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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.

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.

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.

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.

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.

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).

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.

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.

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).

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.

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.

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.

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.

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.

Effective vaccination against cancer, either for prophylaxis or therapy, has been an elusive goal for years. Cytokine gene therapy offers a novel approach to generate immunogenic tumor cell vaccines. To examine the feasibility of cytokine gene transfer into human renal cancer (RC) cells, we introduced the cDNAs for human interleukin-2 (IL-2) or interferon-gamma (IFN-gamma) into various RC cell lines with retroviral vectors. Using the NIH3T3 amplification assay, no replication competent retroviral particles were detectable in cell culture supernatants taken from gene-modified RC cell lines. Efficient expression of both lymphokines was achieved. Depending on the cell line and the vector construct used, lymphokine gene-modified human RC cell lines released 4 to 29 units/10(6) cells of IL-2, or up to 10 units/10(6) cells of IFN-gamma within 48 h. Fluorescence-activated cell sorter analysis of SK-RC-29 cells releasing IFN-gamma showed increased expression of major histocompatibility complex class I antigen, beta 2-microglobulin, and ICAM-1, as well as induction of major histocompatibility complex class II antigen expression [human leukocyte antigen(HLA)-DR, -DP], but no changes in these cell surface markers were observed with SK-RC-29 cells releasing IL-2. Following in vitro gamma-irradiation with 5,000 or 10,000 rad, growth of lymphokine gene-modified RC cells was abrogated, but their capability to release lymphokine and express lymphokine-induced antigenic determinants, such as HLA-DR, was retained. Tumor formation by the human RC cell line SK-RC-29 in BALB/c nude mice was not affected by IFN-gamma secretion, but was inhibited by in vivo release of IL-2 from s.c. injected tumor cells. These studies demonstrate the feasibility of retroviral mediated lymphokine-gene transfer into human RC cells and suggest a means for generating autologous or HLA-matched allogeneic tumor cell vaccines for the treatment of patients with renal cell carcinoma.

The question of whether the antigenic phenotype of human epithelial ovarian cancer varies in a given patient between the primary tumor and metastatic sites or among metastatic sites themselves is an important issue in planning potential therapeutic strategies for ovarian cancer. We have obtained tumor specimens from at least two separate sites during operations on 12 patients with epithelial ovarian cancer, and we have typed these specimens with a group of 18 monoclonal antibodies that react with cell-surface glycoprotein and carbohydrate antigens, including blood group antigens. Antibodies with relative specificity for malignant cells as well as those that detect more widely distributed epithelial antigens were used. A total of 31 specimens from 12 patients with advanced adenocarcinoma (8 serous, 3 undifferentiated, 1 endometrioid) of the ovary were studied, including fresh ascites cells in two patients. Frozen sections of tumor specimens were stained with the antibodies by the indirect immunoperoxidase technique and graded semiquantitatively. Little difference was seen in antigenic expression of tumors that were obtained from various sites in the same patient for either the epithelial cell markers or blood group markers. Intratumoral antigenic heterogeneity was seen, but this was generally quite consistent within a given patient's specimens. As anticipated, variations in antigen expression were seen among specimens from different patients. The antigenic phenotype of the tumor specimens in a given patient, as determined immunohistochemically by our group of antibodies, showed only minor variation among primary and metastatic sites.

We have reviewed our laboratory's efforts to establish continuous human renal cancer cell lines. During the 16-year period of 1972 through 1987, 498 successive attempts resulted in establishment of 63 renal cancer cell lines. Of these lines, 46 were derived from primary kidney tumors and 17 from metastatic sites (lung, brain, bone, and lymph node). Forty-three of these lines have been characterized with regard to morphology, growth kinetics, anchorage-independent growth, tumorigenicity in athymic nude mice, and expression of kidney cell surface antigens. These results were compared with data from primary short term cultures of normal kidney epithelium. The overall success rate of establishing continuous renal cancer cell lines was 12.7%. In general, no significant difference in success was noted based on whether the specimen was derived from a primary or a metastatic lesion. However, all successfully established lines were derived from tumors exhibiting clinically "aggressive" behavior. All cell lines expressed proximal tubular cell differentiation antigens. Significant morphological heterogeneity was observed among normal kidney as well as kidney cancer cell lines in vitro. No significant difference in doubling time was found between cell lines of renal cancer and passage 1 cultures of normal kidney epithelium. Twenty-one of 30 (70%) lines assayed formed clones on soft agar and 26 of 33 (79%) lines grew in athymic mice. Among the 25 lines which were assayed for both soft agar growth and tumorigenicity in nude mice, this pair of phenotypic traits were concordant in 17 lines (60%). Four lines (16%) grew on agar but not in mice, while four other lines (16%) failed to grow in agar but were tumorigenic in mice.

