Improving the identification and diagnostic efficiency of Metagenomic Next-Generation Sequencing for mycobacterial granuloma on postoperative formalin-fixed paraffin-embedded specimens.

OBJECTIVE: Metagenomic Next-Generation Sequencing (mNGS) has been validated to have an important role in the diagnosis of mycobacterium infection. The study aimed to further explore the mycobacteria identification ability of mNGS on formalin-fixed paraffin-embedded（FFPE）tissues from postoperative specimens. METHODS: Patients who underwent surgical biopsy or resection for clarifying the diagnosis and whose initial postoperative pathology indicated granulomatous lesions were included. Fresh tissues were sent for mycobacterium culture and Xpert MTB/RIF (Xpert) to establish the diagnosis. FFPE specimens were sent for mNGS and molecular pathology，the diagnostic values were compared between the two methods. RESULTS: A total of 65 cases with definite diagnoses were finally included in the study. 31 cases were confirmed as mycobacterium granuloma using the fresh specimen etiology as diagnostic criteria. The overall sensitivity and specificity of mNGS on FFPE specimens in the diagnosis of mycobacterium granuloma were 100% and 88.24%, respectively. In 19 cases diagnosed as tuberculous granulomas, the sensitivity (100% vs47.37%) and negative predictive value (NPV, 100%vs 82.14%) of mNGS were both significantly higher than that of molecular pathology on the FFPE section（both p 0.00）while the positive predictive value (PPV) and specificity were not significantly different. In 12 cases diagnosed as Non-tuberculous mycobacterium (NTM）granuloma, the sensitivity of mNGS was also significantly higher than that of molecular pathology on FFPE section (100% vs 66.67%, p 0.00) while the specificity, PPV and NPV were all not significantly different. CONCLUSIONS: The mNGS could be used for one-time detection of pathogens on FFPE sections with high sensitivity. It could be recommended as a supplementary method for the identification of pathogenic bacteria in the diagnosis of postoperative granuloma lesions.

GLSP and GLSP-derived triterpenes attenuate atherosclerosis and aortic calcification by stimulating ABCA1/G1-mediated macrophage cholesterol efflux and inactivating RUNX2-mediated VSMC osteogenesis.

Background and Purpose: Atherosclerosis is the main pathophysiological foundation of cardiovascular disease, which was caused by inflammation and lipid metabolism disorder, along with vascular calcification. Aortic calcification leads to reduced plaque stability and eventually causes plaque rupture which leads to cardiovascular events. Presently, the drug to treat aortic calcification remains not to be available. Ganoderma lucidum spore powder (GLSP) is from Ganoderma lucidum which is a Traditional Chinese Medicine with the homology of medicine and food. It has multiple pharmacological effects, but no research on aortic calcification during atherosclerosis was performed. This study investigated the effects of GLSP on atherosclerosis and aortic calcification and revealed the underlying mechanism. Methods: In vivo, 8-week-aged male LDLR-/- mice were fed a high-fat diet to induce atherosclerosis along with aortic calcification. Simultaneously, the mice were treated with GLSP at the first week of HFD feeding to determine the protection against early and advanced atherosclerosis. Subsequently, the mice tissues were collected to evaluate the effects of GLSP on atherosclerosis, and aortic calcification, and to reveal the underlying mechanism. In vitro, we determined the major components of GLSP triterpenes by HPLC, and subsequently assessed the protective effects of these main active components on lipid metabolism, inflammation, and calcification in RAW264.7 and HASMC cells. Results: We observed GLSP attenuated plaque area and aortic calcification in the mice with early and advanced atherosclerosis. GLSP reduced the number of foam cells by improving ABCA1/G1-mediated cholesterol efflux in macrophages. In addition, GLSP protected against the aortic endothelium activation. Moreover, GLSP inhibited aortic calcification by inactivating RUNX2-mediated osteogenesis in HASMCs. Furthermore, we determined the major components of GLSP triterpenes, including Ganoderic acid A, Ganoderic acid B, Ganoderic acid C6, Ganoderic acid G, and Ganodermanontriol, and found that these triterpenes promoted ABCA1/G1-mediated cholesterol efflux and inhibited inflammation in macrophage, and inactivated RUNX2-mediated osteogenesis in VSMC. Conclusions: This study demonstrates that GLSP attenuates atherosclerosis and aortic calcification by improving ABCA1/G1-mediated cholesterol efflux and inactivating RUNX2-mediated osteogenesis in LDLR-/- mice. GLSP may be a potential drug candidate for the treatment of atherosclerosis and vascular calcification.

