RESUMO
Background: Neoadjuvant anti-PD-1 may improve outcomes for patients with resectable NSCLC and provides a critical window for examining pathologic features associated with response. Resections showing major pathologic response to neoadjuvant therapy, defined as ≤10% residual viable tumor (RVT), may predict improved long-term patient outcome. However, %RVT calculations were developed in the context of chemotherapy (%cRVT). An immune-related %RVT (%irRVT) has yet to be developed. Patients and methods: The first trial of neoadjuvant anti-PD-1 (nivolumab, NCT02259621) was just reported. We analyzed hematoxylin and eosin-stained slides from the post-treatment resection specimens of the 20 patients with non-small-cell lung carcinoma who underwent definitive surgery. Pretreatment tumor biopsies and preresection radiographic 'tumor' measurements were also assessed. Results: We found that the regression bed (the area of immune-mediated tumor clearance) accounts for the previously noted discrepancy between CT imaging and pathologic assessment of residual tumor. The regression bed is characterized by (i) immune activation-dense tumor infiltrating lymphocytes with macrophages and tertiary lymphoid structures; (ii) massive tumor cell death-cholesterol clefts; and (iii) tissue repair-neovascularization and proliferative fibrosis (each feature enriched in major pathologic responders versus nonresponders, P < 0.05). This distinct constellation of histologic findings was not identified in any pretreatment specimens. Histopathologic features of the regression bed were used to develop 'Immune-Related Pathologic Response Criteria' (irPRC), and these criteria were shown to be reproducible amongst pathologists. Specifically, %irRVT had improved interobserver consistency compared with %cRVT [median per-case %RVT variability 5% (0%-29%) versus 10% (0%-58%), P = 0.007] and a twofold decrease in median standard deviation across pathologists within a sample (4.6 versus 2.2, P = 0.002). Conclusions: irPRC may be used to standardize pathologic assessment of immunotherapeutic efficacy. Long-term follow-up is needed to determine irPRC reliability as a surrogate for recurrence-free and overall survival.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/terapia , Pulmão/patologia , Adulto , Antineoplásicos Imunológicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Estudos de Viabilidade , Humanos , Ipilimumab/farmacologia , Ipilimumab/uso terapêutico , Pulmão/imunologia , Pulmão/cirurgia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Terapia Neoadjuvante/métodos , Neoplasia Residual , Nivolumabe/farmacologia , Nivolumabe/uso terapêutico , Pneumonectomia , Prognóstico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Reprodutibilidade dos Testes , Resultado do TratamentoRESUMO
Although mutation of APC or CTNNB1 (beta-catenin) is rare in breast cancer, activation of Wnt signalling is nonetheless thought to play an important role in breast tumorigenesis, and epigenetic silencing of Wnt antagonist genes, including the secreted frizzled-related protein (SFRP) and Dickkopf (DKK) families, has been observed in various tumours. In breast cancer, frequent methylation and silencing of SFRP1 was recently documented; however, altered expression of other Wnt antagonist genes is largely unknown. In the present study, we found frequent methylation of SFRP family genes in breast cancer cell lines (SFRP1, 7 out of 11, 64%; SFRP2, 11 out of 11, 100%; SFRP5, 10 out of 11, 91%) and primary breast tumours (SFRP1, 31 out of 78, 40%; SFRP2, 60 out of 78, 77%; SFRP5, 55 out of 78, 71%). We also observed methylation of DKK1, although less frequently, in cell lines (3 out of 11, 27%) and primary tumours (15 out of 78, 19%). Breast cancer cell lines express various Wnt ligands, and overexpression of SFRPs inhibited cancer cell growth. In addition, overexpression of a beta-catenin mutant and depletion of SFRP1 using small interfering RNA synergistically upregulated transcriptional activity of T-cell factor/lymphocyte enhancer factor. Our results confirm the frequent methylation and silencing of Wnt antagonist genes in breast cancer, and suggest that their loss of function contributes to activation of Wnt signalling in breast carcinogenesis.
