Details of the program have been previously described.7 All patients (or their parents) provided written informed consent. Late effects were detected by physicians through regular visits to reference centers, starting 1 year after HSCT or after completion of chemotherapy and comprised a medical examination and adequate laboratory tests. The following late effects were assessed in the entire cohort: height, weight, body mass index (BMI), gonadal function, hypothyroidism, cardiac function, iron overload, cataract, alopecia, osteonecrosis, diabetes, metabolic syndrome, central nervous system (CNS) complications and viral infection. Health Quality of Life (HQoL) of children and adolescents was assessed using the parents' version of the Vécu et Santé Perçue de l'Adolescent et l'enfant (VSP-Ap) which is completed by the parents of the children and adolescents.8 Adult patients were asked to complete SF-36 questionnaires.9Studies of long-term survivors of childhood leukemia have shown that many patients have significant late effects due to their treatment.Overall, 169 patients treated in the participating centers met eligibility criteria; 113 were enrolled in the L.E.A. cohort  and all of them were included in this analysis. The absence of difference between included and not included patients is detailed in the The median age at diagnosis was 6.35 months (0.03–11.94) and 23 (20.4%) were <3 months of age. The median length of follow-up was 11.71 years (1.89–34.71). Relapse occurred in 18 patients (15.9%).n=33) of cases. The median follow-up was 11.55 years (1.2–24.67) from first transplant. Graft vs host disease occurred in 22 (55%) allografts and was acute for 20 (90.9%) and chronic for four (18.2%). We distinguished transplantations performed in first complete remission (CR1) (n=38) from those performed after CR1 (n=12) to focus on infancy as a risk period for long-term sequelae and to remove the impact of second line chemotherapy on late effects.Patients had been treated according to various French or European multicenter protocols depending on the period of their treatment and the type of leukemia. Sixty-three received chemotherapy only (55.8%), four (3.1%) chemotherapy and CNS irradiation, and 50 (44.2%) HSCT. CNS irradiation doses were 18 and 12 Gy, in three patients and one patient, respectively. Only three patients received total body irradiation (TBI) before HSCT: 2 at 12 Gy and one at 10 Gy. The median age at irradiation was 2.64 years (2.25–3.48) for TBI and 3.02 years (1.16–3.09) for CNS irradiation. Three boys received testis irradiation. Allogeneic transplants were performed in 41/50 (83.7%) patients and were first transplants except for three transplanted twice. The age at diagnosis for this transplanted population was ⩽6 months for 66% (P=0.128) by hypogonadism and all but one received sex hormone replacement therapy. Hypothyroidism was diagnosed in 12 patients (10.6%) but was considered to be overt (elevation of thyroid stimulating hormone associated with decreased levels of free thyroxine) in only two patients (1.8%).At least one late complication was observed in 84 (74.3%) of the 113 patients . There wP=0.01) between 6 and 12 months of age. Conversely, there was no significant difference in the occurrence of late effects according to the type of leukemia (P=0.45), but the mean number of late complications was significantly higher for transplanted children than for those who were not. This was mainly due to HSCT performed after CR1 . In contrast to HSCT performed in CR1, transplantation following a second remission increased the risk of hypogonadism (P=0.027), hypothyroidism (P=0.002), cataract (P=0.01) and CNS complications  and low weight  and CNS complications  or age at diagnosis (P=0.133): 0.24 (−0.19–0.68) and −0.32 (−0.744–0.099) Beta coefficient (95% CI), respectively. In contrast, HSCT performed after CR1 was associated with a significant but moderately higher sequelae risk factor (OR=1.38. P<0.001).Multivariate analyses confirmed the protective role of older age on major (>1 SDS) growth failure . When the HRQoL of our cohort was compared with age- and sex-matched French reference scores, we found similar results (P=0.01), mental health dimension (P=0.02), emotional role dimension (P=0.001) and vitality dimension (P=0.01) were lower in this L.E.A group (10 which suggests that late psychological effects are not specific to infancy.HQoL was reported by 81 parents 92% . Scores  results . Among t results . They re.A group  as in ot6 Those disorders are also described in the literature for survivors of overall childhood cancer and leukemia who showed a moderately increased risk for at least one chronic illness compared with siblings.12The present report is the largest study to evaluate the long-term outcome of infantile leukemia, survivors exhibiting at least one late effect in 75% of cases dominated by growth failure and endocrine dysfunction.15 and that a subgroup of long-term survivors may benefit from psychological assessment and targeted intervention. We also showed that the use of HSCT for infants is feasible given the late effects in this hard to cure population. These data are important not only for designing future therapeutic regimens, but also for identifying areas for late effect surveillance and intervention.The frequent elimination of irradiation from recent protocols for ALL or HSCT and the restricted use of alkylating agents and anthracyclines may have contributed to the low risk for cardiac disease and CNS complications but the relatively short follow-up for some sequelae such as second tumors or metabolic syndrome, justifies continuous, long-term follow up. We demonstrated here that infants ⩽6 months of age at diagnosis require early identification and treatment of growth alterations, including hormone replacement therapy


Objective. To investigate the effects and safety of the aqueous extract of the dried, immature fruit of Poncirus trifoliata (L.) Raf., known as Poncirus fructus (PF), in spinal cord injury (SCI) patients with neurogenic bowel. Methods. Thirty-one SCI patients with neurogenic bowel were recruited. Patients were evaluated based on clinical information, constipation score, Bristol Stool Form Scale, stool retention score using plain abdominal radiograph, and colon transit time. PF was administered in dosages of 800 mg each prior to breakfast and lunch for 14 days. Results. The morphological feature of the stool before and after administration indicated a statistically significant difference from 3.52 ± 1.33 to 4.32 ± 1.44 points (p < 0.05). Stool retention score before and after administration of PF was represented with low significance (7.25 ± 1.60 to 6.46 ± 1.53 points) in the whole colon (p < 0.05), and the colon transit time was significantly shortened (57.41 ± 20.7 to 41.2 ± 25.5 hours) in terms of the whole transit time (p < 0.05). Side effects were observed in 7 people (28.0%) consisting of 2 people with soft stools and 5 people with diarrhea. Conclusion. For SCI patients, PF administration significantly improved defecation patterns, defecation retention, and colon transit time. PF could be an effective aid to improve colonic motility and constipation. Constipation due to a neurogenic bowel after spinal cord injury (SCI) is one of the most common complications and has been reported in 39.1% of patients with SCI . A neuroMost SCI patients use many types of bowel management programs. Successful bowel management is multidimensional. Treatments may be multifaceted, using a mixture of strategies in regard to diet, medicine, electrical stimulation, and/or surgery . Medicat Poncirus trifoliata (L.) Raf. (Rutaceae), known as Poncirus fructus (PF), has been widely used as a traditional medicine in Eastern Asia, especially as an over-the-counter drug in Korea for the treatment of various gastrointestinal disorders [The dried immature fruit ofisorders . Even wiisorders  and an aisorders . Oral adisorders .Previous studies of the effect of PF on gastrointestinal motility have mostly involved animal models or normal adults without constipation. The usual recommended single doses of PF range widely within 2~75 g, with side effects including soft stools, diarrhea, and stomachache. But the types and severity of side effects according to PF dosage and administration are unclear.The present study of SCI patients with neurogenic bowel was undertaken to clarify the change in colon motility and to evaluate the effectiveness and safety of oral PF. We assessed whether oral administration of PF improved the defecation pattern using plain abdominal radiography and altered the colon transit time.n = 4), not recording Kolomark™ dosing time (n = 1), and occurrence of side effects (n = 1). Overall, 25 patients were included as subjects for the study. Patients with congenital abnormality and previous surgery history in the gastrointestinal tract except single appendectomy or cholecystectomy were excluded.Of SCI patients who were admitted to the Department of Rehabilitation Medicine in Wonkwang University Hospital from January 2011 to June 2013, 31 patients were selected for this study after providing informed consent for plain abdominal radiography, for survey of defecation patterns, and for examination of colon transit time. Six individuals were excluded because of arbitrary administration of PF  at Wonkwang University Hospital. All patients received a clear and sufficient explanation of the study purpose, contents, methods, possible PF side effects, and compensation standards and then provided signed informed consent.PF was purchased from Yuil Pharm, a qualified oriental drug store, located in Seoul Kyungdong Market. The purchased product was purified at the Korean Medicine Hospital of Wonkwang University and was used to fill capsules after manufacturing with powder at Hanpoong Pharm. PF (2 kg) was added to 30 L water and boiled at 100°C for 2 hours. The liquid extract was filtered and dried in a stream of hot air to obtain the powder extract followed by rotary evaporation. Recovery was approximately 20%. The powder extract was produced in granules and PF volume per capsule was approximately 400 mg. Each patient consumed 4 capsules daily, with 2 capsules taken prior to breakfast and the other 2 taken prior to lunch, each with 200 mL water. The total daily dose was 1600 mg, which was the lowest dose on the IRB recommendation.Survey of defecation consisted of constipation scores and morphological features of stools. The constipation score was calculated as the sum of the score of each item, ranging within 0~3 points, by adapting Rome Criteria II  for six Stool retention score was estimated using plain abdominal radiography. The radiography images excluded all patient information. The specialists in the Radiology Department evaluated the degree of defecation retention using the Leech method . In evalTo evaluate the colonic mobility, colon transit time was measured. Plain abdominal radiography was carried out 4 days after administering a capsule of Kolomark  for 3 days every morning at 9 AM. The capsule contains 20 rings of radiation nonpenetrating marker. As reported by Arhan et al. , in abdoThe following items were evaluated every day to estimate side effects during PF administration: soft stools, diarrhea, stomachache, abdominal displeasure, headache, vomiting, dizziness, and other effects. The degrees of side effects were scored as 0, 1, 2, and 3 points for no, light, medium, and severe side effects, respectively. The correlations of side effects with PF administration were assessed as “yes,” “possible,” and “no”; hematologic and chemistry tests were conducted every week to check the side effects that come with administration of PF.t-test. Statistical significance level was p < 0.05.Statistical analysis was performed using the Statistical Package for the Social Sciences version 11.0 . The differences in constipation scores, Bristol Stool Form Scale, stool retention score using plain abdominal radiograph, and colon transit times were compared before and after PF administration by paired All 25 patients who were selected as final subjects were given PF with no deviations from the dosing schedule. The study was completed by evaluating surveys, conducting plain abdominal radiography, and measuring colon transit time before and after PF administration. Five of the 6 patients who failed during the study had arbitrarily halted the administration or could not maintain dosing frequency or time. One patient complained of a medium soft stool and requested to stop PF use.The 25 subjects comprised 22 males and 3 females from the range of 18 to 88 years of age (average age 50.9 ± 17.3 years). The period from SCI to study participation was 5.3 ± 6.0 months on average. According to the American Spinal Cord Injury Association Impairment Scale (AIS), 5 people were classified as A, 5 people were classified as B, 4 people were classified as C, and 11 people were classified as D. Fourteen subjects had cervical SCI and 11 had thoracolumbar SCI .p < 0.05). Morphologically, the average stool score before PF administration was 3.52 ± 1.33 points ranging from 1 to 5 points. The postadministration score averaged 4.32 ± 1.44 points ranging from 1 to 6 points. The pre- to postadministration difference was significant . The score in each segment before and after PF administration was 2.45 ± 0.61 and 1.90 ± 0.64 points in the right colon, 2.30 ± 0.86 and 2.20 ± 0.69 points in the left colon, and 1.90 ± 0.85 and 1.40 ± 0.8 points in the rectal colon, respectively. The right colon and the rectal colon exhibited statistical significance . Transit time for each segment before and after PF administration was 14.4 ± 16.2 and 10.1 ± 12.1 hours in the right colon, 21.8 ± 12.3 and 14.8 ± 11.8 hours in the left colon, and 20.8 ± 12.1 and 16.3 ± 14.2 hours in the rectal colon, respectively. The right and the left colon exhibited statistical significance  patients, including 2 patients with soft stools and 7 patients with diarrhea. Two of the patients who complained of diarrhea were diagnosed as pseudomembranous colitis, which is considered to be unrelated to PF administration. The final occurrence for side effects was evaluated in 7 patients (28.0%). Side effects were rated as light in 3 patients and medium in 2 patients. After study termination, patients were monitored for 3 months to identify other side effects. One patient was diagnosed as cardiac arrhythmia thrombus 43 days after study termination. This was considered unrelated to PF administration . In the  Poncirus trifoliata (L.) Raf. and Citrus aurantium var. daidai Mak., belonging to Rutaceae. PF is a traditional medicine used for various gastrointestinal diseases in Southeast Asia including Korea. The Korean pharmacopoeia regulates PF as an immature fruit of Poncirus trifoliata (L.) Raf., with a diameter of 1~2 cm, and its recommended single dosage is 2~75 g [PF is an immature fruit ofs 2~75 g .PF includes over 50 phytochemicals including poncirin, limonene, synephrine, hesperidin, neohesperidin, auraptene, and imperatorin . VariousAdministration of PF promoted peristaltic movements of the small intestine and shortened the colon transit time in experimental animals and normal adults , 8. In aPresently, a 2-week administration schedule of PF significantly improved the constipation score and the morphological features of stools, improved the degree of defecation retention, and showed a decrease in whole colon transit time using plain abdominal radiography. This result is identical to a previous report , 9 that + and Ca2+ influx and via Ca2+ release from internal stores in a mitogen-activated protein (MAP) kinase dependent manner [The prokinetic effects of PF-W are well known but the mechanism of action is still unclear. With the influence of PF on GI tract motility, the action of serotonin receptor subtype 4 (5-HT4R) has been implicated in the prokinetic mechanism of PF-W . Also, tt manner . In addit manner . Considet manner  in bladdThe influence of PF on the GI tract is multifactorial and there may be a difference in the effect based on the type of extract, whether it is aqueous solution, hexane, or methanol. Aqueous extract of PF has been amply linked with prokinetic activity. Several reports described that only hexane extract of PT can dose-dependently increase the low frequency contraction of longitudinal muscle in distal colon strips  and thatThe recommended general over-the-counter dosage of PF is 2~75 g. PF-W was reportedly nontoxic even at a dose of 5 g/kg when orally given to mice [In this study, daily dosages of 1600 mg were administered considering the stability of patients, and the lowest over-the-counter dosage was represented. PF side effects related to GI tract include soft stools, diarrhea, and stomachache. However, the severity and occurrence rates of side effects in relation to dosage have not been reported before. In this study, PF side effects were observed in 7 out of 25 patients (28%). Side effects included soft stools for 2 patients and diarrhea for 5 patients, with the severity being ranked as light in 5 people and medium in 2 people. One patient who had complained of a medium soft stool and asked to halt PF administration showed improved symptoms 2 days after halting PF use. Further study could be instructive in deducing the best administration method and dosing of PF.Generally, the evaluation of neurogenic bowel in patients with SCI depends on subjective symptoms, such as bowel frequency. However, previous reports show patient recall of bowel habits is sometimes inaccurate , so objeFor the clinical application of PF for constipation in the future, the mechanism should be identified to promote GI tract motility and there should be standardization according to extraction methods. In addition, the administration amount should be standardized, which may enhance GI tract movements.In summary, PF could improve defecation and side effects even in SCI patients with neurogenic bowels. There can be further exploration of the difference in effects based on various extraction methods as well as further exploration relating PF dosing and its side effects.In order to evaluate the effect of PF on the colon motility of SCI patients with neurogenic bowel, this study estimated the change of defecation patterns by using constipation score, Bristol Stool Form Scale, stool retention degree through plain abdominal radiography, and colon transit time. Significant improvements after 14 days of PF administration were observed in bowel habits, stool retention, and colon transit time. The data showed PF enhances colon motility and improves constipation symptoms.


P=0.011). Age is a risk factor with the highest SPM incidence observed in transplant non-eligible patients aged >74 years receiving lenalidomide maintenance. The 3-year cumulative incidence in this group was 17.3% (95% CI 8.2–26.4%), compared with 6.5% (95% CI 0.2–12.9%) in observation only patients (P=0.049). There was a low overall incidence of haematological SPM (0.5%). The higher SPM incidence in patients receiving lenalidomide maintenance therapy, especially in advanced age, warrants ongoing monitoring although the benefit on survival is likely to outweigh risk.We have carried out the largest randomised trial to date of newly diagnosed myeloma patients, in which lenalidomide has been used as an induction and maintenance treatment option and here report its impact on second primary malignancy (SPM) incidence and pathology. After review, 104 SPMs were confirmed in 96 of 2732 trial patients. The cumulative incidence of SPM was 0.7%  0.4–1.0%), 2.3% (95% CI 1.6–2.7%) and 3.8% (95% CI 2.9–4.6%) at 1, 2 and 3 years, respectively. Patients receiving maintenance lenalidomide had a significantly higher SPM incidence overall ( A recent meta-analysis of seven trials, totalling 3254 patients, found no difference in the incidence of solid tumours, but rates of haematological malignancies were recorded at 3.1% and 1.4% of patients treated with or without lenalidomide, respectively.14 This increased rate was significant and seemed to be linked to the use of oral melphalan combined with lenalidomide.14 However, there was no increased risk associated with lenalidomide alone or in combination with other agents, including cyclophosphamide or intravenous melphalan-conditioning pre-transplant.In the first trials that addressed the role of lenalidomide maintenance in both transplant eligible (TE) and transplant non-eligible (TNE) populations, an increased risk of second primary malignancy (SPM) was noted.It is clear that more data are required to define the true impact of lenalidomide on SPM development, particularly in relation to the impact of concomitant therapies received, the duration of therapy and the effect of age. We aimed to accurately determine the overall and treatment-specific SPM incidence in patients enrolled to the Myeloma XI trial. The trial comprises both TE and TNE pathways with randomisation to induction and maintenance regimes containing lenalidomide. In addition, we utilised a clinician-led SPM committee review process in which all suspected SPM reported in patients enrolled to the trial are reviewed before they were confirmed as trial-related.Myeloma XI is a phase III, randomised, multi-centre, parallel-group design, open-label trial comparing thalidomide, lenalidomide and bortezomib induction combinations and lenalidomide±vorinostat as maintenance in newly diagnosed myeloma patients aged 18 years or over (NCT01554852). Inclusion and exclusion criteria are outlined . The triAll patients underwent randomisation (1:1) to cyclophosphamide, thalidomide and dexamethasone (CTD), or cyclophosphamide, lenalidomide and dexamethasone (RCD). Patients in the TNE pathway received attenuated dosing regimens. After initial randomisation, patients continued induction treatment until maximum response or intolerance. All patients received a minimum of six cycles before entering either maintenance (TNE) or four cycles before ASCT (TE). Patients continued to ASCT (TE) or maintenance randomisation (TNE) if a very good partial response or better was achieved following induction.15Patients who had a minimal response (MR) or partial response following induction underwent a further 1:1 randomisation to bortezomib, cyclophosphamide and dexamethasone (CVD) or nothing. Patients who had no change or progressive disease (PD) following induction received CVD on protocol. Up to eight cycles of CVD were administered and if an MR or better was observed patients continued to ASCT (TE) or maintenance randomisation (TNE). Patients who had evidence of PD or no change following this were treated off protocol. Response to treatment was assessed using the International Uniform Response Criteria for Multiple Myeloma.One hundred days post high-dose melphalan and ASCT, eligible patients in the TE pathway entered maintenance randomisation. TNE patients were randomised following induction or CVD. Randomisation resulted in entry to one of three regimes; single-agent lenalidomide, lenalidomide and the histone deacetalayse inhibitor vorinostat or observation only .Patients were reviewed at least four weekly during induction. During the maintenance phase monthly follow-up was arranged for those on lenalidomide-based therapy and two monthly for those under active observation. Maintenance lenalidomide±vorinostat was administered for 21 days of a 28-day cycle. After 2 years of maintenance patients under active observation had their monitoring frequency reduced to three monthly whilst those on lenalidomide continued with monthly review .Centres were required to report all SPMs in patients who entered the trial until death irrespective of whether they were still on the trial or not.In all, 2745 patients entered the trial with 2732 receiving at least one dose of protocol treatment between May 2010 and June 2013. Of the 2732 patients that initiated treatment, 1509 and 1223 entered the TE and TNE pathways, respectively. Within the TE pathway 753 patients received CTD induction and 756 received RCD induction. In the TNE pathway 612 patients received CTDa and 611 received RCDa. A total of 1362 patients continued to maintenance randomisation with 832 receiving lenalidomide, 527 as a single agent and 305 in combination with vorinostat . In all, 530 were randomised to active observation. The median follow-up since trial entry and maintenance randomisation is 34.3 and 24.2 months, respectively. The median age of patients enrolled to the TE and TNE pathways was 61 years (range 28–75) and 74 years (range 51–89), respectively .The primary end points were overall survival and progression-free survival, and it is anticipated that these will be reported from 2016 onwards. Here we report an interim analysis of SPM incidence as a key safety end point.All reported SPMs were reviewed and a clinical narrative summarising each case was completed. Additional information was requested from reporting centres, including histological reports, clinic letters and past medical history. All cases were presented to a committee of five clinicians  with a chair independent of the trial management group . Each case was discussed and a decision was made whether to confirm or reject the SPM. Criteria for rejection are outlined . Where a16 and plotted overall, by pathway and by treatment arm. The Pepe-Mori test17 for equality of cumulative incidence functions was used to analyse time to first SPM with unrelated deaths as a competing risk. Fine and Gray competing risks regression18 was used to compare the hazard of SPMs by treatment adjusting for the minimisation factors  with unrelated deaths specified as a competing risk. Person-years on trial was calculated as the sum over all patients receiving at least one dose of study treatment of the time in years from randomisation to death or last date known to be alive. Incidence rates were calculated with the number of events as the numerator and the number of person-years on trial as the denominator. Confidence intervals for incidence rate were calculated using approximations for the Poisson distribution. The analysis of primacy was predefined in the Statistical Analysis Plan to be the Fine and Gray regression. All statistical tests were two-sided and called significant at the 5% level.All statistical analyses were undertaken in SAS  according to the Myeloma XI Trial Statistical Analysis Plan. SPM diagnosed after trial entry was summarised for all patients. Cumulative incidence function curves were estimated by non-parametric maximum likelihood estimationn=14); initial report found to be incorrect (n=4); pre-malignant/benign skin conditions (n=4); recurrence of previous malignancy (n=1); and spontaneous resolution of disease  (n=1). A total of 104 SPMs in 96 patients were confirmed as trial-related. Of those confirmed, 4 patients had developed 2 separate malignancies, 2 patients developed 3 separate malignancies and the remainder a single malignancy. The median age at SPM diagnosis was 72 years and the median time to diagnosis from induction randomisation was 22.3 months (range 3.4–51.8).We identified 128 suspected SPMs in 114 patients. Following review, 24 (18.8%) in 18 patients were rejected. The reasons for rejection were the following: evidence existed to suggest the malignancy was present before enrolment . The cumulative incidence of SPM in the whole trial population was 0.7%  0.4–1.0%), 2.3% (95% CI 1.6–2.7%) and 3.8% (95% CI 2.9–4.6%) at 1, 2 and 3 years, respectively . If non-P=0.016; adjusted hazard ratio (HR) 1.74 (95% CI 0.88–3.45), Fine and Gray P=0.114; P=0.220; adjusted HR 0.86 (95% CI 0.52–1.42), Fine and Gray P=0.548; The 3-year SPM cumulative incidence for patients in the TE pathway who received RCD  and CTD  induction was 2.7% (95% CI 1.4–4.0) and 1.5% (95% CI 0.5–2.5), respectively , P=0.183; adjusted HR for lenalidomide plus vorinostat versus active observation 2.03 (95% CI 1.08–3.79), P=0.027; adjusted HR for lenalidomide±vorinostat 2.14 (95% CI 1.19–3.85), Fine and Gray P=0.011; For all patients randomised to maintenance, the 3-year cumulative incidence for those who received lenalidomide  versus active observation  was 8.9% (5.9–11.8%) and 4.0% (1.8–6.2%), respectively , Fine and Gray P=0.444; adjusted HR for lenalidomide plus vorinostat versus active observation 5.21 (95% CI 1.53–17.2), Fine and Gray P=0.008; adjusted HR for lenalidomide±vorinostat 2.58 (95% CI 0.84–7.86), Fine and Gray P=0.097; For patients enrolled to the TE pathway the 3-year cumulative incidence for those who had been randomised to maintenance and received lenalidomide  versus active observation  was 5.8% (95% CI 2.1–9.4) and 2.0% (95% CI 0.0–4.1), respectively , Fine and Gray P=0.038; adjusted HR for lenalidomide plus vorinostat versus active observation 1.48 (95% CI 0.52–4.20), Fine and Gray P=0.459; adjusted HR for lenalidomide±vorinostat 2.01 (95% CI 0.99–4.07), Fine and Gray P=0.053; Three-year cumulative incidence for lenalidomide maintenance  versus active observation  in the TNE pathway was 12.9% (95% CI 7.9–17.9) and 6.3% (95% CI 2.3–10.2), respectively , Fine and Gray P=0.049; P=0.129, adjusted HR for lenalidomide±vorinostat 3.059 (1.003–9.330), Fine and Gray P=0.511; The median age of the TNE patients was 74 years and, therefore, this was used as a cutoff for determining advanced age. The 3-year SPM cumulative incidence for patients in the TNE pathway aged >74 years who received lenalidomide  maintenance versus active observation  was 17.3% (95% CI 8.2–26.4) and 6.5% (95% CI 0.2–12.9), respectively  and 2273 mg (range 840–17 430 mg) for those ⩽74 and >74, respectively .SPMs were classified as either invasive haematological, invasive solid malignancies, including melanoma skin cancer, or non-invasive, NMSCs. Almost two-thirds (65%) of all SPMs were invasive with 68 cases in 68 patients. Twenty-nine cases were diagnosed in patients enrolled to the TE pathway and 39 in patients enrolled to the TNE pathway giving an incidence of 1.9% and 3.2% for the TE and TNE pathway, respectively .Thirteen confirmed haematological SPMs in 13 patients were observed during the trial. Of the 13, 10 cases were observed in patients enrolled to the TE pathway and 3 cases were observed in patients enrolled to the TNE pathway. In all, 6 patients developed myelodysplastic syndrome, 3 acute myeloid leukaemia, 2 non-Hodgkin lymphoma, 1 Hodgkin lymphoma and 1 chronic myeloid leukaemia. The trial incidence of haematological SPM was 0.48%. Cumulative incidence was 0%, 0.2% (95% CI 0.1–0.4%) and 0.4% (95% CI 0.1–0.7%) at 1, 2 and 3 years, respectively. Of the 10 SPMs in the TE pathway 8 patients had been exposed to lenalidomide and 5 patients received lenalidomide maintenance. Of the 3 SPMs in patients in the TNE pathway all had been exposed to lenalidomide and 2 received lenalidomide maintenance. The median age at the time of SPM diagnosis was 68 years, and median time to diagnosis from induction randomisation was 25.9 months (range 12.4–44.5).A total of 55 solid malignancies in 55 patients were confirmed as trial-related resulting in an incidence of 2.0%. The median time to SPM diagnosis was 20.5 months (range 3.4–45.8). In all, 19 solid SPMs were observed in patients enrolled to the TE pathway and 36 were observed in patients enrolled to the TNE pathway. The median age of SPM diagnosis was 67 years and 75 years for the TE and TNE pathways, respectively. The median time to SPM diagnosis from induction randomisation was 20.3 months for those in the TNE pathway and 21 months for those in the TE pathway. Twenty different types of solid malignancy were diagnosed. Histological confirmation was available for 52 of the 55 SPM with imaging indicating widespread metastatic disease in the other three. The most frequent malignancies observed were breast (7 cases), colon, (7 cases), prostate (6 cases), lung (5 cases) and bladder (4 cases) .Approximately one-third (35%) of cases were low-risk NMSCs with 36 cases diagnosed in 30 patients. Twenty-five NMSCs were observed in 23 patients enrolled to the TNE pathway with a median time to diagnosis from induction randomisation of 25.7 months and a median age of 78 years. Ten cases were basal cell carcinoma and 15 were squamous cell carcinoma. Eleven NMSCs developed in seven patients enrolled to the TE pathway with a median time to diagnosis of 33.7 months and a median age of 71. Eight cases were basal cell carcinoma and three were squamous cell carcinoma.Of the 104 SPM cases diagnosed, 69 cases (66%) in 61 patients occurred before PD. Thirty-eight patients diagnosed with a trial-related SPM have subsequently died (39.6%). Of these, 13 deaths were in patients enrolled to the TE pathway, 5 having received CTD and 8 RCD induction. The remaining 25 deaths occurred in patients enrolled to the TNE pathway with 15 patients having received CTDa and 10 RCDa induction. Nineteen (50%) deaths were observed in patients who had undergone maintenance randomisation, 15 in patients randomised to lenalidomide and 4 in patients being observed. Of the 38 deaths, 6 patients (23.7%) had PD related to myeloma at the time of death. Cause of death due to second malignancy was noted in 27 (71.1%) patients resulting in an overall trial SPM mortality of 1%. Death due to cardiac, respiratory and ‘non-myeloma'-related factors was noted as the cause of death for the remaining 5 patients.20 An effect of age and perhaps comorbidity is suggested by the higher overall SPM incidence in the TNE patients where overall SPM incidence was 5.2% in contrast to 2.7% in TE patients. Lenalidomide as a long-term treatment option in TNE patients has been assessed previously using both continuous and intermittent dosing schedules. In one such study the use of continuous lenalidomide and dexamethasone was associated with a 3-year invasive SPM incidence of 3%, similar to the 3.1% TNE pathway invasive SPM incidence observed here.20 The median ages of patients were also comparable. This perhaps suggests that the use of interrupted lenalidomide has no impact on SPM incidence rates in comparison with continual dosing. The median total dose of drugs administered in the early trials is not reported but in our study the doses were comparable between groups and in the TNE group where a higher SPM incidence was observed there was no evidence of greater drug exposure. This again suggests that other factors such as age, rather than drug exposure alone are factors in SPM development. The link between skin cancer development and immunosuppression is well established and thus the finding that 35% (n=36) of cases were NMSCs is not surprising.21 The 3-year cumulative incidence of SPM falls to 2.9% when NMSCs are excluded.The overall incidence rate of SPM observed on trial is low with a 3-year cumulative incidence of 3.8%, an overall incidence of 3.8% of treated patients and an incidence rate of 1.6 per 100 person-years. This is consistent with other studies in which lenalidomide has been used as a maintenance option in combinations excluding oral melphalan and markedly less than the 6.7 and 6.8% 3 year SPM incidence seen in the early studies where lenalidomide was used in combination with melphalan.n=31), maintenance (n=23) or both (n=19). Thirty-one patients (30%) had received thalidomide only. In the trial 50% of patients receive a lenalidomide-based induction with two-thirds of maintenance eligible patients receiving lenalidomide maintenance. It is, therefore, not unexpected that more patients have been exposed to lenalidomide therapy. This may in part explain the significantly higher SPM incidence observed in patients who received lenalidomide maintenance (P=0.011). However, it is more likely that the higher incidence is due to a combination of factors, including the impact of secondary immunodeficiency developed as a consequence of myeloma, cytotoxic and immunomodulatory therapy, and advancing age. SPM incidence in patients receiving lenalidomide maintenance was not significantly higher when broken down according to pathway , although this is most likely due to insufficient power, illustrated by the significantly higher incidence when the pathways are combined (P=0.011). It is also noted that the type of induction therapy received had no significant impact on SPM incidence. Three-year cumulative incidence of SPM for TE patients who received RCD was 2.7% as compared with 1.5% for those who received CTD (P=0.114). For patients in the TNE pathway 3-year SPM cumulative incidence was 5.9% versus 5.9% for RCD and CTD (P=0.548). This again suggests that treatment duration is likely to be a risk factor for second malignancy development.In relation to lenalidomide exposure 73 SPM (70%) were observed in patients exposed to lenalidomide either during induction , maintenance (n=3) or both (n=1). The significance of this observation is not clear.25 Three cases of acute myeloid leukaemia were confirmed, one in the TE pathway and two in the TNE pathway. We did not see an excess of B-cell malignancies. There were two trial-related diffuse large B-cell lymphoma cases and one Hodgkin lymphoma. The final haematological SPM case diagnosed during the trial was a chronic phase myeloid leukaemia.An increased incidence of haematological SPM was not seen here.P=0.049). Only 43 patients, 19 receiving lenalidomide and 24 being monitored, had reached 3-year post maintenance randomisation in the TNE pathway at the time of data cutoff. Reassuringly, 8 of the 17 SPMs diagnosed in this group were non-invasive NMSCs. Of the remaining 9 SPMs, 7 were solid malignancies and 2 were haematological (both acute myeloid leukaemia).A known risk factor for the development of malignancy in the general population is age, and consistent with this 64 of the 104 (62%) confirmed SPMs were diagnosed in patients enrolled to the TNE pathway giving a higher overall incidence in the TNE pathway of 5.2% versus 2.7% in the TE pathway. We used the median TNE patient age as a cutoff in the group to determine older age and noted an increase in SPM incidence after 2 years in this group. At 3 years the cumulative incidence of SPM in patients >74 years receiving lenalidomide maintenance was 17.3% versus 6.5% in patients randomised to active observation  maintenance to be reviewed every 4 weeks whilst those being observed are seen a maximum of every 8 weeks. Reduced clinic time may result in under-reporting by patients. Patients receiving treatment may also be more vigilant for new symptoms or signs, especially in those informed of previous reports of a link between lenalidomide and SPM development.14Mortality as a consequence of second malignancy was observed in 27 patients resulting in an overall incidence of 1.0%. Only 6 of these patients had progressive myeloma at the time of death. With such a low overall death rate attributable to second malignancies the benefit of lenalidomide therapy is likely to significantly outweigh treatment-related SPM risk. These data support meta-analysis findings, where the SPM death rate observed in patients receiving lenalidomide was 1.0% compared with 0.7% in those not.n=24, 18.8%) were rejected, impacting on the overall incidence of cases. The majority of these cases  were rejected because there was clear evidence that the second malignancy was present before enrolment. We believe that a review process should be incorporated into all trials, where second malignancies are considered a possible risk. The decision to reject or accept SPM as trial-related should be determined by specific pre-determined criteria. On initiation of treatment all patients should undergo a full systems examination, including the skin, to look for signs of possible existing malignancy. In addition, a medical history inclusive of a systems review should be conducted to determine symptoms consistent with possible underlying additional pathologies. Given the incidence of NMSC in this study and others, all patients on active treatment should be asked about the development of new skin lesions on each visit. Skin examination should be conducted at a minimum of three times per year. All patients should be warned of the possible long-term effects of maintenance therapy, irrespective of its nature and followed up appropriately. The majority of solid malignancies were those commonly seen in the ageing population, for example, colon/small bowel, prostate, breast and lung.An important lesson learnt from this study is the timing of second cancer incidence and its relation to trial entry. Many cancers, particularly in the older age group, were diagnosed early in trial follow-up, consistent with them having being present at trial entry. We have demonstrated the importance of a detailed review of all suspected SPM reported in patients enrolled to trials. Almost one-fifth of cases (In summary, overall SPM incidence in association with the trial has been low. Overall, there is an increased incidence of SPM at 3 years in patients receiving lenalidomide compared to observation only. A significantly higher SPM incidence is seen in older patients on maintenance lenalidomide compared with those being observed. An increased incidence of haematological malignancies in association with long-term lenalidomide has not been observed. Although low, there is an increased incidence of haematological malignancies in patients who have undergone stem cell transplantation perhaps secondary to melphalan use. Death as a consequence of second malignancy is very low and the survival benefit provided by myeloma therapy outweighs this risk. The incorporation of an SPM review process has been successful and should be incorporated into future trials in which SPM are deemed a significant risk.


Mmu16) homologous to Hsa21, is well reported to exhibit various typical neuropathological features seen in individuals with DS. This study investigated the role of skeletal muscles and peripheral nerve defects in contributing to muscle weakness in Ts1Cje mice.Down syndrome (DS) is a genetic disorder caused by presence of extra copy of human chromosome 21. It is characterised by several clinical phenotypes. Motor dysfunction due to hypotonia is commonly seen in individuals with DS and its etiology is yet unknown. Ts1Cje, which has a partial trisomy (P<0.0001) greater in the WT mice compared to Ts1Cje mice regardless of gender. The average survival time of the WT mice during the hanging wire test was significantly (P<0.0001) greater compared to the Ts1Cje mice. Also, the WT mice performed significantly (P<0.05) better than the Ts1Cje mice in the latency to maintain a coordinated motor movement against the rotating rod. Adult Ts1Cje mice exhibited significantly (P<0.001) lower nerve conduction velocity compared with their aged matched WT mice. Further analysis showed a significantly (P<0.001) higher population of type I fibres in WT compared to Ts1Cje mice. Also, there was significantly (P<0.01) higher population of COX deficient fibres in Ts1Cje mice. Expression of Myf5 was significantly (P<0.05) reduced in triceps of Ts1Cje mice while MyoD expression was significantly (P<0.05) increased in quadriceps of Ts1Cje mice.Assessment of the motor performance showed that, the forelimb grip strength was significantly (Ts1Cje mice exhibited weaker muscle strength. The lower population of the type I fibres and higher population of COX deficient fibres in Ts1Cje mice may contribute to the muscle weakness seen in this mouse model for DS. Down syndrome (DS) is a genetic disorder due to the presence of an extra copy of the human chromosome 21 (Hsa21), a condition also known as trisomy 21 . IndividThe pathological cause of muscle weakness in individuals with DS is not fully understood. Structural and functional alterations in the central nervous system (CNS) have been implicated as the main cause of muscle weakness in individuals with DS. Previous reports have suggested that there are alterations to the cerebellum, including volume reduction , reductiMmu10, Mmu16, and Mmu 17. Forelimb grip strength was assessed using an automated grip meter  as described by Luca, . The micin vivo was performed as described by Schulz et al., . Two-way ANOVA revealed no significant main effect for gender  = 1.877; P = 0.1797) for the cumulative number of falls in hanging wire test. The relationship between the latency to fall and body weight of the mice  was not statistically significant (P = 0.684). The motor coordination of the mice was assessed using two-way ANOVA and there was no significant difference in the latency to fall between the WT  and Ts1Cje mice  at constant speed of 4 rpm. However, the WT mice  performed significantly greater than the Ts1Cje mice  = 5.343; P = 0.0284) in the latency to maintain a coordinated motor movement against the rotating rod during the accelerated speed of 4–64 rpm  = 0.159; P = 0.6931)  compared to the Ts1Cje mice  . Two-way 0.0016) . Similar 0.0021) . For the<0.0001) . The cum<0.0001) , regardl4–64 rpm . Further 0.6931) .in vivo measurement of the sciatic nerve conduction velocity in adult (but not the ageing group) group showed a significant difference between the WT and Ts1Cje male mice. In the adult mice, the NCV in the WT male mice  was significantly higher as compared to the Ts1Cje male mice  = 13.26; P = 0.0009) (P = 0.1418) . We further assessed whether aging has contributed to the low conduction velocity. Two-way ANOVA analysis revealed no significant main effect on NCV due to age between male adult and aging groups  = 1.001; P = 0.3241).Poor motor performance of Ts1Cje led us to further investigate the health of sciatic nerve by measuring the nerve conduction velocity (NCV) as described previously . The res 0.0009) . In the  0.1418) . We furtP = 0.695) and triceps (P = 0.676) between the Ts1Cje and WT male mice  and triceps (WT = 50.08 ± 0.591 (52.7%); Ts1Cje = 44.87 ± 0.668 (47.3%); n = 248; t = 1.040; P<0.001)  and triceps (WT = 43.16 ± 0.651 (52.0%); Ts1Cje = 39.92 ± 0.602 (48.0%); n = 249; t = 0.809; P<0.01) (P = 0.3) and triceps (P = 0.3) of Ts1Cje male mice was comparable to WT littermates . Comparing to type II fibres, the type I fibres stained more deeply in blue and beige with ATPase and NADH diaphorase, respectively . AnalysiP<0.001)  of WT maP<0.001)  also rev P<0.01)  of WT matermates .C oxidase (COX) stain  and triceps (Ts1Cje = 50.48 ± 0.651 (52.3%); WT = 45.97 ± 0.735 (47.7%); n = 126; t = 4.595; P<0.01) of Ts1Cje male mice as compared with the WT  stain . Muscle X) stain . The resh the WT .P = 0.026; triceps: WT = 0.85± 0.04, Ts1Cje = 0.93 ± 0.10, P = 0.493; soleus: WT = 0.93 ± 0.03, Ts1Cje = 0.87 ± 0.038, P = 0.3365; EDL: WT = 0.61 ± 0.10, Ts1Cje = 0.71 ± 0.17, P = 0.6712]   (The myogenic regulatory factors (MRFs) are involved in muscle formation and differentiation of the myoblasts to myocytes. The development is regulated and controlled by myogenin, Myf5 and MyoD . Structu 0.6712] . Myogeni 0.6978] . SimilarP< 0.05] .Drosophila homologs of Hsa21 [Hypotonia is one of the features seen in individuals with DS and yet there is no clear understanding of what causes muscle weakness. Many studies implicate that CNS contributes to muscle weakness in individuals with DS. Several neuroanatomical defects affecting the CNS and neuromuscular junction have been reported in individuals with DS ,36, and of Hsa21 . This stBehavioural assessments from the present study have supported the suitability of the Ts1Cje mice in the muscle weakness related study, as this mouse model for DS recorded a significantly lower muscle strength compared with the aged matched WT mice. Similarly, due to their weaker muscle strength, the Ts1Cje mice could not stay in the hanging position for long and they fell down more frequently compared to the WT mice, resulting in higher cumulative number of falls which was found to be significant compared to the WT mice. Results from this study are in line with other studies on Ts65Dn and TgDyrk1A transgenic mice ,38.Ts1Cje mice also exhibit impairment in balance and motor coordination, but less severe as compared with other mouse models for DS. Our data shows that the Ts1Cje mice acquired the skill to remain on the stationary rod after a few trial sessions and they performed at the rates nearly similar to the WT mice at a constant speed of 4 rpm. On the other hand, the Ts65Dn mice showed delay in acquiring the skill to stay on the stationary rod and their ability to remain on the rod differs significantly from their control littermates . In the In the present study, the poor performance of Ts1Cje in the rotarod is further supported by the substantial decrease in NCV of Ts1Cje sciatic nerve. Reduced NCV can arise from thinner myeline sheath as well as axonal dysfunction . It is aMRFs play an important role in the process of myogenesis and any alteration in their expression will affect a particular stage or the entire process of myogenesis, leading to defective muscle formation. With limited studies on expression of the MRFs in mouse models for DS and in individuals with DS, their roles warrants further investigation. Alteration in fascicular architecture, organisation and histochemical characteristics of the skeletal muscles may contribute to muscle weakness. The skeletal muscles of the Ts1Cje male mice showed a normal muscle morphology as compared to the WT male mice. No significant fibre morphology difference was observed between the two genotypes. However, the population of type I fibres in both quadriceps and triceps was significantly lower in Ts1Cje male mice as compared with WT male mice and this has been consistent in both ATPase and NADH analyses. Type I muscle fibres have higher resistance to fatigue than type II fibres and this may possibly explain the reason for the poor performance of Ts1Cje mice during the hanging wire test and rotarod test. The Ts1Cje mice could not endure the hanging position and fell down within few seconds of their hanging. Similarly, these mice could not sustain the treadmill rotation and they lost motor coordination much earlier as compared to the WT. On the other hand, the ability of the WT mice to stay longer in the hanging position and perform excellently during the rotarod test may indicate the higher degree of endurance in these mice and it may be associated with higher population of the type I fibres as observed in quadriceps and triceps. Previous study in Ts65Dn mice revealed no significant differences in the population of fibre types between the two genotypes in soleus using immunofluorescence studies .in vivo [The population of the COX deficient fibres was significantly higher in Ts1Cje male mice as compared with the WT male mice and the result of this study further supported the general understanding of the mitochondrial defects in individuals with DS –46. A dein vivo . There iin vivo . The Ts1in vivo . In addiin vivo  leading Ts1Cje mouse model for DS have weaker muscle strength, poorer balance and impaired motor coordination compared with their control littermates and hence this model is suitable to be used for DS research related to muscle weakness. The lower motor conduction velocity of the peripheral nerve has impacted the motor performance of Ts1Cje. The expression of MRFs did not provide a conclusive evidence on their role in causing muscle weakness in Ts1Cje male mice. However, Ts1Cje male mice have significantly lower population of type 1 muscle fibres and higher number of mitochondrial deficient fibres and this may explain their poor motor performance.


In high‐dose buildup region, AAA calculated doses were lower by an average of ‐7.56%(SD=4.73%), while PBC was overestimated by 3.75%(SD=5.70%) as compared to TLD measured doses at 0.2 cm depth. However, at 0.4 and 0.6 cm depth, PBC overestimated TLD measured doses by 5.84%(SD=4.38%) and 2.40%(SD=4.63%), respectively, while AAA underestimated the TLD measured doses by ‐0.82%(SD=4.24%) and ‐1.10%(SD=4.14%) at the same respective depth. In low‐dose buildup region, both AAA and PBC overestimated the TLD measured doses at all depths except ‐2.05%(SD=10.21%) by AAA at 0.2 cm depth. The differences between AAA and PBC at all depths were statistically significant (p<0.05) in high‐dose buildup region, whereas it is not statistically significant in low‐dose buildup region. In conclusion, AAA calculated the dose more accurately than PBC in clinically important high‐dose buildup region at 0.4 cm and 0.6 cm depths. The use of an orfit cast increases the dose buildup effect, and this buildup effect decreases with depth.The purpose of this comparative study was to test the accuracy of anisotropic analytical algorithm (AAA) and pencil beam convolution (PBC) algorithms of Eclipse treatment planning system (TPS) for dose calculations in the low‐ and high‐dose buildup regions. AAA and PBC algorithms were used to create two intensity‐modulated radiotherapy (IMRT) plans of the same optimal fluence generated from a clinically simulated oropharynx case in an in‐house fabricated head and neck phantom. The TPS computed buildup doses were compared with the corresponding measured doses in the phantom using thermoluminescence dosimeters (TLD 100). Analysis of dose distribution calculated using PBC and AAA shows an increase in gamma value in the dose buildup region indicating large dose deviation. For the surface areas of 1, 50 and PACS number: 87.53.Bn Several authors have reported measurement of skin dose on patient and buildup dose on phantom from different treatment techniques. While one study reported increase in skin dose of patients undergoing IMRT treatment, others have reported lesser skin dose as compared to conventional techniques. But most of the studies do not address the comparison of TPS calculated and measured doses. Chung et al. reported large discrepancies in measured dose and dose calculated by commercially available TPS (Pinnacle and Corvus) algorithms. The accurate modeling of dose in the buildup region largely depends on the dose computation algorithm.Accurate calculation of dose distribution in the buildup region still remains a challenge to most of the commercially available photon dose calculation algorithms. This is primarily due to difficulties in modeling the contribution of doses from contaminated electrons originated from flattening filter, collimator assembly and, to a lesser extent, secondary scatter photons from the accelerator head.–5 Th This algorithm uses triple‐source modeling for accurate dose calculation at a point whereby it superimposes the doses from photons of both primary component and secondary scatter photon, and from electron contamination originating from flattening filter, collimator jaws, and accessories. The phase space  parameters are modeled using a Monte Carlo simulation‐derived multiple source model. This consists of a point source for radiation from the primary target, a finite source for extra focal radiation, and a third source to model the electron contamination. It then produces the final dose by superposition and convolution algorithm from these factors. For blocks, beam modifying device and physical wedges, the primary fluence is modified by means of the user‐defined transmission factor. Parameters used to characterize the multileaf collimation (MLC) are the leaf transmission factor and the dosimetric leaf separation. The latter provides the effective dosimetric opening between mechanically closed leaf pairs due to rounded leaf tips. While very limited studies have reported comparison of TPS calculated and measured skin dose in clinical treatment conditions, AAA algorithm has not been tested so far to check its reliability and efficiency in the dose calculation in the dose buildup region. In this study, the accuracy of AAA and PBC algorithms available in Eclipse TPS was extensively investigated in non‐clinical as well as clinical treatment conditions for the IMRT dose calculation in both high‐dose buildup and low‐dose buildup regions.In an attempt to improve the accuracy of dose calculation in tissue interface or inhomogeneous region, Varian Medical System  released a new photon dose calculation algorithm known as anisotropic analytical algorithm (AAA).–,, Th Beam profiles and depth dose curves were measured in a water phantom of RFA 300 Plus with OmniPro Accept software  in slow speed and high precision of 0.5 mm stepping mode at five different depths for a number of square field sizes ranging from 2×2 to 40×40cm2. The five different depths for beam profile measurement were at dmax (depth of dose maximum), 5, 10, 20, and 30 cm. This data of beam profiles and depth dose curves for beam configuration were measured using CC13 ion chambers . An output factor table at 5 cm depth for a series of rectangular field sizes (X and Y ranging from 1 to 40 cm) was also measured using the same ion chamber. These basic beam data measurements were performed at source to skin distance (SSD) = 100 cm. Commissioning and quality assurance for TPS were performed according to International Atomic Energy Agency (IAEA) Technical Report Series (TRS) report number 430 and the recommended guidelines and protocols of Varian Linear accelerator. The Eclipse TPS and Clinac DHX linear accelerator (which is equipped with 40 pairs of multileaf collimator (MLC) each projecting a leaf width of 1 cm at isocenter) were investigated for accurate modeling of dose distribution in the buildup region in clinical IMRT treatment conditions.A commercially available treatment planning system, Eclipse (V 8.6) , was configured for photon pencil beam convolution (PBC) and AAA algorithm using 6 MV X‐rays from Clinac DHX linear accelerator  following manufacturer recommended guidelines and protocols.10 BeaAn acrylic cast of head and neck region was prepared using VISCO VF Perspex molder  from a patient undergoing IMRT treatment of oropharynx. This cast was prepared exactly in the same condition as the thermoplastic immobilization device that was made for actual treatment planning simulation of the same patient. A head and neck phantom Fig.  was fabr(PTV+1.4cm) was defined by growing a uniform margin of 1.4 cm around PTV  algorithm while, in the other plan, PBC (version 8.6) algorithm was used. A calculation grid size of 0.125 cm was used in both plans.CT images of this wax head and neck phantom immobilized in the treatment position were acquired at 0.25 cm slices thickness on VFX‐16 multislice CT scanner . A body contour was generated with ‐550 HU (Hounsfield Unit) to exclude the orfit cast from the phantom. Contours containing clinical target volume (CTV) of the actual patient were copied onto the CT datasets of this phantom on Eclipse TPS, and expanded 0.5 cm isotropically to make the planning target volume (PTV). An arbitrary volume called high‐dose buildup region TV  dose at different locations, three axial planes corresponding to isocenter plane, 5 cm superior and 5 cm inferior to isocentre plane of the head and neck phantom were chosen. Multiple representative points were identified at each plane and at the depth of 0.2, 0.4 and 0.6 cm, respectively. These specific points were defined physically on the phantom by drilling narrow holes perpendicular to the phantom surface. The width of the holes was just sufficient to insert the dosimeter up to a maximum depth of 0.6 cm. These points were localized in the Eclipse TPS, and corresponding doses were calculated using various tools available in the TPS.0.32cm×0.32cm×0.09cm, were placed at each measurement position corresponding to deeper shell at 0.6 cm. In order to preserve their cleanliness and integrity, these TLD chips were kept in small polyethylene bags. The hollow space above the TLD chips was filled with paraffin wax at the same level of the skin to produce the dose buildup effect on these TLDs. After proper alignment of planned isocenter with the machine isocenter, IMRT plan was delivered on the phantom. This procedure was repeated separately with TLDs distributed on all predefined points at shells located at 0.4 cm and 0.2 cm depths, respectively. Thus, three separate measurements were performed for the same plan without orfit cast. Similarly, another three separate measurements were performed with orfit cast, to evaluate the dose buildup effect of the orfit cast. TL chips used in this study could detect doses ranging from 0.005 Gy to 10 Gy, and 50 TLD chips were preselected from the same batch having reproducibility within ±5% (SD) in the select dose region. These TL chips were assigned a permanent individual identification number. The sensitivity , using a lookup function in Microsoft Excel . Two TLD chips of ±1% reproducibility and ±1% variation from the average sensitivity were used as control to apply correction factor for every reading cycle. The exposed TL chips were read using a commercially available TLD reader . Among these 50 TL chips, 14 TL chips of ±1% reproducibility and ±1% variation from the average sensitivity were used for the calibration of this TLD reader using the heat treatment method reported by Yu et al. and Meigooni et al.. A dose calibration curve . The dose measurement using these TLDs at the representative points in head and neck phantom were compared with the corresponding TPS calculated doses.Verification of TPS calculated dose in the buildup region was performed using thermoluminescence dosimeter (TLD). TLD‐100 chips  having dimensions of er et al.20). T in OmniPro IMRT software . Gamma acceptance criteria were set as 3% dose difference and 0.3 cm distance to dose agreement (DTA) tolerances. These dose distribution comparisons were evaluated for three representative transverse planes at isocentre, 5 cm superior and 5 cm inferior to isocentre. Figure (>1.00) were found to be 97.79% and 2.15%, respectively. An increase of gamma values towards the skin of this phantom, represented by the dense red area in Fig. The dose distribution resulting from two separate plans calculated using PBC and AAA algorithms were compared using gamma values23 in 100cm2, PBC overestimated doses as compared to AAA calculated value in the range of 1.34%–3.62% at 0.6 cm depth, 1.74%–2.96% at 0.4 cm depth, and 1.96%–4.06% at 0.2 cm depth, respectively.Figures 100×/TLDmeasureddose. TLD measured doses show better agreement with AAA calculated doses of 0.53%(SD=5.12%) and 0.18%(SD=5.01%) mean differences than the corresponding PBC calculated doses of 4.27%(SD=6.60%) and 1.94%(SD=5.49%) mean differences at 0.4 cm and 0.6 cm depth, respectively (see Table PTV+1.4cm), doses calculated using PBC algorithm overestimate TLD measured doses, whereas AAA underestimates the TLD measured doses at all depths. The percentage differences of calculated doses using AAA and PBC algorithms from TLD measured doses in high‐dose buildup region at three different depths of 0.2, 0.4 and 0.6 cm from skin surface are shown in Table ‐7.56%(SD=4.73%) lower than the TLD measured doses at 0.2 cm depth, whereas PBC overestimates the doses as compared to TLD measurement with an average difference of 3.75%(SD=5.70%), which is significantly larger  as compared to AAA calculated doses (Table ‐0.82%(SD=4.24%) and ‐1.10%(SD=4.14%) for 0.4 and 0.6 cm depth, respectively, the corresponding values from PBC were as large as 5.84%(SD=4.38%) and 2.40%(SD=4.76%), respectively , except for the dose underestimation by AAA at 0.2 cm depth (Table ‐2.05%(SD=10.21%),2.82%(SD=5.38%) and 2.00%(SD=5.73%); those of PBC calculated doses from TLD measured doses were 0.55%(SD=8.0%),2.99%(SD=8.47%) and 1.29%(SD=6.69%) at 0.2, 0.4 and 0.6 cm depths, respectively (Table ‐13.35%(SD=0.846%) to ‐5.74%(SD=5.28%) for AAA, and ‐7.24%(SD=5.28%) to ‐4.01% (6.70%) for PBC with depth (from 0.2 cm to 0.6 cm)  Table .(dmax), whereas PBC calculated dose showed overall maximum deviation of 8.61% (field size of 2×2cm2) from the basic beam data in the dose buildup region and ‐0.48% beyond dmax  whereas the dose overestimation by PBC algorithms was an average dose difference of 2.09% on 0.2 cm strips at 0.2 cm depth  and 0.18%, respectively, as compared with that of 4.27% and 1.94% in the case of PBC at their respective depths. The large variations, represented by standard deviations of AAA (SD=9.17%) and PBC (SD=7.05%) calculated doses from TLD measured doses at 0.2 cm reveal that both AAA and PBC algorithms still have limitations in dose calculation at 0.2 cm depth. The larger mean difference of 4.27% and standard deviation of 6.6% indicates that there is limitation of dose calculation by PBC at 0.4 cm depth. This may be attributed to the fact that AAA calculation takes into account the consideration of electron densities of local neighboring points in 16 lateral directions using the scattering kernels scaled along the depths and lateral directions, which takes into account the contour irregularities. This results in the gradual change of dose gradient in the regions closer to skin contour as well as at a depth, and thus improves the dose uniformity , especially in high‐dose buildup region  and was found to be closer to TLD measured doses as compared to PBC algorithm. PBC was overestimating the doses from TLD measured doses in this high‐dose buildup region. Again, both AAA and PBC doses overestimate the TLD measured doses in low‐dose buildup region, except at 0.2 cm depth, and AAA doses were closer to TLD measured doses. The present study concludes that there is a limitation of dose calculation by both algorithms within 0.2 cm depth in the dose buildup region proximal to PTV as well as farther away from PTV. However, AAA algorithm is found to be more reliable on the points at all depths in high‐dose and low‐dose buildup regions compared with PBC algorithm.


To analyse survival trends and regional variation for very preterm infants admitted to neonatal care.All neonatal units in England.+0–31+6 weeks+daysgestational age (GA) over 2008–2014 and admitted to neonatal care; published data for admitted infants 22+0–25+6 weeks+days GA in 1995 and 2006, and for live births at 22+0–31+6 weeks+days GA in 2013.Infants born at 22We obtained data from the National Neonatal Research Database. We used logistic regression to model survival probability with birth weight, GA, sex, antenatal steroid exposure and multiple birth included in the risk adjustment model and calculated annualpercentage change (APC) for trends using joinpoint regression. We evaluated survival over a 20-year period for infants <26 weeks’ GA using additional published data from the EPICure studies.+0–23+6 weeks (APC 6.03% (95% CI 2.47 to 3.53) p=0.002). Improvement largely occurred in London and South of England (APC: London 1.26% (95% CI 0.60 to 1.96); South of England 1.09% (95% CI 0.36 to 1.82); Midlands and East of England 0.15% (95% CI −0.56 to 0.86); and North of England 0.26% (95% CI −0.54 to 1.07)). Survival at the earliest gestations improved at a similar rate over 1995–2014 , p value for change=0.25).We identified 50 112 eligible infants. There was an increase in survival over 2008–2014  p<0.001). The greatest improvement was at 22Continued national improvement in the survival of very preterm admissions masks important regional variation. Timely assessment of preterm survival is feasible using electronic records. The EPICure studies found survival of extremely preterm infants admitted to neonatal care in England to improve from 1995 to 2006.We identified no nationwide assessment of preterm survival following neonatal care since that time. This is likely to reflect the difficulties and costs of large-scale data collection.Our study shows that survival of preterm infants admitted to neonatal care has continued to improve, particularly for infants of the lowest gestations. However we also identified regional variation that is not explained by patient characteristics.Improvements have not been consistent across the country, warranting further investigation into the reasons for variation.As large, population-based studies are required to detect unusual variation in patient outcomes, electronic patient records provide opportunity to conduct such studies efficiently.Preterm birth is the primary cause of neonatal death worldwide and carries lifelong risks to health.The National Neonatal Research Database (NNRD) is a repository of a predefined set of variables  Information Standard SCCI1595), extracted quarterly from clinician-entered, point-of-care electronic patient records (EPR) for all infants admitted to neonatal units in England, Wales and Scotland.+0–31+6 weeks’ gestation and admitted to neonatal units in England 2008–2014. We assessed regional variation and relationship with socioeconomic deprivation. We examined survival trends over a 20-year period for those born at the earliest gestations by including previously published data. The secondary aims were to examine 28-day survival and postnatal age at death and develop a statistical model to predict survival.We evaluated trends in survival for infants born 22+0–31+6 weeks+days gestational age (GA) and admitted to a neonatal unit in England . The NNRD is approved by the National Research Ethics Service (16/LO/1093) and the Caldicott Guardians of contributing NHS Trusts. Approval is held from the Confidentiality Advisory Group of the Health Research Authority to hold NHS numbers for linkage (ECC8-05(f)/2010).We extracted NNRD data for infants born January 2008–December 2014 from 22Data comprised GA , birth weight (BW), singleton/multiple pregnancy, administration of antenatal steroids, vaginal/caesarean delivery, maternal age, maternal ethnicity, smoking during pregnancy and Index of Multiple Deprivation (IMD) 2010 quintile based on lower super output area (LSOA) rank.To reduce missing data we linked the NNRD to UK Office of National Statistics-Hospital Episode Statistics (ONS-HES) data. ONS-HES data were used for 28-day survival only as we could not ascertain if death occurred in neonatal care. Data extraction and linkage were carried out using SAS V.9.3.We estimated time trends for survival to discharge and 28 days using joinpoint regression.10.1136/archdischild-2017-312748.supp1Supplementary file 1We checked for seasonality by varying the autocorrelation parameter. As the number of neonatal units contributing data increased over time, we analysed complete neonatal networks as a sensitivity analysis. We tested for differences in postnatal age at death using quantile regression.We restricted the regional analysis to 2011 onwards in view of the possibility that lower population coverage in earlier years might bias regional estimates. Infants were assigned to one of the four regions  based on mothers’ residence. We calculated crude and standardised rates of survival to discharge and trends in crude survival; standardised trends by region were not calculated due to low numbers. We calculated crude and standardised rates of survival to discharge for the highest and lowest IMD quintile and computed the risk difference (RD). We added region  and IMD decile (continuous) to the risk adjustment model to test for residual regional variation.+0–25+6 weeks+days GA in March–December 1995 in every maternity unit in the UK and Ireland. EPICure 2+0–25+6 weeks+days GA in England in 2006. Only infants admitted to neonatal care in England were included.We compared NNRD data with published data for England. First we used joinpoint regression to compare recent trends in the NNRD data (2008–2014) with previous estimates from the EPICure studies+0–31+6 weeks+days GA. Data were compared for 2013 due to availability of England-only ONS data.Second, we compared the number of infants at each GA week by 28-day survival status and region of mother’s residence in the NNRD  with published ONS data+0–31+6 weeks GA who were admitted to a neonatal unit in England. We excluded 38 babies with implausible BW for GA, and 317 because BW, sex or multiple birth status was missing, leaving 50 112 infants in the study cohort. Population characteristics were broadly similar across all 7 years  survived to discharge over the whole period. The APC for crude survival was 0.51% , and after standardisation for risk of death, 0.46% . Results were similar for all sensitivity analyses.+0 to 23+6 weeks: APC 6.03% (95% CI 2.47 to 3.53), p=0.002; 30+0 to 31+6 weeks APC 0.01% (95% CI −0.08 to 0.09), p=0.9).+0 to 23+6 weeks to 98.2% at 30+0 to 31+6 weeks). The APC for crude 28-day survival and after standardisation for risk of death were similar (crude: 0.30% (95% CI 0.15 to 0.45), p<0.001; after standardisation: 0.27% (95% CI 0.11 to 0.44), p=0.002). The results were also similar when only neonatal networks where all hospitals contributed data for the whole period were examined (crude APC 0.35% (95% CI 0.19 to 0.52); adjusted APC 0.30% (95% CI 0.14 to 0.47)).Fifty additional deaths were identified by linkage with ONS-HES, of which 20 were within 28 days. There was an increase in the percentage of infants who survived to 28 days from 91.4% in 2008 to 93.5% in 2014. Survival improved with GA  p=0.02); there was no evidence of a change in the median and 25th percentile.Crude survival varied from 89.3% (95% CI 88.6 to 89.9) in the Midlands and East of England to 91.1% (95% CI 90.3 to 91.8) in London; after standardisation the range was 89.2% (95% CI 87.3 to 91.1) to 91.6% (95% CI 89.1 to 94.2). Adjusted survival in the other regions was 90.3 (95% CI 88.0 to 92.5) in the South of England and 89.8 (95% CI 88.0 to 91.8) in the North of England. Only London and the South of England showed improvements in crude survival over 2011 to 2014 (APC: London 1.26% (95% CI 0.60 to 1.96); South of England 1.09% (95% CI 0.36 to 1.82); Midlands and East of England 0.15% (95% CI −0.56 to 0.86); North of England 0.26% (95% CI −0.54 to 1.07)). Infants from the most deprived quintile had lower survival rates compared with those from the least deprived quintile (89.5% (95% CI 88.9 to 90.1) vs 91.1% (95% CI 90.2 to 92.1), RD 1.6% (95% CI 0.5 to 2.7)), but no difference remained after standardisation (89.8% (95% CI 87.9 to 91.5) vs 90.1% (95% CI 87.1 to 93.2), RD 0.3% (95% CI −3.3 to 3.9)). Inclusion of IMD decile in the risk adjustment model did not change results for each region, with evidence of residual variation across regions (p<0.001 from joint test of region indicators).+0–25+6 weeks+days to have continued at a similar rate across 1995 (EPICure), 2006 (EPICure 2) and 2008–2014 (NNRD). The EPICure studies found that survival increased from 40% in 1995, to 53% in 2006, and based on NNRD data, to 66% (654/992) in 2014. The APC for 1995–2014 was 2.73% (95% CI 2.35 to 3.12), with no evidence for a change in the trend (p=0.25). We found improvements in survival to discharge of infants born 22+0–31+6 weeks+days GA recorded in the NNRD for England in 2013 was 6812. This represents 97% (6812/7027) of infants surviving to 28 days recorded by the ONS. There were 538 deaths before 28 days recorded for neonatal admissions in the NNRD, representing 64% (538/845) of deaths among live births in the ONS data. Most of the discrepancy occurred at earlier gestations; there were three survivors and nine deaths among admissions of infants at 22 weeks’ GA in the NNRD, compared with 14 survivors and 130 deaths in the ONS (+0–31+6 weeks+days GA was 89% for the Midlands and East of England, 91% for London, 89% for the South of England and 92% for the North of England in 2013. The number of infants known to have survived to 28 days among admissions of infants born 22Results from the logistic regression model are shown in online We identify continuing improvement in the survival of very preterm infants admitted to neonatal care in England, from 1995 to the present, with the greatest increase in the most immature infants. Of note, there is evidence of a north-south divide, and persisting regional variation after adjustment for infant characteristics and socioeconomic differences.A key strength is that over 50 000 very preterm infants were included, representing almost all neonatal admissions in the country during the period. A novel strength is the use of the NNRD, a repository of point-of-care, EPR-derived data, facilitating up-to-date assessment of neonatal outcomes. The estimated survival probabilities, based on near-contemporaneous data, can help guide discussions with parents, noting however the need to emphasise that these relate not to total live births, but to infants admitted to intensive care, and are valuable information for clinicians, managers and commissioners. Validation of the prediction model using a future cohort would confirm its applicability; such a cohort can be easily established from new admissions in the NNRD. The risk adjustment variables were important, unambiguous clinical characteristics, also obtained from the NNRD. We took several steps to limit or investigate potential bias and conclusions remained valid following a number of sensitivity analyses. Around 3.4% of infants had missing outcome data, which could bias the assessment of survival trends. Outcomes were missing due to transfer to a neonatal unit or specialist surgical provider not contributing data to the NNRD. While the number of neonatal units contributing increased over time, sensitivity analysis including only providers contributing data throughout the period yielded similar results. A limitation is that live-born infants who died before admission to neonatal care were not included. This is illustrated by the lower number of deaths of admitted infants recorded in the NNRD compared with deaths among live births in the ONS, largely at the earliest gestations. This limitation was unavoidable as data capture is triggered by neonatal unit admission. Changes in survival of admitted infants could result from changes in admission practices over time. Although such changes could not be ascertained from the data available, trends persisted after adjustment for key risk factors. However the similarity with ONS data for the number of infants surviving to 28 days provides reassurance on population completeness for admitted infants. Regional variation could be attributable to differences in criteria for active management of extremely preterm infants. If the southern regions have higher survival because the sickest infants are less likely to be admitted for active care, we would expect a lower proportion of live births to be admitted in these regions. Comparison of regional ONS and NNRD data showed no such pattern, although regional ONS data on infants born at 22 weeks’ GA were unavailable.Our study covers the entire population of neonatal admissions in a geographically defined region. This contrasts with previous reports such as those from the US National Institute of Child Health and Human Development Neonatal Research Network that focus on admissions to tertiary neonatal units,Our study has several implications for clinicians, policy makers and researchers. First, although not evidenced by published data to date, continued improvement in survival of very preterm infants may lead in future to a growing number of children and adults with long-term health needs. Opportunity for cost-effective long-term ascertainment of outcomes for all infants admitted to neonatal care is offered by linkage of NNRD data with other national records, such as hospital, general practice and educational data sets. Second, the improvement in survival appears to be largely at lower GA and was inconsistent across the regions. Identifying and reducing inequity in health outcomes are a stated intention of the UK Government and NHS England. Third, we show that NNRD data, derived from EPR, enable timely evaluations of outcomes and eliminate the need for separate data capture by busy clinical teams. The small number of very preterm births and the increasing rarity of death in this population mean that large sample sizes enabled by the national coverage of the NNRD are required to detect variation. There is considerable interest in using EPR for research; we hope our study will serve as a template to advance this approach to improve patient care.


This study investigated the genetic diversity of noroviruses identified from a previous surveillance study conducted at the National Pediatric Hospital in Phnom Penh, Cambodia, from 2004 to 2006. In the previous study, 926 stool samples were collected from children aged 3–60 months with acute diarrhea (cases) and without diarrhea (controls) with reported 6.7% of cases and 3.2% of controls being positive for norovirus. The initial norovirus diagnostic assay was performed with real-time reverse transcription-polymerase chain reaction  which also distinguished between genogroups I and II (GI and GII). Norovirus infection was most commonly detected in children aged 12–23 months in both cases and controls. Norovirus Genotyping Tool and phylogenetic analysis of partial sequences of the 3′ end of the RNA-dependent RNA Polymerase (RdRp) and the capsid domain region were employed to assign genotypes of the norovirus strains. GII.4 was the most predominant capsid genotype detected at 39.5% followed by GII.6 at 14.9%. The GII.4 Hunter 2004 variant was the predominant strain detected. Six RdRP/capsid recombinants including GII.P7/GII.6, GII.P7/GII.14, GII.P7/GII.20, GII.P12/GII.13, GII.P17/GII.16, and GII.P21/GII.3 were also identified. This study of norovirus infection in young children in Cambodia suggests genetic diversity of norovirus as reported worldwide.  Norovirus, a member of the family Caliciviridae, is an important human pathogen and is the leading cause of nonbacterial acute gastroenteritis outbreaks. Norovirus has been increasingly associated with sporadic episodes of acute gastroenteritis in children worldwide. It has been estimated that norovirus infections cause 1 million hospitalizations and 200,000 deaths in children under 5 years of age in the developing world [The norovirus genome is organized into three open reading frames (ORF). ORF1 encodes six nonstructural proteins including the RNA-dependent RNA polymerase (RdRp); ORF2 encodes the capsid; and ORF3 encodes a small, minor structural protein . DespiteGII.4 variants have been reported as the major cause of norovirus gastroenteritis worldwide starting in 1995 with GII.4 variant Asia 2003 as the most widely circulated variant in Asia during 2003–2006 . In a PeA potential mechanism that norovirus utilizes to evade host immunity is genetic recombination at the overlapping regions between the RdRp of ORF1 and the capsid protein encoding gene (ORF2), ORF1/ORF2 junction . MultiplThere have been few reports on norovirus variants circulating in Southeast Asia –14 and hA detailed description of the study design has been reported previously . BrieflyApproximately 3–5 g of stool was collected from subjects. Stool samples were stored at −70°C until processed. A 10% (wt/vol) stool suspension was prepared with distilled sterile water and total nucleic acids were extracted with NucliSens® Magnetic Extraction Kit  following the instructions of the manufacturer.The extracted nucleic acids were screened to identify the genogroup (GI and GII) by real-time RT PCR reactions as described previously . The reaμL of RNA was reverse transcribed with primer G1SKR for GI and G2SKR for GII [The extracted nucleic acids of norovirus positive samples were treated with DNase  to remove DNA prior to reverse transcription reaction. Five  for GII  to generThe ORF1/ORF2 junction was amplified using AmpliTaq Gold® polymerase  containing a mixture of three forward primers  for GI or G2FB  for GII) and reverse primer (G1SKR for GI or G2SKR for GII) , 17. ThePCR products (GI 597 bp and GII 468 bp) were cloned into a TA-Cloning vector . One to three positive clones were sequenced from both forward and reverse directions using a commercial sequencing service . DNA sequencing data were verified for consensus sequence using Sequencher software version 4.1.2 .The identification of norovirus genotype was performed by submitting sequences of the junction between RdRP and capsid genes to the online Norovirus Genotyping Tool (Version 1.0) . PhylogeAll sequences were aligned with ClustalW  in MEGA Simplot version 3.5.1 was used to identify putative recombination breakpoints by comparing query sequence to nonrecombinant and recombinant parental strains . Analysi t-test in IBM® SPSS® Statistics Version 22 .The differences among proportions were analyzed by chi-square test and the difference between means was analyzed byA total of 926 stool samples (580 cases and 346 controls) were previously tested for the presence of norovirus and other enteric pathogens . The preAmplification of the ORF1/ORF2 junction region was successfully performed on 39 (7 cases and 32 controls) out of the 50 real-time PCR norovirus positive samples (2 GI and 37 GII). Repeated attempts to amplify the remaining 11 positive samples were unsuccessful. Identification of norovirus genotypes was achieved by cloning PCR products and performing sequence analysis on positive clones. The cloned PCR product corresponded to a 597 (GI) and a 468 (GII) bp fragment that maps to the overlapping region of ORF1 and ORF2.A total of 43 nucleotide sequences were obtained. Four additional sequences were of mixed norovirus infection from the same sample. GenBank accession numbers of all sequences are KX685457–KX685499. These sequences were then submitted to the online Norovirus Genotyping Tool to identify their genotypes based on partial RdRP and capsid genes. GII.4 was the most predominant capsid genotypes assigned at 39.5% followed by GII.6 at 14.9% . GII.4 cTwo or more consensus genotype sequences were derived from 4 samples suggesting a mixed infection in these samples Figures . Mixed iBoth of the Norovirus Genotyping Tool and phylogenetic analysis revealed 7 different recombinant genotypes from 16 sequences. GII.P7/GII.6 was the predominant recombinant with 6 sequences followed by GII.P7/GII.14 and GII.P7/GII.20 at 3 sequences each. Other recombinants were GI.Pc/GI.5, GII.P12/GII.13, GII.P16/GII.17, and GII.P21/GII.3 at 1 sequence each . GII.P7 All of the 16 recombinant sequences were subjected to Simplot analysis to determine recombination breakpoints. The presence of norovirus in cases of pediatric diarrhea in Cambodia was described previously but little is known about the genetic diversity of the circulating norovirus strains , 29. In Age distribution of norovirus infection in this study was similar to what has been reported elsewhere –35. ChilStudies of sequence and genotypic analysis of norovirus positive samples described GII.4 as the most common genotype detected in both outbreaks and sporadic cases of acute gastroenteritis worldwide . The 16 Recombination between the RNA-dependent RNA polymerase and the capsid genes has been reported worldwide in recent years . The NorAll of the recombinants identified in this study have been reported previously from various geographical locations from samples that were collected prior to, after, or at the same time period as this study. GI.Pc/GI.5 and GII.P21/GII.3 were reported earlier than the rest of recombinants in this study with GII.P21/GII.3 being the most widely detected recombinant , 21, 41.Despite advances in understanding norovirus biology, no norovirus virulence determinants have been identified and there is currently no efficient way to predict which strains will become dominant. Sequence and biochemical studies suggest that norovirus employs at least two mechanisms to persist in the population: antigenic drift and recombination , 42. UndThis study reports on molecular epidemiology of norovirus circulating in young children in Phnom Penh, Cambodia, form 2004 to 2006. The existence of several GII.4 variants and recombinant strains in Cambodia suggests the need for a continued surveillance system that includes molecular aspects to provide a better epidemiological understanding for the development of vaccines against norovirus.