Ann D. Thor

c-erbB-2 Expression and Response to Adjuvant Therapy in Women with Node-Positive Early Breast Cancer

BACKGROUND The role of molecular markers in predicting the response to treatment of breast cancer is poorly defined. The Cancer and Leukemia Group B (CALGB) conducted a randomized adjuvant-chemotherapy trial (CALGB 8541) comparing three doses (high, moderate, and low) of cyclophosphamide, doxorubicin, and fluorouracil in 1572 women with node-positive breast cancer. This study (CALGB 8869) was designed to determine whether the DNA index, the S-phase fraction, c-erbB-2 expression, or p53 accumulation could be used as a marker to identify a subgroup of patients more likely than others to benefit from high doses of chemotherapy. METHODS Tissue blocks were obtained from 442 patients randomly selected from the larger CALGB trial. Paraffin sections from the primary lesions were analyzed for DNA content, S-phase fraction, c-erbB-2 expression, and p53 accumulation. RESULTS Patients randomly assigned to the high-dose regimen of adjuvant chemotherapy had significantly longer disease-free and overall survival if their tumors had c-erbB-2 overexpression. No further information was gained by adding the data on S-phase fraction or p53 accumulation to the analysis. There was no clear evidence of a dose-response effect in patients with minimal or no c-erbB-2 expression. CONCLUSIONS There is a significant dose-response effect of adjuvant chemotherapy with cyclophosphamide, doxorubicin, and fluorouracil in patients with overexpression of c-erbB-2 but not in patients with no c-erbB-2 expression or minimal c-erbB-2 expression. Overexpression of c-erbB-2 may be a useful marker to identify the patients who are most likely to benefit from high doses of adjuvant chemotherapy.

Loss of The Retinoblastoma Tumor-Suppressor Gene in Parathyroid Carcinoma

BACKGROUND The origin and molecular pathogenesis of parathyroid carcinoma are unknown. This life-threatening cause of primary hyperparathyroidism cannot be reliably distinguished from its benign counterpart on the basis of histopathological features alone. Because the PRAD1, or cyclin D1, gene, a cell-cycle regulator, has been implicated in a subgroup of benign parathyroid tumors, we examined the possibility that another cell-cycle regulator with possible functional links to PRAD1, the retinoblastoma tumor-suppressor gene (RB), might be involved in the molecular pathogenesis of parathyroid carcinoma. METHODS Parathyroid carcinomas from 9 patients and adenomas from 21 were studied for evidence of tumor-specific loss of RB gene DNA (allelic loss) by analysis of four DNA polymorphisms and for evidence of altered expression oF RB protein by immunohistochemical staining. RESULTS All of 11 specimens from 5 patients with parathyroid carcinoma and informative DNA patterns and 1 of 19 specimens from 19 patients with parathyroid adenoma and informative DNA patterns lacked an RB allele. Fourteen of 16 specimens (88 percent) from the nine patients with carcinoma had abnormal expression of RB protein (a complete or predominant absence of nuclear staining for the protein). None of the 19 adenomas, including the tumor with loss of an RB allele, had unequivocally abnormal staining for RB protein. CONCLUSIONS Inactivation of the RB gene is common in parathyroid carcinoma and is likely to be an important contributor to its molecular pathogenesis. The presence of such inactivation may help to distinguish benign from malignant parathyroid disease and may have useful diagnostic, prognostic, and therapeutic implications.

Comparative Study of p53 Gene and Protein Alterations in Human Astrocytic Tumors

The p53 gene is a tumor suppressor gene involved in many common malignancies, including astrocytomas. Genetic analysis of the p53 gene and immunohistochemistry of the p53 protein have each been used to screen astrocytomas. To compare these methods, we performed immunohistochemistry with the monoclonal antibody PAb 1801 and single-strand conformational polymorphism (SSCP) with sequence analysis on 34 astrocytic tumors (WHO grades II, III and IV). Seven cases had detectable p53 protein and gene mutations, while twelve cases had neither detectable protein nor gene mutations. Four tumors had frameshift mutations in the p53 gene that were not revealed by immunohistochemistry. One tumor had a genetic polymorphism and no detectable p53 protein. Ten tumors had p53 protein accumulation but no mutations by SSCP; these cases may represent p53 mutations outside of the conserved exons or elevated levels of wild-type p53 protein. Thus, some p53 mutations are missed with PAb 1801 immunohistochemistry alone. p53 immunohistochemistry, however, may reveal p53 accumulation independent of mutations in the conserved portions of the gene. Finally, we suggest that glioblastomas with p53 mutations in the conserved region of the gene may be a subset that are more common in women and in younger patients.

