B-cell Gene Signatures Research Articles

Abstract B010: Tumor B8T expression stratifies overall survival in high risk muscle invasive urothelial carcinoma

Abstract Background and Objective: Determining novel biomarkers to identify patients that benefit and do not benefit from immune checkpoint inhibitor (ICI) therapy is critical to avoid overtreatment. Circulating tumor DNA (ctDNA) has emerged as a biomarker that associates with overall survival (OS) benefit to ICI in patients with muscle invasive urothelial carcinoma (MIUC). However, few studies have examined whether existing immune based biomarkers stratify OS within the high risk, ctDNA(+) population. Methods: To address this, we interrogated ctDNA and RNA sequencing data from the IMvigor010 dataset of patients with high risk MIUC after cystectomy who were randomized to adjuvant atezolizumab versus observation. We correlated OS with ctDNA positivity and expression of a B cell and CD8 T cell gene signature (B8T). Key Findings: In patients who were ctDNA(+), high tumor expression of a B cell gene signature (BCGS) associated with significantly higher OS when compared with patients with low tumor BCGS, regardless of whether patients were treated with atezolizumab. In ctDNA(+) patients with high tumor expression of both BCGS and CD8 T cell gene signature (CD8TGS), e.g. B8T high/high, atezolizumab did not associate with OS benefit. Conversely, in patients with B8T high/low tumors, receipt of adjuvant atezolizumab associated with significantly increased OS (p < 0.001), and this benefit remained significant in multivariable analyses (p = 0.047). Notably, benefit with atezolizumab occurred in patients with B8T high/low tumors who were either ctDNA(+) (p = 0.057) or ctDNA(-) (p = 0.034). Conclusions and Clinical Implications: While tumor B8T high/high expression is a prognostic biomarker for OS in ctDNA(+) patients with high risk MIUC, these results suggest B8T high/low is a predictive biomarker for benefit from ICI. Moreover, in a small subset of patients who are ctDNA(-), tumor B8T high/low associates with benefit to ICI. This supports the utility of the B8T biomarker as a prognostic identifier in high risk MIUC, its ability to identify patients who will benefit from ICI regardless of ctDNA status, and for the first time ascertains a potential cohort of patients who are ctDNA(-) yet benefit from ICI. Citation Format: Roy Elias, Adam K. Aragaki, Jean H. Hoffman-Censits, Noah M. Hahn, David J. McConkey, Burles A. Johnson III. Tumor B8T expression stratifies overall survival in high risk muscle invasive urothelial carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B010.

Clinical and molecular characterization of long-term survivors with extensive-stage small cell lung cancer treated with first-line atezolizumab plus carboplatin and etoposide

ObjectivesIn the Phase I/III IMpower133 study, first-line atezolizumab plus carboplatin and etoposide (CP/ET) treatment for extensive-stage small cell lung cancer (ES-SCLC) significantly improved overall survival (OS) and progression-free survival versus placebo plus CP/ET. We explored patient and disease characteristics associated with long-term survival in IMpower133, and associations of differential gene expression and SCLC-A (ASCL1-driven), SCLC-N (NEUROD1-driven), SCLC-P (POU2F3-driven), and SCLC-inflamed (SCLC-I) transcriptional subtypes with long-term survival. Materials and MethodsPatients with previously untreated ES-SCLC were randomized 1:1 to four 21-day cycles of CP/ET with atezolizumab or placebo. Long-term survivors (LTS) were defined as patients who lived ≥ 18 months post randomization. A generalized linear model was used to evaluate the odds of living ≥ 18 months. Differential gene expression was analyzed using RNA-sequencing data in LTS and non-LTS. OS was assessed by T-effector and B-cell gene signature expression. Distribution of SCLC transcriptional subtypes was assessed in LTS and non-LTS. ResultsMore LTS were in the atezolizumab arm (34%) than in the placebo arm (20%). The odds ratio for living ≥ 18 months in the atezolizumab arm versus the placebo arm was 2.1 (P < 0.03). Enhanced immune-related signaling was seen in LTS in both arms. Exploratory OS analyses showed atezolizumab treatment benefit versus placebo across T-effector and B-cell gene signature expression subgroups. A higher proportion of LTS than non-LTS in both arms had the SCLC-I subtype; this difference was particularly pronounced in the atezolizumab arm. ConclusionThese exploratory analyses suggest that long-term survival is more likely with atezolizumab than placebo in ES-SCLC, confirming the treatment benefit of the IMpower133 regimen.ClinicalTrial.gov Identifier: NCT02763579.

