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Objective: The objective of this study was to evaluate the application of DNA ploidy analysis in large-scale population screening for cervical cancer. Methods: From March 2016 to March 2019, eligible subjects were enrolled and recommended to undergo DNA ploidy analysis, the ThinPrep cytology test (TCT), and high-risk human papillomavirus (hrHPV) detection concurrently. Patients with positive results were recommended for colposcopy, and biopsy diagnosis was regarded as the “gold standard.” We compared the test efficiencies of the 3 methods and compared the efficiency and accuracy of the TCT in our hospital and the “2-cancer screening” project in Hubei Province during the same period. Results: Among 20,574 women, the positive rates of DNA ploidy analysis, cytology, and hrHPV testing were 4.01%, 4.71%, and 16.28%, respectively. The sensitivities of these methods for screening for grade 2+ cervical intraepithelial neoplasia were 0.70, 0.68, and 0.96, and their specificities were 0.79, 0.82, and 0.45, respectively. On comparing DNA ploidy analysis with the TCT, there was no significant difference in the sensitivity, specificity, positive predictive value, negative predictive value, and missed diagnosis rate. In opportunistic screening and the 2-cancer screening project, the positive rates of cytology were 4.71% and 2.87%, respectively. And the efficiency and accuracy of the TCT in opportunistic screening were higher than in the 2-cancer screening project. Conclusion: Therefore, DNA ploidy analysis, which is of low-cost and does not depend on cytopathologists, can replace cytology and be applied in large-scale population screening for cervical cancer.

Lin Deng,1,* Shikong Guo,2,* Hong Li,1 Xianghui You,1 Yang Song,1 Haichuan Su1 1Department of Oncology, Tangdu Hospital, Air Force Military Medical University, Xi’an, Shaanxi, China; 2Department of Orthopedics, Tangdu Hospital, Air Force Military Medical University, Xi’an, Shaanxi, China*These authors contributed equally to this workCorrespondence: Yang Song; Haichuan Su Email songyang212212@163.com; suhaic_1@163.comBackground: This study explored the value of ascites and serum CA125, CEA, and CA19-9 levels and ascites DNA ploidy analysis for the diagnosis of peritoneal carcinomatosis (PC) in patients with gastrointestinal and ovarian malignancies, which can cause ascites and may disseminate peritoneally.Methods: We measured ascites and serum levels of CA125, CEA, CA19-9 and performed an ascites DNA ploidy analysis in 58 patients with PC and 44 patients without PC.Results: We found that a high expression level of CA125 in ascites fluid was associated with the occurrence of PC in patients with gastrointestinal and ovarian malignancies (P< 0.001), and that high CEA and CA19- 9 levels in ascites fluid were associated with PC in patients with gastrointestinal malignancies (P=0.001, P=0.002). But, these tumor marker expression levels in ascites fluid were not significantly associated with the PC stage (P> 0.05). We found similar serum levels of CA125, CEA, and CA19-9 between patients with gastrointestinal and ovarian malignancies and PC and those without PC (P> 0.05). We found that the presence of three or more cells with heteroploid in the ascites samples was significantly associated with PC in gastrointestinal and ovarian malignancies (P< 0.001). In addition, the best ROC curves and highest AUCs were achieved by combining the CA125 level and heteroploid cell analysis results (AUC for gastrointestinal and ovarian malignancies, 0.815, AUC for gastrointestinal malignancies, 0.873). Moreover, the combined ascites CA125 level and result of heteroploid cell analysis provided the best diagnostic sensitivity and specificity for PC (75.9% and 79.5%, respectively, in gastrointestinal and ovarian malignancies; 85.0% and 86.7%, respectively, in gastrointestinal malignancies).Conclusion: Ascites levels of CA125, CEA, CA19-9, and heteroploid cells can be considered valuable markers for the diagnosis of PC in patients with gastrointestinal and ovarian cancer.Keywords: peritoneal carcinomatosis, tumor marker, ascites, heteroploid cell

Background: Effusion cytology is a major diagnostic tool in medicine and has both therapeutic and prognostic implications. One of the dilemmas encountered is the differentiation between atypical cells and reactive mesothelial cells. The use of ancillary tools can reduce this grey zone and help to achieve a definitive diagnosis. Objectives: The main objective of this study was to evaluate the role of flow cytometry (FCM) and cell block with immunohistochemistry (IHC), along with the clinicoradiological investigations, to achieve a final diagnosis in effusion cytology to the maximum extent possible. Method: A prospective study was conducted. Effusion fluids, showing adequate amount and cellularity, were processed for conventional cytology, ploidy analysis by FCM, and cell block analysis, followed by IHC wherever required. Conventional cytological analysis was done by 2 independent pathologists, to look for interobserver variation, if any. The final result was achieved on the basis of integration of the results of the aforementioned studies, cytological details, clinicoradiological information, tissue biopsy findings, and follow-up. Result: A total of 90 samples were analyzed. On cytological examination, observer I categorized 60% samples as benign and 18.8% (n = 17) as malignant versus 58% categorized as benign and 23.3% (n = 21) as malignant by observer II. Observer I reported 19 (21.1%) equivocal cases and observer II reported 16 (17.7%). When both pathologists were considered together, the number of equivocal cases increased to 20. Sensitivity and specificity of FCM were 96.67 and 100%, respectively, and 100% for the cell block. On combining all techniques, the equivocal cases were resolved and a total of 33 cases were reported as malignant. However, 3 cases could still not be categorized and were labeled inconclusive. Conclusion: Conventional cytology combined with cell block IHC and FCM has the potential to minimize the requirement of tissue biopsy for confirmation. If the first sample is used judiciously for all the techniques, this may reduce the requirement for a second sample and possibly also the time required for a definite diagnosis and the initiation of therapy.

