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2.
J Mol Diagn ; 21(4): 658-676, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31055023

RESUMEN

We conducted a multilaboratory assessment to determine the suitability of a new commercially available reference material with 40 cancer variants in a background of wild-type DNA at four different variant allele frequencies (VAFs): 2%, 0.50%, 0.125%, and 0%. The variants include single nucleotides, insertions, deletions, and two structural variations selected for their clinical importance and to challenge the performance of next-generation sequencing (NGS) methods. Fragmented DNA was formulated to simulate the size distribution of circulating wild-type and tumor DNA in a synthetic plasma matrix. DNA was extracted from these samples and characterized with different methods and multiple laboratories. The various extraction methods had differences in yield, perhaps because of differences in chemistry. Digital PCR assays were used to measure VAFs to compare results from different NGS methods. Comparable VAFs were observed across the different NGS methods. This multilaboratory assessment demonstrates that the new reference material is an appropriate tool to determine the analytical parameters of different measurement methods and to ensure their quality assurance.


Asunto(s)
Biomarcadores de Tumor , ADN Tumoral Circulante , ADN de Neoplasias , Biopsia Líquida , Neoplasias/diagnóstico , Neoplasias/genética , Alelos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Humanos , Biopsia Líquida/métodos , Biopsia Líquida/normas , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/normas , Garantía de la Calidad de Atención de Salud , Estándares de Referencia
3.
BMC Biotechnol ; 18(1): 17, 2018 03 20.
Artículo en Inglés | MEDLINE | ID: mdl-29554888

RESUMEN

BACKGROUND: We demonstrate the feasibility of creating a pair of reference samples to be used as surrogates for clinical samples measured in either a research or clinical laboratory setting. The reference sample paradigm presented and evaluated here is designed to assess the capability of a measurement process to detect true differences between two biological samples. Cell-based reference samples can be created with a biomarker signature pattern designed in silico. Clinical laboratories working in regulated applications are required to participate in proficiency testing programs; research laboratories doing discovery typically do not. These reference samples can be used in proficiency tests or as process controls that allow a laboratory to evaluate and optimize its measurement systems, monitor performance over time (process drift), assess changes in protocols, reagents, and/or personnel, maintain standard operating procedures, and most importantly, provide evidence for quality results. RESULTS: The biomarkers of interest in this study are microRNAs (miRNAs), small non-coding RNAs involved in the regulation of gene expression. Multiple lung cancer associated cell lines were determined by reverse transcription (RT)-PCR to have sufficiently different miRNA profiles to serve as components in mixture designs as reference samples. In silico models based on the component profiles were used to predict miRNA abundance ratios between two different cell line mixtures, providing target values for profiles obtained from in vitro mixtures. Two reference sample types were tested: total RNA mixed after extraction from cell lines, and intact cells mixed prior to RNA extraction. MicroRNA profiling of a pair of samples composed of extracted RNA derived from these cell lines successfully replicated the target values. Mixtures of intact cells from these lines also approximated the target values, demonstrating potential utility as mimics for clinical specimens. Both designs demonstrated their utility as reference samples for inter- or intra-laboratory testing. CONCLUSIONS: Cell-based reference samples can be created for performance assessment of a measurement process from biomolecule extraction through quantitation. Although this study focused on miRNA profiling with RT-PCR using cell lines associated with lung cancer, the paradigm demonstrated here should be extendable to genome-scale platforms and other biomolecular endpoints.


Asunto(s)
Biomarcadores de Tumor/genética , Técnicas de Laboratorio Clínico/normas , MicroARNs/genética , ARN Pequeño no Traducido/genética , Análisis de Varianza , Línea Celular Tumoral , Expresión Génica , Humanos , Estándares de Referencia , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas
4.
BMC Genomics ; 19(1): 180, 2018 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-29510677

RESUMEN

BACKGROUND: The potential utility of microRNA as biomarkers for early detection of cancer and other diseases is being investigated with genome-scale profiling of differentially expressed microRNA. Processes for measurement assurance are critical components of genome-scale measurements. Here, we evaluated the utility of a set of total RNA samples, designed with between-sample differences in the relative abundance of miRNAs, as process controls. RESULTS: Three pure total human RNA samples (brain, liver, and placenta) and two different mixtures of these components were evaluated as measurement assurance control samples on multiple measurement systems at multiple sites and over multiple rounds. In silico modeling of mixtures provided benchmark values for comparison with physical mixtures. Biomarker development laboratories using next-generation sequencing (NGS) or genome-scale hybridization assays participated in the study and returned data from the samples using their routine workflows. Multiplexed and single assay reverse-transcription PCR (RT-PCR) was used to confirm in silico predicted sample differences. Data visualizations and summary metrics for genome-scale miRNA profiling assessment were developed using this dataset, and a range of performance was observed. These metrics have been incorporated into an online data analysis pipeline and provide a convenient dashboard view of results from experiments following the described design. The website also serves as a repository for the accumulation of performance values providing new participants in the project an opportunity to learn what may be achievable with similar measurement processes. CONCLUSIONS: The set of reference samples used in this study provides benchmark values suitable for assessing genome-scale miRNA profiling processes. Incorporation of these metrics into an online resource allows laboratories to periodically evaluate their performance and assess any changes introduced into their measurement process.


