Comprehensive analysis of tumoral DNA content reveals clonal ploidy heterogeneity as a marker with prognostic significance in locoregional neuroblastoma

The clinical management of locoregional neuroblastoma (LR NB) is controversial. In a previous study we showed that diploidy was a strong prognostic predictor of outcome and detected the existence of clonal ploidy heterogeneity in a select group of cases. The aims of this study were (1) to assess the frequency of ploidy heterogeneity in LR NB, (2) to ascertain the best method to detect heteterogeneity, and (3) to correlate ploidy populations with clinical outcome. We undertook a comprehensive analysis of tumoral DNA content in 38 LR NBs comparing (1) flow cytometry (FCM), (2) karyotyping, (3) interphase fluorescence in situ hybridization, and (4) laser-scanning cytometry (LSC). Tumor ploidy heterogeneity was found by all methodologies. By FCM, all tumors with aneuploid peaks had detectable diploid DNA peaks. By LSC, all tumors with diploid and hyperploid peaks were GD2-positive in both, consistent with their tumoral origin. A predominant near-triploid clonal population (ratio diploid vs. triploid, <2.5) was observed in most nonprogressing LR NB tumors, and a predominant diploid clone (ratio di- vs. triploid, >2.5) in most progressing LR NB cases. Multivariate analysis was performed to evaluate the prognostic value of tumor ploidy assayed by different methods versus age, INSS (International Neuroblastoma Staging System) stage, and MYCN status. FCM was the most powerful prognostic factor related to poor prognosis (overall survival, P = 0.02; progression-free survival, P = 0.01). These results provide strong evidence for clonal ploidy heterogeneity in LR NB and clonal evolution toward diploidy in progressing LR NB.     

Intratumoral DNA content variability. A study of non-small cell lung cancer.

Twenty-eight resected primary non-small cell lung carcinomas were studied for intratumoral DNA variability by flow cytometry (FCM). Three separate tissue specimens from each resected tumor were divided equally for FCM analysis and histologic evaluation. FCM analysis also was performed on fine-needle aspirates (FNAs) from the center of the resected tumor. Histologically, there were 8 squamous carcinomas, 19 adenocarcinomas, and 1 large cell carcinoma. Twenty-three tumors (82%) were aneuploid, and 5 (18%) were diploid. The DNA index in aneuploid neoplasms ranged from 0.91 to 3.30 (mean, 1.64). All 5 diploid and 19 (83%) of the (23) aneuploid neoplasms manifested intratumoral DNA stability. Four (17%) of the aneuploid tumors showed regional DNA heterogeneity expressed as additional stemlines in at least one sample. The FNA yield was sufficient in 21 cases and inadequate for complete analysis in 7 cases. In general, good correlation between FNA and tissue analysis was obtained. However, in three of the aneuploid neoplasms, FNA materials did not reveal an additional nondominant stemline, as noted in the tissue specimens. The authors attribute this finding to a dilutional factor in the aspiration material. The authors conclude that most non-small cell lung carcinomas express DNA stability; FNA provides adequate cellular material for FCM in most cases.     

改进模糊C-均值分割算法在多发性硬化症MR脑部图像中的应用

背景：脑部MR图像是一种无纹理的图像,未被噪声污染的脑部MR图像的灰度值具有分片为常数的特点。因此,在聚类过程中灰度值有趋向于在同一个分割区域中相对接近的性质。 目的：寻找一个能够自动分割多发性硬化症病灶的模糊C-均值改进方法,为临床对于多发性硬化症的判断提供更方便的工具。 方法：考虑到脑部MR图像相邻象素属于同一分类的概率相近的特性,在迭代过程中对8邻域数据集进行滤波以降低噪声对聚类精度的影响,提出了一种改进的模糊C-均值聚类算法。就是将模糊C-均值聚类算法迭代过程中得到的灰度值看作一个数据集,用每个象素邻域象素的灰度值修正该象素的模糊隶属度取值,从而达到利用空间信息抑制噪声的目的。 结果与结论：选取了10个多发性硬化症患者的脑部MRI图像进行试验。通过对多发性硬化症患者MR T1脑部图像和T2液体衰减反转回复脑部图像的分割实验,结果显示该算法能够有效分割多发性硬化症病灶,与其他方法所做的多发性硬化症病灶分割相比,本算法更易于实现,运算时间短,同时结果与临床医生的勾画比较重叠率较高,对其临床辅助诊断具有重要作用。     

Surgical outcome and pathological effects of bromocriptine preoperative treatment in prolactinomas

The case histories of 40 patients with prolactinomas were retrospectively reviewed. Twenty patients treated preoperatively with bromocriptine were compared to 20 untreated patients. The two groups were comparable according to tumor size and sex. All the patients had an immuno-cytochemically confirmed prolactin adenoma. They were operated on by a transsphenoidal approach. Intraoperative findings were studied. The surgical cure rates (serum PRL level less than 30 micrograms/l) of the bromocriptine-treated group were higher than those of the control group, both in microprolactinomas (87.5% vs 50%) and macroprolactinomas (33% vs 17%). This improvement of surgical success rate may not be due solely to bromocriptine. Surgical cure also depends on preoperative serum PRL levels (less than 500 micrograms/l) and tumoral size (less than grade II0). Tumoral tissue remained in all cases. No increase in fibrosis was observed. By inducing tumoral shrinkage, bromocriptine makes surgical removal of macroprolactinomas easier and safer. This tumoral shrinkage could be explained not only by reduction of cell size, but also by decrease in intratumoral hemorrhage. Preoperative bromocriptine treatment does not adversely affect surgical outcome. Moreover, it may be useful in cases of macroprolactinomas with suprasellar extension.     

Aspiration biopsy cytology of extraabdominal desmoid tumor concurrently occurring in a patient with tumoral calcinosis

Extraabdominal fibromatosis or desmoid tumor (DT) is a slow growing locally aggressive soft tissue tumor that can occur anywhere in the body. We report the aspiration biopsy cytology features of a case of DT of the right neck area in a 35-year-old man who had a long standing history of tumoral calcinosis. The aspirate was interpreted as "benign spindle cell lesion" and confirmed as DT on histologic examination of the resected mass. We discuss the possible differential diagnoses of other benign or malignant lesions on fine-needle aspiration (FNA) biopsy and especially discuss the aspiration cytology features of DT compared with those of tumoral calcinosis. We also discuss the value of immunohistochemical markers that help in differentiating DT from other entities.     

DNA aneuploidy in prostatic adenocarcinoma: A frequent event as shown by fluorescence in situ DNA hybridization

Fluorescence in situ hybridization (FISH) using specific DNA probes for chromosomes 1, 7, 10, and Y was performed on 53 prostatic tissue samples obtained from 33 radical prostatectomy specimens and two benign control specimens. The 53 samples from carcinomatous prostates included 33 cancerous and 20 noncancerous samples. Additionally, four metastatic lymph node specimens were examined. Clonal chromosome abnormalities were observed in 78% of the tumors studied. They were detected in a higher proportion in stage pT2 and pT3 tumors (86% and 88%, respectively) compared with stage pT1 tumors (25%). No stage pT4 tumor was analyzed. There was evidence of remarkable focal intratumoral heterogeneity documented by the study of two samples from the same tumor in three of six cases. Comparing FISH determined ploidy patterns with DNA flow cytometry (FCM) in 22 samples, FISH showed aneuploidy whereas FCM showed none.     

Interphasic analysis of aneuploidy in cancer cell lines using primed in situ labeling.

The primed in situ (PRINS) labeling technique has been adapted to chromosomal screening of interphasic tumoral cells. A panel of ten chromosome-specific alpha-satellite DNA primers was used to evaluate numerical chromosome abnormalities in two colon cancer cell lines (Caco-2 and HT-29) and in three of their subpopulations (PF11, TC7, and HT29-MTX). In each cell line, the copy number distribution for different chromosomes showed different patterns. The observation of significant variations in the chromosome constitutions between subpopulations derived from the same original tumor suggests the common occurrence of chromosome copy number heterogeneity in tumoral cell lines. This study demonstrates that the PRINS procedure offers a simple and reliable method for in situ chromosomal screening, which could be efficiently used for karyotypic analysis of tumoral cells.     

Flow cytometry of transitional cell carcinoma of the urinary bladder: influence of prior local therapy

A review of acridine-orange DNA and RNA flow cytometry (FCM) histograms of 249 bladder irrigation specimens from 129 patients with a previous history of transitional cell carcinoma (TCC) reveals that aneuploidy and tetraploidy (greater than 10% of total cell population) are reliable markers to detect the presence of bladder tumor in patients treated by surgical resection of tumor only. Tetraploidy is unreliable when the patient received intravesical chemotherapy or radiation therapy but aneuploidy remains accurate. A comparison of the reliability of FCM compared with cytology indicates an overall lower sensitivity and specificity for FCM (respectively, 52% and 73%) as opposed to cytology (respectively, 62% and 92%). Sensitivity is improved and raised to 77% if FCM and cytology are used in conjunction and reaches 82% in patients treated by surgery only and 88% in those who received radiation therapy. The lowest sensitivity and specificity obtained with FCM are in patients treated by intravesical chemotherapy (respectively, 44% and 58%) and the highest are in those treated by surgery without additional therapy (56% and 83%). This study demonstrates that FCM criteria for diagnosis of TCC of urinary bladder on bladder irrigation specimens depends on patient's treatment history. It also indicates that sensitivity and specificity of cytology to detect bladder tumor are superior to those obtained with FCM but both methods may be considerably improved if they are used in conjunction.     

Volume and Shape in Feature Space on Adaptive FCM in MRI Segmentation

Intensity non-uniformity (bias field) correction, contextual constraints over spatial intensity distribution and non-spherical cluster’s shape in the feature space are incorporated into the fuzzy c-means (FCM) for segmentation of three-dimensional multi-spectral MR images. The bias field is modeled by a linear combination of smooth polynomial basis functions for fast computation in the clustering iterations. Regularization terms for the neighborhood continuity of either intensity or membership are added into the FCM cost functions. Since the feature space is not isotropic, distance measures, other than the Euclidean distance, are used to account for the shape and volumetric effects of clusters in the feature space. The performance of segmentation is improved by combining the adaptive FCM scheme with the criteria used in Gustafson-Kessel (G-K) and Gath-Geva (G-G) algorithms through the inclusion of the cluster scatter measure. The performance of this integrated approach is quantitatively evaluated on normal MR brain images using the similarity measures. The improvement in the quality of segmentation obtained with our method is also demonstrated by comparing our results with those produced by FSL (FMRIB Software Library), a software package that is commonly used for tissue classification.     

食管鳞状细胞癌DNA倍体异质性研究及其临床病理学意义

为研究食管鳞状细胞癌瘤体内DNA倍体异质性及其意义，采用多点取材法，应用流式细胞术对80例食管鳞状细胞癌DNA倍体进行了研究。结果：（1）DNA指数范围为0．77～1．74，DNA异倍体的检出率为88．8％（71／80例）；（2）80例中，（4．5％38例）瘤体内表现为倍体异质现象；（3）DNA倍体异质性与肿瘤的浸润深度、淋巴结转移及患者预后有关，而与组织学分级及瘤体大小无关。结果揭示，用DNA倍体异质性估价肿瘤的生物学特性和预后可能比单点取材异倍体更为准确。     

Flow cytometry: application for the diagnosis and the follow-up of hematological malignancies

Diagnosis of haematological malignancies is based on multiparametric analysis such as morphology, phenotype and genotype studies. Some entities are only defined by one of these approach. Flow-cytometry (FCM) is useful to determined the normal counterpart of the tumoral process and its differentiation status within the involved lineage. Furthermore, FCM is able to detect clonality in B or T proliferations and criteria for malignancies such as abnormal phenotype. Finally it also specifies prognosis criterias. Among the different haematological malignancies, acute lymphoblastic leukaemia (ALL) can be diagnosed using FCM, whereas acute myeloblastic leukaemia diagnosis is only confirmed by this methodology, which could moreover determine prognosis factors. A scoring system (EGIL) determine the normal counterpart of tumoral cells using a panel of different markers. Immunophenotyping is also useful in chronic lymphoproliferative disorders, such as chronic lymphocytic leukaemia (CLL) by using a similar scoring system (so-called Matutes scoring). Since FCM is able to detect simultaneously numerous cell markers it could be more accurate than immunohistochemistry for the diagnosis of follicular lymphoma, mantle cell lymphoma or hairy cell leukaemia. Finally, during treatment follow-up, minimal residual disease characterised by the detection of rare specific events, may be examined using FCM, in some situations.     

