Timothy C. Greiner

Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.

Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkins lymphoma, is clinically heterogeneous: 40% of patients respond well to current therapy and have prolonged survival, whereas the remainder succumb to the disease. We proposed that this variability in natural history reflects unrecognized molecular heterogeneity in the tumours. Using DNA microarrays, we have conducted a systematic characterization of gene expression in B-cell malignancies. Here we show that there is diversity in gene expression among the tumours of DLBCL patients, apparently reflecting the variation in tumour proliferation rate, host response and differentiation state of the tumour. We identified two molecularly distinct forms of DLBCL which had gene expression patterns indicative of different stages of B-cell differentiation. One type expressed genes characteristic of germinal centre B cells (‘germinal centre B-like DLBCL’); the second type expressed genes normally induced during in vitro activation of peripheral blood B cells (‘activated B-like DLBCL’). Patients with germinal centre B-like DLBCL had a significantly better overall survival than those with activated B-like DLBCL. The molecular classification of tumours on the basis of gene expression can thus identify previously undetected and clinically significant subtypes of cancer.

Molecular Diagnosis of Primary Mediastinal B Cell Lymphoma Identifies a Clinically Favorable Subgroup of Diffuse Large B Cell Lymphoma Related to Hodgkin Lymphoma

Using current diagnostic criteria, primary mediastinal B cell lymphoma (PMBL) cannot be distinguished from other types of diffuse large B cell lymphoma (DLBCL) reliably. We used gene expression profiling to develop a more precise molecular diagnosis of PMBL. PMBL patients were considerably younger than other DLBCL patients, and their lymphomas frequently involved other thoracic structures but not extrathoracic sites typical of other DLBCLs. PMBL patients had a relatively favorable clinical outcome, with a 5-yr survival rate of 64% compared with 46% for other DLBCL patients. Gene expression profiling strongly supported a relationship between PMBL and Hodgkin lymphoma: over one third of the genes that were more highly expressed in PMBL than in other DLBCLs were also characteristically expressed in Hodgkin lymphoma cells. PDL2, which encodes a regulator of T cell activation, was the gene that best discriminated PMBL from other DLBCLs and was also highly expressed in Hodgkin lymphoma cells. The genomic loci for PDL2 and several neighboring genes were amplified in over half of the PMBLs and in Hodgkin lymphoma cell lines. The molecular diagnosis of PMBL should significantly aid in the development of therapies tailored to this clinically and pathogenetically distinctive subgroup of DLBCL.

Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms

Matrix metalloproteinases (MMPs) 9 and 2 are increased in human abdominal aortic aneurysm (AAA) tissue, but their precise role and potential interaction remain unclear. Experimental induction of aortic aneurysms in mice genetically deficient in these peptidases could provide new insight into AAA pathogenesis. Mice deficient in the expression of MMP-9 (MMP-9KO) or MMP-2 (MMP-2KO) and their corresponding wild-type background mice (WT) underwent AAA induction by abluminal application of calcium chloride (CaCl(2)). No aneurysm formation was observed at 10 weeks after treatment in either the MMP-9KO or the MMP-2KO mice, whereas the corresponding WT mice showed an average 74% and 52% increase in aortic diameter, respectively. Reinfusion of competent macrophages from the corresponding WT strains into knockout mice resulted in reconstitution of AAA in MMP-9KO but not MMP-2KO mice. These findings suggest that macrophage-derived MMP-9 and mesenchymal cell MMP-2 are both required and work in concert to produce AAA.

Immunohistochemical Methods for Predicting Cell of Origin and Survival in Patients With Diffuse Large B-Cell Lymphoma Treated With Rituximab

PURPOSE Patients with diffuse large B-cell lymphoma (DLBCL) can be divided into prognostic groups based on the cell of origin of the tumor as determined by microarray analysis. Various immunohistochemical algorithms have been developed to replicate these microarray results and/or stratify patients according to survival. This study compares some of those algorithms and also proposes some modifications. PATIENTS AND METHODS Two-hundred and sixty-two cases of de novo DLBCL treated with rituximab and cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or CHOP-like therapy were examined. RESULTS The Choi algorithm and Hans algorithm had high concordance with the microarray results. Modifications of the Choi and Hans algorithms for ease of use still retained high concordance with the microarray results. Although the Nyman and Muris algorithms had high concordance with the microarray results, each had a low value for either sensitivity or specificity. The use of LMO2 alone showed the lowest concordance with the microarray results. A new algorithm (Tally) using a combination of antibodies, but without regard to the order of examination, showed the greatest concordance with microarray results. All of the algorithms divided patients into groups with significantly different overall and event-free survivals, but with different hazard ratios. With the exception of the Nyman algorithm, this survival prediction was independent of the International Prognostic Index. Although the Muris algorithm had prognostic significance, it misclassified a large number of cases with activated B-cell type DLBCL. CONCLUSION The Tally algorithm showed the best concordance with the microarray data while maintaining prognostic significance and ease of use.

