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More than 1,300,000 prostate needle biopsies are performed annually in the U.S. with up to 16% incidence of isolated high-grade prostatic intraepithelial neoplasia (HGPIN). HGPIN has low predictive value for identifying prostate cancer (PCA) on subsequent needle biopsies in PSA screened populations. In contemporary series, PCA is detected in about 20% of repeat biopsies following a diagnosis of HGPIN. Further, discrete histological subtypes of HGPIN with clinical implication in management have not been characterized. The TMPRSS2-ERG gene fusion that has recently been described in PCA has also been demonstrated to occur in a subset of HGPIN. This may have significant clinical implications given that TMPRSS2-ERG fusion PCA is associated with a more aggressive clinical course.
In this study we assessed a series of HGPIN lesions and paired PCA for the presence of TMPRSS2-ERG gene fusion.
Fusion positive HGPIN was observed in 16% of the 143 number of lesions, and in all instances the matching cancer shared the same fusion pattern. 60% of TMPRSS2-ERG fusion PCA had fusion negative HGPIN.
Given the more aggressive nature of TMPRSS2-ERG PCA, the findings of this study raise the possibility that gene fusion positive HGPIN lesions are harbingers of more aggressive disease. To date, pathological, molecular and clinical parameters do not help stratify which men with HGPIN are at increased risk for a cancer diagnosis. Our results suggest that the detection of isolated TMPRSS2-ERG fusion HGPIN would improve the positive predictive value of finding TMPRSS2-ERG fusion PCA in subsequent biopsies.
In the United States, approximately 1,300,000 prostate biopsies were performed in 2006 with the detection of 234,460 new cases of prostate cancer [American Cancer Society, Cancer Facts & Figures 2006]. The incidence of isolated high-grade prostatic intraepithelial neoplasia (HGPIN) without carcinoma ranges from <1% to 16%1–5, and the risk of finding carcinoma on subsequent biopsies is 10–39% (median risk of 24%6 depending on the time of repeat biopsy and number of cores7–10. A decline in the predictive value of HGPIN for prostate cancer to about 20% in contemporary needle biopsies is most likely due to extended biopsy techniques that yield higher rates of cancer detection11.
Both HGPIN and prostate adenocarcinoma share molecular anomalies including telomere shortening12, RARβ2 hypermethylation13, allelic imbalances14, and several chromosomal anomalies and c-myc amplification15–17. Overexpression of p1618, reduction of annexin I19 and altered proliferation and apoptosis20 in HGPIN and prostate cancer has also been demonstrated. Table 1 summarizes a selection of molecular alterations identified in HGPIN and prostate cancer.
Despite the association with prostate cancer, distinct subtypes of HGPIN with clinical relevance (i.e. greater risk of predicting aggressive cancer) have not been characterized. A recent rearrangement involving the androgen-regulated gene TMPRSS2 and members of the ETS transcription factor family has been identified21 and confirmed by multiple other groups22–28. In particular, the TMPRSS2-ERG gene fusion prostate cancer is associated with higher tumor stage and tumor-specific death or metastasis25, 29–31. Two recent studies have demonstrated the presence of TMPRSS2-ERG gene fusion in approximately 20% of HGPIN lesions22, 26.
The purpose of this study was to assess the TMPRSS2-ERG gene fusion status in a large series of HGPIN lesions with paired prostate cancer. Based on the results, we postulate that TMPRSS2-ERG fusion HGPIN is a distinct molecular subtype and its identification indicates the presence of the same genetic aberration in prostate cancer if present. This may impact clinical management of isolated HGPIN in prostate needle biopsies.
143 HGPIN lesions from equal number of patients were interrogated for the presence of TMPRSS2-ERG gene fusion. This study was conducted under the IRB protocol 2006-P-000715/1 BWH at Brigham and Women’s Hospital. The HGPIN lesions were represented on 22 tissue microarrays (TMA) from prostatectomy specimens (96/143), 34 prostate needle biopsies, and 13 full section prostatectomy samples. Of these, 87% (124/143) had paired prostate cancer. The remaining 19 cases demonstrated isolated HGPIN without evidence of concurrent cancer, and included two cases of HGPIN with adjacent atypical small acinar proliferation10, 32. Clinical and pathologic demographics were available for 93 of the 143 patients. These included 70 of 124 HGPIN lesions with paired prostate cancer as follows: 40 of 96 patients represented in the TMAs, all 34 patients represented in the needle biopsies, and 9 of 13 patients represented in prostatectomy samples. The mean age at presentation was 60 years with a mean pre-operative PSA of 16.5 ng/ml. There were 30% Gleason grade ≤6, 51% Gleason grade 7, and 19% Gleason grade ≥8 prostate cancers.
