Andreas Scorilas

Human kallikrein gene 5 (KLK5) expression is an indicator of poor prognosis in ovarian cancer.

Kallikrein gene 5 (KLK5, also known as KLK-L2), located on chromosome 19q13.4, is one of the newly identified members of the kallikrein gene family, which is a subgroup of the serine protease enzyme family. In normal human tissues, KLK5 is highly expressed in skin, mammary gland and testis. Preliminary RT-PCR analysis has indicated that KLK5 is expressed in a subset of ovarian tumours. We have thus hypothesized that KLK5 may be a new prognostic indicator in ovarian cancer. We have examined the mRNA expression of KLK5 in 142 malignant ovarian tissues. Tumours were pulverized, total RNA was extracted, and cDNA was prepared by reverse transcription. KLK5 was amplified by PCR using gene specific primers, and the identity of the PCR product was verified by sequencing. Ovarian tissues were then classified as KLK5 positive or negative, based on ethidium bromide staining of the PCR product on agarose gels. KLK5 was found to be highly expressed in 58/142 (41%) of ovarian cancer samples while its level of expression was very low in normal ovarian tissues. We found a strong positive relation between KLK5 expression and tumour grade (P = 0.006) and disease stage (P = 0.027). Univariate survival analysis revealed that patients with ovarian tumours positive for KLK5 expression had an increased risk for relapse and death (P = 0.018 and 0.022, respectively). In multivariate analysis, KLK5 expression showed independent prognostic value only in the subset of tumours with lower grade disease (grades I and II). We conclude that KLK5 expression is associated with more aggressive forms of epithelial ovarian carcinoma and has indepdent prognostic value in low grade tumours.

Molecular Cloning of the Human Kallikrein 15 Gene (KLK15) UP-REGULATION IN PROSTATE CANCER

Kallikreins are a subgroup of serine proteases with diverse physiological functions. Growing evidence suggests that many kallikreins are implicated in carcinogenesis. By using molecular cloning techniques, we identified a new human kallikrein gene, tentatively named KLK15 (for kallikrein 15gene). This new gene maps to chromosome 19q13.4 and is located between the KLK1 and KLK3 genes. KLK15 is formed of five coding exons and four introns, and shows structural similarity to other kallikreins and kallikrein-like genes.KLK15 has three alternatively spliced forms and is primarily expressed in the thyroid gland and to a lower extent in the prostate, salivary, and adrenal glands and in the colon testis and kidney. Our preliminary results indicate that the expression ofKLK15 is up-regulated by steroid hormones in the LNCaP prostate cancer cell line. The KLK15 gene is also up-regulated, at the mRNA level, in prostate cancer in comparison to normal prostatic tissue. KLK15 up-regulation was found to be associated with more aggressive forms of prostate cancer. This newly discovered gene has the potential of being used as a diagnostic and/or prognostic marker for prostate cancer.

DECREASED CONCENTRATIONS OF PROSTATE-SPECIFIC ANTIGEN AND HUMAN GLANDULAR KALLIKREIN 2 IN MALIGNANT VERSUS NONMALIGNANT PROSTATIC TISSUE

OBJECTIVES To study quantitatively the relative expression of human glandular kallikrein 2 (hK2) and prostate-specific antigen (PSA) in paired (from the same patient) cancerous and noncancerous prostatic tissue to evaluate whether these proteins are overexpressed or underexpressed in cancer. METHODS We studied 14 patients who underwent radical retropubic prostatectomy for prostate cancer. Cancerous and adjacent normal tissues were excised and then extracted to prepare cytosolic extracts. The extracts were analyzed for total protein, and for hK2 and PSA using sensitive and specific immunofluorometric procedures. RESULTS PSA was present in the prostatic extracts at about 50 to 100 times higher amounts than hK2. The correlation between PSA and hK2 values was good. Both prostate kallikreins were expressed more in noncancerous than in cancerous prostatic tissue. CONCLUSIONS Our results demonstrated that both PSA and hK2 are down-regulated in prostate cancer compared with noncancerous tissue. The degree of down-regulation was higher for PSA than for hK2. The mechanism and physiologic consequences of this down-regulation are unknown.

