Elena Hartmann

Genomic and Gene Expression Profiling Defines Indolent Forms of Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is typically a very aggressive disease with poor outcomes, but some cases display an indolent behavior that might not necessitate treatment at diagnosis. To define molecular criteria that might permit recognition of such cases, we compared the clinicopathologic features, gene expression, and genomic profile of patients who had indolent or conventional disease (iMCL or cMCL). Patients with iMCL displayed nonnodal leukemic disease with predominantly hypermutated IGVH and noncomplex karyotypes. iMCL and cMCL shared a common gene expression profile that differed from other leukemic lymphoid neoplasms. However, we identified a signature of 13 genes that was highly expressed in cMCL but underexpressed in iMCL. SOX11 was notable in this signature and we confirmed a restriction of SOX11 protein expression to cMCL. To validate the potential use of SOX11 as a biomarker for cMCL, we evaluated SOX11 protein expression in an independent series of 112 cases of MCL. Fifteen patients with SOX11-negative tumors exhibited more frequent nonnodal presentation and better survival compared with 97 patients with SOX11-positive MCL (5-year overall survival of 78% versus 36%, respectively; P = 0.001). In conclusion, we defined nonnodal presentation, predominantly hypermutated IGVH, lack of genomic complexity, and absence of SOX11 expression as qualities of a specific subtype of iMCL with excellent outcomes that might be managed more conservatively than cMCL.

High-level expression of the T-cell chemokines CCL3 and CCL4 by chronic lymphocytic leukemia B cells in nurselike cell cocultures and after BCR stimulation

In lymphatic tissues, chronic lymphocytic leukemia (CLL) cells are interspersed with CD68(+) nurselike cells (NLCs), T cells, and other stromal cells that constitute the leukemia microenvironment. However, the mechanism regulating colocalization of CLL and these accessory cells are largely unknown. To dissect the molecular cross talk between CLL and NLCs, we profiled the gene expression of CD19-purified CLL cells before and after coculture with NLCs. NLC coculture induced high-level expression of B-cell maturation antigen and 2 chemoattractants (CCL3, CCL4) by CLL cells. CCL3/CCL4 induction in NLC cocultures correlated with ZAP-70 expression by CLL cells. High CCL3/CCL4 protein levels were found in CLL cocultures with NLCs, and CCL3/CCL4 induction was abrogated by R406, a Syk inhibitor, suggesting that NLCs induce these chemokines via B-cell receptor (BCR) activation. BCR triggering also caused robust CCL3/CCL4 protein secretion by CLL cells. High CCL3 and CCL4 plasma levels in CLL patients suggest that this pathway plays a role in vivo. These studies reveal a novel mechanism of cross talk between CLL cells and their microenvironment, namely, the secretion of 2 T-cell chemokines in response to NLC coculture and BCR stimulation. Through these chemokines, CLL cells can recruit accessory cells and thereby actively create a supportive microenvironment.

Safety and activity of ibrutinib plus rituximab for patients with high-risk chronic lymphocytic leukaemia: a single-arm, phase 2 study

BACKGROUND Ibrutinib, an orally administered covalent inhibitor of Brutons tyrosine kinase (BTK), is an effective treatment for relapsed chronic lymphocytic leukaemia (CLL). We investigated the activity and safety of the combination of ibrutinib with the monoclonal antibody rituximab in patients with high-risk CLL. METHODS In this single-arm phase 2 study, we enrolled adult patients with high-risk CLL at the MD Anderson Cancer Center (Houston, TX, USA). All enrolled participants had high-risk cytogenetic abnormalities (deletion 17p, TP53 mutation, or deletion 11q) or a short progression-free survival (PFS <36 months) after previous first-line chemoimmunotherapy. Patients with symptomatic disease requiring therapy received 28-day cycles of once-daily ibrutinib 420 mg together with rituximab (375 mg/m(2), intravenously, every week during cycle 1, then once per cycle until cycle 6), followed by continuous daily single-agent ibrutinib 420 mg until disease progression or until toxicities or complications precluded further treatment. The primary endpoint was progression-free survival in the intention-to-treat population. This study is registered with ClinicalTrials.gov number NCT01520519, and is no longer accruing patients. FINDINGS Between Feb 28, 2012, and Sept 11, 2012, we enrolled 40 patients with CLL with high-risk disease features, 20 of whom had deletion 17p (del[17p]) or TP53 mutations (16 previously treated, four untreated), 13 had relapsed CLL with deletion 11q (del[11q]), and seven a PFS less than 36 months after first-line chemoimmunotherapy. 18-month PFS in all patients was 78·0% (95% CI 60·6-88·5), whereas in those with a del(17p) or TP53 mutation it was 72·4% (45·6-87·6) Toxicity was mainly mild to moderate in severity (grade 1-2). Diarrhoea occurred in ten (25%) patients (grade 1 in nine patients and grade 2 in one), bleeding events in 14 (33%) patients (eight grade 1 and five grade 2), nausea or vomiting in 15 patients (38%) (ten grade 1 and five grade 2), and fatigue in seven (18%) patients (four grade 1 and three grade 2). Five patients (13%) had grade 3 infections (two lung infections, one upper respiratory tract infection, one sepsis, and one mucositis), and no grade 4 or 5 infections occurred. One patient had grade 4 neutropenia. INTERPRETATION The encouraging safety and activity of ibrutinib and rituximab in this population of patients with high-risk CLL merits further investigation of this combination. FUNDING Pharmacyclics Inc, Cancer Prevention and Research Institute of Texas, Leukemia and Lymphoma Society, National Cancer Institute, MD Anderson Cancer Center.

