Pathobiological implications of the d16HER2 splice variant for stemness and aggressiveness of HER2-positive breast cancer.

We have previously shown that the d16HER2 splice variant is linked to HER2-positive breast cancer (BC) tumorigenesis, progression and response to Trastuzumab. However, the mechanisms by which d16HER2 contributes to HER2-driven aggressiveness and targeted therapy susceptibility remain uncertain. Here, we report that the d16HER2-positive mammary tumor cell lines MI6 and MI7, derived from spontaneous lesions of d16HER2 transgenic (tg) mice and resembling the aggressive features of primary lesions, are enriched in the expression of Wnt, Notch and epithelial-mesenchymal transition pathways related genes compared with full-length wild-type (WT) HER2-positive cells (WTHER2_1 and WTHER2_2) derived from spontaneous tumors arising in WTHER2 tg mice. MI6 cells exhibited increased resistance to anoikis and significantly higher mammosphere-forming efficiency (MFE) and self-renewal capability than the WTHER2-positive counterpart. Furthermore, d16HER2-positive tumor cells expressed a higher fraction of CD29High/CD24+/SCA1Low cells and displayed greater in vivo tumor engraftment in serial dilution conditions than WTHER2_1 cells. Accordingly, NOTCH inhibitors impaired mammosphere formation only in MI6 cells. A comparative analysis of stemness-related features driven by d16HER2 and WTHER2 in ad hoc engineered human BC cells (MCF7 and T47D) revealed a higher MFE and aldehyde dehydrogenase-positive staining in d16HER2- vs WTHER2-infected cells, sustaining consistent BC-initiating cell enrichment in the human setting. Moreover, marked CD44 expression was found in MCF7_d16 and T47D_d16 cells vs their WTHER2 and Mock counterparts. Clinically, BC cases from two distinct HER2-positive cohorts characterized by high levels of expression of the activated-d16HER2 metagene were significantly enriched in the Notch family and signal transducer genes vs those with low levels of the metagene.

Selective axillary dissection after axillary reverse mapping to prevent breast-cancer-related lymphoedema.

BACKGROUND: It has recently been reported that, using axillary reverse mapping (ARM), the lymphatics from the arm can be spared to reduce the incidence of breast-cancer-related lymphoedema (BCRL). The aim of this study was to assess the feasibility of selective axillary dissection (SAD) after using ARM and partially preserving arm drainage, and to assess the occurrence of BCRL. METHODS: Using a radioisotope and lymphoscintigraphy, ARM was performed in 60 patients scheduled for SAD, who were subsequently divided for the purpose of comparing the BCRL rates into: group A, comprising 45 patients who successfully underwent SAD with a residual lymphatic hot spot; and group B with 15 whose hot nodes were removed as is normally the case during complete axillary lymph node dissection (ALND). RESULTS: SAD was feasible in 75% of the 60 patients. SAD was completed successfully in 19 of the first 30 patients, and in 26 of the second 30 patients (p = 0.072). The median follow-up was 16 months (6-36), during which 9 patients developed a BCRL, 4 in group A (9%) and 5 in group B (33%); p = 0.035. None of the patients had nodal relapses during the follow-up. CONCLUSIONS: Using a radioisotope enables an effective and safe SAD in a large proportion of patients. There was evidence of a trend to suggest a learning curve. The rate of BCRL after SAD was less than one third of the rate recorded after ALND, a result that should encourage the development of the former technique.

Effect of adjuvant trastuzumab treatment in conventional clinical setting: an observational retrospective multicenter Italian study.

