DNA Ploidy Analysis Research Articles

A comparison of conventional cytology, DNA ploidy analysis, and fluorescence in situ hybridization for the detection of dysplasia and adenocarcinoma in patients with Barrett's esophagus

New detection methods with prognostic power are needed for early identification of dysplasia and esophageal adenocarcinoma (EA) in patients with Barrett's esophagus (BE). This study assessed the relative sensitivity and specificity of conventional cytology, DNA ploidy analysis with digital image analysis (DIA), and fluorescence in situ hybridization (FISH) for the detection of dysplasia and adenocarcinoma in endoscopic brushing specimens from 92 patients undergoing endoscopic surveillance for BE. FISH used probes to 8q24 (C-MYC), 9p21 (P16), 17q12 (HER2), and 20q13. Four-quadrant biopsies taken every centimeter throughout visible Barrett's mucosa were used as the gold standard. The sensitivity of cytology, DIA, and FISH for low-grade dysplasia was 5%, 5%, and 50%, respectively; for high-grade dysplasia (HGD), 32%, 45%, and 82%, respectively; and for EA, 45%, 45%, and 100%, respectively. FISH was more sensitive (P < .05) than cytology and DIA for low-grade dysplasia, HGD, and EA. The specificity of cytology, DIA, and FISH among patients (n = 14) with tissue showing only benign squamous mucosa was 93%, 86%, and 100% (P = .22), respectively. All patients with a polysomic FISH result had HGD and/or EA within 6 months (n = 33). There was a significant difference between FISH categories (negative, 9p21 loss, gain of a single locus, and polysomy) for progression to HGD/EA (P < .001). These findings suggest that FISH has high sensitivity for the detection of dysplasia and EA in BE patients, with the power to stratify patients by FISH abnormality for progression to HGD/EA. Additional studies are needed to further evaluate the clinical use of FISH.

Aneuploidy Predicts Outcome in Patients with Endometrial Carcinoma and Is Related to Lack of CDH13 Hypermethylation

Many investigators have reported that aneuploidy detected by flow cytometry is a useful prognostic marker in patients with endometrial cancer. Laser scanning cytometry (LSC) is a technology similar to flow cytometry but is more feasible for clinical laboratory use. We evaluated the usefulness of DNA ploidy detected by LSC as a prognostic marker in patients with endometrial cancer and investigated genetic and epigenetic factors related to aneuploidy. Endometrial cancer specimens from 106 patients were evaluated. The methylation status of CDH13, Rassf1, SFRP1, SFRP2, SFRP4, SFRP5, p16, hMLH1, MGMT, APC, ATM, and WIF1 and mutations in the p53 and CDC4 genes were investigated. LSC was carried out to determine DNA ploidy. Fluorescence in situ hybridization was done with chromosome-specific centromeric probes to assess chromosomal instability. Univariate and multivariate analyses revealed that p53 mutation and lack of CDH13 hypermethylation associated positively with aneuploidy. Univariate analysis showed that aneuploidy, chromosomal instability, and lack of CDH13 hypermethylation as well as surgical stage were significantly predictive of death from endometrial cancer. Furthermore, multivariate analysis revealed that stage in combination with either DNA aneuploidy or lack of CDH13 hypermethylation was an independent prognostic factor. These results suggest that analysis of DNA ploidy and methylation status of CDH13 may help predict clinical outcome in patients with endometrial cancer. Prospective randomized trials are needed to confirm the validity of an individualized approach, including determination of tumor ploidy and methylation status of CDH13, to management of endometrial cancer patients.

Flow Cytometric Analysis of DNA Ploidy and S-Phase Fraction in Primary Localized Myxofibrosarcoma: Correlations with Clinicopathological Factors, Skp2 Expression, and Patient Survival

