Association Between Rheumatoid Arthritis and Clonal Hematopoiesis: A Mendelian Randomization Study.

Immunity activation and inflammation are the main characteristics of rheumatoid arthritis and clonal hematopoiesis. However, it remains unclear whether rheumatoid arthritis increase the risk of clonal hematopoiesis. Here, a Mendelian randomization (MR) analysis was conduct to explore the causal effects of rheumatoid arthritis on clonal hematopoiesis. Summary statistics data of rheumatoid arthritis (13,838 cases and 33,742 controls) and clonal hematopoiesis (10,203 cases and 173,918 controls) derived from a genomewide association study were selected to analyze. We selected inverse-variance weighted, MR-Egger, weighted median, simple mode, and weighted mode to evaluate the causal effect of rheumatoid arthritis on clonal hematopoiesis. The two-sample MR analysis suggested a strong causal relationship between rheumatoid arthritis and clonal hematopoiesis by inverse-variance weighted (OR = 1.002311673, 95% CI [1.000110757, 1.004517433], p = .039706) and weighted median (OR = 1.002311673, 95% CI [1.000110757, 1.004517433], p = .039518447) methods. No significant pleiotropy or heterogeneity was found in the sensitivity analysis. These results supported a potentially causal relationship between rheumatoid arthritis and clonal hematopoiesis, and the exposure of rheumatoid arthritis increased the risks of clonal hematopoiesis. Our findings highlight the importance of how chronic inflammation and immune activation induced rheumatoid arthritis enhances the risks of clonal hematopoiesis, and that early intervention with rheumatoid arthritis patients might reduce the clonal hematopoiesis risks in rheumatoid arthritis patients. Moreover, our study provides clues for prediction of risk factors and potential mechanisms of clonal hematopoiesis.

Early proactive monitoring of DNA-thioguanine in patients with Crohn's disease predicts thiopurine-induced late leucopenia in NUDT15/TPMT normal metabolizers.

BACKGROUND: Thiopurine-induced leucopenia significantly hinders the wide application of thiopurines. Dose optimization guided by nudix hydrolase 15 (NUDT15) has significantly reduced the early leucopenia rate, but there are no definitive biomarkers for late risk leucopenia prediction. AIM: To determine the predictive value of early monitoring of DNA-thioguanine (DNATG) or 6-thioguanine nucleotides (6TGN) for late leucopenia under a NUDT15-guided thiopurine dosing strategy in patients with Crohn's disease (CD). METHODS: Blood samples were collected within two months after thiopurine initiation for detection of metabolite concentrations. Late leucopenia was defined as a leukocyte count < 3.5 × 109/L over two months. RESULTS: Of 148 patients studied, late leucopenia was observed in 15.6% (17/109) of NUDT15/thiopurine methyltransferase (TPMT) normal and 64.1% (25/39) of intermediate metabolizers. In patients suffering late leucopenia, early DNATG levels were significantly higher than in those who did not develop late leucopenia (P = 4.9 × 10-13). The DNATG threshold of 319.43 fmol/µg DNA could predict late leucopenia in the entire sample with an area under the curve (AUC) of 0.855 (sensitivity 83%, specificity 81%), and in NUDT15/TPMT normal metabolizers, the predictive performance of a threshold of 315.72 fmol/µg DNA was much more remarkable with an AUC of 0.902 (sensitivity 88%, specificity 85%). 6TGN had a relatively poor correlation with late leucopenia whether in the entire sample (P = 0.021) or NUDT15/TPMT normal or intermediate metabolizers (P = 0.018, P = 0.55, respectively). CONCLUSION: Proactive therapeutic drug monitoring of DNATG could be an effective strategy to prevent late leucopenia in both NUDT15/TPMT normal and intermediate metabolizers with CD, especially the former.

Germline USP36 Mutation Confers Resistance to EGFR-TKIs by Upregulating MLLT3 Expression in Patients with Non-Small Cell Lung Cancer.

