To report a single-centre experience with the novel Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) technique and systematically review the related literature.Since January 2013, patients with extended primary or secondary liver tumors whose future liver remnant (FLR) was considered too small to allow hepatic resection were prospectively assessed for the ALPPS procedure. A systematic literature search was performed using PubMed, Scopus and the Cochrane Library Central.Until July 2014 ALPPS was completed in 9 patients whose mean age was 60±8 years. Indications for surgical resection were metastases from colorectal cancer in 3 cases, perihilar cholangiocarcinoma in 3 cases, intrahepatic cholangiocarcinoma in 2 cases and hepatocellular carcinoma without chronic liver disease in 1 case. The calculated FLR volume was 289±122 mL (21.1±5.5%) before ALPPS-1 and 528±121 mL (32.2±5.7%) before ALLPS-2 (p<0.001). The increase in FLR between the two procedures was 96±47% . Additional interventions were performed in 4 cases: 3 patients underwent Roux-en-Y hepaticojejunostomy, and one case underwent wedge resection of a residual tumor in the FLR. The average time between the first and second step of the procedure was 10.8±2.9 days. The average hospital stay was 24.1±13.3 days. There was 1 postoperative death due to hepatic failure in the oldest patient of this series who had a perihilar cholangiocarcinoma and concomitant liver fibrosis; 11 complications occurred in 6 patients, 4 of whom had grade III or above disease. After a mean follow-up of 17.1±8.5 months, the overall survival was 89% at 3–6 and 12 months. The recurrence-free survival was 100%, 87.5% and 75% at 3-6-12 months respectively. The literature search yielded 148 articles, of which 22 articles published between 2012 and 2015 were included in this systematic review.The ALPPS technique effectively increased the resectability of otherwise inoperable liver tumors. The postoperative morbidity in our series was high in accordance with the data from the systematic review. Age, liver fibrosis and presence of biliary stenting were predisposing factors for postoperative morbidity and mortality. Surgical resection is a potentially curative treatment for patients with primary and secondary malignant liver tumors. Although the liver has the unique capability to regenerate, a remnant of at least 20% of the liver volume must be spared to avoid postoperative liver failure provided that the parenchyma not affected by the tumor is anatomically normal. In the presence of chemotherapy-induced liver injury, the future liver remnant (FLR) should be at least 30% of the total volume; however, in the presence of cirrhosis, a 40% FLR is advisable.Strategies have been developed to increase the resectability of those tumors that are too advanced to be resected leaving a sufficient FLR. These strategies, namely right portal embolization (PVE) and preoperative or intraoperative ligation of the right portal vein (PVL), are based on the occlusion of the flow in one of the main branches of the portal vein (PVO) inducing atrophy in the ipsilateral liver and subsequent hypertrophy of the contralateral lobe; due to the larger volume of the right liver, usually the right branch of the portal vein is occluded to increase the volume of the left liver. In fact, the occlusion of the right portal vein induces compensatory hypertrophy of the left lobe that is on the average of 40% in approximately 4–8 weeks. HoweverRecently, a new technique of hepatic resection that is performed in two stages—called Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS)—has been described. The first step of this procedure associates the intraoperative ligation of the right portal branch to the partition of the liver, usually following the scheme of an extended right hepatectomy. In contrast to a classical hepatectomy, the diseased part of liver is left in situ and remains vascularized by the right hepatic artery only, while the biliary and systemic venous drainage through the right biliary duct and hepatic veins, respectively, are preserved. In the The purpose of this study was to describe our initial experience with extended two-stage liver resections using the ALPPS technique for the treatment of primary or metastatic liver tumors. A systematic review of the literature concerning the ALPPS procedure was also carried out in order to highlight the differences in indications, surgical techniques, increase in FLR and clinical outcome.From February 2005 to September 2014, 553 patients underwent liver resection at the Hepatobiliary and Abdominal Transplantation Surgery of the Polytechnic University of Marche, Ancona, Italy. All patients were evaluated for liver surgery in a multidisciplinary meeting attended by surgeons, radiologists, interventional radiologists and gastroenterologists. Evaluation of the FLR (%) was performed in all cases of planned right extended hepatectomy and in those cases where a right hepatectomy was planned in patients with liver disease. Before January 2013, an FLR <30% was considered a contraindication for surgical resection and an indication for right preoperative portal vein embolization followed by CT after a mean time of 5 weeks. Surgical resection was then performed only when an increase in the FLR to values >30% was observed after PVE. Patients who did not develop sufficient hypertrophy or who showed tumor progression were excluded from surgery and received palliative treatment. Since January 2013, all patients with an FLR <30% were assessed for the ALPPS procedure.Exclusion criteria for the ALPPS procedure were as follows: the presence of distant metastases, age >75 years, chronic liver disease and macrovesicular steatosis greater than 50%. In all cases with diagnosed or suspected liver disease, a liver biopsy and/or the indocyanine green test measured with spectrophotometry were performed preoperatively.The data of patients undergoing ALPPS were prospectively collected. All study enrollment procedures and subsequent data collection and acquisition were approved by the Institutional Review Board at the Ospedali Riuniti Hospital. Written informed consent was obtained from the patients. Postoperative complications were classified according to the Dindo-Clavien classification.The future liver remnant (FLR) was estimated as a fraction of FLR (mL) and the total functioning liver, the latter being the difference between the total liver volume (TLV) and tumor volume (TuV).CT scans were conducted using a 64-row scanner (GE LightSpeed VCT 64). All images were obtained at 120 kV and automated mA. A pre-contrast acquisition and post-contrast tri-phasic protocol were used. After the administration of 1.5 ml/kg body weight of 370 mg/ml iodine contrast material, an arterial phase , a portal venous phase  and a late venous phase  were obtained. All volumetric acquisitions were reconstructed in contiguous axial slices of 2.5 mm.MR scans were performed using a superconductive high-field scanner (GE HdXt 1.5T). For volumetric evaluation, contrast-enhanced breath-hold 3D GRE T1w images were used, obtained after the administration of 0.1 mmol/kg body weight of Gd-DOTA or Gd-EOB-DTPA 0.025 mmol/kg body weight. An arterial phase (15–20 sec after bolus injection), a portal phase (45–55 s after bolus injection), a late venous phase (90–100 sec after bolus injection) and a hepatobiliary phase at 20 min (after Gd-EOB-DTPA) were obtained. All processed axial images had a slice thickness of 4 mm and a slice spacing of 2 mm.Volumetric evaluation was performed using an Advantage Workstation 4.4 . The manual contouring technique on a single axial slice was used, with the total volume calculated as the sum of voxels within areas manually defined. Couinaud’s vascular landmarks were used for virtual resections.All volumes were estimated subtracting portal pedicles until segmental branches, main hepatic trunks, and biliary tree when obstructive jaundice was detected . Tumor vIn all cases, we performed a right subcostal abdominal incision with midline extension. After exploration of the peritoneum, intraoperative ultrasound examination was performed to evaluate the characteristics of the neoplastic nodules and their relationship with the vascular and biliary tree. Hilar lymph node dissection was performed with extemporaneous histological examination. The right liver was detached from the vena cava by ligating and dividing the accessory hepatic veins. The following elements were identified: common bile duct, right and left hepatic artery, right branch of the portal vein, portal vein branch for segment 4 and right hepatic vein. The right portal branch and PV branch for segment 4 were isolated and ligated. Differently from what was described in the first report of the ALPPS technique, the middle hepatic vein was always preserved at step 1, anticipating a recommendation that was subsequently given in a consensus conference.In the case of small lesions in the FLR, these were removed with wedge resections.Liver parenchyma transection was performed using an electric scalpel, the Ligasure and/or Sonoca (Söring GmbH). Liver transection was performed up to the anterior surface of the vena cava.At the end of the hepatic transection, the two cutting surfaces of the liver were separated using an organ bag or a plastic sheet or sheets of Tachosil. In the presence of extrahepatic biliary tumors, a Roux-en-Y hepaticojejunostomy was performed during this first step of the ALPPS . Two abdThe right hepatic vein, right hepatic artery, hepatic artery for segment 4 and right branch of the bile duct were surrounded with vessel loops fixed with titanium clips to allow better identification of these structures at the second step of the procedure.All patients were administered parenteral therapy support and antibiotic therapy. A CT scan or abdominal MRI with volumetric reconstruction was performed to 7±2 days after the first operation. When the FLR increased to at least 30% of the liver volume, the second step of the ALPPS was indicated and performed the following day. When the FLR was lower than 30% of the liver volume, and the patient was in stable clinical conditions, the second step of the ALPPS was delayed.A re-laparotomy was performed on the previous incision line, and the liver was freed from adhesions. The right hepatic vein, right hepatic artery, right branch of the bile duct, and arterial branch to segment 4 were identified, ligated and divided; right trisectionectomy was then completed .Antibiotic prophylaxis and parenteral-enteral nutrition was initiated after the first step and maintained during the first three days after the second step. Deep venous thrombosis prophylaxis was utilized in all patients.Post-hepatectomy liver failure was defined according to 50–50 criteria .After discharge, patients were referred to medical oncologists for the follow-up that included abdominal ultrasound and abdominal CT-scan or MRI 3 and 6–12 months after surgery, respectively. Serum tumor markers were assessed at 3-6-12 months after surgery.http://www.crd.york.ac.uk/PROSPERO) with the following registration number: CRD42014014563.PRISMA statement guidelines to conduct and report systematic reviews were followed. The res“ALPPS” OR “associating liver partition and portal vein ligation for staged hepatectomy” OR “ALTPS” OR “right portal vein ligation” OR”portal vein transection” OR “right portal occlusion” OR "in situ liver transection".A systematic literature search was performed independently by two of the authors (RM and DN) using PubMed, Scopus and the Cochrane Library Central. The search was limited to humans and articles reported in the English language. No restriction was set regarding the type of publication, date or publication status. Participants of any age and sex who underwent ALPPS procedures were considered. The search strategy was based on different combinations of words for each database. For the PubMed database, the following combination was used: The same key words were inserted in the search manager fields of Scopus and the Cochrane Library Central. The search was broadened by extensive cross-checking of reference lists of all retrieved articles fulfilling inclusion criteria. For all databases, the last search was run on April 23, 2015.The same two authors independently screened the title and abstract of the primary studies that were identified in the electronic search. Duplicate studies were excluded. The following inclusion criteria were set for inclusion in this systematic review: 1) studies describing ALPPS procedures; 2) studies reporting at least one perioperative outcome; 3) if more than one study was reported by the same institute, only the most recent or the highest quality study was included.The following exclusion criteria were set: 1) review articles, letters, comments and case reports; and 2) studies where it was impossible to retrieve or calculate data of interest.The same two authors extracted the following main data: 1) first author, year of publication and study type; 2) number and characteristics of patients; 3) increase of FLR between stage 1 and 2, rate of patients who failed to perform ALPPS-2, time (days) between stage 1 and 2, R0 margins (%), indication for ALPPS, overall and severe complications, tumor recurrence up to 12 months, surgical time (minutes), variations to the original technique, recipient morbidity, and in-hospital mortality. Bias of the individual studies was categorized based on study design. All relevant texts, tables and figures were reviewed for data extraction. Discrepancies between the two reviewers were resolved by consensus discussion.Continuous variables normally distributed were described as means±SD, while non-normally distributed variables were described as medians (range). A comparison of pre- and post-liver volumes of ALPPS 1 was performed using Student's t test for paired data. Survival analysis was calculated using the analysis of life tables. Statistical analysis was performed using IBM® SPSS® Statistics ver. 19.0 software.From January 2013 to June 2014, the ALPPS procedure was indicated in 11 cases. Two patients were judged intraoperatively not to be suitable for the procedure and, therefore, are not included in the present analysis: these 2 patients underwent surgery due to perihilar cholangiocarcinoma and were excluded from the planned ALPPS procedure because of severe liver fibrosis diagnosed by intraoperative liver biopsy in one case, and because of intraoperative evidence of a hilar lymph node metastases in the other. The main characteristics of the 9 patients who underwent the ALPPS procedure are described in Perihilar cholangiocarcinoma (pCCA) was the indication for ALPPS in 3 patients of whom 2 had type IV and 1 type IIIa tumor according with the Bismuth–Corlette classification. Two of the 3 patients with pCCA had a biliary stent placed endoscopically before surgery to relieve jaundice.Six patients had undergone previous abdominal surgery, namely resection of colorectal cancer (4 patients), appendectomy and cholecystectomy (1 patient), and wedge hepatic resection for intrahepatic cholangiocarcinoma (1 patient).The liver volumes were calculated before the first surgical step and after 7.4±2 days .The volumetric measurement before ALPPS-1 was based on MRI in 5 cases and on CT in 4 cases. The volumetric study before ALPPS-2 was based on CT in 8 cases and MRI in 1 case .In 5 cases, the liver transection was set for right trisectoniectomy; in 4 cases, the resection was extended to segment I. In one patient, it was possible to preserve segment IVb; in this latter case the middle hepatic vein was spared while in all the other patients the middle hepatic vein was ligated and divided, always at the second step of the operation.Five patients required blood transfusions, with a mean required volume of 650±319 cc. No plasma or platelet transfusions were required.Intermittent hilar clamping was performed in 3 patients, with a total duration of 8.33±2.88 minutes.Additional interventions were performed in 4 cases: 3 patients underwent Roux-en-Y hepaticojejunostomy; in one case, a wedge resection of a residual tumor in the FLR was carried out. In all cases, a cholecystectomy was performed (if not done previously).In the first case performed, an organ bag was used to wrap the right liver and separate the two hemi-livers; in the following 5 cases, we used a sheet of plastic material; in the last three cases, we employed sheets of haemostatic TachoSil.In all patients, it was possible to perform the second step. The second surgery had a surgical time of 198±59 min. Blood transfusions were required in 5 patients with an average volume of 400±141 cc. No plasma or platelets transfusions were required; hilar clamping was not performed.Pathology confirmed the preoperative diagnosis in 8 cases; however, in one case where the preoperative diagnosis was intrahepatic cholangiocarcinoma, metastatic colorectal cancer was revealed. A tumor-free surgical margin was histologically confirmed in all cases (R0 resection = 100%). An average of 4.2 lymph nodes per patient was examined, and one case of intrahepatic cholangiocarcinoma was detected as neoplastic involvement.There were no intraoperative deaths. The main postoperative results are described in Eight patients were kept in the hospital in the interval between the two operations, while one patient was temporarily discharged on postoperative day (POD) 6.Klebsiella pneumoniae and Enterococcum faecium. To try to control the abdominal sepsis, the second step of ALPPS was carried out 8 days after the first, although the increase of the liver volume was not completely satisfactory (FLR = 24%), and the patient developed postoperative liver failure and died on POD 21. Remnant liver biopsy revealed moderate cholestasis, mild steatosis and fibrosis.Overall, 11 complications occurred in 6 patients, 4 of which were grade III or above according to the Dindo-Clavien classification. There was one postoperative death due to liver failure (patient 6). In the latter patient, a 73-year-old woman with type IV perihilar cholangiocarcinoma, two weeks before ALPPS-1, an endoscopic internal biliary stent had been placed due to serum bilirubin >10 mg/dL, the FLR (mL) was 192 mL, and the FLR was 16%; at surgery, liver histology showed micro and macrovesicular steatosis of 10%. After ALPPS-1, the patient developed a biliary leak and sepsis caused by multiresistant Intraabdominal sepsis developed after ALPPS-1 in both patients who had endoscopically-placed biliary stent at the time of surgery, while it was observed only in 1 of the other 7 patients with no biliary stent.Candida tropicalis infection of the peritoneal fluid was identified in one patient and controlled with antifungal therapy.Biliary leak was the most frequent postoperative complication that developed in 3 patients. The high-grade leaks (2 cases) were treated and resolved with placement of an endoscopic stent while the low-grade leak resolved spontaneously. Three patients underwent surgical re-operation. Patient 6 required surgical haemostasis of the wound 5 days after ALPPS-2. Patient 7 underwent exploratory laparotomy 4 days after ALPPS-1 to verify the viability of the liver in the presence of a peak of transaminase . Liver biopsy showed severe necrosis of the right liver. Cytolysis gradually diminished over the following days. Patient 9 required re-operation for haemoperitoneum 2 days after ALPPS-2.After a mean follow-up of 17.1±8.5 months, the overall survival was 89% at 3-6-12 months. Recurrence-free survival was 100%, 87.5% and 75% at 3-6-12 months respectively. Four patients  developed a tumor recurrence after 14.7, 19.6, 3.2 and 6.3 months, respectively but they are currently alive.The characteristics of the 9 patients who underwent the ALPPS procedure are described in The literature search yielded 238 articles; after the removal of duplicates, 148 titles and abstracts were reviewed . Of thesFinally, a total of 22 articles, 22–42 pSeveral variations of the classical ALPPS were described, namely:- Associating Liver Tourniquet and Partial ligation for Staged hepatectomy (ALTPS). The aut- "Left ALPPS": ligation of the left portal vein, multiple resections on the right hemiliver and splitting along the main portal fissure, proposed for bilateral liver metastases where a left hepatectomy is required in addition to multiple resections on the right liver.- "Salvage ALPPS": simple splitting of the liver along the main portal fissure several months after a radiological portal vein embolization that did not allow satisfactory liver hypertrophy, 43, 44.- "Right ALPPS": ligation of the posterolateral branch of the right portal vein, left lateral sectionectomy, multiple resections on the right anterior and left medial section and splitting along the right portal fissure; this technique was proposed to deal with tumors that require simultaneous resection of the lateral segments of the left and right liver.- Laparoscopic ALPPS procedures, 45, 46.