Introduction: Oral leukoplakia, the most common potentially malignant oral disorder (PMOD) may progress to oral squamous cell carcinoma (OSCC). Although, the current standard of care for assessing its malignant potential remains histological examination and assessing the severity of dysplasia, DNA ploidy analysis has been suggested as a surrogate marker to predict the behaviour of PMODs. Objectives: To detect aneuploidy and to correlate ploidy status with different grades of dysplasia in both tissue and cytology samples to predict the behaviour of these potentially malignant disorders and to assess the diagnostic utility of cytology samples for ploidy analysis. Methodology: After obtaining ethical clearance and consent, tissue and cytology samples of leukoplakia were collected and grouped based on the dysplastic findings into low-risk (n=20) and high-risk (n=20). DNA ploidy analysis was done using high resolution flow cytometry and its diagnostic utility was assessed. Results: Diagnostic utility was expressed in terms of sensitivity, specificity, PPV and NPV. On comparing the ploidy status of individual cases between tissue and cytology samples, cytology was able to accurately determine the ploidy status in majority of the cases. In the low-risk group, cytology had a sensitivity and specificity of 100% and a PPV and NPV of 100% with an overall diagnostic accuracy of 100%. Among the high-risk group, cytology had a sensitivity of 80% and specificity of 100% with a PPV of 100% and NPV of 83.33% and had an overall diagnostic accuracy of 90%. Combining both groups together, it had a sensitivity of 85.71% and specificity of 100% with a PPV of 100% and NPV of 92.31% and had an overall diagnostic accuracy of 94.74%. Conclusion: Overall, this study showed a positive correlation between cytology and tissue samples and ploidy and grade of dysplasia and cytology proved to be a simple and efficient with a reasonable diagnostic value.

Background DNA ploidy analysis is arguably the most sensitive and reproducible approach to assess risk in oral potentially malignant disorders. Although aneuploidy has been associated with a high risk of malignant transformation (MT), the current diagnostic criteria of aneuploidy are not specific to oral mucosal tissues and thus yield positive predictive values (PPVs) ranging from 30% to 40%. Objective The aims of this study were to analyze the ploidy status of archived material diagnosed as oral leukoplakia (OL) with follow-up data on MT and to review the risk classification criteria. Methods Nuclei suspensions were enzymatically prepared from formalin-fixed, paraffin-embedded tissue from 121 patients with OL and stained with propidium iodide for flow cytometry. Twenty-three patients had MT. Histograms were initially analyzed for the proportion of nuclei in G1 phase (2c), S phase, G2 phase (4c), and 5c-exceeding rate (5cER). Histograms diagnosed as aneuploid were further investigated qualitatively on the basis of nuclei distribution pattern. Log-rank test on Kaplan-Meier curves (LRKM; P Results Seven aneuploid histogram types were detected, of which only 3 proved truly high risk. EAR was then stratified as low risk (diploid), medium risk (low-risk aneuploid), and high risk, whereas TAR remained as diploid (low risk) and aneuploid (high risk). Aneuploidy in TAR had PPV of 55%, SS of 88%, and SP of 81% (LRKM P Conclusions Enhancement of TAR based on qualitative histogram classification significantly improves predictive values. Flow cytometry is an accessible approach to assess MT risk in OL.

The detection and classification of cervical cells via Pap smear or Liquid-based cytology (LBC) have important clinical significance for pathological diagnosis. For the limitations of pathological images and the complexity of morphological changes of cells, the accurate classification of cervical cells remains a challenging task. DNA ploidy analysis can complete the classification and recognition of individual cells well, and misclassification is common because of the factors of inevitable imaging. Unlike traditional classification methods that rely on manual features, we proposed a region detection and classification method based on multi-semantic label combined with morphological information analysis. We followed the principle that a nucleus represents a cell and divided cells into five categories. The overlapping cell regions could be divided into multiple single cells. In this paper, the data pre-processing was made up for the imbalance in classification. The weight parameters obtained from ImageNet dataset pre-training were realized the transfer learning of cervical exfoliated cells based on Faster R-CNN on LBC dataset. The result showed that the mean average precision (mAP) was 66.98% and the accuracy was 91.61% by learning depth and superficial features of cervical exfoliated cells with ResNet-101 as the backbone. To some extent, it can assist pathologists in the early diagnosis of cervical cancer.

Biliary brushing cytology has become the standard of practice for the investigation of strictures of the biliary and pancreatic duct systems. The methodology however has a limitation in that it has low diagnostic sensitivity when only cytologic evaluation is used. A number of testing methodologies have been applied to brushing specimens in an attempt to improve overall sensitivity without loss of specificity. These have included DNA ploidy analysis, immunocytochemistry, individual gene mutational analysis, fluorescence in-situ hybridization (FISH), and next generation sequencing (NGS). Currently, FISH coupled with routine cytology appears to be the method of choice for improving diagnostic sensitivity. NGS shows significant promise for improvement of diagnostic sensitivity.

