Ensembles Of Tumor-associated Cells Research Articles

Abstract PS3-13: Non-invasive screening for breast cancer risk based on circulating ensembles of tumor associated cells

Abstract Background: Common modalities for breast cancer screening include self-breast examination (SBE) for detection of palpable lumps as well as Mammography scans for detection of suspicious nodules or masses. While both approaches have low specificity, SBE has lower sensitivity for early stage cancers while mammography is associated with radiation exposure risks. A blood-based non-invasive approach for determination of breast cancer risk in asymptomatic individuals can facilitate early detection and improve prognosis and survival. Circulating Ensembles of Tumor Associated Cells (C-ETACs) are heterotypic clusters of malignant cells which originate in a tumor and are ubiquitously detected in peripheral blood of individuals with solid organ cancers. We present findings from two large-cohort prospective observational studies showing the suitability of C-ETACs for non-invasive, non-radiological screening for breast cancer. Methods: 15 ml of peripheral blood was collected from 14,962 female volunteers among whom 832 were suspected cases of breast cancer and 14,962 were asymptomatic individuals with age-associated risk of breast cancer. The 832 suspected cases underwent a foundational (first diagnostic) biopsy following collection of blood while the 14,962 asymptomatic individuals underwent a mammography scan following collection of blood. Peripheral blood mononuclear cells (PBMCs) were isolated from all blood samples and treated with an epigenetically activating treatment medium which exerts selective cytotoxicity towards non-malignant hematolymphoid cells and allows survival of apoptosis resistant malignant cells and their clusters (C-ETACs). C-ETACs were defined as clusters of 3 or more cells which were EpCAM+, PanCK+ and CD45+/-. Results: Among the 832 suspected cases, 779 were eventually diagnosed with breast cancer and 53 with benign breast conditions. C-ETACs were detected in 701 / 779 cases of breast cancer (90.0% sensitivity) with comparable detection rates in metastatic (365/408 = 89.5%) as well as non-metastatic (336/371 = 90.6%). C-ETACs were also detected in 1 / 53 (1.9%) cases of benign breast tumor. Among the asymptomatic cohort of 14,130, C-ETACs were detected in 657 cases (4.7%), which included 509 / 10,859 (4.7%) individuals with BIRADS 1 and in 148 / 3,271 (4.5%) of individuals with BIRADS ≥2. These individuals have been advised clinical follow-up. Conclusions: We show that C-ETACs are ubiquitous in breast cancers and rare in individuals with benign conditions as well as asymptomatic individuals. Being derived from the tumor mass, C-ETACs are specific for cancer and thus provide direct visual evidence of malignancy in cancer cases and risk of malignancy in asymptomatic cases. The non-invasive nature of the approach is well suited for screening of large asymptomatic populations for breast cancer. Citation Format: Dadasaheb Akolkar, Darshana Patil, Pradip Fulmali, Pooja Fulmali, Revati Patil, Archana Adhav, Shoeb Patel, Sachin Apurwa, Sushant Pawar, Harshal Bodke, Vishal Ranjan, Rohit Chougule, Pradyumna Shejwalkar, Shabista Khan, Raja Dhasarathan, Pradip Devhare, Sanket Patil, Vineet Datta, Cynthe Sims, Stefan Schuster, Jatinder Bhatia, Chirantan Bose, Ajay Srinivasan, Rajan Datar. Non-invasive screening for breast cancer risk based on circulating ensembles of tumor associated cells [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 PS3-13.

Abstract A49: Viable circulating ensembles of tumor-associated cells persist in pretreated patients with solid organ cancers showing no radiologically detectable disease

Abstract The success of treatment in solid organ cancers is ascertained by radiologic imaging as per standard-of-care protocol, with PET-CT being the modality of choice. However, even in cases where complete resolution of the disease is noted radiologically, recurrence or emergence of new metastases is not uncommon. To explore the underlying cause of such recurrence, we hypothesized that circulating metastatic disease (CMD) in the nature of viable tumor cells or clusters would be a persistent systemic feature of solid organ cancers, although there may be no overt evidence of disease. We identified 1,217 patients with known and previously treated cases of various solid organ cancers (metastatic and nonmetastatic), where a recent PET-CT scan showed no radiologic evidence of disease. 15 mL peripheral blood was collected from these patients. Peripheral blood mononuclear cells (PBMCs) were harvested by centrifugation. Circulating ensembles of tumor-associated cells (C-ETACs) were enriched from PBMCs using an epigenetically acting stabilization process, which is cytotoxic towards nonmalignant cells but confers survival privilege on apoptosis-resistant cells of tumorigenic origin as well as their heterotypic clusters (C-ETACs). C-ETACs were characterized by immunostaining for EpCAM, pan-CK, and CD45 as well as for organ-specific markers. Among the 1,217 patients with no radiologic evidence of disease, C-ETACs were detected in 1,118 (91.9%) and were confirmed by immunostaining. In a subset analysis, C-ETACs from 80 samples were immunostained for organ- or subtype-specific antigens including those from head and neck (P63), breast (GCDFP15), colorectum (CDX2), liver (Hep Par-1), ovary (CA125), prostate (PSMA), cervical (P63), and lung (TTF-1/Napsin-A). Circulating metastatic disease (CMD) in the form of viable C-ETACs is a potent threat in patients with solid organ cancers despite complete radiologic response to treatment. Citation Format: Sewanti A. Limaye, Timothy Crook, Dadasaheb Akolkar, Darshana Patil, Pradip Devhare, Revati Patil, Shoeb Patel, Shabista Khan, Raja Dhasarathan, Vineet Datta, Cynthe Sims, Ajay Srinivasan, Rajan Datar. Viable circulating ensembles of tumor-associated cells persist in pretreated patients with solid organ cancers showing no radiologically detectable disease [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A49.

