Minna Taskinen

A High Tumor-Associated Macrophage Content Predicts Favorable Outcome in Follicular Lymphoma Patients Treated with Rituximab and Cyclophosphamide-Doxorubicin-Vincristine-Prednisone

Purpose: Tumor-associated macrophage (TAM) content predicts survival in follicular lymphoma (FL) patients treated with chemotherapy. The aim of this study was to determine how combination of rituximab with chemotherapy influences TAM-associated clinical outcome. Experimental Design: Expression of a macrophage marker, CD68, was determined immunohistochemically from FL samples of 96 patients treated with rituximab and cyclophosphamide-Adriamycin-vincristine-prednisone regimen. Of them, 71 received therapy at diagnosis and 25 at relapse. Neutrophil and CD3+ lymphocyte counts were also measured. The median follow-up time for the cohort was 54 months. Fourty-five patients previously treated with chemotherapy served as a control group. Results: Consistent with previous studies, high TAM amount was associated with adverse outcome in chemotherapy-treated patients (P = 0.026). In contrast, after rituximab and cyclophosphamide-doxorubicin-vincristine-prednisone regimen, high TAM content correlated with longer survival rates. According to Kaplan Meier estimates, the median progression free survival (PFS) was not reached for patients with high TAM content compared with 45 months for patients with low TAM scores (P = 0.006). A trend toward a better overall survival (OS) at 5 years was also observed for patients with high TAM content (OS, 97% versus 90%, P = 0.116). The positive prognostic value of TAMs was seen both for the patients treated at diagnosis and at relapse. In multivariate analyses, TAM content remained an independent prognostic factor for OS and PFS. Neutrophil and CD3+ lymphocyte counts did not correlate with outcome. Conclusions: The data suggest that high TAM score is associated with a favorable prognosis in FL patients treated with immunochemotherapy.

Prognostic influence of tumor-infiltrating mast cells in patients with follicular lymphoma treated with rituximab and CHOP

Gene expression profiling and immunohistochemical studies have demonstrated that nonmalignant tumor infiltrating inflammatory cells contribute to clinical outcome in patients with follicular lymphoma (FL). Particularly, tumor-associated macrophage (TAM) content correlates with longer survival rates after immunochemotherapy. Here we investigated the prognostic importance of tumor-associated mast cells (MCs) and their relation to TAMs in patients with FL treated with a combination of rituximab (R) and cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy. Of the 98 patients, 70 received R-CHOP at diagnosis and 28 at relapse. According to Kaplan-Meier estimates, the patients with high MC content had a worse 4-year progression-free survival (PFS) than the ones with low MC content after R-CHOP therapy (34% vs 74%, P = .002). The adverse prognostic value of MCs was seen both for the patients treated at diagnosis and at relapse, whereas no such impact on PFS was observed for the control patients treated with chemotherapy only (P = .4). When the TAM-related PFS was analyzed separately in patients with high and low MC contents, the positive prognostic effect of TAM was seen only in patients with few MCs. Taken together, the data demonstrate that a high MC score is associated with unfavorable prognosis and it eliminates the positive prognostic value of TAMs in patients with FL treated with immunochemotherapy.

Male gender is an adverse prognostic factor in B-cell lymphoma patients treated with immunochemotherapy*

Male gender is an adverse prognostic factor in Hodgkin’s lymphoma, but no such association has yet been established in non‐Hodgkin lymphomas. Here, we have evaluated whether gender has prognostic impact on the survival of patients with B‐cell non‐Hodgkin lymphoma in the postrituximab era of lymphoma therapies. The study populations consisted of 217 diffuse large B‐cell lymphoma (DLBCL) and 110 follicular lymphoma (FL) patients treated with immunochemotherapy. Hundred and sixty chemotherapy‐treated DLBCL patients served as a control group. According to Kaplan–Meier analyses, female patients had a significantly better progression‐free survival than men both in DLBCL (4 yr PFS 75% vs. 60%; P = 0.013) and in FL (4 yr PFS 68% vs. 52%, P = 0.036) patients treated with immunochemotherapy. In chemotherapy‐treated DLBCL patients, no difference in survival between the genders was found. The results support the idea that women seem to respond better to rituximab.

