CD4 T-helper responses to the anaplastic lymphoma kinase (ALK) protein in patients with ALK-positive anaplastic large-cell lymphoma

We have previously shown both humoral and CTL responses to anaplastic lymphoma kinase (ALK) in patients with ALK-positive anaplastic large-cell lymphoma (ALCL). However, because CD4(+) T-helper (Th) cells also play a vital role in developing and maintaining tumor immunity, we investigated the presence of a CD4(+) Th response in ALK-positive ALCL. Using an IFN-gamma ELISPOT assay, we identified two ALK-derived DRB1-restricted 24-mer promiscuous peptides, ALK1(278-301) and ALK2(233-256), as being immunogenic in six ALK-positive ALCL patients but not in two ALK-negative ALCL patients or five normal subjects. A significant interleukin-4 response to the ALK peptides was detected in only one ALK-positive patient. CD4(+) Th cell lines lysed ALK-positive ALCL cell lines in a MHC class II-restricted manner. This first report of a CD4(+) Th response to ALK provides valuable information for developing future immunotherapeutic options for ALK-positive ALCL patients who fail to respond well to conventional therapies.     

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.     

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.     

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.     

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。     

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.     

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。     

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.     

p53 and β-Catenin Expression Predict Poorer Prognosis in Patients With Anaplastic Large-Cell Lymphoma

BackgroundThe Wnt/β-catenin signaling pathway is a major target of p53. β-Catenin/p53 coexpression predicts poorer survival in carcinoma patients. Conversely, CD99 inhibits tumor metastasis through the Wnt/β-catenin pathway. We therefore assessed p53, β-catenin, and CD99 by immunohistochemistry.Patients and MethodsWe studied 45 patients with systemic anaplastic large-cell lymphoma (ALCL), including 20 anaplastic lymphoma kinase (ALK)-positive and 25 ALK-negative ALCL. β-Catenin expression was analyzed using phospho-β-catenin-S552 antibody because its nuclear localization indicates Wnt signaling.ResultsIn this cohort, p53 expression was associated with ALK-negative ALCL. Furthermore, p53 or β-catenin expression alone or β-catenin/p53 double expression showed poorer overall survival and disease-free survival in patients with ALCL overall and in patients with ALK-negative ALCL. CD99 expression was more frequent in ALK-positive ALCL but had no prognostic significance.ConclusionThis is the first study to evaluate phospho-β-catenin-S552 expression in ALCL. The results of this study, although limited by small patient size, suggest that β-catenin and p53 may play a role in pathogenesis and may be helpful in risk stratification of ALCL patients.     

Cyclin D1-specific cytotoxic T lymphocytes are present in the repertoire of cancer patients: implications for cancer immunotherapy

PURPOSE: Cyclin D1, a key cell cycle regulator, is overexpressed in multiple types of cancer. Such tumor-associated genes may be useful targets for cancer immunotherapy. Nevertheless, it had previously been suggested that efficient T cells recognizing cyclin D1-derived epitopes are absent from the repertoire because of thymic deletion. We attempted to induce autologous CTL from healthy donors and patients with cyclin D1-overexpressing tumors using a highly efficient T-cell expansion system based on CD40-activated B cells as antigen-presenting cells. EXPERIMENTAL DESIGN: Cyclin D1-derived, HLA-A*0201-restricted epitopes were predicted by multiple computer algorithms, screened in HLA-A2-binding assays, and used for T-cell stimulation. The generated CTL lines and clones were analyzed by IFN-gamma enzyme-linked immunosorbent spot assay or cytolysis assay. RESULTS: After screening, at least two naturally processed and presented HLA-A*0201-binding cyclin D1 epitopes were identified. CTL specific for these epitopes could be successfully generated from HLA-A2(+) donors. T cells efficiently recognized target cells pulsed with the cognate peptide and cyclin D1-expressing tumor cell lines in an HLA-A*0201-restricted manner. More importantly, HLA-A*0201-matched, primary cyclin D1(+) tumor cells were efficiently recognized by cyclin D1-specific CTL. These CTL could be generated from patients with mantle cell lymphoma and cyclin D1(+) colon cancer. CONCLUSIONS: These results underscore that cyclin D1 needs to be considered as a target for broad-based antitumor immunotherapy.     

