Liquid Biopsy for Detection of Pancreaticobiliary Cancers by Functional Enrichment and Immunofluorescent Profiling of Circulating Tumor Cells and Their Clusters.

Circulating tumor cells (CTCs) have historically been used for prognostication in oncology. We evaluate the performance of liquid biopsy CTC assay as a diagnostic tool in suspected pancreaticobiliary cancers (PBC). The assay utilizes functional enrichment of CTCs followed by immunofluorescent profiling of organ-specific markers. The performance of the assay was first evaluated in a multicentric case-control study of blood samples from 360 participants, including 188 PBC cases (pre-biopsy samples) and 172 healthy individuals. A subsequent prospective observational study included pre-biopsy blood samples from 88 individuals with suspicion of PBC and no prior diagnosis of cancer. CTCs were harvested using a unique functional enrichment method and used for immunofluorescent profiling for CA19.9, Maspin, EpCAM, CK, and CD45, blinded to the tissue histopathological diagnosis. TruBlood® malignant or non-malignant predictions were compared with tissue diagnoses to establish sensitivity and specificity. The test had 95.9% overall sensitivity (95% CI: 86.0-99.5%) and 92.3% specificity (95% CI: 79.13% to 98.38%) to differentiate PBC (n = 49) from benign conditions (n = 39). The high accuracy of the CTC-based TruBlood test demonstrates its potential clinical application as a diagnostic tool to assist the effective detection of PBC when tissue sampling is unviable or inconclusive.

Accurate prostate cancer detection based on enrichment and characterization of prostate cancer specific circulating tumor cells.

BACKGROUND: The low specificity of serum PSA resulting in the inability to effectively differentiate prostate cancer from benign prostate conditions is a persistent clinical challenge. The low sensitivity of serum PSA results in false negatives and can miss high-grade prostate cancers. We describe a non-invasive test for detection of prostate cancer based on functional enrichment of prostate adenocarcinoma associated circulating tumor cells (PrAD-CTCs) from blood samples followed by their identification by immunostaining for pan-cytokeratins (PanCK), prostate specific membrane antigen (PSMA), alpha methyl-acyl coenzyme-A racemase (AMACR), epithelial cell adhesion molecule (EpCAM), and common leucocyte antigen (CD45). METHODS: Analytical validation studies were performed to establish the performance characteristics of the test using VCaP prostate cancer cells spiked into healthy donor blood (HDB). The clinical performance characteristics of the test were evaluated in a case-control study with 160 known prostate cancer cases and 800 healthy males, followed by a prospective clinical study of 210 suspected cases of prostate cancer. RESULTS: Analytical validation established analyte stability as well as acceptable performance characteristics. The test showed 100% specificity and 100% sensitivity to differentiate prostate cancer cases from healthy individuals in the case control study and 91.2% sensitivity and 100% specificity to differentiate prostate cancers from benign prostate conditions in the prospective clinical study. CONCLUSIONS: The test accurately detects PrAD-CTCs with high sensitivity and specificity irrespective of stage, serum PSA or Gleason score, which translates into low risks of false negatives or overdiagnosis. The high accuracy of the test could offer advantages over PSA based prostate cancer detection.

Accurate Screening for Early-Stage Breast Cancer by Detection and Profiling of Circulating Tumor Cells.

BACKGROUND: The early detection of breast cancer (BrC) is associated with improved survival. We describe a blood-based breast cancer detection test based on functional enrichment of breast-adenocarcinoma-associated circulating tumor cells (BrAD-CTCs) and their identification via multiplexed fluorescence immunocytochemistry (ICC) profiling for GCDFP15, GATA3, EpCAM, PanCK, and CD45 status. METHODS: The ability of the test to differentiate BrC cases (N = 548) from healthy women (N = 9632) was evaluated in a case-control clinical study. The ability of the test to differentiate BrC cases from those with benign breast conditions was evaluated in a prospective clinical study of women (N = 141) suspected of BrC. RESULTS: The test accurately detects BrAD-CTCs in breast cancers, irrespective of age, ethnicity, disease stage, grade, or hormone receptor status. Analytical validation established the high accuracy and reliability of the test under intended use conditions. The test detects and differentiates BrC cases from healthy women with 100% specificity and 92.07% overall sensitivity in a case-control study. In a prospective clinical study, the test shows 93.1% specificity and 94.64% overall sensitivity in differentiating breast cancer cases (N = 112) from benign breast conditions (N = 29). CONCLUSION: The findings reported in this manuscript support the clinical potential of this test for blood-based BrC detection.

Circulating tumor cell assay to non-invasively evaluate PD-L1 and other therapeutic targets in multiple cancers.

