Biomedical subjects

D J Slamon

Publications and source records attributed to D J Slamon.

At least 37 records · Page 2Linked to original sources

PubMed1999

Monoclonal antibody to HER-2/neureceptor modulates repair of radiation-induced DNA damage and enhances radiosensitivity of human breast cancer cells overexpressing this oncogene.

The management of human breast cancer frequently includes radiation therapy as an important intervention, and improvement in the clinical efficacy of radiation is desirable. Overexpression of the HER-2 growth factor receptor occurs in 25-30% of human breast cancers and correlates with poor clinical outcome, including earlier local relapse following conservative surgery accompanied by radiation therapy. In breast cancer cells with overexpression of HER-2 receptor, recombinant humanized monoclonal antibodies (rhuMAbs) to HER-2 receptors (rhuMAb HER-2) decrease cell proliferation in vitro and reduce tumor formation in nude mice. Therapy with rhuMAb HER-2 enhances tumor sensitivity to radiation at doses of 1-5 Gy, exceeding remission rates obtained with radiation alone. This benefit is specific to cells with HER-2 overexpression and does not occur in cells without overexpression. Treatment of cells with radiation (2-4 Gy) alone provokes a marked increase in unscheduled DNA synthesis, a measure of DNA repair, but HER-2-overexpressing cells treated with a combination of rhuMAb HER-2 and radiation demonstrate a decrease of unscheduled DNA synthesis to 25-44% of controls. Using an alternate test of DNA repair, i.e., radiation-damaged or undamaged reporter DNA, we introduced a cytomegalovirus-driven beta3-galactosidase into HER-2-overexpressing breast cancer cells that had been treated with rhuMAb HER-2 or control. At 24 h posttransfection, the extent of repair assayed by measuring reporter DNA expression was high after exposure to radiation alone but significantly lower in cells treated with combined radiation and rhuMAb HER-2 therapy. To further characterize effects of rhuMAb HER-2 and the combination of antibody and radiation on cell growth, analyses of cell cycle phase distribution were performed. Antibody reduces the fraction of HER-2-overexpressing breast cancer cells in S phase at 24 and 48 h. Radiation treatment is also known to promote cell cycle arrest, predominantly at G1, with low S-phase fraction at 24 and 48 h. In the presence of rhuMAb HER-2, radiation elicits a similar reduction in S phase at 24 h, but a significant reversal of this arrest appears to begin 48 h postradiation exposure. The level of S-phase fraction at 48 h is significantly greater than that found at 24 h with the combined antibody-radiation therapy, suggesting that early escape from cell cycle arrest in the presence of antireceptor antibody may not allow sufficient time for completion of DNA repair in HER-2-overexpressing cells. Because it is well known that failure of adequate p21WAF1 induction after DNA damage is associated with failure of cell cycle arrest, we also assessed the activity of this critical mediator of the cellular response to DNA damage. The results show induction of p21WAF1 transcripts and protein product at 6, 12, and 24 h after radiation treatment; however, increased levels of p21WAF1 transcript and protein are not sustained in HER-2-overexpressing cells exposed to radiation in the presence of rhuMAb HER-2. Although transcript and protein levels increase at 6-12 h, they are both diminished by 24 h. Levels of p21WAF1 transcript and protein at 24 h are significantly lower than in cells treated by radiation without antibody. A reduction in the basal level of p21WAF1 transcript also occurred after 12-24 h exposure to antibody alone. The effect of HER-2 antibody may be related to tyrosine phosphorylation of p21WAF1 protein. Tyrosine phosphorylation of p21WAF1 is increased after treatment with radiation alone, but phosphorylation is blocked by combined treatment with antireceptor antibody and radiation. This dysregulation of p21WAF1 in HER-2-overexpressing breast cells after treatment with rhuMAb HER-2 and radiation appears to be independent of p53 expression levels but does correlate with reduced levels of mdm2 protein. (ABSTRACT TRUNCATED)

Animals↗

PubMed1999

Amplification and overexpression of the cyclin D1 and epidermal growth factor receptor genes in non-small-cell lung cancer. Lung Cancer Study Group.

