DNA Image Cytometry for Screening the Carcinogenetic Risk of Oral Potential Malignant Disorders.

Background: Oral Submucosal Fibrosis (OSF) and Oral Leukoplakia (OLK) are well-known oral potentially malignant disorders, and cases of Oral Submucosal Fibrosis concomitant Oral Leukoplakia (OSF+OLK) are now being reported clinically. DNA image cytometry is an objective and non-invasive method for monitoring the risk of precancerous lesions in the oral cavity. Methods: A total of 111 patients with clinically characterized oral mucosal lesions underwent simultaneous and independent histopathological and DNA imaging cytometry assessments. Clinical data were also collected for each patient. Results: The frequency of DNA content abnormality was higher in the tongue than in other oral sites (P = 0.003) for OLK. The frequency of DNA content abnormality was higher in the tongue than in other oral sites (P = 0.035) for OSF+OLK. The differences of DNA content abnormality in age, sex, dietary habit, smoking, and alcohol intake were not observed in OLK and OSF+OLK. The study indicates an association between DNA content abnormality and pathological examination in OSF+OLK ( χ2 test, P = 0.007). OLK showed higher sensitivity and specificity than OSF, while the sensitivity and specificity of OSF+OLK are higher than OLK only and OSF only. Conclusion: DNA image cytometry can be utilized as an adjunctive device for the initial detection of oral potentially malignant disorders that require further clinical management.

DNA image cytometry of bronchial washing as a diagnostic adjunct to radial endobronchial ultrasound-guided sampling of peripheral lung lesions: A single center prospective study.

OBJECTIVE: The objective of this study is to study the adjunct role of combining DNA aneuploidy analysis with radial endobronchial ultrasound (R-EBUS)-guided sampling for diagnosis of peripheral lung lesions (PPLs). METHOD: A single-center prospective study was conducted in patients undergoing R-EBUS-guided sampling for PPLs. DNA image cytometry (DNA-ICM) was used to analyze DNA aneuploidy in bronchial washing from the bronchial segment of the PPL. Clinical information, R-EBUS data, pathology, DNA-ICM results, and follow-up data were analyzed. Sensitivity, specificity, and predictive values for R-EBUS-guided sampling, DNA-ICM, and the two methods combined were measured. Binary logistic regression was performed to determine influencing factors on diagnostic positivity rate. Receiver operating characteristic (ROC) curve analysis was used to determine the optimal cutoff point for DNA-ICM. RESULTS: A total of 101 patients were enrolled. Sixty-four (63.4%) patients had confirmed malignant tumor, of whom 33 were confirmed by R-EBUS-guided sampling (biopsy and/or bronchial brush and wash cytology), and 31 by surgery or percutaneous lung biopsy. Thirty-seven patients were finally considered to have benign lesions, based on clinical information and 1-year follow-up. The sensitivity for malignant disease was 51.6% by R-EBUS, and specificity was 100%. DNA-ICM had a sensitivity of 67.2% and a specificity of 86.5%. When combining the two methods, sensitivity increased to 78.1% and specificity was 86.5%. Lesion size and whether the R-EBUS probe was located in the lesion were significantly associated with positivity rate of the combined methods. The optimal cutoff point for DNA-ICM was 5c for max DNA content, and 1 for aneuploid cell count (sensitivity 67.2%, specificity 86.5%, accuracy 63.4%). CONCLUSION: In malignant PPLs, DNA-ICM combined with R-EBUS-guided sampling can improve diagnostic positivity compared with either method alone.

Multiple noninvasive auxiliary tests-assisted photodynamic therapy in actinic cheilitis: A case report.