- “Partial ALPPS” has recently been described preliminarily: in this technique the parenchymal transection during the first step of the procedure is not complete but carried out to obtain a partition of at least 50% of the liver along the resection plane. The authors report lower mortality and rate of complications with this technique as compared to the conventional ALPPS, with a similar degree of hypertrophy of the FLR.Outcomes of interest of each single study are summarized in Tables The median age of the patients who underwent ALPPS ranged between 52.4 and 68 years in the various reports with the youngest patient of 20 and the oldest of 83 years. The median preoperative FLR ranged between 19 and 27%; after a median interval of 6–16 days, all the studies reported an outstanding increase in the volume of the FLR that ranged between 36.1 and 110%. ALPPS procedure achieved a rate of R0 surgical margins that ranged between 86 and 100%. The morbidity was particularly high in all the series (from 36 to 100% of the cases in the single series). Although a few Centers reported mortality-free experiences, the largest series available (ALLPS registry) recorded a 9% postoperative mortality. Median hospital stay ranged between 11 and 56.7 days.No intention-to-treat analysis was available that reported the number of planned ALPPS procedures that were eventually not performed due to intraoperative findings during the first step.The ALPPS technique has recently been described as an alternative to preoperative embolization or ligation of the right branch of the portal vein when extended liver resections are required in the presence of insufficient FLR. In fact, the strategy of PVO has some important limitations: 3 to 8 weeks are necessary to develop sufficient hypertrophy of the FLR; therefoThe ALPPS procedure seems to overcome these limitations as it rapidly induces a consistently more pronounced hypertrophy of the FLR compared with conventional procedures. The presumed mechanism of the quick hypertrophy of the FLR is the interruption of the cross portal circulation between the 2 hemilivers that allows a complete diversion of the portal flow to the FLR. In addiA peculiarity of our series is that the majority of the patients were treated for tumors different from CRLM which was instead the most common indication in the ALPPS registry (70% of the cases) and was associated with better survival. Besides, 3 of our patients had pCCA, a disease that is considered to be at greater risk of complications and perioperative mortality; in the ALPPS registry there were only 11 (5.4%) cases of pCCA and a 27% postoperative mortality was recorded in these patients while in the single-Institution series that included the highest number of pCCAs (5 patients) the operative mortality was 60%, 38.A recent comprehensive meta-analysis on the use of PVE to increase operability of cholangiocellular carcinoma has shown that this is effective when the mean initial FLR is 33.8%; of 836 cases included in that analysis, no one had a FLR below 30%. The efficacy of PVE in cases similar to those reported in the present series, whose FLR was well below 30%, is therefore far from being demonstrated.Although successful ALPPS procedures in patients with hepatocellular carcinoma on cirrhosis have been reported, in our policy chronic liver disease is currently considered a contraindication to surgery , 52, 53.There was a large variability in the increase of FLR, from 24% to 159% and the lowest increase in the liver volume was observed in the only patient who did not survive. Alongside the increase of FLR after ALPPS-1, in four cases an increase in the volume of the liver tumors was observed. This interesting finding may be the result of the arterialization of the right hemiliver that follows the ligation of the right branch of the portal vein. In contrast to the normal parenchyma, neoplastic lesions base their blood supply exclusively on the arterial inflow; consequently, arterialization of the tumor lesions in a deportalized right liver can produce an increased tumor volume.In our experience, sufficient hypertrophy of FLR was obtained in 10.8 days: this time span is significantly lower than the average time required when using PVO (40 days), minimizing the risk of progression of the tumor between the two steps of the procedure , 17.Our experience confirms that a high incidence of postoperative complications can be expected after ALPPS: 67% of our patients experienced complications that were grade 3 or above in 44% of the cases. The main complications reported in the literature were biliary fistulas, sepsis and infection. In our series, the most frequent complication was biliary fistula (3 cases). These data are similar to those reported in other series, however, we treated many more patients with CCA and, in particular, pCCA; consequently, an even higher risk of biliary complications might have been expected.The presence of a biliary stent placed to treat the biliary obstruction caused by pCCA was a predisposing factor for intraabdominal sepsis: this latter complication was observed in 2 of 2 patients with preoperative stenting and in 1 of 7 patients with no preoperative stenting.In our series, all liver resections were oncologically radical, consistent with what was reported in most reports from the literature, 32, 36.Overall, the one-year survival in our study was 89%. This result is relevant because several cases of biliary tumors were offered treatment in contrast to the majority of the other series where biliary cancer was considered a contraindication to ALPPS. As the In the patient who did not survive, several risk factors for postoperative complications (presence of a biliary stent) and poor outcome (age >70 and presence of fibrosis at liver histology) were concomitant. Sepsis that developed after step 1 also contributed to impair the hypertrophy of the FLR in this patient and the attempt to remove the source of the sepsis by completing the ALPPS was unsuccessful: we would therefore recommend to focus on the treatment of the sepsis and delay the completion of the ALPPS in similar circumstances.Based on our experience, we believe that ALPPS should be indicated in patient of less than 70 years of age with no major comorbidities and no chronic liver disease as confirmed by the absence of fibrosis at liver biopsy; liver biopsy should be performed before proceeding to liver partition. In view of the morbidity and mortality that are associated with ALPPS, this procedure should be offered in first instance only when the intrahepatic diffusion of the tumor makes it hazardous to delay its excision in wait for the hypertrophy of the FLR to be achieved through PVO techniques.In our centre, we perform the conventional ALPPS technique. Several variants of the conventional technique have been described: some of these techniques such as “left” and “right” ALPPS can be useful in particular anatomical distributions of the tumors, mainly in case of CRLMs. Among the other variations proposed, “partial ALPPS”, seems to be able to induce an hypertrophy of the FLR similar to that observed in the conventional ALPPS and reduce postoperative mortality and complications by avoiding the complete partition of the liver during step 1; if these findings were confirmed on larger numbers , partial ALPPS could well become the technique of choice.The latest evidence suggests that the use of a plastic bag to separate the two hemi-livers should be avoided because the presence of foreign material in the abdomen seems to correlate with the development of infectious events and adhesions, 54. FurThe main limitations of this study were the low sample size and lack of a comparison with alternative techniques, such as PVE or PVL. Future studies will need to compare this technique with alternative ones, although the excellent results reported in the literature might pose ethical issues. A prospective, randomized comparison among the PVE, PVL and ALPPS techniques is ongoing presently with several participating centres worldwide.The systematic review of the literature has shown a growing worldwide interest in this novel surgical technique. The results of the ALPPS that are reported in the literature are promising however, many of the published articles lack of a detailed report of the postoperative morbidity according with Dindo-Clavien classification and long-terms results of the procedure need to be further analysed and validated through a prospective, multicentre randomized study.In conclusion, our initial experience with liver resections using the ALPPS technique showed that the procedure is effective in patients with insufficient FLR. The morbidity was high in accordance with the data from the literature, and we had one mortality. However, it has to be noted that all the patients in this series had diseases with an ominous prognosis with no alternative effective treatment available.S1 Appendix(DOC)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


Shamo) and the long-crowing chicken (Naganakidori), have been selectively bred for dramatically different phenotypes. The former has been selected exclusively for aggressiveness and the latter for long crowing with an obedient sitting posture. To understand the particular mechanism behind these genetic changes during domestication, we investigated the degree of genetic differentiation in the aforementioned chickens, focusing on dopamine receptor D2, D3, and D4 genes. We studied other ornamental chickens such as Chabo chickens as a reference for comparison. When genetic differentiation was measured by an index of nucleotide differentiation (NST) newly devised in this study, we found that the STN value of DRD4 for Shamo (0.072) was distinctively larger than those of the other genes among the three populations, suggesting that aggressiveness has been selected for in Shamo by collecting a variety of single nucleotide polymorphisms. In addition, we found that in DRD4 in Naganakidori, there is a deletion variant of one proline at the 24th residue in the repeat of nine prolines of exon 1. We thus conclude that artificial selection has operated on these different kinds of genetic variation in the DRD4 genes of Shamo and Naganakidori so strongly that the two domesticated varieties have differentiated to obtain their present opposite features in a relatively short period of time.The chicken domestication process represents a typical model of artificial selection, and gives significant insight into the general understanding of the influence of artificial selection on recognizable phenotypes. Two Japanese domesticated chicken varieties, the fighting cock ( Gallus gallus domesticus) can generally be classified into two categories according to purpose, namely entertainment and consumption. In particular, chickens domesticated for entertainment have attracted people since ancient times with their colorful appearance, masculine posture, and characteristic songs. Lines of evidence also show that chickens were used as religious and political symbols in ancient cultures Domesticated chickens  Kojiki (712 A.D.) and Nihon Shoki (720 A.D.) In our examination of historical records, we found a description of the fighting cock in the classical Chinese book written in 517 B.C. Shamo) and long-crowing chickens (Naganakidori) to study the relationships between human culture and domestication breeding efforts.In the present study, we examined fighting cocks and long-crowing chickens, two extreme examples of domesticated chickens, to study genetic differentiation affected by artificial selection. The former has been bred for cockfighting with