Proliferating cell nuclear antigen and Ki-67 immunohistochemistry in brain tumors: A comparative study

SummaryProliferating cell nuclear antigen (PCNA) is a 36-kDa DNA polymerase-σ auxiliary protein which accumulates in the nucleus during S phase of the cell cycle. Immunohistochemical labeling indices (LI) of PCNA and Ki-67 were compared using an avidin-biotin complex method on frozen sections of 27 nervous system tumors, 3 normal cerebral cortices, and 3 peripheral nerves. In glial tumors, PCNA and Ki-67 LI increased with increasing tumor grade (Daumas-Duport system). In 5 low-grade glial tumors, PCNA and Ki-67 LI were ≤1%, except for one optic nerve glioma (Ki-67 LI=6%). In 7 grade 3 astrocytomas, and 1 mixed glioma, PCNA LI were ≤1–1.5%, while Ki-67 LI were 2%–10%. In 7 grade 4 astrocytomas and 1 metastatic carcinoma, PCNA LI ranged from 6%–15% while Ki-67 LI ranged from 17%–30%. In 5 of 6 schwannomas, focally high PCNA LI (4%–65%) were noted, despite low LI with Ki-67 (≤1.6%). Scattered normal schwann cell nuclei also stained with PCNA, but normal cerebral cortex did not. These data suggest that: (1) in higher-grade gliomas, PCNA may be a more specific S-phase marker, although a less sensitive proliferation marker, than Ki-67; (2) PCNA LI do not distinguish low-grade gliomas from grade 3 astrocytomas; (3) in schwannomas, PCNA may not reflect proliferative activity since it seems to react with an epitope present in normal schwann cells; and (4) the variable PCNA staining pattern introduces greater difficulties in cell counting than with Ki-67. These factors may limit the use of this anti-PCNA antibody in evaluating nervous system tumors.

Monoclonal antibody DF3 correlates with tumor differentiation and hormone receptor status in breast cancer patients.

SummaryThe murine monoclonal antibody (MAb) designated DF3, reacts with a 300-kd human mammary epithelial antigen which is expressed on apical borders of secretory mammary epithelial cells and in the cytosol of less differentiated malignant cells. Human mammary tumors have been evaluated for the level of DF3 antigen as a correlate to clinicopathologic parameters related to degree of tumor differentiation: nuclear grade (NG), histologic grade (HG), and estrogen receptor status (ER). More DF3 antigen was present in breast carcinomas with NG 1 and 2 as compared to tumors with NG 3 (p = .002). Similarly DF3 antigen presence was greater in HG 1 and 2 tumors than in HG 3 (p<.001). The results also demonstrate that quantitative differences in the presence of the DF3 differentiation antigen correlate with estrogen receptor status. Twenty-two of 23 ER positive tumors were also DF3 positive. Only 6 of 23 ER negative tumors were reactive to MAb DF3 (p<.001). There was, however, no correlation between DF3 reactivity and absolute levels of estrogen or progesterone receptor.These findings confirm our hypothesis that MAb DF3 reacts to a differentiation antigen present in some human breast carcinomas. The DF3 antigen phenotype can serve as an independent phenotypic marker with correlations to standard indicators of degree of differentiation and estrogen receptor status of infiltrating ductal carcinomas of the breast, and should thus be evaluated as a prognostic indicator in breast cancer patients. The data also suggests that DF3 histochemistry may be a useful alternative in assessing estrogen receptor status of small breast cancers where there is an insufficient amount of tumor present for biochemical assay of hormone receptor levels.

Ets regulation of the erbB2 promoter.

Evaluating the chromatinized erbB2 gene in nuclei from breast cancer cells expressing varying levels of ErbB2 transcripts, we identified a nuclease-sensitive site within a 0.22 kb region of maximum enhancer activity centered over a conserved 28 bp polypurine(GGA)-polypyrimidine(TCC) mirror-repeat and an adjacent essential Ets binding site (EBS). Promoter footprinting with nuclear extracts reveals an intense Ets hypersensitivity site at the EBS whose degree of intensity correlates with the level of cellular ErbB2 expression. In vitro mapping assays show that the supercoiled erbB2 promoter forms an internal triplex structure (Hr-DNA) at the mirror-repeat element. Mutations preventing Hr-DNA formation can enhance erbB2 promoter activity in human breast cancer cells, a result consistent with previous demonstration that Ets-erbB2 promoter complexes cannot form when the mirror-repeat is engaged in triplex binding, and new results suggesting that Ets binding induces severe promoter bending that may restrict local triplex formation. In addition to previously described erbB2-regulating breast cancer Ets factors (PEA3, ESX/Elf-3), Elf-1 is now shown to be another endogenously expressed Ets candidate capable of binding to and upregulating the erbB2 promoter. Given current strategies to transcriptionally inhibit ErbB2 overexpression, including development of novel erbB2 promoter-targeted therapeutics, an EBS-targeted approach is presented using chimeric Ets proteins that strongly repress erbB2 promoter activity.

p53 gene analysis of ovarian borderline tumors and stage I carcinomas

Mutations of the p53 gene are common in human ovarian carcinomas; however, their role in the early development of ovarian cancer is unclear. Twelve ovarian borderline tumors (BTs; eight of them p53 immunopositive) and 10 stage I carcinomas (four of them p53 immunopositive) were studied for genetic alterations in the p53 gene. The study was based on single-strand conformation polymorphism (SSCP) analysis and DNA sequencing of exons 2 through 11 of the p53 gene using DNA preparations from microdissected tumors. Mutations were found in 40% of the carcinomas (including a borderline component adjacent to carcinoma in one lesion) but in none of the pure BTs. These findings suggest that p53 mutations may not be commonly associated with the borderline phenotype of ovarian epithelial tumors but may occur during malignant transformation.

