Overexpression of cyclin D mRNA distinguishes invasive and in situ breast carcinomas from non-malignant lesions.

The elucidation of molecular alterations that occur during human breast cancer progression may contribute to the development of preventative strategies. Using in situ hybridizations on a cohort of 94 biopsy lesions, quantitatively increased cyclin D mRNA expression levels were observed in only 18% of benign lesions, which confer no or slightly increased breast cancer risk, and 18% of premalignant atypical ductal hyperplasias, which confer a four to fivefold increase in breast cancer risk. The transition to carcinoma was accompanied by frequent cyclin D mRNA overexpression in 76% of low-grade ductal carcinomas in situ, 87% of higher grade comedo ductal carcinomas in situ and 83% of infiltrating ductal breast carcinomas. The data identify a molecular event that may separate benign and premalignant human breast lesions from any form of breast carcinoma.

Advantage of dose fractionation in monoclonal antibody-targeted radioimmunotherapy.

Monoclonal antibody (MAb) B72.3 IgG was radiolabeled with 131I and administered to female athymic NCr-nu mice bearing the LS-174T human colon adenocarcinoma xenograft to determine if fractionation of MAb dose had any advantage in tumor therapy. In the LS-174T xenograft, only approximately 30%-60% of tumor cells express the B72.3-reactive TAG-72 antigen. The LS-174T xenograft was used to reflect the heterogeneity of the TAG-72 antigen often seen in biopsy specimens from patients. In contrast to a single 600-muCi dose of 131I-B72.3 IgG where 60% of the animals died from toxic effects, two 300-muCi doses of 131I-B72.3 IgG (total of 600 muCi) reduced or eliminated tumor growth in 90% of mice, with only 10% of the animals dying from toxic effects. Dose fractionation even permitted escalation of the dose to three doses (each 1 wk apart) of 300 muCi of 131I-B72.3 IgG (for a total of 900 muCi), resulting in even more extensive tumor reduction or elimination and minimal toxic effects. The use of an isotype-matched control MAb revealed a nonspecific component to tumor growth retardation, but the use of the specific B72.3 IgG demonstrated a much greater therapeutic effect. Tumors that had escaped MAb therapy were analyzed for expression of the B72.3-reactive TAG-72 antigen with the use of the immunoperoxidase method; they were shown to have the same antigenic phenotype as the untreated tumors. We verified tumor elimination by killing the test animals after a 7-week observation period and performing histologic examination of tumor sites. We also monitored toxic effects by histologic examination of numerous organs, including bone marrow. These studies thus demonstrate the advantage of dose fractionation of a radiolabeled MAb for tumor therapy. We anticipate that the concept of dose fractionation can be practically applied in radioimmunotherapeutic clinical trials with the development and use of recombinant-chimeric MAbs and modified constructs.

Selective interferon-induced enhancement of tumor-associated antigens on a spectrum of freshly isolated human adenocarcinoma cells.

Freshly isolated cells from patients with pleural or peritoneal effusions cytologically diagnosed as adenocarcinoma (n = 43), malignant nonepithelial neoplasms (n = 10), and benign (n = 8) were analyzed for expression of constitutive levels of the tumor antigens TAG-72 [recognized by monoclonal antibody (MAb) B72.3] and carcinoembryonic antigen (CEA) (recognized by MAb COL-4) as well as the class I and class II major histocompatibility (MHC) antigens, and the ability of human interferons (Hu-IFNs) to enhance cell surface expression of those antigens as measured by MAb binding. Both type I and type II IFNs enhanced the expression of TAG-72 and CEA and altered the level of expression of the MHC antigens. Comparative studies of three different Hu-IFNs (IFN-alpha A, IFN-beta ser, and IFN-gamma) revealed that IFN-gamma was the most potent in augmenting either B72.3 or COL-4 binding. Unlike the IFN-gamma -mediated induction of the class II human leukocyte antigens, the change in tumor antigen expression consisted of enhanced constitutive antigen expression; de novo induction of either TAG-72 or CEA could not be achieved by either type I or type II IFN. Of 43 effusions isolated from different adenocarcinoma patients, 42 (97.7%) expressed either CEA or TAG-72, and treatment with Hu-IFN increased the level of expression of either antigen in 36 of 42 samples (85.7%). These studies demonstrate the augmentation of tumor-associated antigens on human carcinoma cells isolated from serous effusions by Hu-IFNs which may be used to enhance the targeting of conjugated MAbs to human carcinoma lesions.

