Grade I, II and III Follicular Lymphomas Express Ig VH Genes with Different Patterns of Somatic Mutation.

Follicular lymphoma (FL) is an indolent, B-cell, non-Hodgkin's lymphoma with varying cytological appearance and clinical behavior. The genetic hallmark of FL is the t(14;18) translocation, and as a germinal center derived entity it is also characterized by somatic hypermutation of the immunoglobulin heavy chain (IgH) gene. In an attempt to correlate this molecular signature with the cytological grading of FL, we have analyzed the IgH variable (IgVH), regions in all cytological grades of FL. Four FL cases showing t(14;18) translocation were classified into grade I-III categories according to the current WHO guidelines. The IgVH gene segments were PCR-amplified, sequenced, and compared to their respective germline IgVH sequences. The neoplastic cells of grade I and II FLs revealed clonally related, but highly divergent IgVH gene sequences indicating the ongoing nature of somatic hypermutation. Grade III FL also showed extensive presence of somatic hypermutation, but these mutations were not associated with intraclonal divergence. Thus, these results suggest that grade I-II and grade III FL may represent different biological entities. The presence of ongoing somatic hypermutation of IgVH sequences in grade I and II FLs is compatible with direct follicular origin of these tumor cells, contrasting the homogenous, stable clones of grade III FL resembling a post-follicular stage of B-cell development. Our findings demonstrate that contrary to the three tiered cytological grading, molecular features of IgH genes classify FL into two distinct subcategories. These studies also suggest that with progression FL gains post-follicular-like molecular features and becomes independent of the germinal center microenvironment.

Low CD23 expression correlates with high CD38 expression and the presence of trisomy 12 in CLL.

Chronic lymphocytic leukemia (CLL) is characterized by a neoplastic B-cell population coexpressing CD5 and CD23; however, the expression of CD23 is variable. In human, two isotypes of CD23 have been identified and related to different functions. The aim of our study was to investigate the relative expression of the two CD23 isotypes in CLL and find possible correlation with other prognostic factors. The expression of CD23 isotypes was analyzed in 54 cases of CLL by polymerase chain reaction (PCR) and quantitative real-time PCR. The immunophenotype of CLL cells was characterized by flow cytometry. We demonstrated a higher CD23a than CD23b expression of CLL cells. Our results also revealed two subsets of CLL cases with a distinct CD23 isotype expression pattern. Thirty-two percent of the cases (group CLL1) showed both low mRNA level of CD23 isotypes and high protein levels of CD20 and CD38 in contrast to group CLL2 with high CD23 mRNA levels. By correlating these results to the presence of prognostic factors determined by fluorescence in situ hybridization, we found that the majority of the cases of group CLL1 (14/17) carried trisomy 12. In summary, our results confirm a high CD23a/CD23b ratio of the CLL cells and demonstrate that in a subset of CLL cases, low CD23 expression together with high CD20 and CD38 expressions may serve as a surrogate for trisomy 12. Copyright © 2015 John Wiley & Sons, Ltd.

Heparin can liberate high molecular weight DNA from secondary necrotic cells.

The borderline between necrosis and apoptosis is indistinct, but that between types of cell death is important because necrosis may lead to local inflammation, whereas apoptosis usually does not. In certain autoimmune disorders, inhibition of cell death is crucial, since macromolecules released from the dead cells may accelerate the autoimmune processes. We have used various cell death inhibitors to block cell death induced by 4HPR [N-(4-hydroxyphenil)-retinamide] the BL41 and U937 cell lines. VD-FMK, a general caspase inhibitor, inhibited DNA fragmentation induced by 4HPR, but not PI (propidium iodide) uptake and necrosis. Interestingly heparin, a serine-protease inhibitor, lowered the PI fluorescence of the dead cell population and increased the sub-G1 population as measured by flow cytometry. Regarding these changes, we found that heparin failed to increase DNA fragmentation, but merely liberated high molecular mass DNA fragments from dead cells. The exact mechanism is unclear, but heparin during secondary necrosis might enter the cells, bind RNPs (ribonucleoproteins), and pull them out with the attached DNA, where they would be sensitive to enzymatic degradation. Thus, the results suggest that heparin treatment helps in the clearance of cell debris and decreases the immunogenity of secondary necrotic cells.

ROR1 expression is not a unique marker of CLL.

