Optimization and analysis of a quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples.

BACKGROUND: MicroRNAs (miRs) are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE) tissue. RESULTS: Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access) platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p < 0.00001) and outlines the optimal performance conditions of this platform using clinical FFPE samples. We also outline a method of data analysis looking at differences in miR abundance between FFPE and fresh-frozen samples. By dividing the profiled miR into abundance strata of high (Ct<30), medium (30 < or = Ct < or = 35), and low (Ct>35), we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p < 0.001), when examining all miRs, regardless of RNA extraction method used. Examining correlation coefficients between FFPE and fresh-frozen samples in terms of miR abundance reveals correlation coefficients of up to 0.32 (low abundance), 0.70 (medium abundance) and up to 0.97 (high abundance). CONCLUSION: Our study thus demonstrates the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies.

The reliability of lymphoma diagnosis in small tissue samples is heavily influenced by lymphoma subtype.

A specific pathologic diagnosis is important in malignant lymphoma because the diverse disease subtypes require tailored approaches to clinical management. Reliance on small samples obtained with cutting needles has been advocated as a less invasive alternative to using larger, excised samples. Although published studies have demonstrated the safety and apparent sufficiency of this approach in informing clinical care, none have systematically determined the accuracy of pathologic lymphoma subtyping based on very small samples. We used a tissue microarray representing 67 cases of malignant lymphoma and 17 samples of nonneoplastic lymphoid tissue to model lymphoma diagnosis in small samples. Overall, 73.8% of the cases were diagnosed with a level of confidence deemed sufficient for directing clinical management; 85.9% of these diagnoses were accurate. Small cell lymphomas with highly distinctive immunophenotypes, including small lymphocytic, mantle cell, and T-lymphoblastic lymphoma, were recognized most consistently and accurately in the small samples. In contrast, follicular lymphoma and marginal zone lymphoma were especially difficult. Our results indicate that the reliability of lymphoma diagnoses based on small samples is heavily influenced by lymphoma subtype.

Lack of evidence of Epstein-Barr virus infection in patients with Castleman's disease. Molecular genetic analysis.

OBJECTIVE: Epstein-Barr virus (EBV) infection is associated with a diverse group of malignancies and many lymphoproliferative disorders. Castleman's disease (CD) is atypical lymphoproliferative disorder. The role of EBV in the pathogenesis of CD is not clear yet. The objective of this study is to investigate the EBV status in CD. METHODS: We searched medical records for cases of CD at the Toronto General Hospital, Toronto, Canada and King Abdulaziz University Hospital, Jeddah, Saudi Arabia. Twenty cases were found. The presence of EBV was analyzed using polymerase chain reaction. Polymerase chain reaction were performed at the Department of Pathology and Laboratory Medicine, Toronto General Hospital. The study started in 2001 and completed in 2005. RESULTS: The age range was 16-90 years. Seventeen patients manifested the localized form of CD. There were 11 males 9 females. Epstein-Barr virus genome was detected only in 2 cases; both were males and have plasma cell type. One is a localized type and the other is of a multicentric type. One patient revealed clonal rearrangement of the immunoglobulin H. CONCLUSION: The number of cases is small; however it appears that EBV is less likely to play a significant role in the pathogenesis of CD; however, it seems to be associated with clonal progression.

Lack of evidence of Epstein-Barr virus infection in patients with Castleman`s disease. Molecular genetic analysis.

OBJECTIVE: Epstein-Barr virus (EBV) infection is associated with a diverse group of malignancies and many lymphoproliferative disorders. Castleman`s disease (CD) is atypical lymphoproliferative disorder. The role of EBV in the pathogenesis of CD is not clear yet. The objective of this study is to investigate the EBV status in CD. METHODS: We searched medical records for cases of CD at the Toronto General Hospital, Toronto, Canada and King Abdulaziz University Hospital, Jeddah, Saudi Arabia. Twenty cases were found. The presence of EBV was analyzed using polymerase chain reaction. Polymerase chain reaction were performed at the Department of Pathology and Laboratory Medicine, Toronto General Hospital. The study started in 2001 and completed in 2005. RESULTS: The age range was 16-90 years. Seventeen patients manifested the localized form of CD. There were 11 males 9 females. Epstein-Barr virus genome was detected only in 2 cases; both were males and have plasma cell type. One is a localized type and the other is of a multicentric type. One patient revealed clonal rearrangement of the immunoglobulin H. CONCLUSION: The number of cases is small; however it appears that EBV is less likely to play a significant role in the pathogenesis of CD; however, it seems to be associated with clonal progression.

Myeloid leukemia with promyelocytic features in transgenic mice expressing hCG-NuMA-RARalpha.