Clinical trials have shown the efficacy of trastuzumab-based adjuvant therapy in HER2-positive breast cancers, but routine clinical use awaits evaluation of compliance, safety, and effectiveness. Adjuvant trastuzumab-based therapy in routine clinical use was evaluated in the retrospective study GHEA, recording 1,002 patients treated according to the HERA protocol between March 2005 and December 2009 in 42 Italian oncology departments; 874 (87.23 %) patients completed 1-year trastuzumab treatment. In 128 patients (12.77 %), trastuzumab was withdrawn due to cardiac or non-cardiac toxicity (28 and 29 patients, respectively), disease progression (5 patients) or the clinician's decision (66 patients). In addition, 156 patients experienced minor non-cardiac toxicities; 10 and 44 patients showed CHF and decreased LVEF, respectively, at the end of treatment. Compliance and safety of adjuvant trastuzumab-based therapy in Italian hospitals were high and close to those reported in the HERA trial. With a median follow-up of 32 months, 107 breast cancer relapses were recorded (overall frequency, 10.67 %), and lymph node involvement, estrogen receptor negativity, lymphoid infiltration, and vascular invasion were identified as independent prognostic factors for tumor recurrence, indicating that relapses were associated with advanced tumor stage. Analysis of site and frequency of distant metastases showed that bone metastases were significantly more frequent during or immediately after trastuzumab (<18 months from the start of treatment) compared to recurrences in bone after the end of treatment and wash-out of the drug (>18 months from the start of treatment) (35.89 vs. 14.28 %, p = 0.0240); no significant differences were observed in recurrences in the other recorded body sites, raising the possibility that the protection exerted by trastuzumab is lower in bone metastases.

MicroRNAs and future therapeutic applications in cancer.

Every cellular process is likely to be regulated by microRNAs (miRNAs), and an aberrant expression signature of these small non-coding RNAs is a hallmark of several diseases, including cancer. miRNA expression profiling by microarray techniques has provided a powerful tool to reveal the involvement of these tiny molecules in tumor development and progression, showing that they are differentially expressed in tumors as compared to normal tissues. Moreover, specific miRNA signatures have been associated with histopathological and clinical features, suggesting a potential role of these molecules as prognostic and predictive markers. Focusing then on their biological effects and role in cancer, it has been shown that miRNAs can function as potential oncogenes or oncosuppressor genes, depending on the cellular context and on the target genes they regulate. The possibility to modulate miRNA expression either in vitro and in vivo by developing synthetic pre-miRNA molecules or antisense oligonucleotides have at the same time provided a powerful tool to a deeper comprehension of the molecular mechanisms regulated by these molecules, and suggested the intriguing and promising perspective of their possible use in therapy. Herein we review our current knowledge about the involvement of miRNAs in cancer, and their potential as diagnostic, prognostic and therapeutic tools.

Biology, prognosis and response to therapy of breast carcinomas according to HER2 score.

BACKGROUND: The standardization of the HER2 score and recent changes in therapeutic modalities points to the need for a reevaluation of the role of HER2 in recently diagnosed breast carcinoma. PATIENTS AND METHODS: A multicenter, retrospective study of 1794 primary breast carcinomas diagnosed in Italy in 2000/2001 and scored in HER2 four categories according to immunohistochemistry was conducted. RESULTS: Ductal histotype, vascular invasion, grade, MIB1 positivity, estrogen and progesterone receptor expression differed significantly in HER2 3+ tumors compared with the other categories. HER2 2+ tumors almost showed values intermediate between those of the negative and the 3+ subgroups. The characteristics of HER2 1+ tumors were found to be in between those of HER2 0 and 2+ tumors. With a median follow-up of 54 months, HER2 3+ status was associated with higher relapse rates in node-positive and node-negative subgroups, while HER2 2+ only in node positive. Analysis of relapses according to type of therapy provided evidence of responsiveness of HER2-positive tumors to chemotherapy, especially taxanes. CONCLUSIONS: The present prognostic significance of HER2 is correlated to receptor expression level and points to the need to consider HER2 2+ and HER2 3+ tumors as distinct diseases with different outcomes and specific features.

The 67 kDa laminin receptor increases tumor aggressiveness by remodeling laminin-1.