Clinical Significance of Dual-Block Elastic Stain Evaluating Visceral Pleural Invasion in Peripheral Non-Small Cell Lung Cancer.

Visceral pleural invasion (VPI) is a critical component in the staging of peripheral non-small cell lung carcinoma (NSCLC). We aim to investigate whether dual-block elastic stain increases visceral pleural invasion positivity compared with single-block elastic stain. We further analyze the potential predictors of visceral pleural invasion. 8419 peripheral NSCLC patients (including 6008 patients with tumor size≤3 cm in stage I) were divided into a cohort using one paraffin block (single-block group, n = 5184) and a cohort using dual paraffin blocks (dual-block group, n = 3235) for elastic stain. The VPI-positive rate demonstrated by the dual-block elastic stains group was significantly higher than that of the single-block elastic stain group (17.7% (573/3235) versus 9.1% (474/5184), respectively, P < .001). The presence of visceral pleural invasion in T1 (≤3 cm) patients detected by single- and dual-block elastic stain was 6.3% (235/3730) and 12.0% (273/2278), respectively (P < .001). 5.7% of T1 patients (stage IA) were additionally upstaged to T2a (stage IB) by dual-block elastic stain. However, the incidence of visceral pleural invasion in pT2a patients showed no significant difference between the single-block group and the dual-block group (16.8% vs. 17.1%, P = .916). Lymphovascular invasion, lymph node metastasis, dedifferentiated carcinomas, the presence of spread through airspaces (STAS) and a poorly differentiated adenocarcinomatous growth pattern could be significant predictors of visceral pleural invasion (P < .001). Our results indicate that using dual-block elastic stain identifies more visceral pleural invasion positive T1 NSCLC patients who are upstaged to T2a, and who could benefit from optimal management post-operatively. The application of dual-block elastic stain is an efficient and practical method to detect visceral pleural invasion status.

The Correlation Between Histologic, Immunophenotypic, and Molecular Characteristics of Pulmonary Sarcomatoid Carcinoma Reveals That Sarcomatoid Change Is Potentially Derived From Epithelial Carcinoma Cells Undergoing Epithelial-Mesenchymal Transition.

Pulmonary sarcomatoid carcinoma (PSC) is characterized by biphasic tumors with epithelial and mesenchymal phenotype. Little is known about the correlation between histologic, immunophenotypic features and the genetic profile of PSC. We analyzed the expression of epithelial-mesenchymal transition-related markers, adenocarcinoma (ADC) and squamous cell carcinoma lineage-specific markers of 205 PSC cases. The alteration of 5 targeted genes was detected by amplification-refractory mutation system-polymerase chain reaction. The intensity of cytokeratin staining was stronger in epithelial carcinoma (EC) than that of the sarcomatoid component (SC) of pleomorphic carcinoma, while vimentin was positive in only 16.3% (17/104) of EC of pleomorphic carcinoma. There is no significant difference between thyroid transcription factor 1 (TTF-1) expression in the SC (46.5%, 33/71) of pleomorphic carcinoma with ADC components and pure PSC (44.2%, 42/95) without p40 expression ( P =0.858). Four cases with ALK rearrangement were confirmed to co-express ALK fusion protein in both the SC and EC. The incidence of EGFR/ALK/KRAS mutation was similar between pleomorphic carcinoma with ADC components (40.6%, 26/64) and TTF-1 + pure PSC (38.2%, 13/34) ( P =0.583). However, higher proportions of TTF-1 + /p40 - PSC patients (44.8%, 39/87) had EGFR/ALK/KRAS mutation than those with TTF-1 - /p40 - PSC (16.7%, 4/24) ( P =0.031). The incidence of EGFR mutation was significantly higher in TTF-1-positive (18.4%, 16/87) than TTF-1-negative (2.7%, 2/74) PSC ( P =0.002). No EGFR and ALK abnormality were observed in 24 pleomorphic carcinoma cases with squamous cell carcinoma components or pure PSC with p40 expression. Our study reveals a close correlation between SC and EC components of pleomorphic carcinoma in terms of immunophenotypic and genetic features, which suggests that pleomorphic carcinoma is potentially derived from the sarcomatoid change of EC cells undergoing epithelial-mesenchymal transition.

Paradoxical development of pleural-based masses in patients with pleural tuberculosis during treatment: a clinical observational study in China.

