We conducted a pilot open-label randomised controlled trial of combined (oestrogen-progesterone) oral contraceptive pill (COCP)-exposure aimed to examine its effect on BV-recurrence following first-line antibiotics compared to antibiotics alone. Ninety-five women with symptomatic BV were prescribed antibiotic therapy, randomised to COCP-exposure (intervention) or current non-hormonal contraceptive practices (control) and followed monthly for six-months or until BV-recurrence. Modified intention-to-treat methods requiring either ≥1 clinical (primary/Amsel-outcome) or ≥1 microbiological (secondary/Nugent-outcome) BV-recurrence assessment were applied to determine cumulative recurrence rates. Secondary Cox regression analyses assessed factors associated with recurrence in all women. 92/95 women randomised provided baseline requirements. BV-recurrence rates were similar in women randomised to the COCP  compared to controls . In secondary analyses sex with the same pre-treatment regular sexual partner  and BV-history  were associated with increased BV-recurrence. This pilot RCT of COCP-exposure did not improve BV cure but found sex with an RSP and BV-history were associated with recurrence, although impacted by sample size and attrition. These data indicate reinfection from an untreated RSP and persistence of BV-associated bacteria are integral to the pathogenesis of recurrence and may overwhelm potential beneficial effects of hormonal contraception on the vaginal microbiota. Although first-line antibiotics8 have equivalent one-month cure rates of 70–80%9, six-month recurrence rates >50% ensue11. Given the global burden and morbidity associated with BV12, there is a pressing need to improve treatment efficacy to reduce sequelae.Bacterial vaginosis (BV) is the most common vaginal dysbiosis. Heterogeneous diverse bacteria dominate13. However this effect may be due to confounding factors influencing contraceptive choices, including partner type . We aimed to determine by randomised controlled trial (RCT) if COCP-exposure following antibiotic therapy reduces BV-recurrence risk within six-months, compared to antibiotic therapy alone. We hypothesized sustained exogenous sex-hormone exposure may support a healthy vaginal microbiota and reduce recurrence rates. This pilot trial was initially powered for a fully-funded RCT, with ongoing funding expected. The primary objective was to obtain efficacy estimates of the impact of COCP-exposure on recurrence rates and to establish feasibility using the following parameters; recruitment, adherence, adverse effects, and retention. Funding for the full-RCT was not secured and the pilot was terminated without viewing the data, and analyses were then performed.Hormonal contraceptive use, predominantly reflecting combined (oestrogen-progesterone) oral contraceptive pill (COCP)-exposure, is associated with significantly reduced BV prevalence , incidence , and recurrence  by meta-analysisWe conducted modified intention-to-treat (mITT) analyses of women who returned for ≥1 clinical assessment (primary/Amsel-outcome) or returned ≥1 sample for microbiological assessment (secondary/Nugent-outcome) of BV-recurrence within six-months of antibiotics. Secondary analyses assessed characteristics associated with BV-recurrence in all women.From July 2014-March 2016, there were 1644 consultations in which BV was diagnosed, representing 612 women. Clinicians did not refer women to trial nurses if they were known to be ineligible, wanted/did not want to commence hormonal contraception, or declined referral. Of 254 women referred to the nurse for eligibility assessment, 95  were recruited, 93 (37%) were ineligible and 66 declined participation .Two of 95 who consented withdrew immediately post-randomisation and another was found to have a COCP-contraindication; all three were excluded from analyses (no baseline data). The remaining 92 provided baseline characteristics Table , and comThe majority of women eligible for mITT analyses N = 82) were prescribed 7-days of oral metronidazole  with 15  prescribed 7-days of clindamycin  reported possible antibiotic adverse effects including headache/nausea/fatigue (n = 9), mood change (n = 1), mild gastrointestinal symptoms (n = 5), a metallic taste in the mouth (n = 1), thrush/thrush-like symptoms (n = 6), itchiness (n = 4) and bleeding/spotting (n = 2).Twenty-four women reported missing pills during at least one interval of follow-up: 20 missed only 1–2 active pills and four missed 3–5 active pills over at least one interval of follow-up. Reasons for missed pills included forgetting/travelling, losing pills or feeling unwell. Tricycling was practiced by 12 women but not continuously for the full duration of the trial.Forty women with COCP-exposure answered questions on adverse effects ; five controls requested to commence the COCP, three experienced an unplanned pregnancy but wanted to continue participation. One additional participant received a progesterone-implant during follow-up but remained in the control-arm.Ninety-two participants submitted baseline requirements, 66  completed the RCT to primary/Amsel-outcome . Twenty-six women did not return for clinical assessment , of which 16 returned ≥1 home-pack. Reasons for non-attendance included travel (n = 8), withdrawal due to COCP-related adverse effect (n = 2), or attended General Practice for assessment instead (n = 2). Fourteen did not provide reasons for non-attendance. Eighty-two of 92 women  completed the RCT to Secondary/Nugent-outcome, i.e. returned ≥1 pack for microbiological assessment of (1) recurrent-BV (n = 39) or (2) no recurrence (n = 43) by six-months.The primary outcome was assessed by mITT analysis, with BV-recurrence defined as ≥3 Amsel criteria and NS = 4–10 by six-months .vs no sex . COCP-exposure did not significantly reduce recurrence risk assessed as either current-exposure  or any COCP-exposure over follow-up . Condom-use was not associated with recurrence . There was also no association between country of birth, education level, current douching practices, sex work, penile-anal sex or receptive oral sex and BV recurrence measured at either study outcome.The influence of specific characteristics on BV-recurrence in all women at Amsel/Nugent-outcomes was determined using Cox regression adjusting for randomisation Table . Women r14 and there were more intervals of inconsistent condom use reported by women with an ongoing RSP compared to women with a new partner (54% vs 45%), we included condom-use in both models.Variables significantly associated with recurrent BV were included in two multivariate models Table . The firNeither treatment-randomisation nor actual COCP-exposure was associated with recurrence in either model. After adjusting for randomisation, women reporting an ongoing partner had a 3-fold increased risk of recurrence by Amsel/Nugent methods compared to women reporting no sex or a new partner , and BV-history conferred a 2.5–3-fold increased risk of BV-recurrence . The second model, adjusting for COCP-exposure (regardless of randomisation), replicated these findings; an ongoing sexual partner conferred a 3-fold increased risk of recurrence compared to no sex/new sexual partner  and past BV resulted a 2.5–3-fold increased risk of recurrence . There was no significant association between condom-use and BV-recurrence by either outcome or in either model.Attrition was associated with randomisation, with randomisation to COCP-arm associated with a 3-fold increased risk of loss/withdrawal before Amsel-outcome after adjustment , suggesting that COCP-exposure may promote a favourable vaginal microbiota following antibiotics. However, multivariate analyses of all participants showed re-exposure to an ongoing regular sexual partner (suggestive of reinfection) conferred a 3-fold increased risk of BV-recurrence and that women with a BV-history (suggestive of persistence) had a 2.5–3-fold increased risk of recurrence. These data indicate that reinfection from an untreated partner and persistence/re-emergence of BV-associated bacteria following treatment are both playing an important role in the pathogenesis of recurrent BV and may have obscured any beneficial effect of hormonal contraceptives on the vaginal microbiota. Our findings provide compelling evidence for the need to evaluate partner treatment and strategies to eradicate BV-associated bacteria including biofilm disruption as adjunctive therapies.In this open-label pilot RCT of the combined oral contraceptive pill in women treated for BV, COCP-exposure did not significantly reduce BV-recurrence measured by either Amsel or Nugent methods. While this finding was likely influenced by the limited sample size and uneven attrition, particularly affecting the COCP-arm, it may also indicate lack of an effect. Interestingly, the effect sizes observed for COCP-exposure on BV-recurrence were of similar magnitude to that reported by meta-analysis  as reported by meta-analysis (pES = 0.69)13. Our findings may be explained by low acceptability and poor retention, particularly in the COCP-arm, or the lack of a biological effect. As consistent condom-use is associated with reduced BV by meta-analysis, it is also possible that the non-significant increase in condom-use in controls may have obscured any beneficial COCP-related effect. Together our data suggest that the benefits of using randomisation as the gold-standard to reduce confounding may be outweighed by the limitations of this design, as higher attrition in women randomised to the COCP impacted on outcomes. Attrition was not associated with COCP-use if women who switched arms were analysed according to actual exposure. We cannot exclude a biologically favourable effect of COCP-exposure on the vaginal microbiota, but the effect is probably modest, and importantly may not be enough to override persistence of BV-associated bacteria or reinfection from partners.We undertook this pilot to determine whether hormonal contraception following antibiotic therapy reduced BV-recurrence compared to antibiotic therapy alone and to assess the feasibility of the intervention. Women using hormonal contraception have a reduction in BV in a number of observational studies25, illustrating the robustness of this association. Microbiological data from female and male sexual partners of women with BV demonstrate the effect of a regular partner on the vaginal microbiota. Women with a BV-infected female partner have concordant Nugent-scores26 and male partners of women with BV carry BV-associated bacteria28 on their penile-skin and within their distal urethra. Unfortunately, challenges with recruiting couples, attrition, and limited evidence for the optimal male treatment30 all pose challenges for researchers undertaking concurrent-partner treatment trials. Clinical trials of male partner treatment are underway in the US (NCT02209519) and Australia (ACTRN12619000196145).The secondary analyses suggest there are two likely mechanisms involved in post-treatment recurrence: i) BV clearance followed by reinfection from a sexual partner and ii) persistence with re-emergence, perhaps due to specific host factors. Women who have sex with an ongoing RSP post-antibiotics consistently have a 2–3-fold increased risk of BV-recurrence10. Past BV could be associated with BV recurrence for a number of reasons including persistence or re-emergence of BV-associated bacteria and/or biofilm, a host immune-mediated pathway31, or as a result of sexual behaviour, i.e. ongoing exposure to the same partner or inconsistent condom use with partners, as mentioned above. Several lines of evidence suggest persistence of BV-associated bacteria and/or biofilm could be occurring post-antibiotic treatment. This may be due to an inability of antibiotics to penetrate the biofilm matrix to target pathogens, antimicrobial resistance33, ineffective treatment of BV-associated bacteria or high organism loads35. BV-associated biofilms re-emerge after antibiotics37, cohesive Gardnerella vaginalis, which may be integral to the pathogenesis of BV38, is not as susceptible as planktonic G.vaginalis to lactic acid39, and RNA-sequencing shows gene-regulated processes in G.vaginalis biofilm favour bacterial growth40.There was a 2–3-fold increased risk of recurrence in women reporting a BV-history, which has been observed previouslyThis pilot trial was challenging to undertake with attrition related to randomisation. Concern about COCP-related adverse effects may have influenced equipoise regarding the COCP prior to recruitment and contributed to attrition from the COCP-arm. It is important to note that attrition was not related to actual COCP-exposure, demonstrating that women want to be able to select their preferred method of contraception and that the COCP is acceptable to women who chose it. Although equal numbers of women switched arms from both randomisation-groups, the three control-arm pregnancies also demonstrate the ethical challenges regarding randomisation to a group that does not contain a hormonal or highly effective method of