RESUMO
BACKGROUND: Intravitreal injection of chemotherapy in retinoblastoma eyes with vitreous seeds may lead to a risk of extraocular tumour dissemination that has not been assessed so far. AIMS: To develop a sensitive and clinically feasible technique to assess for potential retinoblastoma cell reflux after intravitreal injection of melphalan. METHODS: Filter papers were cut in 6 mm diameter circles and sterilised before use. Eyes with retinoblastoma vitreous seeds (group D, International Classification) received weekly intravitreal melphalan injections (20 µg or 30 µg/dose) followed by cryotherapy as part of local treatment. Immediately after finishing the injection and cryotherapy, filter papers were placed on the injection site and on the cryoprobe tip to assess for the expression of the cone-rod homeobox gene (CRX) by real-time qPCR as a surrogate of retinoblastoma RNA. The assay was developed and validated to determine sensitivity, linearity, recovery, repeatability and reproducibility. RESULTS: The assay for quantitation of CRX expression was linear in the range of 1 to 1000 cells. The lowest limit of detection was one retinoblastoma cell and allowed to recover 100% of the cell load in external supplementation. A total of 14 eyes received 22 cycles of intravitreal melphalan and were evaluated for potential extraocular tumour cell dissemination using the developed technique. None of the cycles were positive for CRX in samples from the scar or from the cryoprobe tip. CONCLUSIONS: A sensitive and simple method of tumour cell assessment has been developed that can be used in the clinics to assess for potential extraocular dissemination after intravitreal injections to assure its performance.
Assuntos
Antineoplásicos Alquilantes/administração & dosagem , Proteínas de Homeodomínio/genética , Melfalan/administração & dosagem , Inoculação de Neoplasia , RNA Neoplásico/análise , Neoplasias da Retina/genética , Retinoblastoma/genética , Transativadores/genética , Biomarcadores Tumorais , Crioterapia , Humanos , Injeções Intravítreas , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Neoplasias da Retina/tratamento farmacológico , Retinoblastoma/tratamento farmacológico , Estudos Retrospectivos , Células Tumorais CultivadasRESUMO
BACKGROUND: Standard surgical practice for colorectal cancer involves resection of the primary lesion and all draining lymph nodes. Accurate intraoperative assessment of nodal status could allow stratified resectional extent. One-step nucleic acid (OSNA) can provide a rapid method of interrogating nodal tissue, whilst near-infrared (NIR) laparoscopy together with indocyanine green (ICG) can identify relevant nodal tissue intraoperatively. METHODS: ICG was administered around the tumour endoscopically prior to the operation. Fluorescent nodes identified by NIR were marked and submitted for whole-node OSNA analysis. Further fresh lymph nodes dissected from the standard resection specimen were examined and analysed by both conventional histology and OSNA. In addition, the status of the fluorescent nodes was compared to that of non-ICG nodes to assess their predictive value. RESULTS: Sixteen patients were recruited with a total final lymph node count of 287. 78 fresh lymph nodes were identified on fresh dissection for both histological and OSNA assessment with an analytical concordance rate of 98.7% (77/78). OSNA sensitivity was 1 (0.81-1, 95% CI) and specificity 0.98 (0.91-1, 95% CI). Six patients had a total of nine nodes identified intraoperatively by ICG fluorescence. Of these nine nodes, one was positive for metastasis on OSNA. OSNA analysis of the ICG-labelled node matched the final histological nodal stage in 3/6 patients (two being N0 and one N1). The final pathological nodal stage of the other three was N1 or N2, while the ICG nodes were negative. CONCLUSION: OSNA is highly concordant with standard histology, although only a minority of nodes identifiable by full pathological analysis were found for OSNA on fresh dissection. OSNA can be combined with NIR and ICG lymphatic mapping to provide intraoperative assessment of nodal tissue in patients with colorectal cancer.
