Testing for EGFR Variants in Pleural and Pericardial Effusion Cell-Free DNA in Patients With Non-Small Cell Lung Cancer.

Importance: Molecular testing in non-small cell lung cancer (NSCLC) is commonly limited by inadequate tumor sample. Plasma cell-free DNA (cfDNA) genotyping as a complementary test is specific but only moderately sensitive. Genotyping of cfDNA in pleural and pericardial effusion (PE-cfDNA) can further optimize molecular diagnostic yield and reduce the need for repeated biopsies. Objective: To prospectively validate droplet digital polymerase chain reaction (ddPCR) for detection of sensitizing EGFR variants and acquired Thr790Met variant (T790M) from PE-cfDNA in patients with NSCLC. Design, Setting, and Participants: This prospective diagnostic validation study was conducted between September 6, 2016, and January 21, 2021 at 2 major Hong Kong cancer centers. Patients with advanced NSCLC with both wild-type and variant EGFR status and exudative PE who underwent thoracocentesis or pericardiocentesis were randomly enrolled. Patients were either EGFR-tyrosine kinase inhibitor (TKI) naive (cohort 1) or EGFR-TKI treated but osimertinib naive (cohort 2). Enrolled patients underwent pleural- or pericardial-fluid and blood sampling for ddPCR EGFR testing. EGFR status results with ddPCR testing of PE-cfDNA and blood were compared with EGFR status in matched tumor biopsy or PE cell block samples. Main Outcomes and Measures: Specificity, sensitivity, and concordance of PE-cfDNA for detection of sensitizing EGFR variants and acquired T790M variation. Results: Among 171 patients (54% female) enrolled, there were 104 in cohort 1 and 67 in cohort 2. In cohort 1, 37% (38/102) were EGFR-variant positive; PE-cfDNA showed 97% sensitivity (95% CI, 92%-100%), 97% specificity (95% CI, 93%-100%), and 97% concordance (ĸ = 0.94, P < .001) for the detection of sensitizing EGFR variants. It was more sensitive than plasma in detecting sensitizing EGFR variants (97% vs 74%, P < .001). In cohort 2, 38% (15 of 40) were positive for the EGFR T790M variant; PE-cfDNA showed 87% sensitivity (95% CI, 69%-100%), 60% specificity (95% CI, 41%-79%), and 70% concordance (ĸ = 0.42, P = .004) for acquired T790M. The EGFR T790M variant was detected in 51% of PE-cfDNA vs 25% of PE cell block samples. Conclusions and Relevance: In this diagnostic study, EGFR variants could be accurately detected from PE-cfDNA in patients with NSCLC. More EGFR T790M was detected in PE-cfDNA than in guideline-recommended PE cell block preparations. These results suggest that PE-cfDNA can complement plasma and tumor genotyping for detecting EGFR variants in patients with advanced NSCLC.

Germline Polymorphisms and Length of Survival of Nasopharyngeal Carcinoma: An Exome-Wide Association Study in Multiple Cohorts.

Germline polymorphisms are linked with differential survival outcomes in cancers but are not well studied in nasopharyngeal carcinoma (NPC). Here, a two-phase association study is conducted to discover germline polymorphisms that are associated with the prognosis of NPC. The discovery phase includes two consecutive hospital cohorts of patients with NPC from Southern China. Exome-wide genotypes at 246 173 single nucleotide polymorphisms (SNPs) are determined, followed by survival analysis for each SNP under Cox proportional hazard regression model. Candidate SNP is replicated in another two independent cohorts from Southern China and Singapore. Meta-analysis of all samples (n = 5553) confirms that the presence of rs1131636-T, located in the 3'-UTR of RPA1, confers an inferior overall survival (HR = 1.33, 95% CI = 1.20-1.47, P = 6.31 × 10-8). Bioinformatics and biological assays show that rs1131636 has regulatory effects on upstream RPA1. Functional studies further demonstrate that RPA1 promotes the growth, invasion, migration, and radioresistance of NPC cells. Additionally, miR-1253 is identified as a suppressor for RPA1 expression, likely through regulation of its binding affinity to rs1131636 locus. Collectively, these findings provide a promising biomarker aiding in stratifying patients with poor survival, as well as a potential drug target for NPC.