Histological assessment for prognostication of myxofibrosarcomas remains challenging. We previously reported independent prognostic value of Skp2, an oncoprotein promoting S-phase progression (Clin Cancer Res 2006;12:487-98). We evaluated S-phase fraction (SPF) and ploidy of myxofibrosarcomas and the association between SPF and Skp2. Flow cytometric findings were analyzed for 75 cases and correlated with clinicopathological factors, Skp2 labeling index (LI), metastasis-free survival (MeFS), and overall survival (OS). Forty-seven and 28 cases were classified as diploid and nondiploid, respectively. High SPF (>or=20%) was detected in 32 of 61 interpretable cases. Skp2 overexpression (LI >or= 10%) was seen in 36 of 72 cases with scoring. Nondiploidy correlated with higher French Federation of Cancer Centers (FNCLCC) grades (P = .006), remarkable necrosis (P = .010), and Skp2 overexpression (P = .018). Noticeably, SPF was significantly related to Skp2 LI (P < .001, r = .458), FNCLCC grade, American Joint Committee on Cancer stage, and mitotic rate. Nondiploidy predicted shorter OS (P = .0045) and MeFS (P = .0489), whereas SPF >or= 20% was only associated with inferior MeFS (P = .0252). In multivariate analyses, nondiploidy independently correlated with both OS (P = .020, RR = 3.337) and MeFS (P = .013, RR = 5.780), together with Skp2 overexpression (P = .014 for OS; P = .017 for MeFS) and disease-positive margins (P = .004 for OS; P = .002 for MeFS). Skp2 promotes S-phase progression in myxofibrosarcomas. SPF provides no independent prognostic usefulness; it is probably overshadowed by Skp2. Nondiploidy adds another predictive value to Skp2 overexpression and disease-positive margins in prognostication.

Genotypic Analysis of Hydatidiform Mole: An Accurate and Practical Method of Diagnosis

Molar gestations are defined at the genetic level by their unique parental chromosomal compositions. Their diagnosis, however, currently relies largely on histologic features and on the occasional support of ancillary immunohistochemical and DNA ploidy analyses. We sought to validate DNA genotyping for the routine diagnosis and subtyping of hydatidiform moles (HMs) by analyzing 52 cases of molar pregnancy and their morphologic mimics. DNA was extracted from microdissected chorionic villi and paired maternal endometrial tissue from unstained paraffin sections and analyzed by AmpFlSTR Identifiler PCR Amplification system (Applied Biosystems, Inc). DNA genotyping was informative in all cases with input template DNA amounts ranging from 1.5 to 2.5 ng. Among 38 cases of HMs confirmed by DNA genotyping, there were 26 complete moles with diandric paternal-only genomes (24 homozygous and 2 heterozygous) and 12 partial moles with diandric, monogynic genomes (11 heterozygous and 1 homozygous). All nonmolar cases, including 10 cases of mimics of HM, demonstrated a balanced, biallelic profile of both maternal and paternal origin. Our study demonstrates the applicability of DNA genotyping, the molecular approach for the diagnosis, and subtyping of molar pregnancy, to the daily clinical practice.

Consistent numerical chromosome aberrations in thecofibromas of the ovary

Sex cord-stromal tumors of the ovary comprise 8% of all ovarian neoplasms. Because they consist of cells that resemble embryonic sex cord and/or specialized ovarian stroma cells, their cytologic and histologic features can be viewed as reflecting a continuum from fibromas to thecomas with thecofibromas in between. Existing cytogenetic knowledge about ovarian thecomas-thecofibromas-fibromas is restricted to 44 cases with chromosomal abnormalities. The most common aberration has been trisomy 12, identified either by karyotyping or using fluorescence in situ hybridization (FISH). We wanted to obtain more information about the genomic composition of these tumors, and, therefore, examined 29 new thecoma-thecofibroma-fibroma tumors of the ovary using karyotyping, comparative genomic hybridization, interphase FISH, and DNA ploidy analysis. We detected aneuploidy in 21 tumors. Trisomy and/or tetrasomy 12 was the most common chromosomal aberration, found in 15 tumors (71.5% of the aneuploid tumors or 51.5% of all analyzed tumors), followed by trisomy for chromosomes 10, 18, 4, and 9. Some monosomies (for chromosomes 4, 9, 10, and 18) were also identified, either as the sole change or in combination with trisomies. The nonrandom occurrence of these aneuploidies in these benign tumors strongly indicates that they play a major pathogenetic role, but how trisomies and other aneuploidies contribute to tumorigenesis remains unknown.

