RESUMO
BACKGROUND: The dual-probe fluorescence in situ hybridization (FISH) assay for human epidermal growth factor receptor 2 (HER2) gene amplification in breast cancer provides an HER2:CEP17 (centromere enumeration probe for chromosome 17) ratio. Copy number alteration (CNA) in CEP17 may skew this ratio. The authors analyzed the impact of the 2013 American Society of Oncology/College of American Pathologists (ASCO/CAP) guidelines and an alternative chromosome 17 probe on HER2 status in tumor specimens with CEP17 CNA. METHODS: Specimens with CEP17 CNA (n = 310) were selected from 3048 tumor samples that were received from January 2013 to June 2015 for testing with the alternative chromosome 17 probe D17S122. Reclassification of HER2 status was assessed using the 2007 and 2013 ASCO/CAP guidelines. RESULTS: The alternative chromosome 17 probe reclassified 82 of 310 (26.5%) and 87 of 310 (28.1%) tumors using the 2007 and 2013 guidelines, respectively. Of the 41 of 310 tumors (13.2%) that were reclassified from nonamplified to amplified according to 2007 guidelines, 28 of 41 (68.3%) had an average HER2 copy number ≥4.0 and <6.0. The 39 of 310 tumors (12.6%) that were reclassified from equivocal to amplified according to 2013 guidelines had a mean HER2 copy number between ≥4.0 and <6.0. Most of these patients had stage I, hormone receptor-positive, lymph node-negative tumors, which is an unusual clinicopathologic profile for HER2-amplified tumors, and most received HER2-targeted therapy in addition to endocrine therapy. CONCLUSIONS: Reflex testing with an alternative chromosome 17 probe using the 2013 ASCO/CAP guidelines reclassified 28.1% of tumor samples that had CEP17 CNA, converting nearly one-half from equivocal to amplified. The benefit of HER2-targeted therapy in this patient population requires further study. Cancer 2017;123:2230-2239. © 2017 American Cancer Society.
Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , Cromossomos Humanos Par 17/genética , Hibridização in Situ Fluorescente/métodos , Sondas Moleculares/genética , Receptor ErbB-2/genética , Antineoplásicos/uso terapêutico , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/classificação , Neoplasias da Mama/tratamento farmacológico , Carcinoma/classificação , Carcinoma/tratamento farmacológico , Progressão da Doença , Feminino , Humanos , Oncologia , Terapia de Alvo Molecular , Recidiva Local de Neoplasia/epidemiologia , Patologia Clínica , Guias de Prática Clínica como Assunto , Estudos RetrospectivosRESUMO
The two clinically validated and Food and Drug Administration approved lung cancer predictive biomarkers (epidermal growth factor receptor mutations and anaplastic lymphoma kinase (ALK) translocations) occur in only about 20 % of lung adenocarcinomas and acquired resistance develops to first generation drugs. Several other oncogenic drivers for lung adenocarcinoma have emerged as potentially druggable targets with new predictive biomarkers. Oncologists are requesting testing for ROS1 translocations which predict susceptibility to crizotinib, already approved for ALK positive lung cancers. Other potential biomarkers which are currently undergoing clinical trials are RET, MET, HER2 and BRAF. Detection of these biomarkers includes fluorescent in situ hybridization and/or reverse transcriptase polymerase chain reaction (ROS1, RET, HER2), mutation analysis (BRAF) and immunohistochemistry (MET). Screening by immunohistochemistry may be useful for some biomarkers (ROS1, BRAF). Targeted next generation sequencing techniques may be useful as well. These five biomarkers are under consideration for inclusion in revised lung cancer biomarker guidelines by the College of American Pathologists, International Association for the Study of Lung Cancer and Association for Molecular Pathology.