Recent Advances in the Development of Biomarkers and Chemoradiotherapeutic Approaches for Nasopharyngeal Carcinoma.

Epstein-Barr virus (EBV) associated nasopharyngeal carcinoma (NPC) is endemic in Southern China, and the prognosis of this cancer has improved in part due to advances in radiotherapy (RT) techniques, broadened therapeutic options, and more precise prognostic stratification of patients. RT is the primary curative treatment of NPC, and the incorporation of chemotherapy (induction, concurrent, adjuvant) to RT has contributed to improved survival in patients with locoregionally advanced NPC. Concurrent chemoradiotherapy (CCRT) in combination with adjuvant or induction chemotherapy is now the standard treatment of locoregionally advanced NPC, but the ideal CCRT therapeutic strategy for NPC remains controversial. Plasma EBV DNA is the archetypal tumor-derived DNA in NPC, and three generations of studies have gradually expanded its clinical applications. Recently, the advent of whole exome/genome sequencing of NPC and the promising clinical activity of immune checkpoint inhibitors have also spurred interest in the development of newer biomarkers. This review will focus on two clinical advances in NPC research that have made substantial impact on the contemporary management of NPC: (1) The integration of plasma EBV DNA in an expanding spectrum of clinical indications, and the development of promising immune-related biomarkers; (2) the current development of CCRT with special emphasis on the use of induction and adjuvant chemotherapy, as well as the potential applications of metronomic chemotherapy and immune checkpoint inhibitors in the treatment of locoregionally advanced NPC.

Correlations of health-related quality of life with serum inflammatory indicators IL-8 and mIBI in patients with hepatocellular carcinoma.

PURPOSE: Health-related quality of life (HRQoL) is a significant prognostic factor for overall survival in hepatocellular carcinoma (HCC) patients, and this is independent of stage and liver function. Inflammation plays a significant role in HCC development and progression. It was hypothesized that the inflammatory status of HCC patients may affect their HRQoL. The relationship between HRQoL and inflammatory status was explored using indicators IL-8 level and modified inflammation-based index (mIBI, based on IL-8, C-reactive protein, and albumin). METHODS: From 2007-2011, HCC patients were enrolled prospectively. Baseline HRQoL assessment utilized the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 and QLQ-HCC18; clinical and laboratory data were collected at diagnosis. Two summary indices, C30 and HCC18 index-scores, were calculated. Correlation analyses were performed between HRQoL and inflammatory markers. RESULTS: In the 445 patients studied, significant correlations were found between IL-8 levels and EORTC QLQ-C30, QLQ-HCC18, C30, and HCC18 index-scores. The strongest correlated factors were those reflective of constitutional symptoms, namely QLQ-C30 "appetite loss" (with Pearson's correlation coefficient, r=0.322, P<0.0001); QLQ-C30 "fatigue" (r=0.311, P<0.0001); QLQ-C30 "role functioning" (r=-0.305, P<0.0001); QLQ-HCC18 "nutrition" (r=0.317, P<0.0001); and QLQ-HCC18 "fatigue" (r=0.306, P<0.0001). In addition, moderate but significant correlations were also observed with HCC18 index score (r=0.321, P<0.0001), and C30 index score (r=0.306, P<0.0001). HRQoL factors were also significantly correlated with mIBI. CONCLUSION: Baseline HRQoL using the conventional assessments of EORTC QLQ-C30 and QLQ-HCC18, as well as C30 and HCC18 index-scores, significantly correlated with inflammatory indicators (IL-8 level and mIBI) in HCC patients. Among the strongest correlations were those between IL-8 level and the two index-scores, as well as HRQoL aspects that represent constitutional symptoms. When paralleled with molecular findings, traditional HRQoL assessment in HCC has gained a new level of understanding: pattern recognition within an HRQoL instrument could potentially identify patients with more severe inflammatory state.

Systematic evaluation of circulating inflammatory markers for hepatocellular carcinoma.

