Accuracy of cell-free Mycobacterium tuberculosis DNA testing in pleural effusion for diagnosing tuberculous pleurisy: a multicenter cross-sectional study.

BACKGROUND: The diagnosis of tuberculous pleurisy (TP) presents a significant challenge due to the low bacterial load in pleural effusion (PE) samples. Cell-free Mycobacterium tuberculosis DNA (cf-TB) in PE samples is considered an optimal biomarker for diagnosing TP. This study aimed to evaluate the applicability of cf-TB testing across diverse research sites with a relatively large sample size. METHODS: Patients suspected of TP and presenting with clinical symptoms and radiological evidence of PE were consecutively enrolled by treating physicians from 11 research sites across 6 provinces in China between April 2020 and August 2022. Following centrifugation, sediments obtained from PE were used for Xpert MTB/RIF (Xpert) and mycobacterial culture, while the supernatants were subjected to cf-TB testing. This study employed a composite reference standard to definite TP, which was characterized by any positive result for Mycobacterium tuberculosis (MTB) through either PE culture, PE Xpert, or pleural biopsy. RESULTS: A total of 1412 participants underwent screening, and 1344 (95.2%) were subsequently enrolled in this study. Data from 1241 (92.3%) participants were included, comprising 284 with definite TP, 677 with clinically diagnosed TP, and 280 without TP. The sensitivity of cf-TB testing in definite TP was 73.6% (95% CI 68.2-78.4), significantly higher than both Xpert (40.8%, 95% CI 35.3-46.7, P < 0.001) and mycobacterial culture (54.2%, 95% CI 48.4-59.9, P < 0.001). When clinically diagnosed TP was incorporated into the composite reference standard for sensitivity analysis, cf-TB testing showed a sensitivity of 46.8% (450/961, 95% CI 43.7-50.0), significantly higher than both Xpert (116/961, 12.1%, 95% CI 10.2-14.3, P < 0.001) and mycobacterial culture (154/961, 16.0%, 95% CI 13.8-18.5, P < 0.001). The specificities of cf-TB testing, Xpert, and mycobacterial culture were all 100.0%. CONCLUSIONS: The performance of cf-TB testing is significantly superior to that of Xpert and mycobacterial culture methods, indicating that it can be considered as the primary diagnostic approach for improving TP detection. Trial registration The trial was registered on Chictr.org.cn (ChiCTR2000031680, https://www.chictr.org.cn/showproj.html?proj=49316 ).

Combined detection of SHOX2 and PTGER4 methylation with serum marker CYFRA21-1 for improved diagnosis of malignant pleural mesothelioma.

AIMS: To investigate the performance of a combined biomarker approach using the methylation status of the short stature homeobox 2 (SHOX2) and prostaglandin E2 receptor EP4 (PTGER4) genes, along with the serum levels of CYFRA21-1, for differential diganosis of malignant pleural mesothelioma (MPM) from benign reactive mesothelial hyperplasia (RMH). METHODS: We analysed 48 MPM tissue or pleural effusion cell block specimens and 42 cases with RMH. Real-time quantitative methylation-specific PCR was used to examine the methylation status of SHOX2, PTGER4, ras association domain family 1 isoform A, septin 9 gene and homeobox gene A9 genes. Additionally, we employed electrochemiluminescence immunoassay to measure nine serum tumour markers commonly used in pan-cancer screening tests. RESULTS: The receiver operating curve indicated that SHOX2, PTGER4 gene methylation and serum biomarker CYFRA21-1 exhibited good diagnostic performance in identifying MPM, with area under curves (AUCs) of 0.761, 0.904 and 0.847, respectively. The combination of SHOX2, PTGER4 methylation and CYFRA21-1 yielded an AUC value of 0.972. The diagnostic sensitivity and specificity of this panel in differentiating MPM from RMH were 91.3% (42/46) and 97.6% (41/42), respectively. Both tissue and cell block specimens can be used in the diagnostic process. Furthermore, elevated CYFRA21-1 levels were associated with poor prognosis (p<0.05). Hypermethylation level of PTGER4 may indicate an unfavourable prognosis of MPM, but the difference was not statistically significant. CONCLUSIONS: The combined detection of SHOX2 and PTGER4 methylation alongside serum CYFRA21-1 level significantly enhances the diagnosis of MPM. Additionally, CYFRA21-1 can serve as a prognostic indicator for MPM.

Performance of SHOX2 and RASSF1A methylation assay in supernatants and matched cell pellets for the diagnosis of malignant pleural effusion.

BACKGROUND: It is difficult to distinguish between malignant pleural effusion (MPE) and benign pleural effusion (BPE). The purpose of this study was to determine the best specimen type by evaluating the DNA methylation status of SHOX2 and RASSF1A in 3 matched PE components. METHODS: In total, 94 patients were enrolled, including 45 MPE, 35 BPE, and 14 undefined PE (UPE) with malignancies. PE samples were processed into supernatants, fresh-cell pellets, and formalin-fixed and paraffin-embedded (FFPE) cell blocks, respectively. A quantitative real-time PCR was used to detect the methylation status of SHOX2 and RASSF1A. RESULTS: SHOX2 and RASSF1A methylation levels were significantly higher in the 3 MPE sample types than those of BPE (P < 0.05). The area under the curve using cell-free DNA (cf-DNA) was the highest. The detection sensitivity of SHOX2 and RASSF1A in fresh-cell DNA, cf-DNA and FFPE cell-block were 71.1% (32/45), 97.8% (44/45) and 66.7% (28/42), respectively, with specificities of 97.1% (34/35), 94.3% (33/35), and 96.9% (31/32). Notably, a combination of the cytological analysis and cf-DNA methylation assay showed an increase in positivity rate from 75.6% to 100%. CONCLUSIONS: The SHOX2 and RASSF1A methylation assay using cf-DNA, the primary recommended specimen type, can excellently increase the diagnostic sensitivity of MPE. A combination of methylation assay with cytological analysis can be used for auxiliary diagnosis of PE.

Simultaneous diagnosis of tuberculous pleurisy and malignant pleural effusion using metagenomic next-generation sequencing (mNGS).

BACKGROUND: Metagenomic next-generation sequencing (mNGS) has become a powerful tool for pathogen detection, but the value of human sequencing reads generated from it is underestimated. METHODS: A total of 138 patients with pleural effusion (PE) were diagnosed with tuberculous pleurisy (TBP, N = 82), malignant pleural effusion (MPE, N = 35), or non-TB infection (N = 21), whose PE samples all underwent mNGS analysis. Clinical TB tests including culture, Acid-Fast Bacillus (AFB) test, Xpert, and T-SPOT, were performed. To utilize mNGS for MPE identification, 25 non-MPE samples (20 TBP and 5 non-TB infection) were randomly selected to set human chromosome copy number baseline and generalized linear modeling was performed using copy number variant (CNV) features of the rest 113 samples (35 MPE and 78 non-MPE). RESULTS: The performance of TB detection was compared among five methods. T-SPOT demonstrated the highest sensitivity (61% vs. culture 32%, AFB 12%, Xpert 35%, and mNGS 49%) but with the highest false-positive rate (10%) as well. In contrast, mNGS was able to detect TB-genome in nearly half (40/82) of the PE samples from TBP subgroup, with 100% specificity. To evaluate the performance of using CNV features of the human genome for MPE prediction, we performed the leave-one-out cross-validation (LOOCV) in the subcohort excluding the 25 non-MPE samples for setting copy number standards, which demonstrated 54.1% sensitivity, 80.8% specificity, 71.7% accuracy, and an AUC of 0.851. CONCLUSION: In summary, we exploited the value of human and non-human sequencing reads generated from mNGS, which showed promising ability in simultaneously detecting TBP and MPE.

[A Real-world Study on the Expression Characteristics of PD-L1 in Patients 
with Advanced EGFR Positive NSCLC and Its Relationship with the 
Therapeutic Efficacy of EGFR-TKIs].

BACKGROUND: Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) is the first choice for first-line treatment of advanced patients with EGFR positive non-small cell lung cancer (NSCLC). For advanced NSCLC patients with negative drive gene and positive programmed cell death ligand 1 (PD-L1) or highly expressed NSCLC patients, the first-line treatment recommends immune checkpoint inhibitors (ICIs) monotherapy or ICIs combined chemotherapy. Therefore, the first-line treatment strategy for advanced NSCLC patients with EGFR positive at different PD-L1 expression levels is worth further exploring. Many previous studies have suggested that the expression of PD-L1 is obviously affected by EGFR mutation, and the expression of PD-L1 is likely to be related to the mechanism of EGFR-TKIs resistance. The purpose of this study was to analyze the expression characteristics of PD-L1 in patients with advanced EGFR positive NSCLC and its relationship with the efficacy of EGFR-TKIs. METHODS: 159 patients with newly diagnosed advanced NSCLC with EGFR positive (including 141 patients with EGFR sensitive mutations) were enrolled to analyze the relationship between clinicopathological characteristics and PD-L1 expression. The factors affecting the therapeutic effect of EGFR-TKIs in 141 patients with EGFR sensitive mutations were also explored. RESULTS: The PD-L1 expression of the included patients was classified according to the tumor promotion score (TPS): negative (TPS<1%) accounted for 47.2%, low expression (1%≤TPS<50%) accounted for 32.1%, and high expression (TPS≥50%) accounted for 20.7%. Among them, patients with solid predominant cancer cell pathomorphology classification are more likely to have high expression of PD-L1, accounting for 52.9% (P<0.0001). The median progression-free survival (mPFS) of patients with PD-L1 negative expression, low expression and high expression who received first generation EGFR-TKIs treatment were 12.4 months, 10.5 months and 3.7 months, respectively, with significant statistical difference (P<0.0001). During the EGFR-TKIs treatment, patients with a history of radiotherapy for lung lesions have a longer PFS benefit than those without a history of radiotherapy (mPFS: 17.0 months vs 9.3 months, P<0.0001). CONCLUSIONS: The expression level of PD-L1 in advanced EGFR positive NSCLC patients was significantly correlated with the pathomorphology classification of cancer cells. The efficacy of EGFR-TKIs in patients with PD-L1 overexpression NSCLC was significantly poor. PFS can be significantly prolonged during targeted therapy combined with radiotherapy of lung lesions.