Aster, Jon Christopher

Abstract 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.

Abstract 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.

NOTCH1 is frequently mutated in adenoid cystic carcinoma (ACC). To test the idea that immunohistochemical (IHC) staining can identify ACCs with NOTCH1 mutations, we performed IHC for activated NOTCH1 (NICD1) in 197 cases diagnosed as ACC from 173 patients. NICD1 staining was positive in 194 cases (98%) in 2 major patterns: subset positivity, which correlated with tubular/cribriform histology; and diffuse positivity, which correlated with a solid histology. To determine the relationship between NICD1 staining and NOTCH1 mutational status, targeted exome sequencing data were obtained on 14 diffusely NICD1-positive ACC specimens from 11 patients and 15 subset NICD1-positive ACC specimens from 15 patients. This revealed NOTCH1 gain-of-function mutations in 11 of 14 diffusely NICD1-positive ACC specimens, whereas all subset-positive tumors had wild-type NOTCH1 alleles. Notably, tumors with diffuse NICD1 positivity were associated with significantly worse outcomes (P=0.003). To determine whether NOTCH1 activation is unique among tumors included in the differential diagnosis with ACC, we performed NICD1 IHC on a cohort of diverse salivary gland and head and neck tumors. High fractions of each of these tumor types were positive for NICD1 in a subset of cells, particularly in basaloid squamous cell carcinomas; however, sequencing of basaloid squamous cell carcinomas failed to identify NOTCH1 mutations. These findings indicate that diffuse NICD1 positivity in ACC correlates with solid growth pattern, the presence of NOTCH1 gain-of-function mutations, and unfavorable outcome, and suggest that staining for NICD1 can be helpful in distinguishing ACC with solid growth patterns from other salivary gland and head and neck tumors.

Abstract Notch signaling plays a role in normal lymphocyte development and function. Activating Notch1-mutations, leading to aberrant downstream signaling, have been identified in human T-cell acute lymphoblastic leukemia (T-ALL). While this highlights the contribution of Notch signaling to T-ALL pathogenesis, the mechanisms by which Notch regulates proliferation and survival in normal and leukemic T cells are not fully understood. Our findings identify a role for Notch signaling in G1-S progression of cell cycle in T cells. Here we show that expression of the G1 proteins, cyclin D3, CDK4, and CDK6, is Notch-dependent both in vitro and in vivo, and we outline a possible mechanism for the regulated expression of cyclin D3 in activated T cells via CSL (CBF-1, mammals; suppressor of hairless, Drosophila melanogaster; Lag-1, Caenorhabditis elegans), as well as a noncanonical Notch signaling pathway. While cyclin D3 expression contributes to cell-cycle progression in Notch-dependent human T-ALL cell lines, ectopic expression of CDK4 or CDK6 together with cyclin D3 shows partial rescue from γ-secretase inhibitor (GSI)-induced G1 arrest in these cell lines. Importantly, cyclin D3 and CDK4 are highly overexpressed in Notch-dependent T-cell lymphomas, justifying the combined use of cell-cycle inhibitors and GSI in treating human T-cell malignancies.