Appearance Of Resistance Mutations Research Articles

Justifying integration of liquid biopsy into routine molecular diagnostics to complement tissue genomic profiling: Experience at Fox Chase Cancer Center.

e15029 Background: Plasma-based liquid biopsy has rapidly evolved from a novel research tool toward translation into clinical use for many diseases including cancer. While there are still some gaps regarding standardization and clinical utility, as tissue next-gen sequencing had previously faced and overcome, there is a growing body of evidence supporting integration of liquid biopsy into standard of care as a diagnostic tool for precision oncology. Selected from a larger cohort, several cases are presented here illustrating the use of the PGDx elio plasma resolve assay to profile circulating tumor DNA (ctDNA) from patients whose tumor and matched normal DNA had also been characterized by a next-generation sequencing panel. Methods: The PGDx elio plasma resolve assay and bioinformatics detects single and multi-nucleotide variants in 33 genes, select amplifications and translocations, as well as microsatellite instability by sequencing on the Illumina NextSeq 500/550 platform. DNA from matched tumor and normal FFPE samples were assessed using the 275 gene Molecular Diagnostics Laboratory’s Comprehensive Cancer Panel (CCP), also on the NextSeq platform. Selected cases represent the range of diagnostic utility that might be expected from ctDNA. Results: Across all cases, there were varying levels of concordance between tissue and plasma as well as novel variants in the plasma that were not present in the tissue DNA demonstrating the benefit of incorporating ctDNA profiling to capture critical genomic information. In one rectal adenocarcinoma case, DNA variants from the primary tumor were detected in ctDNA eight months later during evaluation of a recurrent tumor, suggesting that plasma profiling might have provided early biomarker detection of recurrence. An aggressive and recurrent melanoma, however, showed no concordance between ctDNA and tumor DNA variants, indicating that some tumors may not shed sufficient DNA into the bloodstream to make ctDNA clinically relevant. Another case of melanoma had concordant detection of the driver BRAF V600E mutation in tumor and ctDNA, but ctDNA also contained a pathogenic ATM truncation not observed in the tumor sample. Current clinical trials are investigating the combination of PARP- and ATR-inhibiting drugs, which causes excessive DNA damage and cell death in the context of ATM-deficient cancers. Conclusions: These cases show that combined tissue and ctDNA profiling at the time of initial diagnosis can reveal tumor heterogeneity or distinct metastases not observed in a selected tissue sample, and that profiling during and after therapy may provide early detection of recurrence, appearance of resistance mutations, or monitoring of measurable residual disease.

Treatment response and tumor evolution: lessons from an extended series of multianalyte liquid biopsies in a metastatic breast cancer patient.

Currently, clinical characterization of metastatic breast cancer is based on tissue samples taken at time of diagnosis. However, tissue biopsies are invasive and tumors are continuously evolving, which indicates the need for minimally invasive longitudinal assessment of the tumor. Blood-based liquid biopsies provide minimal invasive means for serial sampling over the course of treatment and the opportunity to adjust therapies based on molecular markers. Here, we aim to identify cellular changes that occur in breast cancer over the lifespan of an affected patient through single-cell proteomic and genomic analysis of longitudinally sampled solid and liquid biopsies. Three solid and 17 liquid biopsies from peripheral blood of an ER+/HER2− metastatic breast cancer patient collected over 4 years and eight treatment regimens were analyzed for morphology, protein expression, copy-number alterations, and single-nucleotide variations. Analysis of 563 single morphometrically similar circulating tumor cells (CTCs) and 13 cell-free DNA (cfDNA) samples along with biopsies of the primary and metastatic tumor revealed progressive genomic evolution away from the primary tumor profiles, along with changes in ER expression and the appearance of resistance mutations. Both the abundance and the genomic alterations of CTCs and cfDNA were highly correlated and consistent with genomic alterations in the tissue samples. We demonstrate that genomic evolution and acquisition of drug resistance can be detected in real time and at single-cell resolution through liquid biopsy analytes and highlight the utility of liquid biopsies to guide treatment decisions.

Ciprofloxacin-Mediated Mutagenesis Is Suppressed by Subinhibitory Concentrations of Amikacin in Pseudomonas aeruginosa.

Resistance to antibiotics is a global health problem. Activation of the SOS response, and the subsequent elevation in mutagenesis, contributes to the appearance of resistance mutations. Among currently used drugs, quinolones are the most potent inducers of the SOS response. In the present study, we show that amikacin inhibits ciprofloxacin-mediated SOS induction and mutagenesis in Pseudomonas aeruginosa.

The HCV Replicase Interactome

Viruses are obligate parasites and can only reproduce within host cells because they lack metabolic pathways to complete their replication cycles. Host factors required in viral replication are mainly those involved in lipid metabolism, cell cycle control and apoptosis, cell-to-cell interactions, immune system regulation, etc. Several inhibitors targeting viral polymerases have been designed. However, the rapid appearance of resistant mutants, as a direct consequence of the viral population structure, diminishes the efficacy of this kind of molecules. To elude the rapid loss of treatment efficiency due to the appearance of resistance mutations, cellular factors have been proposed as a promising therapeutic target to inhibit RNA(+) virus replication. In this review, we focus on those interactions between host factors and HCV replicase, to modulate either cellular metabolism or HCV polymerase activity.

The Three Faces of Riboviral Spontaneous Mutation: Spectrum, Mode of Genome Replication, and Mutation Rate

Riboviruses (RNA viruses without DNA replication intermediates) are the most abundant pathogens infecting animals and plants. Only a few riboviral infections can be controlled with antiviral drugs, mainly because of the rapid appearance of resistance mutations. Little reliable information is available concerning i) kinds and relative frequencies of mutations (the mutational spectrum), ii) mode of genome replication and mutation accumulation, and iii) rates of spontaneous mutation. To illuminate these issues, we developed a model in vivo system based on phage Qß infecting its natural host, Escherichia coli. The Qß RT gene encoding the Read-Through protein was used as a mutation reporter. To reduce uncertainties in mutation frequencies due to selection, the experimental Qß populations were established after a single cycle of infection and selection against RT − mutants during phage growth was ameliorated by plasmid-based RT complementation in trans. The dynamics of Qß genome replication were confirmed to reflect the linear process of iterative copying (the stamping-machine mode). A total of 32 RT mutants were detected among 7,517 Qß isolates. Sequencing analysis of 45 RT mutations revealed a spectrum dominated by 39 transitions, plus 4 transversions and 2 indels. A clear template•primer mismatch bias was observed: A•C>C•A>U•G>G•U> transversion mismatches. The average mutation rate per base replication was ≈9.1×10−6 for base substitutions and ≈2.3×10−7 for indels. The estimated mutation rate per genome replication, μg, was ≈0.04 (or, per phage generation, ≈0.08), although secondary RT mutations arose during the growth of some RT mutants at a rate about 7-fold higher, signaling the possible impact of transitory bouts of hypermutation. These results are contrasted with those previously reported for other riboviruses to depict the current state of the art in riboviral mutagenesis.

HIV-1 Can Persist in Aged Memory CD4+ T Lymphocytes With Minimal Signs of Evolution After 8.3 Years of Effective Highly Active Antiretroviral Therapy

Patients on long-term highly active antiretroviral therapy (HAART) were studied to determine persistence, drug resistance development, and evolution of HIV-1 proviral DNA. Peripheral blood mononuclear cells were obtained by large volume blood drawn (500 mL) from 8 clinically successfully treated patients who had received uninterrupted HAART for up to 8.9 years. HIV-1 load was determined by Taqman real-time polymerase chain reaction. Drug resistance mutations were determined by sequencing and ultrasensitive, allele-specific, reverse transcriptase (RT)-polymerase chain reaction. HIV-1 DNA load was significantly higher in aged memory (CD45RO CD57) when compared with memory (CD45RO CD57) and naive (CD27 CD45RO) CD4 T cells after HAART. Sequencing revealed no major drug resistance mutations in protease in all patients and appearance of resistance mutations in RT in just 1 patient. In 1 of 5 patients with undetectable viremia during treatment, RT M184 substitutions were detected. Phylogenetic analysis showed short genetic distances between patient sequences. During long-term HAART, HIV-1 is able to persist in terminally differentiated CD4 T cells as proviral DNA. Viral evolution was restricted, and in 80% of the patients with undetectable viremia, no sign of viral replication could be detected.

