Cardiovascular magnetic resonance (CMR) myocardial perfusion reserve index (MPRI) has recently shown promise for detecting coronary microvascular dysfunction (CMD) in women with signs and symptoms of ischemia and no obstructive coronary artery disease (CAD). Prior CMR studies in CAD populations and in healthy volunteers have shown good intra and interobserver reproducibility for MPRI. However, interscan reproducibility is more variable. If MPRI is to be considered useful for the detection of CMD in women, the interscan reproducibility in this population must also be understood, such that proposed MPRI cut-off thresholds can be appropriately adjusted. Therefore, the aim of this study was to determine the interscan reproducibility of MPRI in women with suspected CMD.Rest/stress perfusion CMR was performed at 1.5T using a standardized protocol in 27 women with signs and symptoms of ischemia and no obstructive CAD on two separate days. Cardiovascular medications were held for 24-48 hours before each scan. The same pharmacological stress agent (adenosine/regadenoson) was used for both scans. MPRI was calculated from time-intensity curves of the whole myocardium and blood pool at stress and rest . One experienced observer, blinded to clinical data, performed all measurements. Intra-class correlation coefficients (ICC), coefficient of variation (CoV), and Bland-Altman plots were determined.2; 55% had hypertension, 7% diabetes, 63% hyperlipidemia and 41% family history of CAD. The time difference between scans was 107 ± 60 days. There was no significant difference between rest or stress hemodynamic variables (summarized as rate pressure product [RPP]) between scan 1 and 2 . Mean MPRI for the 27 women was higher for scan 2 compared to scan 1 (1.96 ± 0.40 vs. 1.76 ± 0.42); and this relationship persisted even when corrected for resting RPP (1.74 ± 0.5 vs. 1.52 ± 0.35). On Bland-Altman analysis there was a small significant bias for MPRI between scans (bias=0.21 [95% CI: 0.07 to 0.35]) and the 95% limits of agreement were relatively wide (-0.7 to 0.92) (Figure Mean age was 51 ± 20 years old and BMI 28 ± 8 kg/mInterscan reproducibility of CMR-derived MPRI in women with suspected CMD was only modest, with relatively wide limits of agreement. This variability is very similar to that seen in other populations and means that some caution must be exercised when using absolute MPRI cut-offs in isolation for the diagnosis of CMD, or repeated measures of MPRI to track response to therapy. The combination of clinical factors and symptom review with MPRI is therefore likely to be the optimal strategy, rather than its use in isolation.


Overall survival was 8.5 months, progression-free survival 4.33 months and response rate 25%, with disease control rate 57.2%. Grade III-IV neutropenia occurred in 10.7% of the patients, grade III-IV anemia and grade III thrombocytopenia in 10.7% and 7.2%, respectively. Among non haematological toxicity, grade I-II constipation was reported in 14.2% of the patients, without grade III-IV adverse events. No discontinuation for toxicity was observed. This study underlines that Vinfluinine at a dose of 280 mg/m2 associated with primary prophylaxis for neutropenia and constipation is effective and with a favorable toxicity profile.Vinflunine is the only cytotoxic agent tested as a second line therapy in transitional cell carcinoma of the urothelium in a phase III trial. It is not largely employed in clinical practice because of the high incidence of grade 3–4 toxicity. We evaluated efficacy and safety of Vinflunine at the dose of 280 mg/m Transitional cell carcinomas of the urothelium (TCCU) represent more than 90% of all cancers of the urinary tract, among which 90% are localized in the bladder. –2. In EuApproximately half of the patients with muscle-invasive TCCU relapse after radical surgery, the majority of them with distant metastases. At the time of the diagnosis about 15% of patients have advanced disease . SurvivaVinflunine is a microtubule inhibitor  approved2 every 3 weeks for patients with performance status (PS) ECOG 0 and at a dose of 280 mg/m2 every 3 weeks for patients with PS ECOG 0 and a previous pelvic radiation or with PS ECOG 1. Median overall survival was 6.9 months for Vinflunine plus best supportive care versus 4.3 months for best supportive care solely. ORR was 8.6% for Vinflunine vs 0% for BSC, disease control rate 41.1% vs 24.8%, median PFS 3 vs 1.5 months, respectively.In the phase III trial , 20, VinAn overall survival of 7–10 months was reported in other studies on efficacy and tolerability of Vinflunine conducted in Spain , GermanyIn the phase III trial, half of the patients experienced grade 3–4 neutropenia. Other relevant grade 3–4 adverse events were: fatigue in 19.3% of the patients, constipation 16.1%, anemia 19.1% .Overall survival was 8.5 months for the entire population Figure , 8.8 monp = 0.09, HR 0.53 (95% CI 0.22–1.23)].PFS for all patients was 4.33 months  were counterbalanced by a remarkable incidence of adverse events. In particular, 50% of the patients experienced grade III-IV neutropenia, 13.1% grade III-IV anemia, 16.1% grade III-IV constipation, 19.2% grade III-IV fatigue and 51.2% thrombocytopenia. The toxicity of Vinflunine limited its use, in addition to the restricted advantage on survival.2 every 3 weeks after the failure of a previous platinum + Gemcitabine combination.There is a lack of alternative drugs in this setting. In our study, we tested if the reduction of the dose associated with the use of prophylactic G-CSF and laxatives could allow effective results compared to a higher dose with a better toxicity profile. Indeed, we analyzed patients treated with the dose of 280 mg/mOverall survival observed in our study was 8.5 months, PFS 4.3 months, RR 25%, disease control rate 57.2%. These results are similar to those reported in the phase III trial and in other real life studies.The remarkable duration of response (10.6 months) was obtained through the maintenance of dose intensity, to which the lower incidence of adverse events has contributed. The retrospective nature of the study could have influenced this result. The selection of patients with a high probability of prolonged response could be very useful in clinical practice. Unfortunately, in our study the identification of clinical predictive factors was not possible because of the limited number of patients.2, G-CSF as primary prophylaxis for neutropenia and laxative agents allow the reduction of the most common Vinflunine-related adverse events. As a matter of fact, grade III-IV neutropenia was 10.7%, instead of the rate of 50% reported in the phase III trial. Grade III-IV anemia and all grade thrombocytopenia occurred in 10.7% and 17.9%, respectively, compared to 19.1% and 51.2% reported by Bellmunt [The dose of 280 mg/mConcerning non haematological toxicity, in our population only grade I-II constipation and fatigue were observed  whereas Bellmunt  describeThese data confirm a good tolerability of this drug and show a milder toxicity with the dose tested in our study. Prospective clinical trials should be helpful to confirm our observations. They could also represent a source for further pharmacokinetics information and for clinical and pathological predictive factors.2 is effective and has a favorable toxicity profile. Vinflunine as administered in our study could be considered for the second line treatment of advanced TCCU also in the next future, when immunological checkpoint inhibitors [Despite being retrospective and including a limited number of patients, this study underlines that Vinflunine at a dose of 280 mg/mhibitors  might be2 intravenously every 3 weeks for the entire population. Emollient laxatives were used for primary prophylaxis of constipation. Paraffin oil was prescribed fasting at the dose of 15 ml twice a day for 7 days after chemotherapy administration. The timing of administration was chosen based on the nadir of constipation, that occurs 3–5 days after Vinflunine administration. Granocyte-colony stimulating factors (G-CSF) were given as primary prophylaxis of neutropenia: Lenograstim 34 MU was administered for 6 days starting 24 hours after Vinflunine. We collected all demographic data related to patients: gender, age, previous treatments (in neoadjuvant/adjuvant or advanced setting), site of recurrence.Patients with advanced metastatic TCCU, PS (ECOG) 0–1, relapsed within 12 months after neoadjuvant/adjuvant platinum-based chemotherapy or progressed after a first line platinum-based treatment were included in this retrospective analysis. The dose of Vinflunine administered was 280 mg/mThe primary outcomes were overall survival (OS) and toxicity; other outcomes were progression-free survival (PFS) and response rate (RR).p-values were considered significant at the 5% level. Responses were evaluated trough RECIST criteria version 1.1.OS was calculated from the first day of cycle 1 to death and PFS from the first day of cycle 1 to progression or death. The Kaplan and Meier method was used; the comparison of survival between patients’ subgroups was obtained by the log-rank test. A Cox-proportional hazard model with 95% confidence interval (CI) was used to calculate the hazard ratios. All Toxicity was reported using Common Terminology Criteria for Adverse Events (CTCAE) version 4. Data on tolerability were collected considering all the adverse events occurred throughout the treatment period. Clinical evaluations were performed every 3 weeks before Vinflunine administration. Visits were also performed monthly after the end of Vinflunine.Our study was approved by local Ethic Committees and conducted according to the Helsinki Declaration. All the patients released a written informed consent.


