Reverse Transcriptase Mutations Research Articles

Development of HIV Drug-Resistance Mutations and Antiretroviral Efficacy Among Vietnamese Patients After Failure of 5-Year First-Line Therapy.

The emergence of drug-resistant mutations in human immunodeficiency virus (HIV) over time presents a challenge to treatment. We describe the development of drug-resistance mutations and ART efficacy reduction in Vietnamese patients with failure of first-line ART during a 5-year period. This is a 5-year observational cohort study with HIV viral loads of patients evaluated annually for 5 years (2017-2022) at the hospitals in Vietnam. Patients with a viral load ≥ 1000 copies/mL were subjected to identifying mutations in reverse transcriptase, protease, and integrase to evaluate HIV resistance and the efficacy of ART. After 5 years of monitoring the HIV load of 2932 patients on ART, 75 (2.56%) patients had concurrent virological failure at all 5 years. In 2017, only 2/75 HIV strains possessed Protease Inhibitor (PI) resistance mutations, while 75/75 HIV strains had both Nucleoside Reverse Transcriptase Inhibitors (NRTIs) and Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) resistance mutations. Only four PI resistance variants were found, while 40 and 32 mutations were resistant to NRTIs and NNRTIs. After 5 years, the number of HIV PI resistance mutations had increased to 14, with 13 new mutations emerging. There were six novel mutations associated with resistance to NRTIs, NNRTIs, and the proportion of preexisting mutations increased from 1.3% to 13.3%. Furthermore, HIV sensitivity to ART decreased from 2.7% to 18.6%. After 5 years, HIV had increased resistance mutations to PIs, NRTIs, and NNRTIs, with PI resistance mutations increasing the most rapidly, and the decrease in HIV sensitivity to PIs was higher than that to NRTIs and NNRTIs.

HOOK3 Amplification in Bladder Urothelial Carcinoma: Insights from Gene Expression and Survival Analysis.

Bladder urothelial carcinoma (BUC) poses a significant health challenge, ranking as the fourth most common cancer among men in the United States, with a mortality rate of approximately 20%. Genetic abnormalities such as mutations in the telomerase reverse transcriptase (TERT) gene and loss of heterozygosity (LOH) on chromosome 9 are commonly observed in BUC; however, many genes involved remain unidentified. This study aimed to explore the role of HOOK3 in BUC and its impact on survival. Using data from The Cancer Genome Atlas (TCGA) and cBioPortal, we performed differential gene expression and survival analyses to compare the patients with and without HOOK3 amplification. Our findings revealed that 2.2% of genes were up-regulated and 5.3% down-regulated in the HOOK3-amplified group. These changes suggest that HOOK3 amplification is linked to distinct gene expression patterns, with a higher proportion of down-regulated genes. Pathway enrichment related to chromatin remodeling, ion transport, and mitochondrial function suggests that HOOK3 may promote genomic stability and transcriptional regulation, contributing to tumor suppression. The involvement of mitochondrial and ribosomal pathways in protein synthesis and chromosome segregation may also protect against chromosomal abnormalities and uncontrolled cell growth. HOOK3 amplification appears to correlate with improved patient survival. These results suggest a potential protective role of HOOK3 amplification in BUC. Further research is needed to explore the underlying mechanisms and their therapeutic potential for reducing BUC-related mortality.

The role of genetic and epigenetic modifications as potential biomarkers in the diagnosis and prognosis of thyroid cancer.

