SLPI also has antimicrobial and anti-inflammatory properties, but the concentration of SLPI in lung secretions in COPD varies inversely with infection and the concentration of NE. A fall in SLPI concentration is also seen in culture supernatants of respiratory cells exposed to NE, for unknown reasons. We investigated the hypothesis that SLPI complexed with NE associates with cell membranes Respiratory epithelial cells were cultured in the presence of SLPI, varying doses of proteinases over time, and in different experimental conditions. The likely predicted charge of the complex between SLPI and proteinases was assessed by theoretical molecular modelling.We observed a rapid, linear decrease in SLPI concentration in culture supernatants with increasing concentration of NE and cathepsin G, but not with other serine proteinases. The effect of NE was inhibited fully by a synthetic NE inhibitor only when added at the same time as NE. Direct contact between NE and SLPI was required for a fall in SLPI concentration. Passive binding to cell culture plate materials was able to remove a substantial amount of SLPI both with and without NE. Theoretical molecular modelling of the structure of SLPI in complex with various proteinases showed a greater positive charge for the complex with NE and cathepsin G than for other proteinases, such as trypsin and mast cell tryptase, that also bind SLPI but without reducing its concentration.in vitro, which may correlate with changes observed in vivo.These data suggest that NE-mediated decrease in SLPI is a passive, charge-dependent phenomenon  In vitro it has antibacterial [SLPI is an efficient inhibitor of NE and other serine proteinases,2 and isacterial -6 and anacterial  propertiacterial ,9. In adacterial -14. Thesacterial , probablacterial -18 and iacterial ,18. It wacterial , suggestacterial , and lowacterial . This reacterial .in vivo is not understood. The genetics of patients with early-onset COPD have been investigated and no mutations, deletions or disease-associated polymorphisms in the SLPI gene have been described[In general, SLPI and NE concentration appear to be inversely related in sputum in COPD and CB but the described. In our in vitro using tissues ranging from primary nasal and bronchial epithelial cells, through a variety of cell lines, to isolated tracheal submucosal glands [in vivo. Previous in vitro work also included measurement of SLPI gene expression in cells exposed to NE and high levels were found to increase gene expression [A number of studies have described an inverse relationship between SLPI and NE l glands -28. Sincpression -29. The pression .One mechanism that might explain the NE-induced redistribution of SLPI protein hinges on the positive charge of the NE molecule, which may enable it to associate with cell membranes . Once asOther possible mechanisms include failure to secrete, or binding of SLPI to other cell surface proteins exposed by NE. For example, SLPI has been shown to bind specifically to annexin II and to sIn the present study, we have investigated the hypothesis that charge-related association of the SLPI-NE complex with cell membranes is the primary mechanism reducing SLPI concentration in cell culture supernatants from SLPI producing lung epithelial cells in the presence of NE. We have conducted experiments investigating the nature of the effect of NE on SLPI protein levels in cell culture supernatant, and pursued preliminary studies into the localization of SLPI in these cells when exposed to NE.3-p-nitroanilide . Cathepsin G (CG) was similarly obtained from empyema fluid and activity was measured using N-succinyl-phe-pro-phe-p-nitroanilide . Porcine pancreatic elastase (PPE) was obtained from Sigma and the activity was measured using N-succinyl-3-p-nitroanilide. Trypsin was cell culture grade (Invitrogen) and activity was measured using N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) (Sigma). Human mast cell chymase and tryptase were obtained from Elastin Products Co. and activity was measured using N-succinyl-val-pro-phe-p-nitroanilide and Z-gly-pro-arg-p-nitroanilide respectively (both from Bachem). ZD0892, a small synthetic peptidyl trifluoromethylketone NE inhibitor with an affinity for NE similar to that of SLPI[3-p-nitroanilide. Recombinant human SLPI (rhSLPI) was a gift from Amgen and the concentration was confirmed using the SLPI ELISA (see below). Inhibitory activity was determined as for ZD0892.NE was purified from empyema fluid using the method of Martodam et al. Absencet of SLPI, was a gSLPI concentration was measured using the R&D Systems ELISA, a sandwich ELISA consisting of a mouse monoclonal antibody for capture and a horseradish peroxidase-conjugated polyclonal antibody for detection. Total protein was measured in microplates using the Bio-Rad assay . The ELISA was validated using mixtures of SLPI and NE or CG in varying proportions to ensure that the proteinases did not interfere with quantification of SLPI. Briefly, recombinant human SLPI supplied as standard in the ELISA kit was mixed at varying molar ratios with NE and CG, incubated at 37°C for 30 minutes and the amount of SLPI recoverable was measured using the ELISA.A549 cells, a lung epithelial cell line derived from lung carcinoma, were obtained from the ECACC and cultured in 50/50 F10/DMEM  with 10% fetal calf serum (FCS) (Gibco). HepG2 cells, a liver epithelial cell line derived from liver carcinoma, were obtained from the ATCC. They were cultured in DMEM containing 10% FCS, antibiotic antimycotic solution, 150 mmol L-glutamine and MEM non-essential amino acids . Primary human bronchial epithelial cells (PBEC) were obtained from Cambrex (UK) and cultured in the same supplier's recommended basal media with growth supplements according to their instructions. Experiments were carried out with cells between passage 3 and 8, using basal media only.Most experiments were performed on A549 cells and selected experiments were repeated on PBEC or HepG2 cells in standard submerged monolayer culture. All experiments were performed in at least triplicate wells, and results are expressed as mean ± standard error of the mean (SEM) of at least 3 experiments. Cells were cultured to confluence in T75 or T25 flasks (Gibco) and plated out into 12 or 24 well plates (Gibco). Once confluent, the media were changed to serum free media (SFM) . A549 cells and HepG2 cells were cultured in SFM for a further 24 h before experiments were performed. After treatment with the experimental conditions, supernatants were aspirated and stored at -70°C for later analysis for SLPI and protein concentrations. Cells were rinsed with PBS and harvested using trypsin to detach them and viability was assessed where appropriate using trypan blue exclusion. Statistical analysis was performed using SPSS version 12 for Windows.A dose response experiment was undertaken to examine the relationship of NE concentration to the reduction of SLPI concentration in cell culture supernatants. The initial concentration of SLPI in the cell supernatants was not known at the outset of an experiment. Preliminary experimental data showed that the typical concentration of SLPI in supernatants of A549 cells after 24 h culture in SFM was between 0.5 and 2 nM (5–23.4 ng/ml). Cells were therefore incubated in SFM alone as control, or SFM containing NE from 0.5 to 5 nM. Initial experiments also included a control consisting of the buffer used to suspend the NE, in SFM at the same concentration as the highest concentration of NE used. Dose response experiments were repeated with CG and using PBEC with both proteinases.Proteinases such as PPE have, like NE, been shown to increase SLPI expression, althougThe hypothesis of binding of the NE-SLPI complex to cell membranes because of positive charge suggested that addition of NE to conditioned media would cause removal of SLPI that had already been secreted. Confluent PBEC were cultured for 24 h in SFM and at the end of this period an aliquot was removed for baseline analysis and replaced with either SFM or NE at a concentration sufficient to achieve 10 nM in each well. After gentle mixing of the contents of the wells once with a 1 ml pipette, cells were incubated for a further 24 h and aliquots of 25 μl were taken at various time points within this period and stored at -70°C for analysis by ELISA. Similar experiments were repeated on A549 cells using both NE and CG, and with 1 nM rhSLPI in DMEM added to HepG2 cells followed by NE or SFM control. HepG2 cells were used as a control cell line that does not express or secrete SLPI, to demonstrate the effect of proteinases on SLPI concentration without any possibility of secreted SLPI contaminating the results.Addition of NE inhibitors to culture media containing NE has been shown to abrogate the fall in SLPI protein. If bindGiven that many proteins associate non-specifically with charged surfaces such as cell culture plastic, we speculated that the NE-SLPI complex may associate more with cell culture materials than SLPI alone. This was tested by comparing the concentration of SLPI in solutions added to empty tissue culture plates with the concentration of SLPI in solutions added to wells containing confluent cells, with and without NE. SLPI was added to SFM to achieve a concentration of 1 nM, and the solution was incubated in 12 well plates for 10 minutes, a baseline aliquot was taken for measurement of SLPI and replaced with SFM or NE to achieve a final enzyme concentration of 10 nM. After a further period of incubation the contents of the wells were harvested for measurement of SLPI concentration. In order to block the effect of non-specific binding, both 12 well plates containing cells and 12 well plates without cells were also incubated with media containing 1% w/v human serum albumin (HSA) and SLPI at 1 nM. The effect of 10 nM NE was assessed as before in both sets of plates. Plates were also coated with HSA prior to addition of cells, and the above experiments were repeated using plates with or without A549 cells. Finally in cell-free conditions, the effect of 1% Tween 20  was assessed in the same way.in vitro[in vivo epithelial and glandular cells might be exposed to NE release from neutrophils at both sites during neutrophil migration. To examine whether this may influence the secretion of SLPI, a two compartment model was studied. Transwells  were coated with human placental collagen (Sigma) at 50 μg/ml and dried in air. A549 cells were added to the upper compartment in 500 μl media, with 1.5 ml media in the lower compartment. As soon as the cells appeared as a confluent monolayer, media were replaced by SFM at the same volumes. After a further 24 h incubation, reagents were added to upper and lower compartments. Triplicate wells received either SFM alone in both compartments or NE 10 nM in one compartment with SFM in the other. Supernatants were aspirated after a further 10 minutes incubation, and stored at -70°C for later analysis for total protein and SLPI