Arezou A. Ghazani

Ultrasensitive Clinical Enumeration of Rare Cells ex Vivo Using a Micro-Hall Detector

A hybrid microfluidic/semiconductor chip analyzes single, immunomagnetically tagged ovarian cancer cells in unprocessed biological samples. Magnetic Microchip Counts Tumor Cells The idiom “looking for a needle in a haystack” could not be applied more appropriately in medicine than to describe the detection of circulating tumor cells (CTCs). Often, only 10 of these rare cells are present in 10 ml of blood; that’s about 1 CTC for every 1 billion blood cells. Despite their scarcity, these cells may hold a wealth of information to help guide treatment decisions for cancer patients. Undaunted, Issadore and colleagues developed a miniature device that combines microfluidics and magnets to measure CTCs in patient blood at single-cell resolution. The authors designed a micro-Hall detector (μHD) that senses the magnetic moment of particles. In this system, cells were first labeled with magnetic beads conjugated to antibodies directed at a target cell surface molecule. The magnetically labeled cells could then be flowed through the microfluidic channel, where tiny Hall detectors would sense their presence. Issadore et al. first tested the μHD with cells derived from human epithelial cancers (such as breast and brain), looking for three different cancer-related markers: human epidermal growth factor receptor 2 (HER2)/neu, epidermal growth factor receptor (EGFR), and EpCAM. Out of a mixture of blood cells—some labeled, some not—the authors only missed a cancer cell 10% of the time, compared with flow cytometry (the gold standard in the clinic), which had a false-negative rate of 81%. The μHD was also able to detect multiple biomarkers on individual cells simultaneously, which could work toward further refining subpopulations of rare cells according to surface expression. To show use in the clinic, Issadore and coauthors noted elevated numbers of CTCs in the blood of 20 ovarian cancer patients, but not in any of the 15 healthy volunteers. By comparison, CellSearch (the gold standard technology for CTC enumeration) only detected CTCs in 25% of the same patient samples. The μHD appears to be a more sensitive cell counter than existing devices, with the potential to change patient management and disease monitoring in the clinic. The needles are still there, but we now have a rapid way of sorting through the haystack. The ability to detect rare cells (<100 cells/ml whole blood) and obtain quantitative measurements of specific biomarkers on single cells is increasingly important in basic biomedical research. Implementing such methodology for widespread use in the clinic, however, has been hampered by low cell density, small sample sizes, and requisite sample purification. To overcome these challenges, we have developed a microfluidic chip–based micro-Hall detector (μHD), which can directly measure single, immunomagnetically tagged cells in whole blood. The μHD can detect single cells even in the presence of vast numbers of blood cells and unbound reactants, and does not require any washing or purification steps. In addition, the high bandwidth and sensitivity of the semiconductor technology used in the μHD enables high-throughput screening (currently ~107 cells/min). The clinical use of the μHD chip was demonstrated by detecting circulating tumor cells in whole blood of 20 ovarian cancer patients at higher sensitivity than currently possible with clinical standards. Furthermore, the use of a panel of magnetic nanoparticles, distinguished with unique magnetization properties and bio-orthogonal chemistry, allowed simultaneous detection of the biomarkers epithelial cell adhesion molecule (EpCAM), human epidermal growth factor receptor 2 (HER2)/neu, and epidermal growth factor receptor (EGFR) on individual cells. This cost-effective, single-cell analytical technique is well suited to perform molecular and cellular diagnosis of rare cells in the clinic.

Performance of ACMG-AMP Variant-Interpretation Guidelines among Nine Laboratories in the Clinical Sequencing Exploratory Research Consortium

Evaluating the pathogenicity of a variant is challenging given the plethora of types of genetic evidence that laboratories consider. Deciding how to weigh each type of evidence is difficult, and standards have been needed. In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published guidelines for the assessment of variants in genes associated with Mendelian diseases. Nine molecular diagnostic laboratories involved in the Clinical Sequencing Exploratory Research (CSER) consortium piloted these guidelines on 99 variants spanning all categories (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign). Nine variants were distributed to all laboratories, and the remaining 90 were evaluated by three laboratories. The laboratories classified each variant by using both the laboratorys own method and the ACMG-AMP criteria. The agreement between the two methods used within laboratories was high (K-alpha = 0.91) with 79% concordance. However, there was only 34% concordance for either classification system across laboratories. After consensus discussions and detailed review of the ACMG-AMP criteria, concordance increased to 71%. Causes of initial discordance in ACMG-AMP classifications were identified, and recommendations on clarification and increased specification of the ACMG-AMP criteria were made. In summary, although an initial pilot of the ACMG-AMP guidelines did not lead to increased concordance in variant interpretation, comparing variant interpretations to identify differences and having a common framework to facilitate resolution of those differences were beneficial for improving agreement, allowing iterative movement toward increased reporting consistency for variants in genes associated with monogenic disease.

