The administration of vaccines using a combination approach ensures better coverage and reduces the number of injections and cost. The present study assessed liposome-complexed DNA-corresponding proteins of hepatitis E and B viruses (HEV and HBV) as combined vaccine candidates in rhesus monkeys. The HEV and HBV components consisted of 450 bps, neutralizing the epitope/s (NE) region, and 685 bps small (S) envelope gene-corresponding proteins, respectively. Three groups (n = 2 monkeys/group) were intramuscularly immunized with a total of three doses of NE Protein (Lipo-NE-P), NE DNA + Protein (Lipo-NE-DP), and each of NE and S DNA + Protein (Lipo-NES-DP), respectively, given one month apart. All immunized monkeys were challenged with 10,000 fifty percent monkey infectious dose of homologous HEV strain. Post-immunization anti-HEV antibody levels in monkeys were 59.4 and 148.4 IU/mL (Lipo-NE-P), 177.0 and 240.8 IU/mL (Lipo-NE-DP), and 240.7 and 164.9 IU/mL (Lipo-NES-DP). Anti-HBV antibody levels in Lipo-NES-DP immunized monkeys were 58,786 and 6213 mIU/mL. None of the challenged monkeys showed viremia and elevation in serum alanine amino transferase levels. Monkeys immunized with Lipo-NE-DP and Lipo-NES-DP exhibited a sterilizing immunity, indicating complete protection, whereas monkeys immunized with Lipo-NE-P showed limited viral replication. In conclusion, the liposome-complexed DNA-corresponding proteins of HEV and HBV induced protective humoral immune responses to both components in monkeys and are worth exploring further.

We present data on analytical validation of the multigene variant profiling assay (CellDx) to provide actionable indications for selection of targeted and immune checkpoint inhibitor (ICI) therapy in solid tumors. CellDx includes Next Generation Sequencing (NGS) profiling of gene variants in a targeted 452-gene panel as well as status of total Tumor Mutation Burden (TMB), Microsatellite instability (MSI), Mismatch Repair (MMR) and Programmed Cell Death-Ligand 1 (PD-L1) respectively. Validation parameters included accuracy, sensitivity, specificity and reproducibility for detection of Single Nucleotide Alterations (SNAs), Copy Number Alterations (CNAs), Insertions and Deletions (Indels), Gene fusions, MSI and PDL1. Cumulative analytical sensitivity and specificity of the assay were 99.03 (95% CI: 96.54-99.88) and 99.23% (95% CI: 98.54% - 99.65%) respectively with 99.20% overall Accuracy (95% CI: 98.57% - 99.60%) and 99.7% Precision based on evaluation of 116 reference samples. The clinical performance of CellDx was evaluated in a subsequent analysis of 299 clinical samples where 861 unique mutations were detected of which 791 were oncogenic and 47 were actionable. Indications in MMR, MSI and TMB for selection of ICI therapies were also detected in the clinical samples. The high specificity, sensitivity, accuracy and reproducibility of the CellDx assay is suitable for clinical application for guiding selection of targeted and immunotherapy agents in patients with solid organ tumors.

Introduction : Encyclopedic Tumor Analysis (ETA) is multi-analyte, molecular and functional interrogation to identify latent vulnerabilities in solid tumors which can then be targeted in organ- and label-agnostic combination treatment regimens. Case Presentation : We describe here a case of metastatic rectal cancer in a 61-year-old male who was progressed on all prior Standard of Care (SoC) treatment modalities including surgery, chemotherapy and radiotherapy. We addressed disease recurrence via personalized therapy guided by ETA which revealed characteristic molecular heterogeneity in primary and metastatic lesions in terms of single nucleotide variations (SNVs) and gene copy number variations (CNVs). Notably, a novel TBL1XR1 (Exon1) – PIK3CA (Exon 2) gene fusion was identified in the tumor along with gene copy number gains in TERT, IGF-1R, MYC, FGFR1 and EGFR genes. Conclusion : ETA based molecular analysis with synchronous in vitro chemo-sensitivity profiling strategy helped to define de novo combinatorial therapy regimen of targeted and cytotoxic drugs which countered disease progression at each instance and led to the durable regression of primary as well as metastatic lesions.

