ALBERT

Description: Primary mediastinal large B-cell lymphoma (MLBCL) is a clinically distinct entity that typically presents as localized, sclerotic disease in young, female patients. We previously characterized the transcriptional profiles of MLBCLs and identified important shared features with a clinically related disorder, classical Hodgkin lymphoma (cHL) (Blood 102:3871, 2003). Given the documented role of the NFkB survival pathway in Hodgkin Reed-Sternberg cells, we previously assessed NFkB activation in MLBCL by determining the subcellular location of the c-REL subunit of the NFkB heterodimer with a 2-color immunofluoresence assay. In a small pilot MLBCL series, c-REL was localized to the nucleus in the majority of examined cases, consistent with NFkB activation. In the current study, we evaluated c-REL subcellular localization in an additional series of MLBCLs and DLBCLs using a broadly applicable immunoperoxidase method. 100% of MLBCLs exhibited nuclear c-REL staining whereas DLBCL c-REL subcellular localization was more variable. Thereafter, we analyzed the transcription profiles of the 34 MLBCLs and 176 DLBCLs for coordinate expression of NFkB target genes, using literature-curated NFkB target gene lists from three independent sources and gene set enrichment analysis (GSEA). MLBCL signatures exhibited significant enrichment of 2 of the 3 NFkB target gene sets. In addition, 32 NFkB target genes from the combined set were significantly more abundant in MLBCLs than DLBCLs (〉 30% more abundant and 〉 99th percentile in permutation analysis). Similar results were obtained in an independent series of MLBCLs and DLBCLs with available gene expression profiles (J. Exp. Med. 198:851, 2003). To assess the role of c-REL amplification in NFkB activation in our lymphoma series, we compared c-REL amplification, c-REL subcellular localization and coordinate expression of the identified NFkB target genes and classified the DLBCLs according to putative cell of origin. The majority of c-REL amplifications (67%) were found in DLBCLs of germinal center (GC) subtype, consistent with the observation that c-REL is part of the described GC signature. However, most (71%) of the examined GC DLBCLs had cytoplasmic c-REL expression and the GC DLBCLs did not have increased expression of NFkB target genes. Taken together with the MLBCL analyses, these studies indicate that: 1) NFkB is consistently activated in MLBCL; 2) c-REL amplification is not closely associated with NFkB activation in large cell lymphomas (LCLs); and 3) NFkB activation in LCL subtypes does not require amplification of the c-REL locus.

Description: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with recognized variability in clinical outcome, genetic features, and cells of origin. To date, transcriptional profiling has been used to highlight similarities between DLBCL tumor cells and normal B-cell subtypes and associate genes and pathways with unfavorable outcome. Given the genetic heterogeneity in DLBCL, there are likely to be subsets of tumors with different pathogenetic mechanisms and possible treatment targets. To identify DLBCL subtypes that were sufficiently robust to be captured by multiple methods, we analyzed the profiles of 176 newly diagnosed DLBCLs using three different clustering algorithms (hierarchical clustering (HC), self-organizing maps (SOM), and probabilistic clustering (PC)), the top 5% of genes with the highest reproducibility across duplicate samples and largest variation across patient tumors, and a resampling-based method (consensus clustering) that automatically selects the most stable numbers of clusters with each algorithm. Three discrete subsets of DLBCLs -- “Oxidative Phosphorylation” (OxPhos), “B-cell Receptor/Proliferation” and “Host Response” (HR) -- were identified, characterized using gene set enrichment analysis and confirmed in an independent series of newly diagnosed DLBCLs with available array data. There was an association between cluster membership and examined genetic abnormalities in DLBCL. BCL2 translocations were more common in the OxPhos cluster whereas BCL6 translocations were more frequent in the BCR/proliferation cluster. Translocations of either type were uncommon in the HR cluster. Patients with HR DLBCLs were also significantly younger than those with OxPhos or BCR/proliferation tumors; HR patients also had a significantly higher incidence of splenic and BM involvement. The unique characteristics of HR tumors - fewer known genetic abnormalities and prominent host immune and inflammatory cell transcripts -prompted us to assess host immune cells in study tumors using morphologic and immunohistochemical approaches. HR DLBCLs contained significantly higher numbers of morphologically distinct CD2+/CD3+ tumor-infiltrating lymphocytes and interdigitating S100+/GILT+/CD1a-/CD123- dendritic cells. The HR cluster shared features of histologically defined T-cell/histiocyte-richBCL, including fewer known genetic abnormalities, younger age at presentation and frequent splenic and bone marrow involvement. The current study identifies tumor microenvironment and host inflammatory response as defining features in DLBCL, provides insights into the nature of the host immune response in a major DLBCL subtype and suggests rational treatment targets in newly identified tumor groups.

