Prognostic significance of GAGE detection in bone marrows on survival of patients with metastatic neuroblastoma

BACKGROUND: Previously, we reported the utility of GAGE as a molecular marker for neuroblastoma (NB) and malignant melanoma in blood and bone marrow (BM). Among patients with stage III melanoma rendered disease-free by surgery, GAGE expression was a strong prognostic factor for patient survival. PROCEDURE: All patients with advanced NB diagnosed at > 1 year of age initially treated with protocol N6 (n = 24) and N7 (n = 38) at Memorial Sloan-Kettering Cancer Center were included in this study. Their BM cells at 12, 18, and 24 months (median time after diagnosis) were evaluated for the presence of GAGE. RESULTS: GAGE positivity at 12 months (25%), when patients were still on treatment, did not predict progression-free survival (PFS) and overall survival from the time of sampling. Positivity at 18 months (29%) was associated with poorer PFS and survival (but P > 0.05). By 24 months, the presence of GAGE (26%) was a very strong predictor of out-come (P < 0.001). When only remission marrows at 24 months were analyzed, PFS was 4.7-fold lower among GAGE-positive patients. Thirty-seven percent of N6 patients were positive for GAGE, in contrast to 17% of the patients in the more current regimen N7. CONCLUSIONS: The detection of GAGE by RT-PCR in marrow may have utility in molecular staging of patients in clinical remission. It may allow earlier identification of patients at risk, such that appropriate intervention can be given before clinical relapse. GAGE may also serve as a surrogate endpoint for adjuvant treatment strategies, and to determine the duration of therapy.     

Ezrin蛋白、CD44、 E-钙粘素与神经母细胞瘤转移的关系

目的 探讨Ezrin蛋白、CD44、E-钙粘素在神经母细胞瘤中的表达及其与神经母细胞瘤转移的关系.方法 应用免疫组织化学二步法检测40例神经母细胞瘤标本中Ezrin蛋白、CD44、E-钙粘素的表达情况,并应用统计学方法比较其在局灶期、进展期的病例、FH型(组织结构良好型)和UFH型(组织结构不良型)及不同年龄性别病例中的表达程度.结论 Ezrin蛋白在进展期患儿中的表达为20例,明显高于局灶期病例的15例,且有统计学意义(H=6.124,P=0.013).而在进展期病例中CD44的表达为6例,明显低于局灶期的12例,E-钙粘素的表达5例则明显低于局灶期的11例,并且均有统计学意义(H=4.950,P=0.026;H=5.455,P=0.020).Ezrin蛋白和CD44在FH型和UFH型表达的差异具有统计学意义(H=5.182,P=0.023;H=4.496,P=0.034).而 E-钙粘素在FH 中表达为阳性的有11例.在UFH型中为5例,经统计学检验两者之间差异无明显统计学意义(H=2.754,P=0.097).Ezrin蛋白在不同年龄组中的差异具有统计学意义,而CD44和E-钙粘素没有,三者在不同性别组中的表达无明显统计学差异.经统计学分析Ezrin蛋白的表达与CD44的表达呈负相关(χ2=4.675,P=0.031,r=-0.323).Ezrin蛋白的表达与E-钙粘素蛋白的表达呈负相关(χ2=6.077,P=0.014,r=-0.123).E-钙粘素蛋白的表达与CD44的表达呈正相关(χ2=6.077,P=0.014,r=-0.363).结论 Ezrin蛋白、CD44和E-钙粘素的表达与NB的转移有关,其中Ezrin蛋白可促进肿瘤的转移,而CD44、E-钙粘素则对肿瘤的转移有抑制作用.年龄是影响Ezrin蛋白表达的因素之一,但不影响CD44和E-钙粘素的表达,而性别对Ezrin蛋白、CD44和E-钙粘素的表达没有影响.在NB的发生、发展过程中Ezrin蛋白与CD44之间,Ezrin蛋白与E-钙粘素之间可能存在负协同效应,而CD44与E-钙粘素之间可能存在正协同效应.