Identifying the Cells of Origin and Risk-Stratification Strategy By Single-Cell Sequencing in Multiple Myeloma

Background: With the advent of novel agents, poor prognosis remains an unsolved problem for some patients. Identifying patients with super-high-risk and offering novel therapeutic strategy is essential. The aim of our study is to infer the identity of cells of origin in MM and dig out the novel strategy for risk sub-stratification and treatment. Method: We applied single cell RNA sequencing to fresh bone marrow mononuclear cell samples from 7 healthy donors and 12 newly diagnosed MM patients utilizing 10x Chromium platform. Results: Firstly, we separated plasma cells (PCs) in silico and identified 10 cellular subpopulations in total. Via the CNV score and pseudotime trajectory analysis, we distinguished normal PCs and observed the differentiation trajectory from normal PCs to malignant PCs. We identified cluster 4 as the starting point of malignant transition and possessed plasmablast-like gene signature, which displayed high expression of B-cell gene signatures and specifically high level of CD24. Additionally, cluster 4 was featured by high expression of high-risk genes and drug-resistance genes. Gene enrichment analysis implicated that Wnt, Notch, stem cell differentiation and Hedgehog pathway were enriched in cluster4. Notably, we identified the top upregulated differentially expressed genes in cluster 4 including LILRB4, CRIP1, etc. We next explored their biological function in MM cell lines. We found that both LILRB4 and CRIP1 could promote the proliferation of MM cells, indicating their potential novel therapeutic target in MM. Finally, we constructed a 7-Star stratification model, which was validated to better facilitate discrimination of super-high-risk MM patients when combined with International Staging System (ISS). Conclusion: Our study inferred the cells of origin in MM at single cell resolution. We suggested targeting marker genes of cells of origin as potential novel therapeutic strategy, and our 7-Star stratification model as a powerful tool for discrimination of super-high-risk MM patients when combined with ISS stage.

Differential Changes in ACPA Fine Specificity and Gene Expression in a Randomized Trial of Abatacept and Adalimumab in Rheumatoid Arthritis.

IntroductionThe biologics abatacept and adalimumab have different mechanisms of action (MoAs). We analyzed data from patients with rheumatoid arthritis treated in AMPLE (NCT00929864) to explore the pharmacodynamic effects of abatacept or adalimumab on anti-citrullinated protein antibodies (ACPAs) and gene expression.MethodsAMPLE was a phase IIIb, 2-year, randomized, head-to-head trial of abatacept versus adalimumab. Post hoc analyses of baseline anti-cyclic citrullinated peptide-2 (anti-CCP2, an ACPA surrogate) positive (+) status and ACPA fine-specificity profiles over time, as well as transcriptional profiling (peripheral whole blood), were performed.ResultsOf 646 patients treated (abatacept, n = 318; adalimumab, n = 328), ACPA and gene expression data were available from 508 and 566 patients, respectively. In anti-CCP2+ patients (n = 388), baseline fine specificities for most ACPAs were highly correlated; over 2 years, levels decreased with abatacept but not adalimumab. By year 2, expression of genes associated with T cell co-stimulation and antibody production was lower for abatacept versus adalimumab; expression of genes associated with proinflammatory signaling was lower for adalimumab versus abatacept. Treatment modulated the expression of T- and B-cell gene signatures, with differences in CD8+ T cells, activated T cells, plasma cells, B cells, natural killer cells (all lower with abatacept versus adalimumab), and polymorphonuclear leukocytes (higher with abatacept versus adalimumab).ConclusionsIn AMPLE, despite similar clinical outcomes, data showed that pharmacodynamic/genetic changes after 2 years of abatacept or adalimumab were consistent with drug MoAs. Further assessment of the relationship between such changes and clinical outcomes, including prediction of response, is warranted.Trial RegistrationClinicalTrials.gov identifier, NCT00929864.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40744-021-00404-x.

Identification of Myeloma-Initiating Cells By Single-Cell RNA Sequencing

Background: The outcomes of patients with multiple myeloma (MM) have improved due to treatment advances. However, some patients still experience rapid progression, multiple drug resistance or recurrent relapse. Tumor-initiating cells, also known as cancer stem cells (CSC) in some cancer types, have been speculated to induce recurrence of the disease. The aim of this study is to infer the identity of myeloma-initiating cells (MICs) utilizing single-cell transcriptome analysis and explore the unique biological characteristics relating with high-risk and drug-resistance.Method: We applied single cell RNA sequencing to fresh bone marrow mononuclear cell samples collecting from 7 healthy donors and 12 newly diagnosed MM patients utilizing 10x Chromium platform.Results: Firstly, we segregated the patients by tumor cell infiltration at single cell resolution and found that Myc pathway was significantly enriched in patients with high tumor burden (HTB). Next, we performed clustering analysis to tumor cells and identified 13 tumor subpopulations in total. Surprisingly, the distribution pattern of tumor subpopulations presented similarity among HTB patients, whereas we did not find the uniform subpopulation composition among the patients with low (LTB) or medium tumor burden (MTB). Via the tumor subpopulation analysis, we clarified the divergence in biological characteristics of these 13 malignant subpopulations. We identified plasmablasts as displaying high expression of B-cell gene signatures (CD19, CD27, MS4A1 and CD79B) and relatively low expression of plasma cell gene signatures (SDC1 and BCMA). Additionally, we also noted that they showed high level of CD24, which has been validated to be the marker gene for MICs. We next examined proliferative capability and utilized the 70 high-risk gene model and 56 drug resistance-related gene model to further distinguish subpopulations with the most malignant gene expression features. Notably, we found that plasmablasts possessed characteristics of high proliferation, drug-resistance and high-risk gene profiling, indicating their role as the root of myeloma, namely MIC subpopulation. Gene enrichment analysis also implicated that Wnt pathway, Notch pathway, stem cell differentiation pathway and Hedgehog pathway were enriched in MIC subpopulation which were associated with the proliferation, migration and drug resistance of MM. Differentially expressed gene (DEG) analysis showed that common driver genes in myeloma, such as CCND2, ITGB7 and CD74, were upregulated in MIC subpopulation comparing with other subpopulations.Conclusion: Our work presents an integral profiling for tumor cells in myeloma at single cell resolution. We uncovered divergence in the distribution of malignant subclusters across patients and distinct heterogeneity in gene expression across malignant subclusters as well. Plasmablasts expressing high level of CD24, CD27 and dim CD138 presented as the MICs with characteristics of higher proliferation, drug-resistance and high-risk gene profiling. DisclosuresNo relevant conflicts of interest to declare.

High Expression of Programmed Death-Ligand 1 (PD-L1) Correlates with Macrophage Gene Expression and Is Associated with Prolonged Progression-Free Survival (PFS) in Patients (pts) with First-Line (1L) Diffuse Large B-Cell Lymphoma (DLBCL)

▪Introduction PD-L1 and its receptor PD-1 are both expressed on multiple cell types, including antigen-presenting cells, T cells and tumor cells. In DLBCL, PD-L1 expression on non-malignant immune cells is common, whereas tumor-cell PD-L1 expression is restricted to a small percentage of pts. Because PD-L1/PD-1 are expressed on macrophages after in vitro activation, and activated macrophages contribute to antibody-dependent cellular phagocytosis (ADCP), high PD-L1 expression may identify tumors enriched with activated tumor-infiltrating macrophages primed for ADCP. Considering the role of ADCP in clinical responses to anti-CD20 (rituximab/obinutuzumab), we hypothesized that PD-L1 expression could reflect abundance of activated tumor-infiltrating macrophages and may be associated with prolonged PFS in 1L DLBCL. We leveraged 2 large Phase 3 trials and cell-line immune gene expression to better understand the biological and clinical relevance of PD-L1 expression in 1L DLBCL.Methods We used clinical, genomic and pre-treatment immunohistochemistry (IHC) data from 1L DLBCL pts treated in the MAIN (NCT00486759) and GOYA (NCT01287741) trials. Pre-treatment tissue microarray cores from 201 MAIN pts were stained for PD-L1 (clone SP263), with 21 uninvolved lymph node FFPE sections used as controls. PD-L1+ samples were defined as IHC > 1+ (> 1% tissue area occupied by PD-L1+ cells). Tumor cells were identified based on morphological/cytological features, and PD-L1 tumor expression was scored as a percentage. Activated B-cell (ABC), germinal center B-cell (GCB) and unclassified DLBCL prognostic subtypes were determined by the NanoString Lymph2Cx cell-of-origin (COO) assay. We quantified mRNA expression of PD-L1, macrophage and B-cell genes using TruSeq (Illumina) RNAseq among 702 DLBCL pts (GOYA, n = 552; MAIN, n = 150), with 28 DLBCL cell lines and 15 purified normal B-cell samples as comparators. High/low expression of each gene signature was determined by median cutoffs. Median follow-up durations were 24 and 29 mo in MAIN and GOYA, respectively (≈ 30% of PFS events in both cohorts). We used Cox regression to examine associations between these markers and PFS, adjusting for IPI, age, COO, sex and treatment arm.Results Among 201 DLBCL pts in MAIN, PD-L1 was primarily detected on inter-follicular cells with myeloid/dendritic morphology, similar to the PD-L1 staining pattern of normal lymph nodes. In samples with evaluable germinal centers (GCs), a subset of GC cells expressed PD-L1 with a punctate and granular pattern consistent with a myeloid origin. Overall, there were significantly more pts with high PD-L1 expression by IHC (IHC 3+) and mRNA among ABC vs GCB DLBCL pts. A minority of DLBCL pts (10%) had PD-L1+ tumor cells, consistent with low/undetectable CD274 (PD-L1) mRNA in the majority of DLBCL cell lines and purified normal B-cell samples tested. Additionally, among the 702 evaluable pts in both studies, CD274 mRNA inversely correlated with a B-cell gene signature (MAIN, r= −0.55; GOYA, r= −0.32). High PD-L1 expression (IHC 2+/3+) correlated with prolonged PFS (HR, 0.44 [0.24, 0.80]; P < 0.01) among the 201 evaluable pts in MAIN. Among 552 evaluable pts in GOYA, CD274 expression was higher in pts with a PET-CR (P= 0.02), particularly in pts with ABC DLBCL (P= 0.0001), with a trend toward prolonged PFS in the ABC subtype (HR, 0.80 [0.63, 1.0]; P= 0.054). CD274 mRNA positively correlated with macrophage gene expression in both cohorts (MAIN, r= 0.61; GOYA, r= 0.57); pts with high PD-L1 expression (IHC 2+/3+) also demonstrated high mRNA expression of the macrophage marker CD68. Consistent with PD-L1 reflecting abundance of activated tumor-infiltrating macrophages relevant to anti-CD20 response, high expression of a macrophage signature correlated with prolonged PFS (HR, 0.63 [0.45, 0.87]; P= 0.003) in GOYA and a trend toward prolonged PFS among DLBCL pts treated in MAIN (HR, 0.64 [0.35, 1.16]; P= 0.10), although statistical significance was not reached.Conclusions In 1L DLBCL, most pts expressed PD-L1 on immune cells with myeloid/dendritic morphology, potentially reflecting activated macrophages primed for ADCP and supportive of the correlation between high PD-L1 expression and improved clinical outcomes among pts treated with immunochemotherapy. In the context of anti-CD20, the biology of PD-L1 expression on macrophages and the impact of PD-L1/PD-1 blockade on ADCP merit further study. DisclosuresMcCord:Genentech, Inc.: Employment. Bolen:Genentech: Employment, Equity Ownership. Koeppen:Genentech, Inc.: Employment. Kadel:Genentech, Inc.: Employment. Fingerle-Rowson:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Oestergaard:F. Hoffmann-La Roche Ltd: Employment. Venstrom:Genentech, Inc.: Employment.

VP5-2021: IMpower133: Gene expression (GE) analysis in long-term survivors (LTS) with ES-SCLC treated with first-line carboplatin and etoposide (CE) ± atezolizumab (atezo)

