Evelien Rondelez

Susceptibility of Human Immunodeficiency Virus Type 1 to the Maturation Inhibitor Bevirimat Is Modulated by Baseline Polymorphisms in Gag Spacer Peptide 1

ABSTRACT In this study, we evaluated baseline susceptibility to bevirimat (BVM), the first in a new class of antiretroviral agents, maturation inhibitors. We evaluated susceptibility to BVM by complete gag genotypic and phenotypic testing of 20 patient-derived human immunodeficiency virus type 1 isolates and 20 site-directed mutants. We found that reduced BVM susceptibility was associated with naturally occurring polymorphisms at positions 6, 7, and 8 in Gag spacer peptide 1.

Performance of a population-based HIV-1 tropism phenotypic assay and correlation with V3 genotypic prediction tools in recent HIV-1 seroconverters.

Background:Pure X4 and X4R5 dual-tropic viruses may be recognized in ∼15% of drug-naive HIV-1-positive patients. CCR5 antagonists are active against R5 viruses; therefore, HIV tropism should be known before their prescription. Patients and Methods:A population-based phenotypic assay was performed in 61 recent HIV-1 seroconverters. The results were compared with those obtained using 8 different predictor software programs (C4.5, C4.5 with 8 and 12, PART, SVM, Charge Rule, PSSMsinsi, PSSMx4r5, and geno2pheno), which are freely available at 3 different Web sites and use V3 sequences derived from patients viruses. Results:Phenotypic testing reported X4R5 dual-tropic viruses in 10 (16.4%) patients. CD4 cell counts and viral loads were significantly lower in X4R5 dual-tropic (450 cells/μL and 3.9 log HIV RNA copies/mL) than in R5 viruses (629 cells/μL, 4.5 log HIV RNA copies/mL) (P < 0.05). The overall concordance of genotype and phenotype was relatively good (>80%). Although specificity was >90% using all but 1 genotypic predictor (geno2pheno), however, the sensitivity for the detection of X4 variants was low (<30%), except for SVM and geno2pheno (70%). Conclusions:The prevalence of X4 and X4/R5 dual-tropic viruses in recent HIV seroconverters is 16%. Current genotypic algorithms need to be improved for the estimation of HIV-1 coreceptor use before moving to the clinic. This information is crucial for the selection of candidates to receive CCR5 antagonists in places where phenotypic tropism assays may not be feasible.

Cross-Resistance Profile Determination of Two Second-Generation HIV-1 Integrase Inhibitors Using a Panel of Recombinant Viruses Derived from Raltegravir-Treated Clinical Isolates

ABSTRACT The integrase inhibitor raltegravir (RAL) is currently used for the treatment of both treatment-naïve and treatment-experienced HIV-1-infected patients. Elvitegravir (EVG) is in late phases of clinical development. Since significant cross-resistance between RAL and EVG is observed, there is a need for second-generation integrase inhibitors (INIs) with a higher genetic barrier and limited cross-resistance to RAL/EVG. A panel of HIV-1 integrase recombinants, derived from plasma samples from raltegravir-treated patients (baseline and follow-up samples), were used to study the cross-resistance profile of two second-generation integrase inhibitors, MK-2048 and compound G. Samples with Q148H/R mutations had elevated fold change values with all compounds tested. Although samples with the Y143R/C mutation had reduced susceptibility to RAL, they remained susceptible to MK-2048 and compound G. Samples with the N155H mutation had no reduced susceptibility to compound G. In conclusion, our results allowed ranking of the INIs on the basis of the antiviral activities using recombinant virus stocks from RAL-treated patient viruses. The order according to decreasing susceptibility is compound G, MK-2048, and EVG.

Clade-specific HIV-1 integrase polymorphisms do not reduce raltegravir and elvitegravir phenotypic susceptibility.

The contribution of clade-specific polymorphisms in the HIV-1 integrase gene towards integrase inhibitor phenotypic susceptibility was tested on 137 clinical isolates, of which 60 were non-clade B strains. Control Q148R mutant virus showed fold change values of 17.85 ± 2.77 and 88.94 ± 9.02 for raltegravir and elvitegravir, respectively, whereas the average fold change for the clinical samples was 0.91 ± 0.40, and 0.84 ± 0.37. Phenotypic testing proved that clade-specific integrase polymorphisms do not contribute to reduced susceptibility towards integrase inhibitors.

