Detection of rearrangements and transcriptional up-regulation of ALK in FFPE lung cancer specimens using a novel, sensitive, quantitative reverse transcription polymerase chain reaction assay.
BACKGROUND
The approved dual-color fluorescence in situ hybridization (FISH) test for the detection of anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements in non-small-cell lung cancer (NSCLC) is complex and represents a low-throughput assay difficult to use in daily diagnostic practice. We devised a sensitive and robust routine diagnostic test for the detection of rearrangements and transcriptional up-regulation of ALK.


METHODS
We developed a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay adapted to RNA isolated from routine formalin-fixed, paraffin-embedded material and applied it to 652 NSCLC specimens. The reliability of this technique to detect ALK dysregulation was shown by comparison with FISH and immunohistochemistry.


RESULTS
qRT-PCR analysis detected unbalanced ALK expression indicative of a gene rearrangement in 24 (4.6%) and full-length ALK transcript expression in six (1.1%) of 523 interpretable tumors. Among 182 tumors simultaneously analyzed by FISH and qRT-PCR, the latter accurately typed 97% of 19 rearranged and 158 nonrearranged tumors and identified ALK deregulation in two cases with insufficient FISH. Six tumors expressing full-length ALK transcripts did not show rearrangements of the gene. Immunohistochemistry detected ALK protein overexpression in tumors with gene fusions and transcriptional up-regulation, but did not distinguish between the two. One case with full-length ALK expression carried a heterozygous point mutation (S1220Y) within the kinase domain potentially interfering with kinase activity and/or inhibitor binding.


CONCLUSIONS
Our qRT-PCR assay reliably identifies and distinguishes ALK rearrangements and full-length transcript expression in formalin-fixed, paraffin-embedded material. It is an easy-to-perform, cost-effective, and high-throughput tool for the diagnosis of ALK activation. The expression of full-length ALK transcripts may be relevant for ALK inhibitor therapy in NSCLC.
*Department of Clinical Pathology, Robert-Bosch-Krankenhaus, Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, and University of Tübingen, Germany; †Institute of Pathology, University of Würzburg, Würzburg, Germany; ‡Center for Pulmonology and Thoracic Surgery, Klinik Schillerhöhe, Stuttgart-Gerlingen, Germany; and § Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, and Department of Clinical Pharmacology, University Hospital, Tübingen, Germany.
Kim
K
Gruber
Heike
H
Horn
Jörg
J
Kalla
Peter
P
Fritz
Andreas
A
Rosenwald
Martin
M
Kohlhäufl
Godehard
G
Friedel
Matthias
M
Schwab
German
G
Ott
Claudia
C
Kalla
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000077548:Anaplastic Lymphoma Kinase; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D015331:Cohort Studies; D005260:Female; D005500:Follow-Up Studies; D015972:Gene Expression Regulation, Neoplastic; D015321:Gene Rearrangement; D006801:Humans; D007124:Immunoenzyme Techniques; D017404:In Situ Hybridization, Fluorescence; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D016612:Paraffin Embedding; D011379:Prognosis; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D014408:Biomarkers, Tumor; D012333:RNA, Messenger; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases