Regulatory T Lymphocytes and Tumor-Associated Macrophages Have No Impact On Treatment Response and Survival in Brazilian Patients with Classical Hodgkin Lymphoma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4777-4777
Author(s):  
Mariane Cristina Gennari Assis ◽  
Antonio H. F. M. Campos ◽  
José Salvador Rodrigues de Oliveira ◽  
Fernando A Soares ◽  
Joyce M. K. Silva ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Treatment Response ◽  
Hodgkin Lymphoma ◽  
Conflicts Of Interest ◽  
Complete Response ◽  
Public Hospitals ◽  
Response To Treatment ◽  
Classical Hodgkin Lymphoma ◽  
Tumor Associated Macrophages ◽  
Different Populations

Abstract Abstract 4777 Understanding the mechanisms of how tumor microenvironment of classical Hodgkin lymphoma (cHL) fosters immune privilege and survival of Hodgkin-Reed-Sternberg (HRS) cells is crucial for the development of new biomarkers and therapy strategies. Recently, infiltrating regulatory T CD4+CD25+FOXP3+ lymphocytes (Tregs) and tumor-associated macrophages CD68+ (TAMs) have been shown to play a role in HRS immune evasion, disease progression and survival. However, data arising from studies of different populations of cHL patients are conflicting. Purpose: In this study, we evaluated the importance of infiltrating Tregs and TAMs in a subset of 130 cHL patients treated in public hospitals in southeast Brazil and correlated these findings with Epstein-Barr virus (EBV) presence in HRS cells. Material and Methods: Tissue microarrays were constructed using diagnostic biopsies available in 130 patients and stained with CD4, CD8, CD25, FOXP3, CD15, CD30, CD68 e LMP1. Quantification of TAMs and Tregs was performed using automated slide scanning and image analysis (Aperio ScanScope XT Slide Scanner and Aperio ImageScope Software with Aperio Positive Pixel Count Sample Macro algorithm). Immunohistochemical scoring ranged from 1 to 4 for the antibodies tested, with higher scores indicating a greater proportion of positive cells. For Tregs and TAMs quantification, score 1 was considered negative (≤ 25 % of Tregs or TAMs) and scores 2, 3, and 4 (more than 25 % of positive cells) were considered positive. All patients underwent similar chemotherapy protocols. For the present study, only cHL patients whose histology could be confirmed and EBV-association established were studied. Results: From the 130 cHL patients selected for this study, 56 (43%) were classified as EBV related and 74 (57%) EBV non-related cHL. The expression of Tregs (CD4/CD25/FOXP3) was more common in the EBV related cHL group (p=0.02). TAMs did not correlate with EBV presence in HRS cells. Response to treatment, either complete response or partial response, and relapse rate were independent of Tregs and TAMs quantification and EBV status. Increased Tregs and TAMs in the tumor microenvironment did not influence event-free survival (EFS) and overall survival (OS). For further analysis, we stratified our patients into 4 groups, according to Tregs and TAMs quantification and EBV status and we still did not find any difference on EFS and OS. Additionally, stratified survival analysis according to age, stage and IPS-risk group did not identify any impact of Tregs and TAMs quantification on EFS and OS. Conclusion: This study demonstrates that increased Tregs and TAMs in the tumor microenvironment of cHL patients neither correlate with treatment response nor survival. Additionally, increased Tregs correlated with EBV presence in HRS cells. It is well known that the incidence of EBV-related cHL in developing countries is different from that in developed ones, as well as the severity of the disease at presentation, with advanced disease being more common at diagnosis. Our results, although different from those recently published, probably reflect the reality of the Brazilian population enrolled in the public health system, highlighting the importance of studying the same disease and their potential biomarkers within different populations. Disclosures: No relevant conflicts of interest to declare.

