Selection of carbohydrate antigens in human epithelial ovarian cancers as targets for immunotherapy: serous and mucinous tumors exhibit distinctive patterns of expression.

Expression of blood group-related carbohydrate antigens was examined in frozen sections from a series of ovarian carcinomas of different histological types using an indirect immunoperoxidase technique. Antigenic specificities belonging to the O(H) and Lewis blood group families (H-1, H-2, Le(a), sLe(a), Le(x), sLe(x), Le(b) and Le(y)) or the mucin-core family (Tn, sTn and TF) were studied. A distinct difference in antigen expression between mucinous and other ovarian carcinomas (serous and endometrioid) was observed. Specifically, mucinous tumors tended to express sTn, Le(a) and sLe(a) strongly and homogeneously, whereas serous and endometrioid tumors rarely expressed these specificities and, in contrast, expressed Le(y) and H type 2 antigen strongly. When expressed in serous tumors, sTn was usually distributed in a heterogeneous pattern, whereas sTn expression in mucinous tumors was much more homogeneous. The distribution of Le(y) in serous tumors was noticeably homogeneous. H-1, Le(x), sLe(x), Le(b), TF and Tn specificities were rarely expressed in any type of ovarian carcinoma. Our results provide further support for the different histogenesis of mucinous and non-mucinous tumors and indicate alternative differentiation pathways for the 3 pathological subtypes of ovarian tumor. They also provide the basis for the choice of carbohydrate antigens for active and passive immunotherapy of ovarian carcinomas.

Aminopeptidase A expression and enzymatic activity in primary human renal cancers.

The gp160 human kidney differentiation antigen is identical to human aminopeptidase A (APA), a zinc-dependent cell-surface metallopeptidase which hydrolyzes peptides with N-terminal acidic residues. GP160/APA is constitutively expressed by proximal tubule cells, the normal cellular counterpart of most renal cancers (RCs). Immunohistochemical analysis of gp160/APA protein expression in 62 primary renal tumor specimens using monoclonal antibody S4 revealed heterogeneous or homogeneous expression of gp160/APA in 46/51 (90%) of clear cell carcinomas in contrast with 1/8 (13%) papillary renal tumors and 0/3 oncocytomas (p<0.001). Analysis of five primary clear cell carcinomas for gp160 protein expression immunohistochemically and associated APA catalytic activity revealed one tumor which expressed gp160/APA protein which was enzymatically inactive. Direct sequence analysis of DNA derived from this specimen could not detect mutations within the zinc-binding domain which would eliminate gp160/APA catalytic activity. These data indicate that the gp160/APA protein is expressed by primary clear cell but not papillary RCs or oncocytomas, and that alterations in gp160/APA protein including loss of protein expression or enzymatic activity occur in 20% of primary clear cell RCs.

Neutral endopeptidase 24.11 loss in metastatic human prostate cancer contributes to androgen-independent progression.

Neutral endopeptidase 24.11 (NEP) is a cell-surface enzyme expressed by prostatic epithelial cells that cleaves and inactivates neuropeptides implicated in the growth of androgen-independent prostate cancer (PC). We report that NEP expression and catalytic activity are lost in vitro in androgen-independent but not androgen-dependent PC cell lines. In vivo, NEP protein expression is commonly decreased in cancer cells of metastatic PC specimens from patients with androgen-independent but not androgen-dependent PC. Overexpression of NEP in androgen-independent PC cells or incubation with recombinant NEP inhibits PC cell growth. Furthermore, in androgen-dependent PC cells, expression of NEP is transcriptionally regulated by androgen and decreases with androgen withdrawal. These data suggest that decreased NEP expression, common in androgen-independent PCs, is facilitated by the elimination of androgens, and that NEP loss plays an important role in the development of androgen-independent PC by allowing PC cells to use mitogenic neuropeptides as an alternate source to androgen in order to stimulate cell proliferation.

Reversal of the low-affinity neurotrophin receptor stromal-epithelial expression pattern between benign and malignant human prostate.