contraception. Condom-use and emergency contraception were strongly promoted to all participants, as missing pills can also lead to unplanned pregnancies. Although COCP-adherence was self-reported monthly, biomarkers were not collected, so poor adherence may have also undermined findings. Additionally, the trial length (≤six-months) may have been too short to observe a significant benefit on recurrence rates as observational data generally reflects longer periods of COCP-use. Despite the shorter trial length, almost 1/3 did not attend for clinical assessment, which contributed to decreased power in the primary/Amsel-outcome analysis. We used both Amsel and Nugent criteria to comprehensively assess BV and although non-attendance affected the Amsel-outcome, the results between outcomes were consistent. Clearly, approaches to improve retention should be considered. Finally, single-site recruitment may limit generalisability.While this RCT of COCP-exposure did not reduce BV-recurrence, the effect size observed for the COCP was in the order of that reported in observational studies, despite limitations with sample size and disproportional attrition. The most striking finding however was that both BV-history and ongoing exposure to an untreated sex partner were associated with post-treatment recurrence. The robustness of these associations illustrates that reinfection from a sexual partner and persistence of BV-associated bacteria are important drivers of post-treatment recurrence, and it is possible that these factors may have overwhelmed any potential benefit from the COCP. This data supports epidemiological evidence that BV-associated bacteria are sexually transmitted. However, data from well-powered RCTs of partner treatment and further evidence of the mechanisms involved is required. Our inability to make any significant inroads into improving BV cure is likely due to the complex pathogenesis of BV and has led researchers to discuss combination approaches including antimicrobials with biofilm disruptors and partner treatment. Hormonal contraception, particularly containing oestrogen, may still support a favourable microbiota following effective treatment strategies and our ongoing microbiota analyses will explore this further. Well-powered RCTs may further determine the effect of the COCP on BV recurrence. But as attrition was associated with randomisation and not pill-use itself, larger observational studies carefully controlling for confounding factors are likely to be more acceptable to women and to yield valuable data that assist in determining whether there is merit in this adjunctive strategy.Strategies to prevent BV (SToPBV). Melbourne Sexual Health Centre  attendees with vaginal symptoms received a vaginal examination, pH estimation , specimen collection for C. trachomatis, N. gonorrhoeae TMA  and T.vaginalis culture (Trichomonas medium). Women were assessed for BV using both the Amsel method41, which documents the presence of a vaginal discharge, a positive amine test, vaginal pH > 4.5 and clue cells present on vaginal smear, and the Nugent scoring (NS) method42 was applied by experienced microbiologists, which scores the bacteria present on a vaginal smear, with BV = 7–10, intermediate flora = 4–6 and normal flora = 0–3. Women diagnosed with symptomatic BV, defined as ≥3 Amsel criteria with a Nugent score(NS) = 4–10, were prescribed 7-days of oral metronidazole 400 mg bd or, if contraindicated, 7-days of vaginal clindamycin8 and referred to trial nurses. Women aged 18–45 with symptomatic BV were eligible if they were willing to comply with protocol requirements and ineligible if they were (i) already using a hormonal contraceptive; (ii) assessed as having contraindications to the COCP using WHO criteria 43; (iii) pregnant or wishing to conceive within six months; (iv) concurrently diagnosed with pelvic inflammatory disease or HIV; (v) planning substantial travel; or (vi) did not have an Australian Medicare card or did not have reciprocal Medicare rights from an eligible country. Women were then assessed for equipoise (no objection to randomisation to either arm) and read and signed the Participant Information and Consent Form (informed consent) if willing to participate.This was an open-label pilot RCT of COCP-exposure following first-line antibiotics to determine the impact on BV-recurrence, called Women were randomised 1:1 to intervention-arm  or control-arm . The design was open-label as it was considered unethical to provide women with a placebo COCP. Alternative COCPs could be prescribed if clinically indicated or by participant request and any tricycling (skipping placebo pills to skip or shorten their bleeding period in between packs) was captured in questionnaires. A free-call phone number was provided so participants could report adverse effects, withdraw or for any other enquiries. All women received counselling regarding safe-sex practices and condom-use for STI and pregnancy prevention, and advised about indications for emergency contraception. Following enrolment, participants completed a questionnaire capturing demographics and behaviours, and self-collected a high-vaginal swab for future microbial analysis. Throughout follow-up, participants were contacted for clinic appointments or to prompt specimen collection at home. Reminders were sent with non-responders still able to contribute at the following interval. Follow-up involved questionnaires and self-collection of vaginal swabs and smears for NS-assessment at home at day 8 (d8) and months (M) 1, 2, 4, 5, with an additional clinic/Amsel assessment at M3 and 6. Women with a home NS = 7–10 were recalled to determine if they had symptomatic BV (by Amsel method) upon clinical assessment. Antibiotic adherence was recorded in d8 questionnaires and those in the COCP-arm were asked about adherence, missed pills, acceptability and adverse effects monthly. Participants were reimbursed up to AUD$60.44. Approval was obtained from the Alfred Hospital (404/13) and University of Melbourne (1340852) Ethics committees, and all research was performed in keeping with the National Statement on Ethical Conduct in Human Research, with informed consent obtained from all participants.This trial was prospectively registered with the Australian New Zealand Clinical Trials Registry on the 15/10/2013 (ACTRN12613001147774) with CONSORT reportingThe primary outcome was clinically defined BV-recurrence (≥3 Amsel criteria and NS = 4–10) or no recurrence within six-months. No participant with BV-recurrence had a NS < 4. Women were censored from follow-up once either outcome was reached or loss-to-follow-up (LTFU).The secondary outcome was BV recurrence defined as NS = 7–10 or no recurrence within six months. Women were censored from follow-up once secondary endpoint reached or until LTFU.A researcher with no clinical role (JSH) generated and held the random number sequence using randomisation block design (size 6) to ensure that allocation of women to the two arms occurred at a similar rate. Numbered, sealed envelopes that concealed allocation were opened consecutively by the research nurse to determine randomisation-group.Treating clinicians and diagnostic laboratory staff reported Amsel criteria and Nugent score prior to enrolment. Throughout follow-up, one experienced BV trial microbiologist (GF) reported the Nugent score from home samples and MSHC microbiologists reporting the Nugent score, amine and Clue cell criteria from clinical samples were all blinded regarding participant group. The participants, research nurse and treating clinicians were all aware of the allocation as blinding and use of a placebo was deemed unethical and unsafe as: i) women must know if they are at risk of pregnancy and be able to take emergency contraception if indicated, ii) COCP-users experience a regulated menstrual cycle and often mild but distinct initial symptoms such as breast tenderness.45. A sample of 266 gave 80% power to detect a 40% reduction in BV-recurrence from 40% in the control-arm to 24% in the COCP-arm . Assuming 15% LTFU, we aimed to recruit 157 women/arm. The pilot RCT commenced with internal funding, however ongoing funding was not secured and the pilot terminated after 95 recruits (without examining data). Rather than perform retrospective power calculations, we were statistically advised to interpret the likely magnitude of treatment differences using 95% confidence intervals (CIs).The sample size was estimated for a fully-funded trial using previous six-month recurrence rates Analyses were performed using STATA v14.2. The primary analysis was a modified intention-to-treat (mITT) analysis with women analysed as randomised who had ≥1 clinical assessment of BV-recurrence (primary/Amsel-outcome). Secondary analyses included (1) mITT analysis of women analysed as randomised who returned ≥1 microbiological specimen for Nugent scoring (secondary/Nugent-outcome); (2) per-protocol analyses (PPA), which followed women until primary/secondary outcome but censored women who “switched” arms at the point of cross-over; and (3) analyses by COCP-exposure  until primary/secondary outcome. Cumulative BV-recurrence rates per 100 person-years (PY) and Poisson 95%CIs were determined for the whole study population and separately by randomisation-arm within six-months, with differences between arms or COCP-exposure groups depicted by Kaplan-Meier survival curves and assessed by Cox regression. Analyses were adjusted for any baseline discrepancies .Recurrence rates per 100PY were calculated for baseline and longitudinal characteristics, with Poisson 95%CIs for primary/secondary outcomes. Additional Cox regression analyses of all recruited women were performed to calculate Hazard ratios (HRs) for univariate factors associated with BV-recurrence. Multivariate analyses included covariates associated with recurrence by univariate analysis and either randomisation-arm (Model 1) or COCP-exposure (Model 2).To inform feasibility, the proportion recruited, COCP-adherence and adverse effects were described. The attrition rate per 100PY until primary/Amsel-outcome was calculated for selected variables, with Poisson 95%CIs. Cox regression assessed factors associated with attrition.Supplementary Material


A highly accurate and versatile fluorescence polarisation assay for any enzyme adding or removing lipid posttranslational modifications, with the potential to accelerate drug discovery against these targets. S- or N-palmitoylation, N-myristoylation, S-farnesylation or S-geranylgeranylation. Furthermore, it is applicable to transfer and hydrolysis reactions, and we demonstrate its extension to a high-throughput screening format. We anticipate that Acyl-cLIP will greatly expedite future drug discovery efforts against these challenging targets.Posttranslational attachment of lipids to proteins is important for many cellular functions, and the enzymes responsible for these modifications are implicated in many diseases, from cancer to neurodegeneration. Lipid transferases and hydrolases are increasingly tractable therapeutic targets, but present unique challenges for high-throughput biochemical enzyme assays which hinder development of new inhibitors. We present Acylation-coupled Lipophilic Induction of Polarisation (Acyl-cLIP) as the first universally applicable biochemical lipidation assay, exploiting the hydrophobic nature of lipidated peptides to drive a polarised fluorescence readout. Acyl-cLIP allows sensitive, accurate, real-time measurement of  Posttranslational modification (PTM) of proteins with lipids is integral to many cellular processes, and dysregulation is implicated in diseases including cancer and neurodegeneration.Despite this progress, current biochemical assays to measure the activity of target enzymes present limitations that hinder drug discovery efforts. Classical methods for analysing lipidation use radioisotope-labelled lipids;To address the pressing and unmet need for a facile, versatile and robust assay for lipidation, we sought to develop a fluorescence polarisation assay driven by the increase in hydrophobicity upon lipidation which is universally applicable to all lipid PTMs. Fluorescence polarisation assays are commonly used to measure small fluorescent molecules or peptides binding to larger macromolecules, where binding results in a decrease in molecular tumbling and an increase in polarised fluorescence emission. Lipid PTMs are not macromolecular modifications, but we hypothesised that the increase in hydrophobicity could be used to bind the lipidated peptide to lipophilic macromolecular structures, resulting in decreased tumbling and increased polarisation.Here, we report the development of a facile, versatile and robust assay for fatty acylation, which we term Acylation-coupled Lipophilic Induction of Polarisation (Acyl-cLIP). We demonstrate that this method is readily applicable to all lipid PTMs, and overcomes the limitations of all existing biochemical lipidation assays. Acyl-cLIP displays excellent characteristics for high-throughput screening, and provides a powerful method for future investigations and drug discovery programs against this important target class.n-dodecyl β-d-maltoside (DDM), Triton™ X-100 (TX-100), or BSA (50) and detergent CMC  130–170 μM), CMC = 170 μM; TX-100 EC50 = 180 μM (95% CI 140–220 μM), CMC = 200 μM; 50 = 490 nM (95% CI 420–580) to the Kd of BSA for palmitic acid (104 nM) (Vmax = 0.081 pmol min–1 (95% CI 0.076–0.086 pmol min–1) and apparent KM = 170 nM  with palmitic (C16) acid by the enzyme hedgehog acyltransferase (HHAT) was used as a model system to validate the concept of the Acyl-cLIP format . Residue, or BSA . Deterge(104 nM) .19 No in(104 nM) , with th(104 nM) . Calcula(104 nM) . Unlike (104 nM) . The SHHN-myristoyl transferase (NMT) attaches myristic (C14) acid to the N-terminus of proteins, and is an attractive drug target in disease states such as cancer, malaria and the common cold.S-palmitoylation is increasingly appreciated.S-palmitoylation cycle.Legionella effector protein GobX   . ConsistHaving demonstrated that Acyl-cLIP allowed facile access to real-time analysis of transferase and hydrolase enzymes for any type of lipid PTM, we sought to further investigate application to inhibitor discovery and validation using SHH and HHAT as a model system. The ability to generate direct, real-time measurements offers advantages in analysing lipidation kinetics; however, certain situations require stopped-assay conditions, for example during a high throughput screen (HTS). We found that addition of excess non-fluorescently labelled substrate provides a generic means to stop Acyl-cLIP assays. HHAT-catalysed palmitoylation was halted at given time points by addition of non-fluorescent SHH peptide to 20 μM  substrates were employed as competitive inhibitors of SHH-FAM palmitoylation, affording IC50 values of 370 nM (95% CI 300–470 nM) and 440 nM (95% CI 350–570 nM), respectively (50 of 100 nM (95% CI 73–130 nM).A class of 5-acyl-6,7-dihydrothieno[3,2,terature , demonstectively , which c15-CoA), which is incorporated as the native lipid substrate.via bioorthogonal ‘click chemistry’ functionalisation with azido-TAMRA-biotin -catalysed azide–alkyne cycloaddition (CuAAC), and analysed by SDS-PAGE and in-gel fluorescence (IGF).15-CoA or HHAT concentrations did not increase product formation  acylation by HHAT and potent Pal-SHH product inhibition observed in Acyl-cLIP competition experiments, an orthogonal reporter strategy was employed. HHAT was purified to apparent homogeneity and incubated with SHH(FL) and alkyne-tagged Pal-CoA (YnCormation . This suZ-factor (Z′) of an assay is a measure of signal window relative to signal noise, with Z′ > 0.5 indicating an excellent assay for use in an HTS.Z′ = 0.51 under linear reaction progression at 1.7 h  signalling in the formation and maintenance of cancers has driven interest in the therapeutic potential of small-molecule HH-pathway inhibitors.84 wells . Real-ti15 and click-chemistry functionalisation identified two hits, bromocriptine and clomipramine, alongside RUSKI-201 blind controls which showed inhibition of SHH acylation by IGF. However, bromocriptine and clomipramine did not show inhibition of SHH acylation when assessed for increased molecular weight by anti-SHH blotting , in addition to RUSKI-201 . CounterLipid transferases and hydrolases are emerging as attractive and tractable therapeutic targets in a number of disease states; however, drug discovery efforts are hindered by challenges in high-throughput biochemical assays. We report here a new method for monitoring the activity of lipid transferase and hydrolase enzymes, which allows highly accurate real-time and stopped-assay measurement of lipidation. Acyl-cLIP uses an intrinsic property of lipid modification, hydrophobicity, to drive the assay readout, therefore offering advantages over existing assay formats, including enhanced versatility, safety, economy and increased throughput. Furthermore, the method can be readily applied to a range of targets and lipid PTMs thanks to the use of native lipid substrates and synthetically-accessible fluorescent peptides. Bioorthogonal lipid probes, for example incorporating alkyne tags, are equally compatible with the presented assay format. Indeed, we recently reported novel alkyne-farnesyl and -geranylgeranyl probes for global profiling of protein prenylation in live cells, and demonstrated in Prenyl-cLIP assays that the probes reproduce the enzyme and CAAX-box selectivity of Fpp and GGpp.8Km, in contrast to FA binding assays where nM concentrations of fluorescent peptides are typically used. It is therefore important that spectrophotometer settings are adjusted to avoid saturation of the detector. Use of initial velocity conditions (<10% substrate consumption) in enzyme assays means that the magnitude of signal window is smaller than for FA binding assays; however, real-time signal monitoring as presented here allows highly accurate determination of enzyme activity under these conditions.Acyl-cLIP was found to be universally applicable to enzymes involved in processing lipid PTMs. In developing these assays the design of substrate peptides was found to be a critical consideration. Lipophilic or lengthy peptide substrates can result in a decrease or loss of signal window between substrate and product. In developing an Acyl-cLIP assay for a new target both substrate and product peptides should be analysed to identify conditions that afford a maximal assay window. As an enzymatic assay, Acyl-cLIP formats require substrate concentrations defined by the target enzyme N-palmitoylation by HHAT provided insights into both substrate recognition and product inhibition, as well as demonstrating that Acyl-cLIP possesses excellent characteristics to enable future HTS campaigns. A pilot screen of 775 FDA approved drugs was conducted; although no new HHAT inhibitors were identified, the blind positive control compound RUSKI-201 was successfully identified. We note that the potential teratogenic effect of HH pathway inhibition diminishes the likelihood of FDA-approved drugs possessing off-target HHAT inhibition; however, this proof of principle study demonstrated the suitability of Acyl-cLIP and counter-screening assays to identify and triage HHAT inhibitors. We have subsequently successfully completed a full HTS to identify new HHAT inhibitors, the results of which will be disclosed in due course.Investigation of In summary, we present Acyl-cLIP as a versatile and HTS-compatible biochemical assay for lipid PTMs, which overcomes the limitations of existing methods. As enzymes responsible for lipid PTM processing continue to emerge as an increasingly tractable and promising classes of drug targets, we anticipate that this new assay format will greatly expedite future studies and medicinal chemistry programs.There are no conflicts to declare.Supplementary informationClick here for additional data file.


This review focuses on block copolymers featuring different photo-responsive building blocks and self-assembly of such materials in different selective solvents. We have subdivided the specific examples we selected: (1) according to the wavelength at which the irradiation has to be carried out to achieve photo-response; and (2) according to whether irradiation with light of a suitable wavelength leads to reversible or irreversible changes in material properties . Exemplarily, an irreversible change could be the photo-cleavage of a nitrobenzyl, pyrenyl or coumarinyl ester, whereas the photo-mediated transition between spiropyran and merocyanin form as well as the isomerization of azobenzenes would represent reversible response to light. The examples presented cover applications including drug delivery (controllable release rates), controlled aggregation/disaggregation, sensing, and the preparation of photochromic hybrid materials. Stimuli-responsive materials are capable of undergoing (reversible) changes with regard to physico-chemical properties such as solubility, polarity, charge, charge density, size or shape. Often, this is exploited in applications, where either response is translated into a certain action or where changes in environmental conditions are reported as or transformed into an optical or chemical readout. Examples are diagnostics, drug delivery, tissue engineering, or even triboelectric materials . Among tN-isopropylacrylamide))  thymine (tAZOi). Afterwards, the loading could be reversibly released upon irradiation with UV-light  [co-4-vinylbenzyl chloride) copolymers [The Donor–Acceptor Stenhouse Adduct (DASA) is relatively new to the class of photochromic molecules  152], w, w152], co-DMA)] . This wapolymers .block-poly(spiropyranemethacrylate) (PEG-b-PSPMA) diblock copolymers. These amphiphilic materials self-assembled in aqueous media into vesicles, which could be loaded with doxorubicin, gold nanoparticles, or various fluorescence markers. The microstructures of both spiropyrane and merocyanine polymersomes are synergistically stabilized due to hydrophobic interactions, hydrogen bonding, π−π stacking, and electrostatic (zwitterionic) interactions, with the latter two types being exclusively found for MC polymersomes. Moreover, the reversible photo-triggered SP/MC polymersome transition is accompanied by changes in membrane permeability—Thereby shifting from being non-permeable (450 nm) to selectively permeable (420 nm) towards non-charged, charged, and zwitterionic small molecule species below a critical molar mass  . A. Ablock-junction . In bothjunction . VisibleNear infra-red (NIR) light-responsive block copolymers are becoming more and more popular since they represent a promising opportunity to overcome issues that are combined especially with UV light irradiation (poor tissue penetration and toxic side effects) ,3. Receno-nitrobenzyl and coumarinyl esters show also photo-cleavage in the NIR window in terms of two-photon absorption occurring . T. T4:TmYbb-P(NIPAM-co-NBA) adsorbed onto an UCNP was presented in 2014 and again used for Nile red release experiments [b-PS block copolymer with a block junction having both an o-nitrobenzyl moiety and an azobenzene group incorporated was photo-triggered in the presence of UCNPs resulting in disruption of the nanoaggregates [co-spiropyrane methacrylate) and reversible switching of spiropyrane to the merocyanine form was achieved upon irradiation with light at 980 nm [3+ and Tm3+ were encapsulated within mesoporous silica and coated with a methacrylate/methacrylamide terpolymer consisting of spiropyrane and PEG grafted groups as well as side-chain conjugated folic acid functions used as receptors for tumor cell targeting [Similarly, a PEO-eriments . In 2017gregates . Thus fagregates . Howevergregates . These nt 980 nm . In anotargeting . BeforehClearly, the field of photo-responsive block copolymers is still evolving, especially when it comes to materials or examples where irradiation has to be carried out at wavelengths distinctly higher than the far UV regime. However, one thing that becomes evident when comparing different approaches reported so far is that it can be difficult to directly compare photo-response of different reports, as often entirely different irradiation setups are used. In other words, quantitative evaluation of photo-response in nanostructured materials alongside with issues such as complete/incomplete reversibility, determination of the amount of unaffected chromophores, or long-time photo-stability assessment is sometimes difficult to judge. This even translates into entirely different classifications of the term “photo-responsive” as well as a broad variety of light sources being used—Sometimes also without determination of the overall light intensity.Another aspect that is not always considered is whether continuous or repetitive irradiation also leads to photo-damage of the underlying polymer/block copolymer backbone, especially if UV light is used. Nevertheless, many very interesting and promising studies have been reported during the last decade and, although there is a certain variety of applications for such materials, the main research direction with regard to photo-responsive block copolymers in our opinion is towards improved control over spatial and temporal release of encapsulated cargo. Thereby, many examples have been reported where amphiphilic block copolymers containing one segment with covalently or non-covalently attached chromophores self-assembled into micelles within aqueous media and irradiation with light of a suitable wavelength then leads to (burst) release or degradation. In the latter case, irradiation is often accompanied by a sudden increase in solvent quality for the core of such aggregates, leading to swelling and, thus, permeability for guest molecules or even to complete dissolution if the resulting segment then is sufficiently hydrophilic. Nevertheless, most studies still deal with model drugs such as Doxorubicin or even Nile Red and the next step forward, possibly involving methods of upconversion to trigger release, can be anticipated within the next few years.


Cytotoxic neural stem cells (NSCs) have emerged as a promising treatment for Medulloblastoma (MB), the most common malignant primary pediatric brain tumor. The lack of accurate pre-clinical models incorporating surgical resection and tumor recurrence limits advancement in post-surgical MB treatments. Using cell lines from two of the 5 distinct MB molecular sub-groups, in this study, we developed an image-guided mouse model of MB surgical resection and investigate intra-cavity NSC therapy for post-operative MB.Using D283 and Daoy human MB cells engineered to express multi-modality optical reporters, we created the first image-guided resection model of orthotopic MB. Brain-derived NSCs and novel induced NSCs (iNSCs) generated from pediatric skin were engineered to express the pro-drug/enzyme therapy thymidine kinase/ganciclovir, seeded into the post-operative cavity, and used to investigate intra-cavity therapy for post-surgical MB.We found that surgery reduced MB volumes by 92%, and the rate of post-operative MB regrowth increased 3-fold compared to pre-resection growth. Real-time imaging showed NSCs rapidly homed to MB, migrating 1.6-fold faster and 2-fold farther in the presence of tumors, and co-localized with MB present in the contra-lateral hemisphere. Seeding of cytotoxic NSCs into the post-operative surgical cavity decreased MB volumes 15-fold and extended median survival 133%. As an initial step towards novel autologous therapy in human MB patients, we found skin-derived iNSCs homed to MB cells, while intra-cavity iNSC therapy suppressed post-surgical tumor growth and prolonged survival of MB-bearing mice by 123%.We report a novel image-guided model of MB resection/recurrence and provide new evidence of cytotoxic NSCs/iNSCs delivered into the surgical cavity effectively target residual MB foci. Medulloblastoma (MB) is the most common primary brain tumor in children , 2. MoleEngineered neural stem cells (NSCs) are emerging as a promising strategy for treating cancer –12. NSCsHere, we utilized human MB cell lines to create the first mouse model of image-guided MB resection and recurrence. We paired this model with both traditional and novel NSC types to explore multiple aspects of intra-cavity NSC therapy as a new approach to MB treatment. Real-time intra-operative optical imaging allowed resection of 92% of MB volumes. We found post-operative MB exhibited 3-fold faster growth rates compared to pre-operative MB, and observed complete recurrence of the tumor within 5 days post-surgery. Despite the highly aggressive nature of the post-operative cancer, cytotoxic NSCs seeded in the surgical cavity markedly suppressed growth of residual MB volumes and more than doubled the survival of tumor-bearing mice. As a novel approach to personalized therapy in patients, human induced NSCs derived from the skin of pediatric patients (hp-iNSCs) were found to migrate to MB, deliver clinically relevant therapies to slow post-surgical MB recurrence, and prolong median survival. These findings provide the first evidence that intra-cavity NSC therapy is an effective treatment for post-operative MB, and serve as a foundation for advancing this approach towards human patient testing.2 in complete media consisting of DMEM  supplemented with 10% heat-inactivated fetal bovine serum , and penicillin/streptomycin , as previously described.[Human MB  and mouse NSC (C17.2) lines were cultured at 37°C and 5% COescribed. hp-iNSCsescribed.Daoy-Green Fluorescent Protein / Firefly Luciferase (GFPFL) or D283-GFPFL xenografts were established in the right posterior fossa of Nude mice. A craniotomy aided in the visualization of the underlying GFP+ tumor for image-guided tumor resection. Established xenografts were excised using surgical dissection and aspiration methods similar to those previously described. BiolumiNSCs (NSC-mCherry-FLuc (mcF) or hp-iNSC-mcF) were seeded in the presence or absence of MB cells. Cell populations were separately seeded into adjacent wells located 0.5 mm apart in two-chamber culture-inserts , placed in glass bottom microwell dishes , and incubated overnight in complete media. Cells were placed in a VivaView incubator microscope (Olympus) and imaged at 10X every 20 minutes for 24 h.in vivo migratory capability, NSCs were injected into the contralateral cerebellar hemisphere of mice bearing established Daoy xenografts. Mouse brains were harvested three weeks after tumor cell implantation and processed as described[To assess the described.in vitro therapeutic efficacy of thymidine kinase-expressing stem cell therapy, NSCs or human pediatric induced neural stem cells (hp-iNSCtk) were cultured with D283-GFPFL or Daoy-GFPFL, respectively in black walled 96-well plates. Ganciclovir (GCV) was added to the wells at 20 μg/ml and incubated for 72 hr. Media was then aspirated and replaced with 7.5 mg/ml luciferin. After allowing 10 minutes for substrate to react with the enzyme active in living cells, the IVIS kinetic was used to capture bioluminescent signal emanating from surviving cancer cells.To assess the in vivo therapeutic efficacy of thymidine kinase-expressing stem cell therapy, hp-iNSCtk or NSCtk were implanted into the surgical cavity following microsurgical tumor debulking as previously described.[. Mice were monitored and sacrificed upon the development of neurological symptoms.To assess the escribed. Twenty-fIn vitro fluorescence imaging confirmed efficient transduction and robust GFP expression  MB cells into the cerebellum of mice as illustrated in Stem cells have emerged as powerful tools in brain cancer therapy, as the tumor-homing capacity allows these cells to populate distant tumor foci , 28, 29.tk) that converts the pro-drug ganciclovir (GCV) into a toxic agent leads to induce tumor cell death[TK within the surgical cavity, the cells were additionally engineered with firefly luciferase. Established MB were then resected from the cerebellum and NSCtk cells were implanted into the surgical cavity in a fibrin matrix  or the GCV pro-drug and serial BLI was used to monitor the fate of NSCtk over 7 days. We found that levels of NSCtk in control mice did not significantly change over a 1 week period, however NSCtk levels in GCV-treated mice decreased 89% over 7 days  and median survival more than doubled compared to controls (To investigate the NSCiability . To inve-surgery . In conttk). Transduction efficiency was confirmed with fluorescent imaging  that migrated to tumors and delivered therapeutic agents that successfully reduced human glioblastoma xenografts in mice. , 31 Adap imaging , Fig 5C.MB cells , Fig 5E MB cells .tk therapy, we recreated the therapeutic paradigm in a second MB post-surgical model using the more aggressive group 3 tumor cell line D283 [in vitro co-culture experiments, hp-iNSCtk efficiently killed D283 MB tumor cells  and subsequently surgically resected using fluorescence-guided microsurgery. The hp-iNSCtk/fibrin cells were delivered into the post-operative resection cavity. Eleven days after treatment initiation, hp-iNSCtk/fibrin+GCV was found to suppress tumor recurrence by 332% , group 3, and group 4) . The Daoine D283 . Group 3ine D283 . In in vor cells , suggest by 332% , Fig 5I  by 332%  and minireatment . This motk or hp-iNSCtk were co-cultured with both DAOY and D283. Viability assays showed that both NSC drug carriers again induced robust killing of both MB cell lines. However, we found that hp-iNSCtk induced greater killing compared to the NSCtk in both MB cell lines  is the most common childhood brain malignancy with a peak incidence at 7 years of age. Despite Although surgical resection is a universal component of the standard care for MB patients, surgery remains the least studied component of MB therapy. The events following surgical resection of MB have not been well explored, but recent studies using glioma models have reported increased tumor aggressiveness after surgical resection., 26 In tin vitro and in vivo. NSCs administered in the contralateral cerebellar hemisphere migrated towards and co-localized with established MB tumors. Consistent with other reports [MB disseminates into the CSF and spreads to other regions within the CNS. This highly migratory phenotype presents numerous drug delivery obstacles, which must be considered in potential therapeutic approaches. NSCs may be an effective model system for MB therapy as they have inherent tumor-tropic abilities , 20. In  reports , this sutk therapy improved survival of mice with Daoy tumors more than two-fold compared to control mice, representing treatment of the Shh subgroup of MB. The D283 model represents tumors of the group 3 subgroup, which frequently metastasize and lead to the poorest prognosis of all MB subgroups. Implantation of hp-iNSCtk into the post-surgical D283 resection cavity suppressed tumor growth and extended median survival in that aggressive group 3 tumor model. The ability of NSCs to control recurrence of two genetically distinct tumor models demonstrates the potential of NSC therapy in post-operative disease management of both intermediate and aggressive clinical phenotypes.NSCs genetically engineered to express cytotoxic agents are known to significantly diminish tumor burden and extend survival in orthotopic mouse models of malignant gliomas , 37, 38,in vivo teratoma formation. The safety benefit of TD-derived NSCs makes this therapy an attractive method for cell-based MB treatment. Through TD, skin fibroblasts can be used to generate hp-iNSCs in a matter of days [in vitro, suppressed tumor growth, and extended median survival in two genetically distinct MB resection/recurrence mouse models. Though stem cell therapy was effective against post-surgical MB, tumors eventually re-developed and led to death. Future studies on multimodal hp-iNSC therapy may provide an effective method to prolong treatment durability and eliminate therapy-resistant cell populations.To date, the majority of preclinical and clinical studies have focused on allogeneic NSCs, but autologous cells hold potential to evade immune rejection and where the prolonged persistence could enhance treatment durability , 42. Adv of days , 45. Add of days , 47, sugOne of the limitations of our study is the use of established MB cell lines. The DAOY and D283 lines are commonly used and allowed us to complete our initial proof-of-concept testing. Studies have shown that low-passage primary patient-derived (PDX) cells lines or fresh tumor biopsy samples can more accurately recapitulate aspects of the clinical disease. Future In conclusion, our study outlines the development of a novel MB resection model and the first demonstration that intra-cavity NSC/iNSC therapy is suppresses re-growth of post-operative MB. Development of the accurate MB resection/recurrence model presented in this study is vital to the understanding of recurrent MB disease and the subsequent advancement of recurrent MB therapies. This model mimics the aggressiveness of recurrent MB observed in the clinical setting, as resected tumors grew rapidly, spread through the cerebrum, and disseminated through the CSF. Utilizing this accurate pre-clinical model, we observed the ability of both brain-derived NSCs and hp-iNSCs derived from human pediatric fibroblasts to limit post-surgical MB tumor growth. This tumor model, coupled with the effectiveness of cytotoxic NSCs in the recurrent MB setting, provides a foundation for NSC-based therapies in the treatment of pediatric MB.S1 Checklist(PDF)Click here for additional data file.S2 Checklist(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S1 Supplemental methods(DOC)Click here for additional data file.


Cell-to-cell variation in the regenerative potential of mesenchymal stromal cells (MSCs) impedes the translation of MSC therapies into clinical practice. Cellular heterogeneity is ubiquitous across MSC cultures from different species and tissues. This review highlights advances to elucidate molecular profiles that identify cell subsets with specific regenerative properties in heterogeneous MSC cultures. Cell surface markers and global signatures are presented for proliferation and differentiation potential, as well as immunomodulation and trophic properties. Key knowledge gaps are discussed as potential areas of future research. Molecular profiles of MSC heterogeneity have the potential to enable unprecedented control over the regenerative potential of MSC therapies through the discovery of new molecular targets and as quality attributes to develop robust and reproducible biomanufacturing processes. These advances would have a positive impact on the nascent field of MSC therapeutics by accelerating the development of therapies with more consistent and effective treatment outcomes. This poin vivo . MSCs hain vivo , 5, and in vivo , 7. The in vivo , undergoin vivo , exhibitin vivo , regulatin vivo , and secin vivo . The thein vivo .in vivo in the stem cell niche [ex vivo cultivation [A barrier to realize the therapeutic potential of MSCs is their intrinsic heterogeneity. MSCs are a composite of cell progenitors at different states of lineage commitment , 15 and ll niche , is evidll niche , and is tivation . Cell sutivation , 24 and tivation , 14. Celtivation –27. Thistivation , 28, 29.There is a critical need for molecular profiles of MSC heterogeneity to manufacture effective MSC therapies. This review highlights advances to elucidate cell surface markers and global signatures that identify cell subsets with specific regenerative properties in heterogeneous MSC cultures. Molecular profiles of MSC heterogeneity will enable cell enrichment and quality control assessment during the manufacturing of MSC therapies to standardize cell composition. In addition, they will help identify new molecular targets to regulate the regenerative potential of MSCs. Molecular profiles of MSC heterogeneity are expected to make a positive impact on the nascent field of MSC therapeutics by accelerating the development of therapies with more consistent and effective treatment outcomes.ex vivo to obtain a sufficient amount of cells for clinical applications [ex vivo expansion that alters the composition of cell subsets in culture and, in turn, may impact the efficacy of MSC therapies [MSCs are a rare population of progenitors in adult tissue  and are ications . Cell-toherapies . Heterogherapies , 24. We herapies , 32, 33.A focus of ongoing research on MSC heterogeneity is to elucidate an immunophenotype of proliferation potential. Cell surface markers enable noninvasive and nondestructive isolation of specific cell subsets from MSC cultures for research and clinical applications. The International Society for Cellular Therapy has specified that human MSCs must express CD73, CD90, and CD105 . We and + stroma emerges in fetal bone marrow before the onset of hematopoietic activity [+/glycophorin A− fraction of human bone marrow [ex vivo, some isolation markers are rapidly downregulated [Several cell surface markers have been identified to isolate MSCs with high colony-forming efficiency from tissues . Of thesactivity , suggestactivity –39. MSC activity ; howeveractivity . Once ise marrow . When MSegulated , as is tegulated .ex vivo expansion. One category of markers for cultured MSCs is a subset of isolation markers, including CD146 and stage-specific embryonic antigen-4 (SSEA-4), that is expressed in ex vivo cultures of hBM-MSCs [β1 integrins and growth factor receptors in regulating cell proliferation, motility, and survival [Surface markers that detect cellular heterogeneity in cultured MSCs enable quality control of MSC therapies during hBM-MSCs , 35. SurhBM-MSCs . While ChBM-MSCs , SSEA-4 hBM-MSCs  that is hBM-MSCs . NG2 potsurvival . hBM-MSCsurvival . We obseE2F2, PTTG1, and TWIST1 are representative of this common signature [PTTG1 (aka pituitary tumor-transforming gene 1) encodes securin, which participates in synchrony of chromosome separation in the anaphase of mitosis [TWIST1 gene maintained an immature phenotype and exhibited an increased proliferation rate relative to a mock control [The global scope of transcriptomic and proteomic profiling can detect differences in cell populations not evident with immunophenotyping , 52. A gignature . The E2Fignature . PTTG1 ( mitosis . The tra mitosis . hBM-MSC control .SOX2), notch homolog 1 (NOTCH1), and the notch ligand delta-like 3 (DLL3) were among the upregulated genes in fast-growing MSCs [HSPA9) was the most overexpressed, by nearly 10-fold, among the genes upregulated in the slow-growing MSC subset. Overexpression of mortalin in senescent normal human lung fibroblasts increased cumulative population doublings, induced a younger cell morphology, and lowered senescence-associated β-galactosidase activity relative to controls [In another study of fast- and slow-growing subsets of hBM-MSCs from osteoarthritis patients, genes encoding sex-determining region Y-box 2  and tropomyosin (TPM4), in fast-growing MSC populations relative to their slow-growing counterparts . Overexpin vitro high-capacity assay to quantify the clonal heterogeneity in the trilineage potential of hBM-MSCs [Trilineage potential to exhibit adipo-, chondro-, and osteogenesis is one of the minimal criteria for hMSCs established by the International Society for Cellular Therapy . This crhBM-MSCs . By analhBM-MSCs , 64.Surface markers of proliferation potential can isolate cells that are both rapidly dividing and multipotent from heterogeneous MSC cultures , 35, 43;in vitro osteogenesis than TNAP− MSCs from the same culture [+ hBM-MSCs can be divided into a CD56+ fraction that is enriched in chondroprogenitors relative to the CD56− fraction [Much of the research in this area has focused on surface markers of osteogenic and chondrogenic potential given that bone repair and cartilage repair account for nearly 20% of MSC-based clinical trials . Positiv culture . TNAP pl culture , 69. Exp culture . As anotfraction . CD56 isfraction . Like CDfraction .in vitro and in vivo [κB (NF-κB) pathways [+ cells were more prevalent in expanded hBM-MSC cultures from osteoarthritis patients than in control cultures from healthy donors [in vitro relative to the controls [Recently, the receptor tyrosine kinase-like orphan receptor 2 (ROR2) was identified as a predictive marker of chondrogenic potential for hBM-MSCs . Positiv in vivo . Possiblpathways , 73. RORy donors . Consistcontrols .+ CD105+ cells account for as little as 1% of hBM-MSCs and 3-9% of hAD-MSCs [There is a growing body of evidence that MSCs contain a rare population of pluripotent cells, which have been named multilineage-differentiating stress-enduring (MUSE) cells . MUSE cehAD-MSCs , 75. MUShAD-MSCs , 76, andhAD-MSCs . This plhAD-MSCs , 77, 78.hAD-MSCs , which hhAD-MSCs , 75. GivhAD-MSCs , MUSE ceSp7) and its mediator, distal-less homeobox 5 (Dlx5), that are master transcription factors in the bone morphogenetic protein pathway [γ (Pparg) and CCAAT/enhancer-binding protein α (Cebpa) that regulate each other in a positive feedback loop [RNA sequencing elucidated transcriptional signatures that distinguish between single cell-derived colonies of mBM-MSCs with osteogenic and adipogenic potential . The tra pathway . In contack loop , 82. Undack loop . Coexistack loop .SFRP2) and calponin 1 (CNN1) were the most upregulated and downregulated, respectively, in MSC strains that formed a bone/marrow organ relative to those that formed only fibrous tissue [SFRP2 encodes a soluble inhibitor of Wnt signaling [α5β1 complex to inhibit apoptosis [CNN1 gene in bone/marrow-forming MSC strains, CNN1-null mice exhibit increased bone formation [In another study, microarray analysis revealed a transcriptional signature of hBM-MSCs that forms an ectopic ossicle containing both bone and marrow when implanted in immunodeficient mice . Single s tissue . SFRP2 eignaling , but itspoptosis , which mpoptosis . In reguormation .MSCs possess extraordinary immunomodulatory and trophic properties to orchestrate endogenous tissue repair. To resolve inflammation in damaged tissue, MSCs suppress inflammation and may promote clearance of inflammatory stimuli . To prom− and VCAM-1+ subsets, the latter was far more effective in (1) suppressing the secretion of inflammatory cytokines by activated peripheral blood mononuclear cells and (2) stimulating the formation of anti-inflammatory regulatory T cells [+ cells in MSC cultures is tissue specific: more in hCV-MSCs than in hBM-MSCs and none in hAD-MSCs [+ cells in hCV-MSCs may be associated with fetomaternal tolerance. The placenta provides an immune-privileged environment for the fetus [+ MSCs within chorionic villi may help suppress an immune response at the border between maternal and fetal blood.Surface markers that select for colony-forming MSCs, such as CD271 and STRO-1, have been used to enrich MSCs with increased immunosuppressive capacity , 95. In  T cells . The conhAD-MSCs . The highe fetus . VCAM-1+κB to induce an inflammatory response [+ MSCs could potentially have a role in the clearance of viral infections. It is noteworthy that the differentiation potential of tetherin+ hBM-MSCs was compromised relative to tetherin− MSCs from the same culture [A growing body of data suggests that MSCs may exhibit proinflammatory properties in certain instances, perhaps to stimulate pathogen clearance , 102. Reresponse . Tetheri culture , suggest+ hCV-MSCs had more blood vessels at the injection site than controls receiving the CD146− cell fraction [+ hBM-MSCs was evident when they were divided into STRO-1Bright and STRO-1Dim subsets: conditioned medium from the STRO-1Bright MSCs elicited greater cardiac cell proliferation and endothelial tube formation [A few surface markers have been examined for their ability to isolate MSCs with enhanced trophic activity to increase angiogenesis and promote endogenous tissue repair. For instance, murine gastrocnemius muscle injected with CD146fraction . Cell-toormation . Inhibitormation .+ hCV-MSCs provided insight into the enhanced anti-inflammatory potential of this cell subset [− hCV-MSCs, the VCAM-1+ subset had elevated mRNA levels of cyclooxygenase-2 (COX2), indoleamine 2,3-dioxygenase 1 (IDO1), and other key immune modulators, as well as secreted more prostaglandin E2 (PGE2) protein [+ MSCs may have an anti-inflammatory function. Specifically, COX-2 may increase the production of the immunosuppressors PGE2 [Microarray and qPCR analysis of VCAM-1l subset . Relativ protein . While C protein , its incors PGE2  and IDO-ors PGE2  in MSCs.ors PGE2  and reguors PGE2 . Overexpors PGE2 .+ hBM-MSCs revealed a gene signature that is consistent with a possible role for this cell subset in viral clearance [+ hBM-MSCs expressed several antiviral genes, including interferon-stimulated gene 20 kDa protein (ISG20) and oligoadenylate synthetase (OAS), at higher levels than the parental culture and tetherin− strains of hBM-MSCs [Interrogation of the transcriptome of tetherinlearance . Single hBM-MSCs . Both gehBM-MSCs , and OAShBM-MSCs . The anthBM-MSCs  and induhBM-MSCs .Bright MSCs stimulated in vitro endothelial cell proliferation and tube formation and in vivo angiogenesis to a greater extent than ALDHDim MSCs from the same culture [Bright and ALDHDim subsets had similar gene signatures, their secretome was different [Bright MSCs uniquely secreted several proangiogenic cytokines, which included vascular endothelial growth factor β (VEGFB) and platelet derived growth factor α (PDGFA); whereas, ALDHDim MSCs secreted inhibitors of angiogenesis, such as platelet factor 4 (PF4) and plasminogen (PLG) [Proteomic analysis of conditioned medium from hBM-MSCs detected uniquely secreted trophic factors for angiogenesis in cell subsets with elevated expression of aldehyde dehydrogenase (ALDH) . A fluor culture . While tifferent . ALDHBrien (PLG) . The coeen (PLG) . MSC suben (PLG) .in vivo prior to harvesting MSCs from a donor. Repeated cell division during tissue maintenance and repair can cause cellular aging over the life of the donor, particularly in the elderly [ex vivo expansion to produce clinically relevant quantities of MSCs. Second, it may impair the effectiveness of MSC therapies to regenerate damaged tissue. We and others have observed that MSC cultures contain a heterogeneous mixture of cells at different stages of aging, starting at early passage [Cellular aging and replicative senescence compromises stem cell fitness and is an obstacle to the production of effective MSC therapies. Replicative stress caused by rapid MSC expansion during biomanufacturing can induce DNA damage and cellular aging . During  elderly . The neg passage , 17, 124ex vivo expansion [hi MSCs from culture and causes an accumulation of SSEA-4lo cells with diminished regenerative potential [+ cells accounted for less than 10% of hBM-MSCs harvested from elderly donors ages 65 and older. They were able to isolate these high-quality cells and expand them on the extracellular matrix from young-donor MSCs to quantities required for clinical applications [Surface markers for rapidly dividing, multipotent MSCs  are downregulated in aging MSCs during xpansion , 35. Forxpansion  describeotential . Block eotential  reportedications .+ cells accumulates during serial passaging of hBM-MSC cultures: CD264 is initially upregulated at an intermediate passage concurrently with p21 and remains upregulated at late passage as aging progresses to senescence [+ cell content with multiple metrics of stem cell fitness, including colony-forming efficiency as a measure of proliferation potential [A surface marker expressed on aging cells would enable the enrichment of rapidly dividing MSCs by negative selection. Recently, we reported on decoy TRAIL receptor CD264 as the first surface marker of cellular aging for MSCs . The connescence . There iotential . MSCs maotential .Gene and epigenetic signatures have been identified for molecular changes in heterogeneous MSCs associated with replicative senescence , 127, buin vivo survival of the stem cells by attenuating anoikis [The molecular profiles described here provide new insight into MSC biology. This knowledge can be exploited to identify molecular targets to regulate the regenerative potential of MSCs . Targete anoikis . Recent  anoikis . AdjuvanSurface markers and global signatures of MSC heterogeneity have the potential to be effective in predicting treatment outcome in MSC-based clinical trials. Predictive molecular profiles of the regenerative potential of MSCs have utility as quantifiable attributes of cell quality during the manufacturing of MSC therapies . These qMolecular profiles of MSC heterogeneity are far from complete. More research is needed in all categories of MSC function and, in particular, homing. Upon remote delivery of MSC therapies to a patient, the stem cells are capable of homing to the site of injury. Despite its importance to tissue repair, little is known about the cell-to-cell variation in the ability of MSCs to home to injured tissue . To dateThere is limited information on how the molecular profiles of MSC heterogeneity are influenced by variables encountered during the manufacturing of MSC therapies. MSC heterogeneity can be profoundly affected by the choice of stem cell donor , tissue In vitro behavior of MSCs is not a reliable predictor of their in vivo performance. As a case in point, only a fraction of clonal mBM-MSCs, which exhibit in vitro osteogenesis, are capable of in vivo bone formation [in vitro function of MSCs need to be validated first in animal models and then in clinical trials. Molecular profiles that are predictive of clinical outcome are candidates to use as quality attributes for robust and reproducible manufacturing of MSC therapies.Many of the molecular profiles discussed here have yet to be validated in an animal model. ormation . PromisiInconsistencies in the composition of MSC cultures hinder their use in regenerative medicine. There is a critical need for molecular profiles of this heterogeneity to manufacture effective MSC therapies. This review presented cell surface markers and global signatures that isolate cell subsets with specific regenerative properties from heterogeneous MSC cultures. More research is required to ascertain how these molecular profiles are influenced by biomanufacturing conditions. Other areas to be explored include noncoding RNA and epigenetic signatures, in addition to the in vivo validation of many of the molecular profiles. Predictive surface markers and global signatures of regenerative potential will facilitate robust and reproducible manufacturing of MSC therapies, as well as identify new molecular targets to regulate MSC function. This control over MSC composition and function will accelerate the translation of MSC therapeutics into clinical practice.


As pre-exposure prophylaxis (PrEP) effectiveness is strongly linked to adherence, we sought to determine if certain self-report measures could be used to inform objective PrEP adherence. We studied participants from the TAPIR study (a multicenter randomized study of daily text messages to support adherence to PrEP In At-Risk), a 48-week randomized controlled trial of HIV-uninfected men who have sex with men (MSM) randomized to receive text message to support adherence versus standard of care. Self-reported medication adherence was assessed using several validated measures modified for PrEP. Objective PrEP adherence was determined through dried blood spot (DBS) measurement of intracellular tenofovir diphosphate (TFV-DP) and emtricitabine triphosphate (FTC-TP). A summary of adherence was estimated using responses to the seven adherence items at weeks 12 and 48 using confirmatory factor analysis. Correlations between self-report questions and drug concentrations were estimated with Pearson’s correlations for continuous outcomes and point-biserial correlations for dichotomous outcomes. Receiver operating characteristic (ROC) analyses were conducted to assess the performance of self-report measures in predicting protective or perfect TFV-DP concentrations. Of the 369 participants who completed week 12 or 48 visits, the mean age was 35 (standard deviation 9 years), with 79% White, 12% Black, and 29% Hispanic. Correlations between self-report measures of adherence  and quantifiable FTC-TP and continuous TFV-DP concentrations showed that all self-report measures were significantly associated with these objective measures. Compared to a summary measure of self-reported adherence, the 4-week percent taken question medication recall was the only self-report item similarly or more strongly associated with recent adherence and long-term protective and perfect adherence at weeks 12 and 48. ROC analysis also showed that 4-week percent taken question had a reasonable AUC (0.798 at week 12 and 0.758 at week 48) in predicting protective TFV-DP concentrations. All single-item self-report questions assessing PrEP adherence were significantly associated with biomarker quantification, with the 4-week percent taken question performing best. Therefore, in the absence of drug concentration measurements, a 4-week self-report percent taken question may be a good single-item measure of PrEP adherence. Over the last several years, HIV prevention has increasingly included biomedical strategies using pre-exposure prophylaxis (PrEP). Once daily tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) has been shown to greatly reduce the risk of HIV infection in populations at risk for HIV acquisition . Sub-optPresently, no single “gold standard” has been ubiquitously adopted to assess adherence. Research studies have employed biomarker quantification as an objective adherence measure. At present, these pharmacological assays are costly, require specialized laboratory equipment and personnel, and take time to yield results; however, there is research underway to develop point of care immunoassays in urine that could be used as clinical tools . Until rSelf-report adherence measures generally have the lowest concordance with drug concentrations . ClinicaIn this current analysis, we analyze the concordance between several self-report adherence questions with two pharmacologic drug level measures used in a PrEP demonstration project of MSM to identify the most accurate PrEP-appropriate self-report adherence measures.Text Messages to Support Adherence to PrEP In At-Risk for HIV Individuals; NCT01761643)  595 TAPIR study (A Multicenter Randomized Study of Daily 1761643) . ParticiTAPIR participants were MSM, 18 years or older, and HIV-negative as confirmed by an antigen/antibody (Ag/Ab) assay or Ab assay plus HIV nucleic acid test. Additional eligibility criteria included having persistent elevated risk of HIV acquisition through condomless anal intercourse with HIV-positive men and/or partners of unknown status or having a recent sexually transmitted infection (STI) diagnosis. Over a 48-week study period, all participants received once-daily PrEP with TDF/FTC, risk reduction and adherence counseling, safety monitoring, and HIV/STI testing every 3 months. Data were collected at baseline, weeks 4, 12, 24, 36, and 48 by both confidential in-person interviews and computer assisted self-interview (CASI) instruments, which included a survey with self-report adherence measures. All CASI questions were answered and recorded by the participant. At weeks 12 and 48, biologic markers of adherence were measured, described in the next section. In the main study, iTAB participants were more likely to have tenofovir drug concentrations corresponding to near-perfect adherence (∼7 doses per week). This analysis includes the 369 TAPIR participants with completed study visits at weeks 12 or 48 after initiating PrEP.Pre-exposure prophylaxis as a biological marker of adherence was determined through dried blood spot (DBS) measurement of intracellular TFV-DP and FTC-TP (measuring predominately intra-erythrocytic concentrations). Both TFV-DP and FTC-TP were quantified at weeks 12 and 48 using a liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay previously validated . ProtectSelf-reported medication adherence was measured at all study visits after baseline using seven CASI questions, some of which were taken from the ACTG Adherence Instrument  and otheA summary of adherence was additionally estimated using participant responses to the seven adherence items at weeks 12 and 48 using confirmatory factor analysis (CFA). CFA is a structural equation modeling technique of examining the relationship between observed variables and their underlying latent constructs. Similar to other clustering methods, CFA summarizes data and estimates the amount of shared variance between a set of variables . A one-fAssociations between individual self-report questions assessing adherence and objective measures of adherence (FTC-TP and TFV-DP) were assessed using correlation analyses. The adherence factor and objective adherence measures were also correlated to obtain a measure of association between a summary measure of self-reported adherence and DBS measurements. Correlations between continuous variables were estimated as Pearson’s correlations while correlations between continuous self-report adherence measures and dichotomous DBS measurements were estimated as point-biserial correlations using robust maximum likelihood estimation (MLR). To account for the non-independence of observations due to participants’ repeated assessment, standard errors were adjusted using a Huber–White sandwich estimator .z-scores using Fisher Z transformations and tests of the equality of correlation coefficients  were carried out using asymptotic z-tests  analyses were also conducted to assess the performance of these self-report measures in predicting protective or perfect TFV-DP concentrations . Area unOf the 369 participants contributing data to these analyses, the mean age was 35 with a standard deviation of 9 years. The majority of participants were white (79%), with 12% Black and 29% Hispanic. More than half of the participants held a bachelor’s or advanced degree and nearly two-thirds had a monthly income of $2,000 or greater. Half were in the intervention arm of TAPIR Table 2.z-tests suggested that 4-week percent taken was more significantly associated with recent adherence (quantifiable FTC) while 4-week ability, 4-week frequency, 3-month recall, and 1-month difficulty were associated with recent adherence to the same degree as the summary measure. The remaining self-report items had significantly weaker correlations. In terms of longer-term adherence, 4-week percent taken was associated with protective TFV-DP concentrations (>700 fmol/punch) to the same degree as the summary measure; all other measures were significantly less correlated than the summary measure. With regards to perfect TFV-DP adherence , compared with the summary measure, 4-week percent taken, 4-week ability, 4-week frequency, and 3-month recall had a similar correlations. The remaining measures were marginally or significantly less correlated with perfect TFV-DP concentrations than the summary measure , and all adherence items had large and significant factor loadings, suggesting good associations with the latent adherence construct Table 3 Finally, we evaluated the performance of the self-report questions in predicting protective or perfect TFV-DP concentrations using ROC analyses. AUC values were calculated at weeks 12 and 48. Similar to findings above, ROC analyses also showed that 4-week percent taken question had a fairly good AUC (0.798 at week 12 and 0.758 at week 48) in predicting protective TFV-DP concentrations. However, all self-reported measures were not particularly good for predicting perfect TFV-DP . Results from week 48 are displayed in We found that all single-item self-report questions assessing PrEP adherence were significantly associated with biomarker quantification with the 4-week percent taken question performing best. In addition, the 4-week percent taken question had a reasonable AUC value in predicting protective TFV-DP concentrations at both weeks 12 and 48. Therefore, in the absence of drug level measurements, our findings suggest that a 4-week self-report percent taken question may be a good single-item measure of both recent and cumulative adherence.via an interview question assessing missed doses over the last 3 days was only modestly associated with quantifiable drug concentrations of tenofovir (TFV) and FTC quantified in plasma (Several recent studies have similarly examined different combinations of subjective and objective PrEP adherence measures and have found varying degrees of concordance among them. In the TDF2 clinical trial of men and women in Botswana, self-report adherence n plasma . Similarn plasma . Within n plasma . Our stuBecause PrEP efficacy highly depends on medication adherence, it is frequently studied in HIV prevention research and evaluated in PrEP clinical care. Subjective adherence reporting is the most commonly used method to quantify adherence behavior in real time . As is tPrEP delivery in limited resource locations necessitates inexpensive and easy approaches to offer and monitor PrEP. As self-report may be the sole method to measure PrEP adherence, a question that is both sensitive and specific could offer an accurate appraisal of adherence and help direct resources to those needing additional adherence support in well-resourced but high-volume PrEP clinics. Using a single question that elicits a response best reflecting real adherence behavior is an efficient way to gauge adherence. In addition, one adherence question could be included in clinic intake forms that patients can answer more privately, potentially reducing social desirability bias .While our study had several strengths including a large sample size and assessments performed at multiple time points, there are some limitations. Since our study included only MSM in a resource-rich setting, our results may not be generalizable to other at-risk populations or those in resource-limited settings taking PrEP. Study timing may have significantly influenced our findings, as it was conducted soon after the FDA approval of PrEP. We had many early adopters who had high overall adherence based on DBS concentrations and were less likely to overestimate adherence. In addition, we only used single-item adherence questions to evaluate associations with PrEP drug concentrations. Some questions were designed to be asked as part of a group so alone they may have less intrinsic value. Finally, there are other commonly used self-report questions, which may be similarly or better correlated with biological measurements, not included in our study.Self-report questions to measure PrEP adherence are commonly utilized in clinical research and may be the only method deployed to assess medication adherence. It is essential to better understand which subjective measures are most accurate and how they can effectively be integrated into PrEP research and clinical care. Our findings demonstrate that a 4-week percent taken question of medication recall may best reflect true recent and longer-term adherence behavior. In the future, we will explore combinations of different self-report adherence questions that may yield even stronger associations with PrEP drug concentrations.This study was carried out in accordance with the recommendations of the UCSD Human Research Protections Program with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the UCSD Human Research Protections Program.JB, DM, and SM contributed to the conception and design of the study; EE organized the database; SJ and SS performed the statistical analysis; JB wrote the first draft of the manuscript; EP and EE wrote sections of the manuscript. All authors contributed to manuscript revision, and read and approved the submitted version.The work was supported by award EI11-SD-005 from the California HIV/AIDS Research Program, award IN-US-276-D036 from Gilead and 1KL2TR0001444 to Dr. Blumenthal.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


EGFR gene mutations, KRAS gene mutations, EML4-ALK rearrangements and altered MET signaling are widely recognized alterations that play important roles in both the biological mechanisms and the clinical sensitivity to treatment in lung cancer. In this article, we reviewed the discovery of the clinical values of these oncogenic driver mutations and the clinical studies revealing the prognostic and predictive values of these biomarkers for clinical sensitivity and resistance to anti-EGFR therapy or other targeted therapies. These form the basis of personalized treatment in lung cancer based on biomarker profiles of individual tumor, leading to therapeutic advancement and betterment.Lung cancer is a heterogeneous and complex disease. Genomic and transcriptomic profiling of lung cancer not only further our knowledge about cancer initiation and progression, but could also provide guidance on treatment decisions. The fact that targeted treatment is most successful in a subset of tumors indicates the need for better classification of clinically related molecular tumor phenotypes based on better understanding of the mutations in relevant genes, especially in those oncogenic driver mutations. The online version of this article (doi:10.1186/2213-0802-1-6) contains supplementary material, which is available to authorized users. Lung cancer is the leading cause of cancer-related death worldwide. In the past, therapeutic decisions have been based on histological classifications, which distinguish small cell lung cancers (SCLC) and non-small cell lung cancer (NSCLC). The latter comprises three major subtypes: squamous cell carcinoma, large cell carcinoma, and adenocarcinoma . AlthougA diversity of genomic and epigenetic abnormalities has been reported in NSCLC. Oncogenic driver mutations refer to mutations that are responsible for both the initiation and maintenance of the cancer. These mutations are often found in genes that encode for signaling proteins that are critical for maintaining normal cellular proliferation and survival. The presence of mutations on these genes will confer growth advantage on cancer cells, favoring their being selected during tumor progression . NSCLC, EGFR) gene mutations were the first targets for targeted treatment in NSCLC. Clinical efficacy and outcomes of EGFR-tyrosine kinase inhibitors (EGFR-TKI) have been reviewed thoroughly [EGFR are not well defined [Epidermal Growth Factor Receptor  [EGFR mutations at exons 18 – 21, KRAS gene mutation at codons 12 and 13, EML4-ALK fusion genes and deregulation of MET signaling.Apart from r (VEGF) , pemetrer (VEGF) , as wellr (VEGF) . MeanwhiEGFR , ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4) [The epidermal growth factor receptor family of tyrosine kinases consists of four forms: 4 (HER4) . The bin4 (HER4) .EGFR mutations have been extensively studies in NSCLC with about 27% overall incidence . EGFR-mutant NSCLC, which often exhibits adenocarcinoma histology, has been found to be associated with a better prognosis compared to EGFR wild-typed NSCLC in most populations [EGFR mutations have been identified in exons 18 to 21 of the TK domain, about 90% of which are deletions in exon 19  with 230 Japanese advanced, untreated and EGFR-mutant NSCLC patients sustained improved progression-free survival in the first-line gefitinib versus standard chemotherapy [EGFR-mutant patients [ulations –18, exceulations . The actGFR TKIs , 21. Sevotherapy . AccordiEGFR mutations, although they occur in exons 18 to 21, are related to primary resistance to EGFR-TKIs, for example, small insertions or duplications in exon 20. Additionally, the presence of other types of mutations can induce acquired resistance to EGFR inhibitors, such as T790M (exon 20), L747S (exon 19), D761Y (exon 19) and T854A (exon 21) [KARS mutations, PIK3CA mutations, loss of PTEN function, MET amplification and altered EGFR-related signaling [Some exon 21) . Other gignaling . Therefoignaling , 25.EGFR and HER2[EGFR mutations, especially with de novo T790M mutations [EGFR mutations, as well as LUX-Lung 6 comparing afatinib with cisplatin/gemcitabine in the same population as LUX-Lung 3 with more recruitment in China, India and South Korea. Dacomitinib  is an irreversible pan-HER inhibitor, which showed remarkable activity to gefitinib-resistant EGFR T790M mutations or HER2 mutations [Apart from erlotinib and gefitinib, second-generation irreversible EGFR-TKIs have been under extensive investigation, for example, afatinib and dacomitinib. Afatinib  is a highly selective and irreversible inhibitor of both and HER2. It has utations . Severalutations . A phaseKRAS mutation in a squamous cell lung carcinoma with the absence of this point mutation in the corresponding normal bronchial or parenchymal tissue [KRAS mutations have been found frequently in NSCLC, and according to clinical trials, the incidence of KRAS mutations in NSCLC ranges from 8% to 24% [KRAS mutations in NSCLC are located in codons 12 or 13, and are also reported in lung adenocarcinomas [KRAS mutations were analyzed in nearly 500 lung adenocarcinomas showed that KRAS mutations were found in 15%, 22% and 25% of tumors from never smokers, former smokers and current smokers respectively [EGFR mutations tend to occur more frequently in never-smokers with lung cancer, the presence of KRAS mutations cannot be easily predicted based on smoking status alone [KRAS transition mutations were more common in never smokers, whereas former/current smokers were more likely to harbor KRAS transversion mutations. This implies that transversions may be smoking related.A milestone study in 1984 identified a l tissue . Since t% to 24% . Most acrcinomas . The stuectively . Whereasus alone . AnotherKRAS proteins exhibit impaired GTPase activity, resulting in constitutive activation of RAS signaling. Since KRAS is downstream of EGFR, constitutive activation of KRAS renders resistance to anti-EGFR therapy. Many studies have observed lower efficacy of EGFR-TKI therapy in KRAS mutated NSCLC patients [EGFR mutations and KRAS mutations are mutually exclusive in the same tumor. However, there was a report on a case series of three patients with both activating EGFR mutations and KRAS mutations demonstrated that all EGFR+/KRAS+ patients showed a positive response to gefitinib or erlotinib [KRAS mutation to be a significant response predictor to EGFR-TKIs [KRAS positivity cannot be established as a criterion to exclude NSCLC patients from EGFR-TKI therapy.The mutated patients , 36. Therlotinib . SeveralGFR-TKIs –40. As aKRAS signaling is under intensive investigation. An obstacle is that mutant KRAS proteins entail loss-of-function. It is more difficult to inhibit loss-of-function proteins than gain-of-function ones like mutant EGFR. Current approaches try either to inhibit protein synthesis of mutated KRAS or to impede downstream effectors of mutant KRAS.The development of therapeutic agents targeting mutated ALK) protein is a receptor tyrosine kinase in the insulin receptor superfamily. In 2007, Soda et al. identified a small inversion within the short arm of chromosome 2, resulting in the fusion of the N-terminal of the echinoderm microtubule-associated protein-like 4 (EML4) gene with the ALK gene [EML4-ALK variants have been reported, all of which involve the coiled-coil domain of EML4 and the intracellular tyrosine kinase domain of ALK[EML4-ALK variants exhibit dimerization and constitutive activation of the fusion proteins [The anaplastic lymphoma kinase , reverse transcriptase PCR, and immunohistochemistry (IHC). The frequency of EML4-ALK fusions in unselected NSCLC patients, according to previous studies mostly with East Asians, ranges from 1.6% to 11.6% [EML4-ALK rearrangements, lung adenocarcinoma has been reported to be the major type showing EML4-ALK translocations. The majority of Caucasian lung adenocarcinoma harboring EML4-ALK show the signet-ring cell histology, whereas the acinar pattern is pre-dominant in ALK-positive Asian adenocarcinomas [ALK-positivity tends to be found in younger NSCLC patients [EGFR mutations, EML4-ALK fusions are associated with never or light smokers [EML4-ALK translocations and EGFR mutations are mutually exclusive except for rare cases [There is no “gold standard” method to screen the to 11.6% , 44–46. rcinomas . ALK-pos smokers . EML4-ALre cases .et al. study, wild-type NSCLC patients and ALK-positive NSCLC patients displayed a similar response to chemotherapy and no significant differences in overall survival, however, EML4-ALK translocations have been found to be associated with resistance to EGFR-TKIs [EML4-ALK fusion gene was not a significant prognostic factor based on the analysis of 720 resected lung adenocarcinomas [EML4-ALK status did not affect the sensitivity of advanced NSCLC patients to platinum-based combination chemotherapy in terms of response rate and progression-free survival although overall survival of EML4-ALK positive patients tended to be shorter than that of the EGFR mutated cohort nut resembled that in the wild-type cohort [EGFR wild-type and advanced-stage lung adenocarcinoma patients who received either monotherapy or platinum-doublet chemotherapy, ALK positivity conferred superior overall survival [EML4-ALK translocated patients , which may partially explain better outcome. Additionally, in Chinese patients with advanced NSCLC, response rate to EGFR-TKI was similar between ALK rearranged and EGFR mutated patients although median progression-free survival was significantly shorter in those with EML4-ALK gene [et al. may be explained by predominantly Asian population in this study and the limited sample sizes in both studies. The clinicopathological features of EML4-ALK rearrangement in NSCLC patients needs further investigation.In Shaw GFR-TKIs . Similarrcinomas . Consiste cohort . In contsurvival . CompareALK gene . The appALK-positive patients, selective tyrosine kinase inhibitors of ALK are currently under clinical trials, including an ALK/MET inhibitor PF-02341066 (crizotinib) [EML4-ALK-positive tumors. Several secondary mutations in the ALK gene, such as L1196M and C1156Y, have been revealed to contribute to resistance to crizotinib [ALK inhibitor, have exhibited remarkable activity against C1156Y- and L1196M-resistant EML4-ALK mutants [ALK- positive NSCLC [In order to treat zotinib) . Unfortuizotinib , 55. In ve NSCLC .MET gene, which is located on chromosome 7, encodes a receptor tyrosine kinase composed of an extracellular α-chain and a transmembrane β-chain. The natural ligand for MET receptor is hepatocyte growth factor (HGF), also called scatter factor (SF). Since MET interacts with numerous downstream effectors, the activation of MET signaling affects many pathways and regulates various cellular processes, including cell proliferation, cell motility, cell scattering, cell invasion, metastasis, angiogenesis and epithelial to mesenchymal transition (EMT) [The on (EMT) –59. TherMET can be altered via overexpression, genomic amplification, mutations, or alternative splicing [MET activation which can be mediated through HGF-dependent or HGF-independent mechanisms.In transformed cells, splicing , 60, 61.MET overexpression has been observed in NSCLC. In a study using 32 lung cancer tissues, all tumor samples expressed MET with no significant MET staining in corresponding normal lung tissues, and 61% (14 of 23) of NSCLC showed strong MET expression examined by IHC. Furthermore, an increase in MET activity identified by higher levels of phosphorylated MET (p-MET) at sites Y1003 and Y1230/1234/1235 came along with MET overexpression in this study. It was also mentionable that the activated p-MET was preferentially expressed in tumor cells located at the invasive front of NSCLC tissues [MET amplification was observed in 8 (4%) patients with wild-typed EGFR and wild-typed KRAS, indicating that the presence of MET gene amplification might be mutually exclusive with EGFR and KRAS mutations. Phosphorylation of MET at sites Y1234/1235 has been found to be associated with poor survival in patients who have complete resection of lung tumors [ tissues . In anotg tumors .MET gene copy number variations have also been reported in NSCLC. MET status was analyzed with FISH in 447 NSCLC patients and high MET gene copy number (≥5 copies/cell) was observed in 48 cases (11.1%), and patients with high MET gene copy number (MET-positive) exhibited shorter survival than MET-negative patients [patients .MET dysregulation. Mutated MET allows the kinase to overcome inhibitory mechanisms, thus becoming constructively activated or hyperresponsive to stimuli. Mutations could also prolong the duration of stimulating signals by increasing the level of activation or by preventing the degradation of the kinase. One study examined individual exons of semaphorin, juxtamembrane, and tyrosine kinase domains of MET in 141 East Asians, 76 Caucasians and 66 African Americans [MET mutations varied with ethnicity. N375S, occurred within the semaphorin domain, was the most frequent non-synonymous mutation, and the frequency of this mutation was higher in East Asians compared to Caucasians. In both East Asians and Caucasians, the frequency of N375S was higher in squamous cell carcinoma than in other non-small cell lung cancer. Among East Asians, the frequency of N375S in males was much higher compared with females [MET mutations, greater knowledge of this correlation can help us understand incidence, prognosis and treatment of lung cancer.Mutations provide another mechanism for mericans . The res females . Since tMET signaling are under investigation. Among them, a non-ATP competitive MET inhibitor, tivatinib (ARQ197), has been used in combination with erlotinib (EGFR-TKI) as second-line treatment for previously-treated non-small cell lung cancer [KRAS mutations. More MET-targeted agents are under preclinical and clinical studies, making MET the next major biomarkers in lung cancer.Several agents targeting g cancer . The duaERBB3, ERBB4, VEGFR, multiple ephrin receptors genes and NTRK genes [The identification of oncogenic driver mutations reveals the complexity and heterogeneity of NSCLC. A collaborative study investigated 623 candidate cancer genes in 188 lung adenocarcinomas. 26 genes were discovered to be somatically mutated at high frequencies and thus may be related to tumorigenesis, including RK genes .EGFR mutations could be present in early stage NSCLC [Table ge NSCLC , 70, sugAs we further our understanding into the biological mechanisms underlying these oncogenic driver mutations, the clinical relevance of these driver mutations will allow for further advancement into targeted therapeutics in lung cancer.