Immunohistochemical study of neuroblastoma and related tumors with anti-S-100 protein antibody

Histological sections of 36 cases of neuroblastoma and related tumors were studied with anti-S-100 protein antibody (PAP method). Schwann cells in the ganglioneuromas and ganglioneuroblastomas always strongly stained. In addition, varying numbers of spindle-shaped or elongated positively staining cells, which were probably Schwann cells and their precursor cells, were demonstrated in ganglioneuroblastoma and differentiating neuroblastoma. Undifferentiated round cell neuroblastoma showed no reaction. Immunohistochemical findings of these cases were classified into four groups (+ +, +, +/-, -) according to the number of the positive cells and compared with prognosis, histological typing, location of the tumors, stage, and age at surgery. The cases with many positive cells, group (+ +) showed excellent prognosis, and group (-) showed very poor prognosis. The results of this study indicate that S-100 protein staining provides a reliable objective method for evaluation of differentiation of the neuroblastoma cells toward Schwann cells, which appears to be an important factor to predict prognosis.     

Schwann cell-conditioned medium inhibits angiogenesis in vitro and in vivo

BACKGROUND: Neuroblastomas are biologically heterogeneous tumors that consist of two main cell populations: neuroblastic/ganglionic cells and Schwann cells. The amount of Schwannian stroma strongly impacts prognosis. Low tumor vascularity, localized stage, and favorable outcome are associated with tumors that are Schwannian stroma-rich/stroma-dominant. PROCEDURE: To investigate if Schwann cells play a role in inhibiting angiogenesis in neuroblastoma tumors, we examined the ability of human Schwann cell-conditioned medium to affect bFGF- and VEGF-induced endothelial cell proliferation and migration, and in vivo angiogenesis. RESULTS: Schwann cell-conditioned medium significantly inhibited bFGF- and VEGF-induced endothelial cell proliferation and migration. This effect appears to be specific for endothelial cells as smooth muscle cell and fibroblast proliferation were not inhibited by this medium. Schwann cell-conditioned medium also inhibited in vivo angiogenesis in rat corneal assays. CONCLUSIONS: Schwann cells produce a potent inhibitor(s) of angiogenesis that may be responsible for the low level of vascularity and more benign clinical behavior of Schwannian stroma-rich/stroma-dominant neuroblastoma tumors. Studies to identify the inhibitor(s) are ongoing.     

GLUTATHIONE S-TRANSFERASE AND P-GLYCOPROTEIN EXPRESSIONS IN NEUROBLASTOMA

This study was planned to evaluate the prognostic role of glutathione S-transferase pi (GST-pi) and P-glycoprotein (P-gp) expressions in children with neuroblastoma. Sections from formalin-fixed paraffin-embedded tumor blocks from 52 neuroblastoma cases (17 with localized, 35 with advanced disease) were subjected to immunohistochemistry for P-gp and GST-pi expressions. The overall number of tumors positive for P-gp and GST-pi were 19 (36.5%) and 21 (40.4%), respectively. Twenty-two tumors were negative for both GST-pi and P-gp expressions, whereas 10 expressed both proteins. The distribution of staining status of samples in the groups of both proteins showed no significant difference. No relation between the expressions of both proteins and the clinical characteristics of the patients was demonstrable. The differences between the survival rates of patients with positive and negative staining for P-gp expression were not statistically significant. Although 2 common mechanisms of multiple drug resistance, P-gp and GST-pi, might be responsible for drug resistance in neuroblastoma, this complex mechanism has no direct significant impact on prognosis. Multiple mechanisms at cellular levels are responsible for the resistance against antineoplastic therapies in neuroblastoma.     