Prognostic influence of macrophages in patients with diffuse large B-cell lymphoma: a correlative study from a Nordic phase II trial

The prognostic impact of the tumor microenvironment in diffuse large B-cell lymphoma has not been systematically assessed. We analyzed mRNA and antigen expression of monocytes, macrophages, lymphocytes, dendritic and natural killer cells in pretreatment tumor samples of patients with high-risk diffuse large B-cell lymphoma using gene expression microarray and immunohistochemistry. The patients were treated in a Nordic phase II study with dose-dense chemoimmunotherapy and central nervous system prophylaxis. Of the studied markers for non-malignant inflammatory cells, CD68 expression and CD68+ macrophage counts correlated with favorable outcome. Five-year progression-free survival rates were 83% and 43% for the patients with high and low CD68 mRNA levels, respectively (P=0.007), while overall survival rates were 83% and 64%, respectively (P=ns). The patients with high CD68+ macrophage counts had better 5-year progression-free survival (74% versus 40%; P=0.003) and overall survival (90% versus 60%; P=0.009) than the patients with low macrophage counts. Low CD68+ macrophage count retained its prognostic impact on overall survival with age-adjusted International Prognostic Index [RR=5.0 (95% CI 1.024–19.088); P=0.017]. The findings were validated in three independent cohorts of patients treated with chemoimmunotherapy. In contrast, in patients treated with chemotherapy, high CD68+ macrophage count was associated with poor progression-free survival (40% versus 72%; P=0.021) and overall survival (39% versus 72%; P=0.015). Together, the data suggest that macrophages exhibit a dual, treatment-specific role in diffuse large B-cell lymphoma. For the patients treated with chemoimmunotherapy, high pretreatment CD68 mRNA levels and CD68+ macrophage numbers predict a favorable outcome.

Differential gene expression in non‐malignant tumour microenvironment is associated with outcome in follicular lymphoma patients treated with rituximab and CHOP

Rituximab in combination with chemotherapy (immunochemotherapy) is one of the most effective treatments available for follicular lymphoma (FL). This study aimed to determine whether differences in gene expression in FL tissue correlate with outcome in response to rituximab and CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy (R–CHOP). We divided 24 patients into long‐ [time to treatment failure (TTF) >35 months] and short‐term (TTF <23 months) responders, and analysed the gene expression profiles of lymphoma tissue using oligonucleotide microarrays. We used a supervised learning technique to identify genes correlating with outcome, and confirmed the expression of selected genes with quantitative polymerase chain reaction (qPCR) and immunohistochemistry. Among the transcripts with a high correlation between microarray and qPCR analyses, we identified EPHA1, a tyrosine kinase involved in transepithelial migration, SMAD1, a transcription factor and a mediator of bone morphogenetic protein and transforming growth factor‐β signalling, and MARCO, a scavenger receptor on macrophages. According to Kaplan–Meier estimates, high EPHA1, and low SMAD1 and MARCO expression were associated with better progression‐free survival (PFS). Immunohistochemistry showed that EphA1 was primarily localised in granulocytes. In addition, both EphA1 and Smad1 were expressed in vascular endothelia. However, no difference in vasculature was detected between long‐ and short‐term responders. In a validation set of 40 patients, a trend towards a better PFS was observed among patients with high EphA1 expression. We conclude that gene expression in non‐malignant cells contributes to clinical outcome in R–CHOP‐treated FL patients.

Prognostic impact of CD31-positive microvessel density in follicular lymphoma patients treated with immunochemotherapy