Assuntos
Neoplasias da Mama/genética , Epigênese Genética , Proteínas do Olho/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Proteínas Wnt/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular Tumoral , Metilação de DNA , Feminino , Inativação Gênica , Genes Supressores de Tumor , HumanosRESUMO
AIMS: Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a post synaptic density-95/Disk-large/ZO-1 homologous domain-containing protein that is involved in the linkage of integral membrane proteins to the cytoskeleton and plays an important role in cell signalling. To gain insights into its biological relevance, this study examined expression of EBP50 in two cohorts of breast carcinoma. METHODS AND RESULTS: Forty-nine breast carcinoma tissue specimens were first examined by both immunohistochemistry and RNA in situ hybridization. EBP50 expression was correlated with various clinicopathological variables. The relative abundance of EBP50 mRNA in breast carcinomas and their corresponding normal tissue was compared using reverse transcriptase-polymerase chain reaction (RT-PCR). EBP50 immunoreactivity was then further independently validated in 120 breast carcinomas on tissue microarrays. EBP50 immunoreactivity was observed in morphologically normal and cancerous epithelial cells contrasting with the adjacent immunonegative stromal cells. An elevated cytoplasmic accumulation of EBP50 protein was readily detected in 73.5-80% of breast carcinomas. EBP50 immunoreactivity was significantly associated with tumour stage, lymph node and oestrogen receptor status. These immunohistochemical observations were further validated using RNA in situ hybridization and RT-PCR. EBP50 immunoreactivity was significantly correlated with the mRNA expression level. CONCLUSION: Oestrogen-responsive EBP50 may play an important role in tumour progression and might be a potential marker of invasiveness for breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Intraductal não Infiltrante/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , Progressão da Doença , Estrogênios/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica , Fosfoproteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
To find new genes involved in esophageal squamous cell carcinogenesis, we constructed custom cDNA arrays and used the arrays to compare gene expression profiles of 12 matched normal and malignant esophageal samples including seven superficial cancer tissues. The arrays represented nearly 4000 genes, including 1728 that were specifically selected based on pilot studies to find genes that were differentially expressed in esophageal cancers. Expression values for all genes were normalized for each sample and were compared in normal versus tumor tissues. There was a marked decrease in the levels of the transcriptional elongation factor A gene in all 12 of the squamous cell cancer samples compared to matched normal samples. Because the transcription elongation factor A gene has not been previously reported to be involved in cancer development, our results suggest that further investigation of its role in esophageal carcinogenesis is warranted.
Assuntos
Carcinoma de Células Escamosas/genética , Proteínas de Ligação a DNA/genética , Neoplasias Esofágicas/genética , Fatores de Transcrição/genética , Adulto , Idoso , Transformação Celular Neoplásica/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de OligonucleotídeosRESUMO
PURPOSE: E-cadherin (E-cad) and its associated intracellular molecules, catenins, are critical for intercellular epithelial adhesion and are often expressed in non-small-cell lung carcinomas (NSCLCs). We constructed tissue microarrays (TMAs) to investigate the expression of cadherins and catenins and their prognostic significance in NSCLC. PATIENTS AND METHODS: Tumor tissue samples from 193 patients with stages I to III NSCLC were obtained from the University of Colorado Cancer Center and Johns Hopkins Medical Institutions. Viable tumor was sampled in triplicate for the TMAs, and slides were stained by immunohistochemistry with antibodies against E-cad, N-cadherin, alpha (alpha)-, beta (beta)-, and gamma (gamma)-catenin, p120, p27, and adenomatous polyposis coli (APC) gene product. Clinical data were collected by the tumor registries. Patients were followed for a median period of 51 months (range, 18 to 100 months). RESULTS: Absent or severely reduced membranous expression for E-cad, alpha-, beta-, and gamma-catenin, and p120 were observed in 10%, 17%, 8%, 31%, and 61% of the cases, respectively. Tumor cell dedifferentiation correlated with reduced expression for E-cad, beta-catenin, gamma-catenin, and p120 in squamous cell carcinomas but not in adenocarcinomas. There was an inverse correlation between nodal metastasis and expression of E-cad and gamma-catenin. Besides the traditional clinical prognostic variables, E-cad and alpha-, beta-, and gamma-catenin expression were of positive prognostic value in univariate survival analyses. In multivariate analysis, E-cad expression was the only independent prognostic factor for survival in addition to age, node status, tumor status, and pathologic surgical margins. CONCLUSION: Reduced expression of E-cad and catenins is associated with tumor cell dedifferentiation, local invasion, regional metastasis, and reduced survival in NSCLC. E-cad is an independent prognostic factor for NSCLC survival.