Reduced expression of the low-affinity p75 neurotrophin receptor (p75(NTR)) occurs in prostate epithelial cells during malignant transformation. Recent studies indicating that the p75(NTR) can transduce signals that induce apoptosis suggest that diminished p75(NTR) in transformed prostate cells may contribute to immortalization. Mutations in the transmembrane domain of the p75(NTR) gene have been associated with decreased p75(NTR) protein expression and may block the ability of the p75(NTR) to induce apoptosis. Therefore, we used Western blot to analyze prostate cancer (PC) cell lines for p75(NTR) protein expression and gene single strand conformation polymorphism (SSCP) analysis and direct DNA sequencing to analyze mutations in the transmembrane domain of the p75(NTR). p75(NTR) Protein was present in all cell lines, and mutations in the p75(NTR) gene were not detected in cDNA derived from any cell line. To define the expression pattern of p75(NTR) in PCs in vivo, we used immunohistochemical techniques to examine tissue specimens from 20 benign, 19 malignant primary, and 14 metastatic prostate specimens. In benign prostate tissues, expression of p75(NTR) was universally detected in basal cells but not in secretory epithelial or stromal cells. In both primary and metastatic PC tissues, p75(NTR) immunoreactivity could not be detected in malignant prostate epithelial cells. However, in contrast to the benign prostate, p75(NTR) protein was expressed in stromal cells surrounding malignant epithelial cells. Stromal p75(NTR) expression was present in 84% (16 of 19) primary and in 86% (12 of 14) metastatic specimens. These data show that in the benign prostate p75(NTR) protein is expressed by basal cells and not stromal cells whereas in malignant prostate p75(NTR) protein is expressed by stromal cells but not prostatic carcinoma cells. Reversal of the p75(NTR) stromal-epithelial pattern of expression between benign and malignant prostate suggests that p75(NTR) may contribute to the development and maintenance of prostate cancer.

Expression and localization of aminopeptidase A, aminopeptidase N, and dipeptidyl peptidase IV in benign and malignant human prostate tissue.

BACKGROUND: Cell-surface peptidases are ectoenzymes which regulate the access of bioactive peptides to their receptors on cell membranes. Abnormalities in their expression and function result in altered peptide activity which contribute to neoplastic transformation and/or progression. METHODS: Expression of aminopeptidase A (APA), aminopeptidase N (APN, CD13), and dipeptidyl peptidase IV (DPP IV, CD26) was immunohistochemically examined in 20 benign and 33 malignant prostate tissues (19 primaries and 14 metastases). RESULTS: Benign prostatic stroma exhibited no APA, APN, or DPP IV immunoreactivity. Stromal cells surrounding prostatic carcinoma cells demonstrated increased APA expression in 24/33 (73%) of tumors. Benign prostatic epithelial cells strongly expressed APN and DPP IV but not APA. In contrast, APN was expressed in > 80% of tumor cells in 5/33 (15%) of specimens, heterogeneously expressed (20-80% of cells positive) in 4/33 (12%) of specimens, and minimally expressed or absent in 24/33 (73%) of tumor specimens, with a similar pattern of expression in primary and metastatic tumors. DPP IV was expressed by > 80% of tumor cells in 18/19 (95%) of primary prostate cancer specimens, but in only 7/14 (50%) of metastases. CONCLUSIONS: These data show that cell-surface peptidases are differentially expressed by normal prostatic stromal and epithelial cells, with increased expression of APA in the stroma surrounding prostate cancer cells, absent APN expression in most tumor cells, and a decreased frequency of DPP IV expression in metastatic tumors. Further studies will elucidate the biological effects of the presence or loss of cell-surface peptidases in the benign and malignant prostate.

A case-case analysis of factors related to overexpression of p53 in endometrial cancer following breast cancer.