BACKGROUNDS & AIMS: A number of circulating inflammatory factors are implicated in the pathogenesis and prognostication of hepatocellular carcinoma (HCC). We aim to evaluate the prognostication of multiple serum inflammatory factors simultaneously and develop an objective inflammatory score for HCC. METHODS: A prospective cohort of 555 patients with HCC with paired serum samples was accrued from 2009 to 2012. The blood levels of conventional inflammatory markers, namely C-reactive protein (CRP), albumin, neutrophils, lymphocytes and platelet, were determined, and 41 other exploratory markers were measured by a multiplex assay. The prognostication and interaction of markers were determined by univariate and multivarite analyses. RESULTS: The cohort was randomly divided into training cohort (n=139) and validation cohort (n=416). There were no differences in baseline characteristics between the two cohorts. In the training cohort, independent prognostic factors for overall survival included CRP (hazard ratio [HR] 1.107; P=.003), albumin (HR 0.953; P=.032) and interleukin-8 (HR=5.816; P<.001). We have modified the existing inflammation-based index (IBI) by adding serum interleukin-8 level. The modified IBI could stratify patients into four groups with distinct overall survival (P<.001). The results were also validated in the validation cohort. When compared with IBI and other conventional inflammatory markers, the modified IBI had better prognostic performance with higher c-index and homogeneity likelihood ratio chi-square. CONCLUSIONS: Among the conventional and exploratory circulating inflammatory markers, higher CRP, lower albumin and higher interleukin-8 were independent prognosticators. By combining these factors, a simple and accurate inflammatory index could be constructed.

Personalized therapy for hepatocellular carcinoma: Where are we now?

Following the approval of sorafenib, a large number of molecular targeted agents have been tested clinically for advanced hepatocellular carcinoma (HCC), but all have failed to demonstrate significant efficacy in clinical trials. Multiple reasons for this phenomenon have been discussed in the literature, with one reason being the lack of patient selection on the basis of molecular profile in clinical trials. The concept of drug testing in selected populations has been recently suggested by retrospective analyses of HCC clinical trials in which a particular subgroup of patients, either enriched by clinical factors or by tissue biomarkers, derived more benefits from the novel drug. In addition, recent advances in genomic medicine have enhanced the understanding of genetic and epigenetic events occurring in HCC, raising the possibility of personalizing targeted agents in accordance with the genetic make-up of the tumors. The development of 'personalized' treatment for HCC is, however, hindered by the lack of fresh biopsy of advanced HCC, the low incidence of genetic driver mutations in HCC and the tumor heterogeneity. These limitations may be overcome by sequencing cell-free DNA in plasma, frequently known as liquid biopsy, and revolution in the concept of the design of clinical trials. In this review article, we aim to: (1) give a summary of the recent sequencing results of HCC and the related implications for drug development; (2) highlight potential individual targeted agents and existing research on biomarker selection in clinical trials; and (3) discuss future directions, including the potential of liquid biopsy and umbrella clinical trials, to enhance personalized drug testing for HCC.

Rapid identification and differentiation of Gram-negative and Gram-positive bacterial bloodstream infections by quantitative polymerase chain reaction in preterm infants.

OBJECTIVE: To evaluate the usefulness of the Gram-specific probe-based quantitative polymerase chain reaction test for rapid detection and differentiation of Gram-negative and Gram-positive bacterial bloodstream infection in preterm infants. DESIGN: Cross-sectional study. SETTING: University-affiliated Level III neonatal intensive care unit. PATIENTS: Preterm infants with clinical features suggestive of late-onset infection. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: In addition to the full sepsis screen, 0.5 mL of EDTA blood was collected aseptically for Gram-specific quantitative polymerase chain reaction evaluation. The results were analyzed with respect to outcomes of bacterial culture in blood and other body fluids, including peritoneal and cerebrospinal fluids. The diagnostic utilities of the quantitative polymerase chain reaction were determined. A total of 218 suspected infection episodes were investigated, of which 42 episodes were culture positive and 176 were culture negative. For Gram-negative infection, the quantitative polymerase chain reaction test correctly identified 19 of 22 episodes, and the sensitivity and specificity were 86.4% and 99.0%, respectively. For Gram-positive infection, the test correctly identified 14/19 episodes, and the sensitivity and specificity were 73.7% and 98.5%. The remaining one episode was Candida albicans septicemia. None of the episodes with positive quantitative polymerase chain reaction test were classified into the wrong Gram stain category. More importantly, despite negative blood culture in five infants suffering from intra-abdominal sepsis (peritonitis [n = 4] and hepatosplenic abscess [n = 1]), the quantitative polymerase chain reaction test could detect the Gram-specific category of causative organisms in blood. CONCLUSIONS: The Gram-specific quantitative polymerase chain reaction test is reliable and highly specific for rapid identification and differentiation of Gram-negative and Gram-positive bloodstream and intra-abdominal infections. The result could be made available within 5 hrs after the specimen reaches the laboratory. A positive test is able to "rule in" bacterial bloodstream infection before blood culture results become available, and serves as a guide to predict the virulence of the causative organism according to its Gram-specific category so that critical patients can be targeted for intensive treatment.

