Analysis of DNA Ploidy, Cell Cycle and Ki67 Antigen in Nasopharyngeal Carcinoma by Flow Cytometry

Summary: The expression of DNA ploidy, the cell cycle and Ki67 antigen in nasopharyngeal carcinoma (NPC) were studied and their relationship with the clinical biological behaviors and prognosis of NPC was evaluated. Biopsied specimens of NPC were made into cell suspension. By using cytometric double labeling Ki67 and DNA method, the expression of DNA ploidy, the cell cycle and Ki67 antigen were analyzed. The patients were followed-up for about 3 years and the relationship between the above-mentioned parameters and the clinical biological behavior and prognosis of NPC were evaluated. Of the 62 cases of NPC, the DNA aneuploid accounted for 29.03 %. The S phase cells accounted for 0 to 54 % in the cell cycle and the positive expression of Ki67 ranged from 0 to 52 %. There were 40 cases of LPI (64.5 %) including 15 negative cases and 22 cases of HPI (35 5 %) respectively. The DNA anueploid content was positively related to the S phase cells. The patients having a low expression of Ki67 or DNA aneuploid in tumor cells were not sensitive to chemotherapy, liable to metastasis to distant organs and had a poor prognosis, while Ki67 showed no correlation with DNA ploidy and the cell cycle. It was suggested that DNA ploidy and Ki67 could be used as an independent and objective marker to evaluate the radiosensitivity and prognosis of NPC.

Clinical application of DNA ploidy to cervical cancer screening: A review

Screening for cervical cancer with DNA ploidy assessment by automated quantitative image cytometry has spread throughout China over the past decade and now an estimated 1 million tests per year are done there. Compared to conventional liquid based cytology, DNA ploidy has competitive accuracy with much higher throughput per technician. DNA ploidy has the enormous advantage that it is an objective technology that can be taught in typically 2 or 3 wk, unlike qualitative cytology, and so it can enable screening in places that lack sufficient qualified cytotechnologists and cytopathologists for conventional cytology. Most papers on experience with application of the technology to cervical cancer screening over the past decade were published in the Chinese language. This review aims to provide a consistent framework for analysis of screening data and to summarize some of the work published from 2005 to the end of 2013. Of particular interest are a few studies comparing DNA ploidy with testing for high risk human papilloma virus(hrH PV) which suggest that DNA ploidy is at least equivalent, easier and less expensive than hrH PV testing. There may also be patient management benefits to combining hr HPV testing with DNA ploidy. Some knowledge gaps are identified and some suggestions are made for future research directions.

PROGNOSTIC SIGNIFICANCE OF DNA PLOIDY IN PATIENTS WITH STAGE II COLORECTAL CANCER

Objective: To evaluate the relationship between flow cytometric DNA ploidy, biological features and prognosis in patients with Stage II colorectal cancer. Methods: Nuclear DNA content, proliferation index and S-phase fraction were measured in a prospective series of 45 patients with curatively resected Stage II colorectal adenocarcinomas by means of flow cytometry using frozen tumor samples. Results: Of the 45 samples examined, 17 tumors (38%) were diploid and 28 (62%) aneuploid. The diploid tumors were significantly more common in the proximal colon than in the distal colon (67% vs. 23%; P<0.01). There was no correlation between DNA ploidy and the other clinicopathological variables (P>0.05). The proliferation index and S-phase fraction in the distal tumors were higher than those in the proximal tumors, but the difference was not significant (P>0.05). When the 5-year survival rate of patients with Stage II colorectal cancer was compared by the log rank test, a significant relationship between DNA ploidy status and disease free survival was observed in the group of all patients. Patients with DNA diploid tumors had a better disease free survival than those with DNA aneuploid tumors (P<0.05). Conclusion: These findings support that DNA ploidy status, proliferation index and S-phase fraction may differ in the proximal and distal colorectal cancer. Flow cytometric DNA ploidy status might be a useful prognostic factor in patients with Stage II colorectal cancer.

DNA PLOIDY AND p53 EXPRESSION ASSOCIATED WITH TUMOR SITE AND LYMPH NODE METASTASIS IN COLORECTAL CANCER

Objective: To study the association of DNA ploidy abnormality and p53 overexpression with the carcinogenesis of colorectal cancer. Methods: DNA ploidy and p53 expression were measured in a series of 42 colorectal adenocarcinomas by means of flow cytometry and immunohistochemical test. Results: 17 tumors (40%) were diploid and 25 (60%) aneuploid. The aneuploid tumors were significantly more common in the distal colon than in the proximal colon (P<0.01). Aneuploidy was significantly associated with lymph node metastasis (P<0.05). There was no correlation between DNA ploidy and the other clinicopathological variables. Of the 22 samples examined, the positive rate of p53 expression was 59% (13/22). p53 expression was more frequently observed in the distal tumors (11/13) than in the proximal tumors (2/9) (P<0.05). Conclusion: Our data support the hypothesis that the carcinogenesis of colorectal cancer might differ in proximal and distal tumors. DNA ploidy abnormality and p53 overexpression may play an important role in the development of distal colorectal cancer.

A COMPARATIVE STUDY OF DNA FLOW CYTOMETRY AND MICROSPECTROPHOTOMETRY IN GASTRIC CARCINOMAS

Flow cytometric (FCM) and microspectrophotometric (MSP) measurements of cell nuclear DNA content were made in 53 fresh gastric carcinoma specimens and in 30 gastric mucosal specimens with chronic gastritis. DNA aneuploidy was found in 32/53 (60%) of gastric carcinomas, and appeared more frequently in wellor moderately differentiated tubular adenocarcinomas (90%) than in undifferentiated and mucousa cell carcinomas (23.6%) (P【0.001). No aneuploidy was found in chronic gastritis samples, but their proliferative cell fractions were higher than in normal control gastric mucosa samples (P【0.01). A comparison was made between FCM and MSP analyses of DNA content in 29 cases of gastric carcinoma, a high correlation rate (r=0.90) was found. The advantages and limitations of both methods are discussed, they may be used in combination for more precise cytochemical analysis.

Correlated Flow Cytometric Analysis of H－ras p21 and DNA Ploidy in Acute Myelogenous Leukemia

The flow cytometric immunoassay was used to study the correlation between the H-ras oncogene product p21 and the DNA ploidy in 30 de novo cases of acute myelogenous leukemia (AML). The results showed that 17 cases were negative for p21 expression and 13 positive for p21. The patients with positive p21 had higher percentage of bone marrow and peripheral blasts and lower peripheral leukocyte count. The expression of p21 had no influence on the therapeutic effect. Before treatment,DNA diploidy occurred in 18 cases including 13 p21 negative ones,and DNA aneuploidy was revealed in 12 cases including 8 p21 positive ones. Patients with positive p21 or having aneuploidy in complete remission were at risk for early relapse. Our results suggest that p21 may be involved in the process of leukemogenesis and progression in AML.

FLOW CYTOMETRIC ANALYSIS OF DNA CONTENT FOR EARLY DETECTION, ESTIMATING PROGNOSIS IN BLADDER CANCER

Flow cytometry (FCM) was used to analyze the DNA content of epithelial cells in 26 cases of bladder cancer and 10 cases of nonneoplastic disease. Bladder tumor cells can be identified by aneuploidy or hyperdiploidy in histogram and/or a heterogeneity index (HI) greater than 2.30. The percentage of positive FCM in patients with bladder cancer was 84.62%. With respect to histological grading, it was 77.78% in grade 1, 90.91% in grade 2 and 100% in grade 3. While in nonneoplastic cases it was 10% Urinary exfoliative cytology was examined in all 26 patients with bladder cancer, with an accuracy of 61.54%. The DNA content of tumor cells increased with the increase of tumor grade and stage. Follow up showed that of 22 bladder cancer patients treated conservatively by transurethral resection or partial cystectomy, 8 recurred. The HI value in recurrent cases was higher than that in nonrecurrent cases. Among these 8 patients, 6 were correctly predicted by FCM. FCM appears to be an objective, sensitive and quantitative method for the diagnosis and monitoring of bladder cancer, and may be useful in estimating the prognosis of superficial bladder cancer.