BACKGROUND: To explored the clinical, pathological, and bacteriological characteristics of pleural-based masses occurred during anti-tuberculosis (TB) treatment in patients with pleural TB. METHODS: Patients referred with newly diagnosed pleural TB were prospectively enrolled into the study. Patients were followed up throughout the treatment, and clinical data were recorded. Percutaneous biopsy and surgical tissues from pleural-based masses were examined histologically and samples sent for PCR. Cytokines in the pleural effusions and clinical factors were collected and compared between different patients. RESULTS: A total of 122 patients with pleural TB were enrolled, and 34.4% (42/122) displayed newly observed pleural-based mass during the treatment. Twelve cases underwent surgical resection at the 12 ± 0.5 months during the treatment course. Based on the surgical observation, 58.3% (7 /12) were located in pleura, 41.7% (5/12) were located in the lung parenchyma. Pathological observations showed that the pleural-based masses were typed as granulomatous inflammation, fibrous hyperplasia and necrosis. Mycobacterium tuberculosis PCR was positive in 57.1% of the cases (24/42). Any first-line anti-TB drug resistance gene mutations were positive in only 9.5% (4/42). Aside from 12 cases who underwent the surgical operation, 86.7% of the patients (26/30) still had a pleural-based mass at the end of 12 months treatment course. Patients with a pleural-based mass were younger, had a thicker pleural, a higher proportion of pleural adhesive, loculated pleural effusion and residual pleural effusion, and a higher level of LDH, ADA and lower glucose in pleural effusion than those without a pleural-based mass occurrence during the treatment (all Pcorr < 0.05). CONCLUSIONS: Pleural-based masses were observed in about one-third of patients with pleural TB. The masses were in the lung or pleura and were divided into three pathological types.

Early Identification of Fungal and Mycobacterium Infections in Pulmonary Granulomas Using Metagenomic Next-Generation Sequencing on Formalin fixation and paraffin embedding tissue.

BACKGROUND: The purpose of the study is to assess the etiology detection ability of Metagenomic Next-Generation Sequencing (mNGS) on formalin fixation and paraffin embedding (FFPE) tissue from postoperative biopsy specimens. METHODS: We prospectively enrolled specimens from patients undergone surgery biopsy due to undefinite diagnosis and pathologically indicated granulomatous lesions. FFPE tissues were tested by mNGS and histopathology. The etiology detection rate of mNGS was calculated and compared with histopathology.. RESULTS: Among the 69 cases eventually included, 41 (59.42%) were diagnosed with infectious granuloma. The overall fungi and mycobacteria etiology detection rates of mNGS in granuloma lesions was 87.80% (36/41). The mNGS increased the detection rate by 68.29% (28/41) compared with histopathology, the difference was statistically significant (χ2 = 28.97, P = 0.00). The detection rates of mNGS in fungal infections (12/12,100%) and in mycobacterium infections (22/27, 81.48%) were significant higher than those of histopathology (8/12, 66.67% and 0/27, 0.00%; both P = 0.00). Two (2/2.100%) cases of co-infection were detected at one time by mNGS. All mNGS-based clinical decisions were made within 2 days. CONCLUSIONS: The mNGS could accurately and quickly detect fungi and mycobacteria in FFPE specimens from postoperative granuloma specimens and identify the pathogens to the species level. CLINICAL TRIALS REGISTRATION: China Clinical Trial Registry ChiCTR2000035464.

Improving differential diagnosis of pulmonary large cell neuroendocrine carcinoma and small cell lung cancer via a transcriptomic, biological pathway-based machine learning model.

BACKGROUND: Accurately differentiating between pulmonary large cell neuroendocrine carcinomas (LCNEC) and small cell lung cancer (SCLC) is crucial to make appropriate therapeutic decisions. Here, a classifier was constructed based on transcriptome data to improve the diagnostic accuracy for LCNEC and SCLC. METHODS: 13,959 genes mapped to 186 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were included. Gene Set Variation Analysis (GSVA) algorithm was used to enrich and score each KEGG pathway from RNA-sequencing data of each sample. A prediction model based on GSVA score was constructed and trained via ridge regression based on RNA-sequencing datasets from 3 published studies. It was validated by another independent RNA-sequencing dataset. Clinical feasibility was tested by comparing model predicated result using RNA-sequencing data derived from hard-to-diagnose samples of lung neuroendocrine cancer to conventional histology-based diagnosis. RESULTS: This model achieved a ROC-AUC of 0.949 and a concordance rate of 0.75 for the entire prediction efficiency. Of the 27 borderline samples, 17/27 (63.0%) were predicted as LCNEC, 7/27 were predicted as SCLC, and the remainder was NSCLC. Only 8 cases (29.6%) with LCNEC were diagnosed by pathologists, which was significantly lower than the results predicted by the model. Furthermore, cases with predicted LCNEC by the model had a significant longer disease-free survival than those where the model predicted SCLC (P = 0.0043). CONCLUSION: This model was able to give an accurate prediction of LCNEC and SCLC. It may assist clinicians to make the optimal decision for patients with pulmonary neuroendocrine tumors in choosing appropriate treatment.