Assuntos
Adenocarcinoma/genética , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Quinase do Linfoma Anaplásico , Biomarcadores Tumorais/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Terapia de Alvo Molecular , Mutação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismoRESUMO
Increasing use of fine needle aspiration for oncological diagnosis, while minimally invasive, poses a challenge for molecular testing by traditional sequencing platforms due to high sample requirements. The advent of affordable benchtop next-generation sequencing platforms such as the semiconductor-based Ion Personal Genome Machine (PGM) Sequencer has facilitated multi-gene mutational profiling using only nanograms of DNA. We describe successful next-generation sequencing-based testing of fine needle aspiration cytological specimens in a clinical laboratory setting. We selected 61 tumor specimens, obtained by fine needle aspiration, with known mutational status for clinically relevant genes; of these, 31 specimens yielded sufficient DNA for next-generation sequencing testing. Ten nanograms of DNA from each sample was tested for mutations in the hotspot regions of 46 cancer-related genes using a 318-chip on Ion PGM Sequencer. All tested samples underwent successful targeted sequencing of 46 genes. We showed 100% concordance of results between next-generation sequencing and conventional test platforms for all previously known point mutations that included BRAF, EGFR, KRAS, MET, NRAS, PIK3CA, RET and TP53, deletions of EGFR and wild-type calls. Furthermore, next-generation sequencing detected variants in 19 of the 31 (61%) patient samples that were not detected by traditional platforms, thus increasing the utility of mutation analysis; these variants involved the APC, ATM, CDKN2A, CTNNB1, FGFR2, FLT3, KDR, KIT, KRAS, MLH1, NRAS, PIK3CA, SMAD4, STK11 and TP53 genes. The results of this study show that next-generation sequencing-based mutational profiling can be performed on fine needle aspiration cytological smears and cell blocks. Next-generation sequencing can be performed with only nanograms of DNA and has better sensitivity than traditional sequencing platforms. Use of next-generation sequencing also enhances the power of fine needle aspiration by providing gene mutation results that can direct personalized cancer therapy.
Assuntos
Análise Mutacional de DNA/métodos , DNA/análise , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Biópsia por Agulha Fina , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da PolimeraseRESUMO
The American Society of Clinical Oncology/College of American Pathologists ERBB2 testing guidelines address several pre-analytical variables known to affect ERBB2 testing accuracy. According to 2010 updated guidelines, the pre-analytical variable of time to tissue fixation (cold ischemia time) should be kept to <1 h, however, little has been published about cold ischemia time and its significance in ERBB2 testing. To that end, this study evaluated ERBB2 status using two different FDA-approved in-situ hybridization methods and an FDA-approved immunohistochemistry (IHC) assay in the largest cohort to date (n=84) of invasive breast carcinomas with tracked cold ischemia time. Cold ischemia time was stratified into four groups (<1 h (n=45), 1-2 h (n=27), 2-3 h (n=6), and >3 h (n=6)) and ERBB2 status was evaluated in each group by IHC (4B5) and by in-situ hybridization methodologies (PathVysion(®) fluorescence in situ hybridization and the INFORM HER2(®) dual in situ DNA probe assay). Both in-situ hybridization methods were evaluated using three ERBB2 scoring criteria (dual-probe guidelines, single-probe guidelines, and the FDA package insert scoring instructions). Fluorescence in-situ hybridization (FISH) and INFORM HER2(®) demonstrated 100% concordance in the detection of ERBB2 amplification by all three scoring guidelines at all cold ischemia time points. Agreement between in-situ hybridization methodologies and IHC was superior using single-probe guidelines compared with dual probe or FDA scoring instructions. In addition, Inform HER2(®) in-situ hybridization signals were significantly more intense than FISH at all cold ischemia time points, however, no significant loss of either chromosome 17 or ERBB2 signal was detected by FISH or Inform HER2(®) in-situ hybridization in cold ischemia times up to 3 h. On the basis of our findings, cold ischemia time up to 3 h has no deleterious effect on the detection of ERBB2 via in-situ hybridization or IHC.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Isquemia Fria/métodos , Hibridização in Situ Fluorescente/métodos , Receptor ErbB-2/análise , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Citodiagnóstico/métodos , Citodiagnóstico/normas , Feminino , Fixadores , Formaldeído , Humanos , Imuno-Histoquímica , Reprodutibilidade dos Testes , Tempo , Análise Serial de Tecidos , Fixação de TecidosRESUMO
CONTEXT.: Neoplastic cellularity assessment has become an essential component of molecular oncology testing; however, there are currently no best practice recommendations or guidelines for this potentially variable step in the testing process. OBJECTIVE.: To describe the domestic and international practices of neoplastic cellularity assessment and to determine how variations in laboratory practices affect neoplastic cellularity assessment accuracy. DESIGN.: Data were derived from 57 US and international laboratories that participated in the 2019 College of American Pathologists Neoplastic Cellularity Proficiency Testing Survey (NEO-B 2019). NEO-B 2019 included 29 laboratory practice questions and 5 images exhibiting challenging histologic features. Participants assessed the neoplastic cellularity of hematoxylin-eosin-stained digital images, and results were compared to a criterion standard derived from a manual cell count. RESULTS.: The survey responses showed variations in the laboratory practices for the assessment of neoplastic cellularity, including the definition of neoplastic cellularity, assessment methodology, counting practices, and quality assurance practices. In some instances, variation in laboratory practice affected neoplastic cellularity assessment performance. CONCLUSIONS.: The results highlight the need for a consensus definition and improved standardization of the assessment of neoplastic cellularity. We put forth an initial set of best practice recommendations to begin the process of standardizing neoplastic cellularity assessment.
Assuntos
Laboratórios , Ensaio de Proficiência Laboratorial , Coleta de Dados , Hematoxilina , Humanos , Oncologia , Técnicas de Diagnóstico MolecularRESUMO
CONTEXT.: Next-generation sequencing-based assays are being increasingly used in the clinical setting for the detection of somatic variants in solid tumors, but limited data are available regarding the interlaboratory performance of these assays. OBJECTIVE.: To examine proficiency testing data from the initial College of American Pathologists (CAP) Next-Generation Sequencing Solid Tumor survey to report on laboratory performance. DESIGN.: CAP proficiency testing results from 111 laboratories were analyzed for accuracy and associated assay performance characteristics. RESULTS.: The overall accuracy observed for all variants was 98.3%. Rare false-negative results could not be attributed to sequencing platform, selection method, or other assay characteristics. The median and average of the variant allele fractions reported by the laboratories were within 10% of those orthogonally determined by digital polymerase chain reaction for each variant. The median coverage reported at the variant sites ranged from 1922 to 3297. CONCLUSIONS.: Laboratories demonstrated an overall accuracy of greater than 98% with high specificity when examining 10 clinically relevant somatic single-nucleotide variants with a variant allele fraction of 15% or greater. These initial data suggest excellent performance, but further ongoing studies are needed to evaluate the performance of lower variant allele fractions and additional variant types.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/normas , Ensaio de Proficiência Laboratorial , Oncologia/normas , Patologia Clínica/normas , Humanos , Reprodutibilidade dos TestesRESUMO
CONTEXT: -EGFR mutations and anaplastic lymphoma kinase (ALK) translocations have significant biologic and therapeutic implications in lung cancers, particularly lung adenocarcinomas. ALK translocations are less frequent compared with EGFR mutations; interestingly, these two abnormalities are most commonly mutually exclusive. The 2013 College of American Pathologists/Association for Molecular Pathology/International Association for the Study of Lung Cancer molecular testing guideline for lung cancers recommend a testing algorithm in which detection of ALK translocations using fluorescence in situ hybridization (FISH) is to be performed following testing for EGFR mutations. Such an algorithm is cost-effective but potentially slows down turnaround time; and as a secondary test, ALK FISH assay may not be completed because it requires the use of additional tissue, and the small biopsies or cytology specimens may have been exhausted in the extraction of nucleic acid for EGFR mutation screening. OBJECTIVE: -To provide efficient testing of both EGFR and ALK genetic alterations in small biopsies and cytology specimens. DESIGN: -We validated a highly sensitive ALK reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay as a screening tool for ALK translocations and amplifications. RESULTS: -We performed a retrospective review of cases previously tested by FISH and found that all FISH ALK translocation-positive specimens were RT-qPCR positive, and all FISH ALK translocation-negative cases were RT-qPCR negative (the sensitivity and specificity of the ALK RT-qPCR assay were 100%). CONCLUSION: -This assay allows rapid identification of ALK alterations, can be performed in conjunction with EGFR testing, and does not require use of valuable additional tumor tissue.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Amplificação de Genes , Neoplasias Pulmonares/genética , Receptores Proteína Tirosina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Translocação Genética , Quinase do Linfoma Anaplásico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/patologiaRESUMO
