Exploring the Immune Microenvironment of Diffuse Large B Cell Lymphoma in a Tissue Microarray: Predicting Survival with a Score That Incorporates Macrophages, Cytotoxic and Regulatory T Cells
Exploring the Immune Microenvironment of Diffuse Large B Cell Lymphoma in a Tissue Microarray: Predicting Survival with a Score That Incorporates Macrophages, Cytotoxic and Regulatory T Cells
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通过组织微阵列探索弥漫性大 B 细胞淋巴瘤的免疫微环境：通过结合巨噬细胞、细胞毒性和调节性 T 细胞的评分来预测生存期
DOI：
10.1182/blood.v118.21.951.951
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发表时间：
2011
期刊：
影响因子：
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通讯作者：
J. Gribben
中科院分区：
文献类型：
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作者：
R. Coutinho;A. Clear;D. Owen;P. Greaves;Simon Hallam;Andrew D. Wilson;J. Matthews;M. Calaminici;J. Gribben
摘要摘要951弥漫性大B细胞淋巴瘤（DLBCL）的基因表达谱研究已经描述了代表免疫微环境的基因中富集的特征。这些特征与细胞来源分类一起具有预后意义。然而，对炎症和免疫反应在DLBCL中的作用的全面功能理解仍然缺乏。使用免疫组织化学（IHC）来验证和阐述分子结果是有吸引力的。我们的目的是在具有相应临床和生存数据的DLBCL患者的单中心TMA中定量和彻底地评估DLBCL中非恶性群体的细胞组成。基于报道的发现和已知的功能相互作用，选择了包括⑶ 68、F0 XP 3和TIA-1的一组抗体。从1977年至2009年，对来自DLBCL的高质量福尔马林固定石蜡包埋诊断组织活检的TMA进行了IHC。从代表性肿瘤组织的区域制备Trifecta核心，排列在载玻片上并对一组抗体进行染色，旨在表征免疫细胞亚群。TMA包括来自218名患者的组织：128名男性，90名女性，中位年龄为55（18-94）岁; 23.2%为高风险/高风险IPI;中位随访时间为3.1年。33%的患者在利妥昔单抗时代接受治疗。使用自动图像分析系统（Ariol）对所有完整核心中的阳性细胞绝对数量进行计数，由组织病理学家确认，并计算平均值并校正至1 mm 2面积。使用训练验证集方法来产生区分预后组的最佳截止值，并使用卡方检验进行比较。在检查的9种抗体中，使用FOXP 3、TIA-1和CD 68染色提供了统计学显著的预后信息。使用阳性细胞/mm 2的单个截止值来定义低和高标记物密度，针对每种标记物定义两个预后组。预后较好的患者FOXP 3、TIA-1和CD 68的细胞密度最高。为了阐明是否存在加性预后关系，我们开发了一个包含这三个标志物的评分。该评分系统使我们能够区分两个生存参数不同的患者亚组（图1）。标记（临界值高vs低）3年总生存期（高vs低）3年无进展生存期（高vs低）3年疾病特异性生存率（高vs低）FOXP 3（450+细胞/mm 2）64% vs 44%（p 0.005）55% vs 36%（p 0.001）69% vs 54%（0.01）CD68（900+细胞/mm 2）58% vs 28%（p 0.023）50% vs 20%（p 0.05）66% vs 36%（p 0.005）TIA-1（2500+细胞/mm 2）76% vs 45%（p 0.002）66% vs 41%（p 0.01）80% vs 56%（p 0.005）综合评分68% vs 39%（p <0.0002）60% vs 32%（p 0.0003）73% vs 49%（p 0.0008）下载：下载高分辨率图像（22 KB）下载：下载全尺寸图像开发了一个考克斯回归模型，包括年龄、IPI、利妥昔单抗治疗和IHC评分。评分结果与年龄、IPI（OS和DSS）和利妥昔单抗（PFS）一起被验证为OS、PFS和DSS的独立预后标志物。我们证实在诊断DLBCL时免疫细胞浸润存在异质性，并且我们提供了相关的预后数据来提示相应的生物学相关性。免疫细胞标志物的单一截止值是可行的，作为一种临床工具是有吸引力的，并且似乎提供了可靠的预后信息。在我们的单中心队列中，我们发现更高数量的巨噬细胞、T淋巴细胞和细胞毒性T细胞与改善的患者存活率独立相关。CD 68与淋巴恶性肿瘤的不良预后相关，但巨噬细胞的表型多样性可能是本研究结果的原因，并将使用巨噬细胞亚群特异性标志物进行探索。我们的多变量分析显示，免疫组化结果对DLBCL患者的生存率有独立影响。我们目前正在R-CHOP治疗患者的独立TMA中验证这种方法。结合这三种不同的免疫细胞参数，使我们能够区分预后亚组，提供了非恶性免疫细胞在DLBCL生物学中的影响的进一步证据，并验证了它们作为干预的潜在治疗靶点的作用。披露：格里本：吉利德：霍诺拉里亚;蒙迪制药：霍诺拉里亚;葛兰素史克：霍诺拉里亚;新基：霍诺拉里亚;罗氏：霍诺拉里亚; Pharmacyclics：霍诺拉里亚。
Abstract Abstract 951 Gene expression profiling studies in Diffuse Large B cell Lymphoma (DLBCL) have described signatures enriched in genes representing the immune microenvironment. These signatures, together with the cell of origin classification have prognostic significance. However, a comprehensive functional understanding of the role of inflammation and the immune response in DLBCL is still lacking. The use of immunohistochemistry (IHC) to validate and elaborate upon molecular results is appealing. Our objective was to assess the cellular composition of the non-malignant population in DLBCL quantitatively and thoroughly in a single centre TMA of patients with DLBCL with corresponding clinical and survival data. Based on reported findings and known functional interactions a panel of antibodies were selected that included CD68, FOXP3 and TIA-1. IHC was performed on TMAs from high quality formalin-fixed paraffin-embedded diagnostic tissue biopsies of DLBCL from 1977 to 2009. Triplicate cores were made from areas of representative tumour tissue, arrayed onto glass slides and stained for a panel of antibodies, aiming to characterize immune cell subsets. The TMA included tissue from 218 patients: 128 males, 90 females, with a median age of 55 (18-94) years; 23.2% high-int/high risk IPI; with median follow up of 3,1 years. 