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The plasma sample has emerged as a promising surrogate sample for EGFR mutation detection in advanced non-small cell lung cancer (NSCLC). In clinical practice, whether EGFR variants in baseline plasma ctDNA of advanced NSCLC can predict prognosis in addition to guiding targeted therapy remains to be further explored.In total, 315 NSCLC patients were retrospectively enrolled. EGFR mutation data from tissue detected by ARMS-PCR and paired plasma samples within 1 month of admission detected by SuperARMS or ARMS-PCR were collected. The correlation between baseline plasma ctDNA EGFR mutation status and survival was compared.EGFR mutation detection rates in tumor samples and plasma samples were 65.1% (205/315) and 43.8% (138/315). Referred to tissue results, the consistent rate of test ctDNA EGFR alteration by SuperARMS was higher than that detected by ARMS (79.5% vs. 69.0%, p = 0.04), either in stage I-IIIA patients (85.7% vs. 50.0%, p = 0.4) or stage IIIB-IV patients (79.1% vs. 69.4%, p = 0.04). Patients' treatment status and pathological subtype were the two factors that affected plasma ctDNA EGFR alteration detection accuracy. The concordance in non-adenocarcinoma patients was obviously higher than that in adenocarcinoma (p = 0.02), and the concordance in treatment naïve patients was significantly higher than that in relapse patients (p = 0.047). In treatment naïve patients, the median PFS (mPFS) in plasma ctDNA EGFR-positive patients was shorter than that in plasma ctDNA EGFR negative patients (7.0 vs. 10.0 months, p = 0.01). In relapsed patients, the mPFS in plasma ctDNA EGFR-positive patients was 9.0 months versus 11.0 months in plasma ctDNA EGFR negative patients (p = 0.1).A plasma sample could be an alternative for a molecular test when tissue samples was unavailable. The SuperARMS-PCR detection method has high sensitivity in real-world clinical practice. Furthermore, in patients with stage IIIB-IV, baseline plasma ctDNA EGFR mutation positivity not only guides targeted therapy but also predicts a worse prognosis.

Background Circulating tumor DNA (ctDNA) enables rapid and repeat testing of actionable mutations with fewer side effects compared to tissue biopsy. And the use of next-generation sequencing (NGS) based on ctDNA as a diagnostic tool in clinical settings is growing. We developed a hybridization capture massively parallel NGS assay using ctDNA, USCI-CT, across 20 cancer relevant genes of non-small cell lung cancer (NSCLC). Clinical validations of this assay across 17 mutations were presented.Methods Clinical NSCLC samples and simulated negative and positive cell-free DNA (cfDNA) samples were applied to validate the technical performance of this assay. First, 40 simulated negative control cfDNA samples were used to evaluate the systemic error levels to delimit the limit of detection of variant allele frequency (VAF). And, 92 clinical NSCLC samples were genotyped by USCI-CT and ddPCR to decide the proper limit of VAF, depth of coverage, and the average depth of target regions for confidently detecting mutations for USCI-CT. Second, one simulated negative cfDNA sample and two simulated positive cfDNA samples in seven replicates were used to assess the precision of the assay. Finally, 518 clinical NSCLC samples were recruited to evaluate the analytical sensitivity and specificity of USCI-CT.Results The assay-specific systemic error rate was below 0.20% by sequencing sixty-seven simulated negative control cfDNA samples. The proper cutoff of detection of VAF, coverage of depth of mutation loci, and average depth of target regions were decided at 0.20%, 1000×, and 1400×. The analytical sensitivity and specificity achieved a satisfactory level with 98.32% and 99.85% for single nucleotide variant (SNV) detection and 97.30% and 96.86% for EGFR exon 19 deletions, respectively.Conclusions The results indicated that the USCI-CT assay can reliably detect NSCLC related mutations at 0.20% variant frequency level and provided insights into the incorporation of hybridization captured NGS-based ctDNA assay into oncology clinical settings.

ObjectivesThe present study used immunohistochemistry (IHC) to detect antigen Ag85B in tissue sections and aimed to evaluate its validity in histopathologic diagnosis of tuberculosis (TB).MethodsIn total, 204 patients with confirmed TB and 40 other diseases were included in the present study. Ziehl-Neelsen (Z-N) stains, IHC (anti-Ag85B), and quantitative fluorescence polymerase chain reaction were used to detect acid-fast bacilli, Mycobacterium tuberculosis (MTB) antigen, and MTB DNA.ResultsImmunohistochemistry was significantly more sensitive than Z-N stains (93.1% vs 67.2%; P < .001). The sensitivity of Z-N stains significantly correlated with anti-TB treatment history. The sensitivity of Z-N stains was lower in rifampicin (RIF)-resistant TB compared with RIF-sensitive TB (52.8% vs 69.0%; P = .091) and those without treatment history (52.8% vs 84.0%; P = .015). However, IHC was not significantly affected by treatment history (P = .410). Moreover, expression patterns of Ag85B were dependent on treatment history and commonly showed weak scattered spots in RIF-susceptible TB. Conversely, strong brown rods were often found in those with RIF-resistant TB.ConclusionsImmunohistochemistry is a simple, sensitive technique for the diagnosis of TB, especially for those patients with treatment history. The expression pattern of Ag85B is a potential marker for evaluating anti-TB treatment response.

