Tumor microenvironment has a strong effect on the survival of follicular lymphoma (FL) patients. The aim of this study was to determine what are the signaling pathways that mediate the cross-talk ...between lymphoma cells and tumor-infiltrating inflammatory cells and contribute to the clinical outcome of FL patients.
Gene expression profiling and pathway impact analyses were done from pretreatment lymphoma tissue of 24 patients. The findings were validated immunohistochemically in an independent cohort of 81 patients. All patients were treated with the combination of rituximab and cyclophoshamide-doxorubicin-vincristine-prednisone chemotherapy. In addition, microarray was used to screen the genes differentially expressed between control and rituximab-stimulated B-cell lymphoma cells in culture.
Among the transcripts differentially expressed in the FL tissues between the patients with favorable or adverse outcomes, an overrepresentation of genes associated with the signal transducers and activators of transcription (STAT)5a pathway was observed. In a validation set, a better progression-free survival was observed among the patients with high STAT5a protein expression. In the FL tissue, STAT5a positivity was barely detectable in the neoplastic B cells, but a subpopulation of follicular dendritic cells and T lymphocytes showed prominent STAT5a expression. Rituximab was found to induce the expression of STAT5a-associated interleukin-15 in B-lymphoma cells in culture, thereby providing a possible explanation for the cross-talk between rituximab-stimulated FL cells and their microenvironment.
The findings suggest that STAT5a activity in immunologically active nonmalignant cells acts as molecular predictor for rituximab and cyclophoshamide-doxorubicin-vincristine-prednisone-treated FL patients.
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8074
Background: Identification of biological prognostic factors that could be used to define poor risk diffuse large B-cell lymphoma (DLBCL) patients is a main concern. Methods: Study ...population consisted of 38 de novo high risk DLBCL patients less than 65 years old. The patients were treated in the Nordic phase II protocol with six courses of R-CHOEP14 followed by systemic central nervous system prophylaxis with one course of high dose methotrexate and one course of high dose cytarabine. Exon array-based profiling was used to screen signaling pathways and differentially expressed genes between the clinically high risk patients, who had relapsed or remained in remission in response to dose dense chemoimmunotherapy. At the time of the analysis, median follow up was 34 months, progression free survival (PFS) 78% and overall survival (OS) 78%. Results: The screen between relapsed patients and the patients in remission using criteria of p ≤ 0.05 and fold change ≥ 1.6 revealed 566 differentially expressed genes (131 protein coding), of which 24 were likely to be involved in conventional signaling pathways, including those regulating antigen processing and presentation (CIITA, HLA-DQA2, HLA-DQB1, RFXAP), Jak-STAT signaling (SOCS3), Notch signaling (NOTCH1) and Toll-like receptor signalling (IRF5). In cox univariate analysis, 12 of 24 genes were found to associate with PFS (p<0.05). Of these, high expression of CIITA, DLL4, HLA-DQA2, HLA-DQB1, IRF5, NOTCH1, PER1, RFXAP, SEMA4D and ZFP36 had a favorable impact on PFS, whereas high levels of ENPP3 and PRKAR2B were associated with adverse outcome. Differential expression of four genes was confirmed by quantitative PCR, and prognostic value of six genes validated using Lymphoma/Leukemia Molecular Profiling Project microarray data set. Immunohistochemical validation of the findings in a larger patient cohort is ongoing. Germinal centre B-cell signature did not predict survival in this cohort. Conclusions: The results provide evidence that exon-based transcriptome profiling can identify biologically relevant signaling pathways and genes that discriminate the outcome of homogenously treated young high risk DLBCL patients.
