Erica K. Barnell

CIViC is a community knowledgebase for expert crowdsourcing the clinical interpretation of variants in cancer

CIViC is an expert-crowdsourced knowledgebase for Clinical Interpretation of Variants in Cancer describing the therapeutic, prognostic, diagnostic and predisposing relevance of inherited and somatic variants of all types. CIViC is committed to open-source code, open-access content, public application programming interfaces (APIs) and provenance of supporting evidence to allow for the transparent creation of current and accurate variant interpretations for use in cancer precision medicine.

Environmental Enteric Dysfunction Includes a Broad Spectrum of Inflammatory Responses and Epithelial Repair Processes

Background & Aims Environmental enteric dysfunction (EED), a chronic diffuse inflammation of the small intestine, is associated with stunting in children in the developing world. The pathobiology of EED is poorly understood because of the lack of a method to elucidate the host response. This study tested a novel microarray method to overcome limitation of RNA sequencing to interrogate the host transcriptome in feces in Malawian children with EED. Methods In 259 children, EED was measured by lactulose permeability (%L). After isolating low copy numbers of host messenger RNA, the transcriptome was reliably and reproducibly profiled, validated by polymerase chain reaction. Messenger RNA copy number then was correlated with %L and differential expression in EED. The transcripts identified were mapped to biological pathways and processes. The children studied had a range of %L values, consistent with a spectrum of EED from none to severe. Results We identified 12 transcripts associated with the severity of EED, including chemokines that stimulate T-cell proliferation, Fc fragments of multiple immunoglobulin families, interferon-induced proteins, activators of neutrophils and B cells, and mediators that dampen cellular responses to hormones. EED-associated transcripts mapped to pathways related to cell adhesion, and responses to a broad spectrum of viral, bacterial, and parasitic microbes. Several mucins, regulatory factors, and protein kinases associated with the maintenance of the mucous layer were expressed less in children with EED than in normal children. Conclusions EED represents the activation of diverse elements of the immune system and is associated with widespread intestinal barrier disruption. Differentially expressed transcripts, appropriately enumerated, should be explored as potential biomarkers.

A phase II trial of neoadjuvant MK-2206, an AKT inhibitor, with anastrozole in clinical stage II or III PIK3CA-mutant ER-positive and HER2-negative breast cancer

Purpose: Hyperactivation of AKT is common and associated with endocrine resistance in estrogen receptor–positive (ER+) breast cancer. The allosteric pan-AKT inhibitor MK-2206 induced apoptosis in PIK3CA-mutant ER+ breast cancer under estrogen-deprived condition in preclinical studies. This neoadjuvant phase II trial was therefore conducted to test the hypothesis that adding MK-2206 to anastrozole induces pathologic complete response (pCR) in PIK3CA mutant ER+ breast cancer. Experimental Design: Potential eligible patients with clinical stage II/III ER+/HER2− breast cancer were preregistered and received anastrozole (goserelin if premenopausal) for 28 days in cycle 0 pending tumor PIK3CA sequencing. Patients positive for PIK3CA mutation in the tumor were eligible to start MK-2206 (150 mg orally weekly, with prophylactic prednisone) on cycle 1 day 2 (C1D2) and to receive a maximum of four 28-day cycles of combination therapy before surgery. Serial biopsies were collected at preregistration, C1D1 and C1D17. Results: Fifty-one patients preregistered and 16 of 22 with PIK3CA-mutant tumors received study drug. Three patients went off study due to C1D17 Ki67 >10% (n = 2) and toxicity (n = 1). Thirteen patients completed neoadjuvant therapy followed by surgery. No pCRs were observed. Rash was common. MK-2206 did not further suppress cell proliferation and did not induce apoptosis on C1D17 biopsies. Although AKT phosphorylation was reduced, PRAS40 phosphorylation at C1D17 after MK-2206 persisted. One patient acquired an ESR1 mutation at surgery. Conclusions: MK-2206 is unlikely to add to the efficacy of anastrozole alone in PIK3CA-mutant ER+ breast cancer and should not be studied further in the target patient population. Clin Cancer Res; 23(22); 6823–32. ©2017 AACR.