Abstract：

Objective To study Ezrin protein,CD44 and E-cadherin expressions and their association to metastasis of neuroblastoma(NB).Methods The expressions of Ezrin Protein,CD44 and E-cadherin in 40 NB patients were detected by two-step immunohistochemistry staining,and the expression levels of these proteins were analyzed according to local stage and advanced stage,favor able histology(FH) and the unfavorable histology(UFH),age and gender.Results Strong expression of Ezrin Protein was detected only in 15 patients in local stage tumors,and in 20 advanced stage tumors(compared local stage tumors,H=6.124,P=0.013).Strong CD44 staining was found in 12 in local stage tumors,and in 6 advanced stage tumors(H=4.950,P=0.026).Strong E-cadherin staining was found in11 local stage tumors,and in 5 advanced stage tumors(H=5.455,P=0.020).The difference between Ezrin Protein expression in the type of FH and UFH type was significant(H=5.182,P=0.023),and so did the CD44(H=4.496,P=0.034).The expression of Ezrin protein was related to the age.There was negative correlation between the expression of Ezrin and the CD44(χ2=4.675,P=0.031,r=-0.323),and the expression of Ezrin and the E-cadherin(χ2=6.077,P=0.014,r=-0.123).While positive correlation between the CD44 and E-cadherin(χ2=6.077,P=0.014,r=-0.363) was noted.Conclusions Ezrin Protein,CD44 and E-cadherin expressions may be related to metastasis and progression of NB.While Ezrin protein can stimulate tumor metastasis,CD44 and E-cadherin may have an inhibitory effect.Age is one of factors influence the expression of Ezrin protein in neuroblastoma.     

血管内皮生长因子、瘤内血管密度与神经母细胞瘤细胞增殖及转移的关系

目的观察血管内皮细胞生长因子(vascularendothelialgrowthfactor,VEGF)、血管内皮细胞相关抗原荆豆素(UEA)和增殖细胞核抗原(PCNA)在神经母细胞瘤(NB)中表达情况,探讨VEGF、瘤内微血管密度(MDV)与NB细胞增殖和转移的关系.方法应用免疫组化ABC法检测VEGF、UEA、PCNA在27例NB中的表达.应用RT-PCR方法测定患儿外周血、骨髓中微量瘤细胞(MRD).结果VEGF、UEA、PCNA表达均在进展期高于局灶期,预后不良型高于预后良好型,且均具有统计学意义.VEGF表达与MDV和PCNA指数(PCNALI)正相关(r分别为0.69和0.37,P<0.01).MDV与肿瘤转移强度正相关(r=0.73,P<0.01).结论VEGF可通过促进瘤内新生血管形成和细胞增殖在NB浸润转移中具有重要的作用.     

Prenatally detected cystic neuroblastoma

A case of adrenal cystic neuroblastoma (NB) detected by prenatal ultrasonography (US) is presented. The suprarenal mass initially showed pure cystic features on a variety of imaging studies such as US, computed tomography, and magnetic resonance imaging. Tumor markers were negative. The mass was suspected to be an adrenal hemorrhage rather than a NB. Three months later, although the diameter was unchanged, the thickness of the cyst wall seemed to have slightly increased. Surgical exploration revealed an adrenal cystic tumor and histology showed a NB in situ. Forty-five infants with prenatally detected NB were found in the English literature; about one-half of them were cystic NBs, and most had a favorable outcome. Accepted: 29 July 1996     

Natural history and biology of stage A neuroblastoma: a Pediatric Oncology Group Study

