HLA-DR Protein Status Predicts Survival in Patients with Diffuse Large B Cell Lymphoma (DLBCL) Treated with the MACOP-B Chemotherapy Regimen.

Abstract Background: HLA-DR is one of the MHC Class II antigen presenting molecules. Loss of HLA-DR expression on DLBCL tumor cells has been associated with poor survival in patients on Southwest Oncology Group (SWOG) studies as well as the recent Leukemia and Lymphoma Molecular Profiling project (Rosenwald et al, NEJM 2002). This study was conceived to examine the effect of HLA-DR status on a completely different cohort of patients from a single institution treated uniformly with a single chemotherapy regimen, MACOP-B, in an attempt to validate HLA-DR as an important biomarker in DLBCL. Methods: HLA-DR immunostaining was performed using a tissue microarray block containing two 0.6mm cores of paraffin embedded tissues from 97 patients treated on the MACOP-B regimen (1981–86) at the BC Cancer Agency in Vancouver, Canada (Semin Hematol 1988, suppl 2: 41–6). Only cases of B cell lineage were used. We used the HLA-DR antibody Class II DR, (IgG2b) at a 1:50 dilution (Novocastra, UK) with epitope recovery, on an automated immunostainer (Ventana Benchmark System) using a biotin-avidin-diaminobenzidine-based detection system. Two pathologists (LR and DF) scored all slides for positive or negative results. Results: All patients had advanced-stage disease and were treated uniformly with the MACOP-B chemotherapy regimen. The median follow-up of living patients is 17 years. The IPI was predictive of overall survival (OS) in the study group (p = 0.023). Of the 97 B cell cases, 82 had interpretable staining. The other cases were eliminated for lack of tumor (2), necrotic tissue (1), or lack of internal positive control cells in the tissue core (12). Of the remaining 82 cases, 52 were positive for HLA-DR protein with a median OS of 16.2 years while 30 were negative with a median OS of 4.2 years, (p=0.037, figure below). A Cox multivariate model established both IPI (p = 0.031) and HLA-DR status (p = 0.04) as independent predictors of OS. Discussion: The importance of loss of antigen presenting molecules on cells of DLBCL was a key finding of the previous LLMPP work, and is now further confirmed as impacting OS in a separate set of patients treated uniformly with a different chemotherapy regimen. These findings lend credence to the hypothesis that loss of immunogenicity is of key importance in patient response to treatment and OS and suggest that specific therapies focused on this pathway may benefit patients with DLBCL regardless of their treatment regimen. Figure Figure

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Genetic Alterations of the MHC Class II Transactivator CIITA Are Frequent in Primary Mediastinal Large B-Cell Lymphoma and Associated with Diminished MHC Class II Expression

Blood ◽

10.1182/blood.v124.21.3040.3040 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3040-3040

Author(s):

Anja Mottok ◽

Bruce W. Woolcock ◽

Fong Chun Chan ◽

Adele Telenius ◽

Elizabeth A. Chavez ◽

...

Keyword(s):

