Lymphoma Disease Discovery and Defintion

最近，在亚洲和西方国家，在非免疫功能低下的宿主中发现了新型eb病毒（EBV）淋巴增生性疾病（lpd）。其中包括侵袭性t细胞和nk细胞lpd，通常归入慢性活动性EBV感染（CAEBV）的范畴，以及EBV驱动的b细胞lpd，主要影响老年人。为了更好地定义这些疾病的发病机制、分类和治疗，来自亚洲、美洲、欧洲和澳大利亚的参与者在我们小组组织的一次国际会议上提交了临床和实验数据。在我们自己的工作中，我们描述了在没有已知免疫缺陷的西方人群中观察到的ebv相关b细胞LPD的谱。与来自日本的报告一样，大多数患者都是老年人，通常超过60岁。在7年的时间里，116例病例被确定为5种诊断类型：1)淋巴结反应性增生伴ebv阳性b细胞增加，2)ebv阳性淋巴结b细胞淋巴增生类似移植后LPD (PTLD), 3) ebv阳性结外b细胞淋巴增生类似PTLD， 4) ebv阳性弥漫性DLBCLs， 5) ebv阳性b细胞增生类似CHL。28例患者有ebv相关的反应性淋巴样增生，中位年龄为67岁。在大多数患者中，该过程是自限性的，只有一名患者表现为更积极的淋巴增生性过程。所有检测病例均为IgH PCR多克隆。在研究的3例（11%）病例中发现t细胞克隆或限制性t细胞受体基因重排模式。这一发现表明，正如Khanna等人所报道的那样，T细胞受体库的多样性减少。其特征包括保存完好的结构，ebv阳性细胞的细胞大小具有广谱性，并且经常定位于生发中心。ebv阳性DLBCL患者的中位年龄最高（77岁），其中包括11例淋巴结性DLBCL和4例淋巴结或结外浆母细胞淋巴瘤。73例多形性b细胞淋巴瘤在淋巴结和结外的分布大致相等。中位年龄分别为73岁和76岁。7例，中位年龄79岁，组织学和表型与CHL相似（CD30+;CD15+），但表现在CHL不常见的部位，如口腔（腭、牙龈、舌头、嘴唇）和肾上腺。大约20%的多形性b细胞淋巴瘤或DLBCL患者有限制性克隆或寡克隆t细胞应答的证据，这支持了对EBV的限制性t细胞应答可能与免疫应答缺陷相关的概念。我们还描述了一系列与EBV相关的粘膜和皮肤淋巴增生性病变，其病理和临床特征不同。对24例EBV阳性限定溃疡性b淋巴细胞增生（LPs）伴不同类型免疫抑制（IS）的患者进行组织学、免疫表型、EBER原位杂交和PCR克隆性分析。研究组包括9名男性和15名女性患者，中位年龄77岁（42-101岁）。8例IS患者使用硫唑嘌呤（AZA）、甲氨蝶呤（MTX）或环孢素a （CyA）治疗类风湿关节炎（5例）、溃疡性结肠炎（1例）、结节病合并重肌无力（1例）和系统性红斑狼疮（1例）。16例患者由于年龄相关性免疫衰老而被认为免疫抑制。患者表现为孤立的明显边界溃疡，包括口咽粘膜（15）、食管(1)、结肠(1)、直肠(1)和皮肤(6)。组织学显示多形性病变含有具有霍奇金或RS细胞形态的b细胞母细胞。在大量活化t细胞的背景下，b细胞CD20表达降低，CD30和EBER呈强阳性。CD15阳性占48%（10/21）。无论IS的解剖部位或病因如何，其病理特征都是相同的。PCR结果显示，31%（5/16）克隆型IgH重排，克隆型和限制性t细胞型重排分别为43%（6/14）和29%（4/14）。26%的患者（5/19）接受了标准化疗和/或放疗。47%（9/19）的病变在未经治疗的情况下自发消退，16%（3/19）的病变表现为复发和缓解。所有与药物相关的病变（5/5）均对IS的减少有反应。在20.5个月（3-72个月）的中位随访期间，所有患者均获得完全缓解，无疾病相关死亡。EBV阳性粘膜皮肤溃疡（EBVMCU）是一种临床病理实体，经常具有霍奇金样特征，病程自限性，无痛，需要保守治疗。与各种形式的IS相关意味着一个共同的发病机制，通常涉及限制性和克隆t细胞反应。这种疾病的局域性可能是由于对EBV免疫监测的最小和局域性缺失。我们还致力于建立改进的激光捕获显微解剖技术。激光辅助显微解剖（LAM）越来越多地用于分离特定肿瘤细胞群进行分子分析。在某些类型的组织中，常规苏木精-伊红（H&amp；E）染色不足以明确识别靶细胞，因此需要免疫组织化学标记。然而，常规免疫组织化学（IHC）对LAM的应用由于两个因素而变得复杂：LAM膜玻片的特征，以及采用福尔马林固定和石蜡包埋的常规组织处理的影响。在这里，我们描述了一种详细的膜载玻片制备方案，采用254nm紫外线照射和聚l -赖氨酸涂层，使福尔马林固定石蜡包埋（FFPE）标本的抗原检索（AR）和免疫组化（IHC）成功。10例FFPE档案标本[5例原发性纵隔大b细胞淋巴瘤；选择5种经典霍奇金淋巴瘤（结节性硬化症亚型）分别使用CD20或CD30抗体建立方案。测试不同的孵育时间和抗体稀释度，我们可以证明在所有情况下具有高重复性的特异性靶细胞染色。此外，我们在三种不同的LAM显微镜上研究了免疫引导LAM的可行性，并评估了它们集成的自动细胞识别软件。为了进一步验证该方法，我们检测了免疫引导LAM后的DNA质量。对来自同一标本的H&amp；E微解剖肿瘤细胞或免疫组织化学染色组织切片的成对样本进行PCR分析。-珠蛋白基因和CpG岛甲基化阵列。两种染色方法都显示出相同的PCR扩增子模式。在一系列5对样本中，CpG岛甲基化与1505个分析位点有很强的相关性（r = 0.945 ~ r = 0.981）。这些结果证明了我们的AR和IHC方案对装在膜载玻片上的存档FFPE样品的有效性和实用性。这项技术的使用为揭示人类恶性肿瘤的特定基因组改变提供了新的机会。

Recently novel Epstein-Barr virus (EBV) lymphoproliferative diseases (LPDs) have been identified in non-immunocompromised hosts, both in Asia and Western countries. These include aggressive T-cell and NK-cell LPDs often subsumed under the heading of chronic active EBV infection (CAEBV) and EBV-driven B-cell LPDs mainly affecting the elderly. To better define the pathogenesis, classification, and treatment of these disorders participants from Asia, The Americas, Europe, and Australia presented clinical and experimental data at an international meeting organized by our group. In our own work we have described the spectrum of EBV-associated B-cell LPD observed in a western population without known immunodeficiency. As with the reports from Japan, most patients are of advanced age, generally older than 60 years. 