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1.
Blood ; 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38776489

RESUMO

Delays and risks associated with neurosurgical biopsies preclude timely diagnosis and treatment of central nervous system (CNS) lymphoma and other CNS neoplasms. We prospectively integrated targeted rapid genotyping of cerebrospinal fluid (CSF) into the evaluation of 70 patients with CNS lesions of unknown etiology. Participants underwent genotyping of CSF-derived DNA using a qPCR-based approach for parallel detection of single-nucleotide variants in the MYD88, TERT promoter, IDH1, IDH2, BRAF and H3F3A genes within 80 minutes of sample acquisition. Canonical mutations were detected in 42% of patients with neoplasms, including cases of primary and secondary CNS lymphoma, glioblastoma, IDH-mutant brainstem glioma and H3K27M-mutant diffuse midline glioma. Genotyping results eliminated the need for surgical biopsies in 7/33 (21.2%) cases of newly diagnosed neoplasms, resulting in significantly accelerated initiation of disease-directed treatment (median 3 vs 12 days; p = 0.027). This assay was then implemented in a Clinical Laboratory Improvement Amendments (CLIA) environment, with 2-day median turnaround for diagnosis of central nervous system lymphoma from 66 patients across 4 clinical sites. Our study prospectively demonstrates that targeted rapid CSF genotyping influences oncologic management for suspected CNS tumors.

2.
J Clin Pathol ; 74(8): 496-503, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34049977

RESUMO

Developing and deploying new diagnostic tests are difficult, but the need to do so in response to a rapidly emerging pandemic such as COVID-19 is crucially important. During a pandemic, laboratories play a key role in helping healthcare providers and public health authorities detect active infection, a task most commonly achieved using nucleic acid-based assays. While the landscape of diagnostics is rapidly evolving, PCR remains the gold-standard of nucleic acid-based diagnostic assays, in part due to its reliability, flexibility and wide deployment. To address a critical local shortage of testing capacity persisting during the COVID-19 outbreak, our hospital set up a molecular-based laboratory developed test (LDT) to accurately and safely diagnose SARS-CoV-2. We describe here the process of developing an emergency-use LDT, in the hope that our experience will be useful to other laboratories in future outbreaks and will help to lower barriers to establishing fast and accurate diagnostic testing in crisis conditions.


Assuntos
Teste de Ácido Nucleico para COVID-19 , COVID-19/diagnóstico , Serviço Hospitalar de Emergência , Laboratórios Hospitalares , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/genética , COVID-19/virologia , Humanos , Valor Preditivo dos Testes , Reprodutibilidade dos Testes
3.
medRxiv ; 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32909014

RESUMO

Developing and deploying new diagnostic tests is difficult, but the need to do so in response to a rapidly emerging pandemic such as COVID-19 is crucially important for an effective response. In the early stages of a pandemic, laboratories play a key role in helping health care providers and public health authorities detect active infection, a task most commonly achieved using nucleic acid-based assays. While the landscape of diagnostics is rapidly evolving, polymerase chain reaction (PCR) remains the gold-standard of nucleic acid-based diagnostic assays, in part due to its reliability, flexibility, and wide deployment. To address a critical local shortage of testing capacity persisting during the COVID-19 outbreak, our hospital set up a molecular based laboratory developed test (LDT) to accurately and safely diagnose SARS-CoV-2. We describe here the process of developing an emergency-use LDT, in the hope that our experience will be useful to other laboratories in future outbreaks and will help to lower barriers to fast and accurate diagnostic testing in crisis conditions.

