Abstract 437
Escape from immune surveillance has been proposed to play an important role in cancer and in particular in the pathogenesis of a subgroup of lymphomas. Using massively parallel ...sequencing, we have recently identified recurrent rearrangements of the CIITA gene in classical Hodgkin lymphoma (HL) and primary mediastinal B cell lymphoma (PMBCL) (Steidl et al, Nature 2011). Specifically, a gene fusion in the HL cell line KM-H2, involving the genes CIITA and FLJ27352, suppressed expression of CIITA-regulated MHC class II genes in a dominant-negative manner. Therefore, we sought to further investigate if gene alterations targeting the CIITA locus are recurrent somatic events in HL and PMBCL that lead to loss of gene function and thereby reduce immunogenicity of the malignant cells. PATIENTS AND METHODS: To characterize CIITA gene alterations, we comprehensively studied the cell lines DEV (nodular lymphocyte predominance HL-derived) and KARPAS1106P (PMBCL-derived) by whole-transcriptome paired-end sequencing (RNA-seq) and single nucleotide polymorphism arrays (Affymetrix SNP 6.0). To identify single nucleotide mutations in the CIITA coding sequence we studied additional 7 mediastinal biopsy specimens of PMBCL by RNA-seq. Fluorescence in-situ hybridization (FISH) was used to determine and validate structural and copy number alterations of the CIITA locus. 23 samples of PMBCL, 3 PMBCL-derived cell lines and 9 HL cell lines were studied for mutations and small deletions in CIITA intron 1 by long-range PCR and direct sequencing. CIITA and HLA-DR expression levels were determined by quantitative reverse transcriptase PCR. RESULTS: We identified chromosomal rearrangements affecting both alleles of CIITA in DEV cells resulting in expression of CIITA-PDL2, CIITA-SOCS1 and SOCS1-CIITA fusion transcripts. In KARPAS1106P cells chromosome 16 deletions result in the loss of one entire CIITA allele and partial loss of the other allele. As a consequence, in both cell lines wildtype CIITA and HLA-DR expression was undetectable. Genomic breakpoints within the CIITA rearrangements, together with previously identified translocations, defined a 1.6Kb breakpoint cluster region in CIITA Intron 1. Further analysis of this breakpoint cluster region revealed small intronic deletions in 10 of 23 (43%) PMBCL cases while no such deletions were detected in 18 diffuse large B cell lymphoma and 15 reactive lymph node samples. Furthermore, sequencing revealed multiple deletions ranging from 1–1948 bp and a high incidence of single nucleotide mutations in the breakpoint cluster region of the deleted alleles. The base pair changes were enriched for C to T and G to A transitions over other transitions and transversions, indicative of a somatic hypermutation process. We found significantly lower CIITA expression in cases with CIITA rearrangement and/or small intronic deletions compared to cases without CIITA alterations (p=0.044). RNAseq revealed three cases with non-synonymous coding-sequence mutations including a truncating mutation in exon 4. These changes were somatic in 3 cases with available matching normal DNA. DISCUSSION:CIITA is the target of multiple genomic hits including chromosomal translocation, deletion and coding sequence mutations in HL and PMBCL. In two HL and PMBCL cell lines this leads to complete gene inactivation and loss of HLA class II expression. These data characterize CIITA alterations as a recurrent underlying genomic event of the previously described immunophenotype of reduced HLA class II expression in a subset of PMBCL cases. Our data show that an immune escape mechanism utilized by tumor cells can be directly linked to somatically acquired alterations in cancer genomes.
Siebert:Abbott/Vysis: .
Here, we report recurrent focal deletions of the chr14q32.31-32 locus, including
, a negative regulator of NF-κB signaling, in de novo diffuse large B cell lymphoma (DLBCL) (24/324 cases). ...Integrative analysis revealed an association between
copy number loss with accumulation of NIK, the central noncanonical (NC) NF-κB kinase, and increased NC NF-κB pathway activity. Accordingly, TRAF3 genetic ablation in isogenic DLBCL model systems caused upregulation of NIK and enhanced NC NF-κB downstream signaling. Knockdown or pharmacological inhibition of NIK in TRAF3-deficient cells differentially impaired their proliferation and survival, suggesting an acquired onco-addiction to NC NF-κB. TRAF3 ablation also led to exacerbated secretion of the immunosuppressive cytokine IL-10. Coculturing of TRAF3-deficient DLBCL cells with CD8+ T cells impaired the induction of Granzyme B and interferon (IFN) γ, which were restored following neutralization of IL-10. Our findings corroborate a direct relationship between
genetic alterations and NC NF-κB activation, and highlight NIK as a potential therapeutic target in a defined subset of DLBCL.
Primary mediastinal large B-cell lymphoma (PMBCL) is an aggressive malignancy commonly diagnosed in young adult females. In recent years, mutational and gene expression profiling has established ...genotypic and phenotypic similarity of PMBCL with both classical Hodgkin and diffuse large B-cell lymphoma (DLBCL). In-depth analyses of genomes and transcriptomes have highlighted several inactivating mutations (SOCS1, TP53), chromosomal amplifications (2p, 9p, Xp, Xq) and translocations (CIITA) thought to be integral in establishing and/or maintaining the PMBCL phenotype. Programmed death ligands (PDL) 1 (CD274) and 2 (PDCD1LG2), which are located on chromosome 9p24.1, are two emerging genes of interest that have been shown to be altered in PMBCL and can induce T-cell anergy by binding to the receptor, programmed death 1. Here, we describe the recurrence of chromosomal rearrangements of the PDL locus in various B-cell lymphomas and explore the association of these rearrangements with transcript levels.
To establish the frequency of CD274 and PDCD1LG2 aberration, we conducted fluorescence in situ hybridization (FISH) on 551 clinical samples and 20 established cell lines using in-house break-apart probes. Epstein-Barr virus encoded RNA in situ hybridization was also carried out on the clinical cohort. The clinical cases, sourced from the British Columbia Cancer Agency's Centre for Lymphoid Cancer tissue repository, consisted of 125 PMBCLs, 216 DLBCLs, 130 primary DLBCL of the central nervous system (PCNSL), 12 nodular lymphocyte predominant Hodgkin lymphomas (NLPHL) and 68 follicular lymphomas (FL) with diagnoses based on the WHO classification. The DLBCL cohort could be further subdivided into 134 nodal DLBCLs and 82 testicular DLBCLs (T-DLBCL). Quantitative real-time PCR (qRT-PCR) was subsequently conducted on 17 cell lines and a clinical sub-cohort of 76 samples, for which fresh-frozen material was available, to determine the effect of mutations on transcript expression. We then characterized the PDL aberrations of two clinical PMBCL cases and three cell lines (DEV, L-428, L-1236), at base pair resolution, by applying the bioinformatic tools, nFuse, deFuse and destruct to both newly produced and previously published whole genome (WGS) and whole transcriptome (RNA-seq) libraries.
FISH revealed a PDL locus (9p24.1) break-apart frequency of 20% (25/125) in PMBCL. There were no differences in any known clinical parameters or frequency of Epstein-Barr virus positivity between positive and negative PDL break-apart cases. Break-apart frequencies in other malignancies were calculated to be 3% in DLBCL, 7% in T-DLBCL and 1% in PCNSL; no positive cases were identified in either NLPHL or FL. The proportion of break-apart positive cases was significantly higher in PMBCL as compared to the other lymphomas surveyed (P < 0.05). Further, in agreement with the published literature, we observed an amplification frequency of the PDL locus in 36% (45/125) of PMBCLs. qRT-PCR established that PDCD1LG2 transcript levels were significantly higher in cases with 9p24.1 locus rearrangements compared to copy number neutral (P = 0.0003), gain (P = 0.001) and amplified cases (P = 0.005). Likewise, CD274 transcript levels were significantly higher in rearranged cases compared to copy number neutral cases (P = 0.03). Following the analysis of WGS and RNA-seq libraries, we were able to characterize four novel fusion transcripts involving the 9p24.1 locus: PDCD1LG2-NRG1 (PMBCL clinical case), PDCD1LG2-IGHV7-81 (L-1236), CIITA-PDCD1LG2 (DEV) and KIAA1432-CLDN14 (L-428). Aberrations involving both NRG1 and CIITA have previously been implicated in breast cancer and B-cell lymphomas, respectively. We also identified a translocation in another PMBCL clinical case with breakpoints in the intergenic spaces near LRMP and CD274, though this rearrangement did not produce a fusion transcript.