Intratumoral heterogeneity of HER2 gene amplification in breast cancer: its clinicopathological significance

Intratumoral heterogeneity of human epidermal growth factor receptor 2 (HER2) gene amplification has been reported to occur with variable frequencies in breast cancers. However, there have been few studies of its clinicopathological significance. We used tissue microarrays to evaluate two aspects of intratumoral heterogeneity of HER2 gene amplification: regional heterogeneity and genetic heterogeneity. We examined 96 invasive breast cancers in which HER2 amplification had been diagnosed in whole sections, and determined the clincopathological characteristics of those tumors. HER2 regional heterogeneity, defined as the existence of amplification/negative or amplification/equivocal patterns in different tissue microarray cores of a tumor, was present in 17 (18%) of the 96 cases. HER2 genetic heterogeneity, defined as the presence of tumor cells with a HER2/chromosome enumeration probe 17 ratio higher than 2.2 in 5-50% of the tumor cells, was found in 11 cases (11%), all of which showed HER2 regional heterogeneity. The cases with intratumoral heterogeneity of HER2 gene amplification were characterized by low grade or equivocal HER2 amplification and equivocal (2+) HER2 expression in whole sections. The patients with intratumoral heterogeneity of HER2 gene amplification had significantly shorter disease-free survival times than those with homogeneous HER2 gene amplification, and this effect was also evident in subgroup analysis by hormone receptor status. In multivariate analysis, intratumoral HER2 heterogeneity retained its status as an independent prognostic factor for disease-free survival. In conclusion, intratumoral heterogeneity of HER2 gene amplification is present in a subset of HER2-amplified breast cancers, especially in cases with low-grade HER2 amplification and equivocal HER2 expression, indicating a need for HER2 testing on more representative, larger tumor samples for accurate assessment of HER2 status in such cases. The patients with this heterogeneity have decreased disease-free survival, suggesting that genetic instability, and hence aberrant HER2 amplification in subclones of such tumors, may be associated with breast cancer progression.     

Vascular and ductal elastotic changes in pancreatic cancer

This study aims to identify and define the type and frequency of elastotic alterations of vessels and ducts in pancreatic ductal carcinoma (PDAC) and evaluate its diagnostic significance. Representative tissue from 36 Whipple specimens, stained with Verhoeff's Van‐Gieson, was studied focusing on the density and distribution of elastic fibers in walls of vessels and ducts, in perivascular and periductal tissue and in tumor stroma. Vessels and ducts within the carcinoma, at tumor periphery and in non‐tumoral pancreas were grouped and examined separately. Vimentin and α‐SMA immunostains were used for the depiction of fibroblasts and myofibroblasts. Histochemistry revealed mild to severe elastotic changes of vessels and ducts in all examined cases. Vascular and ductal elastosis was more prominent within the tumor and diminished at tumor periphery. In tumor stroma and non‐tumoral pancreatic tissue mild or no elastosis was identified. α‐SMA+ cells were observed in large numbers in tumor stroma and as a ring around carcinomatous structures. There were scant α‐SMA+ cells around elastotic and non‐elastotic vessels. Conclusively, vascular and ductal elastosis is a tumor‐associated phenomenon in PDAC. Its presence is indicative of benignity acquiring a possible diagnostic role.     

Image analysis confirmation of DNA aneuploidy in flow cytometric DNA distributions having a wide coefficient of variation of the G0/G1 peak

With nuclei extracted from paraffin-embedded tissues, resolution of normal diploid DNA and abnormal near-diploid/aneuploid populations by flow cytometry (FCM) is especially difficult. These samples, compared with fresh tissue, tend to show a broader DNA distribution, appearing as a wide (high) coefficient of variation (CV) of the G0/G1 peak. To address the question of whether there may be aneuploid populations hidden in wide CV diploid G0/G1 peaks, the authors measured DNA content in nuclei extracted from paraffin-embedded tumor tissue by morphometric image analysis (IA) in addition to FCM. Of 29 samples showing little evidence of DNA aneuploidy by FCM, in 20 of 20 with G0/G1 CVs greater than or equal to 5.50% there was an aneuploid population when analysis was performed by IA. Of the remaining nine samples with CVs less than or equal to 4.41%, all were diploid in the G0/G1 region by both FCM and IA. The presence of aneuploid populations in FCM distributions with wide CV G0/G1 peaks can be confirmed by IA.     