We studied 54 patients diagnosed with endometrial cancer between 1981 and 1994 following a diagnosis of breast cancer. We used a case-case analysis, comparing tumors with and without overexpression of the p53 gene product to evaluate the association of putative p53 mutations with tamoxifen use and other risk factors for endometrial cancer. Twenty-four % of the tumors showed strong positive staining for the p53 gene product. Tumors in a more advanced stage (stage 2, 3, or 4, compared to stage 1) were more likely to overexpress p53 [odds ratio (OR) = 4.2; 95% confidence interval (CI), 1.1-16.2], as were tumors with serous or clear cell, compared to endometrioid, histology (OR = 5.8; 95% CI, 1.3-26.5). There was a small association between p53 overexpression and treatment with tamoxifen for breast cancer (OR = 2.6; 95% CI, 0.69-9.8). There was a strong relationship between overexpression of p53 and having a first-degree relative with breast cancer (OR = 12.3; 95% CI, 2.6-57.4) and between overexpression of p53 and having an additional cancer, i.e., at sites other than breast or endometrium (OR = 7.9; 95% CI, 1.6-40.1). In this group of women, genetic predisposition to cancer, as reflected in family history of breast cancer and personal history of an additional primary cancer, was strongly associated with overexpression of p53 in endometrial tumors. The results suggest that use of tamoxifen may be associated with an increase in tumors that overexpress p53, although the results could be due to chance.

Distribution of radiolabeled monoclonal antibody MX35 F(ab')2 in tissue samples by storage phosphor screen image analysis: evaluation of antibody localization to micrometastatic disease in epithelial ovarian cancer.

Our objective was to quantify the targeting of the monoclonal antibody (mAb) MX35 F(ab')2 to micrometastatic epithelial ovarian cancer. This mAb detects a Mr 95,000 glycoprotein with homogeneous distribution on 80% of ovarian tumor specimens. Six patients with minimal residual disease from an imaging trial were injected with 2 or 10 mg of 131I- and 125I-labeled mAb MX35 F(ab')2. Biopsied samples were removed at second-look laparotomy 1-5 days post-i.v. or -i.p. infusion of antibody. Serial cryostat sections were stained by indirect immunoperoxidase method for antigen distribution and exposed to storage phosphor screens for quantitative autoradiography. Coregistration of tumor histology, antigen expression, and radionuclide distribution demonstrated specific localization in micrometastatic tumor foci (50 micrometer to 1 mm) found within tissue stroma. The radiolabeled antibody uptake determined by well scintillation counts ranged between 5.2 and 223.5 x 10(-4) percentage of injected dose/g of tumor tissue for 131I. Specific localization of mAb in tumor was determined by tumor:normal tissue (fat) ratios ranging from 0.9:1 to 35.9:1 for 131I. The high resolution and linear response of the storage phosphor screen imager was used to estimate the radionuclide activity localized in each micrometastatic site. Quantitation of phosphor screen response revealed microCi/g values of 0.026-0.341 for normal tissue and 0.184-6.092 for tumor biopsies, evaluated 4 or 5 days post-antibody injection. The tumor:normal tissue (adjacent to tumor) ratios were between 1 and 4 times greater using the phosphor screen method than well counter measurements, but even larger variations of ratios up to 20:1 were observed between tumor cell foci and stromal cells within the same tissue section. This study has demonstrated that mAb MX35 F(ab')2 localizes to the micrometastatic ovarian carcinoma deposits within the peritoneal cavity. The dosimetry results suggest a therapeutic potential for this antibody in patients with minimal residual disease (<5 mm).

Comparison of MUC-1 mucin expression in epithelial and non-epithelial cancer cell lines and demonstration of a new short variant form (MUC-1/Z).

Mucins, including MUC-1, are generally considered to be products of epithelial tissues and of their tumors. To examine the possible expression of MUC-1 in other cell types, a panel of human epithelial and non-epithelial tumor cell lines was studied by reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis, immunocytology and radioimmunoprecipitation. Using the highly sensitive RT-PCR method, products corresponding to the non-repetitive 5' and 3' MUC-1 sequences were detected in all the cell lines examined. Amplified products lacking the tandem repeat region of MUC-1, including a new short form (designated MUC-1/Z) different from the previously reported MUC-1/Y protein, were also detected in most cell lines tested. Northern blot analysis, using a probe to the variable number tandem repeat (VNTR) region, confirmed the presence of MUC-1 mRNA in the astrocytoma, melanoma and neuroblastoma cell lines studied. MUC-1 protein was detected by immunocytology in these cell lines using monoclonal antibody (MAb) 139H2. Immunoprecipitation analysis with [3H]-glucosamine-labeled cell lysates and MAb 139H2 or an antibody to the cytoplasmic domain, CT-1, detected MUC-1 protein in 2 epithelial cell lines, an astrocytoma cell line (SK-MG-4) but not in the melanoma and neuroblastoma cell lines studied. Northern blot analysis using a probe to the 3' end of MUC-1 mRNA, confirmed the presence of MUC-1 mucin and also identified short products corresponding to the size of the short variant forms. Protein products corresponding to the MUC-1/Y and MUC-1/Z variant forms were not observed using either [3H]-glucosamine-labeled OVCAR-3 cells or [3H]-amino acid-labeled MCF-7 cells and either CT-1 antibody or MAb 232A1, detecting an epitope to the C-terminal region. Thus, depending on the sensitivity of the assay used, varying amounts of MUC-1 mRNA and protein could be detected in non-epithelial tumor cell lines. Although the amounts of MUC-1 in these cell lines are much lower than in carcinomas, it is possible that MUC-1 mucin serves a similar function in non-epithelial as in epithelial cells. The possible role of MUC-1/Y and MUC-1/Z variant forms in these cell lines is not understood.