Thyroid cancer (TC) is the most common endocrine cancer, which contributes to more than 43,600 deaths and 586,000 cases worldwide every year. Among the TC types, PTC and FTC comprise 90% of all TCs. Genetic modifications in genes are responsible for encoding proteins of mitogen-associated protein kinase cascade, which is closely related with numerous cellular mechanisms, including controlling programmed cell death, differentiation, proliferation, gene expression, as well as in genes encoding the PI3K (phosphatidylinositol 3-kinase)/protein kinase B (AKT) cascade, which has contribution in controlling cell motility, adhesion, survival, and glucose metabolism, have been associated with the TC pathogenesis. Various genetic modifications including BRAF mutations, RAS mutations, RET mutations, paired-box gene 8/peroxisome proliferator-activated receptor-gamma fusion oncogene, RET/PTC rearrangements, telomerase reverse transcriptase mutations, neurotrophic tyrosine receptor kinase fusion genes, TP53 mutations, and eukaryotic translation initiation factor 1A X-linked mutations can effectively serve as potential biomarkers in both diagnosis and prognosis of TC. On the other hand, epigenetic modifications can lead to aberrant functions or suppression of a range of signalling cascades, which can ultimately result in cancer. Various studies have observed the link between epigenetic modification and multiple cancers including TC. It has been reported that several epigenetic alterations including histone modifications, aberrant DNA methylation, and epigenetic modulations of non-coding RNAs can play significant roles as potential biomarkers in the diagnosis and prognosis of TC. Therefore, a good understanding regarding the genetic and epigenetic modifications is not only essential for the diagnosis and prognosis of TC, but also for the development of novel therapeutics. In this review, most of the major TC-related genetic and epigenetic modifications and their potential as biomarkers for TC diagnosis and prognosis have been extensively discussed.

Clinical and Histopathological Features of Thyroid Cancer with TERT Promoter Molecular Alterations in Isolation Versus with Concurrent Molecular Alterations: A Multicenter Retrospective Study

Background/Objectives: Molecular testing of thyroid nodules enables the detection of genetic alterations, which can help assess the risk of malignancy and tumor behavior. While telomerase reverse transcriptase (TERTp) mutations are known to be associated with aggressive disease, their exact prognostic significance when occurring alone or with other molecular alterations remains underreported. Methods: This study examined patients with thyroid cancer treated at two tertiary care hospitals from 2017 to 2024. We compared tumor behavior in patients with TERTp molecular alterations occurring alone and with concurrent molecular alterations. Aggressive histologic subtypes were defined as tall-cell, hobnail, and columnar variants of papillary carcinoma, as well as poorly differentiated and anaplastic carcinoma. High-risk disease was defined according to the 2015 ATA guidelines as gross extrathyroidal extension, lymph node metastasis >3 cm, postoperative elevated serum thyroglobulin, distant metastases, and/or positive resection margins. Statistical analysis was performed to assess differences between groups. Results: 30 patients with TERTp-positive thyroid malignancies were included. TERTp/BRAF V600E was the most prevalent mutation combination (n = 13, 43.3%), followed by TERTp alone (n = 8, 26.7%) and TERTp/RAS (n = 7, 23.4%). TERTp/EIF1AX/GNAS and TERTp/EIF1AX/PIK3CA were the least common combinations (n = 1, 3.3% each). Nodules with TERTp and concurrent mutations were significantly more likely to be classified as high-risk (p = 0.006) and were more frequently associated with aggressive histologic subtypes (p = 0.003) compared to those with TERTp mutations alone, which tended to exhibit more benign behavior. Conclusions: Thyroid carcinomas harboring both TERTp and concurrent molecular alterations are associated with more aggressive features and a higher likelihood of being classified as high-risk. In contrast, TERTp mutations occurring alone do not confer an elevated risk.

Cell Senescence and the Genetics of Melanoma Development.

Cutaneous malignant melanoma is an aggressive skin cancer with an approximate lifetime risk of 1 in 38 in the UK. While exposure to ultraviolet radiation is a key environmental risk factor for melanoma, up to ~10% of patients report a family history of melanoma, and ~1% have a strong family history. The understanding of causal mutations in melanoma has been critical to the development of novel targeted therapies that have contributed to improved outcomes for late-stage patients. Here, we review current knowledge of the genes affected by familial melanoma mutations and their partial overlap with driver genes commonly mutated in sporadic melanoma development. One theme linking a set of susceptibility loci/genes is the regulation of skin pigmentation and suntanning. The largest functional set of susceptibility variants, typically with high penetrance, includes CDKN2A, RB1, and telomerase reverse transcriptase (TERT) mutations, associated with attenuation of cell senescence. We discuss the mechanisms of action of these gene sets in the biology and progression of nevi and melanoma.

Efficacy of first-line ART regimens based on tenofovir in HIV-infected patients with pre-existing A62V mutation in reverse transcriptase.