4.
FASEB J ; 34(10): 13877-13884, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32856766

RESUMO

The diagnosis of COVID-19 requires integration of clinical and laboratory data. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic assays play a central role in diagnosis and have fixed technical performance metrics. Interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. Our goal was to examine the clinical sensitivity of two most common SARS-CoV-2 diagnostic test modalities, polymerase chain reaction (PCR) and serology, over the disease course to provide insight into their clinical interpretation in patients presenting to the hospital. We conducted a single-center, retrospective study. To derive clinical sensitivity of PCR, we identified 209 PCR-positive SARS-CoV-2 patients with multiple PCR test results (624 total PCR tests) and calculated daily sensitivity from date of symptom onset or first positive test. Clinical sensitivity of PCR decreased with days post symptom onset with >90% clinical sensitivity during the first 5 days after symptom onset, 70%-71% from Days 9 to 11, and 30% at Day 21. To calculate daily clinical sensitivity by serology, we utilized 157 PCR-positive patients with a total of 197 specimens tested by enzyme-linked immunosorbent assay for IgM, IgG, and IgA anti-SARS-CoV-2 antibodies. In contrast to PCR, serological sensitivity increased with days post symptom onset with >50% of patients seropositive by at least one antibody isotype after Day 7, >80% after Day 12, and 100% by Day 21. Taken together, PCR and serology are complimentary modalities that require time-dependent interpretation. Superimposition of sensitivities over time indicate that serology can function as a reliable diagnostic aid indicating recent or prior infection.


Assuntos
Teste de Ácido Nucleico para COVID-19 , Teste Sorológico para COVID-19 , COVID-19/diagnóstico , SARS-CoV-2 , Anticorpos Antivirais/sangue , COVID-19/sangue , Feminino , Hospitais , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sensibilidade e Especificidade
5.
J Mol Diagn ; 21(4): 632-645, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31026600

RESUMO

The use of liquid biopsies to identify driver mutations in patients with solid tumors holds great promise for performing targeted therapy selection, monitoring disease progression, and detecting treatment resistance mechanisms. We describe herein the development and clinical validation of a 28-gene cell-free DNA panel that targets the most common genetic alterations in solid tumors. Bioinformatic and variant filtering solutions were developed to improve test sensitivity and specificity. The panel and these tools were used to analyze commercially available controls, allowing establishment of a limit of detection allele fraction cutoff of 0.25%, with 100% (95% CI, 81.5%-100%) specificity and 89.8% (95% CI, 81.0%-94.9%) sensitivity. In addition, we analyzed a total of 163 blood samples from patients with metastatic cancer (n = 123) and demonstrated a >90% sensitivity for detecting previously identified expected mutations. Longitudinal monitoring of patients revealed a strong correlation of variant allele frequency changes and clinical outcome. Additional clinically relevant information included identification of resistance mutations in patients receiving targeted treatment and detection of complex patterns of mutational heterogeneity. Achieving lower limits of detection will require additional improvements to molecular barcoding; however, these data strongly support clinical implementation of cell-free DNA panels in advanced cancer patients.


Assuntos
Biomarcadores Tumorais , Ácidos Nucleicos Livres , DNA Tumoral Circulante , Testes Genéticos , Biópsia Líquida , Neoplasias/diagnóstico , Neoplasias/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Variações do Número de Cópias de DNA , Progressão da Doença , Feminino , Testes Genéticos/métodos , Testes Genéticos/normas , Humanos , Hibridização in Situ Fluorescente , Biópsia Líquida/métodos , Biópsia Líquida/normas , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Reprodutibilidade dos Testes
6.
JCO Precis Oncol ; 20182018.
Artigo em Inglês | MEDLINE | ID: mdl-30370396

