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.

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.

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.

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.

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.

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.

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.

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.

Efficacy of liquid biopsy for disease monitoring and early prediction of tumor progression in EGFR mutation-positive non-small cell lung cancer.

15-40% of non-small cell lung cancer (NSCLC) patients harbor epidermal growth factor receptor (EGFR)-sensitizing mutations. Tyrosine kinase inhibitors (TKIs) provide significant clinical benefit in this population, yet all patients will ultimately progress. Liquid biopsy can reliably identify somatic tumor-associated EGFR mutations in plasma. This study aimed to assess the feasibility and value of the quantitative assessment of EGFR driver mutations in plasma in EGFR-mutated NSCLC patients treated with EGFR-TKIs as a tool to evaluate therapeutic response to TKIs and monitor for disease progression. The study included 136 patients with tissue biopsy-confirmed EGFR-sensitizing, mutation-positive lung adenocarcinoma with plasma collected prior to TKI treatment and at least two post-initiation TKI treatment/follow-up blood samples. Plasma samples were tested with the cobas® EGFR Mutation Test v2 (cobas EGFR Test), and semi-quantitative index (SQI) values for each identified mutation were reported by the assay software. The most common baseline EGFR mutations detected in tissue were L858R (53.7%) and exon 19 deletion (39.7%). Plasma cell-free DNA analysis detected EGFR mutations in 74% of the baseline samples. Objective response rate by RECIST 1.1 was achieved in 72% of patients, while 93% had a molecular response (defined as disappearance of the EGFR mutation from plasma). 83% of patients had molecular progression (MP; 1.5X SQI increase or new T790M mutation), and 82% who had a clinical response had clinical progression. On average, MP occurred 42 days prior to clinical progression. Patients who progressed while on first-line TKI showed MP of the original EGFR-sensitizing mutations prior to the emergence of a T790M mutation, which was detected in 27% of the EGFR plasma-positive patients. Longitudinal monitoring of EGFR mutational load in plasma is feasible and can predict both response and clinical progression in EGFR-mutated NSCLC patients treated with EGFR-TKIs, as well as detect treatment-emergent EGFR mutations.

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.

Characterization of Collapsin Response Mediator Protein 2 in Colorectal Cancer Progression in Subjects with Diabetic Comorbidity.

BACKGROUND: Common demographic risk factors are identified in colorectal cancer (CRC) and type 2 diabetes mellitus (DM), nevertheless, the molecular link and mechanism for CRC-DM comorbidity remain elusive. Dysregulated glycogen synthase kinase-3 beta under metabolic imbalance is suggested to accelerate CRC pathogenesis/progression via regulating collpasin response mediator protein-2 (CRMP2). Accordingly, roles of CRMP2 in CRC and CRC-DM patients were investigated for elucidating the molecular convergence of CRC and DM. METHODS: CRMP2 profile in tumor tissues from CRC and CRC-DM patients was investigated to explore the link between CRC and DM etiology. Meanwhile, molecular mechanism of glucose to regulate CRMP2 profile and CRC characteristics was examined in vitro and in vivo. RESULTS: CRMP2 was significantly lower in tumor lesions and associated with advanced tumor stage in CRC-DM patients. Physiological hyperglycemia suppressed CRMP2 expression/activity and augmented malignant characteristics of CRC cells. Hyperglycemia promotes actin de-polymerization, cytoskeleton flexibility and cell proliferation/metastasis by downregulating CRMP2 profile and thus contributes to CRC disease progression. CONCLUSIONS: This study uncovers molecular evidence to substantiate and elucidate the link between CRC and T2DM, as well as characterizing the roles of CRMP2 in CRC-DM. Accordingly, altered metabolic adaptations are promising targets for anti-diabetic and cancer strategies.

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.

Whole-exome Sequencing of Epstein-Barr Virus-associated Pulmonary Carcinoma With Low Lymphocytic Infiltration Shows Molecular Features Similar to Those of Classic Pulmonary Lymphoepithelioma-like Carcinoma: Evidence to Support Grouping Together as One Disease Entity.