Quantitative analysis of macrophage inhibitory cytokine-1 (MIC-1) gene expression in human prostatic tissues

Macrophage inhibitory cytokine-1 (MIC-1) gene is a member of transforming growth factor-β superfamily and was reported to be highly overexpressed in human prostate cancer using microarray technology. The aim of this study was to evaluate the quantitative expression of MIC-1 in malignant and benign prostate tissues and to associate expression levels with clinicopathological parameters of prostate cancer. Matched (paired) prostatic tissue samples from the cancerous and noncancerous parts of the same prostates were obtained from 66 patients who underwent radical prostatectomy. Quantitative RT–PCR was performed using SYBR Green I on the Roche LightCyclerTM system. Macrophage inhibitory cytokine-1 gene overexpression in cancerous tissues was observed in 88% of cases, compared to noncancerous tissues (P<0.001). The expression level of MIC-1 in cancerous tissues was significantly higher than in noncancerous tissue (P<0.001). Higher expression of MIC-1 gene was significantly associated with higher Gleason score (P=0.004). The expression of the MIC-1 gene in prostate cancer is significantly higher than in noncancerous tissues, especially in more aggressive forms of the disease (Gleason score>5). This is in contrast to prostate-specific antigen that is downregulated in higher-grade tumours. The upregulation of MIC-1 in prostate cancer and in advanced and more aggressive prostatic tumours suggests that MIC-1 protein should be evaluated as a potential diagnostic and prognostic biomarker.

Immunofluorometric quantitation and histochemical localisation of kallikrein 6 protein in ovarian cancer tissue: a new independent unfavourable prognostic biomarker

Human kallikrein 6 protein is a newly discovered human kallikrein. We determined the amount of human kallikrein 6 in extracts of 182 ovarian tumours and correlated specific activity (ng hK6 mg−1 total protein) with clinicopathological variables documented at the time of surgical excision and with outcome (progression free survival, overall survival) monitored over a median interval of 62 months. Thirty per cent of the tumours were positive for human kallikrein 6 (>35 ng hK6 mg−1 total protein). Human kallikrein 6-specific immunohistochemical staining of four ovarian tissues that included benign, borderline and malignant lesions indicated a cytoplasmic location of human kallikrein 6 in tumour cells of epithelial origin, although the intensity of staining was variable. Tumour human kallikrein 6 (ng hK6 mg−1 total protein) was higher in late stage disease, serous histotype, residual tumour >1 cm and suboptimal debulking (>1 cm) (P<0.05). Univariate analysis revealed that patients with tumour human kallikrein 6 positive specific activity were more likely to suffer progressive disease and to die (hazard ratio 1.71 (P=0.015) and 1.88 (P=0.022), respectively). Survival curves demonstrated the same (P=0.013 and 0.019, respectively). Multivariate analysis revealed that human kallikrein 6 positivity was retained as an independent prognostic variable in several subgroups of patients, namely those with (low) grade I and II tumours (hazard ratio progression free survival 4.3 (P=0.027) and overall survival 4.1 (P=0.023)) and those with optimal debulking (hazard ratio progression free survival 3.8 (P=0.019) and overall survival 5.6 (P=0.011)). We conclude that tumour kallikrein 6 protein levels have utility as an independent adverse prognostic marker in a subgroup of ovarian cancer patients with otherwise apparently good prognosis.