Protein Expression ◽

B Cell ◽

Cell Lines ◽

Mhc Class Ii ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Class Ii ◽

Coding Sequence ◽

Hla Dr ◽

Intron 1

Abstract Introduction: Constitutive MHC class II expression is a hallmark of antigen-presenting cells, including B cells, and is indispensable for the initiation of antigen specific immune responses. It has been shown that certain B cell lymphoma entities are able to evade immune recognition by downregulation of MHC molecules on the tumor cell surface. We have previously identified recurrent chromosomal rearrangements of CIITA, the master regulator of MHC class II transcription, as one possible mechanism to reduce MHC class II expression in primary mediastinal large B-cell lymphoma (PMBCL) and classical Hodgkin lymphoma (cHL) (Steidl et al., Nature 2011). Furthermore, we have recently described a 1.6kb breakpoint cluster region within intron 1 of CIITA and have shown in a small sample set of PMBCL cases that deletions, insertions and single nucleotide variants (SNV) are commonly found within this genomic region (Steidl, ASH abstract # 437, 2011). Therefore, we aimed to explore the frequency of these alterations and the correlation with CIITA and MHC class II protein expression in a larger cohort of PMBCL cases and to further characterize their functional significance. Methods: We have comprehensively analyzed 45 diagnostic PMBCL samples for the presence of coding sequence mutations as well as alterations within the promoter III region and the first 3kb of intron 1 using deep amplicon sequencing (Illumina TruSeq) and/or Sanger sequencing. In addition, we characterized the PMBCL-derived cell lines U2940 and Med-B1 by whole transcriptome paired-end sequencing (RNA-seq). To elucidate the functional consequences of the coding sequence mutations identified in these two cell lines we performed retroviral transductions of wild type CIITA and CIITA mutants in a CIITA and HLA-DR expression-negative cell line (DEV, nodular lymphocyte predominant Hodgkin lymphoma-derived). We subsequently analyzed CIITA mRNA expression using qRT-PCR and HLA-DR surface expression using flow cytometry. Furthermore, we applied immunohistochemistry (IHC) to determine expression levels of CIITA and HLA-DR in a large cohort of PMBCL samples represented on two tissue microarrays (TMA, n=149). The TMAs were also used for fluorescence in-situ hybridization (FISH) to evaluate the presence of copy number alterations or translocations of the CIITA locus. Results: FISH was interpretable in 115 samples with a CIITA break-apart (CIITA-ba) frequency of 33.9% (39/115). Correlative analyses revealed that decreased CIITA protein expression by IHC was significantly correlated with the presence of CIITA-ba (P=0.019), whereas HLA-DR expression was not correlated with CIITA-ba status alone (P=0.219). However, we could demonstrate a positive correlation between protein expression of CIITA and HLA-DR (Pearson r=0.45, P<0.0001). Within the subset of 45 PMBCL cases that were analyzed for the presence of genomic alterations, 39% were CIITA-ba positive (16/41), and in 31.8% (14/44) we observed coding sequence mutations and/or alterations affecting the promoter III region. 45.5% (20/44) of the cases presented indels and/or SNVs in intron 1. Using RNA-seq, we have detected two missense mutations in the Med-B1 cell line affecting both alleles in functionally relevant protein domains. Furthermore, we identified a novel NUBP1-CIITA fusion transcript in U2940 also harboring an SNV on the other allele resulting in the transcription of an elongated protein due to the loss of the original stop codon. Ectopic expression of these CIITA mutants in DEV, which has been shown to have undetectable levels of CIITA and HLA-DR due to a biallelic CIITA inactivation, revealed that these individual SNVs showed a diminished capability to restore HLA-DR surface expression in comparison to wild type CIITA as measured by flow cytometry. Conclusions: Here we show that the presence of CIITA rearrangements is significantly associated with low CIITA protein levels, and we could demonstrate that protein expression of CIITA and HLA-DR are positively correlated in PMBCL. Furthermore, CIITA is frequently targeted by coding sequence mutations and intronic deletions in PMBCL cell lines and clinical samples. Functional studies demonstrate that genomic alterations in CIITA contribute to downregulation of MHC class II expression in malignant lymphomas and therefore represent a potent mechanism of acquired immune privilege and escape from immune surveillance. Disclosures No relevant conflicts of interest to declare.

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Alterations in CIITA constitute a common mechanism accounting for downregulation of MHC class II expression in diffuse large B-cell lymphoma (DLBCL)

Experimental Hematology ◽

10.1016/j.exphem.2008.10.001 ◽

2009 ◽

Vol 37 (2) ◽

pp. 184-194.e2 ◽

Cited By ~ 24

Author(s):

Kelly A. Cycon ◽

Lisa M. Rimsza ◽

Shawn P. Murphy

Keyword(s):

B Cell ◽

Mhc Class Ii ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Class Ii ◽

Common Mechanism ◽

Large B Cell Lymphoma ◽

Large B Cell

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Extensive genetic alterations of the HLA region, including homozygous deletions of HLA class II genes in B-cell lymphomas arising in immune-privileged sites

Blood ◽

10.1182/blood.v96.10.3569.h8003569_3569_3577 ◽

2000 ◽

Vol 96 (10) ◽

pp. 3569-3577 ◽

Cited By ~ 6

Author(s):

Sietske A. Riemersma ◽

Ekaterina S. Jordanova ◽

Roelandt F. J. Schop ◽

Katja Philippo ◽

Leendert H. J. Looijenga ◽

...

Keyword(s):

B Cell ◽

Loss Of Heterozygosity ◽

B Cell Lymphoma ◽

Hla Class I ◽

Class Ii ◽

Genetic Alterations ◽

Class I ◽

B Cell Lymphomas ◽

Hla Dr ◽

Homozygous Deletions

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 < .01), whereas loss of HLA-DR expression was found in 56% and 5%, respectively (P < .01). This was accompanied by extensive loss of heterozygosity within the HLA region in the extranodal DLCLs. In 3 cases, retention of heterozygosity for D6S1666 in the class II region suggested a homozygous deletion. This finding was confirmed by interphase FISH that showed homozygous deletions in the class II genes in 11 of the 18 extranodal lymphomas but in none of the 7 nodal DLCLs (P < .001). Mapping by fiber FISH showed variable deletions that always included HLA-DQ and HLA-DR genes. Hemizygous deletions and mitotic recombinations often involving all HLA genes were found in 13 of 18 extranodal and 2 of 7 nodal lymphomas. In conclusion, a structural loss of HLA class I and II expression might help the B-cell lymphoma cells to escape from immune attack.

