Nonmucinous Ovarian Carcinomas Research Articles

Establishment of a human ovarian clear cell carcinoma cell line mutant in PIK3CB but not PIK3CA

A human ovarian clear cell carcinoma cell line was established from a 46-year-old Japanese woman. That line, designated MTC-22, has proliferated continuously for over 6 months in conventional RPMI 1640 medium supplemented with 10% foetal bovine serum and has been passaged over 50 times. MTC-22 doubling-time is ~ 18 h, which is much shorter than most ovarian clear cell carcinoma lines reported to date. Morphologically, MTC-22 cells exhibit polygonal shapes and proliferate to form a monolayer in a jigsaw puzzle-like arrangement without contact inhibition. Ultrastructurally, cells exhibit numerous intracytoplasmic glycogen granules and well-developed mitochondria. G-band karyotype analysis indicated that cells have a complex karyotype close to tetraploid. We observed that the expression pattern of a series of ovarian carcinoma-related molecules in MTC-22 cells was identical to that seen in the patient’s tumour tissue. Notably, MTC-22 cells, and the patient’s carcinoma tissue, expressed low-sulphated keratan sulphate recognised by R-10G and 294-1B1 monoclonal antibodies, a hallmark of non-mucinous ovarian carcinoma, and particularly of clear cell ovarian carcinoma. Moreover, characteristic point mutations—one in ARID1A, which encodes the AT-rich interaction domain containing protein 1A, and the other in PIK3CB, which encodes the catalytic subunit of phosphoinositide 3-kinase—were seen in the patient’s tumour tissue and retained in MTC-22 cells. Collectively, these findings indicate that MTC-22 cells could serve as a valuable tool for investigating the pathophysiology of ovarian clear cell carcinoma, particularly that harbouring PIK3CB mutations, and for developing and validating new diagnostic and therapeutic approaches to this life-threatening malignancy.

Expression of low-sulfated keratan sulfate in non-mucinous ovarian carcinoma.

Keratan sulfate glycosaminoglycan is composed of repeating N-acetyllactosamine (LacNAc) disaccharide units consisting of galactose (Gal) and N-acetylglucosamine (GlcNAc), both often 6-O-sulfated. Sulfate contents of keratan sulfate are heterogeneous depending upon the origins. In this study, keratan sulfate is classified as either highly sulfated (in which both GlcNAc and Gal residues are 6-O-sulfated) or low-sulfated (in which only GlcNAc residues are 6-O-sulfated). It is reported that highly sulfated keratan sulfate detected by the 5D4 monoclonal antibody is preferentially expressed in normal epithelial cells lining the female genital tract and in their neoplastic counterparts; however, expression of low-sulfated keratan sulfate in either has not been characterized. In the present study, we generated the 294-1B1 monoclonal antibody, which selectively recognizes low-sulfated keratan sulfate, and performed precise glycan analysis of sulfated glycans expressed on human serous ovarian carcinoma OVCAR-3 cells. We found that OVCAR-3 cells do not express highly sulfated keratan sulfate but rather express low-sulfated form, which was heterogeneous in 294-1B1 reactivity. Comparison of mass spectrometry spectra of sulfated glycans in 294-1B1-positive versus -negative OVCAR-3 cells indicated that the 294-1B1 epitope is likely at least 2, and possibly 3 or more, tandem GlcNAc-6-O-sulfated LacNAc units. Then, using the 294-1B1 antibody, we performed quantitative immunohistochemical analysis of 40 specimens from patients with ovarian cancer, consisting of 10 each of serous, endometrioid, clear cell, and mucinous carcinomas, and found that among them low-sulfated keratan sulfate was widely expressed in all but mucinous ovarian carcinoma.

Loss of SMAD4 protein expression in gastrointestinal and extra-gastrointestinal carcinomas.

