Evaluation of genetic clonality of breast cancer and its impact on the therapeutic response

Il carcinoma della mammella rappresenta circa il 30% delle neoplasie della donna e circa l’1% di quelle maschili. La valutazione di Her 2 è di primaria importanza nel carcinoma mammario, poiché legato ad una terapia target. Non è chiaro l’impatto di popolazioni cellulari eterogenee per Her2 sull’andamento della malattia. Nel primo studio abbiamo valutato in FISH (ibridazione in situ fluorescente) l’eterogeneità di Her2 secondo 3 cut-off (30%-20%-10%), in relazione alla presenza di metastasi e recidive. Abbiamo confrontato una popolazione omogenea di 109 casi con una eterogenea simile per età delle pazienti, stadio e grado di malattia. Dai dati ottenuti osserviamo che una sottopolazione non amplificata (≥ 20%) è un fattore prognostico favorevole nei casi FISH positivi; al contrario, in casi FISH negativi, il reperimento di una popolazione di cellule amplificate è indicativo di un andamento più aggressivo della patologia. Il carcinoma della mammella maschile è una patologia poco conosciuta per la bassissima incidenza, che aumenta (25%) in pazienti affetti da sidrome di Klinefelter (47XXY). Pertanto abbiamo valutato la presenza di aneusomie a carico del cromosoma X in 20 casi di carcinoma della mammella maschile rispetto a 10 casi di ginecomastia ed al tessuto cutaneo sano. Si è visto che il carcinoma presenta aneusomia per il cromosoma X in > 20% delle cellule neoplastiche in tutti i casi studiati. Possiamo pertanto ipotizzare un ruolo di X nelle prime fasi della trasformazione neoplastica; ulteriori approfondimenti sono in corso. Abbiamo confrontato 26 casi eterogenei analizzandoli con le tecniche di immunoistochimica (IHC) e FISH standard rispetto alla doppia colorazione IHC e SISH (ibridazione in situ in argento) osservando una concordanza diagnostica significativa tra le metodiche. Infine abbiamo sviluppato (e depositato domanda di brevetto), una metodica in grado di ridurre a poco più di 2 ore il tempo tecnico dell’indagine FISH.Breast cancer is about 30% of female cancers and about 1% of males. The evaluation of Her 2 is of primary importance in breast cancer, because it is linked to a target therapy. Despite updated guidelines, the impact of HER2 heterogeneous cell populations on the disease progression remains unclear. In the first study we evaluated Her2 heterogeneity by FISH (fluorescence in situ hybridization) using 3 cut-off (30% -20% -10%), correlating to the presence of recurrences and metastases. We then compared an homogeneous population of 109 cases with a heterogeneous population similar for age of the patients, stage and grade of the tumours. From the data obtained we observe that an unamplified subpopulation (≥ 20%) is a favorable prognostic factor in FISH positive cases; on the contrary, in FISH negative cases, the detectiong an amplified cells population is indicative of a more aggressive behavior of the disease. Male breast cancer is a poorly known disease due to its very low incidence rate, that increases (up o 25%) in patients with Klinefelter’ sydrome (47XXY). Therefore we evaluated the presence of X chromosome aneusomy in 20 male breast cancer cases compared to 10 cases of gynecomastia and to the healthy skin tissue. Data obtained showed that all the male breast cancers presented X chromosome aneusomy in > 20% of the neoplastic cells. Therefore we can hypothyse the role of X in the early stages of cancer; further investigations are in progress. From a technical perspective, we compared 26 difficult cases analyzing them with standard immunohistochemistry (IHC) and FISH compared to the new double staining IHC and SISH (silver in situ hybridization) observing a significant diagnostic concordance between the methods. Finally we have developed and filed a patent application about a method able to reduce to just over 2 hours the FISH technical time

HER2 isoforms co-expression differently tunes mammary tumor phenotypes affecting onset, vasculature and therapeutic response