high aggressiveness and the latter for long crowing. Although a variety of cultural aspects toward raising and keeping fighting cocks and long-crowing chickens have been observed not only in Japan but also in other Asian countries such as Thailand, Indonesia, and China, Japanese varieties are known to be maintained in a more rigorous fashion. For example, in Japan, breeding stock of these valuable species have never been interbred with other species; moreover, breeders almost never trade their breeding stock of females with each other Shamo and Naganakidori. To accomplish this goal, we performed comparative analyses of the dopamine receptor genes DRD2, DRD3, and DRD4 for three groups of Japanese domesticated chickens: Shamo; Naganakidori; and one other ornamental chicken, the Chabo, as a control. We also analyzed the proline repeat of the DRD4 protein, and predicted the first 3D structures of the receptors of the DRD4 gene.The primary goal of this study is to further elucidate the effects of long-term artificial selection among Dopamine receptors receive dopamine, a catecholamine that has a crucial physiologic role in the regulation of movement, reward, cognition, and emotion STN, which can measure the degree of evolutionary differentiation in populations and can also be applied in subdivided populations that differentiated over a relatively short evolutionary time. By applying this new index to the dopamine receptor genes of Shamo, we could examine the evolutionary differentiation of specific characteristics in other domesticated chickens. To understand the genetic changes that occurred during domestication for specific purposes, we investigated the degree of genetic differentiation in the effects of artificial selection in these two chicken varieties, specifically focusing on dopamine receptor genes.Since we intended to study the effects of artificial selection on the two varieties, we devised a new index, We sampled blood from eight fighting cocks, nine long-crowing chickens, nine other ornamental chickens, one red junglefowl, and one pheasant. For the 28 samples from the three chicken groups we sequenced DRD2 on chromosome 24 from exon 1 to exon 7 and introns (approximately 3100 bps), DRD3 on chromosome 1 from exon 1 to exon 6 and introns (approximately 2800 bps), and DRD4 on chromosome 5 from exon 1 to exon 4 and introns (approximately 2300 bps). The total number of base pairs sequenced thus was approximately 8200 bps. Therefore, we selected regions surrounding each of 41 sites from the DRD2 gene, 20 sites from the DRD3 gene, and 29 sites from the DRD4 gene, giving a total of 90 segregating sites for analysis .STF) STF values were estimated as pairwise STF values between focal population samples and the other samples  was the highest among the populations, we did not find a clear difference in the values between the three populations. Therefore, we devised STN, which can also indicate the evolutionary differentiation between a pair of populations for a nucleotide site as a unit. STN is an updated version of STF in that we now know that a point mutation occurs at a nucleotide site. In addition, while we need to estimate hetero sites  to compute STF, we do not need to do it to compute STN.To measure the degree of the evolutionary differentiation among the three domesticated chicken groups, we computed the fixation index  is distinctly larger than that of the other groups.We applied Shamo for greater aggressiveness in fighting.From our results, it is evident that DRD4 is most responsible for the evolutionary differentiation of STN values between Shamo and the other two groups for the DRD4 gene. A human-rodent comparative study also showed that DRD4 is one of the most rapidly evolving genes in a group of 141 neurotransmitter receptor genes th residue (pro24del) in the nine-proline repeat of exon 1. Our result is in agreement with that of Sugiyama et al. Shamo, red junglefowl, and P. versicolor have nine proline repeats each, the DRD4 protein of Naganakidori (Koeyoshi and Tomaru breeds) has eight repeats. As three prolines each form a unit in the protein 3D structure, eight repeats is considered to be less advantageous than nine repeats. This structure has three residues per turn and many of the repetitive sequences have a periodicity of three. Other extracellular proline-rich proteins appear to act by binding to proteins on the cell surface and influencing cell-cell recognition Our analysis demonstrated that there was a significant difference in the mean 1-adrenergic receptor (PDB code: 2vt4) Naganakidori. The irregular structure prevents the chicken DRD4 from properly reacting with the ligand.To confirm the protein interaction, we predicted the 3D structure of DRD4 for the first time by using the MODELLER function of the Insight II program package version 97.2 (BioSym Technologies). As it required a template, we again obtained the crystal structure of the turkey βShamo, Naganakidori, and other ornamental chickens, because they are typical animals in Japanese culture. The oldest Japanese chronicle, Chôjû GigaShamo already existing at that time. Those ancient records therefore indicate that Japanese people began breeding chickens to produce their favorite breeds more than 1000 years ago The chicken has spread worldwide and differentiated into many varieties to suit specific purposes including food production and entertainment. In this study, we analyzed the evolutionary differentiation of three Japanese domesticated groups, STGSTijN. As mentioned above, even if we could not measure the degree of population diversity at the gene level, we would be able to measure it at the nucleotide site level in introns or non-coding regions. This proved true for the three dopamine receptor genes, as shown in STiN value of DRD4 for Shamo is distinctively larger than that of the others, demonstrating that it has been the most differentiated in evolution among the three receptor genes studied. It is possible that artificial selection has operated most heavily on DRD4 among the three populations, indicating that positive selection has diversified DRD4 more than in the other breeds. STiN of Shamo for DRD4 (0.072) is distinctly larger than that for the other breeds. We took this to imply that DRD4 has a more prominent role than DRD2 and DRD3 in controlling the behavior in chickens. From these results in fighting cocks, artificial increases in aggression might have necessarily led to DNA variations. While there are several genes related with aggressiveness in the animals, we suggest that one of the possible ones is the DRD4 gene examined in this analysis.We investigated difference in selection pressure during artificial selection on the behavior of the chickens in question. For that we chose the three dopamine receptor genes DRD2, DRD3, and DRD4 which are known to be responsible for aggressiveness, among other animal behaviors. Aggressiveness is a very important evolutionary factor for winning mates in order to produce offspring. We then assumed that without knowledge of the mechanics of chicken breeding over the past 1000 years, Japanese people never realized that aggressiveness is adaptive STijN values of DRD4 for Shamo were highest among the three groups; therefore, we can now confirm that the genetic basis for this behavior, particularly the aggressive fighting disposition that is essential for Shamo to attract mates, is in fact the DRD4 gene, although we cannot completely rule out DRD2 or DRD3. This finding suggests that ancient Japanese people performed more artificial selection on fighting cocks than they did on the other breeds of chicken, perhaps because cockfighting was more appealing as entertainment in those days.Our ancestors utilized artificial selection to produce increasingly stronger fighting cocks through repeated breeding without knowing the mechanics of the process. In the present study, the Naganakidori (Koeyoshi and Tomaru breeds) might have been subject to selection according to different proline repeats. Crowing may be controlled more directly by genes other than dopamine receptor genes, because the long-crowing chicken has a deficient 3D structure of DRD4. Our prediction of the 3D structure of DRD4 revealed that a lack of proline repeats in an extracellular region made it less effective at reacting with its ligand, enabling Naganakidori to keep still and quiet except during long crowing.Naganakidori breed, Koeyoshi , long-crowing chickens , other ornamental chickens (nine samples), and one red junglefowl , from the Bird Center of Kurume in Fukuoka prefecture. We also took samples from a pheasant (Phasianus versicolor) from the Nogeyama Zoological Gardens of Yokohama in Kanagawa prefecture. These domesticated chickens had been collected in our previous studies The varieties of chicken examined in this study are given in Escherichia coli (DH5α). Transformants were plated onto LB-agar plates containing the appropriate amounts of ampicillin, X-gal, and IPTG. White recombinant bacteria were grown in 2×LB broth containing ampicillin (100 µg/ml) and the plasmid DNAs were purified using a Montage Plasmid Miniprep Kit . The plasmid DNAs were used as templates and directly cycle-sequenced using M13 (-20) f and M13r primers and a Big Dye Terminator Ver.3.1 Cycle Sequencing Kit (ABI). Last, nucleotide sequences were analyzed on an ABI 3730xl automated DNA sequencer.Blood was taken from each chicken, and each sample was suspended in 400 µl of TNES-8M urea. Twenty microliters of proteinase K (20 mg/ml) and 1M dithiothreitol (DDT) were added to the samples, which were then incubated for 1–5 h at 60°C. Each incubated sample was subsequently mixed with 500 µl of a phenol/chloroform/isoamyl alcohol solution (25∶24∶1) for 3 min; this was repeated twice. After precipitation with 2–2.5 vol. of ethanol, the pellets were rinsed in 70% cold ethanol and dried. The samples were then dissolved in TE buffer . Regions of the dopamine receptor genes were amplified using the polymerase chain reaction (PCR), the primers for which are given in SNT among the three chicken groups. After excluding gaps the total number of nucleotides for each sequence was 8200 bp.The 28 nucleotide sequences surrounding each of DRD2, DRD3, and DRD4 were aligned using CLUSTAL W STF statistics were estimated by the method of Hudson et al. using in-house Perl script DNA haplotypes were estimated using PHASE  1-adrenergic receptor (PDB code: 2vt4) as a template We predicted the 3D structure of the chicken DRD4 protein by using the homology modeling software, MODELLER Table S1List of 90 bp segregation sites of the DRD2, DRD3, and DRD4 genes in each of the three domesticated varieties. The position of segregation sites were referenced No. GCA_000002315.2 into the Galgal 4 database of Ensembl. R:G or A, Y:T or C, M:A or C, K:G or T, S:G or C, W:A or T.(XLS)Click here for additional data file.


