Heikki Seikkula

Loss of PTEN expression in ERG-negative prostate cancer predicts secondary therapies and leads to shorter disease-specific survival time after radical prostatectomy

The clinical course of prostate cancer is highly variable. Current prognostic variables, stage, and Gleason score have limitations in assessing treatment regimens for individual patients, especially in the intermediate-risk group of Gleason score 7. ERG:TMPRSS2 fusion and loss of PTEN are some of the most common genetic alterations in prostate cancer. Immunohistochemistry of PTEN and ERG has generated interest as a promising method for more precise outcome prediction but requires further validation in population-based cohorts. We studied the predictive value of ERG and PTEN expression by immunohistochemistry in two large radical prostatectomy cohorts comprising 815 patients with extensive follow-up information. Clinical end points were initiation of secondary therapy, overall survival, and disease-specific survival. Predictions of clinical outcomes were also assessed according to androgen receptor (AR) activity. PTEN loss, especially in ERG-negative cancers, predicted initiation of secondary treatments and shortened disease-specific survival time, as well as stratifying Gleason score 7 patients into different prognostic groups with regard to secondary treatments and disease-specific survival. High AR immunoreactivity in ERG-negative cancers with PTEN loss predicted worse disease-specific survival. We also observed that in Gleason score 7 ERG-negative cases with PTEN loss and high AR expression have significantly shorter disease-specific survival time compared with ERG-positive cases. Our conclusion is that loss of PTEN is a strong determining factor for shorter disease-specific survival time and initiation of secondary therapies after radical prostatectomy. The predictive value of PTEN immunoreactivity is further accentuated in ERG-negative cancers with high AR expression. Negative PTEN expression, accompanied by ERG status, can be used to stratify patients with Gleason score 7 into different survival groups. Assessment of PTEN and ERG status could provide an additional tool for initial diagnostics when determining the prognosis and subsequent follow-up regimen for patients treated by radical prostatectomy.

Differential Predictive Roles of A- and B-Type Nuclear Lamins in Prostate Cancer Progression

Background Prostate cancer (PCa) is the most common cancer among men in western countries. While active surveillance is increasingly utilized, the majority of patients are currently treated with radical prostatectomy. In order to avoid over-treatment, there is an indisputable need for reliable biomarkers to identify the potentially aggressive and lethal cases. Nuclear intermediate filament proteins called lamins play a role in chromatin organization, gene expression and cell stiffness. The expression of lamin A is associated with poor outcome in colorectal cancer but to date the prognostic value of the lamins has not been tested in other solid tumors. Methods We studied the expression of different lamins with immunohistochemistry in a tissue microarray material of 501 PCa patients undergoing radical prostatectomy and lymph node dissection. Patients were divided into two staining categories (low and high expression). The correlation of lamin expression with clinicopathological variables was tested and the association of lamin status with biochemical recurrence (BCR) and disease specific survival (DSS) was further analyzed. Results Low expression of lamin A associated with lymph node positivity (p<0.01) but not with other clinicopathological variables and low expression had a borderline independent significant association with DSS (HR = 0.4; 95% CI 0.2–1.0; p = 0.052). Similarly, low lamin C expression associated with poorer survival (HR = 0.2; 95% CI 0.1–0.6; p = 0.004). Lamin B1 expression did not associate with clinicopathological variables but high expression independently predicted BCR in multivariable Cox regression analysis (HR = 1.8; 95% CI 1.1–2.9; p = 0.023). Low expression of lamin B2 correlated with lymph node positivity (p<0.01) and predicted unfavorable DSS (HR = 0.4; 95% CI 0.2–1.0; p = 0.047). Conclusions These results suggest differential roles for lamins in PCa progression. Reduced amounts of lamin A/C and B2 increase risk for lymph node metastasis and disease specific death possibly through increased nuclear deformability while high expression of lamin B1 predicts disease recurrence.

Stage-specific mortality and survival trends of prostate cancer patients in Finland before and after introduction of PSA

