Method To Visualize the Intratumor Distribution and Impact of Gemcitabine in Pancreatic Ductal Adenocarcinoma by Multimodal Imaging.

Gemcitabine (dFdC) is a common treatment for pancreatic cancer; however, it is thought that treatment may fail because tumor stroma prevents drug distribution to tumor cells. Gemcitabine is a pro-drug with active metabolites generated intracellularly; therefore, visualizing the distribution of parent drug as well as its metabolites is important. A multimodal imaging approach was developed using spatially coregistered mass spectrometry imaging (MSI), imaging mass cytometry (IMC), multiplex immunofluorescence microscopy (mIF), and hematoxylin and eosin (H&E) staining to assess the local distribution and metabolism of gemcitabine in tumors from a genetically engineered mouse model of pancreatic cancer (KPC) allowing for comparisons between effects in the tumor tissue and its microenvironment. Mass spectrometry imaging (MSI) enabled the visualization of the distribution of gemcitabine (100 mg/kg), its phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP, and the inactive metabolite dFdU. Distribution was compared to small-molecule ATR inhibitor AZD6738 (25 mg/kg), which was codosed. Gemcitabine metabolites showed heterogeneous distribution within the tumor, which was different from the parent compound. The highest abundance of dFdCMP, dFdCDP, and dFdCTP correlated with distribution of endogenous AMP, ADP, and ATP in viable tumor cell regions, showing that gemcitabine active metabolites are reaching the tumor cell compartment, while AZD6738 was located to nonviable tumor regions. The method revealed that the generation of active, phosphorylated dFdC metabolites as well as treatment-induced DNA damage primarily correlated with sites of high proliferation in KPC PDAC tumor tissue, rather than sites of high parent drug abundance.

Quantifying cell cycle-dependent drug sensitivities in cancer using a high throughput synchronisation and screening approach.

BACKGROUND: Chemotherapy and targeted agent anti-cancer efficacy is largely dependent on the proliferative state of tumours, as exemplified by agents that target DNA synthesis/replication or mitosis. As a result, cell cycle specificities of a number of cancer drugs are well known. However, they are yet to be described in a quantifiable manner. METHODS: A scalable cell synchronisation protocol used to screen a library of 235 anti-cancer compounds exposed over six hours in G1 or S/G2 accumulated AsPC-1 cells to generate a cell cycle specificity (CCS) score. FINDINGS: The synchronisation method was associated with reduced method-related cytotoxicity compared to nocodazole, delivering sufficient cell cycle purity and cell numbers to run high-throughput drug library screens. Compounds were identified with G1 and S/G2-associated specificities that, overall, functionally matched with a compound's target/mechanism of action. This annotation was used to describe a synergistic schedule using the CDK4/6 inhibitor, palbociclib, prior to gemcitabine/AZD6738 as well as describe the correlation between the CCS score and published synergistic/antagonistic drug schedules. INTERPRETATION: This is the first highly quantitative description of cell cycle-dependent drug sensitivities that utilised a tractable and tolerated method with potential uses outside the present study. Drug treatments such as those shown to be G1 or S/G2 associated may benefit from scheduling considerations such as after CDK4/6 inhibitors and being first in drug sequences respectively. FUNDING: Cancer Research UK (CRUK) Institute core grants C14303/A17197 and C9545/A29580. The Li Ka Shing Centre where this work was performed was generously funded by CK Hutchison Holdings Limited, the University of Cambridge, CRUK, The Atlantic Philanthropies and others.

Complete loss of ATM function augments replication catastrophe induced by ATR inhibition and gemcitabine in pancreatic cancer models.

BACKGROUND: Personalised medicine strategies may improve outcomes in pancreatic ductal adenocarcinoma (PDAC), but validation of predictive biomarkers is required. Having developed a clinical trial to assess the ATR inhibitor, AZD6738, in combination with gemcitabine (ATRi/gem), we investigated ATM loss as a predictive biomarker of response to ATRi/gem in PDAC. METHODS: Through kinase inhibition, siRNA depletion and CRISPR knockout of ATM, we assessed how ATM targeting affected the sensitivity of PDAC cells to ATRi/gem. Using flow cytometry, immunofluorescence and immunoblotting, we investigated how ATRi/gem synergise in ATM-proficient and ATM-deficient cells, before assessing the impact of ATM loss on ATRi/gem sensitivity in vivo. RESULTS: Complete loss of ATM function (through pharmacological inhibition or CRISPR knockout), but not siRNA depletion, sensitised to ATRi/gem. In ATM-deficient cells, ATRi/gem-induced replication catastrophe was augmented, while phospho-Chk2-T68 and phospho-KAP1-S824 persisted via DNA-PK activity. ATRi/gem caused growth delay in ATM-WT xenografts in NSG mice and induced regression in ATM-KO xenografts. CONCLUSIONS: ATM loss augments replication catastrophe-mediated cell death induced by ATRi/gem and may predict clinical responsiveness to this combination. ATM status should be carefully assessed in tumours from patients with PDAC, since distinction between ATM-low and ATM-null could be critical in maximising the success of clinical trials using ATM expression as a predictive biomarker.

p53 is regulated by aerobic glycolysis in cancer cells by the CtBP family of NADH-dependent transcriptional regulators.