PURPOSE: To study the structure and expression of the cyclin D1 and the epidermal growth factor receptor (EGFR) genes in a cohort of 298 non-small-cell lung cancer (NSCLC) specimens. METHODS: Gene structure was studied by Southern analysis, and gene expression was studied by Northern analysis and immunohistochemical analysis. RESULTS: Amplification of the cyclin D1 locus was found in 14/298 (5%) specimens. All 12/12 specimens with amplification of the cyclin D1 gene for which RNA was available were found to express the cyclin D1 transcript, and 11/12 overexpressed the transcript to levels higher than that of uninvolved lung. The EGFR gene was amplified in 17/286 samples of NSCLC tested, and was overexpressed in 22/169 (13%) cases tested, including 12/13 cases with amplification of the gene for which RNA was available. Cyclin D1 gene amplification was associated with advanced lymph node involvement (P = 0.043), but not with larger tumor size or adverse outcome. Cyclin D1 gene amplification and overexpression occurred independently of retinoblastoma tumor-suppressor gene (RB) inactivation, but tumors with amplification of the cyclin D1 gene were more likely to have EGFR gene amplification (P < 0.005). No correlation of EGFR gene amplification or overexpression with tumor size, lymph node involvement, patient demographic data, or survival was identified. CONCLUSIONS: These data indicate that the cyclin D1 and EGFR genes are amplified and overexpressed in NSCLC, and amplification of the cyclin D1 gene occurs frequently in conjunction with amplification of the EGFR gene.

Adenocarcinoma↗

PubMed1999

Localization of human BRCA1 and its loss in high-grade, non-inherited breast carcinomas.

Although the link between the BRCA1 tumour-suppressor gene and hereditary breast and ovarian cancer is established, the role, if any, of BRCA1 in non-familial cancers is unclear. BRCA1 mutations are rare in sporadic cancers, but loss of BRCA1 resulting from reduced expression or incorrect subcellular localization is postulated to be important in non-familial breast and ovarian cancers. Epigenetic loss, however, has not received general acceptance due to controversy regarding the subcellular localization of BRCA1 proteins, reports of which have ranged from exclusively nuclear, to conditionally nuclear, to the ER/golgi, to cytoplasmic invaginations into the nucleus. In an attempt to resolve this issue, we have comprehensively characterized 19 anti-BRCA1 antibodies. These reagents detect a 220-kD protein localized in discrete nuclear foci in all epithelial cell lines, including those derived from breast malignancies. Immunohistochemical staining of human breast specimens also revealed BRCA1 nuclear foci in benign breast, invasive lobular cancers and low-grade ductal carcinomas. Conversely, BRCA1 expression was reduced or undetectable in the majority of high-grade, ductal carcinomas, suggesting that absence of BRCA1 may contribute to the pathogenesis of a significant percentage of sporadic breast cancers.

Antibodies↗

PubMed1999

Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease.

PURPOSE: Overexpression of the HER2 protein occurs in 25% to 30% of human breast cancers and leads to a particularly aggressive form of the disease. Efficacy and safety of recombinant humanized anti-HER2 monoclonal antibody as a single agent was evaluated in women with HER2-overexpressing metastatic breast cancer that had progressed after chemotherapy for metastatic disease. PATIENTS AND METHODS: Two hundred twenty-two women, with HER2-overexpressing metastatic breast cancer that had progressed after one or two chemotherapy regimens, were enrolled. Patients received a loading dose of 4 mg/kg intravenously, followed by a 2-mg/kg maintenance dose at weekly intervals. RESULTS: Study patients had advanced metastatic disease and had received extensive prior therapy. A blinded, independent response evaluation committee identified eight complete and 26 partial responses, for an objective response rate of 15% in the intent-to-treat population (95% confidence interval, 11% to 21%). The median duration of response was 9.1 months; the median duration of survival was 13 months. The most common adverse events, which occurred in approximately 40% of patients, were infusion-associated fever and/or chills that usually occurred only during the first infusion, and were of mild to moderate severity. These symptoms were treated successfully with acetaminophen and/or diphenhydramine. The most clinically significant adverse event was cardiac dysfunction, which occurred in 4.7% of patients. Only 1% of patients discontinued the study because of treatment-related adverse events. CONCLUSION: Recombinant humanized anti-HER2 monoclonal antibody, administered as a single agent, produces durable objective responses and is well tolerated by women with HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. Side effects that are commonly observed with chemotherapy, such as alopecia, mucositis, and neutropenia, are rarely seen.

