Gene-panel testing of breast and ovarian cancer patients identifies a recurrent RAD51C duplication.

Gene-panel sequencing allows comprehensive analysis of multiple genes simultaneously and is now routinely used in clinical mutation testing of high-risk breast and ovarian cancer patients. However, only BRCA1 and BRCA2 are often analyzed also for large genomic changes. Here, we have analyzed 10 clinically relevant susceptibility genes in 95 breast or ovarian cancer patients with gene-panel sequencing including also copy number variants (CNV) analysis for genomic changes. We identified 12 different pathogenic BRCA1, BRCA2, TP53, PTEN, CHEK2, or RAD51C mutations in 18 of 95 patients (19%). BRCA1/2 mutations were observed in 8 patients (8.4%) and CHEK2 protein-truncating mutations in 7 patients (7.4%). In addition, we identified a novel duplication encompassing most of the RAD51C gene. We further genotyped the duplication in breast or ovarian cancer families (n = 1149), in unselected breast (n = 1729) and ovarian cancer cohorts (n = 553), and in population controls (n = 1273). Seven additional duplication carries were observed among cases but none among controls. The duplication associated with ovarian cancer risk (3/590 of all ovarian cancer patients, 0.5%, P = .032 compared with controls) and was found to represent a large fraction of all identified RAD51C mutations in the Finnish population. Our data emphasizes the importance of comprehensive mutation analysis including CNV detection in all the relevant genes.

A Risk-scoring Model for Predicting Post-recurrence Survival in Patients With Endometrial Carcinoma.

AIMS: The survival time of patients with recurrent endometrial carcinoma is generally short. However, considerable interindividual variation exists. We developed a risk-scoring model for predicting post-recurrence survival in patients with endometrial carcinoma. MATERIALS AND METHODS: Patients with endometrial carcinoma treated at a single institution between 2007 and 2013 were identified. Pearson chi-squared analyses were used to compute odds ratios for the associations between risk factors and short survival after cancer recurrence. The results for biochemical analyses represented values at diagnosis of disease recurrence or values at initial diagnosis for those patients who had a primary refractory disease. Logistic regression models were constructed for the identification of variables that independently predict short post-recurrence survival. The models were used to assign points based on odds ratios for risk factors and risk scores were derived. RESULTS: In total, 236 patients with recurrent endometrial carcinoma were included in the study. Based on overall survival analysis, 12 months was selected as the cut-off for short post-recurrence survival. Factors associated with short post-recurrence survival were platelet count, serum CA125 concentration and progression-free survival. A risk-scoring model with an area under the receiver operating characteristic curve (AUC) of 0.782 (95% confidence interval 0.713-0.851) was developed in patients without missing data (n = 182). When patients with a primary refractory disease were excluded, age and blood haemoglobin concentration were identified as additional predictors of short post-recurrence survival. For this subpopulation (n = 152), a risk-scoring model with an AUC of 0.821 (95% confidence interval 0.750-0.892) was developed. CONCLUSIONS: We report a risk-scoring model that shows acceptable to excellent accuracy in predicting post-recurrence survival in patients with endometrial carcinoma, with primary refractory diseases included or excluded. This model has potential applications in precision medicine in patients with endometrial carcinoma.

Somatic MED12 mutations in uterine leiomyosarcoma and colorectal cancer.

