Proteomic profiling of tumor microenvironment and prognosis risk prediction in stage I lung adenocarcinoma.

OBJECTIVES: With the increasing popularity of CT screening, more cases of early-stage lung cancer are being diagnosed. However, 24.5% of stage I non-small-cell lung cancer (NSCLC) patients still experience treatment failure post-surgery. Biomarkers to predict lung cancer patients at high risk of recurrence are needed. MATERIALS AND METHODS: We collected protein mass spectrometry data from the Taiwan Lung Cancer Moonshot Project and performed bioinformatics analysis on proteins with differential expressions between tumor and adjacent normal tissues in 74 stage I lung adenocarcinoma (LUAD) cases, aiming to explore the tumor microenvironment related prognostic biomarkers. Findings were further validated in 6 external cohorts. RESULTS: The analysis of differentially expressed proteins revealed that the most enriched categories of diseases and biological functions were cellular movement, immune cell trafficking, and cancer. Utilizing proteomic profiling of the tumor microenvironment, we identified five prognostic biomarkers (ADAM10, MIF, TEK, THBS2, MAOA). We then developed a risk score model, which independently predicted recurrence-free survival and overall survival in stage I LUAD. Patients with high risk scores experienced worse recurrence-free survival (adjusted hazard ratio = 8.28, p < 0.001) and overall survival (adjusted hazard ratio = 6.88, p = 0.013). Findings had been also validated in the external cohorts. CONCLUSION: The risk score model derived from proteomic profiling of tumor microenvironment can be used to predict recurrence risk and prognosis of stage I LUAD.

Terminal deoxynucleotidyl transferase expression in different subtypes of childhood B-cell acute lymphoblastic leukemia.

The lack of expression of terminal deoxynucleotidyl transferase (TdT) is frequently associated with KMT2A-rearranged subtype of pediatric acute lymphoblastic leukemia (ALL). However, this association has not been investigated extensively in the Asian population. A retrospective analysis of TdT expression in pediatric B-cell ALL (B-ALL) was performed in patients treated using the Taiwan Pediatric Oncology Group (TPOG) ALL 2002 and 2013 protocols. Among the 331 patients with B-ALL, 12 patients showed TdT negativity at initial diagnosis. Among these, eight patients showed KMT2A rearrangement (66.7%). Other patients showing negative TdT expression had ETV6::RUNX1, MEF2D-rearranged, and other B-ALL subtypes. However, in the context of KMT2A-rearranged B-ALL (n = 20), only eight patients showed TdT negativity. The 5-year event-free survival and overall survival of patients with and without TdT expression were 83.8% versus 46.8% (P <0.001) and 86.3% versus 55.4% (P = 0.004), respectively. Moreover, several aberrant markers, such as CD2, CD56, CD7, and CD117, were rarely expressed in the B-ALL samples, and if expressed, they were enriched in specific genetic subtypes. The results of this study indicate that immunophenotypic features are correlated with specific genetic subtypes of childhood B-ALL.

Low-dose CT screening among never-smokers with or without a family history of lung cancer in Taiwan: a prospective cohort study.

BACKGROUND: In Taiwan, lung cancers occur predominantly in never-smokers, of whom nearly 60% have stage IV disease at diagnosis. We aimed to assess the efficacy of low-dose CT (LDCT) screening among never-smokers, who had other risk factors for lung cancer. METHODS: The Taiwan Lung Cancer Screening in Never-Smoker Trial (TALENT) was a nationwide, multicentre, prospective cohort study done at 17 tertiary medical centres in Taiwan. Eligible individuals had negative chest radiography, were aged 55-75 years, had never smoked or had smoked fewer than 10 pack-years and stopped smoking for more than 15 years (self-report), and had one of the following risk factors: a family history of lung cancer; passive smoke exposure; a history of pulmonary tuberculosis or chronic obstructive pulmonary disorders; a cooking index of 110 or higher; or cooking without using ventilation. Eligible participants underwent LDCT at baseline, then annually for 2 years, and then every 2 years up to 6 years thereafter, with follow-up assessments at each LDCT scan (ie, total follow-up of 8 years). A positive scan was defined as a solid or part-solid nodule larger than 6 mm in mean diameter or a pure ground-glass nodule larger than 5 mm in mean diameter. Lung cancer was diagnosed through invasive procedures, such as image-guided aspiration or biopsy or surgery. Here, we report the results of 1-year follow-up after LDCT screening at baseline. The primary outcome was lung cancer detection rate. The p value for detection rates was estimated by the χ2 test. Univariate and multivariable logistic regression analyses were used to assess the association between lung cancer incidence and each risk factor. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of LDCT screening were also assessed. This study is registered with ClinicalTrials.gov, NCT02611570, and is ongoing. FINDINGS: Between Dec 1, 2015, and July 31, 2019, 12 011 participants (8868 females) were enrolled, of whom 6009 had a family history of lung cancer. Among 12 011 LDCT scans done at baseline, 2094 (17·4%) were positive. Lung cancer was diagnosed in 318 (2·6%) of 12 011 participants (257 [2·1%] participants had invasive lung cancer and 61 [0·5%] had adenocarcinomas in situ). 317 of 318 participants had adenocarcinoma and 246 (77·4%) of 318 had stage I disease. The prevalence of invasive lung cancer was higher among participants with a family history of lung cancer (161 [2·7%] of 6009 participants) than in those without (96 [1·6%] of 6002 participants). In participants with a family history of lung cancer, the detection rate of invasive lung cancer increased significantly with age, whereas the detection rate of adenocarcinoma in situ remained stable. In multivariable analysis, female sex, a family history of lung cancer, and age older than 60 years were associated with an increased risk of lung cancer and invasive lung cancer; passive smoke exposure, cumulative exposure to cooking, cooking without ventilation, and a previous history of chronic lung diseases were not associated with lung cancer, even after stratification by family history of lung cancer. In participants with a family history of lung cancer, the higher the number of first-degree relatives affected, the higher the risk of lung cancer; participants whose mother or sibling had lung cancer were also at an increased risk. A positive LDCT scan had 92·1% sensitivity, 84·6% specificity, a PPV of 14·0%, and a NPV of 99·7% for lung cancer diagnosis. INTERPRETATION: TALENT had a high invasive lung cancer detection rate at 1 year after baseline LDCT scan. Overdiagnosis could have occurred, especially in participants diagnosed with adenocarcinoma in situ. In individuals who do not smoke, our findings suggest that a family history of lung cancer among first-degree relatives significantly increases the risk of lung cancer as well as the rate of invasive lung cancer with increasing age. Further research on risk factors for lung cancer in this population is needed, particularly for those without a family history of lung cancer. FUNDING: Ministry of Health and Welfare of Taiwan.

Whole exome sequencing and MicroRNA profiling of lung adenocarcinoma identified risk prediction features for tumors at stage I and its substages.

OBJECTIVES: About 20% of stage I lung adenocarcinoma (LUAD) patients suffer a relapse after surgical resection. While finer substages have been defined and refined in the AJCC staging system, clinical investigations on the tumor molecular landscape are lacking. MATERIALS AND METHODS: We performed whole exome sequencing, DNA copy number and microRNA profiling on paired tumor-normal samples from a cohort of 113 treatment-naïve stage I Taiwanese LUAD patients. We searched for molecular features associated with relapse-free survival (RFS) of stage I or its substages and validated the findings with an independent Caucasian LUAD cohort. RESULTS: We found sixteen nonsynonymous mutations harbored at EGFR, KRAS, TP53, CTNNB1 and six other genes associated with poor RFS in a dose-dependent manner via variant allele fraction (VAF). An index, maxVAF, was constructed to quantify the overall mutation load from genes other than EGFR. High maxVAF scores discriminated a small group of high-risk LUAD at stage I (median RFS: 4.5 versus 69.5 months; HR = 10.5, 95% CI = 4.22-26.12, P < 0.001). At the substage level, higher risk was found for patients with high maxVAF or high miR-31; IA (median RFS: 32.1 versus 122.8 months, P = 0.005) and IB (median RFS: 7.1 versus 26.2, P = 0.049). MicroRNAs, miR-182, miR-183 and miR-196a were found correlated with EGFR mutation and poor RFS in stage IB patients. CONCLUSION: Distinctive features of somatic gene mutation and microRNA expression of stage I LUAD are characterized to complement the survival prognosis by substaging. The findings open up more options for precision management of stage I LUAD patients.

Stage Shift Improves Lung Cancer Survival: Real-World Evidence.

