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D J Slamon

Publications and source records attributed to D J Slamon.

At least 55 records · Page 3Linked to original sources
PubMed1996

Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization.

Amplification and overexpression of the HER-2/neu gene occurs in 25-30% of human breast cancers. This genetic alteration is associated with a poor clinical prognosis in women with either node negative or node positive breast cancers. The initial studies testing this association were somewhat controversial and this controversy was due in large part to significant heterogeneity in both the methods and/or reagents used in testing archival material for the presence of the alteration. These methods included a number of solid matrix blotting techniques for DNA, RNA and protein as well as immunohistochemistry. Fluorescence in situ hybridization (FISH) represents the newest methodologic approach for testing for this genetic alteration. In this study, FISH is compared to Southern, Northern and Western blot analyses as well as immunohistochemistry in a large cohort of archival human breast cancer specimens. FISH was found to be superior to all other methodologies tested in assessing formalin fixed, paraffin embedded material for HER-2/neu amplification. The results from this study also confirm that overexpression of HER-2/neu rarely occurs in the absence of gene amplification in breast cancer (approximately 3% of cases). This method of analysis is rapid, reproducible and extremely reliable in detecting presence of HER-2/neu gene amplification and should have clinical utility.

Blotting, Northern↗
PubMed1996

186Re-labeled antibodies to p185HER2 as HER2-targeted radioimmunopharmaceutical agents: comparison of physical and biological characteristics with 125I and 131I-labeled counterparts.

Overexpression of the HER2/neu protooncogene has been shown to correlate with poor clinical prognosis. A murine monoclonal antibody (4D5) directed against the extracellular domain (ECD) of p185HER2 has been shown to inhibit in vitro and in vivo growth of carcinomas overexpressing HER2 and has been humanized (rhuMAb HER2). The objective of the study was the identification of an agent which might be useful for in vitro studies, tumor imaging and/or radioimmunotherapy by linking beta-emitting radionuclides to these HER2-targeted antibodies. Murine 4D5 and humanized rhuMAb HER2 were radiolabeled with 125I, 131I or 186Re. Physical characteristics (TCA precipitability, SDS-PAGE, size exclusion chromatography), binding affinities to the HER2 ECD (in an ELISA and on SK-BR-3 cells) and antiproliferative activities of the radiolabeled antibodies were determined. Although 131I-4D5 and 131I-rhuMAb HER2 usually retained > 85% ECD binding, they exhibited increased aggregation and fragment content, drastically reduced antiproliferative activities and poor stability upon storage at 4 degrees C. For these antibody preparations, conservation of binding did not necessarily correlate with preservation of bioactivity indicating the importance of bioactivity determinations in radiolabeled antibody studies. Conversely, 4D5 and rhuMAb HER2 labeled with 125I or 186Re maintained physical properties, ECD binding, antiproliferative activities and were stable upon storage at 4 degrees C for at least 8 days. The superior retention of physical and biological characteristics of 186Re-labeled 4D5 and rhuMAb HER2 compared with their 131I-labeled counterparts suggests the potential for their use as radioimaging and radioimmunotherapeutic agents in the treatment of HER2 overexpressing tumors.

Animals↗
PubMed1995

Expression patterns of immediate early transcription factors in human non-small cell lung cancer. The Lung Cancer Study Group.