Histology impacts the outcome of peripheral T-cell lymphomas after high dose chemotherapy and stem cell transplant

The role of high dose chemotherapy (HDC) and stem cell transplant (SCT) in peripheral T-cell lymphoma (PTCL) was studied in 28 patients, from 1988 to 2002. The aim was to determine if subsets recognized by the REAL/WHO classification have different prognoses. Outcome was compared to 86 patients with diffuse large B-cell lymphoma (DLBCL) transplanted during 1986-2000. The 3-year overall survival (OS) and event free survival (EFS) were 69% and 50%. Patients with anaplastic large cell lymphoma (ALCL) had a better 3-year OS compared to those with non-ALCL histology (86% vs. 47%, P=0.0122). Anaplastic lymphoma kinase (ALK)- positive ALCL patients had a superior EFS compared to ALK-negative ALCL (100% vs. 0; P=0.0228). Patients with cutaneous ALCL (ALK-negative) relapsed, but had an indolent course after SCT. Low International Prognostic Index score at relapse predicted for a better 3-year OS (85% vs. 34%, P=0.0238). When compared to DLBCL, patients with ALCL had a superior OS (86% vs. 36%, P=0.0034) and patients with non-ALCL had a comparable OS. ALCL histology confers better survival compared to non-ALCL and DLBCL histologies. ALK-positive ALCL is associated with the best EFS after relapse with HDC and SCT. The timing of SCT for non-ALCL histology remains to be determined.     

Anaplastic large cell lymphoma--a rare disorder in southern Taiwan

Anaplastic large cell lymphoma (ALCL) is a subgroup of non-Hodgkin's lymphomas with large lymphoma cells expressing CD30 antigen. This entity has rarely been reported in Taiwan. We performed a retrospective clinicopathologic study in a medical center in southern Taiwan during a 13-year period and identified 13 cases. There were 10 males and 3 females with a median age of 49 years old. Seven presented with pure nodal disease and 5 had bony involvement. The staging results were stage I (5 patients), II (1), III (1), and IV (4). The pathologic subtypes were common variant (10), lymphohistiocytic variant (2), and small cell variant (1). Eleven tumors were of T-cell lineage; 2, null-cell. Immunohistochemically, 5 tumors (38.5%) expressed cytotoxic markers, T-cell intracellular antigen-1 and/or granzyme B. Two tumors (15.4%) expressed anaplastic lymphoma kinase (ALK). Long-term follow-up information was available in 8 patients. The 2 patients with ALK-expressing tumors (37 and 49 years old) were free of disease for 61 and 54 months, respectively. The other 6 patients were either died of disease (5 patients) or experienced relapse with progressive disease (1). In conclusion, we reported the largest series of ALCL in Taiwan. We confirmed ALK-expressing ALCL carries favorable prognosis and ALK-negative ALCL has similar poor prognosis as non-anaplastic T-cell lymphoma. As compared to the previous reports from the West, our ALK positive rate was lower and the age of our ALK-positive patients was older. A larger national or multi-institutional study is needed for further characterization of ALCL in Taiwan.     

Identification of an HLA-A*0201 restricted Bcl2-derived epitope expressed on tumors

A large number of human tumor-associated antigen-derived peptides have been identified that are recognized by CTLs in a MHC-I restricted fashion. The apoptosis inhibitory protein Bcl2 is overexpressed in many human cancers as part of their neoplastic phenotype. Since inhibition or loss of Bcl2 expression might impair tumor growth and survival, this protein may serve as a rational target for vaccine-induced CTL responses. By Western blot technique, we screened a panel of established human tumor cell lines for proteins involved in the apoptotic process. Two of eight tumor cell lines, a B lymphoma (Loukes) and a colon carcinoma (CCL220) cell line showed increased Bcl2 protein expression whereas the majority of tumor cell lines expressed proapoptotic proteins. Neither fibroblasts nor peripheral blood mononuclear cells showed Bcl2 expression. An HLA-A*0201 restricted CTL epitope was deduced in silica from the amino acid sequence of the Bcl2 protein and its binding affinity for HLA-A*0201 was confirmed using a biochemical binding assay. We here demonstrate that the 9-mer peptide Bcl2(85-93) induces specific CTL reactivity in immunized C57-A2K(b) or -A2D(b) tg mice. These Bcl2(85-93) specific CTLs react with and lyse Bcl2-expressing human colon carcinoma CCL220 cells which have been transfected with a chimeric HLA-A*0201/H2-K(b) DNA construct similar to that expressed in the transgenic mice. Based on these observations, we suggest that Bcl2(85-93) may be a target for immune therapy.     

