Features | Partner Sites | Information | LinkXpress
Sign In
AB Sciex
RANDOX LABORATORIES
FOCUS DIAGNOSTICS, INC.

New PCR Assay Detects Gene Rearrangements in Preserved Samples from Lung Cancer Patients

By Labmedica International staff writers
Posted on 13 Mar 2014
Image: The ALK gene is located on the short (p) arm of chromosome two at position 23 (Photo courtesy of the [US] National Institutes of Health).
Image: The ALK gene is located on the short (p) arm of chromosome two at position 23 (Photo courtesy of the [US] National Institutes of Health).
A sensitive and robust routine diagnostic test for the detection of rearrangements and transcriptional upregulation of the ALK (anaplastic lymphoma receptor tyrosine kinase) gene was described in a recent publication.

The ALK gene encodes a receptor tyrosine kinase, which belongs to the insulin receptor superfamily. This protein comprises an extracellular domain, a hydrophobic stretch corresponding to a single pass transmembrane region, and an intracellular kinase domain. It plays an important role in the development of the brain and exerts its effects on specific neurons in the nervous system. This gene has been found to be rearranged, mutated, or amplified in a series of tumors including anaplastic large cell lymphomas, neuroblastoma, and non-small-cell lung cancer (NSCLC).

The EML4-ALK fusion gene is responsible for approximately 3%–5% of cases of NSCLC. The standard test used to detect this gene in tumor samples is fluorescence in situ hybridization (FISH), but other techniques such as immunohistochemistry (IHC) and reverse transcriptase PCR (RT-PCR) can also be used to detect lung cancers with an ALK gene fusion.

Formalin fixed, paraffin-embedded (FFPE) tissues are the most widely available specimens for retrospective clinical studies of disease mechanisms. These archived materials provide a valuable source of stable nucleic acids for gene expression analysis, using real-time quantitative reverse transcription-PCR (qRT-PCR) or microarray analysis. As the use of PCR technology has become more prevalent in molecular testing, it has enhanced the clinical utility of FFPE tissues. However, the recovery of quality RNA from FFPE specimens is often problematic, as the fixation process causes cross-linkage between nucleic acids and proteins, and covalently modifies RNA by the addition of monomethyl groups to the bases. As a result, the molecules are rigid and susceptible to mechanical shearing, and the cross-links may compromise the use of RNA as a substrate for reverse transcription. Therefore, in order to utilize FFPE tissues as a source for gene expression analysis, a reliable method is required for extraction of RNA from the cross-linked matrix.

Investigators at the Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology (Stuttgart, Germany) recently described the development of a new RNA-based assay to detect ALK rearrangements in NSCLC. This technique was based on quantitative, real-time PCR ((q)RT-PCR), and included two novel features: an RNA isolation method that was optimized to reverse formaldehyde modification and small RT-PCR amplicons to allow for the use of fragmented nucleic acids for efficient amplification of ALK cDNA. The test measured the expression of the 5' and the 3' portions of the ALK transcript separately.

Analysis of a number of FFPE samples from NSCLC patients revealed that the assay detected unbalanced ALK expression indicative of a gene rearrangement in 24 (4.6%) of 523 interpretable NSCLC specimens and full-length ALK transcript expression in six tumors (1.1%). Both FISH and qRT-PCR testing were done on 182 tumors; qRT-PCR accurately typed 97% of 19 tumors with ALK rearrangements and 158 with no rearrangements.

“The qRT-PCR technique reliably detects ALK-rearranged tumors independently of the fusion partner and also identifies tumors with full-length transcript expression of the gene that is not detectable by FISH but may be relevant for ALK inhibitor therapy as well,” said senior author Dr. Claudia Kalla, a postdoctoral researcher at the Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology. “The technique seems to be a sensitive, easy-to-perform, and high-throughput method suitable for the routine diagnosis of ALK activation not only in lung cancer, but also in other tumor entities where rearrangements with alternative fusion partners or transcriptional upregulation are prevalent.”

The study was published in the March 2014 issue of the Journal of Thoracic Oncology.

Related Links:
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology



VIRCELL
ADVANCED INSTRUMENTS
DiagCor Bioscience
comments powered by Disqus
Life Technologies

Channels

Immunology

view channel
Image: The fluorescence-activated cell sorting FACSCanto II flow cytometer (Photo courtesy of BD Biosystems).

Flow Cytometry Detects Lymphoproliferative Disorders in Fluid Specimens

Immunophenotypic analysis of hematopoietic cell populations by flow cytometry has emerged as a useful ancillary study in the diagnostic evaluation of serous effusions and cerebrospinal fluids (CSFs).... Read more

Lab Tech.

view channel
Image: A technical breakthrough for DNA imaging has been achieved that should quicken diagnosis of diseases for which analysis of DNA from single-cells is critical, such as early stage cancers and various pre-natal conditions (Photo courtesy of McGill University and Génome Québec Innovation Center).

Breakthrough DNA Analysis Technology to Hasten Problem Diagnosis

Researchers have achieved a technical breakthrough that should result in speedier diagnosis of diseases for which analysis of DNA from single-cells is critical, such as early stage cancers and various... Read more

Industry News

view channel

Most IVD Sector Companies Look Outside the EU and USA for Growth

Kalorama Information (New York City, NY, USA) has now published the 9th edition of its biennial market research report “Worldwide Market for In Vitro Diagnostics,” revealing estimates from its survey of the in vitro diagnostics (IVD) industry – presenting the trends, technologies, customer needs, and major suppliers with... Read more
 
Copyright © 2000-2014 Globetech Media. All rights reserved.