Increased Expression and Altered Cellular Localization of Fibroblast Growth Factor Receptor-Like 1 (FGFRL1) Are Associated with Prostate Cancer Progression.

Fibroblast growth factor receptors (FGFRs) 1-4 are involved in prostate cancer (PCa) regulation, but the role of FGFR-like 1 (FGFRL1) in PCa is unclear. FGFRL1 expression was studied by qRT-PCR and immunohistochemistry of patient tissue microarrays (TMAs) and correlated with clinical patient data. The effects of FGFRL1 knockdown (KD) in PC3M were studied in in vitro culture models and in mouse xenograft tumors. Our results showed that FGFRL1 was significantly upregulated in PCa. The level of membranous FGFRL1 was negatively associated with high Gleason scores (GSs) and Ki67, while increased cytoplasmic and nuclear FGFRL1 showed a positive correlation. Cox regression analysis indicated that nuclear FGFRL1 was an independent prognostic marker for biochemical recurrence after radical prostatectomy. Functional studies indicated that FGFRL1-KD in PC3M cells increases FGFR signaling, whereas FGFRL1 overexpression attenuates it, supporting decoy receptor actions of membrane-localized FGFRL1. In accordance with clinical data, FGFRL1-KD markedly suppressed PC3M xenograft growth. Transcriptomics of FGFRL1-KD cells and xenografts revealed major changes in genes regulating differentiation, ECM turnover, and tumor-stromal interactions associated with decreased growth in FGFRL1-KD xenografts. Our results suggest that FGFRL1 upregulation and altered cellular compartmentalization contribute to PCa progression. The nuclear FGFRL1 could serve as a prognostic marker for PCa patients.

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 (Na(V)s/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.

Accuracy of 18F-FDG PET/CT, Multidetector CT, and MR Imaging in the Diagnosis of Pancreatic Cysts: A Prospective Single-Center Study.

UNLABELLED: Accurate diagnosis of the nature of pancreatic cysts is challenging but more important than ever, in part because of the increasing number of incidental cystic findings in the pancreas. Preliminary data suggest that (18)F-FDG PET/CT may have a significant influence on clinical decision making, although its role is still evolving. Our aim was to prospectively compare the accuracy of combined (18)F-FDG PET and contrast-enhanced CT ((18)F-FDG PET/CT), multidetector CT (MDCT), and MR imaging in differentiating malignant from benign pancreatic cysts. METHODS: Thirty-one consecutive patients with pancreatic cysts were enrolled in the study. They underwent a protocol including (18)F-FDG PET/CT, MDCT, and MR imaging combined with MR cholangiopancreatography, all of which were evaluated in a masked manner. The findings were confirmed macroscopically at surgery or histopathologic analysis (n = 22) or at follow-up (n = 9). RESULTS: Of the 31 patients, 6 had malignant and 25 had benign lesions. The diagnostic accuracy was 94% for (18)F-FDG PET/CT, compared with 77% and 87% for MDCT (P < 0.05) and MR imaging, respectively. (18)F-FDG PET/CT had a negative predictive value of 100% and a positive predictive value of 75% for pancreatic cysts. The maximum standardized uptake value was significantly higher in malignant (7.4 ± 2.6) than in benign lesions (2.4 ± 0.8) (P < 0.05). When the maximum standardized uptake value was set at 3.6, the sensitivity and specificity were 100% and 88%, respectively. Furthermore, when compared with MDCT and MR imaging, respectively, (18)F-FDG PET/CT altered the clinical management of 5 and 3 patients, respectively. CONCLUSION: (18)F-FDG PET/CT is an accurate imaging modality for differentiating between benign and malignant pancreatic cysts. We recommend the use of (18)F-FDG PET/CT in the evaluation of diagnostically challenging pancreatic cysts.

Androgen receptor-interacting protein HSPBAP1 facilitates growth of prostate cancer cells in androgen-deficient conditions.

