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Molecular Pathology Abstracts, 6/17

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Editors: Donna E. Hansel, MD, PhD, chief, Division of Anatomic Pathology, and professor, Department of Pathology, University of California, San Diego; John A. Thorson, MD, PhD, associate professor of pathology, director of the Clinical Genomics Laboratory, Center for Advanced Laboratory Medicine, UCSD; Sarah S. Murray, PhD, professor, Department of Pathology, and director of genomic technologies, Center for Advanced Laboratory Medicine, UCSD; and James Solomon, MD, PhD, resident, Department of Pathology, UCSD.

Whole genome single-cell copy number profiling on FFPE tissue samples

Single-cell genomic methods take the concept of analyzing intratumor genetic heterogeneity to its logical conclusion. Traditionally, however, single-cell methods can only be used to analyze fresh or rapidly frozen tissue because formalin fixation and paraffin embedding degrades tumor DNA and cross-links proteins. The authors described and validated a novel technique for performing single-cell whole genome copy number analysis on formalin-fixed, paraffin-embedded (FFPE) tissue. For their study, FFPE tissue blocks were cut into 100-μm-thick sections for microdissection. The tissue was then processed to remove cross-links, treated with enzyme to break down extracellular material, and passed through a fine needle to release nuclei. Next, the nuclei were sorted by flow cytometry to separate single nuclei into individual wells. Once sorted, single nuclei were treated with a cocktail of DNA repair enzymes, and whole genome amplification was performed using a previously reported method shown to work well with poor-quality fragmented DNA. After library preparation, sequencing was performed using Illumina multiplex sequencing, and copy number analysis across the entire genome to a resolution of approximately 700 kb was determined. To compare this technique to those that use rapidly frozen tissue, the authors performed a pilot study in which FFPE and frozen tissue sections were each microdissected and processed as appropriate for each tissue type. The single-cell copy number analyses were compared to each other, as well as to copy number analysis of a FFPE section of bulk tumor that contained about 100,000 cells. Overall, the copy number analyses, both quantitatively and qualitatively, for each of the specimens were highly concordant. The authors showed that the DNA repair step was necessary when using FFPE tissue to significantly improve the whole genome amplification step, as artifacts and significant variability were otherwise seen. A control experiment, where frozen tissue underwent the DNA repair step, showed that no artifacts were introduced. The authors next demonstrated the biologic utility of this groundbreaking technique in a few proof-of-concept experiments. For example, they were able to compare subpopulations of tumor cells in ductal carcinoma in situ (DCIS) versus adjacent invasive cancer in two cases. In one case, the subpopulations of tumor cells seen in the DCIS were distinct from those seen in the invasive components, suggesting that intratumoral heterogeneity developed early in the course of disease, with one of the clones later acquiring alterations that led to invasion. In the other case, the DCIS and invasive components contained similar subpopulations of cells, suggesting that the ability to invade was acquired early in tumor development followed by subsequent intratumoral heterogeneity. Overall, this study presents an exciting method for examining copy number variants in FFPE tissue and demonstrates the importance of a critical DNA repair step. Among the drawbacks are that the integrity of the initial DNA affects the effectiveness of the assay and that the whole genome amplification method, which allows for complete coverage of the genome, is not particularly suited for detecting point mutations and small deletions. Yet the possibility of performing single-cell genomic studies using FFPE tissue presents enormous research and clinical opportunities.

Martelotto LG, Baslan T, Kendall J, et al. Whole-genome single-cell copy number profiling from formalin-fixed paraffin-embedded samples. Nat Med. 2017;23(3):376–385.

Correspondence: Dr. Jorge S. Reis-Filho at reisfilj@mskcc.org, or Dr. Britta Weigelt at weigeltb@mskcc.org, or Dr. James B. Hicks at jameshic@usc.edu

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