Recurrent involvement of the REL and BCL11A loci in classical Hodgkin lymphoma

Comparative genomic hybridization studies have shown gains in chromosome region 2p as the most common imbalance in classical Hodgkin lymphoma (cHL). The minimal region of gain contained 2 candidate oncogenes, REL and BCL11A. This study examined the involvement of REL and BCL11A loci in 44 primary cases of cHL by combined immunophenotyping and interphase cytogenetics (FICTION). A median 2p13 copy number above the tetraploid range was detected in 24 (55%) cases. Adjustment for centromere 2 copy number indicated gains of 2p13 in 11 of 31 cHLs (35%) with 8 (26%) high-level amplifications. One cHL displayed selective amplification of the REL locus not affecting BCL11A; another case studied by FICTION and a cHL with cytogenetic 2p change investigated by fluorescence in situ hybridization showed signal patterns suggesting breakpoints in the region spanned by the REL probe. These data indicate that REL rather than BCL11A may be the target of the 2p13 alterations in cHL.     

Analysis of HIV-1- and CMV-specific memory CD4 T-cell responses during primary and chronic infection

Hodgkin- and Reed-Sternberg (HRS) cells microdissected from 41 classical Hodgkin lymphomas (cHL) of 40 patients comprising 8 lymphocyte-rich (cHL-LR), 16 nodular sclerosis (cHL-NS), 15 mixed-cellularity (cHL-MC), and 2 lymphocyte-depletion (cHL-LD) subtypes were analyzed by comparative genomic hybridization for recurrently imbalanced chromosomal subregions. Chromosomal gains most frequently involved chromosome 2p (54%), 12q (37%), 17p (27%), 9p and 16p (24% each), and 17q and 20q (20% each), whereas losses primarily affected chromosome 13q (22%). Using fluorescence in situ hybridization, amplification of the REL oncogene was demonstrated within a distinct 2p15-p16 amplicon. The high frequency of 2p overrepresentations including REL, particularly in cHL-NS (88%), suggests that an alternative mechanism of constitutive activation of nuclear factor NF-kappaB is a hallmark of HRS cells. Hierarchical cluster analysis of chromosomal imbalances revealed a closer relationship among cHL-NS than other subtypes. Furthermore, there is a tendency for different subtypes of cHL-MC tumors characterized by different ages at the time of tumor onset and gain of chromosome 17p. The imbalance pattern of cHL subtypes suggests that different molecular pathways are activated, with REL or other genes on chromosomal band 2p15-p16 playing a fundamental role in the pathogenesis of classical Hodgkin lymphoma.     

Genetic lesions of the TRAF3 and MAP3K14 genes in classical Hodgkin lymphoma

Hodgkin and Reed/Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) show constitutive activation of nuclear factor (NF)‐κB. Several genetic lesions contribute to this deregulated NF‐κB activity. Here, we analysed two further NF‐κB regulators for genetic lesions, the inhibitory factor TRAF3 and the key signalling component of the alternative NF‐κB pathway, MAP3K14 (NIK). Single nucleotide polymorphism (SNP) array analysis of cHL cell lines revealed a uniparental disomy of the long arm of chromosome 14 associated with a biallelic deletion of TRAF3 located on this chromosome in cell line U‐HO1. Cloning of the deletion breakpoint showed a 123 371 bp deletion. No inactivating mutations of TRAF3 were found in six other cHL cell lines or in microdissected HRS cells from seven cHL. However, in primary cHL samples interphase cytogenetic analyses revealed signal patterns indicating monoallelic deletion of TRAF3 in 3/20 other cases. SNP array analysis revealed a gain of copy number for MAP3K14 in three cHL cell lines. Gains of MAP3K14 were detected in 5/16 cases of primary cHL. In conclusion, in rare instances, HRS cells harbour inactivating mutations of the TRAF3 gene and recurrently show gains of MAP3K14, indicating that more components of NF‐κB signalling show genetic lesions in HRS cells than previously known.     

