Whole-Genome Sequencing of Primary Central Nervous System Lymphoma and Diffuse Large B-Cell Lymphoma

Actionable Genetic Features of Primary Testicular and Primary Central Nervous System Lymphomas

In Silico and Functional Characterization of TBL1XR1 as a Tumor Suppressor in Large B-Cell Lymphomas

Efficacy of Salvage Chemotherapy in Diffuse Large B Cell Lymphoma with Primary Treatment Failure According to Putative Cell of Origin

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), representing around 30% to 40% of all newly diagnosed lymphomas 1. DLBCL is clinically, morphologically and biologically a heterogeneous disease reflected in the highly variable clinical course. The 2008 World Health Organization (WHO) classification of lymphoid malignancies recognizes within the group of DLBCL, not otherwise specified (NOS) several subtypes characterized by unique clinical and pathological features including primary DLBCL of the central nervous system (CNS), primary cutaneous DLBCL, leg type, T-cell histiocyte-rich large cell lymphoma and EBV positive DLBCL of the elderly (Table 1). Nevertheless, most cases of DLBCL fall into the ‘NOS’ category. In the last 15 years our understanding of the genetic changes and biology of DLBCL has increased tremendously 2. Gene expression profiling (GEP) studies have revealed that DLBCL comprises several molecular subgroups that reflect either the stage in B cell development from which the disease originates or the activity of different biological programs 3, 4. The standard initial treatment for DLBCL is combined immunochemotherapy with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) 5. While durable remissions can be achieved in the majority of cases with this combined regimen, over 30% of patients will not respond or will relapse with resistant disease. A possible explanation for such differences in therapeutic success is the considerable biological heterogeneity of DLBCL. Therefore, there is an ongoing effort to tailor therapy based on specific subtypes of DLBCL, and to identify prognostic markers like BCL2 and MYC. The diagnosis of DLBCL needs to integrate, in addition to classic morphology and immunophenotype, all the new genetic and molecular diagnostic tools. This report attempts to review distinctive pathological characteristics of DLBCL and their clinical significance. DLBCL is characterized by monoclonal rearrangement of immunoglobulin heavy (IGH) and light chain (IGκ-IGλ) genes. Analogous to most B-NHL, DLBCL derives from a mature B cell that has experienced the germinal center (GC) reaction. Based on GEP studies DLBCLs have been divided into two main subgroups based on the putative cells of origin 3, 4. GC B cell-like (GCB)-DLBCL (50% cases) exhibits a transcriptional profile that resembles that of GC B-cell with expression of genes normally detected in GC B-cells such as CD10 and the transcriptional repressor BCL6 and harboring highly mutated immunoglobulin genes with ongoing somatic hypermutations (SHM). Activated B cell-like (ABC)-DLBCL shows several features of B cell receptor (BCR) activated B-cells at a plasmablastic stage, just prior to exit the GC with up-regulation of genes required for plasma cell differentiation (IRF4/MUM1) (Figure 1). These tumors downregulate the GC-specific program, activating at the same time, the NF-κB and BCR signaling pathways 2. Consistent with their late GC origin, these tumors do not show evidence of ongoing SHM. Because not all cases of DLBCL can be characterized as GCB or ABC, a less well-characterized group comprising about 15% of the cases remain unclassifiable. Within the group of mature B cell NHL, DLBCL shows the highest degree of genomic complexity including point mutations and copy number aberrations, and less frequently chromosomal translocations and gene amplification 2. Although some of these lesions might be observed in both GCB and ABC subtypes of DLBCL, most of them are preferentially associated with one or the other subtype of DLBCL, suggesting their potential role for diagnostic, prognostic and therapeutic stratification (Figure 2). The frequent mutations found in DLBCL are thought to be the result of an aberrant function of the physiologic SHM mechanism. Accordingly, mutations in CREBBP/EP300 and MLL2 are preferentially seen in GCB-subtype. EZH2 is required for GC formation and mutations in this gene are restricted to GCB-DLBCL. Translocations resulting in deregulated BCL2 and MYC are almost restricted to GCB-DLBCL. Around 30–40% of GCB-DLBCL carry the t(14;18) translocation, whereas MYC translocations have been identified in 5–15% of DLBCL 6. In contrast, translocations involving the BCL6 locus are present in 35% of DLBCL cases, being more frequent in ABC-DLBCL (25%). Another major transformation mechanism that impairs plasma cell differentiation are mutations and deletions in PRDM1/BLIMP1 identified in 25% of ABC-DLBCL. Constitutive activation of the NF-κB transcription factor complex represents the hallmark of ABC-DLBCL 2. The underlying causes of NF-κB signaling pathway activation are diverse and include gain-of-function mutations in signal transduction components of the BCR (CD79a and CARD11) and toll-like receptor (MYD88) signaling pathways. Loss-of-function mutation and/or deletions in the NF-κB negative regulator TNFAIP3/A20 have been reported in up to 30% of ABC-DLBCL. The diagnosis of DLBCL is usually not difficult. Lymph nodes demonstrate a diffuse proliferation of large lymphoid cells that have totally or partially effaced architecture. Cytological, DLBCL can be centroblastic, immunoblastic or anaplastic in appearance 1. Centroblastic morphology is the most common variant composed of medium to large cells with round to oval vesicular nuclei and fine chromatin with two to four nuclear membrane-bound nucleoli. There is moderate amphophilic to basophilic cytoplasm. In the immunoblastic variant greater than 90% of the cells have an immunoblastic appearance with a large centrally located nucleolus and basophilic or amphophilic cytoplasm. This variant has been associated with the non-germinal center type derivation, an adverse prognosis 4 and recently as a major reservoir for MYC-IGH translocations. In most cases, however; the tumors show an admixture of centroblasts and immunoblasts. The distinction of the immunoblastic variant from the centroblastic variant is not always straightforward and has generally met poor intraobserver and interobserver reproducibility. The anaplastic variant is rather rare and is characterized by large cells with bizarre pleomorphic nuclei. These cells may resemble Hodgkin or RS cells and may show sinusoidal and/or cohesive growth pattern and even mimic undifferentiated carcinoma. The anaplastic morphology is independent of ALK expression in B-cell lymphomas. Other rare morphologic variants exist including the signet ring cell, microvillous and spindle cell variants. The neoplastic cells characteristically express pan B-cell markers including CD19, CD20, CD22, PAX5 and CD79a. Surface and/or cytoplasmic immunoglobulin (IgM > IgG > IgA) can be demonstrated in 50–75% of the cases. Other markers commonly used in the characterization of DLBCL include CD10, BCL6, BCL2 and IRF4/MUM1 1. Aberrant phenotypes are not uncommon in DLBCL and may be responsible for confusion in the diagnosis. Lack of one or more B-cell markers may occur. In 10% of the cases the tumor cells express CD5. This CD5 positive DLBCL usually represent de novo cases, and only rarely are transformed CLL cases. Expression of CD5 is associated with worse prognosis even in the rituximab era. Aberrant expression of cytoplasmic CD3 has been also documented mainly in extranodal DLBCL without the expression of other T-cell markers. DLBCL with cyclin D1 expression lack the characteristic t(11;14) translocation of MCL, have a centroblastic morphology and a post-germinal center phenotype with positivity for IRF4/MUM1. These cases should be distinguished from MCL with blastic or pleomorphic morphology. In situ hybridization for EBERs should be performed in cases with geographic necrosis and/or Reed–Stenberg-like cells to exclude the possibility of an EBV+DLBCL of the elderly. Because of the prognostic value of cell of origin and the increasing effort to tailor therapy based on molecular characteristics of DLBCL, a reliable method to identify GCB and non-GCB subtypes is needed. GEP, which is considered the gold standard to assign the molecular subtypes, is not routinely available and is not cost effective in routine diagnosis. Several studies have attempted to recapitulate the molecular subgroups (GCB vs. non-GCB) using a limited panel of antibodies available in most pathology laboratories (Figure 1). The Hans algorithm has been the most widely used in clinical trials. In this classifier three antibodies are used CD10, BCL6 and IRF4/MUM1. According to this algorithm DLBCL with CD10 expression in more than 30% cells belong to the GCB group. Cases that are CD10 negative, BCL6 positive but IRF4/MUM1 negative are also GCB subtype. Cases that are IRF4/MUM1 positive with or without expression of BCL6 are assigned to the non-GCB subtype. There have been other algorithms attempting to improve the Hans classifier. These include the use of FOXP1 and GCET1 by Choi et al., and the use of LMO2 by Natkunam et al. One study comparing different algorithms showed an 87% concordance with GEP for the Choi method and 86% for Hans scheme 7. In this same study, the Tally algorithm based on FOXP1, GCET1, CD10, IRF4/MUM1 and LMO2 antibodies was demonstrated to be the most robust. The use of BCL6 was excluded from the analysis because it was considered a problematic, poorly reproducible stain. Nevertheless, in our experience BCL6 is reliable and helpful in the routine diagnosis. Accordingly, in the report from the international DLBCL rituximab-CHOP consortium program study, an algorithm based on expression of CD10, FOXP1 and BCL6 was used, which had a 92.6% concordance with GEP 8. Although most studies find that immunohistochemical algorithms correlate with prognosis in DLBCL, everybody agrees that these algorithms are an imperfect substitution for GEP. The improvement of molecular techniques is making possible to use paraffin-embedded material with results comparable to fresh-frozen material. The recent development of a 20-gene assay using NanoString technology in paraffin-embedded tissue is a promising methodology with potential to be used as a routine method for determining cell of origin in DLBCL 9. Translocations involving the MYC oncogene are the molecular hallmark of Burkitt lymphoma (BL). Most cases involve the IGH@ gene on chromosome 14. Less commonly, the light chain genes on chromosome 2 and 22 are involved in the translocation. These translocations in BL are characteristically the sole chromosomal aberration identified 10, 11. Approximately 5 to 15% of DLBCL cases have been reported to carry MYC translocations, whereas 19–38% of cases show MYC low copy number gains 6. Most MYC rearranged DLBCL cases fall into the GCB subtype 10, 12. In contrast to BL, MYC rearrangements in DLBCL