Description: Introduction. Primary testicular lymphoma (PTL) and primary central nervous system lymphoma (PCNSL) are large B-cell lymphomas (LBCL) that occur in immune privileged (IP) sites and share certain clinical and molecular features. To date, the treatment of these IP lymphomas is largely empiric and more effective targeted therapies are needed. Methods. To define actionable genetic features of IP lymphomas, we performed comprehensive genomic analyses of 21 PCNSLs and 7 PTLs and validated specific alterations in an independent cohort of 43 additional PTLs. Recurrent copy number alterations (CNAs) were detected using high-density single nucleotide polymorphism (SNP) arrays and the GISTIC algorithm and integrated with transcriptional profiles to identify candidate driver genes. Recurrent somatic mutations were identified using a combination of whole exome sequencing (WES) of paired tumor/normal samples and whole transcriptome sequencing (RNA-Seq) of the additional tumors without paired normal samples. Results. In systemic diffuse large B-cell lymphomas (DLBCLs), multiple low-frequency CNAs and associated target genes decrease p53 activity and perturb cell cycle regulation; infrequent somatic mutations of TP53 also deregulate these pathways (Cancer Cell, 2012; 22:359-372). In contrast, PCNSLs and PTLs primarily exhibit bi-allelic deletion of the upstream regulator of p53 activity and cell cycle, CDKN2A (~70% PCNSLs and ~80% of PTLs) and rarely have copy loss or somatic mutations of TP53 or CNAs of additional pathway components. The most commonly mutated genes in PCNSL and PTL, CD79B and MYD88, are also perturbed in a subset of systemic DLBCLs. However, mutations of these two genes are much more frequent in IP lymphomas (70% MYD88 and 61% CD79B of analyzed PCNSLs and PTLs) and these alterations are commonly found in the same cases (57% of cases in this series). These data indicate that concurrent oncogenic activation of the B-cell receptor (BCR) and the Toll-like receptor (TLR) signaling pathways is a characteristic feature of IP lymphomas with implications for targeted therapies. Among the IP lymphomas, ~20% of PCNSLs and ~40% PTLs exhibit 3q12.3/NFKBIZ copy gain and increased expression of the NFKBIZ protein product, IκB-ζ, an atypical IκB family member induced by TLR signaling. In our PTL series, MYD88 wild-type tumors had the highest 3q12.3/NFKBIZ copy gains, and ~90% of all analyzed PTLs had structural bases for NFκB activation via the TLR pathway. Lentiviral-mediated IκB-ζ knockdown decreased expression of the IκB-ζ target genes, IκB-α and BCL-xL, and induced apoptosis of LBCL cell lines with MYD88 L265P mutations, NFKBIZ gain or both alterations. In addition, enforced expression of NFKBIZ enhanced the growth of LBCLs with normal NFKBIZ copy numbers. Taken together, these data suggest that many IP lymphomas depend upon oncogenic MYD88/NFKBIZ signaling. Although the majority of CNAs and somatic mutations were shared by PCNSLs and PTLs, certain alterations were primarily observed in PTL. In both the initial and independent validation series, 〉 40% of PTLs exhibited copy gain of chromosome 9p24.1/CD274 (PD-L1) / PDCD1LG2 (PD-L2) and associated overexpression of the PD-1 ligands. These observations were of particular interest because 9p24.1 copy gain is a characteristic abnormality in two additional lymphoid malignancies, primary mediastinal LBCL and classical Hodgkin lymphoma, PD-1 signaling promotes tumor immune evasion and the PD-1 pathway is targetable. We also identified one PTL in which a novel translocation juxtaposed the regulatory elements of TBL1XR1 (chromosome 3) to the start codon-bearing exon 2 of PDCD1LG2 (PD-L2) (chromosome 9). This translocation, which was detected by RNA-Seq and confirmed by 5’ RACE and a newly developed split-apart FISH assay, resulted in dramatic overexpression of the PD-L2 protein. These data suggest that PTLs utilize several genetic mechanisms to deregulate the PD-1 ligands and limit anti-tumor immunity. Conclusions. Integrative and comparative genomic studies define PCNSL and PTL as related but unique lymphoid malignancies with targetable