Reverse Transcriptase Mutations Research Articles

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

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.

Concordance of Preferentially Expressed Antigen in Melanoma by Non-Invasively Collected Polymerase Chain Reaction and Immunohistochemistry on Paraffin Embedded Tissue

Background: ​&#x0D; Pigmented lesion evaluation remains a challenging aspect of dermatology. The DermTech Melanoma Test (DMT) is a non-invasive gene-expression 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). This registry study examines the concordance of PRAME detection by polymerase chain reaction (PCR) in samples obtained non-invasively prior to biopsy and PRAME detection by immunohistochemistry (IHC) on the same lesions after biopsy.​&#x0D; Methods: ​&#x0D; 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 DMT. Approximately 8,000 clinically atypical lesions were tested. After receiving the test result, providers followed their clinical judgement for biopsy decision. When lesions expressed genomic markers (LINC, PRAME, and/or TERT) and were biopsied, pathology reports were also submitted to the registry. The presence or absence of PRAME by immunohistochemistry (IHC) was reviewed and compared to the detection of PRAME by PCR from the DMT on the same lesion.​&#x0D; Results: ​&#x0D; At the 1-year mark of the registry, there were roughly 8,000 unique entries. Of those, 1,021 (12.8%) were positive for one or more of the DMT genomic markers. One thousand three lesions (98.2%) had records available. Pathologists used PRAME IHC for 102 lesions (10.2%). Of those, 40 (39.2%) were positive by IHC, and 62 (60.8%) were negative by IHC. PRAME positivity by PCR correlated with PRAME positivity by IHC in 35 of 40 lesions (87.5%). Conversely, PRAME was detected using PCR in 28 of 62 lesions (45.2%) where it was not detected using IHC. ​&#x0D; Conclusions: ​&#x0D; The higher sensitivity of PCR compared to IHC may explain the higher concordance when PRAME is positive by IHC than when it is negative by IHC. In this data set, when PRAME is positive by IHC it is usually also positive by PCR. When PRAME is negative by IHC, it can still be detected by PCR in a substantial percentage of cases. The increased sensitivity of PCR is likely due to several factors, including its detection of the PRAME mRNA and sampling of the entire lesion. As such, PRAME PCR status may aid pathologists in understanding the risk of melanoma even when IHC is negative. Further research is warranted to understand the clinical implications of PRAME PCR versus IHC positivity. ​

Global prevalence and molecular characteristics of three clades within hepatitis B virus subgenotype C2: Predominance of the C2(3) clade in South Korea.

Hepatitis B Virus (HBV) genotypes reflect geographic, ethical or clinical traits and are currently divided into 10 genotypes (A-J). Of these, genotype C is mainly distributed in Asia, is the largest group and comprises more than seven subgenotypes (C1-C7). Subgenotype C2 is divided into three phylogenetically distinct clades, C2(1), C2(2), and C2(3), and is responsible for most genotype C infections in three East Asian nations, including China, Japan, and South Korea, which are major HBV endemic areas. However, despite the significance of subgenotype C2 with regard to clinical or epidemiologic aspects, its global distribution and molecular characteristics remain largely unknown. Here, we analyze the global prevalence and molecular characteristics between 3 clades within subgenotype C2 using 1,315 full genome sequences of HBV genotype C retrieved from public databases. Our data show that almost all HBV strains from South Korean patients infected with genotype C belong to clade C2(3) within subgenotype C2 [96.3%] but that HBV strains from Chinese or Japanese patients belong to diverse subgenotypes or clades within genotype C, suggesting clonal expansion of a specific HBV type, C2(3), among the Korean population. Our genome sequence analysis indicated a total of 21 signature sequences specific to the respective clades C2(1), C2(2), and C2(3). Of note, two types of four nonsynonymous C2(3) signature sequences, sV184A in HBsAg and xT36P in the X region, were detected in 78.9 and 82.9% of HBV C2(3) strains, respectively. In particular, HBV strains C2(3) versus C2(1) and C2(2) show a higher frequency of reverse transcriptase mutations related to nucleot(s)ide analog (NA) resistance, including rtM204I and rtL180M, suggesting an increased possibility of C2(3) infection in those with NA treatment failure. In conclusion, our data show that HBV subgenotype C2(3) is extremely prevalent in Korean patients with chronic HBV infection, which is distinct from two other East Asian nations, China and Japan, where diverse subgenotypes or clades within genotype C coexist. This epidemiologic trait might affect distinct virological and clinical traits in chronic HBV patients in Korea, where exclusively C2(3) infection is predominant.