Correlative Study on the Expression of p53 and DNA Ploidy in Acute Nonlymphocytic Leukemia

We used the flow cytometric immunoassay to study the correlation between the tumor-suppressor gene product p53- and the DNA ploidy in 30 de novo cases of acute nonlymphocytic leukemia (ANLL).The results showed that 15 cases were negative and the other 15 cases were positive expression for p53. As compared with p53 negative (p53) cases, the patients with positive p53 (p53+) had higher percentage of bone marrow blasts and lower peripheral leukocyte and platelet counts,which had no influence on the complete remission rate. Before treatment, DNA diploidy was seen in 18 cases including 12 p53- cases, and DNA aneuploidy in 12 cases including 9 p53+. After therapy, aneuploidy could be transformed into diploidy.Patients with P53+ or having aneuploidy in complete remission were at risk for early relapse. We believe that p53 may be involved in the process of leukemogenesis and progression of ANLL.

基于流式细胞术的蜘蛛抱蛋属植物倍性检测和核DNA含量测定体系的建立

为建立适用于蜘蛛抱蛋属植物的倍性检测及其核DNA含量的流式细胞术方法,以蜘蛛抱蛋属植物成熟叶片为材料,比较了WPB解离液、LB01解离液、Galbraith’s解离液以及改良Galbraith’s解离液制备的细胞核悬液的效果。结果表明:改良Galbraith’s解离液能够更广泛地适用于蜘蛛抱蛋属植物成熟叶片的细胞核悬液的制备且效果较好。利用改良Galbraith’s解离液对12种13个居群的蜘蛛抱蛋属植物进行倍性检测,并通过染色体制片计数法进行验证。结果表明:12种蜘蛛抱蛋属植物的DNA含量的荧光强度峰值范围为1753078.81~5817826.99。其中,11种二倍体植物的峰值范围为1753078.81~2937690.80,四倍体辐花蜘蛛抱蛋的峰值为3892503.69,六倍体泰国蜘蛛抱蛋的峰值为5817826.99。此外,以葱为内标,对以上蜘蛛抱蛋属植物的核DNA含量进行测定和估算,首次估测了11种蜘蛛抱蛋植物的核DNA含量,蜘蛛抱蛋的DNA含量与已有报道的结果相近。其中,二倍体植物的核DNA含量为14.16~18.73 Gb;四倍体辐花蜘蛛抱蛋的核DNA含量为28.33 Gb;六倍体泰国蜘蛛抱蛋的核DNA含量为43.03 Gb。结果可为蜘蛛抱蛋属植物的遗传育种研究、全基因组研究以及喀斯特植物的起源与演化研究提供重要科学依据。

胸水DNA倍体联合细胞角蛋白-19片段检测对良恶性肺部疾病的鉴别诊断意义

目的探讨胸水DNA倍体联合细胞角蛋白-19片段(CYFRA-21-1)检测对良恶性肺部疾病的鉴别诊断意义。方法回顾性分析2021年5月1日至2023年12月1日浙江省荣军医院收治确诊为非小细胞肺癌伴胸水的患者78例作为病例组,同时纳入同期入院存在胸水的肺部良性疾病患者50例作为对照组。对所有患者的胸水及外周血标本进行DNA倍体及CYFRA-21-1阳性率的检测,并统计比较外周血及胸水标本中DNA倍体、CYFRA-21-1单一检测与联合检测的效能。结果病例组胸水、外周血的DNA倍体和CYFRA-21-1阳性率均明显高于对照组,差异均有统计学意义(均P<0.05);病例组胸水DNA倍体和CYFRA-21-1的阳性率均明显高于外周血,差异均有统计学意义(均P<0.05);胸水DNA倍体联合CYFRA-21-1鉴别良恶性肺部疾病的AUC为0.980,大于单一指标胸水DNA倍体的AUC 0.964和胸水CYFRA-21-1的AUC 0.900,差异均有统计学意义(均P<0.05);胸水DNA倍体联合CYFRA-21-1检测鉴别良恶性肺部疾病的灵敏度为1.00,特异度为0.96,准确度为0.98。结论肺癌患者胸水出现DNA异常增殖及CYFRA-21-1阳性率增高且远早于外周血标本,胸水DNA倍体及胸水CYFRA-21-1联合检测有助于提高良恶性肺部疾病的鉴别诊断效能,值得予以重视应用。