DNA analysis of human cholesteatomas.

HYPOTHESIS: The hypothesis tested in this article is that if cholesteatomas are a low-grade squamous cell neoplasm, then evidence of genetic instability, in the form of abnormal or aneuploid amounts of DNA, should be evident. BACKGROUND: Cholesteatoma is a destructive lesion of the middle ear and/or mastoid process that produces complications by erosion of the temporal bone. The clinical hallmarks of cholesteatomas, namely invasion, migration, uncoordinated proliferation, altered differentiation, aggressiveness, and recidivism, are traits typically associated with the neoplastic cell. However, there is little evidence to support or refute the speculation that cholesteatomas are a low-grade squamous cell neoplasm. the existence of defects in the genetic complement of the major cellular constituents comprising a cholesteatoma, fibroblasts and keratinocytes, would support the speculation that cholesteatomas are a neoplasm, since cancers commonly manifest quantitative and qualitative alterations in the normal euploid complement of genetic information, resulting in a cell that has an abnormal or aneuploid amount of DNA. METHODS: DNA content (ploidy) within cholesteatoma tissues was measured by flow cytometry and image analysis. RESULTS: The DNA content of 11 human cholesteatomas and nine postauricular skin specimens was analyzed using flow cytometry, while the DNA content of 10 cholesteatoma specimens was analyzed using image analysis. Interpretable data was obtained from 10 cholesteatoma specimens and six postauricular skin specimens. One cholesteatoma specimen demonstrated an abnormal aneuploid DNA content, whereas the remaining nine cholesteatomas and the six postauricular skin specimens demonstrated a normal euploid DNA content. CONCLUSIONS: We conclude that, due to the lack of overt genetic instability, as evidenced by the presence of a normal euploid DNA content, cholesteatomas are not low-grade neoplasms.

Altered regulation of cell surface peptidases in human cholesteatoma.

Cholesteatoma is a destructive process involving an accumulation of desquamated keratin arising from squamous epithelium that pathologically has invaded the middle ear or mastoid process. The clinical hallmarks of cholesteatomas, namely invasion of healthy tissues, migration, unrestrained proliferation, aggressiveness, recidivism, and uncoordinated differentiation predict the existence of defects in the normal biology and biochemistry of the cellular constituents that compose a cholesteatoma, as well as in the cellular interactions between these cells, the surrounding normal tissue, and the host. In the current report, we analyzed 11 cholesteatomas and matched healthy tissue for altered expression in four different cell surface peptidases, aminopeptidase A, aminopeptidase N, dipeptidyl peptidase IV, and neutral endopeptidase. We suggest that peptidases may modulate cell growth and differentiation by inactivating stimulatory signals (or conversely, by activating inhibitory signals).

Expression of A, B, and H blood group antigens in epithelial ovarian cancer: relationship to tumor grade and patient survival.