Hepatitis E virus (HEV) usually causes self-limiting acute hepatitis, but the disease can become chronic in immunocompromised individuals. HEV infection in pregnant women is reported to cause up to 30% mortality, especially in the third trimester. Additionally, extrahepatic manifestations like neuronal and renal diseases and pancreatitis are also reported during the course of HEV infection. The mechanism of HEV pathogenesis remains poorly understood. Innate immunity is the first line of defense triggered within minutes to hours after the first pathogenic insult. Growing evidence based on reverse genetics systems, in vitro cell culture models, and representative studies in animal models including non-human primates, has implicated the role of the host's innate immune response during HEV infection. HEV persists in presence of interferons (IFNs) plausibly by evading cellular antiviral defense. This review summarizes our current understanding of recognizing HEV-associated molecular patterns by host cell Pattern Recognition Receptors (PRRs) in eliciting innate immune response during HEV infection as well as mechanisms of virus-mediated immune evasion.

Lungs are the most common site of primary malignancy as well as metastasis from primary malignancies in other organs. Radiological imaging scans often present a diagnostic conundrum where suspicious lesions are observed in various organs including the lungs thus necessitating an invasive biopsy for histopathological diagnosis which remains the standard for diagnosis and disambiguation of primary lung malignancy and metastatic deposit. We present a non-invasive means for discerning primary lung malignancy from metastatic deposit based on Immunocytochemistry (ICC) profiling of Circulating Tumor Cells (CTCs) from peripheral blood. We collected peripheral blood from 229 individuals with suspicious findings on radiological scans involving the lung and various other organs such as the liver, chest wall, colon, hepatobiliary tract or the gastroesophageal tract. Patients underwent an invasive biopsy of the lungs to obtain tumor tissue for histopathological examination (HPE) which were initially blinded. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples and treated with an epigenetically activating medium which induces cell death in normal (non-malignant) hematolymphoid cells as well as epithelial cells in peripheral blood, but selectively confers survival privilege on apoptosis resistant tumor-derived Circulating Tumor Cells (CTCs). CTCs were profiled by ICC using IVD approved antibodies against organ and subtype specific (OSS) antigens to determine the tissue of origin (TOO). ICC findings were compared with the HPE results to determine concordance. Among the 229 individuals, 10 each were diagnosed with a primary malignancy of the breast, colon and lungs, 5 each of esophagus and stomach, 4 of liver and 3 of pancreas. ICC profiling of CTCs for OSS antigens indicated a 96.9% overall concordance with HPE findings. ICC profiling of CTCs accurately discerned primary malignancies of the lungs, colon, esophagus, pancreas and stomach with 100% accuracy, of the breast with 90% accuracy and of the liver with 75% accuracy. The findings suggest that ICC profiling of CTCs can non-invasively discern primary malignancy of the lungs from metastatic deposits in the lungs with high accuracy. In the clinical setting, this approach will be useful for diagnostic triaging in individuals with confounding radiological findings, especially where a biopsy of the lungs (or any other organ) is not possible owing to anatomical considerations or patient’s co-morbidities.