High rates of glycolysis in cancer cells are a well-established characteristic of many human tumors, providing rapidly proliferating cancer cells with metabolites that can be used as precursors for anabolic pathways. Maintenance of high glycolytic rates depends on the lactate dehydrogenase-catalyzed regeneration of NAD+ from GAPDH-generated NADH because an increased NADH:NAD+ ratio inhibits GAPDH. Here, using human breast cancer cell models, we identified a pathway in which changes in the extramitochondrial-free NADH:NAD+ ratio signaled through the CtBP family of NADH-sensitive transcriptional regulators to control the abundance and activity of p53. NADH-free forms of CtBPs cooperated with the p53-binding partner HDM2 to suppress p53 function, and loss of these forms in highly glycolytic cells resulted in p53 accumulation. We propose that this pathway represents a "glycolytic stress response" in which the initiation of a protective p53 response by an increased NADH:NAD+ ratio enables cells to avoid cellular damage caused by mismatches between metabolic supply and demand.

Association of Breast and Ovarian Cancers With Predisposition Genes Identified by Large-Scale Sequencing.

Importance: Since the discovery of BRCA1 and BRCA2, multiple high- and moderate-penetrance genes have been reported as risk factors for hereditary breast cancer, ovarian cancer, or both; however, it is unclear whether these findings represent the complete genetic landscape of these cancers. Systematic investigation of the genetic contributions to breast and ovarian cancers is needed to confirm these findings and explore potentially new associations. Objective: To confirm reported and identify additional predisposition genes for breast or ovarian cancer. Design, Setting, and Participants: In this sample of 11 416 patients with clinical features of breast cancer, ovarian cancer, or both who were referred for genetic testing from 1200 hospitals and clinics across the United States and of 3988 controls who were referred for genetic testing for noncancer conditions between 2014 and 2015, whole-exome sequencing was conducted and gene-phenotype associations were examined. Case-control analyses using the Genome Aggregation Database as a set of reference controls were also conducted. Main Outcomes and Measures: Breast cancer risk associated with pathogenic variants among 625 cancer predisposition genes; association of identified predisposition breast or ovarian cancer genes with the breast cancer subtypes invasive ductal, invasive lobular, hormone receptor-positive, hormone receptor-negative, and male, and with early-onset disease. Results: Of 9639 patients with breast cancer, 3960 (41.1%) were early-onset cases (≤45 years at diagnosis) and 123 (1.3%) were male, with men having an older age at diagnosis than women (mean [SD] age, 61.8 [12.8] vs 48.6 [11.4] years). Of 2051 women with ovarian cancer, 445 (21.7%) received a diagnosis at 45 years or younger. Enrichment of pathogenic variants were identified in 4 non-BRCA genes associated with breast cancer risk: ATM (odds ratio [OR], 2.97; 95% CI, 1.67-5.68), CHEK2 (OR, 2.19; 95% CI, 1.40-3.56), PALB2 (OR, 5.53; 95% CI, 2.24-17.65), and MSH6 (OR, 2.59; 95% CI, 1.35-5.44). Increased risk for ovarian cancer was associated with 4 genes: MSH6 (OR, 4.16; 95% CI, 1.95-9.47), RAD51C (OR, not estimable; false-discovery rate-corrected P = .004), TP53 (OR, 18.50; 95% CI, 2.56-808.10), and ATM (OR, 2.85; 95% CI, 1.30-6.32). Neither the MRN complex genes nor CDKN2A was associated with increased breast or ovarian cancer risk. The findings also do not support previously reported breast cancer associations with the ovarian cancer susceptibility genes BRIP1, RAD51C, and RAD51D, or mismatch repair genes MSH2 and PMS2. Conclusions and Relevance: The results of this large-scale exome sequencing of patients and controls shed light on both well-established and controversial non-BRCA predisposition gene associations with breast or ovarian cancer reported to date and may implicate additional breast or ovarian cancer susceptibility gene candidates involved in DNA repair and genomic maintenance.