Assuntos
Caderinas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Musculares , Proteína da Polipose Adenomatosa do Colo/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Cateninas , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Linfonodos/patologia , Metástase Linfática , Masculino , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Fosfoproteínas/metabolismo , Prognóstico , Taxa de Sobrevida , delta CateninaRESUMO
The human genome project has attracted a great deal of attention in recent years among the general public as well as the scientific community. Although it is likely to be a number of years before many of the expected benefits of the genomics revolution are realized, the impact of these scientific breakthroughs on diagnostic pathology is likely to become apparent relatively quickly. In particular, gene array technology, which allows gene expression measurements of thousands of genes in parallel, provides a powerful tool for pathologists seeking new markers for diagnosis. Several recent studies demonstrate how the gene array approach can not only recognize markers for known categories of neoplasia but also lead to recognition of different categories not previously appreciated. Although this approach shows great potential, the successful application of gene arrays to diagnostic problems will require thoughtful interpretation, just as immunochemical technologies require careful planning and analysis.
Assuntos
Perfilação da Expressão Gênica , Genômica , Neoplasias/genética , Patologia/tendências , DNA Complementar/análise , DNA de Neoplasias/análise , Regulação Neoplásica da Expressão Gênica , Projeto Genoma Humano , Humanos , Neoplasias/classificação , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência de DNA/métodosRESUMO
Differences in doublet analysis have the potential to alter DNA cell-cycle measurements. The techniques for doublet determination are often used interchangeably without regard for the complexity in cell shapes and sizes of biological specimens. G(0/1) doublets were identified and quantitated using fluorescence height versus area and fluorescence width versus area pulse measurements, by enumerating the proportion of G(2) + M cells that lack cyclin B1 immunoreactivity, and modeled in the DNA histograms by software algorithms. These techniques were tested on propidium iodide-stained whole epithelial cells or nuclei from asynchronous cultures, or after exposure to chemotherapeutic agents that induced cell-cycle arrest and were extended to human breast tumor specimens having DNA diploid patterns. G(0/1) doublets were easily discernible from G(2) + M singlets in cells or nuclei that are generally homogenous and spherical in shape. Doublet discrimination based on pulse processing or cyclin B1 measurements was nonconcordant in some nonspherical cell types and in cells following cell cycle arrest. Significant differences in G(0/1) doublet estimates were observed in breast tumor specimens (n = 50), with estimates based on pulse width twice those of pulse height and nearly five times greater than computer estimates. Differences between techniques are attributed to difficulties in the separation of the boundaries between G(0/1) doublets and G(2) + M singlet populations in biologically heterogeneous specimens. To improve reproducibility and enhance standardization among laboratories performing cell cycle analysis in experimental cell systems and in human breast tumors, doublet discrimination analysis should best be accomplished by computer modeling. Shape and size heterogeneity of tumor and arrested cells using pulse-processing can lead to errors and make interlaboratory comparison difficult.
