脱氧核糖核酸水平测定对肾母细胞瘤生物学特性评估的价值

目的 探讨小儿肾母细胞瘤组织DNA水平对肿瘤临床生物学特性评估的价值.方法 收集1993年1月-2007年12月临床病理证实为肾母细胞瘤患儿24例,依据美国国家肾母细胞瘤研究组(NWTSG)标准进行分期,其中Ⅰ期12例,Ⅱ期7例,Ⅲ期5例.并根据肿瘤细胞分化程度及有无淋巴结转移分组,肿瘤细胞高分化者10例,中分化者8例,低分化者6例.淋巴结转移者9例,未转移者15例.采用流式细胞仪测定其肿瘤组织DNA水平,采用鳘2检验分析DNA倍体与肿瘤患者临床分期、细胞分化程度及淋巴结转移的关系.结果 小儿肾母细胞瘤组织DNA异倍体主要存在临床分期Ⅱ、Ⅲ期(57.14%,100.0%),与Ⅰ期(25.0%)比较有显著性差异(P<0.05).高分化组肿瘤细胞DNA异倍体率(20.0%)明显低于中、低分化组(75.0%,83.33%),高分化组与低分化组比较有显著性差异(P<0.05).肿瘤组织DNA异倍体率在淋巴结转移组显著高于无淋巴结转移组(77.78% vs 33.33% P<0.05).结论 小儿肾母细胞瘤组织中DNA水平检测对评价肾母细胞瘤生物学特性有重要意义.     

流式细胞仪测定血液病骨髓细胞DNA的临床意义

目的 探讨小儿常见血液病骨髓细胞的异倍体,细胞周期分布及其临床意义。方法 应用流式细胞仪测定４５例血液病患儿骨髓单个核细胞的ＤＮＡ的含量,计算细胞周期各时相比例。结果 白血病组异倍体检出率明显高于非白血病组的异倍体检出率。１２例非白血病中４例为异倍体,其中２例癌变。白血病组骨髓细胞Ｓ％明显低于对照组。３例接受治疗的急性淋巴细胞性白血病。患儿达到完全缓解后,其Ｓ％明显高于治疗前,非白血病组的骨髓细胞     

小儿急性白血病免疫表型和骨髓细胞DNA含量测定的临床意义

目的　分析小儿急性白血病 (AL)的免疫表型、异倍体、细胞周期分布状况的临床意义。方法　应用流式细胞仪检测了 4 2例初诊为AL患儿的免疫表型及骨髓单个核细胞 (MNC)的DNA含量。结果　B -ALL明显多于T -ALL ,髓系抗原在ANLL组中的表达率依次为CD3 3 >CD13 >CD14>CD15 。 4例AL同时表达了淋系和髓系的抗原 ,预后差。AL患儿骨髓细胞周期的S期细胞比例 (S % )明显低于正常对照 ,说明AL患儿白血病细胞的增殖能力低下。ALL组中 ,亚倍体的S %均值与超二倍体、二倍体比较存在显著差异性 ,ANLL组中 ,CD3 3/CD13<1者的S %均值与CD3 3 /CD13 >1者比较亦存在显著差异。结论　AL患儿的免疫表型、倍体关系、细胞周期及其之间的关系 ,对临床诊断、治疗、预后判断具有重要指导意义。     

小儿恶性肿瘤细胞凋亡的形态和超微结构特征及凋亡指数的临床意义

目的：了解细胞凋亡的光镜和超微结构特征，调查小儿常见恶性肿瘤细胞凋亡的程度。方法：光镜观察４２例小儿恶性肿瘤的ＨＥ染色组织切片和电镜观察１１例新鲜肿瘤标本切片，计算凋亡指数和分裂指数。结果：所有肿瘤均有程度不同的细胞凋亡，其形态特征为细胞皱缩，核密度增高，核断裂成几片，可见凋亡小体，凋亡细胞散在分布，无炎症反应。凋亡指数以神经母细胞瘤最高，为９．１３±４．７２，以下依次为肾母细胞瘤、淋巴瘤和横纹肌肉瘤，而肝母细胞瘤最低，仅０．４７±０．３１。结论：细胞凋亡有独特的形态特征，凋亡程度可能与肿瘤类型有关。     

