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.

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.

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.

Draft Genome Sequence of Enterotoxigenic Escherichia coli Strain E24377A, Obtained from a Tribal Drinking Water Source in India.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal disease in humans and animals. Its dissemination can occur through water sources contaminated by it. Here, we report for the first time the draft genome sequence of ETEC strain E24377A, obtained from a tribal drinking water source in India.

Comparative de novo transcriptome analysis and metabolic pathway studies of Citrus paradisi flavedo from naive stage to ripened stage.

Grapefruit (Citrus pardisi) is a popular citrus fruit that is a cross between a sweet orange and pummelo. This research article focuses on an in silico approach for comparative analysis of C. paradisi green flavedo (GF) and ethylene treated flavedo (ETF) transcriptome data. Our pathway analysis provides comprehensive information of genes playing significant role in different stages of ripening in fruit. De novo assembly was carried out using six different assemblers namely GS assembler, SeqMan NGEN, Velvet/Oases, CLC, iAssembler and Cortex followed by subsequent meta-assembly, annotation and pathway analysis. We conclude that de novo transcriptome assembly using meta-assembly approach is used to increase assembly quality in comparison to single assembler.