CONTEXT: -Colorectal carcinoma is the third most common cause of cancer death in males and females in the United States. Rectal adenocarcinoma can have distinct therapeutic and surgical management from colonic adenocarcinoma owing to its location and anatomic considerations. OBJECTIVE: -To determine the oncologic driver mutations and better understand the molecular pathogenesis of rectal adenocarcinoma in relation to colon adenocarcinoma. DESIGN: -Next-generation sequencing was performed on 20 cases of primary rectal adenocarcinoma with a paired lymph node or solid organ metastasis by using an amplicon-based assay of more than 2800 Catalogue of Somatic Mutations in Cancer (COSMIC)-identified somatic mutations. RESULTS: -Next-generation sequencing data were obtained on both the primary tumor and metastasis from 16 patients. Most rectal adenocarcinoma cases demonstrated identical mutations in the primary tumor and metastasis (13 of 16, 81%). The mutations identified, listed in order of frequency, included TP53, KRAS, APC, FBXW7, GNAS, FGFR3, BRAF, NRAS, PIK3CA, and SMAD4. CONCLUSIONS: -The somatic mutations identified in our rectal adenocarcinoma cohort showed a strong correlation to those previously characterized in colonic adenocarcinoma. In addition, most rectal adenocarcinomas harbored identical somatic mutations in both the primary tumor and metastasis. These findings demonstrate evidence that rectal adenocarcinoma follows a similar molecular pathogenesis as colonic adenocarcinoma and that sampling either the primary or metastatic lesion is valid for initial evaluation of somatic mutations and selection of possible targeted therapy.
Assuntos
Adenocarcinoma/genética , Neoplasias do Colo/genética , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Neoplasias Retais/genética , Adenocarcinoma/patologia , Proteína da Polipose Adenomatosa do Colo/genética , Proteínas de Ciclo Celular/genética , Cromograninas/genética , Classe I de Fosfatidilinositol 3-Quinases , Neoplasias do Colo/patologia , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Feminino , GTP Fosfo-Hidrolases/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Neoplasias Retais/patologia , Proteína Smad4/genética , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
CONTEXT: The classification of pulmonary large cell carcinoma has undergone a major revision with the recent World Health Organization (WHO) 2015 Classification. Many large cell carcinomas are now reassigned to either adenocarcinoma with solid pattern or nonkeratinizing squamous cell carcinoma based on immunopositivity for adenocarcinoma markers or squamous cell carcinoma markers, respectively. Large cell carcinomas that are negative for adenocarcinoma and squamous cell carcinoma immunomarkers are now classified as large cell carcinoma with null immunohistochemical features (LCC-N). Although a few studies investigated the mutation profile of large cell carcinomas grouped by immunostain profile before the publication of the new WHO classification, investigation of tumors previously diagnosed as large cell carcinoma and reclassified according to the 2015 WHO classification has not, to our knowledge, been reported. OBJECTIVE: To determine the mutation profiles of pulmonary large cell carcinomas reclassified by WHO 2015 criteria. DESIGN: Archival cases of non-small cell lung carcinoma with large cell carcinoma morphology (n = 17) were reclassified according to 2015 WHO criteria. To determine mutation profile, we employed Ion Torrent (Life Technologies, Carlsbad, California)-based next-generation sequencing (50 genes; more than 2800 mutations) in addition to real-time quantitative reverse transcription polymerase chain reaction for ALK translocation detection. RESULTS: Two of 17 cases (12%) were reclassified as LCC-N, and both had mutations-BRAF D594N in one case and KRAS G12C in the other case. Seven of 17 cases (41%) were reclassified in the adenocarcinoma with solid pattern group, which showed one KRAS G12C and one EGFR E709K + G719C double mutation in addition to mutations in TP53. Eight of 17 cases (47%) were reclassified in the nonkeratinizing squamous cell carcinoma group, which showed mutations in PIK3CA, CDKN2A, and TP53. No ALK translocations or amplifications were detected. CONCLUSIONS: The adenocarcinoma with solid pattern group showed mutations typical of adenocarcinoma, whereas the nonkeratinizing squamous cell carcinoma group showed mutations typical of squamous cell carcinoma. Both LCC-N cases had mutations associated with adenocarcinoma, supporting the hypothesis that LCC-N is related to adenocarcinoma.
Assuntos
Adenocarcinoma/classificação , Biomarcadores Tumorais/genética , Carcinoma de Células Grandes/classificação , Carcinoma Pulmonar de Células não Pequenas/classificação , Carcinoma de Células Escamosas/classificação , Neoplasias Pulmonares/classificação , Adenocarcinoma/genética , Adenocarcinoma/patologia , Idoso , Carcinoma de Células Grandes/genética , Carcinoma de Células Grandes/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Classe I de Fosfatidilinositol 3-Quinases , Estudos de Coortes , Inibidor p16 de Quinase Dependente de Ciclina/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Fosfatidilinositol 3-Quinases/genética , Projetos Piloto , Estudos Retrospectivos , Análise de Sequência de DNA , Proteína Supressora de Tumor p53/genética , Organização Mundial da SaúdeRESUMO
Recently, treatment with bromodomain and extraterminal protein antagonist (BA) such as JQ1 has been shown to inhibit growth and induce apoptosis of human acute myelogenous leukemia (AML) cells, including those expressing FLT3-ITD. Here, we demonstrate that cotreatment with JQ1 and the FLT3 tyrosine kinase inhibitor (TKI) ponatinib or AC220 synergistically induce apoptosis of cultured and primary CD34(+) human AML blast progenitor cells (BPC) expressing FLT3-ITD. Concomitantly, as compared with each agent alone, cotreatment with JQ1 and the FLT3-TKI caused greater attenuation of c-MYC, BCL2, and CDK4/6. Simultaneously, cotreatment with JQ1 and the FLT3-TKI increased the levels of p21, BIM, and cleaved PARP, as well as mediated marked attenuation of p-STAT5, p-AKT, and p-ERK1/2 levels in AML BPCs. Conversely, cotreatment with JQ1 and FLT3-TKI was significantly less active against CD34(+) normal bone marrow progenitor cells. Knockdown of BRD4 by short hairpin RNA also sensitized AML cells to FLT3-TKI. JQ1 treatment induced apoptosis of mouse Ba/F3 cells ectopically expressing FLT3-ITD with or without FLT3-TKI-resistant mutations F691L and D835V. Compared with the parental human AML FLT3-ITD-expressing MOLM13, MOLM13-TKIR cells resistant to AC220 were markedly more sensitive to JQ1-induced apoptosis. Furthermore, cotreatment with JQ1 and the pan-histone deacetylase inhibitor (HDI) panobinostat synergistically induced apoptosis of FLT3-TKI-resistant MOLM13-TKIR and MV4-11-TKIR cells. Collectively, these findings support the rationale for determining the in vivo activity of combined therapy with BA and FLT3-TKI against human AML cells expressing FLT3-ITD or with BA and HDI against AML cells resistant to FLT3-TKI.
Assuntos
Azepinas/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Triazóis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Imidazóis/farmacologia , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/metabolismo , Camundongos , Proteínas Nucleares/antagonistas & inibidores , Piridazinas/farmacologia , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/biossíntese , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
OBJECTIVES: Identification of tumor-specific somatic mutations has had a significant impact on both disease diagnosis and therapy selection. The ability of next-generation sequencing (NGS) to provide a quantitative assessment of mutant allele burden, in numerous target genes in a single assay, provides a significant advantage over conventional qualitative genotyping platforms. METHODS: We assessed the quantitative capability of NGS and a primer extension-based matrix-assisted laser desorption ionization-time-of-flight (PE-MALDI) assay and directly correlated NGS mutant allele burden determination to morphologic assessment of tumor percentage in H&E-stained slides. RESULTS: Our results show a 100% concordance between NGS and PE-MALDI in mutant allele detection and a significant correlation between NGS and PE-MALDI for determining mutant allele burden when mutant allele burden is 10% or more. CONCLUSIONS: NGS-based mutation screening provides a quantitative assessment comparable to that of PE-MALDI. In addition, NGS also allows for a high degree of multiplexing and uses nanogram quantities of DNA, thereby preserving precious material for future analysis. Furthermore, this study provides evidence that H&E-based morphologic assessment of tumor burden does not correlate to actual tumor mutant allele burden frequency.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Neoplasias/genética , Análise Mutacional de DNA , Genes Neoplásicos , Genótipo , Humanos , Reação em Cadeia da Polimerase , Semicondutores , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
