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IntroductionAnalyzing liquid biopsies for tumor-specific aberrations can facilitate detection of measurable residual disease (MRD) during treatment and at follow-up. In this study, we assessed the clinical potential of using whole-genome sequencing (WGS) of lymphomas at diagnosis to identify patient-specific structural (SVs) and single nucleotide variants (SNVs) to enable longitudinal, multi-targeted droplet digital PCR analysis (ddPCR) of cell-free DNA (cfDNA).MethodsIn 9 patients with B-cell lymphoma (diffuse large B-cell lymphoma and follicular lymphoma), comprehensive genomic profiling at diagnosis was performed by 30X WGS of paired tumor and normal specimens. Patient-specific multiplex ddPCR (m-ddPCR) assays were designed for simultaneous detection of multiple SNVs, indels and/or SVs, with a detection sensitivity of 0.0025% for SV assays and 0.02% for SNVs/indel assays. M-ddPCR was applied to analyze cfDNA isolated from serially collected plasma at clinically critical timepoints during primary and/or relapse treatment and at follow-up.ResultsA total of 164 SNVs/indels were identified by WGS including 30 variants known to be functionally relevant in lymphoma pathogenesis. The most frequently mutated genes included KMT2D, PIM1, SOCS1 and BCL2. WGS analysis further identified recurrent SVs including t(14;18)(q32;q21) (IGH::BCL2), and t(6;14)(p25;q32) (IGH::IRF4). Plasma analysis at diagnosis showed positive circulating tumor DNA (ctDNA) levels in 88% of patients and the ctDNA burden correlated with baseline clinical parameters (LDH and sedimentation rate, p-value

Summary: Diffuse large B cell lymphoma (DLBCL) is one of the most common types of aggressive lymphoid malignancies. Here, we explore the contribution of RNA editing to DLBCL pathogenesis. We observed that DNA mutations and RNA editing events are often mutually exclusive, suggesting that tumors can modulate pathway outcomes by altering sequences at either the genomic or the transcriptomic level. RNA editing targets transcripts within known disease-driving pathways such as apoptosis, p53 and NF-κB signaling, as well as the RIG-I-like pathway. In this context, we show that ADAR1-mediated editing within MAVS transcript positively correlates with MAVS protein expression levels, associating with increased interferon/NF-κB signaling and T cell exhaustion. Finally, using targeted RNA base editing tools to restore editing within MAVS 3′UTR in ADAR1-deficient cells, we demonstrate that editing is likely to be causal to an increase in downstream signaling in the absence of activation by canonical nucleic acid receptor sensing.

Abstract Purpose The abundant hepatocyte‐expressed plasma protein histidine‐rich glycoprotein (HRG) enhances antitumor immunity by polarizing inflammatory and immune cells in several mouse models, however, the clinical relevance of HRG in human cancer is poorly explored. The expression and role of HRG in human B‐cell lymphomas was investigated in order to find new tools for prognosis and treatment. Findings Immunohistochemical (IHC) analysis and RNA hybridization of tissue microarrays showed that (i) HRG was expressed by tumor cells in marginal zone lymphoma (MZL), in 36% of 59 cases. Expression was also detected in follicular lymphoma (22%), mantle cell lymphoma (19%), and indiffuse large B‐cell lymphoma (DLBCL;5%) while primary CNS lymphoma (PCNSL) lacked expression of HRG. (ii) MZL patients positive for HRG showed a superior overall survival outcome (HR = 0.086, 95% CI = 0.014‐0.518, P‐value = .007), indicating a protective role for HRG independent of stage, age and sex. (iii) HRG‐expressing MZL displayed significantly increased transcript and protein levels of the host defense peptide alpha defensin 1. In addition, global transcript analyses showed significant changes in gene ontology terms relating to immunity and inflammation, however, infiltration of immune and inflammatory cells detected by IHC was unaffected by HRG expression. Conclusion HRG expression by MZL tumor cells correlates with an altered transcription profile and improved overall survival.

