A smart tele-cytology point-of-care platform for oral cancer screening.

Early detection of oral cancer necessitates a minimally invasive, tissue-specific diagnostic tool that facilitates screening/surveillance. Brush biopsy, though minimally invasive, demands skilled cyto-pathologist expertise. In this study, we explored the clinical utility/efficacy of a tele-cytology system in combination with Artificial Neural Network (ANN) based risk-stratification model for early detection of oral potentially malignant (OPML)/malignant lesion. A portable, automated tablet-based tele-cytology platform capable of digitization of cytology slides was evaluated for its efficacy in the detection of OPML/malignant lesions (n = 82) in comparison with conventional cytology and histology. Then, an image pre-processing algorithm was established to segregate cells, ANN was trained with images (n = 11,981) and a risk-stratification model developed. The specificity, sensitivity and accuracy of platform/ stratification model were computed, and agreement was examined using Kappa statistics. The tele-cytology platform, Cellscope, showed an overall accuracy of 84-86% with no difference between tele-cytology and conventional cytology in detection of oral lesions (kappa, 0.67-0.72). However, OPML could be detected with low sensitivity (18%) in accordance with the limitations of conventional cytology. The integration of image processing and development of an ANN-based risk stratification model improved the detection sensitivity of malignant lesions (93%) and high grade OPML (73%), thereby increasing the overall accuracy by 30%. Tele-cytology integrated with the risk stratification model, a novel strategy established in this study, can be an invaluable Point-of-Care (PoC) tool for early detection/screening in oral cancer. This study hence establishes the applicability of tele-cytology for accurate, remote diagnosis and use of automated ANN-based analysis in improving its efficacy.

Prognostic determinants of locally advanced buccal mucosa cancer: Do we need to relook the current staging criteria?

OBJECTIVES: Current guidelines advocate non-surgical treatment for T4b buccal mucosa carcinoma with surgery preferred in other stages. We investigated oncologic outcomes of this cohort in comparison with T4a cohort, treated by similar multi-modality approach of primary surgery followed by adjuvant treatment and identified prognostic determinants of survival. MATERIALS AND METHODS: Oncologic outcome of prospectively accrued 282 patients with cT4a and cT4b buccal mucosa squamous cell carcinoma were evaluated for overall survival (OS) and disease free survival (DFS) at 2 years of the whole cohort and for the subgroups of T4a and T4b patients. Multivariate Cox proportional hazards regression analysis was performed to identify prognostic determinants. RESULTS: Of 277 eligible patients treated and followed for a median period of 21 months, the OS was comparable between T4a and T4b as 64% vs 58%, (p = 0.354). The DFS between the two subgroups was 64% vs 61%, (p = 0.316). Although there was 47% pathologic down staging from the clinical stage, there was no significant difference in oncologic outcome between pT4a and pT4b (OS, 57% vs 58% for T4a and T4b, p = 0.687; DFS, 58% vs 60% for T4a and T4b, p = 0.776). On multivariate analysis, extra capsular spread (p = 0.042), lateral pterygoid muscle involvement (p = 0.035) and defaulting adjuvant treatment (p < 0.001) were independent predictors of outcome for the T4b cohort when other factors were controlled. CONCLUSIONS: Primary surgery followed by adjuvant chemo-radiotherapy offers comparable results in selected T4b gingiva and buccal mucosal cancer, suggesting the need to relook the staging criteria for oral cavity cancer.

Cancer stem cells and fibroblast niche cross talk in an in-vitro oral dysplasia model.

