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This corrects the article DOI: 10.1038/ncomms14797.
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BACKGROUND: Immunohistochemistry (IHC) and fluorescent-in situ hybridization (FISH) are standard methods to assess human epidermal growth factor receptor 2 (HER2) status in breast cancer (BC) patients. Real-time quantitative polymerase-chain-reaction (qRT-PCR) is able to detect HER2 overexpression. Here we compared FISH, IHC, quantitative PCR (qPCR), and qRT-PCR to determine the concordance rates and evaluate their relative roles in HER2 determination. PATIENTS AND METHODS: We determined HER2 status in 153 BC patients, using IHC, FISH, Q-PCR and qRT-PCR. In discordant cases, we directly measured HER2 protein levels using Western blotting. RESULTS: The overall agreement (OA) between FISH and Q-PCR was 94.1, with a k value of 0.87. Assuming FISH as the standard reference, Q-PCR showed an 86.1% sensitivity and a 99.0% specificity with a global accuracy of 91.6%. OA between FISH and qRT-PCR was 90.8% with a k value of 0.81. Of interest, the disagreement between FISH and qRT-PCR was mostly restricted to equivocal cases. HER2 protein analysis suggested that qRT-PCR correlates better than FISH with HER2 protein levels, particularly where FISH fails to provide conclusive results. SIGNIFICANCE: qRT-PCR may outperform FISH in identifying patients overexpressing HER2 protein. Q-PCR cannot be used for HER2 status assessment, due to its suboptimal level of agreement with FISH. Both FISH and Q-PCR may be less accurate than qRT-PCR as surrogates of HER2 protein determination.
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Neoplasias da Mama/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor ErbB-2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Curva ROC , Receptor ErbB-2/metabolismoRESUMO
Evidence supports a role of 17&-estradiol (E2) in carcinogenesis and the large majority of breast carcinomas are dependent on estrogen. The anti-estrogen tamoxifen (TAM) is widely used for both treatment and prevention of breast cancer; however, it is also carcinogenic in human uterus and rat liver, highlighting the profound complexity of its actions. The nature of E2- or TAM-induced chromosomal damage has been explored using relatively high concentrations of these agents, and only some numerical aberrations and chromosomal breaks have been analyzed. This study aimed to determine the effects of low doses of E2 and TAM (10(&8 )mol L(&1) and 10(&6 )mol L(&1) respectively) on karyotypes of MCF7, T47D, BT474, and SKBR3 breast cancer cells by comparing the results of conventional karyotyping and multi-FISH painting with cell proliferation. Estrogen receptor (ER)-positive (+) cells showed an increase in cell proliferation after E2 treatment (MCF7, T47D, and BT474) and a decrease after TAM treatment (MCF7 and T47D), whereas in ER& cells (SKBR3), no alterations in cell proliferation were observed, except for a small increase at 96 h. Karyotypes of both ER+ and ER& breast cancer cells increased in complexity after treatments with E2 and TAM leading to specific chromosomal abnormalities, some of which were consistent throughout the treatment duration. This genotoxic effect was higher in HER2+ cells. The ER&/HER2+ SKBR3 cells were found to be sensitive to TAM, exhibiting an increase in chromosomal aberrations. These in vitro results provide insights into the potential role of low doses of E2 and TAM in inducing chromosomal rearrangements in breast cancer cells.
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Antineoplásicos Hormonais/toxicidade , Neoplasias da Mama/genética , Aberrações Cromossômicas/induzido quimicamente , Estradiol/toxicidade , Antagonistas de Estrogênios/toxicidade , Tamoxifeno/toxicidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , CariótipoRESUMO
Despite the marked improvement in the understanding of molecular mechanisms and classification of apocrine carcinoma, little is known about its specific molecular genetic alterations and potentially targetable biomarkers. In this study, we explored immunohistochemical and molecular genetic characteristics of 37 invasive apocrine carcinomas using immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), and next-generation sequencing (NGS) assays. IHC revealed frequent E-cadherin expression (89%), moderate (16%) proliferation activity [Ki-67, phosphohistone H3], infrequent (~10%) expression of basal cell markers [CK5/6, CK14, p63, caveolin-1], loss of PTEN (83%), and overexpression of HER2 (32%), EGFR (41%), cyclin D1 (50%), and MUC-1 (88%). MLPA assay revealed gene copy gains of MYC, CCND1, ZNF703, CDH1, and TRAF4 in 50% or greater of the apocrine carcinomas, whereas gene copy losses frequently affected BRCA2 (75%), ADAM9 (54%), and BRCA1 (46%). HER2 gain, detected by MLPA in 38% of the cases, was in excellent concordance with HER2 results obtained by IHC/FISH (κ = 0.915, P < .001). TOP2A gain was observed in one case, while five cases (21%) exhibited TOP2A loss. Unsupervised hierarchical cluster analysis revealed two distinct clusters: HER2-positive and HER2-negative (P = .03 and .04, respectively). NGS assay revealed mutations of the TP53 (2 of 7, 29%), BRAF/KRAS (2 of 7, 29%), and PI3KCA/PTEN genes (7 of 7, 100%). We conclude that morphologically defined apocrine carcinomas exhibit complex molecular genetic alterations that are consistent with the "luminal-complex" phenotype. Some of the identified molecular targets are promising biomarkers; however, functional studies are needed to prove these observations.