The receptor tyrosine kinase orphan receptor 1 (ROR1) is absent in most normal adult tissues but overexpressed in various malignancies and is of importance for tumor cell survival, proliferation, and metastasis. In this study, we evaluated the apoptotic effects of a novel small molecule inhibitor of ROR1 (KAN0441571C) as well as venetoclax (BCL-2 inhibitor), bendamustine, idelalisib (PI3Kδ inhibitor), everolimus (mTOR inhibitor), and ibrutinib (BTK inhibitor) alone or in combination in human MCL primary cells and cell lines. ROR1 expression was evaluated by flow cytometry and Western blot (WB). Cytotoxicity was analyzed by MTT and apoptosis by Annexin V/PI staining as well as signaling and apoptotic proteins (WB). ROR1 was expressed both in patient-derived MCL cells and human MCL cell lines. KAN0441571C alone induced significant time- and dose-dependent apoptosis of MCL cells. Apoptosis was accompanied by decreased expression of MCL-1 and BCL-2 and cleavage of PARP and caspase 3. ROR1 was dephosphorylated as well as ROR1-associated signaling pathway molecules, including the non-canonical WNT signaling pathway (PI3Kδ/AKT/mTOR). The combination of KAN0441571C and ibrutinib, venetoclax, idelalisib, everolimus, or bendamustine had a synergistic apoptotic effect and significantly prevented phosphorylation of ROR1-associated signaling molecules as compared to KAN0441571C alone. Our results suggest that targeting ROR1 by a small molecule inhibitor, KAN0441571C, should be further evaluated particularly in combination with other targeting drugs as a new therapeutic approach for MCL.

Abstract Recognition of nucleic acids by endosomal Toll-like receptors (TLR) is essential to combat pathogens, but requires strict control to limit inflammatory responses. The mechanisms governing this tight regulation are unclear. We found that single-stranded oligonucleotides (ssON) inhibit endocytic pathways used by cargo destined for TLR3/4/7 signaling endosomes. Both ssDNA and ssRNA conferred the endocytic inhibition, it was concentration dependent, and required a certain ssON length. The ssON-mediated inhibition modulated signaling downstream of TLRs that localized within the affected endosomal pathway. We further show that injection of ssON dampens dsRNA-mediated inflammatory responses in the skin of non-human primates. These studies reveal a regulatory role for extracellular ssON in the endocytic uptake of TLR ligands and provide a mechanistic explanation of their immunomodulation. The identified ssON-mediated interference of endocytosis (SOMIE) is a regulatory process that temporarily dampens TLR3/4/7 signaling, thereby averting excessive immune responses.

A subset of hematologic cancer patients is refractory to treatment or suffers relapse, due in part to minimal residual disease, whereby some cancer cells survive treatment. Cell-adhesion-mediated drug resistance is an important mechanism, whereby cancer cells receive survival signals via interaction with e.g. stromal cells. No genome-wide studies of in vitro systems have yet been performed to compare gene expression in different cell subsets within a co-culture and cells grown separately. Using RNA sequencing and species-specific read mapping, we compared transcript levels in human Jeko-1 mantle cell lymphoma cells stably adhered to mouse MS-5 stromal cells or in suspension within a co-culture or cultured separately as well as in stromal cells in co-culture or in separate culture. From 1050 differentially expressed transcripts in adherent mantle cell lymphoma cells, we identified 24 functional categories that together represent four main functional themes, anti-apoptosis, B-cell signaling, cell adhesion/migration and early mitosis. A comparison with previous mantle cell lymphoma and chronic lymphocytic leukemia studies, of gene expression differences between lymph node and blood, identified 116 genes that are differentially expressed in all three studies. From these genes, we suggest a core set of genes (CCL3, CCL4, DUSP4, ETV5, ICAM1, IL15RA, IL21R, IL4I1, MFSD2A, NFKB1, NFKBIE, SEMA7A, TMEM2) characteristic of cells undergoing cell-adhesion-mediated microenvironment signaling in mantle cell lymphoma/chronic lymphocytic leukemia. The model system developed and characterized here together with the core gene set will be useful for future studies of pathways that mediate increased cancer cell survival and drug resistance mechanisms.