concentration.SLPI is secreted both basally and apically by cells in vitro, and in . These structures were then superimposed on the proteinase in the SLPI-proteinase complex using SWISSPDB[. The values for the original proteinase (chymotrypsin) were then deleted and the new complex saved. SLPI alone was obtained by deleting chymotrypsin from the original structure file. Electrostatic surfaces were generated using SWISSPDB with values of -3.00, 2.00 and 8.00 for the red, white and blue extremes of the spectrum.The modelling procedure involved the use of the X-ray crystal structure of SLPI with chymotrypsin (structure file kindly provided by Professor Wolfram Bode as described previously) as a te SWISSPDB. The valThe ELISA confirmed SLPI concentration was fully recoverable at the range of concentrations of NE and CG used in these experiments. However at very high molar ratios of proteinase to inhibitor > 100:1), there was a fall in quantification which was attributed to the effects of unopposed proteinase activity on the antibody used in the ELISA . The concentration of SLPI after 24 h showed a dose related fall with NE treatment from 1.36 ± 0.37 nM at 0.5 nM to 0.20 ± 0.14 nM at 5 nM, whilst media control contained 1.63 ± 0.31 nM  . At these higher proteinase concentrations, SLPI concentration fell sharply. CG caused a dose-dependent fall in SLPI concentration  at 1 nM, 4.7 ± 4.3%, 0.02 ± 0.0% and 0.07 ± 0.1%, (p < 0.001) at 10 nM, 100 nM and 1000 nM respectively), but had no effect at 0.1 nM (96.2 ± 0.8% control) while SLPI concentration fell significantly only at high concentrations for PPE  and trypsin , at which cell morphology was affected. Mast cell chymase caused a significant reduction in SLPI secretion at concentrations from 10–100 nM. This was associated with a marked change in cell morphology: loss of cell-cell contact but no detachment. At lower concentrations, with normal cell morphology, there was no effect on SLPI secretion. Mast cell tryptase did not affect either SLPI concentration or cell morphology at any dose . The concentration of SLPI in NE-treated wells fell to 12.05 ± 2% of control values (p < 0.001 compared to media control). When NE and ZD0892 were pre-incubated, the effect of NE on SLPI was abrogated . Addition of NE after ZD0892 caused some decrease in SLPI concentration  but the fall in SLPI concentration was less than with NE alone (p < 0.05). Addition of NE before ZD0892 caused a fall in SLPI concentration , which was not significantly different from NE alone. The difference between the results for NE and ZD0892 added in either order was not statistically significant. These experiments are summarised in Figure We next explored whether the loss of SLPI in the presence of NE required the presence of cells, or whether it would occur in cell-free conditions. We added SLPI to empty tissue culture plate wells with or without NE. There was a marked loss of SLPI from the empty tissue culture plates as only about 1% of the expected amount was recovered from control wells and therefore an additional effect of NE could not be identified. Further investigation demonstrated that there was a large loss of SLPI when diluted in SFM in standard 20 ml polystyrene tubes, and a further loss in tissue culture plates, which was attributed to charge-related non-specific binding to plastic (data not shown). This may have explained the observed decrease in SLPI in cells treated with high concentrations of trypsin or PPE that caused detachment, since this would have exposed culture plastic which would be capable of binding the previously secreted SLPI, resulting in a fall in SLPI concentration in the supernatant.Use of polypropylene plates and siliconised glass culture systems did not offer more than partial protection against this loss of SLPI (data not shown) and cells did not grow satisfactorily without a substrate on siliconised glass. Addition of HSA 1% w/v to culture media, or coating of tissue culture plates with HSA or human placental collagen prior to plating out cells, partially reduced the non-specific loss of SLPI from culture media (data not shown). Furthermore the presence of HSA in solution partially inhibited the effect of NE in reducing SLPI concentration in cell-free systems Figure , and simSupernatant from the upper compartments of Transwells on which A549 cells were cultured contained about fourteen times as much SLPI as the lower compartments. The difference may be due to polarized secretion of SLPI, or might be accounted for by SLPI binding to the plastic of the cell culture plates in the lower compartment, which had not been treated to prevent non-specific binding and contained no cells. Addition of NE to the upper compartment caused reduction in SLPI concentration to 10.6 ± 0.3% of control values, and in the lower compartment it caused a reduction to 8.6 ± 1.9% of control. SLPI concentrations in the control-treated compartment were not affected . Further studies, using SLPI bound to a fluorescent or radioactive label that does not impede binding to NE, will be required to confirm this.Our initial validation studies demonstrated that in the presence of a molar excess of NE or CG, detection of SLPI by ELISA was not affected. The studies described here did not examine the possibility of cleavage or degradation of SLPI by excess of proteinase, but even if this happened, the cleavage products would appear to be detected fully by the ELISA, suggesting that the monoclonal capture antibody recognises an epitope not affected by proteolysis, and that the polyclonal detection antibody also recognises preserved sites. Incubation with samples containing excessive amounts of NE may however expose the capture antibody to proteolytic degradation, and this may be the explanation for the fall in detection at high concentrations of NE, rather than degradation of SLPI by NE. Since the validation studies used recombinant rather than endogenous SLPI and were conducted in assay buffer rather than conditioned cell culture media, it cannot be guaranteed that the ELISA would perform as reliably in experimental conditions. However in the published studies previously mentioned, the same effect of dose-dependent reduction of SLPI in the cell culture environment was shown by systems using a variety of different antibodies for detection of SLPI, suggesting that the effect is real rather than a systematic measurement error. Possible effects on the ELISA from complex formation or cleavage of SLPI therefore cannot fully explain the complete loss of signal of SLPI in the presence of cells treated with NE. Nevertheless, our experiments do not conclusively demonstrate that SLPI bound to cells in presence of NE is biologically active. Furthermore, it needs to be noted that the difficulties in avoiding non-specific binding of SLPI to the plastic components of the cell culture system are not fully countered in these studies. If the hypothesis that binding of SLPI to NE causes adherence to cells by charge interactions is valid, then the same mechanism could apply to any negatively-charged surface, such as plastic cell culture components which were also shown to bind free SLPI avidly. NE did appear to increase non-specific binding of SLPI to plastic, because when detergent was used as a blocking agent, SLPI concentration fell only in the presence of NE (suggesting that the higher ionic charge of the NE-SLPI complex was able to overcome the concentration of detergent used). The ELISA system contains strong buffering agents that must be presumed to protect against loss of free SLPI. The validation studies suggest that they are sufficient to protect also against loss of the NE-SLPI complex. However it cannot be presumed that there was not loss of SLPI to plastic during the cell culture process, harvesting of supernatant and storing of the samples prior to preparation of the ELISA, and that this loss of SLPI was not enhanced in the presence of NE solely by increased binding to plastic rather than to cell membranes. Proof of the concept of binding of SLPI to cells would require a different experimental approach, and therefore the experiments presented here cannot claim to prove that the effect is biological and relevant to the There is a likely biological relevance of the phenomenon of free SLPI associating with cell membranes and extracellular matrix proteins because this would allow the lung to establish a protective antiproteinase coat over structures that would be at most risk of damage by free NE. The major function of NE is probably intracellular killing of bacteria within the phagolysosome. Where neutrophils are migrating through the lung, some local NE activity (from a released azurophil granule or released from cell membrane association) may possibly be beneficial to this process. Free NE in lung secretions however is believed to be uniformly deleterious. Where this occurs, it would be neutralized to some extent by the presence of SLPI bound to vulnerable structures. There may be some value in the SLPI-NE complex associating with these structures. If the NE-SLPI complex prevented further association of NE with extracellular matrix substrates more effectively than SLPI alone (because of its higher positive charge), it would leave the free NE in epithelial lining fluid to be inactivated by α1 AT, which the latter cannot do once NE is bound to substrate. There aIn summary, the experiments described in this paper support the hypothesis that the association of SLPI in complex with NE to negatively-charged structures leads to its removal from cell culture media, and that this effect is a passive biochemical process, probably dependent on charge. It is likely that this effect occurs with cell membranes as well as non-biological components, but this has yet to be confirmed. Future experiments should include measures to counter the ability of SLPI to bind non-specifically to a variety of structures, particularly in the presence of NE or CG.SLPI: Secretory leukoproteinase inhibitor; NE: Neutrophil elastase; COPD: Chronic obstructive pulmonary disease; CB: Chronic bronchitis, α1 AT: alpha 1 antitrypsin inhibitor; CG: Cathepsin G, PPE: Porcine pancreatic elastase; ELISA: enzyme linked immunosorbent assay; ECACC: European Collection of Cell Cultures; DMEM: Dulbecco's modified Eagle's medium; FCS: Fetal calf serum, ATCC: American Type Culture Collection; MEM: Minimum essential medium; PBEC: primary bronchial epithelial cells; PBS: Phosphate-buffered saline; SEM: Standard error of the mean; SFM: Serum free media, HSA: Human serum albumin, DNA: deoxyribonucleic acid.The authors declare that they have no competing interests.AS carried out the experimental work and wrote the manuscript. TD carried out the molecular modelling studies and produced the illustrations. PH conceived the hypothesis, advised on experimental work and assisted in drafting the manuscript. RS supervised the experimental work and assisted in drafting the manuscript. All authors read and approved the final manuscript.