Understanding the cellular interactions during oral carcinogenesis has the potential to identify novel prognostic and therapeutic targets. This study aimed at investigating the cancer stem cell (CSC)-fibroblast niche interactions using in-vitro dysplastic cell line models developed from different stages of 4NQO-induced oral carcinogenic mice model. The spontaneously transformed epithelial cells (DysMSCTR6, 14 and 16) were developed from three time points (mild/moderate/severe), while two fibroblast cell lines (FibroMSCTR12, 16) were developed from moderate and severe dysplastic tissue. The epithelial (Epcam+/Ck+) and the fibroblast cell lines (Vimentin+/α-SMA+/Ck-) were authenticated and assessment of cells representing progressive grades of dysplastic severity indicated a significant increase in dysplastic marker profile (P < 0.05). Evaluation of the CSC characteristics showed that an increase in expression of Cd133, Cd44, Aldh1a1, Notch1, and Sox2 was accompanied by an increase in migratory (P > 0.05) and colony formation capacity (P > 0.005). Targeting Notch1 (GSI inhibitor PZ0187; 30 µM), showed a significant reduction in cell proliferation capacity (P < 0.05) and in the dysplastic marker profile. Further, Notch1 inhibition resulted in down regulation of Cd133 and Aldh1a 1 (P < 0.05) and a complete abrogation of colony formation ability (P < 0.0001). The effect of niche interactions evaluated using FibroMSCTR12-conditioned media studies, revealed an enrichment of ALDH1A1+ cells (P < 0.05), induction of spheroid formation ability (P < 0.0001) and increased proliferation capacity (3.7 fold; P < 0.005). Although PZ0187 reduced cell viability by ∼40%, was unable to abrogate the conditioned-media induced increase in proliferation capacity completely. This study reports a Notch-1 dependent enrichment of CSC properties during dysplastic progression and a Notch-1 independent dysplastic cell-fibroblast interaction during oral carcinogenesis.

Cancer stem cell and its niche in malignant progression of oral potentially malignant disorders.

OBJECTIVE: The purpose of this study was to determine association between cancer stem cells (CSCs) and their niche with progression of oral potentially malignant disorders. MATERIALS AND METHODS: Patients with histologically confirmed oral potentially malignant disorders, stratified into high/low risk lesions based on the degree of dysplasia and oral cancer were included in this study. Immunohistochemical profiling of markers of CSCs (CD44), endothelial cells (CD31) and CSC-vascular niche cross-talk (CXCR4 and SDF1) were carried out. Statistical analysis was performed to correlate the relationship of markers with histopathology grade (ANOVA, and χ2 test, unpaired t test) using GraphPad InStat v3.06. RESULTS: The study included 550 samples (349 patients) and analysis showed progressive increase in expression levels of CSC and its niche markers with increase in grade of dysplasia as compared to the normal cohort (p < 0.05). Co-expression analysis revealed that, in comparison to the normal cohort, a larger percentage of patients showed increased expression of CD31 and CD44 (CD31high/CD44high; p < 0.05) and of CXCR4 and SDF1 (CXCR4high/SDF1high; p = 0.04), suggesting an association of the CSCs and the vascular niche. Further, distribution of patients with CD44high/CXCR4high (p < 0.05) and CD31high/SDF1high (p = 0.01) was significantly increased in the high-risk group (18%), suggesting a correlation between CD44+/CXCR4+ cells, the vascular niche and progression of oral dysplastic lesions. CONCLUSION: The increased expression of CSCs, the vascular niche and their cross talk markers are associated with increase in severity of dysplasia suggesting their role in the progression of oral potentially malignant disorders and may hence be used in identifying high-risk OPMD.

Mobile microscopy as a screening tool for oral cancer in India: A pilot study.

Oral cancer is the most common type of cancer among men in India and other countries in South Asia. Late diagnosis contributes significantly to this mortality, highlighting the need for effective and specific point-of-care diagnostic tools. The same regions with high prevalence of oral cancer have seen extensive growth in mobile phone infrastructure, which enables widespread access to telemedicine services. In this work, we describe the evaluation of an automated tablet-based mobile microscope as an adjunct for telemedicine-based oral cancer screening in India. Brush biopsy, a minimally invasive sampling technique was combined with a simplified staining protocol and a tablet-based mobile microscope to facilitate local collection of digital images and remote evaluation of the images by clinicians. The tablet-based mobile microscope (CellScope device) combines an iPad Mini with collection optics, LED illumination and Bluetooth-controlled motors to scan a slide specimen and capture high-resolution images of stained brush biopsy samples. Researchers at the Mazumdar Shaw Medical Foundation (MSMF) in Bangalore, India used the instrument to collect and send randomly selected images of each slide for telepathology review. Evaluation of the concordance between gold standard histology, conventional microscopy cytology, and remote pathologist review of the images was performed as part of a pilot study of mobile microscopy as a screening tool for oral cancer. Results indicated that the instrument successfully collected images of sufficient quality to enable remote diagnoses that show concordance with existing techniques. Further studies will evaluate the effectiveness of oral cancer screening with mobile microscopy by minimally trained technicians in low-resource settings.