BACKGROUND: Mediator complex participates in transcriptional regulation by connecting regulatory DNA sequences to the RNA polymerase II initiation complex. Recently, we discovered through exome sequencing that as many as 70% of uterine leiomyomas harbour specific mutations in exon 2 of mediator complex subunit 12 (MED12). In this work, we examined the role of MED12 exon 2 mutations in other tumour types. METHODS: The frequency of MED12 exon 2 mutations was analysed in altogether 1158 tumours by direct sequencing. The tumour spectrum included mesenchymal tumours (extrauterine leiomyomas, endometrial polyps, lipomas, uterine leiomyosarcomas, other sarcomas, gastro-intestinal stromal tumours), hormone-dependent tumours (breast and ovarian cancers), haematological malignancies (acute myeloid leukaemias, acute lymphoid leukaemias, myeloproliferative neoplasms), and tumours associated with abnormal Wnt-signalling (colorectal cancers (CRC)). RESULTS: Five somatic alterations were observed: three in uterine leiomyosarcomas (3/41, 7%; Gly44Ser, Ala38_Leu39ins7, Glu35_Leu36delinsVal), and two in CRC (2/392, 0.5%; Gly44Cys, Ala67Val). CONCLUSION: Somatic MED12 exon 2 mutations were observed in uterine leiomyosarcomas, suggesting that a subgroup of these malignant tumours may develop from a leiomyoma precursor. Mutations in CRC samples indicate that MED12 may, albeit rarely, contribute to CRC tumorigenesis.

Prognostic role of CIP2A expression in serous ovarian cancer.

BACKGROUND: Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein expressed in several solid cancers. Our purpose was to study its role in serous ovarian cancer patients, and the association to clinicopathological variables and molecular markers. METHODS: We collected retrospectively 562 consecutive serous ovarian cancer patients treated at the Helsinki University Central Hospital. We stained tumour tissue microarrays for CIP2A by immunohistochemistry and constructed survival curves according to the Kaplan-Meier method. Associations to clinicopathological and molecular markers were assessed by the χ(2)-test. RESULTS: We found strong cytoplasmic CIP2A immunoreactivity in 212 (40.4%) specimens, weak positivity in 222 (42.4%) specimens, and negative in 90 (17.2%). Immunopositive CIP2A expression was associated with high grade (P<0.0001), advanced stage (P=0.0005), and aneuploidy (P=0.001, χ(2)-test). Cancerous inhibitor of protein phosphatase 2A overexpression was also associated with EGFR protein expression (P=0.006) and EGFR amplification (P=0.043). Strong cytoplasmic CIP2A immunopositivity predicted poor outcome in ovarian cancer patients (P<0.0001, log-rank test). CONCLUSION: Our results show that CIP2A associates with reduced survival and parameters associated with high grade in ovarian cancer patients, and may thus be one of the factors that identify aggressive subtype (type II) of this disease.

A 60-kD protein mediates the binding of transforming growth factor-beta to cell surface and extracellular matrix proteoglycans.

The biological activity of many cytokines is regulated by binding proteins present at the cell surface, in extracellular matrices or in soluble phase. We describe here a TGF-beta binding protein that is both an extracellular matrix and a cell surface protein. When intact extracellular matrices of HEP-G2 cells were affinity cross-linked with 125I-TGF-beta 1, two major binding components were seen: a 250-kD, proteoglycan-like molecule, presumed to be betaglycan, and a 60-kD protein. The 60-kD TGF-beta-binding protein was also present at the cell surface. It could be released from the cell surface by treating cells with high salt, heparin, chondroitin sulfate, heparitinase, or chondroitinase, indicating that it is bound to heparan sulfate and chondroitin sulfate proteoglycans. The 60-kD protein bound TGF-beta 1 with an apparent dissociation constant of 1.6 nM, and there were 30,000 binding sites per cell at the cell surface. In addition to the HEP-G2 cells and another hepatoma cell line, the 60-kD protein was also found in a human colon carcinoma (HT-29) cell line but not in rat kidney (NRK-49F) or human fibroblast (HUT-12) cell lines. The 60-kD protein could be extracted from cells containing it and transferred to the surface of previously negative cells. The 60-kD protein may serve to regulate the binding of TGF-beta to its signal transducing receptors by targeting TGF-beta to appropriate locations in the microenvironment of cells.

Genomic alterations in fallopian tube carcinoma: comparison to serous uterine and ovarian carcinomas reveals similarity suggesting likeness in molecular pathogenesis.