Hormonal therapies targeting androgen receptor (AR) are effective in prostate cancer (PCa), but often the cancers progress to fatal castrate-resistant disease. Improved understanding of the cellular events during androgen deprivation would help to identify survival and stress pathways whose inhibition could synergize with androgen deprivation. Toward this aim, we performed an RNAi screen on 2,068 genes, including kinases, phosphatases, epigenetic enzymes and other druggable gene targets. High-content cell spot microarray (CSMA) screen was performed in VCaP cells in the presence and absence of androgens with detection of Ki67 and cleaved ADP-ribose polymerase (cPARP) as assays for cell proliferation and apoptosis. Thirty-nine candidate genes were identified, whose silencing inhibited proliferation or induced apoptosis of VCaP cells exclusively under androgen-deprived conditions. One of the candidates, HSPB (heat shock 27 kDa)-associated protein 1 (HSPBAP1), was confirmed to be highly expressed in tumor samples and its mRNA expression levels increased with the Gleason grade. We found that strong HSPBAP1 immunohistochemical staining (IHC) was associated with shorter disease-specific survival of PCa patients compared with negative to moderate staining. Furthermore, we demonstrate that HSPBAP1 interacts with AR in the nucleus of PCa cells specifically during androgen-deprived conditions, occupies chromatin at PSA/klk3 and TMPRSS2/tmprss2 enhancers and regulates their expression. In conclusion, we suggest that HSPBAP1 aids in sustaining cell viability by maintaining AR signaling during androgen-deprived conditions.

Prebiopsy multiparametric 3T prostate MRI in patients with elevated PSA, normal digital rectal examination, and no previous biopsy.

PURPOSE: To find the diagnostic accuracy of 3T multiparametric magnetic resonance imaging (mpMRI) and mpMRI targeted transrectal ultrasound (TRUS)-guided biopsy using visual coregistration (TB) in patients with elevated prostate-specific antigen (PSA), normal digital rectal examination, and no previous biopsy. MATERIALS AND METHODS: Fifty-five patients at two institutions underwent mpMRI, consisting of anatomical T2 -weighted imaging (T2 W), diffusion-weighted imaging (DWI), proton magnetic resonance spectroscopy ((1) H-MRS), and dynamic contrast-enhanced MRI (DCE-MRI), followed by TB in addition to 12 core systematic TRUS-guided biopsy (SB). Histopathological scorings of biopsy (n = 38) and prostatectomy (n = 17) specimens were used as the reference standard for calculation of diagnostic accuracy values. Clinically significant prostate cancer (SPCa) was defined as 3 mm core length of Gleason score 3+3 or any Gleason grade 4. RESULTS: The sensitivity, specificity, accuracy, and area under the curve (AUC) values for the detection of SPCa on the sextant level for T2 W+DWI+(1) H-MRS+DCE-MRI were 72%, 89%, 85%, and 0.81, respectively. The corresponding values for T2 wi+DWI were 61%, 96%, 87%, and 0.79, respectively. The overall PCa detection rate per core in 53 patients was 21% (138 of 648 cores) for SB and 43% (33 of 77 cores) for TB (P < 0.001). CONCLUSION: Prebiopsy mpMRI is an accurate tool for PCa detection and biopsy targeting in patients with elevated PSA.

Pharmacologic suppression of JAK1/2 by JAK1/2 inhibitor AZD1480 potently inhibits IL-6-induced experimental prostate cancer metastases formation.