Screening of Individuals for Lung cancer is presently based on radiological evaluation for presence of suspicious thoracic nodules by Low Dose Computed Tomography (LDCT). LDCT is not only associated with risks of exposure to radiation but is also not confirmatory and often necessitates an invasive biopsy in suspicious cases. We present a non-invasive approach for triaging of asymptomatic individuals prior to screening investigations such as LDCT. This approach is based on detection of Circulating Ensembles of Tumor Associated Cells (C-ETACs) which are clusters of malignant cells derived from a tumor mass. We collected peripheral blood from 10625 asymptomatic individuals (6627 females and 3398 males) with no prior diagnosis of cancer and no clinical symptoms indicative of cancer. All individuals underwent LDCT scans following blood collection. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples and treated with an epigenetically activating medium which induces cell death in normal (non-malignant) hematolymphoid cells as well as epithelial cells in peripheral blood, but selectively confers survival privilege on apoptosis resistant tumor-derived Circulating Tumor Cells (CTCs)and their clusters (C-ETACs). Two-way association studies were performed to correlate detection of C-ETACs and LDCT findings (Lung RADS score). Among the 10625 individuals, samples from 467 were positive for C-ETACs while 10158 were negative for C-ETACs. Among the 10625 individuals, 8422 had no abnormal findings on LDCT (LungRADS = 1), 1833 had borderline risk of malignancy (Lung RADS = S / 2), 283 had marginally elevated risk of malignancy (Lung RADS = 2S / 3), while 78 individuals had elevated risk of malignancy (Lung RADS = 3S / 4A / 4B / 4x). Detection of C-ETACs was associated with 5-fold increase in risk of malignancy based on Lung RADS (3.2% vs 0.6%). Conversely, 19.2% of all individuals with elevated risk of malignancy were positive for C-ETACs as compared with 4.1% - 5.3% individuals with no suspicious findings or borderline risk of malignancy suggesting 4.5-fold increased risk. The findings suggest a positive association between detection of C-ETACs and incidence of significant findings in LDCT indicative of higher risk of malignancy. Non-invasive evaluation of asymptomatic individuals for presence of C-ETACs can facilitate efficient triaging prior to LDCT, thus significantly expediting diagnosis and treatment.

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.

Angiokinases (AngK) such as VEGFR, PDGFR and FGFR as well as their ligands (VEGF, PDGF and FGF) are commonly activated in several solid organ cancers and present a viable therapeutic target. However, use of AngK inhibitors in anticancer treatments is large as monotherapy, limited to few cancers and their selection is not based on a molecular indication. It is generally accepted that combination regimens with angiokinase inhibitors may yield improved outcomes as compared to monotherapies. We evaluated a subset of patients in two prospective, interventional, non-randomised studies aimed to determine whether patient-specific combination regimens with AngK inhibitors and other anticancer agents selected on the basis of multi-analyte tumor interrogation (ETA: Encyclopedic Tumor Analysis) yields significant treatment response and survival benefits in advanced or refractory solid organ cancers. We evaluated treatment outcomes in 64 patients with advanced, refractory solid organ cancers where cancer had progressed following failure of 2 or more prior systemic lines. All patients received combination regimens with AngK inhibitors, and other targeted, endocrine or cytotoxic agents as guided by ETA. Patients were followed-up until death, withdrawal of consent or loss to follow-up. Radiological evaluation of treatment response was followed by determination of objective response rate (ORR), disease control rate (DCR), progression free survival (PFS) and overall survival (OS). Among the 64 patients, partial response (PR) was observed in 28 cases (43.8%), stable disease (SD) was observed in 33 cases (51.6%) and outright disease progression (PD, within 60 days) was observed in 3 cases (4.7%). Thus the ORR was 43.8% and DCR was 95.3%. At the most recent follow up, median PFS (mPFS) was 4.5 months and median OS (mOS) was 7.4 months. There were no grade IV therapy related adverse events or treatment related deaths. Patient-specific combination regimens with AngK inhibition and other anti-neoplastic agents, when selected on the basis of multi-analyte molecular and functional profiling of the tumor can yield improved outcomes over AngK inhibitor monotherapy.

