A novel deletion of residue 69 of the HIV-1 reverse transcriptase (RT) gene was detected in combination with mutations V75I/V and F77L/F in a patient with partial virological response to several antiretroviral drug regimens, including stavudine (D4T), didanosine (DDI), lamivudine (3TC), saquinavir (SQV), and nevirapine (NVP). Longitudinal analysis of samples revealed that this deletion emerged upon reinitiation DDI/D4T therapy following a toxicity-induced short discontinuation of all antiretrovirals. Analysis of the resistance phenotype showed a greater than 62-fold increase of the IC50 of NVP, but no significant change in sensitivity to other single nonnucleoside reverse transcriptase inhibitors (NNRTIs). The mutated virus showed only a moderately reduced sensitivity to DDI (6.7-fold) and D4T (4.8 fold). In a subsequent sample 3 months later additional RT mutations were found, including A62V, Y188L, and Q151M, conferring high-level cross-resistance to multiple nucleoside analogs. Our findings provide evidence that the deletion of RT residue 69 selectively confers high-level NVP resistance.
Malignant pleural effusion (MPE) may be diagnosed by cytologic evaluation of pleural fluid, though false negative results can occur. Pleural effusions may provide a source of tumour material for genotyping in lung cancer patients. Detection of MPE may be improved through use of highly sensitive molecular techniques. We identified five patients with non-small cell lung cancer (NSCLC) with initial pleural fluid samples that were non-malignant on cytology, but were subsequently clinically confirmed to have MPE. Tumour mutation status was confirmed via routine testing of diagnostic clinical specimens. Cytologically negative pleural fluid cell-block specimens were analysed by amplicon-based parallel sequencing (APS) for somatic mutations commonly detected in NSCLC, and selected cases by improved and complete enrichment CO-amplification at lower denaturation temperature PCR (ICECOLD PCR) for known mutations. Mutations were detected in three out of three (sensitivity 100%) cytologically non-malignant pleural fluids from patients with a known mutation: two patients with known Kirsten rat sarcoma (KRAS) mutation demonstrated the same KRAS mutation in their pleural fluids by APS, both at approximately 2% mutant allele frequency. In one patient with a known KRAS mutation, ICECOLD PCR detected the same KRAS variant at 0.7% frequency. No mutations were detected in patients with wild-type findings from reference samples (specificity 100%). Sensitive DNA sequencing methods can detect cancer-driver mutations in cytologically non-malignant pleural fluid specimens from NSCLC patients with MPE. Our findings demonstrate the feasibility of sensitive molecular diagnostic techniques for improvement of diagnostic assessment of pleural effusions in patients with lung cancer.
Rates of antiretroviral resistance in recently transmitted virus in Sydney, Australia fluctuated over the past decade, influenced by treatment trends. Current rates of drug resistance are not high in historical terms or compared with those reported. Rates of resistance to reverse transcriptase inhibitors peaked in the mid-1990s, fell dramatically with the introduction of combination therapy and appear to have plateaued at 10–15% over the past 3 years. Primary resistance mutations in the protease gene are still rare. The development of drug resistance mutations is a major obstacle to effective long-term therapy for HIV [1,2]. Several studies have suggested an increasing prevalence of drug-resistant virus among newly HIV-1-infected patients [3–6]. The complete suppression of HIV-1 could be compromised if therapy-naive patients already harbour virus with resistance mutations [7]. Furthermore, the incomplete suppression of viral replication promotes the development of broader drug resistance [2,7]. In order to determine changes in the prevalence of drug-resistant isolates in recently transmitted virus over the past decade, a retrospective study was conducted on the basis of samples collected by the NSW HIV Reference laboratory at St Vincent's Hospital, Sydney. This laboratory diagnoses the majority of cases of newly acquired HIV in the Sydney Metropolitan area. All patients presenting with acute primary HIV-1 infection between January 1992 and November 2001, who had a sterile plasma sample stored at −70°C and who had received no antiretroviral therapy before the first plasma sample was collected, were included in the study. Acute primary infection was defined as the presence of p24 antigenaemia or less than four bands on Western blot. In all 185 subjects were identified. The viral genotype from plasma viral RNA was successfully determined on all subjects using standard methodologies (Visible Genetics, Inc., Toronto, Ontario, Canada). Samples were collected early in primary infection. The median time from the first positive test for acute primary infection and the collection of samples for resistance testing was one day (range 0–13 days). Both viral loads (mean 3 485 781 copies/ml) and CD4 cell counts (mean 537 cells/ml) were high, consistent with acute primary infection. Some 97.3% samples came from men; 86.5% reported homosexual contact as their major risk factor. Their mean age was 37 years. A total of 22% of samples were collected before the introduction of protease inhibitors into this community in 1996. Mutations were designated as primary or secondary according to published criteria [2]. At least one mutation associated with resistance was detected