ALBERT

Description: Introduction Amplification of chromosome 13q31 is a frequent occurrence in lymphoma and solid tumors. The C13orf25 gene at 13q31.3 is the primary miRNA transcript for seven miRNAs. This specific cluster of miRNAs is sequentially related to the homologous miR-106a-92 cluster on chromosome X and the miR-106b-25 cluster on chromosome 7. The miR17-92 cluster has been shown to be over expressed in various non-Hodgkin Lymphoma (NHL). As a specific group of miRNAs which are derived from the same primary miRNA transcript, each miRNA in the cluster may be expected to show a similar expression level. However expression patterns vary markedly in different cancers. Aim of study To compare the expression pattern of the C13orf25 gene and the miRNAs in normal and malignant B-cells. Methods and Materials 51 cases of Ann Abour stage I and II primary diffuse large B-cell lymphoma (DLBCL) were collected together with 29 cases of B-CLL. All samples were examined for expression of miRNA miR-18a, -19, -20a, 17-3p, -17-5p and -92 and the C13orf 25 gene. Expression levels of the mature miRNAs were determined by qRT-PCR using Taqman miRNA assays. The level of C13orf25 was also determined by qRT-PCR using random primers and a Sybergreen probe. Results were compared by using 2−ΔCt and 2−ΔΔCt. Normal B-cell subpopulations were isolated from fresh tonsils obtained during routine pediatric tonsillectomies. B-cell subsets were stained accordingly and sorted by FACS. Results Comparison of the miRNA pattern (2−ΔCt) revealed that DLBCL have the highest expression level of miR-19b and that B-CLL shows a relative high expression level of miR-92. Normal naïve, germinal center and memory B-cells all show a similar expression pattern with miR-92 having the highest expression level. Remarkably a low expression level of miR-19b is seen in all three B-cell populations in contrast to DLBCL. Comparing the relative expression levels (2−ΔΔCt) of both NHL for each individual miRNA shows that B-CLL has a significantly higher level of expression for miR-19a. In DLBCL miR-17-5p, miR-18a and miR-20a have the highest expression levels. Currently 20 Mantle cell lymphoma are also being analyzed for the C13orf25 miRNAs cluster. Conclusion Our results show that both of the NHL B-cell malignancies and the B-cell subsets have different expression patterns of the individual levels of the 7 miRNAs contained in the same C13orf25 cluster. The relative expression levels of certain miRNAs from the same primary transcript are different in various NHL. Although there are no marked differences in the normal B-cell subsets for the C13orf25 cluster, further profiling revealed various different expression levels of other miRNAs. These findings may point towards a difference in processing efficiency or stability of miRNAs in the C13orf25 cluster leading to differences in pathogenesis specific for each malignancy even in cells of similar origin and differentiation stage.

Description: Using DNA fiber fluorescence in-situ hybridization (FISH) and 3-color interphase FISH, 2 cases of follicular lymphoma were identified in which the BCL2 gene was excised from 18q21 and inserted into the immunoglobulin heavy chain (IGH) locus at 14q32. Both the insertion breakpoint at 14q32 and the deletion breakpoint at 18q21 were cloned using inverse polymerase chain reaction. Sequence analysis showed that the JH sequences were juxtaposed to the 5′-side of BCL2, and the DH sequences were juxtaposed to the 3′-side of BCL2. There were breakpoints at both the JH and DH recombination signal sequences, and N-nucleotides were present at all breakpoint junctions. At theBCL2 locus, the 3′-breakpoints in both cases were localized at exactly the same nucleotide position, 6.2 kilobase downstream of the major breakpoint region, directly adjacent to a complete cryptic recombination signal sequence (RSS) consisting of a heptamer, a nonamer, and a 23–base pair (bp) spacer. The BCL25′-breakpoints were approximately 600 bp upstream of the gene, within the CA repeats. Although less evident than for the BCL23′-breakpoints, cryptic RSSs were also identified at these breakpoints, with a 12-bp spacer. On the basis of structural characteristics of these rearrangements, a model is proposed in which the BCL2 gene is deleted from its locus by recombination activation gene-1/-2 (RAG-1/-2)–mediated excision. The gene is subsequently inserted into the recombiningIGH locus, a process involving the formation of hybrid joints between the IGH coding ends and theBCL2 signal ends.

