Biological and clinical implications of BIRC3 mutations in chronic lymphocytic leukemia.

BIRC3 is a recurrently mutated gene in chronic lymphocytic leukemia (CLL) but the functional implications of BIRC3 mutations are largely unexplored. Furthermore, little is known about the prognostic impact of BIRC3 mutations in CLL cohorts homogeneously treated with first-line fludarabine, cyclophosphamide, and rituximab (FCR). By immunoblotting analysis, we showed that the non-canonical nuclear factor-κB pathway is active in BIRC3-mutated cell lines and in primary CLL samples, as documented by the stabilization of MAP3K14 and by the nuclear localization of p52. In addition, BIRC3-mutated primary CLL cells are less sensitive to flu-darabine. In order to confirm in patients that BIRC3 mutations confer resistance to fludarabine-based chemoimmunotherapy, a retrospective multicenter cohort of 287 untreated patients receiving first-line FCR was analyzed by targeted next-generation sequencing of 24 recurrently mutated genes in CLL. By univariate analysis adjusted for multiple comparisons BIRC3 mutations identify a poor prognostic subgroup of patients in whom FCR treatment fails (median progression-free survival: 2.2 years, P<0.001) similar to cases harboring TP53 mutations (median progression-free survival: 2.6 years, P<0.0001). BIRC3 mutations maintained an independent association with an increased risk of progression with a hazard ratio of 2.8 (95% confidence interval 1.4-5.6, P=0.004) in multivariate analysis adjusted for TP53 mutation, 17p deletion and IGHV mutation status. If validated, BIRC3 mutations may be used as a new molecular predictor to select high-risk patients for novel frontline therapeutic approaches.

Diffuse large B-cell lymphoma genotyping on the liquid biopsy.

Accessible and real-time genotyping for diagnostic, prognostic, or treatment purposes is increasingly impelling in diffuse large B-cell lymphoma (DLBCL). Cell-free DNA (cfDNA) is shed into the blood by tumor cells undergoing apoptosis and can be used as source of tumor DNA for the identification of DLBCL mutations, clonal evolution, and genetic mechanisms of resistance. In this study, we aimed at tracking the basal DLBCL genetic profile and its modification upon treatment using plasma cfDNA. Ultra-deep targeted next generation sequencing of pretreatment plasma cfDNA from DLBCL patients correctly discovered DLBCL-associated mutations that were represented in >20% of the alleles of the tumor biopsy with >90% sensitivity and ∼100% specificity. Plasma cfDNA genotyping also allowed for the recovery of mutations that were undetectable in the tissue biopsy, conceivably because, due to spatial tumor heterogeneity, they were restricted to clones that were anatomically distant from the biopsy site. Longitudinal analysis of plasma samples collected under rituximab-cyclophosphamide-doxorubicin-vincristine-prednisone (R-CHOP) chemotherapy showed a rapid clearance of DLBCL mutations from cfDNA among responding patients. Conversely, among patients who were resistant to R-CHOP, basal DLBCL mutations did not disappear from cfDNA. In addition, among treatment-resistant patients, new mutations were acquired in cfDNA that marked resistant clones selected during the clonal evolution. These results demonstrate that cfDNA genotyping of DLBCL is as accurate as genotyping of the diagnostic biopsy to detect clonally represented somatic tumor mutations and is a real-time and noninvasive approach to tracking clonal evolution and the emergence of treatment-resistant clones.

The genetics of nodal marginal zone lymphoma.

Nodal marginal zone lymphoma (NMZL) is a rare, indolent B-cell tumor that is distinguished from splenic marginal zone lymphoma (SMZL) by the different pattern of dissemination. NMZL still lacks distinct markers and remains orphan of specific cancer gene lesions. By combining whole-exome sequencing, targeted sequencing of tumor-related genes, whole-transcriptome sequencing, and high-resolution single nucleotide polymorphism array analysis, we aimed at disclosing the pathways that are molecularly deregulated in NMZL and we compare the molecular profile of NMZL with that of SMZL. These analyses identified a distinctive pattern of nonsilent somatic lesions in NMZL. In 35 NMZL patients, 41 genes were found recurrently affected in ≥3 (9%) cases, including highly prevalent molecular lesions of MLL2 (also known as KMT2D; 34%), PTPRD (20%), NOTCH2 (20%), and KLF2 (17%). Mutations of PTPRD, a receptor-type protein tyrosine phosphatase regulating cell growth, were enriched in NMZL across mature B-cell tumors, functionally caused the loss of the phosphatase activity of PTPRD, and were associated with cell-cycle transcriptional program deregulation and increased proliferation index in NMZL. Although NMZL shared with SMZL a common mutation profile, NMZL harbored PTPRD lesions that were otherwise absent in SMZL. Collectively, these findings provide new insights into the genetics of NMZL, identify PTPRD lesions as a novel marker for this lymphoma across mature B-cell tumors, and support the distinction of NMZL as an independent clinicopathologic entity within the current lymphoma classification.

MicroRNA expression profiling identifies molecular signatures associated with anaplastic large cell lymphoma.

