Pradip Devhare

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.

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.

Https://doi.org/10.18632/oncotarget.27793 Rajnish Nagarkar, Darshana Patil, Sewanti Limaye, Pradip Devhare, Ashwini Ghaisas, Navin Srivastava, Sachin Apurwa, Sanket Patil, Jinumary John, Zarrine...

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.

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.

Diagnosis of suspected cases of gliomas such as intracranial space-occupying lesions (ICSOLs) is based on histopathologic analysis of tumor tissue obtained by invasive biopsies, which are, however, associated with risk of serious complication, high rates of mortality, procedural expenses, sequelae, and other risks. Liquid biopsy (LB)-based noninvasive approaches for diagnosis of gliomas based on circulating tumor biomarkers in peripheral blood have hence gained considerable interest. We describe the discovery of a unique 20 exosomal mRNA signature that can discern gliomas from healthy individuals. We obtained 15 mL of peripheral blood by venous draw from 222 individuals, among whom 47 had confirmed diagnosis of gliomas and 175 were asymptomatic. Exosomes were enriched from peripheral blood and were used for isolation of mRNAs. Exosomal mRNAs were used for expression analysis of 20,802 gene transcripts, including 18,574 coding genes and 2,228 noncoding genes based on University of California Santa Cruz (UCSC) hg19 annotation. Differentially expressed mRNAs were analyzed using a machine learning algorithm to identify transcripts that were unique to or uniquely absent in known cases of gliomas and in healthy individuals. The machine learning algorithm identified a 20-mRNA signature that discerned gliomas from healthy individuals with high sensitivity (87%) and specificity (95%) with an overall accuracy of 94%. The differentially expressed transcripts included MST4, RARRES3, TARP, IL32, PPP3CC, RSL1D1, RSL24D1, EIF4B, ARHGEF3, DENND2D, EVL, SEC11A, RPL22L1, HIST1H1C, IRF8, SOS2, GLTSCR2, GIMAP7, HIST1H4D, and CDK11. These differentially expressed transcripts are potentially involved in various functions such as tumor suppression, protein phosphorylation-dephosphorylation, signal transduction (ligand binding), and cell cycle regulation. Our data indicate that exosomal mRNA profiling is a viable approach for discerning suspected cases of gliomas from healthy individuals. The noninvasive nature of this approach coupled with high sensitivity and specificity favor potential clinical application. Citation Format: Sachin Apurwa, Pooja Fulmali, Dadasaheb Akolkar, Darshana Patil, Sushant Pawar, Sneha Puranik, Vipul Todarwal, Akshay Ainwale, Pradip Devhare, Vineet Datta, Cynthe Sims, Ajay Srinivasan, Rajan Datar. An mRNA signature that accurately discerns gliomas from healthy individuals [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 B17.

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.

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.

E15624 Background: Treatment of advanced refractory cancers face challenges in non-availability of systemic therapy regimens with evidenced benefit. Post failure of two to three lines of systemic treatments, patients with such cancers are usually considered for palliation or clinical trials. Prior attempts at label-agnostic treatment regimens (precision medicine) in such populations were largely based on a single-indication-single-drug paradigm which had limited application. We hypothesized that advanced refractory malignancies have latent vulnerabilities which can be identified by an integrational multi-analyte interrogation of the tumor, and can be targeted using patient-specific combination regimens to yield clinical benefit. Methods: Fresh tumor tissue and blood samples were obtained from 158 patients with solid organ cancers where the disease had progressed following failure of at least two lines of standard of care systemic treatment options. These samples were used for Encyclopedic Tumor Analysis (ETA) which interrogated gene mutations, gene overexpression, pathway dysregulation, immunohistochemistry as well as in vitro chemosensitivity profiling of viable tumor cells. Integration of datasets from the multi-analyte ETA was used to generate patient-specific therapy recommendations. Patients who received ETA-guided treatments were followed up and response to treatment was retrospectively evaluated from radiological scans. Results: All patients received ETA-guided individualized treatments which were combinations of cytotoxic, targeted and endocrine agents. No two patients received the same treatment regimen. Complete or Partial Response (CR or PR) was observed in 76 patients yielding an Objective Response Rate (ORR) of 48.1%. 67 patients showed Stable Disease (SD), thus yielding a Disease Control Rate (DCR) of 90.5%. Median Progression Free Survival (PFS) was 117 days (Range 27 – 379 days). There were no Grade IV therapy related Adverse Events or therapy related deaths. Conclusions: Viable efficacious combination treatment options can be made available for patients with advanced refractory malignancies via ETA, despite perceived non-availability or non-viability of standard of care treatment options.

3544 Background: There are presently no accepted non-invasive means for detection of cancers in asymptomatic individuals or suspected cases. Radiological and serological investigations, though non-invasive, are not confirmatory and necessitate an invasive biopsy to establish malignant status of suspected findings. Invasive biopsies, in turn, face challenges due to non-representative tumor tissue or in cases where biopsy is impossible or unviable. We hypothesized that Circulating Microtumors (also called as C-ETACs: Circulating Ensembles of Tumor Associated Cells) in peripheral blood are universally present in solid organ cancers and their detection can be linked to malignant status. Methods: We obtained peripheral blood from 14,138 patients with various solid organ cancers as well as 10,625 asymptomatic individuals with age associated elevated risk of cancer. Out of the 14,138 patients with cancer, 25.1 % had local (non-metastatic) disease and 56.4% had metastatic disease as confirmed by radiological evaluation. C-ETACs were enriched and harvested from PBMCs using an epigenetically activating medium that is cytotoxic towards non-malignant epithelial and hematolymphoid cells in blood but confers survival benefit on apoptosis resistant circulating cells of tumorigenic origin and their heterotypic clusters (C-ETACs) in peripheral blood. Viable C-ETACs were identified and further characterized by immunocytochemistry (ICC) profiling. Results: C-ETACs were detected in 89.7% of samples from solid organ cancers irrespective of stage, treatment or present radiological status. C-ETACs were rarely detected (3.0%) in asymptomatic individuals. The asymptomatic individuals where C-ETACs were detected are being followed up periodically so as to enable detection of cancer based on clinical or radiological manifestation of symptoms. Conclusions: C-ETACs are ubiquitous in cancers and unexpected in asymptomatic individuals. Detection of C-ETACs in asymptomatic individuals may be indicative of risk of latent undiagnosed malignancy. The non-invasiveness of this approach makes it convenient for screening large populations for cancer.