Actinic cheilitis (AC) is recognized as the most common precursor lesion of squamous cell carcinoma (SCC) of the lip, with a higher risk of invasiveness and metastasis. Early accurate diagnosis and appropriate therapy are essential to prevent carcinogenesis and progression of AC. Topical 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT), a non-surgical and minimally invasive modality, has been proposed as an effective treatment for oral potentially malignant diseases (OPMDs) and oral cancers. Herein, we report a 64-year-old female patient with AC on the lower lip who received 3 sessions of ALA-PDT with an interval of 1 week. Multiple noninvasive auxiliary tests including autofluorescence imaging, toluidine blue staining, and aneuploidy with DNA image cytometry (DNA-ICM) using brushing from screening through diagnosis, treatment, and follow-up. The patient successfully showed a complete response with no adverse effects and no evidence of recurrence at the 20-month follow-up. Noninvasive auxiliary tests assisted PDT is attractive and well-tolerated and may have synergistic effects against AC.

Evaluation of Static DNA Ploidy Analysis Using Conventional Brush Biopsy-Based Cytology Samples as an Adjuvant Diagnostic Tool for the Detection of a Malignant Transformation in Potentially Oral Malignant Diseases: A Prospective Study.

BACKGROUND: The accuracy of DNA image cytometry as an investigation method for potentially malignant disorders of the oral cavity is currently still a subject of controversy, due to inconsistently applied definitions of DNA aneuploidy, small cohorts and different application techniques of the method. The aim of this study was to examine the accuracy of the method as a supplementary diagnostic tool in addition to the cytological examination using internationally consented definitions for DNA aneuploidy. METHODS: A total of 602 samples from 467 patients with various oral lesions were included in this prospective study. Brush biopsies from each patient were first cytologically examined and categorized by a pathologist, second evaluated using DNA image cytometry, and finally compared to either histological biopsy result or clinical outcome. RESULTS: Using the standard definition of DNA aneuploidy, we achieved a sensitivity of 93.5%, a positive predictive value for the detection of malignant cells of 98.0%, and an area under the curve of 0.96 of DNA ploidy analysis for the detection of severe oral epithelial dysplasia, carcinoma in situ or oral squamous cell carcinoma. Importantly, using logistic regression and a two-step model, we were able to describe the increased association between DNA-ICM and the detection of malignant cells (OR = 201.6) as a secondary predictor in addition to cytology (OR = 11.90). CONCLUSION: In summary, this study has shown that DNA ploidy analysis based on conventional specimens of oral brush biopsies is a highly sensitive, non-invasive, patient-friendly method that should be considered as an additional diagnostic tool for detecting malignant changes in the oral cavity.

DNA aneuploidy combined with radial EBUS in the diagnosis of peripheral lung lesions.

OBJECTIVE: To explore the diagnostic value of DNA aneuploidy analysis combined with radial endobronchial ultrasound (R-EBUS)-guided transbronchial biopsy in peripheral lung lesions. METHOD: We performed a retrospective analysis of patients who underwent R-EBUS examination. DNA aneuploidy analysis of bronchial washing from the target bronchial segment were performed. The clinical information, R-EBUS data, pathological results and DNA image cytometry (DNA-ICM) results were collected. For patients who did not have a clear diagnosis after bronchoscopy, follow-up data was recorded. RESULTS: A total of 42 cases were included. Thirty patients had confirmed malignant tumor of the lung, 19 of which were confirmed by pathology after bronchoscopy, and 11 cases were confirmed later by surgery or percutaneous lung puncture. Twelve patients were finally considered to have benign lesions. The sensitivity of R-EBUS is 63.3% and the specificity is 100%. DNA-ICM has a sensitivity of 76.7% and a specificity of 91.7%. When combined, they have a sensitivity of 90%, and specificity 91.7%. As for malignant lesions, we further analyzed smoking, the size and location of lesions on chest CT, the number of aneuploid cells and the maximum value of DNA content. The results indicated that increased number of aneuploid cells or increased max value of DNA content may predict higher probability of malignancy. CONCLUSION: DNA-ICM combined with R-EBUS can improve the diagnostic sensitivity of malignant peripheral lung lesions. Increased number of aneuploid cells or increased max value of DNA content may indicate that the lesions are more likely to be malignant.