In follicular lymphoma, studies addressing the prognostic value of microenvironment-related immunohistochemical markers and tumor cell-related genetic markers have yielded conflicting results, precluding implementation in practice. Therefore, the Lunenburg Lymphoma Biomarker Consortium performed a validation study evaluating published markers. To maximize sensitivity, an end of spectrum design was applied for 122 uniformly immunochemotherapy-treated follicular lymphoma patients retrieved from international trials and registries. The criteria were: early failure, progression or lymphoma-related death 5 years. Immunohistochemical staining for T cells and macrophages was performed on tissue microarrays from initial biopsies and scored with a validated computer-assisted protocol. Shallow whole-genome and deep targeted sequencing was performed on the same samples. The 96/122 cases with complete molecular and immunohistochemical data were included in the analysis. EZH2 wild-type (P=0.006), gain of chromosome 18 (P=0.002), low percentages of CD8+ cells (P=0.011) and CD163+ areas (P=0.038) were associated with early failure. No significant differences in other markers were observed, thereby refuting previous claims of their prognostic significance. Using an optimized study design, this Lunenburg Lymphoma Biomarker Consortium study substantiates wild-type EZH2 status, gain of chromosome 18, low percentages of CD8+ cells and CD163+ area as predictors of early failure to immunochemotherapy in follicular lymphoma treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP [-like]), while refuting the prognostic impact of various other markers.

The cannabinoid receptor type 1 (CB1) antagonist rimonabant has been used as treatment for obesity. In addition, anti-proliferative effects on mitogen-activated leukocytes have been demonstrated in vitro. We have previously shown that rimonabant (SR141716A) induces cell death in ex vivo isolated malignant lymphomas with high expression of CB1 receptors. Since CB1 targeting may be part of a future lymphoma therapy, it was of interest to investigate possible effects on peripheral blood mononuclear cells (PBMC) in patients treated with rimonabant. We therefore evaluated leukocyte subsets by 6 color flow cytometry in eight patients before and at treatment with rimonabant for 4 weeks. Whole-transcript gene expression profiling in PBMC before and at 4 weeks of rimonabant treatment was done using Affymetrix Human Gene 1.0 ST Arrays. Our data show no significant changes of monocytes, B cells, total T cells or T cell subsets in PBMC during treatment with rimonabant. There was a small but significant increase in CD3–, CD16+ and/or CD56+ cells after rimonabant therapy. Gene expression analysis detected significant changes in expression of genes associated with innate immunity, cell death and metabolism. The present study shows that normal monocytes and leukocyte subsets in blood remain rather constant during rimonabant treatment. This is in contrast to the induction of cell death previously observed in CB1 expressing lymphoma cells in response to treatment with rimonabant in vitro. These differential effects observed on normal and malignant lymphoid cells warrant investigation of CB1 targeting as a potential lymphoma treatment.

The cellular microenvironment in follicular lymphoma is of biological and clinical importance. Studies on the clinical significance of non-malignant cell populations have generated conflicting results, which may partly be influenced by poor reproducibility in immunohistochemical marker quantification. In this study, the reproducibility of manual scoring and automated microscopy based on a tissue microarray of 25 follicular lymphomas as compared to flow cytometry is evaluated. The agreement between manual scoring and flow cytometry was moderate for CD3, low for CD4, and moderate to high for CD8, with some laboratories scoring closer to the flow cytometry results. Agreement in manual quantification across the 7 laboratories was low to moderate for CD3, CD4, CD8 and FOXP3 frequencies, moderate for CD21, low for MIB1 and CD68, and high for CD10. Manual scoring of the architectural distribution resulted in moderate agreement for CD3, CD4 and CD8, and low agreement for FOXP3 and CD68. Comparing manual scoring to automated microscopy demonstrated that manual scoring increased the variability in the low and high frequency interval with some laboratories showing a better agreement with automated scores. Manual scoring reliably identified rare architectural patterns of T-cell infiltrates. Automated microscopy analyses for T-cell markers by two different instruments were highly reproducible and provided acceptable agreement with flow cytometry. These validation results provide explanations for the heterogeneous findings on the prognostic value of the microenvironment in follicular lymphoma. We recommend a more objective measurement, such as computer-assisted scoring, in future studies of the prognostic impact of microenvironment in follicular lymphoma patients.

