Identification of fluorescence in situ hybridization assay markers for prediction of disease progression in prostate cancer patients on active surveillance

Prostate cancer (PCa) is the second most common cancer in men with approximately 161,360 men diagnosed annually in the US [1] and 1.1 million men worldwide [2]. Although the lifetime risk of developing PCa is approximately 1 in 6 (~16%), the risk of dying from the disease is only ~2% [3]. Early diagnosis and treatment improved survival in patients with high-risk cancers, however, concerns exist regarding over diagnosis and over treatment of men with lower-risk PCa due to co-morbidities and healthcare costs [4, 5]. Over the last 15–20 years, what was the watchful waiting (WW) approach has evolved into active surveillance (AS), and has gained popularity for managing lower-risk PCa [4, 6, 7]. Men on AS are monitored with periodic biopsies, prostate examination, and prostate-specific antigen (PSA) tests, and treated only when the PCa shows signs of progression.

Clinical parameters such as Gleason score, PSA levels, patient demographics, and combinations of these parameters are used to stratify patients with low-risk (indolent) prostate cancer for AS. Novel imaging and molecular diagnostic tools are emerging to aid in patient risk stratification and monitoring on AS [7‐9]. New genomic biomarkers and biomarker panels, including gene copy number, rearrangements and germline mutations, are being assessed for association with clinically and histologically aggressive disease [10‐12]. However, current methods still lack the precision needed to reliably discriminate men with varying PCa risks. Given that PCa is both a biologically and clinically heterogeneous disease that develops amidst diverse genetic and epigenetic changes [13‐15], identification of molecular biomarkers that can reliably discriminate aggressive vs indolent disease, as well as biomarkers for monitoring of progression during AS is paramount.

Fluorescence in situ hybridisation represents a widely-used molecular technique that allows the detection of numerical and structural abnormalities in tissue and cytology specimens. Multiple chromosomal alterations have been reported in PCa, such as chromosome aneusomy, gain of the 8q24 (MYC) region, loss of 10q23 (PTEN) region, and translocations of ERG and ETV1 genes [13, 16‐22].

In this study, we evaluated FISH biomarkers on a retrospective case-control cohort of 108 PCa patients on WW/AS in order to establish a panel that can differentiate non-aggressive prostate cancer from aggressive prostate cancer.

The natural history of prostate carcinoma is highly variable, and it can be difficult, using current methodologies, to distinguish between patients with aggressive PCa that causes rapid tumour progression and significant clinical outcomes, and patients with indolent PCa. [26]. Undiagnosed, primarily indolent, prostate cancer is a common incidental finding in elderly men at autopsy [27]. This has important implications for management of PCa patients. Prostate specific antigen screening, for example, allows detection of more cases of asymptomatic prostate cancer, however, some of these tumours may not be biologically malignant. Patients with such indolent tumours would have little benefit from medical intervention, in part due to the comorbidities resulting from intervention therapy, such as radical prostatectomy (RP), which remains a preferred option for treatment of apparently localised disease. Thus, overtreatment of low-risk prostate cancer, which still occurs frequently, has significant impact on patient quality of life and health-related costs [28]. Radical prostatectomy represents a worthwhile medical intervention for patients cured of a life-threatening disease, however, not for patients whose tumours are not biologically aggressive, or for those patients who are discovered to have metastases a few months after surgery. This highlights the necessity for discovery and validation of reliable molecular markers to predict the behaviour of individual carcinomas.

FISH is an established molecular platform widely used in single, dual, or multicolour format for the detection of numerical and structural genomic abnormalities [29, 30]. The advantage of multicolour FISH is that this relatively simple technique allows for assessment of several genomic markers simultaneously in the context of the tissue specimen, capturing both genomic and structural heterogeneity of the prostate cancer. With the advent of automation and imaging systems, as well as assay chemistry improvements to reduce time to result, multiplex detection of more than four colours on one tissue specimen slide in 1–2 days has become possible [30, 31].