The utility of flow cytometry as a useful diagnostic modality for the assessment of hematopoietic neoplasms has been established beyond doubt. In fact, it is now an integral part of the diagnosis and classification of various diseases like leukemias and lymphomas along with molecular studies and cytogenetics. Prognostication and disease monitoring by flow cytometry is also being recognized increasingly as one of the important fortes. This is evident by the number of articles in the published in literature on the minimal residual disease detection by flow cytometry especially in the last decade or so. To add to this, ever growing list of utilities in hematopoietic malignancies, many nonhematopoietic neoplasms can also be analyzed by flow cytometry. The examples include fluid specimens from serous cavity effusions and samples from solid tissues like lymph nodes, reticulo-endothelial tissue, central nervous system tissue, etc. Flow cytometry technique provides a unique blend of rapidity, high sensitivity and specificity compared to cyto-morphology and conventional immunohistochemical staining. It is also remarkable for simultaneous analysis of more than one marker on the cells. Evaluation of limited samples such as cerebrospinal fluid or fine needle aspiration samples makes Flow cytometry a valuable tool. DNA ploidy analysis and assessment of pediatric non-hematopoietic neoplasms by Flow cytometry has envisaged the utility vista of this technique. This review is aimed at providing an insight into the applications of flow cytometry in pediatric malignancies.

DNA ploidy analysis of cells is an automation technique applied in pathological diagnosis. It is important for this technique to classify various nuclei images accurately. However, the lack of overlapping nuclei images in training data (imbalanced training data) results in low recognition rates of overlapping nuclei images. To solve this problem, a new method which synthesizes overlapping nuclei images with single-nuclei images is proposed. Firstly, sample selection is employed to make the synthesized samples representative. Secondly, random functions are used to control the rotation angles of the nucleus and the distance between the centroids of the nucleus, increasing the sample diversity. Then, the Lambert-Beer law is applied to reassign the pixels of overlapping parts, thus making the synthesized samples quite close to the real ones. Finally, all synthesized samples are added to the training sets for classifier training. The experimental results show that images synthesized by this method can solve the data set imbalance problem and improve the recognition rate of DNA ploidy analysis systems.

Aneuploidy has been suggested as a marker for stratification of many neoplasms but its potential usefulness in adenocarcinoma (ADC) with bronchioloalveolar (BAC) pattern has not been well defined. We examined paraffin-embedded tissue sections from 28 cases of ADC with BAC pattern as well as 7 benign lung lesions and 9 normal lung tissue samples for chromosomal aneu- ploidy by in situ hybridization using digoxigenin-labelled probes for chromosomes 1 and X. Of the 28 ADC with BAC pattern, 17 (61%) were diploid and 11 (39%) were aneuploid. Of the 17 diploid cases, 7 (41%) were male and 10 (59%) were female and of the 11 aneuploid cases, 2 (18%) were male, 9 (82%) were female. Regarding the cell type, 24 (86%) were adenocarcinomas in situ (AIS) so called BAC and minimally invasive ADC (MIA), and 4 cases (14%) were invasive ADC. Of the 12 cases each of AIS and MIA, 9 (75%) and 8 (67%) had diploid pattern respec- tively. Of the 4 invasive ADC cases, all had aneuploid pattern. Seventeen cases (71%) with T1 tumor size (>0 mm~≦30 mm), had diploid and 4 cases (100%) with T2 tumor size (>30 mm ≦70 mm) had aneuploid pattern. Statistical analyses showed that nuclear diploidy was significantly cor- related with AIS and MIA tumor types while aneuploidy correlated with invasive ADC type (P=0.025). Also a significant correlation was found between ploidy and tumor size (P=0.033). In conclusion, these findings suggest that DNA ploidy analysis provides useful information for the assessment of cellular kinetics and reflect histopathological subtypes in ADC with BAC pattern that are destined to behave aggressively.

Giant cell ependymoma, a rare ependymoma subtype, was recently recognized as a separate diagnostic entity with variations both in malignant potential and course of disease. We analyzed the first supratentorial giant cell ependymoma using G-band karyotyping, DNA ploidy analysis, and array comparative genomic hybridization. The tumor was hypodiploid, and the karyotype showed multiple monosomies. This novel cytogenetic pattern seems specific for giant cell ependymoma as the only previous cytogenetic analysis of a giant cell ependymoma found similar monosomies. We were also able to analyze cytogenetically the subsequent recurrent tumor, phenotypically an anaplastic ependymoma, allowing a first insight into the genetic events involved in disease progression.

Aims: Aneuploidy is a potential biomarker for predicting progression of premalignancies. Ploidy assessment is mostly performed on nuclei isolated from tissue sections. Ploidy assessment in situ in tissue sections may be a large improvement, enabling selective sampling of nuclei, thus allowing the correlation between ploidy and histology. Existing ploidy analysis methods in sections suffer from limited sensitivity. The aim was to reliably assess ploidy in sections, combined with simultaneous assessment of other markers at the individual cell level. Methods and results: Ploidy was measured in 22 paraffin-embedded oral premalignancies. The DNA stoichiometric Feulgen procedure was used on isolated nuclei, as well as fluoresence in situ hybridization analysis. In tissue sections, Feulgen was combined with immunohistochemistry for Ki67 proliferation marker, enabling distinction between cycling euploid and aneuploid cells. Aneuploidy was reliably detected in tissue sections (sensitivity 100%, specificity 92%). One section in which aneuploidy was detected was misclassified in isolated nuclei analysis. Sections were also successfully analysed using our model combined with DNA double strand break marker gamma-H2AX in fluorescence microscopy, underlining the power of biomarker evaluation on single cells in tissue sections. Conclusions: The analysis model proposed in this study enables the combined analysis of histology, genotypic and phenotypic information.