PURPOSE: Although somatic mutations were explored in depth, limited biomarkers were found to predict the resistance of EGFR tyrosine kinase inhibitors (EGFR-TKI). Previous studies reported N6-methyladenosine (m6A) levels regulated response of EGFR-TKIs; whether the germline variants located in m6A sites affected resistance of EGFR-TKIs is still unknown. EXPERIMENTAL DESIGN: Patients with non-small cell lung cancer (NSCLC) with EGFR-activating mutation were enrolled to investigate predictors for response of EGFR-TKIs using a genome-wide-variant-m6A analysis. Bioinformatics analysis and series of molecular biology assays were used to uncover the underlying mechanism. RESULTS: We identified the germline mutation USP36 rs3744797 (C > A, K814N) was associated with survival of patients with NSCLC treated with gefitinib [median progression-free survival (PFS): CC vs. CA, 16.30 vs. 10.50 months, P < 0.0001, HR = 2.45] and erlotinib (median PFS: CC vs. CA, 14.13 vs. 9.47 months, P = 0.041, HR = 2.63). Functionally, the C > A change significantly upregulated USP36 expression by reducing its m6A level. Meanwhile, rs3744797_A (USP36 MUT) was found to facilitate proliferation, migration, and resistance to EGFR-TKIs via upregulating MLLT3 expression in vitro and in vivo. More importantly, MLLT3 and USP36 levels are tightly correlated in patients with NSCLC, which were associated with prognosis of patients. Mechanistically, USP36 MUT stabilized MLLT3 by deubiquitinating MLLT3 in nucleoli and consequently activating its downstream signaling (HIF1α and Snai). Furthermore, inhibition of MLLT3 alleviated USP36 variant-induced EGFR-TKIs resistance in EGFR-mutant NSCLC. CONCLUSIONS: These findings characterized rs3744797 as an oncogenic variant in mediating EGFR-TKI resistance and tumor aggressiveness through deubiquitinating MLLT3, highlighting the variant as a predictive biomarker for EGFR-TKI response in NSCLC.

Safety Concerns with Nusinersen, Risdiplam, and Onasemnogene Abeparvovec in Spinal Muscular Atrophy: A Real-World Pharmacovigilance Study.

BACKGROUND AND OBJECTIVE: Spinal muscular atrophy (SMA) is a genetic disorder with limited treatment options. It is crucial to have a comprehensive understanding of drug safety in order to make informed clinical drug selections for patients with SMA. Assessing the safety profiles of therapeutic drugs for SMA has been challenging due to the limited number of patients included in clinical trials. This study aims to investigate and compare the potential safety concerns associated with three leading SMA therapeutic drugs: nusinersen, risdiplam, and onasemnogene abeparvovec. METHODS: The FDA Adverse Event Reporting System database was used to analyze drug safety, and a case (SMA drug)/noncase (all other drugs in the database) approach was employed to estimate safety signals through disproportionality analysis and reporting odds ratio (ROR). Veen analysis was conducted to compare and select the idiosyncratic adverse events (AEs) associated with each drug. RESULTS: The study included 5324 cases of nusinersen, 1184 cases of risdiplam, and 1277 cases of onasemnogene abeparvovec. Venn analysis revealed 27 common AEs among the three drugs, including cardiac, gastrointestinal, metabolism, musculoskeletal, renal, respiratory disorders, and infections. Additionally, 196 AEs exclusively found in nusinersen included post lumbar puncture syndrome [ROR (95% CI) = 6120.91 (5057.01-7408.64), n = 372], procedural pain [ROR (95% CI) = 54.86 (48.13-62.54), n = 234], idiopathic intracranial hypertension [ROR (95% CI) = 6.12 (2.29-16.33), n = 4], and hypokalemia [ROR (95% CI) = 2.02 (1.24-3.31), n = 16]. Additionally, transient deafness was identified as an unexpected and rare, yet severe, AE for nusinersen [ROR (95% CI) = 23.32 (8.71-62.44), n = 4]. Risdiplam exhibited 50 AEs exclusively, with notable idiosyncratic AEs including diarrhea [ROR (95% CI) = 4.55 (3.79-5.46), n = 121], fatigue [ROR (95% CI) = 2.03 (1.6-2.57), n = 70], photosensitivity reaction [ROR (95% CI) = 9.50 (4.25-21.13), n = 6], rash [ROR (95% CI) = 1.90 (1.36-2.67), n = 34], and [ROR (95% CI) = 4.3 (1.93-9.58), n = 6] in comparison with the other two drugs. Moreover, ileus [ROR (95% CI) = 11.11 (4.14-29.51), n = 4], gastrointestinal hemorrhage [ROR (95% CI) = 2.55 (1.15-5.69), n = 6], and hypoglycemic unconsciousness [ROR (95% CI) = 153.58 (62.98-374.54), n = 5] were rare but severe AEs associated with risdiplam. Onasemnogene abeparvovec had 143 exclusively identified AEs, with significant high signals for troponin I increase [ROR (95% CI) = 627.1 (492.2-798.99), n = 78], troponin T increase [ROR (95% CI) = 233.98 (153.29-357.15), n = 23], blood lactate dehydrogenase increase [ROR (95% CI) = 39.81 (28.88-54.87), n = 38], and transaminases increase [ROR (95% CI) = 36.88 (29.24-46.52), n = 73]. CONCLUSIONS: This study highlights the importance of monitoring injection-related injuries and transient deafness events in patients treated with nusinersen. For onasemnogene abeparvovec, careful monitoring for renal impairment, liver injury, and myocardial damage is necessary. Risdiplam requires attention to the potential risk of rare but severe gastrointestinal damage events and hypoglycemia. Importantly, risdiplam exhibited lower liver and renal toxicity, making it a potential consideration for patients with liver or renal insufficiency or for combined use with other drugs that possess high liver or renal toxicity. These findings can be a reference for drug selection and further prospective studies.

