Detection of Second Primary Lymphoma in Late Diffuse Large B-cell Lymphoma Recurrences.

Approximately one-third of patients with diffuse large B-cell lymphoma (DLBCL) relapse and often require salvage chemotherapy followed by autologous stem cell transplantation. In most cases, the clonal relationship between the first diagnosis and subsequent relapse is not assessed, thereby potentially missing the identification of second primary lymphoma. In this study, the clonal relationship of 59 paired DLBCL diagnoses and recurrences was established by next-generation sequencing-based detection of immunoglobulin gene rearrangements. Among 50 patients with interpretable results, 43 patients (86%) developed clonally related relapsed disease. This was observed in 100% of early recurrences (<2 years), 80% of the recurrences with an interval between 2 and 5 years, and 73% of late recurrences (≥5 years). On the other hand, 7 (14%) out of 50 patients displayed different dominant clonotypes in primary DLBCL and clinical recurrences, confirming the occurrence of second primary DLBCL; 37% of DLBCL recurrences that occurred ≥4 years after diagnosis were shown to be second primary lymphomas. The clonally unrelated cases were Epstein-Barr virus positive in 43% of the cases, whereas this was only 5% in the relapsed DLBCL cases. In conclusion, next-generation sequencing-based clonality testing in late recurrences should be considered in routine diagnostics to distinguish relapse from second primary lymphoma, as this latter group of patients with DLBCL may benefit from less-intensive treatment strategies.

IRF8 is a transcriptional activator of CD37 expression in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) represents the most common form of non-Hodgkin lymphoma (NHL) that is still incurable in a large fraction of patients. Tetraspanin CD37 is highly expressed on mature B lymphocytes, and multiple CD37-targeting therapies are under clinical development for NHL. However, CD37 expression is nondetectable in ∼50% of DLBCL patients, which correlates with inferior treatment outcome, but the underlying mechanisms for differential CD37 expression in DLBCL are still unknown. Here, we investigated the regulation of the CD37 gene in human DLBCL at the (epi-)genetic and transcriptional level. No differences were observed in DNA methylation within the CD37 promoter region between CD37-positive and CD37-negative primary DLBCL patient samples. On the contrary, CD37-negative DLBCL cells specifically lacked CD37 promoter activity, suggesting differential regulation of CD37 gene expression. Using an unbiased quantitative proteomic approach, we identified transcription factor IRF8 to be significantly higher expressed in nuclear extracts of CD37-positive as compared with CD37-negative DLBCL. Direct binding of IRF8 to the CD37 promoter region was confirmed by DNA pulldown assay combined with mass spectrometry and targeted chromatin immunoprecipitation (ChIP). Functional analysis indicated that IRF8 overexpression enhanced CD37 protein expression, while CRISPR/Cas9 knockout of IRF8 decreased CD37 levels in DLBCL cell lines. Immunohistochemical analysis in a large cohort of primary DLBCL (n = 206) revealed a significant correlation of IRF8 expression with detectable CD37 levels. Together, this study provides new insight into the molecular mechanisms underlying differential CD37 expression in human DLBCL and reveals IRF8 as a transcriptional regulator of CD37 in B-cell lymphoma.

High frequency of inactivating tetraspanin C D37 mutations in diffuse large B-cell lymphoma at immune-privileged sites.

Tetraspanin CD37 is predominantly expressed on the cell surface of mature B lymphocytes and is currently being studied as novel therapeutic target for B-cell lymphoma. Recently, we demonstrated that loss of CD37 induces spontaneous B-cell lymphoma in Cd37-knockout mice and correlates with inferior survival in patients with diffuse large B-cell lymphoma (DLBCL). Here, CD37 mutation analysis was performed in a cohort of 137 primary DLBCL samples, including 44 primary immune-privileged site-associated DLBCL (IP-DLBCL) samples originating in the testis or central nervous system. CD37 mutations were exclusively identified in IP-DLBCL cases (10/44, 23%) but absent in non-IP-DLBCL cases. The aberrations included 10 missense mutations, 1 deletion, and 3 splice-site CD37 mutations. Modeling and functional analysis of CD37 missense mutations revealed loss of function by impaired CD37 protein expression at the plasma membrane of human lymphoma B cells. This study provides novel insight into the molecular pathogenesis of IP-DLBCL and indicates that anti-CD37 therapies will be more beneficial for DLBCL patients without CD37 mutations.

Pathways towards indolent B-cell lymphoma - Etiology and therapeutic strategies.

Although patients with indolent B-cell lymphomas have a relatively good survival rate, conventional chemotherapy is not curative. Disease courses are typically characterized by multiple relapses and progressively shorter response duration with subsequent lines of therapy. There has been an explosion of innovative targeted agents in the past years. This review discusses current knowledge on the etiology of indolent B-cell lymphomas with respect to the role of micro-organisms, auto-immune diseases, and deregulated pathways caused by mutations. In particular, knowledge on the mutational landscape of indolent B-cell lymphomas has strongly increased in recent years and harbors great promise for more accurate decision making in the current wide range of therapeutic options. Despite this promise, only in chronic lymphocytic leukemia the detection of TP53 mutations and/or del17p currently have a direct effect on treatment decisions. Nevertheless, it is expected that in the near future the role of genetic testing will increase for prediction of response to targeted treatment as well as for more accurate prediction of prognosis in indolent B-cell lymphomas.