This study assessed whether multicolour FISH could be used to predict progressive PCa. In the preliminary feasibility, radical prostatectomy specimens were used to select FISH probes capable of discriminating patients who would recur within a 15-year follow-up period from those who would not. The hypothesis was that the disease recurrence in radical prostatectomy patients may reflect an aggressive form of prostate adenocarcinoma, with underlying molecular mechanisms that may overlap with those that enhance disease progression in patients on active surveillance. Based on the feasibility results, 12 probes were selected with a potential to discriminate progressive disease. These probes were organised in 3 probe sets and tested on core needle biopsy specimens obtained from patients who were enrolled in Active Surveillance and had a minimum of 10 years follow-up data.

FISH evaluation parameters were derived from enumeration results for each probe, and individual parameters, as well as parameter combinations, were analysed to identify the best combinations capable of discriminating progressive from indolent disease in the AS cohort. Combinations of FISH parameters in this study were selected that were statistically significant in predicting PCa outcome (progressive vs non-progressive), with the highest performance observed in 4–5 parameter combinations. If used in a multicolour FISH assay, these combinations would require 4–6 FISH probes – a level of multiplexing that is can be achieved with automated imaging systems [32].

Current clinical management and risk stratification of localised prostate cancer for enrolment into AS is based upon several clinical parameters including tumour stage, tumour grade as measured by the Gleason score, and the level of PSA assessed at the time of diagnosis [33]. Although these tools undoubtedly have predictive value, detecting progressive disease in a patient considered or selected for AS remains a challenge. It has been shown that many clinically low-risk prostate cancer patients are upgraded to a more aggressive disease at prostatectomy [34, 35]. According to recent estimates, approximately one-third of the patients are reclassified or upgraded as having a higher risk for progression during AS based on annual surveillance biopsy results [36, 37]. On the other hand, there remains a considerable discrepancy in current AS selection criteria, with a notion that some of the criteria may be too strict, thus excluding some patients in whom expectant management would be appropriate and safe [34, 38]. Therefore, it is important to determine whether genomic tissue biomarkers, such as the multicolour FISH panels used in this study, could improve the accuracy of risk stratification when used in combination with the standard of practice clinical parameters for enrolment into the AS. In the logistic regression that combined clinical risk stratification parameters (NCCN Risk Groups) with FISH, FISH parameter combinations were complimentary to clinical parameters and contributed significantly to the prediction of PCa outcome (progressive vs non-progressive). The combination of clinical parameters and FISH outperformed clinical parameters or FISH alone, with a maximum odd ratio of 7.0 achieved in the final model, as compared to 6.2 for the FISH parameters alone. Importantly, multicolour FISH appeared to add most value to risk stratification in the Intermediate risk group, a group of patients that could benefit from improved selection criterial for AS to reduce overtreatment without compromising survival [39]. Although the specimen set tested in this study is relatively small, an encouraging odds ratios of up to 16.5 were achieved in this group. It appears therefore plausible that utilizing multicolour FISH biomarkers could add value if incorporated into the clinical decision making process.

The limitation of this study is in distinguishing patients with the true rapid progression of the disease vs those with aggressive cancer missed on the initial biopsy due to the sampling error. The latter is especially relevant to the studies on archived specimens, approximately half of which have been collected under the original sextant biopsy protocol. However, one of the advantages of FISH is that it allows assessment of genomic biomarkers in the context of the tumour heterogeneity. In our earlier studies, we demonstrated that cytogenetic abnormalities could be observed by FISH within regions of benign histology extending beyond histologically evident tumour margin, indicating a field cancerisation effect in prostate cancer [40]. This characteristic may be beneficial to reduce sampling error and consequently the risk of missing a higher-grade cancer on initial biopsy prior to enrolment in AS, and would need to be addressed in the future studies. Evaluation of the biomarker combinations presented here warrants an additional study to validate their prognostic utility.

Combinations of FISH parameters capable of discriminating progressive from non-progressive disease were selected based on ROC curve analysis. Combination of clinical parameters with FISH demonstrated improved performance when compared to clinical parameters or to FISH alone. Additionally, FISH proved complimentary to clinical parameters (NCCN Risk Groups) in the final model, demonstrating the potential utility of multicolour FISH panels as an auxiliary tool for PCa risk stratification. Further studies with larger cohorts are planned to confirm these findings.