The Effects of Drug Exposure and Single Nucleotide Polymorphisms on Aaptinib-Induced Severe Toxicities in Solid Tumors.

To investigate the value of drug exposure and host germline genetic factors in predicting apatinib (APA)-related toxicities. METHOD: In this prospective study, plasma APA concentrations were quantified using liquid chromatography with tandem mass spectrometry, and 57 germline mutations were genotyped in 126 advanced solid tumor patients receiving 250 mg daily APA, a vascular endothelial growth factor receptor II inhibitor. The correlation between drug exposure, genetic factors, and the toxicity profile was analyzed. RESULTS: Non-small cell lung cancer (NSCLC) was more prone to APA-related toxicities and plasma concentrations of APA, and its main metabolite M1-1 could be associated with high-grade adverse events (AEs) (P < 0.01; M1-1, P < 0.01) and high-grade antiangiogenetic toxicities (APA, P = 0.034; P < 0.05), including hypertension, proteinuria, and hand-foot syndrome, in the subgroup of NSCLC. Besides, CYP2C9 rs34532201 TT carriers tended to have higher levels of APA (P < 0.001) and M1-1 (P < 0.01), whereas CYP2C9 rs1936968 GG carriers were predisposed to higher levels of M1-1 (P < 0.01). CONCLUSION: Plasma APA and M1-1 exposures were able to predict severe AEs in NSCLC patients. Dose optimization and drug exposure monitoring might need consideration in NSCLC patients with CYP2C9 rs34532201 TT and rs1936968 GG. SIGNIFICANCE STATEMENT: Apatinib is an anti-VEGFR2 inhibitor for the treatment of multiple cancers. Though substantial in response, apatinib-induced toxicity has been a critical issue that is worth clinical surveillance. Few data on the role of drug exposure and genetic factors in apatinib-induced toxicity are available. Our study demonstrated a distinct drug-exposure relationship in NSCLC but not other tumors and provided invaluable evidence of drug exposure levels and single nucleotide polymorphisms as predictive biomarkers in apatinib-induced severe toxicities.

FOXO3 mutation predicting gefitinib-induced hepatotoxicity in NSCLC patients through regulation of autophagy.

Hepatotoxicity is a common side effect for patients treated with gefitinib, but the related pathogenesis is unclear and lacks effective predictor and management strategies. A multi-omics approach integrating pharmacometabolomics, pharmacokinetics and pharmacogenomics was employed in non-small cell lung cancer patients to identify the effective predictor for gefitinib-induced hepatotoxicity and explore optional therapy substitution. Here, we found that patients with rs4946935 AA, located in Forkhead Box O3 (FOXO3) which is a well-known autophagic regulator, had a higher risk of hepatotoxicity than those with the GA or GG variant (OR = 18.020, 95%CI = 2.473 to 459.1784, P = 0.018) in a gefitinib-concentration dependent pattern. Furthermore, functional experiments identified that rs4946935_A impaired the expression of FOXO3 by inhibiting the promotor activity, increasing the threshold of autophagy initiation and inhibiting the autophagic activity which contributed to gefitinib-induced liver injury. In contrast, erlotinib-induced liver injury was independent on the variant and expression levels of FOXO3. This study reveals that FOXO3 mutation, leading to autophagic imbalance, plays important role in gefitinib-induced hepatotoxicity, especially for patients with high concentration of gefitinib. In conclusion, FOXO3 mutation is an effective predictor and erlotinib might be an appropriately and well-tolerated treatment option for patients carrying rs4946935 AA.