Contrast-enhanced ultrasound guided pleural biopsy improves diagnostic confidence for pleural based lesions: a 3-year prospective study.

OBJECTIVE: To evaluate the accuracy and safety of contrast-enhanced ultrasound (CEUS) guided biopsy in the diagnosis of radiologically determined pleural based lesions. METHOD: A prospective study was conducted on patients with radiologically determined pleural based lesions. Patients who met the inclusion criteria received pleural biopsy guided by CEUS to obtain specimens, followed by histomathological and microbiological examinations. After treatment and follow-up, surgical thoracoscopy was performed on cases with undefinite diagnosis. RESULT: A total of 460 patients were finally included. CEUS showed internal necrosis in 72.17% cases and obvious peripheral vessels in 55.43% cases, both of which were significantly higher than the conventional ultrasound imaged (p < 0.05). The diagnostic accuracy through CEUS guided biopsy sampling was 98.91% (455/460). The microbiological diagnostic yield achieved 71.88% (225/313) in infectious lesions. In 330 cases combined pleural effusion, CEUS guided biopsy increased the diagnostic yield from 60.30% (199 /330) to 98.36% (325 /330) in all cases (p < 0.05), from 15.56% (14/90) to 94.44% (85/90) in malignant lesions (p < 0.01) and from 77.08% (185/240) to 100% (240/240) in infectious lesions (p < 0.05). No serious adverse events occurred. CONCLUSION: CEUS guided biopsy provides a minimally invasive, effective and safe diagnostic biopsy method for pleural lesions. CLINICAL TRIALS REGISTRATION: Chinese Clinical Trial Registry ChiCTR2000029749 (ChiCTR, www.chictr.org.cn ).

Adenocarcinoma of High-Grade Patterns Associated with Distinct Outcome of First-Line Chemotherapy or EGFR-TKIs in Patients of Relapsed Lung Cancer.

PURPOSE: High-grade patterns (micropapillary/solid/complex gland) are associated with a higher recurrence rate and shorter disease-free survival. Thus far, it remains unclear whether the efficacy of first-line anticancer therapy is different from that of the other adenocarcinoma subgroups for patients with high-grade patterns. The study aimed to investigate the association between an adenocarcinoma with high-grade patterns with the outcomes of first-line treatment in patients with lung cancer. PATIENTS AND METHODS: Patients with a high-grade pattern adenocarcinoma (more than 20% of micropapillary/solid components/complex glandular patterns) were retrospectively analyzed between June 2015 and June 2017. Patients' clinical characteristics and treatment outcomes were compared with those of the remaining control adenocarcinoma subgroups. RESULTS: In total, 239 patients with adenocarcinoma, including 115 (48.1%) high-grade patterns and 124 (51.9%) control groups, were enrolled. Patients' clinical characteristics such as age, sex, smoking status, and stage were similar between the two groups. Among them, 108 patients received first-line chemotherapy, and 131 received epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). In the chemotherapy group, adenocarcinoma of high-grade patterns had a significantly lower objective response rate (ORR; 15.6% vs 36.4%, P=0.045), shorter progression-free survival (PFS; median 4.1 vs 5.4 months, P=0.007) and overall survival (OS, median 19.6 vs 23.8 months, P=0.048) compared with the control group. As for these treated with EGFR-TKIs, a similar ORR (70.7% vs 72.1%, P=0.703), PFS (median 11.3 vs 13.9 months, P=0.065) and OS (median 34.1 vs 29.6%, p=0.575) were observed between these two groups. CONCLUSION: An adenocarcinoma with high-grade patterns is associated with inferior outcomes to first-line chemotherapy in relapsed lung cancer. Patients who received chemotherapy had a significantly shorter PFS and OS and lower ORR than control subjects, while there was no difference in the EGFR-TKI cohort. This study is the first to report the distribution of adenocarcinoma with high-grade patterns.

Metformin attenuates atherosclerosis and plaque vulnerability by upregulating KLF2-mediated autophagy in apoE-/- mice.