Monitoring of HIV-1 Resistance in Tunisia (North Africa) With a Dried Plasma Spot Strategy

Monitoring of HIV-1 Resistance in Tunisia (North Africa) With a Dried Plasma Spot Strategy

Le patient atteint d’hépatite chronique B non contrôlé

The treatment of chronic hepatitis B is now based on the using of pegylated interferon or nucleoside or nucleotide analogs. In the majority of cases, these drugs can control viral replication with an hepatitis B virus (HBV) DNA negativation after approximately 6 months of therapy. In case of primary non response, it is necessary to modify antiviral therapy and if resistance appears to combine a nucleoside and a nucleotide analog. In patients treated by nucleoside analog, if HBV DNA is not negative or do not dramatically decreases at the week 24, it is also necessary to add a nucleotide analog. However, for adefovir therapy, it is usually preferable to wait at week 48. In summary, a regular following every 3 months of HBV DNA detection by a sensitive method (Real Time PCR) allows to evaluate the therapeutic efficacy and to prevent the risk of biochemical and clinical rebound due to appearance of resistance mutations.

Impact of the number of failed therapeutic regimes on the development of resistance mutations to HIV-1 in northeast Brazil

Highly-potent antiretroviral therapy is necessary to avoid viral replication in HIV patients; however, it can favor the appearance of resistance mutations. The mutations 41L, 67N, 70R, 210W, 215Y/F, 219E/Q, 44D and 118I are defined as nucleoside analogous mutations (NAMs), because they affect the efficacy of all nucleoside reverse transcriptase inhibitors (NRTI). The mutation most frequently associated with non-nucleoside reverse transcriptase inhibitors (NNRTIs) is 103N. 33W/F, 82A/F/L/T, 84V and 90M are called protease inhibitor resistance-associated mutations (PRAM), because they are associated with resistance to several protease inhibitors (PI). This study evaluated the development of resistance mutations and examine the susceptibility of HIV with these mutations to antiretrovirals in HIV-1 patients who have failed one or more therapy regimes. Analyses were made of 101 genotypic tests of patients with therapeutic failure to 2 or 3-drug regimens with NRTI, NNRTI or PI. We used the Stanford database to define the susceptibility profile of the viruses. The samples were divided into three treatment-failure groups: first (F), second (S) and multi-failure (MF) to antiretroviral regimens, and we correlated these groups with resistance profiles and principal mutations. There was a significant increase in resistance mutations V82A/F/L/T, I84V, L90M, M41L, K70R, L210W, T215Y/F and K219Q/E in MF. We also found significantly higher resistance to zidovudine, didanosine, stavudine and abacavir in MF. There was no increase in resistance to tenofovir (p=0.28) and lopinavir (p=0.079) in MF. A high degree of resistance to NNRTIs was observed in all groups. Increased resistance mutations will affect future therapeutic options for HIV patients in Brazil because it results in a significant increase in resistance to antiretroviral drugs.

Conflicting Requirements of Plasmodium falciparum Dihydrofolate Reductase Mutations Conferring Resistance to Pyrimethamine-WR99210 Combination

Plasmodium falciparum strains bearing quadruple mutations of dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) at codons 51, 59, 108, and 164 are highly resistant to pyrimethamine (PYR), a diaminopyrimidine, but sensitive to WR99210 (WR), a cycloguanil analog, suggesting different enzyme-inhibitor binding interactions. A combination of these inhibitors to delay the onset of antifolate resistance is proposed. Using error-prone PCR, libraries of random mutants of wild-type PfDHFR and PfDHFR-TS were generated and used to transform Escherichia coli, and transformants were then selected for PYR or WR resistance. Mutants highly resistant to either PYR or WR were also generated from libraries obtained from further random mutagenesis of quadruple mutants (QM) with mutations in PfDHFR or PfDHFR-TS. For reversion mutants carrying altered residues I51N, N108S, and L164I, a further mutation of D54N was required to achieve resistance against WR, but these mutants regained sensitivity to PYR. When a combination of PYR and WR was used, fewer resistant mutants were generated from both mutant libraries using the QM gene templates. The effectiveness of the drug combination in reducing the appearance of resistance mutations is likely due to conflicting requirements for mutations conferring resistance to the two drugs. Thus, a combination of inhibitors from these two drug classes should be effective in impeding the emergence of P. falciparum resistance to antifolates.

