The detection of circulating tumor DNA (ctDNA) in liquid biopsy samples as an oncological marker is being used in clinical trials at every step of clinical management. As ctDNA-based liquid biopsy kits are developed and used in clinics, companies work towards increased convenience, accuracy, and cost over solid biopsies and other oncological markers. The technology used to differentiate ctDNA and cell-free DNA (cfDNA) continues to improve with new tests and methodologies being able to detect down to mutant allele frequencies of 0.001% or 1/100,000 copies. Recognizing this development in technology, the FDA has recently given pre-market approval and breakthrough device designations to multiple companies. The purpose of this review is to look at the utility of measuring total cfDNA, techniques used to differentiate ctDNA from cfDNA, and the utility of different ctDNA-based liquid biopsy kits using relevant articles from PubMed, clinicaltrials.gov, FDA approvals, and company newsletters. Measuring total cfDNA could be a cost-effective, viable prognostic marker, but various factors do not favor it as a monitoring tool during chemotherapy. While there may be a place in the clinic for measuring total cfDNA in the future, the lack of standardization means that it is difficult to move forward with large-scale clinical validation studies currently. While the detection of ctDNA has promising standardized liquid biopsy kits from various companies with large clinical trials ongoing, their applications in screening and minimal residual disease can suffer from lower sensitivity. However, researchers are working towards solutions to these issues with innovations in technology, multi-omics, and sampling. With great promise, further research is needed before liquid biopsies can be recommended for everyday clinical management.

Purpose Samalizumab is a novel recombinant humanized monoclonal antibody that targets CD200, an immunoregulatory cell surface member of the immunoglobulin superfamily that dampens excessive immune responses and maintains self-tolerance. This first-in-human study investigated the therapeutic use of samalizumab as a CD200 immune checkpoint inhibitor in chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). Experimental design Twenty-three patients with advanced CLL and 3 patients with MM were enrolled in an open-label phase 1 study (NCT00648739). Patients were assigned sequentially to one of 7 dose level cohorts (50 to 600 mg/m²) in a 3 + 3 study design, receiving a single dose of samalizumab intravenously once every 28 days. Primary endpoints were safety, identification of the maximum tolerated dose (MTD), and pharmacokinetics. Secondary endpoints were samalizumab binding to CD200, pharmacodynamic effects on circulating tumor cells and leukocyte subsets, and clinical responses. Results Twenty-one patients received > 1 treatment cycle. Adverse events (AEs) were generally mild to moderate in severity. Samalizumab produced dose-dependent decreases in CD200 expression on CLL cells and decreased frequencies of circulating CD200 + CD4+ T cells that were sustained at higher doses. The MTD was not reached. Decreased tumor burden was observed in 14 CLL patients. One CLL patient achieved a durable partial response and 16 patients had stable disease. All MM patients had disease progression. Conclusions Samalizumab had a good safety profile and treatment was associated with reduced tumor burden in a majority of patients with advanced CLL. These preliminary positive results support further development of samalizumab as an immune checkpoint inhibitor. Trial registration ClinicalTrials.gov, NCT00648739 registered April 1, 2008.

Introduction: Prognosis remains dismal for pancreatic ductal adenocarcinoma (PDAC). Genomics and proteomics has depicted heterogeneity in PDAC. Collectively, this information could be useful in improving diagnosis, prognosis, modalities of therapy, treatment responses, deciphering drug resistance and new drug development. Areas Covered: We describe major advances in the cellular and molecular subtypes based on next generation sequencing and their predictive and prognostic value in PDAC patients. We review aberrant genes involving in defined cellular processes in PDAC. Finally, the current state of drug development with novel investigational agents targeting cell fate, cell survival, genomic instability, tumor-stroma and immune checkpoints are discussed. Expert Opinion: Molecular techniques have revealed distinct driver mutations in PDAC. Common genes and cellular processes are dysregulated in the pathogenesis of PDAC. These cellular processes categorized by aberrant pathways include control cell fate, genome maintenance and cell survival. Dysregulation of the tumor microenvironment promotes an intense fibrosis and immune suppression that play a major role in drug resistance. New information on tumor biology has led to the development of targeted/stromal therapies, immunotherapies or combinations with current chemotherapy in PDAC. New drug development targeting multiple hallmarks of PDAC we hope will positively impact the quality and survival of PDAC patients.