Asunto(s)
Encéfalo/metabolismo , Perfilación de la Expresión Génica/normas , Genoma Humano , Hígado/metabolismo , MicroARNs/genética , Placenta/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Embarazo , Estándares de Referencia
5.
Hum Pathol ; 55: 108-116, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27184478

RESUMEN

Hodgkin/Reed-Sternberg (HRS) cells in the setting of chronic lymphocytic leukemia (CLL) exist in 2 forms: type I with isolated HRS cells in a CLL background (Hodgkin-like lesion) and type II with typical classic Hodgkin lymphoma, a variant of Richter transformation (CHL-RT). The clinical significance of the 2 morphological patterns is unclear, and their biological features have not been compared. We retrospectively reviewed 77 cases: 26 of type I and 51 of type II CHL-RT; 3 cases progressed from type I to type II. We examined clinical features, Epstein-Barr virus (EBV) status, and clonal relatedness after microdissection. Median age for type I was 62 years versus 73 years for type II (P=.01); 27% (type I) versus 73% (type II) had a history of CLL. HRS cells were positive for EBV in 71% (55/77), similar in types I and II. Clonality analysis was performed in 33 cases (type I and type II combined): HRS cells were clonally related to the underlying CLL in 14 and unrelated in 19. ZAP-70 expression of the CLL cells but not EBV status or morphological pattern was correlated with clonal relatedness: all 14 clonally related cases were ZAP-70 negative, whereas 74% (14/19) of clonally unrelated cases were ZAP-70 positive. Overall median survival (types I and II) after diagnosis was 44 months. Advanced age was an adverse risk factor for survival, but not histologic pattern, type I versus type II. HRS-like cells in a background of CLL carries a similar clinical risk to that of CHL-RT and may progress to classic Hodgkin lymphoma in some cases.


Asunto(s)
Transformación Celular Viral , Células Clonales , Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4/aislamiento & purificación , Enfermedad de Hodgkin , Leucemia Linfocítica Crónica de Células B , Células de Reed-Sternberg , Adulto , Anciano , Anciano de 80 o más Años , Células Clonales/patología , Células Clonales/virología , Bases de Datos Factuales , Progresión de la Enfermedad , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/mortalidad , Infecciones por Virus de Epstein-Barr/patología , Infecciones por Virus de Epstein-Barr/virología , Femenino , Reordenamiento Génico , Genes de las Cadenas Pesadas de las Inmunoglobulinas , Predisposición Genética a la Enfermedad , Herpesvirus Humano 4/genética , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/mortalidad , Enfermedad de Hodgkin/patología , Enfermedad de Hodgkin/virología , Humanos , Inmunohistoquímica , Hibridación in Situ , Estimación de Kaplan-Meier , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/mortalidad , Leucemia Linfocítica Crónica de Células B/patología , Leucemia Linfocítica Crónica de Células B/virología , Masculino , Persona de Mediana Edad , Fenotipo , Reacción en Cadena de la Polimerasa , Modelos de Riesgos Proporcionales , ARN Viral/genética , Células de Reed-Sternberg/patología , Células de Reed-Sternberg/virología , Estudios Retrospectivos , Factores de Riesgo
6.
Cancer Biomark ; 15(6): 763-73, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26406418

RESUMEN

BACKGROUND: A 3.4kb deletion (3.4kbΔ ) in mitochondrial DNA (mtDNA) found in histologically normal prostate biopsy specimens has been reported to be a biomarker for the increased probability of prostate cancer. Increased mtDNA copy number is also reported as associated with cancer. OBJECTIVE: Independent evaluation of these two potential prostate cancer biomarkers using formalin-fixed paraffin-embedded (FFPE) prostate tissue and matched urine and serum from a high risk cohort of men with and without prostate cancer. METHODS: Biomarker levels were detected via qPCR. RESULTS: Both 3.4kbΔ and mtDNA levels were significantly higher in cancer patient FFPE cores (p= 0.045 and p= 0.070 respectively at > 90% confidence). Urine from cancer patients contained significantly higher levels of mtDNA (p= 0.006, 64.3% sensitivity, 86.7% specificity). Combining the 3.4kbΔ and mtDNA gave better performance of detecting prostate cancer than either biomarker alone (FFPE 73.7% sensitivity, 65% specificity; urine 64.3% sensitivity, 100% specificity). In serum, there was no difference for any of the biomarkers. CONCLUSIONS: This is the first report on detecting the 3.4kbΔ in urine and evaluating mtDNA levels as a prostate cancer biomarker. A confirmation study with increased sample size and possibly with additional biomarkers would need to be conducted to corroborate and extend these observations.