Circulating ensembles of tumor-associated cells in genitourinary cancers.

718 Background: Detection of genitourinary cancers is based on histopathological analysis of tumor tissue obtained by invasive biopsies following manifestation of clinical or radiological symptoms. There is presently no non-invasive blood-based test with high specificity and sensitivity for detection of genitourinary cancers. Considering that unprovoked thromboembolism is a significant risk in multiple cancers, we hypothesized that tumor derived circulating emboli in peripheral blood could comprise cancer cells and would serve as a reliable biomarker for detection of genitourinary cancers. These Circulating Ensembles of Tumor Associated Cells (C-ETACs) are defined as clusters of 3 or more cells of tumorigenic origin (EpCAM+, CK+ and CD45±). Methods: We obtained 15ml of blood from 8828 individuals, including 103 cases of Renal cancer, 79 cases of bladder cancer, 153 cases of prostate cancers as well as from 8493 asymptomatic individuals with age related elevated risk and were negative in various screening investigations (CEA, AFP, CA19-9, CA125, PSA, LDCT, Mammography, PAP Smear). PBMC were isolated by centrifugation. C-ETACs were enriched, harvested and characterized by immunofluorescence staining for EpCAM, CK, CD45 as well as organ specific markers for renal, bladder and prostate cancers. Results: C-ETACs were detected in 87 (84.5%) of 103 renal cancers, 71 (89.9%) of 79 bladder cancers and 138 (90.2%) of 153 prostate cancers respectively irrespective of extent (stage / metastatic status) of disease and prior treatments. Overall sensitivity among 335 cancer patients was 88.4%. Among the 8493 asymptomatic individuals, C-ETACs were detected in 255 individuals (97% specificity). Conclusions: C-ETACs were ubiquitously detected in cancers of Kidney, Bladder and Prostate regardless of stage and treatment status, and pose significant latent risk of metastasis/recurrence. The relative undetectability of C-ETACs in the asymptomatic cohort indicates causative connection with malignancies and are hence suitable for screening for these cancers.

Abstract P4-01-17: Viable circulating ensembles of tumor associated cells persist in patients with no radiologically detectable disease after treatment in breast cancer

Abstract Oncological orthodoxy holds that (metastatic) breast cancer is an incurable disease. Response evaluation in metastatic breast cancer is determined by clinical, biochemical and radiological parameters, FDG PET CT being the imaging modality of choice for most types of breast cancer. However, even in cases where FDG PET CT and/or normalisation of serum tumour markers imply complete response to systemic therapy, durable disease-free survival is uncommon. To explore the mechanistic basis underlying persistent disease in spite of apparently effective systemic therapy, we hypothesized that Circulating Metastatic Disease (CMD) in the form of viable tumor cells or clusters might be a feature of persisting breast cancer. To address this, we obtained 15ml of peripheral blood from 927 known and previously treated cases of breast cancers immediately prior to FDG PET CT restaging. Peripheral blood mononuclear cells (PBMCs) were harvested by centrifugation. Circulating Ensembles of Tumor Associated Cells (C-ETACs), which are clusters of heterotypic apoptosis resistant cells of tumorigenic origin were enriched by a novel process using a combination of commercially available stabilizing agents. C-ETACs were characterized by immunostaining for EpCAM, pan-CK and CD45 as well as GCDFP-15 or GATA3. 927 patients underwent FDG PET CT of whom 731 (78.9%) had evidence of FDG avid residual disease. 196 patients (21.1%) had no metabolically active disease, among whom C-ETACs were detected in 171/196 cases (87.2%). The presence of CMD in a high proportion of cases in patients with no evidence of metabolically active disease implies that the majority of patients in whom conventional parameters of disease are negative have residual disease and are not biologically cured. Citation Format: Ajay Srinivasan, Dadasaheb Akolkar, Darshana Patil, Timothy Crook, Cynthe Sims, Revati Patil, Vineet Datta, Anantbhushan Ranade, Pradip Fulmali, Pradip Devhare, Pooja Fulmali, Shoeb Patel, Akshay Ainwale, Sneha Puranik, Rajan Datar. Viable circulating ensembles of tumor associated cells persist in patients with no radiologically detectable disease after treatment in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-01-17.