Objectives. The differential diagnosis of pleural effusion (PE) is a common but major challenge in clinical practice. This study aimed to establish a strategy based on a PE-cell-free DNA (cfDNA) methylation detection system for the differential diagnosis of malignant pleural effusion (MPE) and benign pleural effusion (BPE). Methods. A total of 104 patients with PE were enrolled in this study, among which 50 patients had MPE, 9 malignant tumor patients had PE of indefinite causes, and the other 45 patients were classified as benign controls. The methylation status of short stature homeobox 2 (SHOX2) and RAS association domain family 1, isoform A (RASSF1A) was detected using PE-cfDNA specimens by real-time fluorescence quantitative PCR. Total methylation (TM) was defined as the combination of the methylation levels of SHOX2 and RASSF1A. The electrochemiluminescence immunoassay was applied to evaluate the levels of multiple serum tumor markers. Results. The PE-cfDNA methylation status of either SHOX2 or RASSF1A was much higher in MPE samples than in benign controls. The combination of SHOX2 and RASSF1A methylation in PE yielded a diagnostic sensitivity of 96% and a specificity of 100%, respectively. When compared with the corresponding serum tumor marker detection results, TM showed the highest diagnostic efficiency (AUC = 0.985). Furthermore, the combination of the SHOX2 and RASSF1A methylation panels using PE-cfDNA could apparently improve the differential diagnostic efficacy of BPE and MPE and could help compensate for the deficiency of cytology. Conclusions. Our results indicated that SHOX2 and RASSF1A methylation panel detection could accurately classify BPE and MPE diseases and showed better diagnostic performance than traditional serum parameters. The SHOX2 and RASSF1A methylation detection of PE-cfDNA could be a potentially effective complementary tool for cytology in the process of differential diagnosis. In summary, PE-cfDNA could be used as a promising non-invasive analyte for the auxiliary diagnosis of MPE.

Background: The detection of epidermal growth factor receptor (EGFR) mutations in patients with non-small cell lung cancer is critical for tyrosine kinase inhibitors therapy. EGFR detection requires tissue samples, which are difficult to obtain for some patients, losing the opportunity for further treatment. To realize EGFR mutation prediction without molecular detection, we aim to build a high accuracy deep learning model with only hematoxylin-eosin-stained slides. Methods: A total of 326 hematoxylin-eosin-stained non-small cell lung cancer slides were collected from Beijing Chest Hospital, China. After digitalized into whole slide images, 226 slides (88 with EGFR mutation) among them were fed to a convolutional neural network for model training. The remaining 100 images (50 with EGFR mutation) were used as the test set. Results: The sensitivity and specificity of the model were 76% and 74%, respectively, with an area under the curve of 0.82. When applying the double threshold approach, the deep learning model could screen out 40% of the patients with a sensitivity and specificity of 96% and 94%, respectively. By further involving adenocarcinoma subtype information, 37.3% of the adenocarcinoma patients could be screened out with 100% sensitivity and specificity. Conclusions: In this research, we show rapid and inexpensive pre-screening potentials of the deep learning-based EGFR mutation prediction model. It could not only act as a high-accuracy complement to current molecular detection techniques, but also provide opportunities for non-small cell lung cancer patients with limited sample to receive further treatment.

Background Pleural effusion from patients with advanced non‐small cell lung cancer (NSCLC) has been proved valuable for molecular analysis, especially when the tissue sample not available. However, simultaneous detection of multiple driver gene alterations especially the fusions is still challenging. Methods In this study, 77 patients with advanced NSCLC and pleural effusion were enrolled, 49 of whom had matched tumor tissues. Supernatants, cell sediments, and cell blocks were prepared from pleural effusion samples for detection of driver alterations by a PCR‐based 9‐gene mutation detection kit. Results Mutations in EGFR, KRAS, and HER2 were detected in DNA and cfDNA, fusions in ALK was detected in RNA and cfRNA. Compared with matched tumor tissue, the supernatant showed the highest overall sensitivity (81.3%), with 81.5% for SNV/Indels by cfDNA and 80% for fusions by cfRNA, followed by cell blocks (71.0%) and the cell sediments (66.7%). Within the group of treatment‐naïve patients or malignant cells observed in the cell sediments, supernatant showed higher overall sensitivity (89.5% and 92.3%) with both 100% for fusions. Conclusions CfDNA and cfRNA derived from pleural effusion supernatant have been successfully tested with a PCR‐based multigene detection kit. Pleural effusion supernatant seems a preferred material for detection of multigene alterations to guide treatment decision of advanced NSCLC., CfDNA and cfRNA derived from pleural effusion supernatant have been successfully tested with a PCR‐based multi‐gene detection kit. Pleural effusion supernatant seems a preferred material for detection of multi‐gene alterations to guide treatment decision of advanced NSCLC.