Exploratory analyses to characterise LTS (patients [pts] who survived ≥18 mo post randomisation) in IMpower133 showed that more LTS were treated with atezo (anti–PD-L1) + CE than placebo (PBO) + CE (Liu et al. ESMO 2020). Here, we report the association of differential GE (single genes, T-effector [Teff] and B-cell signatures) and published SCLC subtypes (Gay et al. Cancer Cell 2021) in IMpower133 LTS. Treatment-naive pts with ES-SCLC were randomised 1:1 to four 21-day cycles of C (AUC 5 mg/mL/min IV, day 1) + E (100 mg/m2 IV, days 1-3) combined with atezo (1200 mg IV, day 1) or PBO, followed by maintenance atezo or PBO until disease progression or toxicity. Co-primary endpoints were PFS and OS. Differential GE was analysed using RNA-sequencing (RNA-seq) data in LTS and non-LTS. OS was assessed by Teff and B-cell gene signature expression in both arms. Distribution of recently identified neuroendocrine, non-neuroendocrine and immune-infiltrated subtypes were also assessed in LTS and non-LTS. 271 pts were in the RNA-seq biomarker-evaluable population, 253 of whom had sufficient follow-up for LTS classification (LTS RNA-seq BEP; LTS, n=64; non-LTS, n=189). Within the LTS RNA-seq BEP, significantly more LTS were in the atezo (n=39, 32%) vs PBO arm (n=25, 19%; P=0.027). In both arms within the LTS RNA-seq BEP, distributions of Teff- and B-cell−related genes and gene signatures (≥ median vs < median) were similar in LTS. Exploratory OS efficacy analyses in the LTS RNA-seq BEP suggest that benefit was observed in favour of atezo vs PBO across all Teff and B-cell expression subgroups (high [≥ median]: Teff, HR, 0.65 [95% CI, 0.43, 0.97]; B-cell, HR, 0.75 [95% CI, 0.51, 1.11], and low [< median]: Teff, HR, 0.75 [95% CI. 0.52, 1.11]; B-cell, HR, 0.66 [95% CI: 0.45, 0.97]). Among the LTS RNA-seq BEP in both arms, GE subtype distribution was broadly similar for LTS. In IMpower133, more LTS were treated with atezo + CE vs PBO + CE. Enhanced immune-related signaling was seen in LTS in both arms. Atezo and PBO had similar GE-defined subtype distribution, suggesting that subtype associations with outcome may be prognostic.

Role of Tumor-Infiltrating B Cells in Clinical Outcome of Patients with Melanoma Treated With Dabrafenib Plus Trametinib.

Although patients with unresectable or metastatic melanoma can experience long-term survival with BRAF- and MEK-targeted agents or immune checkpoint inhibitors over 5 years, resistance develops in most patients. There is a distinct lack of pretherapeutic biomarkers to identify which patients are likely to benefit from each therapy type. Most research has focused on the predictive role of T cells in antitumor responses as opposed to B cells. We conducted prespecified exploratory biomarker analysis using gene expression profiling and digital pathology in 146 patients with previously untreated BRAF V600-mutant metastatic melanoma from the randomized, phase III COMBI-v trial and treated with dabrafenib plus trametinib who had available tumor specimens from screening. Baseline cell-cycle gene expression signature was associated with progression-free survival (P = 0.007). Patients with high T-cell/low B-cell gene signatures had improved median overall survival (not reached [95% confidence interval (CI), 33.8 months-not reached]) compared with patients with high T-cell/high B-cell signatures (19.1 months; 95% CI, 13.4-38.6 months). Patients with high B-cell signatures had high B-cell infiltration into the tumor compartment, corresponding with decreased MAPK activity and increased expression of immunosuppressive markers. B cells may serve as a potential biomarker to predict clinical outcome in patients with advanced melanoma treated with dabrafenib plus trametinib. As separate studies have shown an opposite effect for B-cell levels and response to immunotherapy, B cells may serve as a potential biomarker to facilitate treatment selection. Further validation in a larger patient cohort is needed.

Impact of Anti-HER2 Treatments Combined With Atezolizumab on the Tumor Immune Microenvironment in Early or Metastatic Breast Cancer: Results From a Phase Ib Study