Description: Immunoglobulin class switching usually involves deletion of part of the immunoglobulin CH region. By DNA fiber fluorescence in situ hybridization (FISH) with a barcode of probes covering the DH, JH, and CH genes, the configuration of the entire CH region can be visualized on single DNA molecules. Using this technique, we have studied class switching in three types of B-cell neoplasia, mantle-cell lymphoma (MCL), follicular lymphoma (FL) and hairy cell leukemia (HCL), representing B cells in, respectively, pregerminal center, germinal center, and postgerminal center stages of development. In MCL and FL, simultaneous detection of the t(11;14) and t(14;18) breakpoint with probes for the BCL-1 and BCL-2 loci, respectively, allowed differentiation between productive and nonproductive alleles. In none of 10 MCL cases was class switching detected. In 21 HCL, all nonimmunoglobulin M (IgM) cases had class-switch deletion consistent with the expressed isotype on at least one allele. In FL, however, a peculiar pattern of CH rearrangement was observed. In IgM expressing FL, the translocated alleles had switched in 11 of 13 cases, and the nontranslocated allele showed complex rearrangements downstream from the Cμ-Cδ genes in 9 of 13 cases. These downstream rearrangements may reflect tumor-specific deregulation of the class-switch machinery. All seven immunoglobulin G (IgG) expressing FL showed class switching on both alleles. Fiber FISH analysis also showed several polymorphisms. The most frequent one, present on 38% of all analyzed alleles, consisted of an extra Cγ gene or pseudogene in the 3′ cluster. © 1998 by The American Society of Hematology.

Description: A prompt distinction of Burkitt lymphoma (BL) versus diffuse large B cell lymphoma (DLBCL) has important clinical implications. We analyzed 74 adult grey zone lymphomas (BL/DLBCL) and 10 reference pediatric BL using immunohistochemistry for Ki-67, CD10, bcl2 and bcl6, and Fluorescence In Situ Hybridization (FISH) for MYC, BCL2 and BCL6 breakpoints. Four hematopathologists reached a final (consensus) diagnosis independently in 80% of the reference BL, 24% of the test BL and 69% of the DLBCL. A MYC breakpoint was detected in all reference BL, 95% of the test BL and 35% of the DLBCL. BCL2 and BCL6 breakpoints were infrequent in BL (5 and 6%) and DLBCL (13% and 16%). Three BL and 5 DLBCL contained double breakpoints. A phenotypic shift to BL in the DLBCL group was indicated by the expression of CD10 and bcl6 in 67% and 91% of the cases, respectively. One third of all lymphomas showed a classical genotype and expression pattern of BL (MYC breakpoint+, Ki-67〉90%, CD10+, bcl6+, bcl2-) but only 63% of these cases were classified as BL. Our data indicate that a subgroup of DLBCL shares markers with BL, which is probably due to an overlapping histogenesis of both tumors. Value of immunohistochemistry and additional FISH for BCL2 and BCL6 breakpoints to reach the consensus diagnosis in 38 MYC breakpoint carrying cases of adult grey zone BL / DLBCL lymphomas BCL2 and BCL6 breakpoint no restriction not present no restriction not present 38 / 74 cases carried a MYC/8q24 breakpoint phenotype no restriction no restriction Ki67〉90%, CD10+, bcl6+, bcl2- Ki67〉90%, CD10+, bcl6+, bcl2- N 38 30 24 22 Burkitt (%) 20 (53) 17 (57) 15 (63) 15 (68) DLBCL (%) 18 (47) 13 (43) 9 (37) 7 (32)

Description: In B-cell lymphomas, loss of human leukocyte antigen (HLA) class I and II molecules might contribute to immune escape from CD8+ and CD4+ cytotoxic T cells, especially because B cells can present their own idiotype. Loss of HLA expression and the possible underlying genomic alterations were studied in 28 testicular, 11 central nervous system, and 21 nodal diffuse large B-cell lymphomas (DLCLs), the first two sites are considered as immune-privileged sites. The analysis included immunohistochemistry, loss of heterozygosity analysis, and fluorescent in situ hybridization (FISH) on interphase cells and isolated DNA fibers. Total loss of HLA-A expression was found in 60% of the extranodal cases and in 10% of the nodal cases (P 

