Nanochannel-based biosensor for ultrasensitive and label-free detection of thymidine kinase activity.

Thymidine Kinase 1 (TK1) is a pivotal enzyme in fundamental biochemistry and molecular diagnosis, but recognition and molecule detection is a challenging task. Here, we constructed a DNA-integrated hybrid nanochannel sensor for TK1 activity and inhibition assay. Single-stranded DNA containing thymidine was used as a substrate to functionalize the nanochannels, restricting the ion current through channels. With kinase, the thymidine at the termini of the substrate DNA is phosphorylated, elevating surface charge density and mitigating the pore-obstruction effect by increasing transmembrane ion current. The kinase-induced distinctness can be accurately monitored by this hybrid nanodevice, which benefits from its high sensitivity to the change of surface charge. The excellent analytical performance in both kinase enzyme activity and inhibition analysis resulted in efficient and selective evaluation in human serum. Furthermore, compared to current approaches, it greatly simplifies and offers a direct method of analysis, making it a promising sensor technology for cancer management as well as the activities of multiple types of nucleic acid kinases.

DNA nanosensor based on bipedal 3D DNA walker-driven proximal catalytic hairpin assembly for sensitive and fast TK1 mRNA detection.

Hyperproliferative  diseases are the first step for tumor formation; thymidine kinase 1 (TK1) mRNA is closely related to cell proliferation. Therefore, the risk of malignant proliferation can be identified by sensitively detecting the variance in TK1 mRNA concentration, which can be used for tumor auxiliary diagnosis and monitoring tumor treatment. Owing to the low abundance and instability of TK1 mRNA in real samples, the development of a sensitive and fast mRNA detection method is necessary. A DNA nanosensor that can be used for detecting TK1 mRNA based on bipedal 3D DNA walker-driven proximal catalytic hairpin assembly (P-CHA) was developed. P-CHA hairpins were hybridized to a linker DNA strand coupled with magnetic nanoparticles to increase their local concentrations. The bipedal DNA walking on the surface of NPs accelerates reaction kinetics using the proximity effect. Taking advantage of the signal amplification of P-CHA as well as the rapid reaction rate of the DNA walker in 80 min, the proposed sensor detects TK1 mRNA with a low detection limit of 14 pM and may then be applied to clinical diagnosis.

Nomogram model of serum thymidine kinase 1 combined with ultrasonography for prediction of central lymph node metastasis risk in patients with papillary thyroid carcinoma pre-surgery.

Objective: The aim of this study was to develop a nomogram, using serum thymidine kinase 1 protein (STK1p) combined with ultrasonography parameters, to early predict central lymph node metastasis (CLNM) in patients with papillary thyroid carcinoma (PTC) pre-surgery. Methods: Patients with PTC pre-surgery in January 2021 to February 2023 were divided into three cohorts: the observation cohort (CLNM, n = 140), the control cohort (NCLNM, n = 128), and the external verification cohort (CLNM, n = 50; NCLNM, n = 50). STK1p was detected by an enzyme immunodot-blot chemiluminescence analyzer and clinical parameters were evaluated by ultrasonography. Results: A suitable risk threshold value for STK1p of 1.7 pmol/L was selected for predicting CLNM risk by receiver operating characteristic (ROC) curve analysis. Multivariate analysis identified the following six independent risk factors for CLNM: maximum tumor size >1 cm [odds ratio (OR) = 2.406, 95% confidence interval (CI) (1.279-4.526), p = 0.006]; capsule invasion [OR = 2.664, 95% CI (1.324-5.360), p = 0.006]; irregular margin [OR = 2.922; 95% CI (1.397-6.111), p = 0.004]; CLN flow signal [OR = 3.618, 95% CI (1.631-8.027), p = 0.002]; tumor-foci number ≥2 [OR = 4.064, 95% CI (2.102-7.859), p < 0.001]; and STK1p ≥1.7 pmol/L [OR = 7.514, 95% CI (3.852-14.660), p < 0.001]. The constructed nomogram showed that the area under the ROC curve for the main dataset was 0.867 and that for the validation dataset was 0.830, exhibiting effectivity, and was recalculated to a total score of approximately 383. Through monitoring the response post-surgery, all patients were assessed as tumor-free at 12 months post-surgery, which was significantly associated with a reduction in STK1p to disease-free levels. Conclusion: We demonstrate for the first time that a novel nomogram including STK1p combined with ultrasonography can assist in the clinical prevention of CLNM, by facilitating timely, individualized prophylactic CLNM dissection, thereby reducing the risk of secondary surgery and the probability of recurrence.

Evaluation of Serum Cytokeratines, Thymidine Kinase, and Growth Factors as Cancer Biomarkers in Colorectal Cancer.

BACKGROUND/AIM: Classical serum cancer biomarkers, such as carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA 19-9), remain important tools in colorectal cancer (CRC) management for disease follow up. However, their sensitivity and specificity are low for diagnostic and prognostic evaluation. The aim of this study was to evaluate the potential of biomarkers reflecting biological activity of tumors - tissue polypeptide specific antigen (TPS), cytokeratin fragment 19 (CYFRA 21-1), thymidine kinase (TK), insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGF-BP3) - together with the CEA and CA 19-9 in CRC diagnosis and prognosis. PATIENTS AND METHODS: This is a retrospective study including 148 CRC patients and 68 age-matched healthy subjects. Serum biomarkers were measured in pre-operative serum samples using immunoanalytical methods. The end-point for the diagnostic evaluation was the area under the receiving operating characteristic curve (AUC ROC) of the biomarkers. The end-point for the prognostic evaluation was overall survival. RESULTS: Serum levels of CEA, CA 19-9, TPS, and TK were significantly increased in CRC early-stage patients compared with healthy controls. Each of the studied biomarkers had AUC between 0.6 and 0.7. Analysis of survival demonstrated that the patients with CEA, CA 19-9, cytokeratin, and TK above optimal cut offs had significantly shorter survival. A multivariate analysis performed on all the study biomarkers resulted in the selection of CYFRA 21-1 as the best performing biomarker with hazard ratio 10.413. CONCLUSION: The combination of cytokeratins and thymidine kinase with classical cancer biomarkers enables the prediction of tumor aggressiveness and long-term prognosis.

Integration of Hybridization Chain Reaction and Protein-Binding Amplification for Long-Term Imaging of Intracellular mRNA: Avoiding Signal Fluctuation.

Amplified nanoprobes based on hybridization chain reaction (HCR) have been widely developed for the detection of intracellular low abundance mRNA. However, the formed chain-like assembly decorated with fluorophore would be degraded rapidly by endogenous enzyme, resulting in failure of the long-term fluorescence imaging. To address this issue, herein, a composite signal-amplifying strategy that integrates HCR into protein-binding signal amplification (HPSA) was communicated for the in situ imaging of mRNA by avoiding signal fluctuation. Different from conventional HCR-based nanoprobes (HCR-nanoprobe), the HCR was used as the signal-triggered mode and the amplifying signal generated from in situ fluorophore-protein binding in cells, which can maintain high stability of the signal for a long time. As a proof-of-principle, a nanobeacon based on HPSA (HPSA-nanobeacon) was constructed to detect TK1 mRNA. Taking advantage of the double signal-amplifying mode, the endogenous TK1 mRNA was sensitively detected and the fluorescence signal was maintained for more than 8 h in HepG2 cells. The attempt in this work provides a new option to the current signal-amplifying strategy for sensing nucleic acid targets with high stability, significantly enhancing the acquisition of intracellular molecular information.

Development of a gold nanoparticle-based lateral flow immunochromatographic assay for the rapid and quantitative detection of thymidine kinase 1 in human serum.

