The Ring Study: an international comparison of PD-L1 diagnostic assays and their interpretation in non-small cell lung cancer, head and neck squamous cell cancer and urothelial cancer.

PD-L1 immunohistochemistry has been approved as a diagnostic assay for immunotherapy. However, an international comparison across multiple cancers is lacking. This study aimed to assess the performance of PD-L1 diagnostic assays in non-small cell lung cancer (NSCLC), head and neck squamous cell cancer (HNSCC) and urothelial cancer (UC). The excisional specimens of NSCLC, HNSCC and UC were assayed by Ventana SP263 and scored at three sites in each country, including Australia, Brazil, Korea, Mexico, Russia and Taiwan. All slides were rotated to two other sites for interobserver scoring. The same cohort of NSCLC was assessed with Dako 22C3 pharmDx PD-L1 for comparison. The PD-L1 immunopositivity was scored according to the approved PD-L1 scoring algorithms which were the percentage of PD-L1-expressing tumour cell (TC) and tumour proportion score (TPS) by Ventana SP263 and Dako 22C3 staining, respectively. In NSCLC, the comparison demonstrated the comparability of the SP263 and 22C3 assays (cut-off of 1%, κ=0.71; 25%, κ=0.75; 50%, κ=0.81). The interobserver comparisons showed moderate to almost perfect agreement for SP263 in TC staining at 25% cut-off (NSCLC, κ=0.72 to 0.86; HNSCC, κ=0.60 to 0.82; UC, κ=0.68 to 0.91) and at 50% cut-off for NSCLC (κ=0.64 to 0.90). Regarding the immune cell (IC) scoring in UC, there was a lower correlation (concordance correlation coefficient=0.10 to 0.68) and poor to substantial agreements at the 1%, 5%, 10% and 25% cut-offs (κ= -0.04 to 0.76). The interchangeability of SP263 and 22C3 in NSCLC might be acceptable, especially at the 50% cut-off. In HNSCC, the performance of SP263 is comparable across five countries. In UC, there was low concordance of IC staining, which may affect treatment decisions. Overall, the study showed the reliability and reproducibility of SP263 in NSCLC, HNSCC and UC.

Deciphering Genetic Alterations of Taiwanese Patients with Pancreatic Adenocarcinoma through Targeted Sequencing.

Pancreatic adenocarcinoma (PAC) is the 8th leading cause of cancer-related deaths in Taiwan, and its incidence is increasing. The development of PAC involves successive accumulation of multiple genetic alterations. Understanding the molecular pathogenesis and heterogeneity of PAC may facilitate personalized treatment for PAC and identify therapeutic agents. We performed tumor-only next-generation sequencing (NGS) with targeted panels to explore the molecular changes underlying PAC patients in Taiwan. The Ion Torrent Oncomine Comprehensive Panel (OCP) was used for PAC metastatic lesions, and more PAC samples were sequenced with the Ion AmpliSeq Cancer Hot Spot (CHP) v2 panel. Five formalin-fixed paraffin-embedded (FFPE) metastatic PAC specimens were successfully assayed with OCP, and KRAS was the most prevalent alteration, which might contraindicate the use of anti-EGFR therapy. One PAC patient harbored a FGFR2 p. C382R mutation, which might benefit from FGFR tyrosine kinase inhibitors. An additional 38 samples assayed with CHP v2 showed 100 hotspot variants, collapsing to 54 COSMID IDs. The most frequently mutated genes were TP53, KRAS, and PDGFRA (29, 23, 10 hotspot variants), impacting 11, 23, and 10 PAC patients. Highly pathogenic variants, including COSM22413 (PDGFRA, FATHMM predicted score: 0.88), COSM520, COSM521, and COSM518 (KRAS, FATHMM predicted score: 0.98), were reported. By using NGS with targeted panels, somatic mutations with therapeutic potential were identified. The combination of clinical and genetic information is useful for decision making and precise selection of targeted medicine.

Resistance profiles of anaplastic lymphoma kinase tyrosine kinase inhibitors in advanced non-small-cell lung cancer: a multicenter study using targeted next-generation sequencing.