Asunto(s)
ADN Mitocondrial/genética , Marcadores Genéticos , Próstata/metabolismo , Neoplasias de la Próstata/genética , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , ADN Mitocondrial/sangre , ADN Mitocondrial/orina , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Adhesión en Parafina , Pronóstico , Estudios Prospectivos , Próstata/patología , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/orina , Curva ROC , Reacción en Cadena en Tiempo Real de la Polimerasa , Urinálisis
7.
Expert Rev Mol Diagn ; 13(5): 421-3, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23782249

RESUMEN

Recent discoveries in cancer biology have greatly increased the understanding of cancer at the molecular level, but translating this knowledge into clinically useful diagnostic tests has proved challenging. More efficient transfer of new molecular tests into patient care requires better standardization of laboratory practices, measurement methods and data management. The workshop assembled experts from National Cancer Institute, US FDA, National Institute of Standards and Technology, academia and industry, to address the most efficient approaches to biomarker standardization and validation. The workshop participants described the current state of research in molecular diagnostics standardization and addressed three questions: what has worked? What has not?And what needs improving?


Asunto(s)
Biomarcadores de Tumor/genética , National Cancer Institute (U.S.) , Patología Molecular/normas , United States Food and Drug Administration , Educación , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Estados Unidos
8.
Clin Chem ; 59(1): 68-74, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23193062

RESUMEN

BACKGROUND: The mission of the National Cancer Institute's Early Detection Research Network (EDRN) is to identify and validate cancer biomarkers for clinical use. Since its inception, EDRN investigators have learned a great deal about the process of validating biomarkers for clinical use. Translational research requires a broad spectrum of research expertise, and coordinating collaborative activities can be challenging. The EDRN has developed a robust triage and validation system that serves the roles of both "facilitator" and "brake." CONTENT: The system consists of (a) establishing a reference set of specimens collected under PRoBE (Prospective Specimen Collection Retrospective Blinded Evaluation) design criteria; (b) using the reference set to prevalidate candidate biomarkers before committing to full-scale validation; (c) performing full-scale validation for those markers that pass prevalidation testing; and (d) ensuring that the reference set is sufficiently large in numbers and volumes of sample that it can also be used to study future candidate biomarkers. This system provides rigorous and efficient evaluation of candidate biomarkers and biomarker panels. Reference sets should also be constructed to enable high-quality biomarker-discovery research. SUMMARY: We describe the process of establishing our system in the hope that it will serve as an example of how to validate biomarkers for clinical application. We also hope that this description of the biospecimen reference sets available from the EDRN will encourage the biomarker research community--from academia or industry--to use this resource to advance biomarkers into clinical use.


Asunto(s)
Biomarcadores de Tumor/análisis , Diagnóstico Precoz , Neoplasias/diagnóstico , Humanos , Masculino , Neoplasias de la Próstata/diagnóstico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
9.
Lab Chip ; 12(23): 4972-5, 2012 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-23079718

RESUMEN

We have developed a system to isolate rare cells from whole blood using commercially available components and simple microfluidics. We characterized the capture of MCF-7 cells spiked into whole human blood using this system to demonstrate that enrichment and enumeration studies give results similar to in situ surface-modified devices while reducing fabrication and operation complexity.


Asunto(s)
Anticuerpos/metabolismo , Separación Celular/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Células Neoplásicas Circulantes/metabolismo , Línea Celular Tumoral , Humanos
10.
Blood ; 115(8): 1519-29, 2010 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-20040766

RESUMEN

We identified 18 patients with the distinct clinical phenotype of susceptibility to disseminated nontuberculous mycobacterial infections, viral infections, especially with human papillomaviruses, and fungal infections, primarily histoplasmosis, and molds. This syndrome typically had its onset in adulthood (age range, 7-60 years; mean, 31.1 years; median, 32 years) and was characterized by profound circulating monocytopenia (mean, 13.3 cells/microL; median, 14.5 cells/microL), B lymphocytopenia (mean, 9.4 cells/microL; median, 4 cells/microL), and NK lymphocytopenia (mean, 16 cells/microL; median, 5.5 cells/microL). T lymphocytes were variably affected. Despite these peripheral cytopenias, all patients had macrophages and plasma cells at sites of inflammation and normal immunoglobulin levels. Ten of these patients developed 1 or more of the following malignancies: 9 myelodysplasia/leukemia, 1 vulvar carcinoma and metastatic melanoma, 1 cervical carcinoma, 1 Bowen disease of the vulva, and 1 multiple Epstein-Barr virus(+) leiomyosarcoma. Five patients developed pulmonary alveolar proteinosis without mutations in the granulocyte-macrophage colony-stimulating factor receptor or anti-granulocyte-macrophage colony-stimulating factor autoantibodies. Among these 18 patients, 5 families had 2 generations affected, suggesting autosomal dominant transmission as well as sporadic cases. This novel clinical syndrome links susceptibility to mycobacterial, viral, and fungal infections with malignancy and can be transmitted in an autosomal dominant pattern.


Asunto(s)
Enfermedades Genéticas Congénitas/genética , Predisposición Genética a la Enfermedad/genética , Leucopenia/genética , Infecciones por Mycobacterium/genética , Micosis/genética , Síndromes Mielodisplásicos/genética , Infecciones por Papillomavirus/genética , Linaje , Adolescente , Adulto , Niño , Femenino , Hongos , Enfermedades Genéticas Congénitas/sangre , Enfermedades Genéticas Congénitas/complicaciones , Humanos , Recuento de Leucocitos , Leucopenia/sangre , Leucopenia/complicaciones , Masculino , Persona de Mediana Edad , Mycobacterium , Infecciones por Mycobacterium/sangre , Infecciones por Mycobacterium/etiología , Micosis/sangre , Micosis/etiología , Síndromes Mielodisplásicos/sangre , Síndromes Mielodisplásicos/etiología , Neoplasias/sangre , Neoplasias/etiología , Neoplasias/genética , Papillomaviridae , Infecciones por Papillomavirus/sangre , Infecciones por Papillomavirus/etiología
11.
J Proteome Res ; 8(1): 113-7, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19072545

RESUMEN

Specimen collection is an integral component of clinical research. Specimens from subjects with various stages of cancers or other conditions, as well as those without disease, are critical tools in the hunt for biomarkers, predictors, or tests that will detect serious diseases earlier or more readily than currently possible. Analytic methodologies evolve quickly. Access to high-quality specimens, collected and handled in standardized ways that minimize potential bias or confounding factors, is key to the "bench to bedside" aim of translational research. It is essential that standard operating procedures, "the how" of creating the repositories, be defined prospectively when designing clinical trials. Small differences in the processing or handling of a specimen can have dramatic effects in analytical reliability and reproducibility, especially when multiplex methods are used. A representative working group, Standard Operating Procedures Internal Working Group (SOPIWG), comprised of members from across Early Detection Research Network (EDRN) was formed to develop standard operating procedures (SOPs) for various types of specimens collected and managed for our biomarker discovery and validation work. This report presents our consensus on SOPs for the collection, processing, handling, and storage of serum and plasma for biomarker discovery and validation.


Asunto(s)
Proteínas Sanguíneas/análisis , Proteómica/métodos , Proteómica/normas , Biomarcadores/sangre , Conservación de la Sangre , Ensayos Clínicos como Asunto , Humanos , Luz , Guías de Práctica Clínica como Asunto , Proteómica/tendencias , Reproducibilidad de los Resultados , Manejo de Especímenes/métodos , Manejo de Especímenes/tendencias , Temperatura
12.
Am J Respir Crit Care Med ; 178(10): 1066-74, 2008 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-18703788

RESUMEN

RATIONALE: Pulmonary nontuberculous mycobacterial (PNTM) disease is increasing, but predisposing features have been elusive. OBJECTIVES: To prospectively determine the morphotype, immunophenotype, and cystic fibrosis transmembrane conductance regulator genotype in a large cohort with PNTM. METHODS: We prospectively enrolled 63 patients with PNTM infection, each of whom had computerized tomography, echocardiogram, pulmonary function, and flow cytometry of peripheral blood. In vitro cytokine production in response to mitogen, LPS, and cytokines was performed. Anthropometric measurements were compared with National Health and Nutrition Examination Survey (NHANES) age- and ethnicity-matched female control subjects extracted from the NHANES 2001-2002 dataset. MEASUREMENTS AND MAIN RESULTS: Patients were 59.9 (+/-9.8 yr [SD]) old, and 5.4 (+/-7.9 yr) from diagnosis to enrollment. Patients were 95% female, 91% white, and 68% lifetime nonsmokers. A total of 46 were infected with Mycobacterium avium complex, M. xenopi, or M. kansasii; 17 were infected with rapidly growing mycobacteria. Female patients were significantly taller (164.7 vs. 161.0 cm; P < 0.001) and thinner (body mass index, 21.1 vs. 28.2; P < 0.001) than matched NHANES control subjects, and thinner (body mass index, 21.1 vs. 26.8; P = 0.002) than patients with disseminated nontuberculous mycobacterial infection. A total of 51% of patients had scoliosis, 11% pectus excavatum, and 9% mitral valve prolapse, all significantly more than reference populations. Stimulated cytokine production was similar to that of healthy control subjects, including the IFN-gamma/IL-12 pathway. CD4(+), CD8(+), B, and natural killer cell numbers were normal. A total of 36% of patients had mutations in the cystic fibrosis transmembrane conductance regulator gene. CONCLUSIONS: Patients with PNTM infection are taller and leaner than control subjects, with high rates of scoliosis, pectus excavatum, mitral valve prolapse, and cystic fibrosis transmembrane conductance regulator mutations, but without recognized immune defects.


Asunto(s)
Infecciones por Mycobacterium no Tuberculosas/etiología , Neumonía Bacteriana/etiología , Anciano , Estatura , Estudios de Casos y Controles , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Femenino , Tórax en Embudo/complicaciones , Humanos , Masculino , Persona de Mediana Edad , Mutación , Infecciones por Mycobacterium no Tuberculosas/genética , Infecciones por Mycobacterium no Tuberculosas/inmunología , Fenotipo , Estudios Prospectivos , Factores de Riesgo , Escoliosis/complicaciones , Factores Sexuales , Fumar/efectos adversos , Síndrome , Delgadez/complicaciones
13.
Diagn Cytopathol ; 35(1): 6-11, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17173298

RESUMEN

The effects on morphology and diagnostic interpretation of delayed processing of refrigerated effusion samples have not been well documented. The potential for cellular degeneration has led many laboratories to reflexively fix samples rather than submit fresh/refrigerated samples for cytologic examination. We sought to determine if effusion specimens are suitable for morphologic, immunocytochemical, and DNA-based molecular studies after prolonged periods of refrigerated storage time. Ten fresh effusion specimens were refrigerated at 4 degrees C; aliquots were processed at specific points in time (days 0, 3, 5, 7, 10, 14). Specimens evaluated included four pleural (3 benign, 1 breast adenocarcinoma) and six peritoneal (2 ovarian adenocarcinomas, 1 malignant melanoma, 2 mesotheliomas, 1 atypical mesothelial) effusions. The morphology of the cytologic preparations from the 10 effusions was preserved and interpretable after 14 days of storage at 4 degrees C. The immunocytochemical profile of the samples (AE1/AE3, EMA, calretinin, and LCA) was consistent from day 0 to day 14. Amplifiable DNA was present in all samples tested on day 14. We conclude that cytopathologic interpretation of effusion samples remains reliable with refrigeration at 4 degrees C even if processing is delayed.


Asunto(s)
Artefactos , Líquido Ascítico/patología , Citodiagnóstico/métodos , Neoplasias/diagnóstico , Derrame Pleural Maligno/diagnóstico , Manejo de Especímenes/métodos , Adulto , Líquido Ascítico/química , Biomarcadores de Tumor , ADN de Neoplasias/análisis , Femenino , Humanos , Masculino , Persona de Mediana Edad , Derrame Pleural Maligno/genética , Reacción en Cadena de la Polimerasa , Factores de Tiempo
14.
Pediatr Dev Pathol ; 9(6): 488-95, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-17163788

RESUMEN

Ewing sarcoma family tumors originating in the palate or adrenal gland are extremely rare and may cause difficulty in diagnosis. More common tumors primary to these sites need to be excluded before one arrives at the correct diagnosis. We have recently diagnosed 2 such cases. The 1st case was that of a 24-year-old woman who presented with a swelling in the right side of the hard palate. The 2nd case was diagnosed in a 28-year-old woman who presented with a mass in the right adrenal gland. In both cases, the diagnosis of Ewing sarcoma family of tumors was confirmed by immunohistochemical studies and reverse transcriptase-polymerase chain reaction (RT-PCR). The hard palate case is the 1st and the adrenal gland the 3rd case of Ewing sarcoma family of tumors arising in these sites, in which the diagnosis was confirmed by RT-PCR and/or cytogenetics. Accurate diagnosis of Ewing sarcoma family of tumors is crucial for the management of patients, and when found in such rare locations, diagnosis should be supported by immunohistochemical and/or molecular genetic studies.


Asunto(s)
Neoplasias de las Glándulas Suprarrenales/patología , Neoplasias Palatinas/patología , Paladar Duro/patología , Sarcoma de Ewing/patología , Neoplasias de las Glándulas Suprarrenales/genética , Neoplasias de las Glándulas Suprarrenales/cirugía , Adulto , Biomarcadores de Tumor/análisis , ADN Complementario/análisis , Femenino , Humanos , Inmunohistoquímica/métodos , Imagen por Resonancia Magnética , Proteínas de Fusión Oncogénica/análisis , Neoplasias Palatinas/genética , Neoplasias Palatinas/cirugía , Paladar Duro/cirugía , Embarazo , Proteína Proto-Oncogénica c-fli-1/análisis , ARN Neoplásico/análisis , Proteína EWS de Unión a ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcoma de Ewing/genética , Sarcoma de Ewing/cirugía
15.
Clin Cancer Res ; 12(9): 2804-11, 2006 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-16675574

RESUMEN

Cladribine induces long-term complete remission in hairy cell leukemia (HCL) patients but does not clear minimal residual disease (MRD) according to high-sensitivity PCR assays. To quantify MRD in patients after anti-CD22 recombinant immunotoxin BL22 and other agents, we used a relative quantitative PCR (RQ-PCR) assay using a primer and probe, both patient specific for the immunoglobulin heavy chain rearrangement. Using this method, we were able to detect one Bonna 12 HCL cell in either 10(6) Jurkat cells or in 10(6) normal mononuclear cells. We studied 84 samples from 10 patients, taken before or after treatment with BL22 and other agents. Patient-specific RQ-PCR was much more sensitive than flow cytometry, which in turn was (as recently reported) more sensitive than PCR using consensus primers. RQ-PCR was positive in 62 of 62 (100%) flow-positive samples in 10 patients and in 20 of 22 (91%) flow-negative samples in six patients. The relative level of MRD as quantified by RQ-PCR correlated with disease status and remission. Thus, patient-specific RQ-PCR is the most sensitive test for MRD in HCL patients and could be used to determine maximal response in patients obtaining multiple cycles of nonmyelotoxic biological treatment for this disease.


Asunto(s)
Leucemia de Células Pilosas/sangre , Neoplasia Residual/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Anciano , Antígenos CD/genética , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Leucemia de Células Pilosas/genética , Leucemia de Células Pilosas/inmunología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular
16.
Cancer Immunol Immunother ; 55(9): 1100-10, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16311729

RESUMEN

We previously reported that hairy cell leukemia (HCL) patients have high percentages of CD56+/CD57+/CD3+ large granular lymphocytes consistent with cytotoxic T-lymphocytes (CTLs), and other investigators have reported skewing of the T-cell repertoire. In previous studies of up to seven HCL patients, many of the 22 established T-cell receptor (TCR) beta variable region (TRBV) families showed mono- or oligoclonal restriction. To determine whether percentages of CTLs are correlated with TRBV clonal excess, we studied 20 HCL patients with flow cytometry, PCR of TCR gamma and TRBV regions, and fractional gel electrophoresis of PCR-amplified TRBV CDR3 domains (CDR3 spectratyping). Increased percentages of CD3+/CD8+/CD57+ CTLs correlated with more mono/oligoclonal and fewer polyclonal TRBV families (r=0.53; P=0.016). Age correlated with number of mono/oligoclonal TRBV families (r=0.51; P=0.022). Time since last purine analog therapy correlated with number of polyclonal TRBV families (r=0.46; P=0.040), but treatment with the anti-CD22 recombinant immunotoxin BL22 was not related to clonal excess. We conclude that abnormalities in the T-cell repertoire in HCL patients may represent deficient immunity, and may be exacerbated by purine analogs. Increased CD3+/CD57+ T-cells may be a useful marker of abnormal TRBV repertoire in HCL patients, and might prove useful in deciding whether patients should receive biologic antibody-based treatment rather than repeated courses of purine analog for relapsed disease.


Asunto(s)
Anticuerpos/uso terapéutico , Antineoplásicos/uso terapéutico , Enterotoxinas/uso terapéutico , Inmunotoxinas/uso terapéutico , Leucemia de Células Pilosas/tratamiento farmacológico , Subgrupos de Linfocitos T/clasificación , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Femenino , Citometría de Flujo , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Humanos , Leucemia de Células Pilosas/genética , Leucemia de Células Pilosas/inmunología , Leucocitos Mononucleares/clasificación , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas Recombinantes/uso terapéutico , Subgrupos de Linfocitos T/efectos de los fármacos
17.
Am J Surg Pathol ; 29(11): 1411-21, 2005 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16224207