三个鲫品系DNA含量的比较研究

采用流式细胞术 (FCM)对红鲫、彭泽鲫、异育银鲫进行红血球DNA含量的检测分析比较 ,以鉴定它们的倍性。结果显示 ,红鲫红血球的DNA含量是 3 0pg ,彭泽鲫是 4 7pg ,异育银鲫是 4 8pg。显而易见 ,彭泽鲫的DNA含量是二倍体红鲫的 1 57倍 ,异育银鲫的DNA含量是红鲫的 1 6倍。采用肾细胞直接制作染色体的方法进行红鲫、彭泽鲫、异育银鲫的染色体倍性鉴定 ,结果红鲫的染色体数目是 10 0 ,为二倍体 (2n =10 0 ) ,彭泽鲫的染色体数目是 162 ,为三倍体 (3n =162 ) ,异育银鲫的染色体数目是 156— 162 ,为三倍体 (3n =156— 162 )。研究证明

鼻咽癌DNA倍体、增殖指数对预后的预测意义(英文)

背景与目的:放射治疗是鼻咽癌的基本治疗方法,改变分割次数放疗及化学治疗已成为晚期鼻咽癌综合治疗方案的一部份,但是,已知的预后因素在预测鼻咽癌治疗结果时有较大的偏差,本研究采用流式细胞仪测定鼻咽癌肿瘤细胞DNA含量及增殖指数,确定其能否成为判断预后的参考指标。方法:对1994年至1995年间205例石腊包埋存档的鼻咽癌活检组织块进行DNA倍体、细胞增殖指标包括S-期细胞分数、G2/M期细胞分数及增殖细胞期分数的测定并与相应病人的临床参数及预后进行相关分析。结果:205例检测的标本中,117例肿瘤活检组织符合DNA流式细胞仪会议指南标准,其中35例(30%)为异倍体肿瘤,82例(70%)为二倍体肿瘤,DNA倍体及各增殖指数与年龄、性别、病理类型、T/N分期及临床分期无显著性相关。异倍体肿瘤的S期细胞分数及增殖期细胞分数显著高于二倍体肿瘤,5年无瘤生存率亦存在显著差异(43%verse62%P=0.035)。低、中、高S期细胞分数组及增殖期细胞分数组,5年无瘤生存率分别存在显著性差异(81%verse61%verse21%;77%verse62%verse33%,P=0.000及P=0.048)。低、中、高G2/M期细胞分数组,5年无瘤生存率无明显差异(35%verse58%verse54%,P=0.8617)。结论:DNA倍体、S期细胞分数及增殖细胞分数为鼻咽癌治疗的预后指标,因此,连同临床。

流式细胞术在植物学研究中的应用——检测植物核DNA含量和倍性水平

流式细胞仪作为高效的检测工具,在植物学研究的多个领域都发挥了重要作用。兰州大学干旱与草地生态教育部重点实验室分子生态所通过大量的植物流式细胞术实验,针对检测植物核DNA含量和倍性水平,总结出一套详细通用的实验方法。同时着重阐述了各个实验环节的关键点,分析因碎片过多而导致实验失败的原因,并提供了切实可行的解决方法。对今后检测各种植物具有重大指导意义,同时也促进了流式细胞术在植物学研究中的应用。

利用流式细胞术鉴定甜樱桃砧木细胞核DNA含量和染色体倍性

【目的】研究建立甜樱桃砧木DNA倍性的快速鉴定方法。【方法】以甜樱桃砧木试管苗本溪山樱、‘Colt’、‘兰丁1号’、‘吉塞拉6号’和‘ZY-1’为试材,利用流式细胞术(Flow Cytometry,FCM)检测甜樱桃砧木细胞染色体的倍性,并通过传统的染色体制片的方法对其结果进行验证。【结果】不同砧木品种间细胞核DNA含量差异显著,且随着倍性水平的增加,细胞核DNA相对含量随之成倍增加。流式细胞术检测结果与染色体制片检测结果一致。【结论】利用流式细胞仪测定甜樱桃砧木的细胞核DNA含量,具有样品制备简单,测量快速,准确度高等优点,是进行倍性鉴定的理想方法。

