The effect of genetic variants in the transcription factor TSPYL family on the CYP3A4 mediated cyclosporine metabolism in kidney transplant patients.

CYP3A4 activity shows considerable interindividual variability. Although studies indicate 60%-80% is heritable, common single nucleotide variants (SNVs) in CYP3A4 together only explain ~10%. Transcriptional factors, such as the testis-specific Y-encoded-like proteins (TSPYLs) family, have been reported to regulate the expression of CYP enzymes including CYP3A4 in vitro. Here, we investigated the effect of genetic variants in TSPYL on CYP3A4 activity using data from a clinical study and a human liver bank. Five SNVs (rs3828743, rs10223646, rs6909133, rs1204807, and rs1204811) in TSPYL were selected because of a reported effect on CYP3A4 expression in vitro or suggested clinical effect. For the clinical study, whole blood concentrations, clinical data, and DNA were available from 295 kidney transplant recipients participating in the prospective MECANO study. A multivariate pharmacokinetic model adjusted for body weight, steroid treatment, and CYP3A4 genotype was used to assess the effect of the genetic variants on cyclosporine clearance. In multivariate analysis, homozygous carriers of rs3828743 had a 18% lower cyclosporin clearance compared to the wild-type and heterozygous patients (28.72 vs. 35.03 L/h, p = 0.018) indicating a lower CYP3A4 activity and an opposite direction of effect compared to the previously reported increased CYP3A4 expression. To validate, we tested associations between rs3828743 and CYP3A4 mRNA and protein expression as well as enzyme activity with data from a liver bank (n = 150). No association with any of these end points was observed. In conclusion, the totality of evidence is not in support of a significant role for TSPYL SNV rs3828743 in explaining variability in CYP3A4 activity.

From gene to dose: Long-read sequencing and *-allele tools to refine phenotype predictions of CYP2C19.

Background: Inter-individual differences in drug response based on genetic variations can lead to drug toxicity and treatment inefficacy. A large part of this variability is caused by genetic variants in pharmacogenes. Unfortunately, the Single Nucleotide Variant arrays currently used in clinical pharmacogenomic (PGx) testing are unable to detect all genetic variability in these genes. Long-read sequencing, on the other hand, has been shown to be able to resolve complex (pharmaco) genes. In this study we aimed to assess the value of long-read sequencing for research and clinical PGx focusing on the important and highly polymorphic CYP2C19 gene. Methods and Results: With a capture-based long-read sequencing panel we were able to characterize the entire region and assign variants to their allele of origin (phasing), resulting in the identification of 813 unique variants in 37 samples. To assess the clinical utility of this data we have compared the performance of three different *-allele tools (Aldy, PharmCat and PharmaKU) which are specifically designed to assign haplotypes to pharmacogenes based on all input variants. Conclusion: We conclude that long-read sequencing can improve our ability to characterize the CYP2C19 locus, help to identify novel haplotypes and that *-allele tools are a useful asset in phenotype prediction. Ultimately, this approach could help to better predict an individual's drug response and improve therapy outcomes. However, the added value in clinical PGx might currently be limited.

Why We Need to Take a Closer Look at Genetic Contributions to CYP3A Activity.

Cytochrome P450 3A (CYP3A) subfamily enzymes are involved in the metabolism of 40% of drugs in clinical use. Twin studies have indicated that 66% of the variability in CYP3A4 activity is hereditary. Yet, the complexity of the CYP3A locus and the lack of distinct drug metabolizer phenotypes has limited the identification and clinical application of CYP3A genetic variants compared to other Cytochrome P450 enzymes. In recent years evidence has emerged indicating that a substantial part of the missing heritability is caused by low frequency genetic variation. In this review, we outline the current pharmacogenomics knowledge of CYP3A activity and discuss potential future directions to improve our genetic knowledge and ability to explain CYP3A variability.

Clinical genomic profiling to identify actionable alterations for very early relapsed triple-negative breast cancer patients in the Chinese population.

