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Background: Small molecular inhibitors and immunotherapy has emerged as a novel alternative treatment regime for a variety of epithelial cancers. Large number of clinical trials are in progress to gauge efficacy of tyrosine kinase inhibitors or immune checkpoint inhibitors against actionable targets such as receptor tyrosine kinases and program death ligand 1 (PD-L1). Although highly effective, the outcome of PD-L1 based ICI or TKI against RTKs is vitally contingent on the presence of PD-L1 or RTK expression on circulating tumor cells. We report validation of CTCs with PD-L1, HER2 and EGFR expression in different epithelial cancers. Methods: We retrospectively evaluated 134 carcinoma patients blood for presence of CTCs expressing PD-L1, HER2 or EGFR markers respect. 45 % patients had lung cancer, while 25 % and 20 % were presented with breast and GI and CRC malignancies. Remaining were gallbladder, ovarian, prostate, and head and neck cancers. CTCs were isolated from DCGI approved OncoDiscover platform with immunomagnetic targeting of EpCAM. CTCs were confirmed with expression of CK18, absence of CD45 and presence of DAPI nucleus. Presence/absence of biomarkers was determined using fluorescence imaging. Expression of PD-L1, HER2 or EGFR was detected by fluorescence microscopy using fluorescently labelled anti PD-L1, HER2 or EGFR antibodies respect. Based on fluorescence intensity CTCs were binned as PD-L1, HER2 or EGFR negative for no detectable fluorescence signal or weakly or strongly positive based on low or high fluorescence signal. Results: Among the evaluated cohort, 51% of CTCs showed presence of PD-L1 expression. While 63% showed HER2 positive CTCs (breast cancer). 20% from the PD-L1 positive population showed stronger PD-L1 expression. 78% of CTCs from lung cancer patients showed presence of detectable PD-L1 signal, while 66% breast, GI and CRC patients showed CTCs with PD-L1 expression. CTCs from HNC and gall bladder cancer patients showed low PD-L1 expression (25% and 50% respect.). Among CTCs originating from different cancer types, breast cancer CTCs showed higher mean expression of PD-L1 compared to CTCs from CRC patients. A clear subset of CTCs for PD-L1 and Her2 expression was observed in lung and breast cancer patients respect, suggesting the heterogeneity in expression or presence of different subclones within the same tumor type. CTCs evaluated for EGFR expression, 50% showed presence of detectable EGFR compared to the cut-off signal. Conclusions: CTC can be used as a real-time surrogate for molecular profiling of PD-L1, HER2 and EGFR expression. These CTC cell surface markers offer alternative for immunotherapy or targeted therapies decisions in a adenocarcinomas. Citation Format: Jayant Khandare, Atul Bharde, Sreeja Jayant, Gourishankar Aland, Meghana Garbhe, Sayali Gosavi, Apoorva Janorkar, Purva Tikekar, Mrunmayi Patil, Vikas Jadhav, Ganesh Khutale, Amrut Ashturkar, Aravindan Vasudevan, Kumar Prabhash, Nirmal Raut, Pankaj Chaturvedi. Detection of PD-L1, HER2 and EGFR on circulating tumor cells in carcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6684.

