- Research article
- Open Access
PTEN loss in Gleason grade 7 prostate tumors exhibits intratumoral heterogeneity and is associated with unfavorable pathological features
Applied Cancer Researchvolume 39, Article number: 1 (2019)
PTEN loss is observed in 20–30% of prostate cancers and is associated with a poor outcome, but clinical details of the impact of this biomarker are unclear for intermediate grade tumors.
We investigated 43 radical prostatectomy-derived grade 7 prostate tumors from the Clinics Hospital of Ribeirão Preto. Tissue microarray (TMA) blocks were constructed and PTEN copy number status was determined for all patients through fluorescence in situ hybridization (FISH). To determine the presence of PTEN protein loss in our study cohort, we performed immunohistochemistry (IHC) in TMA sections. We then developed an automated algorithm in HALO™ to identify regions of PTEN protein loss in whole prostate scanned sections from ten patients with known PTEN deletion status by FISH. Clinical analyses were conducted to determine the associations between PTEN loss and patient outcome. All statistical analyses were conducted in R v3.4.3 with P-values below 0.05 being considered statistically significant.
In this study of 43 grade 7 tumors, we found PTEN deletions by FISH in 18.9% of tumors, and PTEN protein loss by IHC in 16.3% of tumors. Both techniques were highly concordant and complementary. Clinical analysis demonstrated that PTEN deletion by FISH was significantly associated with positive margin invasion (P = 0.04) and Gleason score upgrade (P = 0.001). Digital image analysis of ten representative tumors demonstrated distinct intratumoral heterogeneity for PTEN protein loss in four tumors.
This study shows that PTEN loss in Gleason grade 7 tumors can be heterogeneous and that a systematic analysis of this biomarker using a combination of FISH, IHC, and digital imaging may identify patients with a greater risk of poor outcome.
Prostate Cancer (PCa) is the most common solid tumor in men and is the third more common cancer type in the world . Phosphatase and tensin homolog (PTEN) is located in 10q23.31 and is the most frequent somatically mutated tumor suppressor gene in a variety of human malignancies (reviewed in ). Currently, immunohistochemistry and fluorescence in situ hybridization (FISH) assays of PTEN loss in PCa [3,4,5] have shown high specificity to identify tumors that carry the poor prognostic biomarker.
PTEN inactivation in prostate tumors is found in 20–30% of patients and is associated with adverse outcome . Most importantly, PTEN was found to be an independent prognostic indicator of PCa-specific death in both conservatively treated and surgically treated patients and may be employed in the clinical setting as a biomarker for disease stratification (reviewed in ). Although PTEN deficiency carries an unfavorable prognosis, the extent of heterogeneity of loss of the PTEN protein has not been objectively studied by imaging methods.
This study was designed to characterize PTEN deletions and protein loss in Gleason grade 7 (comprising Gleason score 3 + 4 and 4 + 3) to provide more details of the use of loss of PTEN as a biomarker for improved stratification of PCa with intermediate risk. Moreover, we performed whole slide digital image analysis to precisely determine how much intratumoral heterogeneity of PTEN protein loss was present in this study group of Gleason grade 7 tumors.
We evaluated 43 representative PCa cases collected after radical prostatectomy between 2009 and 2010 for this cohort study. Samples were fixed in formalin, embedded in paraffin and were obtained from the archive of the Pathology Service from the Clinics Hospital of Ribeirão Preto (HCRP). From this cohort, 35 tumors were classified as Gleason score 7 (3 + 4) and eight tumors were classified as Gleason score 7 (4 + 3). Gleason score upgrade refers to patients that presented higher Gleason score from radical prostatectomy specimens when compared to the initial Gleason score from preoperative needle core biopsy. Biochemical recurrence was defined as PSA levels > 0.2 ng/mL during the entire patient follow-up of this cohort. Clinical details of the studied cohort are shown in Additional file 1: Table S1.
TMA construction, immunohistochemistry, and FISH analyzes
For the construction of the TMA, we used the Manual Tissue Arrayer (MTA-1 – Beecher Instruments, Silver Spring, MD, USA) to obtain four 1 mm diameter tumor cores per patient. An initial analysis of whole prostate H&E sections was performed by a pathologist (F. P. S.) to identify two regions with the most prevalent Gleason pattern, one region of the second most prevalent Gleason pattern, and one benign adjacent region for each patient. Then, 1 mm needle cores were obtained to build a tissue microarray (TMA). The histological preparations were made as described previously . Four-Color PTEN FISH probes (PTEN del-TECT Four Color FISH probe, Biocare Medical, CA, USA) were used following recently published guidelines . PTEN FISH results were analyzed and interpreted as previously reported .
