Anti-p185HER2 monoclonal antibodies: biological properties and potential for immunotherapy.
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Animals↗
Biomedical subjects
D J Slamon
Publications and source records attributed to D J Slamon.
Transformation mediated by the human HER-2 gene independent of the epidermal growth factor receptor.
Amplification of the HER-2 (c-erbB-2) gene and overexpression of the p185HER-2 gene product is found in approximately one-third of primary human breast and ovarian cancers and is associated with a poor clinical outcome of early relapse and death. The HER-2 gene encodes a cell-surface growth factor receptor with intrinsic tyrosine kinase activity. Wild-type human HER-2 has been shown to act as a potent oncogene when over-expressed in mouse fibroblasts. Recent data suggest that the mechanism by which HER-2 mediates transformation requires the interaction of the epidermal growth factor (EGF) receptor. To test whether overexpression of normal human HER-2 can transform cells independently of the EGF receptor, we have introduced multiple copies of HER-2 into the EGF receptor-negative cell line, NR6, and have performed assays for both transformation and tumorigenicity. Engineered NR6 cells that overexpress the HER-2 gene product display a highly transformed and tumorigenic phenotype as compared with control cells. Additionally, a monoclonal antibody to the extracellular domain of the HER-2 receptor is able to inhibit the proliferation of the overexpressing cells in vitro as well as tumor growth in vivo. This study provides clear evidence that HER-2-mediated transformation can be achieved independently of the EGF receptor.
Cell Transformation, Neoplastic↗
A significant association of Ha-ras p21 in neuroblastoma cells with patient prognosis. A retrospective study of 103 cases.
To evaluate biologic characteristics of neuroblastoma, the authors examined the expression of Ha-ras gene (Ha-ras p21) in 103 primary tumors obtained at the time of diagnosis. Higher expression of the Ha-ras p21 in tumor cells showed a significant association with lower clinical stage of the tumor at diagnosis (chi-square = 35.418, degrees of freedom [df] = 9, P less than 0.001) and survival of the patients (chi-square = 37.111, df = 3, P less than 0.001). Thirty-six (84%) of 43 patients with decreased Ha-ras p21 expression died of aggressive disease. The Ha-ras DNA was examined in the 32 tumors by Southern blot analysis. Neither augmentation nor deletion of the Ha-ras DNA was observed. Amplification of the N-myc DNA was also examined in 43 cases in comparison with Ha-ras p21 expression. N-myc amplification was detected in 12 (55%) of 22 patients who died, and 19 (86%) of the 22 patients showed a low expression of the Ha-ras p21 in tumor cells. Eighteen (86%) of 21 survivors showed a high expression of the Ha-ras p21. The expression of Ha-ras p21 was thought to be a clinically important marker for prognosis in children with neuroblastoma.
Blotting, Southern↗
Expression of the hst-1 and c-kit protooncogenes in human testicular germ cell tumors.
Seventy testicular germ cell tumors were analyzed at the DNA and RNA levels for the c-kit, hst-1, and int-2 oncogenes using Northern and Southern blot analyses, respectively. There were significant differences in oncogene expression between seminomas and nonseminomas with c-kit being expressed in 24 of 30 (80%) seminomas but in only 3 of 40 (7%) nonseminomatous tumors (P = 0.0001, chi 2 test) and hst-1 being expressed in 24 of 38 (63%) nonseminomas but only 1 of 24 (4%) of seminomas (P = 0.0001, chi 2 test), demonstrating an inverse relationship in the expression pattern of these 2 oncogenes in human testicular germ cell tumors. A significant association between tumor stage and hst-1 expression in the nonseminoma group was found (P = 0.0002, chi 2 test). No gross alterations in the c-kit, hst-1, and int-2 loci were found at the DNA level and no int-2 mRNA expression was detected in any of the germ cell tumors examined.
Blotting, Southern↗
Expression of the nm23 gene and breast cancer prognosis.
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Breast Neoplasms↗
Heterotransplantation of human breast carcinomas in nude mice. Correlation between successful heterotransplants, poor prognosis and amplification of the HER-2/neu oncogene.