Taken together, our findings show that rearrangement of the PDL locus is recurrent in PMBCL, characteristic of PMBCL and leads to overexpression of PDL transcripts. Given the well-referenced function of PDLs in repressing the anti-tumor response, these data suggest that targeting the PDL axis in a subgroup of B-cell lymphomas holds clinical promise.
No relevant conflicts of interest to declare.
Classical Hodgkin lymphoma (cHL) is a common malignancy in children and adolescents. Although cHL is highly curable, treatment with chemotherapy and radiation often come at the cost of long-term ...toxicity and morbidity. Effective risk-stratification tools are needed to tailor therapy. Here, we used gene expression profiling (GEP) to investigate tumor microenvironment (TME) biology, to determine molecular correlates of treatment failure, and to develop an outcome model prognostic for pediatric cHL. A total of 246 formalin-fixed, paraffin-embedded tissue biopsies from patients enrolled in the Children's Oncology Group trial AHOD0031 were used for GEP and compared with adult cHL data. Eosinophil, B-cell, and mast cell signatures were enriched in children, whereas macrophage and stromal signatures were more prominent in adults. Concordantly, a previously published model for overall survival prediction in adult cHL did not validate in pediatric cHL. Therefore, we developed a 9-cellular component model reflecting TME composition to predict event-free survival (EFS). In an independent validation cohort, we observed a significant difference in weighted 5-year EFS between high-risk and low-risk groups (75.2% vs 90.3%; log-rank P = .0138) independent of interim response, stage, fever, and albumin. We demonstrate unique disease biology in children and adolescents that can be harnessed for risk-stratification at diagnosis. This trial was registered at www.clinicaltrials.gov as #NCT00025259.
•Gene signatures reflecting tumor microenvironment composition correlate with event-free survival of patients in the COG AHOD0031 trial.•An outcome prognostic model risk stratifies patients according to 5-year event-free survival.
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Abstract 427
Despite modern treatment strategies, about 20% of patients with classical Hodgkin lymphoma (cHL) die due to progressive disease. Little is known about the pathobiology underlying ...treatment failure, in part because the molecular phenotype of the rare malignant Hodgkin Reed Sternberg (HRS) cells is difficult to study. We recently reported associations of the expression profiles of microdissected HRS cells with primary treatment outcome (Steidl et al, ASH abstract 2009). The aim of this study was to explore possible mechanisms and validate these findings. PATIENTS AND METHODS: Twenty-nine cases of cHL treated with curative intent and that had mature follow-up (median 7.8 years) were evaluated by gene expression profiling (GEP) to discover differences between patients experiencing treatment failure, defined as disease progression at any time after initiation of primary therapy (n=14), and success, defined as the absence of progression (n=15). For validation experiments, CSF1R mRNA in-situ hybridization (ISH) was performed on 166 formalin-fixed paraffin-embedded pretreatment lymph node biopsies of cHL on a tissue microarray (TMA). Results were correlated with CD68 IHC available through a previous study (Steidl et al, NEJM 2010), and survival times. All patients received at least 4 cycles of ABVD-type polychemotherapy and stage-dependent radiotherapy. RESULTS: After dichotomizing the gene expression profiles according to the two primary treatment outcome groups, we found 42 up-regulated and 26 down-regulated genes in the treatment failure group (raw p<0.05). Specifically, using Ingenuity pathway analysis, we found genes involved in the developmental process of macrophages over-expressed in treatment failure samples. We chose CSF1R as representative of this overexpression for subsequent validation. For CSF1R ISH, 132 cases were evaluable, 63 cases (48%) stained positively in HRS cells. CSF1R positivity in HRS cells was correlated with non-nodular sclerosis histology (p=0.003, Chi-Square), the number of CD68+ cells in the tumor microenvironment (p=0.024, Chi-Square) and primary treatment failure (p=0.039, Chi-Square). Accordingly, CSF1R+ cases showed inferior progression-free (p=0.0114, log rank) and overall survival (p=0.0468, log rank). By combining CSF1R ISH with CD68 immunohistochemistry (IHC) we were able to define three risk groups: low-risk (CSF1R HRS-negative, CD68 low), high-risk (CSF1R HRS-positive, CD68 high) and an intermediate-risk group (all other patients). 