Retrospective DNA analyses in cervical dysplasia as related to neoplastic progression or regression

Intracellular DNA distribution was measured in cells from two groups of patients with moderate cervical dysplasia. One group consisted of patients who subsequently developed carcinoma in situ; the other consisted of patients whose lesions regressed to normality. Papanicolaou-stained slides were examined cytologically, and dysplastic cells were located. The slides were then destained and restained by means of the Feulgen DNA staining method, after which they were analyzed in a microspectrophotometer. The DNA distribution pattern of both groups was different from that of normal cells and exhibited the same characteristics observed earlier in premalignant cervical cellular atypias. There was no significant difference between the two groups. The results indicate that quantitative DNA determinations in cytomorphologically equivalent dysplastic cervical cells do not offer additional means of predicting the outcome of the lesions.     

Quantitative oral exfoliative cytology. Effect of alcohol on normal buccal mucosa

OBJECTIVE: To assess the effect of chronic alcohol intake on the DNA distribution and cell area of normal oral mucosal cells. STUDY DESIGN: Smears were taken from clinically normal buccal mucosa of 50 patients attending an alcohol-problem service (i.e., chronic alcohol use) and average alcohol units per week recorded. DNA distribution histograms and total cell area values were then compared to those obtained from smears taken from a control group (which included social drinkers) of patients attending for routine dental treatment. Nuclear DNA content was assessed on 100 randomly selected, Feulgen-stained nuclei using a Seescan TV image analysis system, and total cell area was assessed on 50 Papanicolaou-stained cells using the Vids V image analysis system. RESULTS: The DNA distribution histograms were essentially diploid in appearance for the alcohol group, although there was an increase in nuclear DNA content in the occasional nucleus. A highly significant reduction in total cell area was found for the alcohol group when compared to the controls. CONCLUSION: The chronic ingestion of alcohol is associated with a reduction in total cell area but appears to have little effect on nuclear DNA content. Our previous research using the same technique showed that oral cancers are frequently nondiploid. Thus, a nondiploid DNA distribution histogram for smears taken from a clinically suspicious lesion in someone who consumes excessive amounts of alcohol is unlikely to be due to alcohol use alone and should indicate biopsy.     

DNA distribution in biopsy specimens from human cervical carcinoma investigated by flow cytometry.

Flow cytometry was used for the investigation of the DNA distribution in biopsy specimens from 51 patients with cervical carcinoma. Portio biopsy specimens from 9 pregnant women and from 10 patients with cancer of the breast served as controls. The results demonstrate that most specimens from patients suffering from cervical carcinoma contain considerable cell populations with increased DNA as compared with controls. The possible clinical significance of these findings is discussed.     

Cytofluorimetric evaluation of DNA ploidy in lung cancer: a bronchoscopic study

The study of the biological characteristics of lung cancer is gaining more and more interest both because of their potential role as prognostic indicators and for therapeutic reasons. The DNA content estimated by flow cytometry in surgical samples of non-small cell lung cancer (NSCLC) has already been demonstrated to be correlated with survival in these patients. From July 1990 to February 1992 we analyzed the DNA distribution of bronchoscopic biopsies from 88 patients with lung cancer (18 small cell lung cancer, SCLC, and 68 NSCLC, two unspecified histology). Twenty-eight tumors (34.6%) had a diploid DNA distribution, while 53 were aneuploid (65.4%). A correlation was found between DNA ploidy and survival. Evaluation of the DNA content in bronchoscopic samples in a large series of patients could determine the role of this analysis prior to surgery in NSCLC and its value as a marker with respect to prognosis and response to therapy in SCLC.     

Analysis of nuclear chromatin distribution in cervical glandular abnormalities

OBJECTIVE: To measure the intensity of hematoxylin staining for the analysis of chromatin distribution and to define a clear set of standards. STUDY DESIGN: Cervical smears obtained from 12 patients with glandular lesions, 5 with squamous lesions and 3 without cervical lesions were used for NIH image analysis (public domain NIH image program developed at the U.S. National Institute of Health, available through the Internet by anonymous ftp from zippy.nimh.nih.gov or on floppy disk from the National Technical Information Service, Springfield, Virginia). In addition, the same cervical smears were restained with propidium iodide, and the DNA content in the nuclei was compared with that with hematoxylin staining. RESULTS: Chromatin distribution was categorized into 3 patterns. Pattern A was that for which the highest staining density was localized in the periphery of the nucleus, while in pattern C it was localized in the center of the nucleus. Pattern B was the intermediate type between patterns A and C. In patients with adenocarcinoma, pattern B was predominant; pattern C was also relatively frequent in this group. In atypical glandular cells observed in patients with squamous lesions, patterns A and B were predominant and pattern C rarely seen. Analysis of DNA content in the nucleus revealed that nuclei showing pattern B contained significantly higher quantities of DNA than those showing pattern A. CONCLUSION: Nuclear chromatin distribution seems to correlate well with DNA content, and analysis of the chromatin distribution pattern is helpful for the diagnosis of cervical glandular neoplasia.     

