Randy D. Gascoyne

Tumor-Associated Macrophages and Survival in Classic Hodgkin's Lymphoma

BACKGROUNDnDespite advances in treatments for Hodgkins lymphoma, about 20% of patients still die from progressive disease. Current prognostic models predict the outcome of treatment with imperfect accuracy, and clinically relevant biomarkers have not been established to improve on the International Prognostic Score.nnnMETHODSnUsing gene-expression profiling, we analyzed 130 frozen samples obtained from patients with classic Hodgkins lymphoma during diagnostic lymph-node biopsy to determine which cellular signatures were correlated with treatment outcome. We confirmed our findings in an independent cohort of 166 patients, using immunohistochemical analysis.nnnRESULTSnGene-expression profiling identified a gene signature of tumor-associated macrophages that was significantly associated with primary treatment failure (P=0.02). In an independent cohort of patients, we found that an increased number of CD68+ macrophages was correlated with a shortened progression-free survival (P=0.03) and with an increased likelihood of relapse after autologous hematopoietic stem-cell transplantation (P=0.008), resulting in shortened disease-specific survival (P=0.003). In multivariate analysis, this adverse prognostic factor outperformed the International Prognostic Score for disease-specific survival (P=0.003 vs. P=0.03). The absence of an elevated number of CD68+ cells in patients with limited-stage disease defined a subgroup of patients with a long-term disease-specific survival of 100% with the use of current treatment strategies.nnnCONCLUSIONSnAn increased number of tumor-associated macrophages was strongly associated with shortened survival in patients with classic Hodgkins lymphoma and provides a new biomarker for risk stratification.

MYC gene rearrangements are associated with a poor prognosis in diffuse large B-cell lymphoma patients treated with R-CHOP chemotherapy.

Approximately 5% to 10% of diffuse large B-cell lymphomas (DLBCLs) harbor an MYC oncogene rearrangement (MYC+). The prognostic significance of MYC+ DLBCL was determined in an unselected population of patients with newly diagnosed DLBCL treated with rituximab in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy (R-CHOP). Using a Vysis break-apart fluorescence in situ hybridization probe, 12 of 135 (8.8%) cases of MYC+ DLBCL were identified that had no defining high-risk features. MYC+ DLBCL was associated with an inferior 5-year progression-free survival (66% vs 31%, P = .006) and overall survival (72% vs 33%, P = .016). Multivariate analysis confirmed the prognostic importance of MYC for both progression-free survival (hazard ratio = 3.28; 95% confidence interval, 1.49-7.21, P = .003) and overall survival (hazard ratio = 2.98; 95% confidence interval, 1.28-6.95, P = .011). Cases of MYC+ DLBCL also had a higher risk of central nervous system relapse (P = .023), independent of other risk factors. The diagnosis of MYC+ DLBCL is likely underappreciated; and given the lack of defining risk factors, fluorescence in situ hybridization for MYC rearrangements should be performed in all patients with DLBCL. In the R-CHOP treatment era, MYC+ DLBCLs have an inferior prognosis. Treatment regimens similar to those used in Burkitt lymphoma may be more appropriate in this patient population and need to be prospectively tested.

The BCL6 transcriptional program features repression of multiple oncogenes in primary B cells and is deregulated in DLBCL

The BCL6 transcriptional repressor is required for development of germinal center (GC) B cells and when expressed constitutively causes diffuse large B-cell lymphomas (DLBCLs). We examined genome-wide BCL6 promoter binding in GC B cells versus DLBCLs to better understand its function in these settings. BCL6 bound to both distinct and common sets of functionally related gene in normal GC cells versus DLBCL cells. Certain BCL6 target genes were preferentially repressed in GC B cells, but not DLBCL cells. Several such genes have prominent oncogenic functions, such as BCL2, MYC, BMI1, EIF4E, JUNB, and CCND1. BCL6 and BCL2 expression was negatively correlated in primary DLBCLs except in the presence of BCL2 translocations. The specific BCL6 inhibitor retro-inverso BCL6 peptidomimetic inhibitor-induced expression of BCL2 and other oncogenes, consistent with direct repression effects by BCL6. These data are consistent with a model whereby BCL6 can directly silence oncogenes in GC B cells and counterbalance its own tumorigenic potential. Finally, a BCL6 consensus sequence and binding sites for other physiologically relevant transcription factors were highly enriched among target genes and distributed in a pathway-dependent manner, suggesting that BCL6 forms specific regulatory circuits with other B-cell transcriptional factors.

The architectural pattern of FOXP3-positive T cells in follicular lymphoma is an independent predictor of survival and histologic transformation.