SMAD4 (DPC4) is a tumour suppressor gene that is dysregulated in various tumour types, particularly pancreaticobiliary and gastrointestinal carcinomas. Corresponding loss of protein expression has been reported in approximately 50% of pancreatic and 25% of colonic adenocarcinomas. In the evaluation of carcinoma of unknown primary site, immunohistochemical loss of SMAD4 expression is often used to suggest pancreaticobiliary origin, but there are limited data on the spectrum of SMAD4 expression in carcinomas of other sites. This study evaluates the frequency of SMAD4 loss in a large cohort of carcinomas from diverse anatomical sites. Immunohistochemistry for SMAD4 was performed on tissue microarrays or whole tissue sections of 1210 carcinomas from various organs: gastrointestinal tract, liver, pancreas/biliary tract, lung, breast, thyroid, kidney, ovary and uterus. Expression was considered lost when there was complete absence of staining in tumour cell nuclei, in the presence of intact staining in non-neoplastic cells. SMAD4 loss was seen in 58% of pancreatic adenocarcinomas, 27% of appendiceal adenocarcinomas, 19% of colorectal adenocarcinomas, 16% of cholangiocarcinomas, 10% of lung adenocarcinomas and <5% of oesophageal, breast, gastric and mucinous ovarian adenocarcinomas. All papillary thyroid, hepatocellular, non-mucinous ovarian, endometrial and renal cell carcinomas showed intact SMAD4 nuclear expression. In addition to pancreaticobiliary, appendiceal and colonic tumours, SMAD4 loss is also seen in a small subset of other carcinomas, specifically breast, lung, oesophageal and gastric adenocarcinomas, all of which are typically CK7-positive, similar to pancreaticobiliary carcinoma. Awareness of SMAD4 loss in these other carcinoma types is helpful in the evaluation of carcinomas of unknownor uncertain primary site.

Interobserver Agreement for Mismatch Repair Protein Immunohistochemistry in Endometrial and Nonserous, Nonmucinous Ovarian Carcinomas.

Immunohistochemistry (IHC) for mismatch repair (MMR) proteins is an established test to identify Lynch syndrome (LS) in patients with colorectal cancer and is being increasingly used to identify LS in women with endometrial and/or nonserous ovarian cancer (OC). We assessed interobserver agreement in the interpretation of MMR-IHC on endometrial and ovarian carcinomas. The study consisted of 73 consecutive endometrial cancers (n=48) and nonserous, nonmucinous epithelial OCs (n=25). Six pathologists from 2 cancer centers, one with and the other without, previous experience in interpreting MMR-IHC, evaluated MLH1, MSH2, MSH6, and PMS2 stains. Before the study, an experienced pathologist led a review of 9 teaching cases. A decision tool was developed as a guide in MMR-IHC interpretation. Staining was interpreted as intact, deficient, or equivocal for each protein. Interobserver agreement for the patient MMR status was categorized as "almost perfect" with κ=0.919 (95% CI, 0.863-0.976). All observers were in agreement in 66 (92%) tumors. Four of the less experienced pathologists had at least 1 discrepant interpretation. There were 6 discordant cases: 3 MMR-deficient cases and 2 MMR-intact cases by majority opinion were called equivocal by at least 1 observer, and 1 MMR-deficient case by majority opinion was interpreted as MMR intact by 1 pathologist. Only the latter case (1/73 patients, 1.4%) had an unequivocal disagreement that could affect patient management. Issues associated with discordant interpretation included heterogeneous staining, intratumoral lymphocytes, regional reduced internal control tissue staining, and scattered absent/weak staining adjacent to tumor cells with strong nuclear staining.

Frequency of BRCA1 and BRCA2 Germline Variants in Women With Ovarian Cancer in Malaysia

Background: Germline BRCA1 or BRCA2 pathogenic variants in ovarian cancer patients may be informative in risk management and treatment, with the advent of poly(ADP-ribose) polymerase inhibitors. In the era of precision medicine, companion diagnostics for BRCA1 and BRCA2 genes have been featured as a strategy in the Malaysia National Strategic Plan for Cancer Control Program (2016-2020). To facilitate this strategy, frequency data from Malaysia's understudied multiethnic population will be required. Aim: To determine the prevalence of BRCA1 and BRCA2 germline variants in a population-based cohort of ovarian cancer patients in Malaysia. Methods: From August 2016, women with nonmucinous epithelial ovarian, peritoneal or fallopian tube carcinoma are prospectively recruited to the Malaysia-wide population-based MaGiC Observational Study. DNA were tested using a Hi-Plex next generation sequencing method and multiplex ligation-dependent probe amplification to detect &lt; 10 bp alterations and exon deletions or duplications in the BRCA1 and BRCA2 genes. Results: Interim results from 325 patients tested until March 2018 have identified BRCA1 and BRCA2 pathogenic variants in 9.8% (32/325) and 3.1% (10/325) patients, respectively. Variants of uncertain significance were detected in 13.2% (43/325) patients and no pathogenic variants were detected in 73.8% (240/325) patients. Taken together, the frequency of BRCA1/2 pathogenic variants in ovarian cancer patients is approximately 12.9% (42/325). Conclusion: The identification of BRCA1 or BRCA2 carriers across the country have enabled the concentration of efforts from limited genetic counseling resources to high risk families. Results arising from the completion of this study will supplement cancer control programs and genetic services in Malaysia.

BRCA1/2 germline testing in non-mucinous epithelial ovarian carcinoma: changing international practice and implications for service provision.