People with intellectual disability have significantly higher age-adjusted rates of mortality and morbidity (including obesity) than their non-disabled peers. They are also significantly less likely to be physically active.Understanding Society, a new longitudinal study focusing on the life experiences of UK citizens. Interviews were undertaken with 50,994 individuals aged 16 and over in Wave 1 and 54,585 in Wave 2. Of these, 520 participants age 16–49 (1.8% of the unweighted age-restricted sample) were identified at either Wave 1 or Wave 2 as having self-reported intellectual impairments.Secondary analysis of de-identified cross-sectional data from the first two waves of British adults with self-reported intellectual impairments have higher rates of obesity, inactivity, tobacco and alcohol use and poorer nutrition than their non-disabled peers. Adjusting risk estimates for between group differences in age, gender and exposure to material hardship indicated that a significant proportion of their increased risk of obesity, tobacco use and poorer nutrition may be attributable to their poorer living conditions .People with intellectual disabilities should begin to be regarded as a ‘vulnerable’ group in the context of public health policy and practice. Intellectual disability refers to a significant general impairment in intellectual functioning that is acquired during childhood . While eHealth inequalities among adults with intellectual disability are particularly stark in relation to obesity and physical activity levels. There is now substantial evidence to suggest that people with intellectual disabilities are much more likely to be overweight or obese than the general population -28. IncrLess is known about the prevalence of other pertinent health behaviours in people with intellectual disabilities. Studies in the UK and Australia have reported low intake of fruit and vegetables among adults with intellectual disability ,28. FeweCurrent knowledge indicates that the reasons for the poorer health of people with intellectual disability primarily fall within two broad spheres. First, a range of secondary health conditions are associated with some specific causes of intellectual disability . Second, people with intellectual disability are much more likely than their non-disabled peers to be exposed to a range of well-established social determinants of poorer health  .The aims of the present paper are: (1) to describe rates of obesity and pertinent health behaviours in British adults with and without intellectual disability; and (2) to estimate the extent to which any between-group differences in health status may reflect between-group differences in rates of exposure to socio-economic disadvantage.Understanding Society[http://www.data-archive.ac.uk/). Full details of the surveys’ development and methodology are available in a series of reports [We undertook secondary analysis of de-identified cross-sectional data from the first two waves of g Society. Underst reports -44, key Understanding Society is a new longitudinal study focusing on the life experiences of UK citizens. In the first wave of data collection (undertaken between January 2009 and December 2011), random sampling from the Postcode Address File in Great Britain and the Land and Property Services Agency list of domestic properties in Northern Ireland identified 55,684 eligible households. Interviews were completed with 50,994 individuals aged 16 or older from 30,117 households, giving a household response rate of 54% and an individual response rate within co-operating households of 86% [s of 86% ,44. At WData collection was primarily undertaken using Computer Assisted Personal Interviewing. In Wave 2 health data (including height and weight) were collected by nurses for a subsample of 15,591 members of the cohort.Understanding Society does not include information on the formal diagnosis of intellectual disability. As a result, we identified adults with self-reported intellectual impairments on the basis of self-reported long-term impairment or disability associated with difficulties in learning or understanding coexisting with low self-reported educational attainment. Low self-reported educational attainment was used as a selection criterion for two reasons: (1) to exclude from the intellectual disability group respondents with specific learning difficulties such as dyslexia; (2) as evidence that the person’s self-reported difficulty may have originated in childhood. Due to historical changes in educational qualifications and attainment in the UK, we further restricted our analysis to the age range 16–49.Understanding Society uses a two-stage process for identifying participants with impairments or disability. First all participants are asked ‘Do you have any long-standing physical or mental impairment, illness or disability? By ‘long-standing’ I mean anything that has troubled you over a period of at least 12 months or that is likely to trouble you over a period of at least 12 months.’ If participants respond in the affirmative they are then asked ‘Does this/Do these health problem (s) or disability (ies) mean that you have substantial difficulties with any of these areas of your life? Please read out the numbers from the card next to the ones which apply to you.’ One response option is ‘Memory or ability to concentrate, learn or understand’. We initially identified adults in Understanding Society with self-reported intellectual impairments on the basis of their positive response to this routed question. We then excluded from this group participants who did not meet the additional criteria of having a highest level of educational attainment lower than a General Certificate of Secondary Education or equivalent. Overall 22.5% of Understanding Society participants reported lower than this level of attainment. This process identified 520 participants age 16–49 (1.8% of the unweighted age-restricted sample) at Wave 1 or Wave 2 as having self-reported intellectual impairments. In the Wave 2 subsample for which nurse measurements were taken, 142 participants age 16–49 (2.0% of the unweighted age-restricted sample) were identified as having self-reported intellectual impairments.Understanding Society cohort. The interview undertaken at Wave 2 collected self-report information on nutrition , physical exercise , smoking (ever and currently) and alcohol use .In Wave 1 self-reported weight and height was collected and from these data BMI was calculated and obesity determined as BMI > 29. At Wave 2, weight and height were measured by a visiting nurse for a subsample of the Next we have some questions about the sorts of things that some families/people have but which many people have difficulty finding the money for. For each of the following things please tell me the number from the showcard which best explains whether you (and your family/partner) have it or not. Do you (and your family partner) have… (1) A holiday away from home for at least one week a year, whilst not staying with relatives at their home? (2) Friends or family around for a drink or meal at least once a month? (3) Two pairs of all weather shoes for all adult members of the family? (4) Enough money to keep your house in a decent state of repair? (5) Household contents insurance? (6) Enough money to make regular savings of £10 a month or more for rainy days or retirement? (7) Enough money to replace any worn out furniture? (8) Enough money to replace or repair major electrical goods such as a refrigerator or a washing machine, when broken?’ The response options for each item were ‘1 I/We have this, 2 I/We would like to have this but cannot afford this at the moment, 3 I/We do not want/need this at the moment, 4 Does not apply’. A hardship score (0–8) based on the number of items that could not be afforded was derived for each participant.Socio-economic disadvantage was measured by a hardship scale and a measure of self-assessed financial status. The hardship scale, which was only administered at Wave 1, contained eight items: ‘How well would you say you yourself are managing financially these days? Would you say you are… 1 Living comfortably, 2 Doing alright, 3 Just about getting by, 4 Finding it quite difficult or 5 finding it very difficult?’.Self-assessed financial status was assessed at Waves 1 and 2 by a single item: ‘Risk of self-reported intellectual impairments was unrelated to gender, but was significantly related to socio-economic disadvantage , p < 0.001). The prevalence of obesity and health behaviours (for men and women separately and combined) was first summarised through simple descriptive statistics (percentages). Unadjusted estimates of risk associated with self-reported intellectual impairments (odds ratios) are presented followed by estimates adjusted (using multivariate logistic regression) for between-group differences in: (1) age and gender; and (2) age, gender, material hardship and self-assessed financial status. Results are presented in Table As can be seen, unadjusted risk for obesity and all indicators of less healthy behaviours was significantly greater among adults with self-reported intellectual impairments (risk range 1.78 (Wave 1 obesity) to 3.44 (current smoker)). Unadjusted risk for obesity and indicators of less healthy behaviours was significantly greater for both men and women with self-reported intellectual impairments with just two exceptions; obesity was not significantly greater for men, alcohol consumption was not significantly greater for women. Adjusting risk estimates to take account of between-group differences in age and gender had little impact. Further adjusting risk estimates to take account of between-group differences in exposure to material hardship and self-assessed financial status reduced the risk associated with self-reported intellectual impairments for obesity and all indicators of less healthy behaviours with one exception, alcohol use. For 17 of the 30 analyses (57%) this adjustment resulted in statistically significant reductions in risk associated with self-reported intellectual impairments. These effects were most marked in relation to nutrition and smoking.While obesity rates based on nurse measures were markedly higher than obesity rates based one year previously on self-reported height and weight, there was a strong association between the two measures with 90% of people identified as obese at Wave 1 also being identified as obese at Wave 2 , p < 0.001).Our results indicate that: (1) British adults with self-reported intellectual impairments have higher rates of obesity, inactivity, tobacco and alcohol use and poorer nutrition than their non-disabled peers; and (2) that a significant proportion of their increased risk of obesity, tobacco use and poorer nutrition may be attributable to their poorer living conditions .These results add to existing knowledge about the health inequalities faced by people with intellectual disability in four important ways. First, they are based on the analysis of contemporary population-based sampling frames, a relative rarity in this field of study .Second, being based on samples drawn from general households participants are likely to include adults with less severe intellectual disability who may not be in receipt of specialised disability services. Given that most intellectual disability research is based on convenience samples drawn from the users of specialised disability services , very little is currently known about the health or well-being of this group that has been termed the ‘hidden majority’ of adults with intellectual disability ,36,45. TThird, by adjusting risk estimates for between-group differences in exposure to material hardship and self-assessed financial status, the results add to the growing literature suggesting that a significant proportion of the health inequalities experienced by people with intellectual disabilities may be attributable to their poorer living conditions  ,46. ThusFinally, the present study is one of the few in this field to stratify results by gender . ImportaUnderstanding Society are also likely to exclude people with more severe intellectual disability from participating. Consequently, the results are likely to be particularly relevant to understand the health of British adults with less severe intellectual disability. Fourth, whilst obesity was assessed by nurse measures at Wave 2, all other health behaviours were self-reported and it is not possible to assess the accuracy of such self-reports. Finally, while the cross-sectional analyses presented in this paper are consistent with the hypothesis that the poorer health of adults with intellectual disability may be partially attributable to their poorer living conditions, it is not possible to rule out other explanations .However, there are five limitations to the study that should be kept in mind when considering the salience and implications of these results. First, it is not possible to estimate the sensitivity or specificity of the method used to identify participants with potential intellectual disability. Hence our use of the term self-reported intellectual impairments. It should be noted, however, that: (1) the overall prevalence rates lies within the expected boundaries for intellectual disability ,3; (2) tBritish adults with self-reported intellectual impairments have higher rates of obesity, inactivity, tobacco and alcohol use and poorer nutrition than their non-disabled peers. A significant proportion of their increased risk of obesity, tobacco use and poorer nutrition may be attributable to their poorer living conditions . Effectively addressing health inequalities requires policies that are sensitive to the particular personal and social contexts of ‘vulnerable’ groups . People The authors declare that they have no competing interests.JR participated in the design of the study and coordinated the drafting of the manuscript. EE participated in the design of the study, performed the statistical analysis and helped to draft the manuscript. SB and CH participated in the design of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/14/219/prepub


