Touch imprint cytology with massively parallel sequencing (TIC‐seq): a simple and rapid method to snapshot genetic alterations in tumors

Abstract

Identifying genetic alterations in tumors is critical for molecular targeting of therapy. In the clinical setting, formalin‐fixed paraffin‐embedded (FFPE) tissue is usually employed for genetic analysis. However, DNA extracted from FFPE tissue is often not suitable for analysis because of its low levels and poor quality. Additionally, FFPE sample preparation is time‐consuming. To provide early treatment for cancer patients, a more rapid and robust method is required for precision medicine. We present a simple method for genetic analysis, called touch imprint cytology combined with massively paralleled sequencing (touch imprint cytology [TIC]‐seq), to detect somatic mutations in tumors. We prepared FFPE tissues and TIC specimens from tumors in nine lung cancer patients and one patient with breast cancer. We found that the quality and quantity of TIC DNA was higher than that of FFPE DNA, which requires microdissection to enrich DNA from target tissues. Targeted sequencing using a next‐generation sequencer obtained sufficient sequence data using TIC DNA. Most (92%) somatic mutations in lung primary tumors were found to be consistent between TIC and FFPE DNA. We also applied TIC DNA to primary and metastatic tumor tissues to analyze tumor heterogeneity in a breast cancer patient, and showed that common and distinct mutations among primary and metastatic sites could be classified into two distinct histological subtypes. TIC‐seq is an alternative and feasible method to analyze genomic alterations in tumors by simply touching the cut surface of specimens to slides.

Dual-molecular barcode sequencing detects rare variants in tumor and cell free DNA in plasma

“…All tumor tissues and biopsy samples were fixed with 10% neutral buffered formalin and paraffin-embedded. Cytological specimens were fixed with 95% ethanol and stained with Papanicolau staining as previously described 13 .…”

“…and cytotechnologist (K.A.) to check cellular content and characteristics as previously described 13 (Supplemental Table 1). Laser-capture microdissection was performed using an Arcturus XT laser microdissection system (Thermo Fisher Scientific).…”

“…FFPE DNA was treated with uracil DNA glycosylase within the kit. To assess the quality and concentration of FFPE DNA, we used the TaqMan RNase P Detection Reagents Kit and the FFPE DNA QC Assay v2 on a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific) as previously described 13 .…”

Stepwise addition of genetic changes correlated with histological change from “well-differentiated” to “sarcomatoid” phenotypes: a case report

“…1d-g). A panel targeting the exon of 53 lung cancer-associated genes was established in-house , as we previously reported [1–4]. Subsequently targeted sequencing of those 53 lung cancer-related genes was performed.…”

“…The sequencing data were processed using standard Ion Torrent Suite software running on a Torrent Server (Thermo Fisher Scientific). Variants calling were performed using an Ion Reporter Server System (Thermo Fisher Scientific), and peripheral blood DNA was used as a control to detect somatic mutations in tumours using filtration of “confident somatic variants” in a Tumour-Normal pair pipeline [4, 5]. This study was approved by the institutional review board, and the patient provided written informed consent.…”