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Extensive genetic alterations of the HLA region, including homozygous deletions of HLA class II genes in B-cell lymphomas arising in immune-privileged sites

Blood ◽

10.1182/blood.v96.10.3569 ◽

2000 ◽

Vol 96 (10) ◽

pp. 3569-3577 ◽

Cited By ~ 129

Author(s):

Sietske A. Riemersma ◽

Ekaterina S. Jordanova ◽

Roelandt F. J. Schop ◽

Katja Philippo ◽

Leendert H. J. Looijenga ◽

...

Keyword(s):

B Cell ◽

Loss Of Heterozygosity ◽

B Cell Lymphoma ◽

Hla Class I ◽

Class Ii ◽

Genetic Alterations ◽

Class I ◽

B Cell Lymphomas ◽

Hla Dr ◽

Homozygous Deletions

Abstract 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 < .01), whereas loss of HLA-DR expression was found in 56% and 5%, respectively (P < .01). This was accompanied by extensive loss of heterozygosity within the HLA region in the extranodal DLCLs. In 3 cases, retention of heterozygosity for D6S1666 in the class II region suggested a homozygous deletion. This finding was confirmed by interphase FISH that showed homozygous deletions in the class II genes in 11 of the 18 extranodal lymphomas but in none of the 7 nodal DLCLs (P < .001). Mapping by fiber FISH showed variable deletions that always included HLA-DQ and HLA-DR genes. Hemizygous deletions and mitotic recombinations often involving all HLA genes were found in 13 of 18 extranodal and 2 of 7 nodal lymphomas. In conclusion, a structural loss of HLA class I and II expression might help the B-cell lymphoma cells to escape from immune attack.

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HLA-DR protein status predicts survival in patients with diffuse large B-cell lymphoma treated on the MACOP-B chemotherapy regimen

Leukemia & Lymphoma ◽

10.1080/10428190601078605 ◽

2007 ◽

Vol 48 (3) ◽

pp. 542-546 ◽

Cited By ~ 32

Author(s):

Lisa M. Rimsza ◽

Pedro Farinha ◽

Deborah A. Fuchs ◽

Hamid Masoudi ◽

Joseph M. Connors ◽

...

Keyword(s):

B Cell ◽

Chemotherapy Regimen ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Protein Status ◽

Large B Cell Lymphoma ◽

Hla Dr ◽

Large B Cell

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Glatiramer acetate immune modulates B-cell antigen presentation in treatment of MS

Neurology Neuroimmunology & Neuroinflammation ◽

10.1212/nxi.0000000000000698 ◽

2020 ◽

Vol 7 (3) ◽

pp. e698 ◽

Cited By ~ 3

Author(s):

Darius Häusler ◽

Zivar Hajiyeva ◽

Jan W. Traub ◽

Scott S. Zamvil ◽

Patrice H. Lalive ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Antigen Presentation ◽

Glatiramer Acetate ◽

Mhc Class Ii ◽

Class Ii ◽

Cell Antigen ◽

Antigen Presenting

ObjectiveWe examined the effect of glatiramer acetate (GA) on B-cell maturation, differentiation, and antigen presentation in MS and experimental autoimmune encephalomyelitis (EAE).MethodsA cross-sectional study of blood samples from 20 GA-treated and 18 untreated patients with MS was performed by flow cytometry; 6 GA-treated patients with MS were analyzed longitudinally. GA-mediated effects on B-cell antigen-presenting function were investigated in EAE, or, alternatively, B cells were treated with GA in vitro using vehicle as a control.ResultsIn MS, GA diminished transitional B-cell and plasmablast frequency, downregulated CD69, CD25, and CD95 expression, and decreased TNF-α production, whereas IL-10 secretion and MHC Class II expression were increased. In EAE, we observed an equivalent dampening of proinflammatory B-cell properties and an enhanced expression of MHC Class II. When used as antigen-presenting cells for activation of naive T cells, GA-treated B cells promoted development of regulatory T cells, whereas proinflammatory T-cell differentiation was diminished.ConclusionsGA immune modulates B-cell function in EAE and MS and efficiently interferes with pathogenic B cell–T cell interaction.

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