Transforming growth factor-beta and breast cancer risk in women with mammary epithelial hyperplasia.

BACKGROUND: Transforming growth factors-beta (TGF-betas) regulate mammary epithelial cell division. Loss of expression of TGF-beta receptor II (TGF-beta-RII) is related to cell proliferation and tumor progression. Breast epithelial hyperplastic lesions lacking atypia (EHLA) are associated with a mild elevation in breast cancer risk. We investigated the expression of TGF-beta-RII in EHLA and the risk of subsequent invasive breast cancer. METHODS: We conducted a nested case-control study of women with biopsy-confirmed EHLA who did not have a history of breast cancer or atypical hyperplasia of the breast. Case patients (n = 54) who subsequently developed invasive breast cancer were matched with control patients (n = 115) who did not. Formalin-fixed, paraffin-embedded sections of breast biopsy specimens of all 169 patients with EHLA were studied by immunohistochemical analysis with antibodies against TGF-beta-RII. All P values are two-sided. RESULTS: Women with breast EHLA and 25%-75% TGF-beta-RII-positive cells or less than 25% TGF-beta-RII-positive cells had odds ratios of invasive breast cancer of 1.98 (95% confidence interval [CI] = 0.95-4.1) or 3.41 (95% CI = 1.2-10.0), respectively (P for trend =.008). These risks are calculated with respect to women with EHLA that had greater than 75% TGF-beta-RII expression. Women with a heterogeneous pattern of TGF-beta-RII expression in their normal breast lobular units and either greater than 75%, 25%-75%, or less than 25% positive cells in their EHLA had odds ratios for breast cancer risk of 0.742 (95% CI = 0.3-1.8), 2.85 (95% CI = 1.1-7.1), or 3.55 (95% CI = 1.0-10.0), respectively (P for trend =.003). These risks are relative to women with a homogeneous pattern of expression in their normal lobular units and greater than 75% positive cells in their EHLA. CONCLUSION: This study indicates that loss of TGF-beta-RII expression in epithelial cells of EHLA is associated with increased risk of invasive breast cancer.

Enhanced tumor binding using immunohistochemical analyses by second generation anti-tumor-associated glycoprotein 72 monoclonal antibodies versus monoclonal antibody B72.3 in human tissue.

Monoclonal antibody (MAb) B72.3 was generated using a membrane-enriched fraction of a human mammary carcinoma biopsy. It has demonstrated reactivity to the majority of human adenocarcinomas including colorectal, gastric, pancreatic, ovarian, endometrial, mammary, and nonsmall cell lung cancer as well as weak or nondetectable reactivity to the majority of normal adult tissues, with the exception of secretory endometrium. Radiolabeled B72.3 has demonstrated MAb localization of carcinoma in approximately 70% of several hundred colorectal and ovarian carcinoma patients. The B72.3-reactive antigen, tumor-associated glycoprotein 72, has been purified from a human colon cancer xenograft and used as an immunogen to generate second generation MAbs. Twenty-eight of these MAbs, designated CC (colon cancer), were shown to be reactive with tumor-associated glycoprotein 72; direct-binding radioimmunoassays, Western blotting, live cell surface binding assays, liquid competition radioimmunoassays, and affinity constant measurements distinguished CC MAbs from each other and from B72.3. Two of these MAbs, CC49 and CC112, were selected for further immunohistochemical characterization. These MAbs were tested here against a spectrum of normal, benign, and malignant human adult tissues using the avidin-biotin-peroxidase technique, and their reactivity was compared with B72.3. Both CC MAbs were more reactive than B72.3 against a range of tumors. Extensive testing with MAbs CC49 and B72.3 using serial tissue sections demonstrated that both MAbs reacted similarly to most normal adult tissues with MAb CC49 reacting stronger to inflammatory colonic tissue. In 35 of 48 (72%) carcinoma biopsies of the gastrointestinal tract, ovary, breast, and lung in which one of the MAbs reacted to at least 20% of the cells, CC49 reacted to a greater percentage of carcinoma cells and/or tumor-associated mucin than B72.3. The reciprocal was observed in only 2% of the carcinomas. This study thus provides evidence that these second generation anti-tumor-associated glycoprotein MAbs may be more efficient than B72.3 in the further study of human carcinoma cell populations and in the diagnostic and therapeutic procedures presently being pursued with MAb B72.3.

Epidermal growth factor receptor (HER1) tyrosine kinase inhibitor ZD1839 (Iressa) inhibits HER2/neu (erbB2)-overexpressing breast cancer cells in vitro and in vivo.

Aberrrant signaling by the epidermal growth factor receptor [EGFR (HER1, erbB1)] and/or HER2/neu tyrosine kinases is present in a cohort of breast carcinomas. Because HER2 is constitutively phosphorylated in some breast tumors, we speculated that, in these cancers, transmodulation of HER2 may occur via EGFR signaling. To test this possibility, we examined the effect of EGFR-specific kinase inhibitors against the HER2-overexpressing human breast tumor lines BT-474, SKBR-3, MDA-361, and MDA-453. ZD1839 (Iressa) is an ATP-mimetic that inhibits the purified EGFR and HER2 kinases in vitro with an IC(50) of 0.033 and >3.7 microM, respectively. The specificity of ZD1839 against EGFR was confirmed in Rat1 fibroblasts transfected with EGFR or HER2 chimeric receptors activated by synthetic ligands without the interference of endogenous receptors. Treatment of all breast cancer cell lines (except MDA-453) with 1 microM ZD1839 almost completely eliminated HER2 phosphorylation. In contrast, the incorporation of [gamma-(32)P]ATP in vitro onto HER2 receptors isolated from BT-474 cells was unaffected by 1 microM ZD1839. EGFR is expressed by BT-474, SKBR-3, and MDA-361 but not by MDA-453 cells, suggesting that ZD1839-mediated inhibition of the EGFR kinase explained the inhibition of HER2 phosphorylation in vivo. In SKBR-3 cells, ZD1839 exhibited a greater growth-inhibitory effect than Herceptin, a monoclonal antibody against the HER2 ectodomain. In both SKBR-3 and BT-474 cells, treatment with ZD1839 plus Herceptin induced a greater apoptotic effect than either inhibitor alone. Finally, ZD1839 completely prevented growth of BT-474 xenografts established in nude mice and enhanced the antitumor effect of Herceptin. These data imply that EGFR tyrosine kinase inhibitors will be effective against HER2-overexpressing breast tumor cells that also express EGFR and support their use in combination with HER2 antibodies, such as Herceptin, against mammary carcinomas with high levels of the HER2 proto-oncogene.

Serological mapping of the TAG-72 tumor-associated antigen using 19 distinct monoclonal antibodies.

Monoclonal antibody (MAb) B72.3 has been shown to be of potential utility in the management of human carcinoma via its use in (a) the targeting of carcinoma lesions in colorectal and ovarian cancer patients, (b) immunohistochemical analyses of biopsies and effusions, and (c) serum assays to help define the presence of carcinoma. The B72.3-reactive antigen, designated tumor-associated glycoprotein 72 (TAG-72), has been characterized as a high molecular weight glycoprotein with the properties of a mucin. We report here the utilization of MAb B72.3 and 18 second generation MAbs (generated using purified TAG-72 obtained from a colon carcinoma xenograft as immunogen) to construct a serological map of the TAG-72 molecule. The generation and initial characterization of 10 of the second generation MAbs have been described previously; in addition, eight previously unreported MAbs were used. All 19 MAbs produced immune precipitate lines against purified TAG-72 in double immunodiffusion, indicating that each epitope recognized by a single MAb is present at least twice on the TAG-72 molecule. Immunodepletion analyses utilizing 11 of the anti-TAG-72 MAbs indicated that each recognizes the same molecule or population of molecules. Nineteen competition radioimmunoassays were developed and 19 purified competitor immunoglobulins were used in each assay. The patterns of cross-competition indicated the presence of a complex array of tumor-associated epitopes on the TAG-72 molecule. Some of the MAbs recognized epitopes that were structurally or spatially related to one another, but none appeared to recognize identical epitopes. The spectrum of inhibitory reactivities of these MAbs for TAG-72 binding varied from extremely restricted to more broad inhibition. The serological mapping studies reported here provide information as to the range and nature of the epitopes expressed on the TAG-72 molecule, help form the basis for selecting alternative anti-TAG-72 MAbs for use in potential clinical applications, and further define the nature of this oncofetal antigen.

Characterization of the colorectal carcinoma-associated antigen defined by monoclonal antibody D612.

Monoclonal antibody (MAb) D612 recognizes an antigen expressed on the cell surface of normal and malignant gastrointestinal epithelium. It is a murine IgG2a/kappa which has been previously shown to mediate killing of human colon carcinoma cells using human effector cells (which could be enhanced in the presence of interleukin-2). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of MAb D612 immunoprecipitates of extracts of L-[35S]methionine-, L-[3H]leucine-, and D-[3H]glucosamine-labeled human colon carcinoma cells showed that the D612 antigen is a Mr 48,000 glycoprotein. Similar estimates of molecular mass were obtained from SDS-PAGE analyses of MAb D612 immuno-precipitates of radioiodinated extracts of surgically resected colon carcinoma and adjacent normal colonic mucosa. D612 antigen was not detectable in immunoprecipitates of supernatant media from radiolabeled cell cultures, suggesting that the antigen is not readily shed from the surface of cultured cells. The D612 antigen was shown to be clearly distinct from previously described gastrointestinal carcinoma-associated glycoproteins: the D612 antigen shows a migration pattern of SDS-PAGE distinct from those of the antigens recognized by MAbs KS1/4 and GA733, and reciprocal immunodepletion analyses of D-[3H]glucosamine-labeled colon carcinoma cells utilizing MAbs D612 and GA733 revealed no cross-reactivity between these antibodies. Similarly, competitive binding studies between MAbs 17-1A and KS1/4 and MAb D612 revealed no similarity between the epitopes recognized by MAb D612 and MAbs 17-1A and KS1/4. MAbs D612 and 17-1A were also titered in immunoperoxidase staining assays on serial frozen sections of normal and malignant colon. MAb D612 showed a higher titer of immunostaining reactivity with both normal and malignant colon than did MAb 17-1A. MAb D612 showed roughly equivalent immunostaining titers against normal and malignant colon; whereas MAb 17-1A showed higher titer of immunostaining reactivity against the normal colon tissue than against the malignant colon. Flow cytometric analysis of phosphatidylinositol-specific phospholipase C-treated colon carcinoma cells revealed no loss of D612 antigen from the cell surface, suggesting that the mechanisms of attachment of the D612 antigen to the cell surface does not involve linkage to a phosphatidylinositol glycan.(ABSTRACT TRUNCATED AT 400 WORDS)

Mice transgenic for human carcinoembryonic antigen as a model for immunotherapy.