Adult↗

PubMed1999

Combination therapy with trastuzumab (Herceptin) and cisplatin for chemoresistant metastatic breast cancer: evidence for receptor-enhanced chemosensitivity.

The anti-HER-2/neu antibody trastuzumab (Herceptin; Genentech, San Francisco, CA) interferes with DNA repair induced by cisplatin and, as a result, promotes cytotoxicity in HER-2/neu-overexpressing tumor target cells in a synergistic fashion. This effect of trastuzumab, termed receptor-enhanced chemosensitivity, is specific for HER-2/neu-overexpressing cells, having no effect on cells without overexpression. Based on these findings, we conducted phase I and II clinical trials of trastuzumab plus cisplatin to determine the toxicity, pharmacokinetics, response rate, and response duration of this combination in patients with HER-2/neu-overexpressing metastatic breast cancer who had demonstrated disease progression (chemoresistance) while on active chemotherapy just prior to study entry. In phase I, four of 15 patients had objective clinical responses, including one complete response of several years' duration. Of 37 assessable patients enrolled in phase II, nine (24.3%) had objective clinical responses and an additional nine had minor responses or stable disease. The median time to progression among the responders was 8.4 months. The toxicity profile reflected that expected from cisplatin alone, with no apparent increase in toxicity caused by the addition of trastuzumab. Moreover, the pharmacokinetics of trastuzumab were unaltered by coadministration of cisplatin. We conclude that the combination of trastuzumab and cisplatin results in response rates higher than that reported for either single agent alone. Such receptor-enhanced chemosensitivity offers a new approach to target overexpressed growth factor receptors in a variety of cancers, which will lead to new, biologically based therapeutic strategies for clinical intervention.

Antibodies, Monoclonal↗

PubMed1998

Remission of human breast cancer xenografts on therapy with humanized monoclonal antibody to HER-2 receptor and DNA-reactive drugs.

HER-2 oncogene encodes a transmembrane growth factor receptor that is overexpressed in 25-30% of patients with primary breast and ovarian cancer. A murine monoclonal antibody, 4D5, to the extracellular domain of HER-2 receptor elicits cytostatic growth inhibition of tumor cells overexpressing HER-2 protein, but clinical use of this antibody is limited by genesis of human anti-mouse antibodies. To avoid this problem, a recombinant humanized 4D5 monoclonal antibody (rhuMAb HER-2) was developed and tested using a human tumor xenograft model. Human breast and ovarian cancer cells which overexpress HER-2 were inhibited in vivo by the rhuMAb HER-2 antibody. Tumor growth relative to control was reduced at all doses of antibody tested, and the magnitude of growth inhibition was directly related to dose of rhuMAb HER-2. Tumor growth resumed on termination of antibody therapy, indicating a cytostatic effect. To elicit a cytotoxic response, human breast tumor xenografts were treated with a combination of antibody and antitumor drugs, cisplatin or doxorubicin. The combination of antibody with either cisplatin or doxorubicin resulted in significantly greater growth inhibition, with the cisplatin combination demonstrating a greater response. In addition, therapy with cisplatin and antireceptor antibody elicited complete tumor remissions after 2-3 cycles of therapy. The schedule of administration of anti-receptor antibody and cisplatin was critical for occurrence of antibody-induced potentiation in cisplatin cytotoxicity. Enhanced killing of tumor cells was found only if antibody and drug were given in close temporal proximity. Since interference with DNA repair pathways may contribute to this receptor-enhanced chemosensitivity, repair of cisplatin-damaged reporter DNA (pCMV-beta) was determined in human breast cells. As in studies of antibody-enhanced cisplatin cytotoxicity in vivo, treatment with rhuMAb HER-2 blocked the repair of cisplatin-damaged DNA only if the antibody was administered in close temporal proximity to transfection of the drug-exposed reporter DNA. An alternative measure of DNA repair, unscheduled DNA synthesis, was also assessed. Treatment with either cisplatin or doxorubicin led to an increase in unscheduled DNA synthesis that was reduced by combined therapy with antireceptor antibody specific to HER-2-overexpressing breast cancer cells. Using a direct measure of DNA repair, therapy of HER-2-overexpressing cells with rhuMAb HER-2 also blocked the removal of cisplatin-induced DNA adducts. Expression of p21/WAF1, an important mediator of DNA repair, was disrupted in breast cancer cells with HER-2 overexpression, but not in control cells, after treatment with HER-2 antibody, thus suggesting cross-communication between the HER-2 signaling and DNA repair pathways. These data demonstrate an in vivo antiproliferative effect of rhuMAb HER-2 on tumors that overexpress HER-2 receptor and a therapeutic advantage in the administration of the antireceptor antibody in combination with chemotherapeutic agents.

