AInsight ALK Screen assay

The field of molecular diagnostics is becoming increasingly important across health care. Scientific discoveries related to key biomarkers combined with advances in molecular testing have enhanced our ability to diagnose, manage, and treat a range of medical conditions. Perhaps no clinical area has been more altered by molecular diagnostics than oncology.

New diagnostic tests have altered cancer care significantly. Today, we have unprecedented tools to determine an individual’s susceptibilities for certain cancers, identify what cancers they have, decide which course of treatment will best combat their disease, and manage their progress with a given medical therapy. New diagnostic technologies are also vital in the discovery of new cancer treatments.

One of the most exciting diagnostic arenas in oncology today is anaplastic lymphoma kinase (ALK). Discovered by Stephan W. Morris, MD, of St Jude Children’s Research Hospital in Memphis, Tenn, ALK was found to be involved in the t(2;5) chromosomal translocation that occurs in the non-Hodgkin’s lymphoma known as anaplastic large cell lymphoma. The normal ALK gene encodes a receptor tyrosine kinase (RTK) in the insulin receptor superfamily of RTKs and is expressed primarily in the central and peripheral nervous systems.

ALK-1 expression in anaplastic large cell lymphoma.
PATHOLOGY RESIDENT WIKI / PATHINFO.WIKIA.COM

Constitutively activated fusion forms of ALK have been found to have a pathogenic role in more than 250,000 new cancer diagnoses in the United States each year, and detection of ALK mutations and fusions has been considered increasingly important in the diagnosis and therapy selection for many types of cancer, including a subset of diffuse large B-cell lymphoma, the sarcoma known as inflammatory myofibroblastic tumor, non-small cell lung cancer, esophageal squamous cell carcinoma, colorectal cancer, and breast carcinoma. A number of adult and pediatric malignancies have yet to be rigorously analyzed for the presence of ALK abnormalities, supporting the possibility that many additional human cancers could be caused by aberrantly activated forms of ALK.

Because of the potential for ALK-inhibitor therapies to treat so many cancers, at least six pharmaceutical companies have ALK inhibitors in development. Since only patients with ALK fusions are likely to respond to ALK inhibitors, accurate diagnostic screening is essential before prescribing ALK-targeted drugs. The challenge with kinase inhibitors like those targeting ALK is that some patients can develop resistance to them over time, rendering the therapies ineffective. In other cases, these resistance mutations may exist de novo (or at initial diagnosis prior to ALK-targeted therapy). With this in mind, accurate detection of resistance mutations before an ALK inhibitor is prescribed and during ALK-inhibitor therapy is critical.

ALK DIAGNOSTICS

Creating assays for detecting ALK fusions and mutations has been challenging. Methods of ALK detection such as variant-specific PCR, immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH) analysis have well-documented challenges.

David R. Hout, PhD, director of research and development at Insight Genetics, Nashville, Tenn, has extensive experience in the fields of molecular biology and molecular diagnostics. Prior to joining Insight Genetics, he served as senior research microbiologist at Biomune in Lenexa, Kan. Hout holds a PhD in molecular virology from the University of Kansas Medical Center. He completed postdoctoral work in biochemistry and virology at Vanderbilt University.

Variant-specific PCR strategies must incorporate validated primer pairs for a long list of ALK fusions. Because this list continues to grow in each cancer with more than 20 different allele combinations of 5′ fusion partners and variants, the variant-specific PCR approach can be cumbersome and make it difficult to provide accurate coverage for all possible fusion partners and variants.

The HER2 diagnostic experience clearly demonstrated challenges in interlaboratory concordance with the more subjective methods, such as FISH and IHC. Discordance rates were as high as 20%.1 Detection of ALK fusions by FISH and IHC is, in many ways, more challenging than HER2. Some of the ALK-positive cancers, especially lung adenocarcinoma, have exhibited low expression levels and can cause weak staining by IHC. FISH is challenging due to the occasional failure of the 5′ probe to hybridize and also the fact that the signal split can be very subtle.2

Recognizing the need for better methods of not only detecting activating ALK fusions and upregulation across many cancer types but also monitoring for resistance mutations that arise in response to ALK-inhibitor therapy, Insight Genetics, Nashville, Tenn, began to develop advanced qPCR assays designed to address problems of sensitivity, difficulty of interpretation, and turnaround time that were inherent to other methods of ALK fusion and mutation detection. Its Insight ALK Screen assay provides quick, accurate detection of any ALK fusion.

Featuring a unique patent-pending primer design strategy, the Insight ALK Screen offers the accuracy and reliability advantages of a qPCR platform without the need for validated primer pair for each fusion. Unlike a variant-specific multiplex strategy, the Insight ALK Screen can detect the presence of any fusion within the ALK gene. It also can identify ALK upregulation without using a secondary platform.

The company’s Insight ALK Resistance Monitoring assays, which are in development, will assist in monitoring patients for ALK resistance mutations. These assays are based on the same platform as Insight ALK Screen and have the same benefits over other methods of analysis. Insight Genetics holds an exclusive worldwide licensing agreement with St Jude Children’s Research Hospital covering genetic mutations that confer resistance to ALK inhibitors.


David R. Hout, PhD, is a contributing writer for CLP.

References
  1. Wolf AC, Hammond EH, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med. 2007;131(1):18-43.
  2. Sasaki T, Rodig SJ, Chirieac LR, Janne PA. The biology and treatment of EML4-ALK non-small cell lung cancer. Eur J Cancer. 2010;46(10):1773-1780. Epub 2010 Apr 24.