Mice transgenic for the human carcinoembryonic antigen (CEA) gene were prepared for use as a preclinical model for immunotherapy. A 32.6-kb fragment containing the complete human CEA gene and flanking sequences was isolated from a genomic cosmid clone and used to produce transgenic C57BL/6 mice. A homozygous line was established that was designated C57BL/6J-TgN(CEAGe)18FJP. Southern blot analysis showed that this line contained intact copies of the cosmid clone, with approximately 19 integrated copies at one chromosomal location. A mouse-human chimeric anti-CEA monoclonal antibody was used to examine CEA expression by immunohistochemical staining of frozen tissue sections. In the cecum and colon, approximately 20% of the luminal epithelial cells had strong cytoplasmic staining, whereas occasional glands showed intense staining. CEA was also expressed in gastric foveolar cells, whereas small intestine villi had only a few (<1%) positive cells. CEA was not found by immunohistochemistry in other tissues of the digestive tract, nor was it found in a wide range of other tissues or organs. Concordance in results was obtained between immunohistochemistry and analysis of tissue extracts by enzyme immunoassay. The lone exception was the testis, which was positive only by enzyme immunoassay. Expression of human CEA was not observed in tissues derived from nontransgenic mice. The fecal content of CEA in transgenic mice was approximately 100-fold less than that observed for humans. Circulating CEA was not detected. A CEA-transfected syngeneic murine colon carcinoma cell line, MC-38, was prepared that had stable expression of CEA in vitro and in vivo. The molecular size of CEA produced by CEA-transfected MC-38 cells and by the colon of transgenic mice was similar to that obtained with CEA purified from human colon tumors. Anti-CEA antibody appeared in nontransgenic but not transgenic mice bearing transfected MC-38 tumors. These findings demonstrate that CEA distribution and its properties in tissues of mice transgenic for the human CEA gene are similar to that observed in human tissues. As in humans, immune responsiveness to CEA, as reflected by antibody formation, was not detectable in transgenic mice bearing CEA-positive tumors. Thus, CEA transgenic mice may serve as a useful model for studying the efficacy and safety of various immunotherapy strategies directed at this tumor self-antigen.

p27/Kip1 mutation found in breast cancer.

The p27/Kip1 protein belongs to the recently identified family of proteins called cyclin-dependent kinase inhibitors. These proteins play an important role as negative regulators of cell cycle-dependent kinase activity during progression of the cell cycle. Since cyclin-dependent kinase inhibitors can inhibit cell proliferation, they may have a role as tumor suppressor genes. To determine whether p27 alterations may be involved in tumorigenesis, we examined its mutational status in 36 primary breast carcinomas and 9 breast cancer cell lines using PCR-single-strand conformational polymorphism, direct DNA sequencing, and Southern blot analysis. Southern blot analysis showed no homozygous deletions of the p27 gene in either the clinical samples or cell lines. Two point mutations were found in primary tumors. One represents a previously undescribed polymorphism at codon 142; another is a nonsense mutation at codon 104. The latter mutation was absent in the normal matched control sample, and, in addition, it was accompanied with the loss of heterozygosity (LOH) of a microsatellite marker in the vicinity of the p27 gene on chromosome 12p13. These data indicate that p27 mutations are a rare event in breast cancer, but may play an important role in the development of a minority of these cancers. Furthermore, LOH analysis of the 12p13 locus revealed that an additional four of six matched DNA samples had LOH at 12p13 but did not have an alteration of the p27 gene, suggesting that another tumor suppressor gene is located on the short arm of human chromosome 12 which may be frequently involved in the pathogenesis of breast cancers.

Generation and characterization of B72.3 second generation monoclonal antibodies reactive with the tumor-associated glycoprotein 72 antigen.

Monoclonal antibody (MAb) B72.3 was generated using a membrane-enriched fraction of a human mammary carcinoma biopsy. It has demonstrated reactivity to the majority of human adenocarcinomas including colorectal, gastric, pancreatic, ovarian, endometrial, mammary, and non-small cell lung cancer and no or weak reactivity to normal adult tissues, with the exception of secretory endometrium. The B72.3-reactive antigen, termed tumor-associated glycoprotein (TAG)-72, has been purified and used as an immunogen to generate B72.3 second generation MAbs. Since the source of purified TAG-72 was a human colon cancer (CC) xenograft, these MAbs have been given a CC designation. Twenty-eight CC MAbs, all immunoglobulin Gs, have been generated and shown to be reactive with TAG-72 and via both radioimmunoassay and immunohistochemical analyses show differential reactivity to carcinoma versus normal adult tissue biopsies. Nine CC MAbs (CC11, 15, 29, 30, 40, 46, 49, 83, and 92) were selected for further characterization. As a result of analyses using direct-binding radioimmunoassay to a range of human carcinomas, Western blotting, live cell surface binding assays, five liquid competition radioimmunoassays, and Ka measurements, all nine CC MAbs could be distinguished from each other and from B72.3. The Ka of B72.3 was determined to be 2.54 X 10(9) M-1; all the CC MAbs demonstrated higher KaS with MAbs CC92, 49, and 83 having KaS of 14.26, 16.18, and 27.72 X 10(9) M-1, respectively. These studies thus demonstrate that one or more of the anti-TAG-72 CC MAbs may be more efficient than B72.3, or useful in combination with B72.3, toward the further study of human carcinoma cell population and the diagnostic and therapeutic procedures presently utilizing MAb B72.3.

