B and CTL responses to the ALK protein in patients with ALK-positive ALCL

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) has a good prognosis compared to ALK-negative ALCL, possibly as a result of the immune recognition of the ALK proteins. The aim of our study was to investigate the presence of both a B and cytotoxic T cell (CTL) response to ALK in ALK-positive ALCL. We confirmed the presence of an antibody response to ALK in all 9 ALK-positive ALCL patients investigated. An ELISpot assay was used to detect a gamma-interferon (IFN) T cell response after short term culture of mononuclear blood cells with 2 ALK-derived HLA-A*0201 restricted peptides: ALKa and ALKb. A significant gamma-IFN response was identified in all 7 HLA-A*0201-positive ALK-positive ALCL patients but not in ALK-negative ALCL patients (n = 2) or normal subjects (n = 6). CTL lines (>95% CD8-positive) raised from 2 ALK-positive ALCL patients lysed ALK-positive ALCL derived cell lines in a MHC-Class I restricted manner. This is the first report of both a B cell and CTL response to ALK in patients with ALK-positive ALCL. This response persisted during long-term remission. The use of modified vaccinia virus Ankara (MVA) to express ALK is also described. Our findings are of potential prognostic value and open up therapeutic options for those ALK-positive patients who do not respond to conventional treatment.     

原发系统性间变性大细胞淋巴瘤临床病理特征及免疫表型分析

 目的　分析原发系统性间变性大细胞淋巴瘤（ALCL）的临床病理特征和免疫组织化学特点,提高诊治水平。方法　选取22例ALCL患者,均进行分期、国际预后指数（IPI）、乳酸脱氢酶（LDH）检测,应用免疫组织化学SP法检测间变性淋巴瘤激酶（ALK）、Ki-67、Caspase-3、CD30、EMA、Granzyme B等,回顾性分析患者临床、病理形态学资料、免疫表型及生物学特性,并进行预后分析。结果　22例均为原发系统性ALCL,ALK+ 15例（68.2 %）,ALK- 7例（31.8 %）;ALK+患者发病年龄、Ki-67增殖指数较ALK-患者低,Caspase-3表达率高,差异有统计学意义（χ2＝4.618,P＝0.032）;15例ALK+ALCL均表达CD30和EMA。ALCL中ALK的表达与Ki-67、Caspase-3的表达呈负相关（r＝-0.581,P＝0.006;r＝0.458,P＝0.032）。ALK+病例较ALK-病例Granzyme B（χ2＝0.11,P＝0.74）、 TIA-1（χ2＝0.01,P＝0.92）的表达率高,但差异无统计学意义（P＞0.05）。有效率为 54.5 ％（12／22）,其中完全缓解率为18.2 %（4／22）;全组中位生存期12个月,1年生存率为59.1 %（13／22）,2年生存率为50.0 ％（11／22）。Ann Arbor分期、LDH及IPI与疾病预后相关。结论　ALK+较ALK-ALCL患者核增殖低,恶性程度低,临床特征和免疫表型具有一定的特征性;ALK、Ki-67、Caspase-3、分期、血清LDH及IPI对预测ALCL患者的生存和指导治疗有帮助。     

Cytomorphologic examination of anaplastic large cell lymphoma by fine-needle aspiration cytology

BACKGROUND: The cytomorphology of anaplastic large cell lymphoma (ALCL) is distinctive yet variable. To the authors' knowledge, to date only small case series have described the cytologic findings noted in patients with ALCL. The current series is the largest case series presented to date to retrospectively review the cytomorpholgic findings noted in patients with ALCL, with specific attention paid to those with anaplastic lymphoma kinase (ALK)-negative ALCL. METHODS: Over a 13-year period, the available Diff-Quik cytology smears and surgical excision specimens taken from patients with ALCL were evaluated. Different clinical and morphologic parameters were evaluated, including ALK status. RESULTS: A total of 37 cases were retrieved and evaluated, 19 of which had both cytology and surgical pathology specimens available for review. ALK-negative ALCL cytology smears were found to have a high number of anaplastic cells compared with ALK-positive cases. The hallmark cells in the ALK-negative cases were not classic. CONCLUSIONS: ALCL can be diagnosed accurately by fine-needle aspiration cytology (FNAC) alone when aided by immunocytochemistry in ALK-positive cases. Ancillary studies should be anticipated such that material for cell block preparation and molecular studies is taken at the time of FNAC. The results of the current study demonstrate the varied FNAC morphology of ALCL. The presence of severe pleomorphism and anaplasia was found to correlate with ALK-negative status.     