The expression of A, B, and H blood group antigens in epithelial ovarian cancer was evaluated in 137 patients with advanced disease by staining frozen sections with specific monoclonal antibodies using an indirect immunoperoxidase method. Expression of blood group antigens was observed in a proportion of ovarian carcinomas and in some areas of ovarian surface epithelium. Forty-eight percent of the tumors tested from 130 blood group A, B, or 0 individuals showed no expression of the appropriate blood group antigen, 32% had heterogeneous antigen expression, and 20% had strong expression. In the 7 blood group AB patients studied, no expression, heterogeneous expression of both antigens, or absence of one, but not the other antigen, was observed. No tumor showed A, B, or H antigen expression that was not compatible with the patient's blood group type. Histologic Grade 3 tumors showed absence of blood group antigen expression more often than did Grade 2 tumors. The presence or absence of A, B, or H antigen expression did not correlate with survival in this group of patients. This is in contrast to studies in other epithelial tumor types in which the normal epithelium synthesizes blood group antigens and loss of ABH antigen expression is observed in the corresponding tumors.

Serological and immunochemical analysis of Lewis y (Ley) blood group antigen expression in epithelial ovarian cancer.

The expression of Ley blood group antigen in epithelial ovarian cancer tissues and cell lines has been studied using a Ley-specific monoclonal antibody (MAb 3S193). In ovarian cancer specimens, Ley was expressed in 75% of the 140 tumor specimens examined, with strong or moderate expression being observed in 56% of the samples. Seven of the 11 ovarian cancer cell lines studied were Ley-positive. Using immunochemical approaches, Ley epitopes were found to be expressed on 4 types of carrier molecules: CA125 ovarian cancer antigen, MUC-1 mucins, lower m.w. glycoproteins and glycolipids. In cell lines, Ley was more commonly expressed on MUC-1 mucin than on CA125, whereas in tumor specimens Ley was commonly found on both CA125 and MUC-1. The biochemical nature of the smaller Ley glycoproteins was not determined, but it was shown that they were not CEA and LAMP-1, known Ley carriers in some other tumor types. Glycolipids carrying Ley epitopes were detected in both ovarian cancer cell lines and tumor specimens. The presence of Ley epitopes on a number of different molecular carriers, including 2 major ovarian cancer antigens (CA125 and MUC-1), explains the high incidence of Ley in ovarian cancer. The high expression of Ley in ovarian cancer and the availability of specific murine and humanized MAbs make Ley an attractive candidate target for clinical studies.

Major histocompatibility complex class I and class II expression in renal cell carcinoma and modulation by interferon gamma.

PURPOSE: To determine the expression of MHC class I and II in human renal cancer. MATERIALS AND METHODS: We analyzed tissue sections from 22 primary and 28 metastatic renal cell carcinomas (RCC), as well as 31 established RCC cell lines. Tissue specimens from normal kidney and cell cultures of normal kidney epithelium were also studied. In addition, MHC antigen expression on RCC cell lines was assessed both before and after incubation with human recombinant interferon gamma (IFN-gamma). Antigen expression was determined by mixed hemadsorption, indirect immunofluorescence, fluorescence activated cell sorting (FACS) or immunoperoxidase staining using the monoclonal antibodies (mAbs) W6/32 (anti-MHC class I), mAbs NAMB-1 and BBM.1 (anti-beta-2 microglobulin), and mAbs L243 and 13-17 (anti-MHC class II) antibodies. Soluble beta-2 microglobulin in conditioned medium was measured by ELISA. RESULTS: Normal renal epithelial cells, both in vivo and in vitro, showed low level expression of class I antigens. Immunohistochemical staining for MHC class II was limited to some proximal tubular cells, while cultured renal tubular cells were uniformly class II negative. The tumor cell populations in all 22 primary and in 26 of 28 (93%) metastatic RC specimens consisted predominantly of class I positive cells. Half of the samples from primary and metastatic tumors were class II negative. Incubation of RCC cell lines with IFN-gamma enhanced the expression of MHC class I, beta-2 microglobulin and class II. The upregulation of MHC expression was time and dose dependent and associated with increased release of soluble beta-2 microglobulin. CONCLUSIONS: (i) Like normal kidney, virtually all primary human renal cell carcinomas express MHC class I antigens and retain this phenotype even during tumor progression and metastasis; (ii) class II expression on normal and RCC cells appears more limited but occurs frequently in both primary and metastatic lesions; and (iii) in most continuous RCC cell lines expression of MHC class I and II can effectively be stimulated by IFN-gamma. Since expression of MHC molecules might determine the immunogenicity of human RCC, its constitutive expression and augmentation could play an important role for the immunotherapy and prognosis of human renal cancer.

