差示聚合酶链式反应检测骨髓转移的神经母细胞瘤N—…

测定骨髓转移神经母细胞（ＮＢ）Ｎ－ｍｙｃ扩增情况及评价其与预后的关系，采用差示ＰＣＲ法检测１７例骨髓转移ＮＢ的Ｎ－ｍｙｃ拷贝数，并随访１年，结果，１２例（７０．５９％）证实有Ｎ－ｍｙｃ扩增，Ｎ－ｍｙｃ扩增与ＮＢ分期及患者预后均成显著相关（分别为Ｐ〈０．０５，Ｐ〈０．０１）。有Ｎ－ｍｙｃ扩增者预后差，且扩增倍数越高则预后越差，因此，Ｎ－ｍｙｃ扩增对于ＮＢ分期及预后评估均为有价值的指标，它可能与ｃ－ｍ     

神经母细胞瘤眼部转移与N-myc癌基因扩增的临床意义

目的通过定量研究小儿神经母细胞瘤（ＮＢ）Ｎ－ｍｙｃ基因扩增倍数，观察ＮＢ眼部转移以及与预后的关系。方法选择２５例Ⅳ期ＮＢ患儿（有眼部转移１５例、无眼部转移１０例）做骨穿，有眼部转移者并取转移灶组织，用内参照差示聚合酶链反应（ＰＣＲ）的方法确定ＮＢ细胞的Ｎ－ｍｙｃ拷贝数。结果２５例中有２４例（９６％）出现Ｎ－ｍｙｃ扩增，眼部转移组平均拷贝数（５１．３±３２．４）显著高于无眼部转移组的平均拷贝数（４．３±２．８），Ｐ＜０．０５，且前者两年生存率明显低于后者。结论ＮＢ的疗效及预后与癌基因扩增倍数密切相关，扩增倍数高者预后差。应用差示ＰＣＲ方法可及时判断预后，且操作简便快速，适于临床推广应用。     

差示PCR等方法诊断神经母细胞瘤的N－myc扩增

最近对神经母细胞瘤（ｎｅｕｒｏｂｌａｓｔｏｍａ，ＮＢ）的Ｎ－ｍｙｃ癌基固扩增研究较深入。Ｎ－ｍｙｃ扩增对ＮＢ的发生、预后有着很重要作用。常规使用的Ｓｏｕｔｈｅｒｎ杂交分析法较为复杂，随着分子生物学的发展，差示聚合酶链反应（ｄｉｆｆｅｒｅｎｔｉａｌｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ，Ｄ－ＰＣＲ）成为分析基因扩增的手段之一。它根据内参照原理使常规ＰＣＲ法克服了不能定量的局限性，并且具有简便、快速、灵敏和准确等优点，适于临床应用。     

三、用差示聚合酶链式反应检测骨髓转移的神经母细胞瘤N-myc扩增

三、用差示聚合酶链式反应检测骨髓转移的神经母细胞瘤Ｎ－ｍｙｃ扩增张锦华，杨朝晖遗传教研室张学中国医科大学血液研究室，第二临床学院神经母细胞瘤（ＮＢ）是威胁儿童生命的主要肿瘤之一。该肿瘤恶性度极高，易发生骨髓等多部位转移，素有“小儿癌王”之称。由于肿瘤...     

PCR－LOH检测神经母细胞瘤1号染色体短臂缺失

１号染色体短臂（１ｐ）缺失和Ｎ－ｍｙｃ基因扩增是神经母细胞瘤（ＮＢ）的特征性基因改变，特别是１ｐ缺失，Ⅲ、Ⅳ期的ＮＢ８０％～１００％有１ｐ缺失，是评估预后的重要基因标志物。既往的检测方法复杂，不能将１ｐ缺失作为评估预后的常规指标。我们利用ＰＣＲ－ＬＯＨ（聚合酶链反应－杂合性缺失）成功地检测了８例Ⅲ、Ⅳ期Ｎｂ，６例有１ｐ缺失。ＰＣＲ－ＬＯＨ具有简单、快速、特异等优点，为临床从分子水平评估ＮＢ的预后提供有效途径。     

小儿神经母细胞瘤DNA含量,N—myc基因扩增与临床预后的关系

本文比较分析了１８例神经细胞瘤的ＤＮＡ含量，Ｎ－ｍｙｃ基因扩增、临床分期，原发部位等因素与预后的关系，结果表明，有Ｎ－ｍｙｃ基因扩增的死亡率（７／１０）明显高于无扩增的（１／８）；ＤＮＡ含量增高与肿瘤的恶性度成正比，临床分期与Ｎ－ｍｙｃ基因扩增情况相结合与病死率的关系极为密切。     