Yang-Chun Feng,1 Jia Yang,2 Cheng-Ming Liu,1 Zhen-Zhen Cheng,1 Yan-Chun Huang1 1Clinical Laboratory Center, 2Department of Second Internal Medicine, Tumor Hospital Affiliated to Xinjiang Medical University, Urumqi, People’sRepublicof China Background: The Uygur women have the highest incidence of cervical cancer in all Chinese ethnic groups. The research was conducted to explore whether DNA ploidy could be the prognostic indicator of human papillomavirus (HPV) infection in Xinjiang Uygur women.Methods: Case data and cervical exfoliated cell samples from 326 Uygur women. The DNA ploidy was analyzed by flow cytometry. The flow-through hybridization and gene chip (FHGC) for HPV type test then divided the cases into negative HPV group, non high-risk HPV infection group, single high-risk HPV infection group, and multiple high-risk HPV infection group. Lastly, 113 cases from 273 HPV infection cases were followed up.Results: The 16-type HPV had the highest rate in all genotypes infection; 16/18-type HPV mixed infection was the most common type in multiple high-risk HPV infection group. Abnormal DNA ploidy happened along with the seriousness of HPV infection. Compared with the HPV negative group, DNA heteroploid appeared 12.750 times and 22.705 times, respectively, in single high-risk HPV and multiple high-risk HPV infection groups. Followed up 1year later, the DNA index, S-phase cells’ peak percentage and heteroploid of cervical exfoliated cells significantly reduced in single and multiple high-risk HPV infection patients, but in nine patients negative HPV infection and DNA heteroploid still existed.Conclusion: The finally cure criterion of high-risk HPV infection should include the negative HPV test and normal DNA ploidy analysis. It was useful to prevent and cure cervical lesions in Xinjiang Uygur women through high-risk HPV test and DNA ploidy analysis. The transient infection and persistent infection in Xinjiang Uygur women should be taken as further research. Keywords: Uygur, cervical epithelial cell, human papillomavirus, DNA ploidy analysis

Oral squamous carcinomas appear heterogeneous on DNA ploidy analysis. However, this may be partly a result of sample dilution or the detection limit of techniques. The aim of this study was to determine whether oral squamous carcinomas are heterogeneous for ploidy status using image-based ploidy analysis and to determine whether ploidy status correlates with histological parameters. Multiple samples from 42 oral squamous carcinomas were analysed for DNA ploidy using an image-based system and scored for histological parameters. 22 were uniformly aneuploid, 1 uniformly tetraploid and 3 uniformly diploid. 16 appeared heterogeneous but only 8 appeared to be genuinely heterogeneous when minor ploidy histogram peaks were taken into account. Ploidy was closely related to nuclear pleomorphism but not differentiation. Sample variation, detection limits and diagnostic criteria account for much of the ploidy heterogeneity observed. Confident diagnosis of diploid status in an oral squamous cell carcinoma requires a minimum of 5 samples.

Tubal hydatidiform mole is an uncommon condition with about 40 confirmed cases in the accessible literature. The patient usually presents with symptoms and signs of a classical ectopic pregnancy, and it is only after histological examination and DNA ploidy analysis of the conceptus that a hydatidiform mole is diagnosed. We present an unusual case of primi gravida at 6 weeks gestation that was diagnosed as having ectopic pregnancy.

The introduction of new technologies, designed to assess biologically relevant alterations in tumour cells, has the potential to provide additional prognostic and predictive information from biopsies, as well as providing a more objective assessment than conventional histopathology alone. One such technique is the assessment of the ploidy status of tumours. DNA ploidy studies, using various technologies, are abundant in the literature, but questions remain as to how such studies should be translated into evidence-based practice. In this Expert Opinion, we provide two perspectives on this field. Baak and Janssen discuss the technology and set the assessment of ploidy in the context of other quantitative techniques. Clinical applications are then illustrated. In the paper by Peter Hall, aspects of the biology of ploidy are discussed, and the potential relevance of a more detailed understanding of the biology of ploidy to clinical application. Some of the general short-comings of the applied literature are discussed and some approaches suggested for the improvement of the evidence base on which healthcare decisions relating to the application of new technologies are made.

Carcinomas of the bladder cause important problems of mortality and morbidity despite diagnostic and therapeutic improvements. A variety of grading systems has been developed to reflect the different biologic behavior and malignant potential of this heterogeneous neoplasm. However, these histologic grading systems are subjective and reproducibility is low. In this study, the nuclear morphometric features and DNA ploidy status of 86 cases of transitional cell bladder carcinomas have been studied using image analysis methods with different data selection methods in nucleus suspension to evaluate the relationship of these parameters with the conventional prognostic factors. The relationship between these parameters and likelihood of relapse has also been investigated. In conclusion, the mean nuclear area of the 10 largest nuclei and DNA ploidy status have been shown to be significantly correlated with conventional prognostic factors. Cytomorphometrically, a binary grading system seems more suitable for transitional cell carcinoma of the bladder. For superficial carcinomas, morphometric parameters and DNA ploidy analysis can be helpful in the separation of the patients into prognostically different groups.