Glutathione S-transferase and P-glycoprotein expressions in neuroblastoma

This study was planned to evaluate the prognostic role of glutathione S-transferase pi (GST-pi) and P-glycoprotein (P-gp) expressions in children with neuroblastoma. Sections from formalin-fixed paraffin-embedded tumor blocks from 52 neuroblastoma cases (17 with localized, 35 with advanced disease) were subjected to immunohistochemistry for P-gp and GST-pi expressions. The overall number of tumors positive for P-gp and GST-pi were 19 (36.5%) and 21 (40.4%), respectively. Twenty-two tumors were negative for both GST-pi and P-gp expressions, whereas 10 expressed both proteins. The distribution of staining status of samples in the groups of both proteins showed no significant difference. No relation between the expressions of both proteins and the clinical characteristics of the patients was demonstrable. The differences between the survival rates of patients with positive and negative staining for P-gp expression were not statistically significant. Although 2 common mechanisms of multiple drug resistance, P-gp and GST-pi, might be responsible for drug resistance in neuroblastoma, this complex mechanism has no direct significant impact on prognosis. Multiple mechanisms at cellular levels are responsible for the resistance against antineoplastic therapies in neuroblastoma.     

神经母细胞瘤S－100蛋白免疫组化研究及图象分析DNA倍体的预后价值

本文对３２例病理确诊的神经母细胞瘤进行回顾性研究，对所有病理标本重新进行组织学分型，同时进行ＮＳＥ及Ｓ－１００蛋白免疫组化染色，并利用全自动图象分析仪对其中２８例进行了瘤细胞ＤＮＡ定量测定及倍体分析。结果表明：ＮＳＥ及Ｓ－１００蛋白免疫组化染色可反映神经母细胞瘤分化程度，Ｓ－１００蛋白阳性细胞的出现是该肿瘤分化成熟及预后良好的表现，ＤＮＡ异倍体是预后良好的可靠标志。     

Application of fluorescence in situ hybridization to detect N-myc (MYCN) gene amplification on paraffin-embedded tissue sections of neuroblastomas

Fluorescence in situ hybridization (FISH) was applied to neuroblastoma for detection of N-myc (MYCN) oncogene amplification, and the results were compared with Southern blot analysis (Southern). In nine neuroblastomas (formalin-fixed paraffin-embedded tissues were available in seven cases including two cases with touch preparations, and two cell lines), all five cases with N-myc amplification detected by Southern had cells with multiple N-myc signals by FISH, and three cases showed no N-myc amplification either by Southern or FISH procedure. One case, not examined by Southern, showed amplified signals of N-myc by FISH. These data indicate that FISH results for N-myc amplification have close correlation with Southern blot analysis. The chromosome 2-specific repetitive DNA probe was also applied for the analysis of ploidy by FISH. Six cases with N-myc amplification by Southern and/or FISH had diploid tumors and two cases without amplified N-myc showed aneuploidy. The remaining one case consisted of heterogeneous elements showing diploidy in undifferentiated tissue and both aneuploidy (ganglionic cells) and diploidy (Schwann cells) in differentiated area. We conclude that FISH is a practical, useful and reliable method over Southern especially for analysis of N-myc amplification in neuroblastoma, and simultaneous cohybridization with a specific chromosome probe is of great value in predicting the prognosis of patients. Med. Pediatr. Oncol. 29:135–138, 1997. © 1997 Wiley-Liss, Inc.     

先天性巨结肠免疫组织化学研究

为观察神经标志物PGP9．5和S－100蛋白在先天性巨结肠（HD）中的表达,采用一抗为抗PGP9．5和抗S－100蛋白的PAP免疫组织化学方法,探讨其在临床诊断中的意义。结果：（1）在对照组结肠壁神经丛中可见染色深浅不一的PGP9．5免疫反应阳性神经节细胞,神经纤维均匀分布在肠壁各层;神经节细胞胞体不表达S-100蛋白,表现为细胞状“空白区”。（2）HD结肠壁神经发育异常,PGP9．5和S－100蛋白免疫反应性神经纤维明显增生,分布紊乱,未见有PGP9．5阳性神经节细胞;在神经丛中,增生的S－100蛋白阳性神经纤维中偶见有细胞状的“空白”区。提示结肠壁神经发育异常是HD的主要病理生理变化,神经丛中PGP9．5阳性反应的细胞团块和S－100蛋白染色的神经丛中细胞状“空白”区,可特征性地提示神经节细胞的存在,用于HD的临床诊断敏感度高。     