Serous carcinomas of the fallopian tube, uterus, and ovary resemble each other both histologically and in clinical behavior. Comparative genomic hybridization was performed on 20 primary fallopian tube carcinoma specimens to find regions of the genome involved in tubal carcinogenesis and to compare the genomic alterations with those previously detected in serous ovarian and uterine carcinomas. The most frequent changes detected in fallopian tube carcinoma were gains at 3q (70%) and 8q (75%), with high-level amplifications in several cases. Other common gains occurred at 1q, 5p, 7q, 12p, and 20q. The most frequent losses were found at 18q, 8p, 4q, and 5q. The frequency and the pattern of chromosomal changes detected in tubal carcinoma were strikingly similar to those observed in serous ovarian and uterine carcinomas, suggesting common molecular pathogenesis.

Distinct chromosomal imbalances in uterine serous and endometrioid carcinomas.

Endometrial carcinoma shows various histological types that differ in their clinical presentation and prognosis. Comparative genomic hybridization was used to detect gains and losses of DNA sequences along all chromosome arms in 24 uterine serous and 24 uterine endometrioid carcinomas. In serous carcinomas, extensive genetic aberrations were detected in 17 of the 24 specimens, with a mean of 5.7 changes per tumor. The most frequent gains occurred at 3q (50%), 8q (33%), 5p (29%), 6p (29%), and 1q (29%), and the most common losses were located at 4q (17%), 15q (17%), and 18q (17%). Tumors exhibiting DNA copy number changes were associated with shorter overall survival. In endometrioid carcinomas, genetic aberrations were less frequent and simpler than in serous carcinomas. DNA sequence copy number changes were observed in 12 of the 24 cases, with a mean of 1.5 changes per tumor. The most frequent aberrations were gains at 1q (29%), 2q (13%), and 8q (13%). Losses were rarely observed. The diverging pattern of genetic changes observed in uterine serous and endometrioid carcinomas suggests different pathways of carcinogenesis in these tumor types.

Genetic changes in inherited and sporadic ovarian carcinomas by comparative genomic hybridization: extensive similarity except for a difference at chromosome 2q24-q32.

Germ-line mutations in the BRCA1 and BRCA2 genes confer a predisposition to breast as well as ovarian carcinoma. Except for loss of the respective wild-type allele, somatic genetic changes needed for the progression of inherited ovarian tumors are unknown. A genome-wide search for such alterations was performed by comparative genomic hybridization analysis on BRCA1 and BRCA2 mutation-positive (n = 20) ovarian carcinoma specimens. Comparison with sporadic ovarian carcinomas (n = 20) revealed extensive genetic similarity between the inherited and sporadic carcinomas with the sole exception of a frequent gain of 2q24-q32 in the inherited group, suggesting the presence of an oncogene at 2q24-q32 operating in the absence of BRCA1 function. The overall similarity of gains and losses by comparative genomic hybridization suggests a common main pathway in tumor progression of both inherited and sporadic ovarian carcinomas.

Lack of MSH2 and MSH6 characterizes endometrial but not colon carcinomas in hereditary nonpolyposis colorectal cancer.

Hereditary nonpolyposis colorectal cancer syndrome is associated with an inherited predisposition to primarily colorectal cancer (CRC) and endometrial cancer (EC); however, the biological basis of the organ involvement remains unknown. As an attempt to explore whether the expression levels of MLH1, MSH2, and MSH6 may play a role, we used immunohistochemistry to study 42 ECs and 35 CRCs from patients carrying the same predisposing mutations. Among MSH2 mutation carriers, MLH1 was expressed in both tumor types, whereas MSH2 and, in many cases, also MSH6, were absent. Remarkably, among MLH1 mutation carriers, 54% of ECs (21 of 39), but none of the CRCs (0 of 32), lacked the MSH2 and/or MSH6 protein in addition to lacking MLH1 protein expression. These results demonstrate a marked difference between hereditary nonpolyposis colorectal cancer-related CRCs and ECs and suggest that the development of the latter tumors is selectively associated with the MSH2/MSH6 protein complex deficiency.

