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.

High-resolution DNA content analysis of microbiopsy samples in oral lichen planus.

OBJECTIVES: DNA aneuploidy has been reported to be a predictor of poor prognosis in both premalignant and malignant lesions. In oral lichen planus (OLP), this hypothesis remains to be proved. This study aimed to determine the rate of occurrence of DNA aneuploidy in patients with OLP by high-resolution DNA flow cytometry. METHODS: Patients with OLP were consecutively enrolled. Tissue samples were subdivided for formalin fixation and routine histological assessment and for immediate storage at -20°C for later DNA ploidy analysis, which was performed by DAPI staining of the extracted nuclei and excitation with a UV lamp. The DNA aneuploid sublines were characterized by the DNA Index. RESULTS: A DNA aneuploid status was observed in two of 77 patients with OLP (2.6%). When considering the clinical aspect of the OLP lesions, both DNA aneuploid cases had a reticular clinical aspect. CONCLUSIONS: DNA aneuploidy is an uncommon event in OLP and less frequent compared to other non-dysplastic and non-OLP oral potentially malignant disorders. The extremely low rate of DNA aneuploidy could represent an occasional finding or reflect the low rate of malignant transformation observed in patients with OLP even if the real prognostic value of DNA ploidy analysis in patients with OLP remains to be confirmed.

SMFM Statement: clarification of recommendations regarding cell-free DNA aneuploidy screening.

The purpose of this statement is to clarify that the Society for Maternal-Fetal Medicine (SMFM) does not recommend that cell-free DNA aneuploidy screening be offered to all pregnant women, nor does it suggest a requirement for insurance coverage for cell-free DNA screening in women at low risk of aneuploidy. However, SMFM believes, due to the ethics of patient autonomy, that the option should be available to women who request additional testing beyond what is currently recommended by professional societies.

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.

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 aneuploidy identifies a subset of Luminal subtype breast carcinoma patients with worse clinical outcome.

AIM: In breast carcinoma (BC), the relationship between the ploidy pattern and molecular subtyping is still unknown. We aim to investigate the prognostic impact of DNA ploidy within molecular subtypes of a large cohort of BC patients. METHODS: The series involved 520 BC patients with no neoadjuvant therapy and a median follow-up of 115.5 months. Immunohistochemical assessment of hormonal receptors, ERBB2 (HER2) status and Ki67 proliferative activity was the basis of the surrogate molecular subtyping. Ploidy was evaluated by DNA flow cytometry in fresh/frozen tumour samples. Kaplan-Meier (K-M) survival estimation was used for prognostic statistical analysis. RESULTS: Luminal A subtype had the lowest prevalence of disease recurrences (23.6 %) and deaths from disease (18.3 %), while Luminal B (42.2 %/37.9 %), Triple-negative (46.4 %/40.6 %), and HER2-positive (55.9 %/50.0 %) subtypes had the highest. The Triple-positive subtype shows an intermediate/low frequency of adverse events (29.4 % of disease recurrences and 17.6 % of deaths from disease). Luminal A tumours were mostly diploid (55.3 %), while Triple-negative and HER2-positive tumours showed a high incidence of aneuploidy (82.6 % and 88.2 %, respectively). Luminal B and Triple-positive tumours had an intermediate percentage of aneuploidy (63.8 % and 70.6 %, respectively). K-M survival curves showed that DNA aneuploidy is significantly associated with shorter disease-free survival and overall survival in Luminal A and Luminal B molecular subtypes. CONCLUSION: DNA aneuploidy identifies a subset of Luminal BC patients with worse clinical outcome, potentially eligible for more aggressive adjuvant therapy.

Continuous artificial intelligence-assisted DNA aneuploidy cytology for surveilling dysplastic oral leukoplakia treated by photodynamic therapy.

Oral leukoplakia (OLK) is one of the most common potentially malignant disorders. High-risk lesions require early intervention before developing into oral cancer. Photodynamic therapy (PDT) is a noninvasive technique for premalignant lesions. Scalpel biopsy remains a reliable method for monitoring the prognosis of OLK, but it is an invasive procedure with poor reproducibility to suspicious lesions. DNA aneuploidy cytology by oral cytobrush has been proposed as a promising objective and noninvasive tool in screening and diagnosing premalignant and malignant lesions. Here, we discussed the significance of artificial intelligence (AI)-assisted DNA aneuploidy cytology by image cytometry (DNA-ICM) for surveilling non-homogeneous OLK with moderate-to-severe dysplasia that was treated by 5-aminolevulinic acid-mediated PDT (ALA-PDT). The present study provides a scheme of the sequential management and surveillance strategy for OLK.

Development and validation of a risk model for noninvasive detection of cancer in oral potentially malignant disorders using DNA image cytometry.

