Comprehensive Genomic and Transcriptomic Analysis of Sclerosing Pneumocytoma.

Sclerosing pneumocytoma is a rare and distinct lung neoplasm whose histogenesis and molecular alterations are the subject of ongoing research. Our recent study revealed that AKT1 internal tandem duplications (ITD), point mutations, and short indels were present in almost all tested sclerosing pneumocytomas, suggesting that AKT1 mutations are a major driving oncogenic event in this tumor. Although the pathogenic role of AKT1 point mutations is well established, the significance of AKT1 ITD in oncogenesis remains largely unexplored. We conducted comprehensive genomic and transcriptomic analyses of sclerosing pneumocytoma to address this knowledge gap. RNA-sequencing data from 23 tumors and whole-exome sequencing data from 44 tumors were used to obtain insights into their genetic and transcriptomic profiles. Our analysis revealed a high degree of genetic and transcriptomic similarity between tumors carrying AKT1 ITD and those with AKT1 point mutations. Mutational signature analysis revealed COSMIC signatures 1 and 5 as the prevailing signatures of sclerosing pneumocytoma, associated with the spontaneous deamination of 5-methylcytosine and an unknown etiology, respectively. RNA-sequencing data analysis revealed that the sclerosing pneumocytoma gene expression profile is characterized by activation of the PI3K/AKT/mTOR pathway, which exhibits significant similarity between tumors harboring AKT1 ITD and those with AKT1 point mutations. Notably, an upregulation of SOX9, a transcription factor known for its involvement in fetal lung development, was observed in sclerosing pneumocytoma. Specifically, SOX9 expression was prominent in the round cell component, whereas it was relatively lower in the surface cell component of the tumor. To the best of our knowledge, this is the first comprehensive investigation of the genomic and transcriptomic characteristics of sclerosing pneumocytoma. Results of the present study provide insights into the molecular attributes of sclerosing pneumocytoma and a basis for future studies of this enigmatic tumor.

Constant p.L424H Mutation in GTF2I in Micronodular Thymomas With Lymphoid Stroma: Evidence Supporting Close Relationship With Type A and AB Thymomas.

Micronodular thymoma with lymphoid stroma is a rare thymic neoplasm characterized by discrete nodules of epithelial tumor cells separated by abundant lymphoid stroma. The genetic features of micronodular thymoma with lymphoid stroma remain largely unexplored. Owing to the interference of abundant intratumoral, nonneoplastic lymphoid cells, a highly sensitive approach is necessary to study genetic changes in these tumors. In this study, we used a highly sensitive next-generation sequencing assay using the molecular barcoding Ion AmpliSeq HD technology to study the most commonly mutated genes in thymomas, including GTF2I, HRAS, NRAS, KRAS, and TP53. A total of 12 cases of micronodular thymomas with lymphoid stroma were tested, and 2 cases also had areas of type A thymoma in their tumor bed. Two micronodular thymic carcinomas with lymphoid stroma, a histological mimic of micronodular thymoma, were also included for comparison. Recurrent p.L424H mutations in GTF2I were found in all the cases of micronodular thymoma with lymphoid stroma but not in the cases of micronodular thymic carcinomas. In addition, 3 cases of micronodular thymoma with lymphoid stroma also had concomitant HRAS and/or KRAS mutations. Our study showed that p.L424H mutations in GTF2I is a constant genetic feature of micronodular thymoma with lymphoid stroma. This finding strongly suggests that micronodular thymoma with lymphoid stroma is closely related to type A and AB thymomas because they all share p.L424H mutations in GTF2I.

Genomic Profiling With Large-Scale Next-Generation Sequencing Panels Distinguishes Separate Primary Lung Adenocarcinomas From Intrapulmonary Metastases.