Successful desensitization of enfuvirtide after a first attempt failure

In 1989, a 27-year-old white woman was diagnosed with HIV, but only sought treatment in 1994 when her CD4 T-cell count was 230 cells/μl. She was placed on zidovudine monotherapy. Since then she has been treated with nine different combination regimens of nucleoside, non-nucleoside reverse transcriptase inhibitors and protease inhibitors. She has always been asymptomatic, her viral load has rarely been undetectable but has remained below 10 000 copies/ml. Her CD4 T-cell count has been, most of the time, above 200 cells/μl until July 2003, when it was persistently recorded below 200 cells/μl, despite changes in her antiretroviral regimen. In December 2004, she took a genotypic test while on didanosine, lamivudine, tenofovir and lopinavir/ritonavir. The resistance test showed K70E, K101Q, K103N, V118I, M184V, K219R, P225H and L10V, K20R, L33F, M36I, M46I, I54V, D60E, V82A, L90M in the reverse transcriptase and protease regions, respectively. Because of her intolerance to amprenavir, the fact that tipranavir is not yet provided by the public health system, and the Agence Nationale de Recherches sur la Sida algorithm did not show a high level resistance to lopinavir/ritonavir, her antiretroviral regimen was changed to zidovudine, lamivudine, tenofovir, lopinavir/ritonavir, and enfuvirtide was indicated. She waited 8 months to obtain enfuvirtide from the public health system, at which point her CD4 T-cell count was 225 cells/μl, and even though her viral load was undetectable the decision to include enfuvirtide was sustained because of her limited antiretroviral susceptibility. Two weeks after the first injection of enfuvirtide she developed pruritus at the site of the injection, which evolved into itching papules that spread to her arms, trunk and limbs. Enfuvirtide was stopped, and after the complete resolution of her skin lesions she was hospitalized and underwent a desensitization protocol described by DeSimone et al. [1]. After the 10th dose she once again, developed widespread itching papules associated with a general malaise and tachycardia. She was discharged 5 days later, once all her cutaneous lesions subsided, and kept off enfuvirtide. A month later she was re-admitted for a new attempt at desensitization using a second protocol described by Shahar et al. [2], which consists of 18 increasing doses of enfuvirtide diluted 1/1000 administered every 30 min, but we decided to increase the interval between doses to 60 min. She tolerated the protocol without symptoms, and remained well until her last visit in May 2006. Her first viral load, after resuming the full dose of enfuvirtide in March, was 1700 copies/ml and reached an undetectable level (< 80 copies/ml) 2 months later. Injection-site reactions are the most common side-effects of enfuvirtide, and might occur in up to 98% of those receiving it. In contrast, hypersensitivity reactions consisting of rash, fever, nausea, vomiting, chills and hypotension are uncommon, and were reported in only six subjects enrolled in clinical trials of enfuvirtide (< 1%) [3]. In heavily treated patients with very few treatment options, desensitization to enfuvirtide can be an alternative. In the literature, two cases that responded well to two different desensitization protocols were reported [1,2]. In our case, we described a patient in whom a first attempt at desensitization failed, but re-challenge was successful with a longer protocol using a smaller increase in doses. Unfortunately, it is not possible to predict which patient, presenting a systemic allergic reaction, will have life-threatening symptoms when submitted to desensitization. Although a generalization cannot be made, those who do not present life-threatening symptoms may be submitted to a re-challenge provided the patient is closely monitored. Also, late allergic reactions may occur with enfuvirtide. A case of glomerulonephritis, occurring 6 weeks after the introduction of the drug, was reported in the TORO1 clinical trial [4]. We have been monitoring the patient's renal functions since the introduction of tenofovir and have not yet observed alterations. It is unclear why the patient failed one protocol and responded well to a second one. A possibility is that there was a smaller increase of doses in the protocol described by Shahar et al. [2]. One of the main advantages of both protocols is the quick increase in diluted doses, so that patients are exposed to suboptimal doses for less than 12 h, preventing the appearance of resistance mutations. Resistance mutations to enfuvirtide have been seen after 14 days of monotherapy [5], but short protocols, even if ranging for more than 12 h like ours, might not be enough for resistance mutations to develop. Even though we cannot rule out the possibility that resistance has not developed during the extended protocol, the fact that our patient's viral load became undetectable makes this possibility less probable.