In 1995, there will be 172,000 new cases of lung cancer diagnosed and 153,000 deaths from this disease in the United States. While the pathogenesis of the disease process is poorly understood, a growing body of evidence suggests that abnormalities in cellular regulatory genes may play an important role in the induction, maintenance and/or progression of some tumor types. These genes include both growth promoting oncogenes as well as growth inhibitory or suppressor genes. Included among these genetic sequences are several cellular transcription factors. A group of these factors including c-jun, c-fos and EGR1 are members of a class of genes known as immediate early genes whose expression are inducible by a variety of stimuli including mitogenic and differentiation inducing growth factors, indicating a potential important role for these genes in normal growth processes. Since these genes are involved in early regulation of cellular growth properties and at least two (c-jun and c-fos) can act as oncogenes, we wished to determine whether their expression levels were altered in human non-small cell lung cancers (NSCLC) compared to normal lung tissue. To address this, Northern blot analyses were performed using c-fos, c-jun and EGR1 probes on RNA extracted from 101 NSCLC tumor specimens and adjacent uninvolved lung tissue. Analysis of this cohort revealed that 72% of the normal tissues demonstrate significantly greater expression of these transcription factors as compared to adjacent malignant tissue. Moreover, this expression pattern appeared to be coordinate for all three genes in the majority of cases. This differential expression pattern was confirmed at the protein level using an immunohistochemical approach with antibodies directed against the c-jun, c-fos and EGR1 gene products. Southern blot analyses demonstrated no gross alterations of these sequences at the DNA level, indicating that the observed differential expression pattern was not due to gross structural changes in the genes. These data suggest that down-regulation of these genes may be involved in the pathogenesis of lung cancer.

Base Sequence↗
PubMed1995

HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormone-independent growth in human breast cancer cells.

Growth of human breast cells is closely regulated by steroid hormone as well as peptide hormone receptors. Members of both receptor classes are important prognostic factors in human breast cancer. Clinical data indicate that overexpression of the HER-2 gene is associated with an estrogen receptor-negative phenotype. In this study, we demonstrate that introduction of a HER-2 cDNA, converting non-overexpressing breast cancer cells to those which overexpress this receptor, results in development of estrogen-independent growth which is insensitive to both estrogen and the antiestrogen, tamoxifen. Moreover, activation of the HER-2 receptor in breast cancer cells by the peptide growth factor, heregulin, leads to direct and rapid phosphorylation of ER on tyrosine residues. This is followed by interaction between ER and the estrogen-response elements in the nucleus and production of an estrogen-induced protein, progesterone receptor. In addition, overexpression of HER-2 receptor in estrogen-dependent tumor cells promotes ligand-independent down-regulation of ER and a delayed autoregulatory suppression of ER transcripts. These data demonstrate a direct link between these two receptor pathways and suggest one mechanism for development of endocrine resistance in human breast cancers.

Animals↗
PubMed1995

Prognostic classification of breast ductal carcinoma-in-situ.

We present a new prognostic classification designated the Van Nuys classification for ductal carcinoma-in-situ (DCIS). The classification combines high nuclear grade and comedo-type necrosis to predict clinical recurrence. Three groups of DCIS patients were defined by the presence or absence of high nuclear grade and comedo-type necrosis: 1--non-high-grade DCIS without comedo-type necrosis, 2--non-high-grade DCIS with comedo-type necrosis, 3--high-grade DCIS with or without comedo-type necrosis. There were 31 local recurrences in 238 patients after breast-conservation surgery 3.8% (3/80) in group 1, 11.1% (10/90) in group 2, and 26.5% (18/68) in group 3. The 8-year actuarial disease-free survivals were 93%, 84%, and 61%, respectively (all p < or = 0.05). The Van Nuys classification defines three distinct and easily recognisable groups, each of which has a different likelihood of local recurrence if treated with breast conservation.

Bone Neoplasms↗
PubMed1995

Human c-FES is a nuclear tyrosine kinase.

FES is a non-receptor protein tyrosine kinase expressed in hematopoietic progenitors and differentiated myeloid cells. It has recently been implicated in granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and erythropoietin signal transduction. To better understand the role played by FES in normal and neoplastic hematopoiesis, we used cell fractionation techniques to examine the subcellular localization of FES in myeloid cells and cell lines. FES was observed in the nuclear, granular and plasma membrane fractions of primary human neutrophils and the myeloid leukemia cell line, HL-60. The nuclear localization was confirmed by immunocytochemistry of neutrophils.

Animals↗
PubMed1994

Amplification and overexpression of HER-2/neu in carcinomas of the salivary gland: correlation with poor prognosis.