BACKGROUND: Triple-negative breast cancer (TNBC) represents about 19% of all breast cancer cases in the Chinese population. Lack of targeted therapy contributes to the poorer outcomes compared with other breast cancer subtypes. Comprehensive genomic profiling helps to explore the clinically relevant genomic alterations (CRGAs) and potential therapeutic targets in very-early-relapsed TNBC patients. METHODS: Formalin-fixed paraffin-embedded (FFPE) tumour tissue specimens from 23 patients with very-early-relapsed TNBC and 13 patients with disease-free survival (DFS) more than 36 months were tested by FoundationOne CDx (F1CDx) in 324 genes and select gene rearrangements, along with genomic signatures including microsatellite instability (MSI) and tumour mutational burden (TMB). RESULTS: In total, 137 CRGAs were detected in the 23 very-early-relapsed TNBC patients, averaging six alterations per sample. The mean TMB was 4 Muts/Mb, which was higher than that in non-recurrence patients, and is statistically significant. The top-ranked altered genes were TP53 (83%), PTEN (35%), RB1 (30%), PIK3CA (26%) and BRCA1 (22%). RB1 mutation carriers had shorter DFS. Notably, 100% of these patients had at least one CRGA, and 87% of patients had at least one actionable alteration. In pathway analysis, patients who carried a mutation in the cell cycle pathway were more likely to experience very early recurrence. Strikingly, we detected one patient with ERBB2 amplification and one patient with ERBB2 exon20 insertion, both of which were missed by immunohistochemistry (IHC). We also detected novel alterations of ROS1-EPHA7 fusion for the first time, which has not been reported in breast cancer before. CONCLUSIONS: The comprehensive genomic profiling can identify novel treatment targets and address the limited options in TNBC patients. Therefore, incorporating F1CDx into TNBC may shed light on novel therapeutic opportunities for these very-early-relapsed TNBC patients.

Identification and characterization of critical genes associated with tamoxifen resistance in breast cancer.

BACKGROUND: Tamoxifen resistance in breast cancer is an unsolved problem in clinical practice. The aim of this study was to determine the potential mechanisms of tamoxifen resistance through bioinformatics analysis. METHODS: Gene expression profiles of tamoxifen-resistant MCF-7/TR and MCF-7 cells were acquired from the Gene Expression Omnibus dataset GSE26459, and differentially expressed genes (DEGs) were detected with R software. We conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses using Database for Annotation, Visualization and Integrated Discovery. A protein-protein interaction (PPI) network was generated, and we analyzed hub genes in the network with the Search Tool for the Retrieval of Interacting Genes database. Finally, we used siRNAs to silence the target genes and conducted the MTS assay. RESULTS: We identified 865 DEGs, 399 of which were upregulated. GO analysis indicated that most genes are related to telomere organization, extracellular exosomes, and binding-related items for protein heterodimerization. PPI network construction revealed that the top 10 hub genes-ACLY, HSPD1, PFAS, GART, TXN, HSPH1, HSPE1, IRAS, TRAP1, and ATIC-might be associated with tamoxifen resistance. Consistently, RT-qPCR analysis indicated that the expression of these 10 genes was increased in MCF-7/TR cells comparing with MCF-7 cells. Four hub genes (TXN, HSPD1, HSPH1 and ATIC) were related to overall survival in patients who accepted tamoxifen. In addition, knockdown of HSPH1 by siRNA may lead to reduced growth of MCF-7/TR cell with a trend close to significance (P = 0.07), indicating that upregulation of HSPH1 may play a role in tamoxifen resistance. CONCLUSION: This study revealed a number of critical hub genes that might serve as therapeutic targets in breast cancer resistant to tamoxifen and provided potential directions for uncovering the mechanisms of tamoxifen resistance.

Metronomic chemotherapy of cyclophosphamide plus methotrexate for advanced breast cancer: Real-world data analyses and experience of one center.

BACKGROUND: Real-world data of the CM regimen [cyclophosphamide (CTX) plus methotrexate (MTX)] in metronomic pattern for advanced breast cancer is limited to small-sample or retrospective studies. This study was aimed to determine the effectiveness and safety of CM regimen in treating advanced breast cancer and to identify which patients are most likely to benefit from metronomic CM regimen. METHODS: Patients with advanced breast cancer who received the metronomic CM regimen at least once between January 2009 and February 2019 in Sun Yat-sen University Cancer Center were included. Clinicopathological characteristics were collected. Overall survival (OS) and progression-free survival (PFS) were assessed using Kaplan-Meier estimates. Characteristics between patients with PFS < 6 months and ≥6 months were compared using the Chi-square test. Univariate and multivariate Cox regression model was used to estimate the prognostic factors for PFS and OS. RESULTS: A total of 186 patients were included. The median age and follow-up were 49 years and 13.3 months, respectively. Over 50% of the patients were estrogen receptor/progesterone receptor-positive, and 60.8% had been heavily treated (≥3 lines). The objective response rate was 3.8%, the disease control rate at 12 weeks was 41.4%, and the clinical benefit rate at 24 weeks was 31.2% (58/186). The median PFS was 4.0 months [95% confidence interval (CI): 3.6-4.7 months], the median duration of clinical benefit was 9.5 months (95% CI: 8.2-10.8 months), and the median OS was 26.8 months (95% CI: 20.9-37.7 months). Multivariate analysis for PFS revealed the CM regimen as maintenance therapy and no liver metastasis as favorable prognostic factors. Furthermore, patients without liver metastasis were more likely to have a PFS over 6 months than those with liver involvement (P = 0.022). Liver, lymph node, and brain metastases were unfavorable prognostic factors for OS. The CM regimen was well-tolerated without newly reported adverse events. CONCLUSIONS: The CM regimen was effective in selected patients. In clinical practice, it would be better used as maintenance therapy and in patients without liver metastasis. Further follow-up investigation should be performed to examine its effect when used in combination with other treatments and determine predictive biomarkers.