Atherosclerosis is a chronic lipid disfunction and inflammatory disease, which is characterized with enriched foam cells and necrotic core underneath the vascular endothelium. Therefore, the inhibition of foam cell formation is a critical step for atherosclerosis treatment. Metformin, a first-line treatment for Type 2 diabetes, is reported to be beneficial to cardiovascular disease. However, the mechanism underlying the antiatherogenic effect of metformin remains unclear. Macrophage autophagy is reported to be a highly anti-atherogenic process that promotes the catabolism of cytosolic lipid to maintain cellular lipid homeostasis. Notably, dysfunctional autophagy in macrophages plays a detrimental role during atherogenesis. Krueppel-like factor 2 (KLF2) is an important transcription factor that functions as a key regulator of the autophagy-lysosome pathway. While the role of KLF2 in foam cell formation during the atherogenesis remains elusive. In this study, we first investigated whether metformin could protect against atherogenesis via enhancing autophagy in high fat diet (HFD)-induced apoE-/- mice. Subsequently, we further determined the molecular mechanism that whether metformin could inhibit foam cell formation by activating KLF2-mediated autophagy. We show that metformin protected against HFD-induced atherosclerosis and enhanced plaque stability in apoE-/- mice. Metformin inhibits foam cell formation and cellular apoptosis partially through enhancing autophagy. Mechanistically, metformin promotes autophagy via modulating KLF2 expression. Taken together, our study demonstrates a novel antiatherogenic mechanism of metformin by upregulating KLF2-mediated autophagy.

Sublobectomy is a safe alternative for localized cavitary pulmonary tuberculosis.

OBJECTIVE: Surgical resection plays an essential role in the treatment of Pulmonary Tuberculosis (PTB). There are few reports comparing lobectomy and sublobectomy for pulmonary TB with cavity. To compare the advantages between lobectomy and sublobectomy for localized cavitory PTB, we performed a single-institution cross sectional cohort study of the surgical patients. METHODS: We consecutively included 203 patients undergoing lobectomy or sublobectomy surgery for localized cavitary PTB. All patients were followed up, recorded and compared their surgical complication, outcome and associated characteristics. RESULTS: Both groups had similar outcomes after follow up for 13.1 ± 12.1 months, however, sublobectomy group suffered fewer intraoperative blood losses, shorter length of stay, and fewer operative complications than lobectomy group (P <  0.05). Both groups obtained satisfactory outcome with postoperatively medicated for similar period of time and few relapse (P > 0.05). CONCLUSION: Both sublobectomy and lobectomy resection were effective ways for cavitary PTB with surgical indications. If adequate anti-TB chemotherapy had been guaranteed, sublobectomy is able to be recommended due to more lung parenchyma retain, faster recover, and fewer postoperative complications.

The combination of computed tomography features and circulating tumor cells increases the surgical prediction of visceral pleural invasion in clinical T1N0M0 lung adenocarcinoma.

BACKGROUND: Visceral pleural invasion (VPI) is a clinical manifestation associated with a poor prognosis, and diagnosing it preoperatively is highly imperative for successful sublobar resection of these peripheral tumors. We evaluated the roles of computed tomography (CT) features and circulating tumor cells (CTCs) for improving VPI detection in patients with clinical T1N0M0 invasive lung adenocarcinoma. METHODS: Three hundred and ninety-one patients were reviewed retrospectively in this study, of which 234 presented with a pleural tag or pleural contact on CT images. CTCs positive for the foliate receptors were enriched and analyzed prior to surgery. Logistic regression analyses were performed to assess the association of CT features and CTCs with VPI, and the receiver operating characteristic (ROC) curve was generated to compare the predictive power of these variables. RESULTS: Patients mostly underwent either segmentectomies (18.9%) or lobectomies (79.0%). Only 49 of the 234 patients with pleural involvement on CT showed pathologically confirmed VPI. Multivariate logistic regression analysis revealed that CTC level ≥10.42 FU/3 mL was a significant VPI risk factor for invasive adenocarcinoma cases ≤30 mm [adjusted odds ratio (OR) =4.62, 95% confidence interval (CI): 2.05-10.44, P<0.001]. Based on CT features, subgroup analyses showed that the solid portion size was a statistically significant independent predictor of VPI for these peripheral nodules with pleural tag, while the solid portion length of the interface was an independent predictor of pleural contact. The receiver operating curve analyses showed that the combination of CTC and CT features were highly predictive of VPI [area under the curve (AUC) =0.921 for pleural contact and 0.862 for the pleural tag, respectively]. CONCLUSIONS: CTC, combined with CT features of pleural tag or pleural contact, could significantly improve VPI detection in invasive lung adenocarcinomas at clinical T1N0M0 stage prior to the patient's surgery.