Assuntos
Neoplasias Colorretais/cirurgia , Verde de Indocianina/farmacologia , Laparoscopia/métodos , Linfonodos/patologia , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Neoplásico/análise , Biópsia de Linfonodo Sentinela/métodos , Adulto , Idoso , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/secundário , Corantes/farmacologia , Estudos de Viabilidade , Feminino , Fluorescência , Humanos , Período Intraoperatório , Linfonodos/cirurgia , Metástase Linfática/diagnóstico , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Estudos ProspectivosRESUMO
BACKGROUND: RNA extracted from formalin-fixed paraffin-embedded (FFPE) samples is chemically modified and degraded, which compromises its use in gene expression studies. Most of the current approaches for RNA quality assessment are not suitable for FFPE derived RNA. RESULTS: We have developed a single-tube multiplex endpoint RT-PCR assay specifically designed to evaluate RNA extracted from FFPE tissues for mRNA integrity and performance in reverse transcription - quantitative real-time PCR (RT-qPCR) assays. This single-tube quality control (QC) assay minimises the amount of RNA used in quality control. mRNA integrity and the suitability of RNA for RT-PCR is evaluated by the multiplex endpoint RT-PCR assay using the TBP gene mRNA as the target sequence. The RT-PCR amplicon sizes, 92, 161, 252 and 300 bp, cover a range of amplicon sizes suitable for a wide range of RT-qPCR assays. The QC assay was used to evaluate RNA prepared by two different protocols for extracting total RNA from needle microdissected FFPE breast tumour samples. The amplification products were analysed by gel electrophoresis where the spectrum of amplicon sizes indicated the level of RNA degradation and thus the suitability of the RNA for PCR. The ability of the multiplex endpoint RT-PCR QC assay to identify FFPE samples with an adequate RNA quality was validated by examining the Cq values of an RT-qPCR assay with an 87 bp amplicon. CONCLUSIONS: The multiplex endpoint RT-PCR assay is well suited for the determination of the quality of FFPE derived RNAs, to identify which RT-PCR assays they are suitable for, and is also applicable to assess non-FFPE RNA for gene expression studies. Furthermore, the assay can also be used for the evaluation of RNA extraction protocols from FFPE samples.
Assuntos
Formaldeído/química , Inclusão em Parafina , RNA Neoplásico/análise , RNA/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Neoplasias da Mama/genética , Feminino , HumanosAssuntos
Monitoramento de Medicamentos/métodos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucócitos/citologia , Transportador 1 de Cátions Orgânicos/genética , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , RNA Mensageiro/análise , Benzamidas , Monitoramento de Medicamentos/normas , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Contagem de Leucócitos , Leucócitos Mononucleares , Neutrófilos , Piperazinas/farmacocinética , Pirimidinas/farmacocinética , RNA Neoplásico/análiseRESUMO
We performed a technical and cost-effectiveness analysis of quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) for the assessment of HER2 in breast cancer. We evaluated 44 frozen and 55 formalin-fixed paraffin-embedded (FFPE) breast carcinoma specimens by Q-RT-PCR, immunohistochemical analysis, and fluorescent in situ hybridization (FISH). Immunohistochemical and FISH analyses were performed on individual slides and on tissue microarray. Costs of techniques were calculated to study 1 case and 10 or 40 cases. Q-RT-PCR provided reliable data in frozen and FFPE specimens, which were significantly correlated. HER2 messenger RNA levels were significantly stratified in agreement with immunohistochemical data (P < .05). There was complete concordance between Q-RT-PCR and immunohistochemical results for negative and strongly positive (3+) cases. The intermediate immunohistochemical group (2+), including FISH+ and FISH- cancers, could also be stratified by Q-RT-PCR. Cost analysis documented the advantage of Q-RT-PCR in all US Food and Drug Administration-approved assays. Our data support the use of Q-RT-PCR for testing breast cancer specimens to select patients for HER2 inhibitory therapy.