Correlation of minimal residual disease cell frequency with molecular genotype in patients with acute myeloid leukemia.

BACKGROUND: About 70-80 percent of patients with acute myeloid leukemia enter complete remission, but at least half of these patients who achieve remission go on to relapse. Improved treatment is likely to come from increasing the time to relapse, especially for younger patients. With the vastly increasing number of targeted therapies there is a strong need for short-term end-points to efficiently test such therapies for further pursuance. Minimal residual disease assessment may offer such an end-point since it is a strong independent prognostic factor. As proof of principle we examined this concept for FLT3-ITD status at diagnosis. DESIGN AND METHODS: We determined FLT3-ITD status in bone marrow samples from 196 patients with newly diagnosed acute myeloid leukemia. The frequencies of residual leukemic cells of these 196 patients were assessed in 267 follow-up bone marrow samples using immunophenotypic assessment of minimal residual disease. RESULTS: The median frequency of residual leukemic cells after the first cycle of chemotherapy was 8.5-fold higher in patients with FLT3-ITD than in those with wild type FLT3. Such a difference translates into differences in survival, even if other potentially outcome-modulating mutations, such as NPM1, KIT, NRAS, KRAS, FLT3-exon 20 and PTPN11 are included in the analysis. CONCLUSIONS: This study shows that it could be possible to study the efficacy of FLT3 inhibitors using the level of minimal residual disease as a short-term end-point.

High INDO (indoleamine 2,3-dioxygenase) mRNA level in blasts of acute myeloid leukemic patients predicts poor clinical outcome.

Indoleamine 2,3-dioxygenase degrades the amino acid tryptophan which is essential for T cells. Tryptophan depletion causes T-cell cycle arrest and solid tumors that express high levels of indoleamine 2,3-dioxygenase can create immune suppression. Recently, blasts of patients with acute myeloid leukemia were shown to express indoleamine 2,3-dioxygenase. We determined INDO (encoding gene for indoleamine 2,3-dioxygenase) mRNA expression in leukemic blasts of 286 patients with acute myeloid leukemia by gene-expression profiling. Results were validated by quantitative polymerase chain reaction analysis in blasts of an independent cohort of 71 patients. High INDO expression was correlated to significantly shortened overall and relapse-free survival. Correlation of INDO expression to relevant known prognostic factors and survival identified high INDO expression as a strong negative independent predicting variable for overall and relapse-free survival. Inhibition of indoleamine 2,3-dioxygenase expressed by myeloid leukemic blasts may result in breaking immune tolerance and offers new therapeutic options for patients with acute myeloid leukemia.

Concurrent methylation of promoters from tumor associated genes predicts outcome in acute myeloid leukemia.

By assessment of the methylation status of 25 candidate tumor suppressor genes (TSGs) in 119 acute myeloid leukemia (AML) patients and 5 controls, we aimed to determine whether simultaneous methylation of multiple TSGs exerts prognostic impact. Methylation-specific multiplex ligation probe amplification (MS-MLPA) revealed methylation of at least one TSG in 59/119 patients, while no methylation was found in controls. Methylation of different TSGs within patients was substantially correlated (intra-class correlation; 0.38). ESR1 methylation (34/119) strongly predicted concurrent methylation of other genes, OR 7.33 (95%CI 4.13-12.99). A Cox regression model that included the three most frequently methylated TSGs ESR1, CDKN2B/p15 and IGSF4, showed ESR1 to have opposite effects on overall survival (OS) compared with the other two, HR 0.22 (95% CI 0.09-0.53) and HR 1.66 (95% CI 0.73-3.79), HR 1.61 (95%CI 0.66-3.93). By assessment of CDKN2B/p15 and IGSF4 methylation, patients with methylation at multiple loci can be identified. Accumulation of methylation aberrancies is much more pronounced in ESR1 methylated patients. When combined, the methylation status of ESR1, CDKN2B/p15 and IGSF4 enable identification of patient subgroups with large differences in OS (p <0.0001). This study shows that methylation profiling allows risk stratification in AML. In addition, ESR1 methylation may reflect a biological pathway that leads to hypermethylation of multiple genes, which is reflected by methylation of IGSF4 and/or CDKN2B/p15.

Small-molecule XIAP antagonist restores caspase-9 mediated apoptosis in XIAP-positive diffuse large B-cell lymphoma cells.

Clinical outcome in patients with primary nodal diffuse large B-cell lymphomas (DLBCLs) is correlated with expression of inhibitors of the intrinsic apoptosis pathway, including X-linked inhibitor of apoptosis protein (XIAP). XIAP suppresses apoptosis through inhibiting active caspase-3, caspase-7, and caspase-9. In this study, we investigated to see if the small-molecule XIAP antagonist 1396-12 induces cell death in cultured lymphoma cells of patients with DLBCL. Treatment with this XIAP antagonist resulted in relief of caspase-3 inhibition and in induction of apoptosis in 16 of 20 tested DLBCL samples. Sensitivity to the XIAP antagonist was observed in both chemotherapy-refractory and -responsive DLBCL, but did not affect peripheral blood mononuclear cells and tonsil germinal-center B cells from healthy donors. XIAP antagonist-sensitive samples were characterized by high expression levels of XIAP, relatively low expression levels of Bcl-2, and by constitutive caspase-9 activation. These data indicate that the small-molecule XIAP antagonist can induce apoptosis in cultured DLBCL cells and therefore should be considered for possible development as a therapy for these patients. In vitro sensitivity to the XIAP antagonist can be predicted based on biological markers, suggesting the possibility of predefining patients most likely to benefit from XIAP antagonist therapy.

Inhibition of the intrinsic apoptosis pathway downstream of caspase-9 activation causes chemotherapy resistance in diffuse large B-cell lymphoma.

PURPOSE: Inhibition of the apoptosis cascade is an important cause of therapy resistance in diffuse large B-cell lymphomas (DLBCL). In this study, we investigated possible mechanisms and expression levels of apoptosis-related genes in the apoptosis pathway that may be responsible for differences in chemotherapy sensitivity between DLBCL patients. EXPERIMENTAL DESIGN: Twenty-eight DLBCL patient samples were investigated for their expression levels of apoptosis-related genes using reverse transcription-multiplex ligation-dependent probe amplification analysis. Functional analysis of the intrinsic, caspase-9-mediated pathway was done using fluorescence-activated cell sorting analysis, Western blot analysis, and immunohistochemistry. RESULTS: Two DLBCL groups were identified: one with low expression levels of both proapoptotic and antiapoptotic genes and one group with high expression levels of these genes. DLBCL with high expression levels of proapoptotic and antiapoptotic genes frequently seemed to be refractory to clinical chemotherapy. Functional analysis in these latter DLBCL samples and DLBCL cell lines with comparable expression profiles revealed high levels of spontaneous caspase-9 activity without induction of apoptosis, indicating disruption of the apoptosis pathway downstream of caspase-9 activation. This disruption of the apoptosis pathway could be restored using a small-molecule XIAP antagonist. CONCLUSIONS: We conclude that the intrinsic, caspase-9-mediated apoptosis pathway is constitutively activated in part of chemotherapy-refractory DLBCL with concomitant downstream inhibition of the convergence apoptosis pathway and that inhibition of XIAP might be an alternative therapy for chemotherapy-refractory DLBCL.

Activated intrinsic apoptosis pathway is a key related prognostic parameter in acute myeloid leukemia.

PURPOSE: By parallel assessment of multiple apoptosis-related transcripts, we aimed to refine the current concept of apoptosis resistance in acute myeloid leukemia (AML) and identify the combination of genes best predicting overall survival (OS). PATIENTS AND METHODS: The reverse transcriptase multiplex ligation-dependent probe amplification technique was used for simultaneous quantification of 31 apoptosis-related transcripts in viable (7AAD-/AnnexinV-) blasts (CD45dim) from bone marrow aspirates of 120 newly diagnosed AML patients. By forward selection, a prognosis-predicting gene expression profile was constructed. The predictive validity of this profile was assessed by cross validation. RESULTS: High transcript levels were associated with poor OS for seven of 31 genes, three of which were proapoptotic. The average expression of all 12 antiapoptotic genes was associated with poor OS (P = .029). A similar association with poor OS was found for the average expression of all 19 proapoptotic genes (P = .009). Forward selection and cross validation revealed the antiapoptotic gene BIRC3 and the proapoptotic genes BAX-(l) and BMF to optimally predict OS. Three equally sized patient groups, constructed by ranking the cross-validated prognoses of the patients, were clearly distinct (median OS times were 8.2, 16.7, and 85.6 months). CONCLUSION: High expression of both pro- and antiapoptotic genes predicted poor OS, which postulates a mechanism of activation of the apoptosis pathway as a whole. This mechanism, which culminates in a three-gene expression signature, allows accurate clinical outcome prediction in AML and puts efforts to target single antiapoptosis genes in a new perspective.

Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences.

Copy number changes and CpG methylation of various genes are hallmarks of tumor development but are not yet widely used in diagnostic settings. The recently developed multiplex ligation-dependent probe amplification (MLPA) method has increased the possibilities for multiplex detection of gene copy number aberrations in a routine laboratory. Here we describe a novel robust method: the methylation-specific MLPA (MS-MLPA) that can detect changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in a simple reaction. In MS-MLPA, the ligation of MLPA probe oligonucleotides is combined with digestion of the genomic DNA-probe hybrid complexes with methylation-sensitive endonucleases. Digestion of the genomic DNA-probe complex, rather than double-stranded genomic DNA, allowed the use of DNA derived from the formalin treated paraffin-embedded tissue samples, enabling retrospective studies. To validate this novel method, we used MS-MLPA to detect aberrant methylation in DNA samples of patients with Prader-Willy syndrome, Angelman syndrome or acute myeloid leukemia.