Ovarian carcinoma is the fourth most common cancer for women in Republic of Ireland, with an average of 361 cases per year.1 The association with germline mutations in BRCA1/2 genes in non-mucinous ovarian cancer risk is well established.2 Determining the BRCA status of ovarian cancer patients has important prognostic and therapeutic implications for individual patients.3 The frequency of BRCA1/2 germline mutations in non-mucinous ovarian carcinoma is estimated at 14–15%,2 with previous studies finding a higher rate of 16.6% in serous ovarian carcinoma and 17.1% in high-grade serous carcinoma.3 Family history has been shown not to be sufficiently accurate to predict mutation status.2, 3 In Ireland, as in most European countries, and the United States, the current recommendation is that patients with a family history of breast/ovarian cancer should be offered full mutation screening of the BRCA1/2 genes as appropriate following the assessment by a clinical genetics service.4

Increased human epididymis protein 4 in benign gynecological diseases complicated with chronic renal insufficiency patients.

We examined the serum concentration of human epididymis protein (HE4) in patients with benign gynecological diseases complicated with chronic renal deficiency and its significance in the differential diagnosis of benign and malignant gynecological diseases. Serum HE4 and cancer antigen 125 concentrations were detected by chemiluminescence. Clinically or pathologically confirmed gynecological diseases were grouped and retrospectively analyzed, including 50 cases of gynecological benign diseases, 35 cases of non-mucinous epithelial ovarian carcinoma, 36 cases of endometrial adenocarcinoma, 15 cases of gynecological benign diseases patients complicated with chronic renal deficiency, 15 cases of gynecological diseases without chronic renal deficiency, and 30 normal controls. Serum HE4 values in the ovarian cancer group, endometrial cancer group, gynecological benign diseases with chronic renal deficiency group, and chronic renal deficiency group were significantly increased compared with the benign gynecological diseases and normal control groups, showing a significant difference (P < 0.001). A comparison of 4 groups with high HE4 showed that the HE4 level in the 2 groups with renal deficiency were higher than those in the ovarian cancer and endometrial cancer groups, but the difference was not significant (P > 0.05); there was no significant difference between 2 groups with renal deficiency (P > 0.05). Serum concentration of HE4 was high in patients with chronic renal deficiency, which should be distinguished during differential diagnosis of gynecological benign and malignant tumors in patients with chronic renal deficiency to avoid misdiagnosis.

Folate receptor alpha (FRA) expression remains unchanged in epithelial ovarian and endometrial cancer after chemotherapy

ObjectiveBased on its expression profile, folate receptor alpha (FRA) is an attractive candidate for targeted diagnostics and therapeutics. However, applicability of these agents in residual or recurrent disease could be influenced by chemotherapy. We evaluated whether chemotherapy modified FRA expression in non-mucinous epithelial ovarian (EOC) and endometrial carcinoma (EC). MethodsFRA staining was evaluated by immunohistochemistry, using MAb 26B3, in 81 patients (41 EOCs and 40 ECs) and 17 control tissues (5 benign ovarian cysts, 5 normal ovarian, and 7 normal endometrial tissues). Chemotherapy effect was evaluated in 42 patients (30 paired samples at primary and interval debulking surgery and 12 from primary and recurrent disease). FRA expression was assessed using a semi-quantitative staining algorithm, the M-score (range 0–50). ResultsMedian difference in M-score between tumor and control samples was 27.5 for EOC (95% CI 10.0 to 45.0) and 6.7 for EC (95% CI −6.7 to 21.7). Paired samples from both primary and interval debulking surgery did not differ in FRA expression in EOC (median difference of M-score between paired samples of 0.0 [95% CI −2.6 to 2.6]). Recurrent EOC tumors reflected FRA status at diagnosis (median difference of M-score between paired samples of 3.3 [95% CI −7.0 to 13.6]). ConclusionsThis study shows no significant difference in FRA expression after chemotherapy, strengthening the rationale for FRA targeted diagnostics and therapeutics in FRA expressing tumors, whether newly diagnosed or at recurrence.

Folate Receptor-Mediated Drug Targeting: A Possible Strategy for Nonfunctioning Pituitary Adenomas?