High resolution SNP array genomic profiling of peripheral T cell lymphomas,not otherwise specified,identifies a subgroup with chromosomal aberrations affecting the REL locus

Little is known about genomic aberrations in peripheral T cell lymphoma, not otherwise specified (PTCL NOS). We studied 47 PTCL NOS by 250k GeneChip single nucleotide polymorphism arrays and detected genomic imbalances in 22 of the cases. Recurrent gains and losses were identified, including gains of chromosome regions 1q32–43, 2p15–16, 7, 8q24, 11q14–25, 17q11–21 and 21q11–21 (≥5 cases each) as well as losses of chromosome regions 1p35–36, 5q33, 6p22, 6q16, 6q21–22, 8p21–23, 9p21, 10p11–12, 10q11–22, 10q25–26, 13q14, 15q24, 16q22, 16q24, 17p11, 17p13 and Xp22 (≥4 cases each). Genomic imbalances affected several regions containing members of nuclear factor‐kappaB signalling and genes involved in cell cycle control. Gains of 2p15–16 were confirmed in each of three cases analysed by fluorescence in situ hybridization (FISH) and were associated with breakpoints at the REL locus in two of these cases. Three additional cases with gains of the REL locus were detected by FISH among 18 further PTCL NOS. Five of 27 PTCL NOS investigated showed nuclear expression of the REL protein by immunohistochemistry, partly associated with genomic gains of the REL locus. Therefore, in a subgroup of PTCL NOS gains/rearrangements of REL and expression of REL protein may be of pathogenetic relevance.     

Genomic Alterations in Hodgkin’s Lymphoma

Cytogenetic analysis of Hodgkin’s lymphoma (HL) is hampered by the scarcity of neoplastic cells within a sea of reactive cells. There is accumulating evidence that HL represents 2 disease entities, classic HL (cHL) with its morphologic variants and nodular lymphocyte predominant HL (NLPHL). This subdivision, initially worked out in morphologic and immunohisto-chemical studies, has been further substantiated by molecular cytogenetic investigations. Two recurrent chromosomal aberrations, namely gains of 2p13–p16 and 9p24, have been found by comparative genomic hybridization analysis in microdissected cells from cHL patients as well as in cHL cell lines, but not in NLPHL cells. The available cHL cell lines are remarkably heterogeneous in their karyotypes, suggesting profound genomic instability leading to numeric chromosomal aberration and multiple chromosomal breaks and translocations. In this article, we review genomic aberrations that may contribute to the development and maintenance of the morphologic and clinical presentation of these B-cell lymphoma entities. Furthermore, we delineate current data on the genomic changes observed in the neoplastic cells of HL that are created by epigenetic mechanisms, which are alternative mechanisms that regulate the expression of relevant genes.     

Frequent occurrence of BCL6 rearrangements in nodular lymphocyte predominance Hodgkin lymphoma but not in classical Hodgkin lymphoma

We studied the genomic status of BCL6 in 23 cases of nodular lymphocyte predominance Hodgkin lymphoma (NLPHL) and 40 cases of classical Hodgkin lymphoma (cHL), using dual-color interphase fluorescence in situ hybridization (FISH). The BCL6 rearrangement was identified in 48% of NLPHL cases and was not detected in cHL cases. As a confirmation, sequential or simultaneous immunohistochemistry (IHC) and FISH using CD20 or BCL6 antibodies and BCL6 DNA probes was performed in 8 NLPHL cases. The BCL6-associated translocations, t(3;22)(q27;q11), t(3;7)(q27;p12), and the most probable t(3;9)(q27;p13), were identified in 3 cases. A consistent expression of BCL6 protein in popcorn cells with the highest number of intensely stained cells in cases with a genomic BCL6 rearrangement was shown by IHC. These findings support the hypothesis of a germinal center B cell-derived origin of NLPHL, indicate a significant role of BCL6 in the pathogenesis of NLPHL, and provide further evidence of the genetic diversity underlying the pathogenesis of NLPHL and cHL.     

