Application of Circulating Tumor Cells and Circulating Free DNA from Peripheral Blood in the Prognosis of Advanced Gastric Cancer

Objective. To explore the application value of circulating tumor cells (CTCs) and circulating free DNA (cfDNA) from peripheral blood in the prognosis of advanced gastric cancer (AGC). Here, we measured CTCs and cfDNA quantity for predicting the outcome of patients. Patients and Methods. Forty-five patients with advanced gastric cancer who underwent neoadjuvant chemotherapy and surgical treatment were enrolled in this study. All patients received neoadjuvant chemotherapy with paclitaxel + S-1 + oxaliplatin (PSOX) regimen, and CTCs and cfDNA of the peripheral blood were detected before and after neoadjuvant therapy. Relationships between the number/type of CTC or cfDNA and the efficacy of neoadjuvant chemotherapy were analyzed. Results. Among 45 patients, 43 (95.6%) were positive, and the positive rate of mesenchymal CTC was increased with the increase in the T stage. The proportion of mesenchymal CTC was positively correlated with the N stage ( P < 0.05 ), and the larger N stage will have the higher proportion of mesenchymal CTC. Patients with a small number of mesenchymal CTC before neoadjuvant chemotherapy were more likely to achieve partial response (PR) with neoadjuvant therapy. Patients with positive CA-199 were more likely to achieve PR with neoadjuvant therapy ( P < 0.05 ). Patients in the PR group were more likely to have decreased/unchanged cfDNA concentration after neoadjuvant therapy ( P = 0.119 ). After neoadjuvant therapy (before surgery), the cfDNA concentration was higher and the efficacy of neoadjuvant therapy (SD or PD) was lower ( P = 0.045 ). Conclusions. Peripheral blood CTC, especially interstitial CTC and cfDNA, has a certain value in predicting the efficacy and prognosis of neoadjuvant chemotherapy in advanced gastric cancer.

Comparative Evaluation of Diagnostic Performance of Circulating free DNA by Conventional vs. Real Time PCR in Gallbladder Cancer

Background: Circulating free DNA (cfDNA) in serum/plasma has been studied as a promising biomarker in various pathologies, including cancer. However, there is no standardized method for the isolation and quantification of serum cfDNA. An effective and reliable method for isolation and quantification is of utmost importance before any clinical decision. The current study compares the conventional and real-time PCR methods to find any differences and concordance in cfDNA levels between the two methods and the diagnostic accuracy of cfDNA by each method. Methods: Serum sample was collected from 67 subjects, including 17 normal healthy individuals (control, n=17), 19 disease controls (cholecystitis, n=19), and 31 Gallbladder cancer patients (cancer, n=31) before any treatment for cfDNA quantification. Results: The cfDNA level did not differ significantly between two methods in both control and cholecystitis groups. In cancer group, cfDNA level was significantly (P < 0.001) different and higher in real time PCR as compared to conventional PCR. There was no significant correlation between two methods in control (r=0.02, P = 0.937), cholecystitis (r=0.10, P = 0.697), cancer (r=-0.08, P = 0.657) and total cases (control + cholecystitis + cancer) (r=0.06, P = 0.622). The diagnostic accuracy of two methods was found similar (P > 0.05) when assessed between control vs. cholecystitis (Z=0.85, P = 0.397), and control vs. total cases (Z=1.35, P = 0.177). However, the diagnostic accuracy of real time PCR was found significantly different and higher as compared to conventional PCR when assessed between control vs. cancer (Z=2.98, P = 0.003), and cholecystitis vs. cancer (Z=4.41, P < 0.001). Conclusion: Quantitative real-time PCR method is of high accuracy, reproducibility, and time-effectiveness. The diagnostic accuracy of real-time PCR was higher compared to conventional PCR.

Interim Analysis of Mmrf Curecloud Research Initiative Identifies High Prevalence and Patterns of Clonal Hematopoiesis of Indeterminate Potential (CHIP) Mutations in a Real World Myeloma Cohort

