B2M Mutations Research Articles

Emerging therapies provide new opportunities to reshape the multifaceted interactions between the immune system and lymphoma cells.

The acquisition of a complete neoplastic phenotype requires cancer cells to develop escape mechanisms from the host immune system. This phenomenon, commonly referred to as 'immune evasion,' represents a hallmark of cancers and results from a Darwinian selection of the fittest tumor clones. First reported in solid tumors, cancer immunoescape characterizes several hematological malignancies. The biological bases of cancer immunoescape have recently been disclosed and include: (i) impaired human leukocyte antigen-mediated cancer cell recognition (B2M, CD58, CTIIA, CD80/CD86, CD28 and CTLA-4 mutations); (ii) deranged apoptotic mechanisms (reduced pro-apoptotic signals and/or increased expression of anti-apoptotic molecules); and (iii) changes in the tumor microenvironment involving regulatory T cells and tumor-associated macrophages. These immune-escape mechanisms characterize both Hodgkin and non-Hodgkin (B and T cell) lymphomas and represent a promising target for new anti-tumor therapies. In the present review, the principles of cancer immunoescape and their role in human lymphomagenesis are illustrated. Current therapies targeting these pathways and possible applications for lymphoma treatment are also addressed.

Mutations in CD58 and Other Immune System Related Genes in Hodgkin Lymphoma

Hodgkin Lymphoma (HL) is a B cell derived malignancy characterized by a minority of tumor cells, known as Hodgkin Reed-Sternberg (HRS) cells. The background is composed of a wide variety of inflammatory cells with T cells representing the largest population. Chemokines and cytokines produced by HRS cells and by the infiltrating cells shape the environment and provide proliferative and survival signals to the HRS cells. Despite this critical dependence on the microenvironment, HRS cells also need to apply mechanisms to escape from both antigen-dependent and innate immune responses. HRS cells have evolved multiple mechanisms to evade cytotoxic T cell (CTL) and natural killer (NK) cell mediated anti-tumor responses. These mechanisms include secretion of immune-suppressive factors (IL10, TGFβ and others), recruitment of regulatory and helper T cells, expression of PDL1 and CD95 and loss of HLA expression. Recent publications show that mutations in immune system related genes might represent a mechanism of HRS cells to evade detection by immune cells. The aim of this study was to validate whole exome sequencing results of seven HL cell lines focusing on immune system associated genes. We previously showed that B2M mutations affect the ATG start codon in L428 (heterozygous) and DEV (homozygous) cells. B2M mRNA levels were reduced in both cell lines as compared to L1236, whereas HLA-A, HLA-B and HLA-C mRNA levels were in the same range. Consistent with these findings we observed no membranous B2M and HLA class I expression by flow cytometry in the two cell lines with mutated B2M genes. In primary diagnostic HL tissue we showed lack of membranous B2M in 51% of the cases. We now studied two additional genes in more detail. CD58 gene mutations were observed in KMH2 and DEV cells. By manual inspection of the alignments using the Integrative Genomics Viewer (IGV), we also noticed a lack of reads of exons 1, 2 and 3 in SUPHD1. Heterozygous mutations and homozygous loss of exons 1-3 were confirmed for all three cell lines. CD58 mRNA levels were low or absent in SUPHD1 and KMH2 cells and normal in DEV. CD58 protein expression as determined by flow, western blot and IHC was low or absent in all 3 mutated HL cell lines in comparison to four cell lines with wild type CD58. Tumor cells of 36 primary HL cases with good treatment outcome showed a strong CD58 expression in all cases. As HL cell lines are derived from end stage HL patients, we next studied CD58 expression in relapsed HL patients. No or weak CD58 staining was observed in HRS cells in 6 out of 45 patients who experienced a relapse. Our results indicate that mutations in CD58 and loss of CD58 expression are common in HL derived cell lines and that loss of CD58 expression in tumor cells is restricted to relapsed HL patients. Heterozygous CSF2RB mutations in KMH2, SUPHD1, DEV and L1236 were validated by RNA-seq and Sanger-seq. As CSF2RB encodes the common β chain (CD131) shared by the interleukin-3 (IL-3), granulocytic macrophage colony-stimulating factor (GM-CSF) and IL-5 receptors, we also measured the expression of these 3 α chain receptors. We observed the same expression pattern between CD131 and CD116 (GM-CSF α receptor chain) in HL cell lines by flow cytometry suggesting that these mutations mainly affect the GM-CSF receptor. In conclusion, we show that mutations of immune system genes are common in HL. Deleterious mutations in B2M explain the lack of HLA class I expression, indicating that this genetic alteration is responsible for defective antigen presentation. Deleterious mutations or deletions of CD58 exons result in loss of CD58 protein expression. This will lead to loss of binding to CD2 expressed on T cells and will result in a defect in T cell adhesion and activation. Overall these results indicate that mutations are likely to contribute to the immune escape mechanisms applied by the HRS cells. DisclosuresNo relevant conflicts of interest to declare.