Animals↗

PubMed1998

A homologue of Drosophila aurora kinase is oncogenic and amplified in human colorectal cancers.

Genetic and biochemical studies in lower eukaryotes have identified several proteins that ensure accurate segregation of chromosomes. These include the Drosophila aurora and yeast Ipl1 kinases that are required for centrosome maturation and chromosome segregation. We have identified two human homologues of these genes, termed aurora1 and aurora2, that encode cell-cycle-regulated serine/threonine kinases. Here we demonstrate that the aurora2 gene maps to chromosome 20q13, a region amplified in a variety of human cancers, including a significant number of colorectal malignancies. We propose that aurora2 may be a target of this amplicon since its DNA is amplified and its RNA overexpressed, in more than 50% of primary colorectal cancers. Furthermore, overexpression of aurora2 transforms rodent fibroblasts. These observations implicate aurora2 as a potential oncogene in many colon, breast and other solid tumors, and identify centrosome-associated proteins as novel targets for cancer therapy.

3T3 Cells↗

PubMed1998

A B7.1-antibody fusion protein retains antibody specificity and ability to activate via the T cell costimulatory pathway.

We describe the construction and characterization of an Ab fusion protein specific for the tumor-associated Ag HER2/neu linked to sequences encoding the extracellular domain of the B7.1 T cell costimulatory ligand. The Ab domain of the fusion molecule will specifically target HER2/neu-expressing tumor cells, while the B7.1 domain is designed to activate a specific immune response. We show that the B7.1 fusion Ab retained ability to selectively bind to the HER2/neu Ag and to the CTLA4/CD28 counter-receptors for B7.1. Specific T cell activation was observed when the B7.1 Ab fusion protein was bound to HER2/neu-expressing cells. The use of the B7.1 Ab fusion protein may overcome limitations of gene transfer and/or standard Ab therapy and represents a novel approach to the eradication of minimal residual disease.

Amino Acid Sequence↗

PubMed1998

Cytotoxicity and specificity of directed toxins composed of diphtheria toxin and the EGF-like domain of heregulin beta1.

As a step in the design of directed toxins, aimed at cells that overexpress HER receptors, particularly breast carcinoma cells, we studied the properties of a chimera of diphtheria toxin (DT) and heregulin beta1. The EGF-like growth hormone heregulin is a ligand for the HER3 and HER4 receptors and their heterodimers with HER2. The 60-residue EGF-like domain (hrg) of heregulin elicits a biological response and binds to these receptors primarily through its N terminus. We tested a fusion protein in which hrg replaces the C-terminal receptor-binding domain of DT (DT(389)hrg) and an alternative design in which this domain is fused to the N terminus of DT(389). Of those two constructs, the N-terminal fusion was not active as a directed toxin but elicited a growth response. The C-terminal fusion of hrg to DT(389) yielded a functional toxin and showed cell line specific cytotoxicity that is consistent with heregulin specificity. The binding of hrg to its cognate receptor is not impaired as shown by receptor activation, direct binding, and competition with free hrg. Cytotoxicity is dependent on high-affinity binding of DT(389)hrg to HER3 and HER4 receptors and is not mediated by HER2 overexpression alone. For those cell lines exhibiting high-affinity binding sites, the level of cytotoxicity correlates with the rate of internalization. Thus DT(389)hrg chimeras offer a possible avenue toward directed toxins against cells that overexpress HER receptors.

Binding Sites↗

PubMed1998

HER-2/neu as a predictive marker of response to breast cancer therapy.

