ROS1 and ALK fusions in colorectal cancer, with evidence of intratumoral heterogeneity for molecular drivers

Abstract

Activated anaplastic lymphoma kinase (ALK) and ROS1 tyrosine kinases, through gene fusions, have been found in lung adenocarcinomas and are highly sensitive to selective kinase inhibitors. This study aimed at identifying the presence of these rearrangements in human colorectal adenocarcinoma specimens using a 4-target, 4-color break-apart FISH assay to simultaneously determine the genomic status of ALK and ROS1. Among the clinical colorectal cancer specimens analyzed, rearrangement-positive cases for both ALK and ROS1 were observed. The fusion partner for ALK was identified as EML4 and the fusion partner for one of the ROS1-positive cases was SLC34A2, the partner for the other ROS1-positive case remains to be identified. A small fraction of specimens presented duplicated or clustered copies of native ALK and ROS1. In addition, rearrangements were detected in samples that also harbored KRAS and BRAF mutations in two of the three cases. Interestingly, the ALK-positive specimen displayed marked intratumoral heterogeneity and rearrangement was also identified in regions of high-grade dysplasia. Despite the additional oncogenic events and tumor heterogeneity observed, elucidation of the first cases of ROS1 rearrangements and confirmation of ALK rearrangements support further evaluation of these genomic fusions as potential therapeutic targets in colorectal cancer.

Implications: ROS1 and ALK fusions occur in colorectal cancer and may have substantial impact in therapy selection.

Figure 1

A, B) FISH images showing ROS1 rearrangement in two specimens as demonstrated by single 3′ ROS1 (aqua) signals; C) FISH image demonstrating ALK rearrangement based on single 3′ALK (red) signals; D) Sequencing of RT-PCR product from the sample depicted in panel A confirming ROS1 fusion with SLC34A2; E) Sequencing of RT-PCR product from the sample depicted in panel C confirming EML4-ALK fusion.

Figure 2

A, B) Two blocks from the original specimen utilized for TMA demonstrated areas with varying patterns of ALK rearrangement and KRAS mutation status. Red circled areas indicate ALK rearranged and mutated KRAS (ALK+/KRAS+). Blue circled areas indicate ALK wild-type and KRAS mutated (ALK-/KRAS+). Green circled areas indicated ALK rearranged and KRAS wild-type (ALK+/KRAS-). Black areas indicate wild-type for both alterations (ALK-/KRAS-). C, D) Higher magnification of the regions indicated in panel E by yellow arrows demonstrates that some regions are best classified as high-grade dysplasia. FISH analysis in region F was positive for ALK rearrangement and FISH analysis in region G was negative for ALK rearrangement (FISH images not shown).

Figure 3

A) Representative sequencing findings of KRAS mutated area. B) Representative high-resolution melting analysis of regions negative for KRAS mutation by sequencing. Blue curves show overlap of tested regions with wild-type control, red and green show positive controls (2 distinct mutations)

Figure 4

Possible mechanisms to explain findings of different combinations of ALK rearrangement and KRAS mutation status.