BACKGROUND Tumour-infiltrating mast cells (MCs) can remodel tumour microenvironment and growth by suppressing immune responses and potentiating angiogenesis. Furthermore, accumulation of MCs in follicular lymphoma (FL) correlates with unfavourable prognosis after immunochemotherapy. Here we investigated whether tumour vascularity is associated with MC content and outcome in FL patients treated with immunochemotherapy. PATIENTS AND METHODS Microvessel density (MVD) and MC content were determined immunohistochemically from pretreatment samples of 95 FL patients using CD31, CD34 and mast cell tryptase antibodies. Gene expression data from a separate set of 24 FL patients were analysed for comparison. All patients were treated with the combination of rituximab (R) and cyclophoshamide-doxorubicin-vincristine-prednisone (CHOP) chemotherapy. RESULTS Increased CD31+ MVD correlated positively with the number of tumour infiltrating MCs and CD34+ vessels, and negatively with the outcome. Overall survival and progression-free survival were significantly better among patients with low CD31+ MVDs. In multivariate analyses, CD31+ MVD had prognostic value independently of Follicular Lymphoma Prognostic Index but not of MC content. Consistent with the immunohistochemical data, high CD31/PECAM1 mRNA levels were associated with adverse outcome. Conversely, a positive prognostic impact of VEGF mRNA expression on the outcome was found. CONCLUSION Vascularity is associated with MC content and outcome in R-CHOP-treated FL patients.

Deregulation of COMMD1 Is Associated with Poor Prognosis in Diffuse Large B-cell Lymphoma

Background Despite improved survival for the patients with diffuse large B-cell lymphoma (DLBCL), the prognosis after relapse is poor. The aim was to identify molecular events that contribute to relapse and treatment resistance in DLBCL. Methods We analysed 51 prospectively collected pretreatment tumour samples from clinically high risk patients treated in a Nordic phase II study with dose-dense chemoimmunotherapy and central nervous system prophylaxis with high resolution array comparative genomic hybridization (aCGH) and gene expression microarrays. Major finding was validated at the protein level immunohistochemically in a trial specific tissue microarray series of 70, and in an independent validation series of 146 patients. Results We identified 31 genes whose expression changes were strongly associated with copy number aberrations. In addition, gains of chromosomes 2p15 and 18q12.2 were associated with unfavourable survival. The 2p15 aberration harboured COMMD1 gene, whose expression had a significant adverse prognostic impact on survival. Immunohistochemical analysis of COMMD1 expression in two series confirmed the association of COMMD1 expression with poor prognosis. Conclusion COMMD1 is a potential novel prognostic factor in DLBCLs. The results highlight the value of integrated comprehensive analysis to identify prognostic markers and genetic driver events not previously implicated in DLBCL. Trial Registration ClinicalTrials.gov NCT01502982

Primary mediastinal large B-cell lymphoma segregating in a family: exome sequencing identifies MLL as a candidate predisposition gene

Primary mediastinal large B-cell lymphoma (PMBCL) is a subtype of diffuse large B-cell lymphoma (DLBCL) accounting for 2% to 4% of all non-Hodgkin lymphomas. We report a family of 3 siblings with PMBCL and their cousin with extranodal DLBCL. The histopathological characteristics of lymphomas of all 4 patients are similar, implying post-germinal center differentiation and growth deregulation by other mechanisms than BCL2-mediated inhibition of apoptosis and suggesting a shared biological background. We aimed to identify the genetic defect underlying lymphoma susceptibility in this family using exome sequencing and linkage analysis. The only variant segregating in all 4 patients and not reported in genetic databases was 5533C>A (His1845Asn) in the MLL gene. To our knowledge, this is the first time when familial clustering of PMBCL is reported. Although we propose MLL as a candidate predisposition gene for this condition, this finding needs to be validated in additional cases.

Identification of homozygous deletion in ACAN and other candidate variants in familial classical Hodgkin lymphoma by exome sequencing.

