Prognostic impact of kallikrein-related peptidase transcript levels in prostate cancer.

We aimed to study mRNA levels and prognostic impact of all 15 human kallikrein-related peptidases (KLKs) and their targets, proteinase-activated receptors (PARs), in surgically treated prostate cancer (PCa). Seventy-nine patients with localized grade group 2-4 PCas represented aggressive cases, based on metastatic progression during median follow-up of 11 years. Eighty-six patients with similar baseline characteristics, but no metastasis during follow-up, were assigned as controls. Transcript counts were detected with nCounter technology. KLK12 protein expression was investigated with immunohistochemistry. The effects of KLK12 and KLK15 were studied in LNCaP cells using RNA interference. KLK3, -2, -4, -11, -15, -10 and -12 mRNA, in decreasing order, were expressed over limit of detection (LOD). The expression of KLK2, -3, -4 and -15 was decreased and KLK12 increased in aggressive cancers, compared to controls (P < .05). Low KLK2, -3 and -15 expression was associated with short metastasis-free survival (P < .05) in Kaplan-Meier analysis. PAR1 and -2 were expressed over LOD, and PAR1 expression was higher, and PAR2 lower, in aggressive cases than controls. Together, KLKs and PARs improved classification of metastatic and lethal disease over grade, pathological stage and prostate-specific antigen combined, in random forest analyses. Strong KLK12 immunohistochemical staining was associated with short metastasis-free and PCa-specific survival in Kaplan-Meier analysis (P < .05). Knock-down of KLK15 reduced colony formation of LNCaP cells grown on Matrigel basement membrane preparation. These results support the involvement of several KLKs in PCa progression, highlighting, that they may serve as prognostic PCa biomarkers.

Tumor expression of human chorionic gonadotropin beta mRNA and prognosis of prostate cancer treated by radical prostatectomy.

The beta subunit of human chorionic gonadotropin (hCGß) is encoded by six genes (CGB) classified as type I and type II. CGB mRNA is produced in large amounts by trophoblastic tissues and in small amounts by several cancerous tissues including prostate cancer and by a few benign tissues, including the prostate. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) was used to study the expression levels of all CGB mRNAs together (total CGB mRNA) and the two types of CGB mRNA separately in non-cancerous (n = 74) and cancerous prostatic tissue obtained by radical prostatectomy (n = 193). RNA was isolated from formalin-fixed paraffin-embedded (FFPE) samples and mRNA levels of CGB were correlated with disease-specific survival. Total CGB mRNA concentrations were significantly lower (p < .0001) in cancerous than non-cancerous prostatic tissue. Separate analysis of type I CGB and type II CGB mRNA showed that both type I CGB (p < .0001) and type II CGB mRNA (p = .007) are lower in cancerous tissue than in non-cancerous tissue. Low type II CGB mRNA level in cancerous tissue was associated with shorter cancer-specific survival (p = .001) of prostate cancer patients treated by radical prostatectomy.

MAPK inhibitors induce serine peptidase inhibitor Kazal type 1 (SPINK1) secretion in BRAF V600E-mutant colorectal adenocarcinoma.

The mitogen-activated protein kinase (MAPK) pathway plays a central role in colorectal cancers (CRC). In particular, BRAF V600E-mutant tumors, which represent around 10% of CRCs, are refractory to current therapies. Overexpression and secretion of serine peptidase inhibitor Kazal type 1 (SPINK1) are observed in around 50% of CRCs, and its serum level can be used as a biomarker for poor prognosis. Utilizing a recently developed extendable blocking probe assay, we analyzed the BRAF mutation status in a CRC patient cohort (N = 571) using tissue-derived RNA as the starting material. From the same RNA samples, we measured the relative SPINK1 expression levels using a quantitative real-time PCR method. Expression of mutant BRAF V600E correlated with poor prognosis, as did low expression of SPINK1 mRNA. Further, BRAF V600E correlated negatively with SPINK1 levels. In order to investigate the effect of MAPK pathway-targeted therapies on SPINK1 secretion, we conducted in vitro studies using both wild-type and V600E CRC cell lines. BRAF inhibitor vemurafenib, and subsequent MAPK pathway inhibitors trametinib and SCH772984, significantly increased SPINK1 secretion in V600E CRC cell lines Colo205 and HT-29 with a concomitant decrease in trypsin-1 and -2 secretion. Notably, no SPINK1 increase or trypsin-1 decrease was observed in BRAF wild-type CRC cell line Caco-2 in response to MAPK pathway inhibitors. In further mechanistic studies, we observed that only trametinib was able to diminish completely both MEK and ERK phosphorylation in the V600E CRC cells. Furthermore, the key regulator of integrated stress response, activating transcription factor 4 (ATF-4), was downregulated both at mRNA and at protein level in response to trametinib treatment. In conclusion, these data suggest that sustained inhibition of not only MAPK pathway activation, but also ATF-4 and trypsin, might be beneficial in the therapy of BRAF V600E-mutant CRC and that SPINK1 levels may serve as an indicator of therapy response.