The multidrug-resistance gene, MDR1, encodes a plasma membrane glycoprotein termed P-glycoprotein that mediates active cellular efflux of certain chemotherapeutic agents. P-Glycoprotein expression was evaluated in 98 frozen tumor specimens from 57 patients with epithelial ovarian cancer by the indirect immunoperoxidase technique with monoclonal antibodies C219 and JSB-1 used for detection. Tumor specimens were further characterized antigenically with a panel of monoclonal antibodies representing a variety of epithelial cell antigens. Included were 57 specimens from 33 previously untreated patients; 11 specimens were also available from eight patients in this group after chemotherapy. An additional 30 specimens were studied from 24 other patients after chemotherapy. In only four of the 57 patients with ovarian cancer (7%) did one or more of the specimens express P-glycoprotein. Two of these patients had tumors that were considered clinically drug resistant. No increase in P-glycoprotein expression was noted after exposure to chemotherapy, including the eight individuals for whom specimens were available both before and after treatment. Although drug resistance is a major problem in treatment of ovarian cancer, resistance to the drugs most active against these tumors probably occurs through a mechanism other than expression of the MDR1 gene product.

The extent to which the antigenic phenotype of human epithelial ovarian cancer changes during the course of the disease is an issue that must be addressed in order to maximize the potential of antibody-directed imaging and therapy. We have obtained tumor specimens at two separate operations from ten patients with epithelial ovarian cancer and typed these specimens with a panel of 19 monoclonal antibodies to cell surface glycoprotein and carbohydrate antigens including blood group antigens. Antibodies with relatively high specificity for malignant cells as well as those detecting more widely distributed epithelial antigens were used in the study. Mean time between the two operations was 8.5 months. Five patients received intraperitoneal therapy during the interval between the two operations, including platinum-based regimens (four patients) and tumor necrosis factor (one patient). Four patients received intravenous platinum-based chemotherapy; one received no treatment. Frozen sections of specimens were stained with the antibodies by the indirect immunoperoxidase technique. Antigenic phenotypes were found to be unrelated to the patient's age, stage, tumor grade, histologic cell type, prior chemotherapy, and interval between operations. Most significantly, little difference was seen in antigenic expression between tumors obtained at the two operations for either the cell surface or blood group markers. Variations in the staining pattern were seen with antibody B72.3 and, to some extent, with the anti-blood group antibodies, which are known for producing heterogeneous staining. The antigenic phenotype of the tumor specimens in a given patient as determined immunohistochemically by our panel of antibodies showed only minor variation between operations, even under the selective pressures of chemotherapy and immunotherapy.

A panel of seven murine monoclonal antibodies reactive with human hepatocellular carcinoma (HCC) cell line, SK- HEP-1, resulted in the definition of four distinct antigen systems, designated HB4, HB5, HB1 and HJ2. HB4 antigen was found to be expressed specifically on HCC cell lines and fresh HCC specimens but not on normal liver. Immunoprecipitation tests suggest that the HB4 epitope may be a heat-stable carbohydrate determinant on a high molecular mass molecule. HB5 antigen was found to have less-restricted expression on a panel of normal adult tissues and on melanoma, astrocytoma, sarcoma, neuroblastoma and epithelial cancer cell lines. In fetal and adult liver, HB5 antigen localized to bile canaliculi and ducts. Under reducing conditions, three mAbs detected a Mr 140,000 glycoprotein using lysates of [125-I], [3-H]-glucosamine and [35-S]-methionine labeled SK-HEP-1 cells. Under non-reducing conditions an additional component of greater than Mr 200,000 was also detected. HB1 antigen was found on almost all monolayer cell lines and not on most cultured suspension cells. This antigen was also detected on cultured HCC cells inoculated into nu/nu mice. Immunoprecipitation experiments revealed that the HB1 antigen is a bimolecular complex with an Mr 170,000 alpha chain and Mr 130,000 beta chain under non-reducing conditions, and three subunits of Mr 140,000, Mr 30,000 and Mr 130,000 under reducing conditions. Two antibodies reacted with epitopes on the alpha chain. HJ2 antigenic determinant is a heat-stable component which could not be immunoprecipitated. This most widely expressed antigen was found in secreted form in many of the cells and tissues examined. These antibodies introduce new antigens which may serve as useful markers for the diagnosis, classification and investigation of HCC and other liver diseases.