Expression and clinical significance of stem cell marker CD133 in human neuroblastoma

Background  Recent evidences indicate that CD133, a kind of transmembrane protein, can be used as a marker to isolate stem cells from tumors originating from neural crest. This study was undertaken to explore the expression and clinical significance of stem cell marker CD133 in neuroblastoma (NB). Methods  Immunohistochemical staining was used to detect the expression of CD133 in 32 patients with NB and 8 patients with ganglioneuroblastoma (GNB). The relationships were analyzed among CD133 expression, international neuroblastoma staging system (INSS) stages, pathological classification, and postoperative survival time of NB patients. Results  The expression rates of CD133 in NB and GNB were 46.9% (15/32) and 37.5% (3/8) respectively, mainly in cytoplasm of neuroblastoma cells. The expression rates of stage 1–2, stage 3–4 and stage 4S were 30.7%, 57.9% and 37.5%, respectively. The differences in various stages were significant (P<0.05). The positive rate of CD133 in patients with unfavorable histology (52.4%) was significantly higher than that in patients with favorable histology (36.8%) (P=0.007). The survival time of CD133 negative patients was significantly longer than that of CD133 positive patients (P=0.026). Conclusions  CD133 which might be correlated with the development and progression of NB can serve as one of the important indicators for prognosis of NB.     

DETECTION OF TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE (TdT) IN NONHEMATOPOIETIC SMALL ROUND CELL TUMORS OF CHILDREN

In the differential diagnosis of small round cell tumors (SRCTs), terminal deoxynucleotidyl transferase (TdT) is often used as a marker for lymphoblastic lymphomas and leukemias. However, the specificity of TdT using the avidin-biotin-immunoperoxidase (ABC) method is not well documented. To address this issue, we stained paraffin-embedded biopsy specimens of 64 cases of childhood SRCTs using the ABC method with anti-TdT. For any TdT-positive tumors, an additional antibody panel for lymphoid markers was applied. Two patterns of TdT positivity were observed: (1) tumor specific, consisting of strong to moderate nuclear staining, and (2) scattered positive lymphoid cells, usually in a perivascular location and expressing T-cell markers. Analysis showed that 7 of 10 medulloblastomas stained with TdT in a tumor-specific pattern (4 cases moderately to strongly positive in 75-100 of tumor cells, 3 cases weakly to moderately positive in 25-50 of cells). Also, 1 of 19 rhabdomyosarcomas and 1 of 8 Ewing's sarcomas showed moderate to strong tumor-specific TdT staining in 100 and 10 of cells, respectively. Scattered TdT-positive lymphoid cells were observed in 27 of these 64 SRCTs. These findings emphasize that TdT positivity should not be relied upon exclusively for making a diagnosis of lymphoblastic leukemia or lymphoma or ruling out other SRCTs.     

Heterogeneous subgroups in human neuroblastoma for clinically relevant risk stratification

Neuroblastoma is a heterogeneous tumor and that may have a favorable or unfavorable prognosis. In Japan, a nation-wide neuroblastoma mass-screening (MS) project assessed 6-month-old infants between 1985 and 2003, and almost all neuroblastomas, including regressing or maturing tumors were thought to be detected in this period. To evaluate the heterogeneity of neuroblastoma subgroups, we analyzed patients with neuroblastoma who had been diagnosed during this period. The clinical courses of 4,209 patients with neuroblastoma, including 1,560 MS detected patients, whose tumors had been diagnosed between 1971 and 1995 were registered. The 2,520 cases registered between 1985 and 1995 were compared to 1,050 cases registered between 1971 and 1980 and analyzed by a multi-gene target model to determine the age distribution of neuroblastoma incidence. We hypothesized that three target genes were responsible for the progression of neuroblastoma: one pair of tumor suppressor gene alleles, one oncogene, and one gene controlling regression/differentiation. This simulation study revealed that the age distribution at initial diagnosis of neuroblastoma was divided into four groups based on post-fertilization age: 20-40, 40-50, 60-90, and 160-200 weeks. Since neuroblatoma in the first group occurred prenatal, post-natal clinical neuroblastoma can be classified into three age groups: 0-6 months, 1-2 years, and 3-4 years. The 0- to 6-month group consisted of mostly benign tumors, and the two older groups had predominantly malignant phenotypes. Our proposed model could explain qualitatively the distribution of neuroblastoma consisting of one subgroup with a favorable prognosis and two subgroups with unfavorable prognosis. For clinically relevant risk stratification, an age cutoff should be considered by the age distribution of these heterogeneous subgroups.     