There are few reliable prognostic markers of biological aggressiveness for head and neck carcinomas in general. For salivary gland carcinomas, anatomic location, tumor size, histological grade, and extent of disease involvement are considered to be clinically important risk factors for recurrent disease. Molecular genetic alterations in salivary gland carcinomas have not been characterized, and tumor cell proteins have not been shown to be prognostically significant. Here a cohort of mucoepidermoid carcinomas of the major (parotid and submandibular) salivary glands are analyzed for a molecular genetic alteration, HER-2/neu gene amplification, and gene amplification and expression results are compared with long-term clinical follow-up information. Archival tissues resected from 58 patients with mucoepidermoid carcinoma of salivary glands were evaluated for HER-2/neu gene amplification by fluorescence in situ hybridization and for gene expression by immunohistochemistry in a blinded fashion. Clinical follow-up information was compared with the results of these analyses to determine whether there were significant associations. Overexpression, identified as membrane immunostaining by immunohistochemistry, was observed in 22 of 58 (38%) mucoepidermoid carcinomas. Gene amplification, characterized by fluorescence in situ hybridization, was observed in 12 (21%) cases. Eleven of the 12 cases with gene amplification were also immunostained for HER-2/neu. Both gene amplification (P = 0.0001, P < 0.0001) and immunostaining (P < 0.0001, P < 0.0001) were correlated with shorter disease-free interval and poorer overall patient survival, respectively. Multivariate analysis showed that HER-2/neu immunostaining and amplification were markers of poor prognosis independent of histopathological grade, tumor size, and involvement of regional lymph nodes. HER-2/neu is amplified and/or overexpressed in approximately one-third of mucoepidermoid carcinomas of salivary glands. Amplification and/or overexpression appears to be an independent marker of poor prognosis in mucoepidermoid carcinomas of the salivary glands as it is in carcinomas of the breast, ovary, and endometrium.

Adolescent↗
PubMed1994

Sensitivity of HER-2/neu antibodies in archival tissue samples: potential source of error in immunohistochemical studies of oncogene expression.

HER-2/neu oncogene amplification and overexpression of breast cancer tissue has been correlated with poor prognosis in women with both node-positive and node-negative disease. However, several studies have not confirmed this association. Review of these studies reveals the presence of considerable methodological variability including differences in study size, follow-up time, techniques and reagents. The majority of papers with clinical follow-up information are immunohistochemical studies using archival, paraffin-embedded breast cancers, and a variety of HER-2/neu antibodies have been used in these studies. Very little information, however, is available about the ability of the antibodies to detect overexpression following tissue processing for paraffin-embedding. Therefore, a series of antibodies, reported in the literature or commercially available, were evaluated to assess their sensitivity and specificity as immunohistochemical reagents. Paraffin-embedded samples of 187 breast cancers, previously characterized as frozen specimens for HER-2/neu amplification by Southern blot and for overexpression by Northern blot, Western blot, and immunohistochemistry, were used. Two multitumor paraffin-embedded tissue blocks were prepared from the previously analyzed breast cancers as a panel of cases to test a series of previously studied and/or commercially available anti-HER-2/neu antibodies. Immunohistochemical staining results obtained with 7 polyclonal and 21 monoclonal antibodies in sections from paraffin-embedded blocks of these breast cancers were compared. The ability of these antibodies to detect overexpression was extremely variable, providing an important explantation for the variable overexpression rate reported in the literature.

Antibodies↗
PubMed1994

Identification of conserved amino acids in the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit critical for function. Evidence for formation of a heterodimeric receptor complex prior to ligand binding.

A superfamily of growth factor and cytokine receptors has recently been identified, which is characterized by four spatially conserved cysteine residues, a tryptophan-serine motif (WSXWS) in the extracellular domain, and a proline-rich cytoplasmic domain. The high affinity human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (hGM-CSFR) consists of two subunits, alpha (hGM-CSFR alpha) and beta (hGM-CSFR beta), both of which are members of the receptor superfamily. In this study, we prepared mutations in conserved amino acids of the receptor subunit necessary for GM-CSF binding (hGM-CSFR alpha) and analyzed mutant receptors for low affinity binding, internalization, and high affinity binding when complexed with the beta subunit. Mutations in the cytoplasmic domain did not affect GM-CSF binding or receptor internalization. Mutation of a single conserved serine residue within the WSXWS motif diminishes cell surface receptor expression but not ligand binding. Mutation of either the second or third conserved cysteine residue of hGM-CSFR alpha resulted in complete loss of low affinity binding; however, co-expression of the cysteine 2 mutant with hGM-CSFR beta yielded a high affinity receptor complex. Since neither the cysteine 2 mutant nor the beta subunit can bind ligand alone, this result suggests that hGM-CSFR alpha and hGM-CSFR beta exist in a preformed heterodimeric protein complex on the plasma membrane.