DNA-Thioguanine Nucleotides as a Marker for Thiopurine Induced Late Leukopenia after Dose Optimizing by NUDT15 C415T in Chinese Patients with IBD.

Thiopurine dose optimization by thiopurine-S-methyltransferase (TPMT) or nudix hydrolase-15 (NUDT15) significantly reduced early leucopenia in Asia. However, it fails to avoid the late incidence (> 2 months). Although laboratory monitoring of 6-thioguanine nucleotides (6TGN) to optimize thiopurine dose was suggested in White patients the exact association between leucopenia and 6TGN was controversial in Asian patients. In the present study, we aimed to explore whether DNA-thioguanine nucleotides (DNA-TGs) in leukocytes, compared with 6TGN in erythrocytes, can be a better biomarker for late leucopenia. This was a prospective, observational study. Patients with inflammatory bowel disease (IBD) prescribed thiopurine from February 2019 to December 2019 were recruited. Thiopurine dose was optimized by NUDT15 C415T (rs116855232). DNA-TG and 6TGN levels were determined at the time of late leucopenia or 2 months after the stable dose was obtained. A total of 308 patients were included. Thiopurine induced late leucopenia (white blood cells < 3.5 × 109 /L) were observed in 43 patients (14.0%), who had significantly higher DNA-TG concentration than those without leucopenia (P = 4.1 × 10-9 , 423.3 (~ 342.2 to 565.7) vs. 270.5 (~ 188.1 to 394.3) fmol/µg DNA). No difference in 6TGN concentrations between leucopenia and non-leucopenia was found. With a DNA-TG threshold of 340.1 fmol/µg DNA, 83.7% of leucopenia cases could be identified. Multivariate analysis showed that DNA-TG was an independent risk factor for late leucopenia. Quantification of DNA-TG, rather than 6TGN, can be applied to gauge thiopurine therapy after NUDT15 screening in Chinese patients with IBD.

FOXM1 Variant Contributes to Gefitinib Resistance via Activating Wnt/ß-Catenin Signal Pathway in Patients with Non-Small Cell Lung Cancer.

PURPOSE: Although gefitinib prolonged the progression-free survival (PFS) of patients with non-small cell lung cancer (NSCLC), unpredictable resistance limited its clinical efficacy. Novel predictive biomarkers with explicit mechanisms are urgently needed. EXPERIMENTAL DESIGN: A total of 282 patients with NSCLC with gefitinib treatment were randomly assigned in a 7:3 ratio to exploratory (n = 192) and validation (n = 90) cohorts. The candidate polymorphisms were selected with Haploview4.2 in Hapmap and genotyped by a MassARRAY system, and the feature variables were identified through Randomforest Survival analysis. Tanswell and clonogenic assays, base editing and cell-derived tumor xenograft model were performed to uncover the underlying mechanism. RESULTS: We found that the germline missense polymorphism rs3742076 (A>G, S628P), located in transactivation domain of FOXM1, was associated with PFS in exploratory (median PFS: GG vs. GA&AA, 9.20 vs. 13.37 months, P = 0.00039, HR = 2.399) and validation (median PFS: GG vs. GA&AA, 8.13 vs. 13.80 months, P = 0.048, HR = 2.628) cohorts. We elucidated that rs3742076_G conferred resistance to gefitinib by increasing protein stability of FOXM1 and facilitating an aggressive phenotype in vitro and in vivo through activating wnt/ß-catenin signaling pathway. Meanwhile, FOXM1 level was highly associated with prognosis in patients with EGFR-mutant NSCLC. Mechanistically, FOXM1 rs3742076_G upregulated wnt/ß-catenin activity by directly binding to ß-catenin in cytoplasm and promoting transcription of ß-catenin in nucleus. Remarkably, inhibition of ß-catenin markedly reversed rs3742076_G-induced gefitinib resistance and aggressive phenotypes. CONCLUSIONS: These findings characterized rs3742076_G as a gain-of-function polymorphism in mediating gefitinib resistance and tumor aggressiveness, and highlighted the variant as a predictive biomarker in guiding gefitinib treatment.

Development and validation of UHPLC-MS/MS method for simultaneous quantification of escitalopram and its major metabolites in human plasma and its application in depressed patients.