Acute promyelocytic leukemia (APL) is characterized by the accumulation of abnormal promyelocytes in the bone marrow (BM), and by the presence of a reciprocal chromosomal translocation involving retinoic acid receptor alpha (RARalpha). To date, five RARalpha partner genes have been identified in APL. NuMA-RARalpha was identified in a pediatric case of APL carrying a translocation t(11;17)(q13;q21). Using a construct containing the NuMA-RARalpha fusion gene driven by the human cathepsin G promoter (hCG-NuMA-RARalpha), two transgenic mouse lines were generated. Transgenic mice were observed to have a genetic myeloproliferation (increased granulopoiesis in BM) at an early age, and rapidly developed a myeloproliferative disease-like myeloid leukemia. This leukemia was morphologically and immunophenotypically indistinguishable from human APL, with a penetrance of 100%. The phenotype of transgenic mice was consistent with a blockade of neutrophil differentiation. NuMA-RARalpha is therefore sufficient for disease development in this APL model.

MicroRNA signature obtained from the comparison of aggressive with indolent non-Hodgkin lymphomas: potential prognostic value in mantle-cell lymphoma.

PURPOSE: Mantle-cell lymphoma (MCL) has a variable natural history but is incurable with current therapies. MicroRNAs (miRs) are useful in prognostic assessment of cancer. We determined an miR signature defining aggressiveness in B-cell non-Hodgkin lymphomas (NHL) and assessed whether this signature aids in MCL prognosis. METHODS: We assessed miR expression in a training set of 43 NHL cases. The miR signature was validated in 44 additional cases and examined on a training set of 119 MCL cases from four institutions in Canada. miRs significantly associated with overall survival were examined in an independent cohort of 114 MCL cases to determine association with patient outcome. miR expression was combined with current clinical prognostic factors to develop an enhanced prognostic model in patients with MCL. RESULTS: Fourteen miRs were differentially expressed between aggressive and indolent NHL; 11 of 14 were validated in an independent set of NHL (excluding MCL). miR-127-3p and miR-615-3p were significantly associated with overall survival in the MCL training set. Their expression was validated in an independent MCL patient set. In comparison with Ki-67, expression of these miRs was more significantly associated with overall survival among patients with MCL. miR-127-3p was combined with Ki-67 to create a new prognostic model for MCL. A similar model was created with miR-615-3p and Mantle Cell Lymphoma International Prognostic Index scores. CONCLUSION: Eleven miRs are differentially expressed between aggressive and indolent NHL. Two novel miRs were associated with overall survival in MCL and were combined with clinical prognostic models to generate novel prognostic data for patients with MCL.

Negative images of crystalline immunoglobulin in crystal storing histiocytosis: A potential cytologic mimic of mycobacteria in smears.

Two cases are described of crystal storing histiocytosis (CSH) associated with extranodal marginal zone lymphoma, presenting as lung and subcutaneous masses respectively. Fine-needle aspiration of subcutis and smears prepared from the resected lung masses showed negative images. Cytology slides of both cases were reviewed to identify cytomorphological features for the differential diagnosis between immunoglobulin crystals and mycobacteria. The crystals in CSH consist of straight and needle shaped rods with pointed or angular edges and are more variable in thickness than the uniformly thin mycobacteria. Mycobacteria show a haphazard distribution, whereas crystals are frequently present in parallel arrays. Small lymphoid or plasma cells are identified in the background of CSH, whereas a necrotic and inflammatory background is seen in mycobacteriosis. Additional samples for culture in the case of mycobacteriosis, or flow cytometry and molecular clonality testing in the case of CSH can provide critical data for a definitive diagnosis.

Classifying B-cell non-Hodgkin lymphoma by using MIB-1 proliferative index in fine-needle aspirates.

BACKGROUND: MIB-1 proliferation index (PI) has proven helpful for diagnosis and prognosis in non-Hodgkin lymphomas (NHLs). However, validated cutoff values for use in fine-needle aspiration (FNA) samples are not available. We investigated MIB-1 immunocytochemistry as an ancillary technique for stratifying NHL and attempted to establish PI cutpoints in cytologic samples. METHODS: B-cell NHL FNA cases with available cytospins (CS) MIB-1 immunocytochemistry results were included. Demographic, molecular, immunophenotyping and MIB-1 PI data were collected from cytologic reports. Cases were subtyped according to the current World Health Organization classification and separated into indolent, aggressive, and highly aggressive groups. Statistical analysis was performed with pairwise Wilcoxon rank sum test and linear discriminant analysis to suggest appropriate PI cutpoints. RESULTS: Ninety-one NHL cases were subdivided in 56 (61.5%) indolent, 30 (33%) aggressive, and 5 (5.5%) highly aggressive lymphomas. The 3 groups had significantly different MIB-1 PIs from each other. Cutpoints were established for separating indolent (<38%), aggressive (> or =38% to < or =80.1%) and highly aggressive (>80.1%). The groups were adequately predicted in 76 cases (83.5%) using the cutpoints and 15 cases showed discrepant PIs. CONCLUSIONS: MIB-1 immunohistochemistry on CS can help to stratify B-cell NHL and showed a significant increase in PI with tumor aggressiveness. Six misclassified cases had PIs close to the cutpoints. Discrepant MIB-1 PIs were related to dilution of positive cells by non-neoplastic lymphocytes and to the overlapping continuum of features between diffuse large B-cell lymphoma and Burkitt lymphoma. Validation of our approach in an unrelated, prospective dataset is required.