Periampullary adenocarcinomas are rare neoplasm that originates from the pancreatic head, the ampulla of vater, the distal bile duct or the duodenum. Surgical resection followed by adjuvant therapy is considered as the standard of care treatment for these carcinomas. Despite several advances in diagnostics and therapeutics, only 5% of these patients have an overall survival of five years or more. Currently, there is a dearth of viable therapeutic targets for this disease. The role of HER2 in cancer biology has been studied extensively in several tumour subtypes, and HER2 based targeted therapies have shown to have therapeutic benefits on different cancers. In this case report, we present a case of HER2 positive distal common bile duct carcinoma - a subtype of periampullary carcinoma with multiple relapses where multi-analyte testing with Encyclopedic Tumor Analysis (ETA) (Exacta®) identified amplification and over expression of HER2 gene which was used as a potential target to treat the patient with trastuzumab. Synchronous in vitro chemosensitivity profiling on Circulating Tumor Asscociated Cells (C-TACs) isolated from blood aided us to design the personalized chemotherapeutic regimen with cyclophosphamide and methotrexate. The combination of trastuzumab with cyclophosphamide and methotrexate yielded excellent treatment response with the patient remaining in complete response till the last follow-up. Our study suggests HER2 directed therapy as a potent pathway for treatment in the subset of HER-2 amplified distal common bile duct carcinomas.

Background: Diagnosis and treatment of breast cancers is based on histopathological analysis of tumor tissue obtained by invasive biopsies. However invasive biopsies cause pain, anxiety and carry the risk of needle seeding. Though Immunocytochemistry (ICC) profiling of Circulating Tumor Cells (CTCs, defined as EpCAM+, panCK+, CD45-) has been previously attempted for diagnosis, these efforts have been unsuccessful. We used an epigenetically modified medium for enrichment of sufficient viable CTCs. These CTCs were used for diagnosis and characterization. Methods: We obtained 15 ml of venous blood draw from 2500 patients with confirmed diagnosis of breast cancer. CTCs were enriched from PBMCs fraction using an epigenetically acting enrichment process that is lethal towards normal (non-malignant) cells, but confers survival privilege on apoptosis-resistant CTCs. Harvested CTCs were confirmed by immunostaining for EpCAM and pan-CK. Deep ICC profiling of these CTCs was performed with organ specific marker GCDFP15. Theranostic ICC Profiling was performed with ER and Her2. Results: CTCs could be obtained from 2232 samples (89.3%) out of 2500. A subset of 100 samples were characterised by deep ICC profiling, among which 80% of samples were positive for GCDFP15. 14 and 17 samples out of 100 were positive for ER and Her2 respectively. Conclusions: Our results show that ICC based characterization of CTCs can provide necessary diagnostic information non-invasively to substitute conventional procedures dependent on tissue extraction. Citation Format: Dadasaheb Akolkar. Wholesome non-invasive liquid biopsies for pharmacodiagnostic work-up in breast cancer [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 B62.

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.

Activation of the mTOR signalling pathway is ubiquitous in cancers and a favourable therapeutic target. However, presently approved mTOR inhibitor monotherapies have modest benefits in labelled indications while poor outcomes have been reported for mTOR inhibitor monotherapy when administered in a label-agnostic setting based on univariate molecular indications. The present study aimed to determine whether patient-specific combination regimens with mTOR inhibitors and other anticancer agents selected on the basis of multi-analyte tumor interrogation (ETA: Encyclopedic Tumor Analysis) yields significant treatment response and survival benefits in advanced or refractory solid organ cancers. We evaluated treatment outcomes in 54 patients diagnosed with unresectable or metastatic solid organ cancers, of whom 3 were therapy naïve and 51 were pretreated where the cancer had progressed on 2 or more prior systemic lines. Within this cohort, 49 patients received mTOR inhibitor in combination with other targeted, endocrine or cytotoxic agents as guided by ETA and 5 patients received mTOR inhibitor as monotherapy or in combination with physician's choice of anticancer agents (not guided by ETA). Patients were followed-up until death, withdrawal of consent, loss to follow-up or until April 2020, whichever is latest. In the ETA-guided combination regimen group, the objective response rate (ORR) was 57.1%, the disease control rate (DCR) was 91.8%, median progression free survival (mPFS) was 4.9 months and median overall survival (mOS) was 9.4 months. In the Clinician's choice group, ORR was 0%, DCR was 20.0%, mPFS was 1 month and mOS was 5 months. Patient-specific combination regimens with mTOR inhibition and other anti-neoplastic agents, when selected on the basis of multi-analyte molecular and functional profiling of the tumor can yield improved outcomes over mTOR inhibitor monotherapy.

