Histopathology and immunohistochemical analysis of 5-fluorouracil and triamcinolone treated keloids in double-blinded randomized controlled trial.

Keloids are a major complication related to surgical wound healing and very challenging condition to treat. Many treatment options are available, but the efficacy of the treatment is poor in most of cases and some keloids do not respond to the treatment at all. We compared the efficacy of intralesional 5-fluorouracil (5-FU) and triamcinolone (TAC) injections in a double-blind randomized controlled trial (RCT). Forty-three patients with 50 keloid scars were treated with either intralesional TAC or 5-FU-injections over 6 months. We wanted to find out whether biological features (cell density, cell proliferation rate, vascular density, myofibroblast numbers, steroid hormone receptor expression) in keloids could be used to predict the response to therapy and define the biological changes that take place in patients receiving a response. As there was no statistically significant difference in the remission rate between TAC and 5-FU treatments, all patients were combined and analyzed as responders and nonresponders. Although responders have slightly more myofibroblasts than the nonresponders in their keloids in the pretreatment biopsy samples, we could not identify a single predictive factor that could identify those patients that respond to drug injections. The good clinical response to therapy is associated with the simultaneous reduction of myofibroblasts in the keloid. This study demonstrates that myofibroblasts are reduced in number in those keloids that were responsive to therapy, and that both 5-FU and TAC injections are useful for keloid treatment.

Length of prostate biopsies is not necessarily compromised by pooling multiple cores in one paraffin block: an observational study.

BACKGROUND: Individually submitted prostatic needle biopsies are recommended by most guidelines because of their potential advantage in terms of core quality. However, unspecified bilateral biopsies are commonly submitted in many centers. The length of the core is the key quality indicator of prostate biopsies. Because there are few recent publications comparing the quality of 12 site-designated biopsies versus pooled biopsies, we compared the lengths of the biopsies obtained by both methods. METHODS: The material was obtained from 471 consecutive subjects who underwent prostatic needle biopsy in the Tampere University Hospital district between January and June 2013. Biopsies from 344 subjects fulfilled the inclusion criteria. The total number of cores obtained was 4047. The core lengths were measured on microscope slides. Extraprostatic tissue was subtracted from the core length. RESULTS: The aggregate lengths observed were 129.5 ± 21.8 mm (mean ± SD) for site-designated cores and 136.9 ± 26.4 mm for pooled cores (p = 0.09). The length of the core was 10.8 ± 1.8 mm for site-designated cores and 11.4 ± 2.2 mm for pooled cores (p = 0.87). The median length for pooled cores was 11 mm (range 5 mm - 18 mm). For individual site-designated cores, the median length was 11 mm (range 7 mm -15 mm). The core length was not correlated with the number of cores embedded into one paraffin block (r = 0.015). There was no significant difference in cancer detection rate (p = 0.62). CONCLUSIONS: Our results suggest that unspecified bilateral biopsies do not automatically lead to reduced core length. We conclude that carefully embedded multiple (three to nine) cores per block may yield cores of equal quality in a more cost-efficient way and that current guidelines favoring individually submitted cores may be too strict.

ImmunoMembrane: a publicly available web application for digital image analysis of HER2 immunohistochemistry.

AIMS: Assessment of the human epidermal growth factor receptor 2 (HER2) with immunohistochemistry (IHC) is routine practice in clinical pathology laboratories. Visual classification of the staining reaction (usually into 0/1+, 2+ or 3+) is subjective and prone to significant inter- and intra-observer variation. In this study, we describe ImmunoMembrane, an easy-to-use HER2 IHC analysis software, which is freely available as a web application, requiring no download or installation. METHODS AND RESULTS: ImmunoMembrane uses colour deconvolution for stain separation and a customized algorithm for cell membrane segmentation. A quantitative score (IM-score, 0-20 points) is generated according to the membrane staining intensity and completeness. Specimens are classified into 0/1+, 2+ or 3+ based on IM-score cut-offs defined using a training set. The classification and membrane segmentation are presented as a pseudo-coloured overlay image. With a validation set (144 HercepTest(®) -stained whole tissue sections), ImmunoMembrane matched well with the pathologist's visual classification (weighted kappa κ(w) =0.80), as well as fluorescence in-situ hybridization (FISH) (IHC disagreement 3.5%, n=144) and chromogenic in-situ hybridization (CISH) (IHC disagreement 2.8%, n=144). CONCLUSIONS: We anticipate that publicly available web applications, such as ImmunoMembrane, will accelerate the adoption of automated image analysis in clinical diagnostics of HER2 IHC. ImmunoMembrane is freely accessible at: http://jvsmicroscope.uta.fi/immunomembrane/.