Summary
Rituximab in combination with chemotherapy (immunochemotherapy) is one of the most effective treatments available for follicular lymphoma (FL). This study aimed to determine whether ...differences in gene expression in FL tissue correlate with outcome in response to rituximab and CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy (R–CHOP). We divided 24 patients into long‐ time to treatment failure (TTF) >35 months and short‐term (TTF <23 months) responders, and analysed the gene expression profiles of lymphoma tissue using oligonucleotide microarrays. We used a supervised learning technique to identify genes correlating with outcome, and confirmed the expression of selected genes with quantitative polymerase chain reaction (qPCR) and immunohistochemistry. Among the transcripts with a high correlation between microarray and qPCR analyses, we identified EPHA1, a tyrosine kinase involved in transepithelial migration, SMAD1, a transcription factor and a mediator of bone morphogenetic protein and transforming growth factor‐β signalling, and MARCO, a scavenger receptor on macrophages. According to Kaplan–Meier estimates, high EPHA1, and low SMAD1 and MARCO expression were associated with better progression‐free survival (PFS). Immunohistochemistry showed that EphA1 was primarily localised in granulocytes. In addition, both EphA1 and Smad1 were expressed in vascular endothelia. However, no difference in vasculature was detected between long‐ and short‐term responders. In a validation set of 40 patients, a trend towards a better PFS was observed among patients with high EphA1 expression. We conclude that gene expression in non‐malignant cells contributes to clinical outcome in R–CHOP‐treated FL patients.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Abstract 3107
Survival of young high risk diffuse large B-cell lymphoma (DLBCL) is now approaching 80% due to implementation of rituximab and dose-dense chemotherapy protocols but the patients with ...relapsed or refractory disease continue to have a poor prognosis. Such patients could benefit from additional therapies if their clinical outcome could be more accurately predicted at the time of diagnosis. Recently, several gene-expression signatures with prognostic significance in DLBCL have been identified. To date, the accessibility of exon arrays that interrogate exon-level expression has enabled a new, more sensitive method of analysing gene-expression than the traditional 3′ arrays. In the present study, we have tested the utility of exon profiling to define novel prognostic markers for young high risk DLBCL patients.
Study population consisted of 41 patients (36 DLBCL and 5 follicular lymphoma (FL) grade 3) less than 65 years old with high risk (age adjusted International Prognostic Index (aaIPI) Score 2–3) disease. The selection of the patients was based on the availability of freshly frozen lymphoma tissue containing adequate material for mRNA analyses. All tissue samples were taken before treatments. All patients were treated in the Nordic phase II protocol with six courses of R-CHOEP14 followed by systemic CNS prophylaxis with one course of high-dose methotrexate and one course of high-dose cytarabine. In the present report with a median follow-up of 29 months, (range 15–63 months), ten patients had relapsed and nine died. Relapse free survival (RFS) was 74% and overall survival (OS) 77%.
We identified differentially expressed exons between the relapsed patients and the patients in remission using criteria of p ≤ 0.05 and fold change ≥ 1.6. In order to estimate the gene-level expression, and to exclude false positives, the genes were considered as differentially expressed only if at least 20% of all exons were differentially expressed. Accordingly, 646 genes were identified, from which 119 encoded proteins. In a pathway network analysis (Laakso and Hautaniemi, 2010), 23 genes were found likely to be involved in conventional signalling pathways. The identified pathways included important events of lymphoma biology such as antigen processing and presentation, cell adhesion, chemokine signalling as well as TGF-beta, Toll-like receptor, Wnt and MAPK signalling. We also performed a gene level survival analysis with data combined of differentially expressed exons and follow-up information. 12 of the 23 genes were found to associate with RFS (p<0.05). Among these, high expression of HLA-DOA, HLA-DQB1 (both members of MHC class II family), RFXAP (MCH class II transcription regulator), SMAD7 (mediator of TGF-beta signalling), IRF5 (interferon regulatory factor) and CR1 (complement component) had a favourable impact on RFS. In contrast, high IL22 (interleukin 22) and DLG2 (Discs 2) levels were associated with adverse outcome. Similarly, 7 of the 23 genes were predictive for OS (p<0.05). Prognostic impact of one third of the transcripts could be confirmed in an independent gene array based data set of 233 DLBCL patients treated with immunochemotherapy (Lenz et al., 2008).