Quantitative Analysis of Kallikrein 15 Gene Expression in Prostate Tissue

PURPOSE The newly discovered human kallikrein 15 gene KLK15 has been shown in preliminary analysis to be associated with more aggressive types of prostate cancer. We quantitatively measured and compared gene expression of KLK15 in malignant and benign prostate tissues. MATERIALS AND METHODS Matched prostate tissue samples from the cancerous and noncancerous parts of the same prostates were obtained from 90 patients who underwent radical prostatectomy. Quantitative reverse transcriptase-polymerase chain reaction using SYBR Green I and the LightCycler system (Roche Applied Science, Mannheim, Germany) was performed. Associations of KLK15 expression with clinicopathological parameters were analyzed. RESULTS KLK15 over expression in cancerous versus noncancerous tissue was found in 76 of the 90 patient samples (84.4%, p <0.001). The ratio of cancerous-to-noncancerous KLK15 expression tended to be higher in patients with stage pT3/4 versus pT2 tumors (p = 0.1). KLK15 expression tended to be higher in grade 3 than in grade 2 tumors and in Gleason score 7 or greater than in Gleason score less than 7 tumors (p = 0.18 and 0.23, respectively). A 1.7 cutoff at the 40th percentile provided a significant difference in stages pT2 and pT3/4 tumors (p = 0.029). CONCLUSIONS On quantitative real-time polymerase chain reaction KLK15 expression was significantly higher in cancerous than in noncancerous tissue. Up-regulation of the KLK15 gene in advanced and more aggressive tumors may indicate a possible role for KLK15 protein as future serum marker for prostate cancer and for distinguishing tumor aggressiveness.

Human kallikrein gene 13 (KLK13) expression by quantitative RT - PCR: an independent indicator of favourable prognosis in breast cancer

Kallikreins are a group of serine proteases with diverse physiological functions. KLK13 (previously known as KLK-L4) is a novel kallikrein gene located on chromosome 19q13.4 and shares a high degree of homology with other kallikrein family members. Many kallikrein genes were found to be differentially expressed in various malignancies, and their regulation is controlled by steroid hormones in prostate and breast cancer cell lines. We studied the expression of KLK13 by quantitative reverse transcriptase–polymerase chain reaction in 173 patients with epithelial breast carcinoma. An optimal cutoff point equal to the 40th percentile was defined, based on the ability of KLK13 to predict disease-free survival. KLK13 values were then associated with other established prognostic factors and with disease-free survival and overall survival. Higher positivity for KLK13 expression was found in older, oestrogen receptor positive patients. In univariate analysis, KLK13 expression is a significant predictor of improved disease-free survival and overall survival (P<0.001 and P=0.009, respectively). Cox multivariate analysis indicated that KLK13 was an independent prognostic variable in the subgroups of patients with Grade I–II tumours and in patients who were oestrogen receptor and progesterone receptor positive, and node positive. Hazard ratios derived from Cox analysis, related to disease-free survival and overall survival were 0.22 (P=0.001) and 0.24 (P=0.008), respectively, for the Grade I–II group; 0.36 (P=0.008) and 0.44 (P=0.038), respectively, for the node positive group and 0.36 (P=0.008) and 0.18 (P=0.008), respectively, for the oestrogen receptor positive group. The adjusted hazard ratio for progesterone receptor positive patients for disease-free survival was 0.25 (P=0.012). For patients in the node positive and oestrogen receptor positive subgroup (n=51) the adjusted hazard ratio was 0.25 (P=0.006) and for the node positive and progesterone receptor positive subgroup (n=46) the hazard ratio was 0.24 (P=0.008). Taken together, these data suggest that higher KLK13 expression in these subgroups of breast cancer patients is associated with an approximately 55 to 80% reduction in the risk of relapse or death. We conclude that KLK13 expression, as assessed by quantitative reverse transcriptase–polymerase chain reaction, is an independent favourable prognostic marker for breast carcinoma.

The use of kallikrein-related peptidases as adjuvant prognostic markers in colorectal cancer.

Several members of the human tissue kallikrein-related peptidase (KLK) family are emerging cancer biomarkers. The aim of this study was to analyse the expression of a panel of KLKs in colorectal cancer and to find out if the multiparametric combination of them can increase the accuracy of prediction of patients survival beyond the traditional clinical information. Nine KLKs (KLK5-8, KLK10, KLK11, KLK13-15) were measured using ELISA assays in cytosolic extracts of 122 colon cancer tissues and their nearby normal mucosa, obtained during surgery. The mean levels of almost all KLKs in tumour tissues were significantly different from their counterparts of normal tissue (P<0.0001). KLK 5, 6, 7, 13, 14 were significantly associated with overall survival in univariate analysis, but after adjusting for age, TNM and differentiation stage, only KLK5 (HR: 1.24 (95% CI: 1.05–1.47)), KLK7 (HR: 1.57 (95% CI: 1.04–2.37)) and KLK14 (HR: 1.43 (95% CI: 1.05–1.94)) remained significant. Addition of a panel of selected KLK markers to clinical parameters gave an increment in AUC of 0.86 beyond the clinical factors at year 1, showing that it can increase the accuracy of prediction of overall survival beyond the traditional clinical information, particularly the short-term (1 year) survival after surgery.