Prognostic Significance of Immunohistochemical Markers in Classical Hodgkin Lymphoma Response to Treatment A Study of 59 Cases.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 972-972 ◽  
Author(s):  
Danielle Canioni ◽  
Benedicte Deau ◽  
Pierre Taupin ◽  
Jacques Bosq ◽  
Vincent Ribrag ◽  
...  
Keyword(s):  
Treatment Response ◽  
Hodgkin Lymphoma ◽  
Prognostic Significance ◽  
Response To Treatment ◽  
Rank Test ◽  
Classical Hodgkin Lymphoma ◽  
High Power Field ◽  
Mixed Cellularity ◽  
P53 Expression ◽  
Immunohistochemical Markers

Abstract Classical Hodgkin lymphoma (cHL) belongs to the most curable lymphomas in adults. Some cHL however, are primary refractory to usual treatments including anthracyclins regimen. Currently, only clinical factors are considered as relevant for prognosis. In a previous study of a small cohort of patients, we showed that some immunohistochemical markers could help for predicting the treatment response of cHL. In this study, we extended the markers and increased the number of included patients. We performed a retrospective study on pre-treatment biopsy specimen of 59 patients, 18 with primary refractory cHL and 41 responders to chemotherapy and free of disease for at least 3 years. Most refractory cHL had a nodular sclerosis (NS) histological type, except one which was a mixed cellularity type. Thirty six responders had a NS type, 3 patients had a mixed cellularity type and the 2 others an interfollicular cHL. The semi-quantitative immunohistochemical study used CD20, CD3, CD30, bcl2, p53, Ki67, TiA1 and c-kit antibodies. The results were statistically evaluated using a Fisher ’s exact test or a Wilcoxon sum rank test depending on the variable studied. CD30 and Ki67 stained strongly Hodgkin (Hg) and Reed-Sternberg (RS) cells regarless the response status. In contrast, these cells expressed significantly less frequently CD20 in refractory cHL than in responders (p= 0.032) and were never stained with CD3. P53 and bcl2 had a significantly higher expression on Hg or RS cells in refractory cHL (median = 63% & 51%) compared to responders (median = 40% & 12%) (p=0.004 & p=0.015 respectively). The cytotoxic marker TiA1 stained significant higher number of small lymphocytes in refractory cHL (median=42.5 per high power field (hpf)) compared to responders (median= 21 per hpf) (p= 0.0006). C-kit antibody was negative in Hg or RS cells but stained significant more mastocytes in refractory cHL (median=9 per hpf) comparing to responders (median=3.8 per hpf) (p= 0.001). These results indicate that immunohistochemical markers are useful in cHL and should be used in association with clinical parameters for predict the cHL treatment response. The prognostic significance of CD20 expression in cHL is controversial but in this study seems predictive of a better treatment response and is merely a marker of different gene expression program that may be associated with a more favorable outcome. A high bcl2 and p53 expression in refractory cHL supports the notion that an intact apoptosis cascade is essential for cell killing effect of chemotherapy. The increasing of TiA1 and c-kit positive cells raises the importance of the environmental non-neoplastic cells in cHL and suggests that targeted therapy against mast cells could improve prognosis of refractory cHL.

Quantitative Assessment of PD-L1 Expression in Classical Hodgkin Lymphoma Suggests a Critical Role for Tumor Associated Macrophages in Suppressing Anti-Tumor Immunity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1440-1440 ◽  
Author(s):  
Christopher Daniel Carey ◽  
Courtney Connelly ◽  
Evisa Gjini ◽  
Margaretha GM Roemer ◽  
Edward Stack ◽  
...  
Keyword(s):  
Image Analysis ◽  
Tumor Microenvironment ◽  
Board Of Directors ◽  
Hodgkin Lymphoma ◽  
Tumor Immunity ◽  
Research Funding ◽  
Malignant Cells ◽  
Classical Hodgkin Lymphoma ◽  
Tumor Associated Macrophages ◽  
Advisory Committees