genetic alterations, and associated p53 deficiency and cell cycle deregulation, concurrent oncogenic BCR and TLR signaling and PD-1 dependent immune evasion that warrant further clinical investigation. Note: B.C. and M.G.M.R have made equal contributions to this research. Disclosures Feuerhake: Roche Pharma Research and Early Development (pRED) from 2008-2012: Employment. Freeman:Merck: on the PD-1 pathway Patents & Royalties; EMD-Serrono: on the PD-1 pathway Patents & Royalties; Boehringer-Ingelheim: on the PD-1 pathway Patents & Royalties; Amplimmune: on the PD-1 pathway Patents & Royalties; Roche: on the PD-1 pathway Patents & Royalties; Bristol-Myers-Squibb: on the PD-1 pathway Patents & Royalties; Novatis: on the PD-1 pathway, on the PD-1 pathway Patents & Royalties. Shipp:Sanofi: Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers-Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Janssen R&D: Membership on an entity's Board of Directors or advisory committees.

Description: Key Points PCNSLs and PTLs have a defining genetic signature that differs from other LBCLs and suggests rational targeted therapies. PCNSLs and PTLs frequently exhibit 9p24.1/PD-L1/PD-L2 copy number alterations and translocations, likely genetic bases of immune evasion.

Description: INTRODUCTION: A promising rich pipeline of combination therapies targeting checkpoint molecules expressed on T cells and/or tumor cells is currently being developed to abrogate tumor-induced immunosuppression. Novel in vivo models suitable for validating these immunotherapies and predict safety issues are warranted to accelerate their translation to patients. AIM: Epstein Barr virus (EBV) is a type 1 carcinogen that is directly associated with the development of human B cell neoplasms. We modelled EBV infection and tumor progression in long-term humanized mice and investigated the activation of T cells with PD-1 expression. Further, we performed studies evaluating the effects of an anti-PD-1 antibody (pembrolizumab/ keytruda) in on EBV infections and/or tumor growth. METHODS: Humanized mice transplanted with human cord-blood CD34+ stem cells and showing long-term (15 weeks) human T cell reconstitution were infected with an oncogenic recombinant Epstein Barr Virus (EBV), encoding enhanced firefly luciferase (fLuc) and green fluorescent protein (GFP). EBV infections were monitored by optical imaging analyses and PCR. CD8+ and CD4+ T cell subtypes (PD-1+, naïve, central memory, effector memory and terminal effector) were sequentially monitored in blood by longitudinal flow cytometry analyses and in organs at experimental endpoint. Histopathological analyses were performed to characterize EBV infection (EBER+) and PD-1+ T cell-rich infiltrates in tissues and tumors. We used the model to evaluate the effects of pembrolizumab administered after EBV challenge at low dose (first dose 1.65mg/kg and then 3.30 mg/kg, every other week, n=3) or high dose (first dose 5.00 mg/kg and then 10.00 mg/kg every other week, n=3) in respect to EBV infected controls (n=2). RESULTS: EBV-fLuc was detectable one week after infection by non-invasive optical imaging in the spleen, from where it spread rapidly and systemically. Among the EBV-infected mice, 8/18 (=44%) developed macroscopically visible tumors in the spleen. For further analyses of the data, we then compared EBV-infected mice with ("EBV-Tumor") or without ("EBV") macroscopic tumors. At 6 weeks post-infection, the relative CD8+ T cell frequencies increased significantly and constantly (control Vs. EBV p=0.0021, control Vs. EBV-Tumor p=

Description: Introduction: High-dose chemotherapy (HDT) and autologous stem-cell transplantation (ASCT) demonstrated high efficacy in the treatment of newly-diagnosed primary CNS lymphoma (PCNSL) in younger patients (pts.). A 5-year overall survival probability (OS) of 69% could be demonstrated in 30 pts within a phase-II trial on HDT and ASCT with consolidating whole-brain-irradiation (WBRT) (Illerhaus et al. JCO 2006). A subsequent pilot trial on HDT and ASCT without WBRT showed a 5-year OS of 77% (Illerhaus et al. Haematologica 2008). Here we give an update of our two different treatment regimens and future perspectives. Patients and Methods: Thirty pts. ≤65 years were treated within the phase II trial, chemotherapy (CHT) consisted of 3 cycles of high-dose methotrexate (MTX, 8 g/m2), 1 cycle of AraC (2× 3 g/m2) plus thiotepa (TT, 40 mg/m2) followed by rG-CSF and stem-cell-mobilization. Conditioning regimen included BCNU (400 mg/m2) and TT (2×5 mg/kgBW) followed by ASCT. Hyperfractionated WBRT (45 Gy, 2×1Gy/d) was administered as consolidation. In our subsequent pilot trial 13 pts. (age 38–67 years) were treated without consolidating WBRT; CHT was intensified with 4 cycles MTX 8g/m2, 2 cycles AraC (2× 3 g/m2) and TT (40 mg/m2). Dose escalated HDT included BCNU (400 mg/m2) and TT (4×5 mg/kgBW) followed by ASCT. WBRT was reserved for pts. not responding to CHT. Results: Median follow-up of the 30 pts. treated within our phase II trial was extended to 95 months (mo), the updated 5-year OS of all pts. is 66.6% and 82,3% of the subgroup of pts. who underwent HDT and ASCT (n=23), respectively. Three additional deaths occurred due to relapse (n=2) after 45 and 71 mo and due to comorbidity (n=1) after 103 mo. Five of 30 pts. developed severe leukoencephalopathy during follow-up. With a median follow-up of 35 mo in the 13 pts. treated within the pilot-phase without consolidating WBRT 3-year OS of all pts. is 77%. No further relapse or non-relapse mortality occurred in this pilot-group during. Most recent follow-up data will be presented in detail. Conclusion: Sequential systemic application of high-dose cytostatic agents followed by HDT+ASCT is highly effective as initial therapy for pts. with PCNSL. The restriction of WBRT to refractory disease shows similar OS rates and a decrease in neurotoxicity. In an ongoing multicenter phase-II trial immunotherapy with rituximab is combined with HDT and ASCT to further increase remission rates. A future randomized trial should be focused on the efficacy of consolidation with HDT supported by ASCT.

Description: Background: Primary NHL of the CNS (PCNSL) are associated with a dismal prognosis despite initial response to steroids and radiotherapy (RT). Addition of high-dose methotrexate (HD-MTX) to RT has improved the prognosis of patients (pts) with PCNSL. However, the majority of pts eventually relapse. To improve survival we performed a multicenter phase II study with early high-dose chemotherapy (HDT) and autologous stem-cell transplantation (ASCT) followed by hyperfractionated whole-brain radiation (WBRT) for 30 pts under 65yrs. Five-year overall survival rates of 69% for all pts and 87% for 23 pts receiving HDT and ASCT could be reported (Illerhaus et al., J Clin Oncol. 2006). Purpose: Here we present the results of 1) a pilot study for HDT and ASCT with WBRT restricted to residual disease in pts ≤65 years; 2) a multicenter phase II study for MTX-based CT and 3) a pilot-study for chemo-immunotherapy in pts 〉 65 years. Methods and Results: New treatment regimen for pts ≤65 years: CT consists of 4 cycles HD-MTX (8g/m2), 2 cycles AraC (2×3g/m2) and thiotepa (40mg/m2) followed by HDT with BCNU (400mg/m2) and thiotepa (4×5mg/kg) before ASCT. To date, 12 pts have been treated in this single center pilot-study. After HDT and ASCT 7/10 pts (70%) responded with complete remission (CR), 2/10 pts with partial remission (PR), 1 pt showed progressive disease (PD) and died after refusing RT. The 2 pts with PR have been irradiated resulting in continuous CR. Two pts were off study due to refractory disease. After a median follow-up of 17 months (mo) (range 4–41) 9/12 pts are alive in continuous CR. One pt developed a systemic relapse and died 8 months after ASCT. Overall, the treatment was well tolerated without grade IV toxicity. Patients 〉65 yrs, MCP-protocol: Thirty-two pts (17 female, 15 male, median age 71 yrs, range 57–79y) were treated in a phase II trial with 3 repetitive cycles of HD-MTX (3g/m2, d1, 15, 30) combined with procarbazine (60 mg/m2 p.o., d1-10) and CCNU (110 mg/m2 p.o., d 1). There was no lower limit of Karnofsky Performance Status. Thirty-two pts received 1 cycle, 17 pts received 