Abstract P4-01-05: Circulating ensembles of tumor associated cells for detection of breast cancer

Abstract Background: Mammographic screening for breast cancer offers the possibility of reducing morbidity and mortality from the disease but has economic and safety implications. Thromboembolism is a well-recognized, potentially life-threatening complication of cancer. Detection and characterization of tumour emboli comprising Circulating Ensembles of Tumor Associated Cells (C-ETACs) has however remained largely unstudied, essentially due to the difficulties in their isolation. Here we present for the first time an observational study describing the prevalence of C-ETACs in breast cancer patients and in asymptomatic individuals using a novel, label and hypothesis free process. Methods:15 ml of peripheral blood was collected from 7075 female volunteers including 1582 previously diagnosed cases of breast cancer with definite radiological and histopathological evidence of disease of which 361 were non-metastatic, 792 were metastatic, 425 cases were recently diagnosed and treatment naïve and 1006 patients had received prior treatments. The control population comprised 5493 asymptomatic individuals with age-related increased risk of breast cancer. The control patients underwent mammography and had a BI-RADS category score of 1, consistent with absence of malignancy in the breast. Peripheral blood mononuclear cells from the study population were grown in an epigenetically active cell culture medium with paradoxical cytotoxicity that selectively kills normal cells and confers survival and growth advantage on apoptosis-resistant cells of tumorigenic origin and their emboli, viz., C-ETACs. C-ETACs are defined as clusters comprising Epcam+ and CK+ cells irrespective of CD45 expression. In a subset of 100 samples from breast cancer patients we confirmed that the isolated C-ETACs contained cells that were positive for GCDFP. Results: C-ETACs were detected in 1413 / 1582 cases of breast cancer giving a sensitivity of 89.3%. There were no statistically significant differences between metastatic vs non-metastatic status of the disease or prior treatment. Among the 5493 individuals with unremarkable mammography, C-ETACs were detected in 104 (1.89 %) individuals. These individuals are being followed up prospectively for manifestation of clinical or radiological signs of malignancy of the breast as well as in other organs. Conclusions: Our study shows that heterotypic C-ETACs are a common feature of breast cancer irrespective of type, stage and grade and are almost entirely absent in asymptomatic, mammographically normal individuals. Our results therefore identify a potential biomarker for breast cancer which is specific and sensitive and which satisfies the requirements for the non-invasive, radiation-free detection of breast cancer. As such, it is a candidate for development as a modality for population screening. Citation Format: Timothy Crook, Dadasaheb Akolkar, Pradip Fulmali, Revati Patil, Cynthe Sims, Pooja Fulmali, Vineet Datta, Sachin Apurwa, Ajay Srinivasan, Rajan Datar. Circulating ensembles of tumor associated cells for detection of breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-01-05.

Circulating ensembles of tumor-associated cells in gastrointestinal cancers.

808 Background: CEA and CA19-9 are non-specific markers for Gastrointestinal (GI) cancers. Molecular analysis of fecal blood is of limited utility in colorectal cancers. A non-invasive pan-GI-cancer blood-based test with high specificity and sensitivity is an unmet medical need. Considering that unprovoked thromboembolism is a significant risk in multiple cancers, we hypothesized that circulating thrombotic emboli in peripheral blood could comprise cancer cells and would serve as a reliable biomarker for detection of GI cancers. Methods: We obtained 15ml of blood from 7481 individuals, including 181 cases of Esophageal cancer, 125 cases of Gastric cancer, 448 cases of colorectal cancer and from 6727 asymptomatic individuals with age related elevated risk who underwent evaluation of serum CA19-9 and AFP. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation and further processes for negative enrichment and harvesting of circulating tumor cell clusters which were characterized by immunostaining. Circulating Ensembles of Tumor Associated Cells (C-ETACs) were defined as clusters of 3 or more cells which were positive for EpCAM and CK, irrespective of CD45 status. Results: C-ETACs were detected in 86.7% of esophageal cancers, 94.4% of gastric cancers and 91.3% of colorectal cancers respectively irrespective of extent (stage / metastatic status) of disease and prior treatments. Overall sensitivity among 754 cancer patients was 90.7%. Among the asymptomatic individuals, C-ETACs were detected in 31 / 366 (8.5%) individuals with elevated CEA and 10 / 152 (6.2%) individuals with elevated CA19-9. C-ETACs were detected in 160 / 5333 (2.9%) of the asymptomatic individuals who had no abnormal findings in any of the said markers. Conclusions: C-ETACs were ubiquitously detected in cancers of Oesophagus, Stomach and Colorectum regardless of stage and treatment status, and pose significant latent risk of thromboembolism and metastasis/recurrence. The relative undetectability of C-ETACs in the asymptomatic cohort indicates causative connection with malignancies and are suitable for screening for these cancers.