PURPOSE: To prospectively analyze the outcome of patients with Stage A neuroblastoma (NB) treated with surgery alone, especially with regard to the prognostic significance of age, tumor site, MYCN copy number, tumor cell ploidy, and histology. PATIENTS AND METHODS: The clinical course of 329 patients with Stage A disease registered on the POG NB Biology Study #9047 between February, 1990 and October, 1997 were evaluated. Age, tumor site, MYCN copy number, tumor cell ploidy, and histology were analyzed for their impact on event-free survival (EFS) and survival (S). RESULTS: The 5-year estimated EFS and S rates for the 329 patients were 91% (+/-3%) and 96% (+/-2%), respectively. The EFS rate was similar for infants younger than 12 months and children age 12 months or older, but age older than 12 months was predictive of lower S rates (P = 0.044). Patients with adrenal, abdominal non-adrenal, thoracic, and cervical tumors had similar S rates. The majority of patients had tumors with favorable biologic features, and only 3% had MYCN amplification. For infants with diploid tumors, the EFS rate was 82% (+/-16%), but effective therapy yielded an S rate of 100%. Rate of S was 80% (+/-26%) and 64% (+/-27%) for patients with unfavorable tumor histology and MYCN-amplified tumors, respectively. CONCLUSION: The outcome for patients with Stage A NB treated with surgery alone is excellent. Although EFS and S rates were significantly worse for patients with MYCN-amplified tumors, a subset achieved long-term remission after surgery alone. For patients with Stage A and MYCN amplification, additional factors are needed to distinguish the patients who will achieve long-term remission with surgery alone from those who will develop recurrent disease.     

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.     

信号转导子与转录激活子3和缺氧诱导因子-1α在神经母细胞瘤中的表达及其意义

目的检测信号转导子与转录激活子3(STAT3)与缺氧诱导因子-1α(HIF-1α)基因在神经母细胞瘤(NB)中的表达,探讨STAT3 mRNA及HIF-1αmRNA的表达与NB的发生、发展及预后的关系。方法收集30例行手术切除的NB患儿的瘤体组织(研究组)及瘤旁组织(对照组)。其中预后不良型(UFH)20例,预后良好型(FH)10例。应用反转录PCR法,检测STAT3 mRNA、HIF-1αmRNA在瘤体组织及相应的瘤旁组织中的表达情况。结果 STAT3 mRNA水平在对照组、FH组和UFH组的表达分别为0.114±0.002、0.267±0.073、0.529±0.325,3组间比较差异有统计学意义(F=22.00,P=0.001);HIF-1αmRNA水平在对照组、FH组和UFH组的表达分别为0.519±0.251、0.673±0.124、0.779±0.145,3组间比较差异有统计学意义(F=111.82,P=0.001)。且FH组和UFH组STAT3 mRNA和HIF-1αmRNA表达水平分别与对照组比较差异均有统计学意义(Pa<0.05),FH组与UFH组比较差异亦有统计学意义(Pa<0.05)。研究组STAT3 mRNA、HIF-1αmRNA相对表达水平分别为0.530±0.051、0.489±0.032,二者表达呈正相关(r=0.497,P<0.05)。结论 STAT3与HIF-1α在NB中过量表达,NB患者预后较差,STAT3与HIF-1α可相互作用促进肿瘤的发生发展。PCR技术可检测NB中STAT3 mRNA及HIF-1αmRNA表达水平,判断其生物学行为,用于指导临床。     

Recent clinical research on the application of liquid biopsy in neuroblastoma北大核心CSCD

神经母细胞瘤（neuroblastoma,NB）是儿童最常见的颅外实体瘤,具有复发率高和生存率低的特点,因此,早期诊断、疗效评估和复发监测对NB患者至关重要。液体活检是指以血液为主的体液标本中细胞及核酸的检测,具有非侵入性并且可以克服肿瘤异质性,为实现NB的早期诊断及动态监测提供可能。该综述介绍了液体活检在NB中应用的最新临床研究进展。     

Telomerase is a strong indicator for assessing the proneness to progression in neuroblastomas

BACKGROUND: As traditional parameters do not ensure completely accurate prognostic grouping in neuroblastoma (NB), new molecular markers are needed for assessing the individual patient's prognosis more precisely. PROCEDURE, RESULTS, AND CONCLUSIONS: Based on 133 NB, we show that telomerase activity (TA) is a powerful, independent prognostic marker for all stages and is capable of differentiating between good and poor outcome in putative 'favorable' clinical or biological subgroups of NB patients. Analysis of gene and protein expression of telomerase subunits suggests that the presence or absence of TA in NB is strongly correlated with expression levels of both the catalytic subunit hTERT and the internal RNA component (hTR).     