Description: Mantle cell lymphoma (MCL) is a prime example of a well-defined entity based on morphology, phenotype, genetics and also clinical features. Although, most patients have an adverse clinical course, some have a better survival than others. The most consistently reported adverse histopathological prognostic parameter is a high mitotic rate. Recently, it has been shown that hypermutation in the immunoglobulin heavy chain gene occurs in a subset of mantle cell lymphomas. It is, however, unclear, how hypermutation needs to be defined and whether the mutational status is stable over time within a given case. Also it is not clearwhether hypermutation might be influenced by therapy and how it is related to other relevant biological features of MCL, like genetic imbalances, breakpoint of the cyclin D1 gene, and proliferative and apoptotic rate. In the present study we analyzed a series of typical MCL with respect to mutational status, and compared the results with clinicopathological and genetic data to determine whether the presence of mutation does indicate a sub entity with clinical or pathological relevance. For this study 28 specimens from 24 patients were selected and stained for cyclin D1 and CD5 and were reviewed by four experienced pathologists at a multiheaded microscope. Morphological features were assessed, mitotic figures were counted, clonal IGH-VJ gene rearrangements of the MCL were identified according to the Biomed-2-protocol. All, but one case, were clonal by PCR. The negative case was excluded from further molecular studies. Mutation in the VH gene segment was absent in 7 cases, another 6 cases carried 0.5% mutation frequencies; in 10 cases more than 1.5% mutation frequencies were present, in 6 of these more than 2%. We found a preferential usage of VH3-21 (26%) and VH4-34 (17%) in tumor cells of both, mutated and unmutated cases. Of two cases from our series in which sequential biopsies were available tumor cells harbored mutations in the VH genes (1.5% mutation frequency and 2.2% mutation frequency, respectively), but there was no change of mutation status over time, the duration being as long as 7 years in one case. In our study there was a statistically significant difference between MCL with a Ki-67-positivity =35% in regard to a longer survival time in the former whereas mutation status of both groups did not correlate with Ki-67-positivity. Further, no significant correlations were found between mutation status and the other morphologic and genetic features analyzed. In conclusion, our results provide additional evidence that mutation status does not permit definition of sub entities in MCL that are associated invariably with a certain prognosis. Mutation status in MCL is better interpreted as a feature within the spectrum of disease that seems to have little clinical or pathological relevance. Margit Schraders and Sabine Oeschker contributed equally to this work.

Description: Osseous involvement is one the predominant features of patients with multiple myeloma (MM). Whole body X-ray (WBX) is the standard method for detecting skeleton abnormalities but has limited value in relapsing disease since it cannot make a distinction between old and new active skeleton lesions. Therefore changes in metabolic activity might be a more useful method to detect new skeleton lesions in MM. This can be assessed by [F-18] fluorodeoxyglucose positron emission tomography (FDG-PET) imaging. Consequently WBX and FDG-PET was studied in relapsing MM prior to treatment. In addition immunohistochemical staining on bone marrow biopsies was performed for glucose transport protein (GLUT) 3 expression, micro vessel density (MVD), and vascular endothelial growth factor (VEGF) to demonstrate whether these parameters might be predictive for the in vivo scanning results. This study included 44 patients (median age 62 yr (range 48-78)) with a median of 3.5 (range 2-12) relapses. Thus far 86% of the patients were treated with autologous stem cell transplantation in combination with bortezomib (78%) or lenalidomide (65%). New lesions on FDG-PET were more frequently demonstrated than new lesions on WBX (p=0.00001). In 78% of the patients FDG-PET demonstrated a median of 4 lesions (range 0-22) per patient with a median standard uptake value (SUV) max of 5.7 (range 0-24). In 42% of the patients new X-ray lesions (median 0 (range 0-7)) were demonstrated and these were concordant with the results of FDG-PET. In 5 % of the patients new X-ray lesions were observed without FDG-PET lesions. For the relapsing patients treatment consisted of dexamethasone in combination with bortezomib (65%) or lenalidomide (50%). CR was obtained in 27% of the patients, PR in 39% and progressive disease in 22%. Average time to next treatment (TTNT) was 11.7 months (2-43). However FDG-PET positivity was not predictive for PFS (p =0.55) and OS (p=0.40) neither if PET positivity was defined as more than 3 lesions nor if the cut off value of SUV max 〉 4.2 was applied. Immunohistochemical staining on bone marrow biopsies (n=20) with CD34 demonstrated a significant increased MVD of 39 ± 54 (mean ± SD, normal bone marrow MVD 3.5 ± 2.9), in particular when clusters of plasma cells were present. These enhanced MVD was associated with a significant increased expression of VEGF and GLUT-3. However MVD (p = 0.16), VEGF (p = 0.70) or GLUT-3 (p =0.70) expression were not predictive for having a positive or negative FDG-PET. In summary, our results demonstrate that FDG-PET is more informative than WBX for detecting skeleton lesions in relapsing MM patients. However, and in contrast to what was previously shown in patients with newly diagnosed MM, FDG-PET it is not predictive for treatment outcome. This might be due to heterogeneity in studied patients and treatments. Disclosures: No relevant conflicts of interest to declare.