Comparison of miRNA profiles of microdissected Hodgkin/Reed-Sternberg cells and Hodgkin cell lines versus CD77+ B-cells reveals a distinct subset of differentially expressed miRNAs

Classical Hodgkin lymphoma (cHL) is characterized by the presence of malignant Hodgkin and Reed Sternberg (HRS) cells. The scarcity of tumour cells in lymphoma biopsies has hampered genetic analyses of HRS cells, including microRNA (miRNA) expression profiling. We determined the expression of 360 miRNAs in microdissected HRS cells from nine cHL patients. These miRNA profiles were compared to those from four cHL cell lines and CD77⁺ B-cells, yielding a distinct cHL signature of 12 over- and three underexpressed miRNAs. Our data suggest that miRNAs are implicated in the pathogenesis of Hodgkin lymphoma and prompt further investigations concerning their role in cHL.     

Numerical chromosome aberrations are present within the CD30+ Hodgkin and Reed-Sternberg cells in 100% of analyzed cases of Hodgkin's disease [see comments]

In Hodgkin's disease, cytogenetically aberrant clones have been demonstrated in a minority of cases studied. In the remaining cases, only normal metaphases have been found, but it is questionable whether normal karyotypes actually correspond to the pathognomonic Hodgkin and Reed-Sternberg (HRS) cells. Numerical aberrations could be studied by fluorescence in situ hybridization (FISH). However, in Hodgkin's disease, the percentage of tumor cells is mostly below the detection limit of FISH, which is near 1%. With the technique of simultaneous fluorescence immunophenotyping and interphase cytogenetic analysis (FICTION), this problem can be overcome. By FICTION, hybridization signals can selectively be evaluated within the CD30a+ cell population. We have studied 30 cytogenetically analyzed cases of Hodgkin's disease by means of FICTION. In all cases, we found numerical chromosome aberrations within the majority of CD30+ HRS cells. In cases with complex and hyperdiploid karyotypes, the cytogenetic results agreed with the FICTION data. There was considerable variability in the chromosome numbers, demonstrating that karyotype instability is an in vivo phenomenon of HRS cells. Lymphocytes never displayed numerical chromosome changes. Our results indicate that HRS cells regularly exhibit numerical chromosome aberrations and that the chromosome numbers are always in the hyperploid range.     

Report: workshop on mediastinal grey zone lymphoma

Abstract:  There are several indications that classical Hodgkin lymphoma (cHL) and at least a proportion of cases of Primary Mediastinal B cell Lymphoma (PMBL) are derived from B cells at similar stages of differentiation and share common pathogenic mechanisms. The first indication was the existence of mediastinal grey zone lymphomas as identified in the 4th International Symposium on HL, with clinical, histological and immunohistochemical features intermediate between cHL and PMBL. Second, both tumor types resemble a cell that is developmentally situated in-between the germinal center reaction and a plasma cell. Third, cHL and PMBL were found to have similar gene expression profiles, including the lack of immunoglobulin expression and low levels of B cell receptor signalling molecules, and the secretion of molecules like the chemokine TARC and the prominent expression of IL-13 receptors. Fourth, both entities were found to have common genomic aberrancies, notably in 2p15 and 9p24, the sites of the REL oncogene and the tyrosine kinase gene JAK2, respectively. Further comparison of both lymphoma types may provide further insight in the pathogenic mechanisms and allow the design of diagnostic algorithms to sort out the small number of so-called mediastinal grey zone lymphomas, that appear to be intermediate between PMBL and cHL.     

The BCL11 gene family: involvement of BCL11A in lymphoid malignancies.

Many malignancies of mature B cells are characterized by chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus on chromosome 14q32.3 and result in deregulated expression of the translocated oncogene. t(2;14)(p13;q32.3) is a rare event in B-cell malignancies. In contrast, gains and amplifications of the same region of chromosome 2p13 have been reported in 20% of extranodal B-cell non-Hodgkin lymphomas (B-NHL), in follicular and mediastinal B-NHL, and in Hodgkin disease (HD). It has been suggested that REL, an NF-kappaB gene family member, mapping within the amplified region, is the pathologic target. However, by molecular cloning of t(2;14)(p13;q32.3) from 3 cases of aggressive B-cell chronic lymphocytic leukemia (CLL)/immunocytoma, this study has shown clustered breakpoints on chromosome 2p13 immediately upstream of a CpG island located about 300 kb telomeric of REL. This CpG island was associated with a Krüppel zinc finger gene (BCL11A), which is normally expressed at high levels only in fetal brain and in germinal center B-cells. There were 3 major RNA isoforms of BCL11A, differing in the number of carboxy-terminal zinc fingers. All 3 RNA isoforms were deregulated as a consequence of t(2;14)(p13;q32.3). BCL11A was highly conserved, being 95% identical to mouse, chicken, and Xenopus homologues. BCL11A was also highly homologous to another gene (BCL11B) on chromosome 14q32.1. BCL11A coamplified with REL in B-NHL cases and HD lymphoma cell lines with gains and amplifications of 2p13, suggesting that BCL11A may be involved in lymphoid malignancies through either chromosomal translocation or amplification.     