RESUMO

PURPOSE: Targeted therapy is the cornerstone of treatment of advanced EGFR-mutant non-small-cell lung cancer (NSCLC). Next-generation sequencing (NGS), the preferred method for genotyping, typically requires several weeks. Here, we assessed workflows designed to rapidly identify patients with actionable EGFR mutations and reduce time to initiation (TTI) of epidermal growth factor receptor (EGFR)-directed therapy. PATIENTS AND METHODS: We performed rapid testing for EGFR L858R mutations and exon 19 deletions on paraffin-embedded or frozen section biopsy specimens from newly diagnosed patients with metastatic NSCLC by using an EGFR-specific assay (rapid test). To determine clinical utility, we assessed concordance with NGS results, turnaround time, and TTI of EGFR therapy, and we evaluated reimbursement data. RESULTS: Between January 2015 and September 2017, we performed 243 rapid EGFR tests and identified EGFR mutations in 43 patients (18%). With NGS results as a reference, sensitivity and specificity of the rapid EGFR polymerase chain reaction assay were 98% and 100%, respectively. The median turnaround time for NGS was 14 days, compared with 7 days for rapid testing (P < .001). In the rapid group, 95% of patients received an EGFR inhibitor in the first-line setting. The median TTI of EGFR therapy was significantly shorter in the rapid cohort when compared with 121 historical cases (22 v 37 days; P = .01). Escalation of the initiative into an interdisciplinary ultra-rapid next-day frozen-section workflow for highly symptomatic patients (n = 8) resulted in a reduction in the median (± standard deviation) turnaround time to 1 ± 0.4 days and allowed several patients to initiate therapy within 1 week of biopsy. An extended 9-month clinical evaluation phase confirmed operational sustainability (turnaround times: ultra-rapid, 0.81 ± 0.4 days; rapid, 3 ± 1.5 days), and a 63% reimbursement rate indicated financial sustainability. CONCLUSION: Rapid genotyping facilitates earlier initiation of EGFR-directed therapies without compromising NGS workflows.

7.
Acta Neuropathol ; 136(5): 779-792, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30123936

RESUMO

Progressive meningiomas that have failed surgery and radiation have a poor prognosis and no standard therapy. While meningiomas are more common in females overall, progressive meningiomas are enriched in males. We performed a comprehensive molecular characterization of 169 meningiomas from 53 patients with progressive/high-grade tumors, including matched primary and recurrent samples. Exome sequencing in an initial cohort (n = 24) detected frequent alterations in genes residing on the X chromosome, with somatic intragenic deletions of the dystrophin-encoding and muscular dystrophy-associated DMD gene as the most common alteration (n = 5, 20.8%), along with alterations of other known X-linked cancer-related genes KDM6A (n =2, 8.3%), DDX3X, RBM10 and STAG2 (n = 1, 4.1% each). DMD inactivation (by genomic deletion or loss of protein expression) was ultimately detected in 17/53 progressive meningioma patients (32%). Importantly, patients with tumors harboring DMD inactivation had a shorter overall survival (OS) than their wild-type counterparts [5.1 years (95% CI 1.3-9.0) vs. median not reached (95% CI 2.9-not reached, p = 0.006)]. Given the known poor prognostic association of TERT alterations in these tumors, we also assessed for these events, and found seven patients with TERT promoter mutations and three with TERT rearrangements in this cohort (n = 10, 18.8%), including a recurrent novel RETREG1-TERT rearrangement that was present in two patients. In a multivariate model, DMD inactivation (p = 0.033, HR = 2.6, 95% CI 1.0-6.6) and TERT alterations (p = 0.005, HR = 3.8, 95% CI 1.5-9.9) were mutually independent in predicting unfavorable outcomes. Thus, DMD alterations identify a subset of progressive/high-grade meningiomas with worse outcomes.


Assuntos
Distrofina/genética , Deleção de Genes , Neoplasias Meníngeas/genética , Meningioma/genética , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral/patologia , Linhagem Celular Tumoral/ultraestrutura , Estudos de Coortes , Progressão da Doença , Distrofina/metabolismo , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Neoplasias Meníngeas/diagnóstico por imagem , Neoplasias Meníngeas/patologia , Meningioma/diagnóstico por imagem , Meningioma/patologia , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex , RNA Mensageiro/metabolismo , Cromatina Sexual/genética , Telomerase/genética , Telomerase/metabolismo , Sequenciamento do Exoma
8.
Proc Natl Acad Sci U S A ; 115(36): E8388-E8394, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30082399

RESUMO

Aggressive neurosurgical resection to achieve sustained local control is essential for prolonging survival in patients with lower-grade glioma. However, progression in many of these patients is characterized by local regrowth. Most lower-grade gliomas harbor isocitrate dehydrogenase 1 (IDH1) or IDH2 mutations, which sensitize to metabolism-altering agents. To improve local control of IDH mutant gliomas while avoiding systemic toxicity associated with metabolic therapies, we developed a precision intraoperative treatment that couples a rapid multiplexed genotyping tool with a sustained release microparticle (MP) drug delivery system containing an IDH-directed nicotinamide phosphoribosyltransferase (NAMPT) inhibitor (GMX-1778). We validated our genetic diagnostic tool on clinically annotated tumor specimens. GMX-1778 MPs showed mutant IDH genotype-specific toxicity in vitro and in vivo, inducing regression of orthotopic IDH mutant glioma murine models. Our strategy enables immediate intraoperative genotyping and local application of a genotype-specific treatment in surgical scenarios where local tumor control is paramount and systemic toxicity is therapeutically limiting.