The data suggest that exon-based transcriptome profiling of diagnostic tumor tissue can identify biologically relevant genes that discriminate the outcome of homogenously treated young high risk lymphoma patients. Such genes and involved pathways represent markers for improved patient risk stratification and potential targets for novel DLBCL therapies.
Holte:Roche: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Leppa:Roche: Honoraria, Research Funding.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background Despite improved survival for the patients with diffuse large B-cell lymphoma (DLBCL), the prognosis after relapse is poor. The aim was to identify molecular events that contribute to ...relapse and treatment resistance in DLBCL. Methods We analysed 51 prospectively collected pretreatment tumour samples from clinically high risk patients treated in a Nordic phase II study with dose-dense chemoimmunotherapy and central nervous system prophylaxis with high resolution array comparative genomic hybridization (aCGH) and gene expression microarrays. Major finding was validated at the protein level immunohistochemically in a trial specific tissue microarray series of 70, and in an independent validation series of 146 patients. Results We identified 31 genes whose expression changes were strongly associated with copy number aberrations. In addition, gains of chromosomes 2p15 and 18q12.2 were associated with unfavourable survival. The 2p15 aberration harboured COMMD1 gene, whose expression had a significant adverse prognostic impact on survival. Immunohistochemical analysis of COMMD1 expression in two series confirmed the association of COMMD1 expression with poor prognosis. Conclusion COMMD1 is a potential novel prognostic factor in DLBCLs. The results highlight the value of integrated comprehensive analysis to identify prognostic markers and genetic driver events not previously implicated in DLBCL. Trial Registration ClinicalTrials.gov NCT01502982
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Addition of rituximab (R) to chemotherapy (immunochemotherapy) has significantly improved the outcome of B-cell lymphoma patients. However, responses are still unpredictable due to heterogeneous ...nature of these diseases. The aim of this study was to determine if differences in gene expression in follicular lymphoma (FL) and mantle cell lymphoma (MCL) tissue correlate with outcome in response to immunochemotherapy. 24 FL and 15 MCL patients were classified into complete (CR) or other responders after immunochemotherapy, and genes capable of separating the groups were identified using oligonucleotide-based microarray and supervised learning technique. One of the transcripts associated with increased CR rates in FL was signal transducer and activator of transcription 5a (STAT5a), a transcription factor and a mediator of cellular responses after cytokine stimulation. According to Kaplan Meier estimates, high STAT5a mRNA levels were associated with better progression free survival (PFS). Immunohistochemical analyses of FL samples demonstrated that predominant STAT5a activity localized to resident T-lymphocytes but only 20% of the samples were STAT5a positive. In a validation set of 79 FL patients, a better PFS was observed among patients with high STAT5a protein expression. In contrast, high STAT5a mRNA and protein levels were associated with poor outcome in MCL patients. In the MCL tissue, STAT5a expression localized to nonmalignant T-lymphocytes but also to tumor cells. In comparison to FL, T-lymphocytes were more uniformly positive for STAT5a. In sum, the data show that nonmalignant tumor cells have a profound prognostic impact both in FL and MCL, but also suggest that depending on the lymphoma subtype, the activity of a certain signaling pathway can have either positive or negative consequences on the survival of immunochemotherapy-treated lymphoma patients.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
PURPOSE: Germinal centre (GC) and non-GC phenotypes are predictors of outcome in diffuse large B-cell lymphoma (DLBCL), and can be used to stratify chemotherapy-treated patients into low- and ...high-risk groups. Our aim was to determine how combination of rituximab with chemotherapy influences GC-associated clinical outcome.
METHODS: 95 DLBCL patients treated with rituximab in combination with CHOP or CHOEP regimen (immunochemotherapy) were included in the study. The median follow-up time was 27 months. 107 patients previously treated with chemotherapy served as a historical control group. BCL-6, CD10, and MUM1 expression was analyzed immunohistochemically by means of identifying GC and non-GC phenotypes. In addition, BCL-2 expression was determined. Expression data was correlated with survival parameters.