Assuntos
Ciclo Celular/fisiologia , DNA/metabolismo , Citometria de Fluxo/instrumentação , Processamento de Sinais Assistido por Computador , Mama/citologia , Neoplasias da Mama , Carcinoma , Linhagem Celular , Tamanho Celular , Estudos de Avaliação como Assunto , Feminino , Citometria de Fluxo/métodos , Humanos , Processamento de Sinais Assistido por Computador/instrumentaçãoRESUMO
To determine the frequency and distribution of mitochondrial DNA mutations in breast cancer, 18 primary breast tumors were analyzed by direct sequencing. Twelve somatic mutations not present in matched lymphocytes and normal breast tissues were detected in 11 of the tumors screened (61%). Of these mutations, five (42%) were deletions or insertions in a homopolymeric C-stretch between nucleotides 303-315 (D310) within the D-loop. The remaining seven mutations (58%) were single-base substitutions in the coding (ND1, ND4, ND5, and cytochrome b genes) or noncoding regions (D-loop) of the mitochondrial genome. In three cases (25%), the mutations detected in coding regions led to amino acid substitutions in the protein sequence. We then screened an additional 46 primary breast tumors with a rapid PCR-based assay to identify poly-C alterations in D310, and we found seven more cancers with alterations. Using D310 mutations as clonal marker, we detected identical changes in five of five matched fine-needle aspirates and in four of four metastases-positive lymph nodes. The high frequency of D310 alterations in primary breast cancer combined with the high sensitivity of the PCR-based assays provides a new molecular tool for cancer detection.
Assuntos
Neoplasias da Mama Masculina/genética , Neoplasias da Mama/genética , DNA Mitocondrial/genética , Mutação , Biópsia por Agulha , Neoplasias da Mama/patologia , Neoplasias da Mama Masculina/patologia , Feminino , Marcadores Genéticos/genética , Humanos , MasculinoRESUMO
Decreased expression of the epithelial cell adhesion protein E-Cadherin occurs in several forms of human epithelial-derived cancers, including bladder cancers. We investigated the possibility that aberrant methylation of the CpG island flanking the 5' transcriptional start site of the e-cadherin gene is responsible for the decreased expression of this gene in bladder cancer, similar to the relationship previously seen between e-cadherin methylation and gene expression in other types of human cancers. Using methylation-specific polymerase chain reaction, we found methylation of this CpG island in 20 of 47 cases (43%) of bladder neoplasms ranging from low-grade papillary neoplasms to advanced, invasive cancers. When methylation status was compared to immunochemical staining for E-Cadherin, we found significantly diminished levels of E-Cadherin expression in 14 of 15 cases (93%) with methylation of the gene. We also found decreased expression of E-Cadherin, although to a somewhat lesser extent, in a high percentage (77%) of the cases without methylation of the gene. Although these data suggest a relationship between e-cadherin CpG island methylation and decreased gene expression, it evident that other mechanisms also contribute to decreased expression of this gene in bladder neoplasia. Remarkably, we also found low levels of e-cadherin methylation in urothelial cells from three of nine (33%) histologically normal bladders, with all three of the normal bladder samples with methylated e-cadherin being from individuals older than 70 years of age. Thus, methylation of the e-cadherin CpG island may occur normally in this tissue with aging as well as in low-grade papillary neoplasms, and is not specific to cancer in the bladder. This finding of methylation in normal urothelial cells from elderly individuals is provocative with respect to a possible link between aging and increased risk for bladder cancer, but it suggests limitations on the usefulness of using methylation of e-cadherin as a molecular marker for detection of bladder cancer.