Abstract BACKGROUND: The programmed cell death-1 ligands (PD-Ls; PD-L1 and PD-L2) act as negative regulators of anti-tumor immunity by binding their cognate receptor, PD-1, on cytotoxic T-cells and inducing T-cell "exhaustion", a phenotype that is reversible with PD-1 blockade. Human antibodies that block PD-1 induce objective clinical responses in the majority of patients with relapsed / refractory classical Hodgkin Lymphoma (CHL). CHLs include small numbers of malignant Hodgkin Reed-Sternberg (HRS) cells (~5% of total cellularity) within an extensive but ineffective inflammatory and immune cell infiltrate. Over 85% of CHLs express PD-Ls on both the HRS cells and additional non-malignant cells within the tumor microenvironment (Chen et al., CCR 2010). PD-L expression in HRS cells is attributable, in part, to copy gain of chromosome 9p24.1, a region that includes PD-L1, PD-L2, and JAK2 (Green et al., Blood, 2010). However, the contribution of non-malignant cells to the overall PD-L expression within the tumor micro-environment of CHL is poorly defined. METHODS: We analyzed select CHLs (12 EBV+, 8 EBV-) by multiplex immunofluorescence using formalin-fixed, paraffin embedded tissue sections, with successive labeling by primary antibodies (PD-L1, CD30, CD68, pSTAT3, CD163), followed by secondary amplification and tyramide-conjugated fluorophores. For each case 2 large representative areas of tissue, totaling eight 20x fields of view were selected and imaged using a multispectral imaging platform. Two specific image analysis algorithms were designed to accurately identify CD30+ HRS cells and CD68+ macrophages simultaneously, then to threshold PD-L1 by relative fluorescent units (RFU) in each phenotype. Cartesian coordinates for all cells were exported and distance calculations were generated between PD-L1+ and PD-L1- macrophages and their Ônearest neighborÕ CD30+ PD-L1+ HRS cell. RESULTS: The percentages of CD30+ HRS cells and CD68+ macrophages expressing PD-L1 was highly variable across cases (range 9 - 94%, median 46.6% for HRS cells; range 6 - 91.3%, median 48.2% for macrophages). In all cases the majority of PD-L1 protein within the tumor micro-environment was contributed by macrophages (median 77.9%, range 50.4 - 98.5%), although the mean relative intensity of PD-L1 per cell was higher for HRS cells than for macrophages (3.13 +/-0.02 RFU vs 2.85 +/- 0.01 RFU; p < 0.0001 by Welch t-test). Further analysis revealed that the percentage of HRS cells and macrophages expressing PD-L1 was highly correlated (Pearson r = 0.67; 95% CI 0.32 - 0.85; p=0.001) and, in 18/20 tumors, PD-L1+ macrophages were in greater proximity to PD-L1+ HRS cells than PD-L1- macrophages (across 20 cases mean distance of 32.6 µm (SE 5 µm) versus 51.2 µm (SE 6.8 µm), respectively; p < 0.05). CONCLUSIONS: CD68+ tumor-associated macrophages (TAMs) express the majority of PD-L1 in CHLs, which contain rare tumor cells. Image analysis of the distribution of PD-L1 in the tumor microenvironment indicates that PD-L1+ TAMs are significantly enriched in proximity to PD-L1+ HRS cells. These data implicate HRS cells in coordinating PD-L1 induction among TAMs to limit anti-tumor immunity. Figure 1. Figure 1. Disclosures Shipp: Merck: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding. Rodig:Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding.

scholarly journals Tumor Microenvironment in Patients with Relapsed/Refractory Classical Hodgkin Lymphoma before and after Anti-PD-1 Therapy

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 27-28
Author(s):  
Artem A. Gusak ◽  
Kirill V. Lepik ◽  
Natalia B. Mikhailova ◽  
Elena Kondakova ◽  
Yuri R. Zalaylov ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Hodgkin Lymphoma ◽  
Observational Studies ◽  
Principal Investigator ◽  
Immune Checkpoints ◽  
Rank Test ◽  
Classical Hodgkin Lymphoma ◽  
Significance Level ◽  
Cell Counts ◽  
Before And After

