A variety of laboratory methods are available for the detection of deletions of tumor suppressor genes and losses of their proteins. The clinical utility of fluorescence in situ hybridization (FISH) for the identification of deletions of tumor suppressor genes has previously been limited by difficulties in the interpretation of FISH signal patterns. The first deletion FISH assays using formalin-fixed paraffin-embedded tissue sections had to deal with a significant background level of signal losses affecting nuclei that are truncated by the cutting process of slide preparation. Recently, more efficient probe designs, incorporating probes adjacent to the tumor suppressor gene of interest, have increased the accuracy of FISH deletion assays so that true chromosomal deletions can be readily distinguished from the false signal losses caused by sectioning artifacts. This mini-review discusses the importance of recurrent tumor suppressor gene deletions in human cancer and reviews the common FISH methods being used to detect the genomic losses encountered in clinical specimens. The use of new probe designs to recognize truncation artifacts is illustrated with a four-color PTEN FISH set optimized for prostate cancer tissue sections. Data are presented to show that when section thickness is reduced, the frequency of signal truncation losses is increased. We also provide some general guidelines that will help pathologists and cytogeneticists run routine deletion FISH assays and recognize sectioning artifacts. Finally, we summarize how recently developed sequence-based approaches are being used to identify recurrent deletions using small DNA samples from tumors.

Background SB939 is a potent oral inhibitor of class 1, 2, and 4 histone deacetylases (HDACs). These three HDAC classes are highly expressed in castration resistant prostate cancer (CRPC) and associated with poor clinical outcomes. We designed a phase II study of SB939 in men with metastatic CRPC. Methods Patients received SB939 60 mg on alternate days three times per week for 3 weeks on a 4-week cycle. Primary endpoints were PSA response rate (RR) and progression-free survival (PFS). Secondary endpoints included objective response rate and duration; overall survival; circulating tumor cell (CTC) enumeration and safety. Exploratory correlative studies of the TMPRSS2-ERG fusion and PTEN biomarkers were also performed. Results Thirty-two patients were enrolled of whom 88 % had received no prior chemotherapy. The median number of SB939 cycles administered was three (range 1–8). Adverse events were generally grade 1–2, with five pts experiencing one or more grade three event. One patient died due to myocardial infarction. A confirmed PSA response was noted in two pts (6 %), lasting 3.0 and 21.6 months. In patients with measurable disease there were no objective responses. Six patients had stable disease lasting 1.7 to 8.0 months. CTC response (from ≥5 at baseline to <5 at 6 or 12 weeks) occurred in 9/14 evaluable patients (64 %). Conclusion Although SB939 was tolerable at the dose/schedule given, and showed declines in CTC in the majority of evaluable patients, it did not show sufficient activity based on PSA RR to warrant further study as a single agent in unselected patients with CRPC.

Loss of the phosphatase and tensin homolog (PTEN) tumor suppressor gene is a promising marker of aggressive prostate cancer. Active surveillance and watchful waiting are increasingly recommended to patients with small tumors felt to be low risk, highlighting the difficulties of Gleason scoring in this setting. There is an urgent need for predictive biomarkers that can be rapidly deployed to aid in clinical decision‐making. Our objectives were to assess the incidence and ability of PTEN alterations to predict aggressive disease in a multicenter study.

When distinguishing between indolent and potentially harmful prostate cancers, the Gleason score is the most important variable, but may be inaccurate in biopsies due to tumor under-sampling. This study investigated whether a molecular feature, PTEN protein loss, could help identify which Gleason score 6 tumors on biopsy are likely to be upgraded at radical prostatectomy. Seventy one patients with Gleason score 6 tumors on biopsy upgraded to Gleason score 7 or higher at prostatectomy (cases) were compared with 103 patients with Gleason score 6 on both biopsy and prostatectomy (controls). A validated immunohistochemical assay for PTEN was performed, followed by fluorescence in situ hybridization (FISH) to detect PTEN gene deletion in a subset. PTEN protein loss and clinical–pathologic variables were assessed by logistic regression. Upgraded patients were older than controls (61.8 vs 59.3 years), had higher pre-operative PSA levels (6.5 vs 5.3 ng/ml) and a higher fraction of involved cores (0.42 vs 0.36). PTEN loss by immunohistochemistry was found in 18% (13/71) of upgraded cases compared with 7% (7/103) of controls (P=0.02). Comparison between PTEN immunohistochemistry and PTEN FISH showed the assays were highly concordant, with 97% (65/67) of evaluated biopsies with intact PTEN protein lacking PTEN gene deletion, and 81% (13/16) of the biopsies with PTEN protein loss showing homozygous PTEN gene deletion. Tumors with PTEN protein loss were more likely to be upgraded at radical prostatectomy than those without loss, even after adjusting for age, preoperative PSA, clinical stage and race (odds ratio=3.04 (1.08–8.55; P=0.035)). PTEN loss in Gleason score 6 biopsies identifies a subset of prostate tumors at increased risk of upgrading at radical prostatectomy. These data provide evidence that a genetic event can improve Gleason score accuracy and highlight a path toward the clinical use of molecular markers to augment pathologic grading.