Metastatic prostate cancer is lethal and lacks effective strategies for prevention or treatment, requiring novel therapeutic approaches. Interleukin-6 (IL-6) is a cytokine that has been linked with prostate cancer pathogenesis by multiple studies. However, the direct functional roles of IL-6 in prostate cancer growth and progression have been unclear. In the present study, we show that IL-6 is produced in distant metastases of clinical prostate cancers. IL-6-activated signaling pathways in prostate cancer cells induced a robust 7-fold increase in metastases formation in nude mice. We further show that IL-6 promoted migratory prostate cancer cell phenotype, including increased prostate cancer cell migration, microtubule reorganization, and heterotypic adhesion of prostate cancer cells to endothelial cells. IL-6-driven metastasis was predominantly mediated by Stat3 and to lesser extent by ERK1/2. Most importantly, pharmacologic inhibition of Jak1/2 by AZD1480 suppressed IL-6-induced signaling, migratory prostate cancer cell phenotypes, and metastatic dissemination of prostate cancer in vivo in nude mice. In conclusion, we demonstrate that the cytokine IL-6 directly promotes prostate cancer metastasis in vitro and in vivo via Jak-Stat3 signaling pathway, and that IL-6-driven metastasis can be effectively suppressed by pharmacologic targeting of Jak1/2 using Jak1/2 inhibitor AZD1480. Our results therefore provide a strong rationale for further development of Jak1/2 inhibitors as therapy for metastatic prostate cancer.

Cancer-associated changes in the expression of TMPRSS2-ERG, PCA3, and SPINK1 in histologically benign tissue from cancerous vs noncancerous prostatectomy specimens.

OBJECTIVE: To investigate whether messenger ribonucleic acid (mRNA) expression of TMPRSS2-ERG fusion gene, a suggested prostate cancer (PCa) biomarker, was specific to cancerous lesions alone and to study the expression of SPINK1 and PCA3 mRNAs in the same cohort to also explore the proposed mutual exclusivity of TMPRSS2-ERG and SPINK1 expression. METHODS: Levels of 2 TMPRSS2-ERG transcripts, PCA3, and SPINK1 mRNAs were measured with highly standardized reverse transcription quantitative polymerase chain reaction assays in cystoprostatectomy specimens from 19 patients with invasive bladder cancer and 174 radical prostatectomy (RP) samples (88 histologically benign prostate [HBP] tissues and 86 from cancerous lesions) from 87 patients with clinically localized PCa. RESULTS: Expression of TMPRSS2-ERG transcripts was detected in 45 of 88 (51%) HBP tissues from RP specimens and more frequently (57 of 86, 66%) found in cancerous lesions. In contrast, TMPRSS2-ERG expression was detected in only 2 of 19 (11%) cystoprostatectomy specimens, both with incidental PCa foci elsewhere in the gland. Similar trends of changes in the expression of PCA3 and SPINK1 were present in HBP tissue from RP compared with cystoprostatectomy specimens. CONCLUSION: Although the expression of TMPRSS2-ERG, SPINK1, and PCA3 mRNA is higher or more frequently found in cancerous lesions, HBP tissues from patients with clinically localized PCa manifest molecular, mRNA level changes that are absent in cystoprostatectomy specimens lacking incidental PCa foci or infrequent in cystoprostatectomy specimens containing incidental PCa. If this finding is replicated, these molecular assays could be used to inform men with negative biopsy results about the likelihood of cancerous lesions in unsampled regions and hence the need for repeat biopsy.

Lanthanide chelate complementation and hydrolysis enhanced luminescent chelate in real-time reverse transcription polymerase chain reaction assays for KLK3 transcripts.

The requirement for high-performance reporter probes in real-time detection of polymerase chain reaction (PCR) has led to the use of time-resolved fluorometry of lanthanide chelates. The aim of this study was to investigate the applicability of the principle of lanthanide chelate complementation (LCC) in comparison with a method based on hydrolysis enhancement and quenching of intact probes. A real-time reverse transcription (RT) PCR assay for kallikrein-related peptidase 3 (KLK3, model analyte) was developed by using the LCC detection method. Both detection methods were tested with a standard series of purified PCR products, 20 prostatic tissues, 20 healthy and prostate cancer patient blood samples, and female blood samples spiked with LNCaP cells. The same limit of detection was obtained with both methods, and two cycles earlier detection with the LCC method was observed. KLK3 messenger RNA (mRNA) was detected in all tissue samples and in 1 of 20 blood samples identically with both methods. The background was 30 times lower, and the signal-to-background (S/B) ratio was 3 times higher, when compared with the reference method. Use of the new reporter method provided similar sensitivity and specificity as the reference method. The lower background, the improved S/B ratio, and the possibility of melting curve analysis and single nucleotide polymorphism (SNP) detection could be advantages for this new reporter probe.

