Next generation sequencing of prostate cancer from a patient identifies a deficiency of methylthioadenosine phosphorylase, an exploitable tumor target

Abstract

Castrate-resistant prostate cancer (CRPC) and neuroendocrine carcinoma of the prostate are invariably fatal diseases for which only palliative therapies exist. As part of a prostate tumor sequencing program, a patient tumor was analyzed using Illumina genome sequencing and a matched renal capsule tumor xenograft was generated. Both tumor and xenograft had a homozygous 9p21 deletion spanning the MTAP, CDKN2, and ARF genes. It is rare for this deletion to occur in primary prostate tumors, yet approximately 10% express decreased levels of methylthioadenosine phosphorylase (MTAP) mRNA. Decreased MTAP expression is a prognosticator for poor outcome. Moreover, it seems that this deletion is more common in CRPC than in primary prostate cancer. We show for the first time that treatment with methylthioadenosine and high dose 6-thioguanine causes marked inhibition of a patient-derived neuroendocrine xenograft growth while protecting the host from 6-thioguanine toxicity. This therapeutic approach can be applied to other MTAP-deficient human cancers as deletion or hypermethylation of the MTAP gene occurs in a broad spectrum of tumors at high frequency. The combination of genome sequencing and patient-derived xenografts can identify candidate therapeutic agents and evaluate them for personalized oncology.

Figure 1

(A) Immunohistochemical staining of tumour 946 tissue sections shows expression of neuroendocrine markers CD56, Chromogranin A and Synaptophysin, identifying the neuroendocrine origin of the tissue. (B) Synaptophysin staining of xenograft LTL352 tissue.

Figure 2

Comparison of chromosome 9p copy number profiles of the neuroendocrine urethra metastasis from patient #946 and its xenograft, LTL352, as produced by Illumina sequencing (MPS) and Agilent aCGH technologies. Array CGH confirmed that the deletion in the original tumor by MPS was real and not a mapping artifact, and that the patient’s tumour and xenograft had the same deletion. Copy number profiles were visualized using NexusCGH (Biodiscovery, Inc.). The MTAP deletion is expanded in the bottom panel (UCSC). Three additional genes mapping to this deletion are cyclin-dependent kinase inhibitor 2A (CDKN2A), doublesex and mab-3 related transcription factor 1 (DMRTA1) and embryonic lethal, abnormal vision (ELAVL2).

Figure 3

Expression of genes mapping within the 9p21 MTAP deletion. (A) RNA Sequence-derived expression data for MTAP, CDKN2A, DMRTA1, and ELAVL2 – genes located within the minimal deletion at the MTAP locus. Expression levels were normalized to GAPDH. All samples had normal copy numbers of the MTAP locus with the exception of 946 and 972 urethra and penile metastases, respectively. Only the expression of MTAP shows essentially perfect correlation with the copy number. (B) Quantitative RT-PCR was performed to confirm this interpretation of the RNA-sequence data and to confirm concordance between 946 and 352. (C) MTAP expression levels determined in a Sloan-Kettering prostate cancer cohort (35). The cBio Cancer Genomics Portal was used for data access (http://www.cbioportal.org/cgx/?cancer_type_id=pca). The MTAP expression levels correlate well with the reduced copy numbers of MTAP locus. (D) Kaplan-Meier plot showing differences in time to recurrence as measured by PSA for the 26 patients with MTAP expression differing from the mean by a z-score of 2.0 or greater compared to the rest of the cohort. Decreased MTAP expression is significantly correlated with a shorter time to disease recurrence.

Figure 4

(A) Mice carrying LTL352 xenografts were treated (i.p.) with 6-TG (75 mg/kg)+MTA (100 mg/kg) or saline (controls) on days 1, 5 and 9. On day 12, the tumours in the treated group were approximately 64% smaller than those in the untreated control group (a decrease of tumour growth rate by about 74%). Mean values with standard errors of mean are plotted. * indicates significant differences between control and treated tumours (paired t-test, p<0.05). All mice treated with 6-TG in the absence of MTA died within 12 days. (B) Histological and immunohistochemical analysis of LTL352 tissues from control (untreated) and 6-TG+MTA-treated xenografts. Representative fields of H&E and Caspase-3 staining are shown. In the treated mice the tumours had reduced proliferation and increased apoptosis when compared to the control tumours as illustrated by increase of Caspase-3 positive cells from 1.42±0.17% to 4.49±0.26% (mean ± S.D.). (C) The chemical structure of 5′-Deoxy-5′-Methylthioadenosine (MTA). (D) The chemical structure of 6-Thioguanine (6-TG).