儿童原发中枢神经系统间变性淋巴瘤激酶阳性间变性大细胞淋巴瘤一例并文献复习

目的:探讨儿童原发中枢神经系统（CNS）间变性大细胞淋巴瘤（ALCL）的临床特征及预后。方法:回顾性分析福建医科大学附属协和医院收治的1例原发CNS间变性淋巴瘤激酶（ALK）阳性ALCL患儿的临床资料,并复习相关文献。结果:患儿以头痛、发热为主要症状就诊外院,颅脑磁共振成像提示右侧小脑肿块,术前无CNS以外淋巴瘤浸润证据,行小脑肿瘤切除术,术后病理明确诊断为ALK阳性ALCL,未及时化疗。术后第27天转入福建医科大学附属协和医院,化疗前评估肿瘤已扩散至骨髓、睾丸、椎骨等部位,外周血NPM-ALK融合基因阳性。2个疗程化疗后达完全缓解,但最终死于化疗相关并发症。结论:原发CNS的ALK阳性ALCL病例罕见,易误诊,病情进展快,总体预后不良;及时进行活组织病理检查以确诊,早期行以化疗为主的综合治疗可能改善患者预后。     

间变性淋巴瘤激酶阳性和阴性间变性大细胞淋巴瘤分子遗传学研究

目的：研究间变性大细胞淋巴瘤（ALCL）的遗传学特征,探讨其发病机制,寻找有助于ALCL诊断、分类及预后评估的新分子靶标。方法收集ALCL病例石蜡包埋组织10例,其中4例间变性淋巴瘤激酶（ALK）阳性,6例ALK阴性。采用免疫组织化学染色及荧光原位杂交技术分别检测表型及2p23重排,利用OncoScan芯片在全基因组水平上扫描分析10例ALCL的拷贝数变异。结果10例ALCL均存在拷贝数变异,拷贝数获得者多于拷贝数缺失者。拷贝数获得主要累及17q11.2、Xp22.3、Xq28,拷贝数缺失主要累及3q26.1、14q11.2、22q11.23。 ALK阴性者拷贝数变异比ALK阳性者更为复杂：ALK阴性者拷贝数获得主要累及9q24.3-24.1、14q32.33,拷贝数缺失主要累及2p11.2、16p13.3,而ALK阳性者并无此变异。结论 ALCL存在复杂的遗传学不平衡,染色体片段获得多于缺失,ALK阴性ALCL遗传学不平衡更为复杂。 ALCL是异质性明显的一类肿瘤。     

ALK阳性间变性大细胞淋巴瘤临床病理研究进展

约半数的间变性大细胞淋巴瘤（ALCL）病人中存在间变性淋巴瘤激酶（ALK）基因异常，ALK蛋白的异常激活使ALK阳性ALCL具有其典型的临床病理特征，并为ALK阳性ALCL的治疗提供新的靶点，提示ALK阳性ALCL的淋巴瘤可归类为一独立病种。     

Survivin expression predicts poorer prognosis in anaplastic large-cell lymphoma.