The association between expression of the 67 kDa laminin receptor (67LR) and tumor aggressiveness has been convincingly demonstrated although the exact function of this molecule in the metastatic process has remained unclear. In this study, we tested whether the laminin-1, upon interaction with 67LR, promotes tumor cell aggressiveness; the investigation was based on: (i) the previous demonstration that soluble 67LR, as well as a 20-amino-acid peptide corresponding to the 67LR laminin binding site, changes the conformation of laminin upon interaction with this adhesion molecule and (ii) the known relevance of microenvironment remodeling by the tumor, leading to structural modification of extracellular matrix components in tumor progression. MDAMB231 breast carcinoma cells plated on peptide G-treated laminin-1 exhibited a polygonal array of actin filament bundles compared with cells seeded on native laminin-1 which presented the actin bundles organized as multiple cables parallel to margins. Furthermore, in cells seeded on peptide G-treated laminin-1, 67LR was distinct from the alpha6 integrin subunit in filopodia protrusions in addition to colocalizing with this integrin in focal adhesion plaques as it occurs when cells are plated on native laminin-1. In addition to differences in tumor cell adhesion and migration found in cells exposed to peptide G-treated vs native laminin-1, breast carcinoma cells seeded on modified laminin-1 showed a 6-fold increase in invasion capability compared with cells seeded on unmodified laminin-1. Alterations in actin organization as well as adhesion, migration and especially invasion observed in MDAMB231 cells in the presence of peptide G-treated laminin-1 were even found in MDAMB231 cells that, after selection for 67LR high expression, were seeded on native laminin-1. As the 67LR shedding is proportional to its expression level, these findings indicate a role for 67LR in changing laminin structure. Expression analysis of 97 genes encoding proteins that mediate cell matrix interactions, revealed significant differences between cells exposed to modified vs unmodified laminin-1 in 19 genes, 17 of which--including those encoding alpha3 integrin, extracellular matrix protein 1, proteolytic enzymes (such as MT1-MMP, stromelysin-3 and cathepsin L) and their inhibitors--were up-modulated in cells treated with modified laminin-1. Zymogram analysis clearly indicated a significant increase in the activity of the gelatinolytic enzyme MMP-2 in the culture supernatant from cells exposed to modified laminin-1, without an increase in mRNA abundance as observed in microarray analysis. Invasiveness of tumor cells conditioned by modified laminin-1, evaluated as the capability to cross Matrigel basement, was significantly more inhibited by MMPinhibitor TIMP-2 than invasiveness induced by native laminin-1. Taken together, our findings indicate that the role of 67LR in tumor aggressiveness rests in its ability to modify laminin-1 thereby activating proteolytic enzymes that promote tumor cell invasion through extracellular matrix degradation.

Role of HER2/neu in tumor progression and therapy.

HER2 (human epidermal growth factor receptor-2; also known as erbB2) and its relatives HER1 (epidermal growth factor receptor; EGFR), HER3 and HER4 belong to the HER family of receptor tyrosine kinases. In normal cells, activation of this receptor tyrosine kinase family triggers a rich network of signaling pathways that control normal cell growth, differentiation, motility and adhesion in several cell lineages. The first tumor studied for an alteration of the HER2 oncogene is breast carcinoma, and so far the majority of studies have been performed on this oncotype. Although involvement of HER2 as a cause of human cell transformation needs to be further investigated, overexpression of the HER2 oncogene in human breast carcinomas has been associated with a more aggressive course of disease. It has been suggested that this association depends on HER2-driven proliferation, vessel formation and/or invasiveness; however, poor prognosis may not be directly related to the presence of the oncoprotein on the cell membrane but instead to the breast carcinoma subset identified by HER2 overexpression and characterized by a peculiar gene expression profile, as recently identified. HER2-positive tumors were recently shown to benefit from anthracyclin treatment and to be resistant to endocrine therapy. Despite the fact that many pathways interacting with HER2 are still not fully understood, this tyrosine kinase receptor is, to date, a promising molecule for targeted therapy.

Role of hormonal risk factors in HER2-positive breast carcinomas.

Examination of parity, age at menarche and at menopause by HER2 status in a large series of breast carcinomas showed a statistically significant increased-frequency of HER2-positive tumours in lower risk subgroups. The findings suggest a difference in the protective role of hormone-related risk factors between HER2-positive and -negative tumours.