OBJECTIVE: To elucidate whether DNA aneuploidy was an independent discriminator for carcinoma within oral potentially malignant disorders (OPMDs), and further establish and validate a risk model based on DNA aneuploidy for the detection of oral cancer. METHODS: A total of 810 consecutive patients with OPMD were prospectively enrolled from March 2013 to December 2018, and divided into a training set (n = 608) and a test set (n = 202). Brushing and biopsy samples from each patient were processed by DNA-DNA image cytometry and histopathological examination, respectively. RESULTS: DNA aneuploidy of an outside DNA index ≥ 3.5 in OPMD was an independent marker strongly associated with malignant risk [adjusted odds ratio: 13.04; 95% confidence interval (CI): 5.46-31.14]. In the training and test sets, the area under the curve (AUC) was 0.87 (95% CI: 0.82-0.91) and 0.77 (95% CI: 0.57-0.97), respectively, for detecting carcinoma in OPMD patients. The independent risk factors of lateral/ventral tongue and non-homogenous type combined with a risk model built with a multivariate logistic regression revealed a more favorable diagnostic efficacy associated with the training set (AUC: 0.93; 95% CI: 0.91-0.96) and test set (AUC: 0.94; 95% CI: 0.90-0.98). The sensitivity and specificity of carcinoma detection within OPMD was improved to 100% and 88.1%, respectively. CONCLUSIONS: This large-scale diagnostic study established a risk model based on DNA aneuploidy that consisted of a noninvasive strategy with lateral/ventral tongue and non-homogenous features. The results showed favorable diagnostic efficacy for detecting carcinoma within OPMD, irrespective of the clinical and pathological diagnoses of OPMD. Multicenter validation and longitudinal studies are warranted to evaluate community practices and clinical applications.

Current evidence on DNA aneuploidy cytology in noninvasive detection of oral cancer.

DNA-aneuploidy cytology as a promising noninvasive tool in diagnosing oral precancer and cancer has been proposed in 2015. In this letter, we identified 9 studies on DNA aneuploidy cytology with special emphasis on using fresh tissue sample in detection of oral precancer and cancer. Evidence was updated as follows, for detection of OSCC in general oral lesions, the pooled sensitivity and specificity was 84.8 and 99.0 respectively; for discrimination of dysplasia and OSCC form oral lesions, the sensitivity and specificity was 75.7 and 76.8 respectively. On the whole, current evidence on the theme is not robust, and multicenter prospective studies are needed to consolidate the evidence.

Flow cytometry aneuploidy and cell cycle indexing as a possible tool for differentiating between CD10+ diffuse large B-cell lymphoma and follicular lymphoma.

BACKGROUND: Differential diagnosis between diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) becomes a challenge when adequate biopsy is unavailable. The present study aimed to investigate the applicability of DNA cell cycle analysis by flow cytometry (FC) for differentiating between CD10+ DLBCL and FL. METHODS: Data were collected from 57 specimens where CD5- /CD10+ /light chain restricted B cells were detected. DNA staining was performed using the Coulter DNA Prep Kit. Cell cycle fractions were evaluated by automatic analysis using the ModFit LT software. RESULTS: Histopathological analysis confirmed the diagnosis of CD10+ FL in 30 specimens (52.6%), CD10+ DLBCL in 24 specimens (42.1%), and CD10+ Burkitt lymphoma in 3 specimens (5.3%). A significantly higher rate of DNA aneuploidy was detected among CD10+ DLBCL than FL specimens (50 vs. 13.3% respectively, p = .003). Likewise, DNA index was significantly higher in CD10+ DLBCL relative to FL (1.26 ± 0.35 vs. 1.04 ± 0.16 respectively, p = .004). Notably, the proportion of cells in the S-phase and proliferative fraction was significantly higher in CD10+ DLBCL than in CD10+ FL (S-phase: 15.97 ± 13.94 vs. 4.43 ± 4.41 mean ± SD, respectively, p < .0001; proliferative fraction: 18.87 ± 15.17 vs. 5.78 ± 7.04 mean ± SD, respectively, p = .0001). Using a receiver operating characteristic analysis, optimal cutoffs for S-phase ≥7% and proliferative fraction ≥9% were determined. These values could be used to differentiate between CD10+ DLBCL and CD10+ FL. CONCLUSION: Including DNA cell cycle analysis in the FC lymphoma assessment panel may be of diagnostic value in differentiating between CD10+ DLBCL and FL when adequate biopsy is unavailable.

DNA aneuploidy and centrosome amplification in canine tumor cell lines.