INTRODUCTION: Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) crizotinib, ceritinib, alectinib, brigatinib, and lorlatinib are approved for advanced non-small-cell lung cancer (NSCLC) with ALK rearrangement. However, the mechanisms of resistance remain largely unclear. METHODS: This prospective multicenter study analyzed cell-free DNA (cfDNA) and/or cancer tissues of patients with NSCLC after progression on ALK TKI(s), using targeted next-generation sequencing. Patients' clinicopathologic characteristics and treatment outcomes were analyzed. RESULTS: Overall, 88 patients were enrolled; 31 cancer tissues and 90 cfDNA samples were analyzed. Five (16%) ALK mutations (L1196M ×2, I1171T, D1203N, G1269A/F1174L) and 3 possible bypass mutations (NRAS G12V, EGFR R108K, PIK3CA E545K) were found in 32 crizotinib-resistant cancers. Four (22%) ALK mutations (G1128A, G1202R, G1269A, I1171T/E1210K) and 3 possible bypass mutations (KIT D820E, MET E1012∗, EGFR P265_C291del) were found in 18 ceritinib-resistant cancers. Four (17%) ALK mutations (G1202R ×2, W1295C, G1202R/L1196M) and 1 possible bypass mutation (EGFR P753S) were found in 24 alectinib-resistant cancers. Two (11%) ALK mutations (G1202R/G1269A ×2) and 2 possible bypass mutations (BRAF V600E, MET D1246N) were found in 18 lorlatinib-resistant cancers. In patients with simultaneous paired tissue and cfDNA samples (n = 20), mutations were identified in 9 (45%) and 6 (30%) cases, respectively; the concordance rate was 45%. CONCLUSIONS: The mechanisms of ALK TKI resistance were heterogeneous; ALK mutations were found in less than one-third of patients. Compound ALK mutations, which may confer lorlatinib resistance, may occur in crizotinib, ceritinib, and alectinib-resistant lung cancers.

Once-daily tenofovir disoproxil fumarate in treatment-naive Taiwanese patients with chronic hepatitis B and minimally raised alanine aminotransferase (TORCH-B): a multicentre, double-blind, placebo-controlled, parallel-group, randomised trial.

BACKGROUND: Antiviral therapy for patients with non-cirrhotic chronic hepatitis B and minimally raised alanine aminotransferase (ALT) is controversial. We aimed to investigate the efficacy and safety of tenofovir disoproxil fumarate in reducing the risk of disease progression in this patient population. METHODS: TORCH-B is a multicentre, double-blind, placebo-controlled, parallel-group, randomised trial done at six teaching hospitals in Taiwan that enrolled patients with chronic hepatitis B. Eligible patients were aged 25-70 years and had substantial viraemia (viral DNA >2000 IU/mL) and minimally raised serum ALT concentrations more than one-fold but less than two-fold the upper limit of normal (ULN). Exclusion criteria included liver cirrhosis and previous antiviral treatment. Eligible participants were randomly assigned (1:1), stratified by site with a fixed block size of ten, to receive either 300 mg of oral tenofovir disoproxil fumarate or placebo once daily for 3 years. The participants, investigators, research coordinators, pathologists, laboratory personnel, and staff involved in patient care or assessment were masked to treatment assignment. 0·5 mg/day of oral entecavir was added to rescue acute hepatitis flare. The coprimary outcomes were change in necroinflammation severity on the Knodell scale and change in fibrosis stage on the Ishak scale and were evaluated in the modified intention-to-treat population, which comprised all patients with paired liver biopsies. Safety was evaluated in all patients who were randomly assigned. This trial is registered at ClinicalTrials.gov, NCT01522625, and is completed. FINDINGS: From Jan 30, 2012, to Nov 10, 2015, 875 patients were screened and 160 were randomly assigned to receive either tenofovir disoproxil fumarate (n=79) or placebo (n=81). The coprimary outcomes were assessed in 146 patients (73 in each group). Liver fibrosis progressed (an increase of ≥1 stage) in 19 (26%, 95% CI 17-38) of 73 patients in the tenofovir disoproxil fumarate group and in 34 (47%, 35-59) of 73 patients in the placebo group (relative risk [RR] 0·56, 95% CI 0·35-0·88; p=0·013), whereas necroinflammation progressed (an increase of ≥2 points) in five (7%, 95% CI 2-15) patients in the tenofovir disoproxil fumarate group and in 12 (16%, 9-27) patients in the placebo group (RR 0·42, 95% CI 0·15-1·12; p=0·084). Two (3%) of 79 patients in the tenofovir disoproxil fumarate group and 13 (16%) of 81 patients in the placebo group had acute hepatitis flare requiring add-on entecavir (RR 0·16, 95% CI 0·04-0·68; p=0·013). The two groups were otherwise similar in occurrences of adverse events. No patients died. INTERPRETATION: Tenofovir disoproxil fumarate reduces the risk of progression in liver fibrosis in patients with chronic hepatitis B and minimally raised ALT, but its effect on necroinflammation is non-significant. FUNDING: The Taiwan Ministry of Science and Technology, E-Da Hospital, the Taipei Institute of Pathology, Gilead Sciences.

