RESUMO
Preliminary data suggest that allogeneic stem cell transplantation (allo-SCT) may be effective in T-prolymphocytic leukemia (T-PLL). The purpose of the present observational study was to assess the outcome of allo-SCT in patients aged 65 years or younger with a centrally confirmed diagnosis of T-PLL. Patients were consecutively registered with the EBMT at the time of transplantation and followed by routine EBMT monitoring but with an extended dataset. Between 2007 and 2012, 37 evaluable patients (median age 56 years) were accrued. Pre-treatment contained alemtuzumab in 95% of patients. Sixty-two percent were in complete remission (CR) at the time of allo-SCT. Conditioning contained total body irradiation with 6 Gy or more (TBI6) in 30% of patients. With a median follow-up of 50 months, the 4-year non-relapse mortality, relapse incidence, progression-free (PFS) and overall survival were 32, 38, 30 and 42%, respectively. By univariate analysis, TBI6 in the conditioning was the only significant predictor for a low relapse risk, and an interval between diagnosis and allo-SCT of more than 12 months was associated with a lower NRM. This study confirms for the first time prospectively that allo-SCT can provide long-term disease control in a sizable albeit limited proportion of patients with T-PLL.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia Prolinfocítica de Células T , Sistema de Registros , Condicionamento Pré-Transplante , Irradiação Corporal Total , Adolescente , Adulto , Idoso , Aloenxertos , Intervalo Livre de Doença , Feminino , Humanos , Leucemia Prolinfocítica de Células T/mortalidade , Leucemia Prolinfocítica de Células T/terapia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Taxa de SobrevidaRESUMO
Burkitt lymphoma (BL) is a highly aggressive B-cell lymphoma associated with MYC translocation. Here, we describe drug response profiling of 42 blood cancer cell lines including 17 BL to 32 drugs targeting key cancer pathways and provide a systematic study of drug combinations in BL cell lines. Based on drug response, we identified cell line specific sensitivities, i.e. to venetoclax driven by BCL2 overexpression and partitioned subsets of BL driven by response to kinase inhibitors. In the combination screen, including BET, BTK and PI3K inhibitors, we identified synergistic combinations of PI3K and BTK inhibition with drugs targeting Akt, mTOR, BET and doxorubicin. A detailed comparison of PI3K and BTKi combinations identified subtle differences, in line with convergent pathway activity. Most synergistic combinations were identified for the BET inhibitor OTX015, which showed synergistic effects for 41% of combinations including inhibitors of PI3K/AKT/mTOR signalling. The strongest synergy was observed for the combination of the CDK 2/7/9 inhibitor SNS032 and OTX015. Our data provide a landscape of drug combination effects in BL and suggest that targeting CDK and BET could provide a novel vulnerability of BL.
Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Antineoplásicos/farmacologia , Linfoma de Burkitt/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Acetanilidas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Combinação de Medicamentos , Sinergismo Farmacológico , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Oxazóis/farmacologia , Sulfonamidas/farmacologia , Tiazóis/farmacologiaRESUMO
T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive neoplasm of mature T-cells with an urgent need for rationally designed therapies to address its notoriously chemo-refractory behavior. The median survival of T-PLL patients is <2 years and clinical trials are difficult to execute. Here we systematically explored the diversity of drug responses in T-PLL patient samples using an ex vivo drug sensitivity and resistance testing platform and correlated the findings with somatic mutations and gene expression profiles. Intriguingly, all T-PLL samples were sensitive to the cyclin-dependent kinase inhibitor SNS-032, which overcame stromal-cell-mediated protection and elicited robust p53-activation and apoptosis. Across all patients, the most effective classes of compounds were histone deacetylase, phosphoinositide-3 kinase/AKT/mammalian target of rapamycin, heat-shock protein 90 and BH3-family protein inhibitors as well as p53 activators, indicating previously unexplored, novel targeted approaches for treating T-PLL. Although Janus-activated kinase-signal transducer and activator of transcription factor (JAK-STAT) pathway mutations were common in T-PLL (71% of patients), JAK-STAT inhibitor responses were not directly linked to those or other T-PLL-specific lesions. Overall, we found that genetic markers do not readily translate into novel effective therapeutic vulnerabilities. In conclusion, novel classes of compounds with high efficacy in T-PLL were discovered with the comprehensive ex vivo drug screening platform warranting further studies of synergisms and clinical testing.
