Detection of EGFR and KRAS mutations in fine‐needle aspirates stored on Whatman FTA cards

Abstract

BACKGROUND: The aims of this study were to compare the quality of DNA recovered from fine‐needle aspirates (FNAs) stored on Whatman FTA cards with that retrieved from corresponding cell blocks and to determine whether the DNA extracted from the cards is suitable for multiple mutation analyses. METHODS: FNAs collected from 18 resected lung tumors and cell suspensions from 4 lung cancer cell lines were placed on FTA Indicating Micro Cards and further processed to produce paired formalin‐fixed paraffin‐embedded (FFPE) cell blocks. Fragment analysis was used for the detection of EGFR exon 19 deletion, and direct sequencing for detection of EGFR exon 21 L858R mutation and exon 2 deletion of KRAS. Corresponding FFPE tissue sections from 2 resection specimens were also tested. RESULTS: Analyses were successful with all FNAs and lung cancer‐derived cell lines collected on cards. Polymerase chain reaction failed in 2 cell blocks. For FNAs collected on cards, 5 cases showed EGFR and 3 showed KRAS mutations. Eleven cases were wild type. With cell blocks, 4 cases were found to harbor KRAS and 4 harbored EGFR mutations. All lung cancer‐derived cell lines tested positive for their respective mutations, and there was complete agreement between card and cell block FNA samples for EGFR exon 21. For EGFR exon 19, 1 of 18 cases showed discordant results between the card and cell block, and for KRAS 1 of 17. The two resection specimens tested gave concordant results with the FTA card. CONCLUSIONS: Storage of cytologic material on FTA cards can maximize and simplify sample procurement for multiple mutational analyses with results similar to those from cell blocks. Cancer (Cancer Cytopathol) 2010. © 2010 American Cancer Society.

How to Prepare Cytological Samples for Molecular Testing

“…14 In contrast, RT-PCR or quantitative RT-PCR analysis for fusion gene detection is more tolerant of partially degraded RNA because the design can be based on an analysis of smaller regions of RNA. 14 For long-term storage, aliquots can be frozen at 80°C RNA later or similar RNA stabilising solutions and stored in freezers 34 ; fresh cells can also be stored at room temperature for months in Whatman filter paper cards (GE Healthcare Life Sciences, Buckinghamshire, England). The latter method is an easy, fast, inexpensive, operative-friendly procedure and ensures high quality of nucleic acid for molecular testing, but the amount of genetic material that can be extracted from the Flinders Technology Associates (FTA) cards is limited.…”

Lymph Node Fine-Needle Cytology: Beyond Flow Cytometry

“…All these procedures are generally chosen based on the different requests, the availability of the corresponding technologies and the experience of each laboratory. Corresponding technologies have been used on any type of sample, such as paraffin-embedded tissues, fresh cells or tissues, and even cells obtained by FNC and cryopreserved or stored on FTA cards [8,36,37,38,39,40,41,42,43]. …”

“…Numerous studies have shown that FNC samples are suitable for different molecular procedures (fig. 1) [7,8]. Even the drawback of the small amount of material obtained by FNC, which is the main limitation of this procedure, may be overcome by new technologies that are able to maximize the use of a few nanograms of good-quality genetic material (fig.…”

“…Even the drawback of the small amount of material obtained by FNC, which is the main limitation of this procedure, may be overcome by new technologies that are able to maximize the use of a few nanograms of good-quality genetic material (fig. 2) [7,8]. Therefore, once again, FNC may be conveniently used in NHL management, and the new challenge for cytopathologists, starting with FC/FNC and ROSE, will be to acquire updated knowledge and apply new technologies to FNC, exploiting its own basic qualities.…”

Maximizing the yield of lymph node cytology

“…13 Furthermore, a recent study has demonstrated the preservation and recovery of higher molecular weight DNA from archival FNA smears, which was used for high-resolution comparative genomic hybridization array, DNA methylation, and single nucleotide polymorphism genotyping platforms. 14 The use of fresh cells whenever possible may be preferable because DNA will be less degraded.…”