Ductal carcinoma in situ of the breast: a new phenotype classification system and its relation to prognosis.

In a study of invasive breast cancer, multiple correspondence analysis (MCA) revealed clustering of eight pathobiological variables. Two different phenotypes were distinguished by an index calculated on the basis of the variables (histologic grade, necrosis, lymphoid infiltration, number of mitosis and expression of c-erbB-2, p53, progesterone receptor and Bcl-2). Phenotype A lesions share most of the features of normal breast tissue. Phenotype B looks more malignant, has a higher early recurrence rate and is more frequently seen in younger patients. Our aim was to see if ductal breast carcinoma in situ (DCIS) could be divided into the same phenotypes. One hundred and eighty DCIS were investigated. Association between the eight variables was studied in 2 x 2 models. The phenotype index was calculated by summing weights for the variables in the MCA. All variables were associated, except Bcl-2. DCIS was divided in two phenotypes. Thirty-three tumours were Phenotype A and 147 Phenotype B. The mean age at diagnosis was 65.5 and 58.4 years for Phenotypes A and B, respectively (p = 0.0012). No difference regarding local relapse free survival was seen. Two phenotypes were distinguished in DCIS, similar to invasive breast cancer. In an earlier study, 45% of the invasive cancers were classified as Phenotype B. In this study, 82% of DCIS were Phenotype B. This may indicate that invasive breast cancer of Phenotype B is derived from DCIS of Phenotype B. The distribution of DCIS phenotypes with a small proportion of Phenotype A DCIS may be due to that Phenotype A DCIS is less likely to be detected by mammography, or that some invasive breast cancers of Phenotype A progress to invasiveness without passing the in situ phase.

HER2 overexpression in various tumor types, focussing on its relationship to the development of invasive breast cancer.

To date, poor standardization in HER2 status evaluation has precluded reliable comparison of overexpression rates in different tumors. However, standardized methodologies have been introduced recently for these analyses, and have identified frequencies of 51%, 44%, 26% and 25% in Wilm's tumor, bladder, pancreatic and breast carcinoma, respectively. Other tumors tested had frequencies below 20%. The frequency was greater than that predicted by gene amplification data in some tumor types, which may indicate overexpression due to gene deregulation, rather than gene amplification. Analysis of a large retrospective series of breast carcinomas demonstrated an association between HER2 positivity and a number of other prognostic markers. Together, these variables identify a subset of tumors with poor prognosis and early relapse post-surgery. HER2 expression is relatively stable, with 95% concordance between the HER2 status of primary and metastatic lesions. However, contralateral tumors are unrestricted with regard to HER2 status. Preliminary data indicate that the HER2 status of a hormone receptor-positive tumor may fluctuate according to the menstrual cycle. It is anticipated that the emerging wealth of standardized data for HER2 status will help to elucidate the role of HER2 in tumor progression.

Role of p53 in HER2-induced proliferation or apoptosis.

HER2 oncogene overexpression has been associated either with proliferation or differentiation and apoptosis. The role of p53 on these different chances was investigated. Wild type (wt) p53-IGROV1 cells showed growth inhibition and apoptosis after HER2 transfection, whereas no anti-proliferative effect was observed in its mutated p53 sub-line unless wt p53 was cotransfected with HER2. Stable HER2 transfectants derived from wt p53 line treated with heregulin-beta1 or epidermal growth factor showed a decrease in proliferation due to a G(2)/M cell cycle block despite normal mitogen-activated protein kinase activation. In these HER2 transfectants, c-Myc and p53 expression were increased, whereas MDM2 was dramatically down-modulated. By contrast, growth factors stimulation of HER2 transfectants with mutated-p53 induced progression through the cell cycle. Together, our data point to a regulatory role for p53 in HER2 signaling.

Response to cyclophosphamide, methotrexate, and fluorouracil in lymph node-positive breast cancer according to HER2 overexpression and other tumor biologic variables.