Suppressor-type TERT mutations associated with recurrence in ovarian clear cell carcinoma.

Several cancers harbor "enhancer-type" mutations of the telomerase reverse transcriptase (TERT) promoter for immortalization. Here, we report that 8.6% (8/93) of ovarian clear cell carcinomas (OCCCs) possess the "suppressor-type" TERT promoter mutation. The recurrence rate of OCCCs with "suppressor-type" TERT promoter mutations was 62.5% (5/8) and was significantly higher than that of the "unaffected-type" with no mutation (20.8%, 15/72) or "enhancer-type" TERT promoter mutations (7.7%, 1/13). Our findings show that the acquired suppression of TERT is closely associated with OCCC development and recurrence, indicating the need for further research on telomerase suppression in cancers.

No transmitted drug resistance to HIV integrase strand-transfer inhibitors after their scale-up in Estonia in 2017

In Eastern Europe HIV-1 transmitted drug resistance (TDR) data, especially in integrase (IN) region is limited. In Estonia INSTI (Integrase Strand Transfer Inhibitors) TDR has been studied only prior to the INSTI scale-up in late 2010s. Current study aimed to determine the levels of protease (PR), reverse transcriptase (RT) and IN surveillance drug resistance mutations (SDRMs) among newly diagnosed patients in Estonia in 2017. Study included 216 newly diagnosed HIV-1 individuals from January 1 until December 31, 2017 in Estonia. Demographic and clinical data were obtained from the Estonian Health Board, Estonian HIV Cohort Study (E-HIV) and clinical laboratories' databases. The PR-RT and IN regions were sequenced and analyzed for SDRMs and subtype determination. Seventy-one percent (151/213) of available HIV-positive samples were successfully sequenced. The overall level of TDR was 7.9% (12/151; 95% CI 4.4-13.8%); no dual or triple class resistance was detected. No major INSTI mutations were found. The distribution of SDRMs for NNRTI, NRTI and PI was 5.9% (9/151), 1.3% (2/151) and 0.7% (1/151), respectively. The predominant NNRTI mutation was K103N. CRF06_cpx was predominant variant (59%) in Estonian HIV-1 population followed by subtype A (9%) and subtype B (8%). Although no major INSTI mutations were found, close monitoring of INSTI SDRMs is needed considering the extensive use of the first and second-generation INSTIs. PR-RT TDR is slowly rising in Estonia indicating the need for continuous surveillance in the future. Low genetic barrier NNRTIs should be avoided in the treatment regimens.

TERT Mutations in Non-Small Cell Lung Cancer: Clinicopathologic Features and Prognostic Implications.

The associations between the clinical characteristics of non-small cell lung cancer (NSCLC) and mutations in telomerase reverse transcriptase (TERT) gene remain unclear. In this study, we used next-generation sequencing (NGS) to investigate the incidence rate and clinical correlates of TERT mutations in patients with NSCLC. In total, 283 tumor samples from patients with NSCLC were tested using an NGS panel from September 2017 to May 2020. The genetic testing results and clinical data of all patients were collected. TERT mutations were found in 30 patients, which were significantly associated with age, smoking history, sex, and metastasis (P < 0.05). Survival analyses showed that patients who carried TERT mutations had a poorer prognosis. Of the 30 TERT-mutation carriers, 17 harbored epidermal growth factor receptor (EGFR) mutations, which were significantly associated with sex, histopathology type, and metastasis (P < 0.05; overall survival [OS], 21 months; 95% confidence interval [CI], 8.153-33.847 months). Three TERT mutation patients harbored Kirsten rat sarcoma virus (KRAS) mutations, which were significantly associated with metastasis risk (P < 0.05), KRAS mutations carriers had a worse prognosis, with an OS of 10 months (95% CI, 8.153-33.847 months). Multivariate Cox regression analyses showed that age, cancer stage, and TERT mutation carrier status were independent risk factors for NSCLC, and the TERT mutation was 2.731 times higher than that without TERT mutation (95% CI, 1.689-4.418, P < 0.001). TERT mutations were present in 11% of patients with NSCLC. TERT mutations were associated with age, smoking history, sex, and distant metastasis. Co-mutations in TERT and EGFR/KRAS indicated a poor prognosis. The co-mutations of TERT and EGFR differed according to sex, histopathology type, and metastasis, whereas TERT and KRAS co-mutations were only associated with patient metastasis. Age, cancer stage, and TERT mutation carrier status were independent risk factors for poor prognosis in patients with NSCLC.