CIViC: A knowledgebase for expert-crowdsourcing the clinical interpretation of variants in cancer.

CIViC is an expert crowdsourced knowledgebase for Clinical Interpretation of Variants in Cancer (www.civicdb.org) describing the therapeutic, prognostic, and diagnostic relevance of inherited and somatic variants of all types. CIViC is committed to open source code, open access content, public application programming interfaces (APIs), and provenance of supporting evidence to allow for the transparent creation of current and accurate variant interpretations for use in cancer precision medicine.

Growth and HIV-free survival of HIV-exposed infants in Malawi: a randomized trial of two complementary feeding interventions in the context of maternal antiretroviral therapy.

Objective:To compare the growth of HIV-exposed children receiving 1 of 2 complementary foods after prevention of mother-to-child HIV transmission through maternal lifelong antiretroviral therapy (ART). Methods:In rural Malawi, 280 HIV-infected pregnant women were consecutively identified and offered ART, without consideration of their CD4 counts. Mothers were supported to exclusively breast-feed and children tested for HIV status at 1.5 and 5.5 months of age. From this group, 248 HIV-exposed children were enrolled and randomized to receive micronutrients with either whole milk powder or a ready-to-use complementary food (RUF), until the child reached 12 months of age. Children were followed until 18 months of age. Results:HIV-free survival at 12 months was 90% (95% confidence interval: 87% to 94%). Exclusive breast-feeding for the first 6 months of life was practiced in 97% of the children. At 12 months of age, 89% of the children continued to be breast-fed. At 6 months of age, infants had a weight-for-height z score of 0.7 ± 1.1 (mean ± SD) and length-for-age z score of −1.3 ± 1.2. The decrease in length-for-age z score among children receiving RUF at 12 months of age was greater than that seen in those receiving milk powder (−0.3 ± 0.8 vs −0.1 ± 0.7, P = 0.04). Mean weight-for-height z score was >0 at 12 and 18 months of age in both groups. Conclusions:HIV-free survival ≥90% at 12 months was achieved with maternal ART while either milk powder or RUF as a complementary food preserved child anthropometry. Breast-feeding by mothers receiving ART was acceptable.

Standard operating procedure for somatic variant refinement of tumor sequencing data

Purpose: Manual review of aligned sequencing reads is required to develop a high-quality list of somatic variants from massively parallel sequencing data (MPS). Despite widespread use in analyzing MPS data, there has been little attempt to describe methods for manual review, resulting in high inter- and intra-lab variability in somatic variant detection and characterization of tumors. Methods: Open source software was used to develop an optimal method for manual review setup. We also developed a systemic approach to visually inspect each variant during manual review. Results: We present a standard operating procedures for somatic variant refinement for use by manual reviewers. The approach is enhanced through representative examples of 4 different manual review categories that indicate a reviewer’s confidence in the somatic variant call and 19 annotation tags that contextualize commonly observed sequencing patterns during manual review. Representative examples provide detailed instructions on how to classify variants during manual review to rectify lack of confidence in automated somatic variant detection. Conclusion: Standardization of somatic variant refinement through systematization of manual review will improve the consistency and reproducibility of identifying true somatic variants after automated variant calling.

Adapting crowdsourced clinical cancer curation in CIViC to the ClinGen minimum variant level data community-driven standards