The intravitreal anti-vascular endothelial growth factor treatments ranibizumab and aflibercept have proven efficacy in clinical trials, but their real world usage in central retinal vein occlusion (CRVO) has not been assessed. We therefore evaluated the treatment patterns of both drugs in a US claims database.The IMS Integrated Data Warehouse was used to identify the patients with CRVO in the USA with claims for ranibizumab or aflibercept between 24 September 2012 and 31 March 2014 with at least 12 months follow-up. Patients were required to have had no anti-VEGF treatment code for 6 months before index (‘treatment-naive'). Mean numbers of injections and non-injection visits to a treating physician were compared with patients receiving these treatments.n=206) or aflibercept (n=79) at index. The mean (±SD) numbers of injections received by patients treated with ranibizumab or aflibercept were 4.4±2.8 and 4.7±2.9 (P=0.38), respectively; the total number of patient visits to their treating physician was 7.3±3.7 and 7.0±2.9 (P=0.52), respectively. For patients receiving one or more injections (n=238), the mean interval between injections was 55.1 days (ranibizumab) and 54.2 days .Patient characteristics were similar for patients receiving ranibizumab (Our results suggest that, in routine clinical practice, patients receive a comparable number of injections in the first year of treatment with ranibizumab or aflibercept. This may have implications for commissioning and service development of CRVO care pathways. As a result, blood and plasma are discharged into the retina, often causing complications including macular edema and varying degrees of ischemia, potentially leading to severe vision loss. Although occlusion of the central retinal vein ) occurs less frequently than in branch veins, it is associated with severe visual outcomes.Macular edema secondary to retinal vein occlusion (RVO) can cause severe visual impairment owing to obstruction of the retinal vasculature, and is the second most common retinal vascular disease.5 Ranibizumab  is a humanized, affinity-matured VEGF antibody fragment that binds to and neutralizes all isoforms of VEGF. Ranibizumab is recommended to be given monthly based on the evidence from clinical trials.6 The efficacy of ranibizumab for the management of CRVO has been reported in multiple studies including the Randomized Study Comparing Ranibizumab to Sham in Patients with Macular Edema Secondary to CRVO (ROCC)7 and the Ranibizumab for the Treatment of Macular Edema After CRVO Study (CRUISE);9 intravitreal injections of ranibizumab provided rapid improvement in 6-month visual acuity and macular edema following CRVO, with low rates of adverse events.8 These improvements were largely maintained with a subsequent 6 months of dosing as required (pro re nata (PRN)).9 Ranibizumab was approved for treatment of macular edema secondary to CRVO by the US Food and Drug Administration (FDA) in June 2010.10Anti-VEGF therapy is now the standard of care for CRVO, replacing the previous observation-only approach.11 Patients should subsequently be monitored regularly, and treatment should be resumed if visual outcomes deteriorate. Two recent clinical trials (VEGF Trap-Eye: Investigation of Efficacy and Safety in CRVO (GALILEO)13 and VEGF Trap-Eye for macular edema secondary to CRVO (COPERNICUS)15) have shown that monthly intravitreal aflibercept treatment was well tolerated and improved visual acuity after 6 months significantly more than sham injections; these improvements were maintained with subsequent monthly monitoring and PRN dosing.12 Aflibercept was approved for the treatment of macular edema secondary to CRVO in September 2012.16Aflibercept is a fully human, recombinant fusion protein that targets VEGF-A, VEGF-B, and placental growth factor. Aflibercept binds all isoforms of VEGF-A with high affinity—a markedly higher affinity than that of ranibizumab. Like ranibizumab, aflibercept is recommended in the USA to be given as monthly intravitreal injections.Despite promising results from clinical trials as described above, real world usage of aflibercept and ranibizumab in CRVO has not yet been studied. This study therefore aimed to assess the treatment patterns of ranibizumab and aflibercept for the management of macular edema secondary to CRVO in routine clinical practice in the USA using a large, patient-level, physician-entered claims database.This retrospective study was based on the analysis of US physician-level claims data from the Integrated Data Warehouse , a claims database that encompasses ∼1 billion professional fee claims per year, representing ∼80% of practicing eye care specialists  and covering all 50 states. Approximately 95% of claims submitted for payment from these sources are available for analysis within 3 weeks.The study included adult patients with a first medical claim registered in the IDW with a procedure code for intravitreal injection of ranibizumab or aflibercept between 24 September 2012 and 31 March 2014, and with a concomitant diagnosis of CRVO ; this first claim was defined as the patient's index date. Patients were required to have at least 12 months of follow-up data (post index date) within this study period and a minimum of 6 months of available data in the IDW before the index date. The physician administering the index medication was required to have consistently submitted medical claims to the IDW during the 6 months before the index date and during the follow-up period (‘physician stability' criteria). Patients were excluded from the analysis if: their records indicated that they had received an anti-VEGF injection during 6 months before the index date (ensuring ‘naivety'); if they received more than one anti-VEGF drug within 12 months after the index date . The last assumption was relaxed in the sensitivity analysis to assess the number of any anti-VEGF injections received by patients starting on ranibizumab and aflibercept.The primary analysis assessed the number of injections received, non-injection visits made and total visits  made by treatment-naive patients (defined as having received no anti-VEGF treatment claim in the 6 months before the index date) who were treated continuously  with their index therapy for at least 12 months (365 days). Mean dosing intervals (number of days between the injections) were determined for the first year of therapy for patients starting on either treatment and receiving at least two injections.P-values were adjusted for baseline characteristics. Negative binomial regression was used to compare the effect of patient characteristics on injection and visit estimates for those treated continuously with ranibizumab and aflibercept for at least 12 months. A generalized estimating equation (GEE) model applied at the patient level was used to compare the effect of patient characteristics on dosing interval estimates for patients having received two or more injections. The nesting assumption is reviewed in the discussion section. Finally, an autocorrelation of order 1 was used for within-cluster correlation.Differences between the treatment patterns of ranibizumab and aflibercept were assessed, and reported t-tests, with P<0.05 used to define a significant difference. Categorical variables were assessed using Fisher's exact test.Several sensitivity analyses were performed: we assessed the mean number of injections, non-injection visits, and total visits including anti-VEGFs other than that given at index (‘any anti-VEGF'); and we assessed the first 6 months of data to see if there were between-group differences. We also assessed the baseline characteristics of the patients receiving only one injection compared with those receiving multiple injections, to assess whether this patient subset could confound the analyses. For continuous variables, between-group statistical differences were assessed using unpaired Student's n=206; aflibercept, n=79; 17 that occurred in >5% of patients in either group.In total, 285 patients were treated continuously with their index drug over 12 months , respectively, and that of non-injection visits was 2.8±2.6 and 2.2±2.1 (P=0.06), respectively (P=0.52) for ranibizumab and aflibercept, respectively. Patients received an injection on the majority of their visits to their prescribing physician . For patients receiving one injection or more (n=238), the mean interval between the injections was 55.1 days for patients treated continuously with ranibizumab and 54.2 days for patients treated continuously with aflibercept (P=0.44). The details of the GEE model used to adjust for the effects of patient characteristics on mean interval length are shown in For patients treated continuously with ranibizumab or aflibercept, the mean±SD number of injection visits during the first 12 months of treatment , the numbers of all anti-VEGF injections received in the first 12 months of follow-up were 4.7±2.7 and 4.8±2.9 (P=0.59) for patients starting on ranibizumab and aflibercept, respectively (when adjusting for baseline characteristics). The according number of non-injection visits were 3.0±2.6 and 2.3±2.2 (P<0.05) and the total number of visits were 7.6±3.6 and 7.1±2.9 (P=0.25), respectively.When the inclusion criteria were extended to include any additional anti-VEGF treatment claims during follow-up , 78.7% made more than one subsequent non-injection visit to their physician .Of patients receiving only one injection during follow-up , 4.5% of patients were observed to have received bilateral treatment at some point during follow-up .The majority of the 285 patients included in the primary analysis received treatment in only one eye throughout follow-up, with only 3.5% of patients receiving bilateral treatment at any point during follow-up . When the inclusion criteria were relaxed to include any anti-VEGF treatment received during follow-up , diabetic macular edema  or neovascular age-related macular degeneration  in the 6 months before index. One patient had a recorded claim for DME within the first year after index; claims relating to glaucoma associated with vascular disorders and nAMD were not observed during follow-up.This patient-level claims database analysis is, to our knowledge, the first to directly compare the patterns of ranibizumab and aflibercept use when given for treatment of CRVO. The main finding is that the number of injections received and total number of visits made by patients continuously treated with their index therapy was not significantly different regardless of whether patients started treatment with ranibizumab or aflibercept. There were no discernible demographic differences between patients in the ranibizumab and aflibercept groups.1In the USA, both the anti-VEGF treatments assessed presently are recommended to be given as monthly intravitreal injections for the management of macular edema secondary to CRVO. However, the presented results suggest that very few patients receive this regularity of injection throughout the first year of treatment. Potentially, this is due at least in part to improved visual outcomes in the patients receiving these anti-VEGF treatments, as has been seen in clinical trials. However, our time sensitivity analysis shows that even in the first 6 months of treatment most patients do not receive monthly anti-VEGF treatment as recommended by the labels of ranibizumab and aflibercept. Furthermore, we have shown that the likelihood of bilateral treatment is low; less than 5% of patients in both the groups were treated bilaterally in the year after index, compared with reports that ∼10% of those with unilateral CRVO will develop the condition bilaterally.18 Furthermore, when the results were extended to include the number of any intravitreal anti-VEGF injections received during follow-up, the mean number of injections received in the 12 months after index was similar between treatment groups and also similar to those received by patients receiving continuous treatment with their index drug, suggesting that the physicians may be using the two drugs interchangeably.The similarity of the injections given and total visits made by patients in the ranibizumab and aflibercept groups suggest that physicians may be using these treatments similarly in routine clinical practice. These results are in alignment with those observed in other ophthalmic indications, where the number of injections and total visits made were similar whether patients received ranibizumab or aflibercept. Another recent US claims database study in patients with nAMD reported that 5.8 (ranibizumab) and 5.5 (aflibercept) injections were given annually.19 we found no reports of glaucoma associated with vascular disorders in the 6 months before index or during follow-up. Other comorbidities such as DME and nAMD were also rare during the study period, with only one observation of a DME code during the follow-up period. The absence of disease overlap indicates that the treatment patterns are representative of patients with unambiguously diagnosed CRVO.Despite previous reports that a significant proportion of patients with CRVO subsequently experience neovascular glaucoma,20 and that injections constitute the majority of the treatment costs associated with these treatments, the observation that the number of injections administered and physician visits for each treatment is very similar suggests that budgetary considerations for both treatments are likely to be similar in routine clinical practice. Therefore, the findings of this study represent important considerations for payers when evaluating the cost effectiveness of these treatments in the real world. These study findings also highlight that the way new therapies are used in practice may differ from recommendations based on the clinical trials, and emphasize the importance of this type of post approval observational study.Given that current market prices for ranibizumab and aflibercept are similar Owing to the relative recency of aflibercept approval and the number of inclusion/exclusion criteria required to product robust results, a large database with rapid upload of data was essential in order to generate sufficient data for analysis. The IDW is one of the largest claims-based databases in the USA, and 95% of claims are available for analysis within 3 weeks of submission. We believe this to be the largest observational study of its type to directly compare ranibizumab and aflibercept. Our sensitivity analyses support the main findings and suggest that the comparable observations made between these two treatment groups are not confounded by differences between groups in the first 6 months of treatment; of the 55–60% of patients receiving four or more injections in the first year after index treatment, over 40% in each group received these injections in the first 6 months of treatment. As no visual acuity data are available in the claims database, it is not possible to conclude that patients who only received one injection did not need additional ones. However, the fact that the vast majority of those patients had one or more follow-up visits post injection is reassuring.There are several limitations in this study. Like any observational studies comparing two treatments, patient inclusion could be subject to selection bias, particularly if there are differences between patients receiving ranibizumab and those receiving aflibercept. However, the similarities of the available baseline characteristics between both the groups of patients suggest that this is not the case. Physicians' approaches to treating CRVO could differ from each other. One potential way to address the physician-level specific would be to have run a GEE model with multiple levels of cluster nesting (physician and patient). However, in this data set, 147 physicians injected both ranibizumab and aflibercept (average number of patients per physicians: 1.9); 118 (80.3%) of these physicians injected only one or two patients. Therefore, there was not sufficient data to run a meaningful analysis with two levels of nesting. As the aflibercept treatment pool grows, the analysis may become more meaningful. In addition, this study involves a relatively small sample size after application of the inclusion criteria, especially for aflibercept. However, the similarity of the injection and total visit results provide no suggestion that our findings are underpowered, and even with extended analyses the results are unlikely to demonstrate any clinically meaningful differences in injections or total visits. This study uses physician-entered claims codes to assess treatment patterns; as such, a risk of misclassification is inevitable, although we believe any such misclassification applies to both treatment groups equally. For example, the lack of neovascular glaucoma during the study period is unlikely to mean that no such events occurred, and more likely reflects that the diagnosis was unrecorded or missed. Last, we were unable to assess the effect of injection frequency on visual outcomes. This is beyond the scope of this type of study and requires further investigation.In conclusion, this study is the first to directly compare treatment patterns of ranibizumab and aflibercept administered for the management of CRVO in routine clinical practice in the USA. The results suggest that these two therapies are not used as recommended by the labels in the USA and that patients receive similar numbers of injection regardless of the treatment with which they initiate therapy. Further studies are warranted to link these findings to visual outcomes and to evaluate whether the treatment patterns observed in this US study represent those in other countries.


Approximately 20 drugs have been approved by the FDA for breast cancer treatment, yet predictive biomarkers are known for only a few of these. The identification of additional biomarkers would be useful both for drugs currently approved for breast cancer treatment and for new drug development. Using glycoprotein expression data collected via mass spectrometry, in conjunction with statistical models constructed by elastic net or lasso regression, we modeled quantitatively the responses of breast cancer cell lines to ~90 drugs. Lasso and elastic net regression identified HER2 as a predictor protein for lapatinib, afatinib, gefitinib and erlotinib, which target HER2 or the EGF receptor, thus providing an internal control for the approach. Two additional protein datasets and two RNA datasets were also tested as sources of predictor proteins for modeling drug sensitivity. Protein expression measured by mass spectrometry gave models with higher coefficients of determination than did reverse phase protein array (RPPA) predictor data. Further, cross validation of the elastic net models shows that, for many drugs, the prediction error is lower when the predictor data is from proteins, rather than mRNA expression measured on microarrays. Drugs that could be modeled effectively include PI3K inhibitors, Akt inhibitors, paclitaxel and docetaxel, rapamycin, everolimus and temsirolimus, gemcitabine and vinorelbine. Strikingly, this modeling approach with protein predictors often succeeds for drugs that are targeted agents, even when the nominal target is not in the dataset. HER2) predicts sensitivity to pertuzumab, trastuzumab and lapatinib. Additional biomarkers would be of significant value for determining the sensitivity of tumors to drugs already approved for clinical use in breast cancer, as well as for new drug development [The U.S. Food and Drug Administration has approved roughly two dozen drugs for breast cancer treatment, but only a few predictive biomarkers are currently available to guide their use. For example, levels of the estrogen or progesterone receptor have been found to predict sensitivity to compounds that interfere with estrogen receptor signaling, and, the over-expression of human epidermal growth factor receptor 2 . One striking finding is that the cell lines vary greatly in sensitivity to various drugs, in some cases by more than four orders of magnitude. While acquired resistance to chemotherapeutics or targeted agents is well recognized and is the subject of intensive study [Large data sets describing the effects of various drugs on the growth of cancer cell lines have recently been generated for the purpose of accelerating the preclinical evaluation of new compounds –8. One ove study , the varWe applied regularized regression to model the intrinsic sensitivities of cell lines to 90 drugs  using thOur glycoprotein dataset includes 185 glycoproteins obtained from 22 breast cancer cell lines. Glycoproteins were collected using a protocol in which the first step, oxidation of the glycans using periodate, takes place on intact cells . After cProtein analyses were carried out on a Thermo LTQ ion trap mass spectrometer and a Thermo Q Exactive Orbitrap mass spectrometer. Spectral counts were used to determine relative expression levels of a glycoprotein in the various cell lines. Aliquots from the same glycoprotein sample produced similar results when analyzed on the two mass spectrometers, although less protein was required for the analyses carried out on the Orbitrap instrument. To combine samples from the two datasets, the data were plotted in a quantile-quantile plot, and a line was fit. Using the slope and intercept, the inverse transform was applied to the Q Exactive data, forcing it to have the same center and dispersion as the LTQ data. For four of the cell lines  there were only Q Exactive data. Spectral counts for these cell lines were normalized to the LTQ data using household proteins. The seven proteins in the LTQ dataset with the lowest coefficient of variation were P20645, Q9BT09, P62937, Q16563, Q9BVK6 Q08722 and P07602. The Euclidean length of the spectral counts for these seven glycoproteins was calculated for both the LTQ data and the Q Exactive data, and the ratio was used to normalize all Q Exactive spectral count data. After combining LTQ and Q Exactive data, glycoproteins with fewer than 100 spectral counts over all the cell lines were dropped from the dataset. For purposes of regression analysis one spectral count was added to all values, and the base ten logarithm taken.Exon array analysis is described in reference The data (47 cell lines) was used as provided in Additional Files Table 2 of reference The multiple reaction monitoring (MRM) mass spectrometry data (27 cell lines) are based on Table S4 of reference glmnet package in the R statistical programming language. One adjustable parameter, λ, sets the amplitude of the penalty term; a second parameter, α, is a weight that determines the mixture of L1 and L2 norm components in the penalty. Letting α = 1 gives lasso regression, and 0 < α < 1 gives elastic net regression. For elastic net regression we incremented α from 0 to 1 in steps of 0.1. For each value of α, we found the