are often seen to non-IGH@ partners and with complex karyotypes 10. The prognostic significance of MYC rearrangement alone in DLBCL is controversial. Several studies have demonstrated the association of MYC rearrangement with poorer outcome in DLBCL patients treated with R-CHOP 6. However, recent studies have suggested that the impact of MYC is strongly influenced by BCL2 and that MYC alone does not have a worse prognosis 13, 14. MYC rearranged DLBCL may either arise de novo or may represent a high-grade transformation of a low-grade lymphoma, usually follicular lymphoma. In the latter case the MYC translocation is accompanied by a BCL2 rearrangement, so-called ‘double-hit’ (DH) lymphoma. Approximately 40% of all DH lymphomas represent transformed FL cases 15. However, 60–80% of MYC rearranged bona-fide de novo DLBCL are accompanied either by a BCL2 and/or a BCL6 rearrangement, representing DH or triple hit (TH) lymphomas 12, 14. Altogether DLBCL with DH/TH represent around 3–6% of all DLBCL and are generally refractory to standard chemotherapy regimens and have a poor prognosis 13, 14, 16. Importantly, not all patients with DLBCL morphology and DH/TH may have a dismal prognosis. Recently, attention has focused on the apparent importance of the MYC partner because it has been suggested that only cases with an IG/MYC translocation have an adverse prognosis. The development of FISH assays readily performed in paraffin sections and more recently a monoclonal antibody that specifically recognizes MYC protein have facilitated the recognition of MYC alterations in routine diagnosis. Break-apart FISH probes are considered to be the most sensitive assay for identifying chromosomal rearrangements. In addition, the use of MYC/IGH dual fusion probe helps to confirm the MYC partner 6. However, dual fusion probes for MYC/IGκ and MYC/IGλ are not commercially available. MYC protein expression has been proposed as a rapid and cost-effective screening tool to identify MYC rearrangements. Although some studies agree that finding >70% MYC+ cells correlates with MYC rearrangement 6, 13, a considerable number of cases with MYC rearrangement fall into the low MYC expression group (<40%) 12. A combined approach MYC-FISH and IHC seems advisable. More common than MYC translocations is MYC protein overexpression reported to occur in 25–30% of DLBCL cases 13, 14, 16. Most studies used a cut-off of 40% for MYC positivity. Of note, the negative prognostic impact of MYC protein expression is observed only in patients who simultaneously overexpressed BCL2 protein, so-called ‘double-expresser’ (DE). The cut-off for BCL2 protein overexpression varies in different studies, but most studies required at least 50% of tumor cells. MYC and BCL2 DE have been reported to occur in 19–34% of DLBCL patients, and to have a worse prognosis than patients who do not express any or only one protein 13, 14, 16, but better prognosis than DH/TH DLBCL, which have a dismal outcome. Interestingly, the DE cases appear more common in the ABC subtype, and it has been suggested that this may largely contribute to the known inferior survival of the ABC subtype 16. The morphological distinction between BL and DLBCL has been problematic for pathologists. GEP studies have shown that BL has a characteristic signature but that there are cases within the spectrum of DLBCL and aggressive B-cell lymphomas, which have a similar molecular BL signature or fell into an intermediate category 10, 11. The 2008 WHO classification recognized this problematic an added a provisional category of B cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL (BCLU) (Figure 3) 15. BCLU is a disease of older patients presenting with nodal or extranodal disease usually in an advanced clinical stage, high lactate dehydrogenase and frequent bone marrow and CNS infiltration with a dismal prognosis. Interestingly, MYC rearrangements can be detected in 30–50% of the cases usually associated with many more chromosomal aberrations 15. The incidence of DH/TH in BCLU has been reported to be high (32%–78%).The precise morphological boundaries of this category are still not well defined. There is an ongoing discussion about how to classify aggressive lymphomas with DH/TH. One proposal is to continue classifying them based on morphology as DLBCL or BCLU or putting them together as a group of ‘DH/TH lymphomas’ regardless of the morphology of the tumor cells. The understanding of the biology of DLBCL has increased tremendously in the last 15 years. The diagnosis of DLBCL needs to integrate, in addition to standard morphology and immunohistochemistry, all available ancillary techniques. The routine use of FISH and IHC to detect MYC and BCL2 alterations/overexpression is recommended. Patients with DH and double expression of MYC and BCL2 represent poor-risk subsets in which alternative strategies should be explored 5. The distinction of GCB versus ABC-DLBCL has not yet led to differences in primary treatment. The current standard of care for most patients is R-CHOP, which has improved dramatically the outcome of DLBCL. However, for patients who fail R-CHOP, the choice of therapy is very likely to be influenced by the cell of origin 5. For example, bortezomib or BTK inhibitors have been shown to be effective in relapsed ABC-DLBCL but not in GCB subtype. On the contrary treatment with EZH2 inhibitors and BCL6 inhibitors have been shown to be effective in relapsed GCB-DLBCL. Emerging new targeted therapy will certainly influence the diagnosis and treatment of DLBCL in the near future.

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Primary central nervous system lymphoma (PCNSL) is an aggressive non-Hodgkin lymphoma confined to the central nervous system. Whether there is a PCNSL-specific genomic signature and, if so, how it differs from systemic diffuse large B-cell lymphoma (DLBCL) is uncertain.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; We performed a comprehensive genomic study of tumor samples from 19 immunocompetent PCNSL patients. Testing comprised array-comparative genomic hybridization and whole exome sequencing.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; Biallelic inactivation of &lt;i&gt;TOX&lt;/i&gt; and &lt;i&gt;PRKCD&lt;/i&gt; was recurrently found in PCNSL but not in systemic DLBCL, suggesting a specific role in PCNSL pathogenesis. In addition, we found a high prevalence of &lt;i&gt;MYD88&lt;/i&gt; mutations (79%) and &lt;i&gt;CDKN2A&lt;/i&gt; biallelic loss (60%). Several genes recurrently affected in PCNSL were common with systemic DLBCL, including loss of &lt;i&gt;TNFAIP3&lt;/i&gt;, &lt;i&gt;PRDM1&lt;/i&gt;, &lt;i&gt;GNA13&lt;/i&gt;, &lt;i&gt;TMEM30A&lt;/i&gt;, &lt;i&gt;TBL1XR1&lt;/i&gt;, &lt;i&gt;B2M&lt;/i&gt;, &lt;i&gt;CD58&lt;/i&gt;, activating mutations of &lt;i&gt;CD79B&lt;/i&gt;, &lt;i&gt;CARD11&lt;/i&gt;, and translocations &lt;i&gt;IgH-BCL6&lt;/i&gt;. Overall, B-cell receptor/Toll-like receptor/NF-κB pathways were altered in &gt;90% of PNCSL, highlighting its value for targeted therapeutic approaches. Furthermore, integrated analysis showed enrichment of pathways associated with immune response, proliferation, apoptosis, and lymphocyte differentiation.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; In summary, genome-wide analysis uncovered novel recurrent alterations, including &lt;i&gt;TOX&lt;/i&gt; and &lt;i&gt;PRKCD&lt;/i&gt;, helping to differentiate PCNSL from systemic DLBCL and related lymphomas. &lt;i&gt;Clin Cancer Res; 21(17); 3986–94. ©2015 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