Clinically nonfunctioning pituitary adenomas (NFPAs) mostly arise from gonadotroph cells and account for about one-third of all pituitary tumors. Because they are not associated with hormone hypersecretion, these tumors are often diagnosed late when they are large and locally invasive. Consequently, the complete surgical removal of NFPAs is hard to achieve and they tend to recur. Medical treatment of NFPAs is still challenging because they are usually resistant to mainstay peptide receptor therapy with somatostatin (SRIF) analogs (octreotide and lanreotide) or dopamine agonists (bromocriptine and cabergoline). Indeed, although these agents may be useful in some cases to prevent residual tumor growth (1), their efficacy has not been confirmed in larger studies. Recently, SOM230 (pasireotide), a broad-range SRIF analog with affinity for SRIF receptor subtypes 1 to 3 and 5 (2) and BIM-23A760 (dopastatin), a chimeric agent interacting with both SRIF receptor and dopamine receptors were developed (3). However, NFPAs only partially respond to these compounds, at least in vitro (4, 5). After the discovery that NFPAs display overactivation of phosphatidylinositol 3-kinase/protein kinase B (AKT)/mammalian target of rapamycin pathway (6, 7), compounds inhibiting this signaling cascade at various levels have been tested against these tumors. The results are very promising, especially for dual inhibitors such as BEZ235 and XL765, which inhibit both mammalian target of rapamycin and the upstream phosphatidylinositol 3-kinase and exert a potent antiproliferative activity against primary human NFPA cells (8), rodent pituitary cell lines (9), and primary rat gonadotroph adenoma cells in vitro (10). In vivo studies are now required to prove or disprove the efficacy of these agents. Conventional chemotherapy has been one of the major medical advances of the last decades, but it is usually associated with poor specificity for the cancer cells and high toxicity to the normal cells. To overcome these limitations, it would be ideal to identify ways to deliver a biologically effective concentration of anticancer agents to the tumor tissues with very high specificity. To reach this ultimate goal, extensive efforts have been undertaken to develop tumor-selective drugs by conjugating therapeutic agents to hormones, antibodies, and vitamin derivatives. Among them, folic acid holds great promise as a tumor-homing agent (11). Folate (or folic acid) is an essential B vitamin, which plays a pivotal role in cell survival by participating in the biosynthesis of nucleic and amino acids. Folate is internalized into the cells via a low-affinity reduced folate carrier protein or via high-affinity folate receptors. The best studied isoform of these receptors is folate receptor(FR ), a cell surface glycosyl phosphatidylinositol-anchored glycoprotein that can internalize bound folates and folate-conjugated compounds via receptor-mediated endocytosis (12). Whereas FR has a very restricted expression pattern in normal tissues, it is expressed at a high level in various human cancers, particularly epithelial carcinomas, including nonmucinous ovarian carcinoma, cervical carcinomas, and testicular choriocarcinomas, and less frequently in breast, endometrial colon, and renal cell carcinomas (13, 14). Due to its selective expression in tumor but not normal cells, FR has become one of the most investigated cellular surface antigens for targeted delivery of a variety of molecules, including imaging agents, chemotherapeutic agents, oligodeoxynucleotides, and macromolecules. Various types of drug carriers have been conjugated to folate such as liposomes, lipid nanoparticles, polymeric nanoparticles, polymers, and micelles filled with the molecule that needs to be delivered (11). FR -targeted liposomal

Germline BRCA1 and BRCA2 Mutations in Ovarian Cancer

To estimate the frequency of BRCA1 and BRCA2 germline mutations in women with nonmucinous epithelial ovarian carcinoma unselected for a family history of breast or ovarian cancer. From 2004 to 2009, women undergoing surgical staging for nonmucinous epithelial ovarian carcinoma, including fallopian tube and primary peritoneal carcinoma, were invited to participate in tumor banking and genetic counseling for BRCA1 and BRCA2 mutations. Pathology and family history obtained by the gynecologic oncology surgeon and genetic counselors were reviewed. Of 131 women fulfilling entry criteria, germline BRCA1 and BRCA2 mutations were found in 20% (26/131) and were exclusively associated with high-grade serous histology (26/103 [25%]). Restricting BRCA1 and BRCA2 testing to women with family histories of hereditary breast and ovarian cancer, as ascertained by the surgeon, missed 14 mutation carriers, lowering detection rates to 9% (12/131) or 11.6% (12/103) if only considering the patients with high-grade serous histology. This improved to 16% (21/131) or 20.4% (21/103) when ascertained by the genetic counselor; however, 5 of 26 (19%) mutation carriers did not have a family history of hereditary breast or ovarian cancer. Germline BRCA1 and BRCA2 mutations in ovarian (pelvic) cancer are associated with high-grade serous histology. The high incidence (25%) of germline BRCA1 and BRCA2 mutations specific to the high-grade serous subtype suggests that genetic assessment of all women diagnosed with high-grade serous ovarian (pelvic) carcinoma will improve detection rates and capture mutation carriers otherwise missed by referral based on family history alone. II.