Somatic MED12 mutations in prostate cancer and uterine leiomyomas promote tumorigenesis through distinct mechanisms.

BACKGROUND: Mediator is a multiprotein interface between eukaryotic gene-specific transcription factors and RNA polymerase II. Mutations in exon 2 of the gene encoding MED12, a key subunit of the regulatory kinase module in Mediator, are extremely frequent in uterine leiomyomas, breast fibroadenomas, and phyllodes tumors. These mutations disrupt kinase module interactions and lead to diminished Mediator-associated kinase activity. MED12 mutations in exon 26, resulting in a substitution of leucine 1224 to phenylalanine (L1224F), have been recurrently observed in prostate cancer. METHODS: To elucidate the molecular mechanisms leading to tumorigenesis in prostate cancer, we analyzed global interaction profiles of wild-type and L1224F mutant MED12 with quantitative affinity purification-mass spectrometry (AP-MS). Immunoprecipitation and kinase activity assay were used to further assess the interactions between Mediator complex subunits and kinase activity. The presence of L1224F mutation was analyzed in altogether 877 samples representing prostate hyperplasia, prostate cancer, and various tumor types in which somatic MED12 mutations have previously been observed. RESULTS: In contrast to N-terminal MED12 mutations observed in uterine leiomyomas, the L1224F mutation compromises neither the interaction of MED12 with kinase module subunits Cyclin C and CDK8/19 nor Mediator-associated CDK activity. Instead, the L1224F mutation was shown to affect interactions between MED12 and other Mediator components (MED1, MED13, MED13L, MED14, MED15, MED17, and MED24). Mutation screening revealed one mutation in a Finnish (Caucasian) prostate cancer patient, whereas no mutations in any other tumor type were observed. CONCLUSIONS: Specific somatic MED12 mutations in prostate cancer and uterine leiomyomas accumulate in two separate regions of the gene and promote tumorigenesis through clearly distinct mechanisms.

Extendable blocking probe in reverse transcription for analysis of RNA variants with superior selectivity.

Here we provide the first strategy to use a competitive Extendable Blocking Probe (ExBP) for allele-specific priming with superior selectivity at the stage of reverse transcription. In order to analyze highly similar RNA variants, a reverse-transcriptase primer whose sequence matches a specific variant selectively primes only that variant, whereas mismatch priming to the alternative variant is suppressed by virtue of hybridization and subsequent extension of the perfectly matched ExBP on that alternative variant template to form a cDNA-RNA hybrid. This hybrid will render the alternative RNA template unavailable for mismatch priming initiated by the specific primer in a hot-start protocol of reverse transcription when the temperature decreases to a level where such mismatch priming could occur. The ExBP-based reverse transcription assay detected BRAF and KRAS mutations in at least 1000-fold excess of wild-type RNA and detection was linear over a 4-log dynamic range. This novel strategy not only reveals the presence or absence of rare mutations with an exceptionally high selectivity, but also provides a convenient tool for accurate determination of RNA variants in different settings, such as quantification of allele-specific expression.

Technique for strand-specific gene-expression analysis and monitoring of primer-independent cDNA synthesis in reverse transcription.