Previous studies of follicular lymphoma (FL) patients treated heterogeneously have suggested that decreased numbers of regulatory T cells correlates with improved survival. We studied advanced-stage FL patients from a single institution phase 2 trial. All patients were treated uniformly with multiagent chemotherapy and radiation. Tissue microarrays were constructed using diagnostic biopsies available in 105 patients and stained with CD4, CD8, CD25, and forkhead/winged helix transcription factor 3 (FOXP3) antibodies. Both cell content and cell distribution were evaluated. For all antibodies, there were cases with a predominant intrafollicular or perifollicular localization of cells (follicular pattern) while others displayed a diffuse pattern. The median follow-up of living patients was 17.1 years. The International Prognostic Index score predicted overall survival (OS; P = .004) but not risk of transformation (RT). Cell content did not impact survival, while immunoarchitectural patterns of CD4/CD8 were significant for progression-free survival (PFS; P = .056), CD25 for both PFS and OS (P = .002 and P = .024, respectively), and FOXP3(+) predicted PFS, OS, and RT (P = .001, P < .001 and p = .002, respectively). A Cox multivariate model showed both International Prognostic Index score and FOXP3(+) pattern were independent predictors of OS (P = .008 and P < .001, respectively), while only FOXP3(+) pattern predicted RT (P = .004). We conclude that FOXP3(+) cell distribution significantly predicts survival and RT in FL.

Prognostic Factors in Follicular Lymphoma

Follicular lymphoma (FL) is one of the most common types of non-Hodgkins lymphoma. It is usually diagnosed at an advanced stage, for which many treatment options exist, however, no curative standard therapy has been identified. The outcome is highly variable with a median survival of approximately 10 years. The life expectancy of patients with FL has been extended with the use of rituximab, a monoclonal antibody targeting the CD20 antigen on FL cells, but there remains a group of patients who fail to respond to chemoimmunotherapy and die early of their disease. Transformation of FL to an aggressive histology is an important event with high morbidity and mortality. The Follicular Lymphoma International Prognostic Index has become the clinically useful prognostic tool, but gives only a rough estimate of expected outcome. There is a need for useful biomarkers for prediction of the disease course of single patients to individualize therapy, especially in the new era of chemoimmunotherapy.

Genome-wide copy number analysis of Hodgkin Reed-Sternberg cells identifies recurrent imbalances with correlations to treatment outcome

In classical Hodgkin lymphoma (cHL) the mechanisms underlying primary refractory disease and relapse remain unknown. To gain further insight into cHL pathogenesis and genomic changes linked to treatment response, we studied 53 cHL patients by array comparative genomic hybridization, including 23 patients whose primary treatment failed, using DNA from microdissected HRS cells. Copy number alterations found in more than 20% of cases included gains of 2p, 9p, 16p, 17q, 19q, 20q, and losses of 6q, 11q, and 13q. We identified at high resolution recurrent changes defining minimally gained and lost regions harboring genes involved in nuclear factor kappaB signaling, such as REL, IKBKB, CD40, and MAP3K14. Gains of chromosome 16p11.2-13.3 were significantly more frequent in pretreatment and relapse biopsies of unresponsive patients and were associated with shortened disease-specific survival (P = .028). In the therapy-resistant HL cell line KMH2, we found genomic gains and overexpression of the multidrug resistance gene ABCC1 mapping to cytoband 16p13.11. We show that doxorubicin exposure to KMH2 induces ABCC1 expression and that siRNA silencing of ABCC1 sensitizes KMH2 cells to doxorubicin toxicity in vitro, suggesting that overexpression of ABCC1 contributes to the drug resistance phenotype found in KMH2.

Correlations between BCL6 rearrangement and outcome in patients with diffuse large B-cell lymphoma treated with CHOP or R-CHOP.