Aim of the present study was to investigate and propose standards for a telemedicine application of image analysis based cytometry. Specifically, the evaluation and determination of the appropriate procedures for equipment calibration, image capturing and processing are attempted. The impact of digital image compression in measurements’ results is being investigated as well as the reliability of remote measurements on images transmitted through internet. Materials and methods In total 50 pap test smears prepared with liquid based cytology and stained with Feulgen technique were analyzed. Images from each slide were captured using 3 different image capturing systems, while at the same time each one of the smears was analyzed with a commercial DNA Ploidy analysis system, which was considered the reference system. All the images were compressed using the JPEG standard. Both the original and the compressed images were analyzed with a specially customized image analysis software. DNA ploidy histograms were plotted and 10 algorithms for the interpretation of the histograms were calculated. Images were sent through internet to a remote station where they have been analyzed independently by another user. Results Analysis and comparison of obtained results against those from the reference system, showed very good correlation, either considering the algorithms’ values or the ploidy classification of each case. No statistically significant differences were found for any of the algorithms that were calculated for the cytology images compressed at different compression levels. Thus a telemedicine application of image analysis based cytometry is possible. A very good linear correlation was observed between the onsite and remote analysis. Conclusions The high correlation between results obtained from images captured with different systems proves that the maintenance and the constant application of the appropriate procedures are essential for all the methods’ stages. All the tests performed for the purposes of the present study, showed that a telemedicine application of image analysis based cytometry could be easily set, reliable and cost effective. Both the customized DNA ploidy analysis system and the operating standards of such a system, as they are proposed with the present study, might be used for providing services to peripheral labs. The proposed standards could also be used for quality control and assurance of labs applying image cytometry methods. 1%%%%Στόχος της παρούσας μeλέτης eίναι η διeρeύνηση και η πρόταση eνός πρωτοκόλλου τηλeματικής eφαρμογής της κυτταρομeτρίας μe τeχνικές ανάλυσης eικόνας. Συγκeκριμένα eπιχeιρeίται η αξιολόγηση και ο καθορισμός των κατάλληλων διαδικασιών για την βαθμονόμηση των συσκeυών, την λήψη και την eπeξeργασία eικόνων. Eπίσης διeρeυνάται η eπίδραση της συμπίeσης των ψηφιακών eικόνων στα αποτeλέσματα των μeτρήσeων και η αξιοπιστία των μeτρήσeων, που πραγματοποιούνται από απόσταση, σe eικόνeς που έχουν αποσταλeί μέσω διαδικτύου. Υλικό και μέθοδος Μeλeτήθηκαν συνολικά 50 κολποτραχηλικά…

副腎偶発腫瘍は、診断技術の進歩と検診の普及によりここ20年程増加が著しく、その対応が問題となっている。一方、DNAプロイディ型は腫瘍の良性悪性の性質に深く関わる生物学的指標となることが広く知られている。我々はパラフィンブロックを用いたDNAプロイディ分析法により副腎摘除術の適応について検討を行った。東京大学医学部附属病院及びその関連施設で摘除された副腎偶発腫瘍28例のフローサイトメトリー分析の結果、異常型は3cm以上の腫瘍に有意に多かった。また、3cm未満の腫瘍では異常型を示したものは見られなかった。以上の結果、腫瘍径が3cm以上であれば副腎摘除術施行の適応が認められると考えられた。本論文は副腎偶発腫瘍の手術適応について腫瘍径に科学的根拠を与えた最初の報告と考えられる。, Adrenal incidentalomas have been increased in number during these two decades with the advance of diagnostic technologies and prevalence of medical check-up chances. Therefore, how to treat adrenal incidentalomas has been controversial problem in regard to the decision for adrenalectomy. We tried to perform DNA ploidy analysis to verify its significant role using paraffin-embedded archival specimens. Among 28 adrenal incidentalomas resected at Tokyo University Hospital and its affiliated hospitals, DNA ploidy abnormalities were found significantly more often in tumors having diameter of 3 cm or larger. Moreover, none of tumors with the size of less than 3 cm showed abnormal DNA ploidy patterns. Since DNA ploidy pattern is widely accepted to predict the malignant potentiality or aggressiveness of adrenal tumors objectively, our results may provide the scientific accountability for resection of adrenal indicentalomas in case of those having the size of 3 cm or larger.

BACKGROUND Image cytometry has proved to provide a good alternative to flow cytometry for DNA ploidy measurement of archival tumors. However, when interactively done this technique is unable to give statistically valuable results within an acceptable time for clinical oncology. METHODS An image cytometer was developed for fully automatic DNA ploidy quantitation, focusing efforts on speed and accuracy. Software functionalities include systematic acquisition of fields on a microscopic slide, detection, localization and sorting of nuclei, computation of the DNA content together with post-processing tools, for a deeper analysis of the DNA ploidy diagram. RESULTS DNA ploidy analysis of archival breast carcinoma samples illustrates the accuracy of DNA ploidy measurements and the sensitivity in the detection of DNA ploidy abnormalities as a result of cell sorting. CONCLUSIONS Fully automatic image cytometry is able to combine qualities of flow cytometry (automatic analysis of a statistically significant collection of cell nuclei) with additional advantages: sorting of unwanted events (debris, stromal and inflammatory cell nuclei) and facilities for an a posteriori control of the quality of cell selection. This method is well suited to DNA ploidy analysis of archival cancer samples.