Liquid biopsy for detection of EGFR T790M mutation in nonsmall cell lung cancer: An experience of proficiency testing in Taiwan.

BACKGROUND: The use of liquid biopsy to detect epidermal growth factor receptor (EGFR) T790M mutation in nonsmall cell lung cancer (NSCLC) is a promising method to screen patients eligible for third-generation EGFR inhibitors. Proficiency testing (PT) programs involving liquid biopsy are currently lacking. In this study, we conducted a PT program to assess the quality assurance of liquid biopsy tests for detecting EGFR T790M mutation in molecular pathology laboratories in Taiwan. METHODS: Whole blood samples (2 mL) with various concentrations of the EGFR T790M mutation were prepared and analyzed in six participating laboratories using their clinically validated assays. RESULTS: For circulating cell-free DNA (cfDNA) isolation, three of the six participating laboratories used the cobas cfDNA Sample Preparation Kit, and three used the QIAamp Circulating Nucleic Acid Kit. For testing platforms, two of the six participating laboratories used mass spectrometry, three used the cobas EGFR mutation test, and one used a laboratory-developed test. There was 100% concordance in detection of all the given concentrations of EGFR T790M mutation between the participating laboratories and different testing platforms. The testing platforms used by all participating laboratories could successfully detect EGFR T790M mutation to an expected frequency of 1%. CONCLUSION: In this first PT program using liquid biopsy in Taiwan, local clinical laboratories were suitably equipped and proficient in the use of cfDNA to test for the EGFR T790M mutation. Establishing a routine PT system to ensure the reliability and accuracy of liquid biopsy in clinical practice in Taiwan would be helpful.

High PD-L1 expression correlates with primary resistance to EGFR-TKIs in treatment naïve advanced EGFR-mutant lung adenocarcinoma patients.

OBJECTIVES: The main objective was to investigate the relationship between Programmed cell Death-ligand 1 (PD-L1) expression levels and the frequency of primary resistance to Epidermal Growth Factor Receptor (EGFR)-Tyrosine Kinase Inhibitor (TKI) in treatment naïve advanced EGFR-mutant lung adenocarcinoma patients. MATERIALS AND METHODS: From 2012-2017, we enrolled advanced EGFR-mutant lung adenocarcinoma patients who displayed primary resistance to EGFR-TKI therapy, along with patients with disease control, and patients experiencing either stable disease or partial response to EGFR-TKI treatment. RESULTS: Sixty-six patients were enrolled as the primary resistance group, while 57 patients were included as the disease control group. Fifteen-five (22.7%) patients had a PD-L1 Tumor Proportion Score (TPS) ≧50% in the primary resistance group, with only one patient (1.8%) having that score in the disease control group (P＜0.001). Twenty (30.3%) patients had a PD-L1 ≧25% in the primary resistance group, with 2 (3.5%) patients having that level in the disease control group (P＜0.001). Thirty (45.5%) patients had a PD-L1 ≧1% in the primary resistance group, with 7 (12.3%) patients at that level in the disease control group (P = 0.001). Patients with a PD-L1≧1% displayed a higher incidence of primary resistance to EGFR-TKIs than those with a PD-L1＜1% (Odds Ratio (OR), 5.95; 95% Confidence Interval (CI), 2.35-15.05; P＜0.001). The phenomenon existed still when the cutoff value was changed to both 25% (OR, 11.96; 95% CI, 2.65-53.87; P = 0.001) and 50% (OR, 16.47; 95% CI, 2.10-129.16; P = 0.008). The estimated median Progression-free Survival (PFS) rate was 7.3 months in patients with a PD-L1＜1%, 2.1 months in patients with a PD-L1≧1%, 1.8 months in patients with a PD-L1≧25%, and 1.6 months in patients with a PD-L1≧50%. CONCLUSIONS: Treatment for advanced EGFR-mutant lung adenocarcinoma patients displaying a higher PD-L1 expression level experienced a higher frequency of primary resistance to EGFR-TKIs.