Comprehensive CtDNA and CTC profiling of treatment naïve early-stage head and neck cancer patients reveals early signatures of disease progressionBackground: We performed comprehensive ctDNA analysis on early-stage HNC patients in a pilot study to determine the mutational landscape in HNC patients with a known tobacco history. Methods: We analysed ctDNA of 18 early-stage HNC patients for genomic landscapes using the illumina NextSeq 2000 NGS. A custom-designed OncoIndex gene panel was used for the hybrid capture target-enrichment of critical cancer genes. Panel was designed to detect cancer targeting exonic sequence of 600 genes reporting SNVs and indels along fusions and copy number amplification. The gene panel detected genome-wide signatures including bTMB, MSI (microsatellite instability), HRD (homologous recombination deficiency) prediction and calculate cfDNA tumor fraction. Results: 80 % patients showed presence of at least one CTC in peripheral blood, possibly indicating the progressive disease at the time of presentation. Comprehensive genomic profile obtained from plasma cfDNA of early-stage HNC cancer patients predominantly had low bTMB and MSI Scores (99 % patients). However, HRD and LOH matrix was high for 60 % patients indicating highly dysregulated DNA repair activities. Concurring to these observations, 98 % patients had mutations in key tumor suppressor and DNA damage response (DDR) genes possibly resulting in their loss of function. Besides DNA damage pathway, 60% patients harboured alterations in RTK genes including FGFR, EGFR and PDEGFR family and 32% patients showed activating mutations in Erk1 and its upstream regulators. MSH2 was the most prominently mutated gene (37%) followed by FGFR (32%). Surprisingly, unlike HPV positive advanced HNC cases, TP53 mutations were not detected in any patient, though alterations in TS genes were most prevalent in the study population. 51% alterations resulted into truncated proteins possibly impairing their functions, while 42% alterations were point mutations, 6 % were frameshift and 1 % indels. Presence of mutations in BRAF, PDGFR, FGFR and KIT genes suggested for the potential therapy resistance. Tumor fraction representing ctDNA showed elevated range from 20 % to 45 % with a corresponding ploidy between 2 to 4. Conclusions: Comprehensive ctDNA profile showed major gene alterations in TS and DDR response pathway genes besides mutations in proliferative signaling members. TP53 mutation was not detected, although critical tumor suppressor and DDR genes were predominantly mutated, suggesting for a unique mutation pattern associated with early-stage HNC due to tobacco etiology. Our results suggest that comprehensive ctDNA analysis along CTC profiling can predict the disease progression beforehand and may offer new treatment options to early-stage HNC patients. Citation Format: Gowhar Shafi, Atul Bharde, Fauzul Moubeen, Kanchan Hariramani, Alain D’Souza, Trupti Kad, Bhagwat Jadhav, Sangita Prajapati, Aditi Rani, Madhura Basavalingegowda, Mohan Uttarwar, Gourishankar Aland, Sreeja Jayant, Aravindan Vasudevan, Pankaj Chaturvedi, Jayant Khandare. Comprehensive circulating tumor DNA and CTC profiling of treatment naïve early-stage head and neck cancer patients reveals early signature of disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3379.

Background: Genomic profiling has revolutionized precision oncology impacting the diagnosis, prognosis, and therapy decisions. Considering high spatiotemporal diversity and heterogenicity of breast tumor-cell genomes, small-gene panels often fail to capture rare but important genomic alterations. Conversely, comprehensive ctDNA sequencing approaches enable the identification of under characterized ‘long tailed driver’ genomic alterations and capture Intra and inter metastatic heterogeneity. Here, we demonstrate the clinical utility of comprehensive genome profiling with higher sensitivity to predict the possibility of metastasis in early-stage breast cancer patients. Methods: We retrospectively analyzed ctDNA and genomic DNA (gDNA) from FFPE samples as well as circulating tumor cells (CTC) in 10 treatment-naive hormone positive and HER2 negative, primary-stage breast cancer patients [GS1] using the OncoIndx comprehensive 600 gene panel. The panel captures all important cancer-relevant genomic alterations including Tumor Mutation Burden (TMB), Micro Satellite Instability (MSI), homologous recombination deficiency (HRD) prediction, and cfDNA tumor fraction (TF). CTCs were enumerated from 1.5 ml of blood using the OncoDiscover platform approved by the Drug Controller General of India having anti-EpCAM antibody-mediated immunomagnetic nanoparticles. CTCs were confirmed for cytokeratin 18+ and DAPI + markers and the absence of CD45. Results: Comprehensive genomic profile obtained from ctDNA and gDNA from FFPE of early-stage breast cancer patients predominantly exhibited the presence of alterations in PIK3CA and ESR1 signaling pathways. PIK3CA mutations were present in 77% and 44% of baseline ctDNA and gDNA samples, while ESR1 mutations were present in 44% and 22% of baseline ctDNA and gDNA, respectively. In addition, we observed about 70% additional driver mutations in ctDNA samples suggesting shedding of ctDNA together with CTC (80% positive), a likely positive biomarker of metastasis. About 50% of the patients showed higher TMB and HRD. Notably, TF representing ctDNA varied between 13% to 27% in blood samples with a corresponding ploidy range of 2.9 to 4.7. Surprisingly, ~50% of the patient population matched the mutation profile of clinically confirmed metastatic patients. All the patients harboring potential metastatic driver alterations showed the presence of CTCs in peripheral blood. Conclusions: Comprehensive ctDNA genomic profiling showed potential metastasis driving alterations suggesting the role of ctDNA-based liquid biopsy to predict metastasis in early breast cancer patients. We observed enhanced TF at the time of diagnosis, possibly due to the presence of distant metastasis, high disease burden, and aggressive tumor biology. Our results suggest that ctDNA dynamics at the time of disease presentation can predict early metastasis, and may demonstrate the divergent response of tumor heterogeneity to treatment in early-stage breast cancer. Citation Format: Gowhar Shafi, Manoj Dongare, Atul Bharde, Moubeen Fauzul, Kanchan Hariramani, Alain D’Souza, Bhagwat Jadhav, Trupti Kad, Sangeeta Prajapati, Vikas Jadhav, ManojKumar Kumaran, Sumit Haldar, Vatsal Mehra, Sujit Joshi, Gourishankar Aland, Richa Dave, Sreeja Jayant, Aravindan Vasudevan, Mohan Uttarwar, Jayant Khandare. Comprehensive ctDNA profiling reveals potential metastatic genomic signatures in treatment-naive early-stage breast cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR007.