For immunohistochemistry, we used the anti-PTEN antibody D4.3 XP® (Cell Signaling, MA, USA) diluted 1:50 in the TMA samples. More details in the methods can be found elsewhere . The immunohistochemistry data were analyzed by two independent pathologists (F. P. S. and T. J.) based on a previously validated dichotomous scoring system . Briefly, TMA cores having intermingling of tumor with PTEN intact with tumor that has PTEN protein loss were classified as PTEN loss when more than 10% of the neoplastic glands had PTEN negative staining. Benign glands and stromal cells presenting positive PTEN staining were used as internal controls. TMA cores showing heterogeneous patterns of PTEN protein expression were classified using the 10% cut off mentioned above. Moreover, TMA cores lacking internal positive controls or with weak IHC staining were classified as inconclusive and were removed from further analyses. Patients were classified as having PTEN loss when at least one TMA core had PTEN protein loss.
Digital pathological analyses of PTEN protein expression
Since PTEN protein expression in PCa is often heterogeneous , our study was designed to characterize the presence of focal regions of PTEN protein loss in whole prostate glands. We selected ten patient tumors in which PTEN status had previously been determined by FISH based solely on analysis of TMA tumor cores. Based on the results of PTEN deletion status in the sampled TMA cores, we chose a total of ten cases from our cohort, of which three cases had PTEN homozygous deletions, three cases had PTEN hemizygous deletions, and four samples were PTEN intact. We obtained whole tumor sections from all selected ten cases and we performed IHC staining with anti-PTEN antibody following the protocol described above. Whole sections were then scanned using the Aperio Biosystems and analyzed with Aperio Scanscope (Leica Biosystems Inc., ON, Canada). The pathologist in our study (TJ) annotated all tumor glands of each section while excluding regions enriched with stromal cells. The annotated tumor regions were then analyzed using an automated algorithm developed in HALO™ (Indica Labs, NM, USA) for PTEN staining of prostate tumors (see Additional file 1: Supplementary Methods for more details).
Comparison between FISH and IHC for PTEN loss
The analysis of PTEN copy number using FISH was conclusive for 95% (41/43) of the patients. Two cases remained inconclusive due to poor quality probe hybridization. For the IHC, the results were conclusive for 90% (39/43) of the patients. FISH analysis of the TMA showed that PTEN deletion was present in 18.9% (8/41), with PTEN hemizygous deletions in 11.6% (5/41) and PTEN homozygous deletions in 7.3% (3/41) of tumors. Moreover, by IHC, we detected PTEN protein loss in 16.3% (7/39) of the patient tumors (Additional file 1: Table S2). Representative images from tumors with PTEN protein loss and PTEN deletions are shown in Additional file 1: Figure S1.
The comparison between FISH and IHC showed that both assays were highly concordant, with 90% (27/30) showing protein intact and undeleted PTEN by FISH (Additional file 1: Table S3).
Intratumoral heterogeneity of PTEN protein loss
To determine the extent of intratumoral heterogeneity of PTEN protein loss we performed image analysis on ten whole sections in which PTEN status had been previously characterized by FISH and IHC in tumor cores. We found that tumors with PTEN homozygous deletions had regions with complete loss of PTEN protein expression that was evident in the regions from which the pathologist had previously removed the core for TMA construction (Fig. 1a-c). We found that the three tumors with PTEN homozygous deletions have less positively stained cells and more negatively stained cells when compared with tumors with either PTEN hemizygous deletions or with PTEN intact (P = 0.01) (Table 1). These data suggest that digital imaging is able to detect quantitative difference in protein expression.
It can be seen that two (see Fig. 1b and c) of the three tumors with homozygous deletions demonstrate spatially distinct regions of intratumoral heterogeneity for PTEN loss by digital imaging. These likely reflect variation in expression levels of PTEN resulting from clonal regions of tumor that retained a functional copy of the gene. In contrast, the homozygously deleted case shown in Fig. 1a has uniform loss of expression throughout the regions of tumor.
From the three whole sections that presented PTEN hemizygous deletions (Fig. 1d-f), two cases presented a tumor region that had glands with uniform moderate and strong PTEN protein expression in regions containing tumor (Fig. 1d and e). In the third case with a PTEN hemizygous deletion, we observed intratumoral heterogeneity by image analysis evident as two distinct regions of PTEN protein loss surrounded by an extensive region that had retained PTEN protein expression in the tumor (Fig. 1f).