The distinction between different separate primary lung cancers (SPLCs) and intrapulmonary metastases (IPMs) is a challenging but clinically significant issue. Histopathology-based classification is the current practice; however, it is subjective and affected by interobserver variability. Recently, next-generation sequencing (NGS) panels have been used in lung cancer diagnostics. This study aimed to investigate the value of large-scale NGS panels for distinguishing between SPLCs and IPMs. A total of 32 patients with 69 lung adenocarcinomas were included. Comprehensive histopathologic assessments of multiple pulmonary adenocarcinomas were performed independently by 3 pathologists. The consensus of histopathologic classification was determined by a majority vote. Genomic analysis was performed using an amplicon-based large-scale NGS panel, targeting single-nucleotide variants and short insertions and deletions in 409 genes. Tumor pairs were classified as SPLCs or IPMs according to a predefined molecular classification algorithm. Using NGS and our molecular classification algorithm, 97.6% of the tumor pairs can be unambiguously classified as SPLCs or IPMs. The molecular classification was predictive of postoperative clinical outcomes in terms of overall survival (P = .015) and recurrence-free interval (P = .0012). There was a moderate interobserver agreement regarding histopathologic classification (κ = 0.524 at the tumor pair level). The concordance between histopathologic and molecular classification was 100% in cases where pathologists reached a complete agreement but only 53.3% where they did not. This study showed that large-scale NGS panels are a powerful modality that can help distinguish SPLCs from IPMs in patients with multiple lung adenocarcinomas and objectively provide accurate risk stratification.

Dynamic Assessment of Tissue and Plasma EGFR-Activating and T790M Mutations with Droplet Digital PCR Assays for Monitoring Response and Resistance in Non-Small Cell Lung Cancers Treated with EGFR-TKIs.

Assessing tumor EGFR mutation status is necessary for the proper management of patients with advanced non-small cell lung cancer (NSCLC). We evaluated the impact of dynamic analyses of the plasma and tissue EGFR mutation using ultra-sensitive droplet digital PCR (ddPCR) assays to manage NSCLC patients treated with EGFR tyrosine kinase inhibitors (EGFR-TKIs). Paired tumor tissues and plasma samples from 137 EGFR-mutated lung adenocarcinoma patients prior to the first-line EGFR-TKIs treatment (at baseline) and at disease progression were subjected to EGFR mutation analysis using ddPCR, together with the analyses of the clinicopathological characteristics and treatment outcomes. Patients with EGFR-activating mutations detected in baseline plasma were associated with bone metastasis (p = 0.002) and had shorter progression-free survival (12.9 vs. 17.7 months, p = 0.02) and overall survival (24.0 vs. 39.4 months, p = 0.02) compared to those without. Pre-treatment EGFR T790M mutation found in baseline tumor tissues of 28 patients (20.4%; 28/137) was significantly associated with brain metastasis (p = 0.005) and a shorter brain metastasis-free survival (p = 0.001). The presence of EGFR T790M mutations in baseline tumor tissues did not correlate with the emergence of acquired EGFR T790M mutations detected at progression. At disease progression, acquired EGFR T790M mutations were detected in 26.6% (21/79) of the plasma samples and 42.9% (15/35) of the rebiopsy tissues, with a concordance rate of 71.4% (25/35). The dynamic monitoring of tissue and plasma EGFR mutation status at baseline and progression using ddPCR has a clinical impact on the evaluation of EGFR-TKIs treatment efficacy and patient outcomes, as well as the emergence of resistance in NSCLC.

Diffuse Intratumoral Stromal Inflammation in Ovarian Clear Cell Carcinoma is Associated With Loss of Mismatch Repair Protein and High PD-L1 Expression.

Ovarian clear cell carcinoma (OCCC) is an aggressive chemotherapy-resistant cancer with limited treatment options, and some OCCCs have mismatch repair (MMR) deficiency (MMRD). Emerging evidence has revealed that various cancers with MMRD are susceptible to anti-programmed death-1/programmed death ligand-1 (anti-PD-1/PD-L1) immunotherapy, and certain histologic features are associated with MMRD. However, few studies have addressed this in OCCC. We reviewed 76 OCCCs for tumor-associated inflammation (intratumoral stromal inflammation and peritumoral lymphocytes) and performed immunohistochemistry for 4 MMR proteins and PD-L1. MMR-deficient OCCCs were analyzed for microsatellite instability (MSI), and those with MLH1 loss were tested for MLH1 promoter methylation. No patients fulfilled the Amsterdam II criteria for the diagnosis of Lynch syndrome. Four (5.3%) tumors showed diffuse intratumoral stromal inflammation obliterating the tumor-stroma interfaces, and none had peritumoral lymphoid aggregates. MMRD was found in 2 (2.6%) tumors; one had MLH1/PMS2 loss (MSI-high and MLH1 promoter methylation was detected) and the other had MSH2/MSH6 loss (MSI-low). Twenty (26.3%) tumors showed tumoral PD-L1 expression ≥1%. Both MMR-deficient tumors showed diffuse intratumoral stromal inflammation and tumoral PD-L1 expression ≥50%. Three of the 4 (75%) tumors with diffuse intratumoral stromal inflammation also showed tumoral PD-L1 expression ≥50%. None of the tumors without diffuse intratumoral stromal inflammation showed MMRD (P=0.021) or tumoral PD-L1 expression ≥50% (P=0.0001). We identified a strong correlation among diffuse intratumoral stromal inflammation, MMRD, and high tumoral PD-L1 expression in a small but significant subset of OCCCs. Histologic evaluation can facilitate patient selection for subsequent anti-PD-1/PD-L1 immunotherapy.