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Receptor ErbB-2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia , Adulto , Idoso , Idoso de 80 Anos ou mais , Glândulas Apócrinas/metabolismo , Glândulas Apócrinas/patologia , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Lobular/diagnóstico , Carcinoma Lobular/genética , Carcinoma Lobular/metabolismo , Carcinoma Papilar/diagnóstico , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Análise Custo-Benefício , Feminino , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente/economia , Hibridização in Situ Fluorescente/métodos , Pessoa de Meia-Idade , RNA Mensageiro/análise , RNA Neoplásico/análise , Receptor ErbB-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Neoplasias das Glândulas Sudoríparas/genética , Neoplasias das Glândulas Sudoríparas/metabolismo , Neoplasias das Glândulas Sudoríparas/patologia , Análise Serial de Tecidos/economia , Análise Serial de Tecidos/métodosRESUMO
OBJECTIVES: To describe our experience with the optimization and validation of laser-capture microdissection (LCM) for biomarker analysis in prostate tissues. As LCM allows the separation of benign and malignant epithelial structures and stromal elements, it not only allows identification of the source of the biomarker, but might also accentuate gene or protein expression changes by reducing contamination by other cellular elements. MATERIALS AND METHODS: In all, 19 fresh-frozen prostate tissue samples were subjected to LCM, with the cDNA being analysed using quantitative polymerase chain reaction for several genes, to identify the optimum number of cells for capture, as well as gene markers assessing for the purity of the captured cells. The localization was further confirmed by in situ hybridization. RESULTS: Prostate-specific antigen (PSA) and cytokeratin 8, were expressed solely by epithelial cells, whereas hepatocyte growth factor (HGF) and tissue inhibitor of metalloproteinases-3 (TIMP3) were expressed only by stromal cells, and the levels of transcripts of these genes were unaltered between benign and malignant tissues. CONCLUSIONS: These data suggest that PSA, cytokeratin 8, HGF and TIMP3 are reliable gene markers of purity of epithelial and stromal compartments for LCM of prostate tumours. Although this technique is not new and is increasingly used in laboratories, it needs optimization and stringent validation criteria before data analysis. This applies to all tissue types subjected to LCM.
Assuntos
Lasers , Microdissecção/métodos , Próstata/patologia , Neoplasias da Próstata/patologia , RNA Neoplásico/análise , Células Epiteliais/patologia , Expressão Gênica , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Hibridização In Situ , Queratina-8/metabolismo , Masculino , Microdissecção/normas , Antígeno Prostático Específico/metabolismo , Células Estromais/patologia , Inibidor Tecidual de Metaloproteinase-3/metabolismoRESUMO
Gene expression measurement techniques such as quantitative reverse transcriptase (qRT)-PCR require a normalization strategy to allow meaningful comparisons across biological samples. Typically, this is accomplished through the use of an endogenous housekeeping gene that is presumed to show stable expression levels in the samples under study. There is concern regarding how precisely specific genes can be measured in limited amounts of mRNA such as those from microdissected (MD) tissues. To address this issue, we evaluated three different approaches for qRT-PCR normalization of dissected samples; cell count during microdissection, total RNA measurement, and endogenous control genes. The data indicate that both cell count and total RNA are useful in calibrating input amounts at the outset of a study, but do not provide enough precision to serve as normalization standards. However, endogenous control genes can accurately determine the relative abundance of a target gene relative to the entire cellular transcriptome. Taken together, these results suggest that precise gene expression measurements can be made from MD samples if the appropriate normalization strategy is employed.