Assuntos
Envelhecimento/fisiologia , Caderinas/genética , Metilação de DNA , Neoplasias da Bexiga Urinária/genética , Urotélio/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Pessoa de Meia-Idade , Valores de Referência , Neoplasias da Bexiga Urinária/patologia , Urotélio/citologiaRESUMO
Comparative genomic hybridization analysis was performed on 67 non-small-cell lung cancers (NSCLCs), including 32 squamous cell carcinomas (SCCs) and 35 adenocarcinomas (ACs), to identify differences in the patterns of genomic imbalance between these two histologic subtypes. Among the entire tumor set, the chromosome arms most often overrepresented were 1q, 3q, 5p, and 8q, each detected in 50-55% of cases. The most frequently underrepresented arms were 9q, 3p, 8p, and 17p. The number of imbalances was similar in SCCs and ACs (median number/case: 12 and 11, respectively). Moreover, many imbalances, such as gains of 1q, 5p, and 8q, occurred at a high frequency in both histologic subgroups. Several statistically significant differences, however, were found. The most prominent difference was gain of 3q24-qter, seen in 81% of SCCs compared with 31% of ACs (P < 0.0001), with amplification at 3q25-26 being detected in eight of 32 (25%) SCCs but in only two of 35 (6%) ACs. Gain of 20p13 and loss of 4q also were seen at a significantly higher rate in SCCs than in ACs, whereas overrepresentation of 6p was more common in ACs. Gains of 7q and 8q each were associated with higher-stage tumors and either positive nodal involvement or higher tumor grade. These data suggest that genes located in several chromosomal regions, particularly 3q25-26, may be associated with phenotypic properties that differentiate lung SCCs from ACs. Furthermore, certain imbalances, prominent among them gains of 7q and 8q, may be indicative of tumor aggressiveness in NSCLCs.
Assuntos
Adenocarcinoma/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Neoplasias Pulmonares/genética , Aberrações Cromossômicas/genética , Deleção Cromossômica , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 3/genética , Cromossomos Humanos Par 5/genética , Cromossomos Humanos Par 8/genética , Amplificação de Genes/genética , Humanos , Hibridização de Ácido NucleicoRESUMO
We have identified 14-3-3 sigma (sigma) as a gene whose expression is lost in breast carcinomas, primarily by methylation-mediated silencing. In this report, we investigated the timing of loss of sigma gene expression during breast tumorigenesis in vivo. We analysed the methylation status of sigma in breast cancer precursor lesions using microdissection for selective tissue sampling. We found hypermethylation of sigma in 24 of 25 carcinomas (96%), 15 of 18 (83%) of ductal carcinoma in situ, and three of eight (38%) of atypical hyperplasias. None of the five hyperplasias without atypia showed sigma-hypermethylation. Unexpectedly, patients with breast cancer showed sigma hypermethylation in adjacent histologically normal breast epithelium, while this was never observed in individuals without evidence of breast cancer. Also, samples of periductal stromal breast tissue were consistently hypermethylated, underscoring the importance of selective tissue sampling for accurate assessment of 14-3-3-sigma methylation in breast epithelium. These results suggest that hypermethylation of 14-3-3-sigma occurs at an early stage in the progression to invasive breast cancer, and may occur in apparently normal epithelium adjacent to breast cancer. These results provide evidence that loss of expression of sigma is an early event in neoplastic transformation.
Assuntos
Biomarcadores Tumorais , Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Metilação de DNA , DNA de Neoplasias/metabolismo , Exonucleases , Inativação Gênica , Proteínas de Neoplasias/genética , Proteínas/genética , Proteínas 14-3-3 , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/metabolismo , Ilhas de CpG , DNA/sangue , DNA/genética , DNA de Neoplasias/genética , Progressão da Doença , Células Epiteliais/metabolismo , Exorribonucleases , Feminino , Humanos , Hiperplasia , Leucócitos/metabolismo , Invasividade Neoplásica/genética , Reação em Cadeia da Polimerase , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Células Estromais/metabolismoRESUMO
If detected early, breast cancer is curable. We tested cells collected from the breast ducts by methylation-specific PCR (MSP). Methylated alleles of Cyclin D2, RAR-beta, and Twist genes were frequently detected in fluid from mammary ducts containing endoscopically visualised carcinomas (17 cases of 20), and ductal carcinoma in situ (two of seven), but rarely in ductal lavage fluid from healthy ducts (five of 45). Two of the women with healthy mammograms whose ductal lavage fluid contained methylated markers and cytologically abnormal cells were subsequently diagnosed with breast cancer. Carrying out MSP in these fluid samples may provide a sensitive and powerful addition to mammographic screening for early detection of breast cancer.