Addition of Rituximab to Standard Chemotherapy Improves the Survival of Both the Germinal Center B-Cell–Like and Non–Germinal Center B-Cell–Like Subtypes of Diffuse Large B-Cell Lymphoma

PURPOSE Diffuse large B-cell lymphoma (DLBCL) includes at least two prognostically important subtypes (ie, germinal center B-cell-like [GCB] and activated B-cell-like [ABC] DLBCL), which initially were characterized by gene expression profiling and subsequently were confirmed by immunostaining. However, with the addition of rituximab to standard chemotherapy, the prognostic significance of this subclassification of DLBCL is unclear. PATIENTS AND METHODS We studied 243 patient cases of de novo DLBCL, which included 131 patient cases treated with rituximab plus standard chemotherapy (rituximab group) and 112 patient cases treated with only standard chemotherapy (control group). The cases were assigned to GCB or non-GCB subgroups (the latter of which included both ABC DLBCL and unclassifiable DLBCL) on the basis of immunophenotype by using the Hans method. Clinical characteristics and survival outcomes of the two patient groups were compared. RESULTS The clinical characteristics of the patients in the rituximab and the control groups were similar. Compared with the control group, addition of rituximab improved the 3-year overall survival (OS; 42% v 77%; P < .001) of patients with DLBCL. Rituximab-treated patients in either the GCB or the non-GCB subgroups also had a significantly improved 3-year OS compared with their respective subgroups in the control group (P < .001). In the rituximab group, the GCB subgroup had a significantly better 3-year OS than the non-GCB subgroup (85% v 69%; P = .032). Multivariate analyses confirmed that rituximab treatment was predictive for survival in both the GCB and the non-GCB subgroups. CONCLUSION In this retrospective study, we have shown that the subclassification of DLBCL on the basis of the cell of origin continues to have prognostic importance in the rituximab era.

Molecular signatures to improve diagnosis in peripheral T-cell lymphoma and prognostication in angioimmunoblastic T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) is often challenging to diagnose and classify. Gene expression profiling was performed on 144 cases of PTCL and natural killer cell lymphoma and robust molecular classifiers were constructed for angioimmunoblastic T-cell lymphoma (AITL), anaplastic lymphoma kinase-positive (ALK(+)) anaplastic large-cell lymphoma (ALCL), and adult T-cell leukemia/lymphoma. PTCL-unclassifiable was molecularly heterogeneous, but we were able to identify a molecular subgroup with features of cytotoxic T lymphocytes and a poor survival compared with the remaining PTCL-not otherwise specified cases. Many of the pathologic features and substantial components of the molecular signature of AITL are contributed by the follicular dendritic cells, B-cell, and other stromal components. The expression of Th17-associated molecules in ALK(+) ALCL was noted and may represent aberrant activation of Th17-cell differentiation by abnormal cytokine secretion. Adult T-cell leukemia/lymphoma has a homogeneous molecular signature demonstrating high expression of human T-lymphotropic virus type 1-induced genes. These classifiers reflect the biology of the tumor cells as well as their microenvironment. We also constructed a molecular prognosticator for AITL that appears to be largely related to the microenvironmental signature, and the high expression of 2 immunosuppressive signatures are associated with poor outcome. Oncogenic pathways and tumor-host interactions also were identified, and these findings may lead to better therapies and outcome in the future.

BCL2 Translocation Defines a Unique Tumor Subset within the Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed prognostically important subgroups: germinal center B-cell-like (GCB) DLBCL, activated B cell-like (ABC) DLBCL, and primary mediastinal large B-cell lymphoma. The t(14;18)(q32;q21) has been reported previously to define a unique subset within the GCB-DLBCL. We evaluated for the translocation in 141 cases of DLBCL that were successfully gene expression profiled. Using a dual-probe fluorescence in situ hybridization assay, we detected the t(14;18) in 17% of DLBCLs and in 34% of the GCB subgroup which contained the vast majority of positive cases. In addition, 12 t(14;18)-positive cases detected by polymerase chain reaction assays on additional samples were added to the fluorescence in situ hybridization-positive cases for subsequent analysis. Immunohistochemical data indicated that BCL2, BCL6, and CD10 protein were preferentially expressed in the t(14;18)-positive cases as compared to t(14;18)-negative cases. Within the GCB subgroup, the expression of BCL2 and CD10, but not BCL6, differed significantly between cases with or without the t(14;18): 88% versus 24% for BCL2 and 72% versus 32% for CD10, respectively. In the GCB-DLBCL subgroup, a heterogeneous group of genes is overexpressed in the t(14;18)-positive subset, among which BCL2 is a significant discriminator. Interestingly, the t(14;18)-negative subset is dominated by overexpression of cell cycle-associated genes, indicating that these tumors are significantly more proliferative, suggesting distinctive pathogenetic mechanisms. However, despite this higher proliferative activity, there was no significant difference in overall or failure-free survival between the t(14;18)-positive and -negative subsets within the GCB subgroup.