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Ensaios de Triagem em Larga Escala , Leucemia Prolinfocítica de Células T/genética , Mutação , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Ciclo Celular/genética , Linhagem Celular Tumoral , Aberrações Cromossômicas , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Janus Quinases/metabolismo , Leucemia Prolinfocítica de Células T/tratamento farmacológico , Leucemia Prolinfocítica de Células T/metabolismo , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Oxazóis/farmacologia , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição STAT/metabolismo , Tiazóis/farmacologiaRESUMO
Allogeneic stem cell transplantation (alloSCT) is used for treating patients with T-prolymphocytic leukemia (T-PLL). However, direct evidence of GvL activity in T-PLL is lacking. We correlated minimal residual disease (MRD) kinetics with immune interventions and T-cell receptor (TCR) repertoire diversity alterations in patients after alloSCT for T-PLL. Longitudinal quantitative MRD monitoring was performed by clone-specific real-time PCR of TCR rearrangements (n=7), and TCR repertoire diversity assessment by next-generation sequencing (NGS; n=3) Although post-transplant immunomodulation (immunosuppression tapering or donor lymphocyte infusions) resulted in significant reduction (>1 log) of MRD levels in 7 of 10 occasions, durable MRD clearance was observed in only two patients. In all three patients analyzed by TCR-NGS, MRD responses were reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature. Novel clonotypes that could explain a clonal GvL effect did not emerge. In conclusion, TCR-based MRD quantification appears to be a suitable tool for monitoring and guiding treatment interventions in T-PLL. The MRD responses to immune modulation observed here provide first molecular evidence for GvL activity in T-PLL which, however, may be often only transient and reliant on a poly-/oligoclonal rather than a monoclonal T-cell response.
Assuntos
Efeito Enxerto vs Leucemia , Imunomodulação , Leucemia Prolinfocítica de Células T/terapia , Neoplasia Residual/diagnóstico , Receptores de Antígenos de Linfócitos T/análise , Transplante de Células-Tronco/métodos , Adulto , Idoso , Células Clonais/imunologia , Rearranjo Gênico do Linfócito T/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cinética , Leucemia Prolinfocítica de Células T/diagnóstico , Pessoa de Meia-Idade , Neoplasia Residual/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos de Linfócitos T/genética , Transplante HomólogoRESUMO
Clinical information about thiotepa-based autologous stem cell transplantation (auto-SCT) outside the primary central nervous system lymphoma (PCNSL) field is sparse. In this registry-based retrospective study, we evaluated potential risks and benefits of thiotepa-based preparative regimens compared with BEAM (carmustine, etoposide, cytarabine, melphalan) in auto-SCT for diffuse large B-cell lymphoma (DLBCL, excluding PCNSL), follicular lymphoma (FL) or Hodgkin lymphoma (HL). A total of 14 544 patients (589 thiotepa and 13 955 BEAM) met the eligibility criteria, and 535 thiotepa- and 1031 BEAM-treated patients were matched in a 1:2 ratio for final comparison. No significant differences between thiotepa and BEAM groups for any survival end point were identified in the whole sample or disease entity subsets. For a more detailed analysis, 47 TEAM (thiotepa, etoposide, cytarabine, melphalan)-treated patients were compared with 75 matched BEAM patients with additional collection of toxicity data. Again, there were no significant differences between the two groups for any survival end point. In addition, the frequency of common infectious and non-infectious complications including secondary malignancies was comparable between TEAM and BEAM. These results indicate that thiotepa-based high-dose therapy might be a valuable alternative to BEAM in DLBCL, HL and FL. Further evaluation by prospective clinical trials is warranted.