Assuntos
Perfilação da Expressão Gênica/métodos , Microdissecção/métodos , RNA Mensageiro/análise , RNA Neoplásico/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Histocitoquímica , Humanos , Masculino , Neoplasias da Próstata/genética , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: Neuroblastoma (NB) is a well-known malignant disease in infants, which comprises 10% of childhood malignancies. Despite recent advances in understanding the neuro-oncology, NB still accounts for more death in childhood than any other cancer. Research in childhood tumors should not only be focused on the malignant signatures of cancer cells but also novel drug prototypes using phytochemicals. The present study was aimed to determine the role of curcumin against murine neuroblastoma cell line (N2a). METHODS: The in vitro assessment of curcumin against was made in N2a cell line in a dose-dependent manner (group I (control) and group II - IX (10 microM-80 microM). The efficacy of the drug was evaluated by estimating the levels of protein bound carbohydrates, glycoprotein, genomic DNA, total RNA levels, and inhibition of MMP-9 were studied. The gap junctional communication in the cells was also assessed. RESULTS: The levels of protein bound carbohydrates, DNA, RNA levels, glycoprotein were found to be altered on drug supplementation in NB cells. Inhibition of MMP-9 in curcumin-supplemented N2a cells was revealed by zymographic analysis. Assessment of Lucifer yellow dye uptake in curcumin-supplemented N2a cells showed the up-regulation of GJIC. CONCLUSIONS: These observations suggest that the curcumin, the active principle of curcuma longa, could be developed into an effective chemo preventive and chemotherapeutic agent. This selected concentration range needs further studies at molecular level, for conforming its role and its action against uncontrolled proliferation of NB.
Assuntos
Curcumina/farmacologia , Neuroblastoma/patologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA de Neoplasias/análise , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Junções Comunicantes/química , Glicoproteínas/análise , Metaloproteases/análise , Camundongos , RNA Neoplásico/análise , Azul Tripano/farmacologiaRESUMO
Cancer is a leading cause of death in developed countries. For most cancers, a patient's prognosis improves dramatically when the disease is detected at an early stage. Although advancements in imaging technology have dramatically improved early detection, many cancers go undetected until it is too late. As cancer develops, molecular changes occur before many of the current prognostic markers can be detected. Proteomics will be used to interrogate the protein milieu in serum and urine to detect molecular changes indicative of a disease state and will provide a cost-effective alternative to current diagnostic tools.
Assuntos
Neoplasias/diagnóstico , Neoplasias/terapia , Proteômica/métodos , Biomarcadores Tumorais/análise , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Análise Custo-Benefício , Humanos , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/urina , Neoplasias/genética , Prognóstico , Proteômica/economia , RNA Mensageiro/análise , RNA Neoplásico/análiseRESUMO
OBJECTIVES: To quantify gene expression in tumor cells from human head and neck squamous cell carcinomas (HNSCC) using laser capture microdissection (LCM). STUDY DESIGN: Histopathologically identified HNSCC cells were microdissected from frozen sections, RNA was isolated, and vascular endothelial growth factor (VEGF) gene expression was measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR). MATERIALS AND METHODS: Two human HNSCC tumor samples and matched normal mucosal biopsies and five human xenograft tumor specimens were harvested, embedded, and frozen in OCT. The frozen tumors were sectioned to 8 to 10 mum in thickness, and hematoxylin-eosin (H&E) staining was performed before LCM. An estimated 2,000 to 3,000 tumor cells were microdissected from frozen sections and processed for RNA isolation. mRNA for VEGF was analyzed by real time RT-PCR (TaqMan) with commercially available primers and probes. RESULTS: Two thousand to 3000 cells were necessary to obtain a suitable quantity of RNA for subsequent gene expression study by real-time RT-PCR. The gene expression of VEGF, a major tumor angiogenic factor, was tested in microdissected HNSCC and compared with uninvolved normal mucosal controls. A greater than seven-fold increase of VEGF expression in tumor specimens versus mucosal controls was observed. CONCLUSIONS: LCM is a novel sample conserving technique that allows the precise selection of tumor cells from a heterogeneous architecture. The combination of LCM and real-time RT-PCR appears particularly efficacious for studying HNSCC molecular pathogenesis and identifying tissue-specific biomarkers.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias de Cabeça e Pescoço/genética , Lasers , Microdissecção , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Biópsia por Agulha , Carcinoma de Células Escamosas/patologia , Estudos de Coortes , Feminino , Secções Congeladas , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Imuno-Histoquímica , Masculino , Probabilidade , RNA Neoplásico/análise , Sensibilidade e Especificidade , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