INTRODUCTION: Lung cancer is the global leading cause of cancer death. Taiwan initiated several health policies including smoking cessation, precision therapy, and low-dose computed tomography (LDCT) screening in 1997. We aimed to investigate the effect of public policies on lung cancer survival. METHODS: We retrieved the nationwide cancer registry from the Ministry of Health and Welfare to evaluate the smoking prevalence and lung cancer incidence and mortality from 1994 to 2020. We also conducted a retrospective analysis of clinical characteristics and survival on 17,298 patients with lung cancer from 2006 to 2019 using the National Taiwan University Hospital database. RESULTS: Taiwan initiated an anti-smoking campaign in 1997, reimbursed tyrosine kinase inhibitors since 2004, and conducted an LDCT screening trial in 2015. Lung cancer incidence keeps rising but the annual percent change in mortality rate gradually decreased from 0.41% to -2.41%. The National Taiwan University Hospital data revealed that the 5-year survival substantially improved from 22.1% in 2006 to 2011 to 54.9% in 2015 to 2020. Improvement was observed in all stages, especially late stages (stage III: from 17.2% to 35.2%; stage IV: from 7.9% to 16.5%). Furthermore, a remarkable shift in cancer stage was observed (stage 0, I, and IIincreased from 19.3% to 62.8%, and stage III and IV decreased from 70.9% to 33.8%). The prominent improvement in survival was primarily driven by the stage shift from advanced to localized, potentially curable disease. CONCLUSIONS: This real-world evidence suggested an association between improved survival and LDCT screening and the diagnostic shift from late to early-stage of lung cancer, highlighting the importance of early detection for lung cancer control.

Low-Dose Computed Tomography Screening in Relatives With a Family History of Lung Cancer.

INTRODUCTION: The role of a family history of lung cancer (LCFH) in screening using low-dose computed tomography (LDCT) has not been prospectively investigated with long-term follow-up. METHODS: A multicenter prospective study with up to three rounds of annual LDCT screening was conducted to determine the detection rate of lung cancer (LC) in asymptomatic first- or second-degree relatives of LCFH. RESULTS: From 2007 to 2011, there were 1102 participants enrolled, including 805 and 297 from simplex and multiplex families (MFs), respectively (54.2% women and 70.0% never-smokers). The last follow-up date was May 5, 2021. The overall LC detection rate was 4.5% (50 of 1102). The detection rate in MF was 9.4% (19 of 202) and 4.4% (4 of 91) in never-smokers and in those who smoked, respectively. The corresponding rates for simplex families were 3.7% (21 of 569) and 2.7% (6 of 223), respectively. Of these, 68.0% and 22.0% of cases with stage I and IV diseases, respectively. LC diagnoses within a 3-year interval from the initial screening tend to be younger, have a higher detection rate, and have stage I disease; thereafter, more stage III-IV disease and 66.7% (16 of 24) with negative or semipositive nodules in initial computed tomography scans. Within the 6-year interval, only maternal (modified rate ratio = 4.46, 95% confidence interval: 2.32-8.56) or maternal relative history of LC (modified rate ratio = 5.41, 95% confidence interval: 2.84-10.30) increased the risk of LC. CONCLUSIONS: LCFH is a risk factor for LC and is increased with MF history, among never-smokers, younger adults, and those with maternal relatives with LC. Randomized controlled trials are needed to confirm the mortality benefit of LDCT screening in those with LCFH.

Regulation of dendritic cell maturation in osimertinib-treated lung adenocarcinoma patients.

Osimertinib (OSI), a third-generation tyrosine kinase inhibitor (TKI), efficiently benefits lung adenocarcinoma (LUAD) patients with epidermal growth factor receptor (EGFR) mutations. However, combined OSI and immune checkpoint inhibitor in EGFR-mutant patients increases the incidence of interstitial lung disease (ILD), although the mechanism is unknown. Here, we investigated the interaction between dendritic cells (DCs), a potential critical player in ILD, and OSI. Seventeen LUAD patients received TKI therapy, and only the OSI therapy group (N = 10) showed a significant increase in CD40 and CD83 on immature DCs (iDCs), and an elevated trend for both markers on mature DCs (mDCs) during short- and long-term OSI therapy. Our results indicated that OSI therapy may potentially activate DC functions, which might increase the potential immune toxicity when combined with onco-immunotherapy.

Small-molecule PIK-93 modulates the tumor microenvironment to improve immune checkpoint blockade response.