人肾母细胞瘤裸小鼠肾原位移植模型的建立和生物学特性的研究

目的 建立人肾母细胞瘤的裸小鼠肾原位移植模型。方法 直接将未经处理的患儿肾母细胞瘤组织块接种在裸小鼠肾包膜下作原代以及系列传代移植；原、2、3、4代接种裸小鼠数目分别为2、5、5、18只，每代间隔时间均为8周。原、2、3代裸小鼠分别于接种8周后全部处死，4代裸小鼠从接种后3周至8周，每周处死3只至全部，取出移植瘤进行称重，以反映肿瘤生长动力学。采用流式细胞仪检测原代和系列传代肿瘤样本中细胞周期/凋亡分布以及倍体数。光学显微镜下观察肿瘤标本形态学以及角质蛋白18和结蛋白的免疫组化染色特征，聚合酶链反应扩增基因组微卫星序列D14S68、D18S69and D20S199，以确定其人源性。结果 人肾母细胞瘤在原代裸小鼠中形成肾原位移植瘤，随后系列传四代，共30只裸小鼠，其中27只在肾原位可以检测到移植瘤生长；移植瘤生长曲线符合指数模型，其倍体数、细胞周期分布在各代之间相似。所有移植瘤都保留了最初的组织形态学和免疫组化染色特征。而且，移植瘤基因组微卫量DNA序列与人肾母细胞瘤完全一致。结论 该异种原位移植瘤模型，准确地重现所移植的人肾母细胞瘤的形态和恶性生物学特点，具有重要的基础和临床研究应用价值。     

小骨髓间充质干细胞的培养及多向分化潜能的研究

目的研究小儿骨髓间充质干细胞（MSCs）的生物学特性及多向分化能力,为小儿MSCs的临床应用提供实验依据。方法经Percoll梯度分离接种获得小儿MSCs,观察其形态、排列分布情况,用MTT法检测细胞增殖情况,并绘制生长曲线,用流式细胞仪检测细胞周期,诱导剂干预细胞,观察细胞的成骨、成脂肪、成神经等多向分化能力。结果小儿MSCs贴壁容易,呈细长梭形,增殖能力强,融合后旋涡状分布。MSCs分别经各诱导剂诱导后,细胞形态均发生相应的变化,用化学染色、PCR、免疫细胞染色等方法检测成骨标志物（碱性磷酸酶、骨钙素mRNA、钙结节）、脂肪标志物（脂滴、PPARγ-2mRNA）、以及类神经标志物（尼克氏体、NSE、NF-200）有明显的阳性。结论小儿MSCs能稳定增殖、传代,具有成骨、成脂肪、成神经等多方向的定向分化潜能,可以作为多方面组织工程的种子细胞。     

小儿骨髓间充质干细胞的培养及多向分化潜能的研究

目的研究小儿骨髓间充质干细胞(MSCs)的生物学特性及多向分化能力,为小儿MSCs的临床应用提供实验依据。方法经Percoll梯度分离接种获得小儿MSCs,观察其形态、排列分布情况,用MTT法检测细胞增殖情况,并绘制生长曲线,用流式细胞仪检测细胞周期,诱导剂干预细胞,观察细胞的成骨、成脂肪、成神经等多向分化能力。结果小儿MSCs贴壁容易,呈细长梭形,增殖能力强,融合后旋涡状分布。MSCs分别经各诱导剂诱导后,细胞形态均发生相应的变化,用化学染色、PCR、免疫细胞染色等方法检测成骨标志物(碱性磷酸酶、骨钙素mRNA、钙结节)、脂肪标志物(脂滴、PPARγ-2mRNA)、以及类神经标志物(尼克氏体、NSE、NF-200)有明显的阳性。结论小儿MSCs能稳定增殖、传代,具有成骨、成脂肪、成神经等多方向的定向分化潜能,可以作为多方面组织工程的种子细胞。     