In vivo imaging of prostate cancer using [68Ga]-labeled bombesin analog BAY86-7548.

PURPOSE: A novel [(68)Ga]-labeled DOTA-4-amino-1-carboxymethyl-piperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 peptide (BAY86-7548) having high affinity to bombesin receptor subtype II to detect primary and metastatic prostate carcinoma using positron emission tomography/computed tomography (PET/CT) was synthesized and evaluated for prostate cancer. EXPERIMENTAL DESIGN: In this first human study with BAY86-7548, 14 men scheduled for radical prostatectomy (n = 11) or with biochemical recurrence after surgery or hormonal therapy (n = 3) were enrolled. The patients received an intravenous injection of BAY86-7548 followed by over 60-minute dynamic imaging of prostate gland (n = 10) and/or subsequent whole-body imaging (n = 14). The visual assessment of PET/CT images included evaluation of intraprostatic (12 subsextants) and pelvic nodal uptake of BAY86-7548 in 11 surgical patients and detection of potential metastatic foci in all patients. In patients with biochemical recurrence, results were compared with those of either [(11)C]-acetate (n = 2) or [(18)F]-fluoromethylcholine (n = 1) PET/CT. RESULTS: We found a sensitivity, specificity, and accuracy of 88%, 81% and 83%, respectively, for detection of primary PCa and sensitivity of 70% for metastatic lymph nodes using histology as gold standard. BAY86-7548 correctly detected local recurrence in prostate bed and showed nodal relapse in accordance with [(11)C]-acetate PET/CT in 2 patients with biochemical relapse. In the third hormone refractory patient, BAY86-7548 failed to show multiple bone metastases evident on [(18)F]-fluoromethylcholine PET/CT. CONCLUSION: BAY86-7548 PET/CT is a promising molecular imaging technique for detecting intraprostatic prostate cancer.

Association of transcript levels of 10 established or candidate-biomarker gene targets with cancerous versus non-cancerous prostate tissue from radical prostatectomy specimens.

OBJECTIVES: The benefits of PSA (prostate specific antigen)-testing in prostate cancer remain controversial with a consequential need for validation of additional biomarkers. We used highly standardized reverse-transcription (RT)-PCR assays to compare transcript levels of 10 candidate cancer marker genes - BMP6, FGF-8b, KLK2, KLK3, KLK4, KLK15, MSMB, PCA3, PSCA and Trpm8 - in carefully ascertained non-cancerous versus cancerous prostate tissue from patients with clinically localized prostate cancer treated by radical prostatectomy. DESIGN AND METHODS: Total RNA was isolated from fresh frozen prostate tissue procured immediately after resection from two separate areas in each of 87 radical prostatectomy specimens. Subsequent histopathological assessment classified 86 samples as cancerous and 88 as histologically benign prostate tissue. Variation in total RNA recovery was accounted for by using external and internal standards and enabled us to measure transcript levels by RT-PCR in a highly quantitative manner. RESULTS: Of the ten genes, there were significantly higher levels only of one of the less abundant transcripts, PCA3, in cancerous versus non-cancerous prostate tissue whereas PSCA mRNA levels were significantly lower in cancerous versus histologically benign tissue. Advanced pathologic stage was associated with significantly higher expression of KLK15 and PCA3 mRNAs. Median transcript levels of the most abundantly expressed genes (i.e. MSMB, KLK3, KLK4 and KLK2) in prostate tissue were up to 10(5)-fold higher than those of other gene targets. CONCLUSIONS: PCA3 expression was associated with advanced pathological stage but the magnitude of overexpression of PCA3 in cancerous versus non-cancerous prostate tissue was modest compared to previously reported data.

Nuclear Stat5a/b predicts early recurrence and prostate cancer-specific death in patients treated by radical prostatectomy.