‘Conservative’ and ‘variable’ clusters of Alu-family DNA repeats in human chromosomes

The distribution of Alu-family DNA repeats (AFRs) in chromosomes of phytohaemagglutinin-stimulated peripheral blood lymphocytes of four normal donors and non-stimulated bone marrow cells of four patients with acute leukemia (ALL and ANLL) was studied by in situ hybridization using DNA of recombinant phage lambda containing multiple inserts of AFR as a probe. Over some chromosome bands (14cen, 16p13, 16cen) from normal donors and from leukemic patients clusters of silver grains were detected. Over other three bands (3q26, 8p11-p12 and 14q24) the clusters were found only in chromosomes from the four acute leukemia patients, and were absent from chromosomes of healthy donors. The results suggest non-random long-range distribution of AFRs in human chromosomes, and somatic variations in the distribution of the repeats.     

DNA quantification in colorectal carcinoma using flow and image analysis cytometry

Numerous studies using flow cytometry (FCM) have shown that DNA quantification and ploidy classification can provide information of prognostic significance for patients with colorectal carcinoma; recent advances in image analysis cytometry (image cytometry, ICM) provide a new, alternative technique for DNA quantification. This study investigated whether (1) patients with colorectal carcinomas that exhibit a diploid pattern of DNA distribution have improved five-year survival statistics as compared to their non-diploid counterparts and (2) ICM provides quantitative data comparable to that obtained by FCM. DNA quantification and ploidy classification of 27 cases of primary colorectal carcinoma was performed on archival paraffin-embedded tissue by both FCM and ICM; 70% (19) of the tumors were classified as nondiploid by ICM while 56% (15) were similarly classified by FCM. Diploid tumors were associated with Dukes' stage A while nondiploid tumors were associated with Dukes' stage D. The overall five-year survival rate was 75% for patients with ICM diploid tumors and 67% for patients with FCM diploid tumors. The five-year survival was only 53% for patients with nondiploid tumors identified by both techniques. This study confirmed that DNA quantification is an important prognostic indicator for patients with colorectal carcinoma. It also showed that ICM provides data comparable to that of FCM and may be more sensitive.     

Scl 70 autoantibodies from scleroderma patients recognize a 95 kDa protein identified as DNA topoisomerase I

Sera of patients suffering from the autoimmune disease progressive systemic sclerosis (PSS) are known to contain autoantibodies which have been reported to recognize a 70 kDa antigenic protein, designated the Scl 70 antigen. By immunoblotting of nuclear extracts from HeLa cells with sera from scleroderma patients we observed that the size of the antigen present in such cells depends on the conditions of antigen isolation. When protease inhibitors were included in the extraction buffer, a 95 kDa protein was identified instead of a 70 kDa protein. When protease inhibitors were omitted, a number of polypeptides in the size range 66 to 95 kDa was found. Furthermore, antibodies which had been affinity purified on the 95 kDa antigen, crossreacted with the 66 to 95 kDa polypeptides. These results suggest that the smaller proteins were degradation products of the 95 kDa antigen. Immunofluorescence studies on PtK-2 cells with the antibody specific for the 95 kDa protein gave staining of nuclei, nucleoli and of chromosomes and the nucleolar organizer region in mitotic cells. Since this distribution of antigens within the nucleus was reminiscent of the intranuclear distribution of DNA topoisomerase I found by others we probed purified DNA topoisomerase I from calf thymus directly with the autoantibodies from PSS patients, and also the 95 kDa antigens of HeLa cell nuclei with antibodies raised against the bovine DNA topoisomerase I. From the crossreaction pattern observed with the different antigens and antibodies we conclude that DNA topoisomerase I is one of the antigenic components against which autoantibodies are formed in scleroderma patients.     