in 21.6% (40/185) of reverse transcriptase (RT) sequences and 51.4% (95/185) of protease sequences. Mutations associated with resistance to nucleoside analogue reverse transcriptase inhibitors (NRTI) were found in 18.4%, whereas those associated with resistance to non-NRTI were found in 2.7%. No patient had combined NRTI and non-NRTI mutations. Six primary RT mutations were found: M41L (8.1%), T69N (2.2%), K70R (3.2%), K103N/R (2.2%), M184V (0.5%), and T215Y (3.2%). The levels of RT resistance dropped precipitously upon the introduction of highly active antiretroviral therapy into the transmitting community (Fig. 1a). Dividing the population into those presenting pre- and post-January 1996, the date taken to represent the timing of introduction of protease inhibitors [8], revealed a significant decrease in the frequency of primary NRTI resistance mutations from 29.3 to 9.0% (P = 0.008). Similarly, there were significant decreases in the frequency of the individual RT mutations: M41L (17.1 to 5.6%; P < 0.0008), D67N (4.9 to 0%; P < 0.03), K70R (12.2 to 0.7%; P < 0.0008), and T215Y (12.2 to 0.7%; P < 0.0008). Combinations of the three most commonly found mutations (M41L, K70R, and T215Y) confer high level zidovudine resistance [9–12]. Combined M41L and T215Y declined from 12.2 to 0% (P < 0.05). Mutations consistent with possible reversion from resistance mutation T215Y, 215C (n = 2) and 215D (n = 3), [9,10] were all seen post-1995.Fig. 1.: The year by year frequency of primary and secondary mutations in the reverse transcriptase gene (a) and protease gene (b). ―◆― Primary mutation; –░– secondary mutation.Only three primary resistant mutations (D30N, V82I, and L90M) were detected in protease. None were found in combination, all occurred in the past 18 months of the study period. All other resistance mutations in protease were secondary mutations with the most common being L63P (29.2%), V77I (15.7%), and L10I/V (12.3%). Fig. 1b shows the number of primary and secondary mutations seen in protease each year. The frequency of secondary protease resistance mutations remained unchanged despite the introduction of protease inhibitors. There was no change in the proportion of protease resistance mutations represented by any single secondary mutation. These observations add weight to the existing data suggesting that these so-called secondary resistance mutations are more likely to be naturally occurring polymorphisms [7,13–15]. Phylogenetic analysis was performed using software from the program manual for the Wisconsin Package, version 8, (Genetics Computer Group, Madison, WI, USA) on the 131 samples collected up until December 2000. All sequences were subtype B. Fifty-three of the sequences fell into 20 clusters supported by highly significant bootstrap values (> 70%). Fourteen resistant strains occurred as a part of clusters, indicating possible transmission of the same or closely related resistant strain, whereas 19 were sporadic. Seven of the clusters showed evidence of the transmission of RT resistance (data not shown, available from authors on request). The large number of different clusters and the number of unclustered sequences indicate that the population surveyed is probably representative of transmission patterns in Sydney, and is not biased by either multiple transmissions from a single individual or the preferential transmission of particular virus with some advantage in transmission or fitness. Several recent reports have suggested that the transmission of drug-resistant mutants is becoming more frequent [3,6,16–19], raising concern regarding the future effect of treatment of not only individuals but also of populations. Many of the studies were relatively small, based on samples collected up to 36 months after infection, making it impossible to be sure that the viral sequence at the time of sampling reflected transmitted virus. Many were based on data from the mid to late 1990s, so no comparison can be made with pre-existing rates of resistance mutations [7,13,20,21]. Furthermore, no attempt was made to take changes in treatment regimes into account or to show that the sample was representative. This study attempts to address each of these deficiencies. Our samples were taken early in infection, often coincident with the diagnosis of acute primary HIV infection. Approximately a quarter of the samples are from the period before the introduction of protease inhibitors into the Australian population. The phylogenetic analysis suggests that these samples are representative. The rates of antiretroviral therapy use derived from the same communities suggest that the decrease seen in transmitted RT resistance is coincident with the uptake of combination therapy by the transmitting community [22]. Contrary to data published in several other studies, the current rate of resistance in transmitted virus is not high in this population, either in historical terms or in comparison with other populations with similar demographics. Early sampling makes it unlikely that this lower rate is derived from reversion to wild type upon the removal of drug pressure in the newly infected host. The relatively low rates of resistance seen in this study may be explained by attributing most transmission to untreated individuals. This may be in addition to the decreased fitness for transmission associated with certain resistance mutations. The introduction of highly active antiretroviral therapy appears to have reduced the incidence of resistance mutations in the population as a whole, as well as in individuals.