The use of real-time quantitative polymerase chain reaction and comparative genomic hybridization to identify amplification of the REL gene in follicular lymphoma

Using comparative genomic hybridization (CGH), aberrations in DNA copy number were studied before and after transformation of follicular lymphoma to diffuse large B-cell lymphoma in six patients (15 lymph node biopsies in total). The most common and also the most discrete and intense amplification occurring in four out of 15 biopsies from three different patients was of 2p13-16. Using real-time quantitative polymerase chain reaction (RQ-PCR), REL amplification was found to be implicated at this locus. This technique also identified amplified REL in a further two biopsies, presumably below the detection level of CGH. REL amplification was quantified by comparing it, in most cases, with three endogenous reference genes, albumin, beta2-microglobulin and CD8alpha, that lie close to REL on 2p. There was no correlation apparent between 2p13-16 amplification or REL amplification and transformation. This study shows the usefulness of coupling CGH, for detecting recurring abnormalities, with the real-time PCR technique for rapid gene dosage quantification and confirms that the REL gene is a potential candidate in the pathogenesis of a particular subset of follicular lymphomas.     

9p24.1 alterations and programmed cell death 1 ligand 1 expression in early stage unfavourable classical Hodgkin lymphoma: an analysis from the German Hodgkin Study Group NIVAHL trial

High programmed cell death 1 ligand 1 (PD-L1) protein expression and copy number alterations (CNAs) of the corresponding genomic locus 9p24.1 in Hodgkin- and Reed–Sternberg cells (HRSC) have been shown to be associated with favourable response to anti-PD-1 checkpoint inhibition in relapsed/refractory (r/r) classical Hodgkin lymphoma (cHL). In the present study, we investigated baseline 9p24.1 status as well as PD-L1 and major histocompatibility complex (MHC) class I and II protein expression in 82 biopsies from patients with early stage unfavourable cHL treated with anti-PD-1-based first-line treatment in the German Hodgkin Study Group (GHSG) NIVAHL trial (ClinicalTrials.gov Identifier: NCT03004833). All evaluated specimens showed 9p24.1 CNA in HRSC to some extent, but with high intratumoral heterogeneity and an overall smaller range of alterations than reported in advanced-stage or r/r cHL. All but two cases (97%) showed PD-L1 expression by the tumour cells in variable amounts. While MHC-I was rarely expressed in >50% of HRSC, MHC-II expression in >50% of HRSC was found more frequently. No obvious impact of 9p24.1 CNA or PD-L1 and MHC-I/II expression on early response to the highly effective anti-PD-1-based NIVAHL first-line treatment was observed. Further studies evaluating an expanded panel of potential biomarkers are needed to optimally stratify anti-PD-1 first-line cHL treatment.     

Current status of prognostication in classical Hodgkin lymphoma

Classical Hodgkin lymphoma (cHL) is characterized by a paucity of neoplastic Hodgkin/Reed Sternberg (HRS) cells within a complex cellular milieu that is rendered immunologically incapable of reacting against CD30⁺HRS cells due to a plethora of immune escape mechanisms initiated by the neoplastic cells. Accounting for 25% of all lymphomas and nearly 95% of all Hodgkin lymphomas, patients with cHL are typically young adults. Besides traditional prognostic factors, such as the International Prognostic Index (IPI), newer imaging and ancillary biomarkers (CD68, Galectin‐1 and plasma microRNA) have shown promise. Furthermore, the evolution of gene expression profiling (GEP) in recent years has enabled the development of several practically feasible GEP‐based predictors with prognostic relevance. This review discusses the current status of clinical prognostication in cHL, the critical role of histological evaluation in light of several mimicking entities, and the relevance of tissue as well as serum biomarkers pertaining to immune escape mechanisms and recent GEP studies.     