Gene Expression Profile in Primary Tumor Is Associated with Brain-Tropism of Metastasis from Lung Adenocarcinoma.

Lung adenocarcinoma has a strong propensity to metastasize to the brain. The brain metastases are difficult to treat and can cause significant morbidity and mortality. Identifying patients with increased risk of developing brain metastasis can assist medical decision-making, facilitating a closer surveillance or justifying a preventive treatment. We analyzed 27 lung adenocarcinoma patients who received a primary lung tumor resection and developed metastases within 5 years after the surgery. Among these patients, 16 developed brain metastases and 11 developed non-brain metastases only. We performed targeted DNA sequencing, RNA sequencing and immunohistochemistry to characterize the difference between the primary tumors. We also compared our findings to the published data of brain-tropic and non-brain-tropic lung adenocarcinoma cell lines. The results demonstrated that the targeted tumor DNA sequencing did not reveal a significant difference between the groups, but the RNA sequencing identified 390 differentially expressed genes. A gene expression signature including CDKN2A could identify 100% of brain-metastasizing tumors with a 91% specificity. However, when compared to the differentially expressed genes between brain-tropic and non-brain-tropic lung cancer cell lines, a different set of genes was shared between the patient data and the cell line data, which include many genes implicated in the cancer-glia/neuron interaction. Our findings indicate that it is possible to identify lung adenocarcinoma patients at the highest risk for brain metastasis by analyzing the primary tumor. Further investigation is required to elucidate the mechanism behind these associations and to identify potential treatment targets.

AKT1 internal tandem duplications and point mutations are the genetic hallmarks of sclerosing pneumocytoma.

Sclerosing pneumocytoma is a unique benign neoplasm of the lungs. The molecular alterations in sclerosing pneumocytoma are not well understood. In a previous whole-exome sequencing study, recurrent AKT1 point mutation was observed in about half of the cases of sclerosing pneumocytoma. However, in the remaining half, cancer-related mutations have still not been identified. In this study, we first analyzed the raw sequence data from the previous whole-exome sequencing study (PRJNA297066 cohort). Using Genomon-ITDetector, a special software for detection of internal tandem duplications, we identified recurrent internal tandem duplications in the AKT1 gene in 22 of the 44 tumor samples (50%). All the cases positive for AKT1 internal tandem duplications lacked AKT1 point mutations. Next, we performed targeted next-generation sequencing in an independent cohort of sclerosing pneumocytoma from our hospital (VGH-TPE cohort), and again identified recurrent AKT1 internal tandem duplications in 20 of the 40 (50%) tumor samples analyzed. The internal tandem duplications resulted in duplications of 7 to 16 amino acids in a narrow region of the Pleckstrin homology domain of the AKT1 protein. This region contains the interaction interface between the Pleckstrin homology and kinase domains, which is known to play a critical role in the activation of the AKT1 protein. Moreover, we found that AKT1 internal tandem duplications were mutually exclusive of other forms of AKT1 mutations, including point mutations and short indels. Taking all forms of AKT1 mutations together, we detected AKT1 mutations in almost all the sclerosing pneumocytomas in our study (PRJNA297066 cohort: 41 out of 44 cases, 93%; VGH-TPE cohort: 40 out of 40 cases, 100%). Our results suggest that AKT1 mutation is the genetic hallmark of sclerosing pneumocytoma. These results would help in better understanding of the pathogenesis of sclerosing pneumocytoma.

Low-Dose Nicotine Activates EGFR Signaling via α5-nAChR and Promotes Lung Adenocarcinoma Progression.

Nicotine in tobacco smoke is considered carcinogenic in several malignancies including lung cancer. The high incidence of lung adenocarcinoma (LAC) in non-smokers, however, remains unexplained. Although LAC has long been less associated with smoking behavior based on previous epidemiological correlation studies, the effect of environmental smoke contributing to low-dose nicotine exposure in non-smoking population could be underestimated. Here we provide experimental evidence of how low-dose nicotine promotes LAC growth in vitro and in vivo. Screening of nicotinic acetylcholine receptor subunits in lung cancer cell lines demonstrated a particularly high expression level of nicotinic acetylcholine receptor subunit α5 (α 5-nAChR) in LAC cell lines. Clinical specimen analysis revealed up-regulation of α 5-nAChR in LAC tumor tissues compared to non-tumor counterparts. In LAC cell lines α 5-nAChR interacts with epidermal growth factor receptor (EGFR), positively regulates EGFR pathway, enhances the expression of epithelial-mesenchymal transition markers, and is essential for low-dose nicotine-induced EGFR phosphorylation. Functionally, low-dose nicotine requires α 5-nAChR to enhance cell migration, invasion, and proliferation. Knockdown of α 5-nAChR inhibits the xenograft tumor growth of LAC. Clinical analysis indicated that high level of tumor α 5-nAChR is correlated with poor survival rates of LAC patients, particularly in those expressing wild-type EGFR. Our data identified α 5-nAChR as an essential mediator for low-dose nicotine-dependent LAC progression possibly through signaling crosstalk with EGFR, supporting the involvement of environmental smoke in tumor progression in LAC patients.

Molecular typing for detection of high-risk human papillomavirus is a useful tool for distinguishing primary bladder carcinoma from secondary involvement of uterine cervical carcinoma in the urinary bladder.

AIMS: For patients with carcinoma of the urinary bladder and uterine cervix, distinguishing between metastasis and a second primary carcinoma has significant prognostic and therapeutic implications. The aim of this study was to investigate the prevalence of high-risk human papillomavirus (HR-HPV) in cervical carcinoma with secondary involvement of the bladder and primary bladder carcinoma, in order to explore whether the detection of HR-HPV could help to differentiate between the two. METHODS AND RESULTS: Paired bladder and cervix carcinoma specimens from 37 patients with cervical carcinoma with bladder involvement, four patients with bladder carcinoma with uterine cervical involvement and two patients with double primaries were studied with quantitative multiplex polymerase chain reaction and chromogenic in-situ hybridization. Three hundred and seventy-five bladder carcinomas and 220 cervical carcinomas were analysed as controls. All cases of cervical carcinoma with bladder involvement showed concordant HR-HPV-positive patterns. The four cases of bladder carcinoma with uterine involvement were negative for HR-HPV. HR-HPV was detected in the cervical carcinoma but not in the bladder carcinoma of the patients with double primaries. HR-HPV was detected in 91.9% of cervical carcinomas but in none of the bladder carcinomas in the control group. CONCLUSIONS: Molecular typing for HR-HPV detection is useful to distinguish bladder carcinoma from secondary involvement of cervical carcinoma.

MGMT promoter methylation in non-neoplastic brain.

O(6)-methylguanine-DNA-methyltransferase (MGMT) is mainly regulated by cytosine-guanine island promoter methylation that is believed to occur only in neoplastic tissue. The present study was undertaken to investigate whether methylation occurs also in non-neoplastic brains by collecting 45 non-neoplastic brains from autopsies and 56 lobectomy specimens from epileptic surgeries. The promoter methylation status of MGMT was studied by methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ), while protein expression was studied by immunohistochemical stain (IHC). The methylation rates, as determined by MSP and PSQ, were 3.0 % (3/101) and 2.9 % (2/69), respectively. Of note, no case had positive result concomitantly from both MSP and PSQ (3 were MSP+/PSQ- and 2 were MSP-/PSQ+), and all the positive samples were further confirmed by cloning and Sanger sequencing. All the methylated cases, except for those having indeterminate IHC results from autopsy specimens, revealed no loss of MGMT protein expression and similar staining pattern to that of the unmethylated cases. In conclusion, the current study demonstrated that MGMT promoter methylation could occur in a low percentage of non-neoplastic brains but did not affect the status of protein expression, which could be regarded as a normal variation in non-neoplastic brains.

