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1.
Cancers (Basel) ; 15(18)2023 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-37760593

RESUMO

BACKGROUND: Multiplex PCR based on consensus primers followed by capillary electrophoresis and Sanger sequencing are considered as the gold standard method for the evaluation of clonality and somatic hypermutation in lymphoid malignancies. As an alternative, the next-generation sequencing (NGS) of immune receptor genes has recently been proposed as a solution, due to being highly effective and sensitive. Here, we designed a phase III diagnostic accuracy study intended to compare the current gold standard methods versus the first commercially available NGS approaches for testing immunoglobulin heavy chain gene rearrangements. METHODS: We assessed IGH rearrangements in 68 samples by means of both the NGS approach (LymphoTrack® IGH assay, and LymphoTrack® IGH somatic hypermutation assay, run on Illumina MiSeq) and capillary electrophoresis/Sanger sequencing to assess clonality and somatic hypermutations (SHM). RESULTS: In comparison to the routine capillary-based analysis, the NGS clonality assay had an overall diagnostic accuracy of 96% (63/66 cases). Other studied criteria included sensitivity (95%), specificity (100%), positive predictive value (100%) and negative predictive value (75%). In discrepant cases, the NGS results were confirmed by a different set of primers that provided coverage of the IGH leader sequence. Furthermore, there was excellent agreement of the SHM determination with both the LymphoTrack® FR1 and leader assays when compared to the Sanger sequencing analysis (84%), with NGS able to assess the SHM rate even in cases where the conventional approach failed. CONCLUSION: Overall, conventional Sanger sequencing and next-generation-sequencing-based clonality and somatic hypermutation analyses gave comparable results. For future use in a routine diagnostic workflow, NGS-based approaches should be evaluated prospectively and an analysis of cost-effectiveness should be performed.

2.
Int J Mol Sci ; 24(12)2023 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-37373066

RESUMO

The majority of patients with Follicular Lymphoma (FL) experience subsequent phases of remission and relapse, making the disease "virtually" incurable. To predict the outcome of FL patients at diagnosis, various clinical-based prognostic scores have been proposed; nonetheless, they continue to fail for a subset of patients. Gene expression profiling has highlighted the pivotal role of the tumor microenvironment (TME) in the FL prognosis; nevertheless, there is still a need to standardize the assessment of immune-infiltrating cells for the prognostic classification of patients with early or late progressing disease. We studied a retrospective cohort of 49 FL lymph node biopsies at the time of the initial diagnosis using pathologist-guided analysis on whole slide images, and we characterized the immune repertoire for both quantity and distribution (intrafollicular, IF and extrafollicular, EF) of cell subsets in relation to clinical outcome. We looked for the natural killer (CD56), T lymphocyte (CD8, CD4, PD1) and macrophage (CD68, CD163, MA4A4A)-associated markers. High CD163/CD8 EF ratios and high CD56/MS4A4A EF ratios, according to Kaplan-Meier estimates were linked with shorter EFS (event-free survival), with the former being the only one associated with POD24. In contrast to IF CD68+ cells, which represent a more homogeneous population, higher in non-progressing patients, EF CD68+ macrophages did not stratify according to survival. We also identify distinctive MS4A4A+CD163-macrophage populations with different prognostic weights. Enlarging the macrophage characterization and combining it with a lymphoid marker in the rituximab era, in our opinion, may enable prognostic stratification for low-/high-grade FL patients beyond POD24. These findings warrant validation across larger FL cohorts.


Assuntos
Linfoma Folicular , Humanos , Intervalo Livre de Progressão , Linfoma Folicular/genética , Linfoma Folicular/patologia , Estudos Retrospectivos , Recidiva Local de Neoplasia , Rituximab , Microambiente Tumoral
3.
Virchows Arch ; 482(5): 899-904, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36480066

RESUMO

Extranodal T-lymphoproliferative disorders or T-cell lymphomas (TLPD) are classified according to the WHO Classification (4th and upcoming 5th editions) (Swerdlow et al., IARC Press 1; Alaggio et al., Leukemia 36(7):1720-1748, 2) and to the International Consensus Classification Update (Campo et al., Blood 140(11):1229-1253, 3) upon several morphologic, phenotypic, and genetic features. None of those at present included has been characterized by primary pulmonary onset. We herein present two such cases which, to the best of our knowledge, have not been previously reported and that might represent another variant of T-cell proliferation at mucosal sites. The two cases share similar histological and phenotypic features, suggesting an origin from CD4 + effector memory T cells with the expression of a CD279/PD-1 antigen. They are both monoclonal, harbor few mutations, and show no disease progression outside the lung. They only differ concerning the local extension of the process and clinical setting. The two cases are examples of so far unreported primary pulmonary TLDP, with limited stage and low proliferative index. A possible relationship with a local yet unknown inflammatory trigger that might have favored the development of the T-cell clone cannot be ruled out.


Assuntos
Linfoma de Células T , Transtornos Linfoproliferativos , Humanos , Transtornos Linfoproliferativos/genética , Linfoma de Células T/patologia , Linfócitos T/patologia , Mucosa/patologia , Pulmão/patologia
4.
Hum Pathol ; 124: 67-75, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35339566

RESUMO

The differential diagnosis between lymphoplasmacytic lymphoma (LPL) and marginal zone B-cell lymphoma, particularly splenic type (SMZL), can be challenging on onset of bone marrow biopsy (BMB) since morphology and phenotype are not specific and clinical features can overlap or be mildly developed at diagnosis. The LPL-specific L265P mutation in the MYD88 gene is not available in all laboratories, and genetic aberrancies identified in SMZL (del7q, mutations of NOTCH2 and KLF2) are seldom searched in routine practice. The study aim is to investigate the potential role of myeloid nuclear differentiation antigen (MNDA) expression in this specific differential diagnosis. We report MNDA reactivity in 559 patients with small B-cell lymphoma including bone marrow biopsies from 90 LPL and 91 SMZL cases. MYD88 p.Leu265Pro mutation status was assessed and confirmed as positive in 24 of 90 LPL cases, which served as the test set. MNDA staining was negative in 23 of 24 LPL cases in the test set (96%). In the 157 remaining cases (66 LPL, 91 SMZL), which served as the validation set, the MYD88 p.Leu265Pro mutation was unavailable and MNDA was more frequently expressed in SMZL (p < 0.00001). In addition, immunohistochemical features more consistent with SMZL (i.e., presence of CD23+ follicular dendritic cell meshworks, polytypic plasma cells, DBA44 reactivity) were more often present in MNDA-positive cases (statistically significant for 2 such parameters). On the widest case series so far published focusing on LPL and SMZL immunohistochemical diagnosis at onset of BMB, we demonstrated that MNDA expression significantly support the diagnosis of SMZL. This observation may be of particular help in cases where the MYD88 p.Leu265Pro mutational status and/or SMZL-related genetic aberrations are unavailable.


Assuntos
Leucemia Linfocítica Crônica de Células B , Linfoma de Zona Marginal Tipo Células B , Neoplasias Esplênicas , Macroglobulinemia de Waldenstrom , Antígenos de Diferenciação , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Biomarcadores , Biópsia , Medula Óssea/patologia , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma de Zona Marginal Tipo Células B/diagnóstico , Linfoma de Zona Marginal Tipo Células B/genética , Linfoma de Zona Marginal Tipo Células B/patologia , Mutação , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Neoplasias Esplênicas/diagnóstico , Macroglobulinemia de Waldenstrom/diagnóstico , Macroglobulinemia de Waldenstrom/genética , Macroglobulinemia de Waldenstrom/patologia
5.
Diagnostics (Basel) ; 12(2)2022 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-35204381

RESUMO

The spectrum of cutaneous CD30-positive lymphoproliferative disorders encompasses both inflammatory and neoplastic conditions. CD30+ Hodgkin and Reed-Sternberg-like cells have been occasionally reported in primary cutaneous marginal zone lymphoma, where they are thought to represent a side neoplastic component within a dominant background of lymphomatous small B cells. Herein, we describe the histological and molecular findings of three cases of primary cutaneous marginal zone lymphomas with CD30+ H/RS cells, in which next-generation sequencing analysis revealed the clonal population to consist in less than 5% of the cutaneous B-cell infiltrate, providing a thought-provoking focus on a possible main role for CD30+ cells in primary cutaneous marginal zone lymphoproliferations.

7.
Hematol Oncol ; 37(4): 368-374, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31325190

RESUMO

In 2009, the four laboratories of the Fondazione Italiana Linfomi (FIL) minimal residual disease (MRD) Network started a collaborative effort to harmonize and standardize their methodologies at the national level, performing quality control (QC) rounds for follicular lymphoma (FL) and mantle cell lymphoma (MCL) MRD assessment. In 16 QC rounds between 2010 and 2017, the four laboratories received 208 bone marrow (BM) samples (126 FL; 82 MCL); 187 were analyzed, according to the EuroMRD Consortium guidelines, by both nested (NEST) polymerase chain reaction (PCR) and real-time quantitative (RQ) PCR for BCL2/IGH MBR or IGHV rearrangements. Here, we aimed at analyzing the samples that challenged the interlaboratory reproducibility and data interpretation. Overall, 156/187 BM samples (83%) were concordantly classified as NEST+/RQ+ or NEST-/RQ- by all the four laboratories. The remaining 31 samples (17%) resulted alternatively positive and negative in the interlaboratory evaluations, independently of the method and the type of rearrangement, and were defined "borderline" (brd) samples: 12 proved NEST brd/RQ brd, 7 NEST-/RQ brd, 10 NEST brd/RQ positive not quantifiable (PNQ), and 2 NEST brd/RQ-. Results did not change even increasing the number of replicates/sample. In 6/31 brd samples, droplet digital PCR (ddPCR) was tested and showed no interlaboratory discordance. Despite the high interlaboratory reproducibility in the MRD analysis obtained and maintained by the QC round strategy, samples with the lowest MRD levels can still represent a challenge: 17% (31/187) of our samples showed discordant results in interlaboratory assessments, with 6.4% (12/187) remained brd even applying the two methods. Thus, although representing a minority, brd samples are still problematic, especially when a clinically oriented interpretation of MRD results is required. Alternative, novel methods such as ddPCR and next-generation sequencing have the potential to overcome the current limitations.


Assuntos
Exame de Medula Óssea , Medula Óssea/patologia , Ensaio de Proficiência Laboratorial , Linfoma não Hodgkin/patologia , Reação em Cadeia da Polimerase , Exame de Medula Óssea/normas , Células Clonais , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Genes de Imunoglobulinas , Genes bcl-2 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Itália/epidemiologia , Linfoma não Hodgkin/genética , Neoplasia Residual , Proteínas de Fusão Oncogênica/análise , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Garantia da Qualidade dos Cuidados de Saúde , Reprodutibilidade dos Testes , Translocação Genética
9.
Int J Hematol Oncol ; 7(4): IJH08, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30651967

RESUMO

This case report describes the first Italian live birth obtained by cryopreserved ovarian tissue transplantation in a woman affected by non-Hodgkin's lymphoma. Before anticancer treatments, several fertility preservation options were proposed. At 29 years the patient underwent laparoscopy for ovarian tissue cryopreservation. After treatments she experienced premature ovarian failure (POF) and asked for cryopreserved ovarian tissue transplantation. Before transplantation, ovarian samples were analyzed to assess neoplastic contamination and tissue quality. Two subsequent ovarian tissue transplantations were performed 4 and 7 years after cryopreservation. The follicle-stimulating hormone and luteinizing hormone reduction, estradiol increase and first menstrual cycle appeared 2 months after the second transplantation. The woman conceived spontaneously 5 months after the second transplantation. After 39 weeks of uneventful gestation, a healthy male baby was born. Ovarian tissue cryopreservation, thawing and transplantation successfully restored ovarian function and fertility after tissue storage.