The amino acid substitution A62V in reverse transcriptase was identified as a mutation correlated with virologic failure in patients on first-line therapy including tenofovir (TDF) and tenofovir alafenamide (TAF). A62V is a typically polymorphic mutation in HIV-1 sub-subtype A6, which is the most widespread virus variant in Russia. The European EuResist (EIDB) database was queried to form two equivalent groups of patients: group 1 ‒ patients with A62V at baseline treated with TDF or TAF on the first-line therapy, group 2 ‒ patients without A62V at baseline treated with TDF or TAF on the first-line therapy. Each group included 23 patients. There was no statistical difference between the two groups in virologic efficacy in 4, 12, and 24 weeks after the start of antiretroviral therapy (ART) and in the frequency of virologic failures. This study has some limitations, and the exact role of A62V in the efficacy of the first-line ART based on tenofovir deserves further investigation.

DORAVIR: a French national survey of people with HIV-1 treated with an antiretroviral regimen including doravirine.

Doravirine is the latest NNRTI to be approved for the treatment of HIV-1 and has a different resistance profile from first-generation NNRTIs. Our aim was to investigate the virological efficacy of antiretroviral treatment including doravirine in people living with HIV-1 (PLWHIV), the factors associated with virological failure (VF) and those associated with the emergence of reverse transcriptase (RT) mutations in the case of VF. A retrospective national survey of PLWHIV who were either naive or experienced on antiretroviral treatment including doravirine was conducted. VF was defined as two consecutive plasma viral loads (VLs) of ≥50 copies/mL or one VL of ≥200 copies/mL. Genotypic resistance tests were interpreted using the Stanford (v9.4.1) and ANRS (v33) algorithms. Of the 589 PLWHIV treated with a doravirine-containing regimen, 8.5% were naive and 91.5% had prior antiretroviral experience; 56.9% were infected with HIV-1 B subtype. Overall, 88.3% and 85.1% of participants were virologically controlled at Month (M)3 and M6 of doravirine treatment, respectively. In multivariable analysis, CRF02_AG subtype, higher zenith plasma HIV-1 RNA VL, doravirine initiation in the context of failure and baseline V179D mutation presence were associated with VF. Among 88 PLWHIV who experienced virological failure at M6, 15.9% had a median of 2 (IQR 1-3) HIV RT mutations. In multivariable analysis, the only factor associated with the occurrence of mutations was a genotypic sensitivity score that was not fully sensitive. This study is one of the largest to characterize the virological efficacy of doravirine-containing regimens in clinical practice and to identify factors associated with VF or emergence of resistance mutations that should be considered in clinical management.

A systematic analysis with the hierarchical cluster analysis strategy on the complex interaction of TERT and CTNNB1 somatic mutations in Vietnamese hepatocellular carcinoma patients

Telomerase reverse transcriptase (TERT) and β-catenin (CTNNB1) mutations may occur following the hepatocellular carcinoma (HCC) pathway signal. We conducted a Hierarchical cluster analysis study on 408 patients diagnosed with HCC by pathological surgery, identifying TERT promoter and CTNNB1 exon 3 mutations by sequencing. The overall preclinical characteristics, cumulative cut-point values, and the factors associated with these somatic mutations were analyzed in uni/multidimensional scaling model. HBV(+) HCV(−) HCC male patients who were older than 62.74 years old and have TERT promoter mutation as well as AFP > 489.78 ng/ml got a higher risk of HCC grade more than two from 27 % to 200 % with p < 0.05 (RR are from 1.27 [1.09–1.47] to 3.06 [2.04–4.61]). This mutation was a good indicator of grade 2 risk (HR = 0.37 [2.72–0.16], β = −1.00, p = 0.019). TERT promoter and CTNNB1 exon 3 mutations independently influenced tumor size and tumor site status in grade 3 and HBV(−) HCV (−) male HCC patients, where the hazard rates, respectively, were 0.28 [0.09–0.89], 0.023 [0.0023–0.23] and 0.06 [0.012–0.32] (β < 0 and p < 0.01). These two mutations inversely impacted each other the tumor sites status, especially in male HCC patients with grade 2 without B, C hepatitis virus (RRCTNNB1 exon 3 mutate - TERT promoter wildtype = 1.12 [1.04–1.20], p < 0.05). Consequently, the mutations in TERT promoter and CTNNB1 exon 3 may synchronize with other factors or independently impact the hepatocarcinogenesis and are important indicators for HCC prognostic in male patients with very high AFP levels or with moderately as well as poorly differentiated in tumor. Our results serve as the basis for further studies to understand the impact of different factors on the outcome of HCC, especially in monitoring and assessing the cancer risk of patients infect HBV and carry mutations.