Assuntos
Neoplasias da Mama/diagnóstico , Metilação de DNA , Reação em Cadeia da Polimerase/métodos , Mama/citologia , Neoplasias da Mama/genética , Feminino , Humanos , Regiões Promotoras Genéticas , Sensibilidade e Especificidade , Irrigação TerapêuticaRESUMO
Many common human cancer tissues express high levels of fatty acid synthase (FAS), the primary enzyme for the synthesis of fatty acids, and the differential expression of FAS between normal and neoplastic tissues has led to the consideration of FAS as a target for anticancer therapy. To investigate the potential of targeting FAS for the treatment of pleural mesothelioma, we first determined whether FAS is overexpressed in human mesothelioma. By immunohistochemistry, we found 22 of 30 human mesothelioma tissue samples tested to express significantly increased levels of FAS compared with normal tissues, including mesothelium. To further explore FAS as a therapeutic target in mesothelioma, we established a nude mouse xenograft model for human mesothelioma using the H-Meso cell line. The i.p. xenografts of this cell line have high levels of FAS expression and fatty acid synthesis pathway activity and grow along mesothelial surfaces in a manner similar to the growth pattern of human mesothelioma. Growth of these tumor xenografts was essentially abolished in mice treated with weekly i.p. injections of C75, a synthetic, small molecule inhibitor of FAS, at levels that resulted in no significant systemic toxicity except for reversible weight loss. These results suggest that FAS may be an effective target for pharmacological therapy in a high proportion of human mesotheliomas.
Assuntos
4-Butirolactona/análogos & derivados , 4-Butirolactona/uso terapêutico , Antineoplásicos/uso terapêutico , Ácido Graxo Sintases/antagonistas & inibidores , Mesotelioma/tratamento farmacológico , Neoplasias Mesoteliais/tratamento farmacológico , Animais , Antifúngicos/farmacologia , Cerulenina/farmacologia , Modelos Animais de Doenças , Ácido Graxo Sintases/metabolismo , Humanos , Técnicas Imunoenzimáticas , Mesotelioma/enzimologia , Mesotelioma/patologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Mesoteliais/enzimologia , Neoplasias Mesoteliais/patologia , Inclusão em Parafina , Prognóstico , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Methylation of DNA in CpG dense regions of gene promoters (CpG islands) is important for transcriptional inactivation of selective genes in normal and neoplastic cells. Here, we present a spreadsheet-based program adapted from Microsoft Excel that is useful for identifying CpG islands and for assisting in the laboratory analysis of DNA methylation of these regions. Upon execution of the program, a customized workbook analyzes an entered DNA sequence for the total number and percentage cytosine and guanine nucleotides, the total number and percentage of CpG sites, and a CpG:GpC ratio. The program also displays the distribution of CpG sites in a visual format as well as in two different graphical formats. Finally, the program assists in laboratory studies of DNA methylation that employ bisulfite modification of DNA by displaying methylation-dependent effects of bisulfite treatment on DNA sequences.
Assuntos
Metilação de DNA , Análise de Sequência de DNA/métodos , Software , Composição de Bases , Sequência de Bases , Ilhas de CpG/genética , DNA/genética , DNA/metabolismoRESUMO
Loss of expression for both the estrogen receptor-alpha and E-cadherin genes has been linked to disease progression in human ductal breast carcinomas and has been associated with aberrant 5' CpG island methylation. To assess when, during malignant progression, such methylation begins and whether such methylation increases with advancing disease, we have surveyed 111 ductal carcinomas of the breast for aberrant methylation of the estrogen receptor-alpha and E-cadherin 5' CpG islands. Hypermethylation of either CpG island was evident prior to invasion in approximately 30% of ductal carcinoma in situ lesions and increased significantly to nearly 60% in metastatic lesions. Coincident methylation of both CpG islands also increased significantly from approximately 20% in ductal carcinoma in situ to nearly 50% in metastatic lesions. Furthermore, in all cases, the pattern of methylation displayed substantial heterogeneity, reflecting the well-established, heterogeneous loss of expression for these genes in ductal carcinomas of the breast.