Background.Tumor tissue in classical Hodgkin Lymphoma (cHL) contains 1-10% malignant Hodgkin/Reed-Sternberg cells and a significant number of immune cells in the tumor microenvironment that are characterized by expression of inhibitory molecules (PD-1, CTLA-4, LAG-3, TIM-3, TIGIT). Despite overall effectiveness of anti-PD-1 treatment many patients still have relapsed or refractory (r/r) disease, therefore the search for predictive/prognostic biomarkers in patients on immunotherapy is highly demanded. Materials and methods.The study included 39 primary tumor specimens from patients with r/r cHL obtained before starting the treatment with nivolumab (primary biopsies). Specimens from 11 patients were studied before and after treatment (sequential biopsies). Treatment response was evaluated by PET-CT according to LYRIC criteria. Immunohistochemical staining for CD68, CD163, PD-1, LAG-3, TIM-3, CTLA-4, TIGIT was performed with an automated staining system (Bond III; Leica Biosystems). The slides were scanned with Aperio ScanScope XT (AperioTechnologies Inc.) and were analyzed with ImageScope Analysis software (Aperio Technologies) и Qupath (https://qupath.github.io). We explored progression-free survival (PFS) depending on the proportion of cells positive for CD68, CD163, PD-1, LAG-3, TIM-3, CTLA-4, TIGIT in the tumor microenvironment and analyzed the changes of these parameters between primary and sequential biopsies after treatment with nivolumab. Statistical analysis was performed using SPSS software (v.23). Data on sequential biopsies were analyzed with the Wilcoxon signed-rank test. PFS was calculated with the Kaplan-Meier method. The significance level was p ≤ 0.05. Results.A significant correlation was found in primary biopsies group between the value of CD163 and CTLA-4 (correlation coefficient -0,62, p &lt; 0.05). There was no significant association between PFS and proportion of cells positive for CD68, PD-1, TIM-3, CTLA-4, TIGIT, LAG-3 in primary biopsies group. ROC analysis allowed to establish a 9% cut-off value of CD163 expression, dividing these patients into subgroups of CD163high and CD163low. In the CD163low group, the two-year PFS was 19,1% (95% CI 6%-37,7%) with a median PFS of 8,8 months (95% CI 5,7-12) and in the CD163high group - 53,8% (95% CI 28,4%-73,7%) with a median of 24,8 months (95% CI 18,8 - 39,2). In sequential biopsies, a statistically significant increase in numbers of PD-1+ and TIGIT+ T-cells and depletion of CD68+ and CD163+ cells was observed compared to corresponding cell counts in primary biopsies (median PD-1 - 3% vs 10%; median TIGIT - 10% vs 14%; median CD68 - 10% vs 7%; median CD163- 8% vs 3,5%; р &lt;0,05). Conclusion.A comprehensive analysis of expression of CD68, CD163, LAG-3, TIGIT, CTLA4, TIM-3, PD-1 was performed in patients with r/r cHL before and after treatment with nivolumab. Significant association was found between the expression of CD163 and CTLA4. The results of the study indicate inferior PFS among patients with low expression (&lt;9%) of CD163 in lymph node samples before immunotherapy. Biopsies taken after treatment with nivolumab showed a statistically significant increase in the number of PD-1+ and TIGIT+ cells and a decrease in the number of CD68+ and CD163+ cells compared with data from primary biopsies. The results of the study may contribute to our knowledge regarding biology of classical Hodgkin lymphoma and the mechanisms of resistance to therapy with immune checkpoints inhibitors. This study was supported by BMS research grant CA209-8EG Disclosures Ionova: Takeda:Other: principal investigator of the observational studies sponsored by Takeda;BMS:Other: principal investigator of the observational studies sponsored by BMS.

scholarly journals Urine TWEAK level as a biomarker for early response to treatment in active lupus nephritis: a prospective multicentre study

2019 ◽  
Vol 6 (1) ◽  
pp. e000298 ◽  
Author(s):  
Thitima Benjachat Suttichet ◽  
Wonngarm Kittanamongkolchai ◽  
Chutipha Phromjeen ◽  
Sirirat Anutrakulchai ◽  
Thanachai Panaput ◽  
...  
Keyword(s):  
Lupus Nephritis ◽  
Treatment Response ◽  
Induction Therapy ◽  
Characteristic Curve ◽  
Predictive Performance ◽  
Complete Response ◽  
Early Response ◽  
Response To Treatment ◽  
Urine Protein ◽  
Spot Urine