急性白血病细胞凋亡、DNA含量的流式分析

目的分析急性白血病(急淋初治37例、缓解期32例，急非淋9例)细胞凋亡量、DNA含量的变化及临床意义。方法采用流式细胞术(FCM)碘化丙啶插入性DNA定量染色法。结果凋亡百分率在缓解期最高，为19.92±8.05，初治组为8.16±3.12，与正常儿有显著性差异；白血病儿DNA指数(DI值)、异倍体(AN)率、SPF值(S期细胞百分比)均显著性增高，异倍体的发生与肝脾大小、白细胞数有关，与FAB分型无关。结论小儿白血病细胞存在DNA含量的异常，凋亡量、SPF等指标对提示预后有一定的参考价值。     

1312-13急性白血病细胞凋亡、DNA含量的流式分析

目的 分析急性白血病(急淋初治37例、缓解期32例，急非淋 9例）细胞凋亡量、DNA含量的变化及临床意义。方法 采用流式细胞术(FCM)碘化丙啶插入性DNA定量染色法。结果 凋亡百分率在缓解期最高，为19.92±8.05，初治组为8.16±3.12，与正常儿有显著性差异；白血病儿DNA指数（DI值）、异倍体（AN）率、SPF值（S期细胞百分比）均显著性增高，异倍体的发生与肝脾大小、白细胞数有关，与FAB分型无关。结论 小儿白血病细胞存在 DNA含量的异常，凋亡量、SPF等指标对提示预后有一定的参考价值。     

恶性肿瘤患儿自体外周血CD34^＋造血干细胞的动员和检测

目的探讨恶性肿瘤患儿经化疗和细胞因子动员后，外周血CD造血干细胞的含量变化及意义。方法恶性肿瘤患儿6例，经化疗和细胞因子动员后，用CD抗体标记，流式细胞仪检测外周血CD细胞18次，对测定结果进行统计学分析。结果6例患儿的CD细胞的百分率为1．78％～9．72％，较正常儿童外周血的含量增加17～97倍，CFU-GM增加19～74倍。三组不同疾病：NHL、ANLL和ALL之间CD细胞含量有显著性差异，P＜0．01。结论恶性肿瘤患儿经动员后，CD造血干细胞及CFU-GM有明显扩增，CD的检测在自体造血干细胞移植中具有重要的意义。     

Prognostic significance of DNA ploidy in childhood astrocytomas.

The ability to divide subsets of children with astrocytoma into prognostic groups is limited because only a few clinical and pathologic variables are available. This study evaluated DNA ploidy as a potential prognostic factor in 30 children with diagnosed gliomas and examined the correlation of flow cytometric analysis to other parameters such as sex, age at diagnosis, histologic grading, localization of tumor, and completeness of surgical resection. Seventeen children with low-grade glioma and 13 with high-grade glioma were retrospectively reviewed; mean age of the patients was 8.2 years, and mean follow-up of the population was 7.6 years. The tumor was localized to the cerebrum in 19 patients, the cerebellum in 7 patients, the brain stem in 3 patients, and the spine in 1 patient. Fourteen patients underwent complete excision and 16 patients underwent partial excision. DNA diploidy was demonstrated in 21 patients and aneuploidy in 9 patients. Twenty children had no evidence of disease and 10 died of disease. Of the patients with diploid tumors, 81% survived, compared to only 33% survival among patients with aneuploid tumors (p < .011). By Cox regression analysis with age, gender, type of excision, grade, location of tumor, and ploidy as independent variables, ploidy was a statistically significant predictor of survival (p = .043). This investigation provides further evidence that flow cytometry may have prognostic value in children with gliomas. Thus, a larger number of tumors can be studied to extend and validate these observations.     