Performance of the Applied Biosystems HIV-1 Genotyping Kit with Integrase.

HIV genotyping is used to assess HIV susceptibility to antiretroviral drugs. The Applied Biosystems HIV-1 Genotyping Kit with Integrase (AB kit, Thermo Fisher Scientific) detects resistance-associated mutations (RAMs) in HIV protease (PR), reverse transcriptase (RT), and integrase (IN). We compared results from the AB kit with results obtained previously with the ViroSeq HIV-1 Genotyping System. DNA amplicons from the AB kit were also analyzed using next-generation sequencing (NGS). HIV RNA was extracted using the MagNA Pure 24 instrument (Roche Diagnostics; 96 plasma samples, HIV subtype B, viral load range: 530-737,741 copies/mL). FASTA files were generated from AB kit data using Exatype (Hyrax Biosciences). DNA amplicons from the AB kit were also analyzed by NGS using the Nextera XT kit (Illumina). Drug resistance was predicted using the Stanford HIV Drug Resistance Database. The mean genetic distance for sequences from ViroSeq and the AB kit was 0.02% for PR/RT and 0.04% for IN; 103 major RAMs were detected by both methods. Four additional major RAMs were detected by the AB kit only. These four major RAMs were also detected by NGS (detected in 18.1%-38.2% of NGS reads). NGS detected 27 major RAMs that were not detected with either of the Sanger sequencing-based kits. All major RAMs detected with ViroSeq were detected with the AB kit; additional RAMs were detected with the AB kit only. DNA amplicons from the AB kit can be used for NGS for more sensitive detection of RAMs.

Association of ADC of hyperintense lesions on FLAIR images with TERT promoter mutation status in glioblastoma IDH wild type.

Although mutations in telomerase reverse transcriptase (TERT) promoter (TERTp) are the most common alterations in glioblastoma (GBM), predicting TERTp mutation status by preoperative imaging is difficult. We determined whether tumour-surrounding hyperintense lesions on fluid-attenuated inversion recovery (FLAIR) were superior to those of contrast-enhanced lesions (CELs) in assessing TERTp mutation status using magnetic resonance imaging (MRI). This retrospective study included 114 consecutive patients with primary isocitrate dehydrogenase (IDH)-wild-type GBM. The apparent diffusion coefficient (ADC) and volume of CELs and FLAIR hyperintense lesions (FHLs) were determined, and the correlation between MRI features and TERTp mutation status was analyzed. In a subset of cases, FHLs were histopathologically analyzed to determine the correlation between tumor cell density and ADC. TERTp mutations were present in 77 (67.5%) patients. The minimum ADC of FHLs was significantly lower in the TERTp-mutant group than in the TERTp-wild-type group (mean, 958.9 × 10-3 and 1092.1 × 10-3 mm2/s, respectively, P < 0.01). However, other MRI features, such as CEL and FHL volumes, minimum ADC of CELs, and FHL/CEL ratio, were not significantly different between the two groups. Histopathologic analysis indicated high tumor cell density in FHLs with low ADC. The ADC of FHLs was significantly lower in IDH-wild-type GBM with TERTp mutations, suggesting that determining the ADC of FHLs on preoperative MRI might be helpful in predicting TERTp mutation status and surgical planning.

Abstract 1113 Mechanistic studies of incorporation of activated Islatravir by HIV-1 reverse transcriptase mutants

Abstract 1113 Mechanistic studies of incorporation of activated Islatravir by HIV-1 reverse transcriptase mutants

The Impact of Extensive Surgical Resection of Butterfly Glioblastomas on Outcomes in the Presence of TERT Mutation and EGFR Amplification: A Retrospective Cohort Study.