IL-18基因转染对小鼠卵巢癌OVHM体外增殖及体内成瘤的影响

目的:分析转染IL-18基因后,对小鼠卵巢癌OVHM细胞体外增殖、凋亡及体内成瘤的影响,初步探讨IL-18基因转染治疗卵巢癌的应用价值.方法:将逆转录病毒携带的小鼠IL-18基因成功转染至OVHM(OVHM/IL-18),以空载体转染的OVHM(OVHM/LXSN)和野生型(OVHM)作为对照.分别采用MTT、FCM检测体外培养细胞的增殖、凋亡及周期分布.于裸鼠皮下分别接种细胞,观察各组种植瘤生长情况,计算成瘤率;RT-PCR检测瘤组织中IL-18 mRNA表达;FCM和电镜分析肿瘤细胞增殖、凋亡状况.结果:3组细胞的体外增殖未见明显差异,均无明显凋亡现象,增殖指数、细胞周期分布均无明显差异(均P>0.05).接种后,3组裸鼠的成瘤率相同,但OVHM/IL-18组成瘤时间较晚,随瘤龄增长肿瘤生长缓慢,瘤组织中IL-18 mRNA阳性表达.OVHM/IL-18组裸鼠种植瘤细胞可见典型的凋亡现象,G0/G1期细胞明显增多,S期细胞明显减少,增殖指数明显降低,凋亡指数明显增高(P<0.01).结论:IL-18基因成功转染后,虽对体外卵巢癌细胞无直接毒性,但可能在体内借助非直接杀伤的其他途径,通过阻断细胞周期、促进肿瘤细胞凋亡等抑制体内成瘤.     

兔肾缺血再灌注损伤时TNF-α、IL-6和bFGF的变化及当归的影响

目的:探讨当归对兔肾缺血再灌注损伤的防治作用及其机制。方法:健康成年日本大耳白兔25只, 随机均分为假手术对照(control)组、单纯缺血再灌注(IR)组和缺血再灌注+当归(RAS+IR)组。在肾缺血1h再灌注48h后取肾组织作电镜检查, 并测血清肌酐(Cr)、肾组织肿瘤坏死因子(TNF-α)、白细胞介素-6(IL-6)和碱性成纤维细胞生长因子(bFGF)含量。结果:IR组肾组织变性改变显著, RAS+IR组病变轻微;IR组Cr、TNF-α和IL-6含量显著高于control组(P<0.05, P<0.05和P<0.01);RAS+IR组上述指标显著低于IR组。IR组bFGF含量显著低于control组(P<0.01), RAS+IR组bFGF含量显著高于IR组(P<0.01)和control组(P<0.05).结论:当归具有防治肾IR损伤的作用, 其机制可能与其对TNF-α、IL-6和bFGF等细胞因子的调控有关。     

Determination, by in situ hybridization on interphasic nucleus, a cytogenetic DNA index; application to breast cancer; comparison of this DNA index to DNA indexes determined by imaging and flow cytometry

In oncology, flow cytometry (FCM) and image cytometry (ICM) are commonly used to detect DNA aneuploid cell populations in solid tumors. Agreement between these two approaches is good. The use of both techniques in association minimizes the rate of FCM and ICM false negatives and gives better DNA pattern characterization, particularly for detection of any tumoral component in the FCM DNA diploid peak. Nevertheless, discrepancies exist between the FCM and the ICM DNA index values: the ICM DNA index is often greater than the FCM DNA index. The aim of the present study was to establish a cytogenetic DNA index by determining the chromosomal ploidy using a molecular cytogenetic approach and to compare it to the FCM and ICM DNA indexes. We present here the fluorescence in situ hybridization (FISH) technique we have adapted to the study of breast cancer in order to count the number of copies of the 22 + X human chromosomes in interphasic nuclei. This was achieved using a panel of 21 indirect FITC labeled probes which recognize specific chromosomic DNA sequences. Preliminary results obtained from DNA diploid and DNA aneuploid tumors are discussed.     