RESULTS: Consistent with previous studies, chemotherapy-treated patients with GC phenotype displayed a significantly better overall survival (OS) than the non-GC group (70% vs 47% p=0.012). In contrast, GC-phenotype did not predict outcome in immunochemotherapy-treated patients. The OS for GC-group was 82% compared with 80% for those with non-GC phenotype (p=ns). Conversely, the relapse free survival (RFS) for patients with GC and non-GC phenotypes were 74% and 65% (p=ns), respectively. When the patients were grouped according to BCL-2 expression, survival rates among the group of BCL-2 negative patients tended to be better than in BCL-2 positive patients (OS, 94% vs 77%, p=0.097; RFS, 94% vs 66%, p=0.029).
CONCLUSIONS: Rituximab in combination with chemotherapy seems to interfere with the prognostic value of GC- and non-GC phenotypes in DLBCL. However, rituximab may have a positive impact on outcome among the patients with BCL-2 negativity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Rituximab in combination with chemotherapy (immunochemotherapy) is one of the most effective treatments available for follicular lymphoma (FL). This study aimed to determine whether differences in ...gene expression in FL tissue correlate with outcome in response to rituximab and CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy (R-CHOP). We divided 24 patients into long- time to treatment failure (TTF) >35 months and short-term (TTF <23 months) responders, and analysed the gene expression profiles of lymphoma tissue using oligonucleotide microarrays. We used a supervised learning technique to identify genes correlating with outcome, and confirmed the expression of selected genes with quantitative polymerase chain reaction (qPCR) and immunohistochemistry. Among the transcripts with a high correlation between microarray and qPCR analyses, we identified EPHA1, a tyrosine kinase involved in transepithelial migration, SMAD1, a transcription factor and a mediator of bone morphogenetic protein and transforming growth factor- beta signalling, and MARCO, a scavenger receptor on macrophages. According to Kaplan-Meier estimates, high EPHA1, and low SMAD1 and MARCO expression were associated with better progression-free survival (PFS). Immunohistochemistry showed that EphA1 was primarily localised in granulocytes. In addition, both EphA1 and Smad1 were expressed in vascular endothelia. However, no difference in vasculature was detected between long- and short-term responders. In a validation set of 40 patients, a trend towards a better PFS was observed among patients with high EphA1 expression. We conclude that gene expression in non-malignant cells contributes to clinical outcome in R-CHOP-treated FL patients.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Deficiency of adenosine deaminase 2 (DADA2) is caused by biallelic deleterious mutations in CECR1. DADA2 results in variable autoinflammation and vasculopathy (recurrent fevers, livedo reticularis, ...polyarteritis nodosa, lacunar ischemic strokes, and intracranial hemorrhages), immunodeficiency and bone marrow failure. Tumor necrosis factor-α blockade is the treatment of choice for the autoinflammation and vascular manifestations. Hematopoietic stem cell transplantation (HSCT) represents a potential definitive treatment. We present a cohort of 14 patients from 6 countries who received HSCT for DADA2. Indication for HSCT was bone marrow dysfunction or immunodeficiency. Six of 14 patients had vasculitis pre-HSCT. The median age at HSCT was 7.5 years. Conditioning regimens were myeloablative (9) and reduced intensity (5). Donors were HLA-matched sibling (n = 1), HLA-matched unrelated (n = 9), HLA-mismatched unrelated (n = 3), and HLA haploidentical sibling (n = 1). All patients are alive and well with no new vascular events and resolution of hematological and immunological phenotype at a median follow-up of 18 months (range, 5 months to 13 years). Plasma ADA2 enzyme activity normalized in those tested post-HSCT (7/7), as early as day +14 (myeloid engraftment). Post-HSCT hematological autoimmunity (cytopenias) was reported in 4 patients, acute graft-versus-host disease grade 1 in 2, grade 2 in 3, and grade 3-4 in 1, and moderate chronic graft-versus-host disease in 1 patient. In conclusion, in 14 patients, HSCT was an effective and definitive treatment of DADA2.
•HSCT represents an effective and definitive treatment of DADA2.•HSCT can cure the immunological, hematological, and vascular phenotype of DADA2 with 100% survival at median follow-up of 18 months.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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