Prognosis of glioblastoma with faint MGMT methylation-specific PCR product.

Methylation-specific polymerase chain reaction (MSP) for the promoter methylation status of O(6)-methylguanine-DNA-methyltranferase (MGMT) gene theoretically provides a positive or negative result. However, the faint MSP product is difficult to interpret. The aim of this study was to evaluate the significance of faint MSP product in glioblastoma (GBM). Critical concentrations of methylated control DNA, i.e., 100, 1, 0.5 and 0 % were run parallel with 116 newly diagnosed GBMs in order to standardize the interpretation and to distinguish positive (+), equivocal (±), and negative (-; unmethylated) results. Cases with the faint MSP product and its intensity between those of 1 and 0.5 % DNA controls were considered equivocal (±). MGMT methylation quantifications were also determined by quantitative real-time MSP (qMSP) and pyrosequencing (PSQ), and protein expression was detected by immunohistochemistry. There were significant correlations between MSP and all the aforementioned studies. The concordance rates between the MSP+ and qMSP+ cases, as well as the MSP- and qMSP- cases were 100 %, and the MSP± cases comprised 76.5 % of qMSP+ cases and 23.5 % of qMSP- cases. PSQ study showed that heterogeneous methylation was more frequently encountered in the MSP± cases. Multivariate analyses disclosed that although the overall survival of the MSP± cases was indistinct from that of the MSP+ cases, its progression free survival was significantly worse and was indistinct from that of the MSP- cases. In conclusion, GBMs with faint MGMT MSP products should be distinguished from MSP+ cases as their behaviors were different.

Tonsil surface epithelium is ideal for monitoring Ki-67 immunohistochemical staining.

AIMS: To identify an easily obtainable non-neoplastic tissue that can be used as control material for monitoring optimal Ki-67 immunohistochemical staining. METHODS AND RESULTS: Various tissues, including tonsil (60), uterine cervix (31), breast skin (26), oesophagus (15), stomach (15), small intestine (15) and colon (16), were studied in the search for ideal control tissue. Tonsil surface epithelium is superior to other tissues because it displayed an easily recognized Ki-67 staining pattern including high positive (parabasal layer), low positive (intermediate layer) and negative (basal and superficial layers) zones. Moderate to weak staining of the majority of the intermediate cells could serve as a threshold for positive staining. Of the variables potentially affecting staining results that were tested, the pretreatment solution for antigen retrieval had the greatest impact, of which pH 9 EDTA was far better than pH 6 citrate solution. CONCLUSIONS: Tonsil surface epithelium is a useful control for monitoring Ki-67 staining. Achieving optimal staining results could minimize variations in Ki-67 index due to differences in the staining methods used by different laboratories.

Molecular diagnostic algorithm for epidermal growth factor receptor mutation detection in Asian lung adenocarcinomas: comprehensive analyses of 445 Taiwanese patients with immunohistochemistry, PCR-direct sequencing and Scorpion/ARMS methods.

BACKGROUND AND OBJECTIVE: Therapeutic responses of lung adenocarcinoma patients to tyrosine kinase inhibitors (TKIs) of epidermal growth factor receptor (EGFR) are closely associated with activating mutations within the EGFR tyrosine kinase domain. Screening activating EGFR mutations prior to selection for therapeutic strategy has been considered extremely valuable for clinical management of lung adenocarcinoma patients in Asian countries including Taiwan, where the EGFR mutation rate is higher than in the rest of the world. Currently there is no consensus on the method of choice to assess EGFR mutations in tumour tissue. METHODS: We enrolled 445 lung adenocarcinoma patients for analysis of tumour EGFR mutations using polymerase chain reaction (PCR)-direct sequencing, scorpion/amplified refractory mutation system (ARMS) technology and immunohistochemistry with mutation-specific antibodies. RESULTS: Two hundred forty-five patients (245/445; 55%) were found to harbour activating EGFR mutations using PCR-direct sequencing method, with a majority of patients (233/245; 95%) carrying exon 19 deletion or p.L858R point mutations. One hundred three of 200 patients were negative for EGFR mutations from PCR-direct sequencing were further analysed using Scorpion/ARMS technology. Up to 30% of the PCR-direct sequencing negative patients turned out to be positive in the Scorpion/ARMS EGFR mutation tests. For immunohistochemistry analysis of EGFR mutations, the p.E746_A750del specific antibody showed a sensitivity of 57% and a specificity of 100% for exon 19 deletions while the p.L858R point mutation specific antibody showed a sensitivity of 68% and a specificity of 95%. CONCLUSIONS: Based on this study, we proposed an algorithm for comprehensive and efficient testing of EGFR mutations on lung adenocarcinoma patients in Asia.

