Identification of prognostic molecular biomarkers is critical for the clinical management of prostate cancer. With recent improvements in early detection of prostate cancer, studies are now focused on the identification and detection of significant molecular markers that can effectively distinguish men with high risk disease from the majority of indolent tumors. Recurrent gene fusions involving ETS family genes are observed in a majority of human prostate cancers, the most common being TMPRSS2-ERG
fusions occurring in ~50% of localized prostate cancers, and have potential implications for diagnosis, prognosis and therapy (31
In this study we developed dual color immunohistochemical method for the simultaneous detection of ERG-PTEN and ERG-SPINK1 status in prostate cancer. Previous studies have confirmed the diagnostic utility of immunohistochemisty in identifying ERG rearrangement (46
), PTEN deletion status (48
) and SPINK1 overexpression in prostate cancer (17
). This is the first study to report the successful simultaneous evaluation of ERG-PTEN and ERG-SPINK1 status in prostate carcinoma using dual color immunohistochemistry.
In our ERG-PTEN study, 35% of prostate carcinoma stained positive with ERG antibody. Previous studies have reported ERG antibody expression in 40-50% of prostate carcinomas, reflecting the incidence of ERG gene rearrangement in the Caucasian population (52
). We attribute the slightly lower incidence of ERG antibody expression in our cohort to the over-representation of samples with a dominant (index) nodule that did not always include the secondary smaller foci in the tissue microarray evaluated here. ERG rearrangement heterogeneity has been documented in previous studies between discrete tumor nodules within the same prostate gland (57
) and three cases in our study demonstrated heterogeneous ERG expression. Endothelial cells and lymphocytes stained positive with ERG antibody consistent with previous studies (46
In this new immunohistochemisty assay for PTEN, background benign glands showed robust PTEN staining while the fibromuscular stroma was predominantly negative, although some cases demonstrated weak staining of the stroma. The presence of an internal positive control allowed us to apply a simple dichotomous scoring system for malignant glands as either decreased/absent or normal cytoplasmic PTEN staining. Lotan et al. applied similar binary scoring system to assess PTEN status on immunohistochemisty and found this system to be highly reproducible, although their study found that PTEN was expressed in benign prostatic glands as well as the stroma (49
). Sangale et al. evaluated two different scoring systems (59
). The first system was similar to one used in the present study and they reported high reproducibility and concordance between pathologists. The second method was a 3 tier system using 0, 1+ and 2+ scores with endothelial cells as the reference for 2+ staining; however they concluded that endothelial cells were an imperfect control since their intensity varied within the same tissue sample. This system took into account the percent of cells staining and intensity, however the 3 tier scheme was not reproducible amongst pathologists.
A major benefit of our study is that we further validated our PTEN immunohistochemisty results with FISH. We found 87% concordance rate between immunohistochemisty and FISH with a sensitivity of 90% and specificity of 87%. Lotan et al. validated their immunohistochemisty data with FISH on some patients and high resolution copy number SNP microarray analysis for a separate group of patients for a total of 119 of 376 cases, with a sensitivity of nearly 80% for FISH and over 80% for high resolution SNP array(49
). Yoshimoto et al. performed immunohistochemisty and FISH on all of their 35 radical prostatectomy specimens and detected PTEN deletion in 24 of 35 prostate carcinoma patients (54
). All 24 positive cases demonstrated variable weak cytoplasmic and/or nuclear PTEN immunoreactivity thus demonstrating 100% concordance.
Although immunohistochemisty reliably detected homozygous PTEN deletion, in our study we were unable to consistently separate heterozygous from homozygous deletions. Immunohistochemisty was very sensitive in selecting homozygous deletions in cases with absent staining; however there was an overlap of homozygous and heterozygous deletions in cases displaying reduced staining.
gene rearrangement has been described as a prostate cancer-specific alteration, present in 40-50% of prostate cancers (8
rearrangement status has traditionally been detected by FISH or reverse transcriptase polymerase chain reaction techniques that are expensive and require fresh frozen tissues for RNA, specialized equipment and expertise. Recent studies have confirmed high correlation between positive ERG immunohistochemisty staining and FISH for ERG
gene rearrangements (46
). The sensitivity and specificity for prediction of ERG gene rearrangements using anti-ERG antibodies have been calculated at 95.7% and 96.5% (46
genomic loss was identified as a driving molecular aberration in prostate cancer almost 15 years ago (62
) and there is a large body of literature investigating the role of PTEN
in tumorigenesis, cancer progression and response to cancer therapies. The reported frequency of PTEN
deletion is variable between studies. A recent study from our group reported PTEN
deletion in 17% of localized prostate cancers and 54% of metastatic cancers (48
). Similarly, a study by Yoshimoto et al. reported PTEN
genomic deletions in 68% of prostate cancers (54
). Further, they attributed the variation in reported frequency of PTEN
deletion in prostate cancer to differences in tissue preparation, stage of disease and the methodology used to detect molecular aberrations. Cases with intact genomic PTEN
that results in negative PTEN immunohistochemisty may be due to the post-translational inactivation and inversion of PTEN
loss is more common in prostate cancer metastases than in primary tumors as reported by three independent studies, with the incidence of PTEN
loss around 50%. (48
). Loss of PTEN protein expression in prostate cancer has been correlated with poor prognosis and biochemical recurrence (67
). PTEN inactivation has been demonstrated to play an important role in progression to androgen-independence in prostate cancer (30
). Yoshimoto et al. suggested that the acquisition of the deletion and concomitant loss of PTEN functional activity at an earlier phase in prostatic oncogenesis is an important determinant of the molecular pathways that govern a more aggressive tumor phenotype (67
). With biochemical recurrence as the end point, their group identified three patient groups using the following genomic markers: (1) ‘poor genomic grade’ characterized by both PTEN
deletion and TMPRSS2:ERG
fusion; (2) ‘intermediate genomic grade’ with either PTEN
deletion or TMPRSS2:ERG
fusion and (3) ‘favorable genomic grade’ in which neither rearrangement was present (44
As reported by multiple studies, PTEN deletion itself represents an aggressive phenotype and in combination with TMPRSS2: ERG fusion is reported to have a ‘poor genomic grade’. The development of a dual color immunohistochemical assay for rapid, simultaneous evaluation of ERG and PTEN status in prostate cancer would enable better prediction of the course of the disease and identification and treatment of patients at higher risk. Simultaneous ERG and PTEN status detection in biopsies allows early detection in men harboring TMPRSS2:ERG rearrangement and PTEN inactivation with limited cancer on biopsy so they can pursue aggressive therapeutic options. This assay would be particularly advantageous for low-yield biopsy material as both ERG and PTEN status could be detected on a single section.