The aim of this study was to investigate the presence of circulating tumor cells (CTCs) and their correlation with prognostic factors and clinical outcomes in treatment-naive patients with oral squamous cell carcinoma.CTCs were isolated using OncoDiscover technique from presurgically obtained peripheral blood of 152 patients with treatment naïve oral squamous cell carcinoma. Sensitivity analysis was performed by including 40 healthy controls. CTCs cutoff values for clinicopathologic factors were obtained from receiver operating characteristic curves. Multivariate models determined the significance of CTC as independent variables. Kaplan-Meier analysis differentiated in overall survival between CTC values corresponding to the stage.Sensitivity, specificity, and accuracy of CTC detection were 94.32%, 98%, and 95.17%, respectively. Platform differentiated true positives at3.5 CTCs (P.00001). CTCs above 20.5 were suggestive of nodal metastasis (P.0001) with a linear trend for detecting occult metastasis (P = .061). Early and advanced stages could be differentiated by13.5 CTCs (P.0001). Elevated CTCs were significantly associated with extranodal extension (21.45 CTCs, P = .025), perineural invasion (19.35 CTCs, P = .049), and depth of invasion (12.5 CTCs, P = .0038). Median survival was reduced by 19 months when CTCs were13.Preoperative CTC levels demonstrated a strong correlation with adverse clinicopathology factors and suggested its role as a sensitive prognostic marker to predict survival outcome and disease progress.

e15021 Background: To analyze the role of Circulating tumor cells (CTC) as a confirmatory personalized biomarker for monitoring the disease progression, disease burden, and minimal residual disease in epithelial origin cancers. Methods: In this retrospective study, 127 patients with colorectal, breast, and ovarian cancer at stage III and IV were analyzed. The patients were at various stages of intensive chemo and radiotherapy while the CTCs were isolated and enumerated from 1.5 ml of blood. The decision to continue chemotherapy or change to oral metronomic therapy was based on the presence of circulating tumor cells in Stage III. While in stage IV, serial measurement of CTC guided therapy. CTCs were isolated using OncoDiscover platform possessing EpCAM antibody based immunomagnetic targeting of magnetic nanoparticles after RBC lysis. CTCs were imaged and identified as CK18+ and CD45- cells showing a well-defined nucleus using a motorized fluorescence microscope operational with a monochrome camera. CTCs were enumerated using automated image analysis software and counts were expressed as number per 1.5 ml of blood. Results: In this retrospective study we analyzed blood sample from 127 patients with the advanced stage epithelial cancers (breast- 50 %, ovarian -27 %, colorectal- 23 %) for the presence of CTCs. Amongst those, 52 % showed the presence of CTCs (breast- 52 %, ovarian -46 %, colorectal- 58 %). The CTC count ranged between 1-5 / 1.5 ml of blood with mean and median value of 2 and 1. Among the CTC positive population, majority had CTC count of 1 (44.4 %), while more than 2 CTCs were observed in 11 % of population. CTC clusters were detected in 13 % of population which predominantly were stage IV patients. 67 % among the follow up patients showed decrease in CTC count from the baseline due to the prescribed treatment, while 22 % patients showed increase in CTC count from the baseline. 11 % patients did not show change in CTC count from the baseline. When CTCs count was investigated as an independent variable to monitor the therapeutic response, it correlated well with the positive or negative outcome. In few representative cases, the reduction of CTC number from the basal value was indicative of an effective treatment. Exceptionally, in a representative colorectal cancer case, PET showed no primary as well secondary tumor burden, but the presence of CTCs in blood led further investigating an abdominal MRI that indicated multiple liver lesions suggesting micro-metastasis. Subsequent to SIRT treatment, the patient showed complete tumor regression and absence of CTCs in peripheral blood. Conclusions: Our data suggest that CTC can serve as a dynamic intermittent biomarker for monitoring the disease progression in advanced stages and assess the therapeutic response, thus emphasizing the role of CTCs in personalized cancer management.