116 cases were identified over a 7 year period and fell into five diagnostic categories: 1) lymph node based reactive hyperplasia with increased EBV-positive B-cells, 2) EBV-positive nodal B-cell lymphoproliferations resembling post-transplant LPD (PTLD), 3) EBV-positive extranodal B-cell lymphoproliferations resembling PTLD, 4) EBV-positive diffuse DLBCLs, and 5) EBV-positive B-cell proliferations resembling CHL. Twenty-eight patients had EBV-associated reactive lymphoid hyperplasia, with a median age of 67 years. The process was self-limited in most patients, with only one patient showing progression to a more aggressive lymphoproliferative process. All cases tested were polyclonal by IgH PCR. T-cell clonality or a restricted T-cell receptor gene rearrangement pattern was seen in 3 (11%) of the cases studied. This finding suggests a reduction in diversity of the T cell receptor repertoire, as reported by Khanna et al. Features included preserved architecture, and a broad spectrum in cell size of the EBV-positive cells with frequent localization to germinal centers. The median age was highest in patients with EBV-positive DLBCL (77 yrs), which included 11 nodal DLBCL and 4 nodal or extranodal plasmablastic lymphomas. There were 73 polymorphic B-cell lymphomas approximately equally divided between nodal and extranodal sites. Median ages were 73 and 76 years respectively. Seven cases, median age 79, histologically and phenotypically resembled CHL (CD30+;CD15+) but presented in sites unusual for CHL such as oral cavity (palate, gingiva, tongue, lips) and adrenal glands. Supporting the concept that a restricted T-cell response to EBV may be associated with defective immune response, approximately 20% of patients with either polymorphic B-cell lymphoma or DLBCL had evidence of a restricted clonal or oligoclonal T-cell response. We have also described a series of EBV associated mucosal and cutaneous lymphoproliferative lesions characterized by distinctive pathological and clinical features. Histology, immunophenotype, EBER in situ hybridization and clonality by PCR were assessed in 24 EBV positive circumscribed, ulcerative B-cell lymphoproliferations (LPs) associated with various types of immunosuppression (IS). The study group comprised 9 male and 15 female patients, median age 77 years (range 42-101). IS in 8 cases included azathioprine (AZA ), methotrexate (MTX) or cyclosporin-A (CyA) administered for rheumatoid arthritis (5), ulcerative colitis (1), sarcoidosis with myasthenia gravis (1) and systemic lupus erythematosus (1). 16 patients were considered immunosuppressed due to age related immunosenescence. The patients presented with isolated sharply circumscribed ulcers involving oropharyngeal mucosa (15), esophagus (1), colon (1), rectum (1) and skin (6). Histology showed polymorphous lesions harboring B-cell blasts with Hodgkin or Reed-Sternberg (RS) cell morphology. The B-cells showed reduced expression of CD20 with strong CD30 and EBER positivity in a background of abundant activated T-cells. CD15 was positive in 48% of cases (10/21). The pathological features were identical regardless of the anatomical site or cause of IS. PCR revealed 31% (5/16) clonal IgH rearrangements with 43% (6/14) and 29% (4/14) clonal and restricted T-cell patterns respectively. 