BRCA Mutation Frequency and Patterns of Treatment Response in BRCA Mutation–Positive Women With Ovarian Cancer: A Report From the Australian Ovarian Cancer Study Group

The frequency of BRCA1 and BRCA2 germ-line mutations in women with ovarian cancer is unclear; reports vary from 3% to 27%. The impact of germ-line mutation on response requires further investigation to understand its impact on treatment planning and clinical trial design. Women with nonmucinous ovarian carcinoma (n = 1,001) enrolled onto a population-based, case-control study were screened for point mutations and large deletions in both genes. Survival outcomes and responses to multiple lines of chemotherapy were assessed. Germ-line mutations were found in 14.1% of patients overall, including 16.6% of serous cancer patients (high-gradeserous, 17.1%); [corrected] 44% had no reported family history of breast orovarian cancer.Patients carrying germ-line mutations had improved rates of progression-free and overall survival. In the relapse setting, patients carrying mutations more frequently responded to both platin- and nonplatin-based regimens than mutation-negative patients, even in patients with early relapse after primary treatment. Mutation-negative patients who responded to multiple cycles of platin-based treatment were more likely to carry somatic BRCA1/2 mutations. BRCA mutation status has a major influence on survival in ovarian cancer patients and should be an additional stratification factor in clinical trials. Treatment outcomes in BRCA1/2 carriers challenge conventional definitions of platin resistance, and mutation status may be able to contribute to decision making and systemic therapy selection in the relapse setting. Our data, together with the advent of poly(ADP-ribose) polymerase inhibitor trials, supports the recommendation that germ-line BRCA1/2 testing should be offered to all women diagnosed with nonmucinous, ovarian carcinoma, regardless of family history.

Abstract 2516: Immunohistochemical characterization of a novel, highly sensitive monoclonal antibody to folate receptor α

Abstract Background: Folate receptor ≤ (FRα) has restricted expression in normal tissues but high-level expression in a subset of epithelial-derived tumors, including non-mucinous ovarian carcinomas and non-small cell lung (NSCL) adenocarcinomas. FRα has shown significant potential for targeted cancer therapy and several promising agents are in late-stage clinical development. As such, robust reagents are needed that can be used across diverse diagnostic platforms to identify patients who have FRα-expressing tumors. The immunohistochemical (IHC) characterization of a novel, high-affinity monoclonal antibody (MAb) against FRα is described. Methods: MAbs were generated to modified full-length FRα protein using standard methods. One MAb, 26B3.F2, displayed high affinity and the ability to bind FRα on both fresh/frozen and formalin-fixed, paraffin-embedded tissue sections. An optimized IHC protocol was developed and FRα expression was assessed on commercial tissue microarrays (US Biomax). Data were analyzed using an M-score, a newly introduced, weighted-intensity score that incorporates both the proportion of FRα-positive cells and the staining intensity. A board-certified pathologist scored the level of FRα expression. Results: Normal tissue staining for MAb 26B3.F2 demonstrated a limited distribution on focal epithelial surfaces of pancreas, lung, salivary gland, kidney, hypophysis, thyroid, and breast samples. Staining was mainly located in the lumen. The tissue distribution is consistent with the published literature. Strong staining was observed in 100% of serous ovarian samples and a significant proportion of ovarian endometrioid adenocarcinoma samples. FRα expression, as determined by MAb 26B3.F2 in NSCL cancer samples, demonstrated high discrimination between adenocarcinoma (positive) and squamous cell carcinomas (negative) (P &amp;lt; 0.001). Conclusion: MAb 26B3.F2 is a highly sensitive, robust agent suitable for identifying FRα expression by IHC and other diagnostic platforms. In addition to the expected distribution in normal tissue, MAb 26B3.F2 revealed high-level expression of FRα in ovarian cancer and NSCL adenocarcinoma samples, and the ability to discriminate differential expression between two NSCL cancer subtypes, squamous cell carcinoma and adenocarcinoma. Preliminary evidence therefore suggests that MAb 26B3.F2 IHC will be a useful tool to support further development of FRα-targeted therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2516. doi:1538-7445.AM2012-2516

Expression of Pax8 as a Useful Marker in Distinguishing Ovarian Carcinomas From Mammary Carcinomas