Histopathological variables and biomarkers enhancer of zeste homologue 2, Ki-67 and minichromosome maintenance protein 7 as prognosticators in primarily endocrine-treated prostate cancer.

OBJECTIVE: • To evaluate the prognostic value of histopathological variables and immunostainings of biomarkers enhancer of zeste homologue 2 (EZH2), Ki-67 and minichromosome maintenance protein 7 (MCM7) from core biopsies of hormonally treated patients with prostate cancer. PATIENTS AND METHODS: • Biopsies of 247 primarily endocrine-treated patients were analysed for histopathological characteristics and Gleason scores (GS) according to the revised guidelines of International Society of Urologic Pathology (ISUP) consensus conference 2005. • Immunohistochemical stainings were analysed with the aid of digital image analysis. • The prognostic value of the histopathological variables and the biomarkers was analysed with univariate and multivariate Cox regression analysis, with biochemical recurrence as an endpoint. RESULTS: • Biomarkers EZH2 (relative risk [RR] 2.0, 95% confidence interval 1.2-3.3), Ki-67 (3.4, 2.1-5.5) and MCM7 (2.4, 1.5-3.9) were significantly associated with progression-free survival in a univariate analysis. • Ki-67 immunostaining index detected high-risk patients with GS of 7 (9.1, 8.0-10.3). • In a multivariate analysis with non-conventional GS groups 5-7 (3 + 4), 7(4 + 3)-8, and 9-10, the independent prognostic markers were pretreatment GS (2.2, 1.5-3.2), prostate-specific antigen (PSA) level (2.1, 1.1-4.2), perineural invasion (PNI) (1.6, 1.2-2.2), and clinical T-stage (cT) (1.9, 1.0-3.7). • Combination of the independent markers (PSA level > 20 ng/mL or GS >3 + 4 or PNI >3 or cT >2) yielded best risk stratification (RR 11.6, 10.4-12.7). CONCLUSIONS: • GS remains one of the most important prognostic factors in prostate cancer. However, the refined guidelines by ISUP 2005 might have shifted the threshold between low-grade and high-grade cancers from GS 6 vs 7 to GS 3 + 4 vs 4 + 3. • PNI is an independent prognostic marker superior to cT. • Ki-67 is the most useful biomarker in detecting patients with GS = 7 at high risk for progression.

Overall and worst gleason scores are equally good predictors of prostate cancer progression.

BACKGROUND: Gleason scoring has experienced several modifications during the past decade. So far, only one study has compared the prognostic abilities of worst (WGS) and overall (OGS) modified Gleason scores after the ISUP 2005 conference. Prostatic needle biopsies are individually paraffin-embedded in 57% of European pathology laboratories, whereas the rest of laboratories embed multiple (2 - 6) biopsies per one paraffin-block. Differences in the processing method can have a far-reaching effect, because reporting of the Gleason score (GS) is different for individually embedded and pooled biopsies, and GS is one of the most important factors when selecting treatment for patients. METHODS: The study material consisted of needle biopsies from 236 prostate cancer patients that were endocrine-treated in 1999-2003. Biopsies from left side and right side were embedded separately. Haematoxylin-eosin-stained slides were scanned and analyzed on web-based virtual microscopy. Worst and overall Gleason scores were assessed according to the modified Gleason score schema after analyzing each biopsy separately. The compound Gleason scores (CGS) were obtained from the original pathology reports. Two different grade groupings were used: GS 6 or less vs. 7 vs. 8 or above; and GS 7(3 + 4) or less vs. 7(4 + 3) and 8 vs. 9-10. The prognostic ability of the three scoring methods to predict biochemical progression was compared with Kaplan-Meier survival analysis and univariate and multivariate Cox regression analyses. RESULTS: The median follow-up time of the patients was 64.5 months (range 0-118). The modified GS criteria led to upgrading of the Gleason sums compared to the original CGS from the pathology reports 1999-2003 (mean 7.0 for CGS, 7.5 for OGS, 7.6 for WGS). In 43 cases WGS was > OGS. In a univariate analysis the relative risks were 2.1 (95%-confidence interval 1.8-2.4) for CGS, 2.5 (2.1-2.8) for OGS, and 2.6 (2.2-2.9) for WGS. In a multivariate analysis, OGS was the only independent prognostic factor. CONCLUSIONS: All of the three Gleason scoring methods are strong predictors of biochemical recurrence. The use of modified Gleason scoring leads to upgrading of GS, but also improves the prognostic value of the scoring. No significant prognostic differences between OGS and WGS could be shown, which may relate to the apparent narrowing of the GS scale from 2-10 to 5-10 due to the recent modifications.