BackgroundTNF-like weak inducer of apoptosis (TWEAK) is a proinflammatory molecule that plays a key role in active inflammation of lupus nephritis (LN). Urine TWEAK (uTWEAK) levels were found to be associated with renal disease activity among patients with LN. Here, we determined whether serial measurements of uTWEAK during induction therapy could predict treatment response or not.MethodsSpot urine samples were collected from patients with biopsy-proven active LN at time of flare, and 3 and 6 months after flare to assess the uTWEAK levels. All patients received standard immunosuppressive therapy and treatment response was evaluated at 6 months. The performance of uTWEAK as a predictor for treatment response was compared with clinically used biomarkers for patients with LN.ResultsAmong 110 patients with LN, there were 29% complete responders (CR), 34% partial responders (PR) and 37% non-responders (NR). On average, uTWEAK level was consistently low in CR, trended down by 3 months in PR and persistently elevated in NR. uTWEAK levels at month 3 were able to predict complete response at month 6 (OR adjusted for age, sex and creatinine=0.34 [95% CI 0.15 to 0.80], the area under the receiver operating characteristic curve [ROC-AUC]=0.68, p=0.02). The optimal threshold for uTWEAK level at month 3 was 0.46 pg/mgCr, discriminating complete response with 70% sensitivity and 63% specificity. Combining uTWEAK and urine protein at month 3 improved predictive performance for complete response at 6 months (ROC-AUC 0.83, p<0.001).ConclusionsIn addition to urine protein, uTWEAK level at 3 months after flare can improve the accuracy in predicting complete response at 6 months of induction therapy.

scholarly journals Immune and Inflammatory Cells of the Tumor Microenvironment Represent Novel Therapeutic Targets in Classical Hodgkin Lymphoma

2019 ◽  
Vol 20 (21) ◽  
pp. 5503 ◽  
Author(s):  
Eleonora Calabretta ◽  
Francesco d’Amore ◽  
Carmelo Carlo-Stella
Keyword(s):  
Tumor Microenvironment ◽  
Hodgkin Lymphoma ◽  
Checkpoint Inhibitors ◽  
Therapeutic Targets ◽  
Suppressor Cells ◽  
Inflammatory Cells ◽  
Brentuximab Vedotin ◽  
New Drugs ◽  
Classical Hodgkin Lymphoma ◽  
Relevant Role

Classical Hodgkin Lymphoma (cHL) is a B-cell malignancy that, typically, responds well to standard therapies. However, patients who relapse after standard regimens or are refractory to induction therapy have a dismal outcome. The implementation of novel therapies such as the anti-CD30 monoclonal antibody Brentuximab Vedotin and immune checkpoint inhibitors has provided curative options for many of these patients. Nonetheless, responses are rarely durable, emphasizing the need for new agents. cHL is characterized by a unique microenvironment in which cellular and humoral components interact to promote tumor survival and dissemination. Knowledge of the complex composition of cHL microenvironment is constantly evolving; in particular, there is growing interest in certain cell subsets such as tumor-associated macrophages, myeloid-derived suppressor cells and neutrophils, all of which have a relevant role in the pathogenesis of the disease. The unique biology of the cHL microenvironment has provided opportunities to develop new drugs, many of which are currently being tested in preclinical and clinical settings. In this review, we will summarize novel insights in the crosstalk between tumor cells and non-malignant inflammatory cells. In addition, we will discuss the relevance of tumor-microenvironment interactions as potential therapeutic targets.

Hodgkin Lymphoma Outcomes: Can We Expect Ethnic Parity in a Hispanic Prevalent Population?