Biomarker directed selection of targeted anti-neoplastic agents such as immune checkpoint inhibitors, small molecule inhibitors and monoclonal antibodies form an important aspect of cancer treatment. Immunohistochemistry (IHC) analysis of the tumor tissue is the method of choice to evaluate the presence of these biomarkers. However, a significant barrier to biomarker testing on tissue is the availability of an adequate amount of tissue and need for repetitive sampling due to tumor evolution. Also, tumor tissue testing is not immune to inter- and intra-tumor heterogeneity. We describe the analytical and clinical validation of a Circulating Tumor Cell (CTC) assay to accurately assess the presence of PD-L1 22C3 and PD-L1 28.8, ER, PR and HER2, from patients with solid tumors to guide the choice of suitable targeted therapies. Analytically, the test has high sensitivity, specificity, linearity and precision. Based on a blinded case control study, the clinical sensitivity and specificity for PD-L1 (22C3 and 28.8) was determined to be 90% and 100% respectively. The clinical sensitivity and specificity was 83% and 89% for ER; 80% and 94% for PR; 63% and 89% for HER2 (by ICC); and 100% and 92% for HER2 (by FISH), respectively. The performance characteristics of the test support its suitability and adaptability for routine clinical use.

Multi-analyte liquid biopsies for molecular pathway guided personalized treatment selection in advanced refractory cancers: A clinical utility pilot study.

Purpose: The selection of safe and efficacious anticancer regimens for treatment of patients with broadly refractory metastatic cancers remains a clinical challenge. Such patients are often fatigued by toxicities of prior failed treatments and may have no further viable standard of care treatment options. Liquid Biopsy-based multi-analyte profiling in peripheral blood can identify a majority of drug targets that can guide the selection of efficacious combination regimens. Patients and methods: LIQUID IMPACT was a pilot clinical study where patients with advanced refractory cancers received combination anticancer treatment regimens based on multi-analyte liquid biopsy (MLB) profiling of circulating tumor biomarkers; this study design was based on the findings of prior feasibility analysis to determine the abundance of targetable variants in blood specimens from 1299 real-world cases of advanced refractory cancers. Results: Among the 29 patients in the intent to treat (ITT) cohort of the trial, 26 were finally evaluable as per study criteria out of whom 12 patients showed Partial Response (PR) indicating an Objective Response Rate (ORR) of 46.2% and 11 patients showed Stable Disease (SD) indicating the Disease Control Rate (DCR) to be 88.5%. The median Progression-Free Survival (mPFS) and median Overall Survival (mOS) were 4.3 months (95% CI: 3.0 - 5.6 months) and 8.8 months (95% CI: 7.0 - 10.7 months), respectively. Toxicities were manageable and there were no treatment-related deaths. Conclusion: The study findings suggest that MLB could be used to assist treatment selection in heavily pretreated patients with advanced refractory cancers.

Response to pazopanib-based combination regimen in a case of FGFR3 amplified gastric adenocarcinoma.

Angiogenesis inhibitors (AGI) are not presently used for the treatment of gastric cancers. This report demonstrates that angiogenesis inhibitor can be safely and effectively used in combination with cytotoxic anti-cancer agents for treatment of Gastric cancers.

Synthetic lethality-mediated precision oncology via the tumor transcriptome.

Precision oncology has made significant advances, mainly by targeting actionable mutations in cancer driver genes. Aiming to expand treatment opportunities, recent studies have begun to explore the utility of tumor transcriptome to guide patient treatment. Here, we introduce SELECT (synthetic lethality and rescue-mediated precision oncology via the transcriptome), a precision oncology framework harnessing genetic interactions to predict patient response to cancer therapy from the tumor transcriptome. SELECT is tested on a broad collection of 35 published targeted and immunotherapy clinical trials from 10 different cancer types. It is predictive of patients' response in 80% of these clinical trials and in the recent multi-arm WINTHER trial. The predictive signatures and the code are made publicly available for academic use, laying a basis for future prospective clinical studies.

Development and validation of a multigene variant profiling assay to guide targeted and immuno therapy selection in solid tumors.

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.

A case report of androgen receptor inhibitor therapy in recurrent high-grade serous ovarian cancer.

Ovarian cancer is common gynaecological malignancy and a leading cause of death among women. Despite the advances in treatment strategies, majority of patients present with recurrence after first- or second-line treatment. Targeted therapy that has proven to be effective in other advanced or metastatic solid tumors have also demonstrated its efficacy in ovarian cancer. Recent studies have shown that the androgen receptor (AR) signalling is involved in pathogenicity and progression of cancer. Current observations suggest AR could be a potential target in managing the disease. In this case report we present a patient with high grade serous ovarian cancer (HGSOC) with multiple relapses with excellent disease control on AR inhibition with bicalutamide.

Liquid biopsy and multi-analyte testing guided treatment of HER2 positive periampullary adenocarcinoma with durable complete response after trastuzumab based therapy.