肝母细胞瘤各组织类型的免疫表达和预后的关系

目的 探讨肝母细胞瘤各组织类型的免疫表达和预后的关系。方法 对２４例肝母细胞瘤进行临床病理分析和组织学分型,其中１８例作７种标记的免疫组化研究。结果 胎儿型１２例,胚胎型７例,间变型３例和上皮间叶混合型２例;细胞角蛋白,甲胎蛋白,Ｓ－１００蛋白和波形蛋白在肿瘤上皮细胞浆的表达分别为１４例,１０例,９例和６例,癌胚抗原,Ｐ５３和Ｐ１６蛋白在细胞核中表达分别是１１例,９例和７例;     

血管内皮生长因子-C在儿童实体肿瘤生长转移中的表达

目的探讨血管内皮生长因子-C(VEGF-C)在儿童实体肿瘤神经母细胞瘤及肾母细胞瘤中的表达。方法对33例神经母细胞瘤和30例肾母细胞瘤标本进行VEGF—C免疫组织化学分析,神经母细胞瘤Ⅲ期8例,Ⅳ期25例,远处转移25例,其中淋巴结转移5例,组织病理满意型(FH)20例,组织病理不满意型(UFH)13例;肾母细胞瘤Ⅰ Ⅱ期15例,Ⅲ Ⅳ期15例,远处转移12例,肾门、腹主动脉旁淋巴结受累5例,FH22例,UFH8例。结果神经母细胞瘤Ⅲ期VEGF-C阳性率37.5%(3/8),Ⅳ期32%(8/25),FH45%(9/20),UFH30.8%(4/13),远处转移32%(8/25),淋巴结转移66.7%(4/6),肾母细胞瘤Ⅰ Ⅱ期26.7%(4/15),Ⅲ Ⅳ期40%(6/15),FH27.3%(6/22),UFH25%(2/8),远处转移33.3%(4/12),淋巴结转移60%(3/5)。VEGF-C的表达与肿瘤的临床分期及病理类型无显著意义,而与淋巴结的转移存在显著意义(P<0.05)。结论VEGF-C的表达与肿瘤淋巴结的转移存在密切相关。     

神经母细胞瘤组织分型的生物特性与临床意义

目的：探讨不同神经母细胞瘤组织分型的生物特性和预后评估意义。方法：按Shimade分型将61例神经母细胞瘤分为组织结构良好型（FH）和组织结构不良型（UH），应用免疫组化检测Trk-A,N-myc,NSE,CD44表达，统计分析FH和UH型的生物标记表达和生存率差异。结果：61例分型为FH型40例，UH型21例，UH型的Trk-A阳性率（6／14）明显降低于FH型（27／32）、N-myc阳性率（8／14）则高于FH高（10／32），NSE和CD44检测无明显差异。FH型的3年和5年生存率（57．6％和27．3％）均高于UH型（18．8％）。结论：神经母细胞瘤的组织分型对肿瘤生物特性、临床诊治和预后评估有指导意义。，     

Biological characteristics of neuroblastoma with partial deletion in the short arm of chromosome 1