Secondary Integrase Resistance Mutations Found in HIV-1 Minority Quasispecies in Integrase Therapy-Naive Patients Have Little or No Effect on Susceptibility to Integrase Inhibitors

ABSTRACT The goal of this study was to explore the presence of integrase strand transfer inhibitor (InSTI) resistance mutations in HIV-1 quasispecies present in InSTI-naïve patients and to evaluate their in vitro effects on phenotypic susceptibility to InSTIs and their replication capacities. The RT-RNase H-IN region was PCR amplified from plasma viral RNA obtained from 49 HIV-1 subtype B-infected patients (21 drug naïve and 28 failing highly active antiretroviral therapy [HAART] not containing InSTIs) and recombined with an HXB2-based backbone with RT and IN deleted. Recombinant viruses were tested against raltegravir and elvitegravir and for replication capacity. Three-hundred forty-four recombinant viruses from 49 patients were successfully analyzed both phenotypically and genotypically. The majority of clones were not phenotypically resistant to InSTIs: 0/344 clones showed raltegravir resistance, and only 3 (0.87%) showed low-level elvitegravir resistance. No primary resistance mutations for raltegravir and elvitegravir were found as major or minor species. The majority of secondary mutations were also absent or rarely present. Secondary mutations, such as T97A and G140S, found rarely and only as minority quasispecies, were present in the elvitegravir-resistant clones. A novel mutation, E92G, although rarely found in minority quasispecies, showed elvitegravir resistance. Preexisting genotypic and phenotypic raltegravir resistance was extremely rare in InSTI-naïve patients and confined to only a restricted minority of secondary variants. Overall, these results, together with others based on population and ultradeep sequencing, suggest that at this point IN genotyping in all patients before raltegravir treatment may not be cost-effective and should not be recommended until evidence of transmitted drug resistance to InSTIs or the clinical relevance of IN minor variants/polymorphisms is determined.

A combined genotypic and phenotypic human immunodeficiency virus type 1 recombinant virus assay for the reverse transcriptase and integrase genes

With the approval of the first HIV-1 integrase inhibitor raltegravir and a second one in phase III clinical development (elvitegravir), genotypic and phenotypic resistance assays are required to guide antiretroviral therapy and to investigate treatment failure. In this study, a genotypic and phenotypic recombinant virus assay was validated for determining resistance against integrase inhibitors. The assays are based on the amplification of a region encompassing not only HIV-1 integrase, but also reverse transcriptase and RNAseH. The overall amplification success was 85% (433/513) and increased to 93% (120/129) for samples with a viral load above 3 log(10) copies/ml. Both B and non-B HIV-1 subtypes could be genotyped successfully (93%; 52/56 and 100%; 49/49, respectively) and reproducibly. The phenotypic assay showed a high success rate (96.5%; 139/144) for subtype B (100%; 19/19) and non-B subtypes (92%; 45/49), and was found to be accurate and reproducible as assessed using well-characterized integrase mutants. Using both assays, baseline resistance to raltegravir and elvitegravir in subtype B and non-B HIV-1 strains selected at random was not observed, although integrase polymorphisms were present at varying prevalence. Biological cutoff values were found to be 2.1 and 2.0 for raltegravir and elvitegravir, respectively. In summary, a genotypic and phenotypic integrase resistance assay was validated successfully for accuracy, reproducibility, analytical and clinical sensitivity, and dynamic range.

Minor Variant Detection at Different Template Concentrations in HIV-1 Phenotypic and Genotypic Tropism Testing

The clinical trials of maraviroc showed that treatment failure was mostly associated with lack of X4 virus detection at baseline. The detection limit for X4 in tropism assays is ill defined around 10%. In the current study, quantification of X4-tropic minority populations was assessed on artificial mixed samples and 38 clinical isolates. These mixtures were subjected to tropism “clonal genotyping” or “population phenotyping”. The detection of minority variants was dependant on the input of amplifiable copies. At VL > 4 log IU/ml, X4 quantification was deemed reliable. PCR founder effect and clonal resampling might result in misrepresentation of the minority species concentration at VL < 4 log. Fourteen of the clinical isolates contained dual/mixed X4-tropic virus, 5 of which were below 10% of the virus population. Currently, there is no indication what level of X4 would lead to treatment failure. Assays aiming for the detection of minority species should express results in function of VL.

Abstract 4213: BRAF V600 mutation testing on FFPE samples using a novel fully integrated molecular diagnostics platform: A concordance study with reference methods.

Introduction Activating mutations in the BRAF gene are common in skin and colorectal tumors. In more than 90% of the cases, these mutations are located in the BRAF V600 codon (V600E and V600K). Targeted therapies like vemurafenib and dabrafenib have shown objective response rates in up to half of metastatic melanomas harboring these BRAF V600 mutations. Tumor mutation status is usually assessed starting from formalin-fixed, paraffin-embedded (FFPE) tumor tissue shavings. Typically, the test involves shipment of the paraffin block from the local pathology lab to a specialized molecular lab, where several steps need to be performed. Methods The Biocartis molecular diagnostics (MDx) prototype platform is a novel, random access, sample-in result-out automated qPCR system. It uses a disposable cartridge which can simultaneously detect and quantify up to 30 molecular markers from a wide range of solid and liquid sample types, including blood, feces, and sputum. For FFPE-based assays, a single curl or shaving is directly placed into the cartridge. The complete process time for sample preparation, PCR and reporting is less than 90 min, with Results One sample was not eligible for Cobas testing because of low tumor content. Three other samples failed on both platforms. For the remaining 60 samples, results on the Biocartis MDx platform and Cobas were concordant in 58 (96.7%) samples (44 BRAF V600 WT and 14 BRAF V600 mutant samples, of which 36 samples were randomly selected and confirmed by deep sequencing). In 2 (3.3%) samples, no BRAF V600 mutation was detected by Cobas, while a BRAF V600 mutation was detected by both the Biocartis MDx platform and by deep sequencing. Both were melanoma samples (one of which was highly pigmented), excised in 1993 and 2005, and contained 4.6% V600E and 5.6% V600K (as determined by deep sequencing), respectively. Conclusions The new MDx platform is a fast and reliable method for BRAF V600 mutation testing directly on FFPE tumor shavings with superior analytical sensitivity, ease of use, and turnaround time compared to existing diagnostic tests. The BRAF V600 Mutation prototype assay provided excellent concordance with both Cobas (96.7%) and deep sequencing (100%). Citation Format: Benoit Devogelaere, Koen Van Acker, Inky De Baere, Pascale Holemans, Tania Ivens, Bart Claes, Evelien Rondelez, Genevieve Vandercruyssen, Marijke Van der Auwera, Mark Kockx, Isabelle Vanden Bempt, Ina Vandenbroucke, Geert Maertens, Erwin Sablon. BRAF V600 mutation testing on FFPE samples using a novel fully integrated molecular diagnostics platform: A concordance study with reference methods. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4213. doi:10.1158/1538-7445.AM2013-4213

Abstract 1502: A rapid and fully automated multiplex assay for KRAS-BRAF mutations with high mutation sensitivity using novel selective amplification and detection technologies

Introduction The MAPK/ERK pathway is a complex signaling cascade involved in many cancer types. KRAS and BRAF gene mutations are present in a number of cancers, including colon, lung and pancreas, and identification of mutations in these genes is of great importance in clinical diagnostics. Moreover, there is a growing demand for performing multiple tests simultaneously on a single sample and there is an increased need to provide these answers to oncologists in a short timeframe. Methods IdyllaTM is a fully integrated and automated molecular diagnostics platform (1) that combines speed and ease of use with high sensitivity and high multiplexing capabilities. Moreover, it overcomes the current time-consuming step of processing formalin-fixed paraffin-embedded tissue (FFPE) samples. After insertion of a single FFPE slice into the cartridge, the complete process of sample preparation, real-time PCR and reporting is fully automated and takes less than 2 hours. We present here a KRAS-BRAF mutations prototype assay that allows the sensitive detection of 13 KRAS mutations and 5 BRAF mutations in one single assay. The assay discriminates the individual mutations at codons 12, 13 and 61 of KRAS and codon 600 of BRAF using novel “Primer Assisted Sequence Switching” (PASS) primers along with “Multi-component Nucleic Acid enzyme” (MNAzyme) detection. These technologies confer advantages for multiplex mutation analysis; PASS primers selectively amplify the target sequences of interest resulting in enhanced specificity between wild type (WT) and mutant, and between mutants, and MNAzymes allow for efficient detection and discrimination of multiple mutations simultaneously. Results Several performance characteristics of the IdyllaTM KRAS-BRAF prototype assay were examined: specificity, cross-reactivity, sensitivity and performance on clinical samples. Specificity of mutant versus WT as well as cross-reactivity between individual mutations at each codon was evaluated. Results demonstrated excellent specificity and cross-reactivity for all individual targets, with delta Cq values of >7 between mutants and >12 between mutant and WT. Sensitivity was assessed using cell lines embedded in FFPE containing defined ratios of mutants and dilutions of these in FFPE WT background. The results indicated sensitivities of 96 % concordance. Conclusion The new and fully integrated IdyllaTM KRAS-BRAF prototype assay combines extended multiplexing capabilities with excellent specificity, high sensitivity, ease of use, and short turnaround time for mutation analysis on FFPE samples. (1) For research use only Citation Format: Ina Vandenbroucke, Katrien Vermeiren, Elisa Mokany, Lit Yeen Tan, Nicole Lima, Samantha Walker, Genevieve Vandercruyssen, Bart Claes, Inky De Baere, Pascale Holemans, Evelien Rondelez, Alison Todd, Geert Maertens, Erwin Sablon. A rapid and fully automated multiplex assay for KRAS-BRAF mutations with high mutation sensitivity using novel selective amplification and detection technologies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1502. doi:10.1158/1538-7445.AM2014-1502