Background: trucheckTM is a non-invasive micro-biopsy-like approach for diagnostic triaging of symptomatic individuals suspected of Breast Cancer. trucheckTM is based on the detection of Circulating Ensembles of Tumor Associated Cells (C-ETACs: EpCAM+, Pan-CK+, CD45±) of Breast Cancer origin (GCDFP15+, GATA3+); such C-ETACs are ubiquitous in blood samples of patients with Breast Cancer and unexpected in asymptomatic individuals as well as in individuals with Benign Breast conditions. Methods: Analytical validation was performed using control cell lines for EpCAM (SKBR-3), Pan-CK (SKBR-3), CD45 (PBMCs), GCDFP15 (SKBR-3) and GATA3 (MCF-7) respectively. Known amounts of control cells were spiked into healthy donor blood and their recovery rates determined by immunocytochemistry (ICC) to establish Sensitivity, Specificity, Accuracy, Limit of Detection, Linearity and Precision. Clinical Validation was performed using 15 mL peripheral blood collected from 1,527 participants. An initial Retrospective Clinical Pre-validation was performed using blood samples collected from 547 known cases of Breast Cancer and 19 known cases of other (non-Breast) solid organ Cancers. In a subset of 20 Breast Cancer cases with metastases to the Lungs or Liver, C-ETACs were evaluated for markers associated Lung (Napsin-A, TTF-1, p40) and Liver (Hep-Par 1, Glypican-3) primaries. Prospective Clinical Validation was performed on blood samples collected prior to any invasive procedure from 961 symptomatic cases suspected of Breast Cancer. Results: Analytical Validation based on recovery of spiked control cells indicated 94.0% Sensitivity, 100% Specificity, 97.0% Accuracy, 93.2% - 96.7% Precision and significant linearity (R2≥0.99) for all ICC markers. Clinical Pre-validation indicated 89.4% Sensitivity and 100% Specificity. C-ETACs from the known Breast Cancer cases with Lung and Liver metastases were negative for Lung- and Liver-cancer specific ICC markers, respectively, while C-ETACs from non-Breast Cancer samples were negative for Breast Cancer-specific ICC markers. In the Prospective Clinical Validation, histopathological evaluation (HPE) of biopsied tumor tissue indicated Breast Cancer in 848 cases and benign conditions in 113 of the 961 suspected symptomatic cases. C-ETAC-based trucheckTM approach had 91.9% Sensitivity, 98.3% Specificity and 96.9% Accuracy. Conclusion: Analytical and Clinical Validation data establish the viability of C-ETAC-based trucheckTM for diagnostic triaging of symptomatic individuals suspected of Breast Cancer. Individuals positive for Breast Cancer-specific C-ETACs can be prioritized for further clinical procedures whereas C-ETAC negative individuals can be considered for alternate diagnoses.

Citation Format: Dadasaheb Akolkar, Darshana Patil, Pradip Fulmali, Pooja Fulmali, Revati Patil, Kiran Bendale, Archana Adhav, Shoeb Patel, Shabista Khan, Raja Dasarathan, Vishal Ranjan, Rohit Chougule, Pradyumna Shejwalkar, Akshay Ainwale, Mohini Garte, Rahul Sonawane, Madhav Purane, Yogeshwar Chaudhari, Prajakta Sagar, Shubhangi Nerkar, Vineet Datta, Cynthe Sims, Prashant Kumar, Ajay Srinivasan, Rajan Datar. Analytical and clinical validation of the trucheck™ platform for diagnostic triaging of symptomatic cases suspected of breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-40.