Advancing NGS quality control to enable measurement of actionable mutations in circulating tumor DNA.

The primary objective of the FDA-led Sequencing and Quality Control Phase 2 (SEQC2) project is to develop standard analysis protocols and quality control metrics for use in DNA testing to enhance scientific research and precision medicine. This study reports a targeted next-generation sequencing (NGS) method that will enable more accurate detection of actionable mutations in circulating tumor DNA (ctDNA) clinical specimens. To accomplish this, a synthetic internal standard spike-in was designed for each actionable mutation target, suitable for use in NGS following hybrid capture enrichment and unique molecular index (UMI) or non-UMI library preparation. When mixed with contrived ctDNA reference samples, internal standards enabled calculation of technical error rate, limit of blank, and limit of detection for each variant at each nucleotide position in each sample. True-positive mutations with variant allele fraction too low for detection by current practice were detected with this method, thereby increasing sensitivity.

RNA Immune Signatures from Pan-Cancer Analysis Are Prognostic for High-Grade Serous Ovarian Cancer and Other Female Cancers.

Immune cell infiltrates within the tumor microenvironment can influence treatment response and outcome in several cancers. In this study, we developed RNA-based immune signatures from pan-cancer analysis that could serve as potential markers across tumor types and tested them for association with outcome in high-grade serous ovarian cancer (HGSOC) and other female cancers. Pan-cancer RNA-Seq cluster analysis of immune-related gene expression profiles in The Cancer Genome Atlas (TCGA) from 29 different solid tumors (4446 specimens) identified distinct but concordant gene signatures. Among these immune signatures, Cytotoxic Lymphocyte Immune Signature (CLIS), T-cell trafficking (TCT), and the TCT to M2 tumor-associated macrophage (M2TAM) ratio (TCT:M2TAM) were significantly (p < 0.05) associated with overall survival (OS), using multivariable Cox proportional hazards regression models, in a discovery cohort and two independent validation cohorts of HGSOC patients. Notably, the TCT:M2TAM ratio was highly significant (p ≤ 0.000001) in two HGSOC cohorts. Immune signatures were also significant (p < 0.05) in the presence of tumor cytoreduction, BRCA1/2 mutation, and COL2A1 expression. Importantly, the CLIS and TCT signatures were also validated for prognostic significance (p < 0.05) in TCGA cohorts for endometrial and high tumor mutational burden (Hi-TMB) breast cancer. These immune signatures also have the potential for being predictive in other cancers and for patients following different treatment strategies.

Increased expression of neurotensin in high grade serous ovarian carcinoma with evidence of serous tubal intraepithelial carcinoma.

High grade serous ovarian carcinoma (HGSC) without identifiable serous tubal intraepithelial carcinoma (STIC) within the fallopian tube (FT) occurs in approximately 50% of patients. The objective of this study was to use a multisite tumor sampling approach to study HGSC with and without STIC. RNAseq analysis of HGSC samples collected from multiple sites e.g. ovary, FT and peritoneum, revealed moderate levels of intrapatient heterogeneity in gene expression that could influence molecular profiles. Mixed-model ANOVA analysis of gene expression in tumor samples from patients with multiple tumor sites (n = 13) and patients with a single site tumor sample (n = 11) to compare HGSC-STIC to HGSC-NOSTIC identified neurotensin (NTS) as significantly higher (> two-fold change, False Discovery Rate (FDR) < 0.10) in HGSC-STIC. This data was validated using publicly available RNA-Seq datasets. Concordance between higher NTS gene expression and NTS peptide levels in HGSC-STIC samples was demonstrated by immunohistochemistry. To determine the role of NTS in HGSC, five ovarian cancer (OvCa) cell lines were screened for expression of NTS and its receptors, NTSR1 and NTSR3. Increased expression of NTS and NSTR1 was observed in several of the OvCa cells, whereas the NTSR3 receptor was lower in all OvCa cells, compared to immortalized FT epithelial cells. Treatment with NTSR1 inhibitor (SR48692) decreased cell proliferation, but increased cell migration in OvCa cells. The effects of SR48692 were receptor mediated, since transient RNAi knockdown of NTSR1 mimicked the migratory effects and knockdown of NTSR3 mimicked the anti-proliferative effects. Further, knockdown of NTSR1 or NTSR3 was associated with acquisition of distinct morphological phenotypes, epithelial or mesenchymal, respectively. Taken together, our results reveal a difference in a biologically active pathway between HGSC with and without STIC. Furthermore, we identify neurotensin signaling as an important pathway involved in cell proliferation and epithelial-mesenchymal transition in HGSC-STIC which warrants further study as a potential therapeutic target. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Tumor mutational burden is a determinant of immune-mediated survival in breast cancer.