Amino Acid Sequence↗
PubMed1994

Glucocorticoids stabilize HER-2/neu messenger RNA in human epithelial ovarian carcinoma cells.

Recent evidence suggests that HER-2/neu oncogene overexpression may have a direct role in the pathogenesis of ovarian cancer rather than being merely a prognosticator of poor disease outcome. The mechanisms regulating expression of the p185HER-2/neu growth factor receptor protein are poorly understood. Glucocorticoid receptors are present in tumor cells of almost 90% of ovarian cancers, and these hormones inhibit ovarian cancer cell growth. Glucocorticoid regulation of HER-2/neu expression was investigated using the SK-OV-3 human epithelial ovarian cancer cell line in which the HER-2/neu gene is amplified five- to eightfold. Cells cultured in the presence of 10(-9)-10(-5) M dexamethasone or hydrocortisone displayed a dose-dependent increase in HER-2/neu mRNA. To determine if this effect was due to stabilization of existing HER-2/neu transcripts or to new mRNA synthesis, cells were treated with actinomycin D and cycloheximide once steady-state levels of HER-2/neu mRNA had been reached. These studies demonstrated prolongation of the half-life of existing HER-2/neu transcripts in the presence of dexamethasone. No concomitant increase in the p185HER-2/neu receptor protein in response to dexamethasone could be demonstrated by Western blot or immunohistochemical analyses. Cellular proliferation was inhibited approximately 20% by the presence of dexamethasone. These data suggest that post-transcriptional regulatory mechanisms may play a role in modulating some of the biologic effects of the HER-2/neu oncogene.

Cell Division↗
PubMed1994

p53 mutations and MDM-2 amplification in renal cell cancers.

Genomic DNA from 53 primary human renal cell tumors was screened for the presence of mutations in the tumor suppressor gene p53, using the polymerase chain reaction and single-strand conformation polymorphism analysis, followed by direct sequencing of DNA. Five cases showed mobility shifts. Sequencing of these samples revealed two cases of nonsense mutations (codons 182, 192), one case of a missense mutation (codon 285), and two cases of silent mutations at codon 213. The frequency of mutations altering the p53 (3/53 = 6%) was low when compared with the reported frequencies (15% to 65%) of allelic loss of 17p (location of p53) in renal cell cancers, suggesting the possible existence of another tumor suppressor gene in the region of the p53 gene, which when mutated is associated with these cancers. All three tumors with p53 mutations were cases with poor prognosis, i.e., highest pathological grade and/or advanced Robson stage. The human equivalent of the murine double-minute-2 was not amplified in the renal cell tumors. In summary, alterations of p53 may be associated with the development of renal cell carcinoma of higher grade and/or stage.

Base Sequence↗
PubMed1994

Antibody to HER-2/neu receptor blocks DNA repair after cisplatin in human breast and ovarian cancer cells.

