Primary mediastinal large B-cell lymphoma (PMBCL) is a subtype of diffuse large B-cell lymphoma (DLBCL) accounting for 2% to 4% of all non-Hodgkin lymphomas. We report a family of 3 siblings with ...PMBCL and their cousin with extranodal DLBCL. The histopathological characteristics of lymphomas of all 4 patients are similar, implying post–germinal center differentiation and growth deregulation by other mechanisms than BCL2-mediated inhibition of apoptosis and suggesting a shared biological background. We aimed to identify the genetic defect underlying lymphoma susceptibility in this family using exome sequencing and linkage analysis. The only variant segregating in all 4 patients and not reported in genetic databases was 5533C>A (His1845Asn) in the MLL gene. To our knowledge, this is the first time when familial clustering of PMBCL is reported. Although we propose MLL as a candidate predisposition gene for this condition, this finding needs to be validated in additional cases.
•A familial form of PMBCL is reported for the first time.•Exome sequencing identifies MLL 5533C>A (His1845Asn) variant segregating with lymphoma in the reported family.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease both biologically and clinically. Although a number of genetic alterations have been associated with the pathogenesis of DLBCL, ...survival association of most of the identified mutations remains to be shown. Here we have used exome and RNA sequencing (RNAseq) data to identify novel genetic events associated with treatment outcome after chemoimmunotherapy.
Initial screen was performed with eight primary tumor samples and matched normal DNA using exome sequencing. The patients had clinically high risk DLBCL, and were treated in a Nordic phase II protocol with dose dense chemoimmunotherapy and CNS prophylaxis. After stringent filtering (VarScan2 p < 0.05), and removal of known germline variants, 967 somatic point mutations and indels in 561 different genes were identified. Of these, 436 were non-synonymous, and 27 were detected in more than one patient. These included both novel and several previously described DLBCL associated mutations, such as MYD88, CD79B, B2M and PTEN.
To correlate the identified mutations with survival, we used RNAseq and survival data of 92 DLBCL samples from the Cancer Genome Characterization Initiative (CGCI) repository. The mutational status of the 436 genes identified in our screen was assessed with Bambino, which confirmed 405 of these genes also to be mutated in the CGCI set. Kaplan-Meier survival estimates per gene with at least one single nucleotide variant (SNV; with criteria of minimum 3 reads and 10% coverage and known polymorphisms filtered out) revealed 17 genes that were associated with overall survival (OS, p<0.05, minimum 10 samples mutated).
We highlight mutations affecting Deltex1 (DTX1), which functions as an E3 ubiquitin ligase and targets Notch1 intracellular domain for degradation. In the study cohort, 14% of the patients had a non-synonymous DTX1 mutation. Mutations were equally distributed between GCB and ABC DLBCLs and low (0-2) and high (3-5) International Prognostic Index (IPI) risk groups. According to Kaplan-Meier analysis, the 5-year PFS rate for the patients with DTX1 mutations was significantly worse as compared to ones without mutations (46% vs 78%, p=0.010). The corresponding OS values were 54% and 85% (p=0.006). In multivariate analysis with IPI, DTX1 mutation status remained as an independent prognostic factor for both PFS (RR, 2.83; 95% CI, 1.17-6.85, p=0.021) and OS (RR, 3.51; 95% CI, 1.41-8.71, p=0.007). Mutational status was also found to correlate with the expression levels; the patients with mutations had lower DTX1 mRNA levels (p=0.006). When DTX1 mutations were analyzed according to different functional domains, 11 out of 16 (65%) were located in the first exon encoding the WWE 1 domain and a binding site for intracellular domain of Notch1. Interestingly, the patients with mutations in exon 1 had a significantly worse survival in comparison to other patients (Figure 1).
Taken together, the results demonstrate that DTX1 mutations in DLBCL are an independent predictor of survival after chemoimmunotherapy, and mutations enriched in exon 1 identify cases with dismal prognosis.
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No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma in adults. Although 60-70% of the patients can be cured with standard therapeutic regimens, a substantial number of patients die from ...the disease due to treatment resistance. In order to better understand the biological processes behind the resistance to treatment, we have characterized the microRNA (miRNA) expression profiles of matched primary and relapsed DLBCL.