Relationship of DNA aneuploidy with distinctive features of oral potentially malignant disorders: A cytological analysis of 748 cases.

Our previous study reported that clinical features, including the lateral/ventral tongue and non-homogeneous lesions, were associated with increased risk of malignant changes in cytological samples from oral potentially malignant disorders (OPMDs). This cross-sectional study aimed to evaluate the frequency and risk of DNA aneuploidy in the series of 748 patients with OPMD. The cut-off value of aneuploidy was defined as DNA index ≥3.5. We found that the frequency of DNA aneuploidy was higher in OPMD patients >60 years old, and in those with lateral/ventral tongue sites, non-homogeneous lesions, and high-grade dysplasia, than in control group (P < 0.01). Consistently, the risk of aneuploidy occurrence was higher in patients >60 years old (1.69-fold; P = 0.022), in those with lateral/ventral tongue sites (2.35-fold; P < 0.001), and in those with high-grade dysplasia (3.19-fold; P < 0.001). Collectively, increased frequency and risk of DNA aneuploidy occurred in OPMD patients aged over 60 years with high-grade dysplasia located at the lateral/ventral tongue. These patients should be required to intensive management and follow-up.

Use of DNA image cytometry in conducting oral cancer screening in rural India.

OBJECTIVES: Oral cancer screening can assist in the early detection of oral potentially malignant lesions (OPMLs) and prevention of oral cancers. It can be challenging for clinicians to differentiate OPMLs from benign conditions. Adjunct screening tools such as fluorescence visualisation (FV) and DNA image cytometry (DNA-ICM) have shown success in identifying OPMLs in high-risk clinics. For the first time we aimed to assess these technologies in Indian rural settings and evaluate if these tools helped clinicians identify high-risk lesions during screening. METHODS: Dental students and residents screened participants in five screening camps held in villages outside of Hyderabad, India, using extraoral, intraoral, and FV examinations. Lesion and normal tissue brushings were collected for DNA-ICM analysis and cytology. RESULTS: Of the 1116 participants screened, 184 lesions were observed in 152 participants. Based on white light examination (WLE), 45 lesions were recommended for biopsy. Thirty-five were completed on site; 25 (71%) were diagnosed with low-grade dysplasias (17 mild, 8 moderate) and the remaining 10 showed no signs of dysplasia. FV loss was noted in all but one dysplastic lesion and showed a sensitivity of 96% and specificity of 17%. Cytology combined with DNA-ICM had a sensitivity of 64% and specificity of 86% in detecting dysplasia. CONCLUSION: DNA-ICM combined with cytology identified the majority of dysplastic lesions and identified additional lesions, which were not considered high-risk during WLE and biopsy on site. Efforts to follow-up with these participants are ongoing. FV identified most high-risk lesions but added limited value over WLE.

DNA image cytometry parameters to identify high-grade cervical lesions.

OBJECTIVE: Evaluate the performance of different DNA image cytometry (DNA-ICM) ploidy parameters in the categorisation of DNA-ICM results and identification of high-grade cervical intraepithelial neoplasia or worse (≥ CIN2). METHODS: Cervical samples from 232 women were collected for DNA-ICM analysis and biopsy confirmation. Five DNA parameters were used to define DNA aneuploidy: number of cells with exceeding events (EE) over 2.5cEE, 4cEE, 5cEE and 9cEE, and aneuploid stemlines. DNA-ICM results were categorised as normal, suspicious, and abnormal. RESULTS: For individual DNA ploidy parameters, sensitivity values for 50 cells with 2.5cEE, 45 cells with 4cEE, 1 cell with 9cEE and aneuploid stemline were 72.95%. 54.1%, 69.67% and 54.1%, while specificity values were 80.0%, 90.0%, 89.09% and 95.45%, respectively. For the 5cEE parameter, the sensitivity values for 1, 2, 3, 4 and 5 cells were 93.44%, 85.25%, 81.97%, 77.87% and 75.41%, while specificity values were 46.36%, 63.64%, 74.55%, 76.36% and 80.91%, respectively. For categorised DNA-ICM results, a suspicious result showed superior sensitivity than an abnormal result (87.70% vs 82.79%, P = 0.031), but lower specificity (54.55% vs 75.45%, P < 0.001). Both types of DNA-ICM result were statistically significantly different from a normal result (P < 0.05). CONCLUSION: For prognostic purposes, 1 cell with 9cEE, 45 cells with 4cEE and aneuploid stemline are the best parameters with which to categorise an abnormal DNA-ICM result, followed by 50 cells with 2.5cEE and 4 cells with 5cEE. For screening purposes, 10 cells with 2.5cEE, 10 cells with 4cEE, and 2 cells with 5cEE are suitable parameters with which to categorise a suspicious DNA-ICM result.