NPM1 upregulates the transcription of PD-L1 and suppresses T cell activity in triple-negative breast cancer.

Programmed cell death protein-1 (PD-1)/programmed cell death ligand-1 (PD-L1) interaction plays a crucial role in tumor-associated immune escape. Here, we verify that triple-negative breast cancer (TNBC) has higher PD-L1 expression than other subtypes. We then discover that nucleophosmin (NPM1) binds to PD-L1 promoter specifically in TNBC cells and activates PD-L1 transcription, thus inhibiting T cell activity in vitro and in vivo. Furthermore, we demonstrate that PARP1 suppresses PD-L1 transcription through its interaction with the nucleic acid binding domain of NPM1, which is required for the binding of NPM1 at PD-L1 promoter. Consistently, the PARP1 inhibitor olaparib elevates PD-L1 expression in TNBC and exerts a better effect with anti-PD-L1 therapy. Together, our research has revealed NPM1 as a transcription regulator of PD-L1 in TNBC, which could lead to potential therapeutic strategies to enhance the efficacy of cancer immunotherapy.

Administration of Lapatinib with Food Increases Its Plasma Concentration in Chinese Patients with Metastatic Breast Cancer: A Prospective Phase II Study.

LESSONS LEARNED: Administration of lapatinib with food significantly increased its plasma concentration in Chinese patients with metastatic breast cancer. There were no serious adverse events during the study and no significant differences in lapatinib-related adverse events between the fasted and fed states. BACKGROUND: Lapatinib, a small molecular reversible dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR) and human epidermal growth receptor 2 (HER2), was approved for use in combination with capecitabine to treat metastatic HER2-positive breast cancer. Administration of lapatinib in the fasted state was recommended; however, our preliminary phase II trial data showed that administration of lapatinib with food increased its concentration. METHODS: This study was a single-center, open-label, and prospective self-controlled clinical study. Ten Chinese patients with metastatic breast cancer were enrolled from June 2017 to April 2018. They were required to receive lapatinib plus physician's choice of chemotherapy. Patients were required to take lapatinib orally on an empty stomach continually for 10 days, and then take lapatinib with food continually for the next 10 days. Plasma concentration was measured by liquid chromatography on the 9th and 10th day of each state. RESULTS: Area under the concentration-time curve (AUC) of the fasted state and the fed state was 21.23 ± 8.91 mg*h/L (coefficient of variation (CV)% 42%) and 60.60 ± 16.64 mg*h/L (CV% 27%), respectively. The mean plasma concentration in the fasted state was 0.88 ± 0.39 mg/L (CV% 45%), and that in the fed state was 2.53 ± 0.77 mg/L (CV% 30%). Compared with taking lapatinib on an empty stomach, receiving lapatinib with food significantly increased the plasma concentration of lapatinib (Wilcoxon match-paired test, p = .005). In addition, there were no serious adverse events during the study or significant difference in lapatinib-related adverse events between the two states. CONCLUSION: Our study shows that receiving lapatinib with food can increase its plasma concentration with no significantly increased drug-related toxicity. We suggest that a larger-sample-size clinical trial is needed to fully understand the effect of administration of lapatinib with food.

Local treatment for liver oligometastases in breast cancer patients: identification of prognostic factors and exploration of appropriate treatment strategy.

BACKGROUND: The role of local treatment for liver oligometastases in breast cancer patients has been controversial. The aim of the study was to evaluate the prognostic factors for overall survival (OS) and progression-free survival (PFS) after hepatic local treatment and to explore appropriate therapy strategy in breast cancer patients with liver oligometastases. METHODS: A cohort of 91 patients with oligometastatic liver lesions identified from around 34,000 breast cancer patients between 2002 and 2018 were retrospectively reviewed. We collected and analyzed their clinicopathologic and outcome data. RESULTS: With a median follow up of 20.6 months [standard deviations (SD): 35.4], median OS and median PFS were 75.1 months [95% confidence interval (CI): 14.3-135.9 months] and 7.4 months (95% CI: 4.4-10.4 months), respectively. N3 stage, triple-negative breast cancer (TNBC) subtype, progressive disease (PD) after preoperative systemic therapy and older age of operation were associated with worse OS and PFS while postoperative systemic therapy alone, preoperative [partial response (PR)/stable disease (SD)] combined with postoperative systemic therapy indicated improved OS and PFS by univariate analysis. On multivariate analysis, N3 stage [hazard ratios (HR) 23.567; 95% CI: 1.751-317.277; P=0.017] and TNBC subtype (HR 10.758; 95% CI: 1.120-103.301; P=0.040) were related to worse OS. Likewise, N3 stage (HR 3.324; 95% CI 1.604-6.890; P=0.001) and TNBC subtype (HR 3.134; 95% CI: 1.015-9.674; P=0.047) remained associated with worse PFS. The postoperative mortality and morbidity were 0% and 1.099%, respectively. CONCLUSIONS: In well-selected patients, local treatment of breast cancer liver oligometastases is safe and achieves relatively long OS, except patients with advanced N stage or aggressive subtype. Postoperative systemic treatment and effective preoperative systemic therapy combined with postoperative systemic therapy are the recommended treatment strategies.