Assuntos
Neoplasias Encefálicas , Cianetos/farmacologia , Genótipo , Glioma , Guanidinas/farmacologia , Isocitrato Desidrogenase/genética , Terapia de Alvo Molecular/métodos , Mutação , Proteínas de Neoplasias/genética , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Sistemas de Liberação de Medicamentos/métodos , Feminino , Glioma/tratamento farmacológico , Glioma/enzimologia , Glioma/genética , Humanos , Masculino , Camundongos , Camundongos SCID , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Cancer Res ; 76(24): 7118-7129, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27913435

RESUMO

Solid papillary carcinoma with reverse polarity (SPCRP) is a rare breast cancer subtype with an obscure etiology. In this study, we sought to describe its unique histopathologic features and to identify the genetic alterations that underpin SPCRP using massively parallel whole-exome and targeted sequencing. The morphologic and immunohistochemical features of SPCRP support the invasive nature of this subtype. Ten of 13 (77%) SPCRPs harbored hotspot mutations at R172 of the isocitrate dehydrogenase IDH2, of which 8 of 10 displayed concurrent pathogenic mutations affecting PIK3CA or PIK3R1 One of the IDH2 wild-type SPCRPs harbored a TET2 Q548* truncating mutation coupled with a PIK3CA H1047R hotspot mutation. Functional studies demonstrated that IDH2 and PIK3CA hotspot mutations are likely drivers of SPCRP, resulting in its reversed nuclear polarization phenotype. Our results offer a molecular definition of SPCRP as a distinct breast cancer subtype. Concurrent IDH2 and PIK3CA mutations may help diagnose SPCRP and possibly direct effective treatment. Cancer Res; 76(24); 7118-29. ©2016 AACR.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Papilar/genética , Carcinoma Papilar/patologia , Isocitrato Desidrogenase/genética , Fosfatidilinositol 3-Quinases/genética , Biomarcadores Tumorais/genética , Western Blotting , Classe I de Fosfatidilinositol 3-Quinases , Análise Mutacional de DNA , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase
10.
J Mol Diagn ; 18(5): 697-706, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27471182

RESUMO

Next-generation sequencing has evolved technically and economically into the method of choice for interrogating the genome in cancer and inherited disorders. The introduction of procedural code sets for whole-exome and genome sequencing is a milestone toward financially sustainable clinical implementation; however, achieving reimbursement is currently a major challenge. As part of a prospective quality-improvement initiative to implement the new code sets, we adopted Agile, a development methodology originally devised in software development. We implemented eight functionally distinct modules (request review, cost estimation, preauthorization, accessioning, prebilling, testing, reporting, and reimbursement consultation) and obtained feedback via an anonymous survey. We managed 50 clinical requests (January to June 2015). The fraction of pursued-to-requested cases (n = 15/50; utilization management fraction, 0.3) aimed for a high rate of preauthorizations. In 13 of 15 patients the insurance plan required preauthorization, which we obtained in 70% and ultimately achieved reimbursement in 50%. Interoperability enabled assessment of 12 different combinations of modules that underline the importance of an adaptive workflow and policy tailoring to achieve higher yields of reimbursement. The survey confirmed a positive attitude toward self-organizing teams. We acknowledge the individuals and their interactions and termed the infrastructure: human pipeline. Nontechnical barriers currently are limiting the scope and availability of clinical genomic sequencing. The presented human pipeline is one approach toward long-term financial sustainability of clinical genomics.