Use of plasma DNA to predict mortality and need for intensive care in patients with abdominal pain.

BACKGROUND: We investigated the value of plasma deoxyribonucleic acid concentrations in patients presenting with acute abdominal pain to predict need for intensive care or mortality. METHODS: Plasma deoxyribonucleic acid taken from patients with acute abdominal pain was analyzed for the beta-globin gene using the quantitative polymerase chain reaction. The primary outcome measure was the combined 28-day mortality or admission to the intensive care unit. RESULTS: Of 287 consecutive patients with acute abdominal pain recruited, 12 patients were admitted to the intensive care unit and/or died. Median plasma DNA concentrations were higher in patients with cancer and major organ inflammation. Mean plasma DNA concentrations were three-fold higher in patients with systemic inflammatory response syndrome, five-fold higher in patients who died within 28 days, and eight-fold higher in patients admitted to the intensive care unit. The area under the receiver operator curve for plasma DNA concentrations and intensive care unit admission/mortality was 0.804. At a cut-off of 1100 GE/ml, the sensitivity was 67% (95%CI 35-90) and specificity was 89% (95%CI 84-92). At a cut-off of 175 GE/ml, the sensitivity was 100% (95%CI 73-100) and specificity was 30% (95%CI 25-36). Plasma DNA concentration predicted need for intensive care unit admission or death (adjusted odds ratio 1.4; P<0.0001). CONCLUSIONS: Plasma DNA may have a role in patients with acute abdominal pain as a marker for inflammation and cancer, and a predictor of intensive care unit admission/mortality.

Blockade of the epidermal growth factor receptor decreases intimal hyperplasia in balloon-injured rat carotid artery.

HYPOTHESIS: Arterial intimal hyperplasia is induced by injury and is frequently the cause of luminal narrowing after vascular reconstruction. Smooth muscle cells (SMC) respond to injury by proliferating and migrating into the intima. This process is regulated by thrombin, endothelin, and angiotensin II, all ligands of G protein-coupled receptors. Signal transduction from these receptors in cultured cells depends in part on transactivation of epidermal growth factor receptor (EGFR). We hypothesize that EGFR has a substantial role in activation of SMC in vivo and development of intimal hyperplasia. METHODS: Intimal hyperplasia was induced in rat carotid arteries by passage of a balloon catheter. Animals were given a monoclonal blocking antibody to rat EGFR, matched mouse immunoglobulin G (IgG) control antibody, or saline solution. RESULTS: Blocking EGFR antibody inhibited medial SMC proliferation, as determined by 5-bromo-2'-deoxyuridine labeling at 2 days (IgG control, 8.0% +/- 2.0%; anti-EGFR, 3.2% +/- 0.8%) and intimal hyperplasia at 14 days (intimal area: IgG control, 0.07 +/- 0.01 mm(2); anti-EGFR, 0.04 +/- 0.01 mm(2)). CONCLUSION: Activation of EGFR is important for early induction of SMC proliferation and subsequent intimal thickening.

Cell-free nucleic acids in plasma, serum and urine: a new tool in molecular diagnosis.

There has recently been an upsurge of interest in the analysis of circulating nucleic acids (DNA and/or RNA) in blood plasma or serum as a clinical diagnostic tool. Occasional earlier reports suggested the existence of circulating nucleic acids, but the potential clinical implication was not realized until 1996, when DNA with tumour-specific characteristics was demonstrated in the plasma/serum of cancer patients. This finding opened up possibilities for non-invasive cancer diagnosis. Potential applications have been reported in cancer diagnosis, prenatal diagnosis, transplantation and traumatology. Some of the findings are on the verge of being translated into clinical use. DNA is also now being sought in other body fluids such as urine.