Multivalent anions as universal latent electron donors.

Electrodes with low work functions are required to efficiently inject electrons into semiconductor devices. However, when the work function drops below about 4 electronvolts, the electrode suffers oxidation in air, which prevents its fabrication in ambient conditions. Here we show that multivalent anions such as oxalate, carbonate and sulfite can act as powerful latent electron donors when dispersed as small ion clusters in a matrix, while retaining their ability to be processed in solution in ambient conditions. The anions in these clusters can even n-dope the semiconductor core of π-conjugated polyelectrolytes that have low electron affinities, through a ground-state doping mechanism that is further amplified by a hole-sensitized or photosensitized mechanism in the device. A theoretical analysis of donor levels of these anions reveals that they are favourably upshifted from ionic lattices by a decrease in the Coulomb stabilization of small ion clusters, and by irreversibility effects. We attain an ultralow effective work function of 2.4 electronvolts with the polyfluorene core. We realize high-performance, solution-processed, white-light-emitting diodes and organic solar cells using polymer electron injection layers with these universal anion donors, demonstrating a general approach to chemically designed and ambient-processed Ohmic electron contacts for semiconductor devices.

Secretory leukocyte protease inhibitor modulates urethane-induced lung carcinogenesis.

Secretory leukocyte protease inhibitor (SLPI), 11.7 kDa serine protease inhibitor, is produced primarily in the respiratory tract, but it is often elevated in lung, head/neck and ovarian cancers. SLPI expression in relation to cancer progression, metastasis and invasion has been studied extensively in non-small cell lung cancer. However, the role of SLPI during the early stages of carcinogenesis remains unknown. We hypothesized that SLPI is required from the initiation and promotion to the progression of lung carcinogenesis. A skin allograft model using SLPI-knockout (SLPI-KO) mice and short hairpin RNA-treated cells was used to demonstrate that SLPI expression in tumor cells is crucial for tumor formation. Moreover, lung tumorigenesis induced by urethane, a chemical lung carcinogen, was significantly suppressed in SLPI-KO mice in association with decreased nuclear factor-kappaB (NF-κB) activity. SLPI deficiency also resulted in decreased cell numbers and decreased production of inflammatory cytokines in bronchoalveolar lavage fluids. The suppression of NF-κB activation in SLPI-KO mice was associated with lower expression of NF-κB-related survival genes and DNA repair genes. Our findings demonstrate that SLPI plays an important role from the initial stages of lung carcinogenesis to the progression of lung cancer in an NF-κB-dependent manner.

High Proportion of Arsenic Detected in Bronchoalveolar Fluid among Newly Diagnosed Lung Cancer in Jakarta Indonesia.

OBJECTIVE: This study aims to measure arsenic concentration in bronchoalveolar lavage fluid  (BALF ) of newly diagnosed lung cancer and its corelation with clinical profiles. METHODS: This study is a cross-sectional study to identify arsenic levels in newly diagnosed lung cancer patients. Bronchoalveolar lavage fluid was taken during the bronchoscopy. Arsenic concentration was measured using an ICP-EOS spectrometer. RESULTS: Forty-two subjects who met inclusion criteria were recruited in this study. Arsenic metals were detected among 40% of subjects with mean, highest, and lowest values are 0.38 µg/L, 0.5 µg/L, and 0.3 µg/L, respectively. There is no significant difference between arsenic level and patients' demographic and clinical data. CONCLUSION: Arsenic was detected in BALF in majority of newly diagnosed lung cancer patients. Despite the insignificant relationship between arsenic level and patients characteristic, this results is evidence of which arsenic metal exposure in lung cancer during their lifetime and should raise public health awareness regarding mitigating the source of exposure and its potential as lung carcinogenic agent.

Acute exacerbation of idiopathic pulmonary fibrosis model in the rats using bleomycin and lipopolysaccharides.