Immune checkpoint inhibitors (ICIs) targeting PD-L1 immunotherapy are state-of-the-art treatments for advanced non-small cell lung cancer (NSCLC). However, the treatment response of certain patients with NSCLC is unsatisfactory because of an unfavorable tumor microenvironment (TME) and poor permeability of antibody-based ICIs. In this study, we aimed to discover small-molecule drugs that can modulate the TME to enhance ICI treatment efficacy in NSCLC in vitro and in vivo. We identified a PD-L1 protein-modulating small molecule, PIK-93, using a cell-based global protein stability (GPS) screening system. PIK-93 mediated PD-L1 ubiquitination by enhancing the PD-L1-Cullin-4A interaction. PIK-93 reduced PD-L1 levels on M1 macrophages and enhanced M1 antitumor cytotoxicity. Combined PIK-93 and anti-PD-L1 antibody treatment enhanced T cell activation, inhibited tumor growth, and increased tumor-infiltrating lymphocyte (TIL) recruitment in syngeneic and human peripheral blood mononuclear cell (PBMC) line-derived xenograft mouse models. PIK-93 facilitates a treatment-favorable TME when combined with anti-PD-L1 antibodies, thereby enhancing PD-1/PD-L1 blockade cancer immunotherapy.

PM2.5 promotes lung cancer progression through activation of the AhR-TMPRSS2-IL18 pathway.

Particulate matter 2.5 (PM2.5) is a risk factor for lung cancer. In this study, we investigated the molecular mechanisms of PM2.5 exposure on lung cancer progression. We found that short-term exposure to PM2.5 for 24 h activated the EGFR pathway in lung cancer cells (EGFR wild-type and mutant), while long-term exposure of lung cancer cells to PM2.5 for 90 days persistently promoted EGFR activation, cell proliferation, anchorage-independent growth, and tumor growth in a xenograft mouse model in EGFR-driven H1975 cancer cells. We showed that PM2.5 activated AhR to translocate into the nucleus and promoted EGFR activation. AhR further interacted with the promoter of TMPRSS2, thereby upregulating TMPRSS2 and IL18 expression to promote cancer progression. Depletion of TMPRSS2 in lung cancer cells suppressed anchorage-independent growth and xenograft tumor growth in mice. The expression levels of TMPRSS2 were found to correlate with nuclear AhR expression and with cancer stage in lung cancer patient tissue. Long-term exposure to PM2.5 could promote tumor progression in lung cancer through activation of EGFR and AhR to enhance the TMPRSS2-IL18 pathway.

Allele-specific polymerase chain reaction can determine the diplotype of NUDT15 variants in patients with childhood acute lymphoblastic Leukemia.

Mercaptopurine intolerance is an adverse effect of mercaptopurine administration in pediatric patients with acute lymphoblastic leukemia (ALL). NUDT15 variants have emerged as major determinants of mercaptopurine intolerance, especially in the Asian population. Two variants, c.55_56insGAGTCG in exon 1 and c.415C > T in exon 3, were commonly detected in the same allele, named NUDT15*1/*2. Although rare, compound heterozygous mutations also occur, with the two variants on different alleles (NUDT15*3/*6), which may confer tolerance to considerably lesser mercaptopurine dosage. Sanger sequencing or pyrosequencing can determine the NUDT15 variants but not the phase. Here, we designed an allele-specific PCR (AS-PCR) with locked nucleic acid-modified primers. A cohort of 63 patients harboring heterozygous c.55_56insGAGTCG and c.415C > T NUDT15 variations was selected for haplotyping using AS-PCR. Of the 63 patients, 60 harbored the NUDT15*1/*2 variant and three harbored compound heterozygous mutations, including two NUDT15*3/*6 and one NUDT15*2/*7 variants. These findings suggest that AS-PCR can determine NUDT15 diplotype and identify patients with compound heterozygous NUDT15 variants, which may enable precise genetic diagnosis of NUDT15. Nevertheless, a larger clinical trial is required to understand the clinical significance of NUDT15*3/*6 in pediatric patients with ALL because of its low incidence rate and challenges in detecting this variant.

A microRNA signature for clinical outcomes of pediatric ALL patients treated with TPOG protocols.