Downregulation of histone-lysine N-methyltransferase EZH2 inhibits cell viability and enhances chemosensitivity in lung cancer cells.

Histone-lysine N-methyltransferase EZH2 (EZH2) is the principle component of the polycomb repressive complex 2 (PRC2)/embryonic ectoderm development protein-EZH2 complex, which promotes tumorigenesis by repressing transcription of tumor suppressor genes. EZH2 is considered a key marker in several types of cancer, such as colorectal and prostate cancer. However, the molecular mechanisms and clinical value of EZH2 in lung cancer have not yet been fully investigated. The aim of the present study was to investigate the functions of EZH2 in lung cancer progression and to determine whether treatment with an EZH2 inhibitor enhanced the chemosensitivity of lung cancer cells to cisplatin (CDDP). At the logarithmic growth phase, A549 cells were treated with a small interfering (si)RNA-EZH2, and cell viability was detected using an MTT assay. The degree of apoptosis and cell cycle were detected using flow cytometry. Cell migration and invasion were detected via wound healing and Transwell Matrigel assays. According to information from the Gene Expression Omnibus database, the results of the present study demonstrated that EZH2 was upregulated in lung cancer. Furthermore, overexpression of EZH2 was associated with poor patient prognosis, while EZH2 knockdown inhibited cell viability and migration, and enhanced apoptosis and chemosensitivity in a lung cancer cell line. EZH2 knockdown and treatment of A549 cells using EZH2 inhibitor elevated the inhibitory effects of CDDP on cell viability and apoptosis. Western blot and reverse transcription-quantitative PCR analyses were performed to assess the expression levels of relative protein and mRNA, respectively, in A549 cells treated with siRNA-EZH2 or with CDDP. Overall, the results of the present study demonstrated that high EZH2 expression was associated with poor prognosis, accompanied with a potential impairment of migration and viability in lung cancer cells. These findings suggest that EZH2 may act as a candidate molecular target for gene therapy, and treatment with EZH2 inhibitor may be used to increase chemosensitivity to CDDP agents in lung cancer.

Evaluation of liquid based cytology in detection of EGFR mutation in NSCLC by large samples.

BACKGROUND: Cytology samples are the main resources to detect driver oncogene alterations for advanced lung cancer patients. To explore the value of liquid-based cytology in the detection of epidermal growth factor receptor (EGFR) mutation in non-small cell lung cancer (NSCLC), we analyzed data from a large cohort of EGFR mutation-positive patients. METHODS: We analyzed the clinicopathological characteristics of 8,029 NSCLC cases tested for EGFR mutation by liquid-based cytology specimens and 1,934 NSCLC cases tested by formalin-fixed and paraffine-embedded (FFPE) samples in the Shanghai Pulmonary Hospital from September 2015 to December 2019. Before detection, we evaluated the number of tumor cells in the liquid-based cytology slide, and samples with more than 50 tumor cells and visible sediment were selected for DNA extraction after centrifugation. RESULTS: The positive rate of EGFR mutation in liquid-based cytology-tested cases was 47.18%, higher than the 41.37% tested through FFPE sample (P<0.01). Accordingly, the mutation rate of EGFR in adenocarcinoma (AC) and NSCLC was higher than that of the FFPE sample (60.01% vs. 54.15%, P<0.01; 30.54% vs. 21.99%, P<0.01). The positive rate of EGFR mutation in pleural effusion was 62.67%, which was the highest rate among liquid-based cytology sample t (P<0.01). CONCLUSIONS: Using quality control and standard procedure, it was found that liquid-based cytology specimen testing is a convenient and reliable method of EGFR detection, as validated by analysis of a large cohort. EGFR mutation detection should also be carried out in NSCLC patients diagnosed by cytology more than in AC patients.

Cribriform growth pattern in lung adenocarcinoma: More aggressive and poorer prognosis than acinar growth pattern.