Recent studies have identified receptor tyrosine kinase-like orphan receptor 1 (ROR1) on the surface of chronic lymphoid leukaemia (CLL) cells. In order to determine whether ROR1 expression is a suitable surrogate marker for the diagnosis of CLL we analysed the mRNA level of ROR1 in different types of non-Hodgkin lymphomas (NHL), and detected elevated levels of ROR1 compared to control peripheral mononuclear cells in several entities (CLL ≥ mantle cell lymphoma (MCL) > marginal zone lymphoma (MZL) >> diffuse large B-cell lymphoma > follicular lymphoma). ROR1 protein was expressed intensely on the cell surface of lymphoma cells with leukaemic blood count detected by three colour immunofluorescence. Our results indicate that ROR1 expression is not limited to CLL cases, but it is more prevalent in NHLs, mainly in MCL where it is expressed intensely and MZL where it is expressed moderately, suggesting a general role of ROR1 in lymphoma genesis and/or maintenance. Copyright © 2010 John Wiley & Sons, Ltd.

Expression of the follicular lymphoma variant translocation 1 gene in diffuse large B-cell lymphoma correlates with subtype and clinical outcome.

Sphingolipids serve an important role as effector molecules in signaling pathways bearing on apoptosis and cell survival. The balance between proapoptotic ceramide and prosurvival sphingosine-1-phosphate, sometimes termed the "sphingolipid rheostat," has received particular attention. Less well studied is the role of the follicular lymphoma variant translocation 1 (FVT1) gene, which was identified through its involvement in an atypical follicular lymphoma translocation and which encodes an enzyme in the synthetic pathway of ceramide. We investigated the expression of FVT1 in a variety of B-cell non-Hodgkin lymphomas and found that FVT1 is significantly underexpressed by germinal center-type diffuse large B-cell lymphoma (DLBCL) when compared with non-germinal center-type DLBCL, follicular lymphoma, and normal tonsil control samples. Increased expression of FVT1 correlated with decreased survival, suggesting that changes in the expression of FVT1 and in the concentrations of bioactive sphingolipids may be important in the pathogenesis and treatment of some types of DLBCL.

Somatic hypermutation of IGVH genes and aberrant somatic hypermutation in follicular lymphoma without BCL-2 gene rearrangement and expression.

BACKGROUND: Follicular lymphoma is characterized by the t(14;18) translocation resulting in constitutive expression of BCL-2 protein; however approximately 10-15% of follicular lymphomas do not express BCL-2 protein, and a small fraction of these cases does not exhibit translocation of the BCL-2 gene either. It is highly debated whether cases of follicular lymphoma without BCL-2 gene rearrangement and expression represent a separate lymphoma entity with distinct biological characteristics, different from the BCL-2-positive cases. DESIGN AND METHODS: To further characterize follicular lymphomas without BCL-2 gene rearrangement and expression, we analyzed and compared the mutational status of IGVH genes as well as other genes (C-MYC, PAX-5 and RHOH) frequently involved in the specific type of genomic instability called aberrant somatic hypermutation in 11 cases of BCL-2-negative and 7 cases of BCL-2-positive follicular lymphomas. We also determined the levels of expression of activation-induced cytidine deaminase in these cases. RESULTS: The analyzed cases were grade 2 and grade 3A follicular lymphomas. Our findings demonstrate that follicular lymphomas without BCL-2 gene rearrangement and expression are associated with ongoing somatic hypermutation of the IGVH genes, low activity of aberrant somatic hypermutation and elevated activation-induced cytidine deaminase expression. These results were in concordance with the results found in the cases of BCL-2-positive follicular lymphoma. CONCLUSIONS: Although, BCL-2 protein overexpression is considered to be a critical pathogenic event in the development of follicular lymphoma, our findings suggest that follicular lymphomas with the same morphology, immunophenotype, mutational pattern and activation-induced cytidine deaminase expression may develop without the involvement of BCL-2 gene. The present data support the hypothesis that BCL-2-positive and BCL-2-negative follicular lymphomas (grades 1-3A) represent a homogenous group with different initial but several common additional molecular pathways.

DNA and RNA isolated from tissues processed by microwave-accelerated apparatus MFX-800-3 are suitable for subsequent PCR and Q-RT-PCR amplification.

Over the past decade, methods of molecular biology have appeared in diagnostic pathology and are routinely applied on formalin-fixed, paraffin-embedded histological samples, processed via conventional embedding methods. Due to its reagent- and cost-effectiveness, embedding techniques that utilize microwave acceleration in one or more steps of histoprocessing are increasingly used by numerous laboratories. The demand arises that tissues processed this way should also be suitable for the requirements of molecular pathology. In this study, both conventionally embedded and MFX-800-3 machine-processed tissue samples from the same source were used for isolation of DNA and RNA and for performing PCR and real-time PCR. PCR amplification of the beta-globin gene, as well as the real-time PCR amplification of the ABL mRNA was successful in all cases. Our conclusion is that samples processed by the vacuum assisted automatic microwave histoprocessor MFX-800-3 are perfectly applicable for DNA and RNA isolation and provide appropriate templates for further PCR and realtime PCR studies.

Activation of peroxisome proliferator-activated receptor gamma contributes to the survival of T lymphoma cells by affecting cellular metabolism.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a metabolic regulator involved in maintaining glucose and fatty acid homeostasis. Besides its metabolic functions, the receptor has also been implicated in tumorigenesis. Ligands of PPARgamma induce apoptosis in several types of tumor cells, leading to the proposal that these ligands may be used as antineoplastic agents. However, apoptosis induction requires high doses of ligands, suggesting the effect may not be receptor-dependent. In this report, we show that PPARgamma is expressed in human primary T-cell lymphoma tissues and activation of PPARgamma with low doses of ligands protects lymphoma cells from serum starvation-induced apoptosis. The prosurvival effect of PPARgamma was linked to its actions on cellular metabolic activities. In serum-deprived cells, PPARgamma attenuated the decline in ATP, reduced mitochondrial hyperpolarization, and limited the amount of reactive oxygen species (ROS) in favor of cell survival. Moreover, PPARgamma regulated ROS through coordinated transcriptional control of a set of proteins and enzymes involved in ROS metabolism. Our study identified cell survival promotion as a novel activity of PPARgamma. These findings highlight the need for further investigation into the role of PPARgamma in cancer before widespread use of its agonists as anticancer therapeutics.

Molecular monitoring of chronic myelogenous leukemia: identification of the most suitable internal control gene for real-time quantification of BCR-ABL transcripts.

Monitoring breakpoint cluster region-Abelson kinase (BCR-ABL) levels in patients treated for chronic myelogenous leukemia (CML) has become an integral part of patient management. Real-time reverse transcriptase-polymerase chain reaction is the method of choice for this purpose because of its high analytical sensitivity and reproducibility. Given the variation of RNA quality and quantity in clinical specimens, accurate quantitative assessment of BCR-ABL depends on normalization of the BCR-ABL signal to an appropriate internal reference. However, the controls used by different laboratories vary, and there is no clear consensus on an ideal reference due to limited investigations. In this study, we compared nine commonly used control genes for three criteria: mRNA abundance, levels in CML and non-CML cells, and their degradation kinetics in comparison with BCR-ABL. We found that beta-glucuronidase (GUSB) is the most suitable among the nine genes tested. Although ABL is most widely used, our data suggest that the amount of ABL is different in CML and non-CML cells. Moreover, ABL levels are regulated by cellular stress. These findings have a direct impact on current clinical laboratory practice and patient care because the use of a proper control gene affects the reported levels of BCR-ABL transcripts used for patient management decisions.

[Novel method in diagnosis of chronic myeloproliferative disorders--detection of JAK2 mutation]. / Uj lehetoség a krónikus myeloproliferativ betegségek diagnosztikájában--a JAK2 mutaci6ókimutatása.

Chronic myeloproliferative disorders are clonal hematopoietic stem cell disorders characterized by proliferation of one or more myeloid cell lineages in the bone marrow. The WHO classification describes six major groups of chronic myeloproliferative disorders, as follows: chronic myeloid leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, polycythemia vera, essential thrombocythemia and chronic idiopathic myelofibrosis. The diagnosis of chronic myeloid leukemia and certain types of chronic eosinophilic leukemia are based on the detection of fusion genes (in chronic myeloid leukemia the BCR/ABL fusion gene, and in chronic eosinophilic leukemia the FIP1L1-PDGFRalpha gene). On the other hand molecular markers for polycythemia vera, essential thrombocythemia and chronic idiopathic myelofibrosis were lacking, making it difficult to identify these disorders clearly. The authors investigated the incidence of the newly identified somatic point mutation V617F of the Janus-2 tyrosine kinase in patients with polycythemia vera, essential thrombocythemia and myelofibrosis. Janus-2 kinase is a cytoplasmic, non-receptor protein-tyrosine kinase with a key role in signal transduction from multiple hematopoietic growth factor receptors. The mutant protein is constitutively phosphorylated and is able to activate its downstream signaling pathways in the absence of any cytokine, thereby contributing to the pathogenesis of chronic myeloproliferative disorders. The authors investigated DNA samples from 132 patients with chronic myeloproliferative disorders. The V617F mutation was detected by allele-specific polymerase chain reaction, and the patients were genotyped by a DNA tetra-primer amplification refractory mutation system assay. Approximately 73% of polycythemia vera, 60% of essential thrombocythemia and 67% of myelofibrosis showed the JAK2 V617F mutation. Using the amplification refractory mutation system assay, the frequency of homozygotes was 17.5% in polycythemia vera, 5.4% in essential thrombocythemia and 0% in myelofibrosis. The authors established an effective polymerase chain reaction based method for the identification of JAK2 mutation in the routine oncohematologic diagnostics.

Mutational analysis of IgVH and BCL-6 genes suggests thymic B-cells origin of mediastinal (thymic) B-cell lymphoma.

Mediastinal (thymic) large B-cell lymphoma (MBL) has been defined as a subtype of diffuse large B-cell lymphoma (DLBL) arising in the mediastinum with characteristic clinicopathological features. It has been postulated that MBL arise from non-circulating thymic B-cells and represent a distinct lymphoma entity, however, the histogenesis of the disease is not yet fully understood. In order to clarify the histogenetic derivation of MBL and to determine the relationship of MBL to thymic B-cells we have analyzed the nucleic acid sequences of immunoglobulin (Ig) heavy chain variable region (VH) and 5' noncoding region of BCL-6 genes in normal thymic B-cells and six cases of MBL. Thymic B-cells and tumor cells of MBLs displayed hypermutated VH and/or BCL-6 genes but intraclonal divergence did not associate with these mutations. Since somatic mutations of the IgVH and BCL-6 genes are histogenetic markers of B-cell transit through the germinal centre (GC), these results suggest that both thymic B-cells and MBLs derived from GC or an equivalent environment where B-cells underwent somatic hypermutation. The similar pattern of mutations of IgVH and BCL-6 genes found in thymic B-cells and MBLs further supports the theory that MBLs originate from thymic B-cells.

Upregulation of p55 and p75 receptors mediating TNF-alpha transport across the injured blood-spinal cord barrier.

Tumor necrosis factor (TNF-alpha) is involved in the inflammation and tissue regeneration occurring after spinal cord injury (SCI). This study tests the specific role of p55 and p75 receptors in mediating the transport of TNF-alpha across the blood-spinal cord barrier (BSCB) after SCI by compression. Transcytosis of 125I-TNF-alpha across a monolayer of the cerebral endothelial cells that compose the blood-brain barrier was significantly reduced in the absence of functional p55 and p75 receptors. At 3 d after SCI, double receptor knockout mice had a significantly reduced increase in TNF-alpha uptake from blood to injured lumbar spinal cord as compared with their inbred controls, despite the similar extent of BSCB disruption (measured by 99mTc-albumin). The p75 single receptor knockout mice had a reduced increase in 125I-TNF-alpha uptake, whereas the p55 receptor knockout mice had no significant increase of 125I-TNF-alpha uptake after SCI, suggesting that the p55 receptor plays a major role. Hence, the increased uptake of TNF-alpha 3 d after SCI is not explained by nonspecific barrier disruption but by receptor-mediated upregulation of transport. Quantitative RT-PCR analysis further showed that upregulation of TNF-alpha transport was related to increased expression of mRNA for p55 and p75 receptors. The increase of p55 receptor expression was more robust and seen between 12 h and 1 wk after SCI, whereas the increase of p75 receptor expression occurred later and involved fewer regions. Thus, the differential upregulation of p55 and p75 receptors indicates that permeation of TNF-alpha across the injured BSCB remains a regulated process. Knowledge of receptor-mediated regulation could facilitate effective therapeutic manipulation of BSCB permeation of vascular cytokines important to CNS regeneration.

Selective increase in TNF alpha permeation across the blood-spinal cord barrier after SCI.

We generated a novel mouse model of spinal cord injury (SCI) by hemisection of the right L1 lumbar spinal cord, measured the permeability of the blood-spinal cord barrier (BSCB), and tested the hypothesis that tumor necrosis factor alpha (TNF alpha) penetrates the injured BSCB by an enhanced transport system. SCI produced stereotypical sensorimotor deficits resembling the classically described Brown-Seqúard syndrome. Disruption of the BSCB was reflected by increased spinal cord uptake of radiolabeled albumin from blood; this was transient (immediately after SCI) and confined to the lumbar spinal cord. By contrast, specific increase in the entry of TNF alpha was detected in brain, cervical, thoracic, and lumbar spinal cord at 1 week after SCI, in addition to its immediate and transient increase consistent with barrier disruption. Lack of a second peak of increase in the entry of IL1 beta further supported the specificity of the TNF alpha response. Moreover, enhanced uptake of radiolabeled TNF alpha was suppressed by excess non-radiolabeled TNF alpha, indicating competition of entry via the known transport system for TNF alpha. Therefore, upregulation of the transport system after SCI probably mediates the increased permeation of TNF alpha across the BSCB. Enhanced entry of TNF alpha at 1 week after SCI was concurrent with sensorimotor and gait improvement of the mouse. We conclude that SCI by lumbar hemisection activates the transport system for TNF alpha at the BBB and suggest that selective permeation of TNF alpha may facilitate functional recovery.