Analysis of MAGE-3 derived synthetic peptide as a human lung cancer antigen recognized by cytotoxic T lymphocytes

BACKGROUND: The human MAGE-3 gene was originally discovered in melanoma cells that encode tumor antigens, and has been reported to be expressed in various types of tumors, including lung cancer, but not in normal tissues other than testis or placenta. Our aim in this study was to clarify whether HLA-A2 restricted MAGE-3 peptide (FLWGPRALV) could be a lung cancer antigen recognized by cytotoxic T lymphocytes (CTL). METHODS: MAGE-3-derived peptide-specific CTL were induced from the peripheral blood mononuclear cells (PBMC) of HLA-A0201-positive healthy donors and the regional lymph node lymphocytes (RLNL) of HLA-A2-positive patients with lung cancer by multiple stimulations with peptide-pulsed HLA-A0201-positive antigen-presenting cells. RESULTS: Lymphocytes stimulated with MAGE-3 peptide exhibited specific lysis of Epstein-Barr virus-transformed B cells (EBV-B) pulsed with MAGE-3 peptide, but not with control peptide derived from influenza matrix protein, erbB-2, or wild type p53. Specific activity for MAGE-3-presenting targets was found after the second stimulation, and increased depending on the number of stimulations. The peptide-specific activity was inhibited by the addition of monoclonal antibodies against MHC class I and HLA-A2. Such CTL also recognized tumor cell lines expressing both HLA-A2 and MAGE-3 in an MHC class I-restricted manner, but did not recognize tumor cell lines that did not express HLA-A2 or MAGE-3. CONCLUSION: These results suggested the MAGE-3 peptide could be a potential target of specific immunotherapy for HLA-A2 patients with lung cancer.     

More efficient induction of HLA-A*0201-restricted and carcinoembryonic antigen (CEA)-specific CTL response by immunization with exosomes prepared from heat-stressed CEA-positive tumor cells.

PURPOSE: Tumor-derived exosomes are proposed as a new type of cancer vaccine. Heat shock proteins are potent Th1 adjuvant, and heat stress can induce heat shock protein and MHC-I expression in tumor cells, leading to the increased immunogenicity of tumor cells. To improve the immunogenicity of exosomes as cancer vaccine, we prepared exosomes from heat-stressed carcinoembryonic antigen (CEA)-positive tumor cells (CEA+/HS-Exo) and tested the efficacy of these exosomes in the induction of CEA-specific antitumor immunity. EXPERIMENTAL DESIGN: First, we identified the composition of CEA+/HS-Exo and observed their effects on human dendritic cell maturation. Then, we evaluated their ability to induce a CEA-specific immune response in vivo in HLA-A2.1/Kb transgenic mice and CEA-specific CTL response in vitro in HLA-A*0201+ healthy donors and HLA-A*0201+CEA+ cancer patients. RESULTS: CEA+/HS-Exo contained CEA and more heat shock protein 70 and MHC-I and significantly induced dendritic cell maturation. Immunization of HLA-A2.1/Kb transgenic mice with CEA+/HS-Exo was more efficient in priming a CEA-specific CTL, and the CTL showed antitumor effect when adoptively transferred to SW480-bearing nude mice. Moreover, in vitro incubation of lymphocytes from HLA-A*0201+ healthy donors and HLA-A*0201+CEA+ cancer patients with CEA+/HS-Exo-pulsed autologous dendritic cells induces HLA-A*0201-restricted and CEA-specific CTL response. CONCLUSIONS: Our results show that CEA+/HS-Exo has superior immunogenicity than CEA+/Exo in inducing CEA-specific CTL response and suggest that exosomes derived from heat-stressed tumor cells may be used as efficient vaccine for cancer immunotherapy.     

Identification of C-met oncogene as a broadly expressed tumor-associated antigen recognized by cytotoxic T-lymphocytes.