The prognostic significance of DNA measurements in endometrial carcinoma

DNA analysis was performed in 71 cases of endometrial carcinoma, selected from a retrospective series of 445 cases registered at the Department of Gynecology, Radiumhemmet, Karolinska Hospital, Stockholm, during 1973-1975. The histological material from 37 patients surviving more than eight years was compared with that from those who died from cancer within two years. The prognostic value of the DNA distribution pattern of the tumors in relation to the clinical stage and the histological grade of the tumors was evaluated. Patients with near-diploid or -tetraploid tumors were found to have a significantly lower death rate than those with aneuploid tumors. The DNA distribution pattern was also found to correlate better with the survival rate than the clinical stage or the histological grade of the tumors.     

Quantitative cytology in leukemia research

A study was undertaken to determine the usefulness of flow cytometric analysis of bone marrow cells as an objective means for diagnosis, classification and prognosis in patients with leukemia. Abnormal DNA content as a marker of neoplastic disease was found in only 15% of 264 adult patients with acute leukemia (13% in AML, 26% in ALL/AUL). Alternative means of tumor cell detection in heterogeneous marrow samples include determination of nucleolar antigen density and double-stranded RNA content. Phenotypic characterization of leukemia subtypes can be afforded by RNA content analysis of acridine orange-stained cells, demonstrating significantly higher mean RNA content values in AML, compared to ALL/AUL. Cytokinetic parameters amenable to flow cytometric analysis include measurements of cell cycle compartment distribution by DNA content, of cycle traverse rate by BUdR-induced modification of fluorescence intensity of DNA specific dyes and of growth fraction employing the method of in situ DNA denaturation and subsequent acridine orange staining. Determination of cell cycle distribution and RNA content pretreatment and serially during remission induction in 82 patients demonstrated a significantly lower pretreatment biopsy S phase proportion in responding patients with AML compared to individuals failing treatment whereas an opposite trend was noted in patients with ALL/AUL. While of no prognostic impact pretreatment, serial determinations of the RNA content during the first chemotherapy induction course revealed significant differences between responding and failing patients with AML. Also, patients attaining remission demonstrated a rise in marrow biopsy S phase compartment size by day 10 to 14 of treatment, thus, predicting remission during marrow hypoplasia. We conclude that quantitative cytologic examination of marrow cells from patients with acute leukemia provides useful diagnostic and prognostic information that should aid in the stratification of patients with poor prognosis to receive new agents.     

Cytometry of breast carcinoma: significance of plo?dy balance and proliferation index

Image cytometry of DNA distribution in fine needle biopsies of breast carcinomas at first diagnosis was performed to see if there were significant differences in DNA histograms between patients having very different outcome but same tumor histological typing and similar therapy. Two groups of patients were considered retrospectively: the first (20 patients) with survival time shorter than 5 years and the second (20 patients) with survival time longer than 10 years. Seven benign tumors were used as controls. Ten plo?dy classes were defined. The frequencies of cells in those classes were used as independent features in a supervised multivariate analysis. The advantages of this approach was pointed out with respect to the four-type classification of Auer. The scattering of DNA histograms within the feature space showed that a subgroup of patients with poor prognosis was clearly separated from a subgroup of patients with good prognosis but both long survival patients and short survival patients were scattered in between. In order to replace the multivariate classification of histograms by a simpler approach, two parameters were computed which explained most of the scattering in the feature space: the plo?dy balance (difference between the percentages of euploid and aneuplo?d cells) and the proliferation index (percentage of cells between peaks). The scattergram of patients according to these parameters showed again that some DNA distributions were specific for either good or bad prognosis. But the separation was uncertain for seven short-survival patients and six long-survival patients. For six patients, the DNA distributions were very similar between long and short survival times. Those patients thus could not be separated even by means of discriminant analysis. The main conclusion of this study was that, for a significant number of patients, the objective multivariate classification of tumors DNA profiles is of little assistance to the pathologist who has to give a prognosis for the one patient under consideration.     