The bromodomain and extra-terminal (BET) protein family members, including BRD4, bind to acetylated lysines on histones and regulate the expression of important oncogenes, for example, c-MYC and BCL2. Here, we demonstrate the sensitizing effects of the histone hyperacetylation-inducing pan-histone deacetylase (HDAC) inhibitor panobinostat on human acute myelogenous leukemia (AML) blast progenitor cells (BPC) to the BET protein antagonist JQ1. Treatment with JQ1, but not its inactive enantiomer (R-JQ1), was highly lethal against AML BPCs expressing mutant NPM1c+ with or without coexpression of FLT3-ITD or AML expressing mixed lineage leukemia fusion oncoprotein. JQ1 treatment reduced binding of BRD4 and RNA polymerase II to the DNA of c-MYC and BCL2 and reduced their levels in the AML cells. Cotreatment with JQ1 and the HDAC inhibitor panobinostat synergistically induced apoptosis of the AML BPCs, but not of normal CD34(+) hematopoietic progenitor cells. This was associated with greater attenuation of c-MYC and BCL2, while increasing p21, BIM, and cleaved PARP levels in the AML BPCs. Cotreatment with JQ1 and panobinostat significantly improved the survival of the NOD/SCID mice engrafted with OCI-AML3 or MOLM13 cells (P < 0.01). These findings highlight cotreatment with a BRD4 antagonist and an HDAC inhibitor as a potentially efficacious therapy of AML.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Leucemia Mieloide Aguda/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Análise por Conglomerados , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Genes bcl-2 , Genes myc , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Proteínas de Membrana/metabolismo , Camundongos , Células-Tronco Neoplásicas , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/genética , Panobinostat , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cytogenetic alterations are strong outcome prognosticators in uveal melanoma (UVM). Monosomy 3 (-3) and MYC amplification at 8q24 are commonly tested by fluorescence in situ hybridization (FISH). Alternatively, microarray analysis provides whole genome data, detecting partial chromosome loss, loss of heterozygosity (LOH), or abnormalities unrepresented by FISH probes. Nonfixed frozen tissue is conventionally used for microarray analysis but may not always be available. We assessed the feasibility of genomic microarray analysis for high resolution interrogation of UVM using formalin-fixed paraffin-embedded tissue (FFPET) as an alternative to frozen tissue (FZT). Enucleations from 44 patients (clinical trial NCT00952939) yielded sufficient DNA from FFPET (n = 34) and/or frozen tissue (n = 41) for comparative genomic hybridization and select single nucleotide polymorphism analysis (CGH/SNP) on Roche-NimbleGen OncoChip arrays. CEP3 FISH analysis was performed on matched cytology ThinPrep material. CGH/SNP analysis was successful in 30 of 34 FFPET and 41 of 41 FZT samples. Of 27 paired FFPET/FZT samples, 26 (96.3%) were concordant for at least four of six major recurrent abnormalities (-3, +8q, -1p, +6p, -6q, -8p), and 25 of 27 (92.6%) were concordant for -3. Results of CGH/SNP were concordant with the CEP3 FISH results in 27 of 30 (90%) FFPET and 38 of 41 (92.6%) FZT cases; partial -3q was detected in two CEP3 FISH-negative cases and whole chromosome 3, 4, and 6 SNP-LOH in one case. CGH detection of -3, +8q, -8p on FFPET and FZT showed significant correlation with the clinical outcome measures (metastasis development, time to progression, survival). Results of the UVM genotyping by CGH/SNP on FFPET are highly concordant with those of the FZT analysis and with those of the CEP3 FISH analysis, and therefore CGH/SNP is a practical method for UVM prognostication. Genome-wide coverage provides additional data with potential relevance to UVM biology, diagnosis, and prognosis.