Assuntos
Atenção à Saúde , Genômica , Informática Médica/métodos , Software , Atenção à Saúde/economia , Atenção à Saúde/métodos , Atenção à Saúde/organização & administração , Exoma , Genômica/economia , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Informática Médica/economia , Encaminhamento e Consulta , Mecanismo de Reembolso , Pesquisa , Inquéritos e Questionários , Fluxo de Trabalho , Recursos Humanos
11.
Int J Colorectal Dis ; 31(5): 973-982, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27026089

RESUMO

PURPOSE: It is now recognized that Crohn's disease (CD), similar to ulcerative colitis (UC), carries an up to 20-fold higher cancer risk, and the development of colorectal carcinoma (CRC) is a major long-term complication. Once CRC is present, molecular profiling is one of the components in selecting appropriate treatment strategies; however, in contrast to UC, genetic alterations in Crohn's colitis-associated CRC are poorly understood. METHODS: In a series of 227 patients with Crohn's colitis, we identified 33 cases of CRC (~14 %) and performed targeted mutational analysis of BRAF/KRAS/NRAS and determined microsatellite status as well as immunophenotype of the tumors. RESULTS: In the CRC cohort, the median age at time of cancer diagnosis was 58 (range 34-77 vs. 59.5 in sporadic; P = 0.81) and the median CD duration was 29 years (range 6-45). As a group, CRC complicating Crohn's colitis is BRAF (97 %) and NRAS (100 %) wild type and the vast majority is microsatellite stable (94 %); KRAS-mutations were found in six cases (18 %). Stage grouping, anatomic distribution, and overall survival were similar to sporadic CRC; however, long-standing CD (≥25 years) as well as gastric-immunophenotype (MUC5AC+) was associated with significantly shorter overall survival (P = 0.0029; P = 0.036, respectively). CONCLUSION: In summary, the clinicopathological and molecular profile of CD-associated CRC is similar to that observed in sporadic CRC.


Assuntos
Neoplasias Colorretais/complicações , Doença de Crohn/complicações , Adulto , Idoso , Neoplasias Colorretais/genética , Doença de Crohn/patologia , Metilação de DNA/genética , Feminino , Seguimentos , GTP Fosfo-Hidrolases/genética , Estudos de Associação Genética , Humanos , Imunofenotipagem , Masculino , Proteínas de Membrana/genética , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL/genética , Mutação , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de Sobrevida
12.
J Mol Diagn ; 18(1): 124-30, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26596524

RESUMO

Cytological evaluation of pancreatic or biliary duct brushings is a specific, but insensitive, test for malignancy. We compared adjunctive molecular testing with next-generation sequencing (NGS) relative to fluorescence in situ hybridization (FISH) for detection of high-risk neoplasia or malignancy. Bile duct brushings from 81 specimens were subjected to cytological analysis, FISH using the UroVysion probe set, and targeted NGS. Specimens were placed into negative/atypical (negative) or suspicious/positive (positive) categories depending on cytology and negative or positive categories on the basis of FISH and NGS results. Performance characteristics for each diagnostic modality were calculated on the basis of clinicopathologic follow-up and compared in a receiver operating characteristic analysis. There were 33 high-risk neoplasia/malignant strictures (41%) and 48 benign (59%). NGS revealed driver mutations in 24 cases (30%), including KRAS (21 of 24 cases), TP53 (14 of 24 cases), SMAD4 (6 of 24 cases), and CDKN2A (4 of 24 cases). Cytology had a sensitivity of 67% (95% CI, 48%-82%) and a specificity of 98% (95% CI, 89%-100%). When added to cytology, NGS increased the sensitivity to 85% (95% CI, 68%-95%), leading to a significant increase in the area under the curve in a receiver operating characteristic analysis (P = 0.03). FISH increased the sensitivity to 76% (95% CI, 58%-89%), without significantly increasing the area under the curve. These results suggest that ancillary NGS testing offers advantages over FISH, although studies with larger cohorts are needed to verify these findings.