Digital image analysis of the tissue surface areas of site-designated and bilaterally pooled prostate biopsies.

Initial reports about the length of bilaterally pooled biopsies showed alarming tissue loss compared to individual biopsies, but the current understanding of "noodle biopsies" and better embedding techniques may have improved their quality. Here, we implemented digital image analysis to study the differences in tissue surface areas between individual and pooled cores. Prostate biopsy reports from 1242 consecutive patients were reviewed. Urologist-dependent bias on the biopsy quality was eliminated by identifying four urologists who submitted equally individual and bilaterally pooled biopsies. Digital image analysis was applied to the tissue surface areas of 936 virtual slides containing 1440 biopsy cores (12 cores per patient x 120 patients) taken by the four urologists. The median (range) surface areas were 73.8 mm² (40.1-102.5) for the site-designated (n=57) and 77.1 mm² (49.5-119.2) for the bilaterally pooled biopsies (n=63) (p=0.19). For three urologists, the median surface areas were 69.5 mm² (60.4-93.2), 75.5 mm² (48.2-98.7) and 78.2 mm² (47.1-92.7) for the site-designated and 79.2 mm² (49.5-116.4), 69.3mm² (49.6-119.2) and 79.2 mm² (55.1-96.7) for the pooled biopsies, respectively (p=0.58-0.75). For one urologist, the median surface area was marginally higher for the pooled biopsies, 68.1 mm² (40.1-102.5) vs. 81.6 mm² (62.7-108.8) (p=0.03). In conclusion, the histological yields of individual and pooled prostate biopsies were practically equal. The results should not be considered as a recommendation to increasingly submit unspecified bilateral cores but to encourage pathology laboratories to embed and cut all received prostate biopsies with special attention, regardless of submission type.

CIP2A is a candidate therapeutic target in clinically challenging prostate cancer cell populations.

Residual androgen receptor (AR)-signaling and presence of cancer stem-like cells (SCs) are the two emerging paradigms for clinically challenging castration-resistant prostate cancer (CRPC). Therefore, identification of AR-target proteins that are also overexpressed in the cancer SC population would be an attractive therapeutic approach.Our analysis of over three hundred clinical samples and patient-derived prostate epithelial cultures (PPECs), revealed Cancerous inhibitor of protein phosphatase 2A (CIP2A) as one such target. CIP2A is significantly overexpressed in both hormone-naïve prostate cancer (HN-PC) and CRPC patients . CIP2A is also overexpressed, by 3- and 30-fold, in HN-PC and CRPC SCs respectively. In vivo binding of the AR to the intronic region of CIP2A and its functionality in the AR-moderate and AR-high expressing LNCaP cell-model systems is also demonstrated. Further, we show that AR positively regulates CIP2A expression, both at the mRNA and protein level. Finally, CIP2A depletion reduced cell viability and colony forming efficiency of AR-independent PPECs as well as AR-responsive LNCaP cells, in which anchorage-independent growth is also impaired.These findings identify CIP2A as a common denominator for AR-signaling and cancer SC functionality, highlighting its potential therapeutic significance in the most clinically challenging prostate pathology: castration-resistant prostate cancer.

Loss of PTEN is associated with aggressive behavior in ERG-positive prostate cancer.