This study aimed to assess if extensive surgical resection enhances outcomes in wild-type Isocitrate Dehydrogenase (IDH) butterfly glioblastoma (B-GBM) patients, despite the presence of Telomerase Reverse Transcriptase (TERT) mutation and Epidermal Growth Factor Receptor (EGFR) amplification. The study, retrospectively conducted from 2014 to 2022, involved 723 GBM patients, 41 of whom met the criteria for IDH wild-type B-GBM. Exclusion criteria comprised prior diagnoses or treatments for low-grade glial tumors. Surgeons, employing two approaches-partial and extensive surgery-categorized patients based on age, sex, tumor location, corpus callosum involvement, and genetic characteristics. The interval between initial surgery and tumor recurrence/tumor-free period (TR/TFP) and overall survival (OS) were recorded and compared between the partial and extensive resection groups, analyzing the impact of resection width on TR/TFP and OS. Preoperative assessments utilized thin-section cranial computed tomography (CT) and contrast-enhanced magnetic resonance imaging (MRI). Intraoperatively, tumor excision was guided by sodium fluorescein, and margins were delineated via neuronavigation. Genetic alterations (TERT mutations and EGFR amplifications) were correlated with surgical type, TR/TFP, and OS. Karnofsky Performance Scale (KPS) evaluations were performed pre- and post-operatively and at key intervals, comparing outcomes between surgical groups. Standard radiotherapy and chemotherapy regimens were administered to all patients. Extensive resection yielded significantly longer TR/TFP compared to partial resection, despite TERT gene mutation and EGFR amplification being linked to shorter TR/TFP and OS. Its impact on OS, however, was not significant. KPS scores indicated a superior quality of life after extensive resection, with sustained improvement upon recurrence. Extensive resection of B-GBM, even in the presence of adverse genetic alterations, may prolong TR/TFP, offering patients a period of improved comfort with minimal distress.

Major Drug Resistance Mutations on Reverse Transcriptase Gene in Human Immunodeficiency Virus Type-1 in Indonesia: A Systematic Review.

The prevalence of HIV-1 in Indonesia is on a concerning upward trajectory, with a concurrent rise in the development of drug-resistant strains, challenging the efficacy of antiretroviral therapy (ART). Many mutations have been found in the pol gene that makes HIV resistant to ART. We aim to review the major drug resistance mutations (DRMs) of reverse transcriptase (RT) of pol gene in HIV-1 cases in Indonesia. A total of eleven articles reporting DRMs in HIV-1 subjects from various regions between 2015-2020 in Indonesia are included. The prevalence of major DRMs on the RT gene in studies included varies from 3.4% to 34%. The CRF01_AE subtype stands out as the predominant variant. Notably, the prevalence of major DRMs in ART-experienced individuals is 22.1%, while ART-naïve individuals show a lower rate of 4.4%. Among the RT gene mutations, M184I/V emerges as the most prevalent (10.5%) within the nucleos(t)ide reverse transcriptase inhibitors (NRTI) group, while K103N leads among the non-NRTI (NNRTI) group, with a frequency of 6.4%. Regionally, North Sulawesi records the highest prevalence of major DRMs in the RT gene at 21.1%, whereas Riau and Central Papua exhibit the lowest rates at 3.4%. Significant variations in drug resistance mutations within the RT gene across Indonesian regions highlight the importance of closely monitoring and evaluating the effectiveness of current antiretroviral therapy (ART) regimens. Considerably, more studies are needed to understand better and overcome the emergence of DRMs on HIV-1 patients in Indonesia.

Mutation analysis of the TERT gene in ovarian cancer patients of the Turkish population by next generation sequencing method.