Objective: This study was conducted to establish a rat model of acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) using the combination of bleomycin (BLM) and lipopolysaccharides (LPS). Materials and Method: Twenty-four male Sprague Dawley rats were allocated into two equal groups: the sham or the bleomycin and lipopolysaccharides-induced AE-IPF group (BLM-LPS). On Day 7, BLM intratracheally and LPS intraperitoneally were both used to administer AE-IPF. The BLM-LPS group and its respective sham group were terminated on Days 8, 14, or 21. Samples of bronchoalveolar lavage fluid (BALF) and lungs were taken and investigated for cell count and histopathology. Results: On Day 8, histological analysis revealed inflammatory cell infiltration with edema and hyaline membrane, and the BALF differential cell count revealed high neutrophil counts. By having a higher collagen density area and Ashcroft modified score than the sham group on Day 14, the BLM-LPS group displayed significantly lower oxygen saturation, alveolar air area, and a fibrotic appearance. However, there was a spontaneous resolution in inflammation and fibrotic appearance on Day 21 after the BLM administration. Conclusions: By combining BLM and LPS, it was possible to create a successful rat model of AE-IPF. The present model showed the peak exacerbation on Day 8 and the fibrotic peak on Day 14, which gradually improved. The optimal time for the new AE-IPF therapeutic intervention was determined to be between Days 8 and 14.

The Fungal and Bacterial Interface in the Respiratory Mycobiome with a Focus on Aspergillus spp.

The heterogeneity of the lung microbiome and its alteration are prevalently seen among chronic lung diseases patients. However, studies to date have primarily focused on the bacterial microbiome in the lung rather than fungal composition, which might play an essential role in the mechanisms of several chronic lung diseases. It is now well established that Aspergillus spp. colonies may induce various unfavorable inflammatory responses. Furthermore, bacterial microbiomes such as Pseudomonas aeruginosa provide several mechanisms that inhibit or stimulate Aspergillus spp. life cycles. In this review, we highlighted fungal and bacterial microbiome interactions in the respiratory tract, with a focus on Aspergillus spp.

Severe acute respiratory syndrome coronavirus-2 antibody response after Moderna vaccine booster on healthcare providers.

INTRODUCTION: As an endeavor to control SARS-CoV-2 infection, the Moderna vaccine booster was given to healthcare workers to prevent reinfection and reduce the risk of complications from COVID-19. A heterologous booster vaccine is also thought to provide better protection against the current SARS-CoV-2 variants of concern. However, research that evaluates the effectiveness of the Moderna vaccine booster and the resulting SARS-CoV-2 antibody concentration is needed. OBJECTIVE: To evaluate the concentration of SARS-CoV-2 antibodies after the Moderna vaccine booster and the severity of SARS-CoV-2 infection before and after the Moderna vaccine booster. RESULTS: A total of 93 healthcare providers who received Moderna vaccine booster were included in the study. Examination of antibody concentration 3 months after the booster showed an average concentration of 10081.65 U/mL. There was an increase in antibody concentration before the booster and 3 months after, from a median of 1.7 U/mL to 9540 U/mL. Every subject showed a statistically significant increment of antibody concentration 3 months after the booster (p < 0.01). Thirty-seven (39.8%) subjects received two doses of the Sinovac vaccine and were confirmed to have COVID-19 with the Delta variant. After the booster, 26 (28%) subjects were infected with the Omicron Variant. Among the subjects who received two doses of the Sinovac vaccine and were confirmed with COVID-19, 36 (30.1%) had mild symptoms, and 1 (1.1%) was asymptomatic. CONCLUSIONS: Heterologous Moderna vaccine booster effectively increases antibody response against SARS-CoV-2 variants and shows mild symptoms of COVID-19 infection.

Therapeutic bronchoscopy followed by sequential radiochemotherapy in the management of life-threatening tracheal adenoid cystic carcinoma: a case report.