There is an urgent need for reliable markers to identify patients whose prostate cancer (PCa) will recur after initial therapy and progress to lethal disease. Gleason score (GS) is considered the most accurate predictive marker for disease-specific mortality after primary treatment of localized PCa. Most PCas cluster into groups of GS 6 and 7 with considerable variation in the disease recurrence and disease-specific death. In preclinical PCa models, Stat5a/b promotes PCa growth and progression. Stat5a/b is critical for PCa cell viability in vitro and for tumor growth in vivo and promotes metastatic dissemination of cancer in nude mice. Here, we analyzed the predictive value of high nuclear Stat5a/b protein levels in 2 cohorts of PCas: Material I (n = 562) PCas treated by radical prostatectomy (RP), and Material II (n = 106) PCas treated by deferred palliative therapy. In intermediate GS PCas treated by radical prostatectomy, high levels of nuclear Stat5a/b predicted both early recurrence (univariable analysis; P < .0001, multivariable analysis; HR = 1.82, P = .017) and early PCa-specific death (univariable analysis; P = .028). In addition, high nuclear Stat5a/b predicted early disease recurrence in both univariable (P < .0001) and multivariable (HR = 1.61; P = .012) analysis in the entire cohort of patients treated by RP regardless of the GS. Patients treated by deferred palliative therapy, elevated nuclear Stat5a/b expression was associated with early PCa-specific death by univariable Cox regression analysis (HR = 1.59; 95% CI = [1.04, 2.44]; P = .034). If confirmed in future prospective studies, nuclear Stat5a/b may become a useful independent predictive marker of recurrence of lethal PCa after RP for intermediate GS PCas.

High-throughput transcriptomic and RNAi analysis identifies AIM1, ERGIC1, TMED3 and TPX2 as potential drug targets in prostate cancer.

Prostate cancer is a heterogeneous group of diseases and there is a need for more efficient and targeted methods of treatment. In this study, the potential of gene expression data and RNA interference technique were combined to advance future personalized prostate cancer therapeutics. To distinguish the most promising in vivo prevalidated prostate cancer drug targets, a bioinformatic analysis was carried out using genome-wide gene expression data from 9873 human tissue samples. In total, 295 genes were selected for further functional studies in cultured prostate cancer cells due to their high mRNA expression in prostate, prostate cancer or in metastatic prostate cancer samples. Second, RNAi based cell viability assay was performed in VCaP and LNCaP prostate cancer cells. Based on the siRNA results, gene expression patterns in human tissues and novelty, endoplasmic reticulum function associated targets AIM1, ERGIC1 and TMED3, as well as mitosis regulating TPX2 were selected for further validation. AIM1, ERGIC1, and TPX2 were shown to be highly expressed especially in prostate cancer tissues, and high mRNA expression of ERGIC1 and TMED3 associated with AR and ERG oncogene expression. ERGIC1 silencing specifically regulated the proliferation of ERG oncogene positive prostate cancer cells and inhibited ERG mRNA expression in these cells, indicating that it is a potent drug target in ERG positive subgroup of prostate cancers. TPX2 expression associated with PSA failure and TPX2 silencing reduced PSA expression, indicating that TPX2 regulates androgen receptor mediated signaling. In conclusion, the combinatorial usage of microarray and RNAi techniques yielded in a large number of potential novel biomarkers and therapeutic targets, for future development of targeted and personalized approaches for prostate cancer management.

Improved detection of localized prostate cancer using co-registered MRI and 11C-acetate PET/CT.