Ovarian cancer (OC) ranks seventh among malignant tumors worldwide. As one of the most common gynecological malignancies, ovarian cancer has the second-highest mortality rate, after cervical and uterine cancer. Next-Generation Sequencing (NGS) technology has enhanced multi-gene panel analysis and its clinical utility for identifying cancer-causing gene mutations. This study aimed to determine the presence of significant and nonsense mutations in telomerase reverse transcriptase (TERT), alpha-thalassemia/mental retardation, X-linked (ATRX), O-6-methylguanine-DNA methyltransferase (MGMT), and isocitrate dehydrogenase 1 and 2 (IDH1/IDH2) genes using the Next-Generation Sequencing (NGS) method. A cohort of 33 patients diagnosed with ovarian cancer was included in this investigation, and peripheral blood samples were collected from all participants. Significant and nonsense mutations in TERT, ATRX, MGMT, IDH1, and IDH2 genes were detected using the Next-Generation Sequencing method. Bioinformatics analysis was conducted using the QIAGEN Clinical Insight system. Twenty-four patients exhibited seven different TERT mutations, occurring in both exonic and intronic regions. One patient displayed a c.699-3delC deletion in the intronic region of the IDH1 gene, and the c.532G > A (p.V178I) mutation observed in three patients was assessed as potentially harmful. Additionally, novel mutations c.881A > G and c.995A > G were observed in the ATRX gene. The heterozygous novel mutation identified in the ATRX gene was confirmed through Sanger sequencing. These mutations were not previously associated with ovarian cancer and are considered novel candidate markers for ovarian cancer susceptibility. Confirmation of these results through larger cohort studies or functional investigations will contribute to a better understanding of the molecular mechanisms underlying ovarian cancer.

Effect of the TERT mutation on the prognosis of patients with urothelial carcinoma: a systematic review and meta-analysis

BackgroundTelomerase reverse transcriptase (TERT) mutation represents the most prevalent genetic mutation found in urothelial carcinoma (UC) and holds potential as a prognostic indicator for tumor outcomes. However, the association between TERT mutation and prognosis in UC patients remains poorly elucidated due to conflicting findings in existing literature. Therefore, this study aimed to investigate the effect of the TERT mutation on the survival of UC patients.MethodsWe systematically searched the PubMed, Embase, and Cochrane Library databases for studies that investigated the relationship between the TERT mutation and the prognosis of UC patients. Endpoints included the 2-year and 5-year recurrence-free survival (RFS) and overall survival (OS). The Newcastle–Ottawa Scale (NOS) tool was used to assess the risk of bias in the included studies. Review Manager 5.3 was used for the meta-analysis.ResultsNine studies with a total of 1,552 patients were included in the analysis. Two studies were prospective, and seven were retrospective. The TERT promoter mutation was associated with a lower 2-year OS (relative risk [RR] = 0.92, 95% confidence interval [CI] 0.86–0.98; P = 0.007) and a lower 5-year OS (RR = 0.80, 95% CI 0.68–0.94; P = 0.008) compared with the TERT wild type. However, no significantly differences were found between two groups in terms of HR for OS (hazard ratio [HR] = 1.29, 95% CI 0.80–2.08; P = 0.29). Furthermore, we investigated the differences in RFS and disease-specific survival (DSS) between the two groups.ConclusionThe TERT mutation increases the risk of death and decreases the survival time of UC patients. TERT may be a valuable marker with individual prognostic value.

Charge Engineering of the Nucleic Acid Binding Cleft of a Thermostable HIV-1 Reverse Transcriptase Reveals Key Interactions and a Novel Mechanism of RNase H Inactivation

Coupled with PCR, reverse transcriptases (RTs) have been widely used for RNA detection and gene expression analysis. Increased thermostability and nucleic acid binding affinity are desirable RT properties to improve yields and sensitivity of these applications. The effects of amino acid substitutions in the RT RNase H domain were tested in an engineered HIV-1 group O RT, containing mutations K358R/A359G/S360A and devoid of RNase H activity due to the presence of E478Q (O3MQ RT). Twenty mutant RTs with Lys or Arg at positions interacting with the template-primer (i.e., at positions 473–477, 499–502 and 505) were obtained and characterized. Most of them produced significant amounts of cDNA at 37, 50 and 65 °C, as determined in RT-PCR reactions. However, a big loss of activity was observed with mutants A477K/R, S499K/R, V502K/R and Y505K/R, particularly at 65 °C. Binding affinity experiments confirmed that residues 477, 502 and 505 were less tolerant to mutations. Amino acid substitutions Q500K and Q500R produced a slight increase of cDNA synthesis efficiency at 50 and 65 °C, without altering the KD for model DNA/DNA and RNA/DNA heteroduplexes. Interestingly, molecular dynamics simulations predicted that those mutations inactivate the RNase H activity by altering the geometry of the catalytic site. Proof of this unexpected effect was obtained after introducing Q500K or Q500R in the wild-type HIV-1BH10 RT and mutant K358R/A359G/S360A RT. Our results reveal a novel mechanism of RNase H inactivation that preserves RT DNA binding and polymerization efficiency without substituting RNase H active site residues.