新疆维吾尔族与汉族乙型肝炎患者HBVDNA 及肝功能检测结果的比较

目的:探讨新疆乌鲁木齐地区伴有肝功能指标:丙氨酸氨基转移酶(ALT)浓度异常的维吾尔族(维族)及汉族HBeAg阳性乙型肝炎初次就诊患者,乙型肝炎病毒DNA复制载量及ALT浓度是否存在差异及其对患者诊断、预后的意义。方法:回顾性选取门诊伴有ALT浓度异常的汉族、维族初次就诊患者并筛选出HBeAg阳性患者汉族、维族共373例。采用实时荧光定量聚合酶链反应、生化测定及酶联免疫吸附试验法分别测定HBV DNA、ALT浓度及乙肝HBeAg。结果:(1)汉族HBV DNA组秩和8869,维族HBV DNA组秩和10359.36,经Mann-Whitney Test检验两组间尚不能肯定HBVDNA分布有统计学意义,即伴有肝功能损害的汉族、维族初次就诊HBeAg阳性患者HBV DNA复制程度没有差异。(2)汉族ALT组秩和26818.50,维族ALT组秩和22009.50,经Mann-Whitney Test检验两组间ALT分布有统计学意义,即伴有肝功能损害的初次就诊HBeAg阳性患者汉族肝功能损害程度高于维族。(3)HBVDNA低复制组(103-104copy/mL):汉族秩和3771.46,维族秩和4993.2;中复制组(104-106copy/mL):汉族秩和6412.4,维族秩和5088.2;高复制组(>106copy/mL):汉族秩和929.04,维族秩和666.96,经Mann-Whitney Test检验在低复制组两民族间ALT分布无统计学意义,在中、高复制组两民族间ALT具有统计学意义。即:伴有肝功能损害的初次就诊HBeAg阳性患者在HBV DNA低复制组两民族间肝功能损害程度无差异,但在中、高复制组汉族肝功能损害程度高于维族。结论:新疆乌鲁木齐地区伴有肝功能损害的初次就诊的HBeAg阳性的汉族与维族之间HBV DNA的病毒复制无统计学意义(P>0.05),但两民族间的ALT具有统计学意义,可能跟维族的民俗、饮食习惯及生存环境、免疫相关基因HLA基因频率分布差异等因素有关。     

Analysis of DNA distribution in Kaposi's sarcoma in patients with and without the acquired immune deficiency syndrome

While Kaposi's sarcoma in patients with the acquired immune deficiency syndrome (AIDS) may present as a multicentric disease with progressive organ involvement, the classic form of Kaposi's sarcoma is an indolent tumor seldom affecting extracutaneous areas and almost never responsible for the patient's demise. An attempt was made to correlate these clinical differences with the nuclear DNA content of tumor cells in histologic sections from 15 patients (9 with AIDS and 6 without AIDS). All tumors showed a similar DNA distribution pattern, with most cells appearing diploid, indicative of a low malignant potential. These findings indicate that Kaposi's sarcoma of both AIDS and non-AIDS patients is a tumor of intrinsically low malignancy and that lack of immune surveillance is most probably responsible for its aggressive biologic behavior in many AIDS patients.     

碳离子束辐照对质粒DNA的损伤效应

为了证明DNA双链断裂(DSB)片段分布与DNA序列有关的假设,采用32keV／μm的^12C^6 离子和45ke V／μm的^13C^6 离子分别辐照pUCl8质粒,结合限制性内切酶处理,进行琼脂糖凝胶电泳,分析DNA断裂和片段分布。结果表明,除了由一个DSB导致的线性DNA带外,还出现了一条新的、小分子量线性DNA带;限制性内切酶处理后,有另一条线性DNA带产生。证明重离子辐照诱导的DSB是非随机分布的,DNA分子上存在对电离辐射相对敏感的位点。     

Proportion and pattern of dystrophin gene deletions in North Indian Duchenne and Becker muscular dystrophy patients

Population-based variations in frequency and distribution of dystrophin gene deletions have been recognized in Duchenne/Becker (DMD/BMD) muscular dystrophy patients. In the present study, DNA samples from 121 unrelated DMD/BMD patients from North India were analyzed for deletional studies with multiplex PCR and Southern hybridization. A total of 88 (73%) patients showed intragenic deletions in the dystrophin gene. The observed proportion of gene deletions is relatively high, particularly compared with that of Asian counterparts. However, the distribution of breakpoints across the gene does not show significant variations. Received: 5 June 1996 / Revised: 4 September 1996     

Correlation between automated DNA ploidy measurements of Hürthle-cell tumors and their histopathologic and clinical features

The treatment of Hürthle-cell tumors of the thyroid is controversial because of their rarity and the inconsistent histopathologic criteria for their diagnosis. In order to obtain more objective criteria for the management of Hürthle-cell tumors, the nuclear DNA content of cells from 20 cases was measured with the MicroTICAS system and the correlation between the DNA distribution patterns and the clinical and histopathologic findings was evaluated. Three main DNA patterns were found: euploid, polyploid and aneuploid. The euploid or polyploid Hürthle-cell tumors came from patients who did not develop distant metastases or recurrence whereas the aneuploid variants came from patients who died of their disease and/or developed distant metastases and recurrence. Various correlation analyses were performed between DNA ploidy and age, sex, size of tumor, growth pattern, pleomorphism, invasion and metastases. Our data suggests that an aneuploid DNA pattern or one with a large percentage of aneuploid nuclei with DNA content exceeding 5N may predict eventual metastases or recurrence from Hürthle-cell tumor.     