Mounting evidence supports a role for the immune system in breast cancer outcomes. The ability to distinguish highly immunogenic tumors susceptible to anti-tumor immunity from weakly immunogenic or inherently immune-resistant tumors would guide development of therapeutic strategies in breast cancer. Genomic, transcriptomic and clinical data from The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohorts were used to examine statistical associations between tumor mutational burden (TMB) and the survival of patients whose tumors were assigned to previously-described prognostic immune subclasses reflecting favorable, weak or poor immune-infiltrate dispositions (FID, WID or PID, respectively). Tumor immune subclasses were associated with survival in patients with high TMB (TMB-Hi, P < 0.001) but not in those with low TMB (TMB-Lo, P = 0.44). This statistical relationship was confirmed in the METABRIC cohort (TMB-Hi, P = 0.047; TMB-Lo, P = 0.39), and also found to hold true in the more-indolent Luminal A tumor subtype (TMB-Hi, P = 0.011; TMB-Lo, P = 0.91). In TMB-Hi tumors, the FID subclass was associated with prolonged survival independent of tumor stage, molecular subtype, age and treatment. Copy number analysis revealed the reproducible, preferential amplification of chromosome 1q immune-regulatory genes in the PID immune subclass. These findings demonstrate a previously unappreciated role for TMB as a determinant of immune-mediated survival of breast cancer patients and identify candidate immune-regulatory mechanisms associated with immunologically cold tumors. Immune subtyping of breast cancers may offer opportunities for therapeutic stratification.

Clonal lineage of high grade serous ovarian cancer in a patient with neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene encoding neurofibromin, which negatively regulates Ras signaling. NF1 patients have an increased risk of developing early onset breast cancer, however, the association between NF1 and high grade serous ovarian cancer (HGSOC) is unclear. Since most NF1-related tumors exhibit early biallelic inactivation of NF1, we evaluated the evolution of genetic alterations in HGSOC in an NF1 patient. Somatic variation analysis of whole exome sequencing of tumor samples from both ovaries and a peritoneal metastasis showed a clonal lineage originating from an ancestral clone within the left adnexa, which exhibited copy number (CN) loss of heterozygosity (LOH) in the region of chromosome 17 containing TP53, NF1, and BRCA1 and mutation of the other TP53 allele. This event led to biallelic inactivation of NF1 and TP53 and LOH for the BRCA1 germline mutation. Subsequent CN alterations were found in the dominant tumor clone in the left ovary and nearly 100% of tumor at other sites. Neurofibromin modeling studies suggested that the germline NF1 mutation could potentially alter protein function. These results demonstrate early, biallelic inactivation of neurofibromin in HGSOC and highlight the potential of targeting RAS signaling in NF1 patients.

A B-Cell Gene Signature Correlates With the Extent of Gluten-Induced Intestinal Injury in Celiac Disease.

BACKGROUND & AIMS: Celiac disease (CeD) provides an opportunity to study autoimmunity and the transition in immune cells as dietary gluten induces small intestinal lesions. METHODS: Seventy-three celiac disease patients on a long-term, gluten-free diet ingested a known amount of gluten daily for 6 weeks. A peripheral blood sample and intestinal biopsy specimens were taken before and 6 weeks after initiating the gluten challenge. Biopsy results were reported on a continuous numeric scale that measured the villus-height-to-crypt-depth ratio to quantify gluten-induced intestinal injury. Pooled B and T cells were isolated from whole blood, and RNA was analyzed by DNA microarray looking for changes in peripheral B- and T-cell gene expression that correlated with changes in villus height to crypt depth, as patients maintained a relatively healthy intestinal mucosa or deteriorated in the face of a gluten challenge. RESULTS: Gluten-dependent intestinal damage from baseline to 6 weeks varied widely across all patients, ranging from no change to extensive damage. Genes differentially expressed in B cells correlated strongly with the extent of intestinal damage. A relative increase in B-cell gene expression correlated with a lack of sensitivity to gluten whereas their relative decrease correlated with gluten-induced mucosal injury. A core B-cell gene module, representing a subset of B-cell genes analyzed, accounted for the correlation with intestinal injury. CONCLUSIONS: Genes comprising the core B-cell module showed a net increase in expression from baseline to 6 weeks in patients with little to no intestinal damage, suggesting that these individuals may have mounted a B-cell immune response to maintain mucosal homeostasis and circumvent inflammation. DNA microarray data were deposited at the GEO repository (accession number: GSE87629; available: https://www.ncbi.nlm.nih.gov/geo/).

Expression profile of COL2A1 and the pseudogene SLC6A10P predicts tumor recurrence in high-grade serous ovarian cancer.