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4056-4056
Author(s):  
Michelle Janania Martinez ◽  
Prathibha Surapaneni ◽  
Juan F Garza ◽  
Tyler W Snedden ◽  
Snegha Ananth ◽  
...  
Keyword(s):  
Hodgkin Lymphoma ◽  
Conflicts Of Interest ◽  
Relative Survival ◽  
Complete Response ◽  
Statistical Testing ◽  
P Value ◽  
Hispanic Population ◽  
Significant Difference ◽  
The Mean

BACKGROUND It is estimated that 8110 persons will be diagnosed with Hodgkin Lymphoma (HL) in the US during 2019, but the advent of new treatment options has increased the cure rate to at least 80%. It has been reported that the rates of HL are lower in the adolescent and young adult (AYA) Hispanic population but significantly higher in the Hispanic population older than 65. The relative survival estimates are stated to be similar between AYA Hispanics (HI) and non-Hispanics (NH) but for ages 65-84, HI have a significantly higher mortality rate. Pediatric studies have suggested that ethnicity plays a role in outcomes in patients with HL but there is limited data in the adult population. There is an unmet need in the field, where dossiers on underrepresented ethnic minorities need to be carefully considered and compared to existing data. Therefore, our study aims to compare survival outcomes in Hispanics vs Non-Hispanics with HL, who were treated at the only NCI designated cancer center of South Texas. To our knowledge this is the largest cohort of HL patients from a single academic institution that serves primarily Hispanics. METHODS We located and retrospectively analyzed a total of 616 patients with diagnosis of Lymphoma (HL and NHL) by International Classification of Diseases (ICD) codes and identified 116 cases of HL; all the patients received care at UT Health San Antonio, between 2008-2018. Key variables for each patient included age, gender, race/ethnicity, comorbidities, insurance status, stage, BM and extranodal involvement, treatment received, outcome at 3 and 5 years and vitality status in 2018. Continuously distributed outcomes were summarized with the mean and standard deviation and categorical outcomes were summarized with frequencies and percentages. The significance of variation in the mean with disease category was assessed with one way ANOVA and the significance of associations between categorical outcomes was assessed with Pearson's Chi Square or Fisher's Exact test as appropriate. Multivariate logistic regression was used to model binary outcomes in terms of covariates and indicators of disease. All statistical testing was two-sided with a significance level of 5%. R1 was used throughout. The study was approved by the local Institutional Review Board. The findings will be available to patients, funders and medical community through traditional publishing and social media. RESULTS We identified 116 patients with HL, of which 73 were HI (63%), 43 NH (36%) and 1 not specified (1%). In regard to race, 92% identified as Caucasian, 4% as African American, 3% other and 1% Asian. The median age at diagnosis was 37.4, (SD 15.13). There were 49 females (42%) and 67 males (58%). The most common funding source was commercial insurance N=54 (47%), followed by a hospital payment plan N=30 (26%), Medicare N=16 (14%), unfunded N=13 (11%) and Medicaid N=3 (2%). Most prevalent co-morbidities were HTN N=28 (24%) and diabetes mellitus N= 23(20%); 50% of patients had no co-morbidities (N=63).At diagnosis ECOG of 0-1 was seen in 108 patients (93%); 8 were Stage I (7%), 39 stage II (33%), 32 stage III (28%), and 37 stage IV (32%). EBV was positive in 26 patients (22%). There were 15 patients that were HIV positive (13%), 54% with CD4 count <200, and 12 (75%) on antiretroviral therapy at diagnosis. Median PFS was 853.85 days (SD 912.92). We excluded patients who were lost to follow up or had not reached 3/5 years. At 3 year follow up there was: complete response in 37 HI (74%) vs 22 NH (92%); disease progression in 8 (16%) vs 0 (0%); death in 5 (10%) vs 2 (8%), respectively (p-value= 0.094). At 5 year follow up there was: complete response in 30 HI (77%) vs 17 NH (90%); progressive disease in 2 (5%) vs 0 (0); death 7 (18%) vs 2 (11%), respectively (p-value = 0.619). At the end of 2018, 41 HI (84%) were alive compared to 22 NH (88%) [p-value 0.74]. CONCLUSION Within the limitations of sample size, our study demonstrates that in the prevalently Hispanic population of our institution, HI patients with HL have no statistically significant difference in outcome when compared to NH patients. Disclosures No relevant conflicts of interest to declare.