Evidence for Molecular Heterogeneity in Human Ganglioneuroblastoma

Ganglioneuroblastomas are tumors of sympathetic cell origin that contain both primitive neuroblastomatous and mature ganglioneuromatous elements. It is thought that these tumors arise from a single cellular clone and that the morphologically distinct components of ganglioneuroblastomas represent cells in different stages of differentiation. Two pathologic variants of this tumor, composite and diffuse, have been described; metastasis is more commonly seen with composite ganglioneuroblastomas. We analyzed a composite ganglioneuroblastoma for N-myc copy number at initial resection and 2 years later after progressive disease. In the second sample the more differentiated portion of the tumor was analyzed separately from the neuroblastic foci for N-myc copy number and DNA ploidy. The DNA content and N-myc copy number differed in the two morphologically discrete areas of the tumor, suggesting that at least two clones were present. More composite ganglioneuroblastomas need to be examined to determine whether these tumors are largely composed of tumor cell populations with molecular heterogeneity.     

Flow cytometric analysis of DNA ploidy in 62 patients with Wilm's tumor

Flow cytometric analysis of DNA ploidy was done on nuclei extracted from paraffin-embedded tissue blocks from 62 patients with Wilms' tumor, the largest series reported to date. Thirty-eight tumors were aneuploid and 24 were diploid; there were no tetraploids. Sixty-seven percent of the diploids and 44.7% of the aneuploids survived; the difference was statistically significant (P <0.01). The 2 congenital mesoblastic nephromas were diploid; the only 2 adult patients were aneuploid. Aneuploids comprised 75% of those presenting in stage III/IV and 75% of the anaplastic tumors. Genetic heterogeneity, never before reported in Wilm's tumor, was found in 2 patients. Aneuploidy was an ominous prognostic factor, particularly when combined with tumor stage and histological grade. Offprint requests to: M. Rohatgi     

HEPATIC UNDIFFERENTIATED (EMBRYONAL) SARCOMA: A CASE REPORT WITH FINE NEEDLE ASPIRATION CYTOLOGY AND HISTOLOGY

Hepatic undifferentiated (embryonal) sarcoma is an unusual malignant liver tumor with an overwhelming predilection for children. Fine needle aspiration (FNA) cytology of these tumors rarely has been described in the literature. In this report, we described the clinical, radiologic, cytomorphologic, histopathologic, immunohistochemical, and DNA ploidy features of a similar tumor in an 18 year-old patient. The FNA smears showed clusters and isolated tumor cells with marked variation in morphology including spindle cells, large bizarre anaplastic cells, and giant tumor cells. The histopathologic sections recapitulated the varied morphologic patterns within the tumor cells and also showed characteristic eosinophilic intra- and extracytoplasmic globules. DNA ploidy studies, by image analysis, demonstrated this tumor as an aneuploid tumor, indicating poor prognosis. FNA cytology can be utilized as a sensitive screening tool for these rare tumors.     

Comparison Between Flow Cytometric and Cytogenetic Tumor Cell DNA Content Using a Simple Flow Sample Preparation Method: Ploidy Comparison of 86 Fresh Pediatric Tumors

A comparison of tumor ploidy by flow cytometry (FL) and cytogenetics (CYG) was made in 86 fresh pediatric solid (n = 47) and hematopoietic (n = 39) tumors using simple sampling and semiautomated proprietary FL preparation methods and defined histogram interpretive criteria. Tumor karyotypes with 14-48 chromosomes were regarded as CYG diploid and other chromosomal complements CYG aneuploid for comparison purposes. Five histograms were uninterpretable and nine cases failed to produce 15 or more metaphases for karyotyping. Mean G0/G1 peak coefficients of variation of all 86 cases were 2.7 and 3.0 for the diploid and aneuploid populations, respectively. Of the 72 eligible cases, 41 were concordant diploid and 16 concordant aneuploid with an overall concordance of 79%. The DNA index and karyotypic index correlation coefficient was 0.92 for the 16 concordant aneuploid cases. Analysis of the 15 discordant cases highlights the limitations of both methods and of the histogram interpretive criteria and indicates that FL is probably more sensitive for detection of tumor aneuploidy as defined and detected by these methods.     