PURPOSE: Survivin, a member of the inhibitor of apoptosis (IAP) family, is not detected in normal adult tissues but is overexpressed in various cancers, including some types of lymphoma. The frequency and prognostic significance of survivin expression in anaplastic large-cell lymphoma (ALCL) is unknown. Materials and METHODS: We assessed for survivin expression in 62 ALCL tumors (30 anaplastic lymphoma kinase [ALK]-positive and 32 ALK-negative) obtained before doxorubicin-based chemotherapy. Given that survivin is a target of the STAT3 signaling pathway and STAT3 is activated in ALCL, survivin expression was also correlated with STAT3 activation. RESULTS: Survivin was expressed in 34 tumors (55%) and did not correlate with ALK. A significant association between survivin expression and STAT3 activation was observed (P =.007, Fisher's exact test). For the ALK-positive group, the 5-year failure-free survival (FFS) was 34% for patients with survivin-positive ALCL compared with 100% for patients with survivin-negative ALCL (P =.009, log-rank test). For the ALK-negative group, the 5-year FFS was 46% for patients with survivin-positive tumors compared with 89% for patients with survivin-negative tumors (P =.03, log-rank test). Overall survival was similarly worse for patients with survivin-positive tumors in both the ALK-positive and ALK-negative groups. Furthermore, multivariate analysis confirmed the independent prognostic value of survivin expression, along with age older than 60 years and Ann Arbor stage III or IV. CONCLUSION: Survivin is expressed in approximately half of ALCL tumors and independently predicts unfavorable clinical outcome. Modulation of survivin expression or function may provide a novel target for experimental therapy in patients with ALCL.     

Prognostic significance of MUC-1 expression in systemic anaplastic large cell lymphoma.

Systemic anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) and overexpresses anaplastic lymphoma kinase (ALK). MUC-1, a highly glycosylated transmembrane protein, is detected in normal and malignant epithelial cells and has been associated with a poorer patient survival in various human malignancies. We have shown previously that MUC-1 is expressed as a consequence of t(1;14)(q21;32) in a subset of diffuse large B-cell lymphomas. ALCLs are known to express MUC-1, but its clinical significance is undefined. For this study, eligible patients with ALCL were HIV negative, received anthracycline-containing regimens, and had pretreatment archival tissue. Expression of MUC-1 and ALK was determined immunohistochemically after heat-induced antigen retrieval. A 10% cutoff for MUC-1 positivity was used. We identified 63 patients with systemic ALCL (22 ALK+, 41 ALK-) with a median age of 47 years, and 41 were male. MUC-1 was detected in 16 of 22 (73%) ALK-positive and 20 of 41 (49%) ALK-negative ALCL (P = 0.06, chi(2) test). MUC-1 expression was not associated with apoptotic rate as detected by terminal deoxynucleotidyl transferase-mediated nick end labeling assay or proliferation index as evaluated by MIB-1 antibody. For 48 patients with ALCL (16 ALK+, 32 ALK-) and complete clinical follow-up, 5-year progression-free survival (PFS) was 39.7% for patients with MUC-1-positive tumors versus 75.2% (P = 0.027 by Log-rank) for patients with MUC-1-negative tumors. For the ALK-negative ALCL group of 32 patients, the 5-year PFS was 26 versus 70.8% for patients with MUC-1-positive versus MUC-1-negative tumors (P = 0.0096 by Log-rank). For the ALK-positive ALCL group of 16 patients, the 5-year PFS was 52 versus 100% for patients with MUC-1-positive versus MUC-1-negative tumors (P, not significant). In summary, MUC-1 is frequently expressed in systemic ALCL, and its expression is associated with significantly inferior outcome in patients untreated previously with ALK-negative tumors. Future studies should explore the underlying molecular mechanisms of MUC-1 expression in these tumors and its role as a target for novel therapeutic strategies.     

The NPM-ALK oncoprotein abrogates CD30 signaling and constitutive NF-kappaB activation in anaplastic large cell lymphoma

NPM-ALK characterizes anaplastic large cell lymphoma (ALCL), as does the high expression of CD30, a feature shared with H-RS cells of classic Hodgkin's lymphoma. In H-RS cells, ligand-independent signaling by overexpressed CD30 drives constitutive NF-kappaB activation, which is absent in ALCL cells. Here we show that NPM-ALK impedes CD30 signaling and NF-kappaB activation, dependent on both ALK kinase activity and the N-terminal NPM domain. NPM-ALK transduction into H-RS cell lines abrogates recruitment and aggregation of TRAF proteins, inducing an ALCL-like morphology and phenotype. TRAF2 associates with NPM-ALK at a consensus binding motif located in the kinase domain. Thus, NPM-ALK abrogates CD30-driven NF-kappaB activation and can also induce an ALCL phenotype, distinguishing ALCL cells from H-RS cells of T cell origin.     