DNA image cytometric analysis of bronchial washings as an adjunct for the detection of lung cancer in a clinical setting.

BACKGROUND: DNA aneuploidy has a potential to become an adjunct to conventional cytology for diagnosis of lung cancer, but its value in bronchial washings has not been well evaluated. METHODS: We conducted a retrospective study on patients who underwent bronchoscopy and the bronchial washings were submitted for both cytology and DNA image cytometry (DNA-ICM) examination. The sensitivity and specificity of two methods and both in combination were compared. Analysis of clinical subgroups and DNA histogram were also performed. RESULTS: The study included 626 patients (326 patients with lung cancer and 300 patients with benign lung diseases). The sensitivity of cytology, DNA-ICM, and combination test for lung cancer were 53.3%, 62.3%, and 75.8%, respectively, and the sensitivity of DNA-ICM and combination test were superior to that of cytology (p < 0.05). A modest reduction of specificity was found in DNA-ICM compared with cytology (91.3% vs. 98.3%, p < 0.05). Subgroup analysis showed there was no significant difference in sensitivity of DNA-ICM between the visible tumor group and the invisible tumor group (66.5% vs. 56.9%, χ2  = 3.114, p = 0.078). Among 101 patients with invisible endobronchial tumor, the positive rates for DNA-ICM of washing, cytology of washing, brushing and biopsy were 62.4%, 41.6%, 40.6%, and 45.5%, respectively. DNA-ICM in combination with the basic bronchoscopy techniques could increase the sensitivity from 67.3% to 87.1% (p = 0.000). The DNA histogram analysis showed 25.3% washing samples of lung cancer were diploid pattern, 49.4% were scattered aneuploid cells pattern, and 25.3% were aneuploid peaks pattern. Small cell lung cancer had the highest proportion of aneuploid peaks pattern (p < 0.05). CONCLUSIONS: DNA-ICM could be used as an adjunct for the detection of lung cancer. The combination of DNA-ICM and basic bronchoscopy techniques could significantly increase the sensitivity, especially for the patients suspected of peripheral lung cancer, and contribute to select subjects for advanced bronchoscopy.

An improved algorithm using a Health Canada-approved DNA-image cytometry system for non-invasive screening of high-grade oral lesions.