长三角及邻近地区138种草本植物DNA C-值测定及其生物学意义

为了评估DNA C-值和基因组大小(genome size)在植物入侵性评估中的价值,应用流式细胞仪测定了长三角及邻近地区138种草本植物的核DNA含量,其中111种为首次报道。在此基础上比较了不同植物类群这两个值的差异,特别是入侵性与非入侵性植物这两个值的差异。结果表明:(1)138种草本植物平均DNA C-值为1.55 pg,最大者是最小者的37.17倍。127个类群平均基因组大小为1.08 pg,最大者是最小者的34.11倍;(2)统计了菊科(Asteraceae)、禾本科(Poaceae)、石竹科(Caryophyllaceae)、十字花科(Brassicaceae)、玄参科(Scrophulariaceae)、蓼科(Polygonaceae)、唇形科(Labiatae)和伞形科(Umbelliferae)的DNA C-值和基因组大小,发现禾本科植物的这两个值显著地大于其他7个科(P<0.01)。单子叶的DNA C-值和基因组极显著地大于双子叶植物(P<0.01);(3)杂草比非杂草具有更低的DNA C-值(P<0.01)和基因组大小(P<0.001);与DNA C-值相比,基因组大小在这两个类群之间的差异更为明显(P<0.001),这种现象也体现在菊科植物中。随着基因组(X1)和DNA C-值(X2)由大变小,植物的杂草性(入侵性,Y)由弱变强,两者关系分别符合:Y=2.2334-1.2847 ln(X1)(r=0.4612,P<0.01)和Y=2.4421-0.7234 ln(X2)(r=0.2522,P<0.01),DNA C-值和基因组大小可以作为植物入侵性评估的一个指标;(4)多倍体杂草的基因组极明显地小于二倍体杂草(P<0.01),前者为后者的0.63倍。在非杂草中,多倍体基因组比二倍体的略小,前者仅为后者的0.84倍,差异不显著(P>0.5)。菊科植物中多倍体杂草的基因组也显著地小于二倍体杂草(P<0.1)。基因组变小和多倍体化相结合,进一步增强了植物的入侵性。在多倍体植物入侵性评估中,基因组大小比DNA C-值更有价值。

直肠癌远端切除2cm是安全的吗?(41例直肠癌流式细胞DNA分析结果)

用流式细胞技术（FCM）分别对41例直肠癌新鲜标本的癌灶、癌远端（2cm）移行区及近切缘正常组织进行细胞动力学研究。结果：癌灶SPF和PI极显著高于癌远端2cm移行区及近切端正常组织（P＜0．01）。癌远端2cmSPF显著高于近切端组织（P＜0．05），说明癌旁2cm组织的DNA合成期细胞已呈增殖活跃状态，尽管普通光镜下此区尚无癌细胞。结论：直肠癌远端2cm作为切除的距离并不安全，提示直肠癌远端切除距离不够可能是术后复发的重要因素之一。

流式细胞术检测DNA倍体数对鉴别良恶性肿瘤价值的Meta分析

目的探讨流式细胞术（FCM）检测DNA倍体数在良恶性肿瘤鉴别中的应用价值，为临床恶性肿瘤诊断手段的选择提供理论依据。方法由2位研究者独立完成文献筛选，并依据纳入和排除标准，对检索到的所有文献进行二次筛选。使用Meta DiSc1．4对纳入研究的文献数据进行分析，包括异质性检验、敏感度、特异度、诊断比值比（DOR）及受试者工作特征曲线（SROC）等。结果经初筛、补充及二次筛选，共12篇文献纳入研究，包括1340例研究对象，其中恶性肿瘤组516例，良性对照组824例。异质性检验结果显示：灵敏度对数与（1-特异度）对数的Spearman相关系数为-0．343，P=0．275，不存在阈值效应。DOR曲线提示：Cochran．Q=26．49，P=0．0055，表明存在非阈值效应引起的异质性。采用随机效应模型计算合并统计量：敏感度为0．72（95％CI：0．68-0．76，I^2=50．1％）；特异度为0．84（95％CI：0．81～0．86，I^2=65．5％）。绘制DNA倍体数检测良恶性肿瘤的SROC曲线，结果显示：AUC=0．8453，Q＊=0．7768。结论FCM检测DNA异倍体对诊断恶性肿瘤具有较高的准确性，可作为临床良恶性肿瘤鉴别的一项重要辅助手段。