Four hundred and thirty-three human breast carcinomas and 23 cell lines derived from human breast carcinomas were heterotransplanted in nude mice. Twenty-eight tumors and 13 cell lines took and could be serially transplanted. Their human origin was established by isozyme analysis performed on successive passages. Sixteen primary infiltrating duct-cell carcinomas (PIDC) took, from a total of 262 transplanted (6.1%). This is in striking contrast to the greater than 50% rate of takes of most major cancers of epithelial origin. All 16 PIDC growing in nude mice were highly cellular and lacked desmoplastic hyperplasia. The clinical prognosis of the PIDC patients whose tumors were successfully transplanted was poor. Ten of 16 (63%) died of their disease within 3 years, compared to only 49 (20%) of the 246 PIDC patients whose tumors did not take in nude mice. This could not be attributed to later stage disease of the tumors that took, because only 15% of these patients had 4 or more positive axillary lymph nodes as opposed to 28% of the patients whose tumors did not take. Sixty-four percent of the breast carcinomas growing in nude mice exhibited amplification of the HER-2/neu oncogene which is also correlated with poor prognosis in human breast cancer. It is possible that the nude mouse is more susceptible to a population of highly invasive and lethal breast carcinomas.
Adult↗
Human T-cell leukemia virus infection in non-intravenous drug using HIV seropositive men in Los Angeles.
Sera from 634 homosexual men with Western blot-confirmed human immunodeficiency virus (HIV) infection were subjected to radioimmunoprecipation assay (RIPA) using an HTLV-I-infected human T-cell line (SLB-I). Sera obtained from Japanese adult T-cell leukemia patients, noninfected healthy individuals served as positive and negative controls. HIV-infected groups were comprised of asymptomatic homosexuals (n = 131), AIDS-related complex (n = 115), Kaposi's sarcoma (n = 300), AIDS-defining opportunistic infections (n = 76), and high-grade lymphomas (n = 12). Only two patients were known to be intravenous drug users. No instances of dual retroviral infection were detected. As a corollary, no cross reactivity between HTLV and HIV gene products was noted by RIPA. We conclude that HTLV infection is uncommon among select groups of HIV seropositive homosexuals who do not engage in intravenous drug abuse. Additional studies examining the seroprevalence and consequence of HTLV infection in broader based populations at risk for retroviral infection are required.
Adult↗
Hormonal modulation of HER-2/neu protooncogene messenger ribonucleic acid and p185 protein expression in human breast cancer cell lines.
Recent work has suggested that overexpression of the HER-2/neu protooncogene may play a role in the aggressive clinical behavior of some breast tumors. Since hormones are also known to change the proliferation rate and invasiveness of these cells, we have studied the effect of sex steroid hormones and antihormones on levels of the HER-2/neu mRNA and protein in human breast cancer cell lines using complementary DNA and antibody probes. In MCF-7 cells, which contain high levels of estrogen receptor and an estradiol (E2)-inducible progesterone receptor (PR), 1 nM E2 caused a rapid drop in HER-2/neu mRNA (4.8 kilobases), to 40% of control values by 6 h, and a more gradual decrease in HER-2/neu protein, to 50% by 24 h. HER-2/neu protein and mRNA levels remained reduced throughout 1 week of E2 treatment. The effect of E2 was dose dependent, with the maximal effect seen with concentrations of 10(-10) M E2 and above, and antiestrogen partly reversed the E2-induced decrease in HER-2/neu expression. These characteristics suggest that the observed modulation of HER-2/neu is an estrogen receptor-mediated process. In contrast, progestins did not change HER-2/neu mRNA or protein levels in E2-primed MCF-7 cells that contain high levels of PR; in T47D cells, which contain low levels of ER and high levels of PR, addition of E2 or the progestin R5020 or the antiprogestin RU38,486 had no significant effect on HER-2/neu mRNA or protein levels over 6 days of treatment. These results indicate that estrogen but not progestin modulates HER-2/neu protooncogene expression in these breast cancer cell lines and suggest that aggressiveness associated with high levels of HER-2/neu mRNA and protein may be uncoupled from estrogen-stimulated proliferation in these cells.
Breast Neoplasms↗
Expression of the p185 encoded by HER2 oncogene in normal and transformed human tissues.
The human homolog of the rat neu oncogene, HER2 (also termed c-erbB2) has been demonstrated in amplified form in human breast tumors with poor prognosis. Although amplification of the gene correlates with expression of a 185-kDa transmembrane glycoprotein, no extensive information is available regarding the extent of tissue and tumor specificity of this gene product. We have addressed this issue by immunohistochemically evaluating the expression of p185 HER2 in normal tissue and various tumors using monoclonal antibodies (MAbs) to distinct epitopes of its extracellular domain. No detectable levels of p185 HER2 were found in fetal tissues analyzed, with the exception of renal tubules in 2 out of 3 specimens tested and in intestinal epithelium. In adult tissues, detectable levels of this glycoprotein were found in a restricted number of cell types, the expression being heterogeneous among individuals and cell histotypes. Among the neoplasms assayed p185 HER2 was expressed in 46% of primary breast cancers, in 28% of ovarian tumors and in 30% of colon rectum malignancies. No male breast adenocarcinomas were p185-positive. A large number of other tumors tested revealed only a low incidence of expression of the p185. In metastatic breast tumors p185 HER2 was demonstrated homogeneously among multiple autologous lesions and almost invariably (80%) the expression of p185 in the primary lesion correlated with that of the deriving metastases. Our findings indicate that the expression of the p185 HER2 represents a tumor marker of clinical relevance in breast cancer. Whether this holds true for other malignancies remains to be explored.