Assuntos
Neoplasias do Sistema Biliar/diagnóstico , Citodiagnóstico/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Hibridização in Situ Fluorescente/métodos , Neoplasias Pancreáticas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Ductos Biliares , Sistema Biliar/citologia , Sistema Biliar/patologia , Neoplasias do Sistema Biliar/genética , Colangiopancreatografia Retrógrada Endoscópica/métodos , Inibidor p16 de Quinase Dependente de Ciclina/genética , Reações Falso-Negativas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pâncreas/citologia , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de Sequência de DNA/métodos , Proteína Smad4/genética , Proteína Supressora de Tumor p53/genética , Adulto Jovem
13.
Clin Cancer Res ; 19(2): 480-90, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23186780

RESUMO

PURPOSE: Salivary duct carcinomas (SDC) are a rare and aggressive subtype of salivary gland cancers for which cytotoxic chemotherapy has limited efficacy. We investigated whether genotyping analysis could detect novel tumor-specific mutations that would help direct SDC patient treatment using targeted agents. EXPERIMENTAL DESIGN: We genotyped 27 SDC archival specimens from patients followed at Massachusetts General Hospital and Massachusetts Eye and Ear Infirmary (Boston, MA) between 2000 and 2011. These included the tumors of 8 patients who were tested prospectively. Targeted mutational analysis of 13 clinically relevant cancer genes was conducted using SNaPshot multiplexed genotyping. FISH was conducted to detect HER2 gene amplification. Patient medical records and tumor histopathologic features were retrospectively reviewed. RESULTS: Mutually exclusive genetic aberrations were detected in 15 of 27 (56%) tumors, including 2 (7%) mutations in BRAF, 5 (19%) mutations in PIK3CA, and 8 (30%) cases of HER2 gene amplification. To our knowledge, this is the first time that BRAF and PIK3CA mutations have been reported in this tumor type. Prospective clinical testing of 8 patients with SDC identified actionable genetic alterations in 6 tumors and influenced therapeutic decisions for all 6 patients. CONCLUSION: SNaPshot molecular profiling identified novel genetic changes in SDCs, expanded the therapeutic options for patients with this rare tumor, and is changing SDC management at our institution. These findings highlight the importance of using broad-based genetic profiling to expedite the identification of effective-targeted therapies for patients with rare malignancies.


Assuntos
Carcinoma Ductal/genética , Neoplasias das Glândulas Salivares/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Carcinoma Ductal/tratamento farmacológico , Carcinoma Ductal/mortalidade , Carcinoma Ductal/patologia , Classe I de Fosfatidilinositol 3-Quinases , Feminino , Amplificação de Genes , Perfilação da Expressão Gênica , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Fosfatidilinositol 3-Quinases/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Receptor ErbB-2/genética , Neoplasias das Glândulas Salivares/tratamento farmacológico , Neoplasias das Glândulas Salivares/mortalidade , Neoplasias das Glândulas Salivares/patologia , Adulto Jovem
14.
Am J Surg Pathol ; 36(10): 1464-71, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22982889

RESUMO

Potential sources of error in surgical pathology include specimen misidentification, unidentified tissue, and tissue contamination of paraffin blocks and slides. Current molecular approaches to characterize unidentified or misidentified tissue include fluorescence in situ hybridization identification of sex chromosomes (XY FISH) and microsatellite analysis. Polymorphic deletion probe (PDP) FISH, a novel FISH assay based on copy number variants, can distinguish between cells and tissues from 2 individuals in situ, independent of gender. Using a panel of 3 PDPs, we compared the genotypes of potential tissue contaminants (n=19) and unidentified tissues (n=6) with patient tissues to determine the utility of PDP FISH in resolving specimen identity. XY FISH was added to increase the informative potential of the assay, and microsatellite analysis was used as a gold standard to confirm PDP FISH results. PDP FISH distinguished between putative contaminants and patient tissues in 13 of 14 cases and indicated a high likelihood of 2 tissues originating from the same source in 11 of 11 cases. The assay has a sensitivity and specificity of 86% [6/7, exact 95% confidence interval (CI): 42%, 97%] and 100% (9/9, exact 1-sided 97.5% CI: 68%, 100%), respectively, and a positive predictive value and negative predictive value of 100% (6/6, exact 1-sided 97.5% CI: 54%, 100%) and 90% (9/10, exact 95% CI: 55%, 98%), respectively. PDP FISH is an accurate and practical molecular assay for the genetic characterization of potential tissue contaminants and unidentified tissues, especially in the setting of small sample size, and permits concomitant assessment of morphology.