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.

Circulating ensembles of tumor-associated cells: A redoubtable new systemic hallmark of cancer.

Circulating ensembles of tumor-associated cells (C-ETACs) which comprise tumor emboli, immune cells and fibroblasts pose well-recognized risks of thrombosis and aggressive metastasis. However, the detection, prevalence and characterization of C-ETACs have been impaired due to methodological difficulties. Our findings show extensive pan-cancer prevalence of C-ETACs on a hitherto unreported scale in cancer patients and virtual undetectability in asymptomatic individuals. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of 16,134 subjects including 5,509 patients with epithelial malignancies in various organs and 10,625 asymptomatic individuals with age related higher cancer risk. PBMCs were treated with stabilizing reagents to protect and harvest apoptosis-resistant C-ETACs, which are defined as cell clusters comprising at least three EpCAM+ and CK+ cells irrespective of leucocyte common antigen (CD45) status. All asymptomatic individuals underwent screening investigations for malignancy including PAP smear, mammography, low-dose computed tomography, evaluation of cancer antigen 125, cancer antigen 19-9, alpha fetoprotein, carcinoembryonic antigen, prostate specific antigen (PSA) levels and clinical examination to identify healthy individuals with no indication of cancer. C-ETACs were detected in 4,944 (89.8%, 95% CI: 89.0-90.7%) out of 5,509 cases of cancer. C-ETACs were detected in 255 (3%, 95% CI: 2.7-3.4%) of the 8,493 individuals with no abnormal findings in screening. C-ETACs were detected in 137 (6.4%, 95% CI: 5.4-7.4%) of the 2,132 asymptomatic individuals with abnormal results in one or more screening tests. Our study shows that heterotypic C-ETACs are ubiquitous in epithelial cancers irrespective of radiological, metastatic or therapy status. C-ETACs thus qualify to be a systemic hallmark of cancer.

Encyclopedic tumor analysis for guiding treatment of advanced, broadly refractory cancers: results from the RESILIENT trial.

RESILIENT (CTRI/2018/02/011808) was a single arm, open label, phase II/III study to test if label agnostic therapy regimens guided by Encyclopedic Tumor Analysis (ETA) can offer meaningful clinical benefit for patients with relapsed refractory metastatic (r/r-m) malignancies. Patients with advanced refractory solid organ malignancies where disease had progressed following ≥2 lines of systemic treatments were enrolled in the trial. Patients received personalized treatment recommendations based on integrational comprehensive analysis of freshly biopsied tumor tissue and blood. The primary end points were Objective Response Rate (ORR), Progression Free Survival (PFS) and Quality of Life (QoL). Objective Response (Complete Response + Partial Response) was observed in 54 of 126 patients evaluable per protocol (ORR = 42.9%; 95% CI: 34.3%-51.4%, p < 0.0001). At study completion, Disease Control (Complete Response + Partial Response + Stable Disease) was observed in 114 out of 126 patients evaluable per protocol (CBR = 90.5%; 95% CI: 83.9% - 95.0%, p < 0.00001) and Disease Progression in 12 patients. Median duration of follow-up was 138 days (range 31 to 379). Median PFS at study termination was 134 days (range 31 to 379). PFS rate at 90 days and 180 days were 93.9% and 82.5% respectively. The study demonstrated that tumors have latent vulnerabilities that can be identified via integrational multi-analyte investigations such as ETA. This approach identified viable treatment options that could yield meaningful clinical benefit in this cohort of patients with advanced refractory cancers.

Malaria vaccine candidate based on Duffy-binding protein elicits strain transcending functional antibodies in a Phase I trial.

Reticulocyte invasion by Plasmodium vivax requires interaction of the Duffy-binding protein (PvDBP) with host Duffy antigen receptor for chemokines (DARCs). The binding domain of PvDBP maps to a cysteine-rich region referred to as region II (PvDBPII). Blocking this interaction offers a potential path to prevent P. vivax blood-stage growth and P. vivax malaria. This forms the rationale for development of a vaccine based on PvDBPII. Here we report results of a Phase I randomized trial to evaluate the safety and immunogenicity of recombinant PvDBPII formulated with glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE). Thirty-six malaria-naive, healthy Indian male subjects aged 18-45 years were assigned into three cohorts corresponding to doses of 10, 25 and 50 µg of PvDBPII formulated with 5 µg of GLA-SE. Each cohort included nine PvDBPII/GLA-SE vaccinees and three hepatitis B control vaccine recipients. Each subject received the assigned vaccine intramuscularly on days 0, 28 and 56, and was followed up till day 180. No serious AE was reported and PvDBPII/GLA-SE was well-tolerated and safe. Analysis by ELISA showed that all three doses of PvDBPII elicited antigen-specific binding-inhibitory antibodies. The 50 µg dose elicited antibodies against PvDBPII that had the highest binding-inhibitory titres and were most persistent. Importantly, the antibody responses were strain transcending and blocked receptor binding of diverse PvDBP alleles. These results support further clinical development of PvDBPII/GLA-SE to evaluate efficacy against sporozoite or blood-stage challenge in controlled human malaria infection (CHMI) models and against natural P. vivax challenge in malaria endemic areas.