29 Background: Selection of Immune Checkpoint Inhibitor (ICI) therapies in Gastroesophageal cancers are based on IHC based detection of PD-L1 expression in tumor tissue. Invasive biopsy to obtain tumor tissue for IHC is associated with procedural risks, sequelae and expenses. Prior efforts at PD-L1 profiling of Circulating Tumor Cells (CTCs) have been constrained by low CTC yields. We employed a novel approach for harvesting sufficient CTCs from Gastroesophageal cancers that permit PD-L1 profiling by Immunocytochemistry (ICC). Methods: 15 ml peripheral blood was collected from 106 patients among whom 20 were diagnosed with gastric adenocarcinomas (AD) and 86 with either esophageal AD or esophageal squamous cell carcinomas (SCC). CTCs were enriched from PBMCs via an epigenetically acting stabilization process which induced apoptosis in non-malignant cells and conferred survival privilege on apoptosis-resistant CTCs, which were harvested and confirmed by immunostaining for EpCAM and pan-CK positivity. Deep ICC profiling of CTCs was performed with organ-specific markers as well as PD-L1:22c3 and PD-L1:28-8 clones. Results: Viable CTCs could be enriched and harvested in 103 out of 106 samples (97.2%) regardless of metastatic or treatment status. Deep ICC and PD-L1 profiling could be performed in all 103 samples (100%). PD-L1 expression was quantitatively assigned as ‘Low’, ‘Moderate’ or ‘High’. Among the 19 gastric ADs, 5 (26.3%) samples were positive for PD-L1:22c3 and 2 (10.5%) were positive for PD-L1:28-8; all gastric ADs were ‘Low’ for either PD-L1 subtype. Among the 84 esophageal carcinomas (AD+SCC), 32 samples (38.1%) were positive for PD-L1:22c3 (26 Low + 5 Moderate + 1 High) and 16 (19.1%) were positive for PD-L1:28-8 (11 Low + 5 Moderate). Conclusions: CTCs in Gastroesophageal cancers can be considered for evaluating PD-L1 expression in patients where ICI therapies are otherwise viable.

Diagnosis of ovarian adenocarcinomas is based on histopathologic analysis of tumor tissue obtained by invasive biopsies. However, invasive biopsies are associated with procedural expenses, sequelae, and risk of complications. Though immunocytochemistry (ICC) profiling of circulating tumor cells (CTCs, defined as EpCAM+, panCK+, CD45-) has been previously considered for diagnosis in some cancers, the attempts have been unsuccessful. We used a novel approach for enrichment of sufficient viable CTCs that permit meaningful ICC profiling for diagnosis of ovarian cancers in suspected cases. We obtained 15 mL of venous blood draw from 248 patients with confirmed diagnosis of ovarian cancer. PMBCs were isolated by centrifugation. CTCs were enriched from PBMC using an epigenetically acting enrichment process that is lethal towards normal (nonmalignant) cells but confers survival privilege on apoptosis-resistant CTCs. Harvested CTCs were confirmed by immunostaining for EpCAM and pan-CK. Deep ICC profiling of these CTCs was performed with organ-specific markers CA125 and WT1 as well as P63 as a nonspecific marker. Viable CTCs could be obtained from 225 samples (90.7%) out of 248. Among the 100 samples characterized by deep ICC profiling, 70 samples (70%) were positive for CA125 and WT1. None of the samples was positive for P63. Our results show that ICC-based characterization of CTCs can provide necessary diagnostic information noninvasively to substitute conventional procedures dependent on tissue extraction. Citation Format: Dadasaheb B. Akolkar, Darshana Patil, Revati Patil, Pradip Devhare, Pradip Fulmali, Pooja Fulmali, Shoeb Patel, Navin Srivastava, Vineet Datta, Cynthe Sime, Ajay Srinivasan, Rajan Datar. Noninvasive liquid biopsies for guideline-compliant diagnostic assessment in ovarian cancers [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 B16.