RESUMEN

In recent years, overlap in biologic and morphologic features has been identified between classic Hodgkin lymphoma (cHL) and B-cell non-Hodgkin lymphoma. Nevertheless, the therapeutic approaches for these diseases remain different. We undertook a study of "mediastinal gray zone lymphomas" (MGZL), with features transitional between cHL nodular sclerosis (NS) and primary mediastinal large B-cell lymphoma (MLBCL) to better understand the morphologic and immunophenotypic spectrum of such cases. Twenty-one MGZL cases were identified over a 20-year period. We also studied 6 cases of composite or synchronous lymphoma with two distinct components at the same time (cHL-NS and MLBCL) and 9 sequential cases with MLBCL and cHL-NS at different times. All patients had a large mediastinal mass. Immunohistochemical studies focused on markers known to discriminate between cHL and MLBCL, including B-cell transcription factors. VJ-PCR was performed in 8 cases to look at clonality of the immunoglobulin heavy chain gene (IgH). Of the gray zone cases, 11 had morphology reminiscent of cHL-NS, but with unusual features, including a large number of mononuclear variants, diminished inflammatory background, absence of classic Hodgkin phenotype, and strong CD20 expression (11 of 11). Ten cases had morphology of MLBCL, but with admixed Hodgkin/Reed-Sternberg and lacunar cells, absent (3 of 10) or weak (7 of 10) CD20 expression, and positivity for CD15 in 7 cases. B-cell transcription factor expression in the gray zone cases more closely resembled MLBCL than cHL with expression of Pax5, Oct2, and BOB.1 in all but 1 case studied (14 of 15). MAL staining was found in 7 of 10 MGZL, and in at least one component of 6 of 7 evaluable composite or sequential MLBCL/cHL cases. Two cases of sequential lymphoma showed rearrangements of the IgH gene of identical size: one in which MLBCL was the first diagnosis and one in which MLBCL was diagnosed at relapse, indicating clonal identity for the two components of cHL and MLBCL. There is accumulating evidence that MLBCL and cHL are related entities. Further support for a relationship between MLBCL and cHL-NS is provided by composite and metachronous lymphomas in the same patient, as well as the existence of MGZL with transitional morphology and phenotype.


Asunto(s)
Enfermedad de Hodgkin/patología , Linfoma de Células B/patología , Neoplasias del Mediastino/patología , Adolescente , Adulto , Femenino , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/inmunología , Humanos , Linfoma de Células B/genética , Linfoma de Células B/inmunología , Masculino , Neoplasias del Mediastino/genética , Neoplasias del Mediastino/inmunología , Persona de Mediana Edad
18.
Mod Pathol ; 17(10): 1295-301, 2004 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-15195110

RESUMEN

Use of the polymerase chain reaction (PCR) for the detection of B- and T-cell clonality, Epstein-Barr virus (EBV) and Human Herpes Virus 8 (HHV 8) infection is gaining increasing importance as a diagnostic modality. These tests are usually performed on fresh specimens. There are instances when fresh material is not available and there is a clinical utility for the performance of PCR on archival material via slide scrape lysates (SSL). However, the suitability of archival material may be questioned. Records were searched for all archival cytology cases submitted for SSL molecular diagnostics tests since 1998. Results for each case were analyzed for PCR amplification status and individual test results. A randomly chosen control group of equivalent cytologic samples submitted fresh was evaluated for comparison of amplification status. In all, 241 PCR runs were performed on SSL of archival material from 112 cytologic samples (89 cerebrospinal fluids (CSFs), 13 fine-needle aspirates (FNAs), 10 effusions). Out of these samples, 95 (85%) had amplifiable DNA, as assessed by a positive reaction for glyceraldehyde phosphate dehydrogenase (GAPDH). For the control group, 320 PCR runs were performed on 112 fresh cytologic samples (89 CSFs, 13 FNAs, 10 effusions). In total, 102 samples (91%) had amplifiable DNA. There was no statistical difference in the amplification yield between the two groups (P = 0.2177). A morphologic review of 16 of the 17 SSL archival cytologic cases that did not show amplification revealed 11/16 to be of sparse cellularity. Molecular diagnostic tests are performed routinely on fresh cytologic samples with excellent results. At times critical decisions on patient care may need to be made when fresh tissue is not available for molecular diagnostic tests. SSL of archival cytologic material can be used with excellent results for molecular diagnostic tests when fresh material is not available or when the cytologic diagnosis needs further clarification.


Asunto(s)
Citodiagnóstico/métodos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa/métodos , Linfocitos B/citología , Linfocitos B/metabolismo , Biopsia con Aguja Fina , Línea Celular Tumoral , Líquido Cefalorraquídeo , Células Clonales/metabolismo , ADN Viral/líquido cefalorraquídeo , ADN Viral/genética , ADN Viral/aislamiento & purificación , Reordenamiento Génico de Linfocito B/genética , Reordenamiento Génico de Linfocito T/genética , Reordenamiento Génico de la Cadena gamma de los Receptores de Antígenos de los Linfocitos T/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 8/genética , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Derrame Pleural , Linfocitos T/citología , Linfocitos T/metabolismo , Frotis Vaginal
19.
Am J Surg Pathol ; 27(12): 1513-22, 2003 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-14657710

RESUMEN

Coexpression of CD30 and CD15 is typically associated with classic Hodgkin's lymphoma (HL). Peripheral T-cell lymphomas (PTCLs) can often display histologic features that simulate classic HL. However, reports of PTCLs coexpressing both CD30 and CD15 have been infrequently described. We report 11 cases of PTCL in which at least a subset of the neoplastic cells coexpressed CD30 and CD15. The patients included 4 women and 7 men and age ranged from 43 to 83 years (median, 62 years). Nine of 10 patients had advanced stage III or IV disease at presentation. Nodal involvement predominated in 8 of 11 patients, whereas 2 patients presented primarily with skin involvement. Two distinct groups were identified based on morphologic and immunophenotypic features. The first group of 5 cases had histologic features mimicking classic HL with CD30+, CD15+ Reed-Sternberg (RS)-like cells in an inflammatory background of varied extent and composition. The background lymphoid cells showed minimal cytologic atypia. The RS-like cells were negative for CD20 and CD79a in all cases, and CD45 expression was absent in 4 of 5 cases. The RS-like cells expressed CD25 and at least one T-cell-associated marker in all cases. The background T-cell population showed convincing subset predominance in 4 of 5 cases and loss of T-cell-associated antigens in 3 of 5 cases and coexpression of CD30 and CD15 in one case. The second group of 6 cases had morphologic features more in keeping with PTCL than classic HL. The proportion of neoplastic cells coexpressing CD30 and CD15 varied. Loss of T-cell antigens was noted in all cases and CD4 predominated in 4 of 5 cases. Three of the 6 cases expressed CD45. PCR analysis revealed clonal T-cell receptor gamma (TCR-gamma) chain gene rearrangements in 9 of 11 cases, but no immunoglobulin heavy (IgH) chain gene rearrangements. In situ hybridization studies for Epstein-Barr virus were negative in all cases. In some PTCL cases, the overlap with classic HL can be striking, and combined immunophenotypic and molecular studies are often necessary to confirm the diagnosis.


Asunto(s)
Antígeno Ki-1/biosíntesis , Antígeno Lewis X/biosíntesis , Linfoma de Células T Periférico/metabolismo , Linfoma de Células T Periférico/patología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos Virales , Diagnóstico Diferencial , Femenino , Reordenamiento Génico de la Cadena gamma de los Receptores de Antígenos de los Linfocitos T , Enfermedad de Hodgkin/metabolismo , Enfermedad de Hodgkin/patología , Humanos , Inmunofenotipificación , Hibridación in Situ , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa
20.
Am J Surg Pathol ; 27(4): 522-31, 2003 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-12657939

RESUMEN

We describe the clinicopathologic findings of 48 cases of marginal zone B-cell lymphoma (MZL) in children and young adults, a disease that has been recognized previously only rarely in this age group. Patients ranged in age from 2 to 29 years, with pediatric patients (< or =18 years) comprising 52% of the cases. As in adults, both primary nodal (N) and extranodal (E) MZL were observed. However, primary NMZL comprised the majority of the cases (67%) and demonstrated distinctive clinical and histologic features. NMZL occurred most commonly in young males (median 16 years, male/female ratio 5.4:1), with no underlying disease, presenting as localized adenopathy (90% stage I), with excellent prognosis and low rate of recurrence. In contrast, EMZL were much less common, and patients were older (median 24.5 years), with only a slight male predominance (male/female ratio 1.2:1). Most patients had localized disease (73% stage I) with excellent prognosis and infrequent recurrences. In addition, an association with autoimmune disease was observed in 19% of the EMZL. Both primary NMZL and EMZL in young patients shared similar morphologic and immunophenotypic findings to those described in adults and were monoclonal B-cell proliferations with monoclonality demonstrated in 94% of the cases. A common morphologic feature in NMZL was disruption of residual follicles resembling progressive transformation of germinal centers (PTGC), observed in 66% of the cases. Although the precise relationship of primary NMZL and the PTGC-like changes is unclear, it is possible that NMZL arises in a background of PTGC, as florid PTGC often occurs in young males. We conclude that EMZL in children and young adults are similar to EMZL of mucosa-associated lymphoma tissue occurring in older patients. However, pediatric NMZL appear to have distinctive clinical and histologic features.


Asunto(s)
Linfoma de Células B/patología , Adolescente , Adulto , Factores de Edad , Niño , Preescolar , Femenino , Humanos , Inmunofenotipificación , Ganglios Linfáticos/patología , Masculino
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