Claudio Gustavo Stefanoff

Laboratory strategies for efficient handling of paraffin-embedded tissues for molecular detection of clonality in non-hodgkin lymphomas.

We herein present a technical strategy to optimize DNA isolation from paraffin-embedded tissue (PET). This includes the choice of adequate buffers for proteinase K digestion and multiplex PCR amplifications for assessing the appropriateness of DNA extracts for subsequent PCR assays for detecting clonality. We found that the association of proteinase K digestion in nonionic buffer and subsequent extract dilutions accounted for 79% of successful amplifications. A final efficiency of 88% was achieved by additional organic extractions and/or re-extractions. Comparisons were carried out with control DNA extracts from fresh samples to assess the efficiency of each clonality assay. Immunoglobulin CDRIII rearranged region amplification was more efficient for pregerminal center B-cell lymphomas in contrast to CDRII rearrangement detection, which was more effective for germinal and postgerminal lymphomas. T-cell clonality detection by TCR&ggr; PCR was less efficient in PET samples than in fresh tissues showing that DNA integrity is more critical for TCR than for IGH amplification. Two inconclusive cases without phenotypic markers and two other atypical lymphoproliferations masked by reactive T cells were diagnosed as plasmablastic lymphomas and as monoclonal B-proliferations, respectively, due to IGH rearrangements.

Clinical and demographic characteristics of Epstein-Barr virus-associated childhood Burkitt’s lymphoma in Southeastern Brazil: epidemiological insights from an intermediate risk region

This report describes clinical and demographic characteristics of Epstein-Barr virus-associated childhood Burkitt’s lymphoma in Southeastern Brazil. We studied a group of 54 children with Burkitt’s lymphoma from Southeastern Brazil, where epidemiological status of Burkitt’s lymphoma is poorly understood. Epstein-Barr virus association showed an intermediate frequency (~60%) between endemic and sporadic subtypes. Median age was five years. Epstein-Barr virus infection was significantly associated to low age (Epstein-Barr virus+ four years vs. Epstein-Barr virus− eight years). Sex ratio (M:F) was 2:1, with a significantly higher number of males in old age classes. Young age at diagnosis and excess of males at older ages, as well as a causal relationship between low age, epstein-barr virus and Burkitt’s lymphoma risk, may characterize Burkitt’s lymphoma in Brazil.

Hepatosplenic γδ T-cell lymphoma following seven malaria infections

Hepatosplenic γδ T‐cell lymphoma (HSTL) is a clinicopathological entity associated with an immunocompromised status in approximately 25% of patients. Herein is described a case of HSTL in a 53‐year‐old Brazilian man with seven previous malaria infections, initially misdiagnosed as a hyperreactive splenomegaly due to chronic malaria. A characteristic lymphoid infiltrate was observed in spleen, liver and bone marrow sinusoids/sinuses. Neoplastic cells had a CD45RO+, CD2+, CD7+, CD3+, CD5–, CD8+, CD56+, perforin+, FasL‐negative, T‐cell receptor (TCR)αβ‐negative, TCRγδ+ profile. Analyses of γ and δ TCR rearrangements confirmed diagnosis of γδ T‐cell lymphoma by detecting VγI/Vδ1‐Jδ1 clonal rearrangements. Sensitive polymerase chain reaction (PCR) for Plasmodium falciparum, Epstein–Barr virus and herpesvirus‐8 failed to demonstrate infection. The disease progressed to a fatal outcome following cutaneous infiltration and leukemic proliferation. The authors also comment on the association of lymphoma and infection, focusing on PCR diagnosis of TCRγ and δ clonal rearrangements and the presumed pathogenic events leading to HSTL in the context of chronic malaria infection. Initial lymphomagenic stages might not be direct consequences of antigenic stimulation of Vδ1 T‐cells, but might depend on interactions between γδ T and B cells during cooperative or regulatory responses to Plasmodium sp.