Full-length HER2 oncoprotein and splice variant Delta16 are co-expressed in human breast cancer. We studied their interaction in hybrid transgenic mice bearing human full-length HER2 and Delta16 (F1 HER2/Delta16) in comparison to parental HER2 and Delta16 transgenic mice. Mammary carcinomas onset was faster in F1 HER2/Delta16 and Delta16 than in HER2 mice, however tumor growth was slower, and metastatic spread was comparable in all transgenic mice. Full-length HER2 tumors contained few large vessels or vascular lacunae, whereas Delta16 tumors presented a more regular vascularization with numerous endothelium-lined small vessels. Delta16-expressing tumors showed a higher accumulation of i.v. injected doxorubicin than tumors expressing full-length HER2. F1 HER2/Delta16 tumors with high full-length HER2 expression made few large vessels, whereas tumors with low full-length HER2 and high Delta16 contained numerous small vessels and expressed higher levels of VEGF and VEGFR2. Trastuzumab strongly inhibited tumor onset in F1 HER2/Delta16 and Delta16 mice, but not in full-length HER2 mice. Addiction of F1 tumors to Delta16 was also shown by long-term stability of Delta16 levels during serial transplants, in contrast full-length HER2 levels underwent wide fluctuations. In conclusion, full-length HER2 leads to a faster tumor growth and to an irregular vascularization, whereas Delta16 leads to a faster tumor onset, with more regular vessels, which in turn could better transport cytotoxic drugs within the tumor, and to a higher sensitivity to targeted therapeutic agents. F1 HER2/Delta16 mice are a new immunocompetent mouse model, complementary to patient-derived xenografts, for studies of mammary carcinoma onset, prevention and therapy

Early stability and late random tumor progression of a HER2-positive primary breast cancer patient-derived xenograft

We established patient-derived xenografts (PDX) from human primary breast cancers and studied whether stability or progressive events occurred during long-term in vivo passages (up to 4 years) in severely immunodeficient mice. While most PDX showed stable biomarker expression and growth phenotype, a HER2-positive PDX (PDX-BRB4) originated a subline (out of 6 studied in parallel) that progressively acquired a significantly increased tumor growth rate, resistance to cell senescence of in vitro cultures, increased stem cell marker expression and high lung metastatic ability, along with a strong decrease of BCL2 expression. RNAseq analysis of the progressed subline showed that BCL2 was connected to three main hub genes also down-regulated (CDKN2A, STAT5A and WT1). Gene expression of progressed subline suggested a partial epithelial-to-mesenchymal transition. PDX-BRB4 with its progressed subline is a preclinical model mirroring the clinical paradox of high level-BCL2 as a good prognostic factor in breast cancer. Sequential in vivo passages of PDX-BRB4 chronically treated with trastuzumab developed progressive loss of sensitivity to trastuzumab while HER2 expression and sensitivity to the pan-HER tyrosine kinase inhibitor neratinib were maintained. Long-term PDX studies, even though demanding, can originate new preclinical models, suitable to investigate the mechanisms of breast cancer progression and new therapeutic approaches

Transplanted human adipose tissue-derived stem cells engraft and induce regeneration in mice olfactory neuroepithelium in response to dichlobenil subministration

We used immunodeficient mice, whose dorsomedial olfactory region was permanently damaged by dichlobenil inoculation, to test the neuroregenerative properties of transplanted human adipose tissue-derived stem cells after 30 and 60 days. Analysis of polymerase chain reaction bands revealed that stem cells preferentially engrafted in the lesioned olfactory epithelium compared with undamaged mucosa of untreated transplanted mice. Although basal cell proliferation in untransplanted lesioned mice did not give rise to neuronal cells in the olfactory mucosa, we observed clusters of differentiating olfactory cells in transplanted mice. After 30 days, and even more at 60 days, epithelial thickness was partially recovered to normal values, as also the immunohistochemical properties. Functional reactivity to odorant stimulation was also confirmed through electroolfactogram recording in the dorsomedial epithelium. Furthermore, we demonstrated that engrafted stem cells fused with mouse cells in the olfactory organ, even if heterokaryons detected were too rare to hypothesize they directly repopulated the lesioned epithelium. The data reported prove that the migrating transplanted stem cells were able to induce a neuroregenerative process in a specific lesioned sensory area, enforcing the perspective that they could become an available tool for stem cell therapy. \ua9 The Author 2014. Published by Oxford University Press. All rights reserved