Assuntos
Artefatos , Erros de Diagnóstico/prevenção & controle , Técnicas de Diagnóstico Molecular/métodos , Patologia Cirúrgica/métodos , Polimorfismo Genético , Manejo de Espécimes/métodos , Aberrações Cromossômicas , Cromossomos Humanos X , Cromossomos Humanos Y , Sondas de DNA , Feminino , Genótipo , Humanos , Hibridização in Situ Fluorescente , Masculino , Repetições de Microssatélites , Mosaicismo , Inclusão em Parafina , Patologia Cirúrgica/normas , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Manejo de Espécimes/normas
15.
J Clin Oncol ; 30(8): 863-70, 2012 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-22215748

RESUMO

PURPOSE: Chromosomal rearrangements involving the ROS1 receptor tyrosine kinase gene have recently been described in a subset of non-small-cell lung cancers (NSCLCs). Because little is known about these tumors, we examined the clinical characteristics and treatment outcomes of patients with NSCLC with ROS1 rearrangement. PATIENTS AND METHODS: Using a ROS1 fluorescent in situ hybridization (FISH) assay, we screened 1,073 patients with NSCLC and correlated ROS1 rearrangement status with clinical characteristics, overall survival, and when available, ALK rearrangement status. In vitro studies assessed the responsiveness of cells with ROS1 rearrangement to the tyrosine kinase inhibitor crizotinib. The clinical response of one patient with ROS1-rearranged NSCLC to crizotinib was investigated as part of an expanded phase I cohort. RESULTS: Of 1,073 tumors screened, 18 (1.7%) were ROS1 rearranged by FISH, and 31 (2.9%) were ALK rearranged. Compared with the ROS1-negative group, patients with ROS1 rearrangements were significantly younger and more likely to be never-smokers (each P < .001). All of the ROS1-positive tumors were adenocarcinomas, with a tendency toward higher grade. ROS1-positive and -negative groups showed no difference in overall survival. The HCC78 ROS1-rearranged NSCLC cell line and 293 cells transfected with CD74-ROS1 showed evidence of sensitivity to crizotinib. The patient treated with crizotinib showed tumor shrinkage, with a near complete response. CONCLUSION: ROS1 rearrangement defines a molecular subset of NSCLC with distinct clinical characteristics that are similar to those observed in patients with ALK-rearranged NSCLC. Crizotinib shows in vitro activity and early evidence of clinical activity in ROS1-rearranged NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Rearranjo Gênico , Neoplasias Pulmonares/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Adenocarcinoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Quinase do Linfoma Anaplásico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Crizotinibe , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Receptores Proteína Tirosina Quinases/genética , Estudos Retrospectivos , Resultado do Tratamento
16.
PLoS One ; 6(5): e18846, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21602918

RESUMO

The molecular events in chordoma pathogenesis have not been fully delineated, particularly with respect to copy number changes. Understanding copy number alterations in chordoma may reveal critical disease mechanisms that could be exploited for tumor classification and therapy. We report the copy number analysis of 21 sporadic chordomas using array comparative genomic hybridization (CGH). Recurrent copy changes were further evaluated with immunohistochemistry, methylation specific PCR, and quantitative real-time PCR. Similar to previous findings, large copy number losses, involving chromosomes 1p, 3, 4, 9, 10, 13, 14, and 18, were more common than copy number gains. Loss of CDKN2A with or without loss of CDKN2B on 9p21.3 was observed in 16/20 (80%) unique cases of which six (30%) showed homozygous deletions ranging from 76 kilobases to 4.7 megabases. One copy loss of the 10q23.31 region which encodes PTEN was found in 16/20 (80%) cases. Loss of CDKN2A and PTEN expression in the majority of cases was not attributed to promoter methylation. Our sporadic chordoma cases did not show hotspot point mutations in some common cancer gene targets. Moreover, most of these sporadic tumors are not associated with T (brachyury) duplication or amplification. Deficiency of CDKN2A and PTEN expression, although shared across many other different types of tumors, likely represents a key aspect of chordoma pathogenesis. Sporadic chordomas may rely on mechanisms other than copy number gain if they indeed exploit T/brachyury for proliferation.