PU.1-Induced Growth Arrest and Apoptosis in Classical Hodgkin Lymphoma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2628-2628
Author(s):  
Hiromichi Yuki ◽  
Shikiko Ueno ◽  
Hiroaki Niiro ◽  
Hiro Tatetsu ◽  
Hiroyuki Hata ◽  
...  
Keyword(s):  
Cell Growth ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Growth Arrest ◽  
Myeloma Cell ◽  
G1 Arrest ◽  
Classical Hodgkin Lymphoma ◽  
Lymphoma Cells ◽  
Down Regulation

Abstract Abstract 2628 PU.1 is an Ets family transcription factor, which is essential for differentiation of both myeloid and lymphoid lineage cells. We have previously shown that PU.1 is down-regulated in various myeloma cell lines and myeloma cells from a subset of myeloma patients. In such cell lines, the promoter and the upstream regulatory element (URE) located in 17 kb 5'-upstream of the PU.1 gene are highly methylated. Furthermore, conditionally expressed PU.1 induces both cell growth arrest and apoptosis in PU.1-low to -negative myeloma cell lines, U266 and KMS12PE. Therefore, we concluded that the down-regulation of PU.1 is necessary for myeloma cell growth. In another B cell malignancy, classical Hodgkin lymphoma, it has been reported that PU.1 is also down-regulated through methylation of its promoter. To evaluate whether down-regulation of PU.1 is essential for growth of classical Hodgkin lymphoma cells, we conditionally expressed PU.1 in two classical Hodgkin lymphoma cell lines, L428 and KMH2, using the tet-off system (designated as L428tetPU.1 and KMH2tetPU.1 cells, respectively). Up-regulation of PU.1 by tetracycline removal induced complete growth arrest in L428tetPU.1 and KMH2tetPU.1 cells. Annexin V staining revealed that up-regulation of PU.1 induced apoptosis in both cell lines. Furthermore, BrdU staining analysis revealed that PU.1 induced G0/G1 arrest in those cells. L428tetPU.1 and KMH2tetPU.1 cells expressing PU.1 showed morphological changes that included the enlargement cytosol and the appearance of various sizes of vacuoles. We next injected L428tetPU.1 and KMH2tetPU.1 cells to immunodeficiency mice (Rag2−/− Jak3−/− bulb/c) subcutaneously. Tumor formation was observed in all those mice with continuous administration of tetracycline (0.5 g/l) in the drinking water. After enlargement of tumor to 1–2 cm diameter, we removed tetracycline in half of the mice. Tetracyclin withdrawal resulted in tumor regression or stable disease, whereas all the mice continuously receiving tetracycline had continuous tumor growth and finally died. These data strongly suggest that PU.1 induced growth arrest and apoptosis of classical Hodgkin lymphoma cells both in vitro and vivo. We next performed DNA microarray analysis to compare gene expression levels of L428tetPU.1 cells before and after PU.1 expression to elucidate the mechanisms of growth arrest and apoptosis induced by PU.1. Among genes related to cell cycle and apoptosis, p21 (CDKN1A) was highly up-regulated in L428tetPU.1 cells after PU.1 induction, and this was also confirmed by mRNA and protein levels. Finally, to clarify the role of p21 up-regulation by PU.1, we stably introduced p21 siRNA in L428tetPU.1 cells. Such stably expressed p21 siRNA rescued L428tetPU.1 cells from growth arrest induced by PU.1, suggesting that the growth arrest in L428tetPU.1 cells by PU.1 should be at least partially dependent on p21 up-regulation. These data suggested that up-regulation of PU.1 by demethylation agents and/or HDAC inhibitors might serve as a possible treatment modality for classical Hodgkin lymphoma. Disclosures: No relevant conflicts of interest to declare.