Additivo accelerante per reazioni di ibridazione

The present invention relates to the field of molecular analysis of DNA and / or RNA alterations through in situ hybridization techniques, both in the diagnostic and research fields. The accelerating hybridization additive object of the patent application is able, if added to the molecular probe in use, to reduce the analytical times up to 158 minutes compared to the 2/3 days required by the standard method

Prognostic impact of HER-2 Subclonal Amplification in breast cancer

The presence of a limited number of cells with HER-2 amplification (Subclonal Amplification) in breast carcinomas is occasionally encountered, but its prognostic impact is poorly known. The purpose of this study is to evaluate the prognostic impact of HER-2 Subclonal Amplification in a retrospective series of breast cancers. Accordingly, 81 consecutive breast carcinomas showing HER-2 Subclonal Amplification were obtained from the histology files (case series). These cases were subdivided into two groups: (a) those cases in which the HER-2 Subclonal Amplification was consonant to the accepted criteria for amplification, showing clusters of amplified cells, and (b) those cases with rare HER-2 Subclonal Amplification that did not reflect the accepted criteria for amplification, showing scattered amplified cells only. The incidence of metastases and late recurrences of the case series was compared with a series composed of 109 consecutive cases, being HER-2 homogeneous (comprising 14 Amplified and 95 Non-Amplified cases), matched for grade and stage (control series). It appeared that cases showing Subclonal Amplification had an incidence of metastases intermediate between the cases Amplified and Non-Amplified. Specifically, Subclonal Amplification with clustered cells had a lower incidence of metastases than Amplified cases (12.9 versus 21.4%). On the contrary, Subclonal Amplification with scattered cells showed an incidence of metastases higher than Non-Amplified cases (14 versus 9.47%). In addition, patients Subclonal Amplification with clustered cells, who were treated with the specific monoclonal antibody, had a lower incidence of metastases than patients showing Subclonal Amplification with scattered cells, who did not receive target therapy. These data, together with those recently published, indicate that Subclonal Amplification has an impact on prognosis and should be taken into consideration to correctly plan the treatment of breast cancer patients

Preferential expression of NY-BR-1 and GATA-3 in male breast cancer

BACKGROUND: Male breast cancer is an uncommon disease often discovered in advanced stage; thus, in the setting of metastatic adenocarcinoma, breast origin must be taken to account. Breast markers as NY-BR-1, GATA-3, mammaglobin, and BRST-2 are established tools for labelling primary and metastatic female breast cancer; however, none of them has been sufficiently studied in male breast cancer. The aim of this study was to analyze the expression of these markers in male breast cancer. MATERIALS AND METHODS: Thirty consecutive cases of male breast cancer and eight loco-regional metastases were re-revaluated, assembled in tissue micro array (TMA), and stained with immunohistochemistry (IHC) for NY-BR-1, GATA-3, mammaglobin, and BRST-2. The IHC stains were scored either positive or negative. In addition, concordant expression patterns of primary tumors and matched metastasis were noted. RESULTS: 30 of 30 (100%) primary tumors and 8 of 8 (100%) metastases were positive for NY-BR-1. 30 of 30 (100%) primary tumors and 6 of 8 (75%) metastases were positive for GATA-3. 22 of 30 (73.3%) primary tumors and 6 of 8 (75%) metastases were positive for Mammaglobin. 18 of 30 (60%) primary tumors and 5 of 8 (62.5%) metastases were positive for BRST-2. Differences in staining percentage were not significant with Fisher's exact test. CONCLUSION: We found a high sensitivity for all the markers analyzed. Moreover, the expression of NY-BR-1 and GATA-3 seemed the most effective for labelling male breast cancer in primary and metastatic setting

X chromosome gain in male breast cancer

Male breast cancer (MBC) is an uncommon disease whose molecular profile is not well known. X chromosome gain has been described as a marker of aggressive behavior in female breast cancer. The aim of this study is to investigate the role of the X chromosome in male breast cancer. Twenty cases of male breast invasive ductal carcinoma were retrieved and compared with 10 cases of gynecomastia. Cases were tested by fluorescence in situ hybridization to assess a cytogenetic profile for the X chromosome. The X chromosome status was compared with histopathologic features and stage at presentation. All MBC cases harbored an X chromosome gain (100%) in a variable percentage of neoplastic cells, ranging from 31% to 85% (mean, 59%). On the contrary, all cases of gynecomastia showed wild X chromosome asset. The patients' age at surgery and tumor grading showed a statistically significant correlation (P = .0188-.04), with the percentages of neoplastic cells showing an X chromosome gain. These data suggest that this X chromosome gain plays a role in the neoplastic transformation of male breast epithelial cells

HER2 Amplification Status in Feline Mammary Carcinoma: A Tissue Microarray\u2013Fluorescence In Situ Hydridization\u2013Based Study

Human epidermal growth factor receptor 2 (HER2) is a tyrosine kinase receptor overexpressed in a subset of breast cancer due to HER2 gene amplification. HER2 protein is expressed in feline mammary carcinomas, but little is known about its cytogenetic alterations. The aim of this study was to evaluate HER2 gene amplification status and its correlation with HER2 protein expression in feline mammary carcinomas. Feline mammary carcinomas were retrospectively selected and immunohistochemically (IHC) evaluated for HER2 protein expression. All the HER2 IHC-positive (3+) and equivocal (2+) cases and a subset of negative cases (0/1+) were selected for fluorescence in situ hybridization (FISH). Dual-core tissue microarrays were prepared for FISH. IHC and FISH were evaluated according to the 2013 American Society of Clinical Oncology/College of American Pathologists guidelines. The study included 107 feline mammary carcinomas from 88 queens. HER2 protein expression was positive (3+) in 7 cases (6.5%), equivocal (2+) in 48 cases (45%), and negative (0/1+) in 52 cases (48.5%). HER2 status was indeterminate in 8 feline mammary carcinomas (12%), amplified in 3 (4%), equivocal in 4 (6%), and nonamplified in 53 (78%). HER2 gene amplification and protein expression were significantly positively correlated (R = 0.283; P <.0001). HER2 gene is amplified in a subset of feline mammary carcinomas despite the HER2 positive or equivocal protein expression, but it remains to be determined if the HER2 amplification is a gene alteration that drives mammary tumor carcinogenesis or only a bystander passenger mutation

X chromosome gain is related to increased androgen receptor expression in male breast cancer

X chromosome gain has been previously described in male breast cancer (MBC). Androgen receptor (AR) gene is located on X chromosome. The aim of this study was to investigate the role of the X chromosome gain in the development of MBC and its relation with AR gene copy number and expression. The X chromosome status was assessed in 66 cases of male invasive and in situ duct breast carcinoma, in 34 cases of gynecomastia associated with cancer, and in 11 cases of tumor-free gynecomastia. Cases were tested by fluorescence in situ hybridization (FISH) to assess the X chromosome status and AR amplification. AR expression was studied by immunohistochemistry (IHC). In addition, AR methylation status was assessed. X chromosome gain was observed in 74.7% of invasive duct carcinoma, in 20.6% of in situ duct carcinoma, and in 14.6% of gynecomastia when associated with cancer, while all cases of tumor-free gynecomastia showed wild X chromosome asset. AR gene copy number when increased paralleled the number of X chromosomes. AR IHC expression was observed in 100% of MBC tested. AR gene methylation status revealed low level or absence of methylation. These data suggest that X chromosome can play a role in the neoplastic transformation of male breast epithelium. X chromosome gain is paralleled by AR gene polysomy. Polysomic AR genes show low methylation levels and high AR protein expression on IHC. These data should be taken into consideration for MBC treatment planning