We performed next-generation miRNA sequencing of seven primary–relapse sample pairs. A total of 492 known miRNAs were found to be expressed in the DLBCL samples. In addition, we identified 223 potentially novel, previously uncharacterized miRNAs. A majority of the detected miRNAs showed similar expression across primary and relapse samples; we identified 24 high-expressed miRNAs and 177 low-expressed miRNAs in the DLBCL samples as compared to a reference control set of non-malignant cells downloaded from the Gene Expression Omnibus (GSE15229). Interestingly, only 13 miRNAs had differential expression between the primary–relapse sample pairs. Of these, five miRNAs had higher expression and eight miRNAs had lower expression in the relapse samples as compared to the primary samples. In order to identify potential targets for the differentially expressed miRNAs, we integrated the miRNA data with total RNA-sequencing data from the same samples (n=10, or 5 pairs) as well as with the miRNA target predictions from four prediction programs (TargetScan, microCosm, PITA, DIANA microT) and with filtered data of functionally validated miRNA targets from the miRTarBase. This analysis resulted in 1,088 miRNA-transcript pairs representing 787 individual genes inversely correlated with at least one of the 13 miRNAs (r<-0.7, p<0.05), and whose regulatory pairings were supported by at least one prediction program or mirTarBase. Further Gene Ontology annotation and pathway enrichment analyses revealed the putative targets of differentially expressed miRNAs to be significantly enriched for several cancer-associated pathways that include phosphatidyl-inositol signaling (e.g. PIP5K1A, PIK3C2A, PIK3CG, PIK3R1), JAK-STAT signaling (e.g. STAT5A, STAT5B), and B-cell receptor signaling (e.g.SYK, MAPK1), suggesting activation of these pathways in the relapsed DLBCL. In line with this, Kaplan-Meier survival analyses indicated higher expression of genes from the phosphatidyl-inositol signaling and B-cell receptor signaling, such as phosphatidylinositol 4-phosphate 5-kinase (PIP5K1A) and spleen tyrosine kinase (SYK), to be associated with shorter progression-free and overall survival (p<0.001 for both genes) in immunochemotherapy-treated patients (n=92).Validations of the findings are currently ongoing.
In conclusion, our study on the comparison of paired primary and relapsed DLBCL demonstrates that the miRNA expression profile remains relatively constant during the disease progression. However, a small set of differentially expressed miRNAs may contribute to the relapse by regulating key cell survival pathways, thus representing potential novel therapeutic targets.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Alternative splicing (AS) increases dramatically the diversity of the proteome by allowing a single gene to generate multiple transcripts and functionally diverse protein isoforms. Although previous ...studies have provided evidence that AS is often deregulated in cancers, the role of AS events in diffuse large B-cell lymphoma (DLBCL) remains largely unexplored. Here, we design and apply a novel method to analyze the global prognostically significant mRNA and AS variation by comparing differentially expressed exons and splicing variants between diffuse large B-cell lymphoma (DLBCL) patients, who have relapsed (poor prognosis group) or remained in remission (good prognosis group) after chemoimmunotherapy.
We performed genome-wide exon array analysis on RNAs isolated from 38 tumor tissues from young (<65 years) DLBCL patients with high-risk (aaIPI>1) disease. The patients were treated in a Nordic phase II protocol with six courses of R-CHOEP-14 followed by systemic central nervous system prophylaxis with one course of high dose methotrexate and high dose cytarabine. At the time of the analysis, median follow up was 50 months, predicted 5-year progression free survival (PFS) 74% and overall survival (OS) 75%. To identify genes with differential expression or with differential splicing between patients with poor or good prognosis, a novel algorithm based on a splicing index was developed.
With a criteria of p<0.05 and log2 fold change 0.6 we identified 233 differentially expressed genes (DEGs) between the poor and good prognosis subgroups. For the AS analysis we used criteria of p<0.01 and percent splicing index difference (DPSI) of 0.2, resulting in 589 exons (from 378 genes) with differential inclusion level between the groups. Kaplan-Meier analyses identified 118 DEGs and 311 AS genes as survival associated with p<0.05 on PFS time. Interestingly, AS index profile was able to separate poor and good prognosis groups better than differences at the mRNA level. Gene sets from gene and AS level analyses were further found to be enriched in different functional groups. For example, several DEGs targeted Jak-STAT signaling, antigen processing and presentation, and hematopoietic cell lineage pathways, while AS genes were enriched in ABC transporter and phosphatidylinositol signaling systems. Altogether 50% of AS-related exons were protein-coding, and domain prediction showed 33% of such exons to include a functional domain. For example, most of the AS events in ABC transporters occurred in transmembrane helix domains and were predicted to involve phosphorylation sites, which are the key players in signal transduction. PCR and 105 DLBCL samples subjected to RNA-seq from the Cancer Genome Characterization Initiative (CGCI) repository are used for validations.