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Linfoma/mortalidade , Linfoma/terapia , Sistema de Registros , Transplante de Células-Tronco , Tiotepa/administração & dosagem , Adolescente , Adulto , Idoso , Autoenxertos , Carmustina/administração & dosagem , Citarabina/administração & dosagem , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Melfalan/administração & dosagem , Pessoa de Meia-Idade , Podofilotoxina/administração & dosagem , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
Mutations of the tumor suppressor p53 lead to chemotherapy resistance and a dismal prognosis in chronic lymphocytic leukemia (CLL). Whereas p53 targets are used to identify patient subgroups with impaired p53 function, a comprehensive assessment of non-coding RNA targets of p53 in CLL is missing. We exploited the impaired transcriptional activity of mutant p53 to map out p53 targets in CLL by small RNA sequencing. We describe the landscape of p53-dependent microRNA/non-coding RNA induced in response to DNA damage in CLL. Besides the key p53 target miR-34a, we identify a set of p53-dependent microRNAs (miRNAs; miR-182-5p, miR-7-5p and miR-320c/d). In addition to miRNAs, the long non-coding RNAs (lncRNAs) nuclear enriched abundant transcript 1 (NEAT1) and long intergenic non-coding RNA p21 (lincRNA-p21) are induced in response to DNA damage in the presence of functional p53 but not in CLL with p53 mutation. Induction of NEAT1 and lincRNA-p21 are closely correlated to the induction of cell death after DNA damage. We used isogenic lymphoma cell line models to prove p53 dependence of NEAT1 and lincRNA-p21. The current work describes the p53-dependent miRNome and identifies lncRNAs NEAT1 and lincRNA-p21 as novel elements of the p53-dependent DNA damage response machinery in CLL and lymphoma.
Assuntos
Apoptose , Leucemia Linfocítica Crônica de Células B/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Proteína Supressora de Tumor p53/genética , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Proliferação de Células , Imunoprecipitação da Cromatina , Dano ao DNA , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
Chronic lymphocytic leukemia (CLL) is an incurable malignancy of mature B cells. One of the major challenges in treatment of CLL is the achievement of a complete remission to prevent relapse of disease originating from cells within lymphoid tissues and subsequent chemoresistance. In search for novel drugs that target CLL cells in protective microenvironments, we performed a fungal extract screen using cocultures of primary CLL cells with bone marrow-derived stromal cells. A secondary metabolite produced by Penicillium aquamarinium was identified as Chaetoglobosin A (ChA), a member of the cytochalasan family that showed preferential induction of apoptosis in CLL cells, even under culture conditions that mimic lymphoid tissues. In vitro testing of 89 CLL cases revealed effective targeting of CLL cells by ChA, independent of bad prognosis characteristics, like 17p deletion or TP53 mutation. To provide insight into its mechanism of action, we showed that ChA targets filamentous actin in CLL cells and thereby induces cell-cycle arrest and inhibits membrane ruffling and cell migration. Our data further revealed that ChA prevents CLL cell activation and sensitizes them for treatment with PI3K and BTK inhibitors, suggesting this compound as a novel potential drug for CLL.