小儿神经母细胞瘤DNA含量、N－myc基因扩增与临床预后的关系

本文比较分析了１８例神经母细胞瘤的ＤＮＡ含量、Ｎ－ｍｙｃ基因扩增、临床分期、原发部位等因素与预后的关系。结果表明，有Ｎ－ｍｙｃ基因扩增的死亡率（７／１０）明显高于无扩增的（１／８）；ＤＮＡ、含量增高与肿瘤的恶性度成正比；临床分期与Ｎ－ｍｙｃ基因扩增情况相结合与病死率的关系极为密切。     

差示PCR等方法诊断神经母细胞瘤的N—myc扩增

最近对神经母细胞瘤的Ｎ－ｍｙｃ癌基因扩增研究较深入。Ｎｍｙｃ扩增对ＮＢ的发生、预后有着很重要作用。常规使用的Ｓｏｕｔｈｅｒｎ杂文分析法较为复杂，随着分析生物学的发展，差示聚合酶链反应成为分析基因扩增的手段之一。     

CD44在神经母细胞瘤预后评价中的意义

目的：探讨ＣＤ４４在神经母细胞瘤（ＮＢ）中的预后价值。方法：对３０例ＮＢ进行ＣＤ４４免疫组化研究，同时与其他预后因素，如年龄、分期、病理分型、核分裂核碎裂指数（ＭＫＩ）、ＮＳＥ、Ｓ－１００蛋白等作对比分析。结果：①Ⅰ、Ⅱ、Ⅳ－Ｓ期二年生存率７６．７％，Ⅲ、Ⅳ期二年生存率３３．３％，（Ｐ＜０．０５）。②病理分型，生存率随着级别升高而降低，Ｐ＜０．０５。③ＭＫＩ测定结果与生存率无显著性差异，Ｐ＞０．０５。④２０例Ｓ－１００蛋白染色呈阳性，二年生存率（６０．０％）高于Ｓ－１００蛋白阴性组（２０．０％），Ｐ＜０．０５。⑤１４例ＣＤ４４染色阳性，其中≤１岁，Ⅰ、Ⅱ、Ⅳ－Ｓ期，Ｓ－１００蛋白阳性，高分化型，低ＭＫＩＮＢ中ＣＤ４４阳性率显著增高。ＣＤ４４阳性ＮＢ生存率（６８．８％）高于ＣＤ４４阴性者（２１．４％），统计学处理差异有显著性意义（Ｐ＜０．０５）。结论：ＣＤ４４是ＮＢ的一个新的肿瘤标记物，ＣＤ４４阳性是ＮＢ更为可靠的预后良好的指标。     

271例学龄儿童红细胞内转铁蛋白含量及意义的初步研究

本文用ＥＬＩＳＡ法对２７１例儿童的红细胞内转铁蛋白（ＲＢＣ·Ｔｆ）、红细胞内铁蛋白（ＲＢＣ·Ｆ）和血清转铁蛋白（Ｔｆ）进行了检测，同时检测相应的血红蛋白（Ｈｂ）。根据Ｈｂｔ度将２７１例儿童分为两组，正常组：ＲＢＣ·Ｔｆ范围为２０３～１８２８ａｇ／ｃｅｌｌ，Ｘ±Ｓ为４６７±３５０ａｇ／ｃｅｌｌ（ｎ＝２２３），Ｈｂ偏低组中ＲＢＣ·Ｔｆ范围为７３～４６５ａｇ／ｃｅｌｌ，Ｘ±Ｓ为２０４±９７ａｇ／ｃｅｌｌ（ｎ＝４８），两组间差别有显著性意义（Ｐ＜０．００１）．ＲＢＣ·Ｔｆ与Ｔｆ间几无相关性ｒ＝－０．０４１９，Ｐ＞０．５（ｎ＝２４７）；而ＲＢＣ·Ｔｆ与ＲＢＣ·Ｆ问呈正相关ｒ＝０．１５７，Ｐ＜０．０２（ｎ＝２４７）．     

N-myc oncogene amplification in a patient with IV-S neuroblastoma

Neuroblastoma (NB) is a tumor usually arising in children and young adults showing different degrees of malignancy. Recently, the presence of N-myc amplification in neuroblasts has been associated with a poor outcome in the late stages of disease (Evans's Stage IV). Until now no amplification of N-myc gene has been observed in Stage IV-S, usually considered to have a favorable prognosis. In this paper we report a case of a child affected by NB Stage IV-S showing mild N-myc gene amplification. The finding of N-myc amplification in our patient shows that such alteration of the N-myc oncogene is not necessarily correlated with a poor prognosis; in this light the role of N-myc amplification in the neoplastic process should be reconsidered.     

