Susanna Lintula

Tumor-associated trypsin inhibitor in normal and malignant renal tissue and in serum of renal-cell carcinoma patients.

Tumor‐associated trypsin inhibitor (TATI) is a 6‐kDa peptide, which is identical to the pancreatic‐secretory‐trypsin inhibitor (PSTI). TATI is produced by several tumors and cancer cell lines, and is used as a serum marker for mucinous ovarian cancer. Elevated serum levels of TATI have also been observed in renal‐cell carcinoma (RCC). However, it is unclear whether the increase of serum TATI in this disease is caused by production of TATI by the tumor tissue, by the acute‐phase reaction frequently associated with cancer, or by impaired renal function. We examined the expression of TATI in malignant and histologically normal renal tissue by immunohistochemistry, in situ hybridization and reverse‐transcriptase‐polymerase‐chain reaction (RT‐PCR). Furthermore, we measured pre‐operative serum TATI levels in 21 patients with RCC. Immunohistochemically, TATI was detected in 13 of 20 histologically normal renal‐tissue samples, but not in 32 tissue samples from RCC. By RT‐PCR, TATI mRNA was detected in all of 10 histologically normal kidneys and in 6 of 11 RCCs, while in situ hybridization analysis gave negative results. Pre‐operative serum TATI was elevated in 57% of RCC patients. We also studied expression of TATI mRNA and protein in 7 renal‐cancer cell lines, by RT‐PCR and immunofluorometric assay respectively: 6 cancer cell lines were positive for TATI mRNA, while 4 of them also produced TATI protein at low levels. These results indicate that TATI is synthesized by the histologically normal renal tissue and by some renal cancers, and suggest that the elevation of serum TATI associated with renal‐cell carcinoma may be caused by the release of TATI produced by the tumor. Int. J. Cancer 83:486–490, 1999.

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.

Overexpression of Human Chorionic Gonadotropin β 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 β-subunit (hCGβ) is an independent prognostic marker in several nontrophoblastic cancers. hCGβ is encoded by six genes, of which the type II genes (hCGβ 3/9, 5 and 8) are thought to be upregulated in relation to type I genes (hCGβ 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 hCGβ 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 hCGβ 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 hCGβ 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.

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.

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 (p = 0.0014), 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.

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.

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

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.

Expression of luteinising hormone and chorionic gonadotropin beta-subunit messenger-RNA and protein in human peripheral blood leukocytes.

Some pituitary hormones are expressed in leukocytes and are thought to play a role in the regulation of leukocyte function. We studied the expression of the mRNA for the beta-chains of luteinising hormone (LHbeta) and chorionic gonadotropin (CGbeta) and their translation into protein in various leukocyte subsets. Monocytes, granulocytes, B and T-cells from peripheral blood were separated. Lymphocytes were stimulated with various mitogens, prolactin and mixed lymphocyte culture. LHbeta and CGbeta mRNA expression was determined by reverse transcriptase polymerase chain reaction. LH, LHbeta, CG and CGbeta protein were determined in the culture medium by immunofluorometric assays. LHbeta mRNA expression was detected in all cell fractions and cultures and stimulation with prolactin induced LH protein in the culture medium. CGbeta mRNA expression appeared after culture of lymphocytes, but mitogens and prolactin had no clear stimulating effect. The LH expression in leukocytes shown here suggests an autocrine function of this hormone in blood cells.

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 ≈ 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.

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.