BACKGROUND: DNA-image cytometry (DNA-ICM) is able to detect gross alterations of cellular DNA-content representing aneuploidy, a biomarker of malignancy. A Health Canada-approved DNA-ICM system, ClearCyte® in combination with a cytopathologist's review, has demonstrated high sensitivity (89%) and specificity (97%) in identifying high-grade oral lesions. The study objective was to create an improved automated algorithm (iClearcyte) and test its robustness in differentiating high grade from benign reactive oral lesions without a cytopathologist's input. METHODS: A set of 214 oral brushing samples of oral cancer (n = 92), severe dysplasia (n = 20), reactive lesions (n = 52), and normal samples (n = 50) were spun down onto slides and stained using Feulgen-Thionin reaction. Following ClearCyte® scan, nuclear features were calculated, and nuclei categorized into "diploid," "hyperdiploid," "tetraploid," and "aneuploid" DNA ploidy groups by the ClearCyte® software. The samples were randomized into training and test sets (70:30) based on patient's age, sex, tobacco use, and lesion site risk. The training set was used to create a new algorithm which was then validated using the remaining samples in the test set, where sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. RESULTS: The proposed iClearCyte algorithm (>1 "aneuploid" cell or ≥ 1.7% combined "hyperdiploid" and "tetraploid" nuclei frequency) identified high-grade samples with sensitivity, specificity, PPV, and NPV of 100.0%, 86.7%, 89.7%, and 100.0%, respectively, in the test set. CONCLUSION: The iClearCyte test has potential to serve as a robust non-invasive automated oral cancer screening tool promoting early oral cancer detection and decreasing the number of unnecessary invasive biopsies.

DNA ploidy measurement and human papillomavirus in abnormal cervical cytology.

OBJECTIVE: To compare the efficacy of high-risk human papillomavirus (HR-HPV) and DNA image cytometry (DNA-ICM) status for identifying high-grade cervical intraepithelial neoplasia or worse (≥CIN2). METHODS: This cross-sectional study was performed in women undergoing follow-up procedure after a previous abnormal cervical cytology. Cervical cells were collected for HPV detection and DNA ploidy measurement. Biopsy samples were taken for histological confirmation. Sensitivity and specificity values for ≥CIN2 detection with HR-HPV and DNA-ICM were determined. RESULTS: HR-HPV was present in 74.5% of the women. The most frequent HPV infection was HPV 16, followed by HPV 31, 33 and 58. Aneuploidy was observed in 60.6% of all cases. Referral cytology revealed 78.0% sensitivity and 68.6% specificity for detecting a ≥CIN2 lesion. The HR-HPV test alone showed 92.7% sensitivity, albeit it was not statistically different from DNA-ICM (88.1%, P > .05). Positivity for HPV or DNA-ICM resulted in 100% sensitivity. Higher specificity was observed for the combination of HR-HPV and DNA-ICM (88.6%), with no difference from DNA-ICM alone (85.7%, P > .05). CONCLUSION: DNA-ICM or HR-HPV positivity identified all cases of ≥CIN2 in women undergoing follow-up procedure after a previous abnormal cervical cytology. Routine cervical cancer screening could be improved by the incorporation of DNA-ICM as a complementary method to primary screening to identify which women need closer follow-up.

A Comparative Study of Liquid-Based Cytology and DNA Image Cytometry in the Diagnosis of Serous Effusion.

BACKGROUND: Liquid-based cytology is one of the most useful methods to diagnose a patient with serous effusion, especially when malignancy is suspected. As an alternative to the use of liquid-based cytology only, the serous effusion can be further processed using the technique of DNA image cytometry, which may augment diagnostic utility. The aim of this study was to compare the diagnostic yields of liquid-based cytology, DNA image cytometry, and both in combination, regardless of serous-effusion etiology. METHODS: We conducted a descriptive study on patients with serous effusions from July 2016 to June 2018. All samples were submitted for liquid-based cytology and DNA image cytometry techniques. We compared the results of cytopathological studies to the final diagnoses. RESULTS: For a total of 798 samples, final diagnoses included 412 (51.6%) malignancies, 280 (35.1.%) inflammatory diseases, and 106 (13.3%) transudative serous effusions. Liquid-based cytology had a more sensitive diagnostic yield than DNA image cytometry did (38.8% vs 30.7%; P < .05), but the combination of both had a higher yield (43.7%; P < .05) compared with that of liquid-based cytology alone. For the 412 malignant serous effusions, diagnostic yields of liquid-based cytology and DNA image cytometry were 73.8% and 59.5%, respectively. The difference in sensitivity was significant (P < .05). Combined liquid-based cytology + DNA image cytometry improved diagnostic yield to 83.3% (P < .05). However, both liquid-based cytology and DNA image cytometry had low diagnostic yields for inflammatory diseases and transudative serous effusions. CONCLUSION: In serous effusion, liquid-based cytology's diagnostic performance is better than that of DNA image cytometry. Application of both techniques can significantly increase diagnostic yield.