BACKGROUND: Neuroblastoma shows remarkable heterogeneity, resulting in favorable and unfavorable outcomes. It is well known that almost all cases with MYCN amplification have a poor prognosis. We have previously reported that unfavorable tumors show high telomerase activity, whereas favorable tumors show low or nil activity. We also found that the unfavorable neuroblastoma often have a loss of heterozygosity (LOH) at the MYCL locus. PROCEDURE: To clarify the biological and clinical profiles of tumors with genetic abnormalities of the short arm of chromosome 1, we performed deletion mapping on 1p on 92 neuroblastoma tissues and corresponding noncancerous samples obtained from 92 cases for 24 micro- or minisatellite loci. RESULTS: LOH was detected in at least one locus of 1p in 43 (47%) cases. All samples were classified into four groups according to the deleted pattern: interstitial deletion (group I, n = 20), short terminal deletion (group ST, n = 6), large terminal deletion (group LT, n = 17), and without detectable deletion (group N, n = 49). All group I cases, whose SRO (shortest region of overlap) was at 1p36.1-2, survived disease free, and none of them showed MYCN amplification or high telomerase activity except for one case. On the other hand, in group LT cases, who showed a large terminal deletion from D1S162 (1p32-pter), including the SRO of group 1, only 5 out of 17 have survived disease free, and 13 showed MYCN amplification or high telomerase activity. The six group ST cases showed small terminal deletion from 1p36.3 with modest prognosis, similar to the group N. CONCLUSIONS: Thus, we propose three loci, 1p36.1-2, 1p32-34, and 1p36.3, as the candidate loci of neuroblastoma suppressor genes on chromosome 1p responsible for groups I, LT, and ST, respectively. Among them, the 1p32-34 locus may be associated with aggressiveness of tumor progression, possibly due to MYCN amplification and/or telomerase reactivation, while the remaining two loci may not.     

ATRA耐药基因HA117相关蛋白在四种恶性肿瘤的表达及临床意义

目的探讨ATRA耐药基因HA117相关蛋白在肠腺癌、肺癌、乳腺癌及神经母细胞瘤组织中的表达特点及临床意义。方法以肠腺癌标本20例、肺癌标本23例、乳腺癌标本21例以及神经母细胞瘤标本20例手术切除组织为实验组;以巨结肠手术切除扩张段近切H缘的正常结肠组织10例、手术切除的肺癌癌旁组织9例、乳腺癌癌旁组织11例及神经母细胞瘤瘤旁组织14例为对照组;应用免疫组化S-P法检测HA117蛋白在上述组织切片中的表达,并结合临床资料进行回顾性分析。结果HA117在肠腺癌、肺癌、乳腺癌以及神经母细胞瘤组织中阳性表达明显高于非癌组织,差异具有统计学意义（P〈0．05）;HA117蛋白的阳性表达与肠腺癌、肺癌及乳腺癌患者的肿瘤分期、病理分型、淋巴结转移无明显相关性（P〉0．05）,与神经母细胞瘤的病理分型和淋巴结转移有显著相关性（P〈0．05）。结论HA117蛋白在肠腺癌、肺癌、乳腺癌以及神经母细胞瘤组织的高表达提示这四种恶性实体肿瘤对ATRA具有较强的耐药性。     

Opsoclonus-myoclonus-ataxia syndrome in neuroblastoma: histopathologic features-a report from the Children's Cancer Group

BACKGROUND: Opsoclonus-myoclonus-ataxia (OMA) is a paraneoplastic syndrome that occurs in about 2-3% of all cases of neuroblastoma. The histopathologic characteristics of neuroblastoma tumors associated with this syndrome were evaluated in a series of cases and controls. PROCEDURE: Pathology slides from a total of 54 neuroblastoma tumors were reviewed blindly. They included 13 tumors associated with opsoclonus-myoclonus and 41 age- and stage-matched controls. All tumors were classified into either the favorable (FH) or unfavorable histology (UH) group according to the International Neuroblastoma Pathology Classification (the Shimada system). Grade of lymphocytic infiltration was evaluated and presence or absence of lymphoid follicles was recorded in the individual tumor tissues. RESULTS: Twelve of 13 cases with opsoclonus-myoclonus were in the FH group. Twelve of 13 cases had diffuse (found in every section prepared from the multiple portions of the primary tumor) and extensive (occupying more than 50% of a single of multiple microscopic fields with x 100 magnification) lymphocytic infiltration with lymphoid follicles. Of the 41 control cases (27 FH and 14 UH tumors), 18 had focal areas of lymphocytic infiltration and six showed lymphoid follicles, but none had diffuse or extensive infiltration in their primary tumors. CONCLUSIONS: Diffuse and extensive lymphocytic infiltration with lymphoid follicles is a characteristic histologic feature of neuroblastic tumors with opsoclonus-myoclonus. This observation suggests an immune-mediated mechanism for this rare paraneoplastic syndrome.     