Inhibition of HER2/neu (erbB-2) and mitogen-activated protein kinases enhances tamoxifen action against HER2-overexpressing, tamoxifen-resistant breast cancer cells.

HER2/neu (erbB-2) overexpression has been causally associated with tamoxifen resistance in human breast cancer cells. Forced expression of HER2 in MCF-7 breast cancer cells resulted in mitogen-activated protein kinase (MAPK) hyperactivity and tamoxifen resistance. Inhibition of HER2 and MAPKs with AG1478 and U0126, respectively, as well as dominant-negative MEK-1/2 constructs restored the inhibitory effect of tamoxifen on estrogen receptor (ER)-mediated transcription and cell proliferation. Both AG1478 and U0126 also restored the tamoxifen-mediated association of ER with nuclear receptor corepressor (N-CoR) in the antiestrogen-resistant MCF-7 cells. Treatment with a combination of tamoxifen and a HER2 kinase inhibitor reduced tumor MAPK activity and markedly prevented growth of HER2-overexpressing MCF-7 xenografts in athymic mice. Thus, blockade of HER2 and MAPK signaling may enhance tamoxifen action and abrogate antiestrogen resistance in human breast cancer.

Intraperitoneal administration of interferon-gamma to carcinoma patients enhances expression of tumor-associated glycoprotein-72 and carcinoembryonic antigen on malignant ascites cells.

PURPOSE: The study was designed to determine whether in vivo interferon gamma (IFN-gamma) administration could enhance tumor antigen expression on the surface of human tumor cells. MATERIALS AND METHODS: Eight patients (six with ovarian and two with gastrointestinal tumors) with a diagnosis of adenocarcinoma with secondary malignant ascites were given weekly escalating doses of IFN-gamma (ie, 0.1 to 100 MU) intraperitoneally (IP) each week for 8 weeks. Tumor cells were isolated from the patients' ascites samples, which were collected three times per week: before and 24 and 48 hours post-IFN-gamma administration. The level of expression of tumor-associated glycoprotein-72 (TAG-72) and carcinoembryonic antigen (CEA) was measured using flow cytometry and immunocytochemistry. RESULTS: IFN-gamma administered IP dramatically increased TAG-72 (as measured by binding of anti-TAG-72 monoclonal antibodies [MoAbs] B72.3 and CC 49) and CEA (measured by MoAb COL-1) expression on the surface of the carcinoma cells. The ascites-derived tumor cells from seven of the eight patients constitutively expressed TAG-72, and the level of TAG-72 expression was increased by IFN-gamma in all seven patients, as evidenced by the enhanced binding of anti-TAG-72 MoAbs to the tumor-cell surface. In some cases, IFN-gamma treatment increased the percentage of MoAb B72.3-reactive tumor cells from 10% to greater than 90%, and those changes were further corroborated by similar increases in the MoAb staining intensity observed with immunoperoxidase analysis. In addition, ascites-derived tumor cells from two patients with gastrointestinal carcinoma also expressed enhanced CEA levels after IFN-gamma. The increased TAG-72 and CEA expression were observed at low IFN-gamma doses (ie, 0.1 to 1.0 MU), which were well tolerated by all patients. CONCLUSIONS: The ability of IFN-gamma given IP to increase TAG-72 and CEA expression on tumor cells in vivo provides additional argument for the use of the cytokine as an adjuvant to enhance MoAb binding to human carcinoma-cell populations.

Prognostic value of histologic grade and proliferative activity in axillary node-positive breast cancer: results from the Eastern Cooperative Oncology Group Companion Study, EST 4189.