DNA aneuploidy with image cytometry for detecting dysplasia and carcinoma in oral potentially malignant disorders: A prospective diagnostic study.

BACKGROUND: Current evidence on diagnostic value of aneuploidy with DNA image cytometry (ICM) using brushings for oral potentially malignant disorders (OPMD) is limited by sample size and inconsistent classification criteria of aneuploidy. This study aimed to explore the optimal cut-off values of DNA content and evaluate the diagnostic accuracy of DNA-ICM for detecting dysplasia and/or carcinoma in OPMD. MATERIALS AND METHODS: A total of 401 consecutive patients with OPMD were enrolled in this prospective diagnostic study. Brushing and biopsy sample form each patient was processed by DNA-ICM and histological examination respectively. RESULTS: When the optimal cut-off of at least one aneuploid cell with DNA index (DI) ≥2.3, the area under the curves (AUC) was 0.735 and positive predictive value was 92.7% for detecting dysplasia within OPMD. When the optimal cut-off of at least one aneuploid cell with DI ≥ 3.5, the AUC was 0.851 and negative predictive values was 96.8% for detecting carcinoma within OPMD. Importantly, multivariate analysis revealed that aneuploidy with DI ≥ 2.3 in OPMD was significantly associated with dysplasia risk (adjusted OR, 5.52; 95%CI, 2.90-10.51; P < .001), and aneuploidy with DI ≥ 3.5 in OPMD was strongly associated with malignant risk (adjusted OR, 21.05; 95%CI, 9.34-47.41; P < .001). CONCLUSIONS: This largest-scale diagnostic study optimized the criteria of aneuploidy cytology for noninvasive detection of oral dysplasia and carcinoma within OPMD. DNA aneuploidy in OPMD was an independent marker that strongly associated with oral dysplasia/carcinoma. Our findings suggest that DNA-ICM may serve as a useful noninvasive adjunctive tool for oral cancer and OPMD screening.

DNA content abnormality in oral submucous fibrosis concomitant leukoplakia: A preliminary evaluation of the diagnostic and clinical implications.

Oral submucous fibrosis (OSF) is an insidious chronic oral and oropharyngeal potentially malignant disorder. Our previously studies reported that DNA image cytometry (ICM) using brushing added in the diagnosis of high-grade dysplasia and staging of oral leukoplakia (OLK). This preliminary study aimed to investigate the abnormal rate of DNA content in 30 cases of OSF including three cases of OSF concomitant OLK and evaluate their the diagnostic and clinical implications with special emphasis on the OSF concomitant OLK. Brushing and biopsy sample form each patient was processed by DNA-ICM and histological examination, respectively. The results of DNA-ICM analysis showed that all the lesions from the 27 patients with OSF only were identified as normal DNA content. Strikingly, two of three cases of OSF concomitant OLK were identified as abnormal DNA content, and one case of whom progressed to early oral squamous cell carcinoma at the follow-up of 31 months. Collectively, this preliminary evaluation revealed that DNA content abnormality was hardly observed in OSF only; however, it may be usually observed in OSF concomitant OLK, whom should be monitored on a priority basis for early detection of carcinoma. Multicenter large studies are needed to validate the findings of the current study.

DNA aneuploidy and tissue architecture in oral potentially malignant disorders with epithelial dysplasia assessed by a 10 locus FISH panel.