Abstract Background: The early diagnosis and right treatment strategy of localized prostate cancer (PCa) remains problematic. In order to characterize the survival of PCa patients, we compared patients’ all-cause and cancer-specific mortalities between pre- and post-PSA periods by stage in Finland. Material and methods: All PCa cases diagnosed in Finland between 1985 and 2013 (N = 91,329) were identified from the Finnish Cancer Registry (FCR). PCa stage at diagnosis was defined as localized, local node positive or metastasized. Standardized mortality ratios (SMRs), and relative and cause-specific survival were assessed by stage and introduction of PSA testing. The main limitation was the high proportion of men with unknown stage (28%). Results: A clear decreasing trend in the SMR of PCa patients was evident when pre- and post-PSA eras were compared: for localized PCa, the SMR was 1.43 (95%CI 1.38–1.48) in 1985–1989 and 0.98 (95%CI 0.95–1.01) in 2000–2004, and for metastasized PCa, the SMRs were 4.51 (95%CI 4.30–4.72) and 3.01 (95%CI 2.89–3.12), respectively. Difference between cause-specific and relative survival was pronounced in localized PCa in post-PSA period: 10-year relative survival was 94.6% (95%CI 91.4–97.8) and cause-specific 84.2% (95%CI 82.9–85.5%). In metastasized PCa the difference was not that significant. Conclusions: From 1985 to 2009, the SMR among men diagnosed with PCa decreased significantly in Finland. Among men with localized PCa, the SMR decreased even below that of the Finnish male population. This and the increased difference between relative and cause-specific survival reflects most likely selection of men to opportunistic PSA testing. The results highlight the importance of caution in the use of PSA testing in healthy men.

Increased expression of fibroblast growth factor 13 in prostate cancer is associated with shortened time to biochemical recurrence after radical prostatectomy

Fibroblast growth factor homologous factors (FHFs) belong to the fibroblast growth factor (FGF) superfamily, which plays an important role in prostate cancer (PCa). Mining of public database suggests that FGF13 (FHF2) mRNA expression is altered in over 30% of PCa cases. This study examined the FGF13 expression pattern in human PCa specimens and evaluated its potential as a biomarker for patient outcome after radical prostatectomy (RP). Immunohistochemistry (IHC) showed that FGF13 was detectable in the majority of human PCa samples, and FGF13 IHC scores were higher in high‐grade prostatic intraepithelial neoplasia, in primary PCa and in metastatic PCa than in benign prostatic tissue. There was a significant association between high cytoplasmic FGF13 staining and a risk of biochemical recurrence (BCR) after RP. This was also evident in the intermediate to high‐risk patient groups. In contrast, positive nuclear FGF13 staining along with low cytoplasmic FGF13 group showed a decreased BCR risk. Multivariate regression analysis indicated that high cytoplasmic FGF13 staining was associated with BCR and that this could serve as an independent prognostic marker in PCa. Several PCa cell lines showed increased FGF13 expression at the mRNA and protein levels compared to the immortalized prostate epithelial cell line PNT1a. Analysis of co‐labeled cells suggested a possible interaction of FGF13 with α‐tubulin and the voltage‐gated sodium channel proteins (NaVs/VGSCs). Our data indicate that, for PCa patients after RP, FGF13 serves as a potential novel prognostic marker that improves prediction of BCR‐free survival, in particular if combined with other clinical parameters.

Role of ultrasensitive prostate-specific antigen in the follow-up of prostate cancer after radical prostatectomy

OBJECTIVE Prostate-specific antigen (PSA) is an important tool in the follow-up of prostate cancer after radical prostatectomy (RP). However, the relevance of ultrasensitive PSA (uPSA) after RP is not well defined. The aim of this study was to investigate the value of uPSA in follow-up after RP and to determine whether ultrasensitive PSA doubling time (uDT) correlates with traditional PSA doubling time (tDT). PATIENTS AND METHODS In total, 604 consecutive patients undergoing open RP and pelvic lymphadenectomy between 2004 and 2008 (minimum 5y of follow-up) were studied. To evaluate the postsurgical uPSA level, scatter plot statistics were used. To correlate uDT and tDT in patients with a biochemical recurrence (PSA ≥0.2ng/ml), at least 2 uPSA and 2 PSA measurements without salvage treatment were required and a weighted Cohen kappa statistic and receiver operating characteristic curve were used to test agreement across the categories. RESULTS There were 229 patients without biochemical recurrence who did not have 3 rising PSA values after nadir within ultrasensitive area. Their highest uPSA value was between 0.003 and 0.1ng/ml. In 97.4% of patients, the highest uPSA value was less than 0.03ng/ml, and in 89% of these patients, the values were less than 0.02ng/ml. The median uDT and tDT were 10.2 and 11.4 months, respectively. The weighted Cohen kappa statistic between these 2 groups was 0.30 (95% CI:-0.09 to 0.50), demonstrating a poor agreement of PSA doubling time across categories. The predictive capability of uDT was tested with tDT <9 months. A receiver operating characteristic curve area under the curve value was 0.737 (95% CI:-0.577 to 0.897) demonstrating a fair agreement between the groups. CONCLUSIONS uPSA values>0.03ng/ml seems to be valid and can be used in a clinical setting. There was a poor to fair agreement between tDT and uDT. The accuracy of uDT improves when it approaches the traditional PSA threshold of 0.1ng/ml. Also according to our results, there is no prognostic benefit of uDT calculation.

