MP41-06 SPOP MUTATIONS IN PROSTATE CANCER ACROSS DEMOGRAPHICALLY DIVERSE PATIENT COHORTS.

Abstract

BACKGROUND: Recurrent mutations in the Speckle-Type POZ Protein (SPOP) gene occur in up to 15% of prostate cancers. However, the frequency and features of cancers with these mutations across different populations is unknown. OBJECTIVE: To investigate SPOP mutations across diverse cohorts and validate a series of assays employing high-resolution melting (HRM) analysis and Sanger sequencing for mutational analysis of formalin-fixed paraffin-embedded material. DESIGN, SETTING, AND PARTICIPANTS: 720 prostate cancer samples from six international cohorts spanning Caucasian, African American, and Asian patients, including both prostate-specific antigen–screened and unscreened populations, were screened for their SPOP mutation status. Status of SPOP was correlated to molecular features (ERG rearrangement, PTEN deletion, and CHD1 deletion) as well as clinical and pathologic features. RESULTS AND LIMITATIONS: Overall frequency of SPOP mutations was 8.1% (4.6% to 14.4%), SPOP mutation was inversely associated with ERG rearrangement (P < .01), and SPOP mutant (SPOPmut) cancers had higher rates of CHD1 deletions (P < .01). There were no significant differences in biochemical recurrence in SPOPmut cancers. Limitations of this study include missing mutational data due to sample quality and lack of power to identify a difference in clinical outcomes. CONCLUSION: SPOP is mutated in 4.6% to 14.4% of patients with prostate cancer across different ethnic and demographic backgrounds. There was no significant association between SPOP mutations with ethnicity, clinical, or pathologic parameters. Mutual exclusivity of SPOP mutation with ERG rearrangement as well as a high association with CHD1 deletion reinforces SPOP mutation as defining a distinct molecular subclass of prostate cancer. Neoplasia (2014) 16, 14–20 Introduction Prostate cancer is a significant health burden, with 241,740 new diagnoses and 28,170 deaths in the United States in 2012 [1]. The inability to distinguish indolent from aggressive disease is a challenge [2]. Identification of driver lesions for specific subsets of prostate cancer could ultimately lead to the development of biomarkers to improve prognostic ability and risk stratification. Recent advances have uncovered multiple recurrent alterations in prostate cancer. The TMPRSS2-ERG fusion has been observed in nearly 50% of prostate cancers [3,4]. We recently reported somatic mutations in the Speckle-Type POZ Protein (SPOP) gene in 6% to 15% of prostate cancers [5]. SPOPmutations define a distinct subclass of prostate cancer: SPOP mutations and ETS rearrangements are mutually exclusive; SPOP mutant (SPOPmut) prostate tumors generally lack lesions in the phosphatidylinositide 3-kinase (PI3K) pathway, and they are also independent of mutations in the tumor suppressor gene TP53 [5–7]. Although the recurrent nature of SPOP mutations is clear, less is known about the frequency of SPOP mutations across different ethnicities and screening practices, the associations with clinical and pathologic characteristics, and the effects on patient outcomes. This study represents the largest multi-institutional study to date investigating these associations with SPOPmutations across different cohorts. In addition, detection of SPOP mutations in older well-annotated archival prostate cancer samples represents a technical challenge. Formalin fixation of tissue followed by paraffin embedding (FFPE) is widely used; however, analysis of nucleic acid from FFPE material is difficult due to cross-linking between nucleic acid and proteins [8]. The tissue heterogeneity in prostate cancer samples can dilute the signal of tumor-associated mutations with benign wild-type contamination and make the detection of point mutations difficult. In an effort to overcome these challenges, we developed a series of assays employing high-resolution melting (HRM) analysis, which relies on alterations in the melting curve of mutated nucleic acids, and Sanger sequencing [9–11]. We optimized the HRM assay as a high sensitivity pre-screening tool, followed by Sanger sequencing for specific confirmation of mutations. Finally, we employed a nextgeneration sequencing approach on a small subset of samples to determine if massively parallel sequencing could rescue samples considered assay failures using the HRM-Sanger methods. Materials and Methods Patient Populations Table 1 lists the clinical, pathologic, and survival data according to each cohort. In total, prostate cancer samples from 996 patients [radical prostatectomy (RP), transurethral resection of the prostate (TURP), or metastatic biopsies] were examined. Of the 996 patients, 720 samples had DNA of sufficient quality and were screened for their SPOP status. Cohorts included patients from Memorial SloanKettering Cancer Center (MSKCC), cohort from Kyungpook National University School of Medicine, Korea (Korean), African American cohort from New York-Presbyterian Hospital (AA), Weill Cornell Medical College (WCMC) cohorts, and University Hospital of Zurich (USZ), as well as the Swedish watchful waiting cohort (SWWC). A detailed description about each individual cohort can be found in the Supplementary Materials and Methods. Samples were categorized into SPOP wild type (SPOPwt) and SPOPmut and analyzed for correlation Neoplasia Vol. 16, No. 1, 2014 SPOP Mutations in Prostate Cancer Blattner et al. 15