Thymidine kinase 1 (TK1) is a marker of cell proliferation that can be used for early screening, treatment monitoring, and evaluating the prognosis of patients with tumors. The main purpose of this study was to develop clinically applicable TK1 antibodies, establish an appropriate detection method, and provide material and technical support for the research and clinical application for different types of tumors. Experimental mice were immunized with the C-terminal 31 peptide of human TK1 to screen monoclonal cell lines capable of stably secreting specific antibodies. Monoclonal antibodies were then prepared, purified and screened for optimal pairing following the identification of purity and isotype. Finally, based on the principles adopted by the double-antibody sandwich detection method, we constructed a lateral flow immunochromatographic assay (LFIA) to quantify the concentration of TK1 in serum samples when using a gold nanoparticle-labeled anti-TK1 monoclonal antibody as a probe. The limit of detection for TK1 in serum was 0.31 pmol/L with a detection range of 0.31-50 pmol/L. The spiked recoveries ranged from 97.7% to 109.0% with an analytical precision of 5.7-8.2%; there was no cross-reactivity with common proteins in the serum. The established LFIA also exhibited good consistency with commercially available chemiluminescent immunoassay kits for the detection of clinical samples. The LFIA developed in this study has the advantages of high sensitivity, accuracy, reproducibility and strong specificity, and provides a new technical tool for the quantitative detection of TK1.

Therapeutic targeting at genome mutations of liver cancer by the insertion of HSV1 thymidine kinase through Cas9-mediated editing.

BACKGROUND: Liver cancer is one of the most lethal malignancies for humans. The treatment options for advanced-stage liver cancer remain limited. A new treatment is urgently needed to reduce the mortality of the disease. METHODS: In this report, we developed a technology for mutation site insertion of a suicide gene (herpes simplex virus type 1- thymidine kinase) based on type II CRISPR RNA-guided endonuclease Cas9-mediated genome editing to treat liver cancers. RESULTS: We applied the strategy to 3 different mutations: S45P mutation of catenin beta 1, chromosome breakpoint of solute carrier family 45 member 2-alpha-methylacyl-CoA racemase gene fusion, and V235G mutation of SAFB-like transcription modulator. The results showed that the herpes simplex virus type 1-thymidine kinase insertion rate at the S45P mutation site of catenin beta 1 reached 77.8%, while the insertion rates at the breakpoint of solute carrier family 45 member 2 - alpha-methylacyl-CoA racemase gene fusion were 95.1%-98.7%, and the insertion at V235G of SAFB-like transcription modulator was 51.4%. When these targeting reagents were applied to treat mouse spontaneous liver cancer induced by catenin beta 1S45P or solute carrier family 45 member 2-alpha-methylacyl-CoA racemase, the mice experienced reduced tumor burden and increased survival rate. Similar results were also obtained for the xenografted liver cancer model: Significant reduction of tumor volume, reduction of metastasis rate, and improved survival were found in mice treated with the targeting reagent, in comparison with the control-treated groups. CONCLUSIONS: Our studies suggested that mutation targeting may hold promise as a versatile and effective approach to treating liver cancers.

A comprehensive analysis and experimental validation of TK1 in uterine corpus endometrial carcinoma.

Uterine corpus endometrial carcinoma (UCEC) is becoming a main malignant cancer that threaten to women's health. Thymidine kinase 1 (TK1) is considering to be associated with tumorigenesis and development. Nevertheless, the function of TK1 in UCEC is still unclear. Herein, we analyzed the TK1 expression level in pan-cancer and found that TK1 was upregulated in a variety of cancers including UCEC. Patients of UCEC with high expression of TK1 were related to poor outcome. TK1 was also related to clinical stage, histologic grade and lymph node metastasis. Abnormal expression of TK1 in UCEC was related to promoter methylation while gene mutation was not frequent. TK1 and its associated genes appeared to be prominent in cell cycle and DNA replication, according to GO and KEGG analysis. Analysis of immune infiltration revealed a negative correlation between TK1 and CD8 + T cells, macrophages, and dendritic cells. In vitro experiments, TK1 knockdown resulted in the inhibition of proliferation, migration, invasion and EMT in UCEC cell lines.