Assuntos
Cordoma/genética , Aberrações Cromossômicas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Dosagem de Genes , Genes Neoplásicos , PTEN Fosfo-Hidrolase/genética , Cordoma/etiologia , Cordoma/patologia , Hibridização Genômica Comparativa , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Humanos , PTEN Fosfo-Hidrolase/deficiência
17.
J Mol Diagn ; 11(3): 238-47, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19324997

RESUMO

Lynch syndrome is caused by germline mutations in DNA mismatch repair (MMR) genes. Both microsatellite instability (MSI) testing and immunohistochemical analyses (IHC) of colon cancers are valuable diagnostic strategies for Lynch syndrome. We sought to determine whether these markers of MMR deficiency were also detectable in pre-cancerous colorectal adenomas. Fifteen subjects with a germline MMR gene mutation who had 44 adenomas removed during surveillance colonoscopy were identified. MSI testing and IHC for MLH1, MSH2, and MSH6 were performed. MSI was detected in 23 adenomas. There was a significant association between MSI and high-grade dysplasia (P = 0.006) and distal location (P = 0.0008). Loss of MMR protein by IHC was detected in 31 adenomas. A significant association was observed between loss of staining by IHC and high-grade dysplasia (P = 0.04). Among the 40 adenomas in which both MSI tests and IHC were performed, the presence of a germline mutation correlated with an abnormal MSI result in 58% of cases, an abnormal IHC result in 70% of cases, and either an abnormal MSI or IHC result in 73% of cases. The combination of MSI and IHC testing in colorectal adenomas is a sensitive screen for the detection of Lynch syndrome and may be particularly useful when Lynch syndrome is suspected and adenomatous polyps are the only tissues available for analysis.


Assuntos
Pólipos Adenomatosos/complicações , Neoplasias Colorretais Hereditárias sem Polipose/complicações , Reparo de Erro de Pareamento de DNA , Pólipos Adenomatosos/genética , Pólipos Adenomatosos/patologia , Adulto , Idoso , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Feminino , Humanos , Hiperplasia , Imuno-Histoquímica , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade
18.
Genes Chromosomes Cancer ; 46(10): 909-13, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17620295

RESUMO

The recent description of a new X chromosome tumor suppressor gene, WTX, that is commonly inactivated in Wilms' tumor prompted us to examine the possible involvement of WTX in a case of Wilms' tumor containing an apparently balanced reciprocal translocation between chromosomes X and 18 (t(X;18)(q11;p11)). Fluorescence in situ hybridization (FISH) analysis of paraffin tumor sections indeed revealed a deletion of the WTX locus at Xq11. High-resolution array comparative genomic hybridization (array CGH) analysis of tumor DNA revealed a 1.5 Mb chromosome deletion encompassing the WTX gene at Xq11. No loss of genetic material was detected on chromosome 18. Interestingly, unlike most tumors with acquired chromosomal translocations, where a new fusion oncogene or promoter-oncogene fusion is created and drives tumor growth, the t(X;18) in this tumor appears to drive tumorigenesis via deletion of a tumor suppressor. This case demonstrates the importance of array CGH and FISH as adjuncts in tumor cytogenetics and in identifying pathogenic microdeletions in "balanced" translocations that are not truly balanced.


Assuntos
Cromossomos Humanos Par 18/genética , Cromossomos Humanos X/genética , Deleção de Genes , Neoplasias Renais/genética , Translocação Genética , Proteínas Supressoras de Tumor/genética , Tumor de Wilms/genética , Proteínas Adaptadoras de Transdução de Sinal , Perfilação da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Recém-Nascido , Cariotipagem , Neoplasias Renais/metabolismo , Masculino , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Tumor de Wilms/metabolismo
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