MicroRNA (miRNA) expression is reportedly associated with clinical outcomes in childhood acute lymphoblastic leukemia (ALL). Here, we aimed at investigating whether miRNA expression is associated with clinical outcomes in pediatric ALL patients treated with the Taiwan Pediatric Oncology Group (TPOG) protocols. The expression of 397 miRNAs was measured using stem-loop quantitative real-time polymerase chain reaction miRNA arrays in 60 pediatric ALL patients treated with TPOG-ALL-93 or TPOG-ALL-97 VHR (very high-risk) protocols. In order to identify prognosis-related miRNAs, original cohort was randomly split into the training and testing cohort in a 2:1 ratio, and univariate Cox proportional hazards regression was applied to identify associations between event-free survival (EFS) and expressions of miRNAs. Four prognosis-related miRNAs were selected and validated in another independent cohort composed of 103 patients treated with the TPOG-ALL-2002 protocol. Risk score, including the impact of four prognosis-related miRNAs, was calculated for each patients, followed by grouping patients into the high or low risk-score groups. Irrespective of the training, testing, or validation cohort, risk-score group was significantly associated with EFS and overall survival (OS). Risk-score group combining with clinical characteristics including the age onset (≥10 years), white blood cell counts (≥100 × 109/L), cell type (T- or B-cell), sex, and risk groups of the treatment protocols were used as predictors of EFS using the multivariate Cox proportional hazards regression. Results showed that the risk-score group was the strongest predictor. In the validation cohort, hazard ratios (HRs) of the risk-score group were 7.06 (95% CI=1.93-25.84, p-value =0.003) and 14.03 (95% CI=3.34-59.04, p-value =0.003) for EFS and OS, respectively. High risk-score group had higher risk of having poor prognosis and risk of death than that in the low risk group. Accuracy of the prediction model for 5-year EFS could reach 0.76. For the prediction of 5-year OS, accuracy was 0.75. In conclusion, a miRNA signature was associated with clinical outcomes in childhood ALL patients treated with TPOG protocols and might be a suitable prognostic biomarker.

Screening of early-staged colorectal neoplasia by clonal hematopoiesis-based liquid biopsy and machine-learning.

Liquid biopsy test has a better uptake for colorectal cancer (CRC) screening. However, suboptimal detection of early-staged colorectal neoplasia (CRN) limits its application. Here, we established an early-staged CRN blood test using error-corrected sequencing by comparing clonal hematopoiesis (CH) of 63 CRN patients and that of 32 controls. We identified 1,446 variants and classified the uniqueness in CRN patients. There was no significance difference in the amount of variant between CRNs and controls, but the uniqueness of variants with defective DNA mismatch repair-related mutational signature was addressed from peripheral blood in early-staged CRN patients. By machine learning approach, the early-staged CRNs was discriminated from controls with an AUC of 0.959 and an accuracy of 0.937 (95% CI, 0.863 to 0.968). The CRN predictive model was further validated by additional 20 CRNs and 10 controls and showed the accuracy, sensitivity, specificity, positive prediction value (PPV) and negative prediction value (NPV) of 0.933 (95% CI: 0.779 to 0.992), 0.95, 0.90, 0.95 and 0.90, respectively. In summary, we develop a CH-based liquid biopsy test with machine learning approach, which not only increase screening uptake but also improve the detection rate of early-staged CRN.

Integration of immunohistochemistry, RNA sequencing, and multiplex ligation-dependent probe amplification for molecular classification of pediatric medulloblastoma.

BACKGROUND: Medulloblastoma (MB) is commonly classified into four molecular groups, that is, WNT, SHH, group 3, and group 4, for prognostic and therapeutic purposes. METHODS: Here we applied immunohistochemistry (IHC) and RNA sequencing (RNA-seq) for the molecular classification of MB, and utilized multiplex ligation-dependent probe amplification (MLPA) to determine chromosomal alterations and specific gene amplifications. RESULTS: We retrospectively enrolled 37 pediatric MB patients. Twenty-three had genomic material available for gene/RNA analysis. For IHC, ß-catenin, GAB1, and YAP were the biomarkers to segregate MB into three subgroups, WNT (1/23), SHH (5/23), and non-WNT/non-SHH (17/23). However, four cases (17.3%) were found to be misclassified after analysis by RNA-seq. The result of MLPA revealed two group 3 tumors carrying MYC amplification, and three SHH tumors harboring MYCN amplification. While IHC provided rapid subgroup stratification, it might result in incorrect subgrouping. Thus, validation of the IHC result with genomic data analysis by RNA-seq or other tools would be preferred. In addition, MLPA can detect important genetic alterations and is helpful for the identifications of high-risk patients. CONCLUSIONS: Our study revealed that integration of these diagnostic tools can provide a precise and timely classification of MB, optimizing an individualized, risk-directed postoperative adjuvant therapy for these patients. This workflow can be applied in a countrywide fashion to guide future clinical trials for patients with MB.