The role of serum thymidine kinase 1 activity in neoadjuvant-treated HER2-positive breast cancer: biomarker analysis from the Swedish phase II randomized PREDIX HER2 trial.

BACKGROUND: Thymidine kinase 1 (TK1) plays a pivotal role in DNA synthesis and cellular proliferation. TK1 has been studied as a prognostic marker and as an early indicator of treatment response in human epidermal growth factor 2 (HER2)-negative early and metastatic breast cancer (BC). However, the prognostic and predictive value of serial TK1 activity in HER2-positive BC remains unknown. METHODS: In the PREDIX HER2 trial, 197 HER2-positive BC patients were randomized to neoadjuvant trastuzumab, pertuzumab, and docetaxel (DPH) or trastuzumab emtansine (T-DM1), followed by surgery and adjuvant epirubicin and cyclophosphamide. Serum samples were prospectively collected from all participants at multiple timepoints: at baseline, after cycle 1, 2, 4, and 6, at end of adjuvant therapy, annually for a total period of 5 years and/or at the time of recurrence. The associations of sTK1 activity with baseline characteristics, pathologic complete response (pCR), event-free survival (EFS), and disease-free survival (DFS) were evaluated. RESULTS: No association was detected between baseline sTK1 levels and all the baseline clinicopathologic characteristics. An increase of TK1 activity from baseline to cycle 2 was seen in all cases. sTK1 level at baseline, after 2 and 4 cycles was not associated with pCR status. After a median follow-up of 58 months, 23 patients had EFS events. There was no significant effect between baseline or cycle 2 sTK1 activity and time to event. A non-significant trend was noted among patents with residual disease (non-pCR) and high sTK1 activity at the end of treatment visit, indicating a potentially worse long-term prognosis. CONCLUSION: sTK1 activity increased following neoadjuvant therapy for HER2-positive BC but was not associated with patient outcomes or treatment benefit. However, the post-surgery prognostic value in patients that have not attained pCR warrants further investigation. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02568839. Registered on 6 October 2015.

Serum thymidine kinase 1 protein concentrations and presence of its autoantibody as biomarkers for screening dogs with malignant tumors.

BACKGROUND: An accurate and easily accessible method for diagnosing malignancies in local veterinary clinics has not yet been established. OBJECTIVES: To investigate the usefulness of serum thymidine kinase 1 (TK1) protein and its autoantibody as tumor biomarkers in dogs. ANIMALS: Serum samples from 1702 dogs were collected from local animal hospitals and referral animal medical centers in South Korea. METHODS: TK1 protein OD value and TK1 autoantibody ratio (TK1 autoantibody OD/total IgG OD) in serum samples of dogs classified into healthy controls, group with nontumor disease, group with benign and group with malignant tumors were measured using lateral flow immunochromatographic assay methods. RESULTS: TK1 autoantibody levels were significantly higher in malignant tumor group (median 0.71) than in healthy controls (median 0.34), group with nontumor disease (median 0.34), and group with benign tumor (median 0.32, Welch t test, P < .0001). They were also significantly different among dogs with carcinomas (median 0.77), hematopoietic tumors (median 0.71), and sarcomas (median 0.56) than in healthy controls (median 0.34, post hoc Games-Howell test, P < .0001). In the receiver operating characteristic curve of TK1 protein, AUC was 0.633 (95% CI: 0.592-0.675, P < .0001). The AUC of TK1 autoantibody ratio was 0.758 (95% CI: 0.723-0.793, P < .0001). CONCLUSIONS AND CLINICAL IMPORTANCE: TK1 autoantibody is a potentially useful biomarker for differentiating between healthy and tumor-bearing dogs, better than TK1 protein measurement. However, both were inadequate when used as single biomarkers for screening dogs to discover occult malignant tumors.