Definitive diagnosis of solid organ cancers is based on histopathologic analysis of tumor tissue obtained by invasive biopsies. However, invasive biopsies are associated with procedural expenses, sequelae, and risks. Though immunocytochemistry (ICC) profiling of circulating tumor cells (CTCs) has been previously attempted for diagnosis in some cancers, they have been unviable due to limited yields of CTCs. We used a novel approach for enrichment of sufficient viable CTCs that permit meaningful ICC profiling for diagnosis of cancers in suspected cases. We obtained 15 mL of venous blood draw from 965 patients with confirmed diagnosis of various solid organ cancers. CTCs were enriched from PBMCs fraction using an epigenetically activated enrichment process that is lethal towards normal (nonmalignant) cells but confers survival privilege on apoptosis-resistant CTCs. Harvested CTCs were confirmed by immunostaining for EpCAM and pan-CK. Deep ICC profiling of these CTCs was performed with organ-specific marker for various organs such as lungs (TTF-1 or Napsin), breast (GCDFP15 or GATA3), ovarian (CA125 or WT-1), pancreatic (CK19 or CA19-9), cervical (P63), liver (Hep Par-1 or Glypican), colorectal (CK20 or CDX-2), head and neck (P63), kidney (Pax-8 or RCC), and prostate (PSMA or AMACR). CTCs (EpCAM+, pan-CK+) could be obtained from 94.4% of all samples. Organ-specific markers were positive in 100% of lung cancer, 79.3% of breast cancers, 70.0% of ovarian cancers, 93.3% of pancreatic cancers, 100% of cervical cancers, 61% of hepatocellular carcinomas, 92.6% ofcColorectal cancers, 98.8% of head and neck cancers, 100% of renal cancers, and 100% of prostate cancers. Our results show that deep ICC profiling of CTCs can determine tissue of origin in solid organ cancers noninvasively to substitute conventional procedures dependent on tissue extraction. Citation Format: Dadasaheb Akolkar, Revati Patil, Darshana Patil, Pradip Fulmali, Pooja Fulmali, Shoeb Patel, Pradip Devhare, Akshay Ainwale, Vineet Datta, Cynthe Sims, Ajay Srinivasan, Rajan Datar. Circulating tumor cells express tissue specific antigens in multiple cancers [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 B15.

e15623 Background: Tumor tissue profiling following invasive biopsies is presently the standard approach for indication-based therapy management in solid organ cancers. However, challenges in biopsy are traditionally described due to proximity to vital organs, or patients’ co-morbidities or unwillingness for an invasive procedure. Liquid biopsies for evaluation of cancers are also largely restricted to single gene testing for selection of targeted therapy agents. We developed a comprehensive liquid biopsy based multi-analyte (molecular and functional) investigation of the cancer (eLBx: Encyclopedic Liquid Biopsy) for selection and management of individualised treatments in a cohort of advanced refractory cancers. Methods: We obtained 20 mL blood from 65 patients with solid organ cancers where the disease had progressed following failure of at least two lines of systemic therapies and where biopsy to obtain tumor tissue for molecular profiling of tumor was unviable. Cell free tumor DNA (ctDNA) was interrogated for mutations, while exosomal mRNA was profiled for gene expression. Viable circulating tumor associated cells (C-TACs) were tested in vitro for chemoresistance and used to determine expression of cell surface signalling receptors by immunocytochemistry (ICC). The findings were integrated to generate patient-specific treatment regimens. In patients who received treatment, response was determined radiologically. Results: Fifty-one patients received eLBx-guided personalized treatments with combinations of cytotoxic, targeted and endocrine agents. No two patients received the same treatment regimen. Forty-three patients were evaluable for treatment response per protocol among whom Partial Response (PR) was observed in 14 patients yielding an Objective Response Rate (ORR) of 32.6%. Additionally, 23 patients showed Stable Disease thus yielding an overall Disease Control rate of 86.1%. Median Progression Free Survival (PFS) was 108 days. There were no Grade IV therapy related Adverse Events or therapy related deaths. Conclusions: The ability to make informed treatment choices from a convenient blood draw implies a reduced dependence on invasive biopsies for disease management. We demonstrate successful management of advanced refractory solid tumor malignancies using an integrational non-invasive multi-analyte liquid biopsy approach. Clinical trial information: CTRI/2019/02/017548.