TMwas safe and effective in the treatment of MCC as demonstrated by a minimal complication rate and acceptable tumor response.The purpose of this study was to evaluate the patient tolerance and efficacy of delivering locoregional chemotherapy to metastatic colorectal (MC) hepatic metastases via hepatic trans-arterial approach using irinotecan loaded drug eluting beads. This open-label, multi-center, single arm study included 30 MC patients, who had failed first line therapy. Of the 57 total embolization sessions, 12 (21% of sessions) were associated with adverse reactions during or after the treatment. After a median followup of 9 months, response rates by modified RECIST were 75% at 3 months and 66% at 6 months. Hepatic trans-arterial therapy using Irinotecan loaded DC Bead Hepatic metastasis of colorectal cancer (MCC) is quite common occurring at some time in 23% of all of the 190 000 colorectal patients diagnosed each year . There h Patients presenting with initial unresectable metastatic colorectal metastasis either by a number of lesions or extrahepatic metastatic disease have the benefit of systemic 5 fluorouracil-based chemotherapy with a combination of oxaliplatin and/or irinotecan and offers a high rate of response (35%–50%) and a longer median survival (15–20 months) versus historical observation or 5 fluorouracil monotherapy alone –9. HowevIntra-arterial hepatic chemotherapy has the potential to accentuate both the palliation of patients and potentially improve quality of life time. Given that hepatic colorectal metastases are predominantly 95%–100%) fed by the hepatic arterial system has the advantage of delivering high-dose chemotherapy directly to the target lesion with minimal systemic side effects. This technique which has largely been used in the treatment of hepatocellular carcinoma is beginning to be expanded into other liver-dominant metastatic disease %–100% fe. Thus, the aim of this study was to evaluate the initial safety and efficacy of irinotecan-loaded beads delivered by hepatic transarterial approach for the treatment of unresectable metastatic colorectal cancer.An IRB-approved prospective multi-institutional open, noncontrolled repeat treatment registry was evaluated from January 2007 to October 2008 in which 30 patients presenting with liver dominant metastatic colon cancer (MCC) to the liver were treated with irinotecan drug eluting beads. Patients were included for therapy if they were 18 years of age, of any race or sex, who had histologic or radiologic proof of MCC to the liver, who were able to give informed consent and were eligible for treatment. Patients must have had an ECOG performance status score of less than or equal to 2 with a life expectancy of greater than or equal to 3 months, nonpregnant with an acceptable contraceptive in premenopausal women. Exclusion to therapy was contraindication to angiographic and selective visceral catheterization, significant extrahepatic disease, representing an imminent life-threatening outcome, greater than 75% of hepatic parenchymal involvement, severe liver dysfunction, pregnancy, and severe cardiac comorbidities. Only patients with liver dominant  were considered for treatment.Standard pretherapy evaluation of patients with MCC included at least a three-phase CT of the abdomen and pelvis and chest roentgenogram at least one month prior to treatment, with the use of PET scanning depending on the institution and the availability of the technology for use. Prior systemic chemotherapy of any type and duration was allowed and was recorded.Patients were followed for any treatment-related adverse experiences for 30 days after each treatment and monitored for survival for two years. All adverse events were recorded per standards and terminology set forth by the Cancer Therapy Evaluation Program Common Terminology Criteria for Adverse Events, Version 3.0. Followup assessments included a triphase CT scan of the liver within at least one to two months from the treatment completion with the evaluation of the enhancement pattern of the target lesion and tumor response rates measured according to RECIST , EASL 1, and modDiagnostic angiography was performed by the interventional radiologist and consisted of selective celiac and superior mesenteric arteriograms to evaluate the hepatic arterial anatomy. Once it was evaluated for the degree of hepatic tumor perfusion, the next step in evaluation was to limit any type of extrahepatic perfusion of the treatment. The most common branches that will lead to extrahepatic disposition of treatment are the right gastric and the gastroduodenal arteries which are controlled either prior to infusion with coil embolization or at a minimum distal catheter placement. In addition to this defining the amount of liver disease was integral to defining both the number of treatments and the type of catheter position and therapy that would be performed. For finite number of lesions defined as less than four lesions, treatment plan existed for a minimum of two dosing schedules of at least 50 mg of irinotecan to 100 mg of irinotecan loaded in one-two DC/LC bead vials of 100 to 300 microns, 300 to 500 microns, or 500 to 700 microns (every four to eight weeks). For diffuse disease a plan of a minimum of four-dosing schedule again of 50 to 100 mg (depending on the extent of tumor burden and the extent of hepatic parenchyma reserve) again is loaded into one-two DC/LC bead vials of the similar size as above with the plan for at least two treatments per lobe with again every three-to-four week dosing schedule, following toxicity, and extending the interval if toxicity was seen with again planned repeat CT scan three months from the first dose to evaluate tumor response.Periprocedural medications including pain medications, antibiotic prophylaxis, intra-arterial lidocaine, corticosteroids, and proton pump inhibitors were all performed at the physician's discretion.The mixing of loaded DC/LC beads was performed with nonionic contrast (approximately 50/50 dilution) prior to injection. A minimum of 10 cc contrast should be mixed with the loaded DC/LC bead to ensure smooth catheter delivery. After appropriate mixing and removal of the uneluted supernatant, a microcatheter is then placed based again on the extent of liver disease. For a finite number of lesions, the microcatheter is selectively placed (super selective) for the first DC/LC bead vial (a 100 to 300 micron size) with initial infusion with then a more proximal catheter placement into the right or left hepatic artery for the second DC/LC bead vial of the size choice (based on physician discretion). For diffuse disease a more lobar infusion again using a microcatheter into either the right or left artery, depending on the bulk of disease, is performed with either any of the three size DC/LC beads that the physician chooses to evaluate.Slow injection of the irinotecan-loaded DC/LC beads is highly recommended to avoid reflux of embolic material. In addition to that, particular attention into identifying the cystic artery is recommended to ensure that the catheter tip passed this point to avoid extrahepatic infusion into the gallbladder. Additional embolic material is not usually followed after appropriate treatment but was up to the physician's discretion.The saline suspension in the DC/LC bead microsphere  was removed and the beads were mixed with irinotecan solution at a dose of 50 mg per 2 mL at least four hours before the procedure depending on the dose that was planned to be delivered.t-test, and Mann-Whitney's U-test for nominal, continuous, and ordinal variables were used to evaluate the association of independent variables to surgical complications. Proportional hazards analysis was performed on all variables found significant by univariate analysis. Relative risk (RR) with 95% confidence intervals was calculated as a measure of association. Differences of P < .05 were considered significant. Statistical analysis was performed using JMP software .Data was censored at the last recorded patient contact if an endpoint was not reached. Recurrence was also evaluated using serological markers and PET scan. A recurrence was the reoccurrence of viable tumor by radiologic CT criteria of a vascular mass. In the event of subsequent hepatic therapy for recurrence of disease only the first procedure was used for the purposes of this study. Chi-square, Student's A total of 30 patients underwent 57 treatment sessions with the irinotecan drug-eluting beads. There were 11 women and 19 men in this study with a median age of 58 years (range 42–75). Past medical histories were significant for prior cardiac disease in seven patient, prior pulmonary disease in three patients, underlying diabetes in eight patients and prior alcohol abuse in five patients with six patients having a prior smoking history with a pack year median of 60 pack years (range 30–120). All of the study patients' past medical histories were negative for primary breast, carcinoid, renal, ovarian, melanoma, sarcoma, and lung cancers.All patients had had their colon primary resected. Fifteen patients had undergone either prior anatomic hepatic lobectomy or RFA to treat their initial disease.The extent of liver metastasis had a majority of patients with multiple lesions with a median number of lesions being five (range 1–20) with 28 patients having a bilobar lesion with the single largest target lesion being 5.0 cm (range 1–8 cm). The extent of liver involvement had 14 patients with less than 25% liver involvement, 15 patients with 26%–50% liver involvement, and one patient with greater than 50% liver involvement. The total target lesion size (sum of a maximum of five lesions) was 8.8 cm (range 1.0 to 24.0 cm). Sixteen patients had extrahepatic disease with 13 having involvement with small volume lung metastasis, one with two retroperitoneal lymph nodes, one with a bony metastasis at T1, and one with pelvic lymph node and splenic metastases.In review of the 57 treatment sessions, the median number of treatments per patient was one (range 1–5). The right lobe was the most common lobe treated in 42 patients, left lobe alone in 15 patients with the remaining being treated in a bilobar approach. The preprocedural dose planning was a median of 100 mg (range 50 to 200) involving a median of one vial with a range of one to two vials. The most common bead size was 100 to 300 microns in 37 patients, 300 to 500 micron beads in 25 patients, and 500 to 700 micron beads in 18 patients. Forty six of the 57 treatment sessions received 100% of their planned dose with the remaining 11 patients receiving a median of 80% of the dose planned (range 50%–90%) because of early stasis during treatment.The degree of flow occlusion included partial stasis in 22 patients, near stasis in 20 patients, and complete stasis in 15 patients. Four patients did have a gel foam bland embolization following their bead therapy because of small aberrant posterior sector hepatic artery.Periprocedural medications included antibiotics in 52 of 57 patients, most commonly being Kefsol, Levaquin, Flagyl, and Ciprofloxacin. The antiemetic medication was utilized in 57 of 57 patients with the most common medication being Zofran in 42 treatment sessions with the addition of Phenergan and/or Compazine based on the sensitivity of the patient. All patients were given periprocedural and postprocedural proton pump inhibitor or H2 blocker for gastritis prevention. Periprocedural hepatic intra-arterial Lidocaine was utilized prior to bead infusion in 35 of the 57 treatments, with systemic sedation being most commonly Morphine and Versed with a smaller distribution of patients receiving Dilaudid, Demerol, or Fentanyl. Postprocedure anti-inflammatory was utilized in 26 of 57 treatments, most commonly being Decadron or Toradol with a smaller percentage given oral Vicoprofen.In an evaluation of periprocedural outcomes a majority of patients (60%) were treated as an outpatient setting  and on multivariate analysis identified that the risk for admission was found in patients who were receiving greater than or equal to their third treatment or who had embolic product followed in order to get to complete stasis after a successful bead infusion. These results potentially demonstrate that going to a complete stasis endpoint may lead to increase adverse events without potential benefits.Median length of hospital stay for each treatment procedure was 23 hours with a range of 23 hours to 3 days. Pre- and posttreatment WBC, hemoglobin, platelets, total bilirubin, creatinine, albumin, and INR values were obtained. However, some isolated values for particular patients were unable to be obtained. The most significant change between the pre- and posttreatment median lab values involved the WBC count, which increased 25.9%, from 7.2 to 9.1. Pre- and posttreatment median hemoglobin values were 12.1 and 12, respectively—representing a 0.8% decrease from pre- to posttreatment levels. Pre- and posttreatment median platelet values were 241 and 237.5, respectively—representing a 1.5% decrease from pre- to posttreatment levels. Pre- and posttreatment median bilirubin values were 0.6 and 0.7, respectively—representing a 16.7% increase from pre- to posttreatment levels. Pre- and posttreatment median creatinine values were 0.9 and 0.8, respectively—representing an 11.1% decrease from pre- to posttreatment levels. Pre- and posttreatment median albumin values were 4 and 3.8, respectively—representing a 5% decrease from pre- to posttreatment levels. Pre- and posttreatment median INR values did not change and were 1 and 1, respectively.Of the 57 total embolization sessions, 12 (21% of sessions) were associated with adverse reactions during or after the treatment. However, because many patients had more than one treatment session, 9 patients suffered adverse reactions during or after their treatment. 4 of the 9 patients with adverse reactions had reactions that were rated as grade 3, with 1 subsequent death. However, the remaining 5 patients had adverse reactions to treatment that were only grades 1 and 2.The most common adverse event 6 (50%) was postembolic syndrome type symptoms of nausea, vomiting, and, hypertension of either grade 1 or grade 2 in severity that required hospitalization and subsequent treatment. Other treatment-related events included port infection (grade 3), gastritis (grade 1), dehydration (grade 2), cholecystitis (grade 3), and hypertension (grade 2), that all resolved and were felt to be possibly or probably related to the irinotecan treatment.One patient did have an adverse event of liver dysfunction/failure (grade 3 adverse reaction) the day after treatment. More specifically, the patient's bilirubin increased from baseline 2.9 to 3.4 on postoperative day 1. On postoperative day 2, the patient's bilirubin had decreased to 3.0 and subsequently this patient's liver dysfunction resolved. This reaction was thought to be possibly related to the embolization treatment.One patient had an SAE of liver dysfunction, found 28 days after treatment. The patient expired from ongoing liver dysfunction 30 days after the SAE was observed. The treating physician felt that this event was possibly related to the treatment or device. The patient had been treated with 31 total cycles of FOLFOX, 9 cycles of FOLFIRI, and 12 cycles of Avastin and had a prior percutaneous RFA prior to bead therapy. The patient was given a single treatment of 2 vials of DC/LC Bead loaded with 200 mg Irinotecan. The patient had a 3-day hospital stay for nausea and then was discharged home without incident until he returned on day 28. Pt was 52 years of age, had a preoperative bilirubin of 1.9, INR of 2.0, and underwent treatment to the right lobe, with one vial of 300–500 um and one vial of 500–700 um size beads, through a lobar infusion with near stasis after bead infusion. His medications were cipro and flaygy after, with Zofran during the procedure, and an epidural for pain management. His liver involvement was 4 total lesions in seg 5–8, largest lesion 4.2 cm, with 26%–50% liver involvement, with total targets lesion size of 12.9 cm, with extrahepatic disease involving the pancreas, spleen, and lung.After a median followup of nine months on all 30 patients treated, response rate at three months by modified RECIST was seen in 75% of patients and at six months in 66% of patients. Similarly all 30 patients demonstrated a great than or equal 50% drop in their CEA levels at 3 months that was sustain at 6-month evaluation.The multidisciplinary management of metastatic colorectal is becoming far more complex and far more collaborative in the optimal treatment of patients with metastatic colorectal cancer. Given the ever increasing incidence of this disease with an estimated 90 000 to 100 000 new cases of metastatic colorectal cancer being diagnosed each year in the United States, this type of multidisciplinary management and collaboration is integral to the success and quality of life of the patient. These results presented demonstrate that precision hepatic arterial Irinotecan therapy is a safe and effective treatment in the management of patients with metastatic colorectal. These results demonstrate a minimal quality of life side effect and potential optimal response rates and, therefore, improved progression-free survival and overall survival in the management of this challenging disease. This initial pilot evaluation confirms the activity of this device in the management of colorectal cancer.Hepatic-directed therapy in the management of metastatic colorectal cancer is a well-established therapy ranging from hepatic arterial infusion pumps  to conveThe most common adverse events were periprocedural pain, nausea, and hypertension. As demonstrated in this data only a subset of patients received preinfusion hepatic arterial Lidocaine. In a review of the data, this demonstrated to be a significant predictor of reduction or actually suppressing this adverse event when the use of 2 to 4 cc of 1% plain lidocaine is infused into the hepatic arterial system prior to bead infusion. It is well established that the DC/LC Irinotecan bead does lead to a small (5%–10%) chemotherapy burst on initial infusion into the liver and the surrounding parenchyma. The intra-arterial lidocaine has been demonstrated to be effective in reducing the symptoms of this burst, such that patients are able to tolerate the therapy similar to other established hepatic-directed therapies. The intra-arterial lidocaine has now become a standard of pretreatment technique when utilizing the Irinotecan bead for metastatic colorectal or any other type of metastatic disease in which the device is chosen to be utilized. Similar to that, one of other more common adverse events was periprocedural nausea, seen to a greater extent when anecdotally compared to conventional TACE or even prior Yttrium therapy. Effective management of this has now been achieved with now a pretreatment infusion of a higher dose of Zofran that is initiated approximately 15 to 30 minutes prior to procedure with the effective amelioration of this symptom through a more aggressive type of antiemetic treatment. The last, more common, adverse event seen was also hypertension which appeared to be refractory to the more established interventional drugs that are commonly used, those being lopressor and nitroprusside, and is found to be far more sensitive when vasotec is utilized when periprocedural hypertension is seen.Thus in conclusion chemoembolization using Irinotecan-loaded bead was safe and effective in the treatment of MCC as demonstrated by a minimal complication rate, acceptable tumor response, and sustained reduction of CEA levels. Further larger studies are needed to confirm this data and establish where hepatic arterial precision chemotherapy should be utilized in the algorithm of the metastatic colorectal cancer patient.