Interleukin-6 level in serum and ascites as a prognostic factor in patients with epithelial ovarian cancer.

BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine that can be produced by human ovarian cancer cells. Elevated IL-6 levels have been found in the serum and ascites of patients with ovarian cancer, but its role in this disease has not been clearly established. METHODS: The authors studied the relationship between IL-6 levels in serum and ascites, various tumor parameters, and survival in 70 patients with newly diagnosed, untreated epithelial ovarian cancer. Ascites and serum specimens were obtained at the time of initial surgery, and IL-6 levels were determined using the B9 bioassay. RESULTS: All patients underwent platinum-based chemotherapy after initial surgery. The median age of the group was 62 years (range, 28-87 years), and the median follow-up time was 13 months (range, 12-59 months). Significantly higher IL-6 levels were detected in patients' ascites (median, 49,612 pg/ml [range, < 1 to 680,330 pg/ml]) compared with serum (median, 10 pg/ml [range, < 1 to 1221 pg/ml]) (P < 0.0001). IL-6 levels in ascites correlated significantly with the volume of ascites (P < 0.0001) and nearly so with the size of tumor found at initial surgery (P = 0.05). Serum and ascites IL-6 levels did not correlate statistically with overall survival time, tumor stage, grade, histologic findings, residual tumor volume after debulking, and serum CA 125 levels. Although not statistically significant, patients who responded to chemotherapy tended to have lower ascites IL-6 levels (median, 21,102 pg/ml) compared with patients who did not respond to chemotherapy (median, 40,200 pg/ml). CONCLUSIONS: IL-6 is present in very high amounts in the ascites of patients with epithelial ovarian cancer. IL-6 levels in ascites correlated significantly with ascites volume and initial tumor size. IL-6 levels in ascites and serum did not correlate statistically with other tumor parameters or with survival time.

Prevalence and significance of HER-2/neu expression in early epithelial ovarian cancer.

BACKGROUND: Although expression of the HER-2/neu oncogene may be of some prognostic importance in advanced ovarian cancer, its role in early-stage disease has not been established. The current study examined the prevalence and significance of HER-2/neu expression in early epithelial ovarian cancer. METHODS: The authors analyzed the expression of HER-2/neu on frozen tumor specimens from 40 patients with early epithelial ovarian cancer using the indirect immunoperoxidase technique with monoclonal antibodies that detect epitopes on the extracellular domain of the HER-2/neu protein. All patients underwent comprehensive surgical staging. HER-2/neu expression was graded as negative, weak, moderate (1+ to 2+), or strong (3+). Complete clinical data and long-term follow up were available for all patients. RESULTS: The distribution of patients by stage was as follows: Stage IA, 6; IB, 0; IC, 14; IIA, 4; IIB, 6; IIC, 10. The mean patient age was 53 years. Fourteen patients had serous tumors; nine, endometrioid; eight, clear cell; eight, mucinous; and one, undifferentiated. Intratumoral heterogeneity of HER-2/neu expression was observed with most specimens. In eight specimens (20%), some areas of the tumor showed strong (3+) expression, beyond the level that can be seen in normal ovarian epithelium. Twenty-eight specimens (70%) showed moderate (1+ to 2+) staining, whereas four specimens (10%) showed negative or weak staining. At a mean follow-up time among surviving patients of 32 months, 15 patients (37%) have had cancer recurrence. No statistically significant relationship was found between HER-2/neu expression and survival, disease-free survival, stage, or grade. A significant increase was found in 3+ expression of HER-2/neu in clear cell tumors. CONCLUSION: Consistent HER-2/neu overexpression occurs infrequently in early ovarian cancer, making it unlikely that such overexpression is a general early event in ovarian carcinogenesis. HER-2/neu expression does not appear to be a strong prognostic marker in early epithelial ovarian cancer.

Biodistribution and intraoperative evaluation of radiolabeled monoclonal antibody MX35 in patients with epithelial ovarian cancer.