INCIDENTAL PRIMARY PULMONARY DIFFUSE LARGE B-CELL LYMPHOMA

Results from a Phase 1 Study to Evaluate a Memory-Enriched CD19-Specific CAR T Cell Platform in Adult Patients with CD19+ B-Cell Non-Hodgkin Lymphoma (B-NHL)

Differences between Primary Central Nervous System Diffuse Large B-Cell Lymphoma and Intra-Lymph Node Diffuse Large B-Cell Lymphoma Based on Proteomic Research

Mutational Dynamics and Evolutionary Divergence in DLBCL: A Call for Relapse Sampling

Cell of Origin and Genomic Profile of Primary Central Nervous System Lymphoma Determined Using the Nanostring Lst Assay and Ultra Deep Targeted Next Generation Sequencing

Distinctive Genomic Alterations in Testicular Diffuse Large B Cell Lymphoma

Gene expression profiling has shown that diffuse large B-cell lymphoma (DLBCL) clusters into three major subtypes based on similarity in expression patterns to their cell of origin (COO): germinal center B-cell-like (GCB-DLBCL), activated B-cell-like DLBCL (ABC-DBLCL) and primary mediastinal B-cell lymphoma (PMBCL). These subtypes of DLBCLs are associated with distinctly different overall survival rates after standard immunochemotherapy. However, clinical and prognostic heterogeneity remains within COO subsets and strategies are needed to further stratify patients to identify and target high-risk subsets. A comprehensive genomic analysis of COO on a clinically defined set of DLBCL cases has not been performed and the aim of this study was to use whole-exome sequencing (WES) data from 58 paired tumor-normal DLBCL samples to assess association of known DLBCL genomic alterations with cell COO as well as for identification of novel and relevant genetic biomarkers.To investigate genomic alterations associated with DLBCL subsets, we analyzed WES and genome wide copy number data from 58 paired tumor-normal DLBCL tumors. Gene expression profiling or Hans classification was performed to determine DLBCL COO subtype; 31 patients were classified as GCB and 27 as non-GCB. The WES data were used to 1) assess the association of known DLBCL genomic alterations with COO, and 2) identify novel alternations associated with COO. Statistical analysis was performed and the data were ranked by significance (p≤ 0.05) within each DLBCL subtype. In total, 45 genomic abnormalities were analyzed for their association with either GCB, non-GCB or both. Mutations in CREBBP, EZH2, MEF2B, FOXO1 and REL have also been reported as GCB driver mutations and we observed GCB patients with these mutations, but the mutation clustering was not wholly associated with GCB. MYC double-hits were exclusively found in the GCB-subtype group. For the non-GCB cases we found that mutations in MAP2K3 and MYD88 were significantly associated with this subtype(p< 0.05). In addition to mutational patterns, we identified several copy number alterations (CNA) across both groups. Chromosomal losses in GCB patients were found at chromosomes 10q11.21-10q24.23, 4q12-4q35.2, 3q12.1-3q29, 4p12-4p16.3, 10p11.21-10p15.3, and 14q11.2-14q24.3 whereas gains were localized to 7q11.1-7q36.3, 7p11.2-7p22.3, and 1q21-1q32.1 (p < 0.05). No CNA was observed to directly associate with non-GCB patients, however, a loss at 9p21 and gains at 9q24.1 and 18q21.33 trended with the non-GCB subtype, supporting previous reports. Loss at 10q23.31 or a gain in 2p13-2p12 have been reported as being specific for GCB and our data confirmed the association of 10q23.31 with GCB while a gain at 2p13-2p12 (REL)was found in both subtypes. To further understand genomic differences between DLBCL subtypes, we evaluated the relative percentage of each genomic feature. 