PURPOSE: The identification of a subset of patients with axillary lymph node-positive breast cancer with an improved prognosis would be clinically useful. We report the prognostic importance of histologic grading and proliferative activity in a cohort of patients with axillary lymph node-positive breast cancer and compare these parameters with other established prognostic factors. PATIENTS AND METHODS: This Eastern Cooperative Oncology Group laboratory companion study (E4189) centered on 560 axillary lymph node-positive patients registered onto one of six eligible clinical protocols. Flow cytometric (ploidy and S-phase fraction [SPF]) and histopathologic analyses (Nottingham Combined Histologic Grade and mitotic index) were performed on paraffin-embedded tissue from 368 patients. RESULTS: Disease recurred in 208 patients; in 161 (77%), within the first 5 years. Mitotic index and grade were associated with both ploidy and SPF (P

Isolation of large numbers of fully viable human neutrophils: a preparative technique using percoll density gradient centrifugation.

Techniques were developed for the isolation of human neutrophils from large volumes of human blood. This preparative procedure avoids exposure to Ficoll/Hypaque and offers a low cost alternative to centrifugal elutriation. First, leukocyte-enriched suspensions were obtained by treating buffy coats with 2% dextran. The leukocyte suspensions were then fractionated on three steps Percoll density gradients. The gradients were designed to carry large numbers of cells while maintaining resolution of the cell peaks. As a result, at least 320 X 10(6) leukocytes could be processed on a single 12 ml gradient. Extensive testing of the procedure showed that the purity of the neutrophil band was 96.5 +/- 3.16% (n = 50). The band contained 82.4 +/- 14.6% (n = 39) of the neutrophils applied to the gradient. Viability assays (bacterial killing, chemotaxis) demonstrated that neutrophils isolated on Percoll gradients were just as active as those harvested by centrifugal elutriation. Recovery data are presented and compared to the results obtained by others.

A high-risk lesion for invasive breast cancer, ductal carcinoma in situ, exhibits frequent overexpression of retinoid X receptor.

The development of prevention strategies for breast cancer will require a molecular map of carcinogenesis. We have investigated gene expression patterns in premalignant and early carcinomatous human breast lesions that confer to the patient varying risks for developing invasive breast cancer. The relative expression levels of one of the retinoid receptors, retinoid X receptor (RXR), was determined by in situ hybridization to 58 biopsy specimens; RXR mRNA grain density over each lesion was compared to that over the normal ductal/lobular units in each section. Overexpression of RXR mRNA was observed in 66% of noncomedo ductal carcinoma in situ (DCIS), which confer a >8-fold increase in breast cancer risk, and 88% of comedo DCIS lesions, which are associated with a yet higher risk. In contrast, only 8% of lesions that confer little or no increase in breast cancer risk overexpressed RXR mRNA (P = 0.0008). Limited in situ hybridization data using retinoic acid receptor (RAR) riboprobes showed overexpression of RAR alpha, but not RAR beta or -gamma, in only a modest percentage (36%) of cases, suggesting that all members of the retinoid receptor superfamily are not similarly regulated. Immunohistochemistry performed on 52 DCIS specimens for alpha, beta, and gamma isoforms of RXR confirmed its overexpression at the protein level and implicate RXR alpha as the predominant overexpressed form. The data indicate that RXR overexpression is associated with an increased risk for the development of invasive breast cancer in human breast lesions and suggest the hypothesis that it is causally involved in breast oncogenesis. The implications for retinoid chemoprevention are discussed.

The role of pathology in premalignancy and as a guide for treatment and prognosis in breast cancer.

This review emphasizes tissue pathology and its practical relevance to patient management in premalignant breast disease and established breast cancer. The rationale and criteria for recognizing benign lesions that indicate a subsequent increased risk for cancer development are now well established, having been confirmed in several large epidemiologic studies. Our understanding of the heterogeneous nature of ductal carcinoma in situ (DCIS) continues to evolve. Recent efforts to classify DCIS into clinically meaningful categories underscore the central role of histopathology in the management of this disease. Through long term follow up studies, small examples of noncomedo DCIS treated by biopsy alone may predict local recurrence. Adequate surgical excision, however, avoids this possibility in the predominance of such cases. For invasive carcinomas, prognostic issues extend beyond predicting survival after local treatment. Now that the efficacy of systemic chemotherapy is established, the question is whether this therapy will be of use to a particular patient or group of similar patients. The list of possible clinically useful subcategories of prediction is growing and under active development. Prognostic factors that are in general use, having been repeatedly validated, particularly stage and histologic grade, as well as those that are emerging but in need of validation, are reviewed.