Murine monoclonal antibody (MAb) MX 35 shows strong homogeneous reactivity with more than 90% of epithelial ovarian cancers. Twenty-five patients with advanced ovarian cancer were entered into a clinical trial using 125I- or 131I-labeled MX 35 in doses of 2, 10, or 20 mg administered by intravenous (i.v.) or intraperitoneal injection. All patients underwent laparotomy at 7 to 20 days following MAb injection to assess tumor distribution, obtain biopsies of tumor and normal tissue, and evaluate the use of an intraoperative hand-held gamma-detecting device. Following i.v. injection, serum Mab half-life was 36 hr. Tumor biopsies obtained at surgery showed MAb accumulation of from 6.7 x 10(-3) to 4.0 x 10(-5)% injected dose/g of tissue. There was no correlation between absolute MAb accumulation in tumor and MAb dose administered. Regression analysis showed a correlation between MAb accumulation and the interval between MAb injection and surgery (P = 0.008). Specific localization of MAb in tumor was demonstrated by tumor:normal tissue ratios ranging from 2.3:1 to 34:1 (mean, 10.18:1). The tumor:normal tissue ratios were not significantly related to MAb dose, the level of immunohistochemical antigen expression, or the interval between MAb injection and surgery. Due to the relatively long serum half-life, mean tumor:serum ratios were only 1.53 following IV injection. This ratio did not correlate with MAb dose, days from injection, or antigen expression. There was an excellent correlation (P = 0.001) between MAb uptake, as measured by the intraoperative hand-held gamma counter, and direct gamma counting of excised tissues. MAb MX 35 localizes well to tumor in selected patients with ovarian cancer, and MAb uptake can be reliably quantitated in vivo with the hand-held intraoperative gamma counter.

Molecular cloning of the human kidney differentiation antigen gp160: human aminopeptidase A.

gp160 is a cell surface differentiation-related glycoprotein of 160 kDa expressed by epithelial cells of the glomerulus and proximal tubule cells of the human nephron but only by a subset of renal cell carcinomas (RCCs). We have reported that gp160 expression correlates with the resistance of cultured RCCs to the antiproliferative effects of alpha interferon, while lack of expression correlates with sensitivity to alpha interferon. In this study, we have purified gp160 protein, obtained partial sequences of random peptides, and isolated a full-length cDNA. The gp160 cDNA possesses 78% homology to the murine BP-1/6C3 antigen, a B-lymphocyte differentiation protein that exhibits aminopeptidase A (APA; EC 3.4.11.7) activity. Enzymatic assays on human RCC cell lines indicated a 100% concordance between APA activity and gp160 expression. APA activity of gp160-expressing RCC cells was increased or decreased by a panel of APA activators or inhibitors, respectively. Furthermore, anti-gp160 monoclonal antibodies immunoprecipitate APA activity from RCC cell lysates and selectively deplete APA activity from RCC cell extracts. These data indicate that the gp160 human kidney/RCC glycoprotein is human APA.

Comparison of antigen expression on fresh and cultured ascites cells and on solid tumors of patients with epithelial ovarian cancer.

The antigenic phenotype of malignant cells from ascites of patients with epithelial ovarian cancers was examined and compared to that of their primary and metastatic sites. Cell-surface antigens on frozen sections of primary and metastatic tumors and frozen cell pellets from ascites were analyzed with a panel of murine monoclonal antibodies using the indirect immunoperoxidase method. In addition, ascites cells cultured with and without autologous cell-free ascitic fluid were evaluated by immunofluorescence. The pattern of antigen expression detected on fresh and cultured ascitic epithelial cells was shown to be identical to the expression in autologous solid tumor tissues. When placed in culture, malignant epithelial cells generally persisted for a minimum of one, but no more than five, passages. Addition of autologous ascitic fluid to cultures of ascites cells did not alter the phenotype of the epithelial tumor cell population and did not enhance the growth of these cells. From one culture of ascites cells a permanent malignant epithelial ovarian cancer cell line (designated SK-OV-8) was established. The demonstration that epithelial tumor cells found in ascites of patients with epithelial ovarian cancer have the identical antigenic phenotype as their solid tumor counterpart, at least for the panel of antigens studied, may be useful in planning imaging and therapeutic trials with monoclonal antibodies.

High IL-6 levels in ascitic fluid correlate with reactive thrombocytosis in patients with epithelial ovarian cancer.