BACKGROUND: Adenoid cystic carcinoma of the lung is a distinctive salivary-gland-type malignant epithelial neoplasm that rarely presents as a primary tumor of the respiratory tract. Complete surgical resection remains the treatment of choice for adenoid cystic carcinoma. We present a case of large ACC tumors that caused severe central airway obstruction and were effectively treated with therapeutic bronchoscopy followed by radiotherapy and chemotherapy. CASE PRESENTATION: A 31-year-old Malay Indonesian female patient who was a nonsmoker and had no family history of cancer was admitted to the emergency ward because of worsening breathlessness accompanied by stridor since 1 week prior. Chest computed tomography revealed segmental atelectasis of the left lung; a mass on the left main bronchus, with infiltrates in segments 1, 2, and 3 of the left lung; and consolidation in the left inferior lobe, with narrowing of the main left bronchus. Lobulated masses obstructing almost the entire distal trachea up to the carina and the entire left main bronchus were found on bronchoscopy. Owing to the large tumors causing severe central airway obstruction, the medical team decided to perform central airway mass removal through rigid bronchoscopy. A neodymium-doped yttrium-aluminum-garnet laser was used first to facilitate mass shrinkage. After the laser treatment, mechanical mass removal using a rigid scope was performed. The tracheal and carinal lumens were opened to > 50% of their diameter, with the left main bronchus lumen opened only slightly. After the treatment, the patient was stable, and no stridor was found. Adjuvant intensity-modulated radiotherapy and chemotherapy were performed after the therapeutic bronchoscopy. At the end of the entire treatment, reevaluation by thoracic computed tomography scan and bronchoscopy revealed no remaining mass. CONCLUSIONS: In cases of nonresectable large adenoid cystic carcinoma tumors with life-threatening central airway obstruction, therapeutic bronchoscopy followed by sequential radiochemotherapy might achieve a complete response outcome.

The Tuberculosis and COVID-19 coinfection: A report of two cases at a tertiary referral in Indonesia.

COVID-19 cases are still rising globally in the middle of the tuberculosis epidemic. Several countries have reported TB-COVID-19 coinfection that could pose a double burden in the health care facilities in developing countries. We reported two pulmonary tuberculosis patients coinfected with COVID-19 with an overlapping clinical manifestation of tuberculosis and COVID-19 with a good prognosis at the end of COVID-19 treatment. This paper aims to discuss TB patients' susceptibility against SARS-COV-2 infection, the clinical profile of TB-COVID-19 coinfection, and the disease's prognosis. The clinician should be aware of both common disease symptoms that appear in a patient and should be confirmed and treat promptly.

TP53 and EGFR Mutational Status in Thymoma: A Genetic Sequencing Study.

BACKGROUND AND OBJECTIVE: Thymoma is a rare malignant tumor that usually with an indolent presentation, which was falsely assumed to be benign previously. The tumor suppressor P53 (TP53) and EGFR gene mutate most frequently in human cancers. We tried to investigate the presence of TP53 and EGFR mutations among thymoma patients referred to an Indonesian referral respiratory hospital and to discuss its potential role in thymoma management and prognosis. MATERIAL AND METHODS: Surgically resected tumor tissues were collected from thymoma patients and then underwent genomic analysis. PCR was performed on the extracted Paraffinized  DNA to amplify exon 6 of TP53 and exons 18, 19, and 21 of EGFR. The evaluation of mutational status was done using direct sequencing and sequence analysis of purified PCR products. RESULTS: Among 27 collected samples, TP53 exon 6 mutation, namely  missense mutation and nonsense mutation, was observed in two samples (7.4%). EGFR exon 18 mutation, namely E709K and nonsense mutation, was found in 2 samples (7.4%). An intronic mutation in EGFR exon 19 (3.7%) and exon 21 (3.7%) was observed in one sample. CONCLUSION: TP53 and EGFR mutations were not most frequent, so it seems that these genes are not involved in the pathogenesis of thymoma in Indonesian patients. Nevertheless, we found two samples with a significant mutation in p53 and EGFR genes, suggesting further research on thymoma prognostification and targeted therapy.