10-year progression-free survival rates were significantly different (p=0.0008): 75% (n=24, low-risk), 42% (n=56, intermediate-risk) and 19.5% (n=52, high-risk). In a multivariate Cox regression model including the combined score and all factors of the International Prognostic Factors Project Score, the combined ISH/IHC score retained prognostic independence for progression-free (p=0.002) and overall survival (p=0.05). DISCUSSION: Using GEP of microdissected HRS cells we identified a gene signature of macrophage function in HRS cells that was correlated with adverse first line treatment outcome. The correlation of CSF1R expression in HRS cells with numbers of tumor-associated macrophages in the microenvironment suggests a functionally important interaction of HRS cells with macrophages via CSF-1 receptor signaling. These data using clinical samples are in agreement with a recently described functional role of CSF1R-dependent signaling in HL cell lines (Lamprecht et al., Nature Medicine 2010) and suggest CSF1R as a drug target of unfavorable risk cHL. Furthermore, the predictive power of a combined ISH/IHC score, reflecting this underlying biology linked to treatment failure in cHL, might be useful for risk stratification in future clinical trials. Display omitted
Connors:Roche: Research Funding.
Abstract 436
Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma that is characterized by the hallmark t(11;14)(q13;q32) translocation, as well as a high number of secondary ...chromosomal alterations. Further, a small number of genes such as TP53, ATM and CCND1 have been reported to be recurrently mutated in MCL, but do not fully explain the biology and do not adequately account for the wide spectrum of clinical manifestations, response to treatment and prognosis. The aim of this study was to discover new somatic mutations that could contribute to our understanding of the pathogenesis of MCL.
In our discovery cohort, we sequenced the transcriptomes of 18 clinical samples (11 diagnostic and 7 progression biopsies) and 2 mantle cell lymphoma-derived cell lines (Mino and Jeko-1). For this purpose, whole transcriptome shotgun sequencing was performed on RNA extracted from fresh frozen tissue. We assembled an extension cohort of 103 diagnostic patient samples and 4 additional cell lines (Rec-1, Z-138, Maver-1, JVM-2), and performed Sanger sequencing of NOTCH1 exons 26, 27 and 34 on genomic DNA. We further exposed the 6 cell lines to 1 μM of the γ-secretase inhibitor XXI (compound E) for 7 days and measured cellular proliferation with an EdU incorporation assay. Survival analysis was carried out in the 113 patients with diagnostic biopsies and available outcome data.
NOTCH1 mutations were found in 14 out of 121 patient samples (11.6%) and in 2 out of 6 cell lines, Mino and Rec-1 (33.3%). The majority of these mutations (12 out of 14) lie in exon 34 that encodes the PEST domain of NOTCH1 and consist of either small frameshift-causing indels (10 cases) or nonsense mutations (2 cases). These mutations are predicted to cause truncations of the C-terminal PEST domain. To gain further insight into functional relevance, we treated 6 cell lines with compound E, an inhibitor of the γ-secretase complex that plays a critical role in the release of the intracellular domain of NOTCH1 after ligand-induced activation. In Rec-1, that harbours a NOTCH1 mutation, we observed a significant decrease in proliferation (mean percentage of cells in culture incorporating EdU decreasing from 47.5% to 1.4%, p<.001). No effect of compound E was observed in Mino, the other cell line with a NOTCH1 mutation, nor in the 4 cell lines that are wild type for NOTCH1. Outcome correlation analysis showed that NOTCH1 mutations are associated with poor overall survival (1.56 versus 3.86 years respectively, p=.001), but not with significantly shortened progression-free survival (0.88 versus 1.73 years respectively, p=.07).