The predictive value of prognosis based on the histopathological subtype is a critical criterion in the new classification of lung adenocarcinoma published in 2011 by the International Association for the Study of Lung Cancer (IASLC), the American Thoracic Society (ATS), and the European Respiratory Society (ERS) (IASLC/ATS/ERS). In this new classification, the differences of histopathology and prognosis are two considerable parameters to classify the subtypes of lung adenocarcinoma. Cribriform growth pattern is regarded as a variant of acinar growth pattern in lung adenocarcinoma, however, more and more studies pointed out that cribriform growth pattern is associated with more aggressive histopathological structures, higher proportion of recurrence rates, and shorter postoperative survival than acinar growth pattern. These features are similar to solid or micropapillary predominant adenocarcinoma. In this review, we summarized the clinicopathological features, prognosis, and genetic variations of cribriform growth pattern of lung adenocarcinoma, and provided a novel insight into the diagnosis and treatment of cribriform lung adenocarcinoma.

Cell Block as a Surrogate for Programmed Death-Ligand 1 Staining Testing in Patients of Non-Small Cell Lung Cancer.

Introduction: Programmed death-ligand 1 (PD-L1) staining is used in clinical practice to guide the proper use of immune checkpoint inhibitors. This study aimed to investigate the accuracy of PD-L1 staining of non-small cell lung cancer (NSCLC) cytological cell block samples. Methods: Paired cytological cell block and surgical resection samples were consecutively collected from January 2016 to February 2017 in Shanghai Pulmonary Hospital, Tongji University. Two trial-validated PD-L1 assays (28-8 and SP142) were used to quantify PD-L1 expression. Results: A total of 112 pairs of specimens were collected, including 68(60.7%) adenocarcinomas and 28(25.0%) squamous cell carcinomas. Based on a tumor proportion score (TPS) cutoff of 1% for the 28-8 and SP142 assays, PD-L1 expression was positive in 78.6% and 58.9% of surgical samples respectively, while PD-L1 expression was positive in 67.9% and 25.0% of cytological cell block samples. Based on staining by each antibody, fair to substantial concordance of PD-L1 expression was observed for cytological cell block specimens as compared to surgical resection (𝛋 ranges from 0.377 to 0.686). However, as the tumor cells in the cell block specimen increased, the consistency of PD-L1 expression increased. The concordance of PD-L1 expression in cell blocks with abundant cellularity was nearly perfect with various cutoffs (28-8: tumor cells over 400; SP142: tumor cells over 500). Conclusion: Cytological cell block specimens may serve as a surrogate for PD-L1 staining in patients of NSCLC when more than 400-500 cancer cells were contained (over 400 cancer cells for 28-8, over 500 cancer cells for SP142).

Genomic landscape and its correlations with tumor mutational burden, PD-L1 expression, and immune cells infiltration in Chinese lung squamous cell carcinoma.

INTRODUCTION: To depict the genomic landscape of Chinese early-stage lung squamous cell carcinoma (LUSC) and investigate its correlation with tumor mutation burden (TMB), PD-L1 expression, and immune infiltrates. METHODS: Whole-exome sequencing was performed on 189 surgically resected LUSC. TMB was defined as the sum of nonsynonymous single nucleotide and indel variants. CD8+ tumor-infiltrating lymphocyte (TIL) density and PD-L1 expression were evaluated by immunohistochemistry. Six immune infiltrates were estimated using an online database. RESULTS: The median TMB was 9.43 mutations per megabase. Positive PD-L1 expression and CD8+ TILs density were identified in 24.3% and 78.8%. PIK3CA amplification was associated with significantly higher TMB (P = 0.036). Frequent genetic alterations had no impact on PD-L1 expression but PIK3CA amplification and KEAP1 mutation were independently associated with significantly lower CD8+ TIL density (P < 0.001, P = 0.005, respectively). Low TMB and high CD8+ TIL density were independently associated with longer disease-free survival (DFS) while none of them could individually predict the overall survival (OS). Combination of TMB and PD-L1 expression or TMB and CD8+ TIL density could stratify total populations into two groups with distinct prognosis. Classifying tumor-immune microenvironment based on PD-L1 expression and CD8+ TIL density showed discrepant genomic alterations but similar TMB, clinical features, and OS. Notably, patients with different smoking status had distinct prognostic factors. CONCLUSION: The combination of TMB, PD-L1 expression, immune infiltrates, and smoking status showed the feasibility to subgroup stratification in Chinese patients with early-stage LUSC, which might be helpful for future design of personalized immunotherapy trials in LUSC.

Characterization of PD-L1 protein expression and CD8+ tumor-infiltrating lymphocyte density, and their associations with clinical outcome in small-cell lung cancer.