Harmonization of cancer variant representation, efficient communication, and free distribution of clinical variant‐associated knowledge are central problems that arise with increased usage of clinical next‐generation sequencing. The Clinical Genome Resource (ClinGen) Somatic Working Group (WG) developed a minimal variant level data (MVLD) representation of cancer variants, and has an ongoing collaboration with Clinical Interpretations of Variants in Cancer (CIViC), an open‐source platform supporting crowdsourced and expert‐moderated cancer variant curation. Harmonization between MVLD and CIViC variant formats was assessed by formal field‐by‐field analysis. Adjustments to the CIViC format were made to harmonize with MVLD and support ClinGen Somatic WG curation activities, including four new features in CIViC: (1) introduction of an assertions feature for clinical variant assessment following the Association of Molecular Pathologists (AMP) guidelines, (2) group‐level curation tracking for organizations, enabling member transparency, and curation effort summaries, (3) introduction of ClinGen Allele Registry IDs to CIViC, and (4) mapping of CIViC assertions into ClinVar submission with automated submissions. A generalizable workflow utilizing MVLD and new CIViC features is outlined for use by ClinGen Somatic WG task teams for curation and submission to ClinVar, and provides a model for promoting harmonization of cancer variant representation and efficient distribution of this information.

Spontaneous aggressive ERα+ mammary tumor model is driven by Kras activation

The NRL-PRL murine model, defined by mammary-selective transgenic rat prolactin ligand rPrl expression, establishes spontaneous ER+ mammary tumors, mimicking the association between elevated prolactin (PRL) and risk for development of ER+ breast cancer in postmenopausal women. Whole genome and exome sequencing in a discovery cohort (n=5) of end stage tumors revealed canonical activating mutations and copy number amplifications of Kras. The frequent mutations in this pathway were validated in an extension cohort, identifying activating Ras alterations in 79% (23/29) of tumors. Transcriptome analyses over the course of oncogenesis revealed marked alterations associated with Ras activity in established tumors, compared to preneoplastic tissues, in cell-intrinsic processes associated with mitosis, cell adhesion and invasion, as well as in the tumor microenvironment, including immune activity. These genomic analyses suggest that PRL induces a selective bottleneck for spontaneous Ras-driven tumors which may model a subset of aggressive clinical ER+ breast cancers.

Standard operating procedure for somatic variant refinement of sequencing data with paired tumor and normal samples

PurposeFollowing automated variant calling, manual review of aligned read sequences is required to identify a high-quality list of somatic variants. Despite widespread use in analyzing sequence data, methods to standardize manual review have not been described, resulting in high inter- and intralab variability.MethodsThis manual review standard operating procedure (SOP) consists of methods to annotate variants with four different calls and 19 tags. The calls indicate a reviewer’s confidence in each variant and the tags indicate commonly observed sequencing patterns and artifacts that inform the manual review call. Four individuals were asked to classify variants prior to, and after, reading the SOP and accuracy was assessed by comparing reviewer calls with orthogonal validation sequencing.ResultsAfter reading the SOP, average accuracy in somatic variant identification increased by 16.7% (p value = 0.0298) and average interreviewer agreement increased by 12.7% (p value < 0.001). Manual review conducted after reading the SOP did not significantly increase reviewer time.ConclusionThis SOP supports and enhances manual somatic variant detection by improving reviewer accuracy while reducing the interreviewer variability for variant calling and annotation.

Oral Cavity Squamous Cell Carcinoma Xenografts Retain Complex Genotypes and Intertumor Molecular Heterogeneity

SUMMARY Herein, we report an oral cavity squamous cell carcinoma (OCSCC) patient-derived xenograft (PDX) platform, with genomic annotation useful for co-clinical trial and mechanistic studies. Genomic analysis included whole-exome sequencing (WES) and transcriptome sequencing (RNA-seq) on 16 tumors and matched PDXs and additional whole-genome sequencing (WGS) on 9 of these pairs as a representative subset of a larger OCSCC PDX repository (n = 63). In 12 models with high purity, more than 90% of variants detected in the tumor were retained in the matched PDX. The genomic landscape across these PDXs reflected OCSCC molecular heterogeneity, including previously described basal, mesenchymal, and classical molecular subtypes. To demonstrate the integration of PDXs into a clinical trial framework, we show that pharmacological intervention in PDXs parallels clinical response and extends patient data. Together, these data describe a repository of OCSCC-specific PDXs and illustrate conservation of primary tumor genotypes, intratumoral heterogeneity, and co-clinical trial application.