Amplification of the HER-2/neu (c-erbB-2) gene resulting in overexpression of the p185HER-2 growth factor receptor occurs in approximately 25% of early stage breast cancers. HER-2/neu has been established as an important independent prognostic factor in early stage breast cancer in large cohorts of patients and in cohorts with very long (30 year) follow-up duration. New data are emerging to suggest that HER-2/neu may be useful not only as a prognostic factor but also as a predictive marker for projecting response to chemotherapeutics, antiestrogens, and therapeutic anti-HER-2/neu monoclonal antibodies. In this review we highlight recent data on HER-2/neu as a predictive marker of response to breast cancer therapy and discuss the clinical implications of this information. The difficulty in comparing results from different data sets due to the wide variety of reagents and technologies used to detect HER-2/neu amplification/overexpression in clinical specimens is also discussed. Finally, we report results from experimental models of HER-2/neu overexpression which have been used in an effort to understand the relationship between HER-2/neu and response to chemotherapeutics and antiestrogens in breast cancer.

Antibodies, Monoclonal↗

PubMed1998

Ex vivo expansion and differentiation of unselected peripheral blood progenitor cells in serum-free media.

The ability to expand and differentiate unselected PBPC was investigated. Cells were grown in serum-free media containing stem cell factor, GCSF and megakaryocyte growth and development factor (pegylated PEG-rHuMGDF) with or without supplemental serum. Optimal proliferation occurred when PBPC were cultured without prior Ficoll-Paque separation in serum-free media. Cell yields after 17 days of culture were proportional to the percentage of CD34+ cells in the starting population and were 1170+/-302-fold higher than the starting numbers of CD34+ cells. Granulocyte-macrophage colony-forming units increased over 12 days of culture, whereas the numbers of erythroid colony-forming cells peaked between 4 and 7 days. Elimination of PEG-rHuMGDF from cell cultures resulted in significantly lower yields of myeloid and erythroid colony-forming cells and total cell numbers. Cell differentiation into neutrophils was indicated by progressive increases in CD11b, CD15, and CD66b expression. Expanded neutrophils phagocytosed and killed bacteria as efficiently as neutrophils from normal donors. Large-scale expansion studies yielded similar proliferation and differentiation results as parallel small-scale cultures. Therefore, unselected PBPC can be efficiently expanded and differentiated into large numbers of functional mature neutrophils.

Antigens, CD↗

PubMed1998

Phase II study of receptor-enhanced chemosensitivity using recombinant humanized anti-p185HER2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment.

PURPOSE: To determine the toxicity, pharmacokinetics, response rate, and response duration of intravenous (i.v.) administration of recombinant, humanized anti-p185HER2 monoclonal antibody (rhuMAb HER2) plus cisplatin (CDDP) in a phase II, open-label, multicenter clinical trial for patients with HER2/neu-overexpressing metastatic breast cancer. PATIENTS AND METHODS: The study population consisted of extensively pretreated advanced breast cancer patients with HER2/neu overexpression and disease progression during standard chemotherapy. Patients received a loading dose of rhuMAb HER2 (250 mg i.v.) on day 0, followed by weekly doses of 100 mg i.v. for 9 weeks. Patients received CDDP (75 mg/m2) on days 1, 29, and 57. RESULTS: Of 37 patients assessable for response, nine (24.3%) achieved a PR, nine (24.3%) had a minor response or stable disease, and disease progression occurred in 19 (51.3%). The median response duration was 5.3 months (range, 1.6-18). Grade III or IV toxicity was observed in 22 of 39 patients (56%). The toxicity profile reflected that expected from CDDP alone with the most common toxicities being cytopenias (n = 10), nausea/vomiting (n = 9), and asthenia (n = 5). Mean pharmacokinetic parameters of rhuMAb HER2 were unaltered by coadministration of CDDP. CONCLUSION: The use of rhuMAb HER2 in combination with CDDP in patients with HER2/neu-overexpressing metastatic breast cancer results in objective clinical response rates higher than those reported previously for CDDP alone, or rhuMAb HER2 alone. In addition, the combination results in no apparent increase in toxicity. Finally, the pharmacology of rhuMAb HER2 was unaffected by coadministration with CDDP.

Adult↗

PubMed1997

The effect of HER-2/neu overexpression on chemotherapeutic drug sensitivity in human breast and ovarian cancer cells.