Utility of Interrupting Antiretroviral Treatment Before HIV Drug Resistance Testing in Patients With Persistently Detectable Low-Level Viremia

Utility of Interrupting Antiretroviral Treatment Before HIV Drug Resistance Testing in Patients With Persistently Detectable Low-Level Viremia

Lymph Node Human Immunodeficiency Virus RNA Levels and Resistance Mutations in Patients Receiving High‐Dose Saquinavir

The development of resistance mutations and human immunodeficiency virus (HIV) RNA levels were compared in lymph nodes and plasma of patients receiving antiretroviral therapy. Ten HIV-positive patients receiving high-dose saquinavir monotherapy (3600 or 7200 mg/day) underwent 14 lymph node biopsies before and during therapy. HIV RNA levels and appearance of resistance mutations to saquinavir were determined in simultaneous lymph node and plasma samples. HIV RNA levels were found to be consistently higher (mean, 3.16 log RNA copies; SD, 1.04; range, 2.23-5.59) in lymph nodes than in simultaneous plasma samples. Saquinavir therapy resulted in a reduction in HIV RNA levels in both plasma and lymph nodes. The presence or absence of a resistance mutation to saquinavir at codons 48 or 90 of the HIV-1 protease gene was identical in 13 of 14 biopsies, suggesting that resistance mutations to saquinavir appear within close temporal proximity in lymph nodes and plasma.

Ordered accumulation of mutations in HIV protease confers resistance to ritonavir.

Analysis of the HIV protease gene from the plasma of HIV-infected patients revealed substitutions at nine different codons selected in response to monotherapy with the protease inhibitor ritonavir. Mutants at valine-82, although insufficient to confer resistance, appeared first in most patients. Significant phenotypic resistance required multiple mutations in HIV protease, which emerged subsequently in an ordered, stepwise fashion. The appearance of resistance mutations was delayed in patients with higher plasma levels of ritonavir. Early mutants retained susceptibility to structurally diverse protease inhibitors, suggesting that dual protease inhibitor therapy might increase the duration of viral suppression.

Individualization of Therapy Using Viral Markers

Early intervention with a combination of drugs is generally accepted as the way forward in the management of HIV infection. In general, combination therapy should include agents that demonstrate additive or synergistic activity, do not have similar side-effect profiles, and avoid cross-resistance. Use of these criteria will no doubt lead to the development of more effective combinations. However, it is well known that patients respond to therapy in very different ways. A number of trials have provided clear examples of the individualized nature of patient responses to therapy and have also demonstrated that the use of measures such as viral load and CD4 cell count, in conjunction with phenotypic and genotypic characterization of emerging viral strains, may significantly enhance our ability to predict disease progression. A number of methods are becoming available to quantify accurately viral load, and new methodologies such as the Affymetrix chip-sequencing technique will allow quick detection of genotypic alterations known to be associated with disease progression, such as the appearance of resistance mutations. These developments now enable us to conduct clinical trials to assess the impact of an individualized approach to therapy, i.e., the changing of treatment regimens based on the identification of resistance markers, changes in viral load and CD4 cell count, and conversion from non-syncytium-inducing to syncytium-inducing phenotype. It is hoped that studies conducted in the near future will show that an approach of gaining maximal benefit from one treatment regimen before moving on to another, as a result of genotypic or viral load markers, will prevent destruction of the immune system and will ultimately prolong the well-being and survival of patients with HIV infection.