Pneumocystis jirovecii colonization in bronchoalveolar lavage among naïve non-small cell lung cancer from tertiary respiratory hospital in Jakarta, Indonesia.

INTRODUCTION: Pneumocystis jirovecii pneumonia (PJP) is a lung mycosis commonly found in immunocompromised patients (e.g., HIV patients); however, its role in solid cancer remains unclear. This study aims to identify Pneumocystis jirovecii colonization among naïve non-small-cell lung cancer (NSCLC) through bronchoalveolar lavage (BAL) and explore its correlation with clinical parameters. METHODOLOGY: This cross-sectional study recruited newly diagnosed naïve NSCLC patients who had not been given systemic treatments. We tested BAL from patients for P. jirovecii colonization with nested PCR targeting the mtLSU rRNA gene. Demographic and clinical characteristics were obtained from medical records, and the correlation between P. jirovecii colonization and clinicopathological data were analyzed. Kaplan-Meier analyses were done to evaluate survival. RESULTS: Among 56 newly diagnosed, naïve NSCLC patients enrolled, the prevalence of P. jirovecii colonization was 17.9% (10 subjects). There was no statistically significant difference in demographic and clinical characteristics between the P. jirovecii colonization group versus no colonization (p value > 0.05). The overall survival duration for both groups demonstrated no significant difference. CONCLUSIONS: This study demonstrated a relatively high prevalence of P. jirovecii colonization among BAL samples of naïve Indonesian NSCLC patients. Further study is needed to delineate its implications for the potential transmission source, lung cancer pathogenesis, and prognosis.

Bronchoscopy Findings of Severe and Critical COVID-19 Patients Treated in ICU: A Year of Experience in a Developing Country.

Background: Bronchoscopy procedure in patients with COVID-19 poses significant challenges, especially in a developing country with limited resources. Objectives: We aim to describe the clinical characteristics of severe and critical COVID-19 patients treated in an intensive care unit (ICU) and their bronchoscopy findings. Methods: We performed a retrospective analysis of clinical data of ICU patients with COVID-19 treated and received bronchoscopy procedures. This study retrospectively included all consecutive patients who underwent bronchoscopy at a teaching hospital in Depok, Indonesia, from May, 2020, until May, 2021. Results: A total of 57 bronchoscopy procedures in 54 patients were performed in this study. Primary procedure indications were retained mucus (68.4%) and ventilatory support weaning failure (15.8%). Bronchoscopic findings were mostly hyperaemic mucosa (95.00%) and purulent secretion (50.90%). Microbiological findings from bronchoalveolar samples were Acinetobacter baumanii, Klebsiella pneumoniae, and Candida albicans (33.3%, 26.6%, and 10.5%, respectively). The most common fungal isolated were Candida albicans (28%), followed by Candida tropicalis (16%) and Aspergillus sp. (8%). The overall length of hospital stay was 24 days, and the in-ICU stay was 22.06 ± 10.99 days. The patients' survival of 28-days postprocedural outcome was 25.9% (14 subjects). Follow-up found that 20.4% of patients survived after sixty days of hospitalization. Conclusion: Diagnostic and therapeutic bronchoscopy in ICU patients with COVID-19 was safe and feasible to perform in developing countries with limited resources. It could help bronchial mucous clearance and confirm microbiological infection. The procedures should be strictly performed for patients with indications and comply with safety standards.

Possible intraindividual evolution of SARS-CoV-2 in nasopharyngeal and anal swab in an octogenarian: a case report.

Introduction: COVID-19 is an emerging infectious disease that remains to be further investigated. Case report: Here, we describe a case of COVID-19 in an octogenarian woman with comorbidities who slowly recovered during hospitalization, but died due to sudden cardiac death after 2 weeks of hospitalization. Her nasopharyngeal and anal swabs returned positive for SARS-CoV-2 by RT-PCR on day 7 of hospitalization. The NGS showed possible intraindividual evolution of virus. The sample from the nasopharyngeal swab yielded a B.1470 variant classified as clade GH. This variant showed mutation in the spike gene D614G; N gene; NS3 gene; NSP2 gene and NSP12 gene. The sample from the anal swab showed similar mutation but with additional point mutation in spike gene S12F and was classified as B.1.465 variant. Conclusions: The possibility of the gastrointestinal tract that served as reservoir for virus mutation accumulation should also be considered and the potential impact of viral fecal transmission in the environment should be further investigated.