Inulin supplementation prior to mild traumatic brain injury mitigates gut dysbiosis, and brain vascular and white matter deficits in mice.

Introduction: Mild traumatic brain injury (mTBI) has been shown to negatively alter bacterial diversity and composition within the gut, known as dysbiosis, in rodents and humans. These changes cause secondary consequences systemically through decreased bacterial metabolites such as short chain fatty acids (SCFAs) which play a role in inflammation and metabolism. The goal of the study was to identify if giving prebiotic inulin prior to closed head injury (CHI) could mitigate gut dysbiosis, increase SCFAs, and improve recovery outcomes, including protecting cerebral blood flow (CBF) and white matter integrity (WMI) in young mice. Methods: We fed mice at 2 months of age with either inulin or control diet (with cellulose as fiber source) for two months before the CHI and continued till the end of the study. We analyzed gut microbiome composition and diversity, determined SCFAs levels, and measured CBF and WMI using MRI. We compared the results with Naïve and Sham-injury mice at 24 hours, 1.5 months, and 3-4 months post-injury. Results: We found that both CHI and Sham mice had time-dependent changes in gut composition and diversity after surgery. Inulin significantly reduced the abundance of pathobiont bacteria, such as E. coli, Desulfovibrio spp and Pseudomonas aeruginosa, in Sham and CHI mice compared to mice fed with control diet. On the other hand, inulin increased SCFAs-producing bacteria, such as Bifidobacterium spp and Lactobacillus spp, increased levels of SCFAs, including butyrate and propionate, and significantly altered beta diversity as early as 24 hours post-injury, which lasted up to 3-4 months post-injury. The mitigation of dysbiosis is associated with protection of WMI in fimbria, internal and external capsule, and CBF in the right hippocampus of CHI mice, suggesting protection of memory and cognitive functions. Discussion: The results indicate that giving inulin prior to CHI could promote recovery outcome through gut microbiome modulation. As inulin, microbiome analysis, and MRI are readily to be used in humans, the findings from the study may pave a way for a cost-effective, accessible intervention for those at risk of sustaining a head injury, such as military personnel or athletes in contact sports.

The Prognostic Significance of PD1 and PDL1 Gene Expression in Lung Cancer: A Meta-Analysis.

BACKGROUND: Immune checkpoint blockade therapy represents an extraordinary advance in lung cancer treatment. It is important to determine the expression of immune checkpoint genes, such as programmed cell death 1 (PD1) and programmed cell death-ligand 1 (PDL1), to develop immunotherapeutic strategies. The aim of this study was to explore the association between PD1 and PDL1 gene expression and prognoses and outcomes in lung cancer. METHODS: This meta-analysis analyzed 1,251 patients from eight different microarray gene expression datasets and were evaluated for their prognostic implications and verified using another independent research. RESULTS: The mean expression levels of PDL1 in adenocarcinoma (AD) and squamous cell carcinoma (SC) were significantly higher in patients who died than in patients who did not. There was a trend toward incremental increases in PD1 and PDL1 expression significantly decreasing the risk of relapse and death among AD patients (HR = 0.69; 95% CI = 0.53 ~ 0.91; HR = 0.68; 95% CI = 0.54 ~ 0.84, respectively) and SC patients (HR = 0.53; 95% CI = 0.32 ~ 0.89; HR = 0.78; 95% CI = 0.57 ~ 1.00 respectively), as early-stage patients in this study were more likely to have high expression of both PD1 and PDL1 than late-stage patients (P-trend < 0.05). In contrast, late-stage SC patients expressing one or more of the genes at a high level had a significantly elevated risk of relapse (HR = 1.51; 95% CI = 1.07 ~ 2.11) and death (HR = 1.41; 95% CI = 1.08 ~ 1.84). This result was consistent with the validation data set. CONCLUSION: These findings indicate that high expression of PD1 and PDL1 is associated with superior outcome in early-stage lung cancer but an adverse outcome in late-stage lung cancer. The expression levels of PD1 and PDL1 individually or jointly are potential prognostic factors for predicting patient outcomes in lung cancer.

ENO1 Promotes Lung Cancer Metastasis via HGFR and WNT Signaling-Driven Epithelial-to-Mesenchymal Transition.