CD155 is an immune checkpoint protein expressed in tumor cells that interacts with its ligand TIGIT, and inhibition of this point presents a new and novel way for cancer therapy. At present, whether the expression of CD155 affects the response to anti(α)-PD1 treatment in non-small cell lung cancer (NSCLC) patients is unclear. This observational study characterizes the expression of CD155 in NSCLC patients and its responses to PD1 inhibitors. We retrospectively detected the expression of CD155 and tumor-infiltrated lymphocyte (TIL) TIGIT by immunohistochemistry in advanced NSCLC patients who had received αPD1 therapy. The patients with CD155 positive had a significantly worse response to αPD1 therapy compared with CD155-negative patients (ORR: 25.6% vs 54.8%, P < 0.01; median PFS: 5.1 vs 7.1 months, HR = 2.322; 95% CI 1.396–3.861, P = 0.001). This effect is more prominent in PD-L1 positive patients. In PD-L1-positive patients, CD155 expression is associated with a poor response to αPD1 therapy in both LUAC (lung adenocarcinoma) and LUSC (lung squamous cell carcinoma); meanwhile, the expression of CD155 was associated with a poor response to the first-line αPD1 therapy, posterior-line αPD1 therapy, and αPD1 combination therapy. Furthermore, the expression of TIGIT was not correlated with the therapeutic effect of αPD1. Our pilot study suggests that CD155 expression attenuates the therapeutic effect of αPD1 therapy and is associated with a higher risk of progression. The CD155 pathway may be a promising immunotherapeutic target and simultaneously targeting CD155/TIGIT and PD1/PD-L1 can improve the effect of immunotherapy.

Objectives The molecular profiles and prognosis of anaplastic lymphoma kinase (ALK) fusion and resectable non-small cell lung cancer (NSCLC) remain unclear. This study aimed to explore the distribution of ALK fusion variants and prognostic factors in patients with surgically resected NSCLC. Material and methods Among the 93 ALK positive surgical patients screened by immunohistochemistry (IHC) or real-time polymerase chain reaction (RT-PCR), 63 patients were confirmed as ALK rearrangement by next-generation sequencing (NGS), including 55 cases of stage I-III and 8 cases of stage IV. Medical records were retrospectively reviewed, the distribution of ALK fusion variants and prognostic factors were analyzed. Results All of the 55 early stage patients were histological adenocarcinoma. No other fusion types were found except for echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK). EML4-ALK variant 1 (E13:A20; 25/55, 45.5 %) was the predominant variant type, followed by EML4-ALK variant 3 (E6:A20; 19/55, 34.5 %) and variant 2 (E20:A20; 8/55, 14.5 %). Concomitant mutations occurred in 22 patients (22/55, 40.0 %), which involved in 32 co-mutations from 12 kinds of mutated genes. TP53 mutations were most common in coexisting mutations (13/32, 40.6 %). TP53 mutations were less frequently occurred in variant 1 group (3/25, 12.0 %) than in non-variant 1 group (10/30, 33.3 %, P = 0.064). The median disease-free survival (DFS) of the 55 patients was 22.1 months, and the median overall survival (OS) was not mature at the time of analysis. Multivariable analysis showed that stage T3 and EML4-ALK variant 3 were independent prognostic factors for shorter DFS. Neither TP53 mutations nor any coexisting mutations were related to prognosis. Conclusions This study illustrated the patterns of EML4-ALK fusion variants and gene profiles in patients with resected NSCLC. Advanced T stage and EML4-ALK variant 3 were associated with worse prognosis. The role of TP53 mutations in prognosis is worthy of further study.

Background: To understand the association between driver gene variations and age and gender in patients with lung adenocarcinoma, we investigated mutations of the three most important driver genes—epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK) fusion genes and c-ros oncogene 1 (ROS1)—in this retrospective cohort study. Methods: Patients newly diagnosed with lung adenocarcinoma who received EGFR and ALK/ROS1 gene tests at our hospital from September 2014 to May 2019 were enrolled. EGFR mutations and ROS1 fusions were examined by ARMS-PCR and ALK fusions by Ventana-D5F3 IHC assay and ARMS-PCR. Results: Of 2544 eligible subjects, 2539 accomplished EGFR mutation tests. The prevalence of EGFR mutations was 62.1% in females, higher than that of 45.1% in males. In females, the EGFR mutation rate remained relatively stable at 60%–65% across the six age groups. Females showed an increased distribution of EGFR L858R and a decreased distribution of exon 19 deletion (19Del) by age. The incidence of ALK/ROS-1 rearrangements decreased significantly with age. Conclusions: EGFR 19Del mutation is more prevalent in younger males and females, while L858R mutation is prevalent in older females. Both ALK and ROS1 rearrangements are more common in younger lung adenocarcinoma. The young lung adenocarcinoma population is a distinct group rich in targetable genomic alterations, and more research is needed to understand the mechanism.

