Abstract A200: Novel assays for predictive diagnosis of non-small cell lung cancer: Triple-color FISH increases sensitivity and specificity of EML4-ALK translocation detection in pulmonary adenocarcinomas; FGFR1 amplifications are detectable by FISH in 21% of squamous cell carcinomas of the lung.
Authors: Schildhaus Hans-Ulrich , Heukamp Lukas C. , Schmitz Katja , Binot Elke , Buettner Reinhard , Merkelbach-Bruse Sabine 
Published: June 2012

Background: Lung cancer is one of the leading health care issues worldwide. Easy, reliable and standardized tests for predictive diagnoses and targeted therapies are needed. A small subset of pulmonary adenocarcinomas (AC) harbor therapeutically relevant EML4-ALK translocations, however FISH-based diagnosis is sometimes compromised by the different types of chromosome 2p rearrangements and tumor cell aneuploidy. The incidence of squamous cell carcinomas (SC) of the lung increases dramatically but targeted therapeutic options are not well established for this subgroup of NSCLC. Recently, Fibroblast Growth Factor Receptor 1 (FGFR1) amplification in SC was described to be associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients.

Material and Methods: A total of 400 NSCLC samples (routinely processed FFPE material of biopsies and surgical specimens) were included in this study. For detection of EML4-ALK translocations a novel triple color FISH assay was used: two probes labeled orange and green flank the breakpoint region of ALK in telomeric and centromeric direction, and a third probe, labeled in blue, spans the entire EML4 gene. The results of the hybridisation patterns were compared with that of the conventional two color ALK break apart probe. For testing the entire spectrum of ALK translocations also ALK lymphomas and inflammatory myofibroblastic tumors were investigated. Normal tissues and breast carcinomas served as negative controls. A dual color FGFR1/CEN8 probe was used to evaluate the prevalence of FGFR1 amplification in SC and to develop an evaluation strategy for FGFR1 FISH assays.

Results & Conclusions: Using the triple color EML4-ALK probe specific signal patterns were found for different types of ALK rearrangements. An inversion of chromosome 2p results in (1) split (separated) orange and green signals, (2) a split (doubled) blue signal and (3) a colocalisation (fusion) of the separated orange and green signals with blue signals. Additional specific signals patterns were seen in cases with inversions/deletions and interstitial deletions alone. Even ALK translocations without involvement of EML4 were clearly detectable. Aberrant split/fusion signals were not observed in normal tissues and aneuploid negative controls. The novel triple color split/fusion approach decreases the number of questionable or borderline cases at high sensitivity and specificity and allows the diagnosis of specific types of ALK translocations. Thus we present a novel triple color FISH that leads to an improved detection rate of EML4-ALK translocated patients.

Investigating a subset of 254 SC with a two colour FISH assay we found FGFR1 amplifications in 21.3% of SCC but not in AC. We developed an evaluation strategy and observed different FGFR1 amplification patterns. Low amplification levels (as defined by ≥5 FGFR1 signals in ≥50% of tumor cells) were rare with a fequency of 6.3% while high level amplifications (as defined by a FGFR1/CEN8 ratio ≥2.0, or average number of FGFR1 signals per tumor cell nucleus ≥6, or the percentage of tumor cells containing ≥15 FGFR1 signals or large clusters ≥10%) occurred in 15% of all SC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A200.