Mutations within the 5' region of FAS/CD95 gene in nodal diffuse large B-cell lymphoma

CD95 is a cell-surface receptor that mediates apoptosis. A possible association between CD95 mutations and extranodal diffuse large B-cell lymphomas (DLBCL) has been reported. To further elucidate this question, a mutation analysis within the 5′ region and exon 9 of CD95 was performed in a series of 66 DLBCL patients, by polymerase chain reaction, single-strand conformational polymorphism, and sequencing in all cases. Four mutations, all within the 5′ region, were detected in three cases of primary nodal DLBCL (6.3% of primary DLBCL), probably originated as by-products of the somatic hypermutation process. No CD95 mutations in the two analyzed regions were detected in primary extranodal DLBCL, mediastinal large B-cell lymphoma (MLBCL), and DLBCL arising from indolent low-grade lymphomas. Because of our results, a review of published data with respect to the site of mutations was performed, which suggested a different distribution of mutations in nodal and extranodal DLBCL.

Analysis of biological and technical variability in gene expression assays from formalin-fixed paraffin-embedded classical Hodgkin lymphomas

Formalin-fixed paraffin-embedded (FFPE) tissues are invaluable sources of biological material for research and diagnostic purposes. In this study, we aimed to identify biological and technical variability in RT-qPCR TaqMan® assays performed with FFPE-RNA from lymph nodes of classical Hodgkin lymphoma samples. An ANOVA-nested 6-level design was employed to evaluate BCL2, CASP3, IRF4, LYZ and STAT1 gene expression. The most variable genes were CASP3 (low expression) and LYZ (high expression). Total variability decreased after normalization for all genes, except by LYZ. Genes with moderate and low expression were identified and suffered more the effects of the technical manipulation than high-expression genes. Pre-amplification was shown to introduce significant technical variability, which was partially alleviated by lowering to a half the amount of input RNA. Ct and Cy0 quantification methods, based on cycle-threshold and the kinetic of amplification curves, respectively, were compared. Cy0 method resulted in higher quantification values, leading to the decrease of total variability in CASP3 and LYZ genes. The mean individual noise was 0.45 (0.31 to 0.61 SD), indicating a variation of gene expression over ~1.5 folds from one case to another. We showed that total variability in RT-qPCR from FFPE-RNA is not higher than that reported for fresh complex tissues, and identified gene-, and expression level-sources of biological and technical variability, which can allow better strategies for designing RT-qPCR assays from highly degraded and inhibited samples.

Immunoblastic morphology in diffuse large B-cell lymphoma is associated with a nongerminal center immunophenotypic profile

Diffuse large B cell lymphomas (DLCBL) are a group of lymphomas whose biologic and prognostic diversity has been recently well characterized. There is also morphologic heterogeneity, but the relevance of subclassification remains uncertain. The World Health Organization Classification states that pathologists have the choice to use only the term diffuse large B-cell lymphoma or to use one of the specific morphologic variants. The aim of the present study was to evaluate if there is an association between immunoblastic morphology and the immunophenotypic profile in DLBCL. Two observers reviewed 117 DLBCL cases. Cases of immunoblastic lymphoma and cases of centroblastic polymorphic lymphoma with more than 50% immunoblasts were defined as having immunoblastic morphology. Immunohistochemistry was performed on tissue microarray slides to establish the immunophenotypic profile. Patients with immunoblastic morphology more frequently had a non-GCB phenotype (94%vs 6%). This finding suggests that the morphological subclassification of DLBCL does have biological meaning, in line with recent evidence indicating that the immunoblastic morphology should not be overlooked in lymphoma classification.

Burkitt lymphoma/leukaemia transformed from a precursor B cell: clinical and molecular aspects

Burkitt lymphoma/leukaemia (BL/L) is a heterogeneous disease with respect to epidemiological patterns and cell origin. The occurrence of BL/L with an immature phenotype raises the question whether this phenotype might be a consequence of early B‐cell transformation or, alternatively, a secondary feature of transformed, mature B cells. It also poses important clinical questions regarding diagnosis and therapeutic procedures. Here we describe the case of a 4‐yr‐old child with BL/L and FAB L3 morphology, with phenotypic and genotypic characteristics of a CD10+ precursor B‐cell acute lymphoid leukaemia (ALL) associated with t(8;14)(q24;q32). Molecular analysis showed expression of RAG1 and RAG2 and an unmutated VDJCμ immunoglobulin rearrangement coinciding with a lack of AICDA expression, indicating an immature B‐cell origin. His clinical response suggested that FAB L3 ALL with MYC rearrangement and an aberrant precursor B‐cell phenotype is clinically similar to BL/L. Moreover, short, intensive chemotherapeutic protocols seemed to be beneficial. This case also allowed us to refine the description of cellular and molecular variants of BL/L regarding the cell origin and pathogenesis of this biologically heterogeneous disease.

