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Chemical platforms for isolation and enumeration of Circulating Tumor Cells (CTCs) in advancing the relevance of liquid biopsy in cancer diagnostics and treatment.

e15541 Background: Head and neck squamous cell carcinoma is leading cancer in the India with Oral squamous cell carcinoma (OSCC) as the most frequent subtype. OSCC is classified as a locoregional disease and its increased frequency is attributed to lack of good biomarkers compared to other epithelial cancers. At the time of diagnosis, above 50% of cases present the manifestation of advanced-stage disease, and are predisposed to disease failure in spite of appropriate treatment. Thus, early diagnosis of OSCC can significantly reduce the disease burden. Here we describe regulatory approved method to establish Circulating tumor cells (CTCs) presence in OSCC Indian patients and its positive correlation with various clinicopathological parameters, suggesting the potential use of CTCs as a significant parameter to stratify oral cancer with respect to the disease advancement. Methods: In a cross-sectional observational study, 230 OSCC patients at the different pathological stage of the disease and treatment mode were enrolled. CTCs were isolated using approved OncoDiscover liquid biopsy technology (Drug controller general of India approved), platform technology based on immunomagnetic CTC enumeration. CTCs were detected for CK18 presence and well-defined, DAPI-stained nuclei. Enumerated CTC subsequently analyzed for various clinic-pathological parameters such as pstage, extra-capsular spread (ECS), lymphovascular emboli (LVE), perineural invasion (PNI) and depth of invasion (DOI). CTC cut off values were obtained to differentiate early vs advanced stages with respect to different clinical stages and parameters. Results: CTCs of OSCC patients correlated positively with the cancer stages (clinical as well as pathological) as well as aggressive pathological features. The presence of aggressive pathological features that often suggest the poor outcome of the disease, we observed a 25-50 % increase in CTC number. Early stage, treatment naïve patients had lower number of CTCs. Mean CTC number in advanced-stage patients was 50 % higher than early-stage OSCC patients. Conclusions: Considering a positive correlation of CTC number with various pathophysiological features, CTC can be contemplated as a reliable parameter to predict the disease outcome in oral cancer. The consistent presence of CTC across all disease stages also suggests a probable nature of OSCC as a biological systematic disease. Clinical trial information: CTRI/2018/03/012905.

e18011 Background: Tobacco consumption accounts for 1.6 million deaths annually in the South East Asia Region (SEAR). Notably, amongst 10-20% of the global population consuming the betel quid and tobacco, about 81% concentration is in SEAR regions, including India and Bangladesh. The prevalence of HNSCC in these regions is rising alarmingly. For example, HNCs account for 23% of total 156775 cancer incidences in Bangladesh. Liquid biopsy tools are unavailable and expensive for most patients in this region. However, early cancer detection using tumor biomarkers, for example, Circulating Tumor Cells (CTCs) is highly implicated. Furthermore, such biomarkers are being validated and have potential for screening of high-risk patients, such as genetic predisposition, tobacco consumption, etc. We report the first observational study in HNSCC patients in Bangladesh correlating the presence of CTCs to chronic tobacco consumption. Methods: The study involved 70 cancer patients and 10 healthy volunteers (no prior cancer history). 87% of the patients had a specified history of chronic tobacco consumption. CTCs were isolated in 1.5 ml of blood using OncoDiscover Liquid Biopsy Test, which is clinically approved by the Drug Controller General of India, that contains an enriching anti-EPCAM antibody immunomagnetic kit. CTCs are qualified as CK18+, DAPI+and CD45-. Subsequently, CTCs were imaged using Zeiss Axio Observer 7 and quantified for Mean Fluorescence Intensity (MFI) for clinicopathological parameters; age/gender, HNSCC sub-population, and CTC distribution. Results: This is the 1st study on Bangladesh phenotype accounting for the presence of CTCs in HNSCC patients. In this population, 34 males (66%) and 10 females (52%) accounted for 91 CTCs. CTC distribution was 0 to 6 with mean and median ̃ 2.02 and 2, respectively. 25 patients (17 males, 8 females) were negative for any CTCs. Interestingly, 02 patients exhibited CTC clusters indicative of aggressive metastasis in which 01 patient had no prior tobacco usage or family cancer history. There was no correlation between CTC presence in males (66 %) and females (52 %). Healthy volunteer samples exhibited no false positives. The MFI values ranged between 23 and 766, with mean and median MFI values were 157 and 96, respectively, indicative of CK overexpression on CTCs of HNSCC patients. Conclusions: HNSCC patients with a history of chronic tobacco consumption in Bangladesh correlated the presence of CTCs in 64 % of the cases. Prospectively, CTCs may be validated as a biomarker for screening chronic tobacco users in Bangladesh to detect early cancers and HNSCC. Clinical trial information: BMRC/Grants/2018/99 (1-100).

e15043 Background: The presence of circulating tumor cells (CTC) in the vascular system is a tell-tale signature of metastasis in epithelial origin cancers including lung, breast, colorectal and head and neck cancers. Noteworthy, about 90% of cancer deaths are due to the progression of metastasis. Yet, cancer therapy is focussed on inhibiting tumour growth and there is a paucity of options that target metastasis. We demonstrate the POP ‘device’ that removes circulating tumour cells (CTC) from a patient’s blood to reduce the metastatic progression and improve overall survival. Methods: We designed, multi-component glass beads enriched antibody EpCAM conjugate substrates as POP blood fluidic device. We characterized the substrate and accounted for the biocompatibility using whole blood of healthy volunteers. We evaluated, the acute toxicity of substrates using rat (Wistar Albino) whole blood (CPCSEA registration number: 941/PO/Re/S/06/CPCSEA; 31/07/2019) and further studied major histopathological tissues for any toxicity. Finally, we evaluated 06 cancer patients whole blood (1.5 mL) for capturing and for the elimination of CTCs. The captured cells were immuno-stained, and the optimal fluorescence acquisition intensity was critically quantified in accounting CK18 protein over-expression. Results: The multi-component antibody EpCAM based substrate exhibited efficient CTC capture ability with a mean capture efficiency ranging from 40% to 100 % when compared to the OncoDiscover CTC test approved by CDSCO/ drug controller general of India (DCGI). Furthermore, the substrate indicated high biocompatibility primarily exhibited by the absence of haemolysis on whole human blood. Additionally, the preliminary animal experiments in rats showed a 100% survival rate and negligible toxicity to major organs. Conclusions: Removal of circulating tumor cells as a therapeutics is highly implicated in improving the overall survival of epithelial cancer patients.