33% of patients were treated in the rituximab era. Absolute numbers of positive cells were counted across all intact cores using an automated image analysis system (Ariol), confirmed by histopathologists, and means calculated and corrected to a 1 mm2 area. A training-validation set method was used to generate optimal cutoff values discriminating prognostic groups and comparisons performed using the chi-square test. Among nine antibodies examined, statistically significant prognostic information was provided using staining for FOXP3, TIA-1 and CD68. Using single cut-offs of positive cells/mm2 to define low and high marker density, two prognostic groups were defined for each marker. Patients with a more favourable prognosis had the highest cell density for FOXP3, TIA-1 and CD68. To clarify whether there was an additive prognostic relationship we developed a score that incorporated these three markers. This scoring system allowed us to discriminate two subgroups of patients with divergent survival parameters (fig 1). Marker (cutoff value high vs low) Overall Survival at 3yr (high vs low) Progression Free Survival at 3yr (high vs low) Disease Specific Survival at 3yr (high vs low) FOXP3 (450+ cells/mm2) 64% vs 44% (p 0.005) 55% vs 36% (p 0.001) 69% vs 54% (0.01) CD68 (900+ cells/mm2) 58% vs 28% (p 0.023) 50% vs 20% (p 0.05) 66% vs 36% (p 0.005) TIA-1 (2500+ cells/mm2) 76% vs 45% (p 0.002) 66% vs 41% (p 0.01) 80% vs 56% (p 0.005) Combined Score 68% vs 39% (p 0.0002) 60% vs 32% (p 0.0003) 73% vs 49% (p 0.0008) Download : Download high-res image (22KB) Download : Download full-size image A Cox regression model was developed, incorporating age, IPI, rituximab treatment and IHC score. The score results were validated as independent prognostic markers for OS, PFS and DSS, together with age, IPI (for OS and DSS) and rituximab (for PFS). We confirm that there is heterogeneity in immune cell infiltration at diagnosis in DLBCL, and we provide correlative prognostic data to suggest a corresponding biological relevance. Attributing single cut-off values for immune cell markers is feasible, attractive as a clinical tool, and appears to provide robust prognostic information. In our single centre cohort we found that higher numbers of macrophages, Tregs and cytotoxic T cells correlate independently with improved patient survival. CD68 has been associated with an adverse prognosis in lymphoid malignancies but phenotypic diversity of macrophages may account for this study's finding and will be explored using macrophage subset specific markers. Our multivariate analysis revealed that the IHC results have an independent impact on survival on this cohort of DLBCL patients. We are currently validating this approach in an independent TMA of R-CHOP treated patients. Combining these three different immune cell parameters allowed us to discriminate prognostic subgroups providing further evidence of the impact of the non-malignant immune cells in the biology of DLBCL and validating their role as potential therapeutic targets for intervention. Disclosures: Gribben: Gilead: Honoraria; Mundipharma: Honoraria; GSK: Honoraria; Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria.