In this study, we examined the performance of the Hologic AptimaHIV-1QuantDxassay in accurately quantitating plasma viral loads of HIV-1 subtypes from diverse geographic origins, including those currently in widespread circulation as well as var- ious common and unique recombinants. The HIV RNA concen- trations measured in the Aptimaassay for the subtype samples studied were higher than those measured by the CAP/CTM and RealTime assays, by 0.21 and 0.30 log, respectively, on average. Aptima results were higher than CAP/CTM results for subtypes F and G and higher than RealTime results for subtypes C, F, and G and CRF02_AG, by ⬎ 0.4 log. Two outliers (one subtype G and one CRF01_AG specimen) showed more than a log difference between Aptima and RealTime results, while all remaining CAP/ CTM and RealTime results were within 1 log of the Aptima results. Differences observed among assay values could be accounted for by calibration against the WHO standard. According to the man- ufacturers’ package inserts, the Aptimaassay is calibrated at 0.35 copy per WHO international unit, the CAP/CTM assay at 0.59, and the RealTime assay at 0.58, which would transform to Aptima values of 0.23 and 0.22 log higher. Thus, differences in quantita- tion between the three assays can be normalized to nearly equiv- alent levels if the data are expressed in international units, al- though quantitation of subtypes C, F, and G and CRF02_AG would still be 0.20 to 0.25 log lower by RealTime assay than by Aptimaassay. Overall differences in quantitation among the three assays are relatively small and are comparable across diverse group M subtypes, recombinant circulating forms, and group O sam- ples, and the results are consistent with previous studies (30–36). Our study demonstrates the excellent linearity and accuracy of the Aptimaassay in quantifying viral load measurements on diverse HIV-1 subtypes, groups, and circulating recombinant forms, even at very low RNA levels. The Panther instrument features random access of samples to permit simultaneous testing of different pathogens within the same test run, has easy-to-use workflows, requires less stringent technical skills, and can generate up to 320 results in an 8-h period. The linearity and accuracy of the Aptimaassay in the quantitation of HIV-1 on a panel of diverse HIV-1 isolates and clinical specimens collected worldwide, along with
QuantDxassay performed on the Panther system. This assay demonstrated performance comparable to that of the Cobas Am- pliPrep/Cobas TaqMan HIV-1 Test v2.0 on HIV-1 subtype B sam- ples. Aptima provides a suitable alternative for HIV-1 monitoring, particularly for laboratories already using the Panther system for human papillomavirus (HPV), Chlamydia trachomatis, and Neis- seria gonorrhoeae testing. As additional performance data are col- lected for non-subtype B clinical samples, the Panther system’s simple, automated work flow, ease of use, high throughput, and accurate DBS protocol may provide an attractive option for min- istries of health as they consider various approaches to meet the virus load testing demands of the UNAIDS 90-90-90 plan (6). Future time and motion studies (work flow) and comparative cost analyses will be needed to determine the optimal platform, or combination of platforms, to effectively achieve this virus load scale-up. The AptimaHIV-1QuantDxassay and other assays on the Panther system may ultimately deliver the combination of test performance and overall efficiency required to significantly im- pact the global HIV epidemic, as well as improve the diagnosis of other sexually transmitted infections.
The AptimaHIV-1QuantDxassay. The AptimaHIV-1QuantDxassay (Hologic Incorporated, Bedford, MA), run on the fully automated Panther platform, uses speciﬁc target-capture transcription- mediated ampliﬁcation (TMA) and real-time detection technologies to achieve high sensitivity and a broad dynamic range for HIV-1 detection and quantitation (11). In line with other commercial HIV-1 nucleic acid tests developed by Hologic Incorporated (formerly Gen-Probe, San Diego, CA) for HIV-1 diagnosis and blood screening, such as the AptimaHIV-1 RNA qualitative assay (19), the Procleix HIV-1/HCV assay (22, 23), and the Procleix Ultrio and Ultrio Elite (HIV-1&2/HCV/HBV) assays (24), this assay also targets highly conserved regions of HIV-1 polymerase (pol) and long terminal repeat (LTR). Target capture is mediated by oligonucleotides directed at conserved regions of the HIV-1 genome to enable speciﬁc pulldown of HIV RNA species (11). The multiple primers used in this product amplify HIV-1 groups M, N, O, and P along with an internal control. The primer design unique to TMA and the dual target approach ensure accurate detection and quantitation of all groups and subtypes of HIV-1. According to data in the FDA-cleared package insert, the lower limit of quantitation of the assay is 30 copies/ml and the 95% limit of detection is 12 copies/ml using a single analysis of 0.5 ml (11).
The AptimaHIV-1QuantDxassay on the Panther system was approved for European use in the diagnosis and monitoring of HIV- 1 infection in November 2014. It is the ﬁrst commercially available real-time, TMA assay for quantitation of viral RNA levels and the evaluation presented here demonstrated AptimaHIV results are highly correlated with those obtained from real-time PCR technolo- gies. The AptimaHIV test was highly accurate for quantitation of HIV-1 subtype B in the standard panels. Results were similar to those reported by Manak et al. where AptimaHIV quantiﬁcation was comparable to RealTime and CAP/CTM for all major group M HIV-1 subtypes and four group O isolates .
HIV-1 RNA load assays.The AptimaHIV-1QuantDxassay (Hologic, San Diego, CA) was performed according to the manufacturer’s recom- mendations. The Aptimaassay is a TMA-based assay performed on the fully automated Panther system; the assay targets the HIV-1 Pol and long terminal repeat (LTR) regions and is able to quantify with equal efficiency HIV-1 groups M (and subtypes), O, and N, with an LLOQ of 30 copies/ml and a linear range from 30 copies/ml to 10,000,000 copies/ml, using a 0.5-ml reaction mixture volume (12). While plasma was tested according to the manufacturer’s instructions, testing on CVL specimens was carried out based on a protocol developed in our laboratory for research pur- poses. The Abbott RealTime HIV-1assay (Abbott Molecular Inc., Des Plaines, IL), a reverse transcription-PCR (RT-PCR) assay, was performed on the automated Abbott m2000 platform, which consist of the m2000sp instrument for automated extraction of RNA and the m2000rt instrument for real-time PCR analysis, according to the manufacturer’s recommen- dations. The target sequence for the RealTime assay is the highly con- served integrase gene. The linear range of the assay is 40 to 10,000,000 copies/ml when using the protocol for the 0.6-ml version. The assay was performed by using the m2000 0.6 ml HIV-1 RNA 96 version 5 software application (15).
The subtype detection was evaluated using the NRC subtype panel composed of 21 panel members targeted at approximately 100,000 HIV-1 RNA copies/mL. The isolates included subtype A, B, C, D, F, G and H as well as circulating recombinants. Viral load results for the Aptimaassay were slightly higher than those for Roche CAP/CTM but were within 0.5 log copies/mL in all cases with one exception. This isolate which was a re- combinant of G and H had a viral load result from Aptima that was 0.79 log copies/mL higher than Roche CAP/CTM. Abbott RT results were obtained for this panel member which allow a comparison to be made to a third assay. The Aptima result was within 0.27 logs of the Abbott RT result. This suggests that the Roche
H IV-1 viral load testing is an integral part of HIV-1 manage- ment worldwide, both before and during antiretroviral ther- apy (1). Plasma HIV-1 RNA measurements are expected to assess viral copy numbers accurately, to be sensitive and linear with both low and high HIV-1 viral loads, to be applicable to all common HIV-1 subtypes, and to be reproducible and comparable across different platforms. The amount of virus present in the plasma affects clinical decisions; therefore, accurate sensitive viral load assays are particularly important. The current guidelines, which are based on data from assays with limits of quantification ranging between 10 and 40 copies/ml (1 to 1.6 log copies/ml), regard levels of ⬍50 HIV-1 copies/ml (⬍1.7 log copies/ml) in plasma to be the optimal outcomes of highly active antiretroviral therapy. Viral loads exceeding 50 copies/ml trigger further investigation, and ⬎ 1,000 copies/ml ( ⬎ 3 log copies/ml) is considered to be the threshold for resistance testing (2, 3). Decreases or increases in HIV-1 RNA concentrations of 0.5 log copies/ml are considered significant changes that cannot be attributed to testing or normal biological variations and may reflect treatment success or failure (4). In Israel, HIV-1 patients are primarily infected with group M viruses, mainly subtype A1/CRF01_AE (referred to here as sub- type A1), subtype B, subtype C, subtypes CRF02_AG and G (col- lectively referred to here as subtype CRF02_AG/G), and rarer sub- types (5, 6). This pluralism is advantageous for studying the efficiency of different assays to detect various HIV-1 subtypes.
Thus, identiﬁcation and conﬁrmation of true HIV-1 virologic failure are critical for patient management, as patients typically visit physicians only twice per year in the United States or United Kingdom (4 times in Germany). Imprecision in a test result at low VLs (around the 50 and 200 copies/ml cutoffs) could have important medical implications, such as a patient remaining on a failing therapy, possibly leading to resistance, disease progression, and potential increase of transmission. Consequently, current HIV-1 VL assay systems need to deliver precise, reproducible, speciﬁc, and sensitive results, particularly around the clinical relevant benchmarks (50 and 200 copies/ml). Because patient management decisions rely entirely on each test result, the reliability of the HIV VL assay used in the clinical laboratory should be assessed and conﬁrmed through objective rigorous modern analysis methods, such as the sigma metrics (8). The Greek letter sigma is used to represent standard deviations and to show how wide the distribution of values spans. Samples with a high standard deviation are more spread out and show more variability, while samples with a low standard deviation cluster more tightly around the corresponding mean. In quality control processes, it is important to know how many values (standard deviations) ﬁt into a certain deﬁned acceptable range (total analytical error). Therefore, it is recommended that laboratories estimate the total analytical error by combining the estimate of bias from a method comparison study and the estimate of precision from a replication study. Accordingly, we used a multiple of the standard deviation (SD) or coefﬁcient of variation (CV) and total analytical error/bias for a 95% conﬁdence interval, as recently published (8).
In conclusion, we report the evaluation of Aptima HCV performed on the Panther system. This assay demonstrated comparable performance to that of CAP/CTM on serum specimens collected from a U.S. cohort. Furthermore, Aptima HCV showed sufﬁcient analytical and clinical performance on DBSs to be considered by the WHO as a method to facilitate worldwide access to HCV RNA testing, particularly in areas where infrastructure challenges prevent the timely delivery of other, more labile, specimen types for virus load testing. In addition, the availability of Aptima tests for HIV-1 and hepatitis B virus (HBV) is essential given the global burden of HCV coinfections with these blood-borne viral pathogens (36–38). Serum and DBS testing using the Aptima HCV QuantDxassay and other Aptima assays on the Panther system may provide the necessary performance and efﬁciency to contribute to the worldwide elimination of viral hepatitis.
Q uantification of virions is important in HIV-1 management and basic research (1–8). Virions are generally quantified us- ing real-time reverse transcriptase (RT)-PCR amplification of genomic RNA with primers in a conserved region, such as gag (9–11). A limitation is lack of specificity because, in principle, any RNA or DNA containing the relevant sequence can be amplified, including integrated or unintegrated viral DNA, and in transfec- tion studies, plasmids containing HIV-1 sequences. Amplification of DNA templates is apparent from positive no-RT controls but nevertheless complicates quantification. In the analysis of HIV-1 RNA in cells, conventional RT-PCR assays can also detect aber- rant RNAs, such as chimeric host-virus transcripts originating from upstream host promoters (12, 13) and truncated or prema- turely terminated viral transcripts (14–17). Another assay for vi- rions, the enzyme-linked immunosorbent assay (ELISA) detect- ing HIV-1 Gag p24, has low sensitivity (10 3 to 10 6 virus particles/ ml) and can also detect virus-like particles lacking genomic RNA and p24 released from dead cells (18–21).
The ACT, AGC, and AC2 assays are NAATs utilizing target capture specimen processing, TMA technology, and dual kinetic assay detection technology allow- ing the qualitative detection and differentiation of rRNA from C. trachomatis and N. gonorrhoeae in endocervical and male urethral swabs, vaginal swabs, and urine specimens. AC2 amplifies a specific region from the 23S rRNA for C. trachomatis and 16S rRNA for N. gonorrhoeae. ACT and AGC amplify regions located within the 16S rRNA of C. trachomatis and N. gonorrhoeae, which are different from AC2’s target sequences. The AC2 assay was performed as de- scribed in the package insert. ACT and AGC assays were performed identically to AC2, except that the chemiluminescent signal cutoff was determined to be 50,000 relative light units (RLU). The LCx CT and LCx GC assays use the LCR method to amplify C. trachomatis or N. gonorrhoeae plasmid DNA, and the signal emitted by the LCR reaction product is detected by the Abbott LCx analyzer. The assay was performed according to the package insert. DFA was performed by Bartels chlamydiae fluorescent monoclonal antibody test. Cultures for C. trachomatis and N. gonorrhoeae were performed according to the Specialty Lab- oratories standard operating procedures.
served to reduce the between-run variability both within and between laboratories, reflected by the reduction in CVs seen between the data generated by the AMPLICOR HIV-1 MON- ITOR v 1.0 assay for the first set of QC samples (samples QC101 and QC102) and the more recent set of samples (sam- ples QC105 and QC106). In the new version of the AMPLI- COR HIV-1 MONITOR assay, v 1.5, results collected to date suggest that there is no difference in the CV from the previous version of the same assay except when Ultrasensitive sample preparation is used. The high CV observed when the modified sample preparation is used may reflect the inexperience of the operators with the new technique but may also highlight a difficulty obtaining precise results for samples with low viral loads by the AMPLICOR HIV-1 MONITOR assay in conjunc- tion with Ultrasensitive sample preparation. As the new ver- sion does not differ in practical terms from the previous ver- sion, it is not anticipated that the CV will decrease with use of the assay but reflects inherent assay variability. It should be noted that the variability observed with the Quantiplex bDNA 2.0 assay, and now with the Quantiplex bDNA 3.0 assay, has remained consistently less than 35% throughout the program. Each QAP is designed to ask a question specifically of the quality of HIV viral load testing. In the present study, the questions were, “Can laboratories consistently distinguish a 5-fold difference in viral load?,” “What is the within-run re- producibility of the same sample?,” and “Are assays equally effective for detection of different HIV-1 subtypes?” Only 5 of 19 laboratories, using either assay, produced one result for one of the dilution samples (a different one in each case) which fell outside the range of acceptable variability. The reproducibility of the results obtained with replicate samples demonstrated greater variability, particularly in laboratories using the AM- PLICOR HIV-1 MONITOR assay, which may be expected for the PCR technique. Of 17 laboratories, 3 had widely divergent results for replicate samples with high and low viral loads. Only one of these laboratories had problems with reproducibility for samples with both high and low viral loads. The overall results indicate that while variability is important, there are no sys- tematic testing problems with any laboratory in the detection of virus dilutions.
have involved use of denaturing as well as non- denaturing gel electrophoresis conditions. Vinyl polymer -gels have also been used for the diagnosis of mutations within proto – oncogenes. Now sequencing genetic analysis methods can also facilitate study of complex and rapidly involving genetic systems such as RNA viruses. Heteroduplex mobility essays, now used to classify HIV- 1 strains into genetic subtypes. These assays are also applicable to the analysis of other highly variable microorganisms. HMA used to classify HIV-1 strains into genetic subtypes or variation, which has a tremendous impact on vaccine efficiency, designing, development and production. Difficulties in establishing protection against slightly heterogeneous strains in animal vaccine models suggest that the high level of genetic variation found between the major subtypes of HIV-1 affect vaccine efficacy. It is therefore possible that vaccines will induce levels of protection against challenge strains that at least are in part proportional to their level of genetic relatedness to the vaccine strains. The major purpose of this study was to apply a simple reliable and quick method for the subtype identification of large number of samples. HMA fulfills these objectives by passing laborious colony an sequencing studies. A long awaited preventive vaccine remains an elusive target, seemingly in sight but just over the horizon.
Solid-phase EIA for the detection of antibodies to HIV-1 and HIV-2. The UBI HIV1/2 EIA is a solid-phase assay in which synthetic peptide antigens are adsorbed onto the reaction microplate. Aliquots of specimen diluted 1:21 are added to the reaction microplate wells, and the plate is incubated at 37 6 2°C for 30 6 2 min. Antibodies to HIV-1 and/or HIV-2, if present, will bind to the specific peptide antigens. After a thorough washing of the plate has been per- formed to remove unbound antibodies and other specimen components, an aliquot of a standardized preparation of goat anti-human immunoglobulin G antibodies conjugated to horseradish peroxidase is added to each well. The reaction microplate is then returned to the incubator (37 6 2°C) for 15 6 1 min. This enzyme-antibody conjugate will bind to any antigen–anti-HIV-1 and anti- gen–anti-HIV-2 antibody complexes present. The wells are again washed to remove unbound enzyme-antibody conjugate, and a solution of o-phenylenedi- amine (0.67 mg/ml) containing hydrogen peroxide is added to the wells; then the plate is incubated at 37 6 2°C for 15 6 1 min. A yellow-orange color will develop with an intensity proportional to the amount of HIV antibodies present in the specimen. The enzyme–o-phenylenediamine reaction is stopped by the addition of stop solution (1.0 M sulfuric acid). The absorbance value of each microwell is read by an EIA plate reader at a wavelength of 492 6 2 nm. Each run includes wells for two negative reactive controls, two HIV-1 strongly reactive controls, and two HIV-2 weakly reactive controls. The cutoff value used to determine sero- positivity was calculated based on statistical analysis as follows: (0.1 3 mean strongly reactive control value) 1 mean negative reactive control value. The total incubation time for the assay is 60 min, which is less than the 90 to 150 min required for other licensed HIV-1–HIV-2 assays. The total time needed to perform the assay is approximately 110 min.
ES bDNA assay procedure. Plasma specimens, as well as positive and negative controls, were prepared in duplicate for the ES bDNA assay. Aliquots (1.0 ml) were measured into 1.5-ml conical tubes with O-ring screw cap seals (no. 772- 692-005; Sarstedt, Newton, N.C.) and, after the addition of 50 m l of a 0.1% suspension of red polystyrene 0.5- m m-diameter beads (Bangs Laboratories, Car- mel, Ind.) to aid in the visualization of virus pellets, were centrifuged at 23,500 3 g for 1 h in a refrigerated microcentrifuge (centrifuge no. 17RS with rotor no. 3753; Heraeus, South Plainfield, N.J.). Supernatants were aspirated carefully, and the virus pellets either were processed immediately for the ES bDNA assay or were frozen at 2 70 8 C or colder. HIV-1 RNA was liberated from the virus pellets by addition of 220 m l of specimen diluent (100 mM HEPES [N-2-hydroxy- ethylpiperazine-N 9 -2-ethanesulfonic acid] [pH 7.5], 400 mM LiCl, 8 mM EDTA, 1% lithium lauryl sulfate, 12 m g of sonicated salmon sperm DNA per ml, 0.04% sodium azide, 0.04% Proclin 300 [Supelco, Bellefonte, Pa.], 2.2 mg of proteinase K per ml, 0.37 fmol of target probe set 1 per m l, 0.62 fmol of target probe set 2 per m l), followed by vortexing and incubation at 53 8 C for 20 min. Processed virus pellets were vortexed a second time and then held at room temperature for 5 to 15 min.
-copies/ml range, with a correlation coefficient (R) for expected versus observed results of 0.98. Compared to the CAHIM assay, the HPS-CTMHIV assay showed a high correlation (R ⴝ 0.99) across the dynamic range of RNA concentrations that, for the CAHIM assay, requires two different sample preparations. Equivalent performances were also observed for the two systems in the detection and quantification of HIV subtypes A to H. These data indicate that the HPS-CTMHIV assay may be one of the tests of choice for monitoring viral load throughout the course of HIV infection and during highly active antiretroviral therapy.
system were included in this evaluation; 79 of these 521 sam- ples were also tested with a third-generation bDNA method (Chiron Quantiplex 3.0). Samples were processed by the LCx HIV RNA Quantitative assay. Briefly, after addition of an internal standard (IS) that differed slightly from the target sequence, the assay controls and clinical samples were ex- tracted by centrifugation and separation using a QIAAmp col- umn and subjected to amplification with primers targeted at a highly conserved 170-bp sequence within the HIV-1 pol region, which enabled this assay to detect HIV-1 of the M group (A to G subtypes) and the O group. IS and HIV-1 primers were conjugated with two different synthetic capture haptens. Tar- get and IS amplifications were carried out simultaneously in a Perkin-Elmer 4800 thermal cycler in a ready-to-use, closed tube by reverse transcription-PCR (Thermus thermophilus polymerase) that contained all necessary reagents. Two differ- ent specific probes, one for the HIV-1 target and the other for the IS-produced target, conjugated with different haptens from the primers (detection haptens), were added during the last step of amplification. The tubes were then transferred un- opened into an automatic analyzer (LCx assay) for detection and quantification using the Microparticle Enzyme Immuno- assay (MEIA) system (7). Two different conjugates (alkaline phosphatase and beta-galactosidase) are employed; the first one binds to the HIV-1-specific probes, and the second to the IS-specific probes. The two substrates (7-beta galactosidase coumarin-4-acetic [2-hydroxyethylamine] and 4-methylumbel- liferyl phosphate) were added in succession, and the resulting fluorescence was read by the MEIA optic system. A calibration was carried out, with each different lot of LCx-assay-deter- mined HIV-1 RNA, by testing, in duplicate, six calibrators in which the HIV RNA and IS concentrations increased and decreased inversely from calibrator 1 through 6. The results were used with the LCx software to draw a curve on which sample and control results were subsequently calculated; the logarithm (log 10 ) ratio of the fluorescent measure of HIV and
The denaturation process can also affect the size bias observed on Illumina instruments. Denatured libraries are sometimes saved for re-sequencing in the case of a run failure (although Illumina’s best practices recom- mend preparing freshly denatured libraries). To test whether freshly denatured libraries perform differently from frozen previously denatured libraries, we se- quenced a freshly denatured library on a MiSeq, and the same denatured library 1 day later, after a freeze-thaw cycle, on a second MiSeq. The freeze-thaw cycle had a substantial effect on the size bias profile of this library; in particular, there was a dramatic reduction in the fraction of 150-bp molecules observed, resulting in a corresponding upward shift of the curve (Fig. 3e). It is likely that this shift reflects differential re-annealing of 150-bp fragments (which are in molar excess due to the presence of the large number of similarly sized normalization barcodes), or other small library mole- cules in the sequencing pool. This observation sug- gests that some of the difference in clustering size bias observed between the different platforms may be due to differences in denaturation conditions, the amount of time between loading the library and clus- tering, and whether the clustering process takes place in a chilled compartment (such as on the MiSeq) or not (such as the HiSeq 2500 and NextSeq). Consist- ent with this idea, the variation between HiSeq 2500 and HiSeq 4000 flow cells is much larger than the variation between the lanes on the same flow cell (Additional file 1: Figure S6).
Mice carrying a conditionally transcribed firefly luciferase gene in the ROSA26 locus, regulated by a loxP-flanked transcriptional stop element (ROSA-Stop-Luc) (48), can be used to detect Cre recombinase (Cre) activity by photon emission (48, 49). To test whether ROSA-Stop-Luc mice can be used to measure viral infec- tion by HIV-based viruses, we produced Cre-encoding replica- tion-deficient viruses pseudotyped with the envelope protein G of the vesicular stomatitis virus (VSVg-Cre). Viruses pseudotyped with VSVg are pantropic and can infect a broad range of mamma- lian target cells (50). Bioluminescence was readily detected in ROSA-Stop-Luc mice after i.p. injection of VSVg-Cre (75 ng p24), while control mice did not show any specific luciferase activity (Fig. 1A and B). The signal in the peritoneal cavity showed a major hot spot at the site of the omentum that peaked 6 days after injec- tion and that had a signal of 1.5 orders of magnitude above that for the control (Fig. 1B; P ⫽ 0.0159 at day 6). In addition, biolumi- nescence could be detected in the mediastinal region, the site of the primary draining lymph nodes of the peritoneal cavity (51); however, the bioluminescence was at a much lower intensity and had higher levels of variability than in the omentum (data not shown). Cre activity results from viral entry mediated by VSVg, as preincubation of VSVg-Cre with the neutralizing anti-VSVg anti- body I1 (52) results in nearly complete blockage of infection (P ⬍ 0.001), while preincubation with an isotype control did not reduce infection (P ⫽ 0.281) (Fig. 1C). In addition, these results were reflected by bioluminescence in ex vivo tissue samples of the omentum (Fig. 1C) and cells harvested by peritoneal lavage (data not shown). We conclude that HIV-based pseudoviruses encod- ing Cre can be used to detect viral infection in vivo and ex vivo.
selection of adjuvant systemic therapy for patients with estrogen receptor (ER)-positive, lymph node-negative breast cancer. It has ushered in the era of genomic-based personalized cancer care for ER-positive primary breast cancer and is now widely utilized in various parts of the world. Together with several other genomic assays, Oncotype DX has been incorporated into clinical practice guidelines on biomarker use to guide treatment decisions. The Oncotype DX result is presented as the recurrence score which is a continuous score that predicts the risk of distant disease recurrence. The assay, which provides information on clinicopathological factors, has been validated for use in the prognostication and prediction of degree of adjuvant chemotherapy benefit in both lymph node-positive and lymph node-negative early breast cancers. Clinical stud- ies have consistently shown that the Oncotype DX has a significant impact on decision making in adjuvant therapy recommendations and appears to be cost-effective in diverse health care settings. In this article, we provide an overview of the validation and clinical impact studies for the Oncotype DXassay. We also discuss its potential use in the neoadjuvant setting, as well as the more recent prospective validation trials, and the economic and utility implications of studies that use a lower cutoff score to define low-risk disease.