Correlation of DNA Ploidy with Progression of Cervical Cancer

The majority of squamous cell carcinomas of cervix are preceded by visible changes in the cervix, most often detected by cervical smear. As cervical cancer is preceded by long precancerous stages, identification of the high-risk population through detection of DNA ploidy may be of importance in effective management of this disease. Here we attempted to correlate aneuploid DNA patterns and their influence on biological behavior of flow-cytometry analysis of DNA ploidy which was carried out in cytologically diagnosed cases of mild (79), moderate (36), and severe (12) dysplasia, as well as “atypical squamous cells of unknown significance (ASCUS)” (57) along with controls (69), in order to understand its importance in malignant progression of disease. Cytologically diagnosed dysplasias, which were employed for DNA ploidy studies, 39 mild, 28 moderate, and 11 severe dysplasia cases were found to be aneuploid. Out of the 69 control subjects, 6 cases showed aneuploidy pattern and the rest 63 subjects were diploid. An aneuploidy pattern was observed in 8 out of 57 cases of cytologically evaluated ASCUS. The results of the followup studies showed that aberrant DNA content reliably predicts the occurrence of squamous cell carcinoma in cervical smear. Flow cytometric analysis of DNA ploidy may provide a strategic diagnostic tool for early detection of carcinoma cervix. Therefore, it is a concept of an HPV screening with reflex cytology in combination with DNA flow cytometry to detect progressive lesions with the greatest possible sensitivity and specificity.

DNA ploidy analysis as an adjunct for the detection of relapse in B-lineage acute lymphoblastic leukemia

Detection of relapse in acute lymphoblastic leukemia (ALL) is essential for proper management. However, immunophenotypic detection of relapse by flow cytometry in B-lineage ALL can be confounded by several factors, including lack of a unique immunophenotype and modulation of aberrant phenotypes after treatment. We hypothesized that flow cytometric DNA ploidy analysis may detect relapse in aneuploid ALL cases that might be missed by flow immunophenotyping. We retrospectively studied ALL cases at our institution between 1991 and 2003 (n = 114). Aneuploid populations were present at diagnosis in 32% of all patients. Sixty-five percent of all patients had “normal” leukemic immunophenotypes, defined as being similar to normal precursor B-cells, while 35% had “aberrant” immunophenotypes with myeloid or T antigen co-expression. In ALL cases that were originally aneuploid, follow-up ploidy-analysis detected relapsed disease in all cases which were also detected by flow immunophenotyping, suggesting that ploidy analysis is highly sensitive for detecting ALL relapse. However, in 5 cases in which the diagnosis of relapse could not be reliably made by flow immunophenotyping, ploidy analysis successfully detected aneuploid cells, i.e., relapse, in all five; these included 3 patients with normal and 2 with aberrant original immunophenotypes. These results suggest that it may be beneficial to perform ploidy analysis as an adjunct to flow immunophenotyping in following patients with B-lineage ALL who demonstrate aneuploidy at diagnosis.

Usefulness of DNA-ploidy analysis by flow cytometry in patients with malignant lymphoma

Usefulness of DNA-ploidy analysis by flow cytometry in patients with malignant lymphoma

DNA ploidy and cell cycle analyses in the bone marrow cells of patients with megaloblastic anemia using laser scanning cytometry

Megaloblastic anemias are characterized by several hematopoietic cells with dysplastic nuclear morphology. The analyses of DNA ploidy and cell cycle of these cells are important to understand the property of such diseases. As laser scanning cytometry (LSC) is a useful tool to evaluate the morphology of the cells fixed on the slide glass together with the quantitative analysis of the fluorescence information of each cell by rapid scanning of the specimens, the authors examined the DNA ploidy and cell cycle of six cases with megaloblastic anemia using LSC. Giant neutrophilic series such as giant metamyelocytes and giant band cells were found to have extraordinarily higher DNA ploidy, while hypersegmented neutrophils represented the normal diploid pattern like normal neutrophils. As to megaloblasts, cell cycle analysis showed that the proportion of the cells in S phase was increased as compared with the case of normal erythroblasts. The present study clearly demonstrates the abnormal aspects of the hematopoietic cells with megaloblastic anemia from the viewpoint of the DNA ploidy and cell cycle analyzed by the use of LSC.

A Distinct Pattern in the DNA Ploidy Histograms of Hydatidiform Moles and Nonmolar Abortuses Is Caused by Accumulation of Trophoblasts in the Late S-Phase

DNA ploidy analysis is a useful tool to distinguish the partial hydatidiform moles (PMs) from complete hydatidiform moles (CMs) and nonmolar abortuses (NAs). DNA ploidy histograms of hydatidiform moles are sometimes difficult to interpret because of the uneven distribution of nuclei in the S-phase, simulating aneuploid peaks. In this study, we analyzed DNA ploidy histograms of 25 CMs, 16 PMs, and 28 NAs, with special reference to the accumulation of cells in the late S-phase using a high-resolution DNA image cytometry. All the gestational products demonstrated the accumulation of cells in the late part of the S-phase fraction. To objectify the observation, we compared the percentage of cells in the second quarter with that of the third quarter of the S-phase fraction. All the gestational products had significantly lower (P < 0.001) percentage of cells in the second compared with that of the third quarter of the S-phase. The mean ratios of the third quarter to the second quarter in CMs, PMs, and NAs were 1.9, 1.7, and 2.5, respectively. This was significantly different from that of highly proliferative endometrial carcinomas. The knowledge of this specific S-phase fraction distribution in molar and nonmolar pregnancy material is important when interpreting the DNA histograms. The possibility of hypoxia being the cause of this phenomenon is also discussed.