best value of λ by cross validation (cv.glmnet function), using the mean squared error (MSE) to evaluate the fit of the model to the data. Plots of MSE as a function of α showed some instability from run to run, so we used the average of 10 runs. The value of α giving the lowest MSE was selected for the elastic net model. These values differed from drug to drug. We performed cross validation by leaving out all pairwise combinations of cell lines; for the glycoprotein dataset (22 cell lines) this is similar to 10-fold cross validation. We found the correlations between each of the 21 cross validation estimates of drug sensitivities for all cell lines and the observed sensitivity values, and finally averaged these correlations. Optimal values of α and λ were determined for each training set in the cross validation as described above.The methods used are elastic net regression and lasso regression . CalculaQuantitative protein expression data may be more useful than mRNA data for predicting the responses of breast cancer cell lines to drugs. In this study we evaluated the ability of a glycoprotein dataset obtained via mass spectrometry to provide explanatory or predictor variables to fit measured drug sensitivities . The druBoth elastic net and lasso regression reduce the number of predictor variables, but they do so to different extents. Elastic net regression models usually have more predictors than do the lasso models for the same drug, as a result the fits to the data are better. The disadvantage of the elastic net method is that with more variables the model may contain some predictors with little statistical or biological significance.ERBB2, UniProt Accession number P04626), Disintegrin and metalloproteinase domain-containing protein 10  and Junctional adhesion molecule A . These three proteins are also among the 92 predictors identified in the elastic net model. Using the three proteins for ordinary least squares multiple regression gives a model in which the fitted drug sensitivities match the observed ones with a correlation coefficient of 0.91. It should also be noted that lasso regression identified HER2 as a predictor for sensitivity to everolimus and temsirolimus, two derivatives of rapamycin. While the lasso models generally do not fit the data as well as the elastic net models, they select fewer variables. For many drugs in this dataset the sensitivities could be fit well with 1–3 predictor variables (see below).Rapamycin illustrates the differences between the two methods. The breast cancer cell lines in our sample vary in their sensitivity to rapamycin by more than four orders of magnitude. The model constructed using elastic net regression had 92 predictor variables, giving a very tight fit to the observed data. Models constructed using lasso regression showed some variability of results over 1000 separate runs, but three predictor proteins appeared in all models . The thrFive of the drugs we examined were developed with the goal of inhibiting the epidermal growth factor receptor (EGFR), or its constitutively active variant, HER2. HER2 is present in the dataset and is over-expressed in three of the 22 cell lines. Lasso regression identifies HER2 as a predictor for the two HER2 inhibitors, afatinib (BIBW2992) and lapatinib, and also for two of the EGFR inhibitors, AG1478 and gefitinib. This finding serves as a positive control for the application of lasso regression to the glycoprotein data.The quantitative relationships between HER2 expression levels and drug sensitivities can be seen in the scatterplots in ERBB2 or P04626), the lasso model included SLC7A5 , BST2  and A2ML1 ; these are the four predictors identified most often in the lasso models. HER2 expression has a fairly high correlation with afatinib sensitivity, 0.65, but the SLC7A5, BST2 and A2ML1 have lower correlations, 0.61, −0.59 and 0.44, respectively. Part of their contribution is to enable the modeling of the afatinib-sensitive cell lines with normal HER2 expression (blue symbols) correctly. A model using only HER2 as a predictor would mistakenly suggest that the cell lines represented with blue symbols are not sensitive to afatinib.By adding one or more predictor variables to HER2, it is possible to fit the sensitivities of the cell lines to gefitinib, AG1478, afatinib and erlotinib, where the relation between sensitivity and HER2 expression is not linear. The elastic net model for afatinib contained nine predictor variables, compared to four in the lasso model. For both models HER2 was one of the predictor variables. The elastic net model gives a somewhat tighter fit to the observed drug sensitivities . Both mo2 values comparing the observed and fitted drug sensitivities) varied from 0.2 to nearly 0.8 (Lasso regression returned a model for 87 of 90 drugs . For eacarly 0.8 . The freFor each drug we found the best (lowest MSE) model with three predictors using the Leaps and Bounds algorithm . The coeThe magnitude of the improvement may be exaggerated somewhat, due to the possibility of over fitting as the number of predictor variables increases and the best models are selected. Over fitting can occur when the model includes variables that by chance reduce the MSE for the model in the sample under study. A model that overfits would probably perform poorly on cell line data that was not used to construct the model. Predictor variables that contribute to overfitting may have little or no biological relevance to the problem of modeling drug sensitivities.While increasing the number of predictor proteins from one to three may allow some overfitting, the proteins selected by the lasso algorithm as predictors often make biological sense. For example, the model illustrated in We used all available cell line data to create and test the models presented so far. A more demanding and realistic approach would involve building a model based on some training data, then using it to predict drug response to other cell lines not in the training set. Given the time and expense involved in obtaining mass spectrometric data, it is more efficient to use a cross validation approach than to expand the data set further. In cross validation the cell lines are divided into a training set and a test set. A regression model built from the training data is then used to predict the drug sensitivities of the test set cell lines. The coefficient of correlation between the predicted and the observed drug sensitivities gives an idea of how well the modeling performs.We carried out cross validation using elastic net regression on our glycoprotein dataset and on two related mRNA datasets: a microarray gene expression dataset that includes 74 drugs [We tested two other publicly available protein datasets for their ability to predict drug sensitivities: a reverse phase protein array (RPPA) dataset, which depends on antibody binding for quantitation , and a dIn the RPPA dataset, the 70 proteins measured were pre-selected by the investigators on the basis of known linkage of the proteins to cancer, including proteins known to be important in the control of signaling pathways, cell proliferation and DNA repair.In the MRM dataset, targeted assays were devised for 317 proteins in 30 breast cancer cell lines, of which 27 lines overlap with the drug response dataset. The proteins were selected by the authors of that study for differential expression across the cell lines. They are found in many cellular compartments and contribute to a wide range of biological processes. Quantitation was achieved by comparing the test signal intensity to that of a reference peptide labeled with a heavy, stable isotope. Only two proteins, HER2 and cadherin E, are common to the glycoprotein, RPPA and MRM datasets.The sensitivities of the cell lines to EGFR or HER2 blockers  were modeled effectively by both RPPA and MRM data, and these drugs often performed well in cross-validation .Summarizing the results so far, one dataset, the RPPA protein dataset, performed less well than the others in modeling using all cell lines, as judged from the distributions of the coefficients of determination . The arrThere are seven phosphatidylinositol-3-kinase (PI3K) inhibitors among the drugs. Three of them, BEZ235, GSK2126458  and GSK 2119563 performed well in both the modeling  and the Rapamycin, everolimus and temsirolimus are related compounds that block the mammalian target of rapamycin (mTOR); the cell lines varied in sensitivity to these drugs over 4.6, 3.3 and 3.7 orders of magnitude, respectively. mTOR is in the RPPA dataset, but was identified with very low probability as a predictor for these drugs . All thrThe sensitivities of the cell lines to paclitaxel and docetaxel varied over smaller ranges than for rapamycin. For both drugs it was possible to find predictive models with high coefficient of determination . Paclita2) = 0.52 (2 =0.36) than does AKTp473 (R2 =0.20). For the Sigma inhibitor, PDK1 alone gives a model with R2 = 0.48; adding AKTp473 does not improve the model further. Modeling triciribine sensitivity failed with these two predictors. For the two drugs in which modeling succeeded, PDK1 is the more useful predictor even though AKT is the nominal target of the drugs.The drug list includes three AKT1 inhibitors: GSK2141795, Sigma AKT 1,2 inhibitor, and triciribine. The RPPA proteins include AKT (AKT1), AKTp473, and PDK1, a kinase that phosphorylates AKT. AKTp473 and PDK1 (or PDK1p241) were found in lasso regression models for all three drugs. A regression model with AKTp473 and PDK1 as predictors allowed the fitting of the GSK2141795 sensitivities with coefficient of determination (R) = 0.52 . By itse2 = 0.77) appeared in the glycoprotein dataset, with predictors P17900 (Ganglioside GM2 activator), P28799 (Granulins) and P08842 (steryl sulfatase) (2 = 0.85) was found in the MRM dataset, with predictors G6PD (Glucose-6-phosphate 1-dehydrogenase), HRSP12 (Ribonuclease UK114) and TPM4 .These drugs are used for patients who experience recurrence after treatment with the standard of care chemotherapy. As with rapamycin, the sensitivities of the cell lines to gemcitabine and vinorelbine spanned approximately four orders of magnitude. If this variation reflects the situation in patients’ tumors, there are patients who are highly sensitive to these drugs. The best model for gemcitabine (Rlfatase) . The bes2 = 0.79, was found in the MRM protein dataset using mitochondrial thioreduxin-dependent peroxidide reductase (PRDX3), acyl-amino acid releasing enzyme (APEH) and importin subunit alpha (KPNA2).Fascaplysin, NU6102, Olomoucine II and Purvalanol are inhibitors of cyclin-dependent kinases (CDKs) and are in the main drug database studied here. Palbociclib, another CDK inhibitor, has been analyzed elsewhere on breast cancer cell lines . For allFive datasets were compared in their abilities to provide predictors for regression modeling of drug sensitivities in breast cancer cell lines. We used two criteria for evaluating performance: the agreement between observed and predicted sensitivities (coefficient of determination), and prediction error estimated by cross validation. The glycoprotein and MRM datasets, obtained via mass spectrometry, and a RNA seq dataset performed best, with the glycoprotein and MRM datasets giving more consistent results in the cross validation.Drugs that block the EGF receptor or HER2 were modeled well, at least partly because HER2 is in all the datasets. However, it is not necessary that a drug have its target in the data for this approach to work. For example, AKT and mTOR are not present in either the glycoprotein or MRM datasets, yet the Sigma Akt1,2 inhibitor and rapamycin were modeled accurately using predictors from these datasets. It appears that there is information useful for modeling drug sensitivity not just in the nominal targets of the drugs but also in the expression levels of other proteins.These results show that it is possible to predict the responses of breast cancer cell lines to drugs, particularly when mass spectrometry has been used to quantify protein expression. Can the approach be extended to patient samples? One possibility would be to use targeted assays on tumor samples. The MRM dataset targets specific proteins; various methods for targeting proteins by mass spectrometry are currently under development . MethodsSupplementary file