Ovarian endometrioid carcinomas resembling sex cord-stromal tumors: an immunohistochemical study

Ovarian endometrioid carcinomas resembling sex cord-stromal tumors (ECSCSs) may simulate Sertoli cell tumors, Sertoli-Leydig cell tumors (SLCTs), and adult granulosa cell tumors (AGCTs), both clinically and pathologically. Differing clinical features and histologic findings are almost always successful in distinguishing these tumor types, although in some cases the differential diagnosis is difficult. Immunohistochemical staining of 17 ECSCSs, 14 Sertoli cell tumors or SLCTs, and 15 AGCTs was performed with the use of antibodies against cytokeratins (AE1/AE3, 902, and CAM 5.2), epithelial tumor-associated antigens (EMA, OM-1, B72.3, and carcinoembryonic antigen B1.1), vimentin, S-100, neuron-specific enolase, and lysozyme to determine the immunohistochemical profile of each tumor type and to define further the nature of the sex cord-like components in ECSCSs. All 17 ECSCSs, none of the 15 AGCTs, and one of 14 Sertoli cell tumors or SLCTs stained with EMA. Staining for OM-1 was almost as helpful diagnostically, with positive results for of 17 ECSCSs, 0/15 AGCTs, and 1/14 Sertoli cell or SLCTs. Antikeratins were immunoreactive with all the ECSCSs as well as some of the AGCTs and Sertoli cell tumors or SLCTs. The B72.3 and B1.1 were immunoreactive with some ECSCSs and Sertoli cell tumors, but were nonreactive with AGCTs. Neuron-specific enolase was demonstrated in 11 of 17 ECSCSs, two of 14 Sertoli cell tumors or SLCTs, and 0 of 15 AGCTs. Vimentin, S-100, and lysozyme were least helpful in the differential diagnosis. These studies suggest that an immunohistochemical approach may be useful in the differentiation of ECSCSs and sex cord–stromal tumors. Furthermore, it supports the conclusion that the sex cord-like cells in ECSCSs are not Sertoli or granulosa cells, but cells of surface epithelial type growing in architectural patterns similar to those of sex cord–stromal tumors.

pS2 expression in primary breast carcinomas: Relationship to clinical and histological features and survival

SummarypS2 protein expression has been reported to have prognostic significance in human breast carcinomas and to correlate with estrogen receptor positivity, although these findings have not been confirmed by all investigators. pS2 positivity was compared to various clinical and histologic parameters in a retrospective study of 290 patients (median follow-up 7.2 years) and significantly correlated with tumor grade and estrogen receptor content (p=0.001 and p=0.0007, respectively). Significant associations between pS2 positivity and lymph node metastases, T stage, histologic tumor type, and patient age were not observed. Univariate and multivariate analyses (controlling for estrogen receptor content, T and N stage) of the patient population at large showed that pS2 positivity was not predictive of disease-free or overall survival. Univariate analysis of lymph node negative patients demonstrated that both pS2 and estrogen receptor positivity were significantly associated with a better outcome. Multivariate analysis of these patients, however, showed that only estrogen receptor data had independent prognostic significance. This study suggests that immunohistochemical analysis for pS2 protein expression will not contribute additional prognostic information if the estrogen receptor content is known.

Monoclonal antibodies reactive with human breast or ovarian carcinoma: in vivo applications.

Monoclonal antibodies (MoAbs) are unique and useful bioprobes that allow in vivo targeting of membrane-associated or circulating antigens. Most of the clinical trials to date have used low dosages of radiolabeled MoAb given in a single dose. Newer studies have included antibody fragments, repeated injections, intraperitoneal (IP) administration, and other labels such as 90Y. Clinical MoAb trials are often arduous, expensive, and time-consuming to perform. Before human use, animal studies and extensive MoAb characterization are required. The production of pharmaceutical grade, radiolabeled MoAb is technically difficult and costly. Clinical trials require administrative and patient consent as well as extensive written protocols. These studies necessitate interdepartmental and intradepartmental cooperation and coordination. Furthermore, the use of in vivo radiolabeled probes impacts many levels of health care providers from janitorial, nursing, and technical staff to laboratories and physicians. Simple blood tests or disposal of body excretions may concern nursing or technical staff with the possibility of radiation exposure. The responsibility for study design, personnel involvement, and prospective use in patients without a definitive cancer diagnosis ultimately rests with the physician. While many issues have been addressed, additional clinical trials, consideration of safety issues, and standardization between institutions will be necessary before the use of radiolabeled MoAb for diagnosis, management, or therapy of human tumors becomes routine. Continued cooperation and funding should ensure its achievement.