The ovary is a common site of involvement for metastasis and the breast is one of the most common sources. Metastatic breast carcinoma can mimic a primary ovarian carcinoma. Pax8 is a crucial transcription factor for organogenesis of the thyroid gland, kidney, and Müllerian system, and it also regulates Wilms tumor suppressor gene (WT1) expression. A total of 124 cases of ovarian carcinomas (84 serous papillary, 18 endometrioid, 12 mucinous, 10 clear cell) and 243 cases of invasive breast carcinomas (178 ductal, 65 lobular) were immunostained with Pax8 and WT1 by tissue microarrays to see the differential expression. Pax8 reaction was found in 108 of 124 ovarian carcinomas (87.1%) generally in diffuse staining, including 81 of 84 serous papillary carcinomas (96.4%), 16 of 18 endometrioid carcinomas (88.9%), 10 of 10 clear cell carcinomas (100%), and 1 of 12 mucinous carcinomas (8.3%), whereas WT1 expression was seen in 78 of 124 ovarian carcinomas (62.9%), including 73 of 84 serous papillary carcinomas (86.9%), and 5 of 18 endometrioid carcinomas (27.8%), with no expression in all 10 clear cell carcinomas and 12 mucinous carcinomas. All the mammary carcinomas were completely negative for Pax8, but WT1 expression was seen in 5 of 243 cases (2.1%). Pax8 is a useful marker in the differential diagnosis of ovarian and breast carcinomas, and it seems to be superior to WT1 for the diagnosis of all types of nonmucinous ovarian carcinomas, notably clear cell and endometrioid types where WT1 expression is generally negative or only focal.

Flow cytometric method for determining folate receptor expression on ovarian carcinoma cells

The alpha-folate receptor (alpha-FR) is a folate transporter with restricted expression levels in normal tissues. It is over-expressed in several cancers, particularly epithelial carcinomas, including nonmucinous ovarian carcinoma. It offers a novel therapeutic target for selective imaging and cytotoxic agents. Measurement of the receptor could be a valuable tool in selecting patients more likely to respond to new drugs that target the alpha-FR, and monitoring them while on treatment. While tumor samples are often unavailable, a number of patients who relapse develop ascites, which are often rich in tumor cells. We have therefore developed a triple antibody flow cytometric method to assess alpha-FR expression on tumor cells from ascites. An antibody to BerEP4, an epithelial cell marker expressed on >90% ovarian cancers, labeled with fluorescein, and an alpha-FR antibody labeled with antimouse-phycoerythrin have been used to label tumor cells, with a CD45-phycoerythrin-cyanine5 antibody used to exclude white blood cells from the analysis. The method was optimized using human carcinoma cell lines (JEG-3, IGROV-1, and KB cells). Calibrated beads were used to quantify the number of antibodies bound per cell. The triple antibody protocol successfully measured alpha-FR expression levels in cell lines spiked with blood. Tumor cells were obtained from ascites in 25 patients with relapsed ovarian cancer. In each case sufficient cells were harvested to identify an epithelial cell population to estimate the number of binding sites/cell. All the samples contained a single population of BerEP4, alpha-FR positive cells between 5x10(3) and 5x10(5) antibody binding sites/cell. The method can be used to determine the number of anti-alpha-FR antibodies bound per epithelial cell in ascites from patients with ovarian carcinoma. The results obtained were reproducible and the method could be applied to specimens that had been stored at -80 degrees C.

Application of Mesothelin Immunostaining in Tumor Diagnosis

Mesothelin is a differentiation antigen that was first described as the antigenic target of the monoclonal antibody K1. Using this antibody, it was demonstrated that mesothelin is strongly expressed in normal mesothelial cells, mesotheliomas, nonmucinous ovarian carcinomas, and some other malignancies. Immunostaining with the K1 antibody was suggested to be useful in the diagnosis of mesothelioma in the early 1990s. This, however, could not be further explored until recently because of the lack of commercially available anti-mesothelin antibodies. In a recent investigation by this author, all epithelioid mesotheliomas and about 40% of the lung adenocarcinomas reacted with the 5B2 anti-mesothelin antibody, which has only recently become commercially available. It was concluded that immunostaining with this antibody has limited value in discriminating between these conditions. The aim of the current study was to further investigate the potential application of the 5B2 antibody in tumor diagnosis. Mesothelin expression was evaluated in formalin-fixed, paraffin-embedded samples of normal tissues and in 471 tumors of various origins. The carcinomas that most frequently exhibited strong mesothelin reactivity were nonmucinous carcinomas of the ovary (14 of 14 serous, 3 of 3 endometrioid, 6 of 8 clear cell, and 4 of 4 transitional cell carcinoma), and adenocarcinomas of the pancreas (12 of 14), the ampulla of Vater (3 of 3), endometrium (7 of 11), lung (14 of 34), and liver (7 of 19 cholangiocarcinomas). The carcinomas that did not express mesothelin included renal cell carcinomas, hepatomas, carcinomas of the thyroid, adrenal cortical carcinomas, prostatic adenocarcinomas, and carcinoid tumors. All germ cell tumors, with the exception of teratomas, were consistently negative for mesothelin. Because of the strong mesothelin expression in nonmucinous carcinomas of the ovary, but not in a variety of tumors with which these lesions may be confused (eg, clear cell carcinoma of the ovary versus endodermal sinus tumor or renal cell carcinoma, clear cell type; transitional cell carcinoma of the ovary versus TCC of the urinary tract), immunostaining for this marker could be useful in establishing the differential diagnosis. The strong mesothelin expression in the large majority of pancreatic ductal adenocarcinomas (12 of 14), but not in normal pancreas, confirms that this marker may have some diagnostic utility in discriminating between neoplastic and nonneoplastic pancreatic ductal epithelium. The mesothelin expression in about one-third of the cholangiocarcinomas, but not in hepatomas, suggests that this marker may have some utility in distinguishing between these two malignancies when they are poorly differentiated. In the group of small round blue cell tumors, only desmoplastic small round cell tumors exhibited mesothelin positivity (7 of 12). Of the soft tissue tumors, only the epithelial component of biphasic synovial sarcomas (9 of 9) expressed mesothelin. These findings indicate that, in some instances, mesothelin immunostaining can assist in the diagnosis of these tumors. Finally, the strong mesothelin reactivity seen in the adenomatoid tumors (3 of 3) provides further support for a mesothelial derivation for this lesion.