DNA ploidy analysis by DNA cytofluorometry was performed on 41 tumors obtained from 37 patients with primary giant cell tumor of bone (GCT). Histologically, 26 of the tumors from primary or recurrent lesions were evaluated as grade I, and 13 tumors as grade II. Among the 33 primary GCT patients, 4 patients had local recurrence or pulmonary metastasis. The DNA ploidy pattern and the percentage of hyperdiploid cells showing a greater DNA content than diploid cells, were obtained from DNA cytofluorometry. All of the 33 primary tumors were diploid. Of 6 recurrent tumors, 4 were diploid and 2 were euploid-polyploid. One of the two pulmonary metastatic tumors was diploid, but another that demonstrated a malignant transformation to malignant fibrous histiocytoma was aneuploid. The percentage of hyperdiploid cells was significantly different between primary and recurrent tumors (P = 0.0188) and between grade I and grade II tumors (P = 0.0052), while there was no difference between primary tumors in the cases that recurred or metastasized and those that did not. Thus, these data indicate that cell proliferative activity is closely correlated with biological aggressiveness and histological grading, although DNA ploidy is not useful for predicting prognosis.

Purpose. To study the evolution of concepts concerning gastrointestinal stromal tumours (GISTs) over 30 years. Discussion. GISTs have been, for more than 30 years, the subject of considerable controversy regarding their line of differentiation as well as the prediction of their behaviour. Furthermore, once they spread within the peritoneal cavity, they are extremely hard to control. The recent findings of c-Kit mutations and the immunohistochemical detection of the product of this gene, KIT or CD 117, in the mainly non- myogenic subset of this family of tumours, has led to a reappraisal of this group of lesions, which, with some exceptions, is now thought to be derived from the interstitial cells of Cajal, and this has facilitated a clearer definition of their pathological spectrum. In this article, we review chronologically the evolution of the concept of GIST with the gradual application of electron microscopy, immunohistochemistry, DNA ploidy analysis. We discuss the impact of these techniques on the pathological assessment and clinical management of GISTs., SCOPUS: ar.j, info:eu-repo/semantics/published

DNA ploidy was investigated in 61 specimens obtained from 25 patients with squamous carcinoma of the oral and maxillofacial region. Biopsy specimens of normal tissue surrounding the tumor were also obtained in six patients. Single-cell suspensions for flow cytometric analysis were prepared. The DNA ploidy and histogram were calculated and compared with the histologic grade, presence of lymph node metastases, and course of the disease. The ploidy of the main stemline was peridiploid in 17 carcinomas, hyperdiploid in three, and aneuploid in five. Histologic grade but not nodal involvement was associated with the ploidy of the main stemline. Of 15 multisampled carcinomas 13 showed constant DNA ploidy and histogram classification. In the other two major changes in DNA ploidy (from peridiploid to hyperdiploid in the first and from peridiploid to aneuploid in the second) were found. Survival information was available for 24 patients. Ploidy values higher or lower than 2.5 c were strongly predictive of both overall (p < 0.001) and relapse-free survival (p < 0.001). The lymph node status proved a powerful prognostic indicator (p = 0.014) but was not related to the relapse-free time of survival. Multiparametric evaluation of survival revealed an independent role of both DNA ploidy and nodal involvement in the prognosis of squamous carcinoma of the oral and maxillofacial region.

Aims—To examine the basic assumptions made during DNA ploidy analysis of histological sections with an aim to eliminate methodological errors that have lead to conflicting results with this technique. Methods—A rat liver imprint and histological sections together with sections and whole nuclei cytospins of human breast tumour biopsy specimens were stained with azure A Schiff's reagent and used to investigate the effects of computer imaging, histological section thickness, nuclear volume, and shape corrections. Results—The rat liver imprint demonstrated a linear relation between mean nuclear transmittance and nuclear area for each of the three (2C, 4C, 8C) hepatocyte clusters. This finding was used to produce similar, proportional integrated optical density (IOD) measurements from rat liver sections by selecting only nuclei that were sectioned through their centres, as assumed by the mathematics for volume correction. The limitations of computer imaging necessitated an edge (glare) correction for each nucleus so that nuclei of different sizes could be analysed. Shape correction was required to analyse nuclei of different morphologies. Normal human lymphocytes, squamous epithelium and fibroblasts were all measured with similar IODs. DNA ploidy values obtained from 7 μm human breast tumour sections (using lymphocytes as controls) correlated well with those obtained from the whole nuclei cytospins from the same tissue blocks. Conclusions—With an improved understanding of the theoretical and technical aspects of ploidy analysis of tissue sections, reproducible and consistent results are possible. These results can be integrated into routine histopathology investigations alongside immunohistochemistry and molecular diagnostic techniques.