Characteristics and Predictive Value of PD-L1 Status in Real-World Non-Small Cell Lung Cancer Patients.

Immunotherapy targeting the programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) pathway has emerged as an effective treatment for lung cancer patients. It is important to evaluate the practicality of PD-L1 testing in real-world practice. A total of 211 non-small cell lung cancer patients were enrolled to detect 5 driver mutations and PD-L1 status (22C3 and SP263 assays) and to evaluate the characteristics of PD-L1 expression and its predictive value of immunotherapy. The PD-L1 positive (≥1%) and strong positive (≥50%) rate by SP263 assay was 27.0% and 12.8%. The concordance rates between 2 PD-L1 assays while using 1%, 10%, 25%, and 50% positive tumor cells as the cutoffs were 76.8%, 81.5%, 90.5%, and 94.3%, respectively. Smokers and patients without known actionable driver mutation were more likely to present strong positive PD-L1 [adjusted hazard ratio, 5.00 (95% confidence interval-CI, 1.60-15.64); P=0.006 and 3.59 (95% CI, 1.25-10.33); P=0.018, respectively]. Higher levels of smoking were associated with higher PD-L1 expressions. None of the EGFR, ALK, HER2, or BRAF-mutant nonsmokers displayed strong positive PD-L1 expression by SP263 assay. Among patients undergoing PD-1 checkpoint inhibitors therapy, high PD-L1 expression by SP263 was associated with a longer progression-free survival [adjusted hazard ratio, 0.15 (95% CI, 0.03-0.71); P=0.017]. In conclusion, our results suggest that PD-L1 status remains an important predictor of immunotherapy efficacy. The concordance between 22C3 and SP263 assays was greater at a higher cutoff level of positivity. Patients without known actionable driver mutation, along with smokers, particularly those having high smoking pack-years, were more likely to have strong PD-L1 expression.

FAM198B Is Associated with Prolonged Survival and Inhibits Metastasis in Lung Adenocarcinoma via Blockage of ERK-Mediated MMP-1 Expression.

Purpose: The comprehensive understanding of mechanisms involved in the tumor metastasis is urgently needed for discovering novel metastasis-related genes for developing effective diagnoses and treatments for lung cancer.Experimental Design: FAM198B was identified from an isogenic lung cancer metastasis cell model by microarray analysis. To investigate the clinical relevance of FAM198B, the FAM198B expression of 95 Taiwan lung adenocarcinoma patients was analyzed by quantitative real-time PCR and correlated to patients' survivals. The impact of FAM198B on cell invasion, metastasis, and tumor growth was examined by in vitro cellular assays and in vivo mouse models. In addition, the N-glycosylation-defective FAM198B mutants generated by site-directed mutagenesis were used to study protein stability and subcellular localization of FAM198B. Finally, the microarray and pathway analyses were used to elucidate the underlying mechanisms of FAM198B-mediated tumor suppression.Results: We found that the high expression of FAM198B was associated with favorable survival in Taiwan lung adenocarcinoma patients and in a lung cancer public database. Enforced expression of FAM198B inhibited cell invasion, migration, mobility, proliferation, and anchorage-independent growth, and FAM198B silencing exhibited opposite activities in vitro FAM198B also attenuated tumor growth and metastasis in vivo We further identified MMP-1 as a critical downstream target of FAM198B. The FAM198B-mediated MMP-1 downregulation was via inhibition of the phosphorylation of ERK. Interestingly deglycosylation nearly eliminated the metastasis suppression activity of FAM198B due to a decrease of protein stability.Conclusions: Our results implicate FAM198B as a potential tumor suppressor and to be a prognostic marker in lung adenocarcinoma. Clin Cancer Res; 24(4); 916-26. ©2017 AACR.

Oncogenic miR-137 contributes to cisplatin resistance via repressing CASP3 in lung adenocarcinoma.