Reversion of transformed phenotype in ovarian cancer cells by intracellular expression of anti folate receptor antibodies.

The alpha-folate receptor (FR) is selectively overexpressed in 90% of nonmucinous ovarian carcinomas, whereas no expression is detectable in normal ovarian surface epithelium (OSE). Indirect evidence suggests that FR expression is associated with tumor progression and affects cell proliferation. To evaluate better the role of FR, we developed an approach based on intracellular expression of single-chain (sc) antibodies (intrabody) to downmodulate membrane expression of FR in ovary cancer cells. IGROV-1 and SKOV3 ovarian carcinoma cell lines were transfected with an anti-FR intrabody. Transfectants and parental cells were tested for FR, integrins and anti-FR intrabody expression by fluorescence-activated cell sorting (FACS), reverse transcription and polymerase chain reaction (RT-PCR) and/or immunoblotting. Cell growth characteristics and adhesion properties were evaluated in liquid, semisolid and organotypic cultures. The anti-FR scFv inhibited FR expression from 60 to 99%. At physiological concentrations of folate, proliferation varied directly as a function of FR expression. FR downmodulation was accompanied by reduced colony-forming ability in soft agar, morphological change of the cells, significant enhanced adhesion to laminin or Matrigel, a two- to three-fold increase in alpha6beta4 integrin expression, and a marked reduction in laminin production. In three-dimensional organotypic cultures, anti-FR intrabody-transfected IGROV1 cells grew as a single-ordered layer, reminiscent of normal OSE growth in vivo. In conclusion, the anti-FR intrabody reverses the transformed phenotype in ovary cancer cells and may provide an efficient means to inhibit selectively the growth of these cells.

Intraperitoneal cancer dissemination: mechanisms of the patterns of spread.

Well known patterns govern the distribution of hematogenous and lymphatic metastasis of cancer. In the past the distribution of cancer cells free within abdominal cavity received little attention and was thought to be a random event. However, surgical observation led the authors to generate and test hypotheses regarding patterns of spread that vary with tumor type, with the intraperitoneal environment, and with the physiology of the peritoneal surface tissues. The distribution and volume of peritoneal surface malignancy was prospectively recorded in 129 patients with 5 different types of tumors at the time of cytoreductive surgery. The malignancies studied included pseudomyxoma peritonei (PMP) of appendiceal origin, colonic mucinous adenocarcinoma (MA), nonmucinous colonic adenocarcinoma (NMA), ovarian carcinoma (OV) and sarcoma (SA). The abdominal and pelvic cavity was divided into 3 horizontal sectors, 9 regions and 25 sites. The incidences of tumor implants in these designated areas were statistically analyzed for each tumor type and comparisons between tumor types studied. The magnitude of intraperitoneal cancer dissemination was similar for mucinous tumors, including PMP and MA and significantly higher than for non-mucinous tumors. Also the mucinous cancers were more likely to be present in the upper horizontal sector than were non-mucinous. When NMA was compared to PMP and MA the epigastric region was significantly less likely to contain tumor. For all cancer diagnoses the colon, greater omentum and cul-de-sac of Douglas were most often affected. The ileocecal valve region was more likely to have large tumor masses on its surface than small bowel surface or small bowel mesentery. Peritoneal carcinomatosis had a wider distribution when mucinous fluid was present; this cancer distribution by intraperitoneal fluid hydrodynamics occurred regardless of histologic aggressiveness. The organs that have peritoneal fluid resorption (omentum and omental appendages) have a high incidence of implants. Small bowel and its mesentery free to move by peristalsis had a reduced incidence of implants as compared to the ileocecal area, which is fixed to the retroperitoneum. These observations may facilitate efforts to treat peritoneal surface malignancy.