Assuntos
Neoplasias da Mama/genética , Caderinas/genética , Carcinoma Ductal de Mama/genética , Ilhas de CpG/fisiologia , Metilação de DNA , Receptores de Estrogênio/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma in Situ/genética , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patologia , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/fisiologia , Inativação Gênica/fisiologia , Humanos , Metástase Neoplásica , Células Tumorais CultivadasRESUMO
BACKGROUND: Inherited mutations in the BRCA1 gene may be responsible for almost half of inherited breast carcinomas. However, somatic (acquired) mutations in BRCA1 have not been reported, despite frequent loss of heterozygosity (LOH or loss of one copy of the gene) at the BRCA1 locus and loss of BRCA1 protein in tumors. To address whether BRCA1 may be inactivated by pathways other than mutations in sporadic tumors, we analyzed the role of hypermethylation of the gene's promoter region. METHODS: Methylation patterns in the BRCA1 promoter were assessed in breast cancer cell lines, xenografts, and 215 primary breast and ovarian carcinomas by methylation-specific polymerase chain reaction (PCR). BRCA1 RNA expression was determined in cell lines and seven xenografts by reverse transcription-PCR. P values are two-sided. RESULTS: The BRCA1 promoter was found to be unmethylated in all normal tissues and cancer cell lines tested. However, BRCA1 promoter hypermethylation was present in two breast cancer xenografts, both of which had loss of the BRCA1 transcript. BRCA1 promoter hypermethylation was present in 11 (13%) of 84 unselected primary breast carcinomas. BRCA1 methylation was strikingly associated with the medullary (67% methylated; P =.0002 versus ductal) and mucinous (55% methylated; P =.0033 versus ductal) subtypes, which are overrepresented in BRCA1 families. In a second series of 66 ductal breast tumors informative for LOH, nine (20%) of 45 tumors with LOH had BRCA1 hypermethylation, while one (5%) of 21 without LOH was methylated (P =.15). In ovarian neoplasms, BRCA1 methylation was found only in tumors with LOH, four (31%) of 13 versus none of 18 without LOH (P =.02). The BRCA1 promoter was unmethylated in other tumor types. CONCLUSION: Silencing of the BRCA1 gene by promoter hypermethylation occurs in primary breast and ovarian carcinomas, especially in the presence of LOH and in specific histopathologic subgroups. These findings support a role for this tumor suppressor gene in sporadic breast and ovarian tumorigenesis.
Assuntos
Neoplasias da Mama/metabolismo , Genes BRCA1/genética , Perda de Heterozigosidade , Neoplasias Ovarianas/metabolismo , Regiões Promotoras Genéticas/genética , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metilação , Neoplasias Ovarianas/genética , RNA , RNA Neoplásico/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Neoplasms with mixed carcinomatous and sarcomatous growth patterns occur in many organs and tissues. The pathogenesis of these cancers is thought to be either the result of two independent neoplastic processes merging to form a single tumor, or a neoplasm of monoclonal origin that develops phenotypic diversity. To address this issue, we characterized molecular alterations in separately microdissected epithelial and sarcomatous areas in three cases of pancreatic mucinous cystic neoplasms with sarcomatous stroma. Using microsatellite markers for six chromosomal loci commonly deleted in infiltrating ductal adenocarcinomas of the pancreas, we found genetic alterations to be virtually identical between the sarcomatous and epithelial components of two of the three neoplasms. In the third neoplasm, we found allelic losses and retentions to be identical at five of the six chromosomal loci, but at a single locus, we noted allelic loss in the neoplastic epithelial component but not the sarcomatous component. The same neoplasms were also analyzed for activating point mutations in codon 12 of the K-ras gene by using mutant-enriched polymerase chain reaction and allele-specific oligonucleotide hybridization. A K-ras mutation was identified in the epithelial component of one of the three neoplasms (the same tumor with an additional allelic loss in the neoplastic epithelial cells), but the sarcomatous component of this tumor was wild-type at codon 12 of K-ras, as were both components of the other two neoplasms. Overall, these results suggest a monoclonal origin with subsequent divergence of the neoplastic epithelial and sarcomatous portions of these neoplasms.