Assuntos
Biomarcadores Tumorais/genética , Aberrações Cromossômicas , Perfilação da Expressão Gênica , Melanoma/genética , Polimorfismo de Nucleotídeo Único/genética , Neoplasias Uveais/genética , Hibridização Genômica Comparativa , Estudos de Viabilidade , Formaldeído , Humanos , Hibridização in Situ Fluorescente , Melanoma/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Inclusão em Parafina , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Uveais/patologiaRESUMO
CD30 is highly expressed on Hodgkins lymphoma and anaplastic large cell lymphoma, making it an attractive target for therapy. We describe the generation of serum-stabilized ssDNA aptamers that bind CD30 via a hybrid SELEX methodology. The selected aptamer bound CD30 with high affinity and specificity. Further optimization of the aptamer led to a short, truncated variant with a 50-fold higher affinity than its longer counterpart. The multivalent aptamer was able to induce oligomerization of CD30 receptors and, in effect, activate downstream signaling, which led to apoptosis of ALCL cells. Immunotherapy using aptamer-based co-stimulation provides an alternative to antibodies, and has potential to transform cancer treatment.
Assuntos
Imunoterapia/métodos , Antígeno Ki-1/metabolismo , Linfoma Anaplásico de Células Grandes/patologia , Apoptose , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Células HL-60 , Doença de Hodgkin/genética , Doença de Hodgkin/patologia , Humanos , Células K562 , Antígeno Ki-1/genética , Linfoma Anaplásico de Células Grandes/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Análise de Sequência de DNA , Transdução de SinaisRESUMO
Prognostic and predictive markers utilized in invasive breast carcinoma are limited and include ER, PR, Ki67, and ERBB2 (HER2). In the case of HER2, over-expression or amplification serves as eligibility for anti-HER2 based therapy, including trastuzumab (Herceptin®, Genentech). While clinical trials have shown trastuzumab improves overall survival and time to progression, an individual's response to anti-HER2 based therapy is highly variable. This suggests that, in a "uniform" HER2 positive population, additional markers could help in predicting patient outcome to therapy. Here we utilized a recently validated high-specificity HER4 antibody (E200) and generated a standard clinical HER4 scoring algorithm (HER4 H-Score) utilizing two breast carcinoma cohorts: 1) patients receiving neoadjuvant trastuzumab (n=47) and 2) patients receiving trastuzumab for metastatic disease (n=33). Our HER4 H-Score showed significant correlation with high sensitivity RT-qPCR performed on matched patients (p=<0.0001). In addition, patients with HER2/HER4 co-over-expression status showed a significant delay in development of metastasis after neo-adjuvant trastuzumab therapy (p= 0.04) and showed a significant improvement in progression free survival after adjuvant trastuzumab therapy (p=0.03). These findings suggest HER4 IHC, used in conjunction with a standard HER2 testing algorithm, could aid in predicting clinical outcome and help identify patients likely to show improved response to trastuzumab therapy.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Receptores ErbB/biossíntese , Adulto , Algoritmos , Neoplasias da Mama/patologia , Quimioterapia Adjuvante , Receptores ErbB/análise , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Terapia Neoadjuvante , Invasividade Neoplásica , Prognóstico , Receptor ErbB-4 , Trastuzumab , Resultado do TratamentoRESUMO
Patient management based on HER2 status in breast carcinoma is an archetypical example of personalized medicine but remains hampered by equivocal testing and intratumoral heterogeneity. We developed a fully automated, quantitative, bright-field in situ hybridization technique (RNAscope), applied it to quantify single-cell HER2 mRNA levels in 132 invasive breast carcinomas, and compared the results with those by real-time quantitative PCR (qPCR) and Food and Drug Administration-approved methods, including fluorescence in situ hybridization (FISH), IHC, chromogenic in situ hybridization, and dual in situ hybridization. Both RNAscope and qPCR were 97.3% concordant with FISH in cases in which FISH results were unequivocal. RNAscope was superior to qPCR in cases with intratumoral heterogeneity or equivocal FISH results. This novel assay may enable ultimate HER2 status resolution as a reflex test for current testing algorithms. Quantitative in situ RNA measurement at the single-cell level may be broadly applicable in companion diagnostic applications.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma/genética , Carcinoma/patologia , Hibridização In Situ , RNA Mensageiro , Receptor ErbB-2/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama Masculina/diagnóstico , Neoplasias da Mama Masculina/genética , Neoplasias da Mama Masculina/patologia , Carcinoma/diagnóstico , Feminino , Humanos , Hibridização In Situ/métodos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Sensibilidade e EspecificidadeRESUMO