18/45 (40%) were only observed in GCB patients whereas 2/45 (5%) were specific to the non-GCB subtype. The majority (25/45, 55%) overlapped between the two subtypes.Throughout our analysis we noted that 7 non-GCB cases lacked any of the driver mutations analyzed in the study. While all cases carried mutations, they consisted of low frequency mutations that were not specifically associated with COO. 2/7 cases had a gain at 9p24.1 that included CD274 and JAK2. Because 9p24.1 gains have not been fully defined in DLBCL, we reviewed all cases and identified 4 (7%) with a 9p24.1 gain, 3 of which were non-GCB and 1 GCB. One non-GCB case was EBV+ and none of the cases showed evidence of PMBCL. Of the 9p24.1 cases, three had RNAseq data available and we found that PDL1 and JAK2 expression was elevated (12 fold p< 0.01 and 7 fold p< 0.01, respectively) when compared to the 9p24.1 normal cases (n=32). While outcome was not the focus of the study, we did note that 6/7 cases that lacked driver mutations achieved event free survival at 24 months (EFS24).Taken together, this analysis has further characterized the genetic profile of each COO subtype and has identified novel GCB CNAs which require independent replication. Additionally, we identify a subgroup of non-GCB DLBCL patients that do not harbor known driver mutations and require further genomic study to better resolve the biology of these tumors. Together, these data provide insight on the genetic heterogeneity of DLBCL and identify genetic variants that may inform subtype specific therapy. DisclosuresNowakowski:Morphosys: Research Funding; Celgene: Research Funding; Bayer: Consultancy, Research Funding. Rimsza:NCI/NIH: Patents & Royalties: L.M. Rimsza is a co-inventor on a provisional patent, owned by the NCI of the NIH, using Nanostring technology for determining cell of origin in DLBCL..

Treatment with Chimeric Antigen Receptor Modified T Cells Directed Against CD19 (CTL019) Results in Durable Remissions in Patients with Relapsed or Refractory Diffuse Large B Cell Lymphomas of Germinal Center and Non-Germinal Center Origin, "Double Hit" Diffuse Large B Cell Lymphomas, and Transformed Follicular to Diffuse Large B Cell Lymphomas

BackgroundAlthough primary lymphomas of the central nervous system (PCNSL) and extracerebral diffuse large B-cell lymphoma (DLBCL) cannot be distinguished histologically, it is still a matter of debate whether PCNSL differ from systemic DLBCL with respect to their molecular features and pathogenesis. Analysis of the DNA methylation pattern might provide further data distinguishing these entities at a molecular level.MethodsUsing an array-based technology we have assessed the DNA methylation status of 1,505 individual CpG loci in five PCNSL and compared the results to DNA methylation profiles of 49 DLBCL and ten hematopoietic controls.ResultsWe identified 194 genes differentially methylated between PCNSL and normal controls. Interestingly, Polycomb target genes and genes with promoters showing a high CpG content were significantly enriched in the group of genes hypermethylated in PCNSL. However, PCNSL and systemic DLBCL did not differ in their methylation pattern.ConclusionsBased on the data presented here, PCNSL and DLBCL do not differ in their DNA methylation pattern. Thus, DNA methylation analysis does not support a separation of PCNSL and DLBCL into individual entities. However, PCNSL and DLBCL differ in their DNA methylation pattern from non- malignant controls.

Germinal Center B-Cell-like (GCB) Phenotype Predicts Improved Outcomes in Central Nervous System Diffuse Large B-Cell Lymphoma Patients Receiving Stereotactic Radiosurgery (SRS)