DNA ploidy abnormalities in the liver of children with hereditary tyrosinemia type I. Correlation with histopathologic features.

Hereditary tyrosinemia (HT) is an autosomal recessive disorder of tyrosine metabolism that results in cirrhosis and hepatocellular carcinoma early in life, and that may be a useful model of early malignant transformation. This is the first report of DNA ploidy in this disease. The authors studied formalin-fixed liver tissue in three cases (two chronic and one acute) of HT for the presence of DNA aneuploidy by flow cytometric (FCM) analysis and image analysis (IA). In the chronic cases, the authors found DNA aneuploidy by FCM in 2/20 cirrhotic nodules in one case and 1/21 tissue sections in the other. Questionable minor aneuploid peaks were detected by FCM in 2/20 and 2/21 sections in these two cases, respectively. Static DNA measurements by IA were performed on those sections having abnormal histograms as well as in some sections having diploid DNA distribution. By this method, the authors confirmed the results of FCM studies and found additional small aneuploid nodules not detected by FCM, frequently associated with various forms of hepatocellular dysplasia as well as steatosis. In one case of acute HT (autopsy), no definite aneuploid peaks were present in five blocks. By immunohistochemical analysis, the authors found frequent positive staining for alpha-fetoprotein (AFP) in the cirrhotic nodules of the two chronic cases as well as in the steatotic regenerative nodules of the acute case. The expression of AFP may represent disturbed regeneration and maturation of liver cells in this disease. This study shows that DNA ploidy may be a useful marker for early malignant transformation in HT and supports the preneoplastic nature of the hepatocellular dysplasia in this disease.     

Two-Dimensional Chemotherapy Simulations Demonstrate Fundamental Transport and Tumor Response Limitations Involving Nanoparticles

Zheng et al. (2004) developed a multiscale, two-dimensional tumor simulator with the capability of showing tumoral lesion progression through the stages of diffusion-limited dormancy, neo-vascularization (angiogenesis) and consequent rapid growth and tissue invasion. In this paper we extend their simulator to describe delivery of chemotherapeutic drugs to a highly perfused tumoral lesion and the tumor cells' response to the therapy. We perform 2-D simulations based on a self-consistent parameter estimation that demonstrate fundamental convective and diffusive transport limitations in delivering anticancer drug into tumors, whether this delivery is via free drug administration (e.g., intravenous drip), or via 100 nm nanoparticles injected into the bloodstream, extravasating and releasing the drug that then diffuses into the tumoral tissue, or via smaller 1-10 nm nanoparticles that are capable of diffusing directly and targeting the individual tumor cell.Even in a best-case scenario involving: constant ("smart") drug release from the nanoparticles; a homogenous tumor of one cell type, which is drug-sensitive and does not develop resistance; targeted nanoparticle delivery, with resulting low host tissue toxicity; and for model parameters calibrated to ensure sufficient drug or nanoparticle blood concentration to rapidly kill all cells in vitro ; our analysis shows that fundamental transport limitations are severe and that drug levels inside the tumor are far less than in vitro , leaving large parts of the tumor with inadequate drug concentration. A comparison of cell death rates predicted by our simulations reveals that the in vivo rate of tumor shrinkage is several orders of magnitude less than in vitro for equal chemotherapeutic carrier concentrations in the blood serum and in vitro, and after some shrinkage the tumor may achieve a new mass equilibrium far above detectable levels. We also demonstrate that adjuvant anti-angiogenic therapy "normalizing" the vasculature may ameliorate transport limitations, although leading to unwanted tumor fragmentation. Finally, our results suggest that small nanoparticles equipped with active transport mechanisms (e.g., chemotaxis) would overcome the predicted limitations and result in improved tumor response.     

基于形变模型的医学图像分割算法研究

结合形变模型和模糊C-均值（FCM）分割技术，提出了一种基于形变模型的医学图像解剖结构轮廓分割方法，在FCM分类的基础上，利用成员隶属函数定义一种模糊约束力并附加于形变模型的外部约束力中．在该种复合外部约束作用下，使形变模型能更好地收缩于解剖结构的轮廓。图像实验结果表明该方法的有效性和可行性。