Patrizia Casalini

microRNA-205 Regulates HER3 in Human Breast Cancer

An increasing amount of experimental evidence shows that microRNAs can have a causal role in breast cancer tumorigenesis as a novel class of oncogenes or tumor suppressor genes, depending on the targets they regulate. HER2 overexpression is a hallmark of a particularly aggressive subset of breast tumors, and its activation is strictly dependent on the trans-interaction with other members of HER family; in particular, the activation of the PI3K/Akt survival pathway, so critically important in tumorigenesis, is predominantly driven through phosphorylation of the kinase-inactive member HER3. Here, we show that miR-205, down-modulated in breast tumors compared with normal breast tissue, directly targets HER3 receptor, and inhibits the activation of the downstream mediator Akt. The reintroduction of miR-205 in SKBr3 cells inhibits their clonogenic potential and increases the responsiveness to tyrosine-kinase inhibitors Gefitinib and Lapatinib, abrogating the HER3-mediated resistance and restoring a potent proapoptotic activity. Our data describe miR-205 as a new oncosuppressor gene in breast cancer, able to interfere with the proliferative pathway mediated by HER receptor family. Our study also provides experimental evidence suggesting that miR-205 can improve the responsiveness to specific anticancer therapies.

FOXP3 Expression and Overall Survival in Breast Cancer

PURPOSE The transcription factor forkhead box P3 (FOXP3) up- or downregulates a large number of genes and has been recently reported to be expressed in tumor cells. We investigated the prognostic importance of FOXP3 expression in patients with breast cancer. PATIENTS AND METHODS The expression patterns of FOXP3 were characterized by immunohistochemistry in primary breast carcinoma specimens from patients of the Milan 3 and 1 trials. Kaplan-Meier analysis and Cox proportional hazards regression modeling were used to assess the overall survival, distant metastasis-free survival, and local relapse cumulative incidence, according to the presence or absence of FOXP3 expression. RESULTS FOXP3 expression in tumors was associated with worse overall survival probability and the risk increased with increasing FOXP3 immunostaining intensity. FOXP3 was also a strong prognostic factor for distant metastases-free survival but not for local recurrence risk. In multivariate analysis FOXP3 resulted an independent prognostic factor and the hazard ratio of FOXP3 expression and of lymph node positivity were similar. In the Milan 3 trial, the probability of 10-year survival in node-negative subgroup was 100% for FOXP3-negative and 82% for FOXP3-positive patients; in node-positive subgroup 82% for FOXP3-negative and 41% for FOXP3-positive patients. Even in the Milan 1 trial the lack of FOXP3 expression in node-positive subgroup was related to a significantly better prognosis than in FOXP3-positive patients (10-year survival probability, 89% v 59%). CONCLUSION The data identify FOXP3 expression as a new independent prognostic factor in breast carcinoma, which might help to improve the selection of patients for appropriate therapy.

Neutrophil extracellular traps mediate transfer of cytoplasmic neutrophil antigens to myeloid dendritic cells toward ANCA induction and associated autoimmunity

Antineutrophil cytoplasmic antibodies (ANCAs) target proteins normally retained within neutrophils, indicating that cell death is involved in the autoimmunity process. Still, ANCA pathogenesis remains obscure. ANCAs activate neutrophils inducing their respiratory burst and a peculiar form of cell death, named NETosis, characterized by formation of neutrophil extracellular traps (NETs), decondensed chromatin threads decorated with cytoplasmic proteins endorsed with antimicrobial activity. NETs have been consistently detected in ANCA-associated small-vessel vasculitis, and this association prompted us to test whether the peculiar structure of NET favors neutrophil proteins uploading into myeloid dendritic cells and the induction of ANCAs and associated autoimmunity. Here we show that myeloid DCs uploaded with and activated by NET components induce ANCA and autoimmunity when injected into naive mice. DC uploading and autoimmunity induction are prevented by NET treatment with DNAse, indicating that NET structural integrity is needed to maintain the antigenicity of cytoplasmic proteins. We found NET intermingling with myeloid dendritic cells also positive for neutrophil myeloperoxidase in myeloperoxidase-ANCA-associated microscopic poliangiitis providing a potential correlative picture in human pathology. These data provide the first demonstration that NET structures are highly immunogenic such to trigger adaptive immune response relevant for autoimmunity.

Role of HER receptors family in development and differentiation

Members of the epidermal growth factor receptor family of receptor tyrosine kinases play a critical role in both development and oncogenesis. The latter is suggested by the frequent overexpression of HER‐2, EGFR, and HER‐3 in some human carcinomas, primarily breast and squamous cancer. The biological activities of the EGFR family are exerted through various ligand–receptor and receptor–receptor interactions. One receptor that plays a central role in this signaling network is HER‐2/Neu, which is considered the preferred heterodimerization partner for other members of the EGFR family. The role of these receptors and their ligands in development is discussed, with particular emphasis on their ability to mediate a variety of pathways and cellular responses, including proliferation, differentiation, and apoptosis.

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.