Recent studies indicate that oncogenes may be involved in determining the sensitivity of human cancers to chemotherapeutic agents. To define the effect of HER-2/neu oncogene overexpression on sensitivity to chemotherapeutic drugs, a full-length, human HER-2/neu cDNA was introduced into human breast and ovarian cancer cells. In vitro dose-response curves following exposure to 7 different classes of chemotherapeutic agents were compared for HER-2- and control-transfected cells. Chemosensitivity was also tested in vivo for HER-2- and control-transfected human breast and ovarian cancer xenografts in athymic mice. These studies indicate that HER-2/neu overexpression was not sufficient to induce intrinsic, pleomorphic drug resistance. Furthermore, changes in chemosensitivity profiles resulting from HER-2/neu transfection observed in vitro were cell line specific. In vivo, HER-2/neu-overexpressing breast and ovarian cancer xenografts were responsive to different classes of chemotherapeutic drugs compared to control-treated xenografts with no statistically significant differences between HER-2/neu-overexpressing and nonoverexpressing xenografts. We found no instance in which HER-2/neu-overexpressing xenografts were rendered more sensitive to chemotherapeutic drugs in vivo. HER-2/neu-overexpressing xenografts consistently exhibited more rapid regrowth than control xenografts following initial response to chemotherapy suggesting that a high rate of tumor cell proliferation rather than intrinsic drug resistance may be responsible for the adverse prognosis associated with HER-2/neu overexpression in human cancers.

Animals↗

PubMed1997

Differential subcellular localization, expression and biological toxicity of BRCA1 and the splice variant BRCA1-delta11b.

The mechanism of BRCA1 tumor suppression in human breast and ovarian cells is the focus of intense investigation. In this report, full length BRCA1 (230 kDa) introduced into cells with CMV promoter constructs was nuclear when transgene expression was low whereas high expression resulted in cytoplasmic accumulation, aberrant nuclear and cell morphology. A nuclear localization signal (NLS) was mapped to BRCA1 amino acid positions 262-570. We describe a splice variant, BRCA1-delta11b, missing the majority of exon 11 including the NLS. Exogenous BRCA1-delta11b (110 kDa) was cytoplasmic and, unlike the full-length protein, overexpression of the protein encoded by the variant did not appear to be toxic. RNA probe titrations and RT-PCR demonstrated that BRCA1 and delta11b transcripts are coexpressed in a wide variety of cells and tissues. Interestingly, BRCA1-delta11b message was greatly reduced or absent in several breast and ovarian tumor lines relative to exon 11 transcripts and a delta9,10 splice variant. Taken together our results suggest that full-length BRCA1 and BRCA1-delta11b may have distinct roles in cell growth regulation and tumorigenesis.

Alternative Splicing↗

PubMed1997

HER-2/neu signal transduction in human breast and ovarian cancer.

The HER-2/neu proto-oncogene encodes a 185 kDa transmembrane receptor tyrosine kinase with significant sequence homology to other members of the class I receptor tyrosine kinase family. The HER-2/neu gene is amplified and/or overexpressed in 25%-30% of human breast and ovarian cancers, and overexpression of the receptor is associated with poor prognosis. Tyrosine phosphorylation and activation of the HER-2 receptor lead to activation of specific signal transduction pathways in breast and ovarian cancer cells, including the ras/MAP kinase cascade, phosphatidylinositol 3-kinase, and phospholipase C-gamma. HER-2/neu signal transduction pathways ultimately converge on the cell nucleus, where the expression of diverse genes is induced after activation of the receptor. A more complete understanding of HER-2/neu signal transduction pathways may allow the development of specific therapeutics for the treatment of those human breast and ovarian cancers containing this alteration.

Breast Neoplasms↗

PubMed1997

HER-2/neu gene amplification characterized by fluorescence in situ hybridization: poor prognosis in node-negative breast carcinomas.