AS index is a more potential discriminator than conventional gene profiling between good and bad prognosis patients in DLBCL, and may provide a new class of cancer biomarker candidates.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract Background Tumour-infiltrating mast cells (MCs) can remodel tumour microenvironment and growth by suppressing immune responses and potentiating angiogenesis. Furthermore, accumulation of MCs ...in follicular lymphoma (FL) correlates with unfavourable prognosis after immunochemotherapy. Here we investigated whether tumour vascularity is associated with MC content and outcome in FL patients treated with immunochemotherapy. Patients and methods Microvessel density (MVD) and MC content were determined immunohistochemically from pretreatment samples of 95 FL patients using CD31, CD34 and mast cell tryptase antibodies. Gene expression data from a separate set of 24 FL patients were analysed for comparison. All patients were treated with the combination of rituximab (R) and cyclophoshamide-doxorubicin–vincristine–prednisone (CHOP) chemotherapy. Results Increased CD31+ MVD correlated positively with the number of tumour infiltrating MCs and CD34+ vessels, and negatively with the outcome. Overall survival and progression-free survival were significantly better among patients with low CD31+ MVDs. In multivariate analyses, CD31+ MVD had prognostic value independently of Follicular Lymphoma Prognostic Index but not of MC content. Consistent with the immunohistochemical data, high CD31/ PECAM1 mRNA levels were associated with adverse outcome. Conversely, a positive prognostic impact of VEGF mRNA expression on the outcome was found. Conclusion Vascularity is associated with MC content and outcome in R-CHOP-treated FL patients.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Abstract 5206
The prognostic impact of tumor microenvironment on the survival of lymphoma patients has recently been reported. However, early molecular and cellular responses to immunochemotherapy ...are unknown. Here, we have compared the tumor-associated macrophage (TAM) and mast cell (MC) contents in the lymphoma tissue in vivo before and after the first immunochemotherapy course in a small cohort of aggressive B-cell lymphoma patients.
The population of this pilot study consisted of seven diffuse large B-cell lymphoma (DLBCL) and three grade IIIB follicular lymphoma (FL) patients treated with rituximab in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone)-like regimen (immunochemotherapy). Paired tumor samples were collected before and a day after the first course of therapy, and evaluated immunohistochemically for CD68+, CD163+ macrophages and tryptase+ MCs. Freshly frozen lymphoma tissue containing enough material for paired mRNA analyses was available from 8 patients.
Comparing pre- and post-treatment tissue samples, an increase in the number of CD68+ TAMs was observed (p=0.023), whereas no variation in MC contents was found. If the patients were grouped according to response, i.e. remission (n=7) vs relapse (n=3), the most significant increase after therapy was observed in M2-type CD163+ TAM content (p=0.001). In the exon array analyses, the mRNA levels of both CD68 (p=0.052) and CD163 (p=0.023) genes increased after therapy.
Our preliminary data suggest significant changes in macrophage content and their relative subsets in the lymphoma microenvironment after the first course of immunochemotherapy.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract 1557
The aim was to compare differentially expressed exons and splicing variants between diffuse large B-cell lymphoma (DLBCL) patients, who had relapsed or remained in remission after dose ...dense chemoimmunotherapy.
We performed genome-wide exon array analysis from four DLBCL cell lines and 38 tumor tissues from young (<65 years) DLBCL patients with high-risk (aaIPI>1) disease. The patients were treated in a Nordic phase II protocol with six courses of R-CHOEP-14 followed by systemic central nervous system prophylaxis with one course of high dose methotrexate and high dose cytarabine. At the time of the analysis, median follow up was 34 months, predicted 3-year progression free survival (PFS) 78% and overall survival (OS) 79%. RNA for quantitative PCR validation was available from 20 patients. Two DLBCL cell lines and eight patient samples were further analyzed with high throughput RNA sequencing. In addition, microarray data set from 233 DLBCL patients treated with chemoimmunotherapy (Lymphoma/Leukemia Molecular Profiling Project (LLMPP)) was utilized for validation.