Extra copies of ALK gene locus is a recurrent genetic aberration and favorable prognostic factor in both ALK-positive and ALK-negative anaplastic large cell lymphomas

Systemic anaplastic large cell lymphoma (ALCL) is subtyped into ALK-positive ALCL and ALK-negative ALCL based on the presence or absence of ALK protein expression. ALK-positive ALCL is characterized by t(2;5)(p23;q35)/NPM-ALK or variant ALK-involved translocations, while little is known about the genetic changes in ALK-negative ALCL. We investigated the structural and numerical aberrations of the ALK gene using interphase fluorescence in situ hybridization (FISH) in 81 cases with ALCL and analyzed their association with clinical outcome of the patients. ALK gene rearrangement was found in 47 of 50 (94%) ALK-positive ALCLs but in none of 31 ALK-negative ALCLs. Extra copies of the ALK gene locus, representing mainly extra copies of chromosome 2, were seen in 19 ALK-positive (38%) and 15 ALK-negative (48%) cases (P=0.357). In 55 cases with follow-up information, the mean survival time of the 38 ALK positive cases (58 months) was significantly longer than that of 17 ALK-negative cases (22.5 months) (P=0.038). Interestingly, the cases with extra copies of ALK had a significantly longer mean survival time than those without (64.4 months vs 35.3 months) (P=0.023) and this difference was observed in both ALK-positive (72.3 vs 45.9 months) and ALK-negative (34.7 vs 9.9 months) cases. Multivariate analysis showed that both ALK protein expression and extra copies of ALK gene were independent predictors for better survival (P=0.008). Our results suggest that the extra copies of ALK gene locus are a frequent genetic aberration in both ALK-positive and ALK-negative ALCL and is a favorable prognostic marker for the patients.     

Leukotriene B4 receptor inhibitor LY293111 induces cell cycle arrest and apoptosis in human anaplastic large-cell lymphoma cells via JNK phosphorylation.

Anaplastic large-cell lymphoma (ALCL) is a heterogeneous lymphoma category in which a subset of cases carry the t(2;5)(p23;q35) or variant translocations resulting in overexpression of anaplastic lymphoma kinase (ALK). LY293111 (2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]-propoxy]-phenoxy] benzoic acid sodium salt) is a leukotriene B4 receptor antagonist, which was found to be safe and tolerable in Phase I clinical trials. In this study, we investigated the potential therapeutic effects and mechanisms of action of LY293111 in ALCL cell lines. LY293111 inhibited proliferation of both ALK(+) and ALK(-) ALCL cell in a dose-dependent fashion and induced complete G(1)-S cell cycle arrest, which was accompanied by upregulation of p27 and downregulation of cyclin E. Pretreatment with LY293111 for 4 h resulted in profound inhibition of serum-induced phosphorylation of extracellular-regulated kinases-1 and 2 and Akt and a concomitant increase in the phosphorylation of the stress-activated kinase c-jun N-terminal kinases (JNK). Simultaneously, LY293111 induced caspase-dependent apoptosis via activation of the intrinsic pathway, including early loss of mitochondrial inner transmembrane potential and the production of reactive oxygen species (ROS), cleavage of caspases-9, -3, poly ADP-ribose polymerase (PARP) and X-linked inhibitor of apoptosis. The phospho-JNK inhibitor SP600125 partially protected Sup-M2 cells from LY293111-induced apoptosis, PARP cleavage and ROS generation, suggesting a role for JNK in LY293111-induced cell death. These results warrant further studies of LY293111 in ALCL.     

ALK-negative anaplastic large cell lymphoma with extensive peripheral blood and bone marrow involvements manifested as “leukemic phase”

CD30-positive anaplastic large cell lymphoma (ALCL) is a distinctive malignant large cell lymphoma of T-cell lineage, often presenting in lymph node or extranodal sites. ALCL cases with extensive bone marrow and peripheral blood involvement manifested as “leukemic phase” are extremely rare and the most of those cases reported are anaplastic large cell lymphoma kinase (ALK) positive ALCL in childhood population. Here we report four adult cases of ALK-negative ALCL with extensive bone marrow and peripheral blood involvement manifested as “leukemic phase”. Circulating large lymphoma cells varied from 20 to 80% in peripheral blood and bone marrow biopsy showed various nodular or interstitial infiltrates. By reviewing the clinicopathologic data of previously reported ALCL cases with extensive bone marrow and peripheral blood involvement, there appears to be of large variations in regard to the patient's age, morphologic variants, immunophenotypic or genotypic characteristics of the disease. While most cases of ALCL with peripheral blood and bone marrow involvement were ALK-positive or carrying t(2;5) translocation, rare ALK-negative cases were also present. Leukemic ALCL patients usually have unfavourable prognosis, regardless of ALK expression.     