Murine monoclonal antibody (mAb) F31 detects a heat-stable antigen (URO-8) found in the acidic lipid fraction of renal cancer cell extracts. Serological analysis of mAb F31 reactivity was assayed on 176 human cell lines. mAb F31 reacted with 38 of 45 renal cancers, a subpopulation of cells in primary cultures of normal renal epithelia, and two of 13 colon, two of 15 lung, and four of five ovarian cancers. No other epithelial, hematopoietic, or neuroectodermal cell lines tested were reactive. Immunofluorescence and immunoperoxidase analyses of fresh frozen tissue sections revealed mAb F31 reactivity in kidney, gastrointestinal tract, biliary canaliculi, bronchial epithelium, and skin. Within the kidney, mAb F31 immunoreactivity was confined to the straight portion of the proximal tubule. A panel composed of previously characterized mAbs as well as mAb F31 defines the antigenic phenotype of proximal convoluted tubular cells as URO-2+/URO-3+/URO-4+/URO-10+/URO-8-/URO-5-; proximal straight tubular cells as URO-2+/URO-3+/URO-4+/URO-10-/URO-8+/URO-5-; and cells of the descending thin limb of Henle as URO-2-/URO-3+ or -/URO-4+/URO-10-/URO-8-/URO-5+. While adult proximal tubular cells demonstrated reciprocal expression of URO-8 and URO-10, fetal kidney proximal tubule progenitor cells coexpressed both antigens (URO-10+/URO-8+). Fifty renal cancer specimens were typed with these antibodies. Fourteen cases were URO-10+/URO-8-, ten cases were URO-10-/URO-8+, and 25 cases expressed both antigens (URO-10+/URO-8+). These phenotypes are consistent with derivation of these particular subsets from the proximal convoluted tubule, the pars recta, or a proximal tubule progenitor cell, respectively. Only one specimen failed to express either URO-8 or URO-10.

Seven mouse monoclonal antibodies and the lectin from Ulex europaeus, detecting blood group specificities of the ABH and Lewis systems, have been used to define the immunoanatomic distribution of these antigenic structures within the human nephron and urothelium. The reagents employed recognize the following blood group related antigens: A (all variants), B, H, Lewisa (Lea), Lewisb (Leb), X (Lewisx), Y (Lewisy) and type 1 precursor chain. We have analyzed the presence of these antigens in histologically normal kidney and urothelium from 22 adults and 3 fetuses by the immunoperoxidase method. In addition, we simultaneously examined blood group and secretor status in 15 of the 22 adult individuals studied. Immunohistochemical analyses demonstrated that these antigenic systems are differentially expressed in cell types and domains of the human urinary tract. Major differences were observed in secretor as compared to nonsecretor individuals, mainly in the more pronounced expression of precursor, H, Leb, and Y antigens in secretors. In the kidney, all antigens, except X, showed enhanced expression in secretor individuals on epithelial cells of the collecting ducts and urothelium; X antigen was mainly present in the proximal tubules and portions of Henle's loop. The urothelium was particularly rich in blood group antigens and in some cases showed differential expression of Lea/X and Leb/Y on the various cell layers. Secretors could be divided into two groups based on the intensity and pattern of staining; it is suggested that this may be determined by homo- or heterozygosity at the Se locus. Nonsecretor individuals lacked expression of Leb and Y determinants, as well as H antigen, in the urothelium (three of four cases). Comparison of normal fetal and adult tissues suggest that the expression of some of these antigens is related to maturation stages of the human nephron. These studies confirm the importance of blood group antigens as normal differentiation antigens. These reagents have a wide range of applications including typing of blood group and secretory status in body fluids and tissues, studies of histogenesis and organogenesis, and analyses of neoplastic and non-neoplastic diseases.