Although targeted therapy can prolong the survival of non-small cell lung cancer (NSCLC) patients with EGFR mutations, chemotherapy still is the choice for patients with wild-type EGFR or failure in targeted therapy. However, most of the patients will eventually develop chemoresistance. Our previous study showed that miR-137 is a risky microRNA and is associated with poor prognosis in NSCLC patients. Here we investigated the role of miR-137 in cisplatin resistance in lung adenocarcinoma patients. Our data indicated that miR-137 overexpression increases the survival of lung cancer cells exposed to cisplatin and decreases cisplatin-induced apoptosis. Through computational prediction and microarray, we identified caspase-3 (CASP3) as a potential target of miR-137. Luciferase reporter and site-directed mutagenesis assays demonstrated that miR-137 downregulates CASP3 through binding to its 3'-UTR. Moreover, the endogenous CASP3 can be modulated by overexpressing or silencing miR-137 in lung adenocarcinoma cell lines regardless of EGFR status. Suppression of CASP3 by miR-137 provides cancer cells with anti-apoptotic ability, leading to cisplatin resistance. Immunohistochemistry results revealed an inverse correlation between miR-137 and CASP3 expressions in lung adenocarcinoma patients. Together, our data provide a new chemoresistance mechanism in lung adenocarcinoma and a possible target to control chemoresistance in lung adenocarcinoma patients.

The presence of clusters of plasmacytoid dendritic cells is a helpful feature for differentiating lupus panniculitis from subcutaneous panniculitis-like T-cell lymphoma.

AIMS: Both lupus panniculitis (LP) and subcutaneous panniculitis-like T-cell lymphoma (SPTCL) are characterized by subcutaneous lobular lymphocytic infiltrates, and they are sometimes difficult to differentiate. Recently, plasmacytoid dendritic cells (PDCs) were found to be present in various types of cutaneous lupus erythematosus lesions, including LP, and are supposed to play important pathogenetic roles. The aim of this study was to investigate whether PDCs are differentially present in these two diseases and can be utilized to differentiate them. Conventional histopathological features were also compared. METHODS AND RESULTS: Initial biopsies from 21 LP and 11 SPTCL patients were analysed. Our results showed that the presence of lymphoid follicles, dermal mucin deposition and lack of moderate to marked nuclear atypia or adipocyte rimming were more suggestive of LP. Several distinct patterns of fat necrosis, i.e. hyaline/lipomembranous and fibrinoid/coagulative in LP and SPTCL, respectively, were also diagnostically useful. Also, clusters of PDCs were characteristically seen in LP lesions (17/21, 81%) but not in SPTCL lesions (2/11, 18.2%). In LP lesions, but not in SPTCL lesions, the presence of epidermal interface change correlated perfectly with the presence of PDCs in the papillary dermis. CONCLUSIONS: We conclude that the presence of clusters of PDCs and certain histological features are helpful for the differential diagnosis.

Multimodality treatment and long-term follow-up of the primary pulmonary lymphoepithelioma-like carcinoma.

INTRODUCTION: Primary pulmonary lymphoepithelioma-like carcinoma (LELC) is a very rare subtype of non-small-cell lung cancer. Most cases are reported in Southeast Asia and are associated with Epstein-Barr virus infections. Because of its rare incidence, the optimal treatment and the results of long-term follow-up are not well understood. This study is an attempt to discover the multimodality treatment results of the primary pulmonary LELC. METHODS: This retrospective study enrolled 21 patients with primary pulmonary LELC treated at 2 hospitals with a multimodality approach, including surgery, chemotherapy, radiotherapy, and targeted therapy. RESULTS: The median follow-up time is 5.9 years and the median survival is 6.4 years. The median overall survival for patients with stage III and with stage IV disease is 3.4 years. In early-stage primary pulmonary LELC, surgery and adjuvant chemotherapy provided good treatment outcome. Advanced primary pulmonary LELC is relatively more chemosensitive and radiosensitive. CONCLUSION: Patients with primary pulmonary LELC showed better prognosis than those with other types of non-small-cell lung cancer and achieved longer survival under multimodality treatment. This disease character is similar to that of nasopharyngeal carcinoma. Accurate pathologic diagnosis is recommended before the treatment. For advanced diseases, platinum-based doublet chemotherapy can be considered the first-line treatment. Radiation dose should consider tumor location, and 5000 to 7000 cGy is frequently applied for pulmonary LELC.