ENO1 (α-enolase) expression is significantly correlated with reduced survival and poor prognosis in many cancer types, including lung cancer. However, the function of ENO1 in carcinogenesis remains elusive. In this study, we found that high expression of ENO1 is present in metastatic lung cancer cell lines and malignant tumors and is associated with poor overall survival of patients with lung cancer. Knockdown of ENO1 decreased cancer cell proliferation and invasiveness, whereas overexpression of ENO1 enhanced these processes. Moreover, ENO1 expression promoted tumor growth in orthotopic models and enhanced lung tumor metastasis in tail-vein injection models. These effects were mediated by upregulation of mesenchymal markers N-cadherin and vimentin and the epithelial-to-mesenchymal transition regulator SLUG, along with concurrent downregulation of E-cadherin. Mechanistically, ENO1 interacted with hepatocyte growth factor receptor (HGFR) and activated HGFR and Wnt signaling via increased phosphorylation of HGFR and the Wnt coreceptor LRP5/6. Activation of these signaling axes decreased GSK3ß activity via Src-PI3K-AKT signaling and inactivation of the ß-catenin destruction complex to ultimately upregulate SLUG and ß-catenin. In addition, we generated a chimeric anti-ENO1 mAb (chENO1-22) that can decrease cancer cell proliferation and invasion. chENO1-22 attenuated cancer cell invasion by inhibiting ENO1-mediated GSK3ß inactivation to promote SLUG protein ubiquitination and degradation. Moreover, chENO1-22 prevented lung tumor metastasis and prolonged survival in animal models. Taken together, these findings illuminate the molecular mechanisms underlying the function of ENO1 in lung cancer metastasis and support the therapeutic potential of a novel antibody targeting ENO1 for treating lung cancer. SIGNIFICANCE: This study shows that ENO1 promotes lung cancer metastasis via HGFR and WNT signaling and introduces a novel anti-ENO1 antibody for potential therapeutic use in lung cancer.

Vorinostat combined with brigatinib overcomes acquired resistance in EGFR-C797S-mutated lung cancer.

The development of a new generation of tyrosine kinase inhibitors (TKIs) has improved the treatment response in lung adenocarcinomas. However, acquired resistance often occurs due to new epidermal growth factor receptor (EGFR) mutations. In particular, the C797S mutation confers drug resistance to T790M-targeting EGFR TKIs. To address C797S resistance, a promising therapeutic avenue is combination therapy that targets both total EGFR and acquired mutations to increase drug efficacy. We showed that combining vorinostat, a histone deacetylase inhibitor (HDACi), with brigatinib, a TKI, enhanced antitumor effects in primary culture and cell lines of lung adenocarcinomas harboring EGFR L858R/T790M/C797S mutations (EGFR-3M). While EGFR phosphorylation was decreased by brigatinib, vorinostat reduced total EGFR-3M (L858R/T790M/C797S) proteins through STUB1-mediated ubiquitination and degradation. STUB1 preferably ubiquitinated other EGFR mutants and facilitated protein turnover compared to EGFR-WT. The association between EGFR and STUB1 required the functional chaperone-binding domain of STUB1 and was further enhanced by vorinostat. Finally, STUB1 levels modulated EGFR downstream functions. Low STUB1 expression was associated with significantly poorer overall survival than high STUB1 expression in patients harboring mutant EGFR. Vorinostat combined with brigatinib significantly improved EGFR-TKI sensitivity to EGFR C797S by inducing EGFR-dependent cell death and may be a promising therapy in treating C797S-resistant lung adenocarcinomas.

Targeted sequencing to identify genetic alterations and prognostic markers in pediatric T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is caused by the accumulation of multiple genetic alterations. To determine the frequency of common genetic mutations and possible prognostic markers in childhood T-ALL, we performed targeted sequencing of 67 genes across 64 cases treated according to Taiwan Pediatric Oncology Group protocols between January 2002 and December 2015. Together, 302 variants were identified in 60 genes including 233 single nucleotide variants and 69 indels. Sixty-four samples had a median number of six genetic lesions each (range 1-17). Thirteen genes had mutation frequencies > 10%, and 5 were > 20%, with the highest being NOTCH1 (70.31%). Protocadherins FAT1 (32.81%) and FAT3 (17.19%), and the ubiquitin ligase component FBXW7 (28.13%) had higher mutation frequencies than previously reported. Other mutation frequencies (PHF6, DNM2, DNMT3A, CNOT3, and WT1) were within previously reported ranges. Three epigenetic-related genes (KMT2D, DNMT3A, and EZH2) were mutated in our cohort. JAK-STAT signaling pathway genes had mutation frequencies of 3-13% and were observed in 23 cases (35.94%). Changes to genes in the ErbB signaling pathway were detected in 20 cases (31.25%). Patients with NOTCH1/FBXW7 mutations and RAS/PTEN germline exhibited better 5-year overall survival rates.