Differentially expressed exons between relapsed and non-relapsed patients were screened using criteria of p ≤ 0.05 and fold change ≥1.6 converting to 566 differentially expressed genes, of which 131 coded proteins. One of the identified genes with possible alternative splicing was TUBB2B, which encodes therapeutic target of taxanes and vinca alkaloids. The expression of TUBB2B, and specifically the expression of exon 3, was found to be suppressed in relapsed patients in comparison to patients remaining in remission. Differential expression of TUBB2B whole transcript and exon 3 was confirmed with RNAseq and quantitative PCR. Studies in lymphoma cell lines provided further support for the existence of different TUBB2B isoforms. According to Kaplan Meier estimates the patients with high (>median) expression levels of TUBB2B exon 3 had better 3-year PFS and lymphoma related OS rates than the patients with low expression levels (95% vs. 61%, p=0.015 for PFS, 100% vs. 75%, p=0.024 for OS). The prognostic significance of TUBB2B gene expression was validated in LLMPP data set (3-year OS 80% vs. 67%, p=0.040).
The results provide evidence that differential expression and splicing of TUBB2B gene can discriminate the outcome of homogenously treated high risk DLBCL patients.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract 2650
Tumor associated macrophages (TAM) have at least two potential roles in promoting tumor growth: suppression of immune responses and potentiation of angiogenesis. In numerous cancer ...types, including lymphomas, high M2 type TAM content has been associated with worse prognosis. Rarely, high TAM content correlates with better survival. We have recently shown that CD68 positive TAMs in DLBCL contribute to unfavorable survival after high dose chemotherapy. Here we have extended our analyses on M2 type macrophages and questioned how combination of rituximab with chemotherapy influences TAM-associated clinical outcome.
Expression of CD163 and CCL18, which are primarily expressed in M2 type macrophages, were identified immunohistochemically from samples of 101 de novo DLBCL patients treated with rituximab in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone)-like regimen (immunochemotherapy). With a median follow-up of 65 months, (range 16–114 months), 5-year progression free survival (PFS) was 70% and overall survival (OS) 73%. 29 DLBCL patients previously treated with up front high dose chemotherapy served as a control group.
Correlation between CD163 and CCL18 positive TAMs was found (rs=0.427, p<0.001). In the Kaplan-Meier analyses the cutoff level of 67% was found to best discriminate between subgroups with different outcomes. Consistent with previous data, chemotherapy-treated patients with high CD163 or CCL18 positive TAM counts displayed a significantly inferior OS and PFS than the low group (Table). In contrast, after rituximab containing regimen, the patients with high CD163 and CCL18 positive TAM content tended to have favorable survival. Among the patients with low counts in both CD163 and CCL18 positive TAMs, PFS and OS were found to be significantly worse in comparison to others.
SurvivalMarkerImmunochemotherapy groupControl groupLow (%)High (%)P-valueLow (%)High (%)P-value5-year OSCD16370800.23575330.019CCL1869840.15174330.037CD163+CCL1865850.035100380.0035-year PFSCD16366820.09175400.054CCL1868800.20674390.131CD163+CCL1863830.04075420.013
In contrast to data on chemotherapy treated DLBCL or other lymphoma types, M2 type TAM content is associated with favorable prognosis in DLBCL patients after immunochemotherapy.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Tumor infiltrating mast cells have at least two potential roles in remodeling tumor microenvironment and tumor growth: suppression of immune responses and potentiation of angiogenesis. We have ...recently shown that accumulation of mast cells in follicular lymphoma (FL) contributes to unfavorable survival after immunochemotherapy. Interestingly, high mast cell content eliminates the positive prognostic value of tumor-associated macrophages (TAMs). Here we investigated whether tumor vascularity, as determined by CD31 immunostaining, is associated with mast cell content and outcome of primary FL patients treated with combination of rituximab (R) and CHOP chemotherapy. We found that increased microvessel density (MVD) correlates positively with the number of tumor infiltrating mast cells (r=0.266, p=0.011). In a cohort of 95 patients, the patients with high MVD (>the lowest tertile) had a worse 4-year progression free survival (PFS) (44% vs 82%, p=0.001). In multivariate analysis with FLIPI, MVD retained its negative predictive value. Additional prognostic impact was especially provided in patients with low FLIPI scores. When MVD related outcome was estimated separately for patients with high and low mast cell contents, the adverse effect of high MVD on the PFS was seen in both subgroups. Taken together, the data demonstrate that MVD is associated with mast cell content and adverse outcome in R-CHOP treated FL patients. In contrast to TAMs, the predictive value of MVD is independent of tumor infiltrating mast cells.
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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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