Non-Hodgkin's lymphoma involving the breast.

Breast involvement by non-Hodgkin's lymphoma is rare. Differences between primary and secondary breast lymphoma have been reported, and a relationship between primary breast lymphoma and lymphomas of mucosa-associated lymphoid tissue has been suggested. We reviewed 61 cases of breast lymphoma (41 primary, 13 secondary, and 7 unclear) that included 28 right-sided masses at presentation, 17 left-sided, 12 bilateral, and 4 in which the side was not known. A subgroup of bilateral breast lymphomas was identified that occurred in young women, four of which were pregnant or postpartum. A high incidence of intermediate- and high-grade lymphomas were present in both cases of primary and secondary lymphomas as was a high frequency of B-cell phenotype. Additional immunohistochemical studies failed to demonstrate evidence of marginal or mantle cell differentiation in seven of eight cases studied. Lymphoepithelial lesions were identified in a majority of cases, including 67% of primary and 64% of secondary lymphomas. This study failed to demonstrate a morphologic difference between primary or secondary lymphomas of the breast and suggests that breast lymphomas differ from other extranodal lymphomas in that the latter are frequently low grade.

Core biopsy of the breast with atypical ductal hyperplasia: a probabilistic approach to reporting.

The diagnosis of atypical ductal hyperplasia (ADH) at needle core breast biopsy (NCB) is typically regarded as an indication for surgical excision. Although ADH is an intermediate risk nonobligate precursor lesion, the rationale for further therapy is the result of a reported high prevalence of a concomitant more advanced lesion (typically ductal carcinoma in situ) as the index lesion. To assess whether certain histopathologic features of ADH in NCB are predictive of open biopsy outcomes, the authors correlated the extent and pattern of ADH in 47 core biopsies (11-or 14-gauge) with the subsequent surgical specimen. Extent of ADH on NCB was ascertained by determining the number of large ducts and/or terminal duct-lobular units affected, with involvement of one large duct or one terminal duct-lobular unit representing a single focus, involvement of one duct and one terminal duct-lobular unit as two foci, and so on. Of the 47 cases, ADH was restricted to < or =2 foci in 24 cases (51.1%), confined to 3 foci in 8 cases (17.0%), and involved > or =4 foci in 15 cases (31.9%). The corresponding histopathologic findings at excision were benign lesions without atypia (n = 14), focal residual ADH (n = 13), atypical lobular hyperplasia (n = 3), ductal carcinoma in situ (n = 15), and invasive mammary carcinoma (n = 2). When the number of foci of involvement by ADH on NCB (based on an average of 11.6 cores per case) was correlated with the open biopsy results, all cases of ADH limited to < or =2 foci had no worse lesion on excision, whereas ADH present in > or =4 foci was found to be a strong predictor of a more advanced lesion on excision (p <0.0001, chi2). When histologic pattern was evaluated, all cases of pure micropapillary ADH on NCB showed pure micropapillary ductal carcinoma in situ on excision.

Histopathologic effects of tamoxifen on the uterine epithelium of breast cancer patients: analysis by menopausal status.

We evaluated the histopathologic changes of the uterine epithelium in 73 breast cancer patients with tamoxifen stratified by menopausal status. Clinicopathologic data at the time of breast cancer diagnosis and endometrial sampling were analyzed and compared with 122 breast cancer patients not receiving the drug. The incidence of endocervical and/or endometrial polyps was increased in tamoxifen-treated postmenopausal patients compared with untreated patients, 43% (25 of 58) and 24% (16 of 68), respectively (odds ratio=2.46, P=0.02). In contrast, there was no increase in polyps in premenopausal tamoxifen-treated patients. This finding suggests that the effects of tamoxifen on the endometrium may vary with menopausal status.