Childhood acute lymphoblastic leukemia mercaptopurine intolerance is associated with NUDT15 variants.

BACKGROUND: Mercaptopurine-induced neutropenia can interrupt chemotherapy and expose patients to infection during childhood acute lymphoblastic leukemia (ALL) treatment. Previously, six candidate gene variants associated with mercaptopurine intolerance were reported. Herein, we investigated the association between the mean tolerable dose of mercaptopurine and these genetic variants in Taiwanese patients. METHODS: In total, 294 children with ALL were treated at the National Taiwan University Hospital from April 1997 to December 2017. Germline variants were analyzed for NUDT15, SUCLA2, TPMT, ITPA, PACSIN2, and MRP4. Mean daily tolerable doses of mercaptopurine in the continuation phase of treatment were correlated with these genetic variants. RESULTS: Mercaptopurine intolerance was significantly associated with polymorphisms in NUDT15 (P value < 0.0001). Patients with SUCLA2 variants received lower mercaptopurine doses (P value = 0.0119). The mean mercaptopurine doses did not differ among patients with TPMT, ITPA, MRP4, and PACSIN2 polymorphisms (P value = 0.9461, 0.5818, and 0.7951, respectively). After multivariable linear regression analysis, only NUDT15 variants retained their clinically significant correlation with mercaptopurine intolerance (P value < 0.0001). CONCLUSION: In this cohort, the major genetic determinant of mercaptopurine intolerance was NUDT15 in Taiwanese patients. IMPACT: NUDT15 causes mercaptopurine intolerance in children with ALL. The NUDT15 variant is a stronger predictor of mercaptopurine intolerance than TPMT in a Taiwanese cohort. This finding is similar with studies performed on Asian populations rather than Caucasians. Pre-emptive genotyping of the patients' NUDT15 before administering mercaptopurine may be more helpful than genotyping TPMT in Asians.

Antimetabolite pemetrexed primes a favorable tumor microenvironment for immune checkpoint blockade therapy.

BACKGROUND: The immune checkpoint blockade (ICB) targeting programmed cell death-1 (PD-1) and its ligand (PD-L1) has been proved beneficial for numerous types of cancers, including non-small-cell lung cancer (NSCLC). However, a significant number of patients with NSCLC still fail to respond to ICB due to unfavorable tumor microenvironment. To improve the efficacy, the immune-chemotherapy combination with pemetrexed, cis/carboplatin and pembrolizumab (anti-PD-1) has been recently approved as first-line treatment in advanced NSCLCs. While chemotherapeutic agents exert beneficial effects, the underlying antitumor mechanism(s) remains unclear. METHODS: Pemetrexed, cisplatin and other chemotherapeutic agents were tested for the potential to induce PD-L1 expression in NSCLC cells by immunoblotting and flow cytometry. The ability to prime the tumor immune microenvironment was then determined by NSCLC/T cell coculture systems and syngeneic mouse models. Subpopulations of NSCLC cells responding differently to pemetrexed were selected and subjected to RNA-sequencing analysis. The key signaling pathways were identified and validated in vitro and in vivo. RESULTS: Pemetrexed induced the transcriptional activation of PD-L1 (encoded by CD274) by inactivating thymidylate synthase (TS) in NSCLC cells and, in turn, activating T-lymphocytes when combined with the anti-PD-1/PD-L1 therapy. Nuclear factor κB (NF-κB) signaling was activated by intracellular reactive oxygen species (ROSs) that were elevated by pemetrexed-mediated TS inactivation. The TS-ROS-NF-κB regulatory axis actively involves in pemetrexed-induced PD-L1 upregulation, whereas when pemetrexed fails to induce PD-L1 expression in NSCLC cells, NF-κB signaling is unregulated. In syngeneic mouse models, the combinatory treatment of pemetrexed with anti-PD-L1 antibody created a more favorable tumor microenvironment for the inhibition of tumor growth. CONCLUSIONS: Our findings reveal novel mechanisms showing that pemetrexed upregulates PD-L1 expression and primes a favorable microenvironment for ICB, which provides a mechanistic basis for the combinatory chemoimmunotherapy in NSCLC treatment.