Adams, R.J. & Brambilla, D. (2005) Discontinuing prophylactic transfusions used to prevent stroke in sickle cell disease. The New England journal of medicine, 353, 2769–2778. Adams, R.J., McKie, V.C., Hsu, L., Files, B., Vichinsky, E., Pegelow, C., Abboud, M., Gallagher, D., Kutlar, A., Nichols, F.T., Bonds, D.R. & Brambilla, D. (1998) Prevention of a first stroke by transfusions in children with sickle cell anemia and abnormal results on transcranial Doppler ultrasonography. The New England journal of medicine, 339, 5–11. Aygun, B., Wruck, L.M., Schultz, W.H., Mueller, B.U., Brown, C., Luchtman-Jones, L., Jackson, S., Iyer, R., Rogers, Z.R., Sarnaik, S., Thompson, A.A., Gauger, C., Helms, R.W. & Ware, R.E. (2012) Chronic transfusion practices for prevention of primary stroke in children with sickle cell anemia and abnormal TCD velocities. American Journal of Hematology, 87, 428–430. Casella, J.F., King, A.A., Barton, B., White, D.A., Noetzel, M.J., Ichord, R.N., Terrill, C., Hirtz, D., McKinstry, R.C., Strouse, J.J., Howard, T.H., Coates, T.D., Minniti, C.P., Campbell, A.D., Vendt, B.A., Lehmann, H. & Debaun, M.R. (2010) Design of the silent cerebral infarct transfusion (SIT) trial. Pediatric Hematology & Oncology, 27, 69–89. Cohen, A.R., Martin, M.B., Silber, J.H., Kim, H.C., Ohene-Frempong, K. & Schwartz, E. (1992) A modified transfusion program for prevention of stroke in sickle cell disease. Blood, 79, 1657– 1661. Hulbert, M.L., Scothorn, D.J., Panepinto, J.A., Scott, J.P., Buchanan, G.R., Sarnaik, S., Fallon, R., Chu, J.-Y., Wang, W., Casella, J.F., Resar, L., Berman, B., Adamkiewicz, T., Hsu, L.L., Smith-Whitley, K., Mahoney, D., Woods, G., Watanabe, M. & DeBaun, M.R. (2006) Exchange blood transfusion compared with simple transfusion for first overt stroke is associated with a lower risk of subsequent stroke: a retrospective cohort study of 137 children with sickle cell anemia. Journal of Pediatrics, 149, 710–712. Miller, S.T., Jensen, D. & Rao, S.P. (1992) Less intensive long-term transfusion therapy for sickle cell anemia and cerebrovascular accident. Journal of Pediatrics, 120, 54–57. Olujohungbe, A. (ed.) (2008) Standards for the Clinical Care of Adults with Sickle Cell Disease in the UK. Sickle Cell Society, London, UK. Pegelow, C.H., Adams, R.J., McKie, V., Abboud, M., Berman, B., Miller, S.T., Olivieri, N., Vichinsky, E., Wang, W. & Brambilla, D. (1995) Risk of recurrent stroke in patients with sickle cell disease treated with erythrocyte transfusions. Journal of Pediatrics, 126, 896– 899. Ware, R.E. & Helms, R.W. (2012) Stroke With Transfusions Changing to Hydroxyurea (SWiTCH). Blood, 119, 3925–3932.

Signal Transducers and Activators of Transcription 5a–Dependent Cross-talk between Follicular Lymphoma Cells and Tumor Microenvironment Characterizes a Group of Patients with Improved Outcome after R-CHOP

Purpose: Tumor microenvironment has a strong effect on the survival of follicular lymphoma (FL) patients. The aim of this study was to determine what are the signaling pathways that mediate the cross-talk between lymphoma cells and tumor-infiltrating inflammatory cells and contribute to the clinical outcome of FL patients. Experimental Design: Gene expression profiling and pathway impact analyses were done from pretreatment lymphoma tissue of 24 patients. The findings were validated immunohistochemically in an independent cohort of 81 patients. All patients were treated with the combination of rituximab and cyclophoshamide-doxorubicin-vincristine-prednisone chemotherapy. In addition, microarray was used to screen the genes differentially expressed between control and rituximab-stimulated B-cell lymphoma cells in culture. Results: Among the transcripts differentially expressed in the FL tissues between the patients with favorable or adverse outcomes, an overrepresentation of genes associated with the signal transducers and activators of transcription (STAT)5a pathway was observed. In a validation set, a better progression-free survival was observed among the patients with high STAT5a protein expression. In the FL tissue, STAT5a positivity was barely detectable in the neoplastic B cells, but a subpopulation of follicular dendritic cells and T lymphocytes showed prominent STAT5a expression. Rituximab was found to induce the expression of STAT5a-associated interleukin-15 in B-lymphoma cells in culture, thereby providing a possible explanation for the cross-talk between rituximab-stimulated FL cells and their microenvironment. Conclusion: The findings suggest that STAT5a activity in immunologically active nonmalignant cells acts as molecular predictor for rituximab and cyclophoshamide-doxorubicin-vincristine-prednisone–treated FL patients. Clin Cancer Res; 16(9); 2615–23. ©2010 AACR.