1547 Background: Oncology implicates highest precision using next generation diagnostics and progressive therapies assisted by predictive tools. If validated clinically, machine learning (ML) can provide better insights in precision oncology. Furthermore, it longitudinally may stratify the progression of cancer disease burden in a real time. We have developed, Circulating Tumor Cells (CTCs) driven ML model as a predictor for the treatment decision strategy for both surgery and adjuvant therapy in head and neck squamous cell carcinoma (HNSCC) patients. Methods: In this study, a total of 380 HNSCC patients who underwent either surgery alone or surgery plus adjuvant therapy were accounted for. CTCs in patients were stratified based on clinicopathological parameters and using OncoDiscover platform having anti EpCAM antibody system regulated by the Drug Controller of India. Following this, we explored the predictive performance of the ML model on the usefulness of adjuvant therapy in HNSCC patients after the surgery. The available data was randomly divided into two subsets. First, 75%, of the original data was applied for Training the ML, and rest 25% of the data was used as a Test set. Survival curves were generated by Kaplan–Meier method and calculated through the Log rank test. Results: XGBoost machine learning classifier was superior to Random Forest and SVM-based analyses in predicting the usefulness of adjuvant therapy post-surgery using CTC alone or in combination with other clinical parameters in HNSCC patients. Machine learning algorithms were compared for predicting the accuracy of patients stratification. The results for each model were: XGBoost model (Accuracy = 0.84, ROC value = 0.73, Kappa = 0.43); Random Forest model (Accuracy = 0.81 ROC value = 0.70, Kappa = 0.41); SVM model (Accuracy = 0.76, ROC value = 0. 69, Kappa = 0.40). The ROC value of the XGBoost model was highest (0.73) while the ROC value for the SVM model was lower (0.69). We observed that when CTCs were combined with clinicopathological parameters, the accuracy, kappa values and AUC-ROC drastically improved in predicting the usefulness of adjuvant therapy post-surgery. A similar trend was observed when CTCs were combined with clinicopathological parameters in predicting the line of chemotherapy, post-surgery. Conclusions: ML-enabled, CTCs driven predictions can be highly accurate and ascertain the patient treatments. CTCs can be a positive predictor for selecting patient’s treatment regimen in both surgery as well in type of treatment (e.g. surgery alone or surgery + adjuvant therapy). It can also implicate to classify the patients and determine who necessitates an additional adjuvant therapy. Further investigations in this direction are necessary to predict the treatment options based on ML that may improve the overall survival of cancer patients.

3019 Background: Homologous recombination deficient (HRD) tumors are highly responsive to platinum-based chemotherapy and poly (ADP-ribose) polymerase inhibitor (PARPi) therapy. Pathogenic BRCA-1 and BRCA-2 (BRCA1/2) alterations are key members of the HR DNA repair pathway but genomic instability status, including loss of heterozygosity, telomeric allelic imbalance and large scale state transitions across the genome are also predictive of HRD. HRD testing is currently performed by next generation sequencing which can take 2-4 weeks for results, has a high failure rate, requires significant tissue and is costly. We developed and tested the ability of an AI enabled platform to predict HRD status from the analysis of whole slide imaging of the diagnostic H&E slide. This platform, iPREDICT-HRD is rapid, precise, and cost effective. Methods: The AI engine was trained on 120 H&E slides that were used to identify tumor prior to manual microdisseection for HRD assessment by NGS. Histopathological features were extracted, followed by feature mapping to predict HRD status based on the results of NGS testing. ResNet AI algorithm was trained to segment, annotate and predict HRD status. 10 lac tiles of 256x256 size at 40x magnification were generated per pathological class. 70% of the data set was used for training and 30% for validation of the AI model. Results: Using single blinded clinical samples, iPREDICT-HRD tool detected HRD + ve samples with 99.3% accuracy with 100% sensitivity and 99% specificity in the test set. Patch-level predictions of HRD status demonstrated intra-tumor heterogeneity within the H&E slides. Visual inspection of the heatmap suggested the presence of patches with high predictive ability of HRD status and this outperformed an average HRD score for slides with heterogeneity. Conclusions: AI-enabled prediction of HRD status can be accurately performed on diagnostic H&E slides potentially yielding results quickly and afforadably, even when limited tissue is available for testing.