Development of an optimized, non-stem cell line for intranasal delivery of therapeutic cargo to the central nervous system.

Neural stem cells (NSCs) are considered to be valuable candidates for delivering a variety of anti-cancer agents, including oncolytic viruses, to brain tumors. However, owing to the previously reported tumorigenic potential of NSC cell lines after intranasal administration (INA), here we identified the human hepatic stellate cell line LX-2 as a cell type capable of longer resistance to replication of oncolytic adenoviruses (OAVs) as a therapeutic cargo, and that is non-tumorigenic after INA. Our data show that LX-2 cells can longer withstand the OAV XVir-N-31 replication and oncolysis than NSCs. By selecting the highly migratory cell population out of LX-2, an offspring cell line with a higher and more stable capability to migrate was generated. Additionally, as a safety backup, we applied genomic herpes simplex virus thymidine kinase (HSV-TK) integration into LX-2, leading to high vulnerability to ganciclovir (GCV). Histopathological analyses confirmed the absence of neoplasia in the respiratory tracts and brains of immuno-compromised mice 3 months after INA of LX-2 cells. Our data suggest that LX-2 is a novel, robust, and safe cell line for delivering anti-cancer and other therapeutic agents to the brain.

Transcriptomic analysis reveals impact of gE/gI/TK deletions on host response to PRV infection.

BACKGROUND: Pseudorabies virus (PRV) causes substantial losses in the swine industry worldwide. Attenuated PRV strains with deletions of immunomodulatory genes glycoprotein E (gE), glycoprotein I (gI) and thymidine kinase (TK) are candidate vaccines. However, the effects of gE/gI/TK deletions on PRV-host interactions are not well understood. METHODS: To characterize the impact of gE/gI/TK deletions on host cells, we analyzed and compared the transcriptomes of PK15 cells infected with wild-type PRV (SD2017), PRV with gE/gI/TK deletions (SD2017gE/gI/TK) using RNA-sequencing. RESULTS: The attenuated SD2017gE/gI/TK strain showed increased expression of inflammatory cytokines and pathways related to immunity compared to wild-type PRV. Cell cycle regulation and metabolic pathways were also perturbed. CONCLUSIONS: Deletion of immunomodulatory genes altered PRV interactions with host cells and immune responses. This study provides insights into PRV vaccine design.

TK1 expression influences pathogenicity by cell cycle progression, cellular migration, and cellular survival in HCC 1806 breast cancer cells.

Breast cancer is the most common cancer diagnosis worldwide accounting for 1 out of every 8 cancer diagnoses. The elevated expression of Thymidine Kinase 1 (TK1) is associated with more aggressive tumor grades, including breast cancer. Recent studies indicate that TK1 may be involved in cancer pathogenesis; however, its direct involvement in breast cancer has not been identified. Here, we evaluate potential pathogenic effects of elevated TK1 expression by comparing HCC 1806 to HCC 1806 TK1-knockdown cancer cells (L133). Transcriptomic profiles of HCC 1806 and L133 cells showed cell cycle progression, apoptosis, and invasion as potential pathogenic pathways affected by TK1 expression. Subsequent in-vitro studies confirmed differences between HCC 1806 and L133 cells in cell cycle phase progression, cell survival, and cell migration. Expression comparison of several factors involved in these pathogenic pathways between HCC 1806 and L133 cells identified p21 and AKT3 transcripts were significantly affected by TK1 expression. Creation of a protein-protein interaction map of TK1 and the pathogenic factors we evaluated predict that the majority of factors evaluated either directly or indirectly interact with TK1. Our findings argue that TK1 elevation directly increases HCC 1806 cell pathogenicity and is likely occurring by p21- and AKT3-mediated mechanisms to promote cell cycle arrest, cellular migration, and cellular survival.