Background Mesenchymal-epithelial transition (MET) exon14 skipping mutations represent a clinically unique molecular subtype of NSCLC. The prevalence rates of MET exon 14 skipping in lung adenocarcinoma (ADC) range from 0.9% to 4.0% in Asian populations. Since some somatic variants that do not encompass the MET exon 14 splice sites might also induce MET exon 14 skipping, the RNA-based sequencing is speculated as the most accurate method for detecting exon 14 skipping. Patients and Methods A total of 951 NSCLC patients from two hospitals were enrolled in this study. MET exon14 skipping was detected using RNA-based next-generation sequencing (NGS). Also, immunohistochemistry (IHC) was performed in 405 samples simultaneously. Results The overall estimated prevalence of MET exon 14 skipping was approximately 1.8% in ADCs and 1.7% in NSCLCs. The detection rate of MET exon 14 skipping from surgical resection specimen was 2.3% in NSCLCs and 2.0% in ADCs. The MET exon 14 skipping was identified in 6.6% of EGFR/KRAS/ALK/ROS1/RET-negative ADCs. Additionally, PD-L1 was found to be highly expressed in NSCLC patients harboring MET exon 14 skipping (P

BACKGROUND: Epidermal growth factor receptor exon 20 insertions (EGFR ex20ins) occur in about 4–14% of lung adenocarcinoma (LUAD) patients with EGFR mutations. Recently some targeted drugs have been approved for the treatment of LUAD patients with EGFR ex20ins. However, the heterogeneity of EGFR ex20ins mutations and resultant challenges in identifying them have led to the underestimation of their frequency. METHODS: We investigated the molecular and clinicopathologic features of EGFR ex20ins in 3,892 Chinese LUAD patients using next-generation sequencing (NGS). The frequency and distribution of EGFR ex20ins mutations between Chinese and Western LUAD patients were also compared by integrating the data of this study and the data of previous studies. RESULTS: A total of 23 unique EGFR ex20ins were identified in 77 LUAD patients, accounting for 1.98% of all LUAD patients and 3.49% of EGFR mutant LUDA patients. The 2 most common EGFR ex20ins subtypes were S768_D770dup and A767_V769dup, which together accounted for 55.84% of the EGFR ex20ins cases. About 61% (14/23) of the EGFR ex20ins subtypes occurred only once. Additionally, 8 of the EGFR ex20ins subtypes were not recorded in the COSMIC database. These results showed that the EGFR ex20ins mutations were highly heterogeneous. There was no significant difference in the frequency and distribution of EGFR ex20ins mutations between Chinese and Western LUAD patients, but the frequency of EGFR ex20ins mutations was significantly lower in EGFR-mutant Chinese LUAD patients than Western LUAD patients. The co-mutation analysis showed that EGFR ex20ins occurred significantly and exclusively with certain driver genes in LUAD, including ALK fusion (χ(2)=7.133, P=0.008), KRAS (χ(2)=8.468, P=0.004), and PIK3CA (χ(2)=5.792, P=0.016). No gene was observed to be significantly co-mutated with EGFR ex20ins. In general, patients with EGFR ex20ins shared a similar age and gender to patients with other EGFR mutations or without EGFR ex20ins. CONCLUSIONS: Overall, our results revealed the molecular and clinicopathologic features of EGFR ex20ins in Chinese LUAD patients, which will be helpful for drug development and in clinical trials targeting EGFR ex20ins.

BackgroundYoung lung cancer as a small subgroup of lung cancer has not been fully studied. Most of the previous studies focused on the clinicopathological features, but studies of molecular characteristics are still few and limited. Here, we explore the characteristics of prognosis and variation in young lung cancer patients with NSCLC.MethodsA total of 5639 young lung cancer samples (NSCLC, age ≤40) were screened from the SEER and the same number of the old (NSCLC, age ≥60) were screened by propensity score matching to evaluate the prognosis of two groups. 165 treatment-naïve patients diagnosed with NSCLC were enrolled to explore the molecular feature difference between two age-varying groups. CCLE cell line expression data was used to verify the finding from the cohort of 165 patients.ResultsThe overall survival of the young lung cancer group was significantly better than the old. Germline analysis showed a trend that the young group contained a higher incidence of germline alterations. The TMB of the young group was lower. Meanwhile, the heterogeneity and evolutionary degrees of the young lung cancer group were also lower than the old. The mutation spectrums of two groups exhibited variance with LRP1B, SMARCA4, STK11, FAT2, RBM10, FANCM mutations, EGFR L858R more recurrent in the old group and EML4-ALK fusions, BCL2L11 deletion polymorphism, EGFR 19DEL, 20IN more recurrent in the young group. For the base substitution, the young showed a lower fraction of transversion. Further, we performed a pathway analysis and found the EGFR tyrosine kinase inhibitor resistance pathway enriched in the young lung cancer group, which was validated in gene expression data later.ConclusionsThere were significantly different molecular features of the young lung cancer group. The young lung cancer group had a more simple alteration structure. Alteration spectrums and base substitution types varied between two groups, implying the different pathogenesis. The young lung cancer group had more potential treatment choices. Although young lung patients had better outcomes, there were still adverse factors of them, suggesting that the young group still needs more caution for treatment choice and monitoring after the treatment to further improve the prognosis.

Objective: The diagnostic sensitivity and specificity of conventional methods for superficial lymph node tuberculosis (LNTB) are not ideal. We evaluated several novel methods including Xpert Mycobacterium tuberculosis/rifampicin (Xpert MTB/RIF) technology, quantitative fluorescence Polymerase Chain Reaction (qPCR) and High-Resolution Melting Curve (HRMC) in the diagnosis of superficial lymph node TB. Methods: Specimens from eighty-one consecutive patients with suspected LNTB and thirteen cases with other lymph node disease were analyzed by Xpert MTB/RIF, qPCR, and HRMC. Results: Among 81 patients with clinical suspicion of LNTB, there were 74 (91.4%) cases positive Mycobacterium tuberculosis Complex (MTBC) of Xpert MTB/RIF, 60 (74%) positive of qPCR, 24 (29.6%) of positive of BACTEC MGIT960 culture, and 13 (16%) cases positive of Roche culture. 38 cases (46.9%) were diagnosed with LNTB. All test methods showed a diagnostic specificity of 100% for LNTB. The sensitivity of molecular biology techniques was significantly higher than that of the traditional diagnostic methods, and Xpert MTB/RIF was the most sensitive diagnostic assay. On Rifampinresistant detection, Xpert MTB/RIF detected three cases (3.7%) with rpoB gene mutation, and Mycobacterium tuberculosis susceptibility testing detected 2 rifampicin-resistant cases (2.4%) which were consistent with Xpert MTB/RIF results. In the Isoniazid-resistant, 7 cases (8.1) of isoniazid resistance mutations (8.1%) were detected by HNC and 1 case was confirmed by Isoniazid susceptibility test. Conclusion: Molecular detection increased the diagnostic sensitivity of LNTB and improved the detection sensitivity for rifampin and isoniazid resistance strain.

Objectives Tuberculosis meningitis (TBM) is one of the most severe forms of tuberculosis. However, TBM diagnosis is quite challenging due to nonspecific clinical presentation and the paucity of the pathogen in cerebrospinal fluid (CSF) samples. In this study, we report a new method for detecting cell-free Mycobacterium tuberculosis DNA (cf-TB) in CSF and evaluate its clinical value for TBM diagnosis. Methods Of 68 patients prospectively recruited, 46 were diagnosed as having TBM and 22 as non-TBM. We compared the cf-TB method with CSF smear microscopy, mycobacterial culture, and the Xpert MTB/RIF assay (Xpert) using the consensus case definition for TBM proposed in 2009 as a reference standard. Results The sensitivity of the cf-TB test was 56.5% (26/46) in patients with TBM, and it was significantly higher than other methods: microscopy (2.2%, 1/46; P < .001), mycobacterial culture (13.0%, 6/46; P < .001), and Xpert (23.9%, 11/46; P = .001). For specificity, none of the four methods reported false-positive results in the non-TBM group. Conclusions The new method detecting cell-free M tuberculosis DNA in CSF is rapid and accurate for diagnosis of TBM and easily incorporated into regular laboratory tests.

In the era of personalized medicine, lung cancer is a typical disease which can be treated strategically based on the patient's histological and molecular diagnosis. Immunohistochemistry (IHC), fluorescence in-situ hybridization (FISH), Sanger sequencing and real-time PCR are techniques commonly used in clinical laboratories. Many patients are required to use several of the above technologies to get a complete diagnosis, which is expensive and timeconsuming. Next generation of sequencing (NGS) has the advantage to simultaneously analyze multigene mutations. The average cost for each patient is affordable if each run contains a certain number of samples. In this study, we tested a 10-gene, 32-mutation detection NGS method, which was used to test 195 samples from non-small cell lung cancer (NSCLC). Sanger sequencing and Amplification-refractory Mutation System (AMRS) PCR were employed to verify Epidermal Growth Factor Receptor (EGFR) and Anaplastic Lymphoma Kinase (ALK) results. This NGS method was partially proved to have a higher sensitivity to detect mutations with low abundance than Sanger sequencing and even ARMS PCR. Using genomic DNA to detect gene fusions may have some disadvantages to miss low abundance or large fragment fusions. As compared to using a few different technologies to analyze multigene mutations, small NGS analysis panel is a clinically applicable, efficient and affordable choice for NSCLC patients.