PURPOSE: There is considerable interest in biologic markers able to predict the response of cancer patients to therapy. HER2 overexpression is a potential indicator of responsiveness to doxorubicin and paclitaxel and of unresponsiveness to tamoxifen in breast carcinoma patients. However, the significance of HER2 overexpression in responsiveness to cyclophosphamide, methotrexate, and fluorouracil (CMF) has remained unclear. In this study, we investigated this issue in the 386 breast cancer patients in the first CMF controlled clinical trial with a 20-year follow-up. PATIENTS AND METHODS: Node-positive breast carcinoma patients were randomly assigned to receive either no further treatment after radical mastectomy (179 women) or 12 monthly cycles of adjuvant CMF chemotherapy (207 women). Overexpression of HER2 and the status of other tumor variables was assessed by immunohistochemistry in at least 324 (84%) of the 386 patients. Statistical analyses were performed to assess the efficacy of CMF treatment for the subgroups defined by HER2 and the status of other variables using a Bayesian approach. The end points considered were relapse-free survival (RFS) and cause-specific survival (CSS). RESULTS: Bayesian analysis of the treatment effect for HER2 and other variables indicated a clinical benefit from CMF treatment in all subgroups defined according to variables status. In particular regarding HER2 status, Bayesian estimates of RFS hazard ratios were equal to 0.484 and 0.641 and estimates of CSS hazard ratios were equal to 0.495 and 0.730 for HER2-positive and -negative tumors, respectively. CONCLUSION: CMF treatment showed a clinical benefit in the considered subgroups, defined according to HER2 and other tumor variables status. Patients with HER2-positive or HER2-negative tumors benefit from CMF treatment, and the poor prognosis associated with the HER2 overexpression in the untreated group could be completely overcome by the chemotherapy treatment.

Breast cancer patients treated without axillary surgery: clinical implications and biologic analysis.

OBJECTIVE: To evaluate the impact of breast carcinoma (T1-2N0) surgery without axillary dissection on axillary and distant relapses, and to evaluate the usefulness of a panel of pathobiologic parameters determined from the primary tumor, independent of axillary nodal status, in planning adjuvant treatment. METHODS: In a prospective nonrandomized pilot study, 401 breast cancer patients who underwent breast surgery without axillary dissection were accrued from January 1986 to June 1994. At surgery, all patients were clinically node-negative and lacked evidence of distant metastases after clinical or radiologic examination. A precise 4-month clinical and radiologic follow-up was performed to detect axillary or distant metastases. Patients with clinical evidence of axillary nodal relapse were considered for surgery as salvage treatment. Biologic characteristics of primary carcinomas were investigated by immunohistochemistry, and four pathologic and biologic parameters (size, grading, laminin receptor, and c-erbB-2 receptor) were analyzed to determine a prognostic score. RESULTS: The 5-year follow-up of these patients revealed a low rate of nodal relapses (6.7%), particularly for T1a and T1b patients (2% and 1.7%, respectively), whereas T1c and T2 patients showed a 10% and 18% relapse rate, respectively. Surgery was a safe and feasible salvage treatment without technical problems in all 19 cases of progressive disease at the axillary level. The low rate of distant metastases in T1a and T1b groups (<6%) increased to 15% in T1c and 34% in T2 patients. Analyzing the primary tumor with respect to the panel of pathologic and biologic parameters was predictive of metastatic spread and therefore can replace nodal status information for planning adjuvant treatment. CONCLUSIONS: Middle-term follow-up shows that the rate of axillary relapse in this patient population is lower than expected, suggesting that only a minimal number of microembolic nodal metastases become clinically evident. Avoidance of axillary dissection has a negligible effect on the outcome of T1 patients, particularly in T1a and T1b tumors with no palpable nodes, because the rate of axillary node relapse is very low for both. In T1 breast carcinoma, postsurgical therapy should be considered on the basis of biologic characteristics rather than nodal involvement. The authors' prognostic score based on the primary tumor identified patients who required postsurgical treatment, providing a practical alternative to axillary status for deciding on adjuvant treatment. Conversely, in the T2 group, the high rate of salvage surgery for axillary relapses, which is expected in tumors larger than 2.5 cm or 3.0 cm, represents a limit for avoiding axillary dissection. Preoperative evaluation of axillary nodes for modification of surgical dissection in this subgroup would be more useful more than in T1 breast cancer because of the high risk. Complete dissection is feasible without technical problems if precise follow-up detects progressive axillary disease.