HER2 Overexpression and Doxorubicin in Adjuvant Chemotherapy for Resectable Breast Cancer

PURPOSE Human epidermal growth factor receptor 2 (HER2) overexpression was found to predict a good response in breast carcinoma patients treated with doxorubicin (Adriamycin [ADM]). Evidence from our recent study indicates that node-positive patients respond to cyclophosphamide, methotrexate, and fluorouracil (CMF) regardless of HER2 status. We address the issue of whether therapy regimens including CMF and ADM versus CMF alone have the same therapeutic effect in patients with HER2+ and HER2- tumors in terms of relapse-free survival (RFS) and overall survival (OS). METHODS Archival specimens of the primary tumors from 506 patients in a prospective clinical trial were stained with the anti-HER2 monoclonal antibody CB11. Originally, patients were randomly allocated to receive either 12 courses of intravenous CMF or eight courses of the same regimen followed by four cycles of ADM. RFS and OS were analyzed by a Cox model taking into account treatment, HER2 status, and the interaction between treatment and HER2 status, adjusting for the effect of other known clinical and biopathologic factors. RESULTS Analysis of survival rates indicates a possible differential effect of treatment in the patients grouped according to HER2 status. Improved RFS and OS were observed in the HER2+ subgroup after treatment with CMF plus ADM versus CMF alone. With a median follow-up of 15 years, the hazard ratio (HR) for RFS was 0.83 in HER2+ tumors and 1.22 in HER2- tumors. The effect of treatment was more evident on OS in HER2+ patients (HR = 0.61; CI, 0.32 to 1.16) than in HER2- patients (HR = 1.26). CONCLUSION Our data indicate that adding ADM to CMF might be beneficial for patients with HER2+ tumors.

Oncosuppressive role of p53-induced miR-205 in triple negative breast cancer

An increasing body of evidence highlights an intriguing interaction between microRNAs and transcriptional factors involved in determining cell fate, including the well known “genome guardian” p53. Here we show that miR‐205, oncosuppressive microRNA lost in breast cancer, is directly transactivated by oncosuppressor p53.

Lynch Syndrome—Related Endometrial Carcinomas Show a High Frequency of Nonendometrioid Types and of High FIGO Grade Endometrioid Types

Although endometrial cancer (EC) represents the second most common cancer after colonic cancer in patients with Lynch syndrome (LS), the pathologic features and behavior of LS-related EC are not well recognized. ECs from 23 patients (mean age 46.2 years) with MSH2 (16), MLH1 (6), and MLH1/MSH2 (1) constitutional mutations were evaluated for histologic type, FIGO grade, FIGO stage, association with tumors in other sites, and survival. For every LS-associated tumor, 2 same-age cases from patients with no family history of LS-associated cancer were evaluated (sporadic group). In LS-associated EC there were 13 (56.5%) endometrioid (eec) and 10 (43.5%) nonendometrioid carcinomas (neec), and among the sporadic tumors there were 44 (95.7%) eec and 2 (4.3%) neec (P = .001). The eec in women with germline LS mutation had a significantly higher FIGO grade (P = .0378) and more frequent vascular invasion than the controls. The mean survival of the entire group of 23 patients with LS-related EC was 17.326 (14.066 to 20.585). Mean survival according to FIGO stage was significant (P = .010). Difference in mean survival according to age of the patient at the time of the diagnosis (patients >46 years vs <46 years) was not significant. The mean survival of the mutated patients with eec was 20.462 (17.564 to 23.359) and was not significantly different from that of the mutated patients with neec, which was 14.240 (9.119 to 19.361). Log-rank analysis showed that histology did not affect the survival. However, the hazard ratio of neec patients, although not significant, resulted higher. Mean survival of patients with a neec tumor combined with an endometrioid component (14.375 [8.084 to 20.666]) was not different from that of patients with pure neec cancers (14.250 [7.885 to 20.615]). When compared with the control population, LS-related ECs show a wider variety of histologic types; a higher frequency of neec types despite the younger age of the patients, and a higher frequency of high grades among the eec.

Redirected activity of human antitumor chimeric immune receptors is governed by antigen and receptor expression levels and affinity of interaction.

Novel Ab-based immunotherapeutic strategies have exploited T-cell receptor-like chimeric immune receptors (CIR) expressed on the surface of transduced human peripheral blood mononuclear cell (PBMC) to redirect potent non–major histocompatibility complex-dependent cytotoxicity to tumor cells expressing a tumor-associated antigens. We transduced human PBMC with 2 fully human CIRs that trigger through the ζ-chain of CD3 and contain either one of two human scFv specific for the same epitope on the extracellular domain of HER2 but with distinctly different affinities (KD 1616 and 1 nM) for this antigen. Potent direct CIR-mediated killing and in vitro tumor growth inhibition mediated by transduced PBMC were observed against targets expressing different levels of HER2. High-affinity CIR showed stronger ability to bind Ag and retain binding than low-affinity CIR. When lytic potential of the 2 CIRs was evaluated, their efficiency was comparable under conditions of high CIR and Ag expression, whereas low-affinity CIR was more efficient than high-affinity CIR in conditions of limiting Ag and CIR expression levels. When tumor growth inhibition was evaluated, Ag and CIR levels, rather than CIR affinity appeared relevant. Ag-driven CIR activation resulted in the production of soluble factors mediating efficient bystander effect. By carefully defining CIR surface expression and increasing affinity for a specific target antigen, it may be possible to selectively exclude CIR-mediated activity against targets expressing low levels of antigen, as normal cells. On the contrary, low antigen-expressing tumor variants could be eliminated by decreasing CIR affinity. Tuning CIR expression and affinity might help in discriminating different biologic contexts.

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.