799 Background: Definitive diagnosis of gastrointestinal (GI) malignancies is reliant on histopathological examination of tumor tissue obtained by invasive biopsies. However, invasive biopsies are associated with procedural risks, complications and expenses. A non-invasive technique for diagnosis of GI cancers is presently unavailable. Here we present a non-invasive diagnostic approach for GI cancers based on immunocytochemical (ICC) profiling of Circulating Tumor Associated Cells (C-TAC) enriched from peripheral blood. Methods: We collected 15 mL peripheral blood from 1052 patients with known diagnosis of Ca Oesophagus (244), Ca Stomach (170) and Ca Colorectum (638) and with histopathological information available from prior tissue analysis. CTACs were harvested following negative enrichment. C-TACs were identified by immunostaining with EpCAM and panCK. Deep ICC characterization was carried out in a subset of 203 samples (100 colorectal, 19 Gastric and 84 Esophageal) using organ specific markers. A subset of 19 samples from Gastric and 94 samples Esophageal cancers were profiled for Her2 and PD-L1 status. Results: C-TACs could be identified and enriched in 1012 out of 1052 patients (96.2% overall sensitivity). Immunostaining for organ-specific markers was possible in all 203 (100%) samples. Her2 positivity was observed in 2/19 Gastric and 19/84 Esophageal samples. PD-L1 (22C3) positivity was observed in 5/19 Gastric and 32/84 Esophageal samples while PD-L1 (28-8) positivity was observed in 2/19 Gastric and 16/84 Esophageal samples. Conclusions: Our results show that ICC profiling of C-TACs can provide necessary diagnostic information non-invasively to substitute conventional procedures dependent on tissue extraction. This approach fulfils most clinical decision-making requirements in GI malignancies.

Pilomatrical carcinoma is an exceedingly rare, locally aggressive malignancy of the hair follicle.It may arise de novo as a solitary lesion or through malignant transformation from its pre-existing benign counterpart, pilomatrixoma.There are no standard treatment guidelines and wide surgical excision of the primary lesion followed by adjuvant radiotherapy remains principal treatment modality.However, high recurrence rate after primary treatment, presence of nodal and systemic metastases which is non-responsive to chemotherapy are major concerns in disease management.Here, we report a case of a 45-year-old female with recurrent pilomatrical carcinoma who opted for personalized treatment guidance based on Encyclopedic Tumor Analysis (ETA).Molecular analysis of tumor tissue identified a novel EIF3E-RSPO2 gene rearrangement, SETD2 mutation and overexpression of VEGFA and RET genes.These molecular alterations along with synchronous chemo-sensitivity assay on tumor derived cells helped in defining combinatorial off label therapy regimens which countered disease progression at each instance and led to the durable regression of primary as well as metastatic lesions.This personalized treatment management strategy described herein effectively addressed advanced, refractory disease for which current Standard of Care (SoC) options were unviable.

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.