10.
Histopathology ; 71(5): 769-777, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28628241

RESUMO

AIMS: Mantle cell lymphoma (MCL) is characterized by distinctive histological and molecular features. Aberrant expression of BCL6 and CD10 has been reported occasionally, but the biological features of such cases are largely unknown. This study aimed to define the epidemiological, histological and cytogenetic characteristics of BCL6 and CD10-positive MCLs, also investigating possible biological features. METHODS AND RESULTS: A total of 165 cases of cyclin D1 and t(11;14)(q13;q34)-positive MCLs were studied for CD10 and BCL6 immunohistochemical expression, which was documented in 26 of 165 (15.8%) cases (BCL6 17 of 165; CD10 11 of 165; BCL6 and CD10 co-expression two of 165). CD10-positivity was significantly more frequent in females (63.3%; P < 0.01). Either expression correlated significantly with higher mean proliferation index and higher prevalence of MUM1 positivity (P < 0.05). Fluorescence in-situ hybridization (FISH) for BCL6 (3q27) gene derangements was performed on the BCL6- and CD10-positive cases and 98 matched controls: amplifications were documented more frequently in BCL6-positive than -negative cases (50.0% versus 19.4% of cases) (P < 0.05). The mutational status of the variable immunoglobulin heavy chain genes (IGVH) was investigated by Sanger sequencing: five of the six successfully tested cases (83.3%) showed no somatic hypermutations. CONCLUSIONS: Aberrant CD10 and BCL6 expression defines a subset of MCLs with higher mean Ki-67 index and higher prevalence of MUM1 expression. BCL6 protein positivity correlates with cytogenetic aberrations involving the BCL6 gene. Although examined successfully in few cases, the high prevalence of unmutated IGVH genes also points at a pregerminal cell origin for these phenotypically aberrant cases.


Assuntos
Biomarcadores Tumorais/análise , Linfoma de Célula do Manto/patologia , Neprilisina/biossíntese , Proteínas Proto-Oncogênicas c-bcl-6/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/metabolismo , Masculino , Pessoa de Meia-Idade , Adulto Jovem
11.
Mol Cancer Res ; 15(5): 541-552, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28130401

RESUMO

Follicular dendritic cell (FDC) sarcomas are rare mesenchymal tumors with variable clinical, morphologic, and phenotypic characteristics. Transcriptome analysis was performed on multiple FDC sarcomas and compared with other mesenchymal tumors, microdissected Castleman FDCs, and normal fibroblasts. Using unsupervised analysis, FDC sarcomas clustered with microdissected FDCs, distinct from other mesenchymal tumors and fibroblasts. The specific endowment of FDC-related gene expression programs in FDC sarcomas emerged by applying a gene signature of differentially expressed genes (n = 1,289) between microdissected FDCs and fibroblasts. Supervised analysis comparing FDC sarcomas with microdissected FDCs and other mesenchymal tumors identified 370 and 2,927 differentially expressed transcripts, respectively, and on the basis of pathway enrichment analysis ascribed to signal transduction, chromatin organization, and extracellular matrix organization programs. As the transcriptome of FDC sarcomas retained similarity with FDCs, the immune landscape of FDC sarcoma was investigated by applying the CIBERSORT algorithm to FDC sarcomas and non-FDC mesenchymal tumors and demonstrated that FDC sarcomas were enriched in T follicular helper (TFH) and T regulatory (TREG) cell populations, as confirmed in situ by immunohistochemistry. The enrichment in specific T-cell subsets prompted investigating the mRNA expression of the inhibitory immune receptor PD-1 and its ligands PD-L1 and PD-L2, which were found to be significantly upregulated in FDC sarcomas as compared with other mesenchymal tumors, a finding also confirmed in situ Here, it is demonstrated for the first time the transcriptional relationship of FDC sarcomas with nonmalignant FDCs and their distinction from other mesenchymal tumors.Implications: The current study provides evidence of a peculiar immune microenvironment associated with FDC sarcomas that may have clinical utility. Mol Cancer Res; 15(5); 541-52. ©2017 AACR.


Assuntos
Sarcoma de Células Dendríticas Foliculares/imunologia , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Reguladores/metabolismo , Algoritmos , Antígeno B7-H1/genética , Hiperplasia do Linfonodo Gigante/genética , Hiperplasia do Linfonodo Gigante/imunologia , Hiperplasia do Linfonodo Gigante/patologia , Cromatina/genética , Cromatina/patologia , Análise por Conglomerados , Sarcoma de Células Dendríticas Foliculares/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Proteína 2 Ligante de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/genética , Transdução de Sinais , Regulação para Cima
12.
Virchows Arch ; 468(4): 441-50, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26762526

RESUMO

Germinotropic lymphoproliferative disorders were previously described as localized disorders associated with coinfection by human herpes virus 8 and Epstein-Barr virus and characterized by good clinical outcome. We report the clinical, morphological, phenotypical, and molecular features of three cases of a hitherto unreported variant of Epstein-Barr virus (EBV)-positive, human herpes virus 8 (HHV8)-negative large B cell lymphoma with exclusive intrafollicular localization. All cases occurred in elderly individuals (63, 77, and 65 years old; one male, two females) without obvious immunedeficiency, who presented with high stage disease. Lymph nodes showed an effaced nodular architecture with abnormal B follicles colonized by EBV+ large, pleomorphic atypical cells, including Reed-Sternberg-like cells, showing an activated B cell phenotype (CD10-FOXP1-Bcl6-IRF4+ or CD10-FOXP1+Bcl6+IRF4+) and intense expression of CD30. No monoclonal light-chain restriction was detected by immunohistochemistry or in situ hybridization, and IGH rearrangement was polyclonal; notably, EBV clonality was detectable in one case. Lymphoma cells in all cases showed diffuse expression of the c-Myc protein, while Bcl2 was dim or negative; moreover, the strong expression of phosphorylated-STAT3 in tumor cell nuclei suggested activation of the JAK-STAT pathway. FISH analysis was performed in two cases and showed no translocations of BCL2, BCL6, MYC, and PAX5 genes. Response to treatment was poor in 2/3 patients: one died after 18 months, one is alive with disease after 12 months. The intrafollicular EBV-positive large B cell lymphoma expands the spectrum of EBV-associated lymphoproliferative disorders in immunocompetent individuals.


Assuntos
Infecções por Vírus Epstein-Barr/complicações , Centro Germinativo/patologia , Linfoma Difuso de Grandes Células B/patologia , Idoso , Feminino , Humanos , Imuno-Histoquímica , Imunofenotipagem , Hibridização in Situ Fluorescente , Linfoma Difuso de Grandes Células B/virologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Reação em Cadeia da Polimerase
13.
Leuk Lymphoma ; 57(2): 400-410, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25115509

RESUMO

Genomic DNA extraction is a primary component of genomic research and diagnostic routine analysis. Recently, the importance of this process has been highlighted by the necessity to standardize the diagnostic procedure. In this regard, the Minimal Residual Disease (MRD) Network of the Fondazione Italiana Linfomi (FIL MRD Network) has performed a comparative study of four different commercially available kits for DNA extraction, applying them on a panel of cellular pellets, with the aim of defining possible technical recommendations in order to harmonize and standardize diagnostic procedures in the clinical setting. Overall, all four kits usually allowed the recovery of a significant quantity of high-quality DNA (in most conditions), although specific indications could be addressed for cellular pellets of different sizes.

14.
Clin Cancer Res ; 20(24): 6398-405, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25316810

RESUMO

PURPOSE: The role of the minimal residual disease (MRD) in follicular lymphoma is still debated. In this study, we assessed whether the BCL2/IGH rearrangement could have a prognostic role in patients receiving R-CHOP, R-FM, or R-CVP. EXPERIMENTAL DESIGN: DNAs from 415 patients among the 504 cases enrolled in the FOLL05 trial (NCT00774826) were centralized and assessed for the BCL2/IGH at diagnosis, at the end of treatment, and after 12 and 24 months. RESULTS: At diagnosis, the molecular marker was detected in 53% of cases. Patients without molecular marker or with a low molecular tumor burden (<1 × 10(-4) copies) showed higher complete remission (CR) rate and longer progression-free survival (PFS; 3-year PFS 80% vs. 59%; P = 0.015). PFS was significantly conditioned by the PCR status at 12 and 24 months, with 3-year PFS of 66% for MRD(-) cases versus 41% for those MRD(+) at 12 months (P = 0.015), and 84% versus 50% at 24 months (P = 0.014). The MRD negativity at 12 and 24 months resulted in an improved PFS both in CR and in partial remission (PR) patients (3-year PFS = 72% for cases CR/PCR(-) vs. 32% for those CR/PCR(+) vs. 62% for those PR/PCR(-) and 25% for patients in PR/PCR(+); P = 0.001). The prognostic value of MRD at 12 and 24 months of follow-up was confirmed also in multivariate analysis. CONCLUSIONS: In this study, standardized molecular techniques have been adopted and applied on bone marrow samples from a large cohort. Data reported show that the MRD detection is a powerful independent predictor of PFS in patients with follicular lymphoma receiving conventional chemoimmunotherapy.


Assuntos
Linfoma Folicular/mortalidade , Linfoma Folicular/patologia , Neoplasia Residual/diagnóstico , Adolescente , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ensaios Clínicos Fase III como Assunto , Feminino , Dosagem de Genes , Rearranjo Gênico , Genes bcl-2 , Humanos , Cadeias Pesadas de Imunoglobulinas , Linfoma Folicular/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Neoplasia Residual/genética , Prognóstico , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real , Resultado do Tratamento , Adulto Jovem
15.
Front Biosci (Landmark Ed) ; 19(7): 1088-104, 2014 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-24896338

RESUMO

In recent years, DNA-arrays, gene expression profiling and next-generation sequencing have elucidated the high complexity of genomic alterations occurring in lymphoid malignancies. These studies have also contributed to the identification of new diagnostic and prognostic biomarkers, which may represent possible targets for new therapeutic approaches. Such recent advances have significantly expanded the application of molecular tests to routine diagnostic hematopathology. It is thus conceivable that next-generation assays will soon flank traditional clonality tests and chromosomal translocation assays in the diagnostic work-up of difficult cases. This review is focused on the application of molecular biology techniques in the study of lymphoid tumors. Both conventional and next-generation tests will be addressed, with particular attention to their application to clinical practice.


Assuntos
Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Linfoma/diagnóstico , Linfoma/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Biomarcadores Tumorais/genética , Aberrações Cromossômicas , Humanos , Prognóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
16.
Ther Adv Hematol ; 5(2): 35-47, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24688753

RESUMO

Currently, distinguishing between benign and malignant lymphoid proliferations is based on a combination of clinical characteristics, cyto/histomorphology, immunophenotype and the identification of well-defined chromosomal aberrations. However, such diagnoses remain challenging in 10-15% of cases of lymphoproliferative disorders, and clonality assessments are often required to confirm diagnostic suspicions. In recent years, the development of new techniques for clonality detection has allowed researchers to better characterize, classify and monitor hematological neoplasms. In the past, clonality was primarily studied by performing Southern blotting analyses to characterize rearrangements in segments of the IG and TCR genes. Currently, the most commonly used method in the clinical molecular diagnostic laboratory is polymerase chain reaction (PCR), which is an extremely sensitive technique for detecting nucleic acids. This technique is rapid, accurate, specific, and sensitive, and it can be used to analyze small biopsies as well as formalin-fixed paraffin-embedded samples. These advantages make PCR-based approaches the current gold standard for IG/TCR clonality testing. Since the completion of the first human genome sequence, there has been a rapid development of technologies to facilitate high-throughput sequencing of DNA. These techniques have been applied to the deep characterization and classification of various diseases, patient stratification, and the monitoring of minimal residual disease. Furthermore, these novel approaches have the potential to significantly improve the sensitivity and cost of clonality assays and post-treatment monitoring of B- and T-cell malignancies. However, more studies will be required to demonstrate the utility, sensitivity, and benefits of these methods in order to warrant their adoption into clinical practice. In this review, recent developments in clonality testing are examined with an emphasis on highly sensitive systems for improving diagnostic workups and minimal residual disease assessments.

17.
Hematol Oncol ; 32(3): 133-8, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24254547

RESUMO

We compared two strategies for minimal residual disease evaluation of B-cell lymphoproliferative disorders characterized by a variable immunoglobulin heavy chain (IGH) genes mutation load. Twenty-five samples from chronic lymphocytic leukaemia (n = 18) or mantle cell lymphoma (n = 7) patients were analyzed. Based on IGH variable region genes, 22/25 samples carried > 2% mutations, 20/25 > 5%. In the IGH joining region genes, 23/25 samples carried > 2% mutations, 18/25 > 5%. Real-time quantitative polymerase chain reaction was performed on IGH genes using two strategies: method A utilizes two patient-specific primers, whereas method B employs one patient-specific and one germline primer, with different positions on the variable, diversity and joining regions. Twenty-three samples (92%) resulted evaluable using method A, only six (24%) by method B. Method B poor performance was specifically evident among mutated IGH variable/joining region cases, although no specific mutation load above, which the real-time quantitative polymerase chain reaction failed was found. The molecular strategies for minimal residual disease evaluation should be adapted to the B-cell receptor features of the disease investigated.


Assuntos
Genes de Imunoglobulinas , Transtornos Linfoproliferativos/diagnóstico , Transtornos Linfoproliferativos/genética , Mutação , Neoplasia Residual , Frequência do Gene , Rearranjo Gênico , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Reação em Cadeia da Polimerase em Tempo Real
18.
J Clin Oncol ; 31(24): 3019-25, 2013 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-23857971

RESUMO

PURPOSE: The differential diagnosis among the commonest peripheral T-cell lymphomas (PTCLs; ie, PTCL not otherwise specified [NOS], angioimmunoblastic T-cell lymphoma [AITL], and anaplastic large-cell lymphoma [ALCL]) is difficult, with the morphologic and phenotypic features largely overlapping. We performed a phase III diagnostic accuracy study to test the ability of gene expression profiles (GEPs; index test) to identify PTCL subtype. METHODS: We studied 244 PTCLs, including 158 PTCLs NOS, 63 AITLs, and 23 ALK-negative ALCLs. The GEP-based classification method was established on a support vector machine algorithm, and the reference standard was an expert pathologic diagnosis according to WHO classification. RESULTS: First, we identified molecular signatures (molecular classifier [MC]) discriminating either AITL and ALK-negative ALCL from PTCL NOS in a training set. Of note, the MC was developed in formalin-fixed paraffin-embedded (FFPE) samples and validated in both FFPE and frozen tissues. Second, we found that the overall accuracy of the MC was remarkable: 98% to 77% for AITL and 98% to 93% for ALK-negative ALCL in test and validation sets of patient cases, respectively. Furthermore, we found that the MC significantly improved the prognostic stratification of patients with PTCL. Particularly, it enhanced the distinction of ALK-negative ALCL from PTCL NOS, especially from some CD30+ PTCL NOS with uncertain morphology. Finally, MC discriminated some T-follicular helper (Tfh) PTCL NOS from AITL, providing further evidence that a group of PTCLs NOS shares a Tfh derivation with but is distinct from AITL. CONCLUSION: Our findings support the usage of an MC as additional tool in the diagnostic workup of nodal PTCL.


Assuntos
Linfoma de Células T Periférico/diagnóstico , Diagnóstico Diferencial , Feminino , Formaldeído , Humanos , Linfoma de Células T Periférico/genética , Linfoma de Células T Periférico/metabolismo , Linfoma de Células T Periférico/patologia , Masculino , Inclusão em Parafina , Prognóstico , Análise de Sobrevida , Fixação de Tecidos , Transcriptoma
19.
Biomed Res Int ; 2013: 715391, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24392455

RESUMO

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) is the commonest leukemia in adults. Here, we aimed to evaluate hsa-miR-15a/hsa-miR-16-1 expression in CLL tissues by qPCR and correlate it with the other clinicopathological features and clinical outcome. 40 formalin-fixed paraffin-embedded (FFPE) lymph node samples obtained from CLL/SLL patients were classified into two categories, "PCs rich" and "typical." We found a significant common expression level of 4 miRNAs; however, we did not find any significant relationship between PCs presence and miRNAs expression. Moreover, neither the presence of 13q deletion nor the percentage of cells carrying the deletion strictly correlated with miRNAs expression levels, although a significant number of patients with 13q deletion presented hsa-miR-16-1-3p levels below the median value in normal samples (P < 0.05). Finally, although no correlation was found between the expression of each miRNA and other clinicopathological features (Ki67, CD38, ZAP70, and IGVH@ hypermutations), the OS curves showed a positive trend in patients with miRNAs downregulation, though not statistically significant. In conclusion, we showed for the first time that all miRNAs can be successfully studied in FFPE CLL tissues and that del13q and PCs richness do not strictly correspond to miRNAs downregulation; therefore, a specific evaluation may be envisaged at least in patients enrolled in clinical trials.


Assuntos
Transtornos Cromossômicos/genética , Leucemia Linfocítica Crônica de Células B/genética , MicroRNAs/biossíntese , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células , Deleção Cromossômica , Cromossomos Humanos Par 13/genética , Feminino , Regulação Leucêmica da Expressão Gênica/genética , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Linfonodos/metabolismo , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Inclusão em Parafina
20.
Hum Pathol ; 43(12): 2266-73, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22818167

RESUMO

The BCL10 gene encodes for a T-cell receptor signaling downstream protein involved in nuclear factor κB activation. It is expressed in normal lymphoid tissues and in several B-non Hodgkin lymphomas, its aberrant function being related to the pathogenesis of certain subtypes. Conversely, conflicting data are available concerning BCL10 expression in peripheral T cell lymphomas. We analyzed BCL10 expression in peripheral T cell lymphomas and correlated it with NFκB activation, proliferation, phenotypic aberration, and survival. First, gene expression analysis of 40 peripheral T cell lymphomas (28 peripheral T cell lymphomas/not otherwise specified, 6 anaplastic large cell lymphomas, and 6 angioimmunoblastic lymphomas), 4 reactive lymph nodes, and 20 samples of normal T-lymphocytes, showed significantly lower BCL10 gene expression in all tumors in comparison to normal samples, the lowest values being detected in anaplastic large cell lymphoma. Secondly, we studied the immunohistochemical expression of BCL10 in 52 peripheral T cell lymphomas/not otherwise specified on tissue microarrays. BCL10 was expressed in 10/52 cases (19%), not showing any significant correlation with either expression of Ki-67 and the T-cell markers or NFκB activation. Furthermore, BCL10 expression was not associated with peculiar gene expression profiles. Finally, we did not find significant correlations with progression free survival and overall survival, although a favorable trend was recorded in BCL10(+) cases. In conclusion, BCL10 was commonly down-regulated in peripheral T cell lymphomas, suggest the T-cell receptor signaling cascade for future characterization.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Linfoma de Células T Periférico/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Proteína 10 de Linfoma CCL de Células B , Humanos , Linfoma de Células T Periférico/metabolismo , Linfoma de Células T Periférico/mortalidade , Análise de Sobrevida
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