Description: Chromosomal translocations involving t(14;18)(q32;q21) and the chromosome 3q27 region are common in B-cell non-Hodgkin lymphoma of germinal center cell origin. Grade 3B follicular lymphoma (FL), consisting almost exclusively of centroblasts, is a distinct subgroup of follicular lymphomas that has more in common clinically with the aggressive diffuse large B-cell lymphomas than with their indolent FL grade 1 and 2 counterparts. We studied the cytogenetic and molecular genetic aberrations by classic cytogenetics, polymerase chain reaction, Southern blot hybridization, and fluorescence in situ hybridization, with special emphasis on t(14;18), affecting bcl-2, and 3q27 rearrangement, affecting bcl-6, in 32 cases of FL grade 3B. Three distinctive subgroups were identified based upon the existence of breakpoint 3q27, a translocation t(14;18), or the absence of both. Group I involved a t(14;18) and no 3q27 aberrations (n = 13); group II was without a t(14;18) and without 3q27 aberrations (n = 9), but had other cytogenetic aberrations; and group III was without a t(14;18) but with aberrations involving 3q27 (n = 10). None of the FL grade 3B cases harbored both a t(14;18) and 3q27 aberration. These results, in particular the finding of a mutual exclusiveness of bcl-2 and bcl-6 rearrangement, indicate at least 3 different pathways of oncogenesis in FL grade 3B. FL grade 3B with bcl-2 rearrangement probably is part of the same entity as the other follicular lymphomas (1, 2, 3A), whereas the cases with 3q27 abnormalities or other unrelated translocations are more closely related to the majority of diffuse large-cell lymphomas of germinal center cell origin.

Description: In the present study the clinicopathologic and immunophenotypic features of 82 patients with a CD30– peripheral T-cell lymphoma, unspecified, presenting in the skin were evaluated. The purpose of this study was to find out whether subdivision of these lymphomas on the basis of cell size, phenotype, or presentation with only skin lesions is clinically relevant. The study group included 46 primary cutaneous CD30– large cell lymphomas and 17 small/medium-sized T-cell lymphomas as well as 17 peripheral T-cell lymphomas with both skin and extracutaneous disease at the time of diagnosis. Patients with primary cutaneous small- or medium-sized T-cell lymphomas had a significantly better prognosis (5-year-overall survival, 45%) than patients with primary cutaneous CD30– large T-cell lymphomas (12%) and patients presenting with concurrent extracutaneous disease (12%). The favorable prognosis in this group with primary cutaneous small- or medium-sized T-cell lymphomas was particularly found in patients presenting with localized skin lesions expressing a CD3+CD4+CD8– phenotype. In the primary cutaneous T-cell lymphoma (CTCL) group and in the concurrent group, neither extent of skin lesions nor phenotype had any effect on survival. Our results indicate that peripheral T-cell lymphomas, unspecified, presenting in the skin have an unfavorable prognosis, irrespective of the presence or absence of extracutaneous disease at the time of diagnosis, cell size, and expression of a CD4+ or CD8+ phenotype. The only exception was a group of primary cutaneous small- or medium-sized pleomorphic CTCLs with a CD3+CD4+CD8– phenotype and presenting with localized skin lesions.

Description: In many B-cell lymphomas, chromosomal translocations are biologic and diagnostic hallmarks of disease. An intriguing subset is formed by the so-called double- hit (DH) lymphomas that are defined by a chromosomal breakpoint affecting the MYC/8q24 locus in combination with another recurrent breakpoint, mainly a t(14;18)(q32;q21) involving BCL2. Recently, these lymphomas have received increased attention, which contributed to the introduction of a novel category of lymphomas in the 2008 WHO classification, “B cell lymphoma unclassifiable with features intermediate between DLBCL and BL.” In this review we explore the existing literature for the most recurrent types of DH B-cell lymphomas and the involved genes with their functions, as well as their pathology and clinical aspects including therapy and prognosis. The incidence of aggressive B-cell lymphomas other than Burkitt lymphoma with a MYC breakpoint and in particular a double hit is difficult to assess, because screening by methods like FISH has not been applied on large, unselected series, and the published cytogenetic data may be biased to specific categories of lymphomas. DH lymphomas have been classified heterogeneously but mostly as DLBCL, the majority having a germinal center phenotype and expression of BCL2. Patients with DH lymphomas often present with poor prognostic parameters, including elevated LDH, bone marrow and CNS involvement, and a high IPI score. All studies on larger series of patients suggest a poor prognosis, also if treated with RCHOP or high-intensity treatment modalities. Importantly, this poor outcome cannot be accounted for by the mere presence of a MYC/8q24 breakpoint. Likely, the combination of MYC and BCL2 expression and/or a related high genomic complexity are more important. Compared to these DH lymphomas, BCL6+/MYC+ DH lymphomas are far less common, and in fact most of these cases represent BCL2+/BCL6+/MYC+ triple-hit lymphomas with involvement of BCL2 as well. CCND1+/MYC+ DH lymphomas with involvement of 11q13 may also be relatively frequent, the great majority being classified as aggressive variants of mantle cell lymphoma. This suggests that activation of MYC might be an important progression pathway in mantle cell lymphoma as well. Based on clinical significance and the fact that no other solid diagnostic tools are available to identify DH lymphomas, it seems advisable to test all diffuse large B-cell and related lymphomas for MYC and other breakpoints.