Non-haematopoietic malignancies are commonly associated with thrombocytosis. The aetiology of tumour-associated thrombocytosis is still unclear but may be related to tumour-derived thrombopoietin-like factors. Epithelial ovarian tumour cells have been shown to release IL-6 in vitro and high IL-6 levels have been identified in ascites of patients with ovarian cancer. Since IL-6 is a potent stimulator of megakaryocytopoiesis we examined IL-6 production at the tumour site and its relationship to serum IL-6 levels and circulating platelet counts in patients with ovarian cancer. Forty patients undergoing exploratory laparotomy for epithelial ovarian cancer [stage I+II: 6 (15%); stage III: 25 (62.5%); stage IV: 9 (22.5%)] and 24 women with benign ovarian conditions were evaluated. Sera were available from 39 cases with ovarian cancer and from 19 cases with benign ovarian tumours. Ascites was obtained from 35 patients with ovarian cancer. IL-6 activity in serum and ascitic fluid was determined by the standard B9 proliferation assay (detection level: 1 pg/ml). IL-6 bioactivity was detectable in 22 (56%) sera from patients with ovarian cancer, but in only five (26%) of the serum samples obtained from benign cases (P < 0.001). Serum IL-6 levels in patients with ovarian cancer were significantly higher (median 3 pg/ml; range < 1 to 1221 pg/ml) than in patients with benign ovarian conditions (median 0 pg/ml; range < 1 to 4 pg/ml) (P < 0.001). However, much higher concentrations of IL-6 were measured in malignant ascites specimens (median 22,100 pg/ml; range < 1 to 182,600 pg/ml). IL-6 bioactivity in serum and ascites samples was completely inhibited by a neutralizing goat anti-human IL-6 antiserum. Thrombocytosis (platelet counts > 400 x 10(9)/l) occurred in 25 (62.5%) of the 40 patients with ovarian cancer, but in only two (8%) of the 24 cases with benign ovarian tumours. In eight (20%) cases with malignant disease platelet counts ranged between 600 x 10(9)/l and 1060 x 10(9)/l. IL-6 bioactivity in ascitic fluid correlated significantly with circulating platelet counts (r = 0.5916; P < 0.001). Maximum IL-6 bioactivity in ascites and highest platelet counts occurred in patients with undifferentiated ovarian adenocarcinoma or advanced disease. In conclusion, these observations strongly suggest a role for IL-6 in the development of tumour-associated thrombocytosis.

Prognostic significance of HER-2/neu expression in advanced epithelial ovarian cancer: a multivariate analysis.

OBJECTIVE: We tested the hypothesis that there is prognostic significance to the level of expression of the protooncogene HER-2/neu in advanced ovarian cancer, as prior studies have suggested. STUDY DESIGN: We determined expression of HER-2/neu by immunohistochemistry, with monoclonal antibody 9G6 and the indirect immunoperoxidase technique, on frozen tumor specimens from 105 patients with stage III or IV epithelial ovarian cancer. All patients were treated at Memorial Sloan-Kettering Cancer Center, and no patient was lost to follow-up. Median follow-up among surviving patients is 34 months. HER-2/neu expression was scored as negative, weak, 1+, 2+, or 3+. The staining pattern of normal ovarian epithelium was scored negative to 1+. Multivariate analysis was performed to evaluate the prognostic significance of HER-2/neu expression. RESULTS: Twenty-five of the 105 patients (24%) showed strong membrane staining (3+); the other tumor specimens showed weaker membrane staining or no immunoreactivity. There was no correlation of HER-2/neu expression with any of a variety of clinical factors, including stage, grade, cell type, and residual tumor. No significant survival difference was found between patients with levels of staining intensity similar to those of normal ovarian epithelium and those with increased expression (3+). Median survival times were 36 and 27 months, respectively, for the two groups (95% confidence intervals 29 to 45 and 18 to 39 months). Multivariate analysis of possible prognostic factors showed that HER-2/neu overexpression conferred a marginal worsening of survival (p = 0.09) for the subgroup of patients in whom a negative surgical reassessment was not achieved after chemotherapy. CONCLUSION: HER-2/neu expression does not appear to be an important prognostic factor in patients with advanced epithelial ovarian cancer.