Human papillomavirus‐related cellular changes measured by cytometric analysis of DNA ploidy and chromatin texture

Image cytometry has provided two highly sensitive markers for the identification of the malignant potential of squamous lesions. Aneuploidy and chromatin texture have been investigated as quantitative measures of nuclear damage in premalignant lesions and carcinoma. Real-time PCR methods have evolved to yield highly specific measurements of mRNA expression in very sparse cellular samples. Human papillomavirus (HPV) 16 and 18 E7 mRNA expression was measured using quantitative RT-PCR. DNA index and chromatin measures were taken from image cytology samples. The chromatin features, through discriminant analysis, were aggregated into a score, and both measurements were related to mRNA expression. mRNA level and DNA index show an increasing trend over increasing histological grades. However, DNA index and chromatin score were not correlated to mRNA levels in these samples. Chromatin score differed by mRNA type found with HPV 18 infected samples having a higher score than those with HPV 16. Samples infected with HPV 16 and HPV 18 had even higher chromatin scores. DNA index and chromatin score were not directly correlated with mRNA levels. However both mRNA and DNA index were related to histological grade, and chromatin score was associated with HPV type. Therefore, DNA index and mRNA levels could be independent predictors of cervical dysplasia, and chromatin score could be related to the viral integration process in cells infected with HPV 18 or dual infections.

The prognostic significance of determining DNA content in breast cancer by DNA image cytometry: the role of high grade aneuploidy in node negative breast cancer

Aim: To investigate the role of DNA aneuploidy, particularly in patients with node negative breast cancer, in order to identify the different risk profiles within the pool of heterogeneous breast...

DNA ploidy analysis in salivary gland tumours by image cytometry

To determine whether DNA ploidy by image cytometry is a good diagnostic tool to distinguish benign and malignant salivary gland tumours. A total of 62 salivary gland tumours were studied. Cases were histologically diagnosed [haematoxylin and eosin (H&E)]. According to the World Health Organization (WHO) classification, there were 14 mucoepidermoid carcinomas (MEC), 11 adenoid cystic carcinomas (ACC), 10 pleomorphic adenomas (PA), 10 carcinoma ex PA (CEPA), 9 acinic cell carcinomas (ACCa), 3 polymorphous low-grade adenocarcinomas (PLGA), 2 papillary cystadenocarcinomas (PC), 1 myoepithelial carcinoma (MC), 1 undifferentiated carcinoma (UC) and 1 mucinous adenocarcinoma (MA). Paraffin sections (40 microm) were micro-dissected to isolate tumour areas; cell nuclei were extracted and Feulgen-stained cytospin monolayers were analysed using a DNA image cytometry system. For each case, DNA index (DI) was calculated relative to internal controls (lymphocytes; DI=1.0). Cases were categorized as diploid or aneuploid and the proportion of cells over 5c was also calculated. Fifty-three of 62 salivary gland tumours were uniformly diploid. Only nine cases were aneuploid: five CEPA, one low-grade MEC, one PC, one UC and one MA. The vast majority of salivary gland tumours were diploid. High-grade malignancies may be aneuploid, and ploidy may be useful to identify malignant change in atypical PA. Further, larger studies are needed to confirm our results and to further evaluate the usefulness of the technique in high-grade lesions.

P201 Flow cytometric analysis of DNA ploidy in potentially malignant lesions (PML) of the oral mucosa

P201 Flow cytometric analysis of DNA ploidy in potentially malignant lesions (PML) of the oral mucosa

O26 DNA ploidy analysis may predict malignant change in oral epithelial dysplasias

O26 DNA ploidy analysis may predict malignant change in oral epithelial dysplasias

Ploidy Analysis on Brush Biopsy Samples

Introduction. Oral squamous cell carcinomas are the result of an accumulation of genetic alterations, many of which are present in premalignant lesions. DNA ploidy analysis of tissue samples from oral premalignant lesions can detect cells demonstrating a deviation from the normal diploid complement of chromosomes (“aneuploidy”) and which may be a predictive marker for malignant transformation.

