[Aprospective study of detection and clinical significance of bone marrow tumor cells in small cell lung cancer].

Objective: To investigate the detection of bone marrow tumor cells in small cell lung cancer (SCLC) patients and their relationship with clinical features, treatment response and prognosis. Methods: A total of 113patients with newly diagnosed SCLC from January 2018 to October 2022 at Beijing Chest Hospital were prospectively enrolled. Before treatment, bone marrow was aspirated and separately submitted for tumor cells detection by liquid-based cytology and disseminated tumor cells (DTCs) detection by the substrction enrichment and immunostaining fluorescence in situ hybridization (SE-iFISH) platform. The correlation between the detection results of the two methods with patients' clinical features and treatment response was evaluated by Chi-square. Kaplan-Meier method was applied to create survival curves and the Cox regression model was used for multivariate analysis. Results: The positive rate of bone marrow liquid-based cytology in SCLC was 15.93% (18/113). The liver and bone metastases rates were significantly higher (55.56% vs 11.58% for liver metastasis, P＜0.001; 77.78% vs 16.84% for bone metastasis, P＜0.001) and thrombocytopenia was more common (16.67% vs 2.11%, P=0.033) in patients with tumor cells detected in liquid-based cytology than those without detected tumor cells. As for SE-iFISH, DTCs were detected in 92.92% of patients (105/113), the liver and bone metastasis rates were significantly higher (37.93% vs 11.90% for liver metastasis, P=0.002; 44.83% vs 20.23 % for bone metastasis, P=0.010), and the incidence of thrombocytopenia was significantly increased (13.79% vs 1.19%, P=0.020) in patients with DTCs≥111 per 3 ml than those with DTCs＜111 per 3 ml. The positive rates of bone marrow liquid-based cytology in the disease control group and the disease progression group were 12.00% (12/100) and 46.15% (6/13), respectively, and the difference was statistically significant (P=0.002). However, the result of SE-iFISH revealed the DTCs quantities of the above two groups were 29 (8,110) and 64 (15,257) per 3 ml, and there was no statistical difference between the two groups (P=0.329). Univariate analysis depicted that the median progression-free survival (PFS) and median overall survival (OS) of liquid-based cytology positive patients were significantly shorter than those of tumor cell negative patients (6.33 months vs 9.27 months for PFS, P=0.019; 8.03 months vs 19.50 months for OS, P=0.019, P=0.033). The median PFS and median OS in patients with DTCs≥111 per 3 ml decreased significantly than those with DTCs＜111 per 3 ml (6.83 months vs 9.50 months for PFS, P=0.004; 11.2 months vs 20.60 months for OS, P=0.019). Multivariate analysis showed that disease stage (HR=2.806, 95%CI:1.499-5.251, P=0.001) and DTCs quantity detected by SE-iFISH (HR=1.841, 95%CI:1.095-3.095, P=0.021) were independent factors of PFS, while disease stage was the independent factor of OS (HR=2.538, 95%CI:1.169-5.512, P=0.019). Conclusions: Both bone marrow liquid-based cytology and SE-iFISH are clinically feasible. The positive detection of liquid-based cytology or DTCs≥111 per 3 ml was correlated with distant metastasis, and DTCs≥111 per 3 ml was an independent prognostic factor of decreased PFS in SCLC.

[The value of cell free DNA of Mycobacterium tuberculosis in the early diagnosis of tuberculous meningitis].