DNA aneuploidy, the altered DNA content of a cell, is a common feature of canine tumors. However, it is unclear whether aneuploid DNA in canine tumor cells show centrosome amplification (CA), which contributes to numerical and structural chromosome aberrations that result in DNA aneuploidy. Here, we evaluated whether DNA aneuploidy and CA occur concurrently in canine tumor cell lines. Centrosome numbers were evaluated in 18 canine tumor cell lines by immunocytochemistry with anti-γ-tubulin antibody, and DNA content was evaluated by flow cytometry using propidium iodide. A total of 15 cell lines showed DNA aneuploidy, and CA was observed in 5 of these 15 cell lines. Together, our results suggest that DNA aneuploidy in canine tumor cells might be explained at least in part by CA. In addition, cell lines with CA may be useful tools to examine the detailed relationship between CA and DNA aneuploidy and the molecular mechanism of CA in canine tumor.

Intraoperative Flow Cytometry Enables the Differentiation of Primary Central Nervous System Lymphoma from Glioblastoma.

OBJECTIVE: Accurate preoperative and intraoperative differentiation between primary central nervous system lymphoma (PCNSL) and glioblastoma (GBM) is sometimes difficult. Distinguishing between these tumors during surgery is important because surgical treatment is different between the 2 tumors. In this study, we established a new method of intraoperative differentiation between PCNSL and GBM using intraoperative flow cytometry (iFC), and we retrospectively tested whether iFC was useful for the intraoperative diagnosis of PCNSL and GBM. METHODS: We analyzed the iFC data of 250 patients (28 with PCNSL and 222 with GBM) and then evaluated aneuploidy and S-phase population. RESULTS: Aneuploidy was detected in 54.5% of GBM cases but in only 7.14% of PCNSL cases. Aneuploidy indicated GBM, but it was difficult to distinguish PCNSL from GBM when a tumor had a diploid pattern. Thus, for tumors without aneuploidy, we evaluated the S-phase population: S2, the ratio of the average height of the S-phase to the height of the diploid peak. S2 was significantly higher in PCNSL than in GBM. Based on these results, we established an algorithm for differentiating between PCNSL and GBM using DNA aneuploidy and S2. Comparing this new iFC algorithm and the permanent pathologic diagnosis, the sensitivity was 89.3%, the specificity was 93.7%, and the accuracy was 93.2%. CONCLUSIONS: iFC is useful for the intraoperative differentiation between PCNSL and GBM and it aids in intraoperative decision making within a short time. The accuracy of intraoperative diagnosis of these tumors seems to be higher with the combination of iFC and intraoperative rapid pathologic diagnosis.

Evaluation of DNA ploidy with intraoperative flow cytometry may predict long-term survival of patients with supratentorial low-grade gliomas: Analysis of 102 cases.

OBJECTIVE: The objective of the present study was evaluation of the prognostic significance of DNA ploidy assessed with intraoperative flow cytometry (iFC) during resection of low-grade gliomas (LGG). PATIENTS AND METHODS: Retrospective analysis of 102 consecutive cases of newly diagnosed WHO grade II supratentorial gliomas, surgical removal of which was accompanied by iFC, was done. There were 46 diffuse astrocytomas (DA) and 56 oligodendrogliomas (OD). According to iFC, 68 tumors (67%) were considered non-aneuploid and 34 (33%) aneuploid. Median extent of resection (EOR) was 95% (mean, 89.0 ±â€¯14.5%). Postoperative FRT with or without adjuvant chemotherapy was administered in 24 cases (24%). RESULTS: With median follow-up of 29.9 months (range, 9.4-66.9 months), neither median overall survival (OS) nor median progression-free survival (PFS) were reached. The 3-year actuarial OS and PFS rates were 95.8% and 86.3%, respectively. Non-aneuploid DNA histogram type of the tumor was associated with significantly longer OS both in univariate (P = 0.0094) and multivariate (P = 0.0232) analyses, and with longer PFS in univariate analysis (P = 0.0184). Aneuploidy was encountered more frequently in DA, than in OD (43% vs. 25% of cases; P = 0.0488). Tumor progression was noted in 15 DA and 4 patients succumbed to the disease; in this subgroup the main unfavorable prognostic factors for OS and PFS were presence of aneuploidy (P = 0.0510) and MIB-1 index ≧3.9% (P = 0.0141), respectively. Aneuploid DA more frequently progressed to glioblastoma than to anaplastic astrocytoma, but this difference did not reach statistical significance (P = 0.1362). In contrast, only one OD progressed (non-aneuploid neoplasm), and no one patient with such tumor died of the disease. CONCLUSIONS: DNA ploidy assessed with iFC may be effectively used as prognostic indicator in cases of LGG, especially of DA. Aneuploid tumors demonstrate more aggressive clinical course translated into shorter OS of patients. Thus, their detection during surgery may be helpful for decision on the optimal EOR, and for choice of the most appropriate postoperative adjuvant therapy.