Approximately 30% of human breast and ovarian cancers have amplification and/or overexpression of HER-2/neu gene which encodes a cell surface growth-factor receptor. Overexpression of this receptor, p185HER-2/neu, is associated with poor outcome and may predict clinical response to chemotherapy. Antibodies to HER-2/neu receptor have a cytostatic effect in suppressing growth of cells with overexpression of p185HER-2/neu. To elicit a cytocidal effect, therapy with antireceptor antibody was used in combination with the DNA-damaging drug, cisplatin, and this combined treatment produced a synergistic decrease in cell growth. In addition, antibody mediated an increased sensitivity to cisplatin in drug-resistant ovarian carcinoma cells containing multiple copies of HER-2/neu gene. To evaluate the mechanism for this synergy, unscheduled DNA synthesis was measured in cancer cells using incorporation of [3H]thymidine and autoradiography, and formation and repair of cisplatin-induced DNA adducts was also measured. Treatment with cisplatin led to a marked, dose-dependent increase in unscheduled DNA synthesis which was significantly reduced by combined treatment with antireceptor antibody in HER-2/neu-overexpressing cells. Therapy with antibody to HER-2/neu receptor also led to a 35-40% reduction in repair of cisplatin-DNA adducts after cisplatin exposure and, as a result, promoted drug-induced killing in target cells. This phenomenon which we term receptor-enhanced chemosensitivity may provide a rationale for more selective targeting and exploitation of overexpressed growth factor receptors in cancer cells, thus leading to new strategies for clinical intervention.

Animals↗
PubMed1994

Tumor suppressor and immediate early transcription factor genes in non-small cell lung cancer.

Non-small lung cancer (NSCLC) is a disease that exhibits multiple genetic lesions. Lung Cancer Study Group (LCSG) 871 was designed to analyze this group of malignancies for alterations in growth factors and/or their receptors, oncogenes, tumor suppressor genes, and immediate early transcription factor genes. Immunohistochemical analysis showed that 32% of evaluable cases studied contained absent or abnormal Rb expression. Sequence analysis of the p53 gene revealed that 58% of these cancers contained structural alterations of this gene, whereas only 45% of these cases overexpressed p53 by immunohistochemical analysis. Finally, both Northern blot and immunohistochemical analysis showed that these tumors exhibited changes in the mRNA and protein expression levels respectively of the immediate early transcription factor genes c-fos, c-jun, and EGR, in that less expression of these genes was evident in the tumors compared with adjacent normal tissue. Understanding both the biologic and molecular significance of these findings may allow us to explore novel modalities for treatment of this disease.

Carcinoma, Non-Small-Cell Lung↗
PubMed1994

Mutations of the p53 gene are not detectable in human testicular tumors.

The tumor suppressor gene p53 is the most frequently mutated gene in human cancer. We have used polymerase chain reaction-single-strand conformation polymorphism analysis and sequencing to examine the status of the p53 gene in human testicular cancers of various histologies. We were unable to find in 40 samples and four cell lines any mutations in the regions of this gene (exons 5 through 8) that are usually mutated in other cancers. Northern blot analysis showed expression of this gene in most of the samples analyzed, as well as in four human testicular tumor cell lines. The MDM-2 gene is amplified and overexpressed in sarcomas; it binds and functionally inactivates p53. The 44 testicular tumor samples and cell lines were examined for amplification of MDM-2 by dot-blot analysis; none was found. The proto-oncogene c-kit probably plays an important role in normal testicular development. Mutation of the tyrosine phosphorylation site of a closely related member of this family of tyrosine kinase receptors (c-fms) is associated with cellular transformation and cancer. Codon 936 is the analogous tyrosine of c-kit; using polymerase chain reaction-single-strand conformation polymorphism analyses, we were unable to detect mutations at this site in our 44 testicular cancer samples. We conclude from our studies that mutations in the most conserved region of the p53 gene, as well as at codon 936 of the c-kit gene and amplification of MDM-2, are extremely rare in human testicular cancers.

Base Sequence↗
PubMed1993

Correlation of the survival of ovarian cancer patients with mRNA expression of the 60-kD heat-shock protein HSP-60.