Assuntos
Apoptose/efeitos dos fármacos , Citocinese/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Alcaloides Indólicos/farmacologia , Leucemia Linfocítica Crônica de Células B/patologia , Micotoxinas/farmacologia , Células Estromais/efeitos dos fármacos , Actinas/metabolismo , Western Blotting , Estudos de Casos e Controles , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Técnicas de Cocultura , Citoesqueleto/metabolismo , Citometria de Fluxo , Fungos/química , Voluntários Saudáveis , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Estromais/metabolismo , Células Estromais/patologia , Células Tumorais CultivadasRESUMO
Chronic lymphocytic leukemia (CLL) with 17p deletion or mutations of the TP53 gene has a very poor outcome. Optimal treatment of these patients remains a major clinical challenge, and disagreement on the optimal treatment approach exists. Conventional chemo-immunotherapy with rituximab in combination with purine analogues yields lower response-rates and less satisfactory results than for CLL patients with intact p53. Allogeneic stem cell transplantation may allow long-term remissions in this challenging group of patients. In this review, we will discuss current treatment options as well as experimental approaches in clinical trials for CLL patients with deleted or mutated TP53. Particular emphasis will be placed on novel agents with the potential to change clinical practice and future perspectives for the management of these "highest risk" patients.
Assuntos
Cromossomos Humanos Par 17/genética , Genes p53 , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/terapia , Deleção de Sequência , Proteína Supressora de Tumor p53/genética , Antineoplásicos/uso terapêutico , Humanos , Inibidores de Proteínas Quinases/uso terapêutico , Transplante de Células-TroncoRESUMO
Sarcomatoid renal cell carcinoma (SRCC) is an aggressive tumor variant thought to arise predominantly from dedifferentiation of clear cell carcinoma. A few reports of SRCC associated with non-clear cell tumors led to the presumption that SRCC may arise from any renal cell carcinoma, although direct evidence of this is lacking. Cytogenetic studies on 3 previously documented SRCCs associated with papillary renal cancers showed either 3p deletions or absence of trisomy 7, 17 in the sarcomatoid tumors, suggesting origin from a coexistent clear cell tumor. The present case represents the first conclusive evidence of direct progression of non-clear cell carcinoma to SRCC with both tumor components containing multiple copies of chromosomes 7 and 17. Many genetic anomalies, including p53 mutations, frequently recognized in SRCC were not recognized in this case, highlighting the importance of cytogenetic evaluation of all SRCC. The patient is well and without evidence of tumor progression 1 year after surgery, and the sinister outlook of SRCC in association with clear cell carcinoma may not apply in SRCC of non-clear cell origin.
Assuntos
Carcinoma Papilar/patologia , Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Idoso , Carcinoma Papilar/genética , Carcinoma de Células Renais/genética , Aberrações Cromossômicas , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 7/genética , Análise Citogenética , Feminino , Humanos , Cariotipagem , Neoplasias Renais/genética , Sarcoma/patologiaRESUMO
BACKGROUND: Prostate ducts and acini whose lumens are filled with malignant cells represent a well-recognized histological pattern recently termed intraductal carcinoma of the prostate (IDC-P). These tumors are often associated with rapid disease progression, and most recur after radical surgery. Controversy exists as to whether IDC-P should be recognized as a separate entity, an extension of high-grade dysplasia (PIN) or invasive carcinoma as described by the Gleason grading system. This study investigates the use of molecular markers in defining the position of IDC-P in the evolutionary hierarchy of prostate cancer progression. METHODS: IDC-P, high-grade dysplasia, and invasive cancers from a cohort of 20 selected radical prostatectomy specimens were screened for loss of heterozygosity (LOH), using 12 polymorphic microsatellite markers frequently lost in prostate cancer. RESULTS: LOH was absent in Gleason grade 3 cancer, infrequent in high-grade dysplasia (9%) and Gleason grade 4 cancer (29%), but common in IDC-P (60%). In IDC-P, and to a lesser extent Gleason grade 4 cancers, multiple sites of allelic loss in individual cases were usual. CONCLUSIONS: Allelic instability provides further evidence that IDC-P is not a simple extension of dysplasia, nor does it represent invasion of Gleason grade 3 cancers into the ductal/acinar system. IDC-P and Gleason grade 4 cancer represent late but possibly separate events in prostate cancer evolution.