SV40, JC and BK expression in tissue, urine and blood samples from patients with malignant and nonmalignant pleural disease.

BACKGROUND: Polyomaviruses are expressed in both human tumors and immunodepressed patients. Malignant and nonmalignant pleural effusions create an environment that could favor the expression of opportunistic viral infections. We studied if SV40, JC, and BK viral DNA can be amplified from biopsies obtained from different pleural diseases. MATERIALS AND METHODS: DNA was extracted from mesotheliomas (MM), nonspecific inflammatory and tubercular pleural biopsies, blood and urinary sediments from patients with MM, and pleural effusion cytological specimens. SV40, JC and BK viral early regions were amplified by PCR and analyzed by Southern Blot hybridization with specific probes. RESULTS: SV40 was positive in 9/23 MM, 5/18 tubercular and 1/7 nonspecific inflammatory biopsies, and 5/12 pleural effusion cytological specimens. JC was positive in 2/23 MM and in 7/15 urinary sediments. All blood samples were negative and BK was also negative in all samples. CONCLUSIONS: Tissue specific factors, characteristic of MM and TB, may contribute to expression of SV40 in these diseases.

Different susceptibility to polycyclic aromatic hydrocarbons (PAH)-induced DNA damage in lung tissue in male and female non-smokers.

The levels of benzo(a)pyrene diol-epoxide (BPDE)-DNA adducts and polycyclic aromatic hydrocarbons (PAH) were analysed in a limited number of samples of autoptic lung tissue obtained from non-professionally exposed male (n= 13) and female (n= 12) non-smokers in an attempt to evaluate the relationship between gender, lung PAH levels (n= 25) and susceptibility to BPDE-DNA adduct formation (n= 18). Lung concentrations of chrysene, benzo(g,h,i)perylene and benzo(a)pyrene were significantly higher in males than in females (P.

Fluctuation of HER2 expression in breast carcinomas during the menstrual cycle.

The hormonal milieu at time of tumor surgery seems to have a significant impact on survival in premenopausal breast cancer patients. Indeed, surgery performed during the follicular phase of the menstrual cycle was suggested to correlate with a poor prognosis. To investigate the relationship between prognosis and menstrual cycle at time of surgery, we analyzed the expression of some markers associated with tumor aggressiveness, such as the hormone receptors, HER2, p53, Bcl2, and cathepsin D in breast carcinomas obtained from 198 premenopausal women who underwent surgery during different phases of the menstrual cycle. HER2 overexpression was found to fluctuate in hormone receptor-positive tumors. In actual fact, 20% of the tumors removed during the follicular phase scored HER2-positive, versus 8% of those removed during the luteal phase. Similarly, a number of hormone receptor-positive tumor specimens, obtained from the same patients during follicular and luteal phases, were scored HER2-positive when the sample was removed during the follicular phase and HER2-negative when removed in the luteal phase. Southern blot analysis of the HER2 gene indicated that, in hormone receptor-positive cases, the overexpression of HER2 is often not associated with gene amplification. The finding that overexpression of the HER2 gene, associated with tumor aggressiveness, can fluctuate according to the hormonal milieu may explain the increased survival of patients operated during the luteal phase. It is also relevant to the selection and treatment of patients most likely to benefit from anti-HER2 antibody therapy.

Pathobiologic identification of two distinct breast carcinoma subsets with diverging clinical behaviors.