Real-Time Molecular Classification of Diffuse Large B-Cell Lymphoma (DLBCL) By Gene Expression Profiling (GEP): Successful Delivery of a Routine Service for Randomization of Patients Onto the Multicenter Remodl-B Trial (ISRCTN 51837425)

Germinal Center (GCB) and activated (ABC) B-cell subtypes of DLBCL can be identified by Gene expression profiling (GEP). These subgroups are biologically distinct, harboring mutations in different pathways. Patients classified as ABC more often have mutations of the NF-kB pathway and an inferior response to standard R-CHOP therapy. The REMoDL-B trial utilised GEP to stratify patients for the addition of bortezomib to R-CHOP, based on the hypothesis that this agent may selectively improve the outcome of the ABC subtype.GEP was performed on RNA extracted from diagnostic formalin-fixed paraffin-embedded (FFPE) biopsies using Illumina WG-DASLTM during the 1st cycle of R-CHOP. Patients were classified as GCB, ABC or Unclassified before cycle 2 using the cross-platform DAC classifier (Care et al, PLOS ONE 2013) and randomised to continue R-CHOP+/-bortezomib. Work is underway to compare data generated on Affymetrix arrays and targeted RNA-seq (Illumina TRex), as well as validation by targeted mutational analysis of 18 genes associated with DLBCL (TNFAIP3, CARD11, CD79A, CD79B, MYD88, TRAF3, TNFRSF11A, PRDM1, TP53, FAS, B2M, CD58, EZH2, MLL2, MEF2B, EP300, CREBBP, KDM2B) using Fluidigm multiplex PCR and Illumina MiSeq on DNA also from the FFPE blocks.The trial closed to recruitment in May 2015 and 1147 samples have been analysed. One hundred and fifty three (13%) biopsies were unsuitable for GEP (for insufficient tumor tissue, inappropriate block sent). The remaining samples were classified as ABC (n=261, 23%), GCB (n=471, 44%) and Unclassified (n=214, 19%), with only 11 samples (1%) failing to yield a GEP result. GEP was successful in a range of sample types, including needle and endoscopic biopsies, bone marrow trephines and formal biopsies, with results obtained from as little as 40ng of total RNA, all from FFPE samples. Mutational data were available in 199 samples, with 73% of these having a mutation detectable in 1 or more genes (range 0-5) at a AAF (alternative allele frequency) cutoff at 10%. MYD88 was most commonly mutated (in 30% of ABC and 7% of GCB). EZH2 mutations were restricted to the GCB category (26%) and MYD88, CD79a/b and PRDM1 were more commonly associated with the ABC group. MYD88/PRDM1 were the most frequently associated events, with MYD88/CD79a/b and MYD88/NF-kB being mutually exclusive. Where MYD88 was seen in GCB cases, coexisting mutations imply an origin from transformed follicular lymphoma. B2M mutations were commonly identified across all subtypes (n=26), but specifically enriched in Type III (unclassified) cases (25%), which supports the hypothesis that mutational immune escape may be a feature of DLBCL, in common with other tumor types. Cross platform validation is highly concordant using Affymetrix arrays from a pilot series (27/27 gave the same classifier output, with correlating confidences also seen between platforms). RNA-seq analysis is ongoing, however initial analysis shows 86% concordance with the DASL output. Comprehensive cross platform comparison data will be available for presentation at the meeting.This study demonstrates the feasibility of GEP classification of DLBCL at diagnosis in a large international trial. The molecular classification can also be replicated using different technologies. Mutational analysis confirmed the association between DLBCL subtype and specific mutational hotspots. DisclosuresSharon:Johnson & Johnson: Other: Funded the laboratory work for the REMoDL-B trial (ISRCTN 51837425). Davies:Takeda: Honoraria; Seattle Genetics: Research Funding. Jack:Jannsen: Research Funding. Johnson:Johnson & Johnson: Other: Funded the laboratory work for the REMoDL-B trial (ISRCTN 51837425).

Recurrent mutations in epigenetic regulators, RHOA and FYN kinase in peripheral T cell lymphomas.