Genomic Characteristics of the New HIV-1 CRF07_BC K28E32 Variant.

Accompanied with the appearance and prevalence of the new K28E32 variant among men who have sex with men, HIV-1 circulating recombinant form 07_BC (CRF07_BC) was becoming the most predominant subtype circulating in China. The K28E32 variant with five specific mutations in reverse transcriptase coding region appears to have significantly higher in vitro HIV-1 replication ability than the wild-type strain. In this study, we characterized the special mutations/substitutions in the K28E32 variant at the genomic level. Ten specific mutations that rarely appeared in other six main HIV-1 subtypes/CRFs (A-D, CRF01_AE, and CRF02_AG) were identified in the coding genes/regions of the K28E32 variant, including S77L and a novel seven-amino acid detection (32DKELYPL38) (p6Δ7) in p6, I135L in integrase, T189S in Vif, H/Y15L/F in Vpr, I264V/A and LV/LI328-329VG in gp41, and H82C and S97P in Rev. The special locations of the novel p6Δ7, and gp41 mutations I264V/A and LV/LI328-329VG in crucial protein functional domains suggest that these mutations might be functionally important to the K28E32 variant. Furthermore, eight specific substitutions were identified in Rev responsive element (RRE) of the K28E32 variant, and were revealed to increase the stability of RRE structure with a lower minimum free energy. Whether these mutations/substitutions contribute to improved transmissibility of the CRF07_BC K28E32 variant needs to be further confirmed.

Antiviral activity of tenofovir alafenamide (TAF) against HIV-1 clinical isolates harboring K65R.

Tenofovir alafenamide (TAF) is a prodrug of the nucleoside reverse transcriptase (RT) inhibitor tenofovir (TFV). Compared to the earlier TFV prodrug, TFV disoproxil fumarate (TDF), TAF achieves more than fourfold-higher intracellular levels of its active metabolite TFV diphosphate (TFV-DP) in clinical studies, while achieving a significant reduction of TFV systemic exposure. Resistance to TFV has been well established, with the K65R mutation inRTas the signature mutation.Here we evaluated the in vitro activity of TAF and TDF in patient-derived HIV-1 isolates harboring the K65R mutation.Clinical isolates containing K65R were cloned into the pXXLAI construct (n = 42). In vitro phenotypic susceptibility of the constructs to TAF and TDF was evaluated in an MT-2 cell HIV assay and in viral breakthrough assays modeling physiological concentrations of TAF and TDF.TAF and TDF susceptibility were highly correlated in K65R-containing mutants, ranging from 2.7- to 3.0-fold (K65R alone) and 1.2- to 27.6-fold (K65R+ other RT mutations) relative to wild-type. In viral breakthrough assays mimicking differences in physiological concentrations, TAF inhibited breakthrough of 40 of 42 clinical isolates, while the TDF equivalent only inhibited 32 of 42 isolates tested.TAF displayed a higher barrier to resistance than TDF in this panel of K65R-containing clinical isolates.