Escitalopram, one of the Selective Serotonin Reuptake Inhibitors (SSRIs), has been widely used in the patients with major depression. In this study, a simple, sensitive and rapid method was established and validated for simultaneous quantification of Escitalopram (S-CT), desmethyl escitalopram (S-DCT), didemethyl escitalopram (S-DDCT) and escitalopram N-Oxide (S-NOCT) in human plasma by ultra-high performance liquid chromatography-tandem with mass spectrometry (UHPLC-MS/MS). Analytes were extracted from plasma by utilizing protein precipitation and then separated on a Hypersil GOLD C18 column (50 mm × 2.1 mm, 1.9 µm). The mobile phase was water: acetonitrile (70:30, v/v) with 0.25% formic acid at a flow-rate of 0.3 mL/min, within a 5 min run time. The mass analysis used positive electro-spray ionization (ESI) in selection reaction monitoring (SRM). The calibration ranges of the analytes were: S-CT: 2.0-200.0 ng/mL, S-DCT: 1.0-100.0 ng/mL, S-DDCT: 0.5-50.0 ng/mL, S-NOCT: 0.2-20.0 ng/mL. The method has been fully validated for selectivity, linearity, accuracy, precision, matrix effect, recovery, stability and carry over and all the results met the admissible limits according to the the US Food and Drug Administration guidelines. Mean plasma concentration (ng/mL) of S-CT, S-DCT, S-DDCT and S-NOCT in 93 depressed patients were 51.10 ± 45.73, 10.32 ± 15.25, 1.53 ± 1.79 and 0.87 ± 0.94, respectively. it is the first time that a UHPLC-MS/MS method for simultaneous quantification of S-CT and its 3 metabolites in human plasma was established and validated.

Rituximab Concentration Varies in Patients With Different Lymphoma Subtypes and Correlates With Clinical Outcome.

Individual variations in concentrations of rituximab in different B cell non-Hodgkin's lymphoma subtypes and their relevance to efficacy were still unclear. From 2016 to 2021, a prospective clinical trial was conducted, and 510 samples with 6 uncommon subtypes of B-cell lymphoma were enrolled to examine the pharmacokinetic behaviour of rituximab and its impact on clinical outcomes, including complete response (CR), progression-free survival (PFS) and overall survival (OS). Considerable variability was observed in the rituximab trough concentration in the first cycle (C1-trough, 1.16-55.52 µg/ml) in patients with different lymphoma subtypes. Patients with "double-hit" lymphoma (4.01 ± 0.77 µg/ml) or mantle cell lymphoma (MCL; 15.65 ± 16.45 µg/ml) had much lower C1-trough and worse outcomes. Great individual variation in the C1-trough existed among patients with mucosa-associated lymphoma (MALT), and the high C1-trough observed in patients treated with the RB regimen was associated with a better response than was obtained with R-CHOP (38.41 ± 14.13 µg/ml vs 15.49 ± 8.80 µg/ml, p = 0.0029). Despite the high aggressiveness of the cancer, Burkitt lymphoma patients receiving intensive chemotherapy had the highest C1-trough (28.85 ± 9.35 µg/ml) and maintained long-term PFS. The C1-trough in patients with mixed, unclassifiable B-cell lymphoma was close to 20 µg/ml, and these patients had acceptable outcomes. Overall, a low rituximab C1-trough was associated with adverse consequences, including persistent progression, early recurrence and a short OS, however, some high-risk factors appeared to be balanced by the presence of a high C1-trough. Basal levels of circulating CD19+ lymphocytes differed between and within patients with diverse lymphoma subtypes and were negatively correlated with C1-trough. Therefore, the traditional doses of rituximab are inadequate for patients with "double-hit" lymphoma and MCL. Increasing the initial rituximab dose according to the disease, high-risk factors and even the baseline CD19+ lymphocyte count will be new methods to optimize therapeutic regimens for patients with different lymphoma subtypes.

Rational application of gefitinib in NSCLC patients with sensitive EGFR mutations based on pharmacokinetics and metabolomics.

Gefitinib has been available in the market for 20 years, but its pharmacokinetic mechanism of response is little known. In this study, we examined the pharmacokinetic and metabolomic profiles in non-small cell lung cancer (NSCLC) patients with sensitive EGFR mutations. A total of 216 advanced NSCLC patients were enrolled, and administered gefitinib at the standard dosage of 250 mg/day, which was established in heterogeneous subjects with non-sensitive mutations. We identified and quantified three main metabolites (named as M1, M2 and M3) in the plasma of patients, the correlations between the concentration of gefitinib/metabolites and efficacy were analyzed. In exploratory and validation set, gefitinib concentration was not correlated with clinical effects. Considering the result that the therapeutic effects of 250 mg/2-day was better than that of 250 mg/day in a multiple center clinical trial, the standard dose might be higher than that for maximal efficacy according to the hypothetical dose-response curve. Among the three metabolites, the IC50 of M2 in HCC827 and PC9 cell lines was significantly lower, and Conc.brain/Conc.plasma of M2 in mice was significantly higher than those of gefitinib, suggesting its higher potential to penetrate blood-brain barrier and might be more effective in the treatment of brain metastatic tumor than gefitinib. Consistently and attractively, higher M2 plasma concentration was found to be correlated with better clinical outcome in patients with brain metastases (the median PFS of CM2 < 12 ng/mL and CM2 ≥ 12 ng/mL were 17.0 and 27.1 months, respectively, P = 0.038). The plasma concentration of M2 ≥ 12 ng/mL was a strong predictor of the PFS of NSCLC patients. In conclusion, for NSCLC patients with EGFR sensitive mutations, the standard dose is suspectable and could be decreased reasonably. M2 plays an important role in efficacy and may be more effective in the treatment of metastatic tumor than gefitinib.

Rituximab exposure-response in triweekly R-CHOP treatment in DLBCL: A loading dose is recommended to improve clinical outcomes.

Previous exposure-response analyses for rituximab suggest that higher rituximab concentrations were associated with an improvement in efficacy, however, clinical studies investigating a higher rituximab dose had mixed results. To further explore the exposure-response relationship of rituximab, a prospective observational analysis was performed involving 121 newly diagnosed patients with diffuse large B-cell lymphoma treated with triweekly rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). The trough concentration in the first cycle (C1-trough ) was significantly higher in patients achieving complete response (CR) compared with patients that did not achieve CR (22.00 µg/ml vs. 16.62 µg/ml, p = 0.0016), however, this difference between the two groups disappeared in later cycles. The relationship between rituximab C1-trough and achieving a CR was confirmed by matched-pair logistic regression analysis (odds ratio, 0.79; p = 0.0020). In addition, a higher C1-trough (≥18.40 µg/ml) was associated with longer progression-free survival (p < 0.0001) and overall survival (p = 0.0038). The percentages of patients that did not achieve a CR and had recurrence after CR within 24 months were 35% and 22.50%, respectively, for patients with a C1-trough less than or equal to 18.40 µg/ml, compared with 12.35% and 6.17% for patients with C1-trough greater than 18.40 µg/ml. Disease stage was found to be the most significant influencing factor of C1-trough , with 51.02% of patients at stage IV with an observed C1-trough less than 18.40 µg/ml. For these advanced patients, population pharmacokinetic simulations using an established model suggest that a loading dose of 800 mg/m2  may help to improve clinical outcomes.

Novel Clinical Biomarkers for Drug-Induced Liver Injury.

Drug-induced liver injury (DILI) remains a critical clinical issue and has been a treatment challenge today as it was in the past. However, the traditional biomarkers or indicators are insufficient to predict the risks and outcome of patients with DILI due to its poor specificity and sensitivity. Recently, the development of high-throughput technologies, especially omics and multiomics has sparked growing interests in identification of novel clinical DILI biomarkers, many of which also provide a mechanistic insight. Accordingly, in this minireview, we summarize recent advances in novel clinical biomarkers for DILI prediction, diagnosis, and prognosis and highlight the limitations or challenges involved in biomarker discovery or its clinical translation. Although huge work has been done, most reported biomarkers lack comprehensive information and more specific DILI biomarkers are still needed to complement the traditional biomarkers such as alanine aminotransferase (ALT) or aspartate transaminase (AST) in clinical decision-making. SIGNIFICANCE STATEMENT: This current review outlines an overview of novel clinical biomarkers for drug-induced liver injury (DILI) identified in clinical retrospective or prospective clinical analysis. Many of these biomarkers provide a mechanistic insight and are promising to complement the traditional DILI biomarkers. This work also highlights the limitations or challenges involved in biomarker discovery or its clinical translation.

Establishment and application of a predictive model for gefitinib-induced severe rash based on pharmacometabolomic profiling and polymorphisms of transporters in non-small cell lung cancer.