Human granulosa cells synthesize low molecular weight insulin-like growth factor-binding protein.

Human follicular fluid contains insulin-like growth factor I (IGF-I) and its low molecular weight binding protein (IGF-BP). We studied the synthesis of IGF-BP by the granulosa cells obtained after ovarian hyperstimulation for in vitro fertilization. The granulosa cells were cultured for 72 hours in Ham's F-10 medium supplemented with 10% fetal calf serum (FCS). Samples of the culture medium were collected every 24 hours. The IGF-BP concentration in culture medium increased from 1.2 to 2.1 micrograms/l at 48 h and to 3.3 micrograms/l at 72 h. De novo synthesis of IGF-BP was shown by incorporation of labeled methionine into immunoreactive IGF-BP, as detected by SDS polyacrylamide electrophoresis (PAGE) and fluorography. Our results demonstrate synthesis of IGF-BP in the human ovary.

Regulation of insulin-like growth factor-binding protein-1 production in human granulosa-luteal cells.

Human follicular fluid contains the insulin-like growth factor-binding protein (IGFBP-1) synthesized by ovarian granulosa cells. We studied the regulation of IGFBP-1 production by the granulosa-luteal cells from the hyperstimulated follicles of patients attending an in vitro fertilization program. The cells were first allowed to attach and recover from the hyperstimulation for 2 days. Then, a protein kinase-C activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA), and adenylate cyclase activators, gonadotropins, FSH, hCG, cholera toxin, or prostaglandin E2 (PGE2) were added to the cells. The gonadotropins failed to increase IGFBP-1 production, whereas it was enhanced by TPA and to a lesser extent by cholera toxin and PGE2. The maximal response to TPA occurred at the concentration of 1.0 ng/mL, and the enhancing effect of TPA was detected at 24 h. PGE2 stimulated IGFBP-1 production; the lowest effective concentration was 10(-8) mol/L. The mean highest response was 4.3-fold and occurred at the PGE2 concentration of 10(-5) mol/L. The effect of PGE2 on IGFBP-1 production became detectable at 24 h, and it continued to increase up to 72 h. PGE2 also increased granulosa-luteal cell progesterone production in a dose- and time-dependent manner. Incorporation of [35S]methionine into immunoreactive IGFBP-1, as detected by sodium dodecyl sulfate-polyacrylamide electrophoresis and fluorography, was also increased by TPA. This suggests that TPA accelerated the synthesis of IGFBP-1. Our results indicate that the production of IGFBP-1 by human granulosa-luteal cells can be regulated both via protein kinase-C- and adenylate cyclase-dependent pathways.

Transcription factors GATA-4 and GATA-6 and a GATA family cofactor, FOG-2, are expressed in human ovary and sex cord-derived ovarian tumors.

Previous studies have implicated transcription factors GATA-4 and GATA-6 in the regulation of murine ovarian development and function. In rodents, GATA-4 is expressed in granulosa cells of primary and early antral follicles, whereas GATA-6 is expressed in granulosa cells of late antral follicles and luteal glands. Both transcription factors can be detected in lesser amounts in theca cells and interstitial cells. We have now examined the expression of GATA-4 and GATA-6 in human ovaries, human granulosa-luteal (GL) cells and sex cord-derived tumors. We show by in situ hybridization and immunohistochemistry that GATA-4 and GATA-6 messenger RNA (mRNA) and GATA-4 protein are present in granulosa and theca cells in both preantral and antral follicles. Both human ovarian tissue samples and freshly isolated GL cells derived from preovulatory follicles of gonadotropin-treated women express GATA-4, GATA-6, and FOG-2 transcripts, and GATA-6 mRNA expression in GL cell cultures is stimulated by human CG and 8-bromo-cAMP. The vast majority of granulosa and theca cell tumors examined expressed GATA-4 and GATA-6. We also found that mRNA for FOG-2, a recently discovered regulator of GATA-4, is coexpressed with GATA-4 in human ovary samples, normal granulosa cells, and in sex cord-derived tumors. Our results demonstrate that GATA-4, GATA-6, and FOG-2 are expressed in human ovary and in granulosa and theca cell tumors. Our findings support a role for GATA-binding proteins in human ovarian folliculogenesis. Moreover, these data suggest that GATA factors may contribute to the phenotypes of sex cord-derived ovarian tumors.