BACKGROUND: The associations of ERG overexpression with clinical behavior and molecular pathways of prostate cancer are incompletely known. We assessed the association of ERG expression with AR, PTEN, SPINK1, Ki-67, and EZH2 expression levels, deletion, and mutations of chromosomal region 3p14 and TP53, and clinicopathologic variables. METHODS: The material consisted of 326 prostatectomies, 166 needle biopsies from men treated primarily with endocrine therapy, 177 transurethral resections of castration-resistant prostate cancers (CRPC), and 114 CRPC metastases obtained from 32 men. Immunohistochemistry, FISH, and sequencing was used for the measurements. RESULTS: ERG expression was found in about 45% of all patient cohorts. In a multivariate analysis, ERG expression showed independent value of favorable prognosis (P = 0.019). ERG positivity was significantly associated with loss of PTEN expression in prostatectomy (P = 0.0348), and locally recurrent CRPCs (P = 0.0042). Loss of PTEN expression was associated (P = 0.0085) with shorter progression-free survival in ERG-positive, but not in negative cases. When metastases in each subject were compared, consistent ERG, PTEN, and AR expression as well as TP53 mutations were found in a majority of subjects. CONCLUSIONS: A similar frequency of ERG positivity from early to late stage of the disease suggests lack of selection of ERG expression during disease progression. The prognostic significance of PTEN loss solely in ERG-positive cases indicates interaction of these pathways. The finding of consistent genetic alterations in different metastases suggests that the major genetic alterations take place in the primary tumor. IMPACT: Interaction of PTEN and ERG pathways warrants further studies.

Routine dual-color immunostaining with a 3-antibody cocktail improves the detection of small cancers in prostate needle biopsies.

We performed dual-color immunostaining with a 3-antibody cocktail (α-methylacyl coenzyme-A racemase, CK34betaE12, and p63) on prostate biopsies from 200 patients. Current practice (hematoxylin and eosin staining followed by dual-color immunostaining on selected cases) was compared with a protocol in which routine dual-color immunostaining was provided in all cases. In the original pathology reports, adenocarcinoma was diagnosed in 87/200 (43%) patients. Small foci interpreted as putative cancers were detected with dual-color immunostaining in 14/113 patients who were originally diagnosed with a nonmalignant lesion. All of the suggested cancerous foci were independently reevaluated by 5 pathologists. A diagnosis of adenocarcinoma was assessed by consensus in 8 cases, and atypical small acinar proliferation was diagnosed in 1 case. Consensus was not reached in 5 cases. Six of the foci reclassified as cancer were of Gleason score 3 + 3 = 6, while 2 were graded as Gleason score 4 + 4 = 8. The feasibility of routine dual-color immunostaining was also tested by analyzing the time spent on microscopic assessment. Because small, atypical lesions expressing α-methylacyl coenzyme-A racemase (blue chromogen) were easy to detect using dual-color immunostaining, the microscopic analysis of dual-color immunostaining and hematoxylin-eosin staining was faster than that of hematoxylin-eosin staining alone that was later followed by dual-color immunostaining in selected cases (median 251 seconds versus 299 seconds, P < .0001). We concluded that routine dual-color immunostaining of all prostate biopsies would produce better diagnostic sensitivity with a smaller microscopy workload for the pathologist. However, minute foci interpreted as cancer with dual-color immunostaining need to be confirmed with hematoxylin-eosin staining, and minimal criteria for a definitive diagnosis of cancer are still lacking.

Association of SPINK1 expression and TMPRSS2:ERG fusion with prognosis in endocrine-treated prostate cancer.