BackgroundDespite advances, there continues to be unmet need in breast cancer. Combining anti–programmed death-ligand 1 (PD-L1) cancer immunotherapy atezolizumab with other targeted therapies may enhance T-cell–dependent cytolytic antitumor activity. MethodsThis open-label, phase Ib study evaluated the safety of atezolizumab-based combinations with antibody-dependent cellular cytotoxicity or antibody-drug conjugate (ADC) agents. Patients with unresectable human epidermal growth factor receptor 2–positive (HER2+) locally advanced or metastatic breast cancer (mBC) received atezolizumab with trastuzumab/pertuzumab, atezolizumab with the ADC ado-trastuzumab emtansine (T-DM1), or atezolizumab with trastuzumab/pertuzumab and docetaxel. In an early–breast cancer (eBC) “window of opportunity” study, patients with operable HER2+ locally advanced or inflammatory eBC received neoadjuvant atezolizumab with trastuzumab/pertuzumab or atezolizumab/T-DM1, followed by docetaxel, carboplatin, and trastuzumab/pertuzumab. Exploratory outcomes included tumor response and biomarkers. ResultsBy March 15, 2019, 73 patients were enrolled. Safety findings were consistent with the treatment components’ individual profiles. Objective responses were observed in 2 of 6 and 5 of 14 patients in 2 mBC cohorts receiving atezolizumab/T-DM1 and in 6 of 6 patients with mBC receiving atezolizumab, trastuzumab/pertuzumab, and docetaxel. PD-L1 in immune cells was the only biomarker that increased with atezolizumab/T-DM1. In the window of opportunity cohorts, PD-L1 levels and CD8+ T-cell infiltration increased from baseline in HER2+ eBC tumors receiving atezolizumab with trastuzumab/pertuzumab or T-DM1, irrespective of response. Despite increases in T-cell and B-cell gene signatures with trastuzumab/pertuzumab, but not T-DM1, neither combination promoted T-cell receptor clonal expansion. ConclusionAtezolizumab with antibody-dependent cellular cytotoxicity or ADC agents appears safe and may activate the adaptive immune system of patients with HER2+ eBC tumors more than those with mBC tumors.

Changes in Transcript, Metabolite, and Antibody Reactivity During the Early Protective Immune Response in Humans to Mycobacterium tuberculosis Infection.

BackgroundStrategies to prevent Mycobacterium tuberculosis (Mtb) infection are urgently required. In this study, we aimed to identify correlates of protection against Mtb infection.MethodsTwo groups of Mtb-exposed contacts of tuberculosis (TB) patients were recruited and classified according to their Mtb infection status using the tuberculin skin test (TST; cohort 1) or QuantiFERON (QFT; cohort 2). A negative reading at baseline with a positive reading at follow-up classified TST or QFT converters and a negative reading at both time points classified TST or QFT nonconverters. Ribonucleic acid sequencing, Mtb proteome arrays, and metabolic profiling were performed.ResultsSeveral genes were found to be differentially expressed at baseline between converters and nonconverters. Gene set enrichment analysis revealed a distinct B-cell gene signature in TST nonconverters compared to converters. When infection status was defined by QFT, enrichment of type I interferon was observed. A remarkable area under the curve (AUC) of 1.0 was observed for IgA reactivity to Rv0134 and an AUC of 0.98 for IgA reactivity to both Rv0629c and Rv2188c. IgG reactivity to Rv3223c resulted in an AUC of 0.96 and was markedly higher compared to TST nonconverters. We also identified several differences in metabolite profiles, including changes in biomarkers of inflammation, fatty acid metabolism, and bile acids. Pantothenate (vitamin B5) was significantly increased in TST nonconverters compared to converters at baseline (q = 0.0060).ConclusionsThese data provide new insights into the early protective response to Mtb infection and possible avenues to interfere with Mtb infection, including vitamin B5 supplementation.Analysis of blood from highly exposed household contacts from The Gambia who never develop latent Mycobacterium tuberculosis infection shows distinct transcriptomic, antibody, and metabolomic profiles compared to those who develop latent tuberculosis infection but prior to any signs of infection.

A B-Cell Gene Signature Correlates With the Extent of Gluten-Induced Intestinal Injury in Celiac Disease.

Background & AimsCeliac disease (CeD) provides an opportunity to study autoimmunity and the transition in immune cells as dietary gluten induces small intestinal lesions.MethodsSeventy-three celiac disease patients on a long-term, gluten-free diet ingested a known amount of gluten daily for 6 weeks. A peripheral blood sample and intestinal biopsy specimens were taken before and 6 weeks after initiating the gluten challenge. Biopsy results were reported on a continuous numeric scale that measured the villus-height–to–crypt-depth ratio to quantify gluten-induced intestinal injury. Pooled B and T cells were isolated from whole blood, and RNA was analyzed by DNA microarray looking for changes in peripheral B- and T-cell gene expression that correlated with changes in villus height to crypt depth, as patients maintained a relatively healthy intestinal mucosa or deteriorated in the face of a gluten challenge.ResultsGluten-dependent intestinal damage from baseline to 6 weeks varied widely across all patients, ranging from no change to extensive damage. Genes differentially expressed in B cells correlated strongly with the extent of intestinal damage. A relative increase in B-cell gene expression correlated with a lack of sensitivity to gluten whereas their relative decrease correlated with gluten-induced mucosal injury. A core B-cell gene module, representing a subset of B-cell genes analyzed, accounted for the correlation with intestinal injury.ConclusionsGenes comprising the core B-cell module showed a net increase in expression from baseline to 6 weeks in patients with little to no intestinal damage, suggesting that these individuals may have mounted a B-cell immune response to maintain mucosal homeostasis and circumvent inflammation. DNA microarray data were deposited at the GEO repository (accession number: GSE87629; available: https://www.ncbi.nlm.nih.gov/geo/).