We investigated biomarker CEACAM6, a highly abundant cell surface adhesion receptor that modulates the extracellular matrix (ECM) in pancreatic ductal adenocarcinoma (PDA). The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) RNA-Seq data from PDA patients were analyzed for CEACAM6 expression and evaluated for overall survival, association, enrichment and correlations. A CRISPR/Cas9 Knockout (KO) of CEACAM6 in PDA cell line for quantitative proteomics, mitochondrial bioenergetics and tumor growth in mice were conducted. We found CEACAM6 is over-expressed in primary and metastatic basal and classical PDA subtypes. Highest levels are in classical activated stroma subtype. CEACAM6 over-expression is universally a poor prognostic marker in KRAS mutant and wild type PDA. High CEACAM6 expression is associated with low cytolytic T-cell activity in both basal and classical PDA subtypes and correlates with low levels of T-_REG markers. In HPAF-II cells knockout of CEACAM6 alters ECM-cell adhesion, catabolism, immune environment, transmembrane transport and autophagy. CEACAM6 loss increases mitochondrial basal and maximal respiratory capacity. HPAF-II CEACAM6−/− cells are growth suppressed by >65% vs. wild type in mice bearing tumors. CEACAM6, a key regulator affects several hallmarks of PDA including the fibrotic reaction, immune regulation, energy metabolism and is a novel therapeutic target in PDA.

Background: Novel combination therapies to overcome anti-PD-1 resistance are required. Enadenotucirev, a tumor-selective blood stable adenoviral vector, has demonstrated a manageable safety profile and ability to increase tumor immune-cell infiltration in phase I studies in solid tumors. Methods: We conducted a phase I multicenter study of intravenous enadenotucirev plus nivolumab in patients with advanced/metastatic epithelial cancer not responding to standard therapy. Co-primary objectives were safety/tolerability and maximum tolerated dose and/or maximum feasible dose (MTD/MFD) of enadenotucirev plus nivolumab. Additional endpoints included response rate, cytokine responses, and anti-tumor immune responses. Results: Overall, 51 heavily pre-treated patients were treated, 45/51 (88%) of whom had colorectal cancer (35/35 patients with information available were microsatellite instability-low/microsatellite stable) and 6/51 (12%) had squamous cell carcinoma of the head and neck. The MTD/MFD of enadenotucirev plus nivolumab was not reached, with the highest dose level tested (1×10¹² vp day 1; 6×10¹² vp days 3 and 5) shown to be tolerable. Overall, 31/51 (61%) patients experienced a grade 3–4 treatment-emergent adverse event (TEAE), most frequently anemia (12%), infusion-related reaction (8%), hyponatremia (6%), and large intestinal obstruction (6%). Seven (14%) patients experienced serious TEAEs related to enadenotucirev; the only serious TEAE related to enadenotucirev occurring in >1 patient was infusion-related reaction (n=2). Among the 47 patients included in efficacy analyses, median progression-free survival was 1.6 months, objective response rate was 2% (one partial response for 10 months), and 45% of patients achieved stable disease. Median overall survival was 16.0 months; 69% of patients were alive at 12 months. Persistent increases in Th1 and related cytokines (IFNγ, IL-12p70, IL-17A) were seen from ~day 15 in two patients, one of whom had a partial response. Among the 14 patients with matching pre-tumor and post-tumor biopsies, 12 had an increase in intra-tumoral CD8⁺ T-cell infiltration and 7 had increased markers of CD8 T-cell cytolytic activity. Conclusions: Intravenously dosed enadenotucirev plus nivolumab demonstrated manageable tolerability, an encouraging overall survival and induced immune cell infiltration and activation in patients with advanced/metastatic epithelial cancer. Studies of next-generation variants of enadenotucirev (T-SIGn vectors) designed to further re-program the tumor microenvironment by expressing immune-enhancer transgenes are ongoing. Trial registration number: NCT02636036.

Molecular profiling with next generation sequencing (NGS) delivers key information on mutant gene sequences, copy number alterations, gene-fusions, and with immunohistochemistry (IHC), is a valuable tool in clinical decision making for patients entering investigational agent trials. Our objective was to elucidate mutational profiles from primary versus metastatic sites from advanced cancer patients to guide rational therapy. All phase I patients (n = 203) with advanced cancer were profiled by commercially available NGS platforms. The samples were annotated by histology, primary and metastatic site, biopsy site, gene mutations, mutation count/gene, and mutant TP53. A molecular profile of each patient was categorized into common and unique mutations, signaling pathways for each profile and TP53 mutations mapped to 3D-structure of p53 bound to DNA and pre/post therapy molecular response. Of the 171 patients analyzed, 145 had genetic alterations from primary and metastatic sites. The predominant histology was adenocarcinoma followed by squamous cell carcinoma, carcinoma of unknown primary site (CUPS), and melanoma. Of 790 unique mutations, TP53 is the most common followed by APC, KRAS, PIK3CA, ATM, PTEN, NOTCH1, BRCA2, BRAF, KMT2D, LRP1B, and CDKN2A. TP53 was found in most metastatic sites and appears to be a key driver of acquired drug resistance. We highlight examples of acquired mutational profiles pre-/post- targeted therapy in multiple tumor types with a menu of potential targeted agents. Conclusion: The mutational profiling of primary and metastatic lesions in cancer patients provides an opportunity to identify TP53 driver ‘pathways’ that may predict for drug sensitivity/resistance and guide rational drug combinations in clinical trials.