Assuntos
Adenocarcinoma/genética , Carcinoma Intraductal não Infiltrante/genética , Perda de Heterozigosidade , Neoplasia Prostática Intraepitelial/genética , Neoplasias da Próstata/genética , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Alelos , Carcinoma Intraductal não Infiltrante/patologia , Carcinoma Intraductal não Infiltrante/cirurgia , Progressão da Doença , Marcadores Genéticos/genética , Humanos , Masculino , Repetições de Microssatélites/genética , Invasividade Neoplásica , Prostatectomia , Neoplasia Prostática Intraepitelial/patologia , Neoplasia Prostática Intraepitelial/cirurgia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgiaRESUMO
Separation of renal cell tumors into different prognostic groups is an imperative function of the diagnostic pathologist. Recently, chromophobe renal carcinoma has been described as a tumor that is morphologically distinct from conventional "clear cell" carcinoma and that has a low metastatic potential. Identification is based on routine light microscopic features and is confirmed by special stains, immunohistochemistry, and electron microscopy. We present a variant of chromophobe renal carcinoma that did not show the typical cytomorphologic features on light microscopy after formaldehyde fixation. After fixation in Solufix (a commercial fixative), these features were recognized and the diagnosis was confirmed. The tumor also showed an unusual form of calcification and psammoma body formation not previously recognized in chromophobe tumors. Molecular biological assessment was inconclusive, but excluded a chromosome 3p deletion usually found in conventional renal carcinoma. The use of a different primary fixative may provide a cost-effective screening tool to detect variant renal tumors and may have important prognostic implications.
Assuntos
Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Segunda Neoplasia Primária/patologia , Idoso , Calcinose/patologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/cirurgia , Carcinoma de Células Renais/ultraestrutura , Deleção Cromossômica , Cromossomos Humanos Par 3 , Fixadores , Variação Genética , Humanos , Neoplasias Renais/genética , Neoplasias Renais/cirurgia , Neoplasias Renais/ultraestrutura , Masculino , Segunda Neoplasia Primária/genética , Segunda Neoplasia Primária/cirurgia , Nefrectomia , Prognóstico , Neoplasias da Próstata/radioterapia , Fixação de Tecidos/métodosRESUMO
A majority of studies have shown an increase in the risk of breast cancer among women previously diagnosed with fibroadenoma (FA). At present there is conflicting evidence whether some of the chromosome abnormalities frequently found in breast carcinoma, such as loss of heterozygosity (LOH), are already present in FAs and other types of benign breast disease and, if present, whether such abnormalities are associated with the observed increase in risk. Microsatellite instability (MSI) is also recognised as a marker of genetic damage and is thought to occur when there has been damage to the cell's mismatch repair (MMR) system. We have analysed 39 cases of FA obtained from paraffin-embedded tissue for the presence of MSI and LOH at 11 loci to determine if these types of genetic alterations occur in FA. The incidence of MSI and LOH found were 4 of 395 (1.0%) and 5 of 271 (1.8%) informative loci tested respectively. Approximately 8% of cases were positive for MSI and 10% were positive for LOH, with one specimen having multiple occurrences of both MSI and LOH. We conclude that these forms of genetic alteration do occur in FAs but that the incidence is low.
Assuntos
Neoplasias da Mama/genética , Fibroadenoma/genética , Perda de Heterozigosidade , Repetições de Microssatélites , Feminino , HumanosRESUMO
Various approaches can now be taken for amplification of RNA transcripts using the polymerase chain reaction (PCR). Here, we compare three such methods: (i) uncoupled reverse transcription (RT)-PCR (using separate reactions for cDNA synthesis and PCR), (ii) continuous RT-PCR (in which RT and DNA amplification occur in an uninterrupted reaction) using either a single enzyme for both RT and DNA amplification or (iii) using two enzymes, one for each task. We have found that the continuous two-enzyme RT-PCR method is the most sensitive, followed by the uncoupled RT-PCR and then the continuous single-enzyme method. The continuous methods require less sample handling than the uncoupled method, and hence are less labor-intensive and less prone to contamination. The continuous single-enzyme method is the most expensive to perform in terms of reagents due to the quantity of DNA polymerase required; however, it does have advantages over the two enzyme methods in that the use of a thermostable enzyme for RT can alleviate certain problems by allowing RT to occur at higher temperatures than those tolerable by viral reverse transcriptases.