The impact of socioeconomic status on stage specific prostate cancer survival and mortality before and after introduction of PSA test in Finland

Socioeconomic status (SES) has an impact on prostate cancer (PCa) outcomes. Men with high SES have higher incidence and lower mortality of PCa versus lower SES males. PCa cases diagnosed in Finland in 1985–2014 (N = 95,076) were identified from the Finnish Cancer Registry. Information on education level (EL) was obtained from Statistics Finland. EL was assessed with three‐tiered scale: basic, upper secondary and higher education. PCa stage at diagnosis was defined as localized, metastatic or unknown. Years of diagnosis 1985–1994 were defined as pre‐PSA period and thereafter as post‐PSA period. We report PCa‐specific survival (PCSS) and relative risks (RR) for PCa specific mortality (PCSM) among cancer cases in Finland, where healthcare is 100% publicly reimbursed and inequality in healthcare services low. Men with higher EL had markedly better 10‐year PCSS: 68 versus 63% in 1985–1994 and 90 versus 85% in 1995–2004 compared to basic EL in localized PCa. The RR for PCSM among men with localized PCa and higher EL compared to basic EL was 0.76(95%confidence interval (CI) 0.66–0.88) in 1985–1994 and 0.61(95%CI 0.53–0.70) in 1995–2004. Variation in PCSS and PCSM between EL categories was evident in metastatic PCa, too. The difference in PCSM between EL categories was larger in the first 10‐year post‐PSA period than before that but decreased thereafter in localized PCa, suggesting PSA testing became earlier popular among men with high EL. In summary, higher SES/EL benefit PCa survival both in local and disseminated disease and the effect of EL was more pronounced in early post‐PSA period.

Longitudinal modeling of ultrasensitive and traditional prostate-specific antigen and prediction of biochemical recurrence after radical prostatectomy

Ultrasensitive prostate-specific antigen (u-PSA) remains controversial for follow-up after radical prostatectomy (RP). The aim of this study was to model PSA doubling times (PSADT) for predicting biochemical recurrence (BCR) and to capture possible discrepancies between u-PSA and traditional PSA (t-PSA) by utilizing advanced statistical modeling. 555 RP patients without neoadjuvant/adjuvant androgen deprivation from the Turku University Hospital were included in the study. BCR was defined as two consecutive PSA values >0.2 ng/mL and the PSA measurements were log2-transformed. One third of the data was reserved for independent validation. Models were first fitted to the post-surgery PSA measurements using cross-validation. Major trends were then captured using linear mixed-effect models and a predictive generalized linear model effectively identified early trends connected to BCR. The model generalized for BCR prediction to the validation set with ROC-AUC of 83.6% and 95.1% for the 1 and 3 year follow-up censoring, respectively. A web-based tool was developed to facilitate its use. Longitudinal trends of u-PSA did not display major discrepancies from those of t-PSA. The results support that u-PSA provides useful information for predicting BCR after RP. This can be beneficial to avoid unnecessary adjuvant treatments or to start them earlier for selected patients.

Abstract 4951: FGFRL1 in prostate cancer progression

Prostate cancer (PCa) is a disease with high incidence, however, many PCa patients are over-diagnosed and over-treated. Molecular characterization of PCa provides a valid approach to stratify patients, and thus reduce overtreatment. Fibroblast growth factors and their receptor (FGF/FGFR) signaling pathways are involved in various cellular functions such as proliferation, differentiation, migration, and apoptosis of prostate cancer cells. Dysregulated and constitutively activated FGF/FGFR pathways have been shown to be involved in the initiation and progression of prostate cancer. Fibroblast growth factor receptor like 1 (FGFRL1, FGFR5) is the most recently identified member of the FGFR family. FGFRL1 binds several FGFs but its short intracellular part lacks a tyrosine kinase domain. Therefore, the extracellular domain has been suggested to act as a dominant negative regulator of other FGFRs. However, cellular functions of FGFRL1 remain poorly understood. Aberrant FGFRL1 expression has been reported in ovarian, bladder, colon, and other cancers. In silico data analysis indicated altered FGFRL1 mRNA expression in 17% of PCa cases. To date, there have been no systematic studies of FGFRL1 expression and function in prostate and PCa. We studied FGFRL1 expression and function in PCa cell lines, xenografts and in human PCa specimens. FGRL1 was knocked-down in PC-3M cells, by shRNAs, which showed reduced growth as nude mouse xenografts when compared to control. This argues against a dominant negative function. To study FGFRL1 in human PCas, we collected formalin-fixed paraffin-embedded samples from PCa patients undergoing radical prostatectomy (n = 243), metastatic PCa (n = 36) and castration-resistant PCa (n = 21). Samples were prepared for mRNA analysis, and immunohistochemistry. The overall levels of FGFRL1 immunostaining were significantly increased in PCa compared to normal tissue. In normal tissue, FGFRL1 immunostaining localized to the cell membrane and to a lesser extent to the cytoplasm and nuclei. In PCa, protein expression of FGFRL1 was decreased at the cell membrane, while expression in the cytoplasm and nucleus were increased. Low membrane and high nuclear immunostaining of FGFRL1 correlated with high Gleason grade. High nuclear FGFRL1 also correlated with high levels of preoperative serum PSA and an increased proportion of tumors with positive surgical margins. Our results suggest that FGFRL1 may play an active role in PCa cells and in tumor progression and can possibly be used to assess PCa prognosis. Citation Format: Lan Yu, Andrew Erickson, Mervi Toriseva, Teresa Elo, Johanna Tuomela, Heikki Seikkula, Martti Nurmi, Peter Bostrom, Tuomas Mirtti, Kalle Alanen, Markku Kallajoki, Matthias Nees, Pirkko Harkonen. FGFRL1 in prostate cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4951.

