Reality of Single Circulating Tumor Cell Sequencing for Molecular Diagnostics in Pancreatic Cancer Colin M. Court, Jacob S. Ankeny, Shonan Sho, Shuang.

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Presentation transcript:

Reality of Single Circulating Tumor Cell Sequencing for Molecular Diagnostics in Pancreatic Cancer Colin M. Court, Jacob S. Ankeny, Shonan Sho, Shuang Hou, Qingyu Li, Carolyn Hsieh, Min Song, Xinfang Liao, Matthew M. Rochefort, Zev A. Wainberg, Thomas G. Graeber, Hsian-Rong Tseng, James S. Tomlinson The Journal of Molecular Diagnostics Volume 18, Issue 5, Pages 688-696 (September 2016) DOI: 10.1016/j.jmoldx.2016.03.006 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Overview of the single-cell sequencing workflow using NanoVelcro/LCM and stepwise workflow analysis of single-cell sequencing results from cell line studies. After CTC or PC cell line identification by ICC, individual cells are isolated by LCM. Transfer efficiency of LCM was determined based on identification of the cell isolated in the Eppendorf tube cap. Single cells then underwent WGA, and success was assessed by demonstration of a smear band. Next, KRAS PCR was performed and was considered successful if a band of the proper size (295 bp) was found with gel electrophoresis. Finally, Sanger sequencing success was determined by visual inspection of the trace file. A total of 163 single cells were isolated and sequenced, and the success of the individual steps in the workflow was analyzed, revealing that WGA was responsible for most sequencing failures. Data are expressed as n (%). CTC, circulating tumor cell; ICC, immunocytochemistry; LCM, laser capture microdissection; MDA, multiple displacement amplification; PC, pancreatic cancer; Sanger Seq, Sanger sequencing; WGA, whole genome amplification. The Journal of Molecular Diagnostics 2016 18, 688-696DOI: (10.1016/j.jmoldx.2016.03.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Comparison of single cell KRAS sequencing results from different PC cell lines. Cell lines with both heterozygous and homozygous codon 12 mutations were analyzed, and the batch gDNA trace files for the different cell lines are depicted with codon 12 underlined. On the basis of the KRAS sequence base calls, individual cells were classified as demonstrating the mutation only, the wild-type only, or both alleles, indicative of a heterozygous sequence. ADO rate was calculated as the number of single-cell sequences that demonstrated only one of the two alleles in the heterozygous samples. MDR was defined as the percentage of single cells demonstrating a mutant allele over the total number of single cells isolated for sequencing. Homozygous cell lines were used as positive (AsPC-1) and negative (BxPC-3) controls. ADO, allele dropout; het, heterozygous; homo, homozygous; MDR, mutation detection rate; PC, pancreatic cancer; WT, wild-type. The Journal of Molecular Diagnostics 2016 18, 688-696DOI: (10.1016/j.jmoldx.2016.03.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 PC immunocytochemical definitions for CTCs and WBCs using the NanoVelcro/LCM assay. Representative images of the different immunofluorescent channels used to discriminate CTCs from WBCs are shown. Rows 5 to 6 are overlays of the channels above demonstrating the images that are typically used to distinguish CTCs from other circulating cells. Original magnification, ×40. CEA, carcinoembryonic antigen; CK, cytokeratin; CTC, circulating tumor cell; LCM, laser capture microdissection; PC, pancreatic cancer; WBC, white blood cell. The Journal of Molecular Diagnostics 2016 18, 688-696DOI: (10.1016/j.jmoldx.2016.03.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Determination of the number of cells needed to ensure reliable KRAS sequencing. By analyzing the allelic ratio of the sequencing results from different numbers of starting cells, the minimum cellular input for reliable KRAS codon 12 Sanger sequencing can be analyzed. The batch gDNA demonstrates the expected 75:25 G12D mutation-to-wild-type allelic ratio for HPAF-II cells. For each group of differing numbers of cells, the replicate with the best sequence (ie, with an allelic ratio closest to that of batch gDNA) from among the six replicates is depicted. As the number of cells sequenced increases, the allelic ratio becomes more similar to that of batch gDNA. Analysis of all replicates indicates a significant increase in mutation detection reliability above the 5- to 10-cell threshold. The Journal of Molecular Diagnostics 2016 18, 688-696DOI: (10.1016/j.jmoldx.2016.03.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions