In total hip replacement (THR), it is essential to achieve a primary stability to guarantee good long-term results. A novel locking screw hip (LSH)-stem, anchored to the medial cortex of the proximal femur by 5 monocortical locking screws, was developed to overcome the shortcomings of uncemented press-fit and cemented straight stems while simultaneously achieving primary stability. The aim of this study was to investigate the biomechanical competence of the LSH-stem in comparison to an uncemented press-fit stem.P = .05.Six pairs of embalmed human cadaveric femora from donors aged 68 to 84 years were assigned to 2 study groups (n = 6) with equal number of right and left bones. The specimens in each group and pair were implanted with either an uncemented press-fit stem or an LSH-stem and tested biomechanically under progressively increasing cyclic axial loading until catastrophic failure. Axial construct stiffness, failure load, and cycles to failure were detected and statistically evaluated at a level of significance P = .04.Although the axial stiffness was comparable for both prosthesis types, the uncemented press-fit stem showed a significant lower stability in terms of failure load and cycles to failure in comparison to the LSH-stem, Converting our results to percentage of bodyweight (BW) in an assumed adult patient of 80 kg shows that the LSH-stem achieves a primary stability allowing to carry average loads of up to 507% BW, whereas the uncemented press-fit stem carried average loads of up to 404% BW. We conclude that both stems achieve a primary stability strong enough to carry hip joint loads experienced in the immediate rehabilitation period after THR. Therefore, accuracy of host bone preparation and prosthesis design are crucial, whereas implant surface texture and quality of the bone-implant contact determine secondary stabilization.–7Total hip replacement (THR) became a standard procedure not only for the treatment of coxarthrosis but also to treat advanced femoral head necrosis and femoral neck fractures. During the last 5 decades the number of THRs per year has steadily increased. Although THR is recognized as the most successful procedure of the 20th century in orthopedic and trauma surgery, it still bears the risk of failure. Recently, a “locking screw hip” (LSH)-stem  was developed as an alternative to conventional uncemented press-fit and cemented straight stems to achieve an immediate stability after its implantation.Even though locking screw-plate systems have revealed clear advantages in fracture fixation, locking screws have not been considered for the fixation of hip prosthesis stems so far.,11The aim of this study was to investigate the biomechanical behavior of the LSH-stem in comparison to an uncemented stem that claims to enhance primary stability in THR.22.1 were used in the current study. All donors gave their informed consent within the donation of anatomical gift statement during their lifetime. None of the femora showed signs of previous injuries, abnormalities, or diseases.Six pairs human cadaveric femora , embalmed with the method of Thiel,All specimens were stripped of all soft tissue. Bone mineral density (BMD) of each specimen was evaluated in the femoral head by high resolution peripheral quantitative computed tomography using xTreme CT . Scanning was performed at a resolution of 0.082 mm. BMD was calculated as mean value of scanned cortical and cancellous bone content within the volume of interest between 2 slices, perpendicular to the femoral neck axis and located at a distance 5 mm proximally and 5 mm distally to the centre of the femoral head, measured along this axis.The specimen pairs were split to 2 study groups with 6 femora each, with equal numbers of right and left specimens per group, for implantation with either an uncemented press-fit stem  or an LSH-stem .2.2The LSH-stem is made of titanium alloy and has a roughly grit blasted medial side to allow good bone apposition whereas the lateral side is kept smooth to avoid coupling between the medial and lateral cortex Fig. . Primary,14 This stem is made of titanium alloy and has a complex proximal geometry of a double wedge-shaped core in combination with anterior and posterior vertical flutes. The entire stem is with a rough grit-blasted surface with a mean roughness of 3.5 μm (Ra). The stem is straight and achieves a primary stability by contacting the femoral cortex in at least 3 different areas.The uncemented press-fit stem is an equivalent to the Spotorno stem that was developed by Spotorno in 1983 and became one of the most successful endoprosthesis.2.3Digital X-rays were made for preoperative planning to simulate the technique for stem implantation as prescribed for clinical use. The planning for the uncemented press-fit stem implantation was performed with the use of MediCAD software . Since currently there is no existing tool for planning of the LSH-stem implantation, femur size, intramedullary space, and resection line of the femoral neck were detected and measured with the same software for this prosthesis too. Specifically, all femora used for LSH-stem implantation were big enough so that the lateral edge of the stems did not come into contact with the lateral cortex of the femur, as desired by the manufacturer.2.4All implantation procedures were performed by a single surgeon (PS). The femur shafts for the uncemented press-fit stem were prepared according to the current standards for endoprosthetic surgery. The femur shafts for the LSH-stem were prepared according to the surgical technique as prescribed by the manufacturer. Preoperative templates and surgical techniques ensured a proper alignment of the central axis of the stem with the long axis of the femur during implantation.One LSH-stem size was required for the specimens in group 2. The stem size of the uncemented press-fit prosthesis (group 1) was calculated according to the preoperative planning and adjusted to the size of the reamer that achieved a proper grip in the femur during preparation. Intraoperative complications and any obvious femoral cracks were avoided by thorough preparation and careful implantation.2.5All specimens were analyzed postoperatively by computed tomography scanning in order to exclude any femoral damage. No fracture or fissures were detected. X-rays of the implanted prostheses were shot in order to measure the off-set and check the lever arm for each femoral pair using the software tool MediCAD. Center of rotation on the X-rays was detected with the help of Cobalt Chrome prosthesis heads with diameter of 32 mm, offset M (0.0 mm), and a 12/14 conus, which were attached to the prostheses prior to the X-ray shots. Following, the lengths of the femora were measured from the tip of the greater trochanter to the epicondyle line and the distal 3rd was resected. Then the distal end of each specimen was embedded in poly(methyl methacrylate) in preparation for biomechanical testing.2.6,16  with a 25 kN/200 Nm load cell. The specimens were fixed in a specially designed rig for loading in cranio-caudal direction and tilted 8° lateral in the frontal plane and 6° dorsal in the sagittal plane to simulate a single leg stance loading The cyclic biomechanical test started from the final quasistatic loading condition and was performed at a rate of 2 Hz with sinusoidal axial loading. The 1st cycle ranged from 1000 N  to 1500 N (peak). The peak level was then progressively increased by 0.5 N/cycle until construct failure occurred, as described in previous studies.,18Proximally, each specimen was attached to the machine actuator via a ball-and-socket joint. The embedded distal specimen's end was connected to the machine base via an XY table Fig. . The tes2.7Machine data in terms of applied load and actuator displacement were recorded by the system transducers at a rate of 128 Hz. Anteroposterior X-rays were taken at the beginning of the test, then periodically at intervals of 500 cycles and after specimen's failure.Based on the load-displacement curves, axial construct stiffness, failure load, and cycles to failure were defined as parameters of interest for each specimen and considered for statistical evaluation. Axial stiffness was calculated from the gradient of the respective load-displacement curve during the ramped loading in the quasilinear elastic region before plastic construct deformation occurred. Failure load, cycles to failure, and type of failure were defined from the destructive cyclic test.2.8t test. Level of significance was set to P = .05 for all statistical tests.Statistical analysis was performed using SPSS Statistics . Normal distribution of the parameters of interest, namely BMD, axial construct stiffness, failure load, and cycles to failure in each study group was screened with Shapiro-Wilk test. Homogeneity of variances between the groups were checked with Levene test. Significant differences between the 2 groups were checked with paired-samples 3P ≥ .37.All parameters of interest  were normally distributed in each of the study groups and with homogeneity of variances among the groups, 3 [mean ± standard deviation] and LSH-stem, group 2: 161.4 ± 33.6 mg HA/cm3) showed no statistical significant difference, P = .20.BMD  did not differ significantly between the 2 groups, P = .04  and the LSH-stem, group 2 , A similar fracture pattern at catastrophic failure of the bone-implant construct was detected in most of the specimens Fig. . A longi4In the current study, the normal distribution of the parameters of interest within the treatment groups supports the fact that the study sample can be representative for a population which has a normal distribution of those data. Moreover, the application of parametric statistical test for the detection of significant differences between the groups, which is justified by this normal distribution, aimed to achieve higher statistical power during the data analysis.Even though a numerous amount of publications emphasize the good long-term outcomes of THR, endoprosthetic register data shows a high number of hip stems that need revision surgery due to aseptic loosening. One reason for the aseptic loosening is subsequent stress shielding in the trochanteric region occurring years after THR. The reason for stress shielding is not yet completely understood; however, the following facts seem to be in place for it. First, the lateral cortex and lateral cancellous bone of the proximal femur, as well as the gluteal muscles attached at the greater trochanter, are compromised during implantation, especially when lateral or anterolateral approach to the hip joint are used. Second, through the design of the common hip stems, a coupling between the medial and lateral cortex is created, leading to a rather nonphysiological transfer of the loads at the hip joint. Achieving a primary stability of the hip stem can avoid the formation of a fibrous layer at the bone-prosthesis interface and enhance bone in/ongrowth to the prosthesis.Another reason for aseptic loosening of a hip stem is continual micromotion occurring prior to settling of solid prosthesis anchorage via bone in/ongrowth.To overcome these issues, a new hip stem design – the LSH-stem – was developed by Scyon Orthopedics AG. The concept of the LSH-stem targets achievement of primary stability by using 5 monocortical locking screws transferring joint forces directly to the femur through the medial calcar and reproducing a more physiological load situation. In addition, a coupling between the medial and lateral cortex can be avoided by the size of the stem since it does not have to fill the medullary cavity to achieve primary stability.Even though the concept of the LSH-stem seems very promising, it is still not known how well the use of 5 monocortical locking screws, fixing the stem to the medial cortex of the proximal femur, will withstand the hip joint forces that occur until bone remodeling provides a solid prosthesis support.This unknown was the motivation to perform ex vivo experimental investigations to analyze the biomechanical behavior and to compare the biomechanical performance of the LSH-stem to another uncemented stem for THR.By using a human cadaveric model in a combined matched paired design, the influence of anatomical variability in the specimen's parameters such as femur size, curvature, and cortex thickness was considerably neutralized. Thus, a much better comparison of the intrinsic biomechanical performance of the 2 prosthesis types was possible. The 2 following facts became obvious. reported comparable axial construct stiffness and failure load for the LSH-stem and a cemented straight stem. This means that the biomechanical properties of the novel LSH-stem are comparable to the cemented straight stem in terms of primary stability, and even superior to the uncemented press-fit stem.First, no statistically significant difference between the novel LSH-stem and the uncemented press-fit stem was found in terms of axial construct stiffness under single-leg stance loading conditions. Second, the failure load and cycles until failure for the LSH-stem were significantly higher than the uncemented press-fit stem. In a previous study with the same setup for biomechanical testing, Grechenig et al Besides the appropriate surface structure, application of bioceramic coatings can contribute to a good mechanical fixation of an endoprosthetic implant. The LSH-stem is designed with a rough grit-blasted medial side to allow good bone apposition and integration for definitive stability. In a radiostereometric analysis study including 15 patients with implanted LSH-stems, aged 50 years on average, the successful transformation of primary stability to secondary stability could be shown. Taking into account these results and the fact that according to the findings of Bergmann et al,19 the experimental setup we chose seems to be a quite realistic or even processing the worst case scenario to test hip implants with regard to their stability of fixation during the immediate postoperative phase, the clinical application of the LSH-stem may be justified.An uncemented hip stem reaches its maximum stability after bone ingrowth.4.1 detected a reduction of the Young modulus for embalmed cadaver bone.The limitations of this study are similar to those inherent to most cadaveric biomechanical studies with a limited number of tested specimens, small sample size, and scattering of the measured data, thus rendering the statistical power. In addition, the use of embalmed specimens in comparison to fresh-frozen specimens might have influenced the results since Unger et al


In search of improved risk stratification in hypertrophic cardiomyopathy (HCM), CMR imaging has been implicated as a potential tool for prediction of sudden cardiac death (SCD). In follow-up of the promising results with extensive late gadolinium enhancement (LGE), high signal-intensity on T2-weighted imaging (HighT2) has become subject of interest given its association with markers of adverse disease progression, such as LGE, elevated troponin and non-sustained ventricular tachycardia. In lack of follow-up cohorts, we initiated an exploratory study on the association between HighT2 and the internationally defined risk categories of SCD. In a cohort of 109 HCM patients from a multicenter study on CMR imaging and biomarkers, we estimated the 5-year SCD risk (HCM Risk-SCD model). Patients were categorized as low (< 4%), intermediate (≥ 4–<6%) or high (≥ 6%) risk. In addition, risk categorization according to the ACC/AHA guidelines was performed. HighT2 was present in 27% (29/109). Patients with HighT2 were more often at an intermediate-high risk of SCD according to the European  and American guidelines  compared to those without HighT2. The estimated 5-year SCD risk of our cohort was 1.9% (IQR 1.3–2.9%), and projected SCD rates were higher in patients with than without HighT2 . In conclusion, HCM patients with HighT2 were more likely to be intermediate-high risk, with projected SCD rates that were 1.5 fold higher than in patients without HighT2. These pilot findings call for corroborative studies with more intermediate-high risk HCM patients and clinical follow-up to assess whether HighT2 may have additional value to current risk stratification.The online version of this article (doi:10.1007/s10554-017-1252-6) contains supplementary material, which is available to authorized users. Hypertrophic cardiomyopathy (HCM) is the most common inheritable cardiomyopathy and the most frequent cause of sudden cardiac death (SCD) among young athletes –3. UnforAnother imaging feature with potential impact could be high signal intensity using T2-weighted CMR imaging (HighT2). This is based on the reported associations with markers of adverse disease progression, such as LGE, elevated troponin, and non-sustained ventricular tachycardia , 11–13.Whereas LGE is observed in about 60–70% of patients, HighT2 is observed in about one-third of HCM patients. Notably, areas of HighT2 are almost exclusively present in patients with LGE, occurring within the boundaries of LGE –20. ApprIn addition, we have recently reported an independent association with an elevated level of cardiac troponin T, which supports that HighT2 is likely indicative of recently sustained myocyte injury . In viewImportantly, studies with clinical follow-up on HighT2 are lacking. Therefore, we sought to provide the first pilot data on the association of HighT2 with the current SCD risk categorizations according to the ESC and ACC/AHA guidelines. We assessed the proportion of intermediate to high risk patients in relation to the presence or absence of HighT2. In addition, we performed an exploratory analysis on HighT2 and the associated projected SCD rates determined with use of the HCM Risk-SCD model, to provide insight into potential clinical implications .For the present analysis, we studied a series of HCM patients who underwent T2-weighted CMR imaging, as participants of a Dutch multicentre HCM study project . In shorCMR imaging was performed on a 1.5T CMR system  according to local imaging protocols, as previously described in more detail . All imaImages were analyzed with commercially available software  by two observers (FG and JB) unaware of the subjects’ clinical information. All 17 segments of the AHA-model were analyzed for the presence of HighT2 and LGE. HighT2 and LGE were scored visually per segment as either present or absent , 17. In For all participants, the following risk factors were recorded at the day of CMR imaging: age at evaluation; family history of SCD; history of unexplained syncope; NSVT on 24-h Holter monitoring; maximal LV wall thickness, LV outflow tract obstruction gradient (either resting or provocable gradient) and left atrial diameter measured using echocardiography; abnormal blood pressure response during exercise. Missing data was ~ 1%. For a detailed description and analysis of the risk factors, we refer to the appendix.http://doc2do.com/hcm/webHCM.html. An intermediate risk was defined as an estimated 5-year SCD risk of ≥ 4–< 6% and a high risk as ≥ 6% , and were compared between patients with and without HighT2 using a Student’s with LGE , we alsoThe present study population comprised of 109 HCM patients , of whom the majority has previously been described . 59 (58%Twenty-nine out of 109 (27%) were positive for HighT2. HighT2 was mostly observed midwall and co-localized within an area of LGE in hypertrophied segments, as previously described in more detail  Patients with HighT2 tended to be more dyspnoeic and had a higher LV mass indexed to body surface area and a lower LV ejection fraction. Furthermore, cardiac troponin T concentrations were higher in patients with than without HighT2. The proportion of patients with LGE and the extent of LGE were also higher in the former group Table .Patients with HighT2 were more often at an intermediate-high SCD risk according to the ESC guidelines  and ACC/AHA guidelines  . When these findings are confirmed in larger cohorts with a higher proportion of intermediate-high risk patients, HighT2 may prove to be a valuable additive risk modifier or risk factor for future risk stratification schemes.With the increasing use of CMR imaging in HCM, tissue characterization has become a topic of interest to further unravel pathophysiological aspects of the disease, and to determine the additional contribution of these imaging features in risk prediction of sudden death. From these studies we have learned that in almost all patients with HighT2 a substrate for arrhythmias was present in the form of fibrosis –20. In aIn our HCM population, patients with HighT2 were more often at an intermediate-high SCD risk, regardless of whether the European or Northern American guidelines were used. It is evident that in the large subset of our low risk HCM patients, projected SCD rates were significantly higher in patients with HighT2. Whether this can be extrapolated to intermediate-high risk patients remains to be determined.On the one hand, it should be noted that this observed association may be confounded due to the association between HighT2 and some of the conventional risk factors [LV wall thickness in particular Table ]. In addOn the other hand, HighT2 was also associated with the risk modifier extensive LGE (ACC/AHA) and other indicators of disease severity such as higher LV mass, lower LV ejection fraction and higher troponin T concentration , 27, 28.The observed 1.5 fold risk increase for SCD associated with HighT2 is in the same order of magnitude as observed for an extent of LGE of ≥ 15%, which is associated with an almost twofold increased risk , 9.Based on our findings, several hypotheses for both low risk and intermediate-high risk patients could be addressed in future studies as potential implications. Our findings in the large group of low risk patients imply that T2-weighted CMR imaging might be able to increase the negative predictive value of current risk stratification schemes. At present, sudden cardiac death still occurs among low risk patients, and because of the high proportion of low risk patients in the general HCM population absolute numbers of cases with SCD are still considerable . In the As for the impact of HighT2 in intermediate-high risk patients, the small sample size does not allow firm conclusions. Possibly, the presence or absence of HighT2 could help to differentiate between higher and lower risk patients, respectively. As for the former, we demonstrated associations with indicators of advanced disease, such as low ejection fraction and troponin. Moreover, our data suggest that HighT2 seems to differentiate among patients with LGE.It should be acknowledged that approximately half of the patients at intermediate-high risk did not have HighT2. This may be interpreted as an undesirable “missing” of patients at a considerable estimated risk of SCD. However, it has been demonstrated in an independent validation cohort for the HCM-SCD risk model that with a SCD rate of around 5%, about 17 patients need an ICD implantation to prevent one SCD in 5 years . In addiIn search of refinement of the current risk stratification models that are based on echocardiographic and clinical variables, we agree with European and Northern American experts that incorporating myocardial tissue characterization (LGE and HighT2) may be of additional value –35.The positive predictive value of the mere presence of LGE proved to be limited by its prevalence of about 60–70% and the annual risk of SCD of only about 1% in a general HCM population. In response, the extent of LGE has become the topic of interest, with promising results in risk prediction , 36. Of Previously, we have demonstrated that among HCM patients with LGE, those without HighT2 had the lowest troponin concentration, resembling that of patients without any LGE. Moreover, patients without HighT2 had a ninefold lower chance of extensive LGE. Whereas the number of intermediate-high risk patients is limited, our findings in the large cohort of low risk patients are more robust and imply that it is likely that the absence of HighT2 could improve the negative predictive value in these patients.In summary, given the currently suboptimal risk stratification, the addition of HighT2 may be valuable to improve both negative and positive predictive values of the risk models.. Incorporation of the abovementioned CMR variables in (currently running) HCM follow-up studies may provide valuable information with regard to their independent association and additive predictive values , 8.Although this is the largest cohort of HCM patients with T2-weighted imaging information, the main limitation of this study is the low risk profile of the study population. At the time of this study 5-year follow-up was available for 70% of our cohort. With projected 5-year follow-up rates of about 2%, we decided that clinical endpoints rather than estimated sudden death rates would not provide much additional value for the current study question.In this context, we performed exploratory analyses on projected rates of SCD and our findings should therefore be considered as hypothesis generating, and do not validate HighT2 as a risk factor for SCD. Confirmative studies with clinical follow-up in a more intermediate-high risk population are warranted.Furthermore, the technique under investigation is limited by a high signal-to-noise ratio and frequent artifacts. Unfortunately, T2-mapping sequences were not available at the start of our study, but these seem very promising and may lead to more objective data. In analogy to previous studies in HCM, the presence of HighT2 was visually assessed by two independent observers (FG and JB) , 17. A tLastly, we are well aware of the fact that the validity of the HCM Risk-SCD model has recently been challenged for Northern American patients . NonetheIn an era where tissue characterization with CMR imaging has become topic of interest for SCD risk stratification, we are the first to demonstrate that HCM patients with HighT2 are more likely to be at intermediate to high risk of SCD, with projected SCD rates that are 1.5 fold higher than in patients without HighT2. Notably, HighT2 was not only associated with established risk factors, but also with several markers of a detrimental disease course, that are currently not incorporated in the HCM Risk-SCD model.The present findings should be considered as “pilot data”, but do put forward the hypothesis that HighT2 might be valuable for future SCD risk stratification models in HCM.Below is the link to the electronic supplementary material.Supplementary material 1 (DOCX 14 KB)Supplementary material 2 (TIF 1108 KB)Supplementary material 3 (DOCX 12 KB)


JAG2 in muscle fibers. YAP forced-activity in muscle fibers prevents the decrease of JAG2 expression and the number of PAX7+ cells in immobilization conditions. Our results identify a novel mechanism acting downstream of muscle contraction, where YAP activates JAG2 expression in muscle fibers, which in turn regulates the pool of fetal muscle progenitors via NOTCH in a non-cell-autonomous manner.The importance of mechanical activity in the regulation of muscle progenitors during chick development has not been investigated. We show that immobilization decreases NOTCH activity and mimics a NOTCH loss-of-function phenotype, a reduction in the number of muscle progenitors and increased differentiation. Ligand-induced NOTCH activation prevents the reduction of muscle progenitors and the increase of differentiation upon immobilization. Inhibition of NOTCH ligand activity in muscle fibers suffices to reduce the progenitor pool. Furthermore, immobilization reduces the activity of the transcriptional co-activator YAP and the expression of the NOTCH ligand DOI:http://dx.doi.org/10.7554/eLife.15593.001 Skeletal muscle is attached to the skeleton and allows the body to move. Making a new muscle, or repairing an existing one, relies on stem cells that are present inside muscles. A major goal of skeletal muscle research is to understand the signals that regulate the abilities of muscle stem cells to divide and give rise to more stem cells or to become muscle cells. Molecular signals are known to regulate the numbers of stem cells in the muscle. Skeletal muscles become larger if they are exercised, but it is not clear if mechanical forces generated by muscle contractions directly affect the number of muscle stem cells.The NOTCH signaling pathway contributes to maintaining the population of stem cells in muscles by forcing the stem cells to divide and preventing them from becoming muscle cells. Here, Esteves de Lima et al. investigated whether muscle contraction regulates NOTCH signaling during muscle formation in chick fetuses.JAG2. This increases NOTCH signaling activity in the neighboring stem cells and maintains the number of stem cells in the muscle. The next step following this work will be to establish if this mechanism also operates during muscle formation and regeneration in other animals such as mice and zebrafish.The experiments show that muscle contraction stimulates the activity of a protein called YAP in muscle cells, which in turn, activates a gene in the NOTCH signaling pathway known as DOI:http://dx.doi.org/10.7554/eLife.15593.002 Skeletal muscle development, growth and regeneration rely on muscle stem cells. A major goal of muscle research is to understand the signals that regulate the ability of stem cells to self-renew or differentiate.MYF5, MRF4, MYOD, MYOG  promotes satellite cell proliferation and inhibits muscle differentiation in culture . In addiJAG2 in fibers, which maintains the pool of muscle progenitors by activating NOTCH signaling.A link between mechanical forces (provided by muscle contraction) and signaling pathways that regulate fetal myogenesis has not been established. In this study, we show the importance of mechanical forces in the regulation of the number of fetal muscle progenitors. We show that immobilization induces a NOTCH loss-of-function phenotype in muscles. We further provide evidence that, downstream of mechanical forces, YAP positively regulates the expression of the NOTCH ligand PAX7 and MYF5 genes were significantly decreased in DMB-treated limbs compared to control limbs, as early as 12 hr after DMB application and with a more prominent effect at 48 hr    . In addid CYR61) , i.e. coet genes . JAG2 exd cells) . Thus, Y muscles . This sh muscles . Moreove muscles . We concJAG2 transcription, we performed in vivo ChIP experiments using chick limbs as chromatin source. ChIP sequencing data on chick limb cells analyzing promoter-associated histone marks allowed us to characterize the promoter of the chick JAG2 gene , the known binding motif for TEAD complexes  or not containing (−4500 bp −4894 bp) MCAT elements were not occupied by YAP  provides a possible mechanism for the contraction-dependent control of JAG2 expression in fetal muscle fibers.To define whether YAP directly regulates AG2 gene . We idenomplexes . We founomplexes  and RT-qomplexes  analysesd by YAP . This red on PCR  and RT-qd on PCR  analysesnditions . The YAPThe influence of muscle contraction has been extensively studied for skeleton  to muscle atrophy or hypertrophy is a debated issue [reviewed in Spd mutant mice  were incubated at 37.5°C. Chick embryos were staged according to days in ovo.Decamethonium bromide (DMB)  and pancuronium bromide (PB) (Sigma) solutions were freshly prepared before each experiment at 12 mM or 11 mM, respectively, in Hank’s solution (Sigma) with Penicillin-Streptomycin at 1% . The control solution was prepared using Hank’s solution with 1% of Penicillin-Streptomycin. 100 µl of DMB, PB or control solutions were administrated in chick embryos at E7.5 and E8.5. Embryos were fixed at E8 for the 12 hr time point, at E8.5 for the 24 hr time point and at E9.5 for the 48 hr time point.Chick embryonic fibroblasts (CEFs) obtained from E10 chick embryos were transfected with DELTA1/RCAS using the Calcium Phosphate Transfection Kit . Cell pellets of approximately 50–100 µm in diameter were grafted into limb buds of E3.5 embryos as previously described . DELTA1/The pT2AL-MLC-Tomato-2A-DELTA1/DN was designed as follows: the dominant negative form of DELTA1 was defined as a truncated form of DELTA1 that lacks the intracellular domain  and was Primary myoblasts were obtained from hindlimbs of E10 chick embryos, as previously described . DMB andTotal RNAs were extracted from control limbs, experimental limbs or primary fetal myoblast cultures. 500 ng to 1 µg of RNA was reverse-transcribed using the High-Capacity Retrotranscription kit . RT-q-PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems). Primer sequences used for RT-q-PCR are listed in Fifty microliters of EdU (Invitrogen) solution (5 mg/ml) was injected into the circulation in E9.5 chick embryos for 1.5 hr. Embryos were then fixed and immunohistochemistry was performed on limb transverse sections.Forelimbs of control or manipulated  chick embryos were fixed in 4% paraformaldehyde overnight at 4°C and then processed in gelatin/sucrose for 12 µm cryostat sections. The monoclonal antibodies, MF20 that recognizes sarcomeric myosin heavy chains and PAX7 that recognizes muscle progenitors, developed by D.A. Fischman and A. Kawakami, respectively, were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology Iowa City, IA 52242. The Desmin antibody was obtained from Sigma (dilution 1/100). The YAP mouse monoclonal antibody was obtained from Santa Cruz Biotechnology (dilution 1/50). The MEP21 antibody originates from . ProlifeChick forelimbs of control or manipulated  embryos were fixed in Farnoy  and 10% acetic acid) overnight at 4°C, and processed for in situ hybridization on wax tissue sections, as previously described . The digJAG2 coding sequence or by RT-q-PCR to amplify the region 1 . To quantify the number of PAX7+ cells in limb muscles of DMB- and PB-treated fetuses, the number of PAX7+ cells was counted per unit area in dorsal and ventral muscles on three sections of each three different limbs originating from either DMB-treated, PB- treated or control embryos. The quantification of PAX7+ cells and muscle area (delineated with PAX7 and MF20 expression domains) in DELTA1/RCAS-grafted embryos was performed in three sections of each grafted (right) and contralateral (left) limbs of three immobilized and three control embryos. Quantification was performed in ectopic DLL1-expressing muscles of grafted right limbs and compared with equivalent muscles of the contralateral left limbs originating from the same embryos. The quantification of the Tomato+/MF20+ cells, MF20+ cells, PAX7+ cells and muscle area in pT2AL-MLC-Tomato-DELTA1/DN electroporated embryos was performed in five sections of each electroporated (right) and contralateral (left) of four embryos. Quantification was performed in Tomato-expressing muscles of electroporated right limbs and compared with equivalent muscles of the contralateral left limbs originating from the same embryos. The quantification of PAX7+ cells in pT2AL-MLC-Tomato-YapS112A electroporated and immobilized fetuses was performed in four sections of each electroporated (right) and contralateral (left) of four immobilized fetuses. Quantification was performed in Tomato-expressing muscles of electroporated right limbs and compared with equivalent muscles of the contralateral left limbs originating from the same embryos. Hoechst+ nuclei overlapping with MYOD expression were counted in four different muscles over four different sections and compared with the total number of Hoechst+ nuclei in paralyzed (DMB 48 hrs) and control muscles.All cell number and muscle area measurements were performed using the free software ImageJ .Thank you for submitting your article "Muscle contraction is required to maintain the pool of muscle progenitors via YAP and NOTCH during fetal myogenesis" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The regulation of the pool of muscle progenitors during development is a critical determinant of muscle size. In this paper, the authors test the novel hypothesis that muscle contraction is important for regulating the number of muscle progenitors during development. By using drugs that block the contraction of muscles in the chick embryo and by molecular manipulations, they describe the possible role that YAP and NOTCH signaling may play in this process. Altogether this is a nice study with interesting hypotheses put forward which have potentially important implications, however a number of important points need to be addressed before publication can be envisaged.1) In the Introduction, presentation of the role of the Notch pathway in myogenesis is inadequate. This is a complex situation  and presenting it as linear and simple is bound to attract criticism. The authors only refer to the role of Notch in the inhibition of myogenic differentiation of resident progenitors and satellite cells. However, opposite results have been described, e.g. in the Notch3 null mutation or the early differentiation of progenitors from the dermomyotome in the chick embryo . What this variety of responses means is that during myogenesis, Notch has different roles depending on the cellular and molecular context where it acts. This is something that should be clearly presented so that the text is less dogmatic from the start. As it is, it seems that studies that don't fit to the general hypothesis developed here are just not mentioned. In general, there is a bias towards citing much of the authors and co-authors work (and views) on what Notch is doing during myogenesis and missing other important references not only when they do not fit to the picture (see above) but even when they support the general idea .2) The authors show that there is an increase of MyoD or Myog expression after drug treatment, which is paralleled by a decrease of PAX7 expression. They draw the conclusion that PAX7-positive cells have undergone myogenic differentiation. However, there is no definite proof that this is the case. The number of MyoD+ (or Myog+) cells could be the same after treatment, but with the number of PAX7+ cells decreasing, the proportion of MyoD+ or Myog+ cells would automatically increase (in particularly since the PAX7+ population is large). A more direct proof that this a differentiation process would be to demonstrate that the number of PAX7+ cells (per area unit) at t=0 is similar to that of MyoD+ cells per unit area at t=12 or 48 hrs. Since the authors claim there is no apoptosis (see below) the numbers should match. Furthermore a time course on MyoD and PAX7-positive cells would help.3) The authors show that there is no change in EdU labelling in the PAX7+ population, and they conclude that there is no change in the proliferation of resident progenitors, thus supporting the hypothesis of an increase in differentiation (see above). However, the authors show that there is an increase of apoptosis in PAX7-negative cells in the muscle masses. How can they exclude that after drug treatment, the resident progenitors lose PAX7 expression, then undergo apoptosis? It is a concern that the cell population undergoing apoptosis is not identified.4) The Notch ligand Jag2 and the Notch response (Hes5 and HeyL) are more strongly reduced after 12 than 48h, while the phenotype is stronger after 48h . The aut5) The over-expression of Dll1 with RCAS viruses will target both the resident progenitors and the differentiated fibers. Since it is known that Notch is activated in trans and inhibited in cis , what conclusion can be drawn from this experiment? It would be important in these overexpression experiments to check cell type specificity, using the available tools.In addition, since the authors claim it is Jag2 that is the ligand, why not use its cDNA? They could have used electroporation of Jag2 in combination with the muscle-specific promoter they used with the next experiment (DN Dll1) that would drive its expression in differentiated fibers. It would also have been appropriate to use siRNA directed against Jag2. The reviewers do not insist on redoing the experiments with Jag2, although if the authors have these data they should be included. in any case, since Jag2 is membrane bound, they should show that they have an effect only in cells in direct contact with the Jag2/ Dll1 over-expressing fibers.6) It is not clear that Yap activation is downstream of muscle contraction and also that Yap activates Jag2 expression in myofibers. Of course, the most convincing data would be for the authors to show that ectopic activation of YAP in myofibers (via Tol2 MLC electroporation) rescues the loss of PAX7+ progenitors when myofibers have been immobilized. This experiment would considerably strengthen the paper and should be do-able by the authors since they have the Tol2 MLC construct.7) Since YAP is such an ubiquitously expressed molecule, the authors should be careful in the phrasing of the conclusions. 1) In the Introduction, presentation of the role of the Notch pathway in myogenesis is inadequate. This is a complex situation  and presenting it as linear and simple is bound to attract criticism. The authors only refer to the role of Notch in the inhibition of myogenic differentiation of resident progenitors and satellite cells. However, opposite results have been described, e.g. in the Notch3 null mutation or the early differentiation of progenitors from the dermomyotome in the chick embryo . What this variety of responses means is that during myogenesis, Notch has different roles depending on the cellular and molecular context where it acts. This is something that should be clearly presented so that the text is less dogmatic from the start. As it is, it seems that studies that don't fit to the general hypothesis developed here are just not mentioned. In general, there is a bias towards citing much of the authors and co-authors work (and views) on what Notch is doing during myogenesis and missing other important references not only when they do not fit to the picture (see above) but even when they support the general idea .In the original Introduction section describing the NOTCH pathway, we focused on limb foetal myogenesis, which involves the inhibition of muscle differentiation and the maintenance of muscle progenitors. We are aware that additional roles of NOTCH have been described during embryonic and adult myogenesis. We now mention the role of NOTCH during the activation of embryonic myogenesis in axial somites  and during activation, proliferation and quiescence of satellite cells. We also cite the Bjornson et al. (2012) paper, which shows a role for NOTCH in maintaining the number of satellite cells by inhibiting differentiation.2) The authors show that there is an increase of MyoD or Myog expression after drug treatment, which is paralleled by a decrease of PAX7 expression. They draw the conclusion that PAX7-positive cells have undergone myogenic differentiation. However, there is no definite proof that this is the case. The number of MyoD+ (or Myog+) cells could be the same after treatment, but with the number of PAX7+ cells decreasing, the proportion of MyoD+ or Myog+ cells would automatically increase (in particularly since the PAX7+ population is large). A more direct proof that this a differentiation process would be to demonstrate that the number of PAX7+ cells (per area unit) at t=0 is similar to that of MyoD+ cells per unit area at t=12 or 48 hrs. Since the authors claim there is no apoptosis (see below) the numbers should match. Furthermore a time course on MyoD and PAX7-positive cells would help.Unfortunately, there is no good commercial antibody against chick MYOD or MYOG (in contrast to the situation in mice), so we cannot calculate the number of PAX7+ and MYOD+ cells. In order to estimate the number of MYOD-expressing cells, we therefore counted the number of Hoechst+ nuclei of MYOD-expressing cells (based on MYOD in situ hybridization) versus the total number of Hoechst+ nuclei in muscles. We observed an increase in the percentage of MYOD-expressing cells in paralyzed muscles compared to control muscles New . MoreoveWe have added the 24 hour time point for PAX7, MYF5, MYOD and MYOG expression levels in immobilised limbs versus control limbs in 3) The authors show that there is no change in EdU labelling in the PAX7+ population, and they conclude that there is no change in the proliferation of resident progenitors, thus supporting the hypothesis of an increase in differentiation (see above). However, the authors show that there is an increase of apoptosis in PAX7-negative cells in the muscle masses. How can they exclude that after drug treatment, the resident progenitors lose PAX7 expression, then undergo apoptosis? It is a concern that the cell population undergoing apoptosis is not identified.We have looked at apoptosis 24 hours after DMB treatment and there is no increased apoptosis in paralysed muscles compared to control muscles. Since there are no double labeled PAX7+/Tunel+ cells 48 hours after immobilisation, we conclude that muscle progenitors do not undergo apoptosis in paralyzed muscles (48 hours after immobilisation).The apoptosis observed in muscles paralyzed for 48 hours is rarely observed in MF20 + cells or Desmin+ cells (now added in 4) The Notch ligand Jag2 and the Notch response (Hes5 and HeyL) are more strongly reduced after 12 than 48h, while the phenotype is stronger after 48h . The autSince JAG2 is also expressed in vessels, it is difficult to speculate on a stronger effect at 12 h 4 than 48h. We believe that the q-PCR experiments confirm the decrease of JAG2 expression in limbs, which is clearly observed in limb muscles by in situ hybridisation .5) The over-expression of Dll1 with RCAS viruses will target both the resident progenitors and the differentiated fibers. Since it is known that Notch is activated in trans and inhibited in cis , what conclusion can be drawn from this experiment? It would be important in these overexpression experiments to check cell type specificity, using the available tools.DLL1 overexpression experiments with the RCAS virus lead to a NOTCH gain-of-function phenotype in chick limbs  or in chick somites . DLL1-activated NOTCH maintains the expression of muscle progenitor markers including PAX3, MYF5 and FGFR4, while inhibiting the expression of muscle differentiation genes, MYOD, MYOG, DES and MEF2C and Myosins . Based on the RCAS system, DLL1 will be overexpressed only in a subset of proliferating cells (any cell types). Consequently, DLL1 will act in trans to adjacent cells. Therefore, even if a subset of proliferating cells overexpressing DLL1 inhibited NOTCH in cis, the DLL1 signal is sufficient to activateNOTCH in trans according to the phenotypes observed after DLL1 overexpression in muscles, as reported in the papers cited above.In addition, since the authors claim it is Jag2 that is the ligand, why not use its cDNA? They could have used electroporation of Jag2 in combination with the muscle-specific promoter they used with the next experiment (DN Dll1) that would drive its expression in differentiated fibers. It would also have been appropriate to use siRNA directed against Jag2. The reviewers do not insist on redoing the experiments with Jag2, although if the authors have these data they should be included.We used the DLL1/RCAS virus since it has been proven to induce consistently a NOTCH gain-of-function phenotype in limb muscles, i.e. an inhibition of muscle differentiation (based on the inhibition of differentiation muscle markers) and maintenance of progenitors (based on the expression of muscle progenitor markers) . Moreover, we now show that the long-term activation of (DLL1-mediated) NOTCH maintains the number of PAX7+ cells despite the absence of differentiated muscle cells . This reTo block NOTCH ligand activity, we have used an already described dominant negative form of DLL1, which has been shown to block the activity of all NOTCH ligands by preventing NOTCH ligand processing in signal-sending cells and therefore blocked NOTCH activation in signal-receiving cells .The DLL1/RCAS and DLL1/DN tools were already available and have been proven to be efficient to increase NOTCH activity and block NOTCH ligand function, respectively, so we did not repeat the experiments with JAG2.In any case, since Jag2 is membrane bound, they should show that they have an effect only in cells in direct contact with the Jag2/ Dll1 over-expressing fibers.The phenotype observed is a decrease in the number of PAX7+ cells upon loss of NOTCH ligand function in differentiated muscle cells . So it w6) It is not clear that Yap activation is downstream of muscle contraction and also that Yap activates Jag2 expression in myofibers. Of course, the most convincing data would be for the authors to show that ectopic activation of YAP in myofibers (via Tol2 MLC electroporation) rescues the loss of PAX7+ progenitors when myofibers have been immobilized. This experiment would considerably strengthen the paper and should be do-able by the authors since they have the Tol2 MLC construct.We have now performed the YAP rescue experiments in differentiated muscle cells (using the MLC promoter and the Tol2 stable vectors). We indeed found that forced YAP activity (YapS112A) in differentiated muscle cells (using the MLC promoter) prevented the decrease in the number of PAX7+ cells in immobilization conditions. It also prevented the downregulation of JAG2 transcription in muscles. Moreover, YapS112A activated the expression of ANKRD1 in paralyzed muscles. All these data are now included in a new 7) Since YAP is such an ubiquitously expressed molecule, the authors should be careful in the phrasing of the conclusions.We have tried to be careful in our conclusions. Although YAP transcripts and protein are observed in many  cell types, our data nevertheless show that YAP acts downstream of muscle contraction to regulate JAG2 transcription in muscle fibres, which then regulates the pool of muscle progenitors via NOTCH.


Multiple myeloma (MM) is a plasma cell tumour with an approximate annual incidence of 4500 in the UK. Therapeutic options for patients with MM have changed in the last decade with the arrival of proteasome inhibitors and immunomodulatory drugs. Despite these options, almost all patients will relapse post first-line autologous stem cell transplantation (ASCT). First relapse management (second-line treatment) has evolved in recent years with an expanding portfolio of novel agents, driving response rates influencing the durability of response. A second ASCT, as part of relapsed disease management , has been shown to prolong the progression-free survival and overall survival following a proteasome inhibitor-containing re-induction regimen, in the Cancer Research UK-funded National Cancer Research Institute Myeloma X (Intensive) study. It is now recommended that salvage ASCT be considered for suitable patients by the International Myeloma Working Group and the National Institute for Health and Care Excellence NG35 guidance.ACCoRd (Myeloma XII) is a UK-nationwide, individually randomised, multi-centre, multiple randomisation, open-label phase III trial with an initial single intervention registration phase aimed at relapsing MM patients who have received ASCT in first-line treatment. We will register 406 participants into the trial to allow 284 and 248 participants to be randomised at the first and second randomisations, respectively.Con, using high-dose melphalan, or ASCTAug, using high-dose melphalan with ixazomib. All participants achieving or maintaining a minimal response or better, following salvage ASCT, will undergo a second randomisation (1:1) to consolidation and maintenance or observation. Participants randomised to consolidation and maintenance will receive consolidation with two cycles of ixazomib, thalidomide and dexamethasone, and maintenance with ixazomib until disease progression.All participants will receive re-induction therapy until maximal response . Participants who achieve at least stable disease will be randomised (1:1) to receive either ASCTThe question of how best to maximise the durability of response to salvage ASCT warrants clinical investigation. Given the expanding scope of oral therapeutic agents, patient engagement with long-term maintenance strategies is a real opportunity. This study will provide evidence to better define post-relapse treatment in MM.ISRCTN10038996. Registered on 15 December 2016.ISRCTN, The online version of this article (10.1186/s13063-018-2524-8) contains supplementary material, which is available to authorized users. Multiple myeloma (MM) is a plasma cell tumour and has an annual incidence in the UK of approximately 4500 new cases . TherapeIGH, TP53 and MYC rearrangements), the DuR post-salvage ASCT was compromised, despite obtaining similar DoR to those for patients with standard-risk disease [Two clear areas of unmet need arose from the Myeloma X study. Firstly, although superior to non-ASCT consolidation, both the DoR and DuR post-salvage ASCT were inferior to those reported in first-line treatment. This has been reported in phase III collaborative studies and national/international transplant registries. Hence, the question remains of how to utilise novel agents to augment this effect, aiming to achieve similar DoR and DuR to those seen in first-line treatment. Furthermore, in the Myeloma X study, for patients with evidence of molecular high-risk disease  aims to compare salvage ASCT with the IMiD pomalidomide as a maintenance schedule against a non-ASCT strategy of pomalidomide, clarithromycin and dexamethasone. The second trial (NCT01242267) aims to address the augmentation of high-dose melphalan with increasing doses of thalidomide in a phase I/II setting. The third trial (NCT00938626) aims to utilise a bi-specific antibody to CD3 and CD20 to augment T cell activation post-salvage ASCT re-infusion in a phase I/II setting, again not addressing the key issues raised above. These trials will report in the early years of recruitment of this study, and their results will be monitored closely. Each of these studies is USA-based and, hence, may have limited impact on UK practice.Currently, there are three trials registered on the Novel agents including proteasome inhibitors and IMiDs are now routinely utilised as part of the induction regimen prior to ASCT, and this has resulted in substantial improvements in the DoR achieved before transplant in first-line treatment. The only data representing their impact in second-line ASCT is that published by the Myeloma X study group . Given tThe question of how best to maximise the DuR to salvage ASCT warrants clinical investigation. Given the expanding scope of therapeutic agents with differing modes of delivery (intravenous (IV) vs. subcutaneous (SC) vs. oral) combined with the potential for community-based therapy delivery, patient engagement with long-term maintenance strategies becomes a real opportunity. However, an evidence basis for the efficacy and thus the incorporation of further post-ASCT therapy is needed. The second question is to establish which patients benefit and, more importantly, which patients do not benefit from salvage ASCT. There is some evidence that molecular risk stratification at relapse after a prior transplant may delineate subgroups where a second transplant offers no advantage in terms of DuR. This clearly requires a more in-depth examination , as, curIxazomib  is a next generation, small molecule inhibitor of the 20S proteasome that is under development for the treatment of MM and other haematologic and non-haematologic diseases. Inhibition of the 20S proteasome has been validated as a therapeutic target for the treatment of malignancies using VELCADE® (bortezomib) for injection . In an ehttp://www.hovon.nl/studies/studies-per-ziektebeeld/mm.html?action=showstudie&studie_id=103&categorie_id=3), which is currently open to recruitment amongst patients not suitable for high-dose therapy. The use of ixazomib in the maintenance setting was reported by Kumar and colleagues at the American Society of Haematology meeting in December 2014, demonstrating efficacy and tolerability in previously untreated patients . The HOVON 126MM study also includes a maintenance phase randomisation comparing ixazomib and a placebo.It is therefore scientifically appropriate to utilise a proteasome inhibitor and IMiD combination as re-induction therapy to set the platform to assess the impact of conditioning augmentation and post-transplant consolidation/maintenance strategies. In designing the trial, we sought to evaluate the impact of a novel proteasome inhibitor that will facilitate both the clinical benefit and the patient experience with regards to deliverability of the treatment schema. Ixazomib, an orally active boronate-based reversible inhibitor of the proteasome, has shown single agent activity in phase I/II studies alongside combination therapy with dexamethasone and more recently IMiDs –13. MoreThe Myeloma XII (ACCoRd) study aims, firstly, to seek improvement in DoR through augmenting the conditioning regimen and, secondly, to induce more durable responses in the salvage setting via a consolidation/maintenance strategy. Pre-clinical models , 17 and This study will determine whether augmenting the conditioning and adopting a consolidation/maintenance strategy can have a significant impact on the DoR and DuR in all patients deemed suitable for salvage ASCT. The impact on patients with molecular high-risk markers will also be evaluated.The primary objectives of this study are to determine the impact on DoR: < very good partial response (VGPR) vs. ≥ VGPR when salvage ASCT conditioning is augmented by the addition of a proteasome inhibitor, as well as the influence of a consolidation and maintenance strategy on the DuR: PFS. The secondary objectives of this study are to assess OS, time to disease progression , overall response rate to ixazomib, thalidomide, dexamethasone (ITD) re-induction, time to next treatment (TTNT), second progression-free survival (PFS2), DuR, minimal residual disease (MRD) negative rate post re-induction, post-ASCT and conversion after ITD consolidation, as well as to look at engraftment kinetics, toxicity and safety of the treatment regimens and quality of life (QoL).2) indicates that total systemic exposure , following fixed dosing, should be independent of the individual patient’s BSA. Therefore, BSA is not expected to affect maximum plasma drug concentration (Cmax) or AUC after IV administration or oral dosing, and thus fixed dosing is appropriate for both oral and IV routes of administration. The clinical development of ixazomib has therefore transitioned from the use of BSA-based dosing to fixed dosing in all recently initiated Takeda phase I/II studies . Accordingly, the starting dose of ixazomib in the ACCoRd study is a fixed dose of 4.0 mg, on the basis of the recommended dose of 2.23 mg/m2 .The lack of a discernible relationship between body surface area (BSA) and ixazomib clearance over a relatively wide BSA range (1.4–2.6 mBiomarker exploration of prognosis determination: interphase fluorescent in situ hybridisation (iFISH) vs. next generation sequencing (NGS)-based molecular analysisMRD detection: comparative analysis of multi-parameter flow cytometry (MFC) and NGS-based molecular analysisBiomarker discovery for proteasome inhibitor sensitivityImmune biomarker discovery: exploration of immune functional assessment, including immune senescence, in relation to patient outcomes and response to therapy.ACCoRd protocol incorporates four planned translational research studies:The ACCoRd study is a UK-based, individually randomised, multi-centre, multi-stage, open-label phase III trial with an initial single intervention registration phase aimed at patients with relapsed MM who wish to undergo second-line treatment. A total of 406 participants will be registered into the trial to allow 284 participants to be randomised at the first randomisation (R1) and 248 participants to be randomised at the second randomisation (R2).Con, using melphalan, or ASCTAug, using melphalan along with ixazomib. All participants achieving or maintaining a minimal response (MR) or better, following trial ASCT, will undergo a second randomisation to consolidation and maintenance or no further treatment on a 1:1 basis. Participants randomised to consolidation and maintenance will receive consolidation with two 28-day cycles of ITD and maintenance with ixazomib until disease progression. Figure All participants will be registered at trial entry and will receive re-induction therapy with four to six 28-day cycles of ITD in order to reach maximum response. Participants who achieve at least stable disease (SD), as described by IMWG criteria, will be randomised on a 1:1 basis to receive either ASCTThe ACCoRd study aims to investigate the aforementioned treatment strategy within a population of participants with measurable, by IMWG criteria, relapsed MM requiring therapy who had undergone ASCT in first-line treatment and progressed for the first time at least 12 months after undergoing ASCT. The study will be conducted within an adult population (18 years or older), capable of giving written informed consent, with Eastern Cooperative Oncology (ECOG) Performance Status (ECOG PS) 0–2 and adequate full blood count (FBC), as well as satisfactory renal, hepatobiliary, pulmonary and cardiac function.To be eligible for registration into the re-induction phase of the trial, participants must meet all of the inclusion criteria and none of the exclusion criteria outlined in Table At the end of re-induction therapy, patients will be assessed for response. Patients achieving at least SD and fulfilling the eligibility criteria Table  for contAt approximately 100 days post-ASCT, patients will be assessed for response. A minimum of 248 patients achieving or maintaining at least MR and fulfilling eligibility criteria Table  for contThere will be 406 participants registered in the trial to ensure that 284 participants can pass through the first randomisation. This allows for 5% of participants having progressive disease (PD) after re-induction  re-induction in Myeloma X), 20% of participants mobilising insufficient cells for ASCT and 5% dropout overall throughout the trial.Aug arm with 80% power at the two-sided 5% significance level [In Myeloma X, 60% achieved VGPR response or better after ASCT. A total of 91 participants are required in each arm to detect an increase of 20% (from 60 to 80%) in ≥ VGPR rate in the ASCTce level  for firsThe described minimum clinically relevant difference is reasonable given what has been observed in other trials; e.g. an upgrade in complete responses (CRs) of 26% was observed by Roussel and colleagues where high-dose melphalan was augmented by IV bortezomib in a phase II study in the front-line setting  . TherefoThe second randomisation requires the greatest number of participants and hence determines the number of participants required to enter the first randomisation and the number of registered participants entering the trial. A total of 284 participants should be allocated at R1 to ensure 248 participants are available at the second randomisation (R2). Randomising 284 participants at R1 means that we have greater than 95% power to identify the clinically relevant difference of 20% (from 60 to 80%) in ≥ VGPR rate, or 172 participants being randomised to R1 in order to randomise at least 150 participants in R2 to power the trial at 80% to detect a smaller increase in rate of 16% (from 60 to 76%). This calculation accounts for 12.5% of participants suffering PD after ASCT or failing to achieve at least MR after re-induction and ASCT (7.8% had a response of ≤ SD after ASCT in Myeloma X).A median PFS of 24 months was observed in the ASCT arm in Myeloma X amongst those participants achieving ≥ VGPR. R2 in ACCoRd is 3 months later than randomisation in Myeloma X; hence, the study is powered to detect a difference in median PFS of 9 months from R2 where that would be expected to be 21 months after R2 in those participants receiving no further consolidation and maintenance treatment and 30 months after R2 in those participants receiving further consolidation and maintenance. Therefore, 192 events are required for 80% power . As a reThe increase in median PFS of 9 months is within the range (7–10 months) of improvements observed with maintenance therapy in first-line myeloma treatment , 24. In Increased follow-up also allows evaluation of protocol treatment and OS as a key secondary endpoint. The 248 participants randomised at R2 provide almost 80% power at a significance level of 0.05 to detect a 15% increase in OS at 5 years after R2 (from an estimated 59% in Myeloma X up to 74% in ACCoRd). A total of 96 events are required for 79% power at minimum 4 years follow-up for all participants.The 284 participants who undergo R1 will receive randomised ASCT  treatment. At 100 days post-ASCT, these participants will be assessed for response to treatment. In order to proceed to the next stage of the trial, R2, participants must have achieved a minimum of MR (according to the IMWG criteria). Based on experience of Myeloma X, we anticipate that not all participants will maintain at least SD. In order for the study to retain enough power for the statistical analysis, we require a minimum of 248/284 participants to meet the criteria to proceed to R2. This constitutes an approximate dropout rate of 10%.Participants will be recruited from 70 research centres from around the UK, which were identified via a feasibility assessment to determine the most appropriate centres to participate in the trial.Potential participants will be identified by the clinical team at participating centres based on their diagnosis of relapsed MM and subsequently approached during standard clinic visits for management of their disease, as well as being provided with verbal and written details about the trial. Assenting patients will then be invited to provide informed, written consent. Consenting participants will be formally assessed for eligibility, and eligible participants can be registered.Eligibility will be confirmed prior to registration and each randomisation, with the information being recorded in the participant’s medical records and on the relevant Case Report Forms (CRFs). Informed consent must be obtained and the participant must be registered prior to the participant undergoing procedures that are specific to the study and are beyond standard routine care at the participating site.In order to proceed to R1, participants must have achieved at least SD (according to the IMWG criteria). It is foreseen that not all participants will achieve a minimum of SD. Hence, to enable the study to retain enough power for the statistical analysis of the research question associated with the second randomisation (R2), we require a minimum of 284/406 participants to proceed to R1. The anticipated dropout rate is based on previous experience in the Myeloma X trial. Participants who do not show at least SD will cease trial treatment and will receive off-trial treatment at the discretion of the treating clinician. The trial opened to recruitment in March 2017 and is due to close to recruitment in February 2022 or when the planned sample sizes have been reached, whichever is earlier.Following confirmation of written informed consent and eligibility, an authorised member of staff at the trial research site will register participants immediately into the study. Registration will be performed centrally using the Leeds Clinical Trials Research Unit (CTRU) automated 24-h telephone and web registration and randomisation system (Gen24). Following successful registration, participants will undergo four to six cycles of ITD re-induction and will subsequently be assessed for response.Aug) or conventional ASCT (ASCTCon).Following registration and re-induction therapy, an authorised member of staff at the trial research site will randomise participants into the study. Randomisation will be performed centrally using the Gen24 system. At the first randomisation (R1), following completion of ITD re-induction chemotherapy, eligible participants ; length of first remission or plateau ; response to ITD re-induction therapy (< VGPR or ≥ VGPR); cytogenetic risk status at trial entry , as defined by Palumbo et al., in the presence of del(17p) and/or translocation t, and/or translocation t [A Gen24-generated minimisation program that incorporates a random element will be used to ensure treatment groups are well balanced for specific participant characteristics. For R1 these are: βt  and mainAt approximately 100 days following completion of trial ASCT and assessment for response, eligible participants  and response to allocated ASCT (< VGPR or ≥ VGPR).The study is an open-label trial which will use an independent randomisation service, and analyses will be conducted on an intention-to-treat (ITT) basis. The trial is administered by the Chief and Principal Investigators and the CTRU in accordance with Good Clinical Practice (GCP) guidelines. Response and relapse will be assessed using the IMWG International Uniform Response Criteria using blood and urine samples , unless progression of myeloma occurs as an isolated bone lesion, growth of a plasmacytoma or an increase in plasma cells in the bone marrow without a change in M-protein, where a tissue histological examination will be performed.External confirmation of disease progression and response data will be performed on samples of blood and urine at baseline, post re-induction treatment, 100 days post-transplant, post-consolidation (or 8 weeks post-R2 in the no further therapy arm) and at disease progression at a central laboratory. Samples will be forwarded by centres to relevant laboratories for central analysis. All disease progression and response data will be further confirmed by blinded clinical review coordinated by the CTRU.2-microglobulin; estimated creatinine clearance; paraprotein, serum free light chains, serum total (class-specific) immunoglobulins and urinary light chain detection (quantification where available); bone marrow aspirate and trephine (if available) within 4 weeks prior to registration; a pregnancy test; cardiac function (LVEF) within 12 weeks prior to registration; pulmonary function (KCO/DLCO) and an axial skeletal survey , magnetic resonance imaging (MRI) or other investigation when appropriate in accordance with local practice). Additionally, for the central investigation, a set of compulsory samples must be provided to the appropriate lab, to include peripheral clotted blood (10 ml) and random urine sample (10 ml); bone marrow aspirate (2 ml) in ethylenediaminetetraacetic acid (EDTA) and bone marrow trephine (where available) in formalin, embedded in paraffin/wax block or on slides ; ECOG PS and Revised International Staging System (R-ISS) classification; medical history ; Haematopoietic Cell Transplantation (HCT) Comorbidity Index score; FBC; serology of hepatitis B and C; urea and electrolytes, liver function tests, albumin, lactate dehydrogenase, C-reactive protein, calcium, creatinine and βFor the purposes of optional translational research requiring separate consent, the following samples should be taken prior to the start of ITD, for patients who have consented to the translational research investigations: peripheral blood (20 ml) in EDTA; serum (10 ml) and bone marrow aspirate (4–5 ml).Con), using melphalan (200 mg/m2/day on day −1), or augmented ASCT (ASCTAug), using melphalan with ixazomib . Participants will then be assessed for response 100 days post-ASCT and asked to complete the QoL questionnaires. All participants achieving or maintaining MR or better following trial ASCT will undergo R2 to consolidation and maintenance or no further treatment. Participants randomised to R2 will receive consolidation with two 28-day cycles of ITD , thalidomide (100 mg/day on days 1–28) and dexamethasone ) and maintenance with ixazomib  given as a 28-day cycle until disease progression. Participants will be asked to complete QoL questionnaires at 12 and 24 months post-R2 at their clinic visit, unless participants have not consented or have previously withdrawn from the QoL study , thalidomide (100 mg/day on days 1–28) and dexamethasone  (ITD), in order to reach maximum response. At the end of re-induction, participants will be assessed for response and will be asked to complete the pre-randomisation QoL questionnaires, unless participants have not consented or withdrawn from the QoL study element of the trial. Participants will then go on to peripheral blood stem cell (PBSC) mobilisation and harvest, which may be omitted if adequate PBSCs are available from previous mobilisation and harvest. Following this, participants who achieve at least stable disease (SD) will be randomised on a 1:1 basis to receive either conventional ASCT  are any untoward medical occurrences in a participant or clinical trial subject who receives a medicinal product which do not necessarily have a causal relationship with this treatment. AEs can be defined as any unintentional, unfavourable clinical signs or symptoms; any new illness or disease, or the deterioration of existing disease or illness; or any clinically relevant deterioration in any laboratory assessments or clinical tests. Due to the nature of myeloma and its treatment, participants are likely to experience several AEs throughout the course of the disease.All AEs, both related and unrelated to myeloma treatment, occurring from the first dose of ixazomib until 60 days after treatment will be reported and will be evaluated in accordance with the National Cancer Institute Common Terminology Criteria for Adverse Events V4.03 (NCI-CTCAE).Serious adverse events (SAEs) are defined as any untoward medical occurrences or effects that result in death; or are life threatening (at the time of the event); or require in-patient hospitalisation or prolongation of existing hospitalisation; or result in persistent or significant disability or incapacity; or result in a congenital anomaly or birth defect; or any other important medical event. For all participants who undergo R2, SAEs should be reported from the date of first dose of study drug until 60 days after the date of disease progression. For all participants who do not proceed to R2, SAEs should be reported until 60 days after the last dose of study drug. Serious adverse reactions (SARs) are to be reported from registration throughout the trial.SARs are events that are fatal or life-threatening; require or prolong hospitalisation; are significantly or permanently disabling or incapacitating; constitute a congenital anomaly or a birth defect; or jeopardise the participant and require medical or surgical intervention to prevent one of the outcomes listed above. SARs are SAEs that are deemed to be possibly related to any dose administered of any trial treatment. Suspected unexpected serious adverse reactions (SUSARs) are SARs which are not listed in the reference safety information for that medicinal product. SARs and SUSARs should be reported from the start of treatment and for the duration of the trial. An independent Data Monitoring and Ethics Committee (DMEC) will review the safety and ethics of the trial. The CTRU will prepare detailed unblinded reports for the DMEC at approximately yearly intervals. Unblinded safety updates are also prepared for review at 6-monthly intervals whilst participants are receiving trial treatment.Data will be collected using paper CRFs and entered into a validated trial database by the CTRU. A validation check program will be incorporated into the trial database to verify the data, and discrepancy reports will be generated for resolution by the investigator. Priority validations will be incorporated into the validation program to ensure that any discrepancies related to participant rights or safety are expedited to sites for resolution. Data will be monitored for quality and completeness by the CTRU. Missing data will be chased until received, and confirmed as not available, or the trial is at analysis. The CTRU/Sponsor will reserve the right to intermittently conduct source data verification exercises on a sample of participants, which will be carried out by staff from the CTRU/Sponsor. Source data verification will involve direct access to participant notes at the participating hospital sites and the central collection of copies of consent forms and other relevant investigation reports. Data will be held on a secure server at the University of Leeds and paper CRFs stored in a locked unit, both of which are accessible only to authorised trial staff.The CTRU statisticians will be responsible for the statistical analysis, and a final statistical analysis plan will be written before any analyses are undertaken.All analyses will be conducted on the ITT population, where participants will be included according to the treatment to which they were randomised regardless of eligibility, whether they prematurely discontinued treatment or did not comply with the regimen. A per-protocol (PP) analysis, where participants will be included according to the treatment they received, will be considered for the primary endpoints if there are a considerable number of protocol violators. The safety population will consist of all participants who receive at least one dose of the relevant study treatment.An overall two-sided 5% significance level will be used for all efficacy endpoint comparisons. For the primary endpoints, this will be adjusted to account for the formal interim analyses. Interim statistical summaries will be presented to the DMEC in strict confidence at approximately yearly intervals.Safety analyses will summarise all SUSARs, SARs, SAEs, AEs, ARs and treatment-related mortality rates as well as laboratory changes. Safety data will be presented by treatment group for the safety population in addition to relationship to study treatment.Con and ASCTAug is above 1 (where an odds ratio above 1 means that the ASCTAug group is worse), then treatment in this group may be halted. Formal interim analyses for early efficacy will be undertaken when 50% of required participants have completed response assessments at 100 days post-ASCT (91 participants) after R1 and when 50% of required PFS events have been observed (96 events) in follow-up after R2. These interim analyses will be assessed against a pre-specified significance level of 0.005 indicating possible overwhelming evidence of early efficacy. All interim analysis will be advisory with changes to the study at the discretion of study oversight committees. If the interim analysis for R1 leads to changes in the study protocol, study registration would continue to answer the question posed at R2. This would be achieved by extending the registration phase to include the selected treatment option at R1 for all subsequently treated patients before R2. No other formal analysis of the study is planned before the participants have attained the primary endpoints, i.e. all randomised participants have had response evaluated 100 days post-R1 or all randomised participants have been followed up for a minimum of 2 years post-R2. The key secondary endpoint, OS, will be evaluated after all continuing participants have been followed up for a minimum of 4 years post-R2. Analysis of other endpoints will be undertaken alongside these analyses, as appropriate.Formal interim analysis for lack of benefit (or harm in the language of Freidlin and colleagues ) will beCon and ASCTAug will be presented with corresponding 95% CIs.For R1, the number and proportion of participants in each response category  will be summarised by allocated treatment and exact 95% CIs will be calculated. The difference in proportions for each response category in ASCTAug and ASCTCon if the p values for the initial randomisation result odds ratios are ≤ 0.047. Parameter estimates, odds ratios and corresponding 95% CIs, degrees of freedom, test statistics and p values will be presented for each factor in the models. Residuals and predicted values produced from the models will be examined to assess the assumptions of the statistical models.Allocated treatment groups will be compared with respect to the proportion achieving remission (VGPR or CR) using logistic regression to adjust for the minimisation factors. The need for PBSC re-mobilisation will be recorded and adjusted for in these analyses, as appropriate. The significance levels will be adjusted to account for the interim analyses for early efficacy. The O’Brien and Fleming α-spending function  will be p values will be presented for each variable in the model.For R2, causes of progression in all participants will be summarised, and the proportions of participants with each underlying cause will be calculated. To compare ITD consolidation and ixazomib maintenance with no treatment, Cox regression analysis will be used to analyse PFS accounting for the minimisation factors. The significance levels will be adjusted to account for the interim analyses for early efficacy. Participants who are alive and not experiencing progression will be censored at the last date known to be alive and progression-free. Parameter estimates, HRs and corresponding 95% CIs, degrees of freedom, test statistics and The proportional hazards assumptions will be assessed by plotting the hazards over time for each treatment arm and using appropriate statistical tests. If evidence is found to support the violation of the proportional hazards assumption, then alternative appropriate analysis methods will be investigated.Secondary endpoint analysis of OS and other time-to-event endpoints will be analysed using similar methods to those described for the PFS primary endpoint. MRD negativity will be analysed using similar methods to those described for the response primary endpoint. AEs will be summarised using CTCAE categories. QoL will be summarised using mean scores adjusted for baseline and 95% CIs for each European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30 and EORTC QLQ-MY20 module symptom, role and functioning domain at each assessment time point. Similar summaries will be produced for quality-adjusted life years (QALYs), as scored by the EQ-5D questionnaire.A series of subgroup and exploratory analyses will be undertaken. Cytogenetic subgroups will be analysed to explore a number of specific hypotheses, including the effect on PFS, OS, time to progression and response. Some examples of what will be studied include chromosome 14 translocations and abnormalities of chromosomes 1p, 1q, 13q and 17p. In addition, other regions considered to be of interest will be analysed according to the statistical analysis plan. Other subgroup and exploratory analyses may also be performed and will be described in the statistical analysis plan or separate analyses plans related to translational work.The trial opened to recruitment in March 2017 using protocol version 3, dated 26 September 2016. An amendment to protocol version 4 is anticipated to be approved in March 2018, focusing on relaxing exclusion criteria 1 at registration, which stated that if the participant received dexamethasone (up to a maximum of 200 mg within 30 days prior to registration) he/she would not be eligible for the trial. Protocol version 4 will remove the 30-day restriction to allow more participants into the trial whilst maintaining the maximum dose permitted.The trial was developed by the ACCoRd Trial Management Group (TMG), with the support of the UK Myeloma Research Alliance (UK-MRA or NCRI Myeloma Subgroup). The trial is sponsored by the University of Leeds . A core project team, a TMG, a Trial Steering Committee (TSC) and a DMEC have been established. The independent DMEC review the safety and ethics of the trial alongside trial progress, and the overall direction is overseen by the TSC. Six-monthly interim safety reports are presented to the DMEC with a full review annually. The committee members will determine the subsequent frequency of review. The DMEC, in light of the interim data and of any advice or evidence they wish to request, will advise the TSC if there are any concerns or reasons why the trial should not continue. The results of the study will be published in peer-reviewed journals and will be presented at relevant national and international conferences. The CTRU will control the final trial dataset, and any requests for access will be reviewed by the TMG and TSC, subject to existing contractual arrangements with the funders. To maintain the scientific integrity of the trial, data will not be released prior to the end of the trial, either for trial publication or oral presentation purposes, without the permission of the TSC or the Chief Investigator.The study has had an encouraging start to recruitment. The trial has been able to open centres to recruitment at a rapid pace, with 22 centres open to recruitment in the first week and 65 centres open to recruitment within 4 months. The pace of recruitment is likely due to a number of factors.Patient attitudes towards ASCT may have changed , given tA wider contributing factor is potentially the successful recruitment nationally to investigator-initiated trials led by the NCRI, which have provided practice-changing results. Some of the success in recruitment could be explained by considering the excellent rate of recruitment to MRC Myeloma IX (1970 participants) and NCRI Myeloma XI (4420 participants), which have a high overlap of centres with ACCoRd. This benefits from the well-developed relationship with the coordinating CTRU that has evolved over more than 20 years.The ACCoRd trial opened to recruitment in March 2017. At the time of submission, the trial had recruited 140 participants, thus surpassing predicted recruitment for February 2019, 31 participants have gone through R1 and 4 participants have gone through R2.Additional file 1:The ACCoRd Trial  SPIRIT checklist. (DOCX 52 kb)


Although 93% of 12- to 23-month-old children in India receive at least one vaccine, typically Bacillus Calmette–Guérin, only 75% complete the recommended three doses of diphtheria-pertussis-tetanus  vaccine. Determinants can be different for nonvaccination and dropout but have not been examined in earlier studies. We use the three-dose DPT series as a proxy for the full sequence of recommended childhood vaccines and examine the determinants of DPT nonvaccination and dropout between doses 1 and 3.We analyzed data on 75,728 6- to 23-month-old children in villages across India to study demand- and supply-side factors determining nonvaccination with DPT and dropout between DPT doses 1 and 3, using a multilevel approach. Data come from the District Level Household and Facility Survey 3 (2007–08).Individual- and household-level factors were associated with both DPT nonvaccination and dropout between doses 1 and 3. Children whose mothers had no schooling were 2.3 times more likely not to receive any DPT vaccination and 1.5 times more likely to drop out between DPT doses 1 and 3, compared with children whose mothers had 10 or more years of schooling. Although supply-side factors related to availability of public health facilities and immunization-related health workers in villages were not correlated with dropout between DPT doses 1 and 3, children in districts where 46% or more villages had a healthcare subcentre were 1.5 times more likely to receive at least one dose of DPT vaccine compared with children in districts where 30% or fewer villages had subcentres.Nonvaccination with DPT in India is influenced by village- and district-level contextual factors over and above individuals’ background characteristics. Dropout between DPT doses 1 and 3 is associated more strongly with demand-side factors than with village- and district-level supply-side factors. A global review of the grey literature by Favin et al. found that the main reasons for incomplete vaccination were bad experiences at the immunization centre , missed opportunities , fear of side effects, and not knowing whether to return or when. Rainey et al. in their systematic review noted that factors such as parental education, cultural mores, and religious beliefs were more likely to be associated with nonvaccination than with incomplete vaccination A handful of studies have investigated reasons behind dropout after the first dose of DPT or oral polio vaccine (OPV), albeit within a very limited scope. Usman et al. in their cohort study involving 366 mother-infant pairs from six rural immunization centres around Karachi, Pakistan, found that children who received DPT dose 1 in a timely manner and lived closer to the immunization site were more likely to receive the subsequent doses 22.1We use data from the third round of the District Level Household and Facility Survey (DLHS-3), conducted during 2007–08. In this survey, 643,944 ever-married women aged 15–49 years from 720,320 households in 34 Indian states and union territories were interviewed on reproductive services and child health. Receipt of vaccination doses was recorded from children’s vaccination cards shown to the enumerator. If a card was not available, the mother was asked about the vaccines the child had received. Village questionnaires yielded information on availability of health, education, and other facilities in villages, and the facility-level survey gathered information on human resources, infrastructure, and services in the health facilities. Details on the survey methodology, sampling design, and the questionnaires are available elsewhere 2.2Combining data from the immunization cards and maternal recall, we defined three DPT vaccination outcomes: receipt of at least one dose of DPT (denoted by DPT1), receipt of all three DPT doses (denoted by DPT3), and receipt of all three DPT doses versus receipt of only one or two DPT doses (denoted by DPT3|DPT1). Typically, studies have modeled either nonvaccination with DPT or receipt of all three DPT doses. That We included individual- and household-level risk factors such as characteristics of the child, mother, and the household: the child’s sex, birth order, and their interaction; the mother’s and her partner’s years of schooling; her knowledge of diarrhoea management, awareness of immunization-related messages and colostrum feeding practices; religion and caste of the head of the household; and household wealth quintile. In addition, we considered indicators of village-level infrastructure, such as availability of electricity; availability of a subcentre and primary health centre (PHC) in the village and all-weather road connectivity with the subcentre or PHC; and availability in villages of auxiliary nurse midwives (ANMs) and accredited social health activists (ASHAs)—community health workers who help mobilize and vaccinate the rural population in India. We also included variables summarizing the district’s demographic and socioeconomic profile and the health infrastructure: percentages of fourth or higher birth order children, women with six or more years of schooling, households belonging to the richest wealth quintile, and villages with a subcentre. We considered a state-level dummy variable indicating the nine ‘high-focus’ states with poor infrastructure and low public health indicators. Further details on variables are provided in the 2.3MLwiN (version 2.32) for fitting the multilevel models.All analyses were confined to the 75,728 youngest living children, 6–23 months old, from villages across India, except union territories and Goa. We considered children 6 months or older so that all children have had opportunity to receive all three recommended DPT doses and also children less than two years old so that mothers would be able to recollect their vaccination history. For studying the effects of village-level structural determinants on vaccine uptake, we restricted the analyses to children from rural areas only and therefore excluded union territories and Goa, which have very few villages. Coverage for DPT1 and DPT3 was calculated by dividing the number of children receiving at least one DPT dose and all three DPT doses, respectively, by the total number of children aged 6–23 months. Coverage for DPT3|DPT1 was calculated by dividing the number of children receiving all three DPT doses by the number of children who received at least one dose of DPT. Estimates of prevalence and coverage rates were calculated using survey weights to account for unequal selection probabilities, and standard errors were adjusted for clustering at the village level. We performed multivariate logistic regressions to identify determinants of nonvaccination with DPT and dropout between doses 1 and 3. To account for the hierarchical nature of the data, we used multilevel logistic regression  to model the probability of the two binary outcomes—receipt of at least one dose of DPT and receipt of all three DPT doses among infants who received at least one dose of DPT. We conducted the descriptive analyses in R v.3.2.4 and used 3The study sample consists of 75,728 children 6–23 months old from villages across India, except for union territories and Goa. Characteristics of the study sample are summarized in Next, we examine the coverage rates for different DPT vaccination outcomes among infants, by background characteristics . The lasLastly, we examine how individual- and household-level socioeconomic and demographic characteristics, together with village- and district-level variables, differentially affect nonvaccination with DPT and dropout between doses 1 and 3 . We noteCovariates related to the availability of immunization-related workers and public health facilities in the village are not found to be correlated with the completion of the three-dose DPT series among infants who have received at least one DPT dose. However, for receipt of at least one dose of DPT, availability of ASHAs and ANMs in the village is critical. Indicators of village-level general infrastructure, like availability of electricity and all-weather road connectivity with the subcentre or PHC, are associated with both higher chances of receipt of at least one DPT dose and higher chances of completing the three-dose series among infants who have received at least one dose of DPT. We find a strong association between DPT outcomes and the percentage of higher birth order children in the district. The district-level percentage of villages with subcentres is correlated with receipt of at least one dose of DPT but not with completion of the DPT series among those who have received at least one DPT dose.4Our study considers both supply- and demand-side determinants of immunization and allows us to evaluate their relative importance for both DPT outcomes—nonvaccination and dropout between doses 1 and 3. Our multilevel analytical framework helps incorporate the hierarchical structure in the data and, more importantly, study the variation in child’s DPT vaccination status in terms of both individual-level and district- and village-level characteristics.We uncovered important differences in the epidemiology of nonreceipt of and dropout from DPT vaccination. States having more than 90% DPT1 coverage have dropout rates varying between 4%  and 23% (Chhattisgarh). High dropout rates coupled with high DPT1 coverage suggest that many mothers who visit a health facility for dose 1 but do not return for the subsequent ones may be dissatisfied with the services or not made aware that three doses are needed. Studies in other countries have found that designing reminder-type immunization cards Consistent with earlier findings The percentage of fourth or higher birth order children in the district, a proxy for district-level fertility rate, was strongly associated with both DPT outcomes, in line with the finding from a multilevel study in sub-Saharan Africa that children in countries with high fertility rates are less likely to receive all three DPT doses Our study has important limitations. We studied the effect of availability of public health facilities and health workers in villages but were unable to explain the effect of quality of services at these facilities—availability of vaccines and equipment, staffing, knowledge of the workers who perform vaccination, and infrastructure—on vaccination outcomes. Earlier studies have demonstrated that facilities with staffing and infrastructure problems and poor quality of services promote dissatisfaction and decrease demand For the vaccination status of children for whom the enumerator was not able to see the vaccination card, we relied on the mothers’ recall. The accuracy of a mother’s recall of her child’s vaccinations could not be assessed because mothers’ responses were sought only in cases where vaccination cards were not available. Moreover, we treated the DPT outcomes as binary variables by collapsing categories of DPT doses, which may lead to loss of information. Instead, we can retain the ordinal nature of the number of DPT doses variable and fit a random intercepts cumulative logit model, under the proportional odds assumption. Finally, because DLHS-3 was a cross-sectional survey, we could study only associations between risk factors and DPT outcomes and were not able to make causal inferences.5Although immunization strategies in developing countries have often focused on ensuring a reliable supply of health services None of the authors have any competing interests.RL conceived of the study question. AG conducted the statistical analyses, interpreted the data, and drafted the manuscript. RL contributed to interpretation of data, reviewed the manuscript, and helped finalize the draft. All authors approved the version submitted for publication.Wellcome Trust Capacity Strengthening Strategic Award to the Public Health Foundation of India and a consortium of UK universities (WT084754/Z/08/A). This project was supported by a grant by the Bill & Melinda Gates Foundation to the Immunization Technical Support Unit at the Public Health Foundation of India.AG was supported by a