26% of patients (5/19) received standard chemotherapy and/or radiotherapy. 47% of lesions (9/19) regressed spontaneously with no treatment and 16% (3/19) were characterized by relapsing and remitting course. All the medication related lesions (5/5) with available follow up responded to reduction of IS. All patients achieved complete remission with no disease associated deaths over a median follow up period of 20.5 months (range 3-72). EBV positive Mucocutaneous Ulcer (EBVMCU) is a clinico-pathological entity with frequent Hodgkin-like features and a self limited, indolent course, requiring conservative management. Association with various forms of IS implies a common pathogenetic mechanism, frequently involving restricted and clonal T-cell responses. The localized nature of the disease may be due to a minimal and localized lapse in immunosurveillance over EBV. We have also worked to established improved techniques for laser capture microdissection. Laser assisted microdissection (LAM) is increasingly performed to facilitate isolation of specific tumor cell populations for molecular profiling. In certain types of tissue, routine hematoxylin-eosin (H&E) staining is inadequate for clear identification of target cells and therefore immunohistochemical labeling is required. Yet, the use of conventional immunohistochemistry (IHC) for LAM is complicated by two factors, the features of LAM membrane slides, and the effects of routine tissue processing employing formalin fixation and paraffin embedding. Here we describe a detailed protocol for preparation of membrane slides that employs UV irradiation at 254nm and poly-L-lysine coating, enabling successful antigen retrieval (AR) and IHC of formalin-fixed paraffin embedded (FFPE) specimens. Ten archival FFPE specimens [five primary mediastinal large B-cell lymphoma; and five classical Hodgkins lymphoma, nodular sclerosis subtype] were chosen to establish the protocol, using CD20 or CD30 antibodies, respectively. Testing distinct incubation times and antibody dilutions, we could demonstrate specific target cell staining with high reproducibility in all cases. In addition, we investigated the feasibility of immuno-guided LAM on three different LAM microscopes and evaluated their integrated automatic cell recognition software. To further validate the method, DNA quality after immuno-guided LAM was examined. Paired samples of microdissected tumor cells of H&E or immunohistochemically stained tissue sections from the same specimen were analyzed by PCR for β-globin gene and CpG island methylation array. Both staining methods showed identical PCR amplicon patterns for the β-globin gene, and a strong correlation (r = 0.945 to r = 0.981) for CpG island methylation of 1505 analyzed sites in a series of five paired samples. These results demonstrate the validity and utility of our AR and IHC protocol for archival FFPE samples mounted on membrane slides for LAM. The use of this technique offers new opportunities to unravel the specific genomic alterations of human malignancies.