The ATR Inhibitor AZD6738 Synergizes with Gemcitabine In Vitro and In Vivo to Induce Pancreatic Ductal Adenocarcinoma Regression.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers, and overall survival rates have barely improved over the past five decades. The antimetabolite gemcitabine remains part of the standard of care but shows very limited antitumor efficacy. Ataxia telangiectasia and Rad3-related protein (ATR), the apical kinase of the intra-S-phase DNA damage response, plays a central role in safeguarding cells from replication stress and can therefore limit the efficacy of antimetabolite drug therapies. We investigated the ability of the ATR inhibitor, AZD6738, to prevent the gemcitabine-induced intra-S-phase checkpoint activation and evaluated the antitumor potential of this combination in vitro and in vivo In PDAC cell lines, AZD6738 inhibited gemcitabine-induced Chk1 activation, prevented cell-cycle arrest, and restrained RRM2 accumulation, leading to the strong induction of replication stress markers only with the combination. Moreover, synergistic growth inhibition was identified in a panel of 5 mouse and 7 human PDAC cell lines using both Bliss Independence and Loewe models. In clonogenic assays, the combination abrogated survival at concentrations for which single agents had minor effects. In vivo, AZD6738 in combination with gemcitabine was well tolerated and induced tumor regression in a subcutaneous allograft model of a KrasG12D; Trp53R172H; Pdx-Cre (KPC) mouse cancer cell line, significantly extending survival. Remarkably, the combination also induced regression of a subgroup of KPC autochthonous tumors, which generally do not respond well to conventional chemotherapy. Altogether, our data suggest that AZD6738 in combination with gemcitabine merits evaluation in a clinical trial in patients with PDAC. Mol Cancer Ther; 17(8); 1670-82. ©2018 AACR.

Mechanistic Distinctions between CHK1 and WEE1 Inhibition Guide the Scheduling of Triple Therapy with Gemcitabine.

Combination of cytotoxic therapy with emerging DNA damage response inhibitors (DDRi) has been limited by tolerability issues. However, the goal of most combination trials has been to administer DDRi with standard-of-care doses of chemotherapy. We hypothesized that mechanism-guided treatment scheduling could reduce the incidence of dose-limiting toxicities and enable tolerable multitherapeutic regimens. Integrative analyses of mathematical modeling and single-cell assays distinguished the synergy kinetics of WEE1 inhibitor (WEE1i) from CHEK1 inhibitor (CHK1i) by potency, spatiotemporal perturbation, and mitotic effects when combined with gemcitabine. These divergent properties collectively supported a triple-agent strategy, whereby a pulse of gemcitabine and CHK1i followed by WEE1i durably suppressed tumor cell growth. In xenografts, CHK1i exaggerated replication stress without mitotic CDK hyperactivation, enriching a geminin-positive subpopulation and intratumoral gemcitabine metabolite. Without overt toxicity, addition of WEE1i to low-dose gemcitabine and CHK1i was most effective in tumor control compared with single and double agents. Overall, our work provides quantitative insights into the mechanisms of DDRi chemosensitization, leading to the rational development of a tolerable multitherapeutic regimen.Significance: Multiple lines of mechanistic insight regarding DNA damage response inhibitors rationally guide the preclinical development of a tolerable multitherapeutic regimen.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/11/3054/F1.large.jpg Cancer Res; 78(11); 3054-66. ©2018 AACR.

Not All Next Generation Sequencing Diagnostics are Created Equal: Understanding the Nuances of Solid Tumor Assay Design for Somatic Mutation Detection.

The molecular characterization of tumors using next generation sequencing (NGS) is an emerging diagnostic tool that is quickly becoming an integral part of clinical decision making. Cancer genomic profiling involves significant challenges including DNA quality and quantity, tumor heterogeneity, and the need to detect a wide variety of complex genetic mutations. Most available comprehensive diagnostic tests rely on primer based amplification or probe based capture methods coupled with NGS to detect hotspot mutation sites or whole regions implicated in disease. These tumor panels utilize highly customized bioinformatics pipelines to perform the difficult task of accurately calling cancer relevant alterations such as single nucleotide variations, small indels or large genomic alterations from the NGS data. In this review, we will discuss the challenges of solid tumor assay design/analysis and report a case study that highlights the need to include complementary technologies (i.e., arrays) and germline analysis in tumor testing to reliably identify copy number alterations and actionable variants.

Hereditary predisposition to ovarian cancer, looking beyond BRCA1/BRCA2.

OBJECTIVE: Genetic predisposition to ovarian cancer is well documented. With the advent of next generation sequencing, hereditary panel testing provides an efficient method for evaluating multiple genes simultaneously. Therefore, we sought to investigate the contribution of 19 genes identified in the literature as increasing the risk of hereditary breast and ovarian cancer (HBOC) in a BRCA1 and BRCA2 negative population of patients with a personal history of breast and/or ovarian cancer by means of a hereditary cancer panel. METHODS: Subjects were referred for multi-gene panel testing between February 2012 and March 2014. Clinical data was ascertained from requisition forms. The incidence of pathogenic mutations (including likely pathogenic), and variant of unknown significance were then calculated for each gene and/or patient cohort. RESULTS: In this cohort of 911 subjects, panel testing identified 67 mutations. With 7.4% of subjects harboring a mutation on this multi-gene panel, the diagnostic yield was increased, compared to testing for BRCA1 and BRCA2 mutations alone. In the ovarian cancer probands, the most frequently mutated genes were BRIP1 (n=8; 1.72%) and MSH6 (n=6; 1.29%). In the breast cancer probands, mutations were most commonly observed in CHEK2 (n=9; 2.54%), ATM (n=3; 0.85%), and TP53 (n=3; 0.85%). CONCLUSIONS: Although further studies are needed to clarify the exact management of patients with a mutation in each gene, this study highlights information that can be captured with panel testing and provides support for incorporation of panel testing into clinical practice.

Enhanced utility of family-centered diagnostic exome sequencing with inheritance model-based analysis: results from 500 unselected families with undiagnosed genetic conditions.

PURPOSE: Diagnostic exome sequencing was immediately successful in diagnosing patients in whom traditional technologies were uninformative. Herein, we provide the results from the first 500 probands referred to a clinical laboratory for diagnostic exome sequencing. METHODS: Family-based exome sequencing included whole-exome sequencing followed by family inheritance-based model filtering, comprehensive medical review, familial cosegregation analysis, and analysis of novel genes. RESULTS: A positive or likely positive result in a characterized gene was identified in 30% of patients (152/500). A novel gene finding was identified in 7.5% of patients (31/416). The highest diagnostic rates were observed among patients with ataxia, multiple congenital anomalies, and epilepsy (44, 36, and 35%, respectively). Twenty-three percent of positive findings were within genes characterized within the past 2 years. The diagnostic rate was significantly higher among families undergoing a trio (37%) as compared with a singleton (21%) whole-exome testing strategy. CONCLUSION: Overall, we present results from the largest clinical cohort of diagnostic exome sequencing cases to date. These data demonstrate the utility of family-based exome sequencing and analysis to obtain the highest reported detection rate in an unselected clinical cohort, illustrating the utility of diagnostic exome sequencing as a transformative technology for the molecular diagnosis of genetic disease.

Comprehensive genetic analysis of the cystic fibrosis transmembrane conductance regulator from dried blood specimens--implications for newborn screening.

PURPOSE: In the United States, approximately 1/3,700 babies is born with cystic fibrosis each year. The >1,300 documented sequence variants pose a challenge for detection of cystic fibrosis through genetic screening. To investigate whether comprehensive characterization of the cystic fibrosis gene is feasible using dried newborn blood specimens, we modified the whole blood Ambry Test: CF and determined its sensitivity by testing DNA from individuals with cystic fibrosis who still had unknown mutations after commercial mutation panel testing. METHODS: DNA from 42 archived newborn dried blood specimens of affected Hispanic, African-American and Caucasian individuals in California was analyzed by temporal temperature gradient electrophoresis screening and targeted sequencing, and by gross deletion analysis. RESULTS: Excluding two specimens that could not be analyzed due to poor DNA quality, we report a 100% sensitivity and clinical detection rate in the remaining 40 patients. Eighty-three mutations representing 40 different variants were detected, including 8 novel mutations. CONCLUSIONS: This study demonstrates the feasibility of temporal temperature gradient electrophoresis-based full sequence analysis and targeted sequencing from DNA in newborn blood specimens. The Ambry Test: CF, as an additional step in cystic fibrosis newborn screening models, can be used to dramatically reduce the number of cystic fibrosis carrier sweat test referrals.

Diagnostic testing by CFTR gene mutation analysis in a large group of Hispanics: novel mutations and assessment of a population-specific mutation spectrum.

Characterization of CFTR mutations in the U.S. Hispanic population is vital to early diagnosis, genetic counseling, patient-specific treatment, and the understanding of cystic fibrosis (CF) pathogenesis. The mutation spectrum in Hispanics, however, remains poorly defined. A group of 257 self-identified Hispanics with clinical manifestations consistent with CF were studied by temporal temperature gradient electrophoresis and/or DNA sequencing. A total of 183 mutations were identified, including 14 different amino acid-changing novel variants. A significant proportion (78/85) of the different mutations identified would not have been detected by the ACMG/ACOG-recommended 25-mutation screening panel. Over one third of the mutations (27/85) occurred with a relative frequency >1%, which illustrates that the identified mutations are not all rare. This is supported by a comparison with other large CFTR studies. These results underscore the disparity in mutation identification between Caucasians and Hispanics and show utility for comprehensive diagnostic CFTR mutation analysis in this population.