Regulated expression of prostaglandin E(2) receptors EP2 and EP4 in human ovarian granulosa-luteal cells.

Prostaglandins (PGs) have been implicated in regulation of ovarian function. We have previously shown that the expression of cyclooxygenase-2 and the receptor for PGF(2 alpha) are expressed in periovulatory human granulosa cells and upregulated by gonadotropins and cytokines in cultured human ovarian granulosa-luteal (GL) cells. We now show that transcripts for PGE(2) receptor subtypes EP2 and EP4 are expressed in freshly isolated human granulosa cells and in mouse ovaries as detected by Northern blot analysis. However, EP2 and EP4 receptor mRNA levels were low or nondetectable in cultured human GL cells suggesting that these transcripts may be under hormonal and/or cytokine regulation in the ovaries in vivo. Indeed, the proinflammatory cytokine interleukin-1 beta (IL-1 beta) stimulated expression of EP2 and EP4 transcripts in concentration- and time-dependent manner in the GL cells. Furthermore, the transcript for EP2 receptor was localized in the corpus luteum of the mouse ovary by in situ hybridization, and EP2 protein was expressed in human corpus luteum as detected by immunohistochemistry. Our data suggest that IL-1 beta induces expression of EP2 and EP4 receptors in human GL cells, and that EP2 receptor is expressed in both human and murine luteal glands.

Human growth differentiation factor 9 (GDF-9) and its novel homolog GDF-9B are expressed in oocytes during early folliculogenesis.

Growth differentiation factor 9 (GDF-9) is a transforming growth factor-beta family member that is required for normal folliculogenesis in female mice, but its role as a regulator of human fertility is still unclear. We determined here by in situ hybridization and immunohistochemical analyses the localization of the GDF-9 messenger ribonucleic acid (mRNA) and protein during human folliculogenesis. The GDF-9 transcripts were not detected in primordial follicles, but they are abundantly expressed in primary follicles in frozen sections of ovarian cortical tissue material obtained at laparoscopic surgery. We raised antipeptide antibodies against GDF-9 and showed by immunohistochemical studies on paraffin sections of whole human ovaries that the GDF-9 protein is most abundantly expressed in primary follicles. We recently demonstrated that a novel GDF-9-related factor, GDF-9B, is coexpressed with GDF-9 during murine folliculogenesis. We now isolated human GDF-9B complementary DNA and genomic clones and report the unusually restricted expression pattern of human GDF-9B. The human GDF-9B transcript can be detected only in the gonads by RT-PCR analysis, and in situ hybridization studies indicate that it is not expressed in small primary follicles but, rather, in the oocytes of late primary follicles. Functional studies using the Xenopus laeuis embryo model indicate that unlike the transforming growth factor-beta family members activin and bone morphogenetic protein-4, neither GDF-9 nor GDF-9B affects mesoderm induction, suggesting that they may use signaling pathways distinct from those well defined for activin and bone morphogenetic protein-4. We conclude that 1) both GDF-9 mRNA and protein are abundantly expressed in oocytes of primary follicles in human ovary, suggesting that the GDF-9 transcript is translated at this early stage of folliculogenesis; 2) human GDF-9B is specifically expressed in gonads at low levels; and 3) the expression of GDF-9 mRNA begins slightly earlier than that of GDF-9B in the human oocytes during follicular development. Our results are consistent with the suggestion that GDF-9 and GDF-9B may regulate human folliculogenesis in a manner specific to the ovary.