茶多酚对人鼻咽癌细胞株CNE_2细胞DNA损伤和细胞周期的影响

目的 观察茶多酚（ｔｅａ ｐｏｌｙｐｈｅｎｏｌｓ， ＴＰ） 对人鼻咽癌细胞株ＣＮＥ２ 细胞ＤＮＡ 的损伤作用和细胞周期的影响。方法 用琼脂糖凝胶电泳方法测定ＴＰ对细胞ＤＮＡ的断裂作用；用流式细胞术（ＦＣＭ） 观察ＴＰ对ＣＮＥ２ 细胞周期的变化及其诱导凋亡。结果 ＴＰ可引起ＣＮＥ２ 细胞ＤＮＡ 断裂、其断裂程度随剂量的增加和作用时间的延长而增强。ＴＰ不同浓度和不同作用时间可干扰ＣＮＥ２ 细胞在周期中的进程，使Ｇ１ 期细胞明显增多，Ｓ期细胞减少，细胞分裂增殖指数（ＰＩ）亦随之降低。同时，ＴＰ可诱导ＣＮＥ２ 细胞凋亡，其凋亡率随ＴＰ浓度的增加和作用时间的延长在逐渐增加。结论 ＴＰ１９９８１２０４ 收稿，１９９９０７３１ 修回＊ 国家自然科学基金资助课题，Ｎｏ ３９３７０８００作者简介：谢冰芬，女，副研究员，硕士生导师，研究方向为肿瘤药理可引起人鼻咽癌细胞株ＣＮＥ２ 细胞的ＤＮＡ 断裂，使细胞停滞于Ｇ１ 期，阻止Ｇ１ 期细胞进入Ｓ期；同时亦可诱导细胞凋亡。

草海鲫DNA含量与倍性

为研究草海鲫的倍性及核DNA含量特性,以鸡(Gallus sp.)红细胞DNA含量(2.5 pg/N)为标准、鲫(Car-assius auratus auratus)和普安鲫(C.auratus gibelio(Bloch))子一代为参照,采用流式细胞术测定了25尾草海鲫的红细胞核DNA含量。结果显示:25尾草海鲫的核DNA检测直方图明显分为两种类型,其核DNA绝对含量分为3.92 pg/N和5.44 pg/N两种值,DNA指数(DI)分别为0.99和1.37,其核DNA含量与鲫和普安鲫子一代接近,符合二倍体和三倍体特征。综合分析表明草海鲫是由多种倍体组成、具有核DNA含量特性的混合群体,是贵州省已知除普安鲫和正安鲫外的又一种独特的鲫鱼类型,加强其繁殖方式和资源增殖保护研究具有重要意义。

恶性肿瘤细胞DNA倍体分类方法的改进及其临床应用

采用流式细胞术建立一种改良的细胞DNA倍体分类方法,对100例恶性肿瘤患者瘤组织进行了DNA倍体分类,并对其倍体类型和瘤组织细胞生物学特性、患者临床生物学行为之间的关系进行了分析。结果表明瘤组织细胞的DNA倍体类型分为:二倍体、近二倍体、四倍体、非整倍体和多异倍体细胞,后4种倍体类型统称为DNA异倍体。患者瘤组织细胞DNA倍体分类不同,其细胞生物学特性明显不同,患者临床生物学行为也明显不同。改良后的DNA倍体分类方法,不仅能客观地反映肿瘤细胞的生物学特性,而且还能准确地反映患者肿瘤的恶性程度和不同的临床生物学行为特征,可在临床推广应用。