Tumor stromal type is associated with stromal PD-L1 expression and predicts outcomes in breast cancer.

BACKGROUND/AIM: The aim of this study is to determine the relationship between stromal types, PD-L1 status and clinicopathological characteristics in patients with different molecular subtypes of breast cancer. MATERIALS AND METHODS: Protein expression levels of PD-L1 were determined by immunohistochemistry assay. Stromal type was classified based on the maturity of the tumor stroma. RESULTS: Different subtypes of breast cancer had distinct stromal types. Tumors from patients with mature stroma had lower pathological N stage and AJCC stage, more frequent high p53 expression and positive stromal PD-L1 staining. Hormone receptor negative patients had higher frequency of positive stromal PD-L1 staining. Stromal PD-L1 status was also associated with different breast cancer subtypes and EGFR expression level. Importantly, our data revealed that stromal types and stromal PD-L1 status were independent prognostic factors. CONCLUSION: This study highlighted the importance of stromal types and stromal PD-L1 status in determining clinical outcomes in patients with breast cancer, and suggested that stromal type classification might be readily incorporated into routine clinical risk assessment following curative resection or optimal therapeutic design.

Panobinostat (LBH589) inhibits Wnt/ß-catenin signaling pathway via upregulating APCL expression in breast cancer.

Breast cancer is the most common malignant disease among women worldwide and the novel therapeutic agents are urgently needed. Panobinostat (LBH589), a pan-HDACs inhibitor, has shown promising anti-tumor effect in recent years. However, the targets of this compound are largely unclear because of its low selectivity. In consideration of the transcription promoting activity of panobinostat, we speculated that specific tumor suppressor genes might be upregulated after panobinostat treatment. In this study, we verified the inhibition effect of panobinostat in different subtypes of breast cancer cells in vivo and in vitro. We found that panobinostat suppressed proliferation, migration as well as invasion, and induced apoptosis in both TNBC and non-TNBC cells. Consistently, panobinostat inhibited breast cancer growth and metastasis in mouse models. Mechanistically, we found APCL transcription and expression was significantly upregulated in panobinostat treated cells by RNA microarray analysis, while knockdown of APCL resulted in reduced sensitivity to panobinostat in breast cancer cells. APCL is a wnt/ß-catenin pathway regulator that promotes ß-catenin ubiquitylation and degradation. We found that panobinostat inhibited ß-catenin expression by increasing its ubiquitylation and thus reducing its half-life. In addition, the expression of ß-catenin activated targets including c-Jun, c-Myc, Cyclin D1 and CD44 were also decreased by panobinostat treatment in breast cancer cells. These results suggested that panobinostat inhibited tumor growth and metastasis via upregulating APCL expression in breast cancer cells, which was a novel and crucial mechanism of panobinostat.

CDK4/6 inhibitor palbociclib enhances the effect of pyrotinib in HER2-positive breast cancer.

Human epidermal growth factor receptor 2 (HER2) is amplified in about 20% breast cancers. Treat of HER2 positive breast cancers has been greatly promoted in last few years, but the accompany HER2 blockade has hindered the therapeutic effect. Pyrotinib is a pan-HER kinase inhibitor that suppresses signaling through the RAS/RAF/MEK/MAPK and PI3K/AKT pathways. Palbociclib is a CDK4/6 inhibitor that inhibits cell cycle progression and cancer cell proliferation in ER+ breast cancers. We hypothesized that the combination of pan-HER kinase inhibitors and CDK4/6 inhibitors would show synergistic antitumor activity in vivo in vitro. Our data show that a combination of palbociclib and pyrotinib was highly synergistic in inhibiting cancer proliferation and colony formation. The combined treatment also induced significant decreases in pAKT and pHER3 activation, induced G0-G1 cell cycle arrest, and increased rates of apoptosis. In the xenograft model, the combination treatment demonstrated greater antitumor activity than either agent alone, with no apparent increase in toxicity. Our results offer a preclinical rationale clinical investigation of the effectiveness of a combination treatment of palbociclib with pyrotinib for breast cancer treatment.