Serial OX40 engagement on CD4+ T cells and natural killer T cells causes allergic airway inflammation.

RATIONALE: OX40-OX40 ligand (OX40L) interactions have been proposed to support induction of allergic airway inflammation, which may be attributable to OX40 signaling in CD4(+) helper T cells for adaptive immune responses. However, a possible involvement of natural killer T (NKT) cells in the pathogenesis suggests that the underlying mechanisms are not yet fully elucidated. OBJECTIVES: We aimed to characterize the OX40-modulated cellular contribution to allergic airway inflammation in a mouse model of house dust mite (HDM) allergen exposure. METHODS: Mice were sensitized to HDM and, 3 weeks later, challenged with HDM on three consecutive days through the airways. Two days after the last exposure, bronchoalveolar lavage fluids and blood samples and lung tissues were evaluated for the airway inflammation. MEASUREMENTS AND MAIN RESULTS: The development of HDM-induced eosinophilic airway inflammation was dependent on OX40 of both CD4(+) T cells and NKT cells; OX40 engagement on CD4(+) T cells in the sensitization led to pulmonary OX40L augmentation after the allergen challenge, which stimulated pulmonary NKT cells through OX40 to provide the pathogenic cytokine milieu. This was ablated by OX40L blockade by inhalation of the neutralizing antibody during the challenge, suggesting the therapeutic potential of targeting pulmonary OX40-OX40L interactions. Moreover, OX40 expression in CD4(+) T cells, but not in NKT cells, was reciprocally regulated by the helper T cell type 1-skewing transcription factor Runx3. CONCLUSIONS: OX40 on not only CD4(+) T cells but also NKT cells is involved in allergic airway inflammation. Notably, pulmonary blockade of OX40 ligation on NKT cells has therapeutic implications.

Performance of LDBio Aspergillus WB and ICT Antibody Detection in Chronic Pulmonary Aspergillosis.

The detection of Aspergillus antibody has a key role in the diagnosis of chronic pulmonary aspergillosis. Western blot (WB) and immunochromatography (ICT) lateral flow detection of Aspergillus antibody can be used as confirmatory and screening assays but their comparative performance in TB patients is not known. This study investigated the performance of these assays among 88 post-tuberculosis patients with suspected CPA. Sensitivity, specificity, receiver operating curve (ROC), area under-curve (AUC) and the agreement between two assays were evaluated. Both WB and ICT showed good sensitivity (80% and 85%, respectively) for detection of Aspergillus antibodies. Substantial agreement (0.716) between these assays was also obtained. The highest AUC result (0.804) was achieved with the combination of WB and ICT. The global intensity of WB correlated with the severity of symptoms in CPA group (p = 0.001). The combination of WB and ICT may increase specificity in CPA diagnosis.

OX40 ligand expressed by DCs costimulates NKT and CD4+ Th cell antitumor immunity in mice.

The exceptional immunostimulatory capacity of DCs makes them potential targets for investigation of cancer immunotherapeutics. We show here in mice that TNF-alpha-stimulated DC maturation was accompanied by increased expression of OX40 ligand (OX40L), the lack of which resulted in an inability of mature DCs to generate cellular antitumor immunity. Furthermore, intratumoral administration of DCs modified to express OX40L suppressed tumor growth through the generation of tumor-specific cytolytic T cell responses, which were mediated by CD4+ T cells and NKT cells. In the tumors treated with OX40L-expressing DCs, the NKT cell population significantly increased and exhibited a substantial level of IFN-gamma production essential for antitumor immunity. Additional studies evaluating NKT cell activation status, in terms of IFN-gamma production and CD69 expression, indicated that NKT cell activation by DCs presenting alpha-galactosylceramide in the context of CD1d was potentiated by OX40 expression on NKT cells. These results show a critical role for OX40L on DCs, via binding to OX40 on NKT cells and CD4+ T cells, in the induction of antitumor immunity in tumor-bearing mice.

