Abstract 2367: Identification of ALK alternative transcription initiation in BRAF-negative metastatic melanoma patients
Authors: Zhong Shan , Lovly Christine , Malboeuf Christine , Brennan Kristina , Johnson Douglas , Riemenschneider Kelsie , Meador Catherine , White Emily , Otto Geoff A. , Lipson Doron , Stephens Philip , Miller Vincent , Ross Jeffrey , He Jie 
Published: July 2017

Background:

Genomic alterations in the ALK tyrosine kinase, such as copy number gains, point mutations, and ALK gene fusions, have been described in a broad spectrum of malignancies. Recently a novel mechanism of ALK activation in melanoma was discovered in which ALK transcription was initiated from a de novo alternative transcription initiation (ATI) site in ALK intron 19 (ALK-ATI) [1]. In this study, we used whole transcriptome sequencing (WTS) to interrogate a cohort of clinical melanoma specimens in order to identify those that express ALK-ATI.

Method:

RNA-seq libraries of 33 BRAF-negative clinical metastatic melanoma specimens were prepared using the KAPA Stranded RNA-Seq Kit with RiboErase with 200ng RNA as input and sequenced on Illumina HiSeq 4000 at 2x75bp. The specimens were sequenced to an average of 69.4m total read pairs with 11.3m on-target distinct read pairs. Sequencing reads were mapped to the human genome and transcriptome, gene- and exon-level expressions were quantified. An algorithm to identify the ALK-ATI was developed based on assessing 1) transcription of intron 19 putative ATI region, 2) ALK relative RNA expression and 3) ALK 5’ to 3’ differential expression. In addition, immunohistochemistry (IHC) staining for ALK (using D5F3 antibody) was done on 10 of the 33 samples with adequate tumor material.

Results:

High coverage (median 30x) of intron 19 ATI region and high ALK relative expression (log ratio &gt; 2) were observed in 4 of the 33 specimens tested, in which the ATI site is consistent with what had been reported in [1]. Among the 4 potential ALK-ATI positive cases, 3 have shown significant 3’ to 5’ overexpression of ALK. IHC staining were performed on the 4 putative ALK-ATI positive cases and 6 putative negative cases, and has confirmed ALK-ATI calls detected by WTS assay. Two of the ALK IHC positive cases harbor the highest ALK expression, and the other two ALK-ATI likely cases are ALK IHC weak positive. ALK-ATI occurred in cutaneous, mucosal, and unknown primary melanoma, and co-occurred with other driver mutations (NRAS, NF1, KIT). None of the above specimens had ALK rearrangements detected from DNAseq (by FoundationOne) or WTS.

Conclusions:

Our findings confirmed previously reported ALK activation through ALK-ATI in an independent melanoma cohort, and provided direct evidence that such events can be detected using whole transcriptome sequencing and align with IHC results in clinical FFPE tumor specimens. This confirmation together with future functional validation and clinical evidence may provide new therapeutic opportunities for BRAF-negative metastatic melanoma patients.

1. Wiesner, T. et al. Alternative transcription initiation leads to expression of a novel ALK isoform in cancer. Nature 526, 453-457 (2015).

Citation Format: Shan Zhong, Christine Lovly, Christine Malboeuf, Kristina Brennan, Douglas Johnson, Kelsie Riemenschneider, Catherine Meador, Emily White, Geoff A. Otto, Doron Lipson, Philip Stephens, Vincent Miller, Jeffrey Ross, Jie He. Identification of ALK alternative transcription initiation in BRAF-negative metastatic melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2367. doi:10.1158/1538-7445.AM2017-2367