BACKGROUND: Rash is a well-known predictor of survival for patients with gefitinib therapy with non-small cell lung cancer (NSCLC). However, whether patients with more severe rash obtain the more survival benefits from gefitinib is still unknown, and predicted model for severe rash is needed. METHODS: The relationship between gefitinib-induced rash and progression free survival (PFS) was primarily explored in the retrospective cohort. The association between rash and gefitinib/metabolites concentration and genetic polymorphisms were determined by pharmacometabolomic and pharmacogenomics methods in the exploratory cohort and validated in an external cohort. RESULTS: The survival for patients with rash was significantly higher than that of patients without rash (p = 0.0002, p = 0.0089), but no difference was found between grade 1/2 or grade 3/4. Only the concentration of gefitinib, but not its metabolites, was found to be associated with severe rash, and the cutoff value of gefitinib was 204.6 ng/mL conducted by ROC curve analysis (AUC=0.685). A predictive model for severe rash was established: gefitinib concentration (OR = 11.523, 95% CI = 2.898-64.016, p = 0.0016), SLC22A8 rs4149179(CT vs CC, OR = 3.156, 95% CI = 0.958-11.164, p = 0.0629), SLC22A1 rs4709400(CG vs CC, OR = 10.267, 95% CI = 2.067-72.465, p = 0.0087; GG vs CC, OR = 5.103, 95% CI = 1.032-33.938, p = 0.061). This model was confirmed in the validation cohort with an excellent predictive ability (AUC = 0.749, 95% CI = 0.710-0.951). CONCLUSIONS: Our finding demonstrated that the incidence, not the severity, of gefitinib-induced rash predicted improved survival, the gefitinib concentration and polymorphisms of SLC22A8 and SLC22A1 were recommended to manage severe rash.

Low initial trough concentration of rituximab is associated with unsatisfactory response of first-line R-CHOP treatment in patients with follicular lymphoma with grade 1/2.

For follicular lymphoma (FL) with grade 1/2, the complete response (CR) rate of the first-line R-CHOP treatment was significantly low. In this study, we assessed the rationality of the administration of rituximab for FL patients with grade 1/2 based on concentration-response relationship analyses. Thus, we conducted a prospective pharmacokinetic (PK) study in 68 FL patients with grades 1-3 treated with R-CHOP at 21-day intervals. Plasma rituximab concentrations were quantified using ELISA and the population PK modeling was established with Phoenix® NLMETM. The first cycle trough concentration (C1-trough) of rituximab was a significant independent risk factor for achieving CR in matched-pair logistic regression analysis, rather than the concentrations in later cycles; the recommendatory minimum optimal C1-trough was 13.60 µg/mL. Patients with grade 1/2 had significantly lower C1-trough compared with grade 3 (12.21 µg/mL vs. 23.45 µg/mL, P < 0.001), only 30% patients with grade 1/2 could reach 13.60 µg/mL, compared with 91.67% in patients with grade 3, which was in accord with its unsatisfactory CR rates (43.33% vs. 76.32%). The stage indicating the tumor burden (the target) was a crucial influence factor for C1-trough, accounting for 40.70% of its variability, 70% patients with grade 1/2 were stage IV in this study, since the systemic therapy only started at the disseminated disease stage. The initial dose of 1800 mg was recommended by Monte Carlo simulation for patients with grade 1/2. In summary, low C1-trough accounted for low-grade FL's unsatisfactory CR rate, designing the first dosage of rituximab should be a very important component of individualized therapy for FL.

Association of polymorphisms in C1orf106, IL1RN, and IL10 with post-induction infliximab trough level in Crohn's disease patients.

BACKGROUND: Trough levels of the post-induction serum infliximab (IFX) are associated with short-term and long-term responses of Crohn's disease patients to IFX, but the inter-individual differences are large. We aimed to elucidate whether single gene polymorphisms (SNPs) within FCGR3A, ATG16L1, C1orf106, OSM, OSMR, NF-κB1, IL1RN, and IL10 partially account for these differences and employed a multivariate regression model to predict patients' post-induction IFX levels. METHODS: The retrospective study included 189 Crohn's disease patients undergoing IFX therapy. Post-induction IFX levels were measured and 41 tag SNPs within eight genes were genotyped. Associations between SNPs and IFX levels were analysed. Then, a multivariate logistic-regression model was developed to predict whether the patients' IFX levels achieved the threshold of therapy (3 µg/mL). RESULTS: Six SNPs (rs7587051, rs143063741, rs442905, rs59457695, rs3213448, and rs3021094) were significantly associated with the post-induction IFX trough level (P = 0.015, P < 0.001, P = 0.046, P = 0.022, P = 0.011, P = 0.013, respectively). A multivariate prediction model of the IFX level was established by baseline albumin (P = 0.002), rs442905 (P = 0.025), rs59457695 (P = 0.049), rs3213448 (P = 0.056), and rs3021094 (P = 0.047). The area under the receiver operating characteristic curve (AUROC) of this prediction model in a representative training dataset was 0.758. This result was verified in a representative testing dataset, with an AUROC of 0.733. CONCLUSIONS: Polymorphisms in C1orf106, IL1RN, and IL10 play an important role in the variability of IFX post-induction levels, as indicated in this multivariate prediction model of IFX levels with fair performance.