Chronic Pulmonary Aspergillosis in Post Tuberculosis Patients in Indonesia and the Role of LDBio Aspergillus ICT as Part of the Diagnosis Scheme.

Chronic pulmonary aspergillosis (CPA) is a common sequela of pulmonary tuberculosis (TB). The diagnosis of CPA is difficult and often misdiagnosed as smear-negative TB in endemic settings. Aspergillus IgG detection is the cornerstone of CPA diagnosis. There are a lack of studies on the prevalence of CPA in GeneXpert/smear-negative TB patients in Indonesia, despite a high number of TB cases. This study aims to determine the CPA rate in HIV-negative, GeneXpert-negative patients presenting with symptoms following completion of TB therapy and to evaluate the performance of LDBio Aspergillus immunochromatographic technology (ICT) lateral flow assay in the diagnosis of CPA. CPA was diagnosed on the basis of symptoms for ≥3 months, characteristic chest imaging and positive Aspergillus culture. Twenty (22%) out of 90 patients met the criteria for CPA. The LDBio test was positive in 16 (80%) CPA patients and in 21 (30%) non-CPA patients (p < 0.001) with 80% sensitivity and 70% specificity. Logistic regression revealed a positive LDBio Aspergillus ICT result, smoking history and diabetes to be important predictors of CPA diagnosis. Although CPA is an unrecognised disease in Indonesia, this study suggests that more than one in five GeneXpert negative patients with persistent symptoms following completion of TB therapy may have CPA.

Regulation of adenosine 5'-triphosphate (ATP)-gated P2X(4) receptors on tracheal smooth muscle cells.

We examined the effects of extracellular adenosine 5'-triphosphate (ATP) on single airway smooth muscle (ASM) cells from porcine trachea using a patch-clamp technique. ATP induced a sustained inward current. Phospholipase C inhibitor U-73122 failed to inhibit the current, suggesting the involvement of P2X receptor. A specific effecter of P2X(4), ivermectin, augmented the current indicating the existence of P2X(4) receptors. Immunohistochemistry and reverse transcription/polymerase chain reaction analysis and Western blot analysis also showed the distribution of the P2X(4) receptors. The inward current was reduced by SKF-96365, an inhibitor of both voltage-dependent Ca(2+) channels (VDCCs) and voltage-independent Ca(2+) channels, although a VDCC antagonist, verapamil, did not affect the current. SKF-96365 caused complete suppression of both the increase in the intracellular Ca(2+) concentration and the contraction of ASM cells induced by ATP. Our results demonstrate that P2X(4) receptors exist on ASM and that the receptors are responsible for Ca(2+) influx. These findings suggest that the Ca(2+) influx regulated by P2X(4) receptors plays an important role in ASM contraction by a pathway distinct from VDCC.

Efficacy of clarithromycin plus vancomycin in mice with implant-related infection caused by biofilm-forming Staphylococcus aureus.

BACKGROUND: Staphylococcal biofilms pose an important problem, especially after orthopedic surgery using foreign implants. Clarithromycin (CAM) eliminates the biofilms formed by a wide variety of aerobic and anaerobic bacteria. In a previous in vitro study, we showed that treatment with CAM and vancomycin (VCM) eradicated staphylococcal biofilms from surgical implants. To investigate the efficacy of this eradication therapy, we assessed its effects against Staphylococcus aureus on titanium plates implanted in mice. METHODS: A titanium washer covered with S. aureus biofilms was implanted in the muscular tissue around the femoral bone. Mice were given intravenous injections of CAM and intraperitoneal injections of VCM twice daily beginning 72 h after implantation. To confirm eradication of biofilms and S. aureus strains, the resected washer was examined by scanning electron microscopy. RESULTS: Dense colonization and biofilms were seen on the washer implanted in the control mice that received saline, saline plus CAM, or saline plus VCM. Treatment with CAM plus VCM eliminated the biofilms, indicating an S. aureus eradication effect. CONCLUSIONS: Staphylococcal biofilms have demonstrated resistance to most antibiotics, including VCM. Our in vivo data support the hypothesis that combined treatment using CAM plus VCM may effectively eradicate staphylococcal biofilms in patients with implant-related infection.