SOX11 expression is highly specific for mantle cell lymphoma and identifies the cyclin D1-negative subtype

Cyclin D1-negative mantle cell lymphoma is difficult to distinguish from other small B-cell lymphomas. This study shows that SOX11 mRNA and nuclear protein expression is a highly specific marker for both cyclin D1-positive and negative mantle cell lymphoma. See related perspective article on page 1488. Background Cyclin D1-negative mantle cell lymphoma is difficult to distinguish from other small B-cell lymphomas. The clinical and pathological characteristics of patients with this form of lymphoma have not been well defined. Overexpression of the transcription factor SOX11 has been observed in conventional mantle cell lymphoma. The aim of this study was to determine whether this gene is expressed in cyclin D1-negative mantle cell lymphoma and whether its detection may be useful to identify these tumors. Design and Methods The microarray database of 238 mature B-cell neoplasms was re-examined. SOX11 protein expression was investigated immunohistochemically in 12 cases of cyclin D1-negative mantle cell lymphoma, 54 cases of conventional mantle cell lymphoma, and 209 additional lymphoid neoplasms. Results SOX11 mRNA was highly expressed in conventional and cyclin D1-negative mantle cell lymphoma and in 33% of the cases of Burkitt’s lymphoma but not in any other mature lymphoid neoplasm. SOX11 nuclear protein was detected in 50 cases (93%) of conventional mantle cell lymphoma and also in the 12 cyclin D1-negative cases of mantle cell lymphoma, the six cases of lymphoblastic lymphomas, in two of eight cases of Burkitt’s lymphoma, and in two of three T-prolymphocytic leukemias but was negative in the remaining lymphoid neoplasms. Cyclin D2 and D3 mRNA levels were significantly higher in cyclin D1-negative mantle cell lymphoma than in conventional mantle cell lymphoma but the protein expression was not discriminative. The clinico-pathological features and outcomes of the patients with cyclin D1-negative mantle cell lymphoma identified by SOX11 expression were similar to those of patients with conventional mantle cell lymphoma. Conclusions SOX11 mRNA and nuclear protein expression is a highly specific marker for both cyclin D1-positive and negative mantle cell lymphoma.

Landscape of somatic mutations and clonal evolution in mantle cell lymphoma

Significance This is a comprehensive whole-genome/whole-exome analysis of mantle cell lymphoma (MCL). We sequenced 29 MCL cases and validated the findings by target sequencing of 172 additional tumors. We identified recurrent mutations in genes regulating chromatin modification and genes such as NOTCH2 that have a major impact on clinical outcome. Additionally, we demonstrated the subclonal heterogeneity of the tumors already at diagnosis and the modulation of the mutational architecture in the progression of the disease. The identification of new molecular mechanisms may open perspectives for the management of MCL patients. Mantle cell lymphoma (MCL) is an aggressive tumor, but a subset of patients may follow an indolent clinical course. To understand the mechanisms underlying this biological heterogeneity, we performed whole-genome and/or whole-exome sequencing on 29 MCL cases and their respective matched normal DNA, as well as 6 MCL cell lines. Recurrently mutated genes were investigated by targeted sequencing in an independent cohort of 172 MCL patients. We identified 25 significantly mutated genes, including known drivers such as ataxia-telangectasia mutated (ATM), cyclin D1 (CCND1), and the tumor suppressor TP53; mutated genes encoding the anti-apoptotic protein BIRC3 and Toll-like receptor 2 (TLR2); and the chromatin modifiers WHSC1, MLL2, and MEF2B. We also found NOTCH2 mutations as an alternative phenomenon to NOTCH1 mutations in aggressive tumors with a dismal prognosis. Analysis of two simultaneous or subsequent MCL samples by whole-genome/whole-exome (n = 8) or targeted (n = 19) sequencing revealed subclonal heterogeneity at diagnosis in samples from different topographic sites and modulation of the initial mutational profile at the progression of the disease. Some mutations were predominantly clonal or subclonal, indicating an early or late event in tumor evolution, respectively. Our study identifies molecular mechanisms contributing to MCL pathogenesis and offers potential targets for therapeutic intervention.