BRCA1 gene mutation.The purpose of our study was to compare differences in the prognosis of breast cancer (BC) patients at high (H) risk or intermediate slightly (IS) increased risk based on family history and those without a family history of BC, and to evaluate whether ten-year overall survival can be considered a good indicator of BRCA1/2 mutations. The overall survival (OS) and disease free survival (DFS) were the study end-points.We classified 5923 breast cancer patients registered between 1988 and 2006 at the Department of Oncology and Haematology in Modena, Italy, into one of three different risk categories according to Modena criteria. One thousand eleven patients at H and IS increased risk were tested for BRCA1 carriers were identified. A statistically significantly better prognosis was observed for patients belonging to the H risk category with respect to women in the IS and sporadic groups . Comparing only BRCA1 carriers with BRCA-negative and sporadic BC  an advantage in OS was seen.Eighty BRCA1 carriers had a better prognosis than those with sporadic BC. Considering these results, women who previously had BC and had survived ten years could be selected for BRCA1 analysis among family members at high risk of hereditary BC during genetic counselling. Since only 30% of patients with a high probability of having hereditary BC have BRCA1 mutations, selecting women with a long term survival among this population could increase the rate of positive analyses, avoiding the use of expensive tests.Patients belonging to a population with a high probability of being  BRCA1 and BRCA2 were identified in 1994 and 1995, respectively [BRCA1/BRCA2-related cancer have yielded conflicting results. Several reports suggested that women with germline mutations in BRCA1 are more likely to die from their disease than are women with sporadic breast cancer [BRCA2 mutation carriers and non-mutation carriers seem to share a similar prognosis [BRCA1 carriers may be consistent with the histological characteristics usually described for BRCA1-associated breast cancer, which show higher histologic grade and cancers that are more often hormone receptor-negative than sporadic breast cancer cases [BRCA1 mutation carriers compared with non-carriers in a prospective population-based cohort [BRCA1 mutation, those who received chemotherapy had a better survival rate compared with those who did not [The major breast cancer (BC) predisposing genes, t cancer -6, whererognosis ,8. The per cases . Howeverd cohort . Apart fBRCA-positive and BRCA-negativepatients, but none showed a significant survival difference between different risk categories. In case of statistically significant differences in OS between the three groups, a secondary aim was to determine whether patients with a better prognosis were BRCA1 mutation carriers, showing that the outcome could be considered an indicator of BRCA1 inheritance. Additionally, we evaluated whether chemotherapy could play a role in the prognosis of BRCA1 carriers, providing more benefit in this patient population than in patients with sporadic breast cancer.The primary aim of our study was to calculate disease free survival (DFS) and overall survival (OS) of BC patients at high risk (H) or intermediate slightly (IS) increased risk based on family history and those without a family history of breast cancer using the population registered with the Breast Cancer Registry in Modena. Previous studies were aimed at evaluating the outcome in Patients included in our analysis were diagnosed between 1988 and 2006 at the Department of Oncology and Haematology in Modena. All newly-diagnosed, biopsy-proven primary breast cancer patients were evaluated. The family history was collected and classified according to the Modena criteria  and a blAll research regarding the identification, counselling, genetic testing, and clinical data regarding individuals at risk of developing breast cancer were ethically approved by the Ethics Commitee of Modena (reference number 45/00).BRCA1, and analyzed by Direct Automated Sequencing using an ABI Prism 3100 . Subsequently, patients with breast cancer provided consent for genetic testing that was completed for each case.In 1995, DNA started to be extracted from frozen whole blood using the Invisorb Blood Universal kit , amplified by PCR using primers specific for the coding sequence and exon-intron boundaries of BRCA1 mutations were tested for BRCA1 rearrangements using the multiplex ligation-dependent probe amplification assay  following the manufacturer's protocol.Samples negative for 2 test was used to determine differences in clinicopathological features between groups. Survival curves were estimated using the Kaplan-Meier method including the log-rank test group comparison. Patients who were BRCA1 carriers were matched with patients with sporadic breast cancer from the Modena cancer registry. This registry, initiated in 1988, covers an area with approximately 650,000 inhabitants in Northern Italy. A database with a total of 3858 cases of sporadic breast cancer was used to find four matched controls for each case and a randomized matches were assigned between BRCA1 and patients with sporadic BC of the same age at diagnosis , tumour grade , and stage . Multivariate analyses of DFS and OS were conducted using a proportional hazards Cox regression model. All statistical analyses were done with SPSS, version 12.0 .The χp < .0001). The H risk group was more likely to have stage II disease than the sporadic and IS increased risk groups (p < .0001), and to have a significantly lower estrogen receptor (ER) and progesterone receptor (PgR) expression levels (p < .0001) as measured by immunohistochemical analysis  and stained by Ventana Benchmark autostainer. The ER and PgR receptor status was tested by evaluating the percentage of nuclear immunoreactivity with respect to all the nuclei in the neoplastic cells, independently of the staining intensity. No differences were seen in the Ki67 level between the groups (p = .17). The proportion of patients who received adjuvant chemotherapy was greater in the IS increased risk group than in the other two groups (p < .0001). The most frequent chemotherapy strategy was anthracycline-based followed by taxanes and the CMF regimen. The most frequently used first line chemotherapy regimen was platinum based, followed by a CMF scheme. The proportion of patients who received adjuvant hormone therapy was lower in the H risk group than in the other two groups, according to the negative hormonal receptor status (p < .0001).From January 1988 and December 2006, 5923 patients were diagnosed with breast cancer. According to their family history, 4912 were considered sporadic breast cancers, 691 were at IS increased risk, and 320 were considered H risk. The patients' characteristics are detailed in Table BRCA1 mutational analysis was only performed in patients belonging to the H and IS risk groups, with a 22.8% (73/320) and 1.0% (7/691) mutation rate, respectively. In total, 80 patients were BRCA1 carriers.The p < .0001; Fig. p = .12; Fig. BRCA1 carriers and 931 were considered BRCA negative. In the BRCA1 carrier group, 8 deaths (5 caused by a second tumour arising after breast cancer) and 13 relapses  were reported, whereas in the BRCA negative group 220 deaths and 240 relapses occurred. The 10-year overall survival rate was 77% for BRCA1 carriers, 77% for BRCA-negative patients, and 73% for sporadic breast cancer  and 1137 relapses  were reported. The estimated 10-year overall survival rate was 82% for the H risk patients, 75% for the IS increased risk group, and 73% for patients with sporadic breast cancer .The study group of 80 BRCA1 patients had a better OS  compared with the sporadic breast cancer cases. Positive ER and PgR status was associated with a better OS . Also, chemotherapy was an important factor in reducing the mortality rate . Stage > I  and grading III  were significant factors for poorer OS. Younger age (≤ 35 years) represented a risk factor for a shorter OS, but the trend was not statistically significant. In a model that included 2213 patients receiving chemotherapy, BRCA1-positive status  but not BRCA1-negative status  reduced the mortality rate. Positive PgR status  was a favourable factor as was positive ER status , but did not reach statistical significance. Stage > I  and grading III  were factors statistically associated with an increased risk of death. There was a similar but non significant trend in age ≤ 35 years . Conversely, in a model without chemotherapy, the BRCA1-positive status lost statistical significance in reducing mortality , whereas the BRCA1-negative status maintained its favourable impact . Also, positive ER  and PgR status  and age less than 35 years  did not affect OS. Finally, stage > 1  and grading III  were statistically associated with an increased risk of death. Detailed results for multivariate analysis are shown in Table Of the 5923 patients, 5012 were included in multivariate analyses, which included BRCA1 patients who did or did not receive chemotherapy, no difference was seen in terms of OS  of BRCA1 carriers were identified in the H risk group, and may explain the survival advantage in this group of patients, even if other reasons may be involved. Patients who know they are at high risk of developing breast cancer may be more likely to participate in surveillance programs and start at a younger age, receive an earlier diagnosis, and subsequently, experience a better outcome.The results of this large analysis show that patients considered at H risk of being BRCA1 status and was observed in patients treated with chemotherapy. This finding is important when, as happens in a family cancer centre like in ours, we are faced with a patient or a healthy woman who has a family history of breast cancer. By collecting all the information about individuals affected by breast cancer, very long-term survivors can be identified by predicting a predisposition