Pulmonary lymphoepithelioma-like carcinoma (LELC) is a distinct type of Epstein-Barr virus (EBV)-associated non-small cell carcinoma characterized by a syncytial growth pattern with heavy lymphocytic infiltration. We recently identified a group of non-small cell carcinomas, which are also associated with EBV but lack significant lymphocytic infiltration. These EBV-associated pulmonary carcinomas with low lymphocytic infiltration morphologically resemble nonkeratinizing squamous cell carcinoma, but their patient characteristics are more similar to those of LELC, including female sex and nonsmoking status. To clarify the relationships between these disease entities, in this study, we explored the molecular characteristics of the EBV-associated carcinomas with low lymphocytic infiltration using whole-exome sequencing and compared their molecular profiles with those of classic LELC and pulmonary squamous cell carcinoma. We demonstrate that the molecular characteristics of EBV-associated carcinomas with low lymphocytic infiltration are highly similar to those of classic LELC. Both show low tumor mutational burden, lack of commonly mutated driver genes in other types of non-small cell lung cancer, similar mutational signature involving APOBEC-related mutations, and enrichment of CD274 (programmed death-ligand 1) amplification. These molecular characteristics are very different from those of pulmonary squamous cell carcinoma. The unique patient demographics and molecular characteristics shared by EBV-associated carcinomas with low lymphocytic infiltration and classic LELC suggest that these tumors represent one single disease entity defined by EBV association. This study supports the proposal for the usage of the term "EBV-associated pulmonary carcinoma" to encompass the entire morphologic spectrum of this distinct EBV-associated disease entity.

Primary Signet Ring Cell/Histiocytoid Carcinoma of the Eyelid: Somatic Mutations in CDH1 and Other Clinically Actionable Mutations Imply Early Use of Targeted Agents.

Primary signet ring cell/histiocytoid carcinoma of the eyelid is a rare ocular malignancy and its diagnosis is often delayed. This neoplasm presents as an insidious, diffusely infiltrative mass in the periocular area that later infiltrates the orbit. An exenteration is usually indicated; however, nearly one-third of patients develop local recurrence or metastasis. Morphologically, it resembles signet ring cell carcinoma of the stomach and breast, raising the possibility of mutations in CDH1, the gene encoding E-cadherin. To determine whether primary signet ring cell/histiocytoid carcinoma harbors the CDH1 mutation or other actionable mutations, we analyzed the tumor tissue via next-generation sequencing. We identified only one case of primary signet ring cell carcinoma of the eyelid with adequate DNA quality for sequencing from the pathological archive during the period 2000 to 2020. A comprehensive evaluation including histopathology, immunohistochemistry, and next-generation sequencing assay was performed on tumor tissue. Immunohistochemically, the tumor exhibited E-cadherin membranous staining with the aberrant cytoplasmic staining of ß-catenin. Using next-generation sequencing, we demonstrated the mutation in the CDH1 gene. In addition, other clinically actionable mutations including ERBB2 and PIK3CA were also detected. The alterations in other actionable genes indicate a need for larger studies to evaluate the pathogenesis and potential therapies for primary signet ring cell/histiocytoid carcinoma of the eyelid.

Programmed death-ligand 1 (PD-L1)/thyroid transcription factor-1 double immunohistochemical staining facilitates scoring of tumor PD-L1 expression in cytopathology specimens from lung adenocarcinoma patients.

BACKGROUND: Immune checkpoint inhibitor therapy has revolutionized lung adenocarcinoma therapy. Treatment with antibodies against the immune checkpoint molecules programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) can induce a durable response in a subset of patients. Immunohistochemistry characterization of tumor PD-L1 expression using either a histopathology specimen or a cytopathology specimen has been shown to correlate with treatment response. However, the current practice relies on pathologists' visual estimation of tumor PD-L1 staining, which can be variable in certain conditions. Highlighting tumor cells via double immunostaining with PD-L1 and thyroid transcription factor-1 (TTF-1) may improve estimation accuracy. METHODS: We performed PD-L1 single staining and PD-L1/TTF-1 double staining in 42 pairs of cytopathology and histopathology specimens from lung adenocarcinoma patients. An experienced pathologist visually estimated PD-L1 expression in each case and placed tumor PD-L1 expression into 1 of 3 categories: <1%, 1%-49%, or ≥50%. A medical technologist also performed estimations of the same cases based on a count of 200 tumor cells, and the results were compared. RESULTS: PD-L1/TTF-1 double immunohistochemistry could better identify the PD-L1-positive tumor cells in cytopathology specimens compared with PD-L1 single staining. The concordance of PD-L1 expression categorization between the pathologist's visual estimation and the medical technologist's counting was increased by double staining in cytopathology specimens (Cohen's weighted kappa: single stain, 0.784; double stain, 0.880). Double staining reduced possible error in the pathologist's visual estimation of PD-L1 expression from 9.5% to 4.8%. The benefit was not observed in histopathology specimens. CONCLUSION: A simple PD-L1/TTF-1 double immunohistochemistry technique can be applied successfully to cytopathology specimens in better identifying patients who can potentially benefit from immune checkpoint blockade treatment.