Prognosticators of osimertinib treatment outcomes in patients with EGFR-mutant non-small cell lung cancer and leptomeningeal metastasis.

PURPOSE: Leptomeningeal metastasis (LM) is a serious complication of non-small cell lung cancer (NSCLC), particularly in patients with EGFR mutations. In this study, we investigated the survival outcomes of patients with EGFR-mutant NSCLC who have developed LM and explored the factors associated with their survival. METHODS: From April 2018 to November 2021, patients with EGFR-mutant NSCLC who underwent cerebrospinal fluid (CSF) sampling under the clinical suspicion of LM were enrolled. The patients' clinicodemographic characteristics, treatment history including whole-brain radiation therapy (WBRT), overall survival (OS), and intracranial progression-free survival (icPFS) were measured. EGFR mutations in cell-free tumor DNA (ctDNA) of CSF, including T790M mutation, were analyzed. RESULTS: We enrolled 62 patients with NSCLC. The median time form diagnosis to LM was 23.1 months and 16 (25.8%) patients had history of prior third-generation EGFR-TKI use. EGFR mutation in CSF ctDNA was detected in 53 patients (85.5%); of them, 10 (16.1%) had T790M mutation. The patients' icPFS and OS after osimertinib were 6.43 and 9.37 months, respectively, and were comparable among patients with different sensitive EGFR mutations, indicating that EGFR mutation status did not affect osimertinib efficacy. Patients who received WBRT after LM had numerically higher icPFS and OS compared to those without. Multivariate analysis revealed that lack of prior exposure to third-generation EGFR-TKI was associated with better OS. CONCLUSIONS: Osimertinib is effective in patients with EGFR-mutant NSCLC who developed LM and prior third-generation EGFR-TKI use was associated with poor survival in these patients. The role of WBRT warrants further investigation.

Efficacy of different platforms in detecting EGFR mutations using cerebrospinal fluid cell-free DNA from non-small-cell lung cancer patients with leptomeningeal metastases.

BACKGROUND: Cell-free tumor DNA (ctDNA) obtained through liquid biopsy is useful for the molecular analysis of advanced non-small-cell lung cancer (NSCLC). Few studies have directly compared analysis platforms in terms of their diagnostic performance in analyzing ctDNA obtained from the cerebrospinal fluid (CSF) of patients with leptomeningeal metastasis (LM). METHODS: We prospectively analyzed patients with epidermal growth factor receptor (EGFR)-mutant NSCLC who were subjected to CSF analysis for suspected LM. To detect EGFR mutations, CSF ctDNA was analyzed using the cobas EGFR Mutation Test and droplet digital polymerase chain reaction (ddPCR). CSF samples from osimertinib-refractory patients with LM were also subjected to next-generation sequencing (NGS). RESULTS: Significantly higher rates of valid results (95.1% vs. 78%, respectively, p = 0.04) and EGFR common mutation detection (94.3% vs. 77.1%, respectively, p = 0.047) were obtained through ddPCR than through the cobas EGFR Mutation Test. The sensitivities of ddPCR and cobas were 94.3% and 75.6%, respectively. The concordance rate for EGFR mutation detection through ddPCR and the cobas EGFR Mutation Test was 75.6% and that for EGFR mutation detection in CSF and plasma ctDNA was 28.1%. In osimertinib-resistant CSF samples, all original EGFR mutations were detected through NGS. MET amplification and CCDC6-RET fusion were demonstrated in one patient each (9.1%). CONCLUSIONS: The cobas EGFR Mutation Test, ddPCR, and NGS appear to be feasible methods for analyzing CSF ctDNA in patients with NSCLC and LM. In addition, NGS may provide comprehensive information regarding the mechanisms underlying osimertinib resistance.