Our current study parallels a recent publication from our group that identified new methods to risk stratify and detect prostate cancer (70
). The study demonstrated that quantitative measurement of TMPRSS2:ERG
fusion transcript in urine in combination with urine prostate cancer antigen 3 (PCA3
) improved the performance of the multivariate Prostate Cancer Prevention Trial (PCPT) risk calculator in predicting cancer on biopsy. Given that ERG rearrangement is present in a subset of high-grade prostatic intraepithelial neoplasia, the dual immunohistochemical ERG-PTEN assay may be helpful in risk stratifying the high-grade prostatic intraepithelial neoplasia on biopsies in those cases where high-grade intraepithelial neoplasia were the only significant finding. Future studies exploring this group will be required to assess the utility of dual ERG-PTEN status for risk stratification in high-grade intraepithelial neoplasia cases.
outlier expression was observed in ~10% of prostate cancers, is mutually exclusive with ETS gene fusions and was found to be associated with an aggressive outcome (17
). A subsequent study suggested that SPINK1
in ETS-negative prostate cancers may be a promising therapeutic target (18
). Hence, simultaneous detection of ERG
rearrangement and SPINK1
status is important not only for molecular categorization of prostate carcinoma and identification of patients with a more aggressive outcome but also for identifying cancer patients who may benefit from emerging therapeutics. As discussed above, until recently ERG gene rearrangement status was assessed using FISH or reverse transcription polymerase chain reaction techniques. Recent studies utilizing novel anti-ERG monoclonal antibodies established the strong correlation between ERG gene rearrangement and positive immunohistochemisty staining (46
). Considering the combined incidence of about 50-60% of prostate cancer with ERG
rearrangement and SPINK1
overexpression, we developed a rapid, reliable and simple immunohistochemical assay to simultaneously detect ERG and SPINK1 status of prostate cancer. This assay has clinical implications for early detection in biopsy and prostatectomy specimens. We optimized the new protocol on both needle biopsy (data not shown) and prostatectomy samples.
SPINK1 positivity was detected in 9% of the cases (26 of 284 patients) consistent with our previous study (17
). No SPINK1 expression was noted in benign glandular tissue. We confirmed our earlier observation of mutual exclusivity of SPINK1 expression and ETS fusion status (17
). However we report for the first time, two cases with concomitant expression of ERG and SPINK1; in one case in the same focus of tumor and in the other in adjacent foci of same tumor nodule. These cases may represent a rare molecular subtype of prostate cancer and future studies in a large cohort are needed to explore the actual incidence and clinical significance of this subtype. Such observations can be attributed to the advantages of the new dual immunohistochemisty procedure presented in this study.
Although we performed the dual ERG-PTEN and ERG-SPINK1 immunohistochemisty assays using automated protocols, these assays are not restricted to automation; they can also be performed manually. The advantages of automated dual immunohistochemisty assays include simplicity, rapid turnover and consistency. The dual immunohistochemisty procedure takes on an average 4 to 5 hours and would be of great value to high volume laboratories in achieving fast turn-around time. Ours is the first study to report automated dual ERG-PTEN and ERG-SPINK1 immunohistochemisty to simultaneously detect ERG/PTEN and ERG/SPINK1 status in prostate carcinoma. These assays are simple, reliable, reproducible and easily portable to other laboratories. Antibody-based detection of PTEN and ERG shows a high concordance with FISH that offers a reliable alternative method for evaluating their status in prostate cancer. Similarly, we demonstrate that dual ERG-SPINK1 immunohistochemical assay is reproducible and highly sensitive for detecting small foci of SPINK1 expression. These assays will be useful for early screening for prostate cancer to select high risk patients for targeted therapies based on ERG, PTEN and SPINK1status. The dual staining methodology eliminates the need to perform the stain on two separate sections that can be of great value when biopsy samples are limited. Validation studies of this dual immunohistochemisty in prostate needle biopsies are underway. The assays have utility in retrospective or prospective studies for risk stratification of prostate cancer as well as prognostic and therapeutic decision-making purposes. Future studies using this novel dual immunohistochemistry assay will help to identify the incidence of the newly identified rare molecular subsets of prostate cancer with ERG-SPINK1 expression in the same or independent foci.