Importancia pronóstica de las mutaciones del gen promotor de la transcriptasa inversa de la telomerasa en los meningiomas de alto grado

Estimar la importancia pronóstica de las mutaciones de la transcriptasa inversa de la telomerasa en pacientes colombianos con meningiomas de grados II y III. Materiales y métodos. Es un estudio de cohorte, retrospectivo y multicéntrico, que incluyó pacientes con diagnóstico de meningioma persistente o recidivante, de grados II y III, según la clasificación de la OMS, reclutados entre el 2011 y el 2018, con tratamiento sistémico (sunitinib, everolimus con octreótido o sin él, y bevacizumab). El estado de la mutación del promotor de la transcriptasa inversa de la telomerasa se determinó por medio de la PCR. Se incluyeron 40 pacientes, en 21 (52,5 %) de los cuales se encontraron mutaciones en la transcriptasa inversa de la telomerasa, siendo las variantes más frecuentes la C228T (87,5 %) y la C250T (14,3 %). Estas fueron más frecuentes entre los pacientes con meningiomas anaplásicos (p=0,18), en aquellos con más de dos recurrencias (p=0,04), y en los que presentaron lesiones en la región parasagital y la fosa anterior (p=0,05). Los sujetos caracterizados por tener alteraciones puntuales fueron tratados con mayor frecuencia con la serie de medicamentos everolimus, sunitinib y bevacizumab (p=0,06). Tras el inicio del tratamiento médico, la supervivencia global fue de 23,7 meses (IC95% 13,1-34,2) en los pacientes con mutaciones y, de 43,4 meses (IC95% 37,5-49,3), entre aquellos sin mutaciones (p=0,0001). Los resultados del análisis multivariado demostraron que, únicamente, el número de recurrencias y la presencia de mutaciones en el gen de la transcriptasa inversa de la telomerasa, fueron factores que afectaron negativamente la supervivencia global. Las mutaciones en el gen promotor de la transcriptasa inversa de la telomerasa permiten identificar los pacientes con alto riesgo, cuya detección podría ser de utilidad para seleccionar el mejor esquema terapéutico.

HIV-1 resistance against dolutegravir fluctuates rapidly alongside erratic treatment adherence: a case report

We report a case of incomplete HIV-1 suppression on a dolutegravir, lamivudine, and abacavir single-tablet regimen with the emergence of the H51Y and G118R integrase resistance mutations. Integrase sequencing was performed retrospectively by Sanger and next-generation sequencing. Rates of emergence and decline of resistance mutations were calculated using next-generation sequencing data. Dolutegravir plasma concentrations were measured by ultra-performance liquid chromatography-tandem mass spectrometry. The effects of H51Y and G118R on infectivity, fitness, and susceptibility to dolutegravir were quantified using cell-based assays. During periods of non-adherence to treatment, mutations were retrospectively documented only by next-generation sequencing. Misdiagnosis by Sanger sequencing was caused by the rapid decline of mutant strains within the retroviral population. This observation was also true for a M184V lamivudine-resistant reverse transcriptase mutation found in association with integrase mutations on single HIV genomes. Resistance rebound upon treatment re-initiation was swift (>8000 copies per day). Next-generation sequencing indicated cumulative adherence to treatment. Compared to WT HIV-1, relative infectivity was 73%, 38%, and 43%; relative fitness was 100%, 35%, and 10% for H51Y, G118R, and H51Y+G118R viruses, respectively. H51Y did not change the susceptibility to dolutegravir, but G188R and H51Y+G118R conferred 7- and 28-fold resistance, respectively. This case illustrates how poorly-fit drug-resistant viruses wax and wane alongside erratic treatment adherence and are easily misdiagnosed by Sanger sequencing. We recommend next-generation sequencing to improve the clinical management of incomplete virological suppression with dolutegravir.

Frontiers in Breast Pathology.

Frontiers in Breast Pathology.

A New HIV-1 K28E32-Reverse Transcriptase Variant Associated with the Rapid Expansion of CRF07_BC among Men Who Have Sex with Men.

ABSTRACTHIV-1 CRF07_BC originated among injection drug users (IDUs) in China. After diffusing into men who have sex with men (MSM), CRF07_BC has shown a rapid expansion in this group; however, the mechanism remains unclear. Here, we identified a new K28E32 variant of CRF07_BC that was characterized by five specific mutations (E28K, K32E, E248V, K249Q, and T338S) in reverse transcriptase. This variant was mainly prevalent among MSM, and was overrepresented in transmission clusters, suggesting that it could have driven the rapid expansion of CRF07_BC in MSM, though founder effects cannot be ruled out. It was descended from an evolutionary intermediate accumulating four specific mutations and formed an independent phylogenetic node with an estimated origin time in 2003. The K28E32 variant was demonstrated to have significantly higher in vitro HIV-1 replication ability than the wild type. Mutations E28K and K32E play a critical role in the improvement of in vitro HIV-1 replication ability, reflected by improved reverse transcription activity. The results could allow public health officials to use this marker (especially E28K and K32E mutations in the reverse transcriptase (RT) coding region) to target prevention measures prioritizing MSM population and persons infected with this variant for test and treat initiatives.IMPORTANCE HIV-1 has very high mutation rate that is correlated with the survival and adaption of the virus. The variants with higher transmissibility may be more selective advantage than the strains with higher virulence. Several HIV-1 variants were previously demonstrated to be correlated with higher viral load and lower CD4 T cell count. Here, we first identified a new variant (the K28E32 variant) of HIV-1 CRF07_BC, described its origin and evolutionary dynamics, and demonstrated its higher in vitro HIV-1 replication ability than the wild type. We demonstrated that five RT mutations (especially E28K and K32E) significantly improve in vitro HIV-1 replication ability. The appearance of the new K28E32 variant was associated with the rapidly increasing prevalence of CRF07_BC among MSM.

Compassionate use of long-acting cabotegravir plus rilpivirine for people living with HIV-1 in need of parenteral antiretroviral therapy.

Physicians could request compassionate use of oral and long-acting (LA) cabotegravir + rilpivirine for people living with HIV-1 under a single-patient request programme supported by ViiV Healthcare and Janssen. Outcomes are reported. Eligibility criteria included need for parenteral therapy, no primary resistance mutations to cabotegravir or rilpivirine, and established retention in care. Demographic, efficacy, and safety data were obtained from standardized programme applications and quarterly clinical updates. Individuals received a loading dose of LA cabotegravir 600 mg + rilpivirine 900 mg, followed by LA maintenance doses of 400 mg/600 mg every 4 weeks; some received lead-in oral cabotegravir and rilpivirine. Through July 2020, 35 people living with HIV-1 had data available. The most frequent reason for compassionate use request was chronic non-adherence due to psychological conditions (n=15). Of 35 people living with HIV-1, 28 had detectable viremia (median viral load 60 300 copies/mL) and seven were virologically suppressed at programme entry; 16/28 and 6/7 achieved or maintained virological suppression at data cutoff, respectively. Seven people living with HIV-1 discontinued for incomplete virological response, six with detectable viremia at initiation; six and four had new reverse transcriptase and integrase mutations at discontinuation, respectively. Six non-fatal serious adverse events were reported, two considered possibly treatment related. Four deaths were reported; none were treatment related. One individual reported two pregnancies and continued LA dosing. Most people living with HIV-1 had advanced disease and achieved (16/28) or maintained (6/7) virological suppression with LA therapy. Cabotegravir LA + rilpivirine LA as compassionate use provided a valuable treatment option for individuals with adherence issues with oral therapy and advanced disease.