The main objective of the present study is to define the expression and/or modulation of antigenic phenotypes in cells of the normal human kidney and urothelium according to cell type. Fourteen antibodies detecting differentiation and structural antigens expressed in the human urinary tract have been used to define the immunoanatomic distribution of these antigenic systems. They include urinary tract antigens (Tamm-Horsfall glycoprotein and prostate-specific antigen), tissue-associated antigens (epithelial membrane antigen, Factor VIII antigen, and Protein S-100), and cytoskeletal antigens of the intermediate filament classes (cytokeratins, vimentin, desmin, glial fibrillary acidic protein, and neurofilaments. Immunofluorescence and immunoperoxidase analyses performed on normal human fetal and adult tissue sections have demonstrated that these antigens are expressed by different cell types and domains of the nephron. Studies correlating normal fetal and adult tissues reveal that some of the antigens appear at distinct stages of maturation, representing early and late antigenic expression events. These antibodies offer a wide range of potential applications that include studies of embryogenesis of the human urinary tract and immunopathologic analyses of neoplastic and nonneoplastic diseases of the human kidney and urothelium.

Nine murine monoclonal antibodies which detect differentiation antigens of the human kidney are described. Immunofluorescence and immunoperoxidase studies demonstrate that these antigens are expressed by different cell types comprising the nephron. Monoclonal antibody MA99 detects a glycoprotein complex of the glomerular basement membrane. Monoclonal antibody S4 detects a glycoprotein of 160,000 daltons (gp160) expressed by glomerular and proximal tubular epithelial cells. Monoclonal antibodies S23, S27 and S6 immunoprecipitate a glycoprotein of 120,000 daltons (gp120) found on cells of the proximal tubule and portions of Henle's loop. Monoclonal antibody C26 identifies a glycoprotein of 40,000 daltons (gp40) expressed by cells of the distal and collecting tubules. Monoclonal antibodies M2 and S8 are specific for A and B blood group antigens, respectively, found on cells of the collecting tubule in individuals of the respective blood type. This panel of antibodies is useful in the study of normal renal embryogenesis, microanatomy and physiology as well as pathological processes including tumors.

Seven mouse monoclonal antibodies and the lectin Ulex europaeus, detecting blood group related antigens of the ABH and Lewis systems, have been used to determine the immunophenotype of human renal cell carcinomas. Immunohistochemical analyses have demonstrated that these antigenic systems are differentially expressed by distinct normal cell types and domains of the human nephron. In the present study we analyzed the immunophenotype of 29 primary and 15 metastatic renal carcinomas by the immunoperoxidase method. Blood type was known in all of the cases and secretor status in nine cases. ABH specificities were not detected in tumor cells of the primary tumors studied, although two of the metastases showed heterogeneous expression of H and A antigens, respectively. Lewisx (Lex) determinant was detected in 76% of primary renal cell carcinomas; however, Lex was only expressed by occasional cells in 20% of the metastatic tumors analyzed. Lewisa (Lea) was detected with a heterogeneous pattern of expression in 31% of the primary and 26% of the metastatic renal tumors studied. Lewisy (Ley) antigen expression was found in 17% of the primary and 20% of the metastatic tumors analyzed. Detection of precursor type 1 structure was observed in 28% of primary and 20% of metastatic renal cell carcinomas. The present study suggests the histogenesis of renal cell carcinoma in the proximal nephron, based on the expression of Lex and Lea antigens. It also shows: (a) an apparent deletion, downregulation or structural modification of Lex determinant in most of the metastatic tumors; (b) undetectable levels of ABH specificities in tumor cells of primary renal cell carcinoma; and (c) enhanced expression and/or neosynthesis of precursor type 1 structure and Ley determinant in some renal cell carcinomas.