Abstract 3454: High expression of fibroblast growth factor 13 (FGF13) in prostate cancer is associated with a shortened time to biochemical recurrence after radical prostatectomy

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Fibroblast growth factor homologous factors (FHF1-4) belong to the FGF family involved in prostate cancer. Based on the cBioPortal database (MSKCC, Cancer Cell 2010), FGF13 (FHF2), one of the FHF subfamily members, shows altered mRNA expression in prostate cancer. In this study we aimed to analyze FGF13 expression and functions in prostate cancer. We collected prostate cancer specimens from 234 patients who underwent radical prostatectomy in Turku University Hospital. Frozen tissue was used for qRT-PCR to measure FGF13 mRNA in 76 samples in which glandular or cancer area covered at least 50% of the specimen (non-cancer, n = 31; cancer, n = 45). Tissue microarrays (TMA) containing cancer (n = 152) and adjacent non-cancer (n = 203) samples were examined for FGF13 protein by immunohistochemical staining. Total FGF13 staining score of each TMA core was calculated using HistoScore system. Chi-square test, Kaplan-Meier and Cox proportional hazards regression model were used to analyze the association between FGF13 staining and clinicopathological parameters. Immunofluorescence was used to detect FGF13 localization in prostate cancer cells in vitro. The level of FGF13 mRNA was significantly higher in cancer tissues than in non-cancer tissues (p = 0.0028). FGF13 mRNA levels did not show significant association with the Gleason score, pathologic stage, pre-operation PSA value or PSA failure time. In the TMA study, cancer cells showed weak, moderate or strong cytoplasmic staining for FGF13 in over 99% of the samples whereas luminal epithelial cells were positive in only 53% of the adjacent non-cancer samples. Correspondingly, the cytoplasmic staining of FGF13 presented higher scores in cancer than in non-cancer areas (p<0.001). Moreover, nuclear staining of FGF13 was also observed, and it was more frequent in cancer samples compared to non-cancer samples (59% and 12%, respectively). Especially, basal cells showed positive immunoreactivity sporadically in non-cancer tissues. Only very weak or negative immunostaining of FGF13 was found in stromal cells but interestingly, prominent expression of FGF13 was observed in mononuclear cells. In terms of time to biochemical recurrence after prostatectomy, patients with high FGF13 cytoplasmic staining had a shorter PSA failure free time compared to patients who had lower cytoplasmic FGF13 expression (p = 0.009). Multivariate analysis revealed that high cytoplasmic FGF13 staining (HR = 3.76, 95%CI [1.5-9.3], p = 0.004) was significantly associated with a shorter PSA failure time. Immunofluorescence staining in PC3M cells showed that FGF13 was located to the cytoplasm and nuclei. Overall, our study demonstrates, for the first time, expression and location of FGF13 protein in prostate cancer and suggests that FGF13 could have prognostic value in primary prostate cancer. Citation Format: Lan Yu, Miikka Tuomala, Mervi Toriseva, Teresa Elo, Johanna Tuomela, Heikki Seikkula, Martti Nurmi, Peter Bostrom, Tuomas Mirtti, Kalle Alanen, Markku Kallajoki, Pirkko Harkonen. High expression of fibroblast growth factor 13 (FGF13) in prostate cancer is associated with a shortened time to biochemical recurrence after radical prostatectomy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3454. doi:10.1158/1538-7445.AM2015-3454