for being a BRCA1-mutation carrier in the descendants. Furthermore, BRCA1 carriers may be more likely to be found among women who have a high probability of hereditary breast cancer and who have a long term survival. If BRCA1 carriers could be identified through these characteristics, the use of expensive tests could be avoided and the rate of positive analyses increased.One of the most important findings of our study was that the survival difference was attributable, in a multivariate analysis, to the patient's BRCA1 status. The most common weakness of previous studies includes the small number of patients or selection bias, such as a retrospective analysis for BRCA1 status. Owing to the large number of subjects, we could calculate OS differences between risk groups, and even in subgroup analyses.Our study involves a very large number of Caucasian patients with breast cancer (N = 5923) who were first evaluated for their family history of breast cancer and subsequently for BRCA1 status was not related to the DFS. This finding shows that the H risk and BRCA1 patients have the same DFS as the other two groups, but seem to respond better to chemotherapeutic agents. This finding is in contrast with the results of Rennert et al. [BRCA carriers and non carriers and between BRCA1 carriers and BRCA-negative patients treated with chemotherapy. Our argument is that, in BRCA1 patients, 41% of relapses were local recurrences, whereas in patients with sporadic BC the local recurrence rate was 25%, and distant metastases accounted for 75%. Consequently, it is not surprising that a difference in OS was found and could be explained by the use of alkylating agents, such as platinum-derived drugs, in metastatic disease that are well known to be more effective in BRCA-related tumours [BRCA1 patients maintain a statistically significant OS advantage, even after matching each case to four controls for age and tumour grade and disease stage, this result is very important in terms of its practical value, because all confounders have been removed.Unexpectedly, the survival difference according to H risk and t et al.  who show tumours . FurtherBRCA1 mutation was an independent predictor of breast cancer mortality in a multivariate analysis of a group of women who did not receive chemotherapy, but not in women who received adjuvant chemotherapy.Robson et al  suggesteBRCA1-positive status was an independent predictor for a better survival in all patients and also in the subgroup of patients who received chemotherapy, but not in patients who did not receive chemotherapy. No differences are shown for BRCA1 patients, independently of chemotherapy or not. Also, this result might be explained by the fact that deaths in the treated group of patients were all related to a second tumour, while in the non treated group one of three deaths was caused by breast cancer. In conclusion, chemotherapy has a greater protective effect in BRCA1 mutation carriers compared with patients who are BRCA-negative and those with sporadic breast cancer. Furthermore, since BRCA1-related tumours are more likely to be triple negative, the greatest advantage was shown for ER-negative breast cancer, were chemotherapy is the most active. Hence, increasing the number of BRCA1 carriers identified by correctly selecting patients with a high probability of having hereditary breast cancer through a 10-year survival analysis could improve the benefit derived from specific chemotherapy agents .In a multivariate analysis, we found that chemotherapy is a prognostic factor for better survival in all patients combined; furthermore, we found that a BRCA1 and BRCA2 proteins participate with RAD51 in the repair of double-stranded DNA breaks induced by DNA-damaging agents [BRCA proteins, which confer substantial cellular sensitivity to the inhibition of poly (ADP-Ribose) polymerase enzyme (PARP). This polymerase is a key enzyme in the repair of single-stranded DNA damage via the base excision repair pathway. The loss of PARP activity in BRCA mutant cells might lead to the persistence of DNA lesions normally repaired by homologous recombination, resulting in increased chromosome instability and programmed cell death specifically in tumour cells [BRCA-positive and treated with anthracycline-based chemotherapy regimens. In this study, all patients received anthracycline-based neoadjuvant chemotherapy, and 10 of 11 patients with BRCA mutations had a clinical complete response compared with only 8 of 27 BRCA-negative patients with sporadic breast tumours. From this study, it was inferred that tumours with BRCA1 mutations are highly sensitive to anthracycline-based chemotherapy regimens.In fact, the chemotherapy benefit is well known because normal g agents -19. The ur cells ,21. Therur cells  found a In our study, patients were considered part of a hospital-based population and were not selected for age, tumour stage, or treatment, which could influence survival rates. We were successful in obtaining blood samples from all patients and were able to genotype all the samples we received. The patients were followed for a median of 72 months. Treatment regimens were chosen on the basis of disease staging. The Modena Cancer Registry captures outcome data from almost all patients with cancer who are treated in the province. Data on pathological diagnosis are verified by a pathologist, and incomplete information is retrieved from medical records when possible. All patients were Caucasian, and it is possible that the chemotherapy sensitivity can be associated with specific modifier genes not found in other populations.BRCA1 carriers in the sporadic group would be very low. Since we identified only 80 mutation carriers, the subgroup analysis relied on a small number of subjects.Our study has a number of limitations. Tumour stage and hormone receptor status were not routinely recorded, particularly during the first period. Since we tested only a subpopulation of patients with a positive family history of breast cancer, it is possible that some hereditary cases were misclassified; but we found that only 1% of mutations occurred in the IS increased risk group suggesting that the number of BRCA1 analysis as a predictor of mutations. We feel that randomized studies need to be designed to further address the relationship between BRCA1 mutations and sensitivity to chemotherapy, and further clinical investigations are required to determine whether the BRCA1 status can be used to predict treatment outcomes.In conclusion, our study is the first to evaluate a relationship between familial risk of breast cancer and a genetic assessment in terms of DFS and OS. We propose that the increased survival associated with a family history of breast cancer, suggesting hereditary breast cancer according to the Modena criteria, should be considered with respect to BRCA1 carriers have a better prognosis than other risk groups, and this could be considered an indicator of BRCA1 inheritance. This paper provides, for the first time, evidence-based proof that women at high risk of being BRCA1 carriers have a favorable prognosis after 10 years follow-up.In conclusion, our data show that patients belonging to a population with a high probability of being The authors declare that they have no competing interests.LC participated in the design of the study and drafted the manuscript. CM acquired the data and performed the statistical analysis. CC participated in the statistical analysis. VM performed the genetic testing. IM acquired the data from the centre. GC participated in the collection of patient data from Mantua Hospital. GP participated in the collection of patient data from Rimini Hospital.DT participated in the collection of patient data from Bologna Hospital. MF designed the study and revised the final manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/90/prepub


To evaluate treatment satisfaction, efficacy and functional ability of the rapid release formulation of sumatriptan 100 mg tablets (sumatriptan RT 100 mg) in an early intervention paradigm in patients who were dissatisfied with low-dose sumatriptan and not completely satisfied with their current migraine regimen.Experienced migraineurs who reported a mild migraine pain phase, dissatisfaction with the previous sumatriptan treatment and some dissatisfaction with their current treatment regimen had no experience with sumatriptan at the 100 mg dose were enrolled in an open-label, single group study. Subjects were instructed to treat four migraine attacks within 30 min of the onset of mild pain. Treatment satisfaction was measured with the Patient Perception of Migraine Questionnaire Revised version (PPMQ-R) questionnaire.More than half of the subjects were either very satisfied or satisfied with the efficacy of early intervention sumatriptan RT 100 mg after each attack and at the follow-up study visit. The mean total PPMQ-R score was 75.2 out of 100. Between 63% and 73% of subjects were pain-free within 4 h of dosing. Between 79% and 90% of subjects reported an ability to function normally within 4 h of taking the study medication.Subjects who were previously unsatisfied with lower doses of sumatriptan and less than very satisfied with their current treatment regimen were more likely to be satisfied or very satisfied with sumatriptan RT 100 mg in an early intervention paradigm. Results were consistent across four migraine attacks and at a follow-up visit. The treatment satisfaction results corresponded with positive results on efficacy measures and a functional status measure. Sumatriptan RT 100 mg tablets effectively treat migraine headaches in an early treatment paradigm.Patients who were previously unsatisfied with lower doses of conventionally formulated and other formulations of sumatriptan therapy are more likely to report that they are satisfied with sumatriptan RT 100 mg treatment when used in an early intervention paradigm.Migraine is a chronic condition characterised by episodic attacks of