Kristina Hotakainen

The role of SPINK1 in ETS rearrangement-negative prostate cancers

ETS gene fusions have been characterized in a majority of prostate cancers; however, the key molecular alterations in ETS-negative cancers are unclear. Here we used an outlier meta-analysis (meta-COPA) to identify SPINK1 outlier expression exclusively in a subset of ETS rearrangement-negative cancers ( approximately 10% of total cases). We validated the mutual exclusivity of SPINK1 expression and ETS fusion status, demonstrated that SPINK1 outlier expression can be detected noninvasively in urine, and observed that SPINK1 outlier expression is an independent predictor of biochemical recurrence after resection. We identified the aggressive 22RV1 cell line as a SPINK1 outlier expression model and demonstrate that SPINK1 knockdown in 22RV1 attenuates invasion, suggesting a functional role in ETS rearrangement-negative prostate cancers.

Expression of cyclooxygenase-2 in human transitional cell carcinoma of the urinary bladder.

Recent studies suggest that expression of cyclooxygenase-2 (Cox-2) is elevated in transitional cell carcinoma (TCC) of the urinary bladder and that inhibition of Cox-2 activity suppresses bladder cancer in experimental animal models. We have investigated the expression of Cox-2 protein in human TCCs (n = 85), in in situ carcinomas (Tis) of the urinary bladder (n = 17), and in nonneoplastic urinary bladder samples (n = 16) using immunohistochemistry. Cox-2 immunoreactivity was detected in 66% (67 of 102) of the carcinomas, whereas only 25% (4 of 16) of the nonneoplastic samples were positive (P: < 0.005). Cox-2 immunoreactivity localized to neoplastic cells in the carcinoma samples. The rate of positivity was the same in invasive (T1-3; 70%, n = 40) and in noninvasive (Tis and Ta; 65%, n = 62) carcinomas, but noninvasive tumors had a higher frequency (32%) of homogenous pattern of staining (>90% of the tumor cells positive) than the invasive carcinomas (10%) (P: < 0.05). However, several invasive TCCs exhibited the strongest intensity of Cox-2 staining in the invading cells, whereas other parts of the tumor were virtually negative. Finally, strong Cox-2 positivity was also found in nonneoplastic ulcerations (2 of 2) and in inflammatory pseudotumors (2 of 2), in which the immunoreactivity localized to the nonepithelial cells. Taken together, our data suggest that Cox-2 is highly expressed in noninvasive bladder carcinomas, whereas the highest expression of invasive tumors associated with the invading cells, and that Cox-2 may also have a pathophysiological role in nonneoplastic conditions of the urinary bladder, such as ulcerations and inflammatory pseudotumors.

The free beta-subunit of human chorionic gonadotropin as a prognostic factor in renal cell carcinoma.

The free β-subunit of human chorionic gonadotropin β is expressed in several nontrophoblastic tumours and this is usually associated with aggressive disease. Little is known about human chorionic gonadotropin β expression in renal cancer. We determined the pretreatment levels of human chorionic gonadotropin β in serum of patients with renal cell carcinoma, and studied whether elevated levels predicted the clinical outcome. Serum samples were collected before surgery from 177 patients with renal cell carcinoma and from 84 apparently healthy controls. Human chorionic gonadotropin β in serum was measured by a highly sensitive time-resolved immunofluorometric assay. The prognostic value of human chorionic gonadotropin β, and of usual clinical and pathological variables was analyzed by the Kaplan-Meier method, the log rank test and Cox multiple hazard regression. The serum concentrations of human chorionic gonadotropin β were increased in 23% of the renal cell carcinoma patients and they were significantly higher in patients with renal cell carcinoma than in controls (P<0.0001). The concentrations did not correlate with clinical stage and histopathological grade, but patients with increased human chorionic gonadotropin β levels had significantly shorter survival time than those with levels below the median (cut-off 1.2u2009pmolu2009l−1, P=0.0029). In multivariate analysis human chorionic gonadotropin β, tumour stage and grade were independent prognostic variables. The serum concentration of human chorionic gonadotropin β is an independent prognostic variable in renal cell carcinoma. The preoperative value of human chorionic gonadotropin β in serum may be used to identify patents with increased risk of progressive disease.

Gonadotropins in doping: pharmacological basis and detection of illicit use

Parenteral administration of human chorionic gonadotropin (hCG) or luteinizing hormone (LH) stimulates the production of testosterone in males and these gonadotropins can therefore be used by athletes to enhance muscle strength. However, they are more expensive and less efficient than testosterone and anabolic steroids. Therefore their main use is probably to stimulate gonadal testosterone production during and after self‐administration of testosterone or anabolic steroids. A positive effect of hCG on muscle strength has not been demonstrated in women and elevated concentrations of hCG in females are often caused by pregnancy. The use of gonadotropins is therefore prohibited only in males but not in females. HCG occurs at low but measurable concentrations in plasma and urine of healthy males and can be measured by sensitive methods. However, the characteristics of the method to be used for doping control have not been defined. Virtually all commercially available hCG assays have been designed for determination of hCG in serum rather than urine, which is used for doping control. Methods based on mass spectrometric detection of fragments derived from hCG extracted from urine by immunoadsorption have been developed but their suitability for doping control remains to be determined. The concentrations of LH in serum and urine are variable and more then 10‐fold higher than those hCG. It is therefore difficult to detect illicit use of LH. The characteristics and reference values for hCG and LH assays used in doping control and the cutoff values need to be defined.

High expression of tumour-associated trypsin inhibitor correlates with liver metastasis and poor prognosis in colorectal cancer

Increased expression of tumour-associated trypsin inhibitor (TATI) in tumour tissue and/or serum has been associated with poor survival in various cancer forms. Moreover, a proinvasive function of TATI has been shown in colon cancer cell lines. In this study, we have examined the prognostic significance of tumour-specific TATI expression in colorectal cancer, assessed by immunohistochemistry (IHC) on tissue microarrays (TMAs) with tumour specimens from two independent patient cohorts. Kaplan–Meier analysis and Cox proportional hazards modelling were used to estimate time to recurrence, disease-free survival and overall survival. In both cohorts, a high (>50% of tumour cells) TATI expression was an independent predictor of a significantly shorter overall survival. In cohort II, in multivariate analysis including age, gender, disease stage, differentiation grade, vascular invasion and carcinoembryonal antigen (CEA), high TATI expression was associated with a significantly decreased overall survival (HR=1.82; 95% CI=1.19–2.79) and disease-free survival (HR=1.56; 95% CI=1.05–2.32) in curatively treated patients. Moreover, there was an increased risk for liver metastasis in both cohorts that remained significant in multivariate analysis in cohort II (HR=2.85; 95% CI=1.43–5.66). In conclusion, high TATI expression is associated with liver metastasis and is an independent predictor of poor prognosis in patients with colorectal cancer.

Expression of the free β-subunit of human chorionic gonadotropin in renal cell carcinoma: Prognostic study on tissue and serum

Expression of the free β‐subunit of human chorionic gonadotropin (hCGβ) in malignant tumors is frequently associated with aggressive disease. We have shown previously that the pretreatment serum concentration of hCGβ is an independent prognostic variable in patients with renal cell carcinoma (RCC). We now compare the serum levels with the expression of hCGβ antigen and mRNA in tumor tissue and studied whether these are associated with the clinical outcome. Serum samples were collected before surgery from patients with RCC (n = 256) and from 84 apparently healthy controls. HCGβ in serum was measured by a time‐resolved immunofluorometric assay. Tissue expression was detected by immunohistochemical staining of a tissue microarray (TMA) comprising 229 samples, and in selected cases by reverse transcription polymerase chain reaction (RT‐PCR) of hCGβ mRNA (n = 20) from tumor tissue. The prognostic value of hCGβ in serum and tissue and the association with usual clinicopathological variables was analyzed by the Kaplan‐Meier method, the log‐rank test, Cox multiple hazard regression, Mann‐Whitney U‐test or Kruskal‐Wallis test. The serum concentrations of hCGβ were increased in 27% of the RCC patients and patients with increased hCGβ levels had significantly shorter survival time than those with levels below the median (cut‐off 1.2 pmol l−1, p = 0.0044). HCGβ antigen was detected in 15% (35 of 229) of the tumors by immunohistochemistry, and hCGβ mRNA in 8 of 20 samples (40%) by RT‐PCR. Tissue positivity for hCGβ antigen was not associated significantly with mRNA expression, serum concentrations of hCGβ or survival. In multivariate analysis tumor stage, grade, size and serum hCGβ were independent prognostic variables. The serum concentration of hCGβ is an independent prognostic variable in RCC. Tissue expression of hCGβ detected by immunohistochemistry occurs in 15% of RCCs but it is not significantly associated with prognosis. Expression at the mRNA level seems to be associated with other predictors of adverse outcome.