INTRODUCTION: Primary cutaneous B-cell lymphoma (1 degrees CBCL) accounts for 25% of all lymphomas. The difficulty in distinction of reactive from neoplastic B-cell infiltrates prompts the use of molecular diagnostic adjuncts. While T-cell clonality can be seen in various reactive states, clonal B-cell infiltrates are often neoplastic; standard assays employed include polymerase chain reaction (PCR) or Southern blot analysis to assess heavy chain rearrangement. We sought to assess the utility of kappa (kappa) and lambda (lambda) mRNA expression using the Ventana automated assay (Ventana Medical Systems, Tucson, AZ, USA) in the analysis of atypical cutaneous B-cell lymphoid infiltrates. MATERIALS AND METHODS: Multiple 4 micro m sections of paraffin-embedded, formalin-fixed skin biopsies from 31 patients with CBCL were placed on silane-coated slides, deparaffinized, then digested in pepsin (5 mg/ml) for 30 min at 37 degrees C. Fluorescein-tagged oligoprobes and tissue mRNA were denatured at 80 degrees C for 5 min, hybridized for 2 h at 37 degrees C, and incubated with antifluorescein alkaline phosphatase conjugates. Detection of the probe target complex employed nitroblue tetrazolium and bromochloroindolyl phosphate conjugates with a nuclear fast red counterstain. A kappa : lambda ratio > 3 : 1 was held to represent kappa light chain restriction and a kappa : lambda ratio = 1 : 1 to indicate lambda light chain restriction. RESULTS: The diagnosis in each case was determined by careful integration of clinical, histologic, and phenotypic data. The diagnoses included: pseudolymphoma (PL), marginal zone lymphoma (MZL), 1 degrees CBCL of the trunk, scalp or leg, 2 degrees lymphoma, and plasma cell dyscrasia. All but one case of lymphoma were light chain restricted. All cases of PL were proven to be polyclonal by this methodology. In non-plasmacytic small cell lymphomas, only 5-10% of the infiltrate expressed kappa or lambda, with clonality established through the abnormal kappa : lambda ratio. Interpretations were most difficult in the 2 degrees small cell-dominant follicular center cell lymphomas and easiest in cases with significant plasmacytic differentiation (i.e. MZL, immunocytomas, or plasma cell dyscrasias). CONCLUSION: The Ventana kappa/lambda assay is a reliable, quick, and inexpensive way to determine B-cell clonality in cutaneous lymphoid infiltrates in paraffin-embedded formalin-fixed tissue.
Assuntos
Linfócitos B/patologia , Cadeias Leves de Imunoglobulina/genética , Infiltração Leucêmica , Linfoma de Células B/patologia , RNA Mensageiro , Pele/patologia , Linfócitos B/metabolismo , Células Clonais , Humanos , Cadeias Leves de Imunoglobulina/metabolismo , Hibridização In Situ , Linfoma de Células B/genética , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , RNA Neoplásico/análiseRESUMO
The presence of lymph node metastases is the major determinant for prognosis in head and neck squamous cell carcinoma (HNSCC). It is at present unknown whether the same holds true for the presence of histologically undetectable micrometastases. We analyzed 456 histologically tumor-negative lymph nodes of 23 HNSCC patients without (pN0) and 18 patients with one or two tumor-positive lymph nodes (pN+) in their neck dissection specimens at histopathologic examination. To detect the presence of disseminated tumor cells and micrometastases in these lymph nodes, we used real-time quantitative RT-PCR with E48 (Ly-6D) transcripts as a squamous cell-specific molecular marker. The results were compared with histopathologic examination and clinical outcome. E48 transcripts were detected in lymph nodes of 5 (22%) of 23 patients in the pN0 group, and in histologically negative lymph nodes of 10 (56%) of 18 patients in the pN+ group. In the pN0 group, the presence of E48-positive lymph nodes was significantly associated with a distinctly poor cause-specific survival as compared with those with E48-negative lymph nodes. Our results indicate that E48 real-time quantitative RT-PCR is a suitable method for the detection of micrometastases in lymph nodes of patients with HNSCC. Moreover, detection of micrometastases seems clinically relevant but should be confirmed in a large multicenter trial.