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.

Establishing diagnostic algorithms for SARS-CoV-2 nucleic acid testing in clinical practice.

BACKGROUND: Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic. Our laboratory initially used a two-step molecular assay, first reported by Corman et al, for SARS-CoV-2 identification (the Taiwan Center for Disease Control [T-CDC] method). As rapid and accurate diagnosis of COVID-19 is required to control the spread of this infectious disease, the current study evaluated three commercially available assays, including the TaqPath COVID-19 Combo kit, the cobas SARS-CoV-2 test, and the Rendu 2019-nCoV Assay kit, to establish diagnostic algorithms for clinical laboratories. METHODS: A total of 790 clinical specimens, including nasopharyngeal swabs, throat swabs, sputum, saliva, stool, endotracheal aspirate, and serum were obtained from patients who were suspected or already confirmed to have COVID-19 at the Taipei Veterans General Hospital from February to May 2020. These specimens were tested for SARS-CoV-2 using the different assays and the performance variance between the assays was analyzed. RESULTS: Of the assays we evaluated, the T-CDC method and the TaqPath COVID-19 Combo kit require lots of hands-on practical laboratory work, while the cobas SARS-CoV-2 test and the Rendu 2019-nCoV Assay kit are fully automated detection systems. The T-CDC method and the TaqPath COVID-19 Combo kit showed similar detection sensitivity; however, the T-CDC method frequently delivered false-positive signals for envelope (E) and/or RNA-dependent RNA polymerase (RdRP) gene detection, thus increasing the risk of reporting false-positive results. A manual test-based testing strategy combining the T-CDC method and the TaqPath COVID-19 Combo kit was developed, which demonstrated excellent concordance rates (>99%) with the cobas and Rendu automatic systems. There were a few cases showing discrepant results, which may be due to the varied detection sensitivities as well as targets among the different platforms. Moreover, the concordance rate between the cobas and Rendu assays was 100%. CONCLUSION: Based on our evaluation, two SARS-CoV-2 diagnostic algorithms, one focusing on the manual assays and the other on the automatic platforms, were proposed. Our results provide valuable information that allows clinical laboratories to implement optimal diagnostic strategies for SARS-CoV-2 testing based on their clinical needs, such as test volume, turn-around time, and staff/resource limitations.

The clinical manifestations and interval changes of reverse-transcriptase quantitative polymerase chain reactions among different specimens of coronavirus disease 2019 patients.

BACKGROUND: Since December 2019, a number of cases and deaths due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic have been reported worldwide. In spite of clinical manifestations similar to the SARS-CoV epidemic in 2003, affected organs and severity are yet to be defined. Moreover, viral load alterations and viral shielding among different specimens remained scarce. Therefore, clarifying clinical presentations and correlations among viral loads, disease severity, and viral shielding of SARS-CoV-2 infection is crucial in the disease prevention. METHODS: The clinical courses of SARS-CoV-2 cases were presented through Gantt charts. Laboratory examinations and reverse-transcriptase quantitative polymerase chain reactions (RT-qPCR) among different specimens were tested periodically. Cycle thresholds (CT) were recorded and presented as viral loads. RESULTS: From March 2020 to April 2020, 4 SARS-CoV-2 cases were presented, of which, cases 1 and 2 manifested the symptoms severer than cases 3 and 4, along with higher serum lactate dehydrogenase levels and graded for lymphocytopenia. Case 4 initially exhibited anosmia but recovered within a short period. Curves of the CT of all the cases, except case 2, concaved upward after prescribing hydroxychloroquine (HCQ) and azithromycin. Except for case 4, the CT in most stool specimens remained undetectable; however, none of the cases presented gastrointestinal symptoms. Surprisingly, the CT values of the saliva specimens were inconsistent with those of the nasopharyngeal swabs and sputum. CONCLUSION: SARS-CoV-2 manifests various symptoms. Sudden onset of central nervous system symptoms should be considered. The timing of HCQ and azithromycin administration might be a key factor in the viral load reduction. Positive prediction values of RT-qPCR of different specimens should be tested carefully to prevent false-negative results.