Assuring safety of fecal microbiota transplantation in the COVID-19 era: A single-center experience.

Background and Aim: Fecal microbiota transplantation (FMT) is used to treat recurrent or refractory Clostridioides difficile infection (CDI). In the past, screening of fecal donors required surveillance of personal behavior, medical history, and diseases that could be transmitted by the blood or fecal-oral route. In addition, the exclusion of multidrug-resistant organisms (MDROs) has been recommended since 2018. This task has become more complicated in the era of the coronavirus disease-2019 (COVID-19) pandemic. To prevent fecal transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is crucial to commence screening for SARS-CoV-2, alongside other traditional tests. Our aim was to investigate whether hidden carriers of SARS-CoV-2 were enrolled for stool donation, and the status of the presence or incidence of MDRO during fecal donation in Taiwan. Methods: Fecal products collected from March 2019 to December 2022 were tested for MDRO and nucleic acid amplification tests for SARS-CoV-2 using the pooling method. The period of fecal product collection crossed the time before and during the COVID pandemic in Taiwan. Results: A total of 151 fecal samples were collected. The fecal products were tested using polymerase chain reaction (PCR) to detect SARS-CoV-2. The results were negative for all stocks. This was similar to the results of MDRO testing. The safety of FMT products has been guaranteed during the pandemic. Conclusion: Our FMT center produced MDRO-free and COVID-19-free products before and during the COVID-19 outbreak in Taiwan. Our protocol was effective for ensuring the safety of FMT products.

Proliferative ability of circulating tumor cells is a prognostic factor in Early-Stage lung adenocarcinoma.

INTRODUCTION: Circulating tumor cells (CTCs) and their proliferative ability in lung adenocarcinoma (LUAD) were not well-investigated. We developed a protocol combining an efficient viable CTC isolation and in-vitro cultivation for the CTC enumeration and proliferation to evaluate their clinical significance. METHOD: The peripheral blood of 124 treatment-naïve LUAD patients were processed by a CTC isolation microfluidics, DS platform, followed by in-vitro cultivation. LUAD-specific CTCs were defined by immunostaining of DAPI+/CD45-/(TTF1/CK7)+ and were enumerated upon isolation and after 7-day cultivation. The CTC proliferative ability was evaluated by both the cultured number and the culture index, a ratio of cultured CTC number to the initial CTC number in 2 mL of blood. RESULT: All but two LUAD patients (98.4%) were detected with at least one CTC per 2 mL of blood. Initial CTC numbers did not correlate with metastasis (75 ± 126 for non-metastatic, 87 ± 113 for metastatic groups; P = 0.203). In contrast, both the cultured CTC number (mean: 28, 104, and 185 in stage 0/I, II/III, and IV; P < 0.001), and the culture index (mean: 1.1, 1.7 and 9.3 in stage 0/I, II/III, and IV; P = 0.043) were significantly correlated with the stages. Overall survival analysis within the non-metastatic group (N = 53) showed poor prognosis for patients with elevated cultured counts (cutoff ≥ 30; P = 0.027). CONCLUSION: We implemented a CTC assay in clinical LUAD patients with a high detection rate and cultivation capability. Cultured CTC count and proliferative ability, rather than the crude CTC numbers, highly associated with cancer prognosis.

Deep exploration of immune function in EGFR wild-type and mutated lung adenocarcinomas by gene expression profiling: role of TRAIL-R2 (TNFRSF10B) in patient treatment and outcome.