BRCA1 and BRCA2 mutations among 233 unselected Finnish ovarian carcinoma patients.

Germline mutations of BRCA1 and BRCA2 predispose to hereditary breast-ovarian cancer syndrome. In Finland, 20 different BRCA1/2 mutations have been identified, and 13 of them are founder mutations that account for the vast majority of Finnish BRCA1/2 families. The purpose of our study was to determine the prevalence of BRCA1/2 mutations in unselected Finnish ovarian carcinoma patients and to evaluate the relationship between mutation carrier status and personal/family history of cancer. Two hundred and thirty-three patients were screened for all the 20 BRCA1/2 mutations known in the Finnish population. Additionally, a subgroup of patients with personal history of breast cancer and/or family history of breast and/or ovarian cancer was screened for novel BRCA1/2 mutations. Thirteen patients (5.6%) had mutations: eleven in BRCA1 and two in BRCA2. All the mutation-positive patients were carriers of the previously known Finnish BRCA1/2 mutations, and seven recurrent founder mutations accounted for 12 of the 13 mutations detected. A logistic regression analysis was used to determine the odds of mutation for ovarian carcinoma patients. The most significant predictor of a mutation was the presence of both breast and ovarian cancer in the same woman, but family history of breast cancer was also strongly related to mutation carrier status. Although BRCA1/2 mutation testing is not warranted in the general Finnish ovarian cancer patient population, patients who have also been diagnosed with breast cancer or have family history of breast or breast and ovarian cancer could benefit from referral to genetic counselling and mutation testing.

Multiple founder effects and geographical clustering of BRCA1 and BRCA2 families in Finland.

In the Finnish breast and ovarian cancer families six BRCA1 and five BRCA2 mutations have been found recurrently. Some of these recurrent mutations have also been seen elsewhere in the world, while others are exclusively of Finnish origin. A haplotype analysis of 26 Finnish families carrying a BRCA1 mutation and 20 families with a BRCA2 mutation indicated that the carriers of each recurrent mutation have common ancestors. The common ancestors were estimated to trace back to 7-36 generations (150-800 years). The time estimates and the geographical clustering of these founder mutations in Finland are in concordance with the population history of this country. Analysis of the cancer phenotypes showed differential ovarian cancer expression in families carrying mutations in the 5' and 3' ends of the BRCA1 gene, and earlier age of ovarian cancer onset in families with BRCA1 mutations compared with families with BRCA2 mutations. The identification of prominent and regional BRCA1 and BRCA2 founder mutations in Finland will have significant impact on diagnostics in Finnish breast and ovarian cancer families. An isolated population with known history and multiple local founder effects in multigenic disease may offer distinct advantages also for mapping novel predisposing genes.

Regulation of the production of placental protein 5 by human endometrial stromal cells; the role of prostaglandins E2 and F2 alpha.

Cultured human endometrial stromal cells were found to release placental protein 5 (PP5), a glycoprotein with properties of a serine protease inhibitor. Progesterone had no effect on PP5 release, but cholera toxin and 12-O-tetradecanoylphorbol 13-acetate stimulated PP5 release in a time- and concentration-dependent fashion. Prostaglandin E2 (PGE2) caused a parallel increase in cAMP and PP5 release in a time- and concentration-dependent fashion. The lowest PGE2 concentration which increased cAMP and PP5 release was 1 X 10(-9) M. Maximal increase in cAMP (42-fold) and PP5 (25-fold) release was obtained by 10(-5) M PGE2. Stimulation of cAMP by PGE2 was detectable at 15 min and was followed by an increased PP5 release at 24 h. The concentrations of prostaglandin F2 alpha (PGF2 alpha) which stimulated cAMP and PP5 release were pharmacological suggesting that this effect is nonspecific. The results indicate that the activation of cAMP- and protein kinase C-dependent pathways in endometrial stromal cells increases the production of PP5. PGE2 could be one of the physiological ligands employing the cAMP-dependent pathway in endometrial stromal cells.