Fluoxetine enhances the antitumor effect of olfactory ensheathing cell-thymidine kinase/ganciclovir gene therapy in human glioblastoma multiforme cells through upregulation of Connexin43 levels.

Glioblastoma multiforme (GBM) is the most invasive form of primary brain astrocytoma, resulting in poor clinical outcomes. Herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy is considered a promising strategy for GBM treatment. Since Connexin43 (Cx43) expression is reduced in GBM cells, increasing Cx43 levels could enhance the effectiveness of gene therapy. The present study aims to examine the impact of fluoxetine on HSV-TK/GCV gene therapy in human GBM cells using human olfactory ensheathing cells (OECs) as vectors. The effect of fluoxetine on Cx43 levels was assessed using the western blot technique. GBM-derived astrocytes and OECs-TK were Cocultured, and the effect of fluoxetine on the Antitumor effect of OEC-TK/GCV gene therapy was evaluated using MTT assay and flow cytometry. Our results showed that fluoxetine increased Cx43 levels in OECs and GBM cells and augmented the killing effect of OECs-TK on GBM cells. Western blot data revealed that fluoxetine enhanced the Bax/Bcl2 ratio and the levels of cleaved caspase-3 in the coculture of OECs-TK and GBM cells. Moreover, flow cytometry data indicated that fluoxetine increased the percentage of apoptotic cells in the coculture system. This study suggests that fluoxetine, by upregulating Cx43 levels, could strengthen the Antitumor effect of OEC-TK/GCV gene therapy on GBM cells.

Combined cytotoxic and immune-stimulatory gene therapy using Ad-TK and Ad-Flt3L: Translational developments from rodents to glioma patients.

Gliomas are the most prevalent and devastating primary malignant brain tumors in adults. Despite substantial advances in understanding glioma biology, there have been no regulatory drug approvals in the US since bevacizumab in 2009 and tumor treating fields in 2011. Recent phase III clinical trials have failed to meet their prespecified therapeutic primary endpoints, highlighting the need for novel therapies. The poor prognosis of glioma patients, resistance to chemo-radiotherapy, and the immunosuppressive tumor microenvironment underscore the need for the development of novel therapies. Gene therapy-based immunotherapeutic strategies that couple the ability of the host immune system to specifically kill glioma cells and develop immunological memory have shown remarkable progress. Two adenoviral vectors expressing Ad-HSV1-TK/GCV and Ad-Flt3L have shown promising preclinical data, leading to FDA approval of a non-randomized, phase I open-label, first in human trial to test safety, cytotoxicity, and immune-stimulatory efficiency in high-grade glioma patients (NCT01811992). This review provides a thorough overview of immune-stimulatory gene therapy highlighting recent advancements, potential drawbacks, future directions, and recommendations for future implementation of clinical trials.

Therapeutic effects against high-grade glioblastoma mediated by engineered induced neural stem cells combined with GD2-specific CAR-NK.

PURPOSE: High-grade glioblastoma is extremely challenging to treat because of its aggressiveness and resistance to conventional chemo- and radio-therapies. On the contrary, genetic and cellular immunotherapeutic strategies based on the stem and immune cells are emerging as promising treatments against glioblastoma (GBM). We aimed to developed a novel combined immunotherapeutic strategy to improve the treatment efficacy using genetically engineered PBMC-derived induced neural stem cells (iNSCs) expressing HSV-TK and second-generation CAR-NK cells against GBM. METHODS: iNSCs cells expressing HSV-TK (iNSCsTK) and GD2-specific CAR-NK92 (GD2NK92) were generated from PBMC-derived iNSCs and NK92 cell lines, respectively. The anti-tumor effect of iNSCsTK and the combinational therapeutics of iNSCsTK and GD2NK92 were evaluated by GBM cell line using in vitro and in vivo experiments. RESULTS: PBMC-derived iNSCsTK possessed tumor-tropism migration ability in vitro and in vivo, which exhibited considerable anti-tumor activity via bystander effect in the presence of ganciclovir (GCV). iNSCsTK/GCV could slow GBM progression and prolong median survival in tumor-bearing mice. However, the anti-tumor effect was limited to single therapy. Therefore, the combinational therapeutic effect of iNSCsTK/GCV and GD2NK92 against GBM was investigated. This approach displayed a more significant anti-tumor effect in vitro and in xenograft tumor mice. CONCLUSIONS: PBMC-derived iNSCsTK showed a significant tumor-tropic migration and an effective anti-tumor activity with GCV in vitro and in vivo. In addition, combined with GD2NK92, iNSCsTK therapeutic efficacy improved dramatically to prolong the tumor-bearing animal model's median survival.