Pathogenesis of Mantle-Cell Lymphoma: All Oncogenic Roads Lead to Dysregulation of Cell Cycle and DNA Damage Response Pathways

Mantle-cell lymphoma (MCL) is a well-defined subtype of B-cell non-Hodgkins lymphomas (B-NHL), accounts for approximately 6% of all lymphoid neoplasms, and has a median survival of 3 to 4 years. The genetic hallmark of MCL is the chromosomal translocation t(11;14)(q13;q32) that leads to deregulation and upregulation of Cyclin D1, an important regulator of the G1 phase of the cell cycle. This genetic event is present in virtually all cases of MCL, whereas additional genetic alterations that occur in subsets of MCL have been described. Most of these alterations appear to disturb the cell cycle machinery/interfere with the cellular response to DNA damage, thus making MCL a paradigm for cell cycle and DNA damage response dysregulation in cancer in general. In particular, Cyclin D1 upregulation, genomic amplification of the cyclin-dependent kinase (CDK) -4, deletions of the CDK inhibitor p16(INK4a) and overexpression of BMI-1, a transcriptional repressor of the p16(INK4a) locus, are associated with dysregulation of the cell cycle machinery in MCL. The DNA damage response pathway is affected by frequent alterations of the ataxia-telangiectasia mutated (ATM) kinase as well as occasional inactivation of checkpoint kinase (CHK)-1 and CHK2 that are kinases that act downstream of ATM in response to detection of DNA damage. Moreover, p53 is frequently targeted by alterations in MCL. A recent gene expression profiling study defined the proliferation signature, a quantitative measure of gene expression of proliferation-associated genes as the strongest survival predictor available to date allowing the definition of prognostic MCL subgroups that differ in median survival by more than 5 years.

Specific Secondary Genetic Alterations in Mantle Cell Lymphoma Provide Prognostic Information Independent of the Gene Expression–Based Proliferation Signature

PURPOSE To compare the genetic relationship between cyclin D1-positive and cyclin D1-negative mantle cell lymphomas (MCLs) and to determine whether specific genetic alterations may add prognostic information to survival prediction based on the proliferation signature of MCLs. PATIENTS AND METHODS Seventy-one cyclin D1-positive and six cyclin D1-negative MCLs previously characterized by gene expression profiling were examined by comparative genomic hybridization (CGH). RESULTS Cyclin D1-negative MCLs were genetically characterized by gains of 3q, 8q, and 15q, and losses of 1p, 8p23-pter, 9p21-pter, 11q21-q23, and 13q that were also the most common alterations in conventional MCLs. Parallel analysis of CGH aberrations and locus-specific gene expression profiles in cyclin D1-positive patients showed that chromosomal imbalances had a substantial impact on the expression levels of the genes located in the altered regions. The analysis of prognostic factors revealed that the proliferation signature, the number of chromosomal aberrations, gains of 3q, and losses of 8p, 9p, and 9q predicted survival of MCL patients. A multivariate analysis showed that the gene expression-based proliferation signature was the strongest predictor for shorter survival. However, 3q gains and 9q losses provided prognostic information that was independent of the proliferative activity. CONCLUSION Cyclin D1-positive and -negative MCLs share the same secondary genetic aberrations, supporting the concept that they correspond to the same genetic entity. The integration of genetic information on chromosome 3q and 9q alterations into a proliferation signature-based model may improve the ability to predict survival in patients with MCL.