A B-Cell Gene Signature Correlates With the Extent of Gluten-Induced Intestinal Injury in Celiac Disease

A B-Cell Gene Signature Correlates With the Extent of Gluten-Induced Intestinal Injury in Celiac Disease

FRI0101 The value of gene signatures in the diagnosis of pre-clinical ra

BackgroundDiagnosis of the preclinical phase of rheumatoid arthritis (RA) allows timely start of treatment with the potential to prevent disease progression. It is known that antibodies against citrullinated proteins (ACPA)...

Horizontal transmission of malignancy: in-vivo fusion of human lymphomas with hamster stroma produces tumors retaining human genes and lymphoid pathology.

We report the in-vivo fusion of two Hodgkin lymphomas with golden hamster cheek pouch cells, resulting in serially-transplanted (over 5–6 years) GW-532 and GW-584 heterosynkaryon tumor cells displaying both human and hamster DNA (by FISH), lymphoma-like morphology, aggressive metastasis, and retention of 7 human genes (CD74, CXCR4, CD19, CD20, CD71, CD79b, and VIM) out of 24 tested by PCR. The prevalence of B-cell restricted genes (CD19, CD20, and CD79b) suggests that this uniform population may be the clonal initiating (malignant) cells of Hodgkin lymphoma, despite their not showing translation to their respective proteins by immunohistochemical analysis. This is believed to be the first report of in-vivo cell-cell fusion of human lymphoma and rodent host cells, and may be a method to disclose genes regulating both organoid and metastasis signatures, suggesting that the horizontal transfer of tumor DNA to adjacent stromal cells may be implicated in tumor heterogeneity and progression. The B-cell gene signature of the hybrid xenografts suggests that Hodgkin lymphoma, or its initiating cells, is a B-cell malignancy.

P2-12-11: Clinical Relevance of a IL-8/B-Cell Gene Signature Identified from Triple Negative Breast Cancer (TNBC) in Intrinsic Breast Cancer Subtypes.

Abstract Background: As presented recently (SABCS 2010, #S5-5) a ratio of high B-cell and low IL-8 metagenes using gene expression analysis identified 32 % of triple negative breast cancers with good prognosis and was the only significant predictor in multivariate analysis including routine clinicopathological variables. However, the clinical relevance of this signature within the intrinsic breast cancer subtypes still remains unclear and is analyzed here. Methods: Affymetrix gene expression data from n=2417 breast cancer patients have been assembled. We performed an unsupervised analysis to define metagenes that distinguish molecular subsets within TNBC (SABCS 2010, #S5-5). A high expression of B- cell metagenes was associated with good and high expression of IL-8-related metagenes were associated with poor prognosis. To identify intrinsic subtypes we used the method previously described by Hu et al. (2006) and the prognostic value within those subtypes was assessed by analyzing the event free survival of patients as function of high and low B-cell/IL-8 metagene ratio. Results: Comparing ER positive with ER negative patients the B-ceh7LL-8 ratio showed only in ER negative breast cancer a significant prognostic value (log rank p-value &amp;lt;.0001). Within the entire cohort 37.8 % of patients could be assigend to luminal A, 35.2 % to luminal B, 7,4 % to erbB2 and 19.6 % to basal-like subtypes. Event free survival of patients with good or poor B-cell/IL-8 ratio showed only in basal-like breast cancer patients a statistical significant difference (p&amp;lt;.0001). However, we could not observe any difference in prognosis in luminal A and B, as well as erbB2 tumors. No difference in the expression of the proliferation metagene was observed when samples of the intrinsic subtypes were stratified according to the prognostic predictor based on high expression of the B-Cell metagene and low expression of the IL-8 metagene. Conclusion: The B-cell/IL-8 ratio is highly prognostic in basal-like/TNBC and shows no association with proliferation status. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-11.

