RESUMO
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.
Assuntos
Encéfalo/metabolismo , Perfilação da Expressão Gênica/normas , Genoma Humano , Fígado/metabolismo , MicroRNAs/genética , Placenta/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Gravidez , Padrões de ReferênciaRESUMO
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.
Assuntos
Biomarcadores Tumorais/genética , Técnicas de Laboratório Clínico/normas , MicroRNAs/genética , Pequeno RNA não Traduzido/genética , Análise de Variância , Linhagem Celular Tumoral , Expressão Gênica , Humanos , Padrões de Referência , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normasRESUMO
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.
Assuntos
Biomarcadores Tumorais/análise , Diagnóstico Precoce , Neoplasias/diagnóstico , Humanos , Masculino , Neoplasias da Próstata/diagnóstico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
Assuntos
Doenças Genéticas Inatas/genética , Predisposição Genética para Doença/genética , Leucopenia/genética , Infecções por Mycobacterium/genética , Micoses/genética , Síndromes Mielodisplásicas/genética , Infecções por Papillomavirus/genética , Linhagem , Adolescente , Adulto , Criança , Feminino , Fungos , Doenças Genéticas Inatas/sangue , Doenças Genéticas Inatas/complicações , Humanos , Contagem de Leucócitos , Leucopenia/sangue , Leucopenia/complicações , Masculino , Pessoa de Meia-Idade , Mycobacterium , Infecções por Mycobacterium/sangue , Infecções por Mycobacterium/etiologia , Micoses/sangue , Micoses/etiologia , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/etiologia , Neoplasias/sangue , Neoplasias/etiologia , Neoplasias/genética , Papillomaviridae , Infecções por Papillomavirus/sangue , Infecções por Papillomavirus/etiologiaRESUMO
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.
Assuntos
Infecções por Mycobacterium não Tuberculosas/etiologia , Pneumonia Bacteriana/etiologia , Idoso , Estatura , Estudos de Casos e Controles , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Tórax em Funil/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/imunologia , Fenótipo , Estudos Prospectivos , Fatores de Risco , Escoliose/complicações , Fatores Sexuais , Fumar/efeitos adversos , Síndrome , Magreza/complicaçõesRESUMO
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.
Assuntos
Biomarcadores Tumorais , DNA Tumoral Circulante , DNA de Neoplasias , Biópsia Líquida , Neoplasias/diagnóstico , Neoplasias/genética , Alelos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Biópsia Líquida/métodos , Biópsia Líquida/normas , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Garantia da Qualidade dos Cuidados de Saúde , Padrões de ReferênciaRESUMO
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.
Assuntos
Leucemia de Células Pilosas/sangue , Neoplasia Residual/diagnóstico , Reação em Cadeia da Polimerase/métodos , Idoso , Antígenos CD/genética , Sequência de Bases , Clonagem Molecular , Primers do DNA , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Leucemia de Células Pilosas/genética , Leucemia de Células Pilosas/imunologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência MolecularRESUMO
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.
Assuntos
Artefatos , Líquido Ascítico/patologia , Citodiagnóstico/métodos , Neoplasias/diagnóstico , Derrame Pleural Maligno/diagnóstico , Manejo de Espécimes/métodos , Adulto , Líquido Ascítico/química , Biomarcadores Tumorais , DNA de Neoplasias/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Derrame Pleural Maligno/genética , Reação em Cadeia da Polimerase , Fatores de TempoRESUMO
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.
Assuntos
Transformação Celular Viral , Células Clonais , Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4/isolamento & purificação , Doença de Hodgkin , Leucemia Linfocítica Crônica de Células B , Células de Reed-Sternberg , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Clonais/patologia , Células Clonais/virologia , Bases de Dados Factuais , Progressão da Doença , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/mortalidade , Infecções por Vírus Epstein-Barr/patologia , Infecções por Vírus Epstein-Barr/virologia , Feminino , Rearranjo Gênico , Genes de Cadeia Pesada de Imunoglobulina , Predisposição Genética para Doença , Herpesvirus Humano 4/genética , Doença de Hodgkin/genética , Doença de Hodgkin/mortalidade , Doença de Hodgkin/patologia , Doença de Hodgkin/virologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Estimativa de Kaplan-Meier , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/mortalidade , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia Linfocítica Crônica de Células B/virologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Reação em Cadeia da Polimerase , Modelos de Riscos Proporcionais , RNA Viral/genética , Células de Reed-Sternberg/patologia , Células de Reed-Sternberg/virologia , Estudos Retrospectivos , Fatores de RiscoRESUMO
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.
Assuntos
Doença de Hodgkin/patologia , Linfoma de Células B/patologia , Neoplasias do Mediastino/patologia , Adolescente , Adulto , Feminino , Doença de Hodgkin/genética , Doença de Hodgkin/imunologia , Humanos , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Masculino , Neoplasias do Mediastino/genética , Neoplasias do Mediastino/imunologia , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
DNA Mitocondrial/genética , Marcadores Genéticos , Próstata/metabolismo , Neoplasias da Próstata/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , DNA Mitocondrial/sangue , DNA Mitocondrial/urina , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Inclusão em Parafina , Prognóstico , Estudos Prospectivos , Próstata/patologia , Neoplasias da Próstata/sangue , Neoplasias da Próstata/patologia , Neoplasias da Próstata/urina , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real , UrináliseRESUMO
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.
Assuntos
Antígeno Ki-1/biossíntese , Antígenos CD15/biossíntese , Linfoma de Células T Periférico/metabolismo , Linfoma de Células T Periférico/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos Virais , Diagnóstico Diferencial , Feminino , Rearranjo Gênico da Cadeia gama dos Receptores de Antígenos dos Linfócitos T , Doença de Hodgkin/metabolismo , Doença de Hodgkin/patologia , Humanos , Imunofenotipagem , Hibridização In Situ , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
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.
Assuntos
Linfoma de Células B/patologia , Adolescente , Adulto , Fatores Etários , Criança , Pré-Escolar , Feminino , Humanos , Imunofenotipagem , Linfonodos/patologia , MasculinoRESUMO
T-cell/histiocyte-rich large B-cell non-Hodgkin's lymphoma (THRLBCL) is an unusual morphologic variant of diffuse large B-cell lymphoma. We reviewed 30 cases of THRLBCL to evaluate its heterogeneity based on morphologic, immunophenotypic, and genetic features. Cases were classified according to the appearance of the large neoplastic B cells into three morphologic variants: 1) lymphocytic and histiocytic (L&H-like) (resembling the L&H cells of nodular lymphocyte predominance Hodgkin's lymphoma (14 cases); 2) centroblast (or immunoblast)-like (10 cases), and 3) Reed-Sternberg cell-like (resembling the neoplastic cells of classic Hodgkin's lymphoma) (6 cases). We used a panel of immunohistochemical stains, including those with specificity for germinal center B cells: CD20, CD79a, CD30, CD15, epithelial membrane antigen, BCL-2, BCL-6, and CD10. The /JH polymerase chain reaction assay was further performed to investigate a relationship to follicular lymphoma. The results were correlated with Epstein-Barr virus status as determined by staining for latent membrane protein and EBER-1 in situ hybridization. All cases were of B-cell immunophenotype with strong surface CD20 reactivity in the neoplastic large lymphoid cells, although CD79a was more inconsistently and weakly expressed (10 of 17). Nuclear positivity for the BCL-6 protein was detected in the tumor cells in 26 of 29 (90%) cases. However, differences in expression of other antigens were encountered in the histologic subtypes. Epithelial membrane antigen positivity, a feature often seen in nodular lymphocyte predominance Hodgkin's lymphoma, was observed in 11 of 30 (37%) cases and was most commonly seen in cases with L&H cell morphology (8 of 14; 57%). CD30 expression was observed in 9 of 30 (30%) cases but was most frequent in cases with Reed-Sternberg-like morphology (3 of 6 [50%]). CD10 expression was infrequent overall (3 of 29; 10%), with 2 of 3 positive cases identified in the centroblastic group. The overall rarity of positivity for CD10, BCL-2 (3 of 22; 13%), and -2 JH rearrangement (1 of 28; 4%) indicates a lack of connection to follicular lymphoma for all subtypes. The three cases that were negative for BCL-6 protein were LMP-1 positive and EBER-1 positive by in situ hybridization, and 2 of 3 had neoplastic cells with Reed-Sternberg-like morphology. These results demonstrate that although a large proportion of THRLBCL represent tumors of germinal center B cell derivation, they exhibit a diversity of morphologic and immunophenotypic features. A subset of THRLBCL may be related to nodular lymphocyte predominance Hodgkin's lymphoma. A small percentage show features closely resembling classic Hodgkin's lymphoma and could be considered a variant of grey zone lymphoma.
Assuntos
Linfócitos B/patologia , Centro Germinativo/patologia , Histiócitos/patologia , Linfoma de Células B/patologia , Linfoma Difuso de Grandes Células B/patologia , Linfócitos T/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/metabolismo , Biomarcadores Tumorais/metabolismo , DNA de Neoplasias/análise , Proteínas de Ligação a DNA/metabolismo , Feminino , Centro Germinativo/metabolismo , Histiócitos/metabolismo , Humanos , Técnicas Imunoenzimáticas , Imunofenotipagem , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6 , Células de Reed-Sternberg/metabolismo , Células de Reed-Sternberg/patologia , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismoRESUMO
We studied 86 specimens from 24 patients with hairy cell leukemia (HCL) to determine the sensitivity of routine flow cytometry (FC) and consensus primer PCR (cpPCR) in this disease. FC was more sensitive, detecting HCL in 48 (56%) of 86 specimens, while clonal B-cell populations were detected by cpPCR in only 23 (27%) of 86 specimens. FC and cpPCR were both more sensitive than morphologic examination. A positive cpPCR result is associated with higher tumor cell numbers than a negative cpPCR result, as determined by FC (P = .0017). We determined cutoff values for number of tumor cells at which cpPCR is consistently positive. At 6.8 tumor cells per microliter, cpPCR would be expected to be positive in at least 90% of the samples. FC was adequate in 86 cases (100%), while cpPCR was adequate in 74 cases (86%). FC is superior to cpPCR for detecting minimal residual HCL. It is more sensitive and more specific and permits quantitation of tumor cell number.
Assuntos
Citometria de Fluxo/métodos , Leucemia de Células Pilosas/diagnóstico , Neoplasia Residual/diagnóstico , Reação em Cadeia da Polimerase/métodos , Contagem de Células , Células Clonais , Sequência Consenso , DNA de Neoplasias/genética , Rearranjo Gênico de Cadeia Pesada de Linfócito B/genética , Humanos , Imunofenotipagem , Leucemia de Células Pilosas/genética , Leucemia de Células Pilosas/imunologia , Neoplasia Residual/genética , Neoplasia Residual/imunologia , Sensibilidade e EspecificidadeRESUMO
Follicular lymphoma (FL) is a low-grade lymphoma that typically lacks CD5 antigen expression. We report 3 cases of FL with unusual expression of CD5. All cases showed histologic features of FL, including effaced nodal architecture, follicular growth pattern, and a spectrum of grades from 1 to 3 using World Health Organization criteria. In flow cytometric studies, all 3 cases showed a light chain-restricted, CD19+, CD20+ B-cell population coexpressing CD10 and low-level CD5. Immunohistochemical studies demonstrated an identical B-cell immunophenotype with weak expression of CD5 and coexpression of bcl-2 protein and the germinal center-associated markers, CD10 and bcl-6 protein. None of the cases showed expression of CD43, cyclin D1, or IgD. By molecular analysis, immunoglobulin heavy chain gene rearrangements were demonstrated in all 3 cases, and 2 of 3 cases had a t(14;18). These cases highlight the difficulty classifying these lymphomas by flow cytometric studies alone and emphasize the importance of recognizing FL in the differential diagnosis of CD5+ B-cell lymphomas.
Assuntos
Antígenos CD5/metabolismo , Linfoma Folicular/metabolismo , Linfoma Folicular/patologia , Adulto , Linfócitos B/metabolismo , Linfócitos B/patologia , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 18 , DNA de Neoplasias/análise , Proteínas de Ligação a DNA/metabolismo , Feminino , Citometria de Fluxo , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Humanos , Cadeias Leves de Imunoglobulina/metabolismo , Linfoma Folicular/genética , Masculino , Pessoa de Meia-Idade , Neprilisina/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6 , Fatores de Transcrição/metabolismo , Translocação GenéticaRESUMO
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?
Assuntos
Biomarcadores Tumorais/genética , National Cancer Institute (U.S.) , Patologia Molecular/normas , United States Food and Drug Administration , Educação , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Estados UnidosRESUMO
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.
Assuntos
Anticorpos/metabolismo , Separação Celular/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Células Neoplásicas Circulantes/metabolismo , Linhagem Celular Tumoral , HumanosRESUMO
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.