The tumor microenvironment is important in the initiation and progression of lung adenocarcinoma (LUAD). In this study, we aim to analyze the expression profile of immune-related genes in LUADs, examine the differential expression of immune-related genes in epidermal growth factor receptor (EGFR) wild-type and mutant LUADs, and the clinicopathologic significance of these differentially expressed genes. We used the NanoString PanCancer Immune Profiling Panel to examine 34 cases of LUADs (18 EGFR wild-type, 16 EGFR mutant). In EGFR wild-type LUADs, the macrophage and neutrophil signatures are significantly higher, and significantly higher expression of chemokines, interleukins, leukocyte, macrophage, natural killer cell, pathogen defense, Tumor necrosis factor superfamily, and transporter function signatures are also observed. TNFRSF10B mRNA was preferentially expressed in EGFR wild-type LUADs (P = 6.15e-6, adjusted P = .0244). Immunohistochemical staining for TRAIL-R2 (encoded by TNFRSF10B) on 134 tissue microarray LUAD cases demonstrated strong, moderate, and weak staining in 75 (56.0%), 46 (34.3%), and 13 (9.7%) cases, respectively. Strong TRAIL-R2 expression was significantly associated with poor overall survival (OS) in all stages and EGFR wild-type LUADs, but not in EGFR-mutant tumors. Furthermore, strong TRAIL-R2 expression (P = .004) was an independent risk factor for poor OS. In summary, TNFRSF10B mRNA revealed significantly higher expression in EGFR wild-type LUADs, and strong TRAIL-R2 expression predicts an unfavorable prognosis for these tumors. These patients may benefit from additional treatment with TRAIL-R2-targeted therapies.

Programmed Death-Ligand 1 Expression in Lymphovascular Tumor Emboli in Lung Cancer.

Introduction: Programmed death-ligand 1 (PD-L1) expression determined by immunohistochemistry is the most widely used biomarker for predicting response to immune checkpoint inhibitors. The characteristics of PD-L1 expression in tumor cells inside lymphovascular spaces are largely unknown. Although PD-L1 expression in circulating tumor cells within vascular spaces had been studied, results were conflicting due to lack of standardized PD-L1 expression assessment. Methods: We investigated PD-L1 expression in lung cancer primary tumor tissue, lymphovascular tumor emboli, and lymph node metastasis using the standard PD-L1 immunohistochemistry 22C3 pharmDx assay. PD-L1 expression was scored in the primary tumor, lymphovascular emboli, and lymph node metastasis by a pathologist using the tumor proportion score (TPS). Results: We collected and analyzed surgical specimens from 36 patients with lung cancer with lymph node metastasis. In the primary tumor, 64% of cases were PD-L1 negative (TPS < 1%), 25% were PD-L1 low (TPS 1%-49%), and 11% were PD-L1 high (TPS ≥ 50%). In contrast, in lymphovascular tumor emboli, 89% of cases were PD-L1 negative, 11% were PD-L1 low, and none were PD-L1 high. In lymph node metastases, 72% of cases were PD-L1 negative, 17% were PD-L1 low, and 11% were PD-L1 high. Conclusions: We observed a significant decrease of PD-L1 expression in lymphovascular tumor emboli compared with that in primary tumors (p = 0.002). Whether such differences are related to intrinsic tumor cell heterogeneity or extrinsic factors such as the microenvironment warrants further investigation.

Real-world Evaluation of a Sample Pooling Strategy for Large-Scale Rapid COVID-19 Testing.

BACKGROUND: The worldwide outbreak of COVID-19 has become a public health crisis of unprecedented proportions. The fast spread of emerging variants increases the needs of rapid diagnostic and screening testing. Sample pooling efficiently expands the testing capacity under limited resources. OBJECTIVES: We evaluated the performance of sample pooling on the Point-of-Care (POC) Liat® and cobas® 6800 systems and provided real-world experiences for implementing these systems in large-scale screenings. METHODS: Positive nasopharyngeal (NP) specimens with Ct values < 25, 25∼30 or > 30 were tested individually and in pools to optimize the POC Liat® and cobas® 6800 systems, which were then implemented in community screenings. RESULTS: The 5-sample pooling strategy did not affect the positive detection rates on Liat® or cobas® 6800 in samples with Ct values <25 or 25∼30. However, in samples with low viral loads (Ct values >30), five-sample pooling has a higher positive detection rate on POC Liat® (20/20; 100%), compared to cobas® 6800 (9/20; 45%). Five-sample pooled on POC Liat® and two-sample pooled on cobas® 6800 appear to be appropriate for SARS-CoV-2 detection. By implementing the pooling strategies in two large-scale community screenings, 7,606 NP specimens was tested within 36 h; the average turn-around time was 4.8 h for cobas® 6800 and 1.3 h for POC Liat®. Eight positive specimens (0.11%; 8/7,606) were identified, with Ct values ranging from 18.85 to 37.68. CONCLUSION: The performance of sample pooling on POC Liat® was demonstrated to be an effective, accurate, and economical approach for large-scale community screenings for COVID-19.