Design and activity of AP endonuclease-1 inhibitors.

Apurinic/apyrimidinic endonuclease-1/redox effector factor-1 (APE-1) is a critical component of base excision repair that excises abasic lesions created enzymatically by the action of DNA glycosylases on modified bases and non-enzymatically by hydrolytic depurination/depyrimidination of nucleobases. Many anticancer drugs generate DNA adducts that are processed by base excision repair, and tumor resistance is frequently associated with enhanced APE-1 expression. Accordingly, APE-1 is a potential therapeutic target to treat cancer. Using computational approaches and the high resolution structure of APE-1, we developed a 5-point pharmacophore model for APE-1 small molecule inhibitors. One of the nM APE-1 inhibitors (AJAY-4) that was identified based on this model exhibited an overall median growth inhibition (GI50) of 4.19 µM in the NCI-60 cell line panel. The mechanism of action is shown to be related to the buildup of abasic sites that cause PARP activation and PARP cleavage, and the activation of caspase-3 and caspase-7, which is consistent with cell death by apoptosis. In a drug combination growth inhibition screen conducted in 10 randomly selected NCI-60 cell lines and with 20 clinically used non-genotoxic anticancer drugs, a synergy was flagged in the SK-MEL-5 melanoma cell line exposed to combinations of vemurafenib, which targets melanoma cells with V600E mutated BRAF, and AJAY-4, our most potent APE-1 inhibitor. The synergy between AJAY-4 and vemurafenib was not observed in cell lines expressing wild-type B-Raf protein. This synergistic combination may provide a solution to the resistance that develops in tumors treated with B-Raf-targeting drugs.

Synthesis and characterization of DNA minor groove binding alkylating agents.

Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases, the N-terminus was appended with an O-methyl sulfonate ester, while the C-terminus group was varied with nonpolar and polar side chains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) versus major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is >10-fold higher than that of the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells overexpressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to the expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and the diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization.

Identification and characterization of human apurinic/apyrimidinic endonuclease-1 inhibitors.

The repair of abasic sites that arise in DNA from hydrolytic depurination/depyrimidination of the nitrogenous bases from the sugar-phosphate backbone and the action of DNA glycosylases on deaminated, oxidized, and alkylated bases are critical to cell survival. Apurinic/apyrimidinic endonuclease-1/redox effector factor-1 (APE-1; aka APE1/ref-1) is responsible for the initial removal of abasic lesions as part of the base excision repair pathway. Deletion of APE-1 activity is embryonic lethal in animals and is lethal in cells. Potential inhibitors of the repair function of APE-1 were identified based upon molecular modeling of the crystal structure of the APE-1 protein. We describe the characterization of several unique nanomolar inhibitors using two complementary biochemical screens. The most active molecules all contain a 2-methyl-4-amino-6,7-dioxolo-quinoline structure that is predicted from the modeling to anchor the compounds in the endonuclease site of the protein. The mechanism of action of the selected compounds was probed by fluorescence and competition studies, which indicate, in a specific case, direct interaction between the inhibitor and the active site of the protein. It is demonstrated that the inhibitors induce time-dependent increases in the accumulation of abasic sites in cells at levels that correlate with their potency to inhibit APE-1 endonuclease excision. The inhibitor molecules also potentiate by 5-fold the toxicity of a DNA methylating agent that creates abasic sites. The molecules represent a new class of APE-1 inhibitors that can be used to probe the biology of this critical enzyme and to sensitize resistant tumor cells to the cytotoxicity of clinically used DNA damaging anticancer drugs.

Small-molecule inhibitors of DNA damage-repair pathways: an approach to overcome tumor resistance to alkylating anticancer drugs.

A major challenge in the future development of cancer therapeutics is the identification of biological targets and pathways, and the subsequent design of molecules to combat the drug-resistant cells hiding in virtually all cancers. This therapeutic approach is justified based upon the limited advances in cancer cures over the past 30 years, despite the development of many novel chemotherapies and earlier detection, which often fail due to drug resistance. Among the various targets to overcome tumor resistance are the DNA repair systems that can reverse the cytotoxicity of many clinically used DNA-damaging agents. Some progress has already been made but much remains to be done. We explore some components of the DNA-repair process, which are involved in repair of alkylation damage of DNA, as targets for the development of novel and effective molecules designed to improve the efficacy of existing anticancer drugs.