Assuntos
Adenocarcinoma Mucinoso/genética , Carcinossarcoma/genética , Genes ras/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma Mucinoso/patologia , Carcinossarcoma/patologia , Células Clonais , Cistos/patologia , Primers do DNA/química , DNA de Neoplasias/análise , Humanos , Perda de Heterozigosidade , Repetições de Microssatélites , Neoplasias Pancreáticas/patologia , Reação em Cadeia da Polimerase , Células Estromais/patologiaRESUMO
Carcinosarcomas of the uterus, ovaries, and fallopian tubes are highly aggressive neoplasms with incompletely understood histogenesis. Although recent immunohistochemical, cell culture, and molecular genetic studies all favor these cancers to be monoclonal in origin, the extent of intratumoral genetic heterogeneity in these tumors with divergent histology has not been reported previously. For this study, we microdissected a total of 172 carcinomatous or sarcomatous foci from 17 gynecological carcinosarcomas and analyzed allelic status with 41 microsatellite markers on chromosomal arms 1p, 1q, 3p, 4q, 5q, 6q, 8p, 9p, 10q, 11p, 11q, 13q, 16q, 17p, 17q, 18q, and 22q. With the exception of a single case with microsatellite instability, we found shared allelic losses and retentions among multiple individually dissected foci of each case, strongly supportive of the concept of a monoclonal origin for these neoplasms. In eight of these cases, we also found heterogeneous patterns of allelic loss at limited numbers of chromosomal loci in either the carcinomatous or sarcomatous components of the neoplasms. These heterogeneous patterns of allelic losses were consistent with either genetic progression or genetic diversion occurring during the clonal evolution of these neoplasms. In two cases, we found the specific patterns of genetic progression to be consistent with sarcomatous components of the neoplasms arising from carcinomatous components. We conclude that most of the gynecological carcinosarcomas have a monoclonal origin, and that genetic progression and diversion parallel the development of divergent phenotypes in these tumors. Because phenotypically divergent areas of the tumors share numerous genetic alterations, this divergence most likely occurs relatively late in the evolution of these tumors.
Assuntos
Carcinossarcoma/genética , Neoplasias das Tubas Uterinas/genética , Perda de Heterozigosidade , Neoplasias Ovarianas/genética , Neoplasias Uterinas/genética , Carcinossarcoma/patologia , Progressão da Doença , Neoplasias das Tubas Uterinas/patologia , Feminino , Variação Genética , Humanos , Repetições de Microssatélites , Neoplasias Ovarianas/patologia , Fenótipo , Neoplasias Uterinas/patologiaRESUMO
The c-myc oncogene is frequently amplified in cells grown from lung tumors and has been linked to the malignancy of these cancers. In support of this, c-myc transfection enhances the in vitro proliferation and soft agar cloning of human small cell lung cancer (SCLC) cells. In this study, we surprisingly found that c-myc expression suppressed the formation of tumors by SCLC cells in athymic nude mice. c-myc expression down-regulated the protein and transcript for vascular endothelial growth factor (VEGF) in these SCLC cells, as well as VEGF transcript in rat fibroblasts manipulated for c-myc expression and in liver cells of c-myc-transgenic mice. Finally, bivariate and multivariate analyses demonstrated that the probability of tumor formation from lung cancer cell lines was negatively correlated with the relative expression of c-Myc, positively correlated with the relative expression of VEGF, and that the latent time to tumor formation was increased by the expression of c-Myc and decreased by the expression of VEGF. We hypothesize that, for lung cancer cells, c-Myc suppresses the formation of tumors in vivo by down-regulating VEGF, and that the amplification of c-myc seen in cells grown from lung tumors with a poor prognosis is an artifact of selection for growth in vitro.