Many different pathological and biological variables which characterize breast carcinomas have been found to be associated. The aim of this work was to analyze the complex relationship among these parameters. The pathologic, biologic, and clinical characteristics of a series of primary breast carcinomas from 676 patients were retrospectively investigated. Multiple correspondence analysis of 13 factors revealed clustering of eight pathobiologic variables, that is histologic grade, necrosis, lymphoid infiltration, number of mitoses, c-erbB-2 overexpression, p53, progesterone receptor, and bcl2 expression. An index for each tumor calculated on the basis of these eight factors served to distinguish two different tumor phenotypes, designated A and B. Phenotype A is represented by tumors sharing most of the biologic features of normal breast tissues: indeed, these tumors are characterized by a relatively high degree of differentiation, low proliferation, no necrosis or leukocyte infiltration, and no gene alterations. By contrast, phenotype B is quite divergent from the normal tissue because of its poor differentiation, high proliferation, frequent gene alterations and evidence of a host immune reaction. As regards the disease progression, these two subsets showed marked differences: phenotype A tumors had a low recurrence rate per year that remained constant over time and affected more frequently elderly patients, whereas group B tumors showed high aggressivity in the first years after surgery followed by a low long-term recurrence rate and were more frequently seen in younger patients. These data suggest that breast carcinoma consists of two different subsets that can be identified on the basis of pathobiologic features.

Sequences homologous to the mouse mammary tumor virus env gene in human breast carcinoma correlate with overexpression of laminin receptor.

We previously reported that a 660-bp sequence that is homologous to the env gene of the mouse mammary tumor virus (MMTV) but not to endogenous retroviruses or to other known genes was present in 38% of human breast cancers and in some breast cancer cell lines studied (Y. Wang et al., Cancer Res., 55: 5173-5179, 1995). Here, we have investigated whether the MMTV-like sequences were associated with the clinical, pathological, and molecular parameters that have been reported to define two subsets of human breast cancers. Archival breast carcinoma samples were analyzed for four clinical parameters, obtained from patients' records, and for six pathological characteristics. Expression of c-erbB-2, p53, bcl-2, progesterone receptor, laminin receptor, and cathepsin D was detected by immunochemistry using monoclonal antibodies. PCRs were used to amplify 250 bp of the MMTV env gene-like sequence. The chi2, log-rank, and generalized Wilcoxon tests were used to analyze the data. The MMTV env gene-like sequence was detected in 37.7% of the samples. The presence of this sequence was not significantly associated with any of the pathological clinical or biological parameters studied. It did correlate, however, with expression of the laminin receptor, a marker for invasiveness and poor prognosis. This is the first phenotypic characterization of human breast cancers containing retroviral sequences.

Increased expression of c-erbB-2 in hormone-dependent breast cancer cells inhibits cell growth and induces differentiation.

c-erbB-2, a member of the tyrosine kinase oncogene family, is overexpressed in about 30% of human breast tumors where it correlates with poor prognosis. In vitro studies have suggested that increased expression of the receptor plays an important role in malignant progression. To better understand the direct effects of p185HER2 overexpression, a human c-erbB-2 expression vector was transfected into the hormone-dependent MCF-7 human breast carcinoma cell line and cell growth was analysed. Unexpectedly, colony formation assay revealed a reduction in the number and size of colonies as compared with mock-transfected cells. In hormone-deprived medium, c-erbB-2 transfected cells acquired growth capability, consistent with previous reports. By contrast, two c-erbB-2-transfected clones grown in complete medium showed a reduced proliferation rate despite the activation of a fully functional oncoprotein capable of autophosphorylation and induction of the MAPK pathway. The number of c-erbB-2-overexpressing cells in the S phase of the cell cycle was about one-half the number of control and mock-transfected cells. Also, overexpression of c-erbB-2 induced overexpression of p21WAF1, pRB hypophosphorylation and a mature differentiated cell phenotype with production of lipid droplets. Functional inactivation of p185HER2 by means of a specific single chain antibody indicated the c-erbB-2-dependence of the observed alterations. These data show that the exogenous overexpression of the c-erbB-2 gene in hormone-dependent breast cancer cells inhibits proliferation and induces differentiation.