Objective: To detect the cell-free DNA of Mycobacterium tuberculosis (Cf-TB) in the cerebrospinal fluid (CSF) of patients with tuberculous meningitis (TBM), and to assess the diagnostic value of this method for TBM. Methods: We prospectively included patients with suspected meningitis from the Department of Tuberculosis, Beijing Chest Hospital, Department of Neurology, Beijing Chaoyang Hospital and Department of Neurology, 263 Hospital of the People's Liberation Army from September 2019 to March 2022. A total of 189 patients were included in this study. Among them, 116 were male and 73 were female, aged from 7 to 85 years, with an average of (38.5±19.1) years. The CSF specimens of the patients were collected for Cf-TB, MTB culture and Xpert MTB/RIF. SPSS 20.0 was used for statistical analysis and the difference was statistically significant with P<0.05. Results: Among the 189 patients, there were 127 patients in the TBM group and 62 patients in the non-TBM group. The sensitivity of Cf-TB was 50.4% (95%CI 41.4%-59.3%), the specificity was 100% (95%CI 92.7%-100.0%), the positive predictive value was 100% (95%CI 92.9%-100.0%), and the negative predictive value was 49.6% (95%CI 40.6%-58.6%). Using clinical diagnosis as the gold standard, the sensitivity of Cf-TB was 50.4% (64/127), which was significantly higher than that of MTB culture (8.7%, 11/127) and Xpert MTB/RIF (15.7%,20/127) (all P<0.001). Using etiology as the gold standard, the sensitivity of Cf-TB was 72.7% (24/33), which was significantly higher than that of MTB culture [33.3%, 11/33, (χ2=10.28, P=0.001)] and was similar to Xpert MTB/RIF (60.6%, 20/33) (χ2=1.091, P=0.296). Conclusion: The sensitivity of the Cf-TB test was significantly higher than that of CSF MTB culture and Xpert MTB/RIF. Cf-TB may provide evidence for earlier diagnosis and treatment of TBM.

[Clinical value of the MeltPro MTB assays in detection of drug-resistant tuberculosis in paraffin-embedded tissues].

Objective: To evaluate the clinical value of the MeltPro MTB assays in the diagnosis of drug-resistant tuberculosis. Methods: A cross-sectional study design was used to retrospectively collect all 4 551 patients with confirmed tuberculosis between January 2018 and December 2019 at Beijing Chest Hospital, Capital Medical University. Phenotypic drug sensitivity test and GeneXpert MTB/RIF (hereafter referred to as "Xpert") assay were used as gold standards to analyze the accuracy of the probe melting curve method. The clinical value of this technique was also evaluated as a complementary method to conventional assays of drug resistance to increase the detective rate of drug-resistant tuberculosis. Results: By taking the phenotypic drug susceptibility test as the gold standard, the sensitivity of the MeltPro MTB assays to detect resistance to rifampicin, isoniazid, ethambutol and fluoroquinolone was 14/15, 95.7%(22/23), 2/4 and 8/9,respectively; and the specificity was 92.0%(115/125), 93.2%(109/117), 90.4%(123/136) and 93.9%(123/131),respectively; the overall concordance rate was 92.1%(95%CI:89.6%-94.1%),and the Kappa value of the consistency test was 0.63(95%CI:0.55-0.72).By taking the Xpert test results as the reference, the sensitivity of this technology to the detection of rifampicin resistance was 93.6%(44/47), the specificity was100%(310/310), the concordance rate was 99.2%(95%CI:97.6%-99.7%), and the Kappa value of the consistency test was 0.96(95%CI:0.93-0.99). The MeltPro MTB assays had been used in 4 551 confirmed patients; the proportion of patients who obtained effective drug resistance results increased from 83.3% to 87.8%(P<0.01); and detection rate of rifampicin, isoniazid, ethambutol, fluoroquinolone resistance, multidrug and pre-extensive drug resistance cases were increased by 3.2%, 14.7%, 22.2%, 13.7%, 11.2% and 12.5%, respectively. Conclusion: The MeltPro MTB assays show satisfactory accuracy in the diagnosis of drug-resistant tuberculosis. This molecular pathological test is an effective complementary method in improving test positivity of drug-resistant tuberculosis.

[Mycobacterium xenopi pulmonary disease:report of 3 cases and literature review].

Objective: To analyze the clinical manifestations, radiographic characteristics and prognosis of Mycobacterium xenopi pulmonary disease, in order to improve diagnosis and treatment of the disease. Methods: Using "Mycobacterium xenopi, pulmonary disease" as the search term, from February 15, 2007 to February 21, 2021, a total of 1 264 cases were retrieved in the PubMed database. In the Wanfang database, using "Mycobacterium xenopi, pulmonary disease" as the search term, from February 15, 2007 to February 21, 2021, no related document was retrieved. In the CNKI database, "Mycobacterium xenopi, pulmonary disease" was used as the search term, and one relevant case report was retrieved, but did not meet the diagnostic criteria of Mycobacterium xenopi pulmonary disease issued by American Thoracic Society in 2007. The 1 264 cases from the literature and 3 cases of our institution were used for review. Results: Our 3 cases were elderly males complaining of cough and expectoration, and had underlying lung diseases. The imaging examination showed cavitary lesions. All of them had positive sputum smear for acid-fast bacillus and negative Xpert MTB/RIF examination. Mycobacterium xenopi was isolated at least 2 times from sputum samples. Although prescribed with chemotherapy, case 1 and case 2 died 4 years and 2 years later, respectively, after the diagnosis. Case 3 got sputum conversion, symptom improvement and radiographic responses after 30-month chemotherapy. Conclusions: The clinical manifestations of Mycobacterium xenopi pulmonary disease are atypical. For patients with positive sputum smear for acid-fast bacillus and negative Xpert MTB/RIF examination and conventional mycobacterial culture, Mycobacterium xenopi pulmonary disease should be considered. The disease deserves further attention from clinicians due to poor prognosis.

[Pathological diagnosis of lung cancer based on deep transfer learning].

Objective: To establish an artificial intelligence (AI)-assisted diagnostic system for lung cancer via deep transfer learning. Methods: The researchers collected 519 lung pathologic slides from 2016 to 2019, covering various lung tissues, including normal tissues, adenocarcinoma, squamous cell carcinoma and small cell carcinoma, from the Beijing Chest Hospital, the Capital Medical University. The slides were digitized by scanner, and 316 slides were used as training set and 203 as the internal test set. The researchers labeled all the training slides by pathologists and establish a semantic segmentation model based on DeepLab v3 with ResNet-50 to detect lung cancers at the pixel level. To perform transfer learning, the researchers utilized the gastric cancer detection model to initialize the deep neural network parameters. The lung cancer detection convolutional neural network was further trained by fine-tuning of the labeled data. The deep learning model was tested by 203 slides in the internal test set and 1 081 slides obtained from TCIA database, named as the external test set. Results: The model trained with transfer learning showed substantial accuracy advantage against the one trained from scratch for the internal test set [area under curve (AUC) 0.988 vs. 0.971, Kappa 0.852 vs. 0.832]. For the external test set, the transferred model achieved an AUC of 0.968 and Kappa of 0.828, indicating superior generalization ability. By studying the predictions made by the model, the researchers obtained deeper understandings of the deep learning model. Conclusions: The lung cancer histopathological diagnostic system achieves higher accuracy and superior generalization ability. With the development of histopathological AI, the transfer learning can effectively train diagnosis models and shorten the learning period, and improve the model performance.

The significance of Siglec-15 expression in resectable non-small cell lung cancer.

Siglec-15 (S15) is another important mechanism of tumor immune escape besides the PD-L1/PD-1 pathway and represents a new kind of immune checkpoint inhibitor. However, the associations of tumor Siglec-15 expression with clinicopathological characteristics and outcomes of non-small cell lung cancer (NSCLC), and tumor-infiltrating lymphocytes (TILs) in a tumor microenvironment (TME) have so far been unclear. A total of 324 NSCLC surgical samples on tumor microarray were used in this study for investigating the association of S15 expression with clinicopathological characteristics and overall survival (OS) as well as correlation with TILs using multiplex immunofluorescence staining and PD-L1. Results showed that the expression of S15 in adenocarcinoma was significantly higher than that in squamous cell carcinoma. S15 expression was positively correlated with CD8+ T cell density in the stroma. The expression rate of PD-L1 in lung squamous cell carcinoma was higher than that in lung adenocarcinoma. S15 expression was not associated with the prognosis of early NSCLC. The pathological mechanism of the co-expression of S15 and PD-L1 in resectable NSCLC remains to be further studied.

[Detection of tuberculosis genes associated with drug-resistance in paraffin-embedded tissue specimens using next generation sequencing technology].

Objective: To evaluate the use of multiplex PCR amplicon sequencing (mPCR-NGS) technology in detecting gene mutations related to drug resistance of Mycobacterium tuberculosis (MTB) in formalin-fixed paraffin-embedded tissue specimens, and to explore its clinical value in the diagnosis of drug-resistant tuberculosis. Methods: Fifty clinical MTB strains isolated in the Changping District Tuberculosis Control Institute of Beijing from April 2013 to October 2015 with drug susceptibility test (DST) results of rifampicin, isoniazid, ethambutol, streptomycin, ofloxacin, capreomycin, kanamycin and amikacin available were recovered, including 42 drug-resistant strains and 8 drug-sensitive strains. The mPCR-NGS test was established to detect genes related to the 8 anti-tuberculosis drugs according to the previously published studies and databases. Fifty-five paraffin-embedded tissue specimens from drug-resistant tuberculosis patients were collected in the Department of Pathology, Beijing Chest Hospital, Capital Medical University during November 2017 to September 2018. All the specimens showed no less than one mutation in the gene regions related to drug resistance of any of the 4 drugs (rifampicin, isoniazid, ethambutol or fluoroquinolones) by probe melting curve assay. The effectiveness of mPCR-NGS test was evaluated on clinical MTB isolates using phenotypic DST as the reference. Clinical evaluation of mPCR-NGS test on formalin-fixed paraffin-embedded specimens from TB patients was performed using probe melting curve assay as the reference. The sensitivity, specificity and coincidence of mPCR-NGS were analyzed. Results: Using phenotypic DST as the reference, the sensitivities of the mPCR-NGS for detecting drug-resistance of rifampicin, isoniazid, streptomycin, and ethambutol were 95% (38/40), 93% (27/29), 93% (27/29), and 72% (13/18), respectively; and the specificities were 100% (10/10), 95% (20/21), 100% (21/21), and 94% (30/32), respectively. The sensitivities for capreomycin, kanamycin and amikacin were all 100% (2/2, 3/3, 3/3), and the specificities were 98% (47/48), 100% (33/33) and 100% (47/47), respectively. The sensitivity and specificity of ofloxacin were 70% (7/10) and 100% (40/40), respectively. The total coincidence rate for the 8vdrugs was 94%, and the Kappa value was 0.87. The 55 paraffin-embedded tissue specimens included in this study were all tested by probe melting curve assays. Among them 28 were resistant to rifampicin, 37 resistant to isoniazid, 13 resistant to ethambutol, and 17 resistant to fluoroquinolones. Using the probe melting curve assay as the reference, the sensitivities of the mPCR-NGS for detecting resistant to rifampicin, isoniazid, ethambutol, and fluoroquinolones were 100% (28/28), 95% (35/37), 100%, and 100%, respectively; and the specificities were all 100% (42/42, 38/38). The total coincidence rate of the two methods was 99%, and the Kappa value was 0.98. Conclusions: mPCR-NGS showed good sensitivities and specificities in detecting drug-resistant gene mutations both in clinical MTB isolates and paraffin-embedded tissue specimens. mPCR-NGS has the potential to be an accurate and rapid molecular pathological technology for diagnosis of drug-resistant tuberculosis.

The joint impact of smoking plus alcohol drinking on treatment of pulmonary tuberculosis.

Tuberculosis, smoking, and alcohol drinking are major public health and social issues worldwide. We investigated the joint effect of smoking plus alcohol drinking on TB treatment. Retrospective study was conducted among TB patients in 49 units from eight provinces in China. All patients enrolled were classified into four groups according to their smoking and/or alcohol status. Current smokers plus drinkers belonged to group 1; ex-smokers plus ex-drinkers were in group 2; current smokers and ex-drinkers, current smokers and never drinkers, ex-smokers and current drinkers, ex-smokers and never drinkers, never smokers and current drinkers, and never smokers and ex-drinkers belonged to group 3; while the never smokers plus never drinkers were in group 4. We used a chi-square test to compare adverse drug reaction, lesions absorption and cavities of lung, sputum culture at the end of the second month, and treatment outcomes among the four groups. Among the 1256 participants enrolled in the study, 6.1% (76/1256) were current smokers plus drinkers; 25.9% (325/1256) were ex-smokers plus drinkers; 29.1% (366/1256) were current/never/ex-smokers and/or drinkers, and 38.9% (489/1256) were never smokers plus drinkers, respectively. Compared to the never smokers and drinkers, smoker plus drinker TB patients were more likely to experience adverse drug reaction (x2 = 8.480, P = 0.037), less proportion of lesions absorption in lungs (x2 = 10.330, P = 0.016), lower proportion of culture conversion (x2 = 18.83, P = 0.04), and more unfavorable outcomes. Smoking plus alcohol drinking adversely affect response against TB treatment, which increase adverse drug reactions, sputum culture-positive rate at the end of the second month, and failure rate of pulmonary tuberculosis patients.