Impact of smoking on frequency and spectrum of K-RAS and EGFR mutations in treatment naive Indonesian lung cancer patients.

Background: Indonesia has the highest cigarette consumption in the world. We explored the clinical impact of smoking on the prevalence of EGFR and K-RAS mutations and survival in this prospective study. Methods: 143 treatment naive lung cancer patients were recruited from Persahabatan Hospital, a national tertiary hospital. DNA from cytological specimens had been extracted and genotyped for both EGFR and K-RAS mutations using a combination of PCR high resolution melting, restriction fragment length polymorphism (RFLP) and direct DNA sequencing. Results: EGFR mutation frequency in never smokers (NS) and ever smokers (ES) were 75% and 56% (p = 0.0401), respectively. In this cohort, the overall K-RAS mutation rate was 7%. Neither gender nor smoking history were associated with K-RAS mutation significantly. However, K-RAS transversion mutations were more common in male ES than transition mutations. Smoking history did not affect EGFR and K-RAS mutation frequencies in women. Concurrent EGFR/K-RAS mutation rate was 2.8% (4 of 143 patients). Four out of 91 EGFR mutation positive patients (4.4%) had simultaneous K-RAS mutation. Conclusions: In region where cigarette consumption is prevalent, smoking history affected frequencies of EGFR and K-RAS mutations, mainly in males.

Evaluation of PCR-HRM, RFLP, and direct sequencing as simple and cost-effective methods to detect common EGFR mutations in plasma cell-free DNA of non-small cell lung cancer patients.

BACKGROUND: Lung cancer patients with mutations in epidermal growth factor receptor (EGFR) gene are treated with tyrosine kinase inhibitor (TKI). AIMS: We aimed to evaluate polymerase chain reaction (PCR)-high-resolution melting (HRM), restriction fragment length polymorphism (RFLP), and direct sequencing (DS) to detect EGFR mutations in cell-free DNA (cfDNA) before and after TKI treatment in real-world settings of a developing country. METHODS: Paired cytology and plasma samples were collected from 116 treatment-naïve lung cancer patients. DNA from both plasma and cytology specimens was isolated and analyzed using PCR-HRM (to detect exon 19 insertion/deletion), RFLP (to genotypes L858R and L861Q), and DS (to detect uncommon mutations G719A, G719C, or G719S [G719Xaa] in exon 18 and T790M and insertion mutations in exon 20). RESULTS: EGFR genotypes were obtained in all 116 (100%) cfDNA and 110/116 (94.82%) of cytological specimens of treatment-naïve patient (baseline samples). EGFR-activating mutations were detected in 46/110 (40.6%) plasma samples, and 69/110 (63.2%) mutations were found in routine cytology samples. Using cytological EGFR genotypes as reference, we found that sensitivity and specificity of baseline plasma EGFR testing varied from 9.1% to 39.39% and 83.12% to 96.55%, respectively. In particular, the sensitivity and specificity of this assay in detecting baseline T790M mutations in exon 20 were 30% and 89.58%, respectively. Three months after TKI treatment, plasma T790M and insertion exon 20 mutations appeared in 5.4% and 2.7% patients, respectively. CONCLUSIONS: Despite low sensitivity, combined DS, RFLP, and PCR-HRM was able to detect EGFR mutations in plasma cfDNA with high specificity. Moreover, TKI resistance exon 20 insertions mutation was detected as early as 3 months post TKI treatment.