e21069 Background: Plasma sample has emerged as a promising surrogate sample for EGFR mutation detection in advanced non-small cell lung cancer (NSCLC). In clinical practice, whether EGFR variants in pretreatment plasma ctDNA of advanced lung cancer can predict prognosis in addition to guiding targeted therapy remains to be further explored. Methods: In this study, we retrospectively enrolled 315 NSCLC patients from Beijing Chest Hospital, 94.9% of whom were in stage IIIB-IV. EGFR gene mutation data from tissue detected by ARMS-PCR (Amoy Diagnostics, Xiamen, China) and paired plasma samples within one month of admission detected by SuperARMS-PCR (Amoy Diagnostics, Xiamen, China) were collected. The patients’ clinicopathological characteristics and treatment information were also collected for further analysis. Differences in PFS of targeted therapy between patients with positive and negative pretreatment plasma ctDNA EGFR mutation were compared. Results: EGFR-positive rate of tissue and plasma ctDNA was 65.1% (205/315) and 43.8% (138/315), respectively. L858R, 19-DEL, T790M, G719X, 20-INS, and L861Q mutations were detected in both samples. The sensitivity, specificity, PPV, NPV and concordance of ctDNA detection was 65.4%,96.4%, 97.1%, 59.9%, and 76.2%, respectively. In the study, patients’ pathological subtype was the factor affected plasma ctDNA EGFR alteration detection accuracy. The concordance in adenocarcinoma patients was obviously higher than that in non-adenocarcinoma (p = 0.02). 92 treatment naïve advanced patients with tissue EGFR mutation were received first and second-generation EGFR tyrosine kinase inhibitors (TKIs) therapies. 59.8% (55/92) patients also had EGFR mutation in plasma ctDNA, 40.2% (37/92) patients had no detectable EGFR mutations in plasma ctDNA. The median PFS in plasma ctDNA EGFR positive patients was 7.0 months versus 14.0 month in plasma ctDNA EGFR negative patients, with significantly difference (p = 0.004). Conclusions: Plasma sample could be a surrogate sample for molecular test when tissue sample was unavailable. The poor prognosis was observed in treatment naïve advanced NSCLC patients with baseline plasma EGFR mutation.

Objectives The MeltPro MTB assays for detection of resistance to antituberculosis (TB) drugs perform well in genotypic drug susceptibility testing (DST) of clinical samples, but their effectiveness with formalin-fixed, paraffin-embedded (FFPE) tissues is unknown. Methods FFPE tissues were obtained from 334 patients with TB. Susceptibility to rifampicin (RIF), isoniazid (INH), and fluoroquinolones was examined using the MeltPro MTB assays, with Xpert MTB/RIF (Xpert) and/or phenotypic DST (pDST) results as references. Samples with discordant results were analyzed by multiplex polymerase chain reaction–targeted amplicon sequencing (MTA-seq). Results With pDST as the reference, the MeltPro MTB assays sensitivity for RIF, INH, levofloxacin (LVX), and moxifloxacin (MXF) was 95.00%, 96.00%, 100%, and 100%, respectively, and the specificity was 95.15%, 95.92%, 94.69%, and 89.92%, respectively. Concordance was 99.08% between the MeltPro MTB and Xpert (κ = 0.956) for RIF and 95.12% (κ = 0.834), 95.93% (κ = 0.880), 95.12% (κ = 0.744), and 90.24% (κ = 0.367) between the MeltPro MTB and pDST for RIF, INH, LVX, and MXF, respectively. MTA-seq confirmed the discordancy between the MeltPro MTB and pDST for 26 (89.66%) of 29 samples. Conclusions The MeltPro MTB assays rapidly and efficiently predict Mycobacterium tuberculosis resistance to the main first- and second-line anti-TB drugs in FFPE tissues.

Introduction: Lung cancer is the leading cause of cancer-related morbidity and mortality in the People’s Republic of China. Targeted therapies for patients with lung cancer, which depend on accurate identification of actionable genomic alteration, have improved survival compared with previously available treatments. However, data on the types of molecular testing often used in the People’s Republic of China, and how they have changed over time, are scarce. We explored the overall landscape of molecular testing of lung cancer in mainland People’s Republic of China in the past decade. Methods: We distributed a stratified random sampling survey of molecular testing to 49 hospitals from members of the Molecular Pathology Collaboration Group of Chinese Anti-Cancer Association which was weighted by the numbers of lung cancer cases in seven different geographic regions in mainland People’s Republic of China from 2010 to 2019. The questionnaire contained four parts for all respondents. The questionnaire ascertained the use of approved in vitro diagnostic (IVD) devices published by the Center for Medical Device Evaluation, National Medical Products Administration of the People’s Republic of China. Results: A total of 226,227 NSCLC specimens were tested from 2010 to 2019 in the selected hospitals. The annual number of initiated molecular tests increased over time (p < 0.0001), with an average annual growth rate of 31.8%. A notable increase in the number of molecular tests occurred during 2014 and 2016, which coincided with the approval of the National Medical Products Administration to IVD devices. For the diagnosis of molecular subtypes, EGFR mutation testing was first conducted in year 2007, followed by ALK translocation testing in 2010 and ROS1 in 2011. For other rare genetic variations in NSCLC, BRAF mutation testing was first launched in 2012, MET exon 14 skipping mutation in 2014, HER2 exon 20 mutations in 2017, and RET translocation in 2015. A markedly uneven distribution was also observed in the geography of leading units with the largest number of leading units located in east People’s Republic of China (34.7%, 17 of 49) and the smallest number located in northwest People’s Republic of China (6.1%, 3 of 49). The growth trends we observed illustrate the progress and increasing capability of molecular testing of lung cancer achieved in mainland People’s Republic of China in the decade from 2010. Conclusions: In the decade 2010 to 2019, progress and increased capability of molecular testing of lung cancer were achieved in mainland People’s Republic of China. Further efforts should address the clinical application of next-generation sequencing technology, rare genomic aberrations, and the balance between novel genomic testing techniques and the approval of IVD products.