ALK gene rearrangements in advanced non-small cell lung carcinomas (NSCLC) are an indication for targeted therapy with crizotinib. Fluorescence in situ hybridization (FISH) using a recently approved companion in vitro diagnostic class FISH system commonly assesses ALK status. More accessible IHC is challenged by low expression of ALK-fusion transcripts in NSCLC. We compared ultrasensitive automated IHC with FISH for detecting ALK status on 318 FFPE and 40 matched ThinPrep specimens from 296 patients with advanced NSCLC. IHC was concordant with FFPE-FISH on 229 of 231 dual-informative samples (31 positive and 198 negative) and with ThinPrep-FISH on 34 of 34 samples (5 positive and 29 negative). Two cases with negative IHC and borderline-positive FFPE-FISH (15% and 18%, respectively) were reclassified as concordant based on negative matched ThinPrep-FISH and clinical data consistent with ALK-negative status. Overall, after including ThinPrep-FISH and amending the false-positive FFPE-FISH results, IHC demonstrated 100% sensitivity and specificity (95% CI, 0.86 to 1.00 and 0.97 to 1.00, respectively) for ALK detection on 249 dual-informative NSCLC samples. IHC was informative on significantly more samples than FFPE-FISH, revealing additional ALK-positive cases. The high concordance with FISH warrants IHC's routine use as the initial component of an algorithmic approach to clinical ALK testing in NSCLC, followed by reflex FISH confirmation of IHC-positive cases.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Neoplasias Pulmonares/diagnóstico , Receptores Proteína Tirosina Quinases/genética , Algoritmos , Quinase do Linfoma Anaplásico , Carcinoma Pulmonar de Células não Pequenas/genética , Crizotinibe , Rearranjo Gênico , Humanos , Neoplasias Pulmonares/genética , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Sensibilidade e Especificidade , Manejo de EspécimesRESUMO
OBJECTIVES: To assess the feasibility of using a novel ultrasensitive bright-field in situ hybridization approach (BRISH) to evaluate κ and λ immunoglobulin messenger RNA (mRNA) expression in situ in B-cell non-Hodgkin lymphoma (NHL). METHODS: A series of 110 semiconsecutive clinical cases evaluated for lymphoma with historic flow cytometric (FCM) results were assessed with BRISH. RESULTS: BRISH light chain restriction (LCR) results were concordant with FCM in 108 (99%) of 109 evaluable cases. Additional small B-cell lymphoma cohorts were successfully evaluated. CONCLUSIONS: BRISH analysis of κ and λ immunoglobulin mRNA expression is a sensitive tool for establishing LCR in B-cell NHL when FCM results are not available.
Assuntos
Linfócitos B/patologia , Cadeias kappa de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/genética , Transtornos Linfoproliferativos/genética , RNA Mensageiro/metabolismo , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Células Clonais , Humanos , Cadeias kappa de Imunoglobulina/metabolismo , Cadeias lambda de Imunoglobulina/metabolismo , Hibridização In Situ/métodos , Transtornos Linfoproliferativos/metabolismo , Transtornos Linfoproliferativos/patologia , RNA Mensageiro/análiseRESUMO
Early recognition of BK viral nephropathy is essential for successful management. Our aim in this study was to evaluate a novel fluorescence in situ hybridization (FISH) assay for detection of BK virus in renal transplant biopsies in the context of standard detection methods. Renal allograft biopsies (n = 108) were analyzed via H&E, immunohistochemistry (IHC) for simian virus 40, and FISH for BK virus. BK virus was detected in 16 (14.8%) cases by H&E, 13 (12%) cases by IHC, 18 (16.6%) cases by FISH, and 19 (17.6%) cases by real-time PCR; 24 of 108 showed a discrepancy in ≥1 testing modalities. Comparison of H&E, IHC, and FISH showed no statistical difference in detection of BK virus. However, performing comparisons between the different tissue-based assays in the context of plasma or urine real-time PCR results showed significant improvement in detection of BK by FISH over H&E (P = 0.02) but not IHC (P = 0.07). This novel FISH-based approach for BK virus identification in renal allograft biopsy tissue mirrored real-time PCR results and showed superior performance to detection of inclusions by H&E. Therefore, use of FISH for BK virus detection in the setting of renal allograft biopsy is a useful and sensitive detection method and could be adopted in any laboratory that currently performs FISH analysis.