PURPOSE: Ovarian carcinomas express the 60-kD heat-shock protein HSP-60 at widely varying levels in different tumors. The aim of this study was to determine whether there was a relationship between expression of HSP-60 and survival in patients with ovarian carcinoma. MATERIALS AND METHODS: Total RNA and DNA were prepared from 51 epithelial ovarian cancer tissue samples. The expression and structure of the HSP-60 gene were examined by Northern and Southern blot analyses using the carboxyl-terminal portion of this gene as a probe (0.89 kilobases [kb]). HSP-60 expression was correlated with overall survival by the Kaplan-Meier method. RESULTS: The 2.3-kb HSP-60 message was detected in all samples, but there was marked variation from tumor to tumor. Patients were classified into two groups on the basis of HSP-60 expression: group 1 (n = 25) included patients with low expression, and group 2 (n = 26) consisted of patients with high expression. There were no significant differences between the groups with respect to age, cell type, pathologic grade, clinical stage, and previous treatment. After a median follow-up period of 17 months, Kaplan-Meier plots demonstrated a much better survival for group 1 (median, 46.8 months; 41% at 4 years) than group 2 (median, 22.1 months; 16% at 3.9 years), a difference that was highly significant by the Mantel-Haenszel test (P = .00183). Southern blot analysis of these samples showed no amplification or rearrangement of the gene. CONCLUSION: The level of HSP-60 mRNA expression is a valuable prognostic factor in epithelial ovarian cancer. Variation in the level of expression is not due to amplification of this gene.

Adult↗
PubMed1993

Evidence for nm23 RNA overexpression, DNA amplification and mutation in aggressive childhood neuroblastomas.

Reduced expression of nm23 RNAs/proteins has been associated previously with high tumor metastatic potential. In contrast, we report that regional (state III) and metastatic (stage IV) childhood neuroblastomas exhibit elevated nm23 RNA levels as compared with localized tumors. Elevated neuroblastoma nm23 RNA levels were associated with significant reductions in patient survival in the overall (n = 75) and N-myc non-amplified (n = 61) portion of the cohort. Amplification of the chromosomal nm23-H1 gene was observed in 6/18 stage III and IV tumors; amplification of nm23-H2 was not demonstrated. Genomic amplification of nm23-H1 was associated with increased tumor nm23 RNA expression and reduced patient survival. Single-strand conformational polymorphism (SSCP) analysis was performed on seven neuroblastomas. Minor subpopulations of cDNAs exhibiting altered mobility were apparent in both nm23-H1 and nm23-H2 translated regions of stage III and IV tumors, suggestive of mutations. Confirmation of the SSCP data was provided by direct sequencing of nm23-H2 in a stage IV tumor, revealing a leucine to valine mutation at position 48. The data indicate that molecular alterations to nm23 other than its reduced expression can be associated with tumor aggressiveness, and provide the first evidence for nm23 mutation in a human cancer.

Amino Acid Sequence↗
PubMed1993

Inactivation of the retinoblastoma susceptibility gene in non-small-cell lung cancer. The Lung Cancer Study Group.

Mutations that prevent the normal expression of the retinoblastoma susceptibility gene (RB) have been linked to the pathogenesis of several human malignancies. Mutational inactivation of this tumor-suppressor gene appears to initiate the development of retinoblastoma, and may also contribute to the pathogenesis of osteosarcomas, soft-tissue sarcomas, small-cell lung cancer and other malignancies. In cooperation with the Lung Cancer Study Group, we studied the structure and expression of the RB gene in a cohort of 219 primary non-small-cell lung cancers (NSCLCs). RB gene structure was studied at the DNA level by Southern blot and chromosome 13 restriction fragment length polymorphism analyses. Expression of the RB gene was evaluated by Northern analysis of the transcript and immunohistochemical analysis of the protein (p105RB). Immunohistochemistry of the RB protein proved to be the most sensitive method for the detection of Rb gene inactivation. Absent or abnormal RB protein staining was detected in 53/163 (32%) evaluable cases studied, while Northern analysis showed 22/219 cases to have an altered or absent RB transcript. Southern analysis revealed only two cases of structural alteration of the gene, but loss of heterozygosity from chromosome 13 was common in tumors that failed to express the protein. Analysis of the clinical outcomes of the patients whose tumors were studied did not show any correlation of RB inactivation with time to relapse or death. The data from this study indicate that the RB gene is inactivated in a significant number of NSCLCs. The role that these mutations may play in the development of NSCLC remains to be defined.

Base Sequence↗