The impact of tumor profiling approaches and genomic data strategies for cancer precision medicine

BackgroundThe diversity of clinical tumor profiling approaches (small panels to whole exomes with matched or unmatched germline analysis) may engender uncertainty about their benefits and liabilities, particularly in light of reported germline false positives in tumor-only profiling and use of global mutational and/or neoantigen data. The goal of this study was to determine the impact of genomic analysis strategies on error rates and data interpretation across contexts and ancestries.MethodsWe modeled common tumor profiling modalities—large (n = 300 genes), medium (n = 48 genes), and small (n = 15 genes) panels—using clinical whole exomes (WES) from 157 patients with lung or colon adenocarcinoma. We created a tumor-only analysis algorithm to assess germline false positive rates, the impact of patient ancestry on tumor-only results, and neoantigen detection.ResultsAfter optimizing a germline filtering strategy, the germline false positive rate with tumor-only large panel sequencing was 14 % (144/1012 variants). For patients whose tumor-only results underwent molecular pathologist review (n = 91), 50/54 (93 %) false positives were correctly interpreted as uncertain variants. Increased germline false positives were observed in tumor-only sequencing of non-European compared with European ancestry patients (p < 0.001; Fisher’s exact) when basic germline filtering approaches were used; however, the ExAC database (60,706 germline exomes) mitigated this disparity (p = 0.53). Matched and unmatched large panel mutational load correlated with WES mutational load (r2 = 0.99 and 0.93, respectively; p < 0.001). Neoantigen load also correlated (r2 = 0.80; p < 0.001), though WES identified a broader spectrum of neoantigens. Small panels did not predict mutational or neoantigen load.ConclusionsLarge tumor-only targeted panels are sufficient for most somatic variant identification and mutational load prediction if paired with expanded germline analysis strategies and molecular pathologist review. Paired germline sequencing reduced overall false positive mutation calls and WES provided the most neoantigens. Without patient-matched germline data, large germline databases are needed to minimize false positive mutation calling and mitigate ethnic disparities.

Molecular characterization of scant lung tumor cells using iron-oxide nanoparticles and micro-nuclear magnetic resonance

UNLABELLED Advances in nanotechnology and microfluidics are enabling the analysis of small amounts of human cells. We tested whether recently developed micro-nuclear magnetic resonance (μNMR) technology could be leveraged for diagnosing pulmonary malignancy using fine needle aspirate (FNA) of primary lesions and/or peripheral blood samples. We enrolled a cohort of 35 patients referred for CT biopsy of primary pulmonary nodules, liver or adrenal masses and concurrently obtained FNA and peripheral blood samples. FNA sampling yielded sufficient material for μNMR analysis in 91% of cases and had a sensitivity and specificity of 91.6% and 100% respectively. Interestingly, among blood samples with positive circulating tumor cells (CTC), μNMR analysis of each patients peripheral blood led to similar diagnosis (malignant vs benign) and differential diagnosis (lung malignancy subtype) in 100% and 90% (18/20) of samples, respectively. μNMR appears to be a valuable, non-invasive adjunct in the diagnosis of lung cancer. FROM THE CLINICAL EDITOR The authors of this study established that recently developed micro-nuclear magnetic resonance (μNMR) technology can be leveraged for diagnosing pulmonary malignancy using fine needle aspirate (FNA) of primary lesions and/or peripheral blood samples derived from 35 patients, suggesting practical clinical applicability of this technique.

Erratum: Performance of ACMG-AMP Variant-Interpretation Guidelines among Nine Laboratories in the Clinical Sequencing Exploratory Research Consortium (American Journal of Human Genetics (2016) 98(6) (1067–1076) (S0002929716300593) (10.1016/j.ajhg.2016.03.024))

Laura M. Amendola,1,16 Gail P. Jarvik,1,16,* Michael C. Leo,2 Heather M. McLaughlin,3 Yassmine Akkari,4 Michelle D. Amaral,5 Jonathan S. Berg,6 Sawona Biswas,7 Kevin M. Bowling,5 Laura K. Conlin,7 Greg M. Cooper,5 Michael O. Dorschner,8 Matthew C. Dulik,9 Arezou A. Ghazani,10 Rajarshi Ghosh,11 Robert C. Green,3,12,15 Ragan Hart,1 Carrie Horton,13 Jennifer J. Johnston,14 Matthew S. Lebo,3,12 Aleksandar Milosavljevic,11 Jeffrey Ou,1 Christine M. Pak,4 Ronak Y. Patel,11 Sumit Punj,4 Carolyn Sue Richards,4 Joseph Salama,1 Natasha T. Strande,6 Yaping Yang,11 Sharon E. Plon,11 Leslie G. Biesecker,14 and Heidi L. Rehm3,12,15,*

A survey of current practices for genomic sequencing test interpretation and reporting processes in US laboratories