From the four samples that were classified as PTEN intact by FISH (Fig. 1g-j) all tumors had moderate to strong protein expression from regions cored for TMA analysis. However, in one tumor, we observed a complete PTEN protein loss (negative staining) in two small distant regions, both > 5 mm away from the sampled core region (arrows in Fig. 1h). Moreover, as illustrated in Fig. 1h the tumor region adjacent to core 1 was primarily characterized as intraductal carcinoma of the prostate. This case also presented a distant region adjacent to core 2 that had glands with PTEN loss surrounded by PTEN intact glands (moderate and strong staining).
Prognostic impact of PTEN gene and protein loss
We evaluated the effect of PTEN loss by FISH, IHC and both techniques in clinical features of the patients with PCa (Table 2). PTEN loss by FISH was associated with aggressive pathological features, such as significant presence of positive surgical margins (P = 0.04) and a trend towards extraprostatic extension (P = 0.09) (Table 2). We also found that 71% (5/7) of the cases with Gleason score 7(4 + 3) had PTEN deletions (P = 0.001). Concordantly, 57% (4/7) of patients that had Gleason score upgrade to 4 + 3 had PTEN deletions (P = 0.02). Furthermore, Log Rank analysis showed no significant association between PTEN deletions by FISH and PTEN loss by IHC and biochemical recurrence (P = 0.65 and P = 0.20, respectively) (Additional file 1: Figure S2).
Deciding the best treatment of newly diagnosed grade group 2/3 PCa is still challenging for urologists. In our study, we observed that PTEN deletion was detected in 18.9% for the patients of the HCRP cohort. Picanço-Albuquerque et al. (2016) showed that 17.2% of the Gleason 7 patients harbored PTEN deletions by FISH. PTEN protein analysis by IHC showed that 16.3% of the patients harbored protein loss, which is highly concordant for another Gleason score 7 cohort study by IHC (18.3%) .
Our findings demonstrate that PTEN deletions in grade group 2/3 PCa are associated with invasive pathological features and with Gleason score upgrade. A study conducted with 260 Gleason score 3 + 4 = 7 biopsies showed PTEN-deficient tumors are more likely to have non-organ confined disease at radical prostatectomy . Furthermore, several reports have shown significant associations between PTEN deletions found in needle core biopsies and Gleason score upgrade [4, 13] and other invasive features found at the time of surgery .
Digital image analysis of PTEN protein using ten whole sections identified distinct regions of intratumoral heterogeneity of PTEN protein loss in four of the ten tumors studied. Interestingly, we found that the presence of homozygous deletions of PTEN in a representative TMA cores was sufficient to predict that non-sampled region of the tumor will also show PTEN protein loss. In addition, we found that 25% (1/4) of the cases that were PTEN intact by FISH also had regions in the whole gland that presented with complete PTEN protein loss. We also found that one tumor with hemizygous PTEN gene deletion exhibited distinct regions with complete protein loss, suggesting that a second somatic inactivating mutation or epigenetic loss of function of the remaining intact PTEN took place during progression .
Our study demonstrates that PTEN loss in Gleason grade 7 tumors may be heterogeneous and that a systematic analysis of deletion or protein loss of this tumor suppressor gene using a combination of FISH, IHC, and digital imaging can help identify those patient tumors more likely to have a worse prognosis. Our findings also suggest that defining PTEN copy number status by FISH in TMA cores may be useful in predicting when whole pathological sections of PCa specimens have PTEN protein loss. Further investigation using the described approach should be carried out in larger cohorts to precisely determine how widespread PTEN inactivation is in PCa.
Fluorescence in situ hybridization
Clinics Hospital of Ribeirão Preto
Phosphatase and tensin homolog
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We acknowledge the São Paulo Research Foundation (FAPESP) for funding JAS and TV and the National Council for Scientific and Technological Development (CNPq) for funding CGPA. The authors also would like to thank Lee Boudreau for helping with immunohistochemistry staining and Shakeel Virk for scanning the stained TMAs on the Aperio Scanscope and developing HALO algorithm for protein analysis.
This research was supported by São Paulo Research Foundation (FAPESP) grants no. 2015/09111–5 (to JAS) and no. 2015/22785–5 and 2017/08614–9 (to TV). CGPA was funded by CNPq.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate
This study was approved by the Ethical Committee for Research from the Clinics Hospital of Ribeirão Preto. A consent form was signed by all the patients that provided pathological specimens for this study.
Consent for publication
The authors declare that they have no competing interests.
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Additional file containing supplementary methods, two figures and three tables. (PDF 666 kb)