Non-Invasive Genomic Profiling in Pediatric Patients

Background:Pigmented lesions with clinical features suspicious for melanoma are not uncommon in pediatric patients. Evaluation of these lesions is complicated by the low prevalence of pediatric melanoma and challenges associated with performing biopsies in children. The DermTech Melanoma Test (‘the test’) is a non-invasive genomic test designed to rule out melanoma. It consists of the Pigmented Lesion Assay (PLA), which detects RNA gene expression of long intergenic non-coding RNA 00518 (LINC00518, or LINC) and preferentially expressed antigen in melanoma (PRAME), and an add-on assay for DNA promoter mutations in telomerase reverse transcriptase (TERT), which is performed if ordered and if sufficient genomic material is available. Patients younger than 18 years were not included in initial validation studies. In this analysis, we sought to compare genomic biomarker results between uncertain pigmented lesions from adult and pediatric patients.&#x0D; Methods:De-identified samples submitted to the clinical lab for the test from May through October 2022 were used for this analysis. Genomic results were available for 36,377 samples. The anatomic locations of the lesions were categorized as head/neck, trunk, or extremities and compared between adult and pediatric patients. Positivity rates for the PLA (and each individual marker) and the TERT add-on assay were calculated for adults and patients younger than 18 years of age.&#x0D; Results:The PLA was performed on 34,050 skin samples from adults and 2,327 samples from pediatric patients. There were no differences between adults and pediatric patients in anatomic location of the lesions tested. The proportion of PLA-positive samples was similar between adult (7.0%, n=2,393) and pediatric (8.0%, n=187) patients. Rates of biomarkers detected among PLA-positive adult and pediatric patient samples, respectively, were as follows: LINC only (31.4% vs 69.5%), PRAME only (45.5% vs 14.4%), LINC and PRAME (23.0% vs 16.0%). The TERT add-on assay was performed in 11,084 samples from adults and 613 samples from pediatric patients. Of these, TERT was positive in 830 (7.5%) adult and 3 (0.5%) pediatric patient samples.&#x0D; Conclusions:This analysis provides new information about genomic profiling of uncertain pigmented lesions from pediatric patients. While overall PLA positivity rates were similar across adult and pediatric samples, the proportion positive only for LINC was more than two times higher in pediatric samples. Additionally, TERT promoter mutations were rarely detected in pediatric samples. Further investigation of the significance of LINC, PRAME, and TERT abnormalities in lesions from pediatric patients is ongoing.

Genomic Profiling of Lentigo Maligna within an Interim Registry Analysis​

Background: Pigmented lesion analysis remains a challenging aspect of dermatology. The DermTech Melanoma Test (‘the test’) is a non-invasive genomic test designed to rule-out melanoma. It consists of the pigmented lesion assay, which detects RNA products of Long Intergenic Non-Coding RNA 00518 (LINC00518) and Preferentially Expressed Antigen in Melanoma (PRAME), and an add-on assay for DNA promoter mutations in telomerase reverse transcriptase (TERT). In previous studies, the test was found to have a negative predictive value ≥99%. This interim analysis of a registry study examines the genomic patterns of the Lentigo Maligna (LM) subtype of melanoma. Methods: Between April 2021 and March 2022, multiple geographically diverse sites throughout the US submitted data to a registry to assess real-world use of the test. Approximately 8,000 clinically atypical lesions were tested. After receiving the test result, providers followed their clinical judgement for biopsy decision. Histopathologic diagnoses for biopsied lesions were correlated with test results and all melanomas were sorted into LM subtype vs non-LM subtype. In addition, lesions that were noted to be on sun exposed skin and/or noted to have solar elastosis and called atypical melanocytic hyperplasia were included. Results: At the 1-year mark of the registry, there were roughly 8000 unique entries. Of those, 1003 expressed one or more genomic markers from the DMT and had records available, and 134 (13.2%) were found to be melanoma or melanoma in-situ. More than a third (n=46, 34.3%) of the melanomas were of the Lentigo Maligna sub-type, with 7 of those being Lentigo Maligna Melanoma (LMM). Seven additional lesions were called atypical junctional melanocytic hyperplasia (AJMH) on sun-damaged skin. This group of 53 LM, LMM, and AJMH lesions were all evaluated for correlations to the DMT markers. LINC was the most commonly expressed genomic marker (n=45, 84.9%), with PRAME (n=36, 67.9%) and TERT (n=24, 45.3%) following. Most invasive tumors (LMM) expressed all three markers (n=4) and all 7 expressed LINC. Conclusion: While the original validation study included the LM subtype, this interim registry analysis demonstrates real-world use of the DMT in assessing pigmented macules concerning for LM. Over 1/3 of the melanomas reported in the registry were LM subtypes. LINC had a higher correlation with the lentigo maligna subtype of melanoma and was present in all invasive tumors. While AJMH is considered borderline, it may be worthwhile in the clinical context to group AJMH lesions with LM due to similarities in treatment.