Primer-independent cDNA synthesis during reverse transcription hinders quantitative analysis of bidirectional mRNA synthesis in eukaryotes as well as in cells infected with RNA viruses. We report a simple RT-PCR-based assay for strand-specific gene-expression analysis. By modifying the cDNA sequence during reverse transcription, the opposite strands of target sequences can be simultaneously detected by postamplification melting curve analysis and primer-initiated transcripts are readily distinguished from nonspecifically primed cDNA. We have utilized this technique to optimize the specificity of reverse transcription on a panel of 15 target genes. Primer-independent reverse transcription occurred for all target sequences when reverse transcription was performed at 42°C and accounted for 11%-57% of the final PCR amplification products. By raising the reaction temperature to 55°C, the specificity of reverse transcription could be increased without significant loss of sensitivity. We have also demonstrated the utility of this technique for analysis of (+) and (-) RNA synthesis of influenza A virus in infected cells. Thus, this technique represents a powerful tool for analysis of bidirectional RNA synthesis.

Developing biomarkers for improved diagnosis and treatment outcome monitoring of bladder cancer.

IMPORTANCE OF THE FIELD: A non-invasive marker for the follow-up and diagnosis of bladder cancer is highly needed. Several markers have been studied with regard to sensitivity and specificity in detecting bladder cancer. Comparison of studies is complicated by limited data on tumor characteristics and treatment details. Many studies do not differentiate between primary and recurrent tumors, nor is the performance of the studied marker assessed separately in superficial and invasive or high- versus low-grade tumors. AREAS COVERED IN THIS REVIEW: The field of bladder cancer biomarker research from the past 15 years. WHAT THE READER GAIN: A summary of the current field of bladder biomarker research with concluding remarks on some specific challenges in developing biomarkers for improved diagnosis and monitoring the disease. TAKE HOME MESSAGE: In general, the best new markers give higher sensitivity than urinary cytology, but specificity is usually lower. By using new markers, the intervals between follow-up cystoscopies can be increased and the detection of relapse can be improved. But to date no non-invasive biomarker has proven to be sensitive and specific enough available to replace cystoscopy, neither in the diagnosis nor in the follow-up of bladder cancer. However, new marker combinations and algorithms for risk assessment hold promise for the future.

Overexpression of human chorionic gonadotropin beta genes 3, 5 and 8 in tumor tissue and urinary cells of bladder cancer patients.

OBJECTIVE: Human chorionic gonadotropin (hCG) is a marker of trophoblastic tumors, and the serum concentration of the free beta-subunit (hCGbeta) is an independent prognostic marker in several nontrophoblastic cancers. hCGbeta is encoded by six genes, of which the type II genes (hCGbeta 3/9, 5 and 8) are thought to be upregulated in relation to type I genes (hCGbeta 6/7) in cancer. METHOD: We developed a novel quantitative RT-PCR method for the quantification of the relative expression levels of the two groups of hCGbeta genes and analyzed 28 bladder tumors and 15 urine samples. RESULTS: We found a higher relative expression level of type II genes in malignant compared with benign urothelia (p = 0.016) and in exfoliated urinary cells from cancer patients compared with those from benign controls (p = 0.026). The expression level was increasing with higher stage (p = 0.014) and grade (p = 0.001) and tended to be higher in relapsing tumors (p = 0.059). CONCLUSION: The increased hCGbeta concentrations in body fluids of patients with aggressive bladder cancer may be due to overexpression of type II genes. Quantification of the relative mRNA expression levels of the hCGbeta type I and II genes in urine cells should be further studied as a potential noninvasive tool for the diagnosis and follow-up of bladder cancer.

Expression of human chorionic gonadotropin beta-subunit type I genes predicts adverse outcome in renal cell carcinoma.