disabling headaches that are generally pulsatile in nature and often associated with gastrointestinal disturbances, photophobia, phonophobia and neurological disruptions in cognition and mood . In someFor patients who seek migraine treatment through their healthcare providers, a member of the triptan drug class may be prescribed for the acute treatment of migraine attacks . The effIn an early intervention paradigm, the optimal dose and rapid delivery of the medication may improve effectiveness and treatment satisfaction by preventing progression in the migraine cycle. Multiple studies of conventional sumatriptan administered in an early intervention paradigm have demonstrated that the 100 mg dose is more efficacious than lower doses at providing pain-free response in a clinical trial setting ,18,19,23The objectives of this study were to evaluate treatment satisfaction, efficacy and functional ability associated with the rapid release formulation of sumatriptan 100 mg tablets (sumatriptan RT 100 mg) in an early intervention paradigm (within 30 min of onset of pain and while still mild) among patients who were dissatisfied with the previous low-dose sumatriptan treatment and who were not completely satisfied with their current migraine treatment regimen. Patient reported outcomes such as treatment satisfaction are increasingly considered end-points that encompass the overall treatment experience . The FedThe study population included female and male migraineurs aged 18–65 years with a diagnosis of migraine, with or without aura (2004 IHS Criteria ICHD-II diagnosis 1.2.1 and 1.1)  and one at study exit (follow up). Follow-up visits were conducted within 2 weeks of the last treated attack or at 8 weeks after the screening visit.At the screening visit, subjects were provided with four tablets of sumatriptan RT 100 mg for the initial treatment of mild migraine headaches and five tablets of sumatriptan RT 100 mg for rescue use during the 2–24 h after the initial dose of study medication, if needed. Other rescue medications  were permitted when taken at least 2 h after the initial dose of study medication and documented in the patient diary. Subjects were not required to meet a minimum level of pain severity as a prerequisite for taking rescue medications. Ergotamine or ergot-type medications (e.g. dihydroergotamine and methysergide) and other triptans were not allowed during the 24 h before or after administration of the study medication.Subjects were instructed to treat four consecutive migraine attacks within 30 min of the onset of mild pain and to refrain from using study medication to treat attacks with moderate or severe pain at onset. Subjects were allowed to continue migraine prophylactic medications provided the medication had been included in their previous regimen for at least 1 month prior to screening.Subjects were instructed to complete a patient diary entry for each attack treated with study medication. The patient diary required subjects to record the date and time migraine pain started, the date and time of the first dose of study medication, the pain severity level, the presence or absence of symptoms and the level of functional impairment. The treatment satisfaction questionnaire was included in the patient diary. The diary also captured the date, time and the type of rescue medication; the pain severity level at the time of rescue; and all medications, excluding study medications and daily medications, taken 24 h before and after the study medication doses.In the patient diary, subjects recorded their pain severity level, associated symptoms and functional ability at the time of dosing and 30 min, 1, 2, 4 and 24 h after dosing. Pain severity level was measured on a 4-point scale . Subjects reported the presence or absence of nausea, vomiting, photophobia and phonophobia. Subjects recorded their ability to function on a 5-point scale .Treatment satisfaction with sumatriptan RT 100 mg was assessed using the Patient Perception of Migraine Questionnaire Revised version (PPMQ-R). Subjects were instructed to complete the PPMQ-R at 24 h after taking the first dose of study medication for each treated attack. Subjects also completed the PPMQ-R at the follow-up visit.The PPMQ-R is a validated patient satisfaction instrument containing 32 items that contribute to four scales: Tolerability (10 items), Efficacy (11 items), Functionality (four items) and Ease of Use (two items). The items contributing to the Efficacy, Functionality and Ease of Use scales were scored on a scale ranging from 1 (very dissatisfied) to 7 (very satisfied). The items contributing to the Tolerability scale were scored on a scale ranging from 1 (extremely bothersome) to 5 . A total score was calculated based on the average of scores for Efficacy, Functionality and Ease of Use ,30.In addition to the items contributing to the Efficacy, Functionality, Ease-of-Use and Tolerability scales, the PPMQ-R contains three global satisfaction items – one each measuring overall satisfaction, satisfaction with medication effectiveness and satisfaction with side effects. The global satisfaction items were scored on a scale ranging from 1 (very dissatisfied) to 7 (very satisfied) ,30.Two PPMQ-R items designed to measure satisfaction with drug cost were excluded from the study questionnaire because study medication was provided as part of the clinical trial. The PPMQ-R has demonstrated reliability and validity in measuring patient satisfaction with acute migraine treatment ,30.At the follow-up visit, subjects returned unused study drug and completed diaries. Subjects also completed follow-up questions and the PPMQ-R. For the PPMQ-R, subjects were instructed to consider their experience with the study drug across all attacks treated during the study.All subjects who took at least one dose of sumatriptan RT 100 mg were included in the safety population. The intention to treat (ITT) population included subjects who treated at least one attack according to the treatment instructions (i.e. migraines were treated within 30 min of the onset of the migraine headache pain and during a mild pain phase). Data from attacks in which subjects did not comply with the protocol with respect to treatment instructions were not included in the analyses. As a result, summaries on a per-attack basis will have different denominators based on the number of observations from subjects who treated the specific attack according to the treatment instructions.For the PPMQ-R, mean scores were calculated from the item scores for each of the four scales . Each scale score ranges from 0 to 100, with higher scores indicating greater satisfaction or better tolerability. A total score, a composite of the scale scores for Efficacy, Functionality and Ease of Use, was also computed. Previous research suggests that a 5-point difference in the Efficacy, Functionality and Ease-of-Use scale scores and the Total score is clinically meaningful ,27. GlobDescriptive statistics were calculated for efficacy and functional ability end-points. The percent of patients who were pain-free at each time point was calculated. The percent of patients reporting the presence of nausea, photophobia and phonophobia was calculated at dosing and each time point. The percent of patients reporting normal functioning at dosing and 30 min, 1, 2, 4 and 24 h after dosing was calculated.A total of 140 subjects from six sites in the United States were enrolled and 134 (96%) returned their patient diaries. The safety population consisted of 120 subjects who were randomised and treated. The ITT population included 105 subjects who treated at least one migraine according to the study instructions. In the safety population, 9% withdrew prematurely. Three were lost to follow up, one because of an adverse event, one withdrew consent, one for an unspecified protocol violation and four for unknown or other reasons.A majority of subjects in the ITT population were women (88%) and Caucasian (88%). The average age of study subjects was 41 years . The meaOf the subjects who had discontinued previous sumatriptan therapy at a dose lower than 100 mg, more patients had taken sumatriptan in the 50 mg tablet form (65%) than any other form . The mosIn the 3 months prior to study enrolment, 93% of subjects reported using any pharmacological treatment. Pharmacological treatments included other triptans (55%), over-the-counter analgesics (46%), narcotics (22%) and prescription NSAIDS (19%). As per the protocol, no subject reported that he/she was very satisfied with the current treatment regimen. Three percent (3 of 101) were satisfied with their current migraine mediations and 37% were somewhat satisfied with their current migraine medications. Five percent of subjects were neither satisfied nor dissatisfied with their current treatment. A total of 35% of subjects were somewhat dissatisfied, 13% were dissatisfied and 8% were very dissatisfied with their current migraine treatment regimen.Based on the results from the PPMQ-R overall satisfaction question measured at the follow-up visit, 26% of subjects who were not very satisfied with their previous treatment regimen were very satisfied with sumatriptan RT 100 mg in an early intervention paradigm . More thMore than half of all subjects were either very satisfied or satisfied with the efficacy of early intervention sumatriptan RT 100 mg after each attack and at the follow-up study visit . With thThe mean PPMQ-R scores are presented in Between 53% (attack 1) and 61% (attack 4) of subjects reported being pain-free within 2 h of taking the first dose of study medication . Within When compared with symptoms at the time of dosing, reports of nausea, photophobia and phonophobia were lower at 2, 4 and 24 h postdose for all four attacks . Across Between 79% (attacks 1 and 2) and 90% (attack 4) of subjects reported an ability to function normally within 24 h of taking the study medication . FollowiOf the subjects in the ITT population, 65% of subjects in