Array comparative genomic hybridization reveals similarities between nodular lymphocyte predominant Hodgkin lymphoma and T cell/histiocyte rich large B cell lymphoma

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and T cell/histiocyte rich large B cell lymphoma (THRLBCL) usually affect middle‐aged men, show tumour cells with a B cell phenotype and a low tumour cell content. Whereas the clinical behaviour of NLPHL is indolent, THRLBCL presents with advanced stage disease and an aggressive behaviour. In the present study, array comparative genomic hybridization was performed in seven typical NLPHL, four THRLBCL‐like NLPHL variants, six THRLBCL and four diffuse large B cell lymphomas (DLBCL) derived from NLPHL. The number of genomic aberrations was higher in THRLBCL compared with typical and THRLBCL‐like variant of NLPHL. Gains of 2p16.1 and losses of 2p11.2 and 9p11.2 were commonly observed in typical and THRLBCL‐like variants of NLPHL as well as THRLBCL. Gains of 2p16.1, affecting the REL locus were confirmed in an independent cohort. Expression of the REL protein was observed at similar frequencies in typical and THRLBCL‐like variant of NLPHL as well as THRLBCL (33–38%). In conclusion, the present study reveals further similarities between NLPHL and THRLBCL on the genomic level, confirming that these entities are part of a pathobiological spectrum with common molecular features, but varying clinical presentations.     

Off-targeting oft-targeted CD20 in cHL

We now think of classical Hodgkin lymphoma (cHL) as derived from "crippled" germinal center B cells that have frequently acquired rearranged and somatically mutated Ig genes.1,2 Despite their B-cell origin, the malignant Hodgkin and Reed-Sternberg (HRS) cells have lost most of the superficial trappings of B cells and therefore have been hidden from investigators' lenses for decades, prompting an arduous but persistent race to uncover the mystery of the HRS cell.     

Transformation of follicular lymphoma to diffuse large cell lymphoma is associated with a heterogeneous set of DNA copy number and gene expression alterations

Genomic aberrations in a series of paired biopsy samples from patients who presented initially with follicle center lymphoma (FCL) and subsequently transformed to diffuse large B-cell lymphoma (DLBCL) were measured by array comparative genomic hybridization (CGH). The consequences of these aberrations on gene expression were determined by comparison with expression analysis on these specimens using cDNA microarrays. A heterogeneous pattern of acquired genomic abnormalities was observed upon transformation, some of which were recurrent in small subsets of patients. Some of the genomic aberration acquired upon transformation, such as gain/amplification of 1q21-q24, 2p16 (REL/BCL11A gene loci), 3q27-q29 (including the BCL6 locus), 7q11.2-q22.1, 12pter-q12, 18q21 (including the BCL2 locus) and Xq, and deletion of 6q22-q24, 13q14-q21 and 17p13 (P53 locus) have been previously implicated in the FCL/DLBCL pathogenesis. In addition, novel genomic imbalances not previously reported in association with FCL transformation, such as overrepresentation of 4p12-pter, 5p12-p15, 6p12.3-p21, 9p23, 9q13-q31, 16q, 17q21, and loss of 1p36.3, 4q21-q23, 5q21-q23, 9q31-qter, 11q24-q25, and 15q23, were identified. We observed a differential expression profile of many genes within regions of gain and deletion upon transformation, including novel target genes associated with FCL transformation. However, other genes did not show deregulated expression despite their location within these areas. In summary, the combination of array CGH and expression analysis provides a more comprehensive picture of the transformation of FCL to DLBCL. This process is associated with the acquisition of a variable spectrum of genomic imbalances affecting recurrent chromosomal areas that harbor overexpressed or underexpressed genes targeted upon transformation.