Lacking immunocytological GD2 expression in neuroblastoma: report of 3 cases

Immunocytological bone marrow assessment for contamination with neuroblastoma cells is based on their characteristic GD2 surface staining. Neuroblastoma without GD2 expression have been rarely and only after antibody therapy reported. Conventional cytology was performed using Pappenheim staining. For immunocytology, the APAAP method was utilized with the 14G2a anti-GD2 mouse monoclonal antibody. 7 x 10(5) cells on cytospin preparations were investigated. In 2003, 288 bone marrow samples from 191 neuroblastoma patients were investigated by cytology and immunocytology. Three cases demonstrated GD2 negativity on cytologically unambiguous neuroblastoma cells. Two female cases (94 and 37 months of age) with stage 4 neuroblastoma had GD2 expressing neuroblastoma cells in bone marrow at diagnosis. At 2nd relapse 25 and 23 months after diagnosis and 8 months and 12 months after anti-GD2 antibody treatment (ch14.18), the bone marrow infiltrating neuroblastoma cells lacked GD2 staining. The third patient, a 63-month-old girl with bone marrow replacement by neuroblastoma cells showed at diagnosis a mixture of GD2-unstained tumor clumps and very weakly stained neuroblastoma cells. Neuroblastoma cells may lack GD2 expression at diagnosis and at recurrence. This observation has diagnostic and therapeutic implications.     

Neuronal Differentiation of Ewing's Sarcoma Induced by Cholera Toxin B and Bromodeoxyuridine—Establishment of Ewing's Sarcoma Cell Line and Histochemical Study—

An Ewing's sarcoma (ES) cell line was established from a metastatic bone marrow specimen in a patient with advanced disease, and some histochemical characteristics were investigated by neuronal differentiation induced with cholera toxin B (CTB) and bromodeoxyuridine (BrdU). Neuronal differentiation was investigated by the expression of neurofilament and Leu-7, and glial differentiation was observed by expression of S-100 protein. Neurofilament (NF) and Leu-7 were positive in ES cells and these were expressed more intensively by induction with CTB than with BrdU. There was no expression of S-100 protein in untreated or differentiated ES cells. ES cells became differentiated to neuronal cells with CTB and BrdU, but it was not observed, that ES cells had the potential to differentiate to glial cells. It appears that ES is of more primitive neural origin than neuroblastoma, primitive neuroectodermal tumors and other related neural tumors.     

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??Objective To detect the expression of UBE2C in neuroblastoma??NB?? tissue and analyze its association with the clinical features of NB patients. Methods Paraffin-embedded surgical tissue specimens from 51 NB patients kept by the Department of Pathology?? Xinhua Hospital??from January 2012 to January 2015??were collected. The expression of UBE2C protein was detected by immunohistochemistry??and the clinicopathological characteristics of these patients were analyzed retrospectively. The survival curve was established with Kaplan-Merier analysis. Results Of 51 cases of NB?? the male-to-female ratio was 1.8??1. The median age at diagnosis was 36 months??ranging 3.3-156 months?? and median duration of follow-up was 25.6 months??ranging 5.5-42.7 months??. The results of immunohistochemistry showed that the UBE2C protein positive expression rate in stage ?? and ?? group ??90.0%?? was significantly higher than that in stage ???? and ??s group ??47.6%????P??0.001?? ?? the UBE2C protein positive expression rate in recurrence group??91.3%?? was also higher than that in the non-recurrence group??57.1%????P??0.001??. The survival curve based on Kaplan-Merier analysis revealed that patients with UBE2C positive expression had poor prognosis??P??0.006??. Conclusion The expression of UBE2C protein is closely related to the stage and the relapse of NB patients. Positive expression of UBE2C protein implies the poor prognosis for NB. UBE2C may play an important role in the invasion??metastasis and relapse of NB. It might become a new biomarker and potential drug target to NB.