The androgen-regulated gene human kallikrein 15 (KLK15) is an independent and favourable prognostic marker for breast cancer

Many kallikrein genes were found to be differentially expressed in various malignancies, and prostate specific antigen (encoded by the KLK3 gene) is the best tumour marker for prostate cancer. Prostate specific antigen has recently been shown to be an independent favourable prognostic marker for breast cancer. KLK15 is newly discovered kallikrein gene that is located adjacent to KLK3 on chromosome 19q13.4. KLK15 has 41% similarity to KLK3 and the encoded protein, hK15, can activate pro-prostate specific antigen. We studied the expression of KLK15 by real-time quantitative reverse transcriptase–polymerase chain reaction in 202 tissues from patients with breast carcinoma of various stages, grades and histological types. KLK15 expression was found to be a significant predictor of progression-free survival (hazard ratio of 0.41 and P=0.011) and overall survival (hazard ratio of 0.34 and P=0.009). When all other known confounders were controlled in the multivariate analysis, KLK15 retained its prognostic significance. Higher concentrations of KLK15 mRNA were found more frequently in node negative patients (P=0.042). No association was found between KLK15 expression and any other clinicopathological variable. Further, KLK15 is an independent prognostic factor of progression-free survival and overall survival in the subgroup of patients with lower grade and those with oestrogen receptor and progesterone receptor negative tumours in both univariate and multivariate analysis. KLK15 levels of expression were slightly higher (although not statistically significant) in the oestrogen receptor negative and progesterone receptor negative subgroups of patients. KLK15 is up-regulated by androgens in breast cancer cell lines. Time-course and blocking experiments suggest that this regulation is mediated through the androgen receptor.

Quantitative analysis of human kallikrein gene 14 expression in breast tumours indicates association with poor prognosis

KLK14 (formerly known as KLK-L6) is a recently identified member of the human kallikrein gene family. This family harbours several genes aberrantly expressed in various cancers as well as established (PSA/hK3, hK2) and potential (hK6, hK10) cancer markers. Similar to other kallikrein genes, KLK14 was found to be regulated by steroid hormones, particularly androgens and progestins, in breast and ovarian cancer cell lines. Preliminary studies indicated that KLK14 is differentially expressed in breast, ovarian, prostatic and testicular tumours. Given the above, we determined the prognostic significance of KLK14 expression in breast cancer. We studied KLK14 expression in 178 histologically confirmed epithelial breast carcinomas by quantitative reverse transcription–polymerase chain reaction and correlated with clinicopathological variables (tumour stage, grade, histotype etc.) and with outcome (disease-free survival and overall survival), monitored over a median of 76 months. KLK14 mRNA levels ranged from 0 to 1219 arbitrary units in breast cancer tissues, with a mean±s.e. of 136±22. An optimal cutoff value of 40.5 arbitrary units was selected, to categorise tumours as KLK14-positive or negative. Higher concentrations of KLK14 mRNA were more frequently found in patients with advanced stage (III) disease (P=0.032). No statistically significant association was found between KLK14 and the other clinicopathological variables. KLK14 overexpression was found to be a significant predictor of decreased disease-free survival (hazard ratio of 2.31, P=0.001) and overall survival (hazard ratio of 2.21, P=0.005). Cox multivariate analysis indicated that KLK14 was an independent prognostic indicator of disease-free survival and overall survival. KLK14 also has independent prognostic value in subgroups of patients with a tumour size ⩽2 cm and positive nodal, oestrogen receptor and progestin receptor status. We conclude that KLK14 expression, as assessed by quantitative reverse transcription–polymerase chain reaction, is an independent marker of unfavourable prognosis for breast cancer.