2 cycles and 10 pts received 3 cycles. Best documented response in 25 evaluable pts were CR in 13/32 (41%), PR in 7/32 (22%) and PD 5/32 (16%) pts. Five of 32 pts developed severe renal impairment after MTX and were treated off-study. One patient died due to neutropenic fever. With a median follow-up of 64 mo (range 0–82 mo), the 5-year overall survival probability currently is 30.5%, the median survival is 15 mo. As of July 2006 9/32 (28%) pts are alive, 8 without evidence for leukoencephalopathy. New treatment regimen for pts 〉65 years, R-MCP-Protocol: In a subsequent pilot-phase rituximab has been added before each MTX-application. In a single center pilot-phase, 9 pts were treated within the protocol. The response rates were CR in 4/7 (57%) evaluable pts, PR, SD and PD, each in one pt, respectively. One patient received only one dose of MTX due to liver toxicity and developed CR with rituximab as single agent. To date, after a median follow-up of 4 mo (range 0–11mo) 8 of 9 pts are alive. Conclusion: The protocols presented here are safe and show high efficacy in treating patients with PCNSL in both age-groups. The addition of rituximab to MTX-based chemotherapy is promising and warrants further investigation.

Description: The proteasome inhibitor, bortezomib (VELCADE®, formerly PS341), has significant anti-tumor activity in several lymphoid malignancies. Reported targets of this broad-based inhibitor include the NF K B pathway (I K B A). Recently defined subtypes of large B-cell lymphoma (LBCL) exhibit constitutive activation of NF K B, prompting us to analyze the efficacy of bortezomib in a panel of 10 DLBCL cell lines. Six of the diffuse LBCL cell lines were sensitive to bortezomib treatment at doses below 10 nM (range IC50 = 2.9 to 6.9 nM) whereas 4 cell lines were resistant at 10 nM (IC50 = 14.8 to 70.2 nM). Baseline proteasomal function, as defined by cleavage of the 20S proteasome-specific fluorogenic peptide LLVY-AMC, was similar in sensitive and resistant DLBCLs; however, the IC50 for bortezomib proteasomal inhibition was somewhat lower in sensitive vs. resistant lines (sens. vs res., p = .04, one-sided t test). Baseline NF K B activity varied widely in the DLBCL cell lines and did not differ in cell lines that were sensitive vs. resistant to bortezomib. Ten nM bortezomib did not inhibit NF K B activity in resistant DLBCL cell lines whereas the same dose reduced NF K B activity in sensitive DLBCL cell lines (sens. vs. res., p 〈 .005, rank test [Mann-Whitney]). However, 5 of 6 sensitive DLBCL cell lines had very low baseline NF K B levels (〈 0.5 relative absorbance units) suggesting that NF K B inhibition was not a major factor in bortezomib response and prompting further analysis of additional bortezomib targets. Three sensitive and 1 resistant DLBCL cell line were selected for detailed analyses of transcriptional profiles following bortezomib treatment. We developed an algorithm for identifying genes that were significantly up- or down-regulated in the bortezomib-sensitive cell lines but unchanged in the resistant line. In addition, we utilized gene set enrichment analysis (GSEA) and gene ontogeny (GO) termed enrichment to interpret the molecular signatures of response. Genes down-regulated in response to bortezomib included critical B-cell transcription factors, components of the B-cell receptor signaling cascade and genes regulating mitosis and cell cycle control; up-regulated genes included heat shock proteins (HSP) and multiple proteasomal components. Consistent with the functional data, down-regulation of NF K B target genes was not a common feature in all bortezomib-sensitive cell lines. In contrast, target genes of the c-MYC transcription factor were significantly down-regulated and c-MYC activity was decreased in sensitive (but not resistant) DLBCL cell lines following bortezomib treatment (sens. vs. res., p 〈 .005, rank test). Taken together, the results provide insights into likely mechanisms of action of bortezomib in DLBCL, highlighting c-MYC as a potentially important target and identifying HSP as a complementary target to overcome bortezomib resistance.