OBJECTIVES: We aimed to study the ability of contrast enhanced MRI at 1.5 T and 11C-acetate PET/CT, both individually and using fused data, to detect localized prostate cancer. METHODS: Thirty-six men with untreated prostate cancer and negative for metastatic disease on pelvic CT and bone scan were prospectively enrolled. A pelvic 11C-acetate PET/CT scan was performed in all patients, and a contrast enhanced MRI scan in 33 patients (6 examinations using both endorectal coil and surface coils, and 27 examinations using surface coils only). After the imaging studies 10 patients underwent prostatectomy and 26 were treated by image guided external beam radiation treatment. Image fusion of co-registered PET and MRI data was performed based on anatomical landmarks visible on CT and MRI using an advanced in-house developed software package. PET/CT, MRI and fused PET/MRI data were evaluated visually and compared with biopsy findings on a lobar level, while a sextant approach was used for patients undergoing prostatectomy. RESULTS: When using biopsy samples as method of reference, the sensitivity, specificity and accuracy for visual detection of prostate cancer on a lobar level by contrast enhanced MRI was 85%, 37%, 73% and that of 11C-acetate PET/CT 88%, 41%, 74%, respectively. Fusion of PET with MRI data increased sensitivity, specificity and accuracy to 90%, 72% and 85%, respectively. CONCLUSIONS: Fusion of sequentially obtained PET/CT and MRI data for the localization of prostate cancer is feasible and superior to the performance of each individual modality alone.

Immunoassay for the discrimination of free prostate-specific antigen (fPSA) forms with internal cleavages at Lys(145) or Lys(146) from fPSA without internal cleavages at Lys(145) or Lys(146).

Total levels of circulating prostate-specific antigen (tPSA) are strongly associated with prostate cancer (PCa) risk and outcome but benign prostate disease is the most frequent cause of a moderately elevated PSA level. Free PSA (fPSA) forms are independently associated with PCa risk and contribute modest diagnostic enhancements above and beyond tPSA alone. We developed an immunoassay for fPSA subfractions containing internal cleavages at Lys(145) or Lys(146) (fPSA-N). The assay was based on blocking intact single-chain fPSA (fPSA-I) with antibody 4D4 which does not detect PSA containing internal cleavages at Lys(145) or Lys(146). We also measured fPSA-N in blood from healthy volunteers and in anti-coagulated plasma from 76 men with or without evidence of PCa at biopsy. The analytical and functional detection limits of this assay were 0.016 ng/mL and 0.10 ng/mL, respectively. The median recovery of male fPSA-N from female plasma was 95.0%. All 12 female samples (average age 28 years) had fPSA-N concentrations at or below the analytical detection limit. The median fPSA-N concentration (0.050 ng/mL) in 9 healthy male volunteers (age<40 years) was below the functional detection limit, 0.420 ng/mL in 27 patients with benign prostate conditions and 0.239 ng/mL in 49 patients with PCa. Deming regression analysis of the patient samples showed that the measured fPSA-N concentrations were generally 23% lower than the previously calculated (fPSA minus fPSA-I) concentrations, likely due to differences in the antibody combinations used. In conclusion, we have developed a sensitive, specific and direct immunoassay for fPSA-N which can be used to study the clinical relevance of this PSA isoform.

Significance of site-specific prognosis of cancer stem cell marker CD44 in head and neck squamous-cell carcinoma.

In several recent studies, CD44 expression has been associated with aggressive behavior in cancers of different types. CD44 expression is also linked to cancer stem cells, which have been shown to play a significant role in tumor progression and poor prognosis in head and neck squamous cell carcinoma (HNSCC), as well as in other cancers. Although CD44 is a potential prognostic marker, it has not been adopted to wider clinical use as a part of treatment planning in HNSCC patients. The aim of this research was to study whether CD44 overexpression is associated with 5year overall survival in HNSCC. We also studied site-specific associations between increased CD44 expression and 5year overall survival. Associations between relative tumor CD44 expressions and smoking, heavy alcohol consumption, histological grade of cancer, TNM staging and HNSCC staging were also studied. In total, 135 paraffin-embedded blocks from HNSCC patients were stained immunohistochemically with a CD44 antibody and were classified by the anatomic location of the tumor. CD44 overexpression had statistically significant association with decreased 5year survival rates when all HNSCC samples were studied (p<0.001). Significant association between intense CD44 expression and poor 5year survival rates was found in the patients with SCC of the oro- and hypopharynx (p<0.001) and the larynx (p=0.042). In patients suffering from HNSCC in the oral cavity, CD44 overexpression did not have a significant effect on overall 5year survival rates. Heavy smoking of over 10 pack years had a significant association with tumor CD44 overexpression (p=0.009). Only pharyngeal (p=0.046) and laryngeal (p=0.047) SCC, but not oral-cavity SCC, had statistically significant associations between heavy smoking and CD44 overexpression when HNSCC was studied in regional groups. Alcohol consumption and tumor grade did not have a significant association with the tumor's CD44 expression. Our results suggest that CD44 overexpression could be used as a sign of aggressiveness, in addition to the HNSCC staging, as a prognostic factor in pharyngeal and laryngeal HNSCC and to assist in treatment selection.