Antibodies, Monoclonal↗
Studies of the HER-2/neu proto-oncogene in human breast cancer.
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Breast Neoplasms↗
HER-2/neu oncogene expression and DNA ploidy analysis in breast cancer.
This study was performed to evaluate the correlation between HER-2/neu gene expression and DNA ploidy patterns. Forty-five cases of breast-cancer were analyzed. Immunohistochemical staining of HER-2/neu protein on frozen sections was used to detect the HER-2/neu protein, and the Feulgen DNA staining method was used to assess DNA amounts in the same tumor cells. Positive HER-2/neu overexpression was evaluated visually, and quantitation of the HER-2/neu protein was measured by image analysis. Twenty-two of the 45 cases were visually scored to be positive for the overexpression of the HER-2/neu protein, and these cases also contained above 10% HER-2/neu protein compared with a standard control cell line. All 22 of these cases had near-tetraploid DNA content. In contrast, cells, derived from the 23 cases that did not overexpress the HER-2/neu protein, contained DNA amounts that ranged from euploid (diploid) to varying degrees of aneuploid. The results of this study indicated that tumors that overexpress the HER-2/neu protein have tetraploid or near-tetraploid DNA content. This pattern could relate to the biological behavior of these tumors.
Adenocarcinoma↗
Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues.
The HER-2/neu proto-oncogene is homologous with, but distinct from, the epidermal growth factor receptor. Current evidence indicates that this gene is frequently amplified and/or overexpressed in some human breast and ovarian cancers and that these alterations may be clinically important; however, little is known about the expression pattern of the gene in normal tissues. Using immunohistochemistry and northern blot analyses to identify the HER-2/neu protein and transcript respectively, we have evaluated a variety of normal adult and fetal tissues for HER-2/neu expression. HER-2/neu protein was identified on cell membranes of epithelial cells in the gastro-intestinal, respiratory, reproductive, and urinary tract as well as in the skin, breast and placenta. Northern hybridization confirmed the presence of the 4.5 kb transcript encoding the protein in these tissues. The amount of HER-2/neu message and protein was generally higher in fetal tissues than in the corresponding normal adult tissues. HER-2/neu expression levels in these normal tissues were similar to the levels found in non-amplified, non-overexpressing breast cancers and breast cancer cell lines. Southern hybridization of extracted DNA showed that none of the normal tissues expressing HER-2/neu had amplification of the gene. These results confirm that HER-2/neu is normally a membrane constituent of a variety of epithelial cell types.
Blotting, Northern↗
HER-2/neu oncogene amplification and expression in breast and ovarian cancers.
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Breast Neoplasms↗
Detection of anti-HTLV-I Tax antibodies in HTLV-I enzyme-linked immunosorbent assay-negative individuals.
The HTLV-I tax gene protein (Tax) is not packaged within the mature viral particle from which the proteins for the commercially available enzyme-linked immunosorbent assay (ELISA) are derived. Screening of 162 individuals within a cohort of white intravenous (IV) drug abusers, previously identified as having an increased incidence of HTLV-I infection, demonstrated that seven of them had antibodies to the HTLV-I Tax protein but tested negative in HTLV-I ELISAs and Western blots prepared from purified virion proteins. Three out of 35 individuals in other behaviorally defined high-risk groups also displayed this limited pattern of reactivity to HTLV-I proteins. The presence of the anti-HTLV-I p40/Tax antibodies was determined by radioimmunoprecipitation assay (RIPA), which also revealed low levels of anti-env reactivity. The specificity of the anti-p40 reactivity was confirmed on specific Tax ELISAs and Western blots prepared from recombinantly produced Tax. In vitro gene amplification by the polymerase chain reaction (PCR) was used to establish the presence of sequences homologous to HTLV-I proviral DNA in four/four of these HTLV-I ELISA negative, Tax ELISA/Tax western blot/RIPA positive individuals. These data suggest that the true incidence of HTLV-I infection within high-risk cohorts is greater than previously reported.
Animals↗
Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer.