The effect of ascites, mass volume, and peritoneal carcinomatosis on serum CA125 levels in patients with ovarian carcinoma

The aim of this study is to investigate the effects of ascites, ovarian mass volume, and peritoneal carcinomatosis on serum CA125 levels in patients with nonmucinous epithelial ovarian carcinoma. Serum CA125 levels were determined by a commercial enzyme immunoassay kit in a series of 98 patients with stage I-IV nonmucinous epithelial ovarian carcinoma. Amounts of ascites were determined in each patient with ascites. Ovarian mass volumes were calculated in 22 patients with stage I disease without ascites. Peritoneal carcinomatosis was detected in 35 ovarian cancer patients. Serum CA125 levels were compared among the patients with different clinical conditions.Serum CA125 levels were significantly higher in cases of ovarian cancer with ascites when compared with those without ascites (P&lt; 0.01). Abnormal levels of serum CA125 were found in 80% of all patients; these abnormal levels were detected in 92% and 97% of the patients with ascites and with peritoneal carcinomatosis, respectively. A positive correlation between serum CA125 levels and ascites amounts was found in patients with ascites (P&lt; 0.01,r= 0.74). However, there was no correlation between ovarian mass volumes and levels of serum CA125 in patients having stage I disease but no ascites (P= 0.5,r= 0.15). Our results showed that serum CA125 levels might be affected by the amount of ascites and the presence of peritoneal carcinomatosis but not with ovarian mass volume. However, these findings need to be confirmed in more and larger studies. These results may be beneficial in the management of ovarian carcinoma patients with elevated CA125 levels.

Ovarian mixed-epithelial carcinomas with a microcystic pattern and signet-ring cells.

Primary ovarian carcinomas with unusual histologic patterns can be difficult to differentiate from metastases. In this study, we reviewed 15 cases of mixed-epithelial carcinoma (12 serous, 1 serous and endometrioid, 1 endometrioid, 1 undifferentiated) with a predominant microcystic pattern and signet-ring cells. The patients' ages ranged from 31 to 78 (mean 58) years. The microcystic component in 11 patients had features of high-grade carcinoma and in 4 patients had features of low-grade carcinoma associated with areas of borderline tumor. The tumors in all 15 patients showed a predominant microcystic growth pattern composed of small cysts that were variable in size and shape. Signet-ring cells were also present in all cases (diffusely in nine cases, focally in six cases) within the neoplastic epithelial proliferation. Mucin was present in the lumina of some of the microcysts and in the cytoplasm of most of the signet-ring cells. A microcystic pattern and mucin-containing signet-ring cells can be seen as small foci or as a predominant component in primary epithelial nonmucinous ovarian carcinomas. It is important for pathologists to recognize these unusual findings in ovarian neoplasms, because they may produce a confusing apperance, even potentially suggesting a metastasis.

Functional effect of point mutations in the alpha-folate receptor gene of CABA I ovarian carcinoma cells.

The alpha-folate receptor (alpha FR) is overexpressed in 90% of nonmucinous ovarian carcinomas. In addition to the known role of alpha FR binding and mediating the internalization of folates, functional interaction of alpha FR with signaling molecules was recently shown. To identify a model to study the role of alpha FR in ovarian carcinoma, we characterized the alpha FR gene in the ovarian carcinoma cell line CABA I in comparison to a reference line, IGROV1. In CABA I cells, Northern blot analysis revealed an alpha FR transcript of the expected length and FACS analysis indicated receptor expression on the cell membrane; however, RNase protection assay revealed no specific signals. Southern blot and genomic PCR analysis suggested the presence of a rearrangement(s) involving the 5' region of the gene in CABA I cells as compared to IGROV1 cells. Cloning and sequencing of CABA I alpha FR cDNA revealed several point mutations. The partitioning of alpha FR in membrane microdomains from CABA I cells and its association with regulatory molecules was comparable to that of IGROV1 cells. By contrast, the alpha FR expressed on the CABA I cell membrane bound folic acid with lower affinity, and ectopic expression of the corresponding cDNA in CHO cells confirmed impaired folic acid binding. Thus, CABA I cells may provide a tool to delineate functional domains of the alpha FR.