Anaplastic Lymphoma Kinase Rearrangement in Digestive Tract Cancer: Implication for Targeted Therapy in Chinese Population

Abstract

Background: Anaplastic lymphoma kinase (ALK) rearrangements define a subgroup of lung cancer which is eligible to targeted kinase inhibition. The aim of this study is to observe the incidence rate of ALK fusion in a large cohort of Chinese digestive tract cancer patients.

Patients and methods: Tissue microarray (TMA) was constructed from 808 digestive tract cancer cases, including 169 esophageal squamous cell carcinoma, 182 gastric cancer and 457 colorectal cancer (CRC) cases. We tested all cases for ALK expression via a fully automated immunohistochemistry (IHC) assay. The IHC-positive cases were subjected to fluorescence in situ hybridization (FISH), real-time polymerase chain reaction (qRT-PCR), target gene enrichment and sequencing for confirmation of ALK gene rearrangement and discovery of novel fusion partner.

Results: Among the tested cases, 2 (0.44%) CRC cases showed positive both by IHC and FISH. By qRT-PCR, EML4-ALK fusion was found in one IHC-positive CRC case. In another IHC-positive CRC case, target gene enrichment and sequencing revealed ALK was fused to a novel partner, spectrin beta non-erythrocytic 1 (SPTBN1). One gastric cancer case showed partially positive IHC result, but no fusion was found by FISH and gene sequencing.

Conclusions: The incidence rate of ALK gene fusion in Chinese CRC patients was 0.44%,but not detectable in gastric and esophageal cancers. The novel SPTBN1 -ALK fusion, together with other ALK fusion genes, may become a potential target for anti-ALK therapy.

Fig 1. ALK fusion in colorectal cancer (Case one).

A-B, Immunohistochemistry showed cytoplasmic immunoreactivity for ALK protein expression (A, 20×; B, 200×). C, No immunoreactivity was found in normal tissue (200×). D, fluorescence in situ hybridization (FISH) performed with Vysis LSI ALK Dual color Break-Apart FISH probes detected ALK fusion as split red and green signals (arrows) (1000×).

Fig 2. ALK fusion in colorectal cancer (Case two).

A-B, Immunohistochemistry showed cytoplasmic immunoreactivity for ALK protein expression (A, 20×; B, 200×). C, fluorescence in situ hybridization (FISH) performed with Vysis LSI ALK Dual color Break-Apart FISH probes detected ALK fusion as split red and green signals (arrows) (1000×). D, Real-time PCR detection of EML4-ALK fusions. Graph from the real-time PCR showed change in the normalized reporter signal (delta Rn) against PCR cycle number. The grey curve stands for internal control and the blue curve stands for the EML4-ALK fusion.

Fig 3. ALK fusion in gastric cancer (Case three).

A, Immunohistochemistry showed cytoplasmic immunoreactivity for ALK protein expression in a proportion of cancer cells (20×). B-C, ALK protein was only expressed in tumor cells with neuroendocrine differentiation, but not expressed in gastric adenocarcinoma cells, indicating intratumoral heterogeneity (A, 20×; B, 200×). D, fluorescence in situ hybridization (FISH) performed with Vysis LSI ALK Dual color Break-Apart FISH probes showed FISH-negative result as intact fused signals. (1000×).

Fig 4. A novel fusion gene SPTBN1-ALK.

A, Targeted sequencing analysis revealed a novel fusion gene SPTBN1-ALK which created by invertion between two breakpoints in the intron 7 of SPTBN1 gene and the intron 19 of ALK gene. Sanger sequencing of reverse transcription-PCR product confirmed the fusion of SPTBN1-ALK gene, as showed in the lower panel. B, Functional domain analysis of SPTBN1, ALK, and SPTBN1–ALK fusion protein sequences. ALK, anaplastic lymphoma kinase; SPTBN1, spectrin, beta, non-erythrocytic 1; CH, calponin homology domain; PH, pleckstrin homology domain; TM, transmembrane domain.