Arachidonic acid pathway members PLA2G7, HPGD, EPHX2, and CYP4F8 identified as putative novel therapeutic targets in prostate cancer.

The arachidonic acid and prostaglandin pathway has been implicated in prostate carcinogenesis, but comprehensive studies of the individual members in this key pathway are lacking. Here, we first conducted a systematic bioinformatic study of the expression of 36 arachidonic acid pathway genes across 9783 human tissue samples. The results showed that the PLA2G7, HPGD, EPHX2, and CYP4F8 genes are highly expressed in prostate cancer. Functional studies using RNA interference in prostate cancer cells indicated that all four genes are also essential for cell growth and survival. Clinical validation confirmed high PLA2G7 expression, especially in ERG oncogene-positive prostate cancers, and its silencing sensitized ERG-positive prostate cancer cells to oxidative stress. HPGD was highly expressed in androgen receptor (AR)-overexpressing advanced tumors, as well as in metastatic prostate cancers. EPHX2 mRNA correlated with AR in primary prostate cancers, and its inhibition in vitro reduced AR signaling and potentiated the effect of antiandrogen flutamide in cultured prostate cancer cells. In summary, we identified four novel putative therapeutic targets with biomarker potential for different subtypes of prostate cancer. In addition, our results indicate that inhibition of these enzymes may be particularly powerful when combined with other treatments, such as androgen deprivation or induction of oxidative stress.

Intact and internally cleaved free prostate-specific antigen in patients with prostate cancer with different pathologic stages and grades.

OBJECTIVES: To measure the concentration levels of free prostate-specific antigen (PSA) isoforms in patients with prostate cancer selected for curative treatment using radical prostatectomy and to study the association between the isoforms and the pathologic cancer stage and grade. METHODS: Preoperative plasma samples were obtained from 309 consecutive patients scheduled to undergo radical prostatectomy at Turku University Hospital. The pathologic TNM stage, Gleason score, and World Health Organization grade of the tumors were recorded. The total, free, and intact PSA (tPSA, fPSA, and fPSA-I, respectively) concentrations of the archived samples were measured with in-house immunoassays, and the nicked PSA (fPSA-N) concentrations (fPSA minus fPSA-I) and ratios of different PSA forms were calculated. These were compared with the prostate cancer stage, Gleason score, and World Health Organization grade. RESULTS: The median fPSA-I and fPSA-N concentrations in the patients with prostate cancer was 0.42 and 0.28 ng/mL, constituting an average of 60% and 40% of fPSA, respectively. The nicked/total PSA and free/total PSA ratios had the strongest negative correlations with a higher pathologic stage, Gleason score, and World Health Organization grade (Spearman rho -0.205 to -0.262, P < .05). Within a patient subgroup with tPSA <10 ng/mL, fPSA-N as a single marker had a negative correlation with a higher Gleason score (rho -0.160, P = .021). CONCLUSIONS: Lower proportions of fPSA-I and fPSA-N to total PSA were associated with a more advanced cancer stage and grade. A long-term follow-up study and a comparison with currently used clinical methods are needed to evaluate the usefulness of the analytes as prognostic markers for cancer aggressiveness in individual patients.

Detection of melanoma-derived cancer-testis antigen CT16 in patient sera by a novel immunoassay.