Association of N-myc amplification with neuroblastoma: The Australian and New Zealand experience

Tumour samples from 38 patients with neuroblastoma were analysed for the presence of N-myc amplification. N-myc gene copy number in tumour DNA was determined by Southern blotting, and by dilution analysis where appropriate. Available clinical data, obtained at tissue collection and by subsequent questionnaire included patient age at diagnosis, catecholamine, ferritin and neuron-specific enolase levels, treatment and disease status. This study was designed to investigate the use of N-myc amplification data as an additional indicator for determination of prognosis. Patients with amplified N-myc had more rapid disease progression than those without amplification (P less than 0.005). Stratification of Stage III and IV patients using N-myc amplification permitted identification of a subgroup with poorer prognosis. The results demonstrate that determination of N-myc amplification is important in assessment of prognosis and subsequent treatment in patients with neuroblastoma.     

Competitive polymerase chain reaction for the determination of N-myc amplification in neuroblastoma: report of clinical cases.

If an unfavorable prognosis is suspected in neuroblastoma, decision on a treatment protocol should be based on the N-myc copy number (12). We already demonstrated that the newly developed competitive polymerase chain reaction (competitive PCR) is a promising method for the determination of the N-myc copy number (6), and have started to use this competitive PCR procedure in neuroblastoma patients, together with fine-needle biopsy in selected cases. Seven children were studied. In one infant of 5 months of age whose tumor was diagnosed before undergoing mass screening for neuroblastoma, the competitive PCR procedure was performed with a fine-needle biopsy, and after obtaining a negative report on N-myc amplification within 48 hours, a regular protocol of treatment could be started without delay. We report that competitive PCR is a rapid and accurate method for the determination of the N-myc copy number, requiring only a small amount of material, and anticipate that competitive PCR will become the procedure of choice for the determination of N-myc copy number in neuroblastoma.     

Expression of N-myc and MRP genes and their relationship to N-myc gene dosage and tumor formation in a murine neuroblastoma model

BACKGROUND: Although the association between N-myc gene amplification and poor clinical outcome in neuroblastoma is well established, the mechanism by which amplification influences prognosis is not well defined. PROCEDURE: We used a human N-myc transgenic mouse model to investigate the role of N-myc in neuroblastoma, including its relationship to the multidrug-resistance-associated protein (MRP) gene. We developed a rapid real-time PCR method to distinguish homozygous and hemizygous N-myc mice that is comparable to Southern analysis. RESULTS: A highly significant correlation (P < 0.0001) between N-myc and MRP expression was demonstrated in murine tumors. Amplification of the transgene was observed in the majority of tumors, highlighting the clinical relevance of this model. However, no correlation between N-myc expression and transgene dosage or tumor latency was observed. CONCLUSIONS: The data suggest that increased N-myc dosage contributes to increased tumor incidence and decreased latency by mechanisms independent of N-myc expression.     

新生儿神经母细胞瘤：发病特点、治疗刍议以及临床转归

目的 总结新生儿神经母细胞瘤的发病特点、随访结果,针对治疗方案提出本中心的观点。方法 对2003～2009年我院收治的14例新生儿神经母细胞瘤患儿进行回顾性分析,14例新生儿神经母细胞瘤中,男8例,女6例。发病年龄最大29d,最小2d,平均(19±3.5)d,中位数16d。Ⅰ期5例,Ⅱ期1例,Ⅲ期1例,Ⅳ期1例,4S期6例。腹膜后占位8例,纵隔4例,盆腔2例。分析每个患儿的发病特点,临床分期、生物学特征、治疗方案、及其与预后的关系。结果 8例患儿接受手术切除,其中6例完整切除,2例部分切除后辅以化疗。4例患儿活检后化疗,2例患儿单纯行经验性化疗,未手术/活检。所有患儿中只有4例患儿出现尿VMA的增高(28.6％),Mycn扩增3例,不扩增9例,UH:FH为4:8。两年的总体生存率(OS)是92.9％,局限性神经母细胞瘤患儿的生存率是100％。单纯手术组的生存率(7/8,87.5％),化疗组生存率(5/6,83.3％) (P=0.065);Mycn扩增组的生存率为50％,不扩增组的生存率为100％ (P=0.021);VMA阳性生存率为50％,阴性者生存率为100％(P=0.021)。死亡2例,其中1例死于化疗后的并发症,另1例死于肿瘤的复发。结论 N-myc不扩增和VMA不增高的新生儿神经母细胞瘤的预后很好,单纯手术组和化疗组生存率没有差别。手术、化疗以及其他的相关的并发症要比疾病本身所致的死亡例数要多,手术、化疗应仔细考量。手术治疗提倡改变既往观念,建议短期内密切观察和随访,尽量避免在新生儿期手术。     