Curcumin and metformin-mediated chemoprevention of oral cancer is associated with inhibition of cancer stem cells.

Effective chemoprevention is critical for improving outcomes of oral cancer. As single agents, curcumin and metformin are reported to exhibit chemopreventive properties, in vitro as well as in patients with oral cancer. In this study, the chemopreventive efficacy of this drug combination was tested in a 4-nitro quinoline-1-oxide (4NQO) induced mice oral carcinogenesis model. Molecular analysis revealed a cancer stem cell (CSC)-driven oral carcinogenic progression in this model, wherein a progressive increase in the expression of CSC-specific markers (CD44 and CD133) was observed from 8th to 25th week, at transcript (40-100-fold) and protein levels (P ≤ 0.0001). Chemopreventive treatment of the animals at 17th week with curcumin and metformin indicated that the combination regimen decreased tumor volume when compared to the control arm (0.69+0.03 vs 6.66+2.4 mm3 ; P = 0.04) and improved overall survival of the animals (P = 0.03). Assessment of the molecular status showed an overall downregulation of CSC markers in the treatment arms as compared to the untreated control. Further, in vitro assessment of the treatment on the primary cells generated from progressive stages of 4NQO-induced mice tissue showed a concordant and consistent downregulation of the CSC markers following combination treatment (P < 0.05). The treatment also inhibited the migratory and self-renewal properties of these cells; the effect of which was prominent in the cultures of early dysplastic tissue (P < 0.002). Collectively, our observations suggest that the combination of curcumin and metformin may improve chemopreventive efficacy against oral squamous cell carcinoma through a CSC-associated mechanism.

Improving Bethesda Reporting in Thyroid Cytology: A Team Effort Goes a Long Way and Still Miles to Go .

CONTEXT: Fine-needle aspiration cytology is the first step in evaluation of thyroid nodules. Although the Bethesda classification for reporting thyroid cytology has been purported that this uniformity in reporting cytology thereby facilitating clinical decision-making, there are also studies indicating that the reporting percentage and the rates of malignancy in each category vary considerably from center to center making the clinical decision more difficult. AIM AND MATERIALS AND METHODS: We looked at our retrospective cytology and histopathology data of thyroid nodules operated between 2012 and 2014 and then prospectively collected data during 2015-2016. In the prospective arm, for every thyroid nodule that was sampled, there was a discussion between the endocrinologist and the cytopathologist on the risk of thyroid cancer (based on the patient's history, examination findings, sonographic pattern, and the cytological appearance). RESULTS: We noted that there was considerable improvement in reporting standards with the rates of nondiagnostic cytology dropping from 11% to 5%, an increased reporting of Bethesda Category 2 and 6 which are the definitive strata of benign and malignant nodules (38% to 41% in Category 2 and 7% to 11% in Category 6) with a high specificity (100%). There was a decline in numbers of Category 4 and 5 (13% to 9% in Category 4 and 12% to 3% in Category 5). The reporting prevalence of Category 3 increased from 19% to 27%. CONCLUSIONS: We conclude that a team approach between the clinician who performs the ultrasound and the reporting cytopathologist improves Bethesda reporting, its predictive value, and thus potentially avoiding unnecessary thyroidectomies in benign thyroid nodules and hemithyroidectomies in thyroid cancers.