The ALLOCATE study was designed as a pilot to demonstrate the feasibility and clinical utility of real-time targeted molecular profiling of patients with recurrent or advanced ovarian cancer for identification of potential targeted therapies.A total of 113 patients with ovarian cancer of varying histologies were recruited from two tertiary hospitals, with 99 patient cases suitable for prospective analysis. Targeted molecular and methylation profiling of fresh biopsy and archived tumor samples were performed by screening for mutations or copy-number variations in 44 genes and for promoter methylation of BRCA1 and RAD51C.Somatic genomic or methylation events were identified in 85% of all patient cases, with potentially actionable events with defined targeted therapies (including four resistance events) detected in 60% of all patient cases. On the basis of these findings, six patients received molecularly guided therapy, three patients had unsuspected germline cancer-associated BRCA1/2 mutations and were referred for genetic counseling, and two intermediate differentiated (grade 2) serous ovarian carcinomas were reclassified as low grade, leading to changes in clinical management. Additionally, secondary reversion mutations in BRCA1/2 were identified in fresh biopsy samples of two patients, consistent with clinical platinum/poly (ADP-ribose) polymerase inhibitor resistance. Timely reporting of results if molecular testing is done at disease recurrence, as well as early referral for patients with platinum-resistant cancers, were identified as factors that could improve the clinical utility of molecular profiling.ALLOCATE molecular profiling identified known genomic and methylation alterations of the different ovarian cancer subtypes and was deemed feasible and useful in routine clinical practice. Better patient selection and access to a wider range of targeted therapies or clinical trials will further enhance the clinical utility of molecular profiling.
Abstract Background: The Australian Ovarian Cancer Assortment Trial (ALLOCATE) was designed as a pilot study to demonstrate feasibility of molecularly profiling patients with recurrent ovarian cancer with the aim of allocating patients to targeted therapies based on the genomic profile of their tumors. Materials and Methods: Two next-generation sequencing (NGS) panels, as well as a BRCA1 methylation assay, were used for molecular profiling of most common subtypes of ovarian cancer. A custom Illumina TruSeq Amplicon Low Input (v2) panel with dual-strand coverage was designed to target 38 genes commonly mutated and clinically important in ovarian cancer. The second assay was a NGS modification of the Multiplex Ligation-dependent Probe Amplification (MLPA) assay that was designed to target 11 genes with common copy number alterations (CNA) in ovarian cancer, including extensive BRCA1/2 coverage for large exonic deletions (Kondrashova et al., 2015). A thorough analytic validation was performed to ensure that both tests were fit for diagnostic use. Patients with recurrent epithelial ovarian cancer were eligible for the study. Where feasible, patients underwent biopsies of recurrent tumor that were snap frozen. Otherwise, archival FFPE tumor blocks were retrieved. Sequencing was performed using Illumina Miseq and HiSeq 2500 with target median coverage of 2000x (amplicon panel) and 800x (MLPA-Seq). Data were analyzed using an internally built pipeline, an upgraded version of AmpliVar (Hsu et al 2015), with Variant Effect Predictor (Mclaren et al., 2016) used for variant annotation. Results: Between December 2013 and October 2016, 113 patients with recurrent ovarian cancer were recruited from two tertiary hospitals, with 15 cases (13%) excluded due to insufficient tumor material or poor-quality DNA. Ninety-eight cases (87%) were analyzed and reports issued back to the referring clinician. Fifty-six patients (61%) in the study had recurrent high-grade serous ovarian cancer (HGSC). Of these, TP53 mutations were identified in 91%. Events in genes other than TP53 were detected in 44 cases, most commonly MYC and CCNE1 amplifications and BRCA1/2 mutations. BRCA1/2 reversions were identified in two cases, explaining their lack of response to platinum/PARPi. Fifteen patients (16%) had recurrent low-grade serous ovarian cancer (LGSC), with KRAS or BRAF mutations identified in four cases. Two HGSC tumors were reclassified as LGSC on the basis of a lack of TP53 mutation, presence of KRAS mutation, and subsequent pathology review. Other cases in the study included mucinous, clear cell, and mixed-histology carcinomas and a metastatic carcinosarcoma. In terms of clinical utility, 6 patients (7%) received a matched therapy. Three HGSC patients with somatic BRCA1/2 mutations were treated with PARP inhibitors. Another HGSC patient with ERBB2 amplification was treated with trastuzumab. One LGSC patient with a BRAF mutation was enrolled on a BRAF inhibitor clinical trial. A second LGSC patient was enrolled in a trial of anastrazole. Furthermore, 7 patients (14%) with HGSC who were previously untested were found to have a germline BRCA1/2 mutation and were subsequently referred to a familial cancer clinic for further management and cascade testing. The limitations in the study included the turnaround time and advanced stage of disease at enrolment, which significantly affected the clinical utility of the test. Conclusion: We demonstrated that molecular profiling of recurrent ovarian cancer using the ALLOCATE panel was feasible and reflected the known genomic characteristics of the different subtypes. However, challenges remain, including appropriate patient selection and efficient turnaround time for reporting. Furthermore, improved access to targeted therapies or clinical trials will also enhance the clinical utility of the ALLOCATE panel. Citation Format: Olga Kondrashova, George Au-Yeung, Leakhena Leas, Gwo-Yaw Ho, Sebastian Lunke, Kathryn Alsop, Clare Scott, Anne Hamilton, Sumitra Ananda, Alison Freimund, Michael Quinn, Orla McNally, Nadia Traficante, Tiffany Cowie, Matthew Wakefield, Arthur Hsu, Alex Dobrovic, Michael Christie, Graham Taylor, David Bowtell, Linda Mileshkin, Paul Waring. Australian Ovarian Cancer Assortment Trial–Allocating ovarian cancer patients into clinical trials based on molecular profiling. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B35.
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