Tumor recurrence, following initial response to adjuvant chemotherapy, is a major problem in women with high-grade serous ovarian cancer (HGSOC). Microarray analysis of primary tumors has identified genes that may be useful in risk stratification/overall survival, but are of limited value in predicting the >70% rate for tumor recurrence. In this study, we performed RNA-Seq analysis of primary and recurrent HGSOC to first identify unique differentially expressed genes. From this dataset, we selected 21 archetypical coding genes and one noncoding RNA, based on statistically significant differences in their expression profile between tumors, for validation by qPCR in a larger cohort of 110 ovarian tumors (71 primary and 39 recurrent) and for testing association of specific genes with time-to-recurrence (TTR). Kaplan-Meier tests revealed that high expression of collagen type II, alpha 1 (COL2A1) was associated with delayed TTR (HR = 0.47, 95% CI: 0.27-0.82, p = 0.008), whereas low expression of the pseudogene, solute carrier family 6 member 10 (SLC6A10P), was associated with longer TTR (HR = 0.53, 95% CI: 0.30-0.93, p = 0.027). Notably, TTR was significantly delayed for tumors that simultaneously highly expressed COL2A1 and lowly expressed SLC6A10P (HR = 0.21, 95% CI: 0.082-0.54, p = 0.0011), an estimated median of 95 months as compared to an estimated median of 16 months for subjects expressing other levels of COL2A1 and SLC6A10P. Thus, evaluating expression levels of COL2A1 and SLC6A10P at primary surgery could be beneficial for clinically managing recurrence of HGSOC.

Dysregulation of the epigenome in triple-negative breast cancers: basal-like and claudin-low breast cancers express aberrant DNA hypermethylation.

A subset of human breast cancer cell lines exhibits aberrant DNA hypermethylation that is characterized by hyperactivity of the DNA methyltransferase enzymes, overexpression of DNMT3b, and concurrent methylation-dependent silencing of numerous epigenetic biomarker genes. The objective of this study was to determine if this aberrant DNA hypermethylation (i) is found in primary breast cancers, (ii) is associated with specific breast cancer molecular subtypes, and (iii) influences patient outcomes. Analysis of epigenetic biomarker genes (CDH1, CEACAM6, CST6, ESR1, GNA11, MUC1, MYB, SCNN1A, and TFF3) identified a gene expression signature characterized by reduced expression levels or loss of expression among a cohort of primary breast cancers. The breast cancers that express this gene expression signature are enriched for triple-negative subtypes - basal-like and claudin-low breast cancers. Methylation analysis of primary breast cancers showed extensive promoter hypermethylation of epigenetic biomarker genes among triple-negative breast cancers, compared to other breast cancer subclasses where promoter hypermethylation events were less frequent. Furthermore, triple-negative breast cancers either did not express or expressed significantly reduced levels of protein corresponding to methylation-sensitive biomarker gene products. Together, these findings suggest strongly that loss of epigenetic biomarker gene expression is frequently associated with gene promoter hypermethylation events. We propose that aberrant DNA hypermethylation is a common characteristic of triple-negative breast cancers and may represent a fundamental biological property of basal-like and claudin-low breast cancers. Kaplan-Meier analysis of relapse-free survival revealed a survival disadvantage for patients with breast cancers that exhibit aberrant DNA hypermethylation. Identification of this distinguishing trait among triple-negative breast cancers forms the basis for development of new rational therapies that target the epigenome in patients with basal-like and claudin-low breast cancers.

The MicroArray Quality Control (MAQC)-II study of common practices for the development and validation of microarray-based predictive models.

Gene expression data from microarrays are being applied to predict preclinical and clinical endpoints, but the reliability of these predictions has not been established. In the MAQC-II project, 36 independent teams analyzed six microarray data sets to generate predictive models for classifying a sample with respect to one of 13 endpoints indicative of lung or liver toxicity in rodents, or of breast cancer, multiple myeloma or neuroblastoma in humans. In total, >30,000 models were built using many combinations of analytical methods. The teams generated predictive models without knowing the biological meaning of some of the endpoints and, to mimic clinical reality, tested the models on data that had not been used for training. We found that model performance depended largely on the endpoint and team proficiency and that different approaches generated models of similar performance. The conclusions and recommendations from MAQC-II should be useful for regulatory agencies, study committees and independent investigators that evaluate methods for global gene expression analysis.

DNMT3b overexpression contributes to a hypermethylator phenotype in human breast cancer cell lines.