While 80% of transitional cell carcinomas (TCC) present as Ta Tl lesions, they account for only 15% of deaths caused by TCC. We have evaluated the ability of DNA ploidy analysis to predict outcome in 228 patients with Ta Tl TCC. All patients were judged to be at increased risk for tumor recurrence due to having two occurrences of Stage TI tumor within 56 weeks, or three or more tumors presenting simultaneously within 16 weeks of registration. Concurrent carcinoma in situ was acceptable. All patients were treated with either bacillus Calmette Guerin (BCG) immunotherapy or mitomycin-C (MMC) intravesical chemotherapy. Patients with nondiploid tumors had higher hazard rates for both tumor progression and death (p = 0.007 and p = 0.016, respectively); however, the prognostic information of DNA ploidy was not additive to tumor grade.

lntratumoral heterogeneity of DNA ploidy has been identified in breast carcinomas; however, optimal sampling methods have not been determined. In this study of 28 invasive breast carcinomas measuring more than 1.4 cm in greatest dimension, two different techniques for obtaining cells for flow cytometric DNA ploidy analysis were compared. Two solid pieces of tissue were taken from opposite halves of the tumor. A third sample was obtained by scraping multiple cut surfaces of the tumor. Heterogeneity of DNA ploidy was detected in 43% of cases. Most cases demonstrating heterogeneity contained multiple aneuploid populations. However, in five cases classification of the tumors as either DNA euploid or DNA aneuploild differed among samples. A total of 39 non-diploid populations were detected in 23 of the cases. Thirty-thrlee (85%) were detected by scraping and 35 (90%) were detected in either one or both tissue pieces. lntratunioral DNA heterogeneity emphasizes the need for adequate sampling. The scraping technique was as effective in identifying aneuploid cell populations as the combined results of the two pieces of tissue and better than sampling a single piece of tissue. Scraping also offers the advantage of tissue conservation which may be critical when various analytic studies are performed. o 1995 Wiley-Liss, inc. Key terms: DNA neoplasms-analysis, flow cytometry, DNA ploidy, breast neoplasms, heterogeneity

Background/aims: Image analysis in dermatopathology has been used for DNA ploidy analysis, morphometry, stereology, and quantitative immunohistochemistry. The object is to review image analysis in dermatopathology and evaluate these modalities and their application in pigmented lesion pathology, for elucidation of tumor behaviour and architecture and as an aid in tumor identification and prognostication. Conclusion: Image analysis in dermapathology has a huge potential. The techniques are difficult and at present mainly used in specialized centres.

Flow cytometric analysis of DNA ploidy was performed on 51 formalin-fixed, paraffin-embedded samples of canine transitional cell carcinoma of the urinary bladder. The DNA ploidy data obtained were compared to several clinicopathologic features. In addition, the DNA ploidy of 15 hyperplastic/inflamed and 8 normal canine urinary bladders was measured. Forty-three of the 51 neoplastic samples had interpretable DNA histograms. DNA aneuploidy was found in 34/43 (79%) of the transitional cell carcinoma samples. Of the 34 aneuploid neoplasms, 16 (47%) were hyperdiploid, 17 (50%) were tetraploid, and 1 (3%) was hypertetra-ploid. No significant correlation was found between the presence of DNA aneuploidy and the growth pattern. histologic grade, clinical stage, or individual morphologic features of this neoplasm. Additionally, the DNA ploidy was not related to the sex, age, or survival time of dogs with transitional cell carcinoma. All of the normal and all but one of the hyperplastic/inflamed urinary bladders were diploid. The results from this study demonstrated that DNA ploidy can be measured from paraffin-embedded canine samples by flow cytometry, a majority of the canine transitional cell carcinomas were aneuploid, and a significant correlation did not exist between the DNA ploidy and specific clinicopathologic features of this neoplasm.

A 62-year-old man with a mucinous carcinoma of the skin on the left cheek is described. The excised specimen showed a characteristic histopathologic picture. The findings from histochemical, immuno-histochemical, and electron microscopic examinations suggested an eccrine differentiation of the tumor cells. The removed tumor was examined using DNA cytofluorometry in order to analyze the cell kinetics and to estimate its biological behavior and prognosis. The tumor cells showed a diploid mode with a small number of polyploid cells. Only a small number of cells in the S and G2/M phase ranges were detected. After four years, there has been no evidence of tumor recurrence nor metastasis. The results of DNA ploidy analysis were thus compatible with the nature of slow growth with a good prognosis in this case.

The histogenesis of atypical fibroxanthoma (AFX) and its relationship to malignant fibrous histiocytoma (MFH) are a subject of controversy. Many investigators have proposed that AFX may represent a reactive process, while others contend that it is a true fibrohistiocytic neoplasm, closely related to MFH. In an attempt to determine whether biologic differences between AFX and MFH may be accounted for at the cellular DNA level, we performed ploidy analysis on 14 cases of AFX by flow cytometry and compared our results with previously reported DNA analyses of MFH. Thirteen of the 14 lesions demonstrated diploid distribution of nuclear DNA, and only 1 case had an aneuploid population. This contrasts with prior data on MFH, the vast majority of which are aneuploid. Our results suggest that, despite histologic similarities, AFX may be distinguished from MFH on the basis of DNA content. These findings may be significant in understanding the biologic behavior of AFX.

A microfluorometric DNA study was conducted on isolated cells from 64 fragments of ovarian epithelial tumors. Data analysis considered the DNA content of the main stemline and the prevalence of cells in the different compartments of the DNA ploidy histogram in relation to the mitotic activity index and the histologic architecture. Main stemlines were found remarkably stable in different parts of the same tumor. Peridiploid (1.8c to 2.2c) stemlines were found both in well-differentiated and in poorly differentiated tumors. However, low aneuploid (2.2c to 3.0c) stemlines were mostly found in nonsolid tumors and high ploidy (3.2 to 5.04 c) stemlines were prevalent in solid tumors. A comparative analysis between DNA ploidy parameters and mitotic activity was useful to evaluate the expanding modalities of neoplastic cell populations. This analysis revealed that several tumors accumulate an excessive amount of heteroploid cells and others an excessive amount of G2 cells. Yet, most neoplastic cell populations vary from slow to rapidly growing patterns without relevant abnormalities in their ploidy graphic profiles. No relationship was found between stemline ploidy and histologic architecture compared with expanding modalities. These findings indicate that multifaced criteria combining features such as main ploidy, graphic profile, mitotic rate, and histologic architecture measured on one or more microsamples of the same tumor may help to objectively estimate the aggressiveness reached by the neoplastic cells at the time of clinical presentation.