Expression of the free beta-subunit of human chorionic gonadotropin (hCGbeta) in malignant tumors is frequently associated with aggressive disease. The pretreatment serum concentration of hCGbeta is an independent prognostic variable in renal cell carcinoma (RCC). The three so-called type II genes (hCGbeta 3/9, 5, and 8) have been shown to be up-regulated in relation to type I genes (hCGbeta 6/7) in some malignant tumors. We developed a reverse transcription-polymerase chain reaction method for quantification of relative levels of the mRNAs for the two types of hCGbeta genes and studied the association between the expression in RCC tissue (n = 104) and clinical outcome. hCGbeta mRNA expression was detected in 40% (42 of 104) of the tumors, and in 40 of these (93%), this consisted of hCGbeta type I mRNA only, whereas type II hCGbeta mRNA was detected in two samples. hCGbeta mRNA expression was significantly associated with a shorter disease-specific (log-rank P = 0.023; median survival 1.4 versus 7.9 years) and overall survival (log-rank P = 0.011). In a Cox regression model, stage (P < 0.0001) and hCGbeta mRNA expression (P < 0.0001) were independent prognostic variables. We conclude that expression of type I hCGbeta genes indicates adverse prognosis in RCC.

Relative concentrations of hK2/PSA mRNA in benign and malignant prostatic tissue.

BACKGROUND: Prostate-specific antigen (PSA/KLK3) and human kallikrein 2 (hK2/KLK2) belong to the human kallikrein gene family. These two highly homologous genes are specifically expressed in the prostate under androgen control. Expression of these is regulated by similar mechanisms but changes in their relative expression have been observed in prostate cancer. METHODS: We determined the relative levels of PSA and hK2 mRNA in benign and malignant prostate tissue using a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. The mRNA of PSA and hK2 are reverse transcribed and amplified in one reaction with the same primers. RESULTS: The variation in the ratio of hK2/PSA mRNA was remarkably small, the difference between the highest and lowest values being three-fold. The ratio was significantly higher in WHO grade 2 compared to normal or benign prostatic hyperplasia tissue (P = 0.032 and P = 0.035, respectively) and in grade 3 compared to normal or benign prostatic hyperplasia tissue (P = 0.006 in both). CONCLUSIONS: The new quantitative RT-PCR technique facilitates very accurate quantitation of the relative mRNA levels of homologous genes. Using this method we have shown that the ratio of hK2/PSA mRNA is higher in cancerous than in benign prostatic tissue.

Expression of vascular endothelial growth factor in peripheral blood cells of preeclamptic women.

OBJECTIVE: Preeclampsia is associated with platelet and endothelial dysfunction. Vascular endothelial growth factor (VEGF) is found in peripheral blood leukocytes and released from platelets on activation. We analyzed the content of (VEGF) in peripheral blood cells of preeclamptic women. METHODS: The VEGF content of platelets, mononuclear white blood cells, and granulocytes of peripheral blood were analyzed from 12 women with preeclampsia, 19 healthy pregnant women, and 20 nonpregnant women. Protein released from lysed cells was analyzed by enzyme-linked immunoassay (ELISA). RESULTS: Platelet VEGF content of preeclamptic women (0.081 ng/109 cells, 0.016 to 2.7 ng/109 cells; median, range) was similar to that of healthy pregnant women (0.31 ng/109 cells, 0.013 to 0.92 ng/109 cells) and that of nonpregnant (0.073 ng/109 cells, 0.012 to 0.76 ng/109 cells) women. Likewise, the VEGF content of granulocytes was similar in preeclamptic (18.5 ng/109 cells, 1.2 to 193 ng/109 cells), healthy pregnant (25.3 ng/109 cells, 0.8 to 441 ng/109 cells), and nonpregnant (29 ng/109 cells, 0.25 to 200 ng/109 cells) women. In mononuclear cells, the VEGF content of healthy pregnant women was higher (4.4 ng/109 cells, 0.13 to 13.7 ng/109 cells) than in nonpregnant women (1.7 ng/109 cells, 0.15 to 11.4 ng/109 cells, P < 0.05). Also, the mononuclear cell VEGF content of preeclamptic women (8.2 ng/109 cells, 0.04 to 23 ng/109 cells) tended to be higher than in nonpregnant women (P approximately 0.07). CONCLUSION: Uncomplicated pregnancy is associated with an elevated VEGF content of mononuclear cells. Preeclampsia does not seem to affect the VEGF content of maternal peripheral blood mononuclear cells, granulocytes, or platelets.