PURPOSE: C-Met proto-oncogene is a receptor tyrosine kinase that mediates the oncogenic activities of the hepatocyte growth factor. Using a DNA chip analysis of tumor samples from patients with renal cell carcinoma and sequencing of peptides bound to the HLA-A*0201 molecules on tumor cells a peptide derived from the c-Met protein was identified recently. EXPERIMENTAL DESIGN: We used this novel HLA-A*0201 peptide for the induction of specific CTLs to analyze the presentation of this epitope by malignant cells. RESULTS: The induced CTL efficiently lysed target cells pulsed with the cognate peptide, as well as HLA-A*0201-matched tumor cell lines in an antigen-specific and HLA-restricted manner. Furthermore, the induced c-Met-specific CTLs recognized autologous dendritic cells (DCs) pulsed with the peptide or transfected with whole-tumor mRNA purified from c-Met-expressing cell lines. We next induced c-Met-specific CTLs using peripheral blood mononuclear cells and DC from an HLA-A*0201-positive patient with plasma cell leukemia to determine the recognition of primary autologous malignant cells. These CTLs lysed malignant plasma cells while sparing nonmalignant B- and T-lymphocytes, monocytes, and DCs. CONCLUSION: Our results demonstrate that c-Met oncogene is a novel tumor rejection antigen recognized by CTL and expressed on a broad variety of epithelial and hematopoietic malignant cells.     

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。     

Detection of Wilms' tumor antigen--specific CTL in tumor-draining lymph nodes of patients with early breast cancer.

PURPOSE: The Wilms' tumor antigen (WT1) is overexpressed in approximately 90% of breast tumors and, thus, is a potential target antigen for the immunotherapy of breast cancer. We have tested the working hypotheses that WT1 can be immunogenic in patients with breast cancer and can stimulate CTL of sufficient avidity to kill tumor cells. EXPERIMENTAL DESIGN: Paired tumor-draining lymph node and peripheral blood samples were analyzed from five HLA-A2-positive patients with stage I/II breast cancer. Fluorescent HLA-A*0201/WT1 tetramers were used to quantify WT1-specific CTL and the functional capacity of the CTL was assessed using cytotoxicity assays and intracellular cytokine staining. RESULTS: WT1 tetramer-binding T cells expanded from all lymph node samples but none of the corresponding peripheral blood samples. Functional assays were carried out on T cells from the patient who had yielded the highest frequency of HLA-A*0201/WT1 tetramer-positive cells. The cytotoxicity assays showed WT1 peptide--specific killing activity of the CTL, whereas intracellular cytokine staining confirmed that the tetramer--positive T cells produced IFN-gamma after stimulation with WT1 peptide. These WT1-specific T cells killed HLA-A2-positive breast cancer cell lines treated with IFN-gamma but no killing was observed with untreated tumor cells. CONCLUSIONS: These results show that WT1-specific CTL can be expanded from the tumor-draining lymph nodes of breast cancer patients and that they can display peptide-specific effector function. However, the CTL only killed IFN-gamma-treated tumor targets expressing high levels of HLA-A2 and not tumor cells with low HLA expression. This suggests that induction of autologous WT1-specific CTL may offer only limited tumor protection and that strategies that allow a high level of peptide/MHC complex presentation and/or improve CTL avidity may be required.     

Identification of a new HLA-A*0201-restricted cryptic epitope from CYP1B1

Cytochrome P450 1B1 (CYP1B1) was recently shown to be a candidate tumor antigen broadly expressed in solid and hematologic malignancies. Nevertheless, use of such self-antigens as targets for immune intervention can be limited because of loss of high-avidity T cells during negative selection in the thymus. Recent data suggest that targeting of cryptic epitopes may represent a way to circumvent such self-tolerance and induce efficient antitumor CTL responses. Here, we present the identification and characterization of a novel, cryptic HLA-A*0201-binding peptide from CYP1B1. The nanomer CYP246 was identified by epitope deduction using algorithms to predict HLA-A*0201-binding peptides. CYP246 is characterized by strong initial HLA-A*0201 binding but a short MHC/peptide binding half-life. Expansion of high-avidity CTL was readily possible using autologous CD40-activated B cells from normal donors and cancer patients as antigen-presenting cells, suggesting that an intact T-cell repertoire can be expanded for this epitope. Lysis of CYP1B1-expressing, HLA-A*0201+ tumor cell lines and primary tumor cells confirmed that sufficient levels of CYP246 are presented by tumor cells for effector CTL killing. These findings indicate that CYP246 is a candidate cryptic epitope for immune interventions in which tumor CYP1B1 is targeted.