Background: Studies about CD8(+) FOXP3(+) T cells as a subtype of regulatory T cells (Treg cells) in non-small cell lung cancer (NSCLC) are few. Associations among the clinicopathologic factors of NSCLC and tumor-infiltrating lymphocytes (TILs) such as CD8(+) FOXP3(+) T cells, CD8(+) T cells, FOXP3(+) T cells and tumor PD-L1 expression are unclear. Methods: We retrospectively enrolled 192 patients who underwent resections for NSCLC. We used tissue microarrays (TMA) with multiplex immunofluorescence and immunohistochemistry staining to evaluate the expression of CD8, FOXP3, cytokeratin, DAPI and PD-L1. We then used Wilcoxon test, Kaplan-Meier method, and Cox hazard proportion model to analyze their relationships with clinicopathologic factors and prognosis. Results: Density of tumor CD8(+) FOXP3(+) T cells was significant by univariate analysis, and positively associated with tumor CD8(+) T cells and FOXP3(+) T cells. Density of tumor CD8(+) T cells was higher in lung adenocarcinoma (LUAD) than squamous cell carcinoma (LUSC), and was an independent prognostic factor for NSCLC. The density of tumor FOXP3(+) T cells decreased with tumor size. Tumor PD-L1 expression was higher in LUSC than LUAD. Cox hazard proportion model analysis correlated being younger than 65 years, early TNM stage, early T stage, high tumor CD8(+) T cell density, and adjuvant chemotherapy with longer overall survival. Conclusion: Infiltration of CD8(+) FOXP3(+) T cells, CD8(+) T cells, and FOXP3(+) T cells is important in non-small cell lung cancer microenvironment, and needs to be investigated more.

Supplemental material, figure_legend_-R3 for Driver gene alterations in lung adenocarcinoma: Demographic features of 2544 Chinese cases by Mingming Hu, Tongmei Zhang, Yuan Yang, Nanying Che, Jie Li, Zichen Liu and Baolan Li in The International Journal of Biological Markers

Nuclei segmentation is an essential step in DNA ploidy analysis by image-based cytometry (DNA-ICM) which is widely used in cytopathology and allows an objective measurement of DNA content (ploidy). The routine fully supervised learning-based method requires often tedious and expensive pixel-wise labels. In this paper, we propose a novel weakly supervised nuclei segmentation framework which exploits only sparsely annotated bounding boxes, without any segmentation labels. The key is to integrate the traditional image segmentation and self-training into fully supervised instance segmentation. We first leverage the traditional segmentation to generate coarse masks for each box-annotated nucleus to supervise the training of a teacher model, which is then responsible for both the refinement of these coarse masks and pseudo labels generation of unlabeled nuclei. These pseudo labels and refined masks along with the original manually annotated bounding boxes jointly supervise the training of student model. Both teacher and student share the same architecture and especially the student is initialized by the teacher. We have extensively evaluated our method with both our DNA-ICM dataset and public cytopathological dataset. Without bells and whistles, our method outperforms all existing weakly supervised entries on both datasets.

Background Mesenchymal–epithelial transition (MET) exon14 skipping mutations represent a clinically unique molecular subtype of NSCLC. The prevalence rates of MET exon 14 skipping in lung adenocarcinoma (ADC) range from 0.9% to 4.0% in Asian populations. Since some somatic variants that do not encompass the MET exon 14 splice sites might also induce MET exon 14 skipping, the RNA-based sequencing is speculated as the most accurate method for detecting exon 14 skipping. Patients and Methods A total of 951 NSCLC patients from two hospitals were enrolled in this study. MET exon14 skipping was detected using RNA-based next-generation sequencing (NGS). Also, immunohistochemistry (IHC) was performed in 405 samples simultaneously. Results The overall estimated prevalence of MET exon 14 skipping was approximately 1.8% in ADCs and 1.7% in NSCLCs. The detection rate of MET exon 14 skipping from surgical resection specimen was 2.3% in NSCLCs and 2.0% in ADCs. The MET exon 14 skipping was identified in 6.6% of EGFR/KRAS/ALK/ROS1/RET-negative ADCs. Additionally, PD-L1 was found to be highly expressed in NSCLC patients harboring MET exon 14 skipping (P