Purpose:While the diagnostic success of genomic sequencing expands, the complexity of this testing should not be overlooked. Numerous laboratory processes are required to support the identification, interpretation, and reporting of clinically significant variants. This study aimed to examine the workflow and reporting procedures among US laboratories to highlight shared practices and identify areas in need of standardization.Methods:Surveys and follow-up interviews were conducted with laboratories offering exome and/or genome sequencing to support a research program or for routine clinical services. The 73-item survey elicited multiple choice and free-text responses that were later clarified with phone interviews.Results:Twenty-one laboratories participated. Practices highly concordant across all groups included consent documentation, multiperson case review, and enabling patient opt-out of incidental or secondary findings analysis. Noted divergence included use of phenotypic data to inform case analysis and interpretation and reporting of case-specific quality metrics and methods. Few laboratory policies detailed procedures for data reanalysis, data sharing, or patient access to data.Conclusion:This study provides an overview of practices and policies of experienced exome and genome sequencing laboratories. The results enable broader consideration of which practices are becoming standard approaches, where divergence remains, and areas of development in best practice guidelines that may be helpful.Genet Med advance online publication 03 Novemeber 2016

Assigning clinical meaning to somatic and germ-line whole-exome sequencing data in a prospective cancer precision medicine study

Purpose:Implementing cancer precision medicine in the clinic requires assessing the therapeutic relevance of genomic alterations. A main challenge is the systematic interpretation of whole-exome sequencing (WES) data for clinical care.Methods:One hundred sixty-five adults with metastatic colorectal and lung adenocarcinomas were prospectively enrolled in the CanSeq study. WES was performed on DNA extracted from formalin-fixed paraffin-embedded tumor biopsy samples and matched blood samples. Somatic and germ-line alterations were ranked according to therapeutic or clinical relevance. Results were interpreted using an integrated somatic and germ-line framework and returned in accordance with patient preferences.Results:At the time of this analysis, WES had been performed and results returned to the clinical team for 165 participants. Of 768 curated somatic alterations, only 31% were associated with clinical evidence and 69% with preclinical or inferential evidence. Of 806 curated germ-line variants, 5% were clinically relevant and 56% were classified as variants of unknown significance. The variant review and decision-making processes were effective when the process was changed from that of a Molecular Tumor Board to a protocol-based approach.Conclusion:The development of novel interpretive and decision-support tools that draw from scientific and clinical evidence will be crucial for the success of cancer precision medicine in WES studies.Genet Med advance online publication 26 January 2017

Real-time Genomic Characterization of Advanced Pancreatic Cancer to Enable Precision Medicine

Clinically relevant subtypes exist for pancreatic ductal adenocarcinoma (PDAC), but molecular characterization is not yet standard in clinical care. We implemented a biopsy protocol to perform time-sensitive whole-exome sequencing and RNA sequencing for patients with advanced PDAC. Therapeutically relevant genomic alterations were identified in 48% (34/71) and pathogenic/likely pathogenic germline alterations in 18% (13/71) of patients. Overall, 30% (21/71) of enrolled patients experienced a change in clinical management as a result of genomic data. Twenty-six patients had germline and/or somatic alterations in DNA-damage repair genes, and 5 additional patients had mutational signatures of homologous recombination deficiency but no identified causal genomic alteration. Two patients had oncogenic in-frame BRAF deletions, and we report the first clinical evidence that this alteration confers sensitivity to MAPK pathway inhibition. Moreover, we identified tumor/stroma gene expression signatures with clinical relevance. Collectively, these data demonstrate the feasibility and value of real-time genomic characterization of advanced PDAC.Significance: Molecular analyses of metastatic PDAC tumors are challenging due to the heterogeneous cellular composition of biopsy specimens and rapid progression of the disease. Using an integrated multidisciplinary biopsy program, we demonstrate that real-time genomic characterization of advanced PDAC can identify clinically relevant alterations that inform management of this difficult disease. Cancer Discov; 8(9); 1096-111. ©2018 AACR.See related commentary by Collisson, p. 1062This article is highlighted in the In This Issue feature, p. 1047.

Approaches to carrier testing and results disclosure in translational genomics research: The clinical sequencing exploratory research consortium experience

Clinical genome and exome sequencing (CGES) is primarily used to address specific clinical concerns by detecting risk of future disease, clarifying diagnosis, or directing treatment. Additionally, CGES makes possible the disclosure of autosomal recessive and X‐linked carrier results as additional secondary findings, and research about the impact of carrier results disclosure in this context is needed.

Secondary findings from clinical genomic sequencing: prevalence, patient perspectives, family history assessment, and health-care costs from a multisite study

PurposeClinical sequencing emerging in health care may result in secondary findings (SFs).MethodsSeventy-four of 6240 (1.2%) participants who underwent genome or exome sequencing through the Clinical Sequencing Exploratory Research (CSER) Consortium received one or more SFs from the original American College of Medical Genetics and Genomics (ACMG) recommended 56 gene–condition pair list; we assessed clinical and psychosocial actions.ResultsThe overall adjusted prevalence of SFs in the ACMG 56 genes across the CSER consortium was 1.7%. Initially 32% of the family histories were positive, and post disclosure, this increased to 48%. The average cost of follow-up medical actions per finding up to a 1-year period was