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Glândulas Apócrinas/química , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Neoplasias da Mama/diagnóstico , Carcinoma/diagnóstico , Imuno-Histoquímica , Técnicas de Diagnóstico Molecular , Neoplasias das Glândulas Sudoríparas/diagnóstico , Glândulas Apócrinas/patologia , Neoplasias da Mama/química , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma/química , Carcinoma/genética , Carcinoma/patologia , Análise por Conglomerados , Feminino , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Reação em Cadeia da Polimerase Multiplex , Fenótipo , Valor Preditivo dos Testes , Neoplasias das Glândulas Sudoríparas/química , Neoplasias das Glândulas Sudoríparas/genética , Neoplasias das Glândulas Sudoríparas/patologiaRESUMO
Merkel cell carcinoma of the skin is a malignant neuroendocrine tumour, whose prognostic criteria are a matter of dispute. Specifically, no predictor is presently available in stage I-II tumours. We collected clinical and follow-up data from 70 Merkel cell carcinomas of the skin. The same cases were studied for p63 expression by immunohistochemistry, by reverse-transcription PCR (RT-PCR) and TP63 gene status by FISH and for presence of Merkel cell polyomavirus by PCR. Stage emerged as a significant prognostic parameter (P=0.008). p63 expression, detected in 61% (43/70) of cases by immunohistochemistry, was associated with both decreased overall survival (P<0.0001) and disease-free survival (P<0.0001). Variable expression patterns of the different p63 isoforms were found only in cases immunoreactive for p63. In these latter lesions, at least one of the N-terminal p63 isoforms was detected and TAp63α was the most frequently expressed isoform. TP63 gene amplification was observed by FISH in only one case. Presence of Merkel cell polyomavirus DNA sequences was detected in 86% (60/70) of Merkel cell carcinomas and did not emerge as a significant prognostic parameter. Merkel cell carcinoma cases at low stage (stage I-II) represented over half (40/70 cases, 57%) of cases, and the clinical course was uneventful in 25 of 40 cases while 15 cases died of tumour (10/40 cases) within 34 months or were alive with disease (5/40 cases) within 20 months. Interestingly, a very strict correlation was found between evolution and p63 expression (P<0.0001). The present data indicate that p63 expression is associated with a worse prognosis in patients with Merkel cell carcinoma, and in localised tumours it represents the single independent predictor of clinical evolution.
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Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Carcinoma de Célula de Merkel/química , Carcinoma de Célula de Merkel/genética , Neoplasias Cutâneas/química , Neoplasias Cutâneas/genética , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/análise , Proteínas Supressoras de Tumor/genética , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Célula de Merkel/mortalidade , Carcinoma de Célula de Merkel/patologia , Carcinoma de Célula de Merkel/terapia , Carcinoma de Célula de Merkel/virologia , DNA Viral/análise , Intervalo Livre de Doença , Feminino , Amplificação de Genes , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Itália , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Polyomavirus/genética , Modelos de Riscos Proporcionais , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medição de Risco , Fatores de Risco , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Neoplasias Cutâneas/virologia , Taxa de Sobrevida , Fatores de Tempo , Resultado do TratamentoRESUMO
Approximately 8% of breast cancers show increased copy numbers of chromosome 17 centromere (CEP17) by fluorescence in situ hybridization (FISH) (ie average CEP17 >3.0 per nucleus). Currently, this pattern is believed to represent polysomy of chromosome 17. HER2-amplified cancers have been shown to harbour complex patterns of genetic aberrations of chromosome 17, in particular involving its long arm. We hypothesized that aberrant copy numbers of CEP17 in FISH assays may not necessarily represent true chromosome 17 polysomy. Eighteen randomly selected CEP17 polysomic cases and a control group of ten CEP17 disomic cases, as defined by dual-colour FISH, were studied by microarray-based comparative genomic hybridization (aCGH), which was performed on microdissected samples using a 32K tiling-path bacterial artificial chromosome microarray platform. Additional FISH probes were employed for SMS (17p11.2) and RARA (17q21.2) genes, as references for chromosome 17 copy number. Microarray-based comparative genomic hybridization revealed that 11 out of the 18 polysomic cases harboured gains of 17q with involvement of the centromere, one displayed 17q gain sparing the centromeric region, and only one could be defined as polysomic. The remaining five cases displayed amplification of the centromeric region. Among these, one case, showing score 2+ by immunohistochemistry and 8.5 HER2 mean copy number, was classified as not amplified by HER2/CEP17 ratio and as amplified by HER2/SMS ratio. Our results suggest that true chromosome 17 polysomy is likely to be a rare event in breast cancer and that CEP17 copy number greater than 3.0 in FISH analysis is frequently related to gain or amplification of the centromeric region. Larger studies investigating the genetic profiles of CEP17 polysomic cases are warranted.