Bifidobacterium infantis-Mediated Herpes Simplex Virus-TK/Ganciclovir Treatment Inhibits Cancer Metastasis in Mouse Model.

Previous studies have found that Bifidobacterium infantis-mediated herpes simplex virus-TK/ganciclovir (BF-TK/GCV) reduces the expression of VEGF and CD146, implying tumor metastasis inhibition. However, the mechanism by which BF-TK/GCV inhibits tumor metastasis is not fully studied. Here, we comprehensively identified and quantified protein expression profiling for the first time in gastric cancer (GC) cells MKN-45 upon BF-TK/GCV treatment using quantitative proteomics. A total of 159 and 72 differential expression proteins (DEPs) were significantly changed in the BF-TK/GCV/BF-TK and BF-TK/GCV/BF/GCV comparative analysis. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis enriched some metastasis-related pathways such as gap junction and cell adhesion molecules pathways. Moreover, the transwell assay proved that BF-TK/GCV inhibited the invasion and migration of tumor cells. Furthermore, immunohistochemistry (IHC) demonstrated that BF-TK/GCV reduced the expression of HIF-1α, mTOR, NF-κB1-p105, VCAM1, MMP13, CXCL12, ATG16, and CEBPB, which were associated with tumor metastasis. In summary, BF-TK/GCV inhibited tumor metastasis, which deepened and expanded the understanding of the antitumor mechanism of BF-TK/GCV.

Acyclovir-resistant HSV-1 isolates among immunocompromised patients in southern Taiwan: Low prevalence and novel mutations.

Herpes simplex virus type 1 (HSV-1) can establish latency in humans and easily relapse in immunocompromised patients, with significant mortality. Treatment with acyclovir (ACV) can result in the emergence of HSV resistance. A total of 440 frozen HSV-1 isolates collected from 318 patients from January 2014 to July 2019 were obtained from National Cheng Kung University Hospital in southern Taiwan. These 440 isolates were subjected to phenotypic studies for ACV-resistance by initial screening with the plaque reduction assay (PRA) and further validation by the DNA reduction assay (DRA). The ACV-resistant strains were further investigated by Sanger sequencing for the full-length UL23 and UL30 genes, which encode thymidine kinase and DNA polymerase, respectively. Hematological malignancies or hematopoietic stem-cell transplantation patients accounted for 56.9% (124/218) among the immunocompromised patients (218/318) in this study. Repeated sampling for HSV testing was 50% (109/218) in immunocompromised patients. Only 1.38% (3/218) of immunocompromised patients and 0.9% (3/318) of all patients developed ACV-resistant HSV-1 as measured by phenotypic screening assays. It is noteworthy that a novel Y248D mutation in the UL23 gene from an immunocompromised patient was found by both PRA and DRA. In 3D protein predicting analysis, uncharged Y248 was located at an alpha-helix and substituted by negative-charged D248, which may alter the function of viral thymidine kinase. Besides, three unreported mutations related to natural polymorphism were found in virus isolates from two immunocompetent patients, including 683-688 deletion, R227H, and A351D in the UL30 gene. These data show that the prevalence of ACV-resistant HSV-1 among immunocompromised patients in southern Taiwan is low. These results will be helpful for the clinical management and treatment of HSV infections.