We have identified recurrent mutations in NOTCH1 in a subset of patients with MCL (11.6%). The frequency and the pattern of mutations are strikingly similar to what has recently been reported in chronic lymphocytic leukemia, the other major CD5 positive B-cell malignancy (Nature, 2011 Jun 5, 475:101–105 and J Exp Med, 2011 Jul 4, 208:1389–1401). NOTCH1 mutations are associated with adverse prognosis as evidenced by shortened overall survival. This latter finding, however, should ideally be validated in a larger and uniformly treated cohort. Finally, the sensitivity of the Rec-1 cell line to compound E suggests that NOTCH1 mutations could serve as the target for tailored therapy in mantle cell lymphoma.
Sehn:Roche/Genentech: Consultancy, Honoraria, Research Funding. Connors:Roche: Research Funding.
Abstract 268
Classical Hodgkin lymphoma (cHL) is unique among lymphomas due to the scarcity of the malignant Hodgkin Reed Sternberg (HRS) cells, which are derived from clonal germinal center B cells. ...Investigations using laser capture microdissection permit more detailed analysis of these cells. However, most recent studies were limited by low case numbers and lack of available clinical data.
We studied 29 cases of cHL and the 5 HL lines KMH2, HDLM2, L428, L540, and L1236 by gene expression profiling. All patients were treated at the BC Cancer Agency between 1984 and 2006 and received at least 4 cycles of polychemotherapy and stage-dependent radiotherapy. The cohort also included 5 biopsies taken at relapse. Treatment failure was defined as disease progression or relapse at any time (n=14) after initiation of treatment; treatment success as absence of progression (n=15). We used laser microdissection (Molecular Machines & Industries Cellcut with Nikon Eclipse TE2000-S microscope) to study the enriched HRS cell compartment separately from the microenvironment. RNA extraction was performed on pools of 1000 microdissected HRS cells in each case. Gene expression profiles were generated using Affymetrix HG UA133 2.0 Plus arrays using two-cycle labeling reactions. HRS cell profiles were compared to microdissected germinal centers (GC), and HL cell line profiles compared to enriched tonsillar CD77+ centroblasts (MACS cell separation, Miltenyi). Furthermore, we compared gene expression profiles of treatment failures to those of treatment successes.
We identified 1342 differentially expressed probesets (fold change >5, False Discovery Rate (FDR) adjusted p value <0.001) between HRS and GC cells. Using overrepresentation analysis we found genes involved in NFκB, JAK/Stat, IL-6, IL-9 signaling, cytotoxic T lymphocyte-mediated apoptosis and IL-15 production to be significantly over-expressed in HRS cells and genes involved in B cell, T cell receptor signaling and many transcriptional regulators such as FOXO1, E2F5, IRF8, NFATC1, NFYB, POU2AF1 to be significantly under-expressed in HRS cell. Comparison of these data to differentially expressed genes in the HL cell lines (1004 genes, fold change >5, FDR-adjusted p value <0.001) showed significant overlap of genes involved in proliferation, apoptosis, IL15 signaling and B cell receptor signaling, while overrepresentation of metabolism genes was unique to the cell lines. Hierarchical clustering of all 29 primary HL cases identified 3 separate clusters characterized by 1) a cytotoxic signature, 2) TNF/TGFB receptor signaling or 3) a residual B cell signature. Dichotomizing the profiles into the two treatment outcome groups demonstrated that NFγB signaling, complement system genes and genes involved in the developmental process of hematopoietic progenitor cells, macrophages and blood vessels were overexpressed in treatment failure samples.
Using microdissection of HRS cells in a large number of cases we were able to further characterize the unique expression program of HL and refine the data inventory about dysregulated cellular functions and pathways in this disease. Overexpression of genes associated with NFκB, complement and hematopoietic progenitor cells proliferation correlate strongly with treatment failure. Further study using immunohistochemistry is currently ongoing to validate these findings and to develop clinically useful biomarkers.
Gascoyne:Roche Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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