Comparison of Chromosome Analysis to DNA Content by Flow Cytometry for Pediatric Tumors

Chromosome analyses in a series of 50 pediatric tumor samples showed abnormal chromosome number, ranging from hypodiploid to tetraploid. Aliquots of the same solid tumor samples were analyzed by flow cytometry (FCM). Material from spontaneous abortions and 12 tumor samples with normal chromosomes were also compared in a control series of 83 samples. Tumors or abortuses with 44-48 chromosomes could not be differentiated from material with normal diploid complement by FCM. However, 3-4 extra chromosomes produced detectable differences in mean DNA index. Triploidy and tetraploidy were readily identified by FCM. It is concluded that FCM could identify an important group of hyperdiploid pediatric tumors, as well as 3N and 4N tumor complements.     

The genetics of retinoblastoma. Relevance to the patient

The understanding of the molecular biology of human cancer has advanced rapidly in the last decade, in part due to discoveries in the rare, pediatric ocular tumor, retinoblastoma. RB studies have led to recognition of a class of human genes, the tumor suppressor genes, that are critical in the initiation and progression of the malignant process. Mutations in the RB1 gene initiate RB and other specific tumors. They may also contribute to progressive stages of many other malignancies. The protein product of RB1 (p110RB1) is a basic regulator of the cell cycle. In the absence of normal protein, the cell proceeds to the next cell division without the potential to become quiescent. Understanding the genetics of RB has benefited the patients, as the precise identification of the RB1 mutations in families has led to accurate prediction of individuals at risk for RB tumors. It seems unlikely, in the foreseeable future, that direct genetic manipulation of mutant RB1 genes will play a role in therapy, but complete understanding of the function of p110RB1 may eventually allow exploitation of its powerful antiproliferative effect. Other molecular genetic events in addition to RB1 mutations are documented in RB tumors, and may play a critical role in the full malignant phenotype. The oncogene, N-myc, is amplified in some RB tumors and is expressed in normal fetal retina. The cytogenetic abnormality, i(6p), is almost unique to RB tumors. The molecular and tissue-specific roles of these abnormalities are not yet known. Many RB tumors also acquire excessive expression of the cell surface membrane glycoprotein, p170, linked to multidrug resistance, whether or not the RB tumor has been exposed to chemotherapy. We anticipate that ways to avoid or counteract the drug resistance of excessive p170 expression will be developed for other pediatric tumors and eventually will be applied to chemotherapy for RB patients.     

Comparison of chromosome analysis to DNA content by flow cytometry for pediatric tumors

Chromosome analyses in a series of 50 pediatric tumor samples showed abnormal chromosome number, ranging from hypodiploid to tetraploid. Aliquots of the same solid tumor samples were analyzed by flow cytometry (FCM). Material from spontaneous abortions and 12 tumor samples with normal chromosomes were also compared in a control series of 83 samples. Tumors or abortuses with 44-48 chromosomes could not be differentiated from material with normal diploid complement by FCM. However, greater than or equal to 3-4 extra chromosomes produced detectable differences in mean DNA index. Triploidy and tetraploidy were readily identified by FCM. It is concluded that FCM could identify an important group of hyperdiploid pediatric tumors, as well as 3N and 4N tumor complements.     

DNA CONTENT AND PROLIFERATIVE ACTIVITY IN CHILDREN WITH NEUROBLASTOMA

Many studies have reported that neuroblastoma patients with aneuploid DNA content and a low cellular proliferative activity have better outcome; other studies have reported contradictory results. Formalin-fixed, paraffin-embedded archival pretreatment specimens of 56 neuroblastomas were studied. Thick sections from paraffin blocks were deparaffinized, and rehydrated. Following enzymatic digestion and filtration, cellular suspensions were analyzed by flow cytometry. Six tumors were aneuploid (13.3%) and 39 samples were diploid (86.7%). S-phase fraction (SPF) ranged from 1 to 78% with a median of 31%. DNA ploidy and proliferative activity results showed no correlation with the prognostic variables. There was no significant difference between the 5-year overall and event-free survival rates of the aneuploid and the diploid neuroblastomas or between the neuroblastomas with a high and low proliferative activity. The results revealed the prognostic significance of neither DNA ploidy nor the cellular proliferative activity in neuroblastoma in contrast to other studies.