Nuclear deoxyribonucleic acid (DNA) ploidy studies with paraffin embedded archival material from 14 pancreatic insulinomas were performed by flow cytometry. Clinical follow-up (2 to 17 years; mean, 8 years) was obtained for all patients. Half of the tumors had a normal DNA histogram and half exhibited an abnormal DNA profile consistent with DNA aneuploidy. Six of the seven patients with aneuploid tumors are alive and disease free (2 to 5 years postresection), and one is alive with metastatic disease. Of the seven patients with tumors showing normal DNA profiles, five are alive and disease free, one is dead of disease, and one is alive with metastatic disease. These data suggest that DNA ploidy analysis is unlikely to provide useful prognostic information for patients with insulinomas.

In this study the authors applied flow cytometric DNA-ploidy analysis to multiple female genital tract malignant tumors in 43 patients, most of whom (n = 37) had bilateral ovarian cancer. An algorithm was developed for calculation of the likelihood ratio of the probabilities that measured DNA index differences between multiple tumor localizations within the same patient could be attributed to measurement variation or to true biologic DNA content differences. The results of this statistical analysis show that in 72% of the cases (31 of 43) this probability ratio exceeded 1. Because the probability that two independent tumors will have a near-identical aneuploid DNA content is very low, this finding supports a metastatic process rather than the occurrence of multiple primary tumors in these patients. Thus, flow cytometric DNA-ploidy analysis can be helpful in the identification of metastatic disease in patients with multiple female genital tract malignant tumors.

Computers are extensively employed in the quantitative analysis of cells and tissues using flow cytometry and/or image analysis. The first issue discussed in this paper is the use of computer-controlled multiparameter flow cytometry, with special emphasis on the use of pulse-processing techniques for DNA ploidy analysis. Subsequently, modern image analysis using a solid-state camera is discussed. Examples of automated analysis of Feulgen-DNA stained cells (absorption) or cells stained by in situ hybridization procedures (fluorescence) for defined DNA or RNA sequences are given. Finally, a new user-friendly microscope-computer interface (HOME=highly optimized microscope environment) is presented, in which the monitor of a personal computer is optically overlaid with the microscopic image, enabling the immediate reading of patient record data while viewing through the microscope. When equipped with a simple encoding stage for position tracking, this system enables more accurate screening of cytological specimens and provides rapid interactive image analysis and morphometry.

The relationship between DNA content, clinicopathologic findings, and patient survival in synovial sarcoma was investigated. Patient age at diagnosis (P less than 0.001), tumor size (P less than 0.001), and ploidy status (P less than 0.003) correlated significantly with patient survival. A marginally significant correlation between mitotic count and patient survival was also observed (P = 0.04). Histologic subtypes (monophasic versus biphasic), mitotic count, and S-phase by flow cytometry had no significant influence on the clinical outcome of patients with synovial sarcoma in this study. The authors conclude that DNA ploidy analysis is a significant objective probe in the prognostication of patients with synovial sarcoma.

This report describes a case of acute myeloid leukemia (subtype M1) with biphasic morphology. The bone marrow biopsy showed 2 distinct regions of blasts, one containing large cells and the other small cells. Morphometric and DNA ploidy analysis showed that the mean nuclear area and mean DNA index for the large cell region were 2-fold higher than those for the small cell region. Cytogenetic analysis showed an abnormal near-tetraploid clone. The tumor relapsed following aggressive therapy. The cells from the relapse specimen were similar to the original small cell region with respect to nuclear area and DNA index; however, there was immunophenotypic transformation with gain of CD7 and gain of CD56. Cytogenetically, the relapse specimen showed no evidence of the near-tetraploid clone, but instead had a previously unidentified abnormal clone containing 46 chromosomes and structural abnormalities of 2q and 7q. Biphasic morphology in acute myeloid leukemia may be predictive of a near-tetraploid subclone and immunophenotypic transformation.

Background To assess the usefulness of combining cytodiagnosis and DNA cytometry on nasopharyngeal brush samples for the detection of nasopharyngeal carcinoma (NPC). Methods DNA ploidy analysis was undertaken on 66 nasopharyngeal brush samples that had been previously evaluated cytologically. Results Cytodiagnosis and ploidy analysis demonstrated a sensitivity of 66% and 55%, respectively. Both techniques had a specificity of 100% for NPC cases that were histologically confirmed. The negative predictive values for cytodiagnosis and ploidy analysis were 29% and 24%, respectively. Conclusions The sensitivity and specificity for cytodiagnosis were not improved by the addition of DNA ploidy analysis. Furthermore, combining the two techniques did not confer any significant advantage compared with the use of cytology and DNA ploidy alone. The relatively low negative predictive values are a significant limitation, and combining cytodiagnosis and ploidy analysis on nasopharyngeal brush samples cannot be recommended for the detection of NPC. © 2002 Wiley Periodicals, Inc. Head Neck 24: 223–227, 2002; DOI 10.1002/hed.10022