Assuntos
Antígenos Ly/metabolismo , Carcinoma de Células Escamosas/metabolismo , Moléculas de Adesão Celular/metabolismo , Glicoproteínas/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Linfonodos/patologia , Metástase Linfática/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/secundário , Primers do DNA/química , Sondas de DNA/química , Proteínas Ligadas por GPI , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Linfonodos/metabolismo , Metástase Linfática/genética , Esvaziamento Cervical , RNA Mensageiro/metabolismo , RNA Neoplásico/análise , Taxa de SobrevidaRESUMO
Genetic testing for cancer predisposing mutations in BRCA1 and BRCA2 has been of benefit to many individuals from breast and ovarian cancer-prone kindreds. However, a function has not been assigned to many of the domains that make up these complex proteins and hence, the significance of many sequence variants, including missense mutations, splice-site mutations, and in-frame deletions/insertions, remains unclear. We identified a putative splice site mutation (IVS6-2delA) in BRCA1 in a family attending a Familial Cancer Centre that had a significant history of both breast and ovarian cancer. This sequence variant was not novel but the exact effect on mRNA splicing and hence the biological impact of this sequence variation was unclear and therefore the finding was unable to be used in genetic counseling of the family. Via the construction of novel GFP-based expression fusion constructs, we demonstrated that this sequence variation prevented normal splicing of the BRCA1 transcript. By combining these data with an assessment of the histopathological features of the breast carcinomas in this family and mutation penetrance estimate we were able to conclude that this BRCA1 variant conveyed an increased risk of breast cancer.
Assuntos
Proteína BRCA1/genética , Proteínas Luminescentes/biossíntese , Mutação , Penetrância , Sítios de Splice de RNA/genética , Adulto , Idoso , Processamento Alternativo/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , DNA Complementar/genética , Feminino , Genes BRCA1 , Triagem de Portadores Genéticos/métodos , Predisposição Genética para Doença/genética , Testes Genéticos/métodos , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Masculino , Pessoa de Meia-Idade , Neoplasias Ovarianas/genética , Linhagem , RNA Neoplásico/análiseRESUMO
BACKGROUND: About 30-40% of men with localized prostate cancer undergoing radical prostatectomy will have cancer recurrence. It is estimated that one third recur locally and two thirds develop distant metastases with or without local recurrence. METHODS: In the present study we investigate the detection of prostate-specific antigen (PSA) mRNA in peripheral blood samples (n=200 patients) and pelvic lymph nodes (n=154 patients) by PSA reverse transcriptase polymerase chain reaction (RT-PCR) and compare these results to standard histological and immunohistochemical staging. RESULTS: We have observed a statistically significant correlation of lymph node PSA RT-PCR with standard pathologic risk factors, such as Gleason score (p=0.011), the presence of Gleason patterns 4 or 5 (p=0.005), lymph node metastasis (p<0.001) and a nearly significant correlation with the pT category (p=0.087). 39.5% (57/145) of the pN0 patients had PSA mRNA detectable in their lymph nodes. Blood PSA RT-PCR showed no correlation with the aforementioned factors and was even inversely correlated with preoperative serum PSA and lymph node status. Immunohistochemistry did not detect unsuspected prostate micrometastases in any pN0 patient. CONCLUSIONS: Lymph node PSA RT-PCR correlates with the Gleason score and the presence of Gleason patterns 4 or 5. Further clinical follow-up and correlation of RT-PCR status with overall outcome is required to allow validation of lymph node RT-PCR as a predictor of distant disease recurrence.