PURPOSE: The HER-2/neu gene codes for a membrane receptor protein that is homologous, but distinct from the epidermal growth factor receptor. This investigation was performed to validate fluorescence in situ hybridization (FISH) as a sensitive and specific method for assessing HER-2/neu gene amplification in archival tissue and to test whether this alteration is associated with poor prognosis. MATERIALS AND METHODS: HER-2/neu gene amplification was determined by FISH in 140 archival breast cancers, previously characterized for gene amplification by Southern hybridization or dot-blot hybridization, and for gene expression by Northern hybridization, Western immunoblot, or immunohistochemistry. A separate cohort of 324 node-negative breast cancers was assessed for amplification by FISH to determine the utility of HER-2/neu gene amplification. RESULTS: Relative to solid-matrix blotting procedures, FISH analysis of HER-2/neu gene amplification showed a sensitivity of 98% and a specificity of 100% in 140 breast cancers. Among patients treated by surgery only, the relative risks (relative hazard) of early recurrence (recurrent disease within 24 months of diagnosis), recurrent disease (at any time), and disease-related death were statistically significantly associated with amplification. The prognostic information contributed by HER-2/neu amplification was independent of the other markers studied. CONCLUSION: FISH was an alternative technique for determining gene amplification and had some distinct advantages over Southern hybridization. Our results demonstrate that HER-2/neu gene amplification in the absence of adjuvant therapy is an independent predictor of poor clinical outcome and is a stronger discriminant than tumor size. Women with small tumors that had gene amplification were at increased risk of recurrence and disease-related death.

Aged↗

PubMed1996

DNA sequence analysis of exons 2 through 11 and immunohistochemical staining are required to detect all known p53 alterations in human malignancies.

p53 mutations are the most common genetic alterations found in human malignancies. However current estimates of p53 alterations in cancers may be inaccurate because there is evidence that current approaches do not detect all p53 alterations. In this study we determine the status of the p53 gene by complete DNA sequencing of exons 2 through 11 as well as immunohistochemical staining in cohorts of primary human breast, ovarian and non small cell lung cancer. Overall, 24 of 93 (26%) breast cancers, 62 of 108 (57%) ovarian cancers and 88 of 154 (57%) non small cell lung cancers contained DNA sequence mutations, whereas positive immunohistochemical staining was detected in 15 of 64 (23%) breast, 35 of 94 (37%) ovarian, and 63 of 137 (46%) lung cancers. Of those tumors that contained mutations, the mutation occurred outside the 'hot-spot' region in 19% of breast, 18% of ovarian and 17% of lung tumors, indicating that a substantial number of mutations remain undetected in studies that are restricted to exons 5 through 9. We observed a high concordance between the presence of p53 missense mutations and positive immunohistochemical staining, but a poor concordance between other types of mutations and staining in all three types of malignancies. We conclude that a combination of DNA sequence analysis of exons 2 through 11 and immunohistochemical staining are required to detect all known alterations in the p53 gene in human malignancies.

Breast Neoplasms↗

PubMed1996

Prk, a cytokine-inducible human protein serine/threonine kinase whose expression appears to be down-regulated in lung carcinomas.

We have cloned and characterized a putative protein serine/threonine kinase termed prk through a combination of polymerase chain reaction and conventional cDNA library screening approaches. There are apparently two distinct domains within prk protein deduced from its nucleotide sequences. The amino-terminal portion has the feature of the catalytic domain of a serine/threonine kinase and shows strong homology to mouse fnk and other polo family kinases including mouse snk, human and murine plk, Drosophila polo, and yeast Cdc5. The carboxyl-terminal portion, presumably the regulatory domain, shares extensive homology to mouse fnk. Northern blotting analyses reveal that prk expression is restricted to a very limited number of tissues with placenta, ovaries, and lung containing detectable amounts of prk mRNA. prk mRNA expression is also detected at a low level in the megakaryocytic cell line Dami, MO7e, and three brain glioma cell lines. In addition, refeeding of serum-deprived MO7e, Dami, and K562 cells of hematopoietic origin and GMOO637D of lung fibroblasts rapidly activates prk mRNA expression with its peak induction around 2 h after serum addition. prk gene activation by the serum requires no new protein synthesis. The recombinant cytokines such as interleukin-3 and thrombopoietin also activate prk mRNA expression in MO7e cells. Furthermore, a survey of RNAs isolated from the tumor and the uninvolved tissues from 18 lung cancer patients reveals that prk mRNA expression is significantly down-regulated in tumor tissues. Southern blotting analysis indicates that the prk gene is present in a single copy in the genome of tumors and normal cells. Taken together, these results suggest that prk expression may be restricted to proliferating cells and involved in the regulation of cell cycle progression. The molecular cloning of prk cDNA will facilitate the study of its biological role as well as its potential role in tumorigenesis.

Amino Acid Sequence↗