Background BCL6 gene rearrangement is the most frequent chromosomal abnormality in diffuse large B-cell lymphoma, a malignancy characterized by genetic heterogeneity and wide variability in clinical outcome. The prognostic significance of BCL6 rearrangement has not been evaluated in the context of rituximab therapy for diffuse large B-cell lymphoma. We analyzed the effect of the BCL6 rearrangement on survival in patients with diffuse large B-cell lymphoma treated with CHOP and CHOP plus rituximab (R-CHOP). Design and Methods BCL6 rearrangement status was analyzed by fluorescence in situ hybridization with break-apart probes in 164 patients with diffuse large B-cell lymphoma treated with CHOP (n=65) or R-CHOP (n=99). Cell-of-origin immunophenotype including BCL6 protein expression were determined by immunohistochemistry on a tissue microarray. Results BCL6 rearrangement was detected in 19.5% of cases. The presence of the gene rearrangement was associated with a non-germinal center B-cell immunophenotype (P=0.006), and showed no correlation with BCL6 protein expression. A trend toward inferior overall survival was observed in association with the BCL6 rearrangement among patients treated with R-CHOP (P=0.08), but not among patients treated with CHOP (P=0.64). However, BCL6 rearrangement also correlated with a high International Prognostic Index score (P=0.02), and did not demonstrate independent prognostic value by multivariate analysis. Conclusions The introduction of rituximab may have altered the prognostic impact of BCL6 gene rearrangement in patients with diffuse large B-cell lymphoma. However, prospective analysis within large randomized clinical trials will be needed to clarify the prognostic significance of this biomarker in the rituximab era.

Vascularization predicts overall survival and risk of transformation in follicular lymphoma

Follicular lymphoma patients display heterogeneous overall survival and variable risk of transformation. Recent studies have highlighted the role of the microenvironment. The contribution of microvessel density to follicular lymphoma survival remains controversial. We used a quantitative tumor mapping approach to determine whether the degree of vascularization correlated with outcome in a uniformly treated cohort. Whole-tissue sections of diagnostic biopsies from 84 cases were stained for CD34 and tumor-to-vessel-distance that encompassed 90% of the tumor (TVD90) was determined using image analysis. Twenty-one cases with lower TVD90 showed inferior overall survival (P=0.0001) and high risk of transformation (P=0.01). These cases significantly correlated with increased Lymphoma-Associated Macrophages (χ2=0.025). In multivariate analysis macrophages content, IPI and TVD90 were independent predictors of overall survival (P=0.05, P=0.001 and P=0.01, respectively) and IPI and TVD90 predicted risk of transformation (P=0.008 and P=0.08, respectively). Increased angiogenesis is an independent marker of inferior survival and may promote transformation.

Lymphoma cell VEGFR2 expression detected by immunohistochemistry predicts poor overall survival in diffuse large B cell lymphoma treated with immunochemotherapy (R-CHOP)

Diffuse large B cell lymphoma (DLBCL) is clinically and biologically heterogeneous. In most cases of DLBCL, lymphoma cells co‐express vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2, suggesting autocrine in addition to angiogenic effects. We enumerated microvessel density and scored lymphoma cell expression of VEGF, VEGFR1, VEGFR2 and phosphorylated VEGFR2 in 162 de novo DLBCL patients treated with R‐CHOP (rituximab, cyclophosphamide, vincristine, doxorubicin and prednisone)‐like regimens. VEGFR2 expression correlated with shorter overall survival (OS) independent of International Prognostic Index (IPI) (Pu2003=u20030·0028). Phosphorylated VEGFR2 (detected in 13% of cases) correlated with shorter progression‐free survival (PFS, Pu2003=u20030·044) and trended toward shorter OS on univariate analysis. VEGFR1 was not predictive of survival on univariate analysis, but it did correlate with better OS on multivariate analysis with VEGF, VEGFR2 and IPI (Pu2003=u20030·036); in patients with weak VEGFR2, lack of VEGFR1 coexpression was significantly correlated with poor OS independent of IPI (Pu2003=u20030·01). These results are concordant with our prior finding of an association of VEGFR1 with longer OS in DLBCL treated with chemotherapy alone. We postulate that VEGFR1 may oppose autocrine VEGFR2 signalling in DLBCL by competing for VEGF binding. In contrast to our prior results with chemotherapy alone, microvessel density was not prognostic of PFS or OS with R‐CHOP‐like therapy.

Expression pattern of intracellular leukocyte-associated proteins in primary mediastinal B cell lymphoma

Two microarray studies of mediastinal B cell lymphoma have shown that this disease has a distinct gene expression profile, and also that this is closest to the pattern seen in classical Hodgkins disease. We reported previously an immunohistologic study in which the loss of intracellular B cell-associated signaling molecules in Reed–Sternberg cells was demonstrated, and in this study we have investigated the expression of the same components in more than 60 mediastinal B cell lymphomas. We report that these signaling molecules are frequently present, and in particular that Syk, BLNK and PLC-γ2 (absent from Reed–Sternberg cells) are present in the majority of mediastinal B cell lymphomas. The overall pattern of B cell signaling molecules in this disease is therefore closer to that of diffuse large B cell lymphoma than to Hodgkins disease, and is consistent with a common cell of origin as an explanation of the similar gene expression profiles.