Determination of apatinib and its three active metabolites by UPLC-MS/MS in a Phase IV clinical trial in NSCLC patients.

Aim: To develop and validate a simple method using UPLC-MS/MS for determination of apatinib and its three active metabolites in a Phase IV clinical trial. Materials & methods: All compounds were separated on a Hypersil GOLD™ aQ C18 Polar Endcapped LC column (50 × 2.1 mm, 1.9 µm, Thermo) using 5 mmol/l ammonium acetate with 0.1% formic acid:acetonitrile (20:80, v/v) as the mobile phase after a rapid liquid-liquid extraction. This method was validated over the linear concentration range of 1.00-1000 ng/ml for each compound. Results: The interassay precision and accuracy were less than ±15%. The validated method was successfully applied to determine concentrations of clinical samples in non-small-cell lung cancer patients.

Development and validation of a sensitive LC-MS/MS method for determination of gefitinib and its major metabolites in human plasma and its application in non-small cell lung cancer patients.

Gefitinib, the first approved oral epidermal growth factor receptor (EGFR) inhibitor, has been demonstrated effective in cancers with EGFR active mutations. In this study, we established and validated a method for determining gefitinib and its main metabolites, M605211, M387783, M537194 and M523595 in patients with non-small cell lung cancer (NSCLC) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The mobile phase was water: acetonitrile (35:65, v/v) with 0.1% formic acid at a flow-rate of 0.35 mL/min, within a 3 min run time. Gefitinib and its main metabolites were separated on a X-Terra RP18 column (50 × 2.1 mm, 3.5 µm) at 40 ℃ and subjected to mass analysis using positive electro-spray ionization (ESI). The calibration ranges of gefitinib and M523595 were 0.5-1000 ng/mL, and other compounds were 0.05-100 ng/mL with the correlation coefficients (r2) ≥ 0.99. Accuracies ranged from 92.60%-107.58 and the inter- and intra-assay precision were less than 15% for all analytes in quality control samples. There was no significant matrix effect. The ranges of extraction recoveries were 86-105% for all analytes and IS. Thirty plasmas were obtained from Sun Yat-sen university cancer center. The mean plasma concentration of (± SD) of gefitinib M537194, M523595, M387783 and M605211 were 247.18 (± 140.39) ng/mL, 7.78 (± 6.74) ng/mL, 101.09 (± 93.44) ng/mL, 1.6 (± 0.9) ng/mL and 11.63 (± 4.98) ng/mL, respectively. The validated LC/MS/MS method was effectively used in the determination of gefitinib and its four metabolites in NSCLC patients.

Potent In Vitro Synergism of Fluconazole and Osthole against Fluconazole-Resistant Candida albicans.

Osthole is a natural coumarin that exhibits wide biological and pharmacological activities such as neuroprotective, osteogenic, immunomodulation, antitumor, and anti-inflammatory effects. In this study, we investigated the antifungal effects of osthole in vitro A checkerboard microdilution assay showed that osthole has significant synergistic effect with fluconazole against fluconazole-resistant Candida albicans Similar results were obtained from a growth curve assay. Meanwhile, XTT reduction assay demonstrated the synergism of fluconazole and osthole against C. albicans biofilm formation. Microarray results showed that the expression of genes involved in the oxidation-reduction process, energy metabolism, and transportation changed significantly after the combined treatment with fluconazole and osthole, and further results showed that endogenous reactive oxygen species (ROS) was significantly increased in the combination group. In conclusion, these results demonstrate the synergism of fluconazole and osthole against fluconazole-resistant C. albicans and indicate that endogenous ROS augmentation might contribute to the synergism of fluconazole and osthole.