acetaminophen exposure has been associated with an increased risk of childhood asthma phenotypes. Demonstration of biologically plausible interactions between these exposures and maternal and child antioxidant gene polymorphisms would strengthen causal inference. OBJECTIVE: To explore potential interactions between prenatal and infant acetaminophen exposure and antioxidant genotypes on childhood asthma. METHODS: In the Avon Longitudinal Study of Parents and Children, we typed a functional nuclear erythroid 2 p45-related factor 2 (Nrf2) polymorphism and glutathione S-transferase (GST) M1, T1, and P1 polymorphisms. Effects of prenatal and infant acetaminophen exposure on asthma phenotypes at 7 years were stratified by genotype in >4000 mothers and >5000 children. RESULTS: Risk of asthma and wheezing associated with early gestation acetaminophen exposure was increased when maternal copies of the minor T allele of Nrf2 were present (P interactions, .02 and .04, respectively). Risk of asthma associated with late gestation exposure was higher when maternal GSTT1 genotype was present rather than absent (P interaction, .006), and risk of wheezing was increased when maternal GSTM1 was present (P interaction, .04). Although acetaminophen use in infancy was associated with an increased risk of atopy, child antioxidant genotype did not modify associations between infant acetaminophen use and asthma phenotypes. However, the increased risk of asthma and wheezing associated with late gestation acetaminophen exposure in the presence of maternal GSTM1 was further enhanced when GSTM1 was also present in the child. CONCLUSION: Maternal antioxidant gene polymorphisms may modify the relation between prenatal acetaminophen exposure and childhood asthma, strengthening evidence for a causal association. In contrast, relations between infant acetaminophen use and asthma and atopy were not modified by child genotype and may be confounded by pre-existing wheeze or allergy.

V-ATPase Inhibition Decreases Mutant Androgen Receptor Activity in Castrate-resistant Prostate Cancer.

Prostate cancer is critically dependent on androgen receptor (AR) signaling. Despite initial responsiveness to androgen deprivation, most patients with advanced prostate cancer subsequently progress to a clinically aggressive castrate-resistant prostate cancer (CRPC) phenotype, typically associated with expression of splice-variant or mutant AR forms. Although current evidence suggests that the vacuolar-ATPase (V-ATPase), a multiprotein complex that catalyzes proton transport across intracellular and plasma membranes, influences wild-type AR function, the effect of V-ATPase inhibition on variant AR function is unknown.Inhibition of V-ATPase reduced AR function in wild-type and mutant AR luciferase reporter models. In hormone-sensitive prostate cancer cell lines (LNCaP, DuCaP) and mutant AR CRPC cell lines (22Rv1, LNCaP-F877L/T878A), V-ATPase inhibition using bafilomycin-A1 and concanamycin-A reduced AR expression, and expression of AR target genes, at mRNA and protein levels. Furthermore, combining chemical V-ATPase inhibition with the AR antagonist enzalutamide resulted in a greater reduction in AR downstream target expression than enzalutamide alone in LNCaP cells. To investigate the role of individual subunit isoforms, siRNA and CRISPR-Cas9 were used to target the V1C1 subunit in 22Rv1 cells. Whereas transfection with ATP6V1C1-targeted siRNA significantly reduced AR protein levels and function, CRISPR-Cas9-mediated V1C1 knockout showed no substantial change in AR expression, but a compensatory increase in protein levels of the alternate V1C2 isoform.Overall, these results indicate that V-ATPase dysregulation is directly linked to both hormone-responsive prostate cancer and CRPC via impact on AR function. In particular, V-ATPase inhibition can reduce AR signaling regardless of mutant AR expression.

Rapid microfluidic isolation of virally infected primary bronchial epithelial cells for single-cell RNA sequencing.

Single-cell RNA sequencing (scRNA-seq) of the bronchial epithelium enables examination of cellular subtypes and their responses to viral infections. Here, an optimized method for the isolation of virally infected primary bronchial epithelial cells using a commercially available microfluidic device is presented. Using this method single cells can be rapidly isolated with minimal equipment available in most laboratories. Isolation can be carried out inside biological safety cabinets, permitting the use of virally infected cells. Both cell-line and primary cells isolated using the device retained sufficient RNA integrity for the generation of short-read sequencing-compatible cDNA libraries to facilitate scRNA-seq.

CD8+CD103+ tissue-resident memory T cells convey reduced protective immunity in cutaneous squamous cell carcinoma.

BACKGROUND: Tumor infiltrating lymphocytes play a key role in antitumor responses; however, while several memory T-cell subtypes have been reported in inflammatory and neoplastic conditions, the proportional representation of the different subsets of memory T cells and their functional significance in cancer is unclear. Keratinocyte skin cancer is one of the most common cancers globally, with cutaneous squamous cell cancer (cSCC) among the most frequent malignancies capable of metastasis. METHODS: Memory T-cell subsets were delineated in human cSCCs and, for comparison, in non-lesional skin and blood using flow cytometry. Immunohistochemistry was conducted to quantify CD103+ cells in primary human cSCCs which had metastasized (P-M) and primary cSCCs which had not metastasized (P-NM). TIMER2.0 (timer.cistrome.org) was used to analyze TCGA cancer survival data based on ITGAE expression. Immunofluorescence microscopy was performed to determine frequencies of CD8+CD103+ cells in P-M and P-NM cSCCs. RESULTS: Despite intertumoral heterogeneity, most cSCC T cells were CCR7-/CD45RA- effector/resident memory (TRM) lymphocytes, with naive, CD45RA+/CCR7- effector memory re-expressing CD45RA, CCR7+/L-selectin+ central memory and CCR7+/L-selectin- migratory memory lymphocytes accounting for smaller T-cell subsets. The cSCC CD8+ T-cell population contained a higher proportion of CD69+/CD103+ TRMs than that in non-lesional skin and blood. These cSCC CD69+/CD103+ TRMs exhibited increased IL-10 production, and higher CD39, CTLA-4 and PD-1 expression compared with CD103- TRMs in the tumor. CD103+ cells were more frequent in P-M than P-NM cSCCs. Analysis of TCGA data demonstrated that high expression of ITGAE (encoding CD103) was associated with reduced survival in primary cutaneous melanoma, breast carcinoma, renal cell carcinoma, kidney chromophobe cancer, adrenocortical carcinoma and lower grade glioma. Immunofluorescence microscopy showed that the majority of CD103 was present on CD8+ T cells and that CD8+CD103+ cells were significantly more frequent in P-M than P-NM cSCCs. CONCLUSION: These results highlight CD8+CD103+ TRMs as an important functional T-cell subset associated with poorer clinical outcome in this cancer.

Dual dean entrainment with volume ratio modulation for efficient droplet co-encapsulation: extreme single-cell indexing.

The future of single cell diversity screens involves ever-larger sample sizes, dictating the need for higher throughput methods with low analytical noise to accurately describe the nature of the cellular system. Current approaches are limited by the Poisson statistic, requiring dilute cell suspensions and associated losses in throughput. In this contribution, we apply Dean entrainment to both cell and bead inputs, defining different volume packets to effect efficient co-encapsulation. Volume ratio scaling was explored to identify optimal conditions. This enabled the co-encapsulation of single cells with reporter beads at rates of ∼1 million cells per hour, while increasing assay signal-to-noise with cell multiplet rates of ∼2.5% and capturing ∼70% of cells. The method, called Pirouette coupling, extends our capacity to investigate biological systems.

Genomic Analysis of Response to Neoadjuvant Chemotherapy in Esophageal Adenocarcinoma.

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.