间变性大细胞淋巴瘤临床病理分析

 目的　研究间变性大细胞淋巴瘤（ALCL）的临床病理特征、免疫表型，以提高诊断水平。方法　回顾性分析19例ALCL的临床、病理形态学资料和免疫组织化学，CD30、ALK、CD43、CD45RO、EMA和TIA-1检测结果。结果　19例均为原发系统型ALCL，其中男性11例，女性8例，年龄2～71岁，平均26岁。16例（84.2 %）出现B症状，14例（73.7 %）结外侵犯。肿瘤细胞沿淋巴窦生长或散在分布，均可见标志性肿瘤细胞，CD+30 19例（100 %），ALK（+）15例（78.9 %），EMA（+）10例（52.6 %），TIA-1（+）14例（73.7 %），ALK、EMA和TIA-1同时阴性2例（10.5 %）。重新诊断17例（89.5 %）正确。结论　ALCL表现多样，大部分患者经临床、组织病理学和免疫标记特征综合判断可作出相应诊断，CD30、ALK、EMA、TIA-1对鉴别诊断有帮助。     

间变性大细胞淋巴瘤的ALK和c-myc基因研究

 目的　探讨间变性大细胞淋巴瘤（ALCL）中间变性淋巴瘤激酶（ALK）基因与c-myc基因的分子遗传学改变。方法　收集原发系统性ALCL石蜡包埋组织标本72例,利用间期荧光原位杂交（FISH）技术检测ALCL肿瘤组织中ALK和c-myc基因结构与数目的变化。结果　72例ALCL中,ALK阳性者42例,40例存在涉及ALK基因的染色体易位,其中17例同时伴有ALK基因的多拷贝;ALK阴性的30例均未发现ALK基因的易位,但其中14例存在ALK基因的多拷贝。ALK基因多拷贝的发生率在ALK阳性与 阴性组中的差异无统计学意义（P＞0.05）。72例病例中,均未发现涉及c-myc基因的染色体易位,但其中24例存在c-myc基因的多拷贝。结论　大部分ALCL伴有ALK基因的异常（75.0 %）。以涉及ALK基因的染色体易位最为多见（55.6 %）,ALK基因多拷贝也是ALCL较为常见的遗传学改变（43.1 %）。前者只出现于ALK阳性ALCL中,后者既可出现在ALK阳性也可出现在ALK阴性的ALCL中。ALCL中不见或罕见涉及c-myc基因的染色体易位,但c-myc基因多拷贝的现象较为常见（33.3 %）。     

系统型间变性大细胞淋巴瘤30例临床分析

 目的　探讨系统型间变性大细胞淋巴瘤（S-ALCL）的临床特征和预后相关因素。方法　回顾性分析30例S-ALCL患者的临床资料。30例患者均以联合化疗为主,配合局部病灶野放疗8例。化疗方案主要为 CHOP、EPOCH、Hyper-CVAD,以CHOP方案为主。结果　30例S-ALCL患者中位年龄36岁,男女比例为1.5∶1,有B症状、Ⅲ～Ⅳ期和结外侵犯者分别占60.0 %（18／30）、73.3 %（22／30）和60.0 %（18／30）;乳酸脱氢酶（LDH）升高者占46.7 %（14／30）;间变性大细胞淋巴瘤激酶（ALK）+ 18例（60.0 %）,其发病年龄小于ALK- 者（u＝3.92,P＝0.001）。单因素分析显示ALK-及LDH升高是重要的预后不良因素。结论　S-ALCL患者发病年龄较轻,预后较好。但ALK-、LDH升高者预后不良。治疗以联合化疗为主,对于有不良预后因素的患者,大剂量治疗可能获益。