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Neoplasias da Mama/genética , Centrômero , Cromossomos Humanos Par 17 , Hibridização Genômica Comparativa , Feminino , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Análise em Microsséries , Poliploidia , Receptor ErbB-2/genética , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido RetinoicoRESUMO
In humans, renal resident stem cells were identified within the interstitium, the tubular cells, and the Bowman's capsule. The aim of the present study was to investigate whether multipotent stem cells are present also in the adult human-decapsulated glomeruli and whether they represent a resident population. We found that human glomeruli deprived of the Bowman's capsule contain a population of CD133+CD146+ cells and a population of CD133-CD146+ cells expressing mesenchymal stem cell (MSC) markers and renal stem cell markers CD24 and Pax-2. The CD133+CD146+ cells differed from those previously isolated from Bowman's capsule as they co-expressed endothelial markers, such as CD31 and von Willebrand factor (vWF), were CD24-negative and were not clonogenic, suggesting an endothelial commitment. The glomerular mesenchymal CD133-CD146+ population (Gl-MSC) exhibited self-renewal capability, clonogenicity, and multipotency. In addition to osteogenic, adipogenic, and chondrogenic differentiation, these cells were able to differentiate to endothelial cells and epithelial cells expressing podocytes markers such as nephrin, podocin, and synaptopodin. Moreover, Gl-MSC when cultured in appropriate conditions, acquired mesangial cell markers such as alpha-smooth muscle actin (alpha-SMA) and angiotensin II (AT-II) receptor I. The expression of the embryonic organ-specific PAX-2 gene and protein and of donor sex identity when isolated from glomeruli of a renal allograft suggested these cells to be a tissue resident population. In conclusion, these results indicate the presence of a multipotent mesenchymal cell population resident in human glomeruli that may have a role in the physiological cell turnover and/or in response to glomerular injury.
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Glomérulos Renais/citologia , Células-Tronco Mesenquimais/citologia , Diferenciação Celular , Linhagem da Célula , Separação Celular , Forma Celular , Células Cultivadas , Células Endoteliais/citologia , Imunofluorescência , Humanos , Fatores Imunológicos/metabolismo , Células Mesangiais/citologia , Fenótipo , Podócitos/citologiaRESUMO
Primary olfactory neurons located in the olfactory neuroepithelium project to the ipsilateral olfactory bulb and undergo a continuous process of neurogenesis and differentiation. We describe, in the adult rat, the kinetics of proliferation, differentiation and survival of primary olfactory neurons either in the presence or absence of their target, the olfactory bulb. The experimental design included unilateral bulbectomy, coupled with a single bromodeoxyuridine pulse 35 days after surgery. The rate of proliferation and survival of olfactory neurons was then examined by immunohistochemistry for bromodeoxyuridine, and the differentiation status by in situ hybridization for calmodulin messenger RNA in immature and mature olfactory neurons and immunohistochemistry for the dipeptide carnosine in mature olfactory neurons. We show that primary olfactory neurons can synthesize carnosine in the absence of the olfactory bulb. However, the number of carnosine-immunopositive neurons in the absence of their target is dramatically reduced to less than one-fourth, whereas the number of olfactory neurons expressing calmodulin messenger RNA is only slightly reduced. The numeric reduction of carnosine-positive neurons in the target-deprived neuroepithelium is correlated with a dramatic reduction in the survival rate of olfactory neurons, since newly generated olfactory neurons are completely lost 35 days after the bromodeoxyuridine pulse. In contrast, in the normal olfactory neuroepithelium almost one-third of newly generated olfactory neurons survive 35 days after the bromodeoxyuridine pulse. On the whole, these data indicate that most of the primary olfactory neurons have a short lifespan but that once they have connected with the olfactory bulb they may persist longer, and suggest that throughout adulthood olfactory neurons are overproduced, differentiate independently from their target, and then undergo a process of target-induced neuronal selection.