The distribution of interspersed repetitive DNA sequences in the human genome

The distribution of interspersed repetitive DNA sequences in the human genome has been investigated, using a combination of biochemical, cytological, computational, and recombinant DNA approaches. "Low-resolution" biochemical experiments indicate that the general distribution of repetitive sequences in human DNA can be adequately described by models that assume a random spacing, with an average distance of 3 kb. A detailed "high-resolution" map of the repetitive sequence organization along 400 kb of cloned human DNA, including 150 kb of DNA fragments isolated for this study, is consistent with this general distribution pattern. However, a higher frequency of spacing distances greater than 9.5 kb was observed in this genomic DNA sample. While the overall repetitive sequence distribution is best described by models that assume a random distribution, an analysis of the distribution of Alu repetitive sequences appearing in the GenBank sequence database indicates that there are local domains with varying Alu placement densities. In situ hybridization to human metaphase chromosomes indicates that local density domains for Alu placement can be observed cytologically. Centric heterochromatin regions, in particular, are at least 50-fold underrepresented in Alu sequences. The observed distribution for repetitive sequences in human DNA is the expected result for sequences that transpose throughout the genome, with local regions of "preference" or "exclusion" for integration.     

LINE-1 Hypermethylation in Serum Cell-Free DNA of Relapsing Remitting Multiple Sclerosis Patients

Concentrations of cell-free DNA (cfDNA) circulating in blood and its epigenetic variation, such as DNA methylation, may provide useful diagnostic or prognostic information. Long interspersed nuclear element-1 (LINE-1) constitutes approximately 20% of the human genome and its 5’UTR region is CpG rich. Due to its wide distribution, the methylation level of the 5’UTR of LINE-1 can serve as a surrogate marker of global genomic DNA methylation. The aim of the current study was to investigate whether the methylation status of LINE-1 elements in serum cell-free DNA differs between relapsing remitting multiple sclerosis (RRMS) patients and healthy control subjects (CTR). Serum DNA samples of 6 patients and 6 controls were subjected to bisulfite sequencing. The results showed that the methylation level varies among distinct CpG sites in the 5’UTR of LINE-1 repeats and revealed differences in the methylation state of specific sites in this element between patients and controls. The latter differences were largely due to CpG sites in the L1PA2 subfamily, which were more frequently methylated in the RRMS patients than in the CTR group, whereas such differences were not observed in the L1HS subfamily. These data were verified by quantitative PCR using material from 18 patients and 18 control subjects. The results confirmed that the methylation level of a subset of the CpG sites within the LINE-1 promoter is elevated in DNA from RRMS patients in comparison with CTR. The present data suggest that the methylation status of CpG sites of LINE repeats could be a basis for development of diagnostic or prognostic tests.     

Altered nucleic acid contents of mononuclear cells in blood of renal allograft recipients

The DNA and RNA contents of blood mononuclear cell populations of 29 cadaver renal allograft recipients and 49 blood donors (controls) were estimated by acridine orange flow cytometry (AO FCM) to assess their cell cycle status. All patients received azathioprine and prednisone for immunosuppression. The patients represented three clinical categories: clinically stable patients, those with acute rejections (clinically overt or impending), and those with infections. Three cell cycle compartments were analyzed for percentage (%) and RNA content (R) of cells: G0/1, consisting of all cells with diploid DNA content; 2 S.D., consisting of cells with diploid DNA content and RNA content 2 standard deviations above the mean RNA content of controls; and SG2M, consisting of cells with a DNA content higher than that of G0/1 cells. The relative coefficient of variation (rCV) of the DNA distribution of G0/1 cells was also determined. In such cell cycle evaluations, the means of rCV and SG2M% of stable recipients were significantly different from those of blood donors. Multivariate analysis of the variables of the three categories of patients resulted in the tentative formulation of two simple logistic equations: one that differentiates stable patients from those with impending or overt rejections based on 2SD% and another one that distinguishes infected patients from those with impending or overt rejections based on SG2M% and RG0/1.