Head and neck squamous cell carcinoma is leading cancer in the Indian subcontinent with Oral squamous cell carcinoma (OSCC) as the most frequent subtype. Conventionally 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 a novel, regulatory approved method to establish Circulating tumor cells (CTCs) presence in peripheral blood of 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 cytokeratin 18 (CK18 marker) 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 obtained from peripheral blood of OSCC patients correlated positively with the cancer stages (clinical as well as pathological) as well as aggressive pathological features such as presence or absence of ECS, LVE, PNI and DOI. Interestingly, with the presence of aggressive pathological features that often suggest the poor outcome of the disease, we observed a 25-50 % increase in CTC number. On the other hand early stage, treatment naïve patients had a lower number of CTCs. Mean CTC number in advanced-stage patients was 50 % higher than early-stage OSCC patients. Conclusions: Efficient and rapid isolation of CTCs from peripheral blood of OSCC patients using proprietary technology suggested a positive correlation between CTC number and pathological stages of the disease. 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. Citation Format: Jayant Khandare, Burhanuddin Nuruddin Qayyumi, Atul Bharde, Gourishankar Aland, Sreeja Jayant, Swati Tripathi, Nitin Singh, Reecha Badwe, Alain D'Souza, Balram Singh, Smriti Arora, Narendra Kale, Aravindan Vasudevan, Kumar Prabhash, Pankaj Chaturvedi. CTCs positively correlate clinical manifestations in Indian oral cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-148.

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.

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.

e14516 Background: ‘Liquid biopsy’ technologies are unaffordable and unavailable in developing countries despite having highest cancer burden and mortality rates. Current Circulating Tumor Cell (CTCs) technologies sustain clinical concerns of a) non-specificity b) low efficiency c) high blood volume requirement d) long turn-around time, and d) exorbitant cost (~$900-1400). We report, an extremely low cost, innovative nanosystem for rapid enumeration of CTS with higher specificity and efficiency. Methods: We designed a nanosystem mediated by conjugation of anti-EpCAM through multi-reactive glutathione spacer, carbon allotrope and amine terminated dendrimer. The platform was evaluated for enhanced aqueous dispersibility and increased interaction with CTCs for rapid isolation and enumeration in 100 Head and Neck Cancer (HNC) patients having primary tumor sub-sites-oral cavity, larynx, hypopharynx, oropharynx, nasopharynx, salivary gland, and thyroid. The captured cells were immuno-stained and the optimal fluorescence acquisition intensity was validated in accounting CTCs with CK18 protein expression. There was complete elimination of the false positive normal cells (NC) count to CTCs by our method. The analysis was performed with only 1.5 ml of collected blood samples. Results: The CTC distribution in cohort study ranged from 1 - 85 cells per 1.5 mL of blood. In more than 80% of patient’s CTCs, the quantitative estimation of anti-CK18 protein over-expression indicated ~10-fold higher intensity over to NCs. As compared to treatment naive, recurrent, and disease-free patients, the spread of CTC number across the clinical range appeared to be tight (close to mean value). The CTC enumeration sensitivity linearity was ~99.2%, and the complete enumeration process time was under 03 hours/1.5 ml of blood. Consequently, efficient, rapid and yet affordable CTC platform was designed and clinically validated. Conclusions: ‘OncoDiscover’ liquid biopsy technology for CTC enumeration is poised to revolutionize the liquid biopsy due to its high sensitivity and affordability (~ $120) and shall resolve a major unmet medical need in impoverished world.