The morphological diagnosis was confirmed on H&E stained paraffin sections by two pathologists (J-MM and SP) prior to assessment of gene fusion by fluorescent in-situ hybridization (FISH) on a step section, corresponding to one unstained section at identical level obtained at the time of initial tissue sectioning. HGPIN lesions were differentiated into four morphological subtypes: tufting, flat, micropapillary, and cribriform33, 34. In a subset of cases with equivocal diagnosis, immunohistochemistry (IHC) for prostatic basal cells was performed. These were 6 needle biopsy cases with atypical small acinar proliferation (ASAP) for which IHC helped to confirm the diagnosis of prostate cancer. For that purpose, additional unstained slides were deparaffinized in xylene and rehydrated in graded ethanols. The tissue level of the immunohistochemical study was identical to the original H&E. Pressure-cooking was applied as the antigen retrieval method. Primary antibodies against p63 (1:50 dilution of clone 4A4, NeoMarkers, Fremont, CA) and high molecular weight cytokeratin (1:200 dilution of clone 34βE12, DAKO, Carpinteria, CA) for the detection of basal cells were applied with over night incubation at 4°C in a humid chamber. Immunohistochemistry was performed with the avidin-biotin peroxidase technique.
We have previously described a dual-color interphase break-apart FISH assay to indirectly assess the fusion of TMPRSS2-ERG25, 26, 29. Briefly, two differentially labeled probes were designed to span the telomeric and centromeric neighboring regions of the ERG locus. Using this break-apart probe system a nucleus without ERG rearrangement demonstrates two pairs of juxtaposed red and green signals, forming yellow fusion signals. A nucleus with an ERG break-apart (reflecting a TMPRSS2-ERG fusion) shows split-apart of one juxtaposed red-green signal pair resulting in a single red and green signal for the translocated ERG allele, and a still combined (yellow) signal for the non-translocated ERG allele in each nucleus. The samples were analyzed under a 60× oil immersion objective using an Olympus BX-51 fluorescence microscope equipped with appropriate filters, a CCD (charge-coupled device) camera (Olympus, Center Valley, PA), and the CytoVision FISH imaging and capturing software (Applied Imaging, San Jose, CA). Evaluation of the cases was independently performed by two pathologists (J-MM and SP), both with expertise in analyzing interphase FISH experiments. For each case, we attempted to score at least 50 nuclei. Cases with significant differences between the results of both pathologists were refereed by a third pathologist (MAR).
Of the 143 HGPIN cases, 16% (23/143) demonstrated TMPRSS2-ERG gene fusion. All cases shared the same fusion status with the paired prostate cancer (22/22). There was a single case of TMPRSS2-ERG fusion HGPIN without concurrent adenocarcinoma. The follow-up biopsy of this isolated HGPIN on prostate needle biopsy had not been performed at the time of preparing this manuscript. Of 120 TMPRSS2-ERG fusion negative HGPIN cases, 85% (102/120) had matching adenocarcinoma, and in 32% of these (33/102) the paired prostate cancer demonstrated TMPRSS2-ERG fusion (Figure 1).
Two cases of HGPIN also demonstrated adjacent small atypical glands10, 32. One was fusion positive in both areas (Figure 2A), whereas the other one showed fusion negative HGPIN with adjacent fusion positive atypical glands. Neither case had follow-up re-biopsy at the time of preparing this manuscript. Interestingly, we could identify two cases that showed presence of TMPRSS2-ERG gene fusion HGPIN and adjacent normal epithelium (with no fusion), within the same gland (Figure 2B). Among the morphological subtypes, 31% (44/143) were tufting HGPIN, 4% (6/143) showed flat HGPIN, 2% (3/143) were micropapillary HGPIN, 1% (1/143) cases had cribriform HGPIN morphology, and 62% (89/143) combined more than one of the above subtypes.
Several suggested protocols for management of isolated HGPIN in prostate needle biopsies exist. They vary from repeat biopsy at three to six months, at six to twelve months, or at three years35–37. The most aggressive protocol suggests repeat biopsies at three to six-month intervals for two years, thereafter every year for life7. Recent data suggest that the incidence of prostatic adenocarcinoma after the initial diagnosis of isolated HGPIN in needle biopsies is lower than previously reported10, 11, and despite molecular data on HGPIN, biomarkers with direct clinical application have not been used to stratify the risk for subsequent detection of adenocarcinoma. In addition, morphologic features and extent of HGPIN show inconsistent data to predict risk of consecutive prostate cancer. Therefore, the clinical management of patients with isolated HGPIN is problematic and to date, no treatment is indicated after this diagnosis is rendered.
It is valid to speculate that stratification of different subtypes of HGPIN at the molecular level (i.e. TMPRSS2-ERG fusion HGPIN) may be needed for potential prognostic implications, and in view of clinical trials for chemoprevention of prostate cancer where the inclusion criteria is the diagnosis of isolated HGPIN38, 39.
Our results may help in prognostication of a subset of isolated HGPIN lesions, that is, those harboring the TMPRSS2-ERG gene fusion. We have recently postulated that the TMPRSS2-ERG gene fusion is a clonal, early pathogenic event in prostate cancer26, 40. Evidence supporting this hypothesis is that in most instances the gene fusion is homogenously present throughout the cancer within a tumor nodule, is not identified in benign prostatic tissue, and is detected only in a subset of HGPIN lesions. Another group has also confirmed the presence of TMPRSS2-ERG gene fusion in HGPIN using polymerase chain reaction (PCR) technique22. Interestingly, both studies show approximately 20% gene fusion positivity among a small series of HGPIN.