Carcinoma of the breast and ovary account for one-third of all cancers occurring in women and together are responsible for approximately one-quarter of cancer-related deaths in females. The HER-2/neu proto-oncogene is amplified in 25 to 30 percent of human primary breast cancers and this alteration is associated with disease behavior. In this report, several similarities were found in the biology of HER-2/neu in breast and ovarian cancer, including a similar incidence of amplification, a direct correlation between amplification and over-expression, evidence of tumors in which overexpression occurs without amplification, and the association between gene alteration and clinical outcome. A comprehensive study of the gene and its products (RNA and protein) was simultaneously performed on a large number of both tumor types. This analysis identified several potential shortcomings of the various methods used to evaluate HER-2/neu in these diseases (Southern, Northern, and Western blots, and immunohistochemistry) and provided information regarding considerations that should be addressed when studying a gene or gene product in human tissue. The data presented further support the concept that the HER-2/neu gene may be involved in the pathogenesis of some human cancers.
Animals↗
Expression of protooncogene c-myb in normal human hematopoietic cells.
Expression of the protooncogene, c-myb, in various subpopulations of normal human hematopoietic cells was characterized. Cells expressing the immature cell surface marker, CD34 (My10), were isolated by immune adherence with the "panning" technique or immunomagnetic microspheres and were shown to be strongly positive for c-myb protein expression in an immunoperoxidase assay. The CD34+ progenitor cell population was further separated into myeloid plus erythroid progenitors (CD34+, CD10-) v B-lymphoid precursors (coexpressing CD34 and CD10) by two-color FACS. Both CD34+ progenitor cell subsets strongly expressed c-myb protein by the immunoperoxidase assay. A flow cytometric assay was then developed which permitted simultaneous detection of a cell-surface antigen (to characterize lineage and stage of maturation) and the nuclear oncoprotein. This assay confirmed that CD34+ cells were strongly positive for c-myb expression and also allowed quantitative comparisons of c-myb expression in selected populations of other normal hematopoietic cells. Most human bone marrow cells appear to express some level of c-myb protein, although the CD34+ progenitor cell population expresses the highest amount.
Antigens, CD34↗
A polyclonal CD4+ and CD8+ lymphocytosis in a patient doubly infected with HTLV-I and HIV-1: a clinical and molecular analysis.
HTLV-I is associated with adult T-cell leukemia/lymphoma (ATL) characterized by monoclonal expansions of CD4+ T-lymphocytes. In this report we describe a histologically benign, polyclonal HTLV-I infection in a patient exhibiting both an absolute CD4+ and CD8+ lymphocytosis. Three T-cell lines containing integrated HTLV-I proviral copies established from this patient were initially polyclonal, but with time all grew out the same two clones as determined by analysis of their T-cell antigen receptor beta chain gene rearrangements. The patient subsequently developed pulmonary and nasopharyngeal nodules containing HTLV-I infected cells. Restriction analysis of the patient's HTLV-I provirus revealed no differences from prototype HTLV-I and the tax gene was normally expressed in vivo and in vitro. The patient's T-lymphocytosis and HTLV-I+ pulmonary tract nodules were put into a complete clinical remission by treatment with alkylating agents and steroids. Subsequently, the patient developed a severe immunodeficiency state and expired. Retrospective serologic and gene amplification assays for HIV-1 demonstrated that he had been doubly infected from the time of presentation. Postmortem analysis by polymerase chain reaction revealed the presence of both HTLV-I and HIV-1 in lymphatic tissues and the testes; HIV-1 was also detected in brain tissue.
Acquired Immunodeficiency Syndrome↗
Studies of the retinoblastoma gene in human sarcomas.
The retinoblastoma susceptibility gene, (RB), is a tumor suppressor gene which, when deleted is associated with the development of retinoblastoma. The observation that children with heritable retinoblastoma frequently develop second malignancies, principally sarcomas, led to the detection of similar RB gene deletions in some osteosarcomas. We studied 44 unselected sarcomas from patients with no antecedent retinoblastoma to determine the prevalence and nature of RB gene alterations. DNA and RNA were extracted from fresh tumors and analysed by Southern and Northern blotting. Three of nine osteosarcomas and 4 of 29 soft-tissue sarcomas had deletions of the RB gene. Four of these were full-length, and three were partial deletions of the gene. RNA from 5 of the 7 deleted tumors was studied, and 4 cases completely lacked the RB transcript. Transcripts were found in 19 of 20 tumors with an apparently intact RB gene, and in all the normal tissues studied. An additional tumor lacked the RB transcript, but the gene appeared intact at the DNA level. In total, 8 of 38 sarcomas were found to have alterations of the RB gene. These data indicate that the RB gene is inactivated in a significant number of sarcomas unrelated to retinoblastoma, and that the potential role for the gene in the pathogenesis of human malignancy may not be limited to retinoblastoma.
Adolescent↗