t(11;18)(q21;q21) is the most common translocation in MALT lymphomas

BACKGROUND Low grade malignant lymphomas arising from mucosa associated lymphoid tissue (MALT) represent a distinct clinicopathological entity. The cytogenetic findings and molecular genetics of MALT lymphomas remain minimally defined. Cytogenetic studies infrequently constitute part of the diagnostic work-up of MALT lymphomas, most commonly due to small biopsy size and their extranodal localization. Only 28 MALT cases with a clonal karyotype have been published to date. A number of chromosomal abnormalities have been observed with the majority of the cases featuring trisomy of chromosome 3 which is present in up to 78% of the cases. MATERIALS AND METHODS A total of 116 cases of MALT lymphoma were diagnosed at BCCA between 1988 and 1997. Eleven cases of pathologically confirmed MALT lymphomas were subjected to cytogenetic analysis at the time of the initial evaluation. Eight of 11 cases yielded successful cultures and the presence of a clonal karyotype using standard cytogenetic methodology. In addition, a single case of orbital MALT lymphoma with a clonal karyotype has been obtained through our consultative practice from University of Nebraska Medical Center. These nine cases of MALT lymphoma with a clonal karyotype are the subject of this report. RESULTS AND CONCLUSION In this study we report nine cytogenetically studied MALT lymphomas, three of which feature a novel t(11;18)(q21;q21) translocation which has also been observed in five other MALT cases described in the literature. This recurrent translocation is the most common translocation associated with MALT lymphomas being present in 33% (three of nine) of our cases and 18% (five of 28) of the previously published cases. The results suggest that a potentially important gene located at one of these breakpoints may be involved in the pathogenesis of MALT lymphomas.

BCL2 Expression Is a Prognostic Marker for the Activated B-Cell–Like Type of Diffuse Large B-Cell Lymphoma

BACKGROUND The role of BCL2 as a predictor of survival in diffuse large B-cell lymphoma (DLBCL) is controversial. DLBCL is heterogeneous, and the expression of BCL2 is variable within the two major subgroups of DLBCL, germinal center B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL, as well as primary mediastinal DLBCL. PATIENTS AND METHODS In this study, we investigated the correlation of BCL2 expression with survival in the two major subgroups of DLBCL, as well as the mechanisms of BCL2 expression. RESULTS There was no significant correlation between BCL2 protein expression and overall survival within the GCB subgroup, but BCL2 expression had a significant adverse effect on overall survival within the ABC subgroup (P = .008). This correlation was also observed at the mRNA level (P < .04). The difference remained significant when the analyses were performed at different cutoff values. The t(14;18) was frequently observed in the GCB subgroup and was highly associated with BCL2 expression. Patients with ABC DLBCL did not exhibit t(14;18) but had a markedly higher frequency of chromosome 18q21 amplification, on which BCL2 resides. Thus, alternative mechanisms such as 18q21 amplification or activation of the nuclear factor-kappa B pathway, as reported previously, seem to be mainly responsible for the upregulation of BCL2 expression in the ABC subgroup. CONCLUSION Treating all DLBCL as a single entity ignores the mechanistic differences in BCL2 upregulation and obscures the prognostic significance of BCL2 expression. Hence, the significance of BCL2 and other biomarkers should be assessed in the context of DLBCL subgroups in future studies.

Determining cell-of-origin subtypes of diffuse large B-cell lymphoma using gene expression in formalin-fixed paraffin-embedded tissue

The assignment of diffuse large B-cell lymphoma into cell-of-origin (COO) groups is becoming increasingly important with the emergence of novel therapies that have selective biological activity in germinal center B cell-like or activated B cell-like groups. The Lymphoma/Leukemia Molecular Profiling Projects Lymph2Cx assay is a parsimonious digital gene expression (NanoString)-based test for COO assignment in formalin-fixed paraffin-embedded tissue (FFPET). The 20-gene assay was trained using 51 FFPET biopsies; the locked assay was then validated using an independent cohort of 68 FFPET biopsies. Comparisons were made with COO assignment using the original COO model on matched frozen tissue. In the validation cohort, the assay was accurate, with only 1 case with definitive COO being incorrectly assigned, and robust, with >95% concordance of COO assignment between 2 independent laboratories. These qualities, along with the rapid turnaround time, make Lymph2Cx attractive for implementation in clinical trials and, ultimately, patient management.