Subjectivity in oral dysplasia grading has prompted evaluation of molecular­based tests to predict malignant transformation. Aneuploidy detected by DNA image­based cytometry (ICM) is currently the best predictor but fails to detect certain high risk lesions. A novel multiplex fluorescence in situ hybridization (FISH) panel was used to explore possible explanations by detecting aneuploidy at the single cell level. FISH was compared to reference standard DNA ICM in 19 oral lesions with epithelial dysplasia and used to characterize the cellular architecture. Copy number variation at 3q28, 7p11.2, 8q24.3, 11q13.3 and 20q13.12 and matched chromosome specific loci were assessed by dual­color FISH to assess numerical and spatial patterns of copy number increase and gene amplification. FISH revealed wide variation in copy number at different loci. Only low level copy number gain was present and often in only a small proportion of cells, although usually with all or all but one locus (9/12). Four cases showed gene amplification, one at two loci. Some probes revealed an internal presumed clonal structure within lesions not apparent in routine histological examination. Both methods produced similar diagnostic results with concordance in detection of aneuploidy by both methods in 17 out of 19 samples (89%). We have shown that oral dysplastic lesions may contain very few aneuploid cells at a cellular level, high copy number gain is rare and changes appear to arise from large chromosomal fragment duplications. Single stem lines are relatively homogeneous for loci with copy number gain but there is a subclonal structure revealed by gene amplification in some lesions.

The role of DNA image cytometry in screening oral potentially malignant lesions using brushings: A systematic review.

It is believed that the majority of oral cancers develop from oral potentially malignant lesions (OPML). Though they can be easily detected during screening, risk stratification is difficult. During screening clinicians often find it difficult to distinguish OPMLs from benign lesions, and predicting OPML at risk of malignant transformation is particularly challenging. DNA aneuploidy has been known to be a marker of malignancy in a number of sites including the oral cavity. We performed a systematic review to evaluate the effectiveness of DNA-ICM using brushings in differentiating OPMLs from benign/inflammatory lesions during screening and in predicting malignant transformation. MEDLINE, Pubmed, EMBASE electronic databases were systematically searched using a combination of keywords and subject headings. A total of 11 articles satisfied our inclusion criteria. These studies reported a wide range of sensitivity (16-96.4%) and specificity (90-100%) due to the differences in study design, definitions of high risk or low risk lesions and DNA-ICM protocol used. No long-term longitudinal studies were identified to assess the role of DNA-ICM using brushings in predicting malignant transformation. No studies evaluated the role of DNA-ICM in community screening settings. A number of studies combined DNA-ICM with other techniques like cytology or argyrophilic nucleolar organizer region counts leading to improved test results. In spite of DNA aneuploidy being accepted as a marker of malignancy, there is limited evidence of DNA-ICM using brushings being successful as an adjunct oral cancer screening tool. Longitudinal studies and large community screening studies need to be undertaken to draw stronger conclusion.

Assessment of the cancerization risk for oral potentially malignant disorders by clinical risk model combined with autofluorescence and brush biopsy with DNA-image cytometry.

PURPOSE: To explore the feasibility of assessing the cancerization risk of oral potentially malignant disorders (OPMD) through a clinical risk model combined with autofluorescence and brush biopsy with DNA-image cytometry. METHODS: We collected the baseline clinical data of 269 patients; then, performed autofluorescence, brush biopsy with DNA-image cytometry and histopathological examination. Then, we obtained the significant factors by univariate logistic analysis, constructed the clinical risk model by multiple logistic regression and selected the optimal cutoff value according to the maximum Youden index. Finally, we calculated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the clinical risk score ≥ cutoff value, autofluorescence and brush biopsy with DNA-image cytometry, and plotted the receiver-operating characteristic (ROC) curves and decision curve analysis (DCA). RESULTS: The clinical risk model is represented by the formula: 1 × gender + 1.6 × age group + 1 × lesion site + 1.4 × local stimulus + 1.5 × drink. The area under the curve (AUC) was 0.83, and the optimal cutoff score was 3. The AUC indicated that the clinical risk score ≥ 3 (0.74) and autofluorescence (0.77) had a certain diagnostic values, while brush biopsy with DNA-image cytometry (0.92) displayed a good value. Besides, the DCA showed that all three tests had clinical significance. CONCLUSIONS: The cancerization risk of patients can be assessed by the clinical risk model combined with sequence application of autofluorescence and brush biopsy with DNA-image cytometry, to decide whether histopathological examination or other intervention measures should be selected.