Background Pleural effusion from patients with advanced non-small cell lung cancer has been proved valuable for molecular analysis, especially when the tissue sample not available. However, simultaneous detection of multiple driver gene alterations especially the fusions is still challenging. Methods In this study, 77 patients with advanced NSCLC and pleural effusion were enrolled, 49 of whom had matched tumor tissues. Supernatants, cell sediments, and FFPE cell blocks were prepared from pleural effusion samples for detection of driver alterations by a PCR-based 9-gene mutation detection kit. Results In addition to mutations in EGFR, KRAS and HER2 detected in genomic DNA from cell sediments, FFPE cell blocks, and in cfDNA from supernatants, and fusions in ALK detected in RNA from cell sediments and FFPE cell blocks, fusions in ALK were also successfully detected in cfRNA from supernatants. Compared with matched tumor tissue, the supernatant showed the highest overall sensitivity (81.3%), with 81.5% for SNV/Indels by cfDNA and 80% for fusions by cfRNA, followed by FFPE cell blocks (71.0%) and the cell sediments (66.7%). Within the group of treatment-naïve patients or malignant cells observed in the cell sediments, supernatant showed higher overall sensitivity (89.5%, 92.3%) with both 100% for fusions. Based on the results, an optimized driver gene mutations detection procedure of pleural effusion is proposed. With the proposed procedure, an overall sensitivity 85.3% was achieved, with 82.1% for SNV/Indels and 100% for fusions. Conclusions CfDNA and cfRNA derived from pleural effusion supernatant (PES) have been successfully tested with a PCR-based multi-gene detection kit. An optimized procedure could maximize clinical value of testing pleural effusion samples and has good potential for routine clinical application. Citation Format: Xuejing Chen, Kun Li, Zichen Liu, Fei Gai, Guanshan Zhu, Shun Lu, Nanying Che. Muti-gene ARMS PCR using both cfDNA and cfRNAin the supernatant of pleural effusion achieves rapid and accuracy driver gene mutations detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 558.

Objective The clinicopathological significance of Mucin5AC (MUC5AC) in lung adenocarcinoma with mucin production is still unclear. This study aimed to explore MUC5AC expression in lung adenocarcinoma with mucin production and its correlation with histological subtypes, common driver mutations and its impact on prognosis. Methods MUC5AC and thyroid transcription factor 1 immunohistochemistry was performed on surgical samples from 90 patients with lung adenocarcinoma with mucin production. Common driver mutations including EGFR and KRAS mutations and ALK rearrangement were detected by established methods. Results MUC5AC was significantly associated with lymphovascular invasion (P = 0.023) and tumors with intra-cytoplasmic mucin (P Conclusions The clinicopathological features of non-pure mucinous subtype of lung adenocarcinoma with mucin production were distinct and should be distinguished from pure mucinous subtype. MUC5AC was associated with poor prognosis and could be a potential therapeutic target for this distinct type of lung adenocarcinoma that has few effective treatments.

Next-generation sequencing (NGS)-based assays to understand various mutations and co-occurrence of genomic alterations in non-small cell lung cancer (NSCLC) have enabled understanding of treatment impact on clinical outcomes. This retrospective study was conducted in 1353 formalin-fixed paraffin-embedded (FFPE) tissues from surgically resected, pre-TKI-treated NSCLC patients with identified gene alterations. Genomic DNA and RNA extraction was followed by NGS library preparation and sequencing using the Ion Ampliseq Colon and Lung Cancer Gene Panel V2 and the AmpliSeq RNA Lung Cancer Research Fusion Panel. A total of 2328 alterations in 25 genes were detected from the 1293 patients. DNA mutations and RNA fusions co-occurred in 27 patients with TP53 being the most common co-occurring DNA mutation (43.8%) with concurrent ALK fusions. Analysis of the 975 patients with EGFR mutations revealed that the incidence of dual EGFR L858R/T790M mutations was higher compared to EGFR 19del/T790M, and the mean allele fraction (MAF) of T790M was lower compared to 19del in dual EGFR 19del/T790M patients. NSCLC patients represented genetically heterogeneous subgroup with a high frequency of co-occurring mutations in cancer-associated pathways. This diverse mutational profile may have key clinical and research implications for understanding the variability of treatment outcome in pre-TKI-treated NSCLC population. The differences in the MAF of EGFR T790M may determine different responses to TKI therapy in patients harboring dual mutations.

In this study, we aimed to assess the performance of Xpert in fresh tissue and formaldehyde-fixed and paraffin embedded (FFPE) specimens from suspected lymphatic tuberculosis for the diagnosis of Mycobacterium tuberculosis (MTB). A total of 52 suspected lymphatic tuberculosis (TB) samples and 10 non-tuberculous lymph nodes samples were collected from outpatients. Using the comprehensive diagnostic criteria as the gold standard, the specificity in fresh and FFPE samples was 100% and the sensitivity was 82.7% and 67.3%, respectively. The majority of fresh tissue specimens had medium and low MTB content, while the low and very low MTB content were noted in 42.9% and 54.3% of FFPE tissue specimens, respectively. There were statistical differences in the MTB content between the two specimen groups detected by Xpert. Three rifampicin-resistant cases in FFPE samples were noted as rifampicin-susceptible in fresh tissue samples. Notably, all three cases with contradictory results of rpoB gene mutation test in fresh and FFPE samples had very low MTB content in FFPE samples. Fresh tissue specimens are more likely to yield Xpert results with high greater MTB content than FFPE specimens from lymphatic TB. The false detection of rpoB mutants is associated with the low bacterial content in the specimens.