Differential regulation of hCG and progesterone secretion by cholera toxin and phorbol ester in human cytotrophoblasts.

The secretion of hCG and progesterone (P) by human cytotrophoblasts was studied in response to cholera toxin (CT), which activates adenylate cyclase, and 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C stimulator. During a 24 h incubation CT and TPA increased hCG and P secretion in a concentration-dependent manner. At maximal effective concentrations, CT (1.0 ng/ml), TPA (10 ng/ml) and CT plus TPA stimulated hCG production by 5.7-, 2.7- and 10.0-fold, respectively, and P production by 1.8-, 1.8- and 2.0-fold, respectively. Time-course studies indicated that these effects became detectable after 12 h and increased up to 48 h of incubation. During a 24 h culture TPA potentiated CT-induced cAMP formation by 1.4-fold indicating that its effects on hCG production may be, at least partly, mediated through cAMP. In conclusion, CT and TPA are potent stimulators of human cytotrophoblast hCG and P production. Simultaneous stimulation by these agents results in potentiation of hCG production whereas P production remains at the same level as with CT or TPA alone. The results suggest that the endocrine activity of human cytotrophoblasts is under multihormonal control and hCG and P secretion are differentially regulated.

Endothelial cell KIT expression in human tumours.

Receptor tyrosine kinases expressed in endothelial cells are potential targets for therapy with specific tyrosine kinase inhibitors. Endothelial cell KIT expression has not been systematically evaluated in human cancer. In the present study, endothelial cell KIT expression was assessed in 345 tumours consisting of 34 different histological types using a tissue microarray technique. Marked KIT expression occurred in the tumour endothelial cells only in primary glioblastomas in the microarray. Moderate to strong KIT and phosphorylated KIT expression was detected in the tumour endothelial cells in six (16%) and seven (19%) of the 37 primary glioblastomas examined, respectively. In whole tissue sections, KIT and phosphorylated KIT were expressed in tumour endothelial cells in 13 (59%) and 11 (50%) of the 22 glioblastomas examined, respectively. RNA in situ hybridization showed KIT mRNA expression in most glioblastomas both in tumour vessel endothelial cells and in perinecrotic palisading glioblastoma cells, whereas little KIT mRNA was found in the endothelial cells of colon or pancreatic carcinomas. Phosphorylated KIT, its ligand stem cell factor, and the downstream signalling molecules phosphorylated Akt and mTOR were often expressed in glioblastoma cells located in the perinecrotic tumour areas that often also contained abundant HIF-1alpha. It is concluded that marked KIT and phosphorylated KIT expression is frequently present in the endothelial cells of glioblastomas, which are known to harbour florid microvascular proliferation with characteristic morphological features. Glioblastomas also express phosphorylated KIT and its activated downstream signalling molecules in the tumour cells. Lower levels of KIT and phosphorylated KIT are present in endothelial cells of other tumour types and in normal tissues. Endothelial cell and tumour cell expression of activated KIT might explain in part the responsiveness of glioblastomas to the combination of imatinib (an inhibitor of KIT) and hydroxyurea.

The human fallopian tube contains placental protein 5.

Placental protein 5 (PP5), a glycoprotein with properties of a serine protease inhibitor, was found in human Fallopian tubes removed during proliferative (n = 6) and secretory (n = 6) phases of the menstrual cycle. The content of PP5 did not differ in fimbrial, ampullar and isthmic parts of the tube. In gel filtration, PP5-immunoreactivity from Fallopian tube extracts eluted as one major peak corresponding to a mol. wt of 36 kd. In radioimmunoassay, the dose--response curves of purified placental PP5 and Fallopian tubal extracts were parallel. In immunofluorescence staining, utilizing polyclonal and monoclonal antibodies, PP5 was localized to the epithelium of the Fallopian tube, but weaker staining was observed also in the stroma. As a potential serine protease inhibitor, PP5 might play a part in the implantation of the fertilized ovum.