Assuntos
Adenocarcinoma/patologia , Linfonodos/patologia , Estadiamento de Neoplasias/métodos , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/patologia , RNA Neoplásico/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Adenocarcinoma/cirurgia , Idoso , Análise de Variância , Biópsia por Agulha , Estudos de Coortes , Humanos , Imuno-Histoquímica , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Probabilidade , Prognóstico , Estudos Prospectivos , Neoplasias da Próstata/cirurgia , RNA Mensageiro/análise , Sensibilidade e Especificidade , Ressecção Transuretral da PróstataAssuntos
Perfilação da Expressão Gênica/métodos , Leucemia de Células B/genética , Leucemia de Células T/genética , Linfoma não Hodgkin/genética , RNA Mensageiro/análise , RNA Neoplásico/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sistemas Computacionais , Humanos , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economiaRESUMO
Synovial sarcoma is the most common nonrhabdomyosarcomatous soft tissue sarcoma in children and adolescents and is characterized by a reciprocal t(X;18)(p11;q11) which results in the fusion of the SYT gene on chromosome 18q11 to either of two closely related genes, SSX1 (Xp11.23) or SSX2 (Xp11.21). Detection of this translocation or its resultant gene fusion by molecular methods is helpful in the pathologic diagnosis of synovial sarcoma, especially in poorly differentiated tumors. This study was designed to evaluate the utility of a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect and distinguish SYT-SSX1 and SYT-SSX2 fusions in fresh and archival specimens of synovial sarcoma in pediatric patients seen at St. Jude Children's Research Hospital. In addition, the clinicopathologic features of the tumors with SYT-SSX1 vs. SYT-SSX2 fusions were compared. The 25 patients studied had a median age of 13 years 9 months (range 5 to 19 years). Estimates of survival and event-free survival at 5 years were 78.7 +/- 10.5% and 56.2 +/- 13.2%, respectively. Seventeen (68%) tumors were monophasic, eight (32%) were biphasic. Seven tumors contained poorly differentiated areas. Positive results for either SYT-SSX1 or SYT-SSX2 were obtained in 21/25 (84%) cases. Three cases did not have a detectable gene fusion and one had no amplifiable RNA. SYT-SSX1 transcripts were found in 18/24 (75%) of the tumors while SYT-SSX2 transcripts were identified in 3/24 (12.5%). All of the poorly differentiated tumors and seven out of eight tumors from patients who developed lung metastases had an SYT-SSX1 fusion transcript. Real-time PCR is useful in detecting and distinguishing SYT-SSX1 from SYT-SSX2 gene fusions in synovial sarcoma. Valuable aspects of this methodology are the applicability to both frozen and formalin-fixed samples, decreased labor costs, and the rapidity of results. In addition, distinguishing SYT-SSX1 from SYT-SSX2 fusions with these methods allow for prospective collection of information that may clarify issues of prognostic relevance.
Assuntos
Proteínas de Fusão Oncogênica/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sarcoma Sinovial/genética , Neoplasias de Tecidos Moles/genética , Adolescente , Adulto , Biomarcadores Tumorais/genética , Criança , Pré-Escolar , Custos e Análise de Custo , Primers do DNA/química , Feminino , Hospitais Pediátricos , Humanos , Masculino , RNA Neoplásico/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia , Sarcoma Sinovial/mortalidade , Sarcoma Sinovial/patologia , Neoplasias de Tecidos Moles/mortalidade , Neoplasias de Tecidos Moles/patologia , Taxa de Sobrevida , Tennessee/epidemiologiaRESUMO
Ultrastructural studies have shown that Clara cell-type is a more common type of adenocarcinoma than alveolar type II cell-type, and that both types may provide better prognosis than other types, indicating an importance of differentiation toward peripheral airway cells. Pulmonary surfactant protein (SP)-A is a specific marker for both alveolar type II cells and Clara cells in peripheral lung tissues, while SP-C and Clara cell 10 kD protein (CC10) may be particularly and highly specific to alveolar type II cells and Clara cells, respectively. The aims of this study were to assess the differentiation of adenocarcinoma cells in pleural effusions by evaluating the expression of these cell markers and to evaluate their values as diagnostic tools for judging the cause of pleural effusion. We examined pleural effusions from 52 patients; 20 with primary lung adenocarcinomas, 6 with small cell lung carcinomas, 11 with metastatic malignant tumors and 