Detection of squamous‐cell carcinoma antigen‐expressing tumour cells in blood by reverse transcriptase‐polymerase chain reaction in cancer of the uterine cervix

We used a reverse transcriptase‐polymerase chain reaction method for squamous‐cell carcinoma (SCC) antigen mRNA to detect circulating tumour cells in patients with carcinoma of the uterine cervix. The sensitivity of the method, as determined by cell spiking experiments, was 10 cultured A431 cells among 106 white blood cells. Circulating tumour cells were detected in 6 of 15 patients. In our control group of 24 women, SCC antigen mRNA was detected in 2 pregnant women at term. We followed up the patients for 24 months after sampling and evaluated the outcome. Three out of 6 patients positive for SCC antigen mRNA have relapsed. Additionally, 1 patient has developed breast cancer. In the group of 9 patients negative for SCC antigen mRNA there has been 1 relapse and 1 case of progression of disease. These results suggest that detection of SCC antigen mRNA in peripheral blood by RT‐PCR could be useful for staging and evaluation of prognosis in epidermoid carcinoma of the uterine cervix. Int. J. Cancer 74:75‐80.

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.

Chorionic Gonadotropin β-Subunit and Core Fragment in Bladder Cancer: mRNA and Protein Expression in Urine, Serum and Tissue

OBJECTIVESnMany transitional cell carcinomas (TCC) of the bladder express the beta-subunit (CGbeta) of chorionic gonadotropin (CG), and elevated serum levels occur especially in advanced disease. We have compared the diagnostic utility of various methods for detecting CG and CGbeta expression at the protein and mRNA level.nnnMETHODSnWe used RT-PCR to detect CGbeta mRNA in urinary cells and highly sensitive immunoassays to determine CG and CGbeta in serum and the core fragment of CGbeta (CGbetacf) in urine from patients under follow-up for bladder cancer. Tissue expression was studied by immunohistochemistry.nnnRESULTSnCGbeta mRNA was detected in urinary cells in 50% (n=84) of the cancer cases and in none of the healthy controls (n=15). Positive staining for CGbeta in tissue samples was observed not only in 30% (n=96) of the TCC cases, but also in 5 of 20 histologically benign samples from TCC patients, and in 10 of 21 samples from benign bladder diseases. Serum and urinary concentrations of CGbeta were elevated in 29% (n=66) and 8% (n=72), respectively, while serum CG was elevated in 18% of the TCC patients. Urinary CGbetacf concentrations were higher in invasive (T1-T4) than superficial (T in situ and Ta) tumors (p=0.037), in cases positive for CGbeta mRNA (p=0.03) and cases with suspicious or malignant urinary cytology (p=0.002). The ratio of urinary to serum concentration of CGbeta showed the strongest correlation with tumor stage (p<0.00001), grade (p<0.00001), and staining for CGbeta (p=0.019).nnnCONCLUSIONSnAlthough CGbeta expression may occur in benign bladder epithelium, CGbeta mRNA in urinary cells is a potential marker of bladder cancer. Urinary and serum CGbeta have low sensitivity in early disease, but the urine/serum ratio appears to indicate local release of CGbeta into urine. Further studies are needed to evaluate the clinical usefulness of different forms of CGbeta expression.

Detection of messenger RNA for the β-subunit of chorionic gonadotropin in urinary cells from patients with transitional cell carcinoma of the bladder by reverse transcription-polymerase chain reaction

We studied whether detection of messenger‐RNA (mRNA) for the beta‐subunit of chorionic gonadotropin (CGβ) in urinary cells from bladder cancer patients could be used as a marker of disease activity. Sixty‐eight urine samples from patients under follow‐up for bladder cancer and 23 samples from patients with other malignancies and non‐malignant surgical conditions, as well as 14 samples from healthy controls were analyzed. RNA was isolated from urinary cells collected by centrifugation. Reverse transcription‐polymerase chain reaction (RT‐PCR) was used to detect CGβ mRNA. The results were compared to those obtained by cystoscopy and urinary cytology. For comparison, we determined CG and CGβ in serum and urine and the core fragment of CGβ (CGβcf) in urine by immunofluorometric assays. CGβ mRNA was detected in 29 of 68 urine samples from patients with a history of bladder cancer, whereas all 14 samples from healthy controls tested negative. Elevated levels of CGβ were observed in serum in 18 of 45 bladder cancer patients, but the association with CGβ mRNA was weak. However, CGβ mRNA expression in the absence of detectable cancer also occurred in some conditions associated with cellular atypia such as urinary tract infection, instrumentation and certain therapies. There was a highly significant association between histologically verified transitional cell carcinoma of the bladder and CGβ mRNA in urine (pu2009=u20090.0014), implying CGβ mRNA expression in tumor tissue. We conclude that CGβ mRNA is a potential new marker for monitoring of bladder cancer. Further studies are needed to evaluate whether it provides independent clinical information. Int. J. Cancer (Pred. Oncol.) 84:304–308, 1999.