Bortezomib Resistance in Mantle Cell Lymphoma Is Associated with Expression of a Plasmacytoid Differentiation Program.

Abstract 287Mantle cell lymphoma (MCL) is a lymphoproliferative disorder of mature B-cells with an aggressive course and short survival. The proteasome inhibitor bortezomib (BZM) induces clinical responses in up to 50% of patients. Conversely, in half of the cases the lymphoma cells are intrinsically resistant or rapidly develop resistance to BZM. To investigate the mechanisms of BZM resistance, we generated HBL2 and JEKO bortezomib resistant (HBL2-BR, JEKO-BR) derivative lines by continuous culture in sub-lethal concentrations of BZM. After several months, clones of HBL2-BR and JEKO-BR were obtained showing BZM IC50 at 48h of 41.6 and 44.6 nM, compared to 6 and 4.9 nM for the respective parental lines. Acquired resistance to BZM remained stable over months but gradually decreased with extended passages in the absence of BZM, suggesting adaptive changes rather than a single gene mutation as the basis of BZM resistance. BR cells exhibited higher proteasome activity, which was dose-dependently inhibited by higher concentrations of BZM. However, BR cells were able to survive with lower proteasome activity than the parental cells, indicating that BR cells had acquired additional changes. To investigate these changes, we use gene expression profiling (GEP) on Affymetrix U133A plus 2 arrays to compared HBL2-BR (in triplicate) and JEKO-BR (in duplicate) subclones to the corresponding parental lines. Unexpectedly, Gene Set Enrichment Analysis (GSEA) of microarray data revealed reduced expression of the mature B-cell gene signature (including genes for CD19, BLNK, SPIB, SYK) and increased expression of plasma cell differentiation signatures (including genes for CD38, IRF4, BLIMP, CD138) in both HBL-2 BR and JEKO-BR. BR lines also expressed higher protein levels of the master plasma cell regulators BLIMP and IRF4, but did not show enhanced expression of the secretory program controlled by XBP1. Flow cytometry analysis confirmed that BR cells had dramatically reduced expression of B-cell surface markers, including CD19, CD24 and CD52, and expressed plasma cell markers, such as CD38 and CD138. Consistent with a partial plasmacytoid phenotype, BR cells tended to be somewhat larger and more granular than parental cells. Loss of BZM resistance over months of culture in the absence of BZM was paralleled by the recovery of CD19 and CD24 expression and down-regulation of CD38, supporting a mechanistic link between the acquisition of a plasmacytoid phenotype and BZM resistance. We have previously shown that the MCL cell lines Mino and REC-1 are less sensitive to BZM than HBL-2, JEKO and most other MCL cell lines. Here we found that these constitutively resistant cells also showed plasmacytoid features including CD38 and CD138 surface expression, increased granularity and size, and an enlarged endoplasmic reticulum (ER). Combined these changes may enhance the ability of the cells to deal with an increased protein load due to bortezomib inhibition. In addition, we also observed higher expression of IRF4 and its target genes in the constitutively resistant cells, as well as higher IRF4 and CD38 expression in primary tumor cells of patients with poor response to BZM. Given the important role of IRF4 as a survival factor in multiple myeloma, we tested whether BZM treatment could decrease IRF4 expression in MCL cells. Indeed, within 24 hours BZM dose-dependently decreased IRF4 expression and the degree of downregulation of IRF4 correlated with the induction of apoptosis. Knockdown of IRF4 expression by shRNA has been shown to be toxic to myeloma cells (Shaffer et al, Nature 2008). Surprisingly, we found a similar toxic effect of IRF4 knockdown using the same inducible shRNA system in the MCL cell lines HBL2, JEKO and REC, which was more prominent in the latter BZM resistant cell line. These results identify loss of IRF4 expression as an additional mechanism by which BZM may induce cell death. However, overexpression of IRF4 in MCL cells is not sufficient to induce bortezomib resistance, indicating that several components of the plasma cell program cooperate to protect cells from BZM induced apoptosis. Furthermore, we have identified markers of BZM resistance that may be clinically relevant predictors of outcome. Disclosures:No relevant conflicts of interest to declare.