The role of liquid-based cytology in the investigation of colorectal lesions: a cytohistopathological correlation and evaluation of diagnostic accuracy

The role of cytologic techniques is not widely accepted even if it is well documented that the cytologic investigation of colorectal tract could complement the biopsy and increase the correct diagnosis of carcinomas. This study aims to evaluate the role of Thin-Prep(R) liquid-based cytology in the investigation of colorectal lesions. We compared the diagnostic accuracy of Thin-Prep(R) with that of biopsy in 158 patients with signs and symptoms of the lower gastrointestinal tract. Each patient underwent colonoscopy, followed by tissue biopsy and brush cytology. Brushing material was obtained and prepared according to the operating manual of Thin-Prep(R). The rate of unsatisfactory smears was 5% (8/158) with liquid-based technique and appeared to be slightly higher than the respective of biopsy [3.2% (5/158) (5 vs 3.2%, P = 0.18)]. Based on the final diagnosis, 89 out of 93 total malignant cases and 53 out of 53 total benign cases were correctly diagnosed with Thin-Prep(R) technique (four false negatives). Accurate diagnosis with biopsy was performed in 87 out of 93 total malignant cases and 53 out of 53 total benign cases with biopsy (six false negatives). Neither in Thin-Prep(R) technique nor in biopsy were false-positive cases observed. The sensitivities of detecting malignancy by Thin-Prep(R) and biopsy were 95.7, and 93.5%, respectively (no significant difference, P = 0.239). The sensitivity was augmented (98.9%) when the two techniques were combined, and this difference was found to be statistically significant (98.9 vs 92.9, P = 0.01, and 98.9 vs 95.7, P = 0.039). Liquid-based cytology appears to be an easy, highly accurate, and reliable cytologic method for the diagnostic approach of colorectal diseases and could be applied as complementary to biopsy for the improvement of the diagnosis. Moreover, could be used for DNA ploidy analysis and immmunohistochemical studies.

Laparoscopic management of a cornual ectopic pregnancy associated with persistant gestational throphoblastic disease: a case report

A 30-year-old woman underwent laparoscopy for diagnosis and treatment of ectopic pregnancy. A dark-red bulging mass was observed in the right uterine horn. Laparoscopic hysterotomy was performed. The histological examination revealed a hydatidiform mole, which was confirmed by DNA ploidy analysis showing triploidy (69 XXY) as apartial hydatidiform mole after the cytogenetic examination. On the third postoperative day, the thoracic computed tomography scan revealed punctuate lesions. These lesions disappeared after single-agent chemotherapy with methothrexate. To the best of our knowledge, this is the first case of cornual persistent gestational trophoblastic neoplasia managed by laparoscopic surgery.

Segregation Analysis of p53, c-myc and DNA Ploidy Using Flow Cytometry Among Egyptian Families with Childhood Leukemia

Segregation Analysis of p53, c-myc and DNA Ploidy Using Flow Cytometry Among Egyptian Families with Childhood Leukemia

DNA ploidy in proliferative verrucous leukoplakia

Proliferative verrucous leukoplakia (PVL) is a clinicopathologically distinctive form of oral leukoplakia presenting with multifocal flat, nodular and verrucous lesions that progress inexorably to squamous carcinoma. The aims of this investigation were to describe the clinical and histopathological features of six cases of PVL and to determine whether lesional epithelium demonstrates DNA ploidy anomalies prior to malignant transformation. The clinical and pathological features of six patients were reviewed and all biopsy specimens were subjected to image-based DNA ploidy analysis. The female:male ratio was 5:1 and the average age on first biopsy was 66 years. Only one patient reported both tobacco smoking and alcohol intake. The most frequently affected sites were alveolar ridge and/or gingiva (6/6), buccal mucosa (3/6), palate (3/6), tongue (2/6), buccal sulcus (2/6), and lip (1/6). Three patients developed multiple primary carcinomas, either invasive or verrucous. A ploidy anomaly at any oral site would have predicted malignant transformation in four cases and probably in a fifth for whom DNA ploidy failed to meet diagnostic criteria but was suspicious of aneuploidy. The site of transformation was predicted by ploidy and histopathology for three carcinomas and a further carcinoma showed severe dysplasia and a suspicious ploidy result in adjacent tissue. Both conventional histopathology and DNA ploidy proved effective in predicting the site of transformation in this limited series.