Assuntos
Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , DNA Polimerase Dirigida por DNA/metabolismo , Eletroforese em Gel de Ágar , Humanos , Melanoma , RNA Neoplásico/análise , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
Optimal cutting temperature (OCT) is a widely used embedding medium for tissues for histopathologic analysis. This investigation examined the effects that OCT storage can have upon the ability to perform subsequent molecular biological analyses. Tumor material was dissected into small pieces and stored at approximately 20 degrees C both with and without OCT. DNA and RNA were then extracted from the tissue fragments and analyzed by the polymerase chain reaction (PCR), using primer sets designed to amplify a range of product sizes, and also by reverse transcriptase-PCR (RT-PCR). The storage of pathological specimens in OCT compound was found to affect significantly and irreversibly the ability to amplify DNA in the PCR, particularly as the size of the amplified fragment increased. This effect appeared to occur as a result of greater degradation of DNA extracted from tissue embedded in OCT compared to DNA extracted from tissue stored without OCT. RNA quality appeared unaffected, which may be because of the extraction protocol employed. Our results suggest that OCT-embedded frozen-tissue samples may be used for RNA isolation for subsequent RT-PCR and for the in vitro amplification of DNA targets of approximately < 300 base pairs only. We strongly advise against the routine storage of any tissue biopsy material in OCT if molecular analyses may be required.
Assuntos
Secções Congeladas , Reação em Cadeia da Polimerase , Inclusão do Tecido/métodos , Marcadores Genéticos , Humanos , Linfoma não Hodgkin/genética , Linfoma não Hodgkin/patologia , Reação em Cadeia da Polimerase/métodosRESUMO
We describe a method using an inexpensive craft glue to routinely isolate specific areas of tissue as small as 1 mm2 from paraffin sections. The tissue may be digested to release nucleic acid suitable for PCR or reverse transcription PCR. The use of this procedure obviates the requirement for manual microdissection or ultraviolet light irradiation. Tissue remaining on the slide can be stained and analyzed, allowing the precision of the extraction to be determined. The slide can be stored as a permanent record of the material taken for analysis.
Assuntos
DNA de Neoplasias/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , RNA Neoplásico/isolamento & purificação , Adesivos/química , Sequência de Bases , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , DNA de Neoplasias/efeitos da radiação , Feminino , Gengiva/química , Técnicas Histológicas , Humanos , Dados de Sequência Molecular , Inclusão em Parafina , Reação em Cadeia da Polimerase/instrumentação , Amido/química , Raios UltravioletaRESUMO
Detection of viral RNA by polymerase chain reaction (PCR) requires the prior reverse transcription of the viral RNA. In order to minimise the number of manual manipulations required for processing large numbers of samples, we attempted to design a system whereby all the reagents required for both reverse transcription and amplification can be added to one tube and a single, non-interrupted thermal cycling program performed. Whilst attempting to set up such a one-tube system with Taq polymerase (Taq; Biotech International) and avian myoblastosis virus (AMV) reverse transcriptase (RT), we noticed a substantial decrease in the sensitivity of detection of viral RNA. Investigation of this phenomenon has revealed direct interference of RT with Taq polymerase. Evidence supporting this conclusion includes the following observations: (1) increasing the ratio of Taq to RT improves sensitivity; (2) adding non-homologous RNA improves sensitivity; (3) RT that has been heat inactivated prior to Taq addition does not exert this effect; (4) the effect is not sequence restricted; (5) the Mg2+ ions are not sequestered by RT. In addition, the effect is not limited to AMV RT, Moloney murine leukaemia virus RT also affects Taq activity.