Abstract Fluorescence in situ hybridization (FISH) is a robust technique when used with appropriate interpretative guidelines, and the assay can yield consistent results that provide diagnostically and prognostically useful information to help guide patient therapy. The establishment of quality control standards in clinical laboratories using routine FISH analysis of formalin fixed paraffin embedded (FFPE) sections in different tumor types for fusion and break-apart strategies and gene amplifications have been developed for both analysis and reporting, but at the present time there are no comprehensive guidelines for the analysis of genomic deletions using FFPE sections. The use of FISH on archival FFPE samples is technically demanding and becomes more challenging when applied to paraffin-embedded tissue microarrays. The evaluation of FISH signals in interphase nuclei of FFPE sections is affected by truncation and overlapping of the nuclei due to varying cell density, tissue architecture differences and histological forms. In this study we report a generalizable four-color deletion FISH approach to assist interpretation problems arising when evaluating FISH signals. The guidelines will help address interpretative dilemmas associated with overlapping and truncated nuclei in FFPE prostate cancer sections. The four-color FISH approach was developed using the PTEN tumor suppressor gene deletion model in prostate cancer. The PTEN assay was based on a robust bioinformatics analysis of 311 published human genome array datasets and comprises a centromeric “chromosome enumeration” probe, a specific PTEN gene probe, and control flanking probes either side of the target probe. The sensitivity and specificity parameters of the four-color PTEN probe set were further characterized using a large number of well-characterized tumors and stringent scoring criteria. The incorporation of flanking control probes allowed the analysts to determine if the chromosomal region was subject to truncation loss. A minimum threshold for apparent deletion frequency was set to address the heterogeneous and homogeneous nature of tumor histology. In addition the approach facilitated analysis of genotypic heterogeneity and varying clonality within different foci of tumor in the prostate. Overall the approach provided robust and highly reproducible results that minimized inter- and intra-assay variability. The four-color FISH deletion assay reduced the frequency of misinterpretation and improved both the quality and throughput of FISH analyses using clinical samples. Moreover the use of established controls and conservative cut-offs for assigning deletions will facilitate more coherent approach to developing reporting standards for deletion assays as more tumor suppressor genes of clinical importance are discovered by next generation sequencing methods. Citation Format: Maisa Yoshimoto, Olga Ludkovski, Jennifer Good, Robert J. Gooding, Jean McGowan-Jordan, Alexander Boag, Andrew Evans, Ming-Sound Tsao, Paulo Nuin, Jeremy A. Squire. Incorporation of flanking probes reduces truncation losses for fluorescence in situ hybridization analysis of recurrent genomic deletions in tumor sections. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 63. doi:10.1158/1538-7445.AM2013-63

ABSTRACT Background SB939 is a potent oral inhibitor of class 1, 2, and 4 histone deacetylases (HDACs). These 3 HDAC classes are highly expressed in CRPC and associated with poor clinical outcomes. HDAC inhibition abrogates androgen receptor signaling via inactivation of HSP90, and may have particular relevance in prostate cancer with the TMPRSS2-ERG (T2-E) fusion. Methods Pts with locally recurrent or metastatic CRPC received SB939 60mg every other day 3 times per week x 3 weeks on a 28-day cycle. Primary endpoints were PSA response rate (RR) (>50% decline from baseline for > 4 weeks) and progression-free survival (PFS). Secondary endpoints included objective response rate and duration; overall survival; circulating tumor cell (CTC) enumeration at baseline, 6 and 12 weeks; T2-E fusion analysis; correlative analysis of archival tissue; and safety. In a 2-stage design, a planned maximum of 29 response evaluable patients were to be enrolled. Results 32 pts were enrolled and have completed study treatment. Median age was 70; 88% of pts had received no prior chemotherapy; and ECOG performance status was 0/1 in 44%/56%. Bone/lymph node/visceral metastases were present in 91%/75%/12%. The median number of SB939 cycles administered was 3 (range 1-8). Adverse events were generally grade 1-2, with 5 pts experiencing one or more grade 3 events (fatigue (5), vomiting (1), dyspnea (1)). One pt died due to myocardial infarction. A confirmed PSA response was noted in 2 pts (6%), lasting 3.0 and 9.4 mo. In 16 pts with measurable disease, there were no objective responses, 7 pts had stable disease lasting 1.6 to 8.0 months. CTC response (from ≥5 at baseline to 5 at 6 weeks correlated with PSA progression. Correlative studies to evaluate T2-E from whole blood as well as T2-E fusion and PTEN deletion status on archival tissue are ongoing. Conclusion SB939 is tolerable at the dose/schedule given, but does not merit further study as a single agent in CRPC based on PSA RR. Activity was observed in terms of CTC declines however. Study of SB939 with cytotoxic or AR targeted therapies may be warranted. Supported by grants from the Canadian Cancer Society and the Ontario Institute for Cancer Research. Disclosure All authors have declared no conflicts of interest.