The value of DNA image cytometry combined with brush routine cytology in diagnosing indeterminate biliary strictures: A large sample size retrospective study.

BACKGROUND AND AIM: Brush cytology is widely applied to diagnosis indeterminate biliary stricture but suffer from low sensitivity. Changes in DNA content are a character of malignant cell and can be detected by DNA image cytometry (DNA-ICM). The study aimed to estimate the value of routine cytology (RC), DNA-ICM, and their combination in diagnosing indeterminate biliary strictures. METHODS: A total of 362 patients who underwent both RC and DNA-ICM tests were analysed. Their results were retrospectively applied to final diagnoses. Diagnostic values were compared among RC, DNA-ICM, and their combination based on the location of strictures. RESULTS: The DNA-ICM and combination of two methods had higher diagnostic accuracy than RC in all strictures (63.3% vs 42.3%, P < 0.001, 64.36% vs 42.3%, P < 0.001) and in distal strictures (65.36% vs 42.81%, P < 0.001, 66.01% vs 42.81%, P < 0.001). But in proximal strictures, DNA-ICM showed no superior (51.8% vs 42.81%, P = 0.184). Combination of two methods was not fully significant superior to RC in proximal strictures (55.36% vs 39.29%, P = 0.089). After classification of "suspicious for malignancy" as positive for malignancy, the diagnostic accuracy of DNA-ICM was still higher than that of RC in all strictures (63.3% vs 51.9%, P = 0.002) and in distal strictures (65.36% vs 52.29%, P = 0.001). Combination of two methods was no superior to DNA-ICM alone (64.36% vs 63.3%, P = 0.757). The utilization of DNA-ICM was more accurate in distal strictures than in proximal strictures (65.36% vs 51.8%, P = 0.017). CONCLUSION: DNA-ICM is an objective and effective addition tool with RC, especially in distal strictures. The combination of DNA-ICM and RC showed no superior to DNA-ICM alone but could improve diagnostic accuracy to RC in proximal strictures although not fully significant.

Pathologic Features of Tumor Activity and Stability in Uveal Melanoma Specimens after Fractionated CyberKnife Radiosurgery.

To evaluate uveal melanoma cell activity and pathologic features after stereotactic CyberKnife radiosurgery in specimens from five patients. Specimens from five patients treated by CyberKnife radiosurgery in three fractions were included in this study. Because of persistent retinal detachment in 3 patients, tumour endoresection was performed at four, seven and ten month after CyberKnife radiosurgery. At nine and twelve months after treatment, enucleation of the eye globe was performed in 2 patients because of secondary tumour bleeding and missing regression. After histomorphological analysis and determination of Ki67-proliferation index, DNA cytophotometry, fluorescence in-situ hybridization evaluation for chromosome 3 loss, GNA11and GNAQ mutation analysis were performed. Four of the five tumours included in this study showed variable radiation-induced morphologic changes in the form of enlargement of cells and nuclei, cytoplasmic vacuolisation and nuclear fragmentation. The DNA content of a large fraction of tumour cells was hypoploid. On the other hand, single strikingly hyperchromatic melanoma cells showed marked aneuploidy. The proliferation fraction in the three endoresected tumours was very low (<1%), but it was elevated in the enucleation cases. Monosomy 3 was detected in two of the endoresection cases, but none of the enucleation cases. None of the patients experienced a local tumour recurrence, but two of the patients developed liver metastasis. Many melanoma cells seemed to be vital within the first 6 months after CyberKnife radiosurgery, but obvious radiation-induced morphologic changes, including tumour necrosis, hypoploid DNA content plus low Ki-67 index could indicate sublethal cell damage.