In the current study, the incidence of TMPRSS2-ERG gene fusion HGPIN is 16%, in 143 cases. Given that all TMPRSS2-ERG gene fusion HGPIN lesions share the same fusion pattern with matching cancer, and no fusion positive HGPIN lesions were associated with paired TMPRSS2-ERG fusion negative prostate cancer, we demonstrate that the presence of TMPRSS2-ERG gene fusion HGPIN is always indicative of a prostate cancer bearing the same genetic aberration. Conversely, TMPRSS2-ERG fusion prostate cancer may present with fusion negative HGPIN. Possible scenarios that could explain this finding are that fusion negative HGPIN does either not precede TMPRSS2-ERG fusion prostate cancer, or that TMPRSS2-ERG fusion HGPIN was not sampled if we consider the presence of gene fusion heterogeneity in HGPIN as a possibility. In our previous work26, 41 we had made these observations. However, in the series reported by Cerveira et al22, PCR assessment yielded two cases where the fusion transcript was detected in HGPIN, but not in the concurrent cancer of the same gland. In the present study we have screened a significantly larger number of HGPIN lesions using FISH, the gold standard method to detect these molecular alterations, and we have not observed such combination. This discrepancy could be due to artifact in the PCR assay, or as a consequence of TMPRSS2-ERG heterogeneity in prostate cancer, where the fusion positive area of tumor may have not been sampled. Although TMPRSS2-ERG gene fusion heterogeneity in prostate cancer is out of the scope of the current study, it is pertinent to mention that in our most recent study, 41% of radical prostatectomy high stage cases (at least pT2c) demonstrated interfocal clonal heterogeneity40, also described by Mehra et al42 and Furusato et al43. This fact may have significant clinical implications for follow-up biopsy and treatment strategies, in the context of isolated TMPRSS2-ERG fusion HGPIN.
Taking these results together, we consider that TMPRSS2-ERG fusion HGPIN is a true precursor of a subset of TMPRSS2-ERG prostate cancer, and the presence of the former is always indicative of the latter. Remarkably, we identified two cases where TMPRSS2-ERG fusion HGPIN was showing either early invasion (see Figure 2A) or coexistence with normal epithelium in the same gland (see Figure 2B). This morphologic/gene fusion status correlation further supports our statement, as well as the hypothesis of HGPIN to cancer progression (in this case, of those lesions harboring the TMPRSS2-ERG fusion). These observations are clinically relevant since there is emerging data supporting that TMPRSS2-ERG fusion prostate cancer is associated with worse prognosis, namely, higher tumor stage and tumor-specific death or metastasis24, 25, 29, 31, 44, 45. Hence, the finding of isolated TMPRSS2-ERG fusion HGPIN in needle biopsies may have the highest predictive value for further detection of fusion positive prostate cancer with the significant clinical implication noted above.
Based on the results of our recent work on morphological features associated with TMPRSS2-ERG fusion prostate cancer46, we also considered a potential correlation between the morphology of HGPIN and the TMPRSS2-ERG fusion status. However, 62% of HGPIN cases combined two or more of the morphologic subtypes, hampering a significant association.
Although prospective studies with follow-up of isolated TMPRSS2-ERG gene fusion HGPIN are needed to modify the current approach of management of isolated HGPIN, our results show convincing evidence that fusion positive HGPIN lesions are consistently associated with TMPRSS2-ERG prostate cancer. To further support our findings, studies with follow-up of patients with isolated TMPRSS2-ERG fusion HGPIN or TMPRSS2-ERG fusion HGPIN with adjacent small atypical glands like one or our cases, are underway as part of an Early Detection Research Network (EDRN) protocol. Further, evaluation of the status of TMPRSS2-ERG fusion could also modify inclusion criteria in the aforementioned clinical trials. Moreover, the development of non-invasive (i.e. urine based) diagnostic tests for fusion transcripts could also help in these protocols47.
In summary, we have assessed the largest series of HGPIN lesions for TMPRSS2-ERG fusion status to date and confirmed a prevalence of 16%, similar to previously reported series. In all instances, fusion positive HGPIN is associated with concurrent TMPRSS2-ERG prostate cancer. Given the worse prognosis linked to the latter, detection of isolated TMPRSS2-ERG fusion HGPIN may help us stratify patients into a discrete risk group.
Research supported by the NIH Prostate SPORE at the Dana-Farber/Harvard Cancer Center NCI P50 CA090381(M.A.R.), R01AG21404 (M.A.R), German Research Foundation PE1179/1-2) and Department of Defense Grant (PC050965) (S.P.), NIH Grant UO1 CA 113913 for the BID EDRN (Harvard/Michigan Prostate Cancer Clinical Center), and UCSF Prostate Cancer SPORE, NIH Grant P50CA89520 (P.L.P., J.S.).
The authors are grateful to Chungdak Namgyal of the DFHCC TMA core facility, and Laura A. Johnson and Christopher LaFargue for technical support critical to this study.