BACKGROUND: DNA hypermethylation events and other epimutations occur in many neoplasms, producing gene expression changes that contribute to neoplastic transformation, tumorigenesis, and tumor behavior. Some human cancers exhibit a hypermethylator phenotype, characterized by concurrent DNA methylation-dependent silencing of multiple genes. To determine if a hypermethylation defect occurs in breast cancer, the expression profile and promoter methylation status of methylation-sensitive genes were evaluated among breast cancer cell lines. RESULTS: The relationship between gene expression (assessed by RT-PCR and quantitative real-time PCR), promoter methylation (assessed by methylation-specific PCR, bisulfite sequencing, and 5-aza-2'deoxycytidine treatment), and the DNA methyltransferase machinery (total DNMT activity and expression of DNMT1, DNMT3a, and DNMT3b proteins) were examined in 12 breast cancer cell lines. Unsupervised cluster analysis of the expression of 64 methylation-sensitive genes revealed two groups of cell lines that possess distinct methylation signatures: (i) hypermethylator cell lines, and (ii) low-frequency methylator cell lines. The hypermethylator cell lines are characterized by high rates of concurrent methylation of six genes (CDH1, CEACAM6, CST6, ESR1, LCN2, SCNN1A), whereas the low-frequency methylator cell lines do not methylate these genes. Hypermethylator cell lines coordinately overexpress total DNMT activity and DNMT3b protein levels compared to normal breast epithelial cells. In contrast, most low-frequency methylator cell lines possess DNMT activity and protein levels that are indistinguishable from normal. Microarray data mining identified a strong cluster of primary breast tumors that express the hypermethylation signature defined by CDH1, CEACAM6, CST6, ESR1, LCN2, and SCNN1A. This subset of breast cancers represents 18/88 (20%) tumors in the dataset analyzed, and 100% of these tumors were classified as basal-like, suggesting that the hypermethylator defect cosegregates with poor prognosis breast cancers. CONCLUSION: These observations combine to strongly suggest that: (a) a subset of breast cancer cell lines express a hypermethylator phenotype, (b) the hypermethylation defect in these breast cancer cell lines is related to aberrant overexpression of DNMT activity, (c) overexpression of DNMT3b protein significantly contributes to the elevated DNMT activity observed in tumor cells expressing this phenotype, and (d) the six-gene hypermethylator signature characterized in breast cancer cell lines defines a distinct cluster of primary basal-like breast cancers.

DNA methylation-dependent silencing of CST6 in human breast cancer cell lines.

Cystatin M (CST6) is a candidate breast cancer tumor suppressor that is expressed in normal and premalignant breast epithelium, but not in metastatic breast cancer cell lines. CST6 is subject to epigenetic silencing in MCF-7 breast cancer cells related to methylation of the CpG island that encompasses the CST6 proximal promoter region and exon 1. In the current study, CST6 CpG island methylation and expression status was examined in a panel of breast cancer cell lines. Seven of 12 (58%) cell lines lack detectable expression of CST6 and treatment of these cells with 5-aza-2'-deoxycytidine resulted in a significant increase in CST6 expression, suggesting that the loss of expression may be related to methylation-dependent epigenetic silencing. Bisulfite sequencing of CST6 in a subset of breast cancer cell lines revealed CpG island hypermethylation in CST6-negative cells, and an absence of CpG island methylation in cells that express CST6. The extent of regional methylation was strongly associated with the lack of expression of CST6 among these cell lines. In particular, hypermethylation of the proximal promoter was significantly associated with CST6 gene silencing, and methylation of a number of individual CpGs was found to be statistically correlated with extinction of gene expression. These results establish a strong link between CST6 promoter hypermethylation and loss of CST6 expression in breast cancer cell lines, and suggest that methylation-dependent epigenetic silencing of CST6 may represent an important mechanism for loss of CST6 during breast carcinogenesis in vivo.

DNA methylation-dependent epigenetic regulation of gene expression in MCF-7 breast cancer cells.

To identify epigenetically-regulated genes in breast cancer, MCF-7 cells were exposed to 250 nM 5-aza or 5-aza + 50 nM TSA for three weeks followed by a five week recovery period after treatment withdrawal and gene expression patterns were examined by microarray analysis. We identified 20 genes that are associated with a > or = 2-fold increase in expression in response to the demethylating treatment but returned to control levels after treatment withdrawal. RT-PCR verified that the genes identified were expressed at low or undetectable levels in control MCF-7 cells, but increased expression in treated cells. Most of these putative epigentically-regulated genes in MCF-7 cells do not contain CpG islands. In fact, these genes could be classified based upon their promoter CpG features, including genes with: (1) typical CpG features (CpG islands), (2) intermediate CpG features (weak CpG islands), and (3) atypical CpG features (no CpG islands). Prototype genes from each class (including CpG-deficient genes) were shown to be methylation-sensitive (subject to CpG methylation and responsive to demethylating agents), suggesting that not all gene targets of DNA methylation in breast cancer will contain a CpG island. Based upon the results of the current study and observations from the literature, we propose expansion of the current model for methylation-dependent regulation of gene expression to include genes lacking typical CpG islands. The expanded model we propose recognizes that all promoter CpG dinucleotides represent legitimate targets for DNA methylation and that the methylation of specific CpG dinucleotides in critical domains of regulatory regions can result in gene silencing.