Low NDRG1 mRNA expression predicts a poor prognosis in neuroblastoma patients

Purpose

N-myc downstream regulated gene 1 (NDRG1) markedly reduces metastasis of numerous tumors. However, NDRG1’s function in malignant tumors has not been fully determined. Therefore, we investigated the association of NDRG1 expression with clinical outcomes in neuroblastoma (NB) patients.

Methods

We obtained total RNA from residual cancer cells using microdissection from NB patients. Furthermore, we examined the expression of NDRG1 in NB patients using immunohistochemical staining.

Results

Of the 48 patients observed, low NDRG1 expression was associated with poor prognostic factors such as primary tumor size and MYCN amplification. Low expression of NDRG1 was associated with a poor prognosis (p = 0.001) and multivariate analysis identified low expression of NDRG1 as an independent risk factor for predicting poor prognosis in NB patients. Furthermore, in the MYCN non-amplification group (n = 33), low expression of NDRG1 was associated with a poor prognosis (p = 0.001). Immunohistochemical analysis showed NDRG1 expression at the plasma membranes of NB cells. NDRG1 expression levels were also correlated with expression of NDRG1 mRNA.

Conclusion

We confirmed that low NDRG1 expression is a significant and independent prognostic indicator in NB by multivariate analysis. Furthermore, NDRG1 may be a novel prognostic marker in MYCN non-amplification NB patients.     

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.     

Assessment of fine needle aspiration cytology samples for molecular genetic analysis in neuroblastoma

Purpose

To assess the utility of fine needle aspiration cytology (FNAC) samples for molecular genetic analysis of neuroblastoma.

Methods

The case files from the pediatric solid tumor clinic were reviewed to identify 20 neuroblastoma patients whose pre-treatment FNAC slides were preserved in the cytology laboratory. The FNAC slides were destained, air dried and hybridisation with fluorescence in situ hybridisation (FISH) probes was performed as per protocol. All slides were screened and analyzed carefully under the fluorescent microscope. Over four-fold increase of the N-myc signal numbers was defined as N-myc amplification. Focal occurrence of cells (at least 50) showing N-myc amplification surrounded by non-amplified tumor cells was defined as focal N-myc amplification. Presence of three or more signals for the long arm of chromosome 17 was defined as 17q gain.

Results

FISH analysis gave informative results for all the FNAC smears in our study. FISH analysis of FNAC smears showed N-myc amplification in 5 (25 %) out of 20 patients and 15 (75 %) showed normal N-myc copy number. Three out of these five patients had homogenous amplification and two patients had focal N-myc amplification, indicating tumor heterogeneity. On investigation of chromosome 17q status, 5 (25 %) out of 20 patients demonstrated gain of 17q and 15 (75 %) patients showed normal 17q status. Four out of the five patients with 17q gain also showed N-myc amplification.

Conclusions

The current study indicates that FNAC is a rapid and atraumatic diagnostic method for neuroblastoma which provides sufficient material for molecular genetic analyses by means of FISH.     

N-myc status of neuroblastoma from bone marrow cells

The analysis of the N-myc gene in bone marrow specimens at the time of initial diagnosis and as well at the time of relapse from patients with Neuroblastoma stage IV and bone marrow infiltration could give some informations about the N-myc status of these patients. In stage IV neuroblastoma patients with bone marrow infiltration an estimation of the N-myc gene amplification should be attempted, if otherwise no information about the tumor content of the N-myc gene could be gathered. In our investigation we could demonstrate a Southern-blot-analysis of 27 bone marrow specimens with respect to the N-myc gene status which correlated qualitatively well to the N-myc amplification detected later on in the corresponding tumor tissue. In six cases the tumor infiltrated bone marrow showed a clear amplification of the N-myc gene. Because of the contamination by non malignant cells in bone marrow there was a quantitative difference in the calculated N-myc gene copies between the examined bone marrow specimens and corresponding tumor tissue.