Background: In order to gain a deeper understanding of the clinical, molecular and immune parameters involved in multiple myeloma (MM) disease initiation, progression and response to treatment, the Multiple Myleoma Research Foundation (MMRF) and its partners launched the CureCloud Research Initiative (NCT03657251), a Direct-to-Patient (DTP) effort aimed at enrolling 5,000 individuals from whom comprehensive molecular and immune analyses are generated from blood specimens and the resulting data aggregated with the correlating clinical information. Methods: To support the molecular characterization of liquid biopsies for CureCloud, a set of myeloma-specific blood-based approaches were developed. The first of such assays is a hybrid selection panel that captures 70 commonly altered MM and Clonal Hematopoiesis of Indeterminate Potential (CHIP) genes detecting somatic variants present in a patient's circulating-free DNA (cfDNA). This MM-70 assay was thoroughly validated to &gt;90% sensitivity for events at 1% variant allele frequency with a specificity of &lt;0.2 FP/Mb when sequenced at 60,000x. A clinical-grade (CLIA) pipeline was established enabling the return of results. Several quality control measures were used to select for CHIP related mutations including filtering out known myeloma driver events, removing high frequency events, and overlapping with previously published CHIP related mutations. Results: 580 subjects were enrolled to the study at abstract submission, including 127 smolderers, 173 newly diagnosed and 265 relapsed participants. 430 cases have been analyzed on the MM-70 assay. A mean cfDNA amount of 31 ng was recovered for input. Mean raw coverage depth of 176,000x was attained, with a mean duplex depth of 1271 (517-2406). 417 cases passed the minimum CLIA validation thresholds. Some of the most frequently mutated genes observed were in the MAPK, DNA repair, epigenetic, cell cycle, and NF-kappaB pathways in agreement with MM retrospective genomic research studies performed on bone marrow aspirates. Of interest, CHIP mutations were detected in a large proportion of cases (174) with their prevalence increasing with age (Figure 1A). 117 of patients had only one CHIP mutation while 57 had two or more with the same genomic loci were recurrently mutated in DNMT3A, TP53, ASXL1, and PPM1D. No significant differences in CHIP were observed between smoldering and overt myeloma subjects (Figure 1B). Interestingly, a greater (&gt;0.1) CHIP Variant Allelic Fraction (VAF) was seen in individuals with higher risk genomic alterations (Figure 1C). Conclusion: This genomic characterization of one of the largest cohorts to date of individuals with myeloma and precursor conditions using a cfDNA-based liquid biopsy assay was able to recapitulate known myeloma-specific mutations and reinforced the importance of CHIP alterations in MM. CHIP mutations are indeed frequent in myeloma, increase with age, and higher burden might be related to disease aggressiveness. Figure 1 Figure 1. Disclosures Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy.

Implementation of Next Generation Sequencing-Based Liquid Biopsy for Clinical Molecular Diagnostics in Non-Small Cell Lung Cancer (NSCLC) Patients

Genetic screening of somatic mutations in circulating free DNA (cfDNA) opens up new opportunities for personalized medicine. In this study, we aim to illustrate the implementation of NGS-based liquid biopsy in clinical practice for the detection of somatic alterations in selected genes. Our work is particularly relevant for the diagnosis and treatment of NSCLC. Beginning in 2020, we implemented the use of Roche’s Avenio ctDNA expanded panel in our diagnostic routine. In this study, we retrospectively review NGS-based clinical genetic tests performed in our laboratory, focusing on key analytical parameters. Avenio ctDNA kits demonstrated 100% sensitivity in detecting single nucleotide variants (SNVs) at >0.5% variant allele frequency (VAF), and high consistency in reproducibility. Since 2020, we performed cfDNA genotyping test in 86 NSCLC patients, and we successfully sequenced 96.5% (83/86) of samples. We observed consistency in sequencing performance based upon sequencing depth and on-target rate. At least one gene variant was identified in 52 samples (63%), and one or more actionable variants were detected in 21 out of 83 (25%) of analysed patients. We demonstrated the feasibility of implementing an NGS-based liquid biopsy assay for routine genetic characterization of metastatic NSCLC patients.

Neutrophil extracellular traps and neutrophil-derived mediators as possible biomarkers in bronchial asthma

Neutrophils (PMNs) contain and release a powerful arsenal of mediators, including several granular enzymes, reactive oxygen species (ROS) and neutrophil extracellular traps (NETs). Although airway neutrophilia is associated with severity, poor response to glucocorticoids and exacerbations, the pathophysiological role of neutrophils in asthma remains poorly understood. Twenty-four patients with asthma and 22 healthy controls (HCs) were prospectively recruited. Highly purified peripheral blood neutrophils (> 99%) were evaluated for ROS production and activation status upon stimulation with lipopolysaccharide (LPS), N-formylmethionyl-leucyl-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA). Plasma levels of myeloperoxidase (MPO), CXCL8, matrix metalloproteinase-9 (MMP-9), granulocyte–monocyte colony-stimulating factor (GM-CSF) and vascular endothelial growth factor (VEGF-A) were measured by ELISA. Plasma concentrations of citrullinated histone H3 (CitH3) and circulating free DNA (dsDNA) were evaluated as NET biomarkers. Activated PMNs from asthmatics displayed reduced ROS production and activation status compared to HCs. Plasma levels of MPO, MMP-9 and CXCL8 were increased in asthmatics compared to HCs. CitH3 and dsDNA plasma levels were increased in asthmatics compared to controls and the CitH3 concentrations were inversely correlated to the % decrease in FEV1/FVC in asthmatics. These findings indicate that neutrophils and their mediators could have an active role in asthma pathophysiology.

cfDNA in pancreatic neuroendocrine carcinoma management with Cushing’s syndrome

We report a case of metastatic pancreatic neuroendocrine carcinoma associated with paraneoplastic Cushing’s syndrome, successively treated with five lines of treatment (platin-etoposide, LV5FU2-dacarbazine, Folfirinox, pembrolizumab, and paclitaxel) and anti-secretory treatment. Circulating-free DNA (cfDNA) was analysed at each morphological evaluation starting from the second-line treatment. cfDNA changes were well-correlated with the disease course, and cfDNA may be used as a predictive marker and/or an early marker of response. In addition, absolute count of atypical cells was elevated upon disease progression.