A Validation Study for the Use of ROS1 Immunohistochemical Staining in Screening for ROS1 Translocations in Lung Cancer

INTRODUCTION: The presence of ROS proto-oncogene 1, receptor tyrosine kinase gene (ROS1) rearrangements in lung cancers confers sensitivity to ROS kinase inhibitors, including crizotinib. However, they are rare abnormalities (in ∼1% of non-small cell lung carcinomas) that are typically identified by fluorescence in situ hybridization (FISH), and so screening using immunohistochemical (IHC) staining would be both cost- and time-efficient.

METHODS: A cohort of lung tumors negative for other common mutations related to targeted therapies were screened to assess the sensitivity and specificity of IHC staining in detecting ROS1 gene rearrangements, enriched by four other cases first identified by FISH. A review of published data was also undertaken.

RESULTS: IHC staining was 100% sensitive (95% confidence interval: 48-100) and 83% specific (95% confidence interval: 86-100) overall when an h-score higher than 100 was used. Patients with ROS1 gene rearrangements were younger and typically never-smokers, with the tumors all being adenocarcinomas with higher-grade architectural features and focal signet ring morphologic features (two of five). Four patients treated with crizotinib showed a partial response, with three also showing a partial response to pemetrexed. Three of four patients remain alive at 13, 27, and 31 months, respectively.

CONCLUSION: IHC staining can be used to screen for ROS1 gene rearrangements, with patients herein showing a response to crizotinib. Patients with tumors that test positive according to IHC staining but negative according to FISH were also identified, which may have implications for treatment selection.

De novo methylation of tumor suppressor gene p16/INK4a is a frequent finding in multiple myeloma patients at diagnosis.

The p16 gene competes with cyclin D for binding to CDK4/CDK6 and therefore inhibits CDK4/6 complex kinase activity, resulting in dephosphorylation of pRb and related G1 growth arrest. Inactivation of this gene has been involved in a variety of tumors by different mechanisms: homozygous/hemyzygous deletions, point mutations and methylation of a 5' CpG island into exon E1alpha of the p16 gene. Homozygous deletions have been rarely found in multiple myeloma (MM) and no point mutations have been reported. Two recent studies have reported a high prevalence of methylation in the exon E1alpha of the p16 gene, but included only a small number of cases. We have analyzed the methylation pattern of exon E1alpha of the p16 gene in 101 untreated MM and five primary plasma cell leukemias (PCL). A PCR assay, relying on the inability of some restriction enzymes to digest methylated sequences, was used to analyze the methylation status. Southern blot analysis was used to confirm these results. Forty-one of 101 MM patients (40.5%) as well as four of the five (80%) primary PCL patients had shown methylation of the exon E1alpha. Our study confirms that hypermethylation of the p16 gene is a frequent event in MM. Leukemia (2000) 14, 183-187.