Myc plays an important role in tumor development, including acute myeloid leukemia (AML). However, MYC is also a powerful inducer of apoptosis, which is one of the major failsafe programs to prevent cancer development. To clarify the relative importance of the extrinsic (death receptor-mediated) versus the intrinsic (mitochondrial) pathway of apoptosis in MYC-driven AML, we coexpressed MYC together with anti-apoptotic proteins of relevance for AML; BCL-X(L)/BCL-2 (inhibiting the intrinsic pathway) or FLIP(L) (inhibiting the extrinsic pathway), in hematopoietic stems cells (HSCs). Transplantation of HSCs expressing MYC into syngeneic recipient mice resulted in development of AML and T-cell lymphomas within 7-9 weeks as expected. Importantly, coexpression of MYC together with BCL-X(L)/BCL-2 resulted in strongly accelerated kinetics and favored tumor development towards aggressive AML. In contrast, coexpression of MYC and FLIP(L) did neither accelerate tumorigenesis nor change the ratio of AML versus T-cell lymphoma. However, a change in distribution of immature CD4(+)CD8(+) versus mature CD4(+) T-cell lymphoma was observed in MYC/FLIP(L) mice, possibly as a result of increased survival of the CD4+ population, but this did not significantly affect the outcome of the disease. In conclusion, our findings provide direct evidence that BCL-X(L) and BCL-2 but not FLIP(L) acts in synergy with MYC to drive AML development.

The SRY (sex determining region Y)-box 11 (SOX11) gene, located on chromosome 2p25, encodes for a transcription factor that is involved in tissue remodeling during embryogenesis and is crucial for neurogenesis. The role for SOX11 in hematopoiesis has not yet been defined. Two genes under direct control of SOX11 are the class- III β-tubulin gene (TUBB3) in neural cells and the transcription factor TEA domain family member 2 (TEAD2) in neural and mesenchymal progenitor cells. Normal, mature lymphocytes lack SOX11 but express SOX4, another member of the same group of SOX transcription factors. We and others recently identified SOX11 as aberrantly expressed in mantle cell lymphoma (MCL). Since SOX11 is variably expressed in MCL it may not be essential for tumorigenesis, but may carry prognostic information. Currently, no specific functional effects have been linked to SOX11 expression in MCL and it is not known which genes are under influence of SOX11 in lymphoma. In this study we found variable expression of SOX11, SOX4 and SOX12 mRNA in mantle cell lymphoma cell lines. Downregulation of SOX11 expression by siRNA verified that SOX11 controlled the expression of the gene TUBB3 in the MCL cell line Granta 519. Furthermore we identified, by global gene expression analysis, 26 new target genes influenced by siRNA SOX11 downmodulation. Among these genes, DBN1, SETMAR and HIG2 were found to be significantly correlated to SOX11 expression in two cohorts of primary mantle cell lymphomas. Chromatin immunoprecipitation (ChIP) analysis showed that these genes are direct targets of the SOX11 protein. In spite of almost complete downregulation of the SOX11 protein no significant effects on Granta 519 cell proliferation or survival in short term in vitro experiments was found. In summary we have identified a number of genes influenced by SOX11 expression in MCL cell lines and primary MCL. Among these genes, DBN1, SETMAR and HIG2 are direct transcriptional targets of the SOX11 protein.

In 231 diffuse large B-cell lymphomas, the expression of cyclin D1 and CD5 was evaluated. All cases were CD5-. Ten (4.3%) were positive for cyclin D1 and were subjected to fluorescence in situ hybridization at the CCND1 locus. One case showed the t(11;14). In another case, the telomeric probe signal for cyclin D1 was lost in most tumor cells, and in a small proportion of the cells, there were fluorescence signals indicative of the t(11;14). Two other cases displayed additional cyclin D1 signals in the absence of the t(11;14). All cases but 1 were positive for bcl-6 or MUM1, disfavoring the possibility of misdiagnosed blastoid variants of CD5- mantle cell lymphomas. Thus, contrary to the current view, there seems to exist a certain number of cyclin D1+ and CD5- diffuse large B-cell lymphomas, some of which have structural aberrations at the CCND1 locus, including the t(11;14). [ABSTRACT FROM AUTHOR]