Cancer-testis antigens (CTAs) are expressed mainly in various cancer tissues and in testis or placenta. Because of their restricted expression pattern, the CTAs can be potentially used for vaccine development and diagnostic applications. CTA CT16 has been found to be expressed in lung and renal cancers as well as in melanomas. Detection of CT16 protein directly from patient serum could facilitate monitoring of tumor growth and response to therapy in CT16-positive patients. A highly sensitive time-resolved fluorescence-based immunoassay measuring CTA CT16 in serum was developed. Generally, CTAs have not been measured directly from body fluids. CT16 level was detectable in 14 of 23 (61%) patients with metastatic melanoma, whereas none of the nine healthy volunteers collected by us had measurable CT16 level. For an unknown reason, 1 of 20 commercial control serum samples gave a positive result. The Wilcoxon-Mann-Whitney exact test showed statistically significant difference when patients with metastatic melanoma were compared to our control group (p = 0.006) or to the commercial set (p < 0.001). Four melanoma patients had exceptionally high serum CT16 level. CT16 did not correlate either with S100B, a recognized marker of progressing melanoma, or with unspecific serum marker lactate dehydrogenase. Elevation of CT16 titers preceded or followed the clinical diagnosis of disease progression in four patients with metastatic melanoma. As a conclusion, our results show that CT16 protein can be measured directly from patient serum, and the developed assay has a potential for clinical use.

Cancer-associated fibroblasts, a parameter of the tumor microenvironment, overcomes carcinoma-associated parameters in the prognosis of patients with mobile tongue cancer.

Mobile tongue squamous cell carcinoma (MTSCC) is known for its strong propensity for regional metastasis and poor patient survival despite aggressive treatment, thus calling for new and reliable markers for predicting prognosis and guiding therapeutic management. Towards this end, three classes of markers were investigated: cancer-associated fibroblasts (CAFs; α-SMA positivity) as a representative of the tumor microenvironment, maspin (mammary serine protease inhibitor) as a tumor marker likely to be modulated by factors within the tumor microenvironment, and DNA content and Ki-67 labeling index as inbuilt tumor markers in 128 cases of MTSCC using immunohistochemistry and image cytometry. Of these markers, only CAF density was independently and relatively strongly associated with elevated mortality from MTSCC. The hazard ratio in the CAF-rich type of tumor microenvironment was 4.85 (95% CI 1.41-16.6, versus the CAF-poor) when adjusted by proportional hazards modeling for the center where the patient was managed, gender, tumor stage, presence of neck metastasis and age at diagnosis. CAF density was unrelated to non-MTSSC mortality. Given the strong association between increased CAF density and higher mortality in MTSCC, routine assessment of CAF density for disease course prognosis and inclusion as an integral part of treatment protocols are recommended.

Activating mutation (V617F) in the tyrosine kinase JAK2 is absent in locally-confined or castration-resistant prostate cancer.

BACKGROUND: Transcription factor Stat5a/b is highly critical for the viability of human prostate cancer cells in vitro and for prostate tumor growth in vivo. Stat5 is constitutively active in clinical prostate cancers but not in the normal human prostate epithelium. Moreover, Stat5a/b activation in prostate cancer is associated with high histological grade of prostate cancer. However, the molecular mechanisms underlying constitutive activation of Stat5a/b in prostate cancer are unclear. The receptor-associated tyrosine kinase Jak2 is a known key activator of Stat5a/b in prostate cancer cells in response to ligand stimulation. Recently, a single gain-of-function point mutation of JAK2 was described in myeloproliferative diseases leading to constitutive Jak2 kinase activity, subsequent Stat5a/b activation and involvement of V617F Jak2 in the pathogenesis of myeloproliferative disorders. MATERIALS AND METHODS: We determined whether JAK2 undergoes the V617F activating mutation during clinical progression of human prostate cancer using a highly sensitive assay (amplification refractory mutation system) and a unique material of fresh specimens from organ-confined or castration-resistant prostate cancers. RESULTS: The JAK2 V617F mutation was not found in any of the normal or malignant prostate samples analyzed in this study. CONCLUSIONS: Future work should focus on determining the molecular mechanisms other than V617F mutation of Jak2 resulting in continuous Stat5 activation in clinical prostate cancers.