BURKITT-LIKE LYMPHOMA IN AN INFANT: A CASE REPORT

Childhood non-Hodgkins lymphomas, including Burkitt and Burkitt-like, are rarely diagnosed in infants. A case of B-cell lymphoma in a 13-month-old girl with extensive abdominal disease, ascites, pleural effusion, and tumor lysis syndrome is reported. Phenotypic analysis showed a germinal center B-cell phenotype, and a B-cell clonality was confirmed by polymerase chain reaction. There was no evidence of Epstein-Barr and HIV infection. The case herein reported emphasizes the need for considering the diagnosis of lymphoma even in very young children.

Use of V H, D and J H immunoglobulin gene segments in Brazilian patients with chronic lymphocytic leukaemia (CLL)

Chronic lymphocytic leukaemia (CLL) is a haematological malignancy for which reliable prognostic markers are needed in view of its clinical heterogeneity. In approximately 50% of CLL patients, immunoglobulin (Ig) rearrangements are modified by somatic hypermutation (SHM), a process that represents a reliable prognostic indicator of favourable progression. In this study, we investigated SHM in 37 Brazilian CLL patients and identified the preferential involvement of specific immunoglobulin gene families and segments through PCR-amplified fragments or subcloned fragments. Forty-one rearrangements were observed and 37 of them were functional. A 98% homology cut-off with germinal sequences showed 18 patients (48.7%) with SHM. Unmutated cases showed a poorer clinical outcome. VH3 was the most frequent VH family, followed by VH4. The VH4-39 segment was the most frequently used, mainly in unmutated cases, while the VH3 family was predominant in mutated cases. The D3 and JH4/JH6 families were the most frequently observed.

Pathway-focused gene expression profiles and immunohistochemistry detection identify contrasting association of caspase 3 (CASP3) expression with prognosis in pediatric classical Hodgkin lymphoma

The search for clinically relevant molecular markers in classical Hodgkin lymphoma (cHL) is hampered by the histopathological complexity of the disease, resulting from the admixture of a small number of neoplastic Hodgkin and Reed‐Sternberg (H‐RS) cells with an abundant and heterogeneous microenvironment. In this study, we evaluated gene expression profiles of 11 selected genes previously proposed as a molecular score for adult cHL, aiming to validate its application in the pediatric setting. Assays were performed by RT‐qPCR from formalin‐fixed paraffin‐embedded (FFPE) lymph nodes in 80 patients with cHL. Selected genes were associated with cell cycle (CENPF, CDK1, CCNA2, CCNE2, and HMMR), apoptosis (BCL2, BCL2L1, and CASP3), and monocytes/macrophages (LYZ and STAT1). Despite using controlled preanalytical and analytical strategies, we were not able to validate the 11‐gene score to be applied in pediatric cHL. Principal component analysis (PCA) disclosed 3 components that accounted for 65.7% of the total variability. The second PC included microenvironment and apoptosis genes, from which CASP3 expression was associated with a short time of progression‐free survival, which impact was maintained in the unfavorable risk group, Epstein‐Barr virus‐negative cases, and multivariate analysis (P < .05). Because this is a counterintuitive association, CASP3 active expression was assessed at the protein level in H‐RS cells by double immunohistochemistry. In contrast to the association of mRNA levels with a poor therapeutic response, a high number of cleaved CASP3+ cells were associated with longer progression‐free survival (P = .03) and overall survival (P = .002). Our results demonstrate the feasibility of using FFPE samples as RNA source for molecular prognostication, but argue against the concept of direct and wide applicability of molecular scores in cHL. We reinforce the potential of CASP3 as an interesting target to be explored in adult and pediatric cHL, and alert for its dual biological role in H‐RS cells and tumor microenvironment.