Although sickle cell disease (SCD) children are highly susceptible to bacterial infections (BIs), there is a dreadful lack of data related to the burden and spectrum of BIs in sub-Saharan Africa (SSA), the highest affected region with SCD. This study aimed to determine the burden and spectrum of BIs among SCD children hospitalized in a pediatric reference hospital in Cameroon, a SSA country.We conducted a retrospective analysis of records of children hospitalized from November 2012 to August 2015 in the SCD unit of the Mother and Child Centre of the Chantal Biya Foundation, Cameroon. We enrolled all known SCD children aged 15 years or less, hospitalized for a suspicion of BI and who presented a positive culture of a body specimen.Haemophilus influenzae type b antigens; 58 children (84.1%) had received no vaccine. The specimen yielding positive cultures were: blood (70.7%), urine (13.1%), pus (9.1%), synovial fluid (4.1%), cerebrospinal fluid (2.0%), and bone fragment (1.0%). The different types of infection included: urinary tract infections (13.5%), myositis (8.3%), arthritis (6.3%), osteomyelitis (4.2%), and meningitis (2.1%); the site of infection was unidentified in 65.6% of cases. The main bacteria included: Salmonella sp. (28.1%), Staphylococcus sp. (18.8%), Klebsiella pneumoniae (17.7%), Escherichia coli (10.4%), Enterobacter sp. (5.2%), Acinetobacter sp. (4.2%), Streptococcus sp. (4.2%) and Serratia sp. (4.2%).A total of 987 SCD children were hospitalized during the study period. Cultures were positive for 96 patients (9.7%) among whom 60.4% males. Ages ranged from 6 to 192 months with a median of 53 (Interquartile range (IQR) 21–101) months. For children no more covered by the Expanded Programme on Immunization, only 13 (18.8%) had received the Pneumo 23® and Meningo A&C® antigens, and 12 (17.4%), the Typhim vi® and the Salmonella sp. (28.1%), Staphylococcus sp. (18.8%), Klebsiella pneumoniae (17.7%), and Escherichia coli (10.4%).This retrospective analysis revealed 9.7% cases of BIs, mainly caused by  Sickle Cell disease (SCD) also referred to as Sickle Cell Anemia, is the most widespread and severe monogenetic disorder in the world, specifically in Africa south of the Sahara where it constitutes a real public health issue with 1–4% of babies born affected . In CameSCD is particularly disastrous, mainly due to its acute and chronic complications, including painful vaso-occlusive events, cerebral vasculopathy, priapism, chronic kidney disease, acute chest syndrome, pulmonary hypertension and bacterial infections (BIs) among others –7. IndeeStreptococcus pneumoniae, non-typhi Salmonella sp., Haemophilus influenzae type b, Escherichia coli, Acinetobacter sp. and Klebsiella sp. [Streptococcus pneumoniae, non-typhi Salmonella sp. and Haemophilus influenzae type b [The organisms most commonly isolated from children with SCD are: ella sp. , 11. Evie type b . Moreovee type b , 14.While sub-Saharan Africa (SSA) is the highest affected region with SCD, there is a paradoxical dearth of data regarding the spectrum of BIs among SCD patients to guide the proper and efficient management of these threats across the region, especially in Cameroon, a SSA country. Willing to fill this critical gap, the present study was undertaken, the aim of which was to assess the burden and spectrum of BIs which have occurred among SCD children hospitalized in a pediatric reference hospital in Cameroon.We conducted a retrospective analysis of records of children hospitalized in the SCD unit of the Mother and Child Centre of the Chantal Biya Foundation from November 2012 to August 2015. This Centre, which has been well described elsewhere , is a peDuring the study period, we consecutively and exhaustively included all known SCD children aged 15 years or less, who have been hospitalized for a suspicion of BI and presented a positive result after culture of a body specimen . Samples were collected in the unit and transported without delay to the Centre Pasteur of Cameroon where they were analyzed. Bacterial isolations were conducted using standard culture milieus such as Cystine Lactose Electrolyte Deficient, Chapman, Mac Conkey, chocolate (agar cooked/fresh blood), or polyvitex chocolate, depending on the type of specimen to be analyzed. Identification of the bacteria used different specific antibiotic galleries. Poly-microbial cultures were not considered in the present work, neither were contaminations .Haemophilus influenzae type b and Typhim vi® antigens), clinical signs and symptoms at presentation , site of infection and type of sampling performed, isolated bacterium, treatment and outcome in the course of hospitalization.We used a preconceived and standardized questionnaire for data abstraction. Data were collected on age, sex, vaccination status after the Expanded Programme on Immunization (EPI) . Results are presented as frequency (percentage) for qualitative variables, and median (interquartile range (IQR)) for quantitative ones. Comparison between qualitative variables used the Chi-2 test, and results were considered statistically significant when Before commencing this study, authorizations were obtained from administrative authorities of the Mother and Child Centre, and an ethical clearance was granted by its Ethical Review Board. All the procedures used in the present study were in keeping with the current revision of the Helsinki Declaration. As the study was retrospective, patients’ consents could not be obtained. However, the study was approved by the association of parents of SCD children of the Mother and Child Centre which includes the majority of parents of these diseased children formerly followed-up or hospitalized in the SCD unit.Haemophilus influenzae type b antigens; 58 children (84.1%) had received no vaccine . Patients’ ages ranged from 6 to 192 months, with a median of 53 (IQR 21–101) months. There were 58 males (60.4%), hence a M/F sex ratio of 1.53/1. Twenty seven children (28.1%) were aged less than 24 months (Table p = 0.779), and between the under-5 and 5+ years old (p = 0.291) with regard to the type of infection.The specimen yielding positive cultures were: blood (70.7%), urine 13.1%), pus (9.1%), synovial fluid (4.1%), cerebrospinal fluid (2.0%), and bone fragment (1.0%). The different types of infection were: urinary tract infections (13.5%), myositis (8.3%), arthritis (6.3%), osteomyelitis (4.2%), and meningitis (2.1%). Besides, the site of infection was unidentified  in 63 patients , and the under-5 and 5+ years (p = 0.221). Table Salmonella sp. (28.1%), Staphylococcus sp. (18.8%), Klebsiella pneumoniae (17.7%), Escherichia coli (10.4%), Enterobacter sp. (5.2%), Acinetobacter sp. (4.2%), Streptococcus sp. (4.2%), and Serratia sp. (4.2%). The majority of cases of septicemias were caused by Staphylococcus sp. (24.6%), Salmonella sp. (24.6%) and Klebsiella pneumoniae (16.9%). Escherichia coli was responsible for the majority of cases of urinary tract infections (53.8%) and Salmonella sp., for all cases of myositis, The two cases of meningitis were caused by Streptococcus pneumoniae , with no difference between males and females . The outcome was favorable for 82 patients (85.4%), but worse for the other ones among whom 4 patients ending up with osteo-articular sequelae (4.2%); 7 patients (7.3%) were transferred to undergo surgery (for osteomyelitis or myositis), and 3 patients (3.1%) died. Duration of hospitalization in the SCD unit varied between 1 and 21 days with a median of 9 (IQR 8–12) days.Bacterial infections (BIs) are a major cause of morbidity and mortality among SCD children and adults , but theIn Cameroon indeed, newborn hemoglobinopathy testing is neither available nor implemented universally compared to developed countries. Only parents of SCD children who are being followed in specialized centers will be educated and counselled on infection prophylaxis with oral penicillin (to be paid by the parent), and to bring their children to the hospital when the temperature rises. Since 2011, the Prevenar 13® vaccine was introduced in the Cameroon Expanded Programme on Immunization, which is administered free of charge to all children at 6, 10 and 14 weeks of age. Additionally, a 23-valent pneumococcal vaccine as well as some others  are proposed at 24 months to SCD children who are followed-up, but it is not given free of charge; hence only parents who can afford will vaccinate their kids.This explains perhaps why we observed in this study, a low vaccination coverage after the EPI, around 15.9%. Similarly, previous African reports have bolstered a low vaccination coverage after the EPI in the young pediatric population, around 6.2–32.5% in the Gabonese public sector with pneumococcal antigen coverage equaling 0.8%, and around 10.8–13% in Cameroon , 18. It Streptococcus pneumoniae especially [S. pneumoniae infections were recorded; these two children who presented meningitis had neither received the Prevenar 13® nor the Pneumo 23® antigens.Most of bacteria (71.4%) were identified through blood culture, blood being easily accessible to be sampled. Besides, a great number of infections including localized ones, have a bacteremic phase during which the bacterium can be picked up through culture of blood , 16. Thepecially . Our obsSalmonella sp. (28.1%), corroborating previous reports [Klebsiella pneumoniae (17.7%), not often found in these proportions according to previous reports [K. pneumoniae was the second bacterium responsible for urinary tract infections (30.8%), in line with results from Asinobi et al. (18.9%) and Mava et al. (24.6%) [Escherichia coli was responsible for 10.4% of infections, mainly urinary tract infections (53.8%) still concurring with the literature [The bacterial ecology was dominated by  reports , 16, 19.lla sp. 2.1%, corr reports , 15, 16. reports , 15, 16. (24.6%) , 20. Escterature , 13, 20.Acinetobacter sp. was the fourth bacterium reported by Williams et al., found in 7% of cases [Staphylococcus sp. was responsible for 18.8% of BIs, corroborating previous studies [H. influenzae infections in the present report may be explained by the introduction (in 2008) of three doses of the Haemophilus influenzae type b antigen in the Cameroon EPI, notably at 6, 10 and 14 weeks of age, and perhaps abusive use of antibiotics as self-medication.of cases , hence a studies , 21, 22.Streptococcus pneumoniae, and a 5 years old male child at 48 h from a severe sepsis caused by a non-typhi Salmonella sp. The third case was a female child of 7 years who was suffering from a severe sepsis with multifocal sites caused by a non-hospital acquired multi-resistant Enterobacter cloacae; she was transferred to the emergency care unit where she died after 3 months of hospitalization.We recorded 5 cases of infectious recurrence reflecting the high susceptibility of SCD patients to BIs, driven by a functional asplenia, a default in complement activation, micronutrient deficiencies, and mechanical and genetic factors . Three pUnfortunately, this study presents a number of flaws. First, the retrospective analysis of this work precluded us from obtaining all the information necessary, and having a control group to investigate some factors driving the occurrence of BIs in our milieu. Second, parents’ financial constraints and lack of funding impeded the realization of more bacteriological explorations which could have permitted to depict and confirm more cases of BI; for the same reasons, only one blood culture (instead of at least three per patient) was performed for most patients, hence diminishing considerably our probability of catching up the causal agent. Moreover, a free access to non-prescribed antibiotics in our context may have constituted a source of bias towards our results. Third, our analyses are based only on known SCD children, in a context where newborns are not systematically screened for SCD; this could have led to an underestimation of the real burden of BIs. Fourth, SCD children were recruited from only one hospital, perhaps hindering the generalization of results obtained. Nonetheless, the study was conducted in a reference hospital with patients coming from all over the country and the Central African sub-region. Besides, the study period of almost 3 years permitted to record a great number of cases.Salmonella sp. (28.1%), Staphylococcus sp. (18.8%), Klebsiella pneumoniae (17.7%), Escherichia coli (10.4%), Enterobacter sp. (5.2%), Streptococcus sp. (4.2%), Acinetobacter sp. (4.2%), and Serratia sp. (4.2%). Substantial efforts to vaccinate SCD children after 12 months of age (when they leave the EPI) and increase the vaccination coverage in this group of vulnerable children could help to reduce the burden of BIs in SCD children. Patient’s parents should be continuously sensitized and educated on the importance of vaccinating their kids, observe the penicillin antibio-prophylaxis and bring their kids to the hospital as soon as the temperature rises. Our clinicians must be adequately trained to manage these infections, bearing in mind that broad spectrum antibiotherapy should be started without waiting for bacteriological results. On the other hand, we believe it is high time our Governments think of subsidizing the management of SCD children for those parents who cannot pay. Cost-benefit studies are warranted in this regard.This 3-years retrospective analysis permitted to record 96 cases (9.7%) of bacterial infections among SCD children living in Cameroon. The bacterial ecology was mainly represented by


To evaluate the diagnostic and prognostic value of the immunohistochemical expression of WT1, p53 and p16 in low- (LGSOCs) and high-grade serous ovarian carcinomas (HGSOCs).p = 0.32). Focal and/or complete absence of p53 expression with negative p16 expression was found in 90.5% of LGSOCs, in contrast to the 88.1% of HGSOCs with diffuse or complete absence of p53 expression with positive p16 expression (<0.001). The IHC p53/p16 index and the morphological classification were closely matched (k = 0.68). In the univariate analysis, FIGO stage, post-surgery residual disease and histological grade were significantly associated with progression-free survival (PFS) and overall survival (OS). The IHC p53/p16 index was associated only with PFS. WT1 was not associated with PFS or OS. According to the multivariate analysis, advanced FIGO stage and presence of post-surgery residual disease remained independent prognostic factors for worst PFS, however these features had only a trend association with OS.HGSOC had a significantly higher proportion of advanced stage disease, higher CA125 levels, higher proportion of post-surgery residual disease and higher recurrence or disease progression. WT1 was expressed in 71.4% of LGSOCs and in 57.1% of HGSOCs  criteria. Immunohistochemistry (IHC) was performed in tissue microarray slides. IHC p53/p16 index was compared with the morphological classification.The IHC p53/p16 index was a good marker for the differentiation of LGSOC and HGSOC, but the morphologic classification showed a better association with survival. FIGO stage and post-surgery residual disease remained the only independent prognostic factors for survival. High-grade serous ovarian carcinomas (HGSOCs) comprise the majority of serous ovarian carcinomas (SOCs), as they account for 80%–90% of these tumours and are the most aggressive –3. AdvanWT1) gene, located on chromosome 11p13, was first identified as the gene responsible for the development of a childhood malignancy [WT1 expression has also been demonstrated in various adult cancers [The Wilms Tumor 1  is suggestive of the absence of mutations in HGSOC [TP53 mutations are almost never present in these tumours [A damage . Mutatioin HGSOC , 15, 16. tumours .CDKN2A tumour suppressor gene. The p16 protein performs an important role in cell cycle regulation by decelerating cell progression from G1 to S phase [p16 is a protein encoded by the  S phase . Approxi S phase , 20 for  S phase , 21.et al. [Recently, Köbel et al.  proposedIn the present study, we aimed to examine the diagnostic value of these markers, because interobserver variability occurs in clinical practice, in the distinction between LGSOC and HGSOC . We wereet al. [For this retrospective cohort study, we retrieved consecutive formalin-fixed paraffin-embedded (FFPE) tissue samples and the accompanying clinical files of 138 women who were diagnosed and treated at the Women’s Hospital of Campinas State University, Campinas, Brazil, from 1994 to 2013 and who were followed-up until 2016. The local institutional ethics committee (CEP 1086/2009) approved this study. All pathological specimens collected during primary surgery or before neoadjuvant chemotherapy were analysed by an expert gynaecological pathologist (L.A.) according to the guidelines of the World Health Organization (WHO) International Classification of Ovarian Tumours . Stage wet al. .Slides from the original paraffin blocks were stained with haematoxylin and eosin (H&E) and were reviewed so that representative areas of the tumour could be identified. Tissue microarray blocks  were constructed using two samples from each case. Sections were obtained from each TMA and were placed on electrically charged slides for all IHC procedures.® histology V-Kit, clone E6H4, Roche mtm laboratories AG, Germany). The peroxidase-labelled polymer ADVANCE™ HRP Detection System (Dako) was applied for 30 minutes at room temperature. DAB chromogen substrate  was applied, at a proportion of 0.06 g to 100 mL of PBS, 500 μL hydrogen 3% peroxide and 1 mL dimethyl sulfoxide at 37° C for 5 minutes. The slides were subsequently washed in water, counterstained in haematoxylin, dehydrated and mounted. Tissue samples with adequate immunoreactivity were used as positive controls for each antibody. Negative controls were produced by omission of the primary antibodies.After initial deparaffinization, endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide. The sections were then microwaved in 10 mM citrate buffer (pH 6.0) or Tris-EDTA to unmask the epitopes. The slides were incubated with the following primary antibodies according to optimized protocols: monoclonal mouse anti-human Wilms Tumor 1 (WT1) protein , monoclonal mouse anti-human p53 protein , and monoclonal mouse anti-p16  with expertise in ovarian cancer, who was blinded to the clinical and pathological data, scored the samples. Two TMA sets of each tumour component were used for each marker, i.e., each tumour area was assessed twice. The reactions were evaluated according to the percentage of positive cells. In a Nuclear WT1 protein expression was analyzed in each case, and the percentages of cells with nuclear staining were estimated independently of intensity. Cases with ≥1% positive tumour nuclei were considered positive, and those with zero or less than 1% were considered negative . Nuclearet al [We also performed a combined IHC classification proposed by Köbel et al ., the IHp ≤ 0.10 in the univariate Cox analysis were included in the multivariate Cox analysis. Differences were significant when p < 0.05.Differences between groups were analysed using the Chi-square test or Fisher’s exact test. Continuous data were analysed using the Mann-Whitney test. Weighted Kappa was calculated to evaluate the IHC p53/p16 index with the morphological classification of the tumors. Progression-free survival (PFS) was measured from the time of diagnosis until relapse, progressive disease, or last follow-up, and overall survival (OS) from the time of diagnosis until any cause of death or last follow-up. PFS and OS probabilities were estimated by the Kaplan-Meier method, and curves were compared by the log-rank test. The Cox hazards model was used to identify variables that predicted PFS and OS. Variables for which p < 0.001), presented with higher CA125 levels , had a higher rate of post-surgery residual disease  and had higher recurrence/progression rates  compared with women with LGSOC. No association was observed between LGSOC and HGSOC and age, menopausal status or response to platinum-based chemotherapy  and 21 (19.8%) LGSOCs. Women with HGSOC accounted for a significantly higher proportion of advanced stage disease . p53 expression was diffuse in 68.2% of cases, was completely absent in 30.6%  and was focal in 1.2% of HGSOCs, compared with LGSOCs, which demonstrated diffuse expression in 9.5%, complete absence in 81.0% and focal expression in 9.5% (p < 0.0001). p16 was expressed in 58.5% of HGSOC samples compared with 9.5% of LGSOC samples (p < 0.001). Table p < 0.0001). Only two samples that were morphologically classified as LGSOC (9.5%) were also classified as HGSOC by the IHC p53/p16 index. On the contrary, 11.9% of HGSOCs that were classified as such on the basis of morphology, were reclassified as LGSOCs by the IHC p53/p16 index. When the IHC p53/p16 index and morphologic classification were compared, the kappa Cohen coefficient was moderate (k = 0.68). A higher expression of p16 was seen in FIGO stage III and IV disease compared with FIGO stage I and II disease, and no difference was observed in p53 and WT1 expression according to stage (data not shown).WT1 was expressed in 71.4% of LGSOCs and in 57.1% of HGSOCs, and no significant difference was found in the expression of WT1 in these two tumour types p = 0.3. p53 expFor the entire cohort, the median follow-up duration was 56 months (range: 1–213 months). At 60 months of follow-up, the PFS and OS were 37.1% and 50.9%, respectively. The PFS and OS were 73.0% and 83.1%, respectively, in women with LGSOC and 29.1% and 43.8%, respectively, in women with HGSOC (data not shown).p = 0.06) and post-surgery residual disease  with OS in a multivariate model. The IHC p53/p16 index was not an independent prognostic factor for either PFS or OS  were significantly associated with worse PFS and OS. The IHC p53/p16 index was associated with worse PFS  but only marginally with OS   [In our study, p53 expression was diffuse in 68.2% and was completely absent (null type) in 30.6% of women with HGSOCs . Our results are similar to those of a previous study, which considered p53 expression by IHC ( HGSOCs) , 34. p16 HGSOCs) .et al. [Recently, Köbel et al.  concludeet al. . In our TP53 alterations are associated with high rates of tumour cell proliferation, but the association between p53 expression and patient prognosis remains controversial [oversial . In malioversial . In our n = 61) and 2004 to 2013 (n = 45). No difference was observed in terms of the clinical features or survival (data not shown). Finally, our ratio of LGSOC to HGSOC is higher than that reported in the literature. This might be expected to occur once the majority of HGSOC cases present with advanced FIGO stages and are treated with neoadjuvant chemotherapy; these cases would thus be excluded from the study sample.Some important limitations of our research should be highlighted. First, our IHC evaluation was performed in TMA samples, and it is known that large ovarian cancers can show regional variability in the expression of protein markers. Indeed, IHC analyses were performed after a long period of time which means that the quality of the paraffin-embedded tissue might have been compromised. Second, the same cases that were included in our study were diagnosed almost 20 years ago. Herein, we divided our casuistry based on the diagnostic data: 1994 to 2003 (The IHC p53/p16 index and the morphological classification are closely matched, and the IHC p53/p16 index seems to be a good marker for the differentiation of LGSOC and HGSOC. However, the WHO morphologic classification showed a better association with survival. FIGO stage and post-surgery residual disease remained an independent prognostic factors for PFS but only a trend association with OS. However, the IHC p53/p16 index was not an independent prognostic factor for either PFS or OS.In the evaluation of a new case of SOC, the initial assessment should be performed according to WHO morphologic criteria. In doubtful cases, IHC should be performed to determine the IHC p53/p16 index. Thus, p53 and p16 expression have an important role in the routine differential diagnosis of ovarian carcinoma.


To determine the prevalence of anemia and its association with disease severity in children and adolescents with IBD.  CEDATA-GPGE is a registry for pediatric patients with IBD in Germany and Austria from 90 specialized centers. As markers of disease severity, analysis included patient self-assessment on a Likert scale  and physicians' general assessment (0 = no activity to 4 = severe disease) and the disease indices. Anemia was defined as hemoglobin concentration below the 3rd percentile. p < 0.0001; UC: 2.9 ± 0.9 versus 2.3 ± 0.9, p < 0.0001). Accordingly, physicians' general assessment (PGA) was significantly worse in anemic than in nonanemic patients in CD (p < 0.0001) and UC (p < 0.0001). PCDAI in anemic CD, p < 0.0001, and PUCAI in anemic UC patients, p < 0.0001, were significantly higher than in nonanemic patients. 40.0% of anemic CD and 47.8% of anemic UC patients received iron during follow-up.  Prevalence of anemia was 65.2% in CD and 60.2% in UC. Anemic CD and UC patients showed significantly worse self-assessment than patients without anemia (average ± standard deviation; CD: 3.0 ± 0.9 versus 2.5 ± 0.9,  Almost 2/3 of pediatric IBD patients are anemic. Patients' self-assessment and disease severity as determined by PGA and activity indices are worse in anemic patients. Contrastingly, only a minority received iron therapy. Pediatric onset inflammatory bowel disease (IBD) is a group of chronic, relapsing disorders of the gastrointestinal tract, which affect an increasing number of children and adolescents. While incidence in adult populations seems to stabilize, pediatric onset is increasing. In recent decades, there has been a general increase in the incidence and prevalence of IBD in both industrialized and emerging countries . A shiftAnemia can occur in pediatric IBD patients for different reasons: gastrointestinal blood loss, insufficient dietary intake of iron, decreased absorption of dietary iron, and decreased activity of bone marrow because of chronic inflammation . Anemia This interferes with the aims of therapy in pediatric IBD: relief of symptoms, normal growth and pubertal development, improved quality of life, but minimization of drug toxicity . DiagnosPrevious studies on anemia in pediatric IBD included relatively small patient numbers . The aimCEDATA-GPGE is a registry, currently based at the University Children's Hospital Giessen for pediatric IBD patients (0–18 years of age) in German-speaking countries from over 90 pediatric gastroenterology centers in Germany and Austria . The datAnemia was defined as hemoglobin below the 3rd percentile for age and gender in recent population-based pediatric Hb reference values in Germany as published by the KiGGS Study . To evalPatient self-assessment was quantified on a Likert scale from 1 to 5 (1 = very good and 5 = very bad). Disease activity was measured by physicians' general assessment on a 4 point scale (1 = no activity/remission and 4 = severe activity) and by calculation of the Pediatric Crohn's Disease Activity Index (PCDAI) for patients with CD and the Pediatric Ulcerative Colitis Activity Index (PUCAI) , 19. ThePrevalence of anemia were calculated once at the time of diagnosis and subsequently in a cross section fashion in all patients after one to five years follow-up of their disease course. The registry does not hold data on ferritin or transferrin. A decrease in mean corpuscular volume (MCV) was considered as suggestive for iron deficiency anemia. Since azathioprine can increase MCV, azathioprine therapy was compared in patients with anemia and without anemia to correct for potential bias.t-test was performed when comparing continuous or pseudonormal data; otherwise, Mann–Whitney U Test was performed where appropriate. Statistical significance was assumed when p < 0.05. The database and reporting procedure were examined and approved by the relevant ethics committees and data protection employees of the three institutions that maintained the registry during the time period covered by the research  of Technische Universität Dresden; the Institute of Epidemiology at the Helmholtz Zentrum, LMU Munich; and the Department of General Pediatrics and Neonatology, JLU Giessen). The Austrian hospitals that participated also obtained local ethics approval.Analysis was performed in SAS  and R . Some of the graphics and tables were redrawn in Microsoft Excel. Distribution was assessed for normality, and 3554 patients were initially included in the study, with a total of 25,765 patient contact documentations. 109 patients were excluded because of incomplete initial data, 51 for missing date of primary diagnosis, 22 for implausible data or typing errors, and 616 patients for missing the second follow-up. 2756 patients ) were available for analysis.p = 0.013) and in IBD-U .Of 2756 children and adolescents with IBD, 1743 (63.2%) suffered from anemia at any time during the disease course—referred to standard values for hemoglobin from the KiGGS study . Anemia p < 0.0001). Similar significant differences were found in anemic versus nonanemic UC patients , p < 0.0001) and in anemic versus nonanemic IBD-U  versus median 2.0, IQR; p < 0.001 Mann–Whitney U test)  .p < 0.0001), UC , and IBD-U . In all three disease groups, anemic patients had higher disease activity with a median between low and medium activity  than nonanemic, PUCAI was significantly higher in anemic versus nonanemic UC patients .Anemic CD patients showed a significantly higher PCDAI .The Pearson correlation coefficient for disease activity indices  and hemoglobin concentration in CD patients and UC patients was moderately negative with −0.66 and −0.45 , 77.29 ± 7.54 versus 81.67+ −7.76; UC (n = 550 versus 331), 80.06 ± 7.53 versus 84.24 ± 7.76; and IBD-U (n = 90 versus 31), 77.27 fl ± 7.51 versus 84.27 fl ± 6.28, p < 0.0001 for all). 60.1% of patients on azathioprine and 65.3% without azathioprine presented with anemia ). While 38% of anemic patients on azathioprine had low MCV, the share in patients without azathioprine was 56% ). When correcting for an estimated +4 fl effect of azathioprine on MCV, this difference was not significant (57% versus 56%), so the residual share of microcytic anemia, a correlate of iron deficiency anemia, can be estimated at about 50%. The correlation between MCV and azathioprine dosage was weak to moderate depending on group selection (r = 0.14–0.30).Average MCV was significantly below normal in all patient groups , 46% of CD, 53% of UC, and 38% of IBD-U patients were anemic.p = 0.0005, OR 2.34 (95% CI 1.45–3.79)), and all other differences were not significant.In the group 6 to <10 years, 60% of CD patients, 61% UC patients, and 42% of IBD-U patients showed anemia. In the oldest age group ≥ 10 years, CD patients were anemic in 67% of the cases, UC in 61%, and IBD-U in 58%. Anemia prevalence was significantly higher in adolescents with CD in comparison to CD patients < 6 years .56.9% of CD patients from the middle age group (6 to <10 years) suffered from mild anemia, 3.6% from severe, and in UC, the percentage was 54.4% and 6.4% (IBD-U 38.4% and 3.8%). The adolescents ≥ 10 years of age had a mild anemia in 65.3% and severe in 1.8% when CD was the diagnosis, 55.7% and 5.1% when UC was the diagnosis, and 52.4% and 5.9% when IBD-U was the diagnosis, respectively.p = 0.0365), and this trend did not reach statistical significance in CD or IBD-U (43% (196) versus 38.1% (262); 46.2% (12) versus 38.1% (16)). Data on route of iron supplementation  or dosing regiments were not recorded in the registry. CD and UC patients with severe anemia received iron supplementation more often than patients with mild anemia: 41% versus 18.9% of UC patients, 22.2% versus 14.4% of CD patients and 20.4% versus 20% of IBD-U patients.Only 740 (42.5%) of 1743 anemic patients received iron therapy at the time anemia was present and documented. While in UC, a larger population of female anemic patients received iron supplementation compared to their male counterparts (52.2% (145) versus 43.1% (109), We found in our study that almost two-thirds of pediatric IBD patients were anemic at the time of diagnosis. So far, there are very few studies that aim at anemia and iron deficiency in pediatric IBD, most of them in rather small cohorts and with short follow-up , 15, 16.Previously reported data from smaller studies have shown similar prevalence of anemia with 70% in children and 42% in adolescents compared to 40% in adults , 16.At presentation, we found a relatively high percentage of anemic patients , but less than in other studies reported like the 75% observed by Wiskin et al. .When comparing age groups 0 to <6 years, 6 to <10 years, and ≥10 years, we found an increase in anemia rate at presentation with age . However, the proportion of severe anemic patients decreases . A potential explanation could be the prolonged diagnostic latency in early and very early onset IBD that has been described previously for the German population .Wiskin et al. also reported that 30% of patients with pediatric IBD are anemic two years after their initial diagnosis . In our Looking at the long-term data, in CD patients the anemia rate remains surprisingly stable at about 50%, while prevalence decreases for UC (from 53.4% at diagnosis to 37.9% after 5 years) and IBD-U (from 50% at diagnosis to 35.4% after 5 years).The impact of anemia on adult patients' quality of life has previously been described. Typical symptoms include not only headache, fatigue, and dizziness but also impairment of cognitive function, stomatitis, and esophageal webs . Our datBesides the multifactorial etiology of anemia in IBD, the vast majority of anemic IBD patients suffer from iron deficiency anemia , 21.Wiskin et al. reported prevalences of iron deficiency in 90%, respectively, 95% and 70% in CD and UC patients, respectively, 65% at follow-up after two years .Our data can only provide indirect information on the etiology of anemia from hemoglobin and MCV, since no parameters of iron metabolism like ferritin, transferrin saturation, or soluble transferrin receptor were obtained by the registry.Decreased MCV as a surrogate marker for iron deficiency anemia has to be interpreted with caution since medication used in pediatric IBD can significantly change MCV. Several studies have suggested a correlation with 6-thioguaninnucleotide (6-TGN) levels, and some even suggested MCV as a surrogate marker instead of measuring metabolites for therapeutic drug monitoring .From our data, correction for azathioprine exposure was difficult to estimate, since metabolism differs significantly between individuals even receiving the same dose of azathioprine or 6-MP  and metaIn our patient cohort, we confirmed a significantly higher MCV in the patient groups with azathioprine compared to patients without, but the difference was less pronounced . When corrected for this difference, the residual share of microcytic anemia, therefore estimated share of iron deficiency anemia was around 50%.Only about 1/3 to half of anemic patients received iron supplementation . This isPels et al. showed that expectant management of anemia in pediatric IBD patient leads to a slower hematologic recovery . PersistTreatment with iron supplementation seems mandatory for patients with proven iron deficiency anemia. For physicians from our registry considering all limitations, an undertreatment of highly prevalent microcytic anemia, potential iron deficient anemia has to be suspected. Possible causes are suspected side effects of iron supplementation orally or parenterally or a suspected cause for anemia in chronic inflammation. A possible explanation could be seen in the high number of patients with relatively mild anemia (less than 2 g/dl Hb below 3rd percentile), but even when looking at severe anemic patients alone, the proportion of patients that receive iron supplementation only increases slightly to 41% in UC, 22.2% in CD, and 20.4% in IBD-U.The strength of this study is the large number of children and adolescents and the number of follow-up data from our pediatric IBD registry since well-powered studies and registry analysis are scarce in pediatric patients. Limitations are the collection method and study design, which is not tailored to reflect individual courses of disease but rather group characteristics with a cross-sectional instead of longitudinal approach, corresponding to the initial question. To answer questions about treatment and individual patients' benefit, a longitudinal approach has to be taken and is on the way in a future analysis.There is a high prevalence of anemia in pediatric patients with IBD. Even more patients with CD are anemic than patients with UC. The rate is highest in adolescents at the onset of disease, and in young children, the rate of patients with severe anemia is higher. Anemia is associated with impairment in patient self-assessment of general well-being, physician assessment of disease activity, and higher disease activity as determined by activity indices. During the disease course, the prevalence of anemia remains high. In contrast, only one-third of patients are treated by iron supplementation. This undertreatment should be addressed by pediatric gastroenterologists.


Adults of the peri-urban Peruvian shantytown of Lomas de Zapallal have a high prevalence of risk factors for developing cardiovascular disease (CVD)—likely due to behavioral choices established during childhood and adolescence. To guide the development of community-based risk reduction programs, we assessed the prevalence of risk factors for developing CVD among adolescents.We collected cross sectional data from adolescents of Peruvian peri-urban shantytown to evaluate four domains of CVD risk factors: (1) clinical , (2) anthropometric , (3) behavioral , and (4) psychosocial .p = 0.002). Total cholesterol was elevated (>170 mg/dL) in nearly half (45.5%) of the adolescents, and 71% had impaired fasting blood glucose (>100 mg/dL). Females were less likely to exercise daily (95.4%) than males (84.2%) (p = 0.002) but reported higher rates of depression (66.4%), anhedonia (67.6%), and self-harm behavior (37.9%) .We enrolled 275 adolescents . Prevalence of overweight or obese status was 27.8%. High blood pressure was more common in males (37.4%) than females (20.5%) (Adolescents living in the peri-urban population of Puente Piedra had high prevalence of risk factors for future development of CVD; preventative efforts focused on improving nutrition, increasing physical inactivity, and addressing mental health conditions could reduce such risk factors. Cardiovascular disease (CVD) is a major cause of mortality and morbidity throughout the world , 2. ObesEarly detection of risk factors may offer opportunities for lifestyle change to reduce morbidity and mortality . AdolescFew studies have examined risk factors for developing CVD in shantytown populations, and even fewer have examined adolescents living in low-income settings –24. To gpueblo joven (“shantytown”) community in the Northern Lima district of Puente Piedra, Peru. The majority of houses in the area are made of cement with corrugated metal roofs. A sanitation system was installed in 2009 that provides inconsistent portable water to 90% of inhabitants [Lomas de Zapallal is a small abitants , 25. ManA cross-sectional study was conducted between November 2015 and April 2016 to evaluate the prevalence of risk factors for CVD: anthropometric and clinical , as well as behavioral and psychosocial . Approval was obtained from the Institutional Review Board of the Universidad Nacional Mayor de San Marcos. Exempt status was granted from the Institutional Review Board of the University of Washington as the present study did not meet their definition of research.All adolescents in second, third, and fourth grade of secondary school at Colegio Pitágoras 8183 were invited to participate. Student grade level was classified by their 2015 status (first to fourth grade). Only students with parental consent and who provided voluntary informed assent were enrolled.A team of five trained Peruvian health students and professionals obtained anthropometric and clinical data. Data was collected at four stations: (1) weight and height to calculate BMI and overweight/obesity status, (2) waist circumference (WC), (3) blood pressure (BP), and (4) capillary blood draw. Students were instructed to remove their shoes, hair buns, and jackets prior to measuring weight and height. Two nurses drew capillary blood for three point-of-care (POC) assays: total cholesterol , fasting glucose , and hemoglobin .BP was measured using an automated sphygmomanometer  with an appropriately sized cuff on the participant’s left upper arm, per guidelines and study protocol . If readData were collected and managed using REDCap, a secure web-based electronic data capture tool, hosted at the University of Washington  and analAll adolescents attending Colegio Pitágoras 8183 in second, third, or fourth grade of secondary school during the 2015 or 2016 school years were invited to participate. Of the 275 participants enrolled, one male participant did not complete the behavioral questionnaire and nine participants  chose not to participate in the clinical data collection.Of the 266 participants who completed the clinical data collection, 252 were given a numeric score for total cholesterol. Glucose samples were attained for all 266 participants; however, only 183  reported being in “fasting state.” Analysis of glucose data excluded participants who denied a fasting state. The process of enrollment and data collection are described in Fig. The majority (56.4%) of the study population was female, and the median age of participants was 14 years of age range 12−18 years). Seventy-four (26.9%) participants were in first grade, 26.5% in second grade, 22.6% in third grade, and 24% in fourth grade of secondary school in the 2015 school year. Average household size was six persons, and 38.2% of participants reported having visited a health clinic in the last year. Although the majority of participants denied knowledge of a family history (FH) of medical conditions, 16.9% reported a FH of diabetes, 13.6% reported hypertension, 23.2% reported high cholesterol, and 9.9% reported heart disease . The range of abdominal circumference was 61–109 cm (SD 9) for males, and 57–98 cm (SD 7) for females. Based on this circumference, 15.7% of males and 15.2% of females were at high risk of developing CVD, and 10.4% of males and 8.6% of females were at very high risk [p = 0.67). Abnormal systolic or diastolic BP were detected more frequently in males than females; 37.4% of males were categorized as either pre-hypertensive (18.3%) or hypertensive (19.1%) and 20.5% of females as pre-hypertensive (11.9%) or hypertensive (8.6%) (p = 0.002). According to the capillary puncture data, 23.7% males and 32.5% of females had hemoglobin within mild  or moderate  anemia (p = 0.12). The lowest hemoglobin reading was 8.5 g/dL in a female participant. Nearly half of adolescents of both genders (45.5%) had an abnormal total cholesterol  with a range of 151–257 mg/dL. A majority (71.0%) of adolescents presented impaired FBG levels ; 5.5% of whom had a FBG >126 mg/dL .Over one-quarter (27.8%) of adolescents were overweight or obese with no gender differences . Adolescents also reported high levels of sedentary behavior, with a daily average of 3.4 h of television (SD 3.6), 3.9 h of cellular phone use (SD 5.5) and 2.1 h of internet (SD 4.1), with an average daily screen time of 9.4 h. Daily consumption of vegetables was less frequent (32.1%) than fruits (42.3%) and 11.4% of adolescents reported “often or always” adding salt to their food.The most prevalent behavioral risk factor was inadequate physical activity (90.6%); more females (95.4%) denied engaging in intense daily physical activity than males (84.2%) (Alcohol use was reported more frequently than tobacco use (49.4 vs. 16.3%) or marijuana use (4.4%). Of the 125 participants who reported having tried alcohol, the majority noted drinking with friends (39.8%) or family (30.9%); 69.9% endorsed drinking once annually; however, 18.6% reported drinking more than three times per month. The mean age for first-time alcohol experimentation was 11.9 years (SD 2.82) vs. 11.7 years (SD 2.53) for tobacco experimentation. Forty-two adolescents (17.1%) reported living or having lived with a family member who abused alcohol, and 14.2% reported having a family member who used tobacco. No major differences were noted by sex.p < 0.001). Similarly, anhedonia was reported in 67.6% of females and 50% of males (p = 0.006). Females were more than twice as likely to report past or present thoughts of self-harm than males (37.9 vs. 15.7%); p < 0.001) and were nearly three times as likely to report a family history of mental illness as compared to their male counterparts . Females also reported more frequent physical abuse at home  but less at school . Six females and one male adolescent endorsed a history of sexual abuse.Depression within the last 2 weeks was prevalent in both genders (55.3%) with higher prevalence among females than males  and nearly twice as likely to have impaired fasting blood glucose (FBG) . However, males were significantly less likely to be physically inactive , be depressed , or conduct self-harm behavior . The relationships between clinical and behavioral risk factor and sex are described in Table In unadjusted logistic regression models, males were more than twice as likely to have hypertension  to have high BP or an impaired FBG . High BP was also more often found among male adolescents  and in those with elevated cholesterol .The interrelationship between major CVD outcomes  with related risk factor exposures is described in Table Adolescence is a formative stage of development when poor health choices are developed and can form into lifelong habits . Our finThe observed prevalence of risk factors was higher than expected among our adolescent Peruvian population and varied by gender. Males and females of Lomas de Zapallal were comparably overweight or obese to adolescents living in developed countries but much more overweight than adolescents living in developing countries . In addiAs exemplified by our data, obesity was a key independent risk factor for future development of co-morbid hypertension, metabolic , body image dissatisfaction , and othOur biologic measures were imperfect. For instance, for the fasting blood glucose, some participants exerted themselves prior to participation and 31% did not fast prior to blood collection. A capillary puncture was used instead of a venous draw to collect cholesterol, glucose, and hemoglobin data. However, each point-of-care device was chosen for the high correlation with laboratory-based diagnostic assays , 45 and Due to the unique socio-environmental factors of Lomas de Zapallal, the current data limit the generalizability of CVD risk factors to other Peruvian adolescents. It does, however, serve as an example of the increased risk for other youth living in peri-urban slum populations in Lima, Peru, as well as other impoverished areas of the world. Expansion of the study to include additional Peruvian secondary schools with varied socio-economic status could provide a more comprehensive picture of the CVD risk factors of adolescents living in South America.Our study provides a comprehensive snapshot of the CVD risk factor profile among adolescents living in a northern Lima shantytown population. When compared to other local and global adolescent populations, adolescents living in Lomas de Zapallal had comparable or higher prevalence of risk factors associated with development of CVD , 46–49.


There were no statistically significant differences of bond strength decrease after one month water aging among the tested groups (p > 0.05). Microbial-aging method yield more drop of bond strength than water aging in all groups except 5% DMADDM (p < 0.05). Meanwhile, 5% DMADDM had the same matrix metalloproteinases (MMPs) inhibitory effects as the other two agents (p > 0.05), but much stronger antibacterial capability than 0.2% CHX (p < 0.05). This indicated that a cavity disinfectant with 5% DMADDM is promising for improving the stability of resin–dentin bonds in appearance of saliva biofilm; and the saliva microbial-aging method is more promising for studying the durability of resin–dentin bonds than water aging.This study was set to assess the possible benefits of novel cavity disinfectants with 5% dimethylaminododecyl methacrylate (DMADDM); and compare the effectiveness of saliva microbial-aging method with water-aging in measuring the changing of resin–dentin bond strength. Three cavity disinfectants were tested: 0.2% Chlorhexidine (CHX); 5% DMADDM; and 5% DMADDM + 0.2% CHX. Microtensile bond strength (μTBS) test was performed after microbial-aging with saliva microbial or water aging for one month. Hydroxyproline (HYP), the production of collagen degradation, was measured spectrophotometrically. Additionally, the antibacterial effects of each reagent were evaluated. The 5% DMADDM exerted the least percentage of resin–dentin bond strength loss after one month microbial-aging ( Resin composites are widely used as filling materials . HoweverThe resin–dentin bond strength will deteriorate over time due to the hydrolysis of collagen matrix of the hybrid layers ,11. DemiCavity disinfectants are used to deal with above problems. The ideal dentin disinfectant should combine the possession of a potent antimicrobial action and inhibitory effect of the MMPs.It has been demonstrated that Chlorhexidine (CHX) have broad spectrum antibacterial activity and can inhibit the catalytic activity of MMPs, so CHX is recommended used as cavity disinfectant to improve resin–dentin bond durability ,17. HoweDimethylaminododecyl methacrylate (DMADDM) is a new quaternary ammonium methacrylates (QAMs) monomer. It has excellent antibacterial and MMPs-inhibitory effects . PreviouWater aging is one of the most popular ways to study resin–dentin bond strength. This method has not taken the influence of bacteria into consideration. To deal with this drawback, the authors applied a saliva microbial-aging method: the microtensile specimen was covered by saliva biofilm, incubating in a modified artificial saliva medium with cysteine (Mcbain medium).Accordingly, the objectives of this study were to investigate the possibility of using novel cavity disinfectant containing DMADDM to improve the stability of resin–dentin bond strength, as well as compare the effectiveness of saliva microbial-aging method with water aging in measuring the changing of resin–dentin bond strength for the first time.n = 6). The percentage of bond strength loss due to microbial aging was much higher than water aging in all groups except DMADDM (p < 0.05). Moreover, after one-month water aging, there were no differences among the four groups (p > 0.05), for microbial aging, the percentage of decreased bond strength in DMADDM group was the lowest (p < 0.05).n = 6). Dentin incubated in complete storage medium (CM) without any inhibitor lost 18.16% (7 d) and 45.46% (30 d) of the dry mass. Dentin mass losses of the last three groups were significantly less than control group (p < 0.05) and not significantly different to each other (p > 0.05), yielding a loss of 1.00% (7 d) and 13.19% (30 d) for CHX group, 1.00% (7 d) and 12.99% (30 d) for DMADDM group, and 0.25% (7 d) and 3.67% (30 d) for DMADDM + CHX group.The percentage of dentin mass loss at seven and 30 days is shown in n = 6). Hydroxyproline dissolution weights (μg) per 1 mg dry weight of collagen matrix of the three groups were significantly lower than the control group (p < 0.05): 0.8 (7 d) and 1.18 (30 d) for CHX group, 0.30 (7 d) and 1.64 (30 d) for DMADDM group, 0.20 (7 d) and 1.45 (30 d) for DMADDM + CHX.The dissolution of collagen peptides determined by hydroxyproline (HYP) analysis is shown in n = 10). The CFU was the highest in the control dentin blocks without treatment. DMADDM and DMADDM + CHX eliminate almost all of the bacteria in dentin blocks. The group of CHX showed the least bactericidal activity, reducing the CFU by no more than two orders of magnitude.These qualitative observations were corroborated with quantification of bacteria colony-forming units (CFU) in dentin blocks in To improve the durability of resinous restorations, cavity disinfectants should possess antimicrobial action, MMPs inhibitory effect and durability.CHX is one of the gold standard antimicrobial agents and matrix metalloproteinase inhibitors . CHX hasDemineralized dentin collagen matrix acts as a scaffold for resin infiltration during the resin–dentin bonds procedure . DegradaHowever, CHX pre-treatment can be problematic, even though 0.002% CHX is sufficient to prevent host-derived MMPs from degrading exposed collagen fibrils , and itsDMADDM is a kind of quaternary ammonium methacrylates. It has potent antimicrobial activity for its positive charge, thus can change the membrane permeability or surface electrostatic balance of the negatively charged bacteria, leading to cytoplasmic leakage ,40. BesiCHX and DMADDM were physically mixed in the cavity disinfectant. However, the combination showed no extra benefit when compared with DMADDM alone. DMADDM is in the state of monomers before polymerization. In a study by Hiraishi et al., it is speculated that CHX could interfere with the function of adhesive monomers . TherefoAssessment of hydroxyproline, collagen fragments that are produced through the action of MMPs on type I collagen, is a quantitative way to measure MMP-inhibitory ability of materials . Loss ofIn comparison, DMADDM achieved antibacterial effects much stronger than CHX alone, and the same as the combination of DMADDM and CHX. All four CFU counts in dentin disk with DMADDM or dual agents were six orders of magnitude lower than that of CHX alone. The results of SEM also showed the same trend. Therefore, it would be more beneficial to use DMADDM alone or combine DMADDM with CHX to obtain a greatly increased antibacterial potency compared to CHX.Microtensile bond strength (μTBS) methodology was introduced in 1994, and is widely used to measure bond strength ,47. Via It is well known that in vitro artificial aging techniques are carried out to simulate in service conditions . They arHowever, the present study was performed in a static environment. Clinically, numerous other factors affect bond strength such as thermal, mechanical, chemical and fatigue stresses during function. Thus, further in vitro studies that simulate the real clinical environment are needed.The extracted human caries-free third molars used in this study were obtained with patient’s informed consent under a protocol approved by the West China Hospital of Stomatology, Sichuan University . These teeth were stored in 0.01% thymol solution for no more than 1 month before use. The following experiments were performed at least three times.DMADDM was synthesized as described in previous studies ,38, and The enamel and superficial dentin of the third molars were removed from the crown with a diamond saw to prepare a flat mid-coronal dentin surface. The surfaces were further polished with a diamond bur for 60 s in order to standardize the smear layer ,54.3 composite dentin sticks.The teeth were distributed into four groups according to the cavity disinfectants used: (1) no disinfection treatment; (2) 0.2% CHX, a cotton pellet saturated with 0.2% CHX, with a dwell time of 20 s, after that the dentin surface was blown dry; (3) 5% DMADDM; and (4) 5% DMADDM + 0.2% CHX. For the last two groups, the dentin surface was disinfected with respective solution in the same way as Group 2. After pre-treatment of the dentin, the primer of SE Bond  was applied with a brush-tipped applicator and rubbed for 20 s and dried with a stream of air for 5 s. Adhesive was spread on the dentin surface and light-cured for 10 s. Then, composite blocks were prepared on the teeth surface. The bonded specimens were stored in water at 37 °C for 24 h. Thereafter, the bonded teeth were sectioned occlusogingivally into 1 × 1 × 10 mmn = 6): (1) subjected to microtensile test immediately; (2) stored in water at 37 °C for 1 month; and (3) stored in McBain broth with saliva biofilm at 37 °C for 1 month, changing the incubating media once a day. Saliva for incubation was obtained from seven healthy female and three male volunteers, ranging in age from 23 to 25. The volunteers had no history of extant periodontal disease and not taken antibiotics for the previous 3 months [Seventy-two resin–dentin sticks in each group were treated in three different ways . Radiography was used to ensure the absence of residual minerals [The dentin beams were submerged in 10% phosphoric acid for 18 h at 25 °C to demineralize them. In order to remove phosphoric acid, the dentin beams were incubated in deionized water for 72 h at 4 °C under constant stirring, and the water was changed every 16 h. Then, they were dried over anhydrous calcium sulfate for 8 h, and the mass of each dentin beam was measured by a digital microbalance. The beams were rehydrated for 24 h in 0.9% NaCl containing 10 U·mLminerals .n = 10): (1) a calcium- and zinc-containing complete storage medium CM, containing 5 mM HEPES, 2.5 mM CaCl2·H2O, 0.05 mM ZnCl2, and 0.3 mM NaN3 (pH 7.4); (2) 0.2% CHX in CM (pH 7.4); (3) 5% DMADDM in CM (pH 7.4); and (4) 0.2% CHX + 5% DMADDM in CM (pH 7.4) [Forty-eight dentin beams were distributed into four groups and placed in Eppendorf tube, incubated with 500 μL different types of media at 37 °C for 7 or 30 days ((pH 7.4) . After tStreptococus mutans , Veillonella parvula , and Lactobacillus acidophilus  [3 were cut from the crown of the extracted third molar, using silicon carbide abrasive. The dentin blocks were treated with 37% phosphoric acid for 3 min, and then rinsed with distilled water for 3 min. They were sterilized with ethylene oxide for 12 h and de-gassed for 7 days to remove the ethylene oxide gas.We use the method described in Cheng’s study to judge the effects of the experimental agents on bacteria impregnated in human dentin blocks . The useVA, USA) , which aStreptococus mutans, Veillonella parvula, and Lactobacillus acidophilus suspension in BHI were prepared to 1010 CFU/mL. A 2 μL aliquot of the above bacteria suspension was placed on the surface of the dentin blocks, and the suspension was left to infiltrate into the dentin for 10 min to simulate bacterial colonization in the dentin. After impregnation of bacteria, 10 μL disinfection agents were daubed on the dentin surface according to the group criteria, for 20 s. These dentin blocks were divided into 2 groups: (1) Immersed treated and untreated blocks in 1% glutaraldehyde in PBS for 4 h at 4 °C, and then dehydrated with graded ethanol, and rinsed them with 100% hexamethyldisilazane. Sputter-coating gold was performed. Next, examined using SEM; (2) Place into 2 mL CPW in a tube, with ten blocks for each group. In order to collect the bacteria, sonication was used at a frequency of 40 kHz for 5 min, and the bacteria suspension in CPW was vortexed at the maximum speed for 20 s by a vortex mixer. The collected bacteria suspension was serially diluted in CPW, and plated on blood agar (BA) plate and two selective media: BA plate was used for measuring the whole number of the harvested live bacteria, mitis sucrose bacitracin agar (MSB) was for evaluating the number of Streptococus mutans, and Lactobacillus selective agar (LBS) for Lactobacillus acidophilus. The plates were incubated at 5% CO2 and 37 °C for 3 days, and the number of colonies was counted. The numbers of Streptococus mutans and Lactobacillus acidophilus were directly calculated by the counted CFU number, while the quantity of Veillonella parvulathe was determined by subtracting the number of Streptococus mutans and Lactobacillus acidophilus from total number of bacteria.Twenty-four-hour cultures of p = 0.05.Kruskal–Wallis test, followed by Dunn’s multiple comparison was performed to detect the significant effects of the variables. Statistical significance was preset at The present study investigated the effect of novel cavity disinfectants with 5% DMADDM, and compared the difference between microbial-aging method with water-aging method in measuring the stability of resin–dentin bond strength for the first time. This study demonstrated that: (1) the cavity disinfectant with 5% DMADDM might be able to improve the bond strength durability; (2) 5% DMADDM had the same MMPs inhibitory capability as 0.2% CHX and 0.2% CHX + 5% DMADDM; (3) 5% DMADDM alone or in conjunction with 0.2% CHX had antibacterial effects much stronger than 0.2% CHX alone; and (4) when compared with water-aging method, microbial-aging method was more effective for detecting the difference of stability of bond strength between groups, which makes it a promising method for studying the durability of resin–dentin bonds.


Guidelines for frequency of Type 2 diabetes mellitus (DM) screening remain unclear, with proposed screening intervals typically based on expert opinion. This study aims to demonstrate that HbA1c screening intervals may differ substantially when considering individual risk for diabetes.This was a multi-institutional retrospective open cohort study. Data were collected between April 1999 to March 2014 from one urban and one rural cohort in Japan. After categorization by age, we stratified individuals based on cardiovascular disease risk (Framingham 10-year cardiovascular risk score) and body mass index (BMI). We adapted a signal-to-noise method for distinguishing true HbA1c change from measurement error by constructing a linear random effect model to calculate signal and noise of HbA1c. Screening interval for HbA1c was defined as informative when the signal-to-noise ratio exceeded 1.Among 96,456 healthy adults, 46,284 (48.0%) were male; age (range) and mean HbA1c (SD) were 48 (30–74) years old and 5.4 (0.4)%, respectively. As risk increased among those 30–44 years old, HbA1c screening intervals for detecting Type 2 DM consistently decreased: from 10.5 (BMI <18.5) to 2.4 (BMI > 30) years, and from 8.0 (Framingham Risk Score <10%) to 2.0 (Framingham Risk Score ≥20%) years. This trend was consistent in other age and risk groups as well; among obese 30–44 year olds, we found substantially shorter intervals compared to other groups.HbA1c screening intervals for identification of DM vary substantially by risk factors. Risk stratification should be applied when deciding an optimal HbA1c screening interval in the general population to minimize overdiagnosis and overtreatment. Type 2 diabetes mellitus (DM) affects approximately 7.2 million adults in Japan, with a worldwide prevalence of 387 million in 2013 [In Japan, for example, a large majority of the population, including virtually all employed adults, receive free annual health checkups mandated by the 1972 Industrial Safety and Health Act, which typically include annual DM screening. In the United States, the American Diabetes Association recommends that screening for Type 2 DM in adults occur roughly every 3 years, though this is level IV evidence (expert opinion) . In the 2 had an OR of 3.66 for being diagnosed with DM [The risk of developing Type 2 DM differs with age, obesity, and lack of physical activity . A CDC r with DM . However with DM , 16, desIn order to demonstrate that screening intervals may be substantially different when considering risk stratification, our study uses two clinically feasible risk stratification strategies, Framingham (10-year cardiovascular) risk  and BMI,This was a retrospective open cohort study, combining an urban and rural population in Japan. The urban cohort was collected from St. Luke’s International Hospital Center for Preventive Medicine  between January 2005 and December 2014. Approximately 80% of participants were either employees or dependents of various companies and local government organizations in metropolitan Tokyo, health screening costs for whom were paid by the employer. The remaining 20% of participants were residents of Tokyo, independently registering and paying for the program. The rural cohort was collected from the Yamanashi Koseiren Health Care Center  between April 1999 and March 2009 as part of a private health check-up service. Data in both cohorts included individuals presenting for health screening at least twice, with no previous history of Type 2 DM or cardiovascular events at first visit, and with complete data on risk equation covariates for Framingham Risk Score. Consistent with previous studies, patients were classified into 3 age groups  . ClinicaTo distinguish true change in HbA1c progression, we adapted a statistical method for distinguishing the properties of tests from the variability of measurements; the methodology has been described in detail elsewhere , 26. BriAll models were fitted in Stata software version 12.1 .When patients presented to both medical facilities, they were provided with a document explaining that their anonymous data may be used for research purposes. While written informed consent was not provided, both institutions provided opt-out policy information in paper form and all patients had the opportunity to refuse use of their information from the electronic medical record. Ethical approval was obtained from two committees: the Research Ethics Committee of St. Luke’s International Hospital  as well as the Research Ethics Committee of University of Yamanashi .2 in the urban cohort and 22.7 (3.0) kg/m2 in the rural cohort. Mean (SD) age of the rural cohort was roughly 5 years older than that of the urban cohort at 51.2 (10.2) versus 46.5 (10.4) years, respectively. Mean (SD) Framingham Risk Score was slightly higher in rural than urban cohort, at 9.0% (0.08) and 6.0% (0.07), respectively. The proportion of current smokers in the rural cohort was twice that of the urban cohort. We merged the two cohorts into a single population for subsequent analyses to increase the generalizability of this study. Table 2): 71.0% for ages 30–44, 72.4% for ages 45–59, and 74.0% for those 60–74 years old. Patients were similarly concentrated in the low risk (0–10%) group of Framingham Risk Score: 97.7% for those 30–44 years old, 73.3% for 45–59 years old, and 36.7% for those 60–74 years old.Of the 149,191 adults in both cohorts, 96,456 healthy adults with an average age (SD) of 48.0 (10.6) years, comprising 46,284 (48.0%) males, were eligible for inclusion in this study. Exclusions are shown in the flow chart in Fig. Figure Figure Meaningful screening intervals varied substantially by BMI, ranging from as long as 10.5 (8.9–12.9) years to as short as 2.4 (1.6–4.2) years. When Framingham cardiovascular risk was used for stratification, monitoring intervals were similarly varied, from 8.0 7.0–9.3) to 2.0 (1.4–4.0) years. Regardless of the stratification method employed, analysis of higher risk groups consistently resulted in shorter screening intervals compared to those at lower risk. While existing guidelines .0–9.3 to, 16 and Despite a growing body of evidence that population-level DM screening may not be useful, we anticipate that it will remain unpalatable from a societal perspective to do away with screening entirely, especially in light of recent reports of the alarmingly high prevalence of DM and its risk factors, including obesity . As demoThis study has some limitations. We relied on self-reports for some clinical criteria, such as use of DM medication, DM and cardiovascular event history, as well as current smoking status for calculating Framingham Risk Scores. Differences in data collection technique may have existed between the two cohorts: participants in the urban cohort received a personalized interview by a trained preventive health nurse, in order to maximize accuracy of data, while there was no nurse interview system in the rural cohort. Despite this, clinically relevant differences were not seen between the two cohorts. Second, this was a multi-institutional open cohort study of data collected from urban and rural Japan. Generalizability to other countries warrants further exploration. Finally, under current Japanese occupational health law, contents of health exams may differ based on employee age; those under 39 or over 75 years old are not required to have blood tests, thus raising the possibility of selection bias via missing data, especially for those 30–39 years old. However, most companies choose to include screening blood tests for all employees; in the urban cohort, only 0.03% of participants failed to have a blood test panel.When stratifying by risk for DM development, HbA1c screening intervals varied substantially. Taking into account differences of true HbA1c change, to avoid overdiagnosis and overtreatment, risk stratification should be applied when deciding an optimal HbA1c screening interval in a specific population. Annual screening appears to be unwarranted in any risk group, while those at low risk may not warrant re-screening for a decade or longer.


The ITACa trial was designed to define the role of cetuximab (Cet) and bevacizumab (Bev) in combination with standard chemotherapy  as first- and second-line treatment in metastatic colorectal cancer. All patients with WT KRAS tumors who had been enrolled in the first-line trial were randomized onto two independent second-line trials: CT or CT + Cet (study 2A) and CT + Bev or CT + Bev + Cet (study 2B). Patients with mutated KRAS were not eligible for randomization and were treated with CT alone (study 2A) or CT + Bev (study 2B). The primary endpoint was progression-free survival (PFS). 48 and 56 KRAS WT patients were randomized while 31 and 40 KRAS mutated patients were treated without randomization. Study 2A: median PFS was 3.4 (95%CI 2.3–4.6) and 6.2 (95%CI 4.3–7.8) months for the CT and CT + Cet arms, respectively, with a hazard ratio (HR) = 0.64 . Study 2B: median PFS was 7.7 (95%CI 4.1–10.1) and 4.9 (95%CI 3.2–7.0) months for CT + Bev and CT + Cet + Bev arms, respectively, with a HR = 1.31 . Notwithstanding limitations due to the small sample size, among patients with WT KRAS the addition of Cet to second-line CT increased PFS, whereas the addition of Cet to CT + Bev was associated with worse PFS. Combinations of fluorouracil (FU), leucovorin (LV) and oxaliplatin (FOLFOX), and of FU, LV, and irinotecan (FOLFIRI) are considered standard regimens for metastatic CRC (mCRC). Most patients with mCRC receive first-line chemotherapy (CT) and approximately 70% of those who progress after one line of CT will undergo at least one further line of systemic treatment5 consisting in CT in combination with molecular-targeted agents directed against epidermal growth factor receptor (EGFR) or vascular endothelial growth factor (VEGF).Colorectal cancer (CRC) is the third most commonly diagnosed cancer among males and the second among females worldwide10. Similarly, anti-EGFR agents  have been shown to improve outcomes compared to CT alone in the second line setting in patients with wild type (WT) Kirsten rat sarcoma viral oncogene homolog (KRAS) mCRC15. Given the number of randomized trials demonstrating the efficacy of different second-line treatments in mCRC, strategy trials are needed to define the optimal use and best sequencing of all available agents.The use of antiVEGF agents  in combination with second-line CT is supported by the positive results of several randomized phase III clinical trials16. The present paper reports the results of the second-line randomized studies, designed to determine the effect of adding Cet to standard CT or to CT plus Bev on progression-free survival (PFS). This study is registered on ClinicalTrials.gov (NCT01878422).The ITACa trial, supported by the Italian Medicines Agency (AIFA), was designed to define the role of Cet and Bev in combination with standard chemotherapy (FOLFIRI or FOLFOX4) as first- and second-line treatment of mCRC Fig. . ResultsIn the first-line trial, all eligible patients were randomized to receive either CT plus Bev (arm A) or CT alone (arm B). CT was FOLFIRI or FOLFOX4, at the discretion of the clinician, for either arm. Upon progression, all eligible patients could be randomized onto one of two independent second-line trials, Study 153 01/2A and 153 01/2B. In study 2A, arm A patients with WT KRAS were randomized to the other CT regimen (FOLFIRI or FOLFOX4) or the other CT plus Cet. Arm A patients with mutated KRAS were not randomized and were treated with the other CT regimen alone. In study 2B, arm B patients with wild type KRAS were randomized to the other CT plus Bev or the other CT plus Bev plus Cet. Arm B patients with mutated KRAS were not randomized and were treated with the other CT plus Bev  performance status of ≤2 (≤1 if aged ≥70 years), and an estimated life expectancy of at least 12 weeks were enrolled onto the trials. KRAS mutational status evaluation was mandatory after the protocol amendment no.1 of May 2009. Adequate hematologic, hepatic, and renal functions were required, as well as D-dimer within normal range . Chemoembolization and radical or cytoreductive or palliative surgery at the end of first-line therapy were allowed if there was evidence of progressive disease. Exclusion criteria included pregnancy or breast-feeding; clinically significant cardiovascular disease; known CNS metastases; interstitial pneumonia or extensive symptomatic fibrosis of the lungs; uncontrolled hypertension; pulmonary embolism or any arterial thromboembolism, deep vein thrombosis or other significant thromboembolic events, bleeding diathesis or coagulopathy, clinically significant peripheral vascular disease; chronic use of aspirin (>325 mg/day), anti-platelet agents or anticoagulants; proteinuria .The ITACa trial was approved by the local ethics committee (Comitato Etico Area Vasta Romagna) on September 19th, 2007 and was registered in our National Clinical Trials Observatory (Osservatorio delle Sperimentazioni Cliniche) and in the European Clinical Trials Database (EudraCT no. 2007–004539–44) before patient recruitment began. Registration on ClinicalTrials.gov (NCT01878422) was not mandate but was carried out at a later date (07/06/2013). All patients provided written informed consent and the studies were carried out in accordance with the Declaration of Helsinki under good clinical practice conditions and after full ethics committee approval of all participating centers .4. Cet was administered on day 1 at an initial dose of 400 mg/m2 as a 2-hour intravenous infusion, and then at 250 mg/m2 intravenously over one hour on a weekly basis. Bev was administered after FU bolus as a 30- to 90-minute intravenous infusion at a dose of 5 mg/kg on day 1 if combined with CT alone, or on day 2 if combined with CT plus Cet. Treatment was to be continued until disease progression (PD), withdrawal of consent or unacceptable toxicity, whichever came first. Pre-specified dose modifications of CT were provided after the occurrence and resolution of severe hematologic or non-hematologic toxicity. No dose reductions of Bev were made in any patient. Bev was suspended in the event of grade 3 venous thromboembolism or incidentally discovered pulmonary embolus (until a stable dose of anticoagulant was administered), proteinuria >2 g protein/24 hours (until improvement to ≤2 g/24 hours), and uncontrolled hypertension. Bev was permanently discontinued in the presence of any grade 4 toxicity. The dose of Cet was reduced for recurrent Cet-related grade 3 toxicities only and at the second or third occurrence of grade 3 skin toxicity. Cet was discontinued after grade 3–4 allergic/hypersensitivity reactions, if more than two consecutive infusions were withheld or a fourth occurrence of a grade 3 skin toxicity occurred despite appropriate dose reduction. If one of the regimen components was discontinued due to toxicity, treatment could be continued with the remaining components.The FOLFIRI and FOLFOX4 regimens were as previously describedThe primary objective was to determine, separately for each study, whether the addition of Cet to CT (Study 153 01/2A), or to CT plus Bev (Study 153 01/2B), would improve efficacy in terms of progression-free survival (PFS). The secondary objectives were objective response rate (ORR), overall survival (OS) and safety profile of the treatments administered.nd line studies to the date of death due to any cause or the last date the patient was known to be alive (censored observation). Adverse events were graded according to the National Cancer Institute Common Toxicity Criteria (NCI-CTC) for Adverse Events, Version 3.PFS was defined as the time from the date of randomization in the second-line study to the date of the subsequent observation of documented PD or death due to any cause. Patients without PD at the time of analysis were censored at their last date of tumor evaluation. Patients undergoing curative metastasectomy were censored at the time of surgery. Response Evaluation Criteria in Solid Tumors (RECIST) guidelines were used to define all responses. An independent central review of patient scans was not carried out. OS was measured from the date of randomization in the 216, the ITACa trial of first- and second-line management strategies was initially conceived to enrol 1000 candidates for first-line treatment to compare the efficacy of the two strategies in terms of overall survival. In the original design, hypothesizing that about 60% of the first-line population would be eligible for second-line studies, and that about 60% of patients would have a wild type KRAS tumor, we estimated that a population of about 400 patients (200/study) would be needed for the study to have sufficient power.As previously mentionedAfter the study was amended and the sample size of the first-line trial was reduced to 376 patients due to slower-than-expected patient recruitment, the primary endpoint of the first-line study became PFS and the statistical design of the second-line studies was also modified. According to literature data, the median time to progression after second-line treatment ranged from 4 to 6 months. Assuming a 48-month accrual period, a further 12-month follow-up and a 5% significance level (2-sided), we calculated that 80 patients randomized to each study would achieve an 80% power for PFS analysis under various assumptions of progression rate and true underlying reduction in HR by adding Cet to CT or to CT plus Bev.KRAS status .Patients were randomized in a 1:1 allocation ratio. Separate randomization lists using a permuted block balanced procedure were generated for each participating center, stratified by the CT regimen and by -values were based on two-sided testing and statistical analyses were performed using SAS statistical software version 9.4 .Safety analysis was based on the population of all treated patients (at least one cycle). Time to event data  were described using Kaplan-Meier curves and compared by the logrank test (at a significance level of 5%). Ninety-five percent confidence intervals (95% CI) were calculated by non-parametric methods. Estimated hazard ratios (HRs) and their 95% CI were calculated using the Cox proportional-hazard model adjusted by center and CT regimen (FOLFOX4 or FOLFIRI). The ORR  was calculated with an exact 95% CI using standard methods based on binomial distribution. Non-interim efficacy analysis was planned. All pAll data generated or analyzed during this study are included in this published article (and its Supplementary Information files).Between April 2008 and December 2015, 48 and 56 WT KRAS patients were randomized onto Studies 153 01/2A and 153 01/2B, respectively, while 31 and 40 KRAS mutated patients were treated without randomization in the former and latter study . The median number of treatment cycles per patient was higher for the Cet and CT combination  than for CT alone , and the total number of treatment cycles was also superior in the experimental arm (219 vs. 144). Reductions in CT dose were more frequent in the CT plus Cet arm (34.7% vs. 24.3%), while cycle delays were comparable.Study 2B (WT KRAS patients). Although the patients in either treatment arm received a median of 6 cycles, the total number of treatment cycles was higher in the CT plus Bev group (352 vs. 263). Reductions in the CT dose were slightly more frequent in the CT plus Bev arm (20.2 vs. 12.6%), while cycle delays were comparable.Treatment with Cet was provided for patients in the control arm of both studies after disease progression. A Cet-based third-line regimen was administered to 10/25 (40%) and 10/31 (32.3%) patients in studies 2A and 2B, respectively.Table The data cut-off for efficacy analysis of both studies was April 2016 when the median duration of follow-up was 46 months (range 1–61 months).At the data cut-off, 48 (100%) events had occurred for PFS. All patients had progressed or died. Median PFS was 3.4 (95% CI 2.3–4.6) and 6.2 (95% CI 4.3–7.8) months for the CT and CT plus Cet arms, respectively, with a HR of 0.64  Fig.  adjustedOverall, 45 patients died, 23 (92%) in the CT arm and 22 (95.7%) in the CT plus Cet arm. Median OS was 9.3 (95% CI 3.8–12.0) and 11.1 (95% CI 5.9–14.4) months in the CT and CT plus Cet groups, respectively, with a HR of 1.30  Fig.  adjustedAt the data cut-off, 55 (98.2%) events had occurred for PFS. Median PFS was 7.7 (95% CI 4.1–10.1) and 4.9 (95% CI 3.2–7.0) months for the CT plus Bev and CT plus Cet plus Bev arms, respectively, with a HR of 1.31  Fig.  adjustedOverall, 51 patients died, 28 (90.3%) in the CT plus Bev arm and 23 (92%) in the CT plus Cet plus Bev arm. Median OS was 15.1 (95% CI 12.0–17.5) and 8.9 (95% CI 5.9–13.9) months for the CT plus Bev and CT plus Cet plus Bev groups, respectively, with a HR of 1.31  Fig.  adjustedAt the data cut-off, all patients in the study 2A with mutated KRAS had progressed and 30 (96.8%) had died. Median PFS was 4.2 months (95% CI 2.4–7.9), median OS was 8.7 months (95% CI 5.3–12.9) and ORR was 25.8%. 39 (97.5%) events for PFS and 37 (92.5%) for OS were reported among study 2B patients with mutated KRAS. Median PFS was 8.6 months (95% CI 6.8–11.2), median OS was 15.1 months (95% CI 12.9–18.0) and ORR was 25%.WT KRAS were further evaluated for mutations in NRAS and BRAF and in other KRAS exons. Among 2A patients, 7 RAS mutations and 7 BRAF mutations were detected, while 8 RAS mutations and 3 BRAF mutations were detected among 2B patients.In the light of recent literature data, patients with Median PFS was 7.0 months (95% CI 2.7–8.9) and 3.3 (95% CI 1.9–4.6) months for CT plus Cet and CT, respectively, with an HR of 0.56  adjusted by center and CT regimen (FOLFOX4 or FOLFIRI). Median OS was 11.1 (95% CI 5.0–14.4) in the CT plus Cet arm and 7.8 (95% CI 1.9–16.7) in the CT alone arm, with an adjusted HR of 1.27 .Median PFS was 7.5 months (95% CI 4.3–9.6) and 3.4 months (95% CI 2.3–4.6) for CT plus Cet and CT, respectively, with an adjusted HR of 0.48 . Median OS was 13.1 months (95% CI 5.0–14.4) in the CT plus Cet arm and 9.6 months (95% CI 1.9–16.7) in the CT alone arm, with an adjusted HR of 1.29 .Median PFS was 6.0 months (95% CI 3.7–8.7) and 7.3 months (95% CI 4.1–9.9) for CT plus Cet plus Bev and CT plus Bev, respectively, with an HR of 1.04  adjusted by center and CT regimen (FOLFOX4 or FOLFIRI). Median OS was 10.7 months (95% CI 4.5–17.8) in the CT plus Cet plus Bev arm and 15.1 months (95% CI 10.3–17.2) in the CT plus Bev arm, with an adjusted HR of 1.03 .Median PFS was 6.0 months (95% CI 3.2–8.7) and 7.3 months (95% CI 4.1–9.9) for CT plus Cet plus Bev and CT plus Bev, respectively, with an adjusted HR of 1.19 . Median OS was 11.5 months (95% CI 3.7–17.8) in the CT plus Cet plus Bev arm and 15.1 months (95% CI 10.3–17.2) in the CT plus Bev arm, with an adjusted HR of 1.20 .17. Recently, several studies investigating the predictive and prognostic role of RAS mutations other than in KRAS exon 2 reported that patients with these mutations did not respond to therapy. Consequently the use of anti-EGFR drugs has been further limited to all WT RAS tumors18.The ITACa second-line trial is, to our knowledge, the first randomized trial to prospectively investigate the role of cetuximab in a WT KRAS population in combination with second-line CT (Study 2A) or CT plus Bev (Study 2B). Since 2008, the use of anti-EGFR agents has been restricted to WT KRAS patients as it has been shown that KRAS exon 2-mutated tumors are resistant to this therapyRAS/BRAF WT patients showed a significant improvement in PFS in the experimental arm .The study 2A compared the activity of Cet plus second-line CT with that of CT alone. Our results suggest that Cet improved PFS by almost 3 months and OS by almost 2 months, in addition to doubling ORR. Although median OS was higher in the CT plus Cet arm than in the CT arm, the HR shows an increased risk of death for the CT plus Cet arm due to the longer survival of CT arm patients after the 15-month time point Fig. . A possi11. No differences in terms of OS were seen in a subsequent analysis of a small subset of patients with WT KRAS tumors (15% of the entire population)12. Similar results were observed in a randomized phase III study of second-line panitumumab in combination with FOLFIRI compared to FOLFIRI alone in patients with metastatic colorectal cancer. A significant improvement in PFS was observed in the WT KRAS subpopulation when panitumumab was added to CT , together with a non-significant trend towards increased OS 13.The efficacy of Cet in combination with CT after first-line treatment failure was investigated in the multicenter, open-label, phase III EPIC trial in which 1298 patients unselected for KRAS were randomly assigned to Cet plus irinotecan or irinotecan alone. The median OS (primary endpoint) was similar in both arms: 10.7 and 10.0 months for CT plus Cet and CT alone, respectively . However, CT significantly improved PFS  and ORR  in the intention-to-treat population20. The ITACa trial was ongoing when the results from both studies were presented and the Steering Committee evaluated the idea of amending the study by eliminating the CT plus Bev plus Cet arm. However, following a review of available literature data, it was decided not to proceed with any modification because both PACCE and CAIRO2 trials were conducted before KRAS mutations were identified as a predictor of poor response to anti-EGFR monoclonal antibodies, therefore patients were not selected. Moreover, both were first-line trials and the CT regimens analyzed differed from those of the ITACa trial (panitumumab in PACCE instead of Cet and capecitabine in CAIRO2 instead of 5FU). All ITACa investigators were nonetheless instructed to monitor carefully for toxicity. Our results of a reduction of almost 3 months in PFS and a 50% decrease in median OS and ORR would appear to confirm the detrimental effect of adding Cet to CT plus Bev, even in WT KRAS patients.A ‘head to head’ comparison between VEGF and EGFR inhibitors in combination with second-line chemotherapy should be considered in future randomized trials as this would help to identify the best second-line regimen in the RAS WT population. The study 2B investigated the role of Cet added to second-line CT plus Bev. Two large randomized phase 3 trials investigated the efficacy and safety of treatment regimens incorporating CT in combination with Bev and an EGFR inhibitor: the Panitumumab Advanced Colorectal Cancer Evaluation (PACCE) study analyzed CT plus Bev with or without panitumumab, while the CAIRO2 study assessed the efficacy and safety of capecitabine plus oxaliplatin and Bev with or without Cet. In either trial, the combination of the 2 monoclonal antibodies was associated with increased toxicity and decreased efficacyIn conclusion, our findings indicate that the addition of Cet to second-line CT increased PFS in mCRC patients with WT KRAS, whereas the addition of Cet to CT + Bev was associated with shorter PFS. Whilst we are aware that the small population of both second-line studies does not enable definitive conclusions to be drawn, the clinical relevance of our results is high. The novelty of the work stems from the fact that it is a follow-on of a study in which first- and second-line strategies were jointly evaluated and all patients received the same first-line treatment, thus limiting the biases due to possible interactions, resistance or long-term effects that can occur when the study population has received heterogeneous treatments.