BACKGROUND: This study aimed to characterize programmed death ligand-1 (PD-L1) expression and CD8+ tumor-infiltrating lymphocytes (TILs) density, and their impact on survival in patients with surgically resected small-cell lung cancer (SCLC). METHODS: Fifty-six patients with surgically resected SCLC were included. PD-L1 protein expression and CD8+ TILs were tested by immunohistochemistry. A meta-analysis of 15 articles with 1,505 patients that investigated the prevalence and prognostic significance of PD-L1 expression in SCLC was conducted. RESULTS: Twenty-two (39.3%) patients had positive PD-L1 protein expression and 42 (75.0%) had high CD8+ TILs density. PD-L1 expression level was not associated with CD8+ TILs density (P=0.528). No any association between clinicopathological features and PD-L1 expression level or CD8+ TILs density was observed. Positive PD-L1 expression [hazard ratio (HR) =0.374, P=0.002] and high CD8+ TILs density (HR =0.429, P=0.008) were independently associated with significantly longer overall survival (OS), which remain the statistical significance in multivariate analyses (P=0.007, P=0.002; respectively). Meta-analysis showed that the prevalence of positive PD-L1 expression was 0.35 [95% confidence interval (CI), 0.22-0.48] and positive PD-L1 expression was correlated with markedly longer OS (HR =0.61; 95% CI, 0.31-0.91) in patients with SCLC. CONCLUSIONS: The prevalence of PD-L1 expression in surgically resected SCLC is lower than that published for NSCLC. There was no association between PD-L1 expression or CD8+ TILs density and clinicopathological parameters. PD-L1 expression and CD8+ TILs density was independently correlated with better outcome in patients with SCLC.

Predictive value of oncogenic driver subtype, programmed death-1 ligand (PD-L1) score, and smoking status on the efficacy of PD-1/PD-L1 inhibitors in patients with oncogene-driven non-small cell lung cancer.

BACKGROUND: This multicenter, retrospective study explored the value of oncogene driver subtype, programmed death-1 ligand (PD-L1) status, and smoking status for predicting which patients with oncogene-driven non-small cell lung cancer (NSCLC) would benefit from treatment with programmed death-1 (PD-1)/PD-L1 inhibitors. METHODS: The clinical features, PD-L1 tumor proportion scores, and PD-1/PD-L1 inhibitor (PDi) outcomes (objective response rate and progression-free survival) of patients who had advanced NSCLC with Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) mutations or common, actionable oncogenic drivers were captured. RESULTS: In total, 189 oncogene-positive patients were analyzed. Of these, 104 received a PDi, and 108 had undergone prior PD-L1 testing. The frequency of PD-L1 positivity (≥1%) was higher in patients who had KRAS mutations (P = .031), smokers (P = .006), and non-Asian patients (P = .002). Multivariable analysis indicated that smoking status (P < .001) was the only factor associated significantly with KRAS mutation. The objective response rate to PDi treatment was 16.9% (11 of 65 patients) among smokers (17.3% in the KRAS-mutant and 15.4% in the non-KRAS-mutant smoker subgroups), which was significantly higher than the 0% rate (0 of 26 patients; P = .019) among never-smokers. In subgroup analyses, progression-free survival was influenced by KRAS mutation status (median, 4.57 vs 1.63 months; P = .004), smoking status (4.07 vs 1.73 months; P = .004), PD-L1 positivity (3.8 vs 1.2 months; P = .040), and non-Asian race (3.0 vs 1.97 months; P = .046). In multivariable analysis, only smoking status (P = .008) remained a significant predictor when a PD-L1 level ≥1% was used. However, both smoking status (P = .001) and PD-L1 status (P = .028) were independent predictors when a PD-L1 level ≥50% was used. CONCLUSIONS: Among associated clinical features among patients who have NSCLC with oncogenic drivers, smoking status potentially was the most important, easily available predictor of single PDi efficacy.

A lung squamous carcinoma patient with ROS1 rearrangement sensitive to crizotinib.

It is widely known that ROS1 rearrangement mostly occurs in the adenocarcinoma subtype of non-small-cell lung cancer. Patients with squamous cell carcinoma harboring ROS1 rearrangement are extremely rare. This is a case report of a squamous cell carcinoma patient with ROS1 rearrangement. An 84-year-old Chinese woman with a about 6.6 cm mass in the right middle lobe and right pleural effusion, enlarged mediastinal and hilar lymph nodes was diagnosed with stage IIIB lung squamous cell carcinoma harboring ROS1 rearrangement is highly sensitive to crizotinib in the first treatment setting. This is the first time to report lung squamous carcinoma patient with ROS1 rearrangement sensitive to crizotinib. We hope that oncologists will notice this phenomenon and it will help the lung squamous cell carcinoma patient to get longer overall survival time.