Strong HLA Class I Expression Is Positively Correlated with the Number of PML Nuclear Bodies and Negatively with the Percentage of SATB1 Positive HRS Cells in EBV+ Classical Hodgkin Lymphoma (cHL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3633-3633
Author(s):  
Yuxuan Liu ◽  
Lydia Visser ◽  
Rianne Veenstra ◽  
Bea Rutgers ◽  
Anke Van Den Berg ◽  
...  
Keyword(s):  
Hodgkin Lymphoma ◽  
Conflicts Of Interest ◽  
Malignant Neoplasm ◽  
Hla Class I ◽  
Nuclear Bodies ◽  
Class I ◽  
Positive Staining ◽  
Classical Hodgkin Lymphoma ◽  
Pml Nuclear Bodies ◽  
Significant Difference

Abstract Abstract 3633 Introduction: Classical Hodgkin lymphoma (cHL) is a malignant neoplasm of the immune system, characterized by the presence of an abundant reactive infiltrate and a minority of Hodgkin Reed-Sternberg cells (HRS cells). HRS cells retain their professional antigen presenting phenotype in most cases. In EBV- cHL, membranous HLA class I expression is retained in HRS cells in approximately 30% of the cases, whereas in EBV+ cHL HLA class I expression is retained in HRS cells in 79% of the cases. Moreover, a proportion of the EBV+ cHL cases shows an enhanced HLA class I expression as compared to the surrounding infiltrating cells. The mechanism of the enhanced or lost HLA class I expression is unknown. Special AT-rich region binding protein 1 (SATB1) and promyelocytic leukemia protein (PML) are two proteins that have been shown to regulate HLA class I expression. Downregulation of SATB1 in Jurkat cells results in an enhanced expression of HLA-A, HLA-G, HLA-H and HCG4P6, whereas downregulation of PML results in a reduced HLA-A and HLA-G expression. PML is the main component of nuclear bodies (NBs) that organize the chromatin structure into loops by anchoring matrix attachment regions to the nuclear matrix. SATB1 has been shown to be associated with the PML-NBs in the HLA region. Aim: To investigate the possible role of SATB1 and PML-NBs in the regulation of HLA class I expression in EBV+ cHL. Methods: We analyzed 64 EBV+ cHL cases and as a control included 29 EBV- cHL cases. HLA class I membranous staining (HC10 antibody) by HRS cells was scored as positive or strongly positive, cases that lacked membrane staining were scored negative. ß2-microglobulin served as an additional marker for membranous HLA class I expression. For SATB1 (14/SATB1), we scored the percentage of HRS cells with nuclear SATB1 staining. For PML (PG-M3), we scored HRS cells based on the number of PML nuclear bodies in two categories; 10 or less NBs per cell or >10 NBs per cell (per 4 um tissue section). Results: In the EBV+ group 24 cases stained strongly positive, 22 positive and 18 negative for HLA class I. In the EBV- group, none of the cases showed a strong positive staining, 7 cases stained positive and 22 cases were negative for HLA class I. HLA class I staining results were consistent with the ß2-microglobulin staining results in all cases. The percentage of SATB1 positive HRS cells varied from 0–100% in both EBV+ and EBV- cHL. The number of PML-NBs was between 0–10 in 46 and >10 in 18 EBV+ cHL cases and between 0–10 in 23 and >10 in 6 EBV- cHL cases. We observed no correlation between HLA class I staining in the EBV- cHL cases and the percentage of SATB1 positive HRS cells or the number of PML-NBs in the HRS cells. In EBV+ cHL cases we observed significant differences in the percentages of SATB1 positive cells between the HLA class I negative, normal and strongly positive groups (p=0.0412). The cases with normal HLA class I staining had significantly higher percentages of SATB1 positive cells as compared to the cases with strong HLA class I staining pattern (p<0.05) (Figure 1). There was no significant difference between negative and normal or negative and strongly positive HLA class I groups with respect to the percentage of SATB1. The percentage of EBV+ cHL cases with >10 PML-NBs significantly increased from HLA class I negative (1 out of 18, i.e. 5%) to normal (4 out of 22, i.e. 18%) and strong (13 out of 24, i.e. 54%) positive cHL cases (p=0.0011). Conclusion: We found an inverse correlation between the percentages of SATB1 positive HRS cells in the HLA class I strong and normal EBV+ cHL groups. The number of cases with >10 PML-NBs were significantly increased in HLA class I strong as compared to the HLA class I normal and negative EBV+ cHL groups. Thus SATB1 and PML may play an important role in the regulation of HLA class I expression levels in EBV+ cHL. Disclosures: No relevant conflicts of interest to declare.

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