15 with non-neoplastic diseases. The cell pellets from effusions were subjected to immunocytochemical staining for SP-A, proSP-C, a precursor of SP-C, and CC10. By this immunocytochemical study for SP-A and proSP-C, 10 (50%) and 6 (30%) of 20 adenocarcinomas, respectively, showed a positive immunoreactivity in their effusion cells, while none of them expressed CC10. Alveolar type II cells therefore may be the main progenitor cells of some adenocarcinomas. In pleural effusions from patients with primary lung adenocarcinomas, reverse transcriptase-polymerase chain reaction (RT-PCR) for SP-A mRNA showed a sensitivity of 83%, while, in all remaining patients, these assays were negative. In conclusion, we demonstrated that lung adenocarcinomas, which are partially differentiated toward alveolar type II cells, are not as rare as previously thought, and that both the RT-PCR and immunocytochemical analyses for SP-A and pro-SP-C could be worthy indicators of differential diagnosis.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias Pulmonares/metabolismo , Derrame Pleural Maligno/metabolismo , Adenocarcinoma/diagnóstico , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/diagnóstico , Proteínas Associadas a Surfactantes Pulmonares/análise , RNA Neoplásico/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não ParamétricasRESUMO
The inv(16)(p13q22) chromosomal rearrangement associated with FAB M4Eo acute myeloid leukemia (AML) subtype is characterized by the presence of the CBFbeta/MYH11 fusion transcript that can be used to detect minimal residual disease (MRD). However, qualitative RT-PCR studies of MRD have so far produced conflicting results and seem of limited prognostic value. We have evaluated retrospectively MRD in a large series of CBFbeta/MYH11-positive patients employing both qualitative and quantitative (real-time PCR) approaches. 186 bone marrow samples from 36 patients were examined with a median follow-up of 27.5 months; 15 patients relapsed during follow-up. In qualitative studies, carried out by 'nested' RT-PCR assay, all patients in complete remission (CR) immediately after induction/consolidation therapy were found to be PCR positive. However, follow-up samples at later time points were persistently negative (except one case) in patients remaining in continuous CR (CCR) for more than 12 months. 16 patients were evaluated by quantitative real-time PCR assay: CBFbeta/MYH11 transcript copy number was normalized for expression of the housekeeping gene ABL, expressed as fusion gene copy number per 10(4) copies of ABL. A 2-3 log decline in leukemic transcript copy number was observed after induction/consolidation therapy. After achieving CR, the mean copy number was significantly higher in patients destined to relapse compared to patients remaining in CCR (151 vs 9, P < 0.0001 by Mann-Whitney test). Moreover, in CCR patients, the copy number dropped below the detection threshold after the treatment protocol was completed and remained undetectable in subsequent MRD analysis in accordance with results obtained by qualitative RT-PCR. On the contrary, in the seven patients who relapsed, the copy number in CR never declined below the detection threshold; thus a cut-off value discriminating these two groups of patients could be established. The findings of our study, if confirmed, might confer an important predictive value to quantitative real-time PCR determinations of MRD in patients with inv(16) leukemia.
Assuntos
Proteínas de Fusão Oncogênica/análise , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adolescente , Adulto , Idoso , Criança , Estudos de Coortes , Feminino , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Neoplasia Residual , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , RNA Neoplásico/análise , Indução de Remissão , Estudos Retrospectivos , Resultado do TratamentoRESUMO
We describe a simple system of tissue arraying with multiple tissue fragments obtained with a biopsy punch from selected areas of paraffin blocks. The new blocks thus constructed allow multiple tissue sections in which the uniform shape of the fragments coupled with a geometrical display and a significant amount of tissue per case allows a dependable, cost-effective way to screen tumors or other kinds of tissues with techniques such as immunohistochemistry. This system avoids the disadvantages of previous laborious methods of tissue arraying, such as expensive equipment and scarce tissue sampling, and it can be implemented in any institution with minimal cost and elaboration.