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous and poorly understood group of non Hodgkin lymphomas1,2. Here we combined whole exome sequencing of 12 tumor-normal DNA pairs, RNAseq analysis and targeted deep sequencing to identify new genetic alterations in PTCL transformation. These analyses identified highly recurrent epigenetic factor mutations in TET2, DNMT3A and IDH2 as well as a new highly prevalent RHOA p.Gly17Val (NM_001664) mutation present in 22/35 (67%) of angioimmunoblastic T-cell lymphomas (AITL) and in 8/44 (18%) not otherwise specified PTCL (PTCL NOS) samples. Mechanistically, the RHOA Gly17Val protein interferes with RHOA signaling in biochemical and cellular assays, an effect potentially mediated by the sequestration of activated Guanine Exchange Factor (GEF) proteins. In addition, we describe new and recurrent, albeit less frequent, genetic defects including mutations in FYN, ATM, B2M and CD58 implicating SRC signaling, impaired DNA damage response and escape from immune surveillance mechanisms in the pathogenesis of PTCL.

Polymorphisms of Neonatal Fc Receptor in Cynomolgus and Rhesus Macaques

Neonatal Fc receptor (FcRn), a heterodimer of MHC class I-like protein and β2-microglobulin, encoded by FCGRT and B2M, respectively, is important for recycling immunoglobulin G (IgG) antibodies by binding with the Fc region of IgG. Cynomolgus macaques are important animal species used in the evaluation of therapeutic antibodies, largely due to sequence similarities of target proteins to those of humans. Because the function of FcRn could be modified by mutations in FCGRT or B2M, 71 cynomolgus and 24 rhesus macaques were analyzed in the present study. A total of 21 variants were identified, of which 4 were non-synonymous in FCGRT. Fifteen variants were unique to cynomolgus macaques, of which 3, 2, and 5 were unique to cynomolgus macaques bred in China (MacfaCHN), Cambodia (MacfaCAM), and Indonesia (MacfaIDN), respectively. Five variants were shared by MacfaCHN and MacfaCAM, but not by MacfaIDN. In B2M, only 5 variants were found, including 2 non-synonymous variants. Tissue expression analysis showed that cynomolgus FCGRT and B2M were widely expressed in the 10 tissue types analyzed. None of the non-synonymous variants of FCGRT or B2M found changes in the amino acid residues known to be important for FcRn function, suggesting that substantial inter-animal variability of FcRn is not expected for the cynomolgus macaques analyzed.

Three Kinships with ALAS2 P520L Mutations, Two in Association with Severe Iron Overload, and One with Sideroblastic Anemia and Severe Iron Overload.

Aminolevulinate synthase 2 (ALAS2) is an erythroid-expressed gene located on chromosome Xp11.21. Mutations in ALAS2 have been shown to be associated with sideroblastic anemia. We have identified a novel mutation in ALAS2, P520L, in three kinships. The P520L mutation was not found in 316 white male control subjects. The proline in this position is highly conserved across species from humans to zebrafish.In the C kinship, the P520L mutation was first identified in a white man who presented with severe iron overload at an early age and was a HFE C282Y homozygote. Genetic analyses of members of the C kinship suggest that the presence of P520L alone in hemizygous males, or simple heterozygosity in females, is not associated with anemia or iron overload. Females heterozygous for both HFE C282Y and ALAS2 P520L also had normal ferritin levels. Only subjects homozygous for HFE C282Y and hemizygous or heterozygous for ALAS2 P520L had severe iron overload. Sequencing studies revealed that the propositus did not have missense mutations in HAMP, HJV, FPN1, ABC7, IL6, or RAG1.In the H kinship, the propositus was a white woman with severe iron overload who was heterozygous for ALAS2 P520L; she had a wildtype HFE genotype. The pedigree of the H kinship identified 5 additional females who were heterozygous for the P520L ALAS2 mutation. Only the propositus had iron overload and none of the subjects with P520L had sideroblastic anemia. The propositus did not have missense mutations in FPN1, HAMP, HJV, TFR2, B2M, IRP2, ABC7, or SFT. We speculate that the propositus in the H kinship has a mutation in a currently unknown gene that contributes to her severe iron overload. In the S kinship, the P520L mutation was identified in a white man with sideroblastic anemia and severe iron overload. The patient had a second mutation in ALAS2, R560H, a mutation previously described in two brothers with sideroblastic anemia of variable penetrance (Blood 100:4236–4238, 2002). It is not possible to determine the effect of the P520L mutation on the penetrance of the R560H-associated sideroblastic anemia and iron overload.We conclude that the ALAS2 P520L occurs at a very low allele frequency in the white population. The present observations also suggest that there is no distinctive phenotype associated with the P520L mutation alone, but that P520L may act as a modifier of iron overload in the presence of HFE homozygosity, other missense mutations of ALAS2, or mutations of uncharacterized iron regulatory genes.