PURPOSE: The aim of the study was to examine whether TMPRSS2:ERG fusion or SPINK1 protein expression is associated with hormone responsiveness of prostate cancer and can thus be used as a biomarker. EXPERIMENTAL DESIGN: Diagnostic needle biopsies from prostate cancer patients primarily treated by endocrine therapy were evaluated for TMPRSS2:ERG fusion with fluorescence in situ hybridization and SPINK1 protein expression with immunohistochemistry. RESULTS: The frequency of TMPRSS2:ERG fusion in 178 biopsies of hormonally treated patients was 34%. Of the fusion-positive cases, 71% showed deletion between the two genes, and 23% showed gain of the fusion. The fusion was associated with high Ki-67 staining (P=0.001), age at diagnosis (P=0.024), and tumor area (P=0.006), but not with Gleason score, T stage, M stage, prostate-specific antigen (PSA), or progression-free survival. Strong positive SPINK1 expression was found in 11% (21 of 186) of the biopsies. SPINK1-positive cases had significantly shorter progression-free survival compared with SPINK1-negative cases (P=0.001). The expression was not associated with any other clinicopathologic variables studied. In a multivariate analysis, SPINK1 expression showed independent prognostic value, with a relative risk of 2.3 (95% confidence interval, 1.1-4.6). SPINK1 expression and the fusion were not associated with each other. CONCLUSIONS: There was no association between TMPRSS2:ERG fusion and prognosis, suggesting that TMPRSS2:ERG rearrangement does not implicate hormone dependence of the cancer. SPINK1 expression, found in approximately 10% of prostate cancers, was associated with aggressive form of the disease and could serve as a biomarker in endocrine-treated prostate cancer.

Bax and Bcl-2 are focally overexpressed in the normal epithelium of cancerous prostates.

OBJECTIVE: Development of prostate cancer is connected with a disturbance of apoptosis. Prostate cancer is multifocal, suggesting that the control of apoptosis is impaired at multiple foci. We wanted to know whether apoptosis is generally disturbed in cancerous prostates and if changes in apoptotic control could be detected even in the absence of any morphologically visible changes. Therefore, we compared expression of two common apoptotic markers, Bax and Bcl-2, in normal epithelium of cancerous prostates and controls. We also evaluated the expression of these proteins in hyperplasia, prostatic intraepithelial neoplasia (PIN), carcinomas of different Gleason grades and capsular perineural invasion. MATERIAL AND METHODS: The tissue material was obtained from radical prostatectomies, transurethral resection chips and autopsies. Individual tissue arrays were done for each patient. The intensity of Bax and Bcl-2 immunostaining was estimated semiquantitatively. The data were analyzed using a linear mixed-models analysis as well as dichotomized staining indices. RESULTS: Normal epithelium of cancerous prostates contained foci with high expression of Bax and Bcl-2. The expression of Bax in Gleason grades 3-5 carcinoma was significantly higher than that in Gleason grade 2, and was highest in foci with perineural invasion. The expression of Bcl-2 was strongest in PIN foci. CONCLUSIONS: Expression of Bax and Bcl-2 in normal epithelium of cancerous prostates suggests that increases in these indirect markers may reflect altered apoptotic control in these foci. Further studies are needed to show whether these changes represent the earliest step of the multifocal carcinogenetic process. Control of apoptosis seems to be involved and modulated during local progression of prostate cancer.

Dual-specificity phosphatase 1 and serum/glucocorticoid-regulated kinase are downregulated in prostate cancer.

Inactivation of tumor suppressor genes through deletion, mutation and epigenetic silencing has been shown to occur in cancer. In our study, we combined DNA demethylation and histone deacetylation inhibition treatments with suppression subtraction hybridization (SSH) and cDNA microarrays to identify potentially epigenetically downregulated genes in PC-3 prostate cancer cell line. We found 11 genes whose expression was upregulated after relieving epigenetic regulation. Expression of 3 genes [dual-specificity phosphatase 1 (DUSP1), serum/glucocorticoid regulated kinase (SGK) and spermidine/spermine N1-acetyltransferase (SAT)] was subsequently studied in clinical sample material using real-time quantitative RT-PCR and immunohistochemistry. The DUSP1 and SGK mRNA expression was lower in hormone-refractory prostate carcinomas compared to benign prostate hyperplasia (BPH) or untreated prostate carcinomas. BPH, normal prostate and high-grade prostate intraepithelial neoplasia (PIN) expressed high levels of DUSP1 and SGK proteins. Ninety-two percent and 48% of the prostate carcinomas showed almost complete lack of DUSP1 and SGK proteins, respectively, indicating common downregulation of these genes. The genomic bisulphite sequencing did not reveal dense hypermethylation in the promoter regions of either DUSP1 or SGK. In conclusion, the data suggest that downregulation of DUSP1 and SGK is an early event and could be important in the tumorigenesis of prostate cancer.