Anaplastic large-cell lymphomas (ALCLs) encompass at least 2 systemic diseases distinguished by the presence or absence of anaplastic lymphoma kinase (ALK) expression. We performed genome-wide microRNA (miRNA) profiling on 33 ALK-positive (ALK[+]) ALCLs, 25 ALK-negative (ALK[-]) ALCLs, 9 angioimmunoblastic T-cell lymphomas, 11 peripheral T-cell lymphomas not otherwise specified (PTCLNOS), and normal T cells, and demonstrated that ALCLs express many of the miRNAs that are highly expressed in normal T cells with the prominent exception of miR-146a. Unsupervised hierarchical clustering demonstrated distinct clustering of ALCL, PTCL-NOS, and the AITL subtype of PTCL. Cases of ALK(+) ALCL and ALK(-) ALCL were interspersed in unsupervised analysis, suggesting a close relationship at the molecular level. We identified an miRNA signature of 7 miRNAs (5 upregulated: miR-512-3p, miR-886-5p, miR-886-3p, miR-708, miR-135b; 2 downregulated: miR-146a, miR-155) significantly associated with ALK(+) ALCL cases. In addition, we derived an 11-miRNA signature (4 upregulated: miR-210, miR-197, miR-191, miR-512-3p; 7 downregulated: miR-451, miR-146a, miR-22, miR-455-3p, miR-455-5p, miR-143, miR-494) that differentiates ALK(-) ALCL from other PTCLs. Our in vitro studies identified a set of 32 miRNAs associated with ALK expression. Of these, the miR-17∼92 cluster and its paralogues were also highly expressed in ALK(+) ALCL and may represent important downstream effectors of the ALK oncogenic pathway.

STAT3-mediated activation of microRNA cluster 17~92 promotes proliferation and survival of ALK-positive anaplastic large cell lymphoma.

Systemic anaplastic large cell lymphoma is a category of T-cell non-Hodgkin's lymphoma which can be further subdivided into two distinct entities (ALK(+) and ALK(-)) based on the presence or absence of ALK gene rearrangements. Among several pathways triggered by ALK signaling, constitutive activation of STAT3 is strictly required for ALK-mediated transformation and survival. Here we performed genome-wide microRNA profiling and identified 48 microRNA concordantly modulated by the inducible knock-down of ALK and STAT3. To evaluate the functional role of differentially expressed miRNA, we forced their expression in ALK(+) anaplastic large cell lymphoma cells, and monitored their influence after STAT3 depletion. We found that the expression of the microRNA-17~92 cluster partially rescues STAT3 knock-down by sustaining proliferation and survival of ALK(+) cells. Experiments in a xenograft mouse model indicated that forced expression of microRNA-17~92 interferes with STAT3 knock-down in vivo. High expression levels of the microRNA-17~92 cluster resulted in down-regulation of BIM and TGFßRII proteins, suggesting that their targeting might mediate resistance to STAT3 knock-down in anaplastic large cell lymphoma cells. We speculate that the microRNA-17~92 cluster is involved in lymphomagenesis of STAT3(+) ALCL and that its inhibition might represent an alternative avenue to interfere with ALK signaling in anaplastic large cell lymphomas.

IRF4 Mediates the Oncogenic Effects of STAT3 in Anaplastic Large Cell Lymphomas.

Systemic anaplastic large cell lymphomas (ALCL) are a category of T-cell non-Hodgkin's lymphomas which can be divided into anaplastic lymphoma kinase (ALK) positive and ALK negative subgroups, based on ALK gene rearrangements. Among several pathways aberrantly activated in ALCL, the constitutive activation of signal transducer and activator of transcription 3 (STAT3) is shared by all ALK positive ALCL and has been detected in a subgroup of ALK negative ALCL. To discover essential mediators of STAT3 oncogenic activity that may represent feasible targets for ALCL therapies, we combined gene expression profiling analysis and RNA interference functional approaches. A shRNA screening of STAT3-modulated genes identified interferon regulatory factor 4 (IRF4) as a key driver of ALCL cell survival. Accordingly, ectopic IRF4 expression partially rescued STAT3 knock-down effects. Treatment with immunomodulatory drugs (IMiDs) induced IRF4 down regulation and resulted in cell death, a phenotype rescued by IRF4 overexpression. However, the majority of ALCL cell lines were poorly responsive to IMiDs treatment. Combination with JQ1, a bromodomain and extra-terminal (BET) family antagonist known to inhibit MYC and IRF4, increased sensitivity to IMiDs. Overall, these results show that IRF4 is involved in STAT3-oncogenic signaling and its inhibition provides alternative avenues for the design of novel/combination therapies of ALCL.

Convergent mutations and kinase fusions lead to oncogenic STAT3 activation in anaplastic large cell lymphoma.

A systematic characterization of the genetic alterations driving ALCLs has not been performed. By integrating massive sequencing strategies, we provide a comprehensive characterization of driver genetic alterations (somatic point mutations, copy number alterations, and gene fusions) in ALK(-) ALCLs. We identified activating mutations of JAK1 and/or STAT3 genes in ∼20% of 88 [corrected] ALK(-) ALCLs and demonstrated that 38% of systemic ALK(-) ALCLs displayed double lesions. Recurrent chimeras combining a transcription factor (NFkB2 or NCOR2) with a tyrosine kinase (ROS1 or TYK2) were also discovered in WT JAK1/STAT3 ALK(-) ALCL. All these aberrations lead to the constitutive activation of the JAK/STAT3 pathway, which was proved oncogenic. Consistently, JAK/STAT3 pathway inhibition impaired cell growth in vitro and in vivo.