Expression and clinical significance of heparanase in neuroblastoma

Background  Previous studies indicate that heparanase (HPA), an endoglycosidase involved in tumor angiogenesis and metastasis, is up-regulated in a variety of malignancies. However, the expression of HPA in neuroblastoma (NB), one of the most common extra cranial solid tumors in children, remains unknown. This study was undertaken to explore the expression and clinical significance of HPA in NB. Methods  Immunohistochemical staining was applied to detect the expression of HPA in 42 cases of NB. The relationships among HPA expression, international neuroblastoma staging system (INSS) stages, histopathological classification, and postoperative survival of the NB patients were analyzed. Results  The expression rate of HPA in NB was 61.9% (26/42), mainly in the cytoplasm of neuroblastoma cells. The expression rates of stage 1–2, stage 3—4 and stage 4S were 35.7%, 80.0% and 62.5%, respectively. The differences between stage 1–2 and stage 3–4 were significant (P<0.01). The expression of HPA was significantly higher in the NB cases that had one of the histopathological factors: age more than 1 year (P<0.01), poorer differentiation (P<0.01), and higher mitosis karyorrhexis index (P<0.01). The survival time of HPAnegative patients was significantly longer than that of HPA-positive patients (P<0.05). Conclusion  Although these results indicate that heparanase might be correlated with development and progression of NB, a larger series of patients with a longer follow-up are probably needed to strengthen its role in assessment of NB prognosis.     

神经母细胞瘤及胚胎肾上腺组织端粒酶基因表达的研究

目的 观察神经母细胞瘤和胚胎肾上腺组织中端粒酶基因（hTR/hTRT)的表达,以及神经母细胞瘤细胞在药物诱导分化时端粒酶基因表达的变化,探讨端粒酶在神经母细胞瘤形成和发展中的作用。方法 体外转录法制备生物素标记的hTR、hTRTcRNA探针。神经母细胞瘤存档石蜡标本20例,2-5个月胎儿肾上腺组织石蜡标本12例,应用原位杂交组织化学（ISHH）方法检测hTR及hTRT表达,用维甲酸类似物R9158诱导人神经母细胞瘤细胞系SH-SY-5Y及SK-N-SH,细胞（90.0%)阳性。各月龄胎儿肾上腺组织均有hTR及hTRT表达,神经嵴细胞和继发皮质细胞内信号强于原发皮质细胞,神经嵴细胞形态极似神经母细胞瘤细胞,维甲酸敏感细胞SH-SY-5Y在R9158作用5d后出现明显形态学变化,表达hTR的细胞明显减少,维甲酸耐受细胞SK-N-SH玩明显变化。结论 端粒酶基因在神经母细胞瘤和胚胎肾上腺组织中高表达,神经母细胞瘤细胞在药物诱导下分化时端粒酶基因hTR表达明显降低。端粒酶在神经母细胞瘤形成和发展中有重要作用。     

Diagnosis of neuroblastoma metastasis in bone marrow with a panel of monoclonal antibodies

Neuroblastoma, along with rhabdomyosarcoma, Ewing's sarcoma, and acute lymphoblastic leukemia/lymphoma, is one of the small, round-cell tumors of childhood. All of these malignancies show a propensity to metastasize to bone marrow. Occasionally when the clinical picture is unclear and the tumor is particularly anaplastic, it can be difficult to arrive at a diagnosis by conventional histological and biochemical procedures. In the present study, a panel of nine monoclonal antibodies was used to undertake a detailed analysis of seven bone marrows contaminated with tumor cells: six cases of stage IV neuroblastoma, and one case of stage IV-S neuroblastoma. The antibody profiles obtained were compared with those deduced from the studies of over 20 marrows from patients with acute lymphoblastic leukemia. A comparison of these data with those obtained from the studies of rhabdomyosarcoma and Ewing's sarcoma cell lines and tissues suggests that when high levels of tumor cells are present in the marrow, it is possible to obtain a confident diagnosis of either neuroblastoma or acute lymphoblastic leukemia. In addition, the immunocytological identification of neuroblasts in bone marrow enables accurate staging without histological examination.     

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.