Evolution of an adenocarcinoma in response to selection by targeted kinase inhibitors

Steven Jm Jones, Janessa Laskin, Yvonne Y Li, Obi L Griffith, Jianghong An, Mikhail Bilenky, Yaron S Butterfield, Timothee Cezard, Eric Chuah, Richard Corbett, Anthony P Fejes, Malachi Griffith, John Yee, Montgomery Martin, Michael Mayo, Nataliya Melnyk, Ryan D Morin, Trevor J Pugh, Tesa Severson, Sohrab P Shah, Margaret Sutcliffe, Angela Tam, Jefferson Terry, Nina Thiessen, Thomas Thomson, Richard Varhol, Thomas Zeng, Yongjun Zhao, Richard A Moore, David G Huntsman, Inanc Birol, Martin Hirst, Robert A Holt, Marco A Marra, Steven Jm Jones, Janessa Laskin, Yvonne Y Li, Obi L Griffith, Jianghong An, Mikhail Bilenky, Yaron S Butterfield, Timothee Cezard, Eric Chuah, Richard Corbett, Anthony P Fejes, Malachi Griffith, John Yee, Montgomery Martin, Michael Mayo, Nataliya Melnyk, Ryan D Morin, Trevor J Pugh, Tesa Severson, Sohrab P Shah, Margaret Sutcliffe, Angela Tam, Jefferson Terry, Nina Thiessen, Thomas Thomson, Richard Varhol, Thomas Zeng, Yongjun Zhao, Richard A Moore, David G Huntsman, Inanc Birol, Martin Hirst, Robert A Holt, Marco A Marra

Abstract

Background: Adenocarcinomas of the tongue are rare and represent the minority (20 to 25%) of salivary gland tumors affecting the tongue. We investigated the utility of massively parallel sequencing to characterize an adenocarcinoma of the tongue, before and after treatment.

Results: In the pre-treatment tumor we identified 7,629 genes within regions of copy number gain. There were 1,078 genes that exhibited increased expression relative to the blood and unrelated tumors and four genes contained somatic protein-coding mutations. Our analysis suggested the tumor cells were driven by the RET oncogene. Genes whose protein products are targeted by the RET inhibitors sunitinib and sorafenib correlated with being amplified and or highly expressed. Consistent with our observations, administration of sunitinib was associated with stable disease lasting 4 months, after which the lung lesions began to grow. Administration of sorafenib and sulindac provided disease stabilization for an additional 3 months after which the cancer progressed and new lesions appeared. A recurring metastasis possessed 7,288 genes within copy number amplicons, 385 genes exhibiting increased expression relative to other tumors and 9 new somatic protein coding mutations. The observed mutations and amplifications were consistent with therapeutic resistance arising through activation of the MAPK and AKT pathways.

Conclusions: We conclude that complete genomic characterization of a rare tumor has the potential to aid in clinical decision making and identifying therapeutic approaches where no established treatment protocols exist. These results also provide direct in vivo genomic evidence for mutational evolution within a tumor under drug selection and potential mechanisms of drug resistance accrual.

Figures

Figure 1
Figure 1
Identified regions of chromosomal copy number variation (CNV) and loss of heterozygosity (LOH) in both the pre-treatment (T1) and post-treatment (T2) tumor samples and matched normal patient DNA (R) plotted in Circos format [52]. CNV values are the hidden Markov model (HMM) state. Δ indicates the degree in change of HMM state between the two cancers.
Figure 2
Figure 2
Cancer signaling pathways affected within the tumor. (a) Pre-treatment: overall, the down-regulation of PTEN and up-regulation of the RET signaling pathway appear to be driving tumor proliferation. Increased signaling independent of EGFR is consistent with the observed erlotinib insensitivity of the tumor. (b) Post-versus pre-treatment: after treatment with the RET inhibitors sunitinib and sorafenib, there is a marked increase in the signaling of pathway constituents, increasing tumor proliferation. Black and red pathway arrows represent activation and inhibition, respectively. Dotted arrows represent indirect interactions. The number of arrows denoting significantly over- or under-expressed genes are quantified using fold change of tumor versus compendium in (a), and primary tumor versus the tumor recurrence in (b): 1 arrow is FC ≥2; 2 arrows is FC ≥10; and 3 arrows is FC ≥50. CNV, copy number variation.
Figure 3
Figure 3
Fluorescent in situ hybridization (FISH) and immunohistochemical analysis of the sublingual adenocarcinoma. (a) Hematoxylin and eosin stained section of tumor (20× objective). (b) Striking amplification of RBBP8 (40×, with RBBP8 probe in red). (c) Focal nuclear and cytoplasmic expression of PTEN (20×) is associated with (d) a missing red signal indicating monoallelic loss of PTEN (100×; the orange gene-specific probe signals are decreased in number compared to the centromeric probe). (e) Diffuse, strong cytoplasmic expression of RET (20×) is associated with (f) amplification of the RET gene (40× with bacterial artificial chromosomes flanking the RET gene labeled in red and green).
Figure 4
Figure 4
PET-CT scans of the patient. (a) 1 October 2008, 1 month before sunitinib initiation. (b) 29 October 2008, baseline before sunitinib initiation on 30 October 2008. (c) 9 December 2008, 4 weeks on sunitinib.

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