Uniparental disomies, homozygous deletions, amplifications, and target genes in mantle cell lymphoma revealed by integrative high-resolution whole-genome profiling

Mantle cell lymphoma (MCL) is genetically characterized by the t(11;14)(q13;q32) translocation and a high number of secondary chromosomal alterations. However, only a limited number of target genes have been identified. We have studied 10 MCL cell lines and 28 primary tumors with a combination of a high-density single-nucleotide polymorphism array and gene expression profiling. We detected highly altered genomes in the majority of the samples with a high number of partial uniparental disomies (UPDs). The UPD at 17p was one of the most common, and it was associated with TP53 gene inactivation. Homozygous deletions targeted 4 known tumor suppressor genes (CDKN2C, BCL2L11, CDKN2A, and RB1) and 6 new genes (FAF1, MAP2, SP100, MOBKL2B, ZNF280A, and PRAME). Gene amplification coupled with overexpression was identified in 35 different regions. The most recurrent amplified regions were 11q13.3-q13.5, 13q31.3, and 18q21.33, which targeted CCND1, C13orf25, and BCL2, respectively. Interestingly, the breakpoints flanking all the genomic alterations, including UPDs, were significantly associated with genomic regions enriched in copy number variants and segmental duplications, suggesting that the recombination at these regions may play a role in the genomic instability of MCL. This integrative genomic analysis has revealed target genes that may be potentially relevant in MCL pathogenesis.

IgVH mutational status and clonality analysis of Richter's transformation: diffuse large B-cell lymphoma and Hodgkin lymphoma in association with B-cell chronic lymphocytic leukemia (B-CLL) represent 2 different pathways of disease evolution.

Approximately 5% of B-cell chronic lymphocytic leukemia (B-CLL) patients develop a secondary aggressive lymphoma, usually of diffuse large B-cell type (DLBCL), termed Richters transformation (RT). Rarely, classic Hodgkin lymphoma (HL) is observed. Published small series suggest that tumor cells in DLBCL and HL can be clonally identical to the B-CLL clone or arise as an independent, secondary lymphoma. We describe the morphology, immunophenotype, and clinical features of 34 classic RT patients with DLBCL, 6 cases of B-CLL with HL, and 8 cases with scattered CD30-positive Hodgkin and Reed-Sternberg (HRS)-like cells. The clonal relationship of the 2 components was analyzed using sequencing analysis of immunoglobulin heavy chain variable region (IgVH) genes. In classic RT, 18/23 B-CLL cases (78%) showed clonal progression to DLBCL with identical IgVH sequences in both lymphoma components, whereas in 5 cases (22%) the DLBCL was clonally unrelated. Among clonally related RT samples, 73% carried unmutated IgVH genes, whereas 4/5 unrelated cases were mutated. Immunophenotypically, most cases of DLBCL irrespective of clonal relatedness showed significant differences in phenotype compared with the B-CLL, with common loss of CD5 and CD23. Using immuno-laser capture microdissection, sequencing of the IgVH CDR3 region of isolated HRS cells showed that 2/2 cases with HL were clonally unrelated, whereas they were clonally identical in 1/2 cases of B-CLL with scattered HRS-like cells. HRS or HRS-like cells in all 3 unrelated cases showed evidence of Epstein-Barr virus infection. Of interest, 5/6 cases of B-CLL with HL, and 5/6 cases of B-CLL with HRS cells showed mutated IgVH genes.

Follicular lymphomas with and without translocation t(14;18) differ in gene expression profiles and genetic alterations

Follicular lymphoma (FL) is genetically characterized by the presence of the t(14;18)(q32;q21) chromosomal translocation in approximately 90% of cases. In contrast to FL carrying the t(14;18), their t(14;18)-negative counterparts are less well studied about their immunohistochemical, genetic, molecular, and clinical features. Within a previously published series of 184 FLs grades 1 to 3A with available gene expression data, we identified 17 FLs lacking the t(14;18). Comparative genomic hybridization and high-resolution single nucleotide polymorphism (SNP) array profiling showed that gains/amplifications of the BCL2 gene locus in 18q were restricted to the t(14;18)-positive FL subgroup. A comparison of gene expression profiles showed an enrichment of germinal center B cell-associated signatures in t(14;18)-positive FL, whereas activated B cell-like, NFkappaB, proliferation, and bystander cell signatures were enriched in t(14;18)-negative FL. These findings were confirmed by immunohistochemistry in an independent validation series of 84 FLs, in which 32% of t(14;18)-negative FLs showed weak or absent CD10 expression and 91% an increased Ki67 proliferation rate. Although overall survival did not differ between FL with and without t(14;18), our findings suggest distinct molecular features of t(14;18)-negative FL.