the ITT population reported that they would use this medication again. A total of 85% of subjects reported that the medication was very easy or easy to use, and 94% would treat future migraine attacks within 30 min of onset.The results of this study suggest that experienced migraineurs who were previously unsatisfied with lower doses of sumatriptan in any form  and less than very satisfied with their current treatment regimen are more likely to be satisfied or very satisfied with sumatriptan RT 100 mg in an early intervention paradigm. In this study, the mean scale scores for treatment satisfaction when measured by a validated treatment satisfaction questionnaire indicate that subjects tended to be satisfied with the Efficacy, Ease Of Use, Functionality and side effects of sumatriptan RT 100 mg when treating migraine in an early intervention paradigm. The treatment satisfaction results corresponded with positive results on efficacy measures and a functional status measure.The percentage of patients who were very satisfied with treatment following the first attack was lower than the percentage at attacks 2, 3, 4, and at the follow-up visit. With any new treatment or intervention, experience impacts the outcome. Global satisfaction may have been slightly lower after the first attack because it was the subjects’ first experience using this intervention (EI paradigm and sumatriptan RT 100 mg) after having reported dissatisfaction with sumatriptan. That is, because these subjects were previously dissatisfied with sumatriptan, they may have had lower expectations at the beginning of the study.Additionally, the percent of patients who were satisfied and very satisfied with side effects is higher for attacks 3 and 4 than for attacks 1 and 2. While it is possible that patients who remained in the study had fewer or less severe side effects compared with those who dropped out, patients may have become more familiar with the drug and its side effects over the course of the study. In our experience, it is not uncommon for subjects to report fewer adverse events (AEs) over time in long-term studies.Treatment satisfaction, defined as the subject’s evaluation of important attributes associated with the process and outcomes of treatment, is becoming a more common end-point in clinical trials . In an iOther studies have demonstrated that treatment satisfaction for migraineurs is closely linked with clinical efficacy and that patient preferences are impacted by previous treatment experience –36. ThisMultiple studies of conventional sumatriptan administered in an early intervention paradigm have demonstrated that the 100 mg dose is more efficacious than lower doses in a clinical trial setting ,18,19,23Improvement in functional status is an important end-point for migraineurs because the majority of the costs associated with migraine results from missed time and productivity loss rather than from direct healthcare costs . A produThe results of this study should be interpreted in the context of the limitations. Although it is desirable to evaluate treatment outcomes in a real-world environment, certain biases are more likely to be present in an open-label, uncontrolled study compared with a randomised, controlled study. To reduce the impact of selection bias, we restricted the study population to experienced migraineurs who reported dissatisfaction with the study drug in the previous treatment settings and less than complete satisfaction with their current medication regimen. Although these subjects were less likely to represent migraineurs with a mild condition, their experience with the condition and treatment provided a specific perspective on treatment satisfaction. Lost to follow up can be a significant source of selection bias. In this study, the drop out rate was < 10%, which suggests that the study medication and treatment regimen were well tolerated.A potential source of recall bias was that subjects were required to remember their previous experiences with sumatriptan to be eligible for the study. The impact of this was minimised by providing potential subjects with only two options: satisfied or dissatisfied. To reduce the recall bias associated with the study measures, subjects were required to complete the patient diary within 24 h of each attack as opposed to waiting to report their experiences until a study visit. This study was designed to mimic clinical use by allowing subjects to continue with their established prophylactic medication and to use the rescue medication of their choice. The mean results from all four attacks and at the follow-up visit were similar, which suggests that the results are valid.A limitation in this study was that the global satisfaction question asked at enrolment and the follow-up global question were worded slightly differently. The PPMQ-R includes a global satisfaction question that asks subjects to consider their overall satisfaction with the ‘study medication’. For screening, subjects were asked to consider their overall satisfaction with their current treatment regimen. The same 7-point scale was used for measuring subject responses.Studying an early intervention paradigm presents a challenge because mild migraines may be self-limiting and other types of headache may be mistaken for mild migraines . HoweverObtaining information about the level of satisfaction with pharmaceutical products is reflective of the desire to consider the perspective of the patient in medical decision making. However, gathering information directly from patients presents methodological challenges. The act of asking questions about patients’ medication may impact their responses. To minimise the impact of examiner influence on the results of this study, we used a validated treatment satisfaction questionnaire.Additional studies should be conducted to measure the relative importance of the impact of improvement in satisfaction related to dose, treatment paradigm or formulation. Although this study does not weigh the relative contribution of early intervention, appropriate dosing and rapid release formulation, the results suggest that patients who believe that sumatriptan was ineffective under different circumstances could be rechallenged under new treatment conditions before sumatriptan is declared suboptimal for the patient.In conclusion, the results of this study suggest that for patients who are not completely satisfied with their current medication regimen, physicians have an opportunity to optimise a rapid and complete response by encouraging patients to treat migraine attacks using an early intervention paradigm and by prescribing the most effective dose and a faster dispersing, disintegrating and absorbing formulation.


Logistic regression analysis revealed that EGFR mutations in cytology specimens represented an independent predictor of the gefitinib response. The overall and progression-free survivals were significantly longer in the EGFR mutation group than in the wild-type EGFR group (P<0.05). In conclusion, cytology specimens could be useful for analysing the EGFR status in the majority of patients with non-small-cell lung cancer to determine whether they are likely to benefit from gefitinib treatment.We conducted a feasibility study to examine whether small numbers of cancer cells could be utilised for analysis of the EGFR gene status using the loop-hybrid mobility shift assay, which is a modified heteroduplex technique. Cytology specimens obtained by transbronchial abrasion were successfully used for analysis of the EGFR gene status in 50 of 52 (96.2%) patients diagnosed with class V non-small-cell carcinoma. Furthermore, the relationship between the EGFR gene status and clinical outcome was analysed in 25 patients treated with gefitinib. Overall, 10 of 11 patients with EGFR mutations in exon 19 or 21 showed tumour regression with gefitinib treatment, compared to only two of 14 patients with wild-type EGFR. The response rate was significantly higher in the EGFR mutation group than in the wild-type EGFR group (90.9  Non-small-cell lung cancer (NSCLC) is the leading cause of cancer deaths in Japan. Current chemotherapy regimens for metastatic NSCLC are not particularly effective, and the disease cannot be cured even with the most effective platinum and new combination chemotherapies. Recent progress in lung cancer biology has led to the development of small-molecule inhibitors of target proteins involved in proliferation, apoptosis and angiogenesis. The epidermal growth factor receptor (EGFR) superfamily was identified early on as a potential therapeutic target in solid tumours. Given the biological importance of the EGFR molecular network in carcinomas, several molecules that can inhibit the EGFR tyrosine kinase domain have been synthesised. These inhibitors include gefitinib and erlotinib, both of which are orally active and can produce an objective response in previously treated or untreated advanced NSCLC  patients  was used to extract DNA from the cells in accordance with the manufacturer's instructions. Briefly, an appropriate amount of a viscous solution  was applied over the area of cancer cells on the slide and air-dried to a thin film, together with the underlying cells. The film was then lifted with a blade, transferred to a tube containing a solution of proteinase K and digested at 55°C for 4 h. The tube was further incubated at 98°C for 10 min to inactivate the enzyme and then immediately quenched on ice. After vigorous vortexing, the tube was centrifuged and part of the supernatant was directly subjected to the following polymerase chain reaction (PCR)-based analyses.M. The mixture was then subjected to an LH cycle consisting of denaturation at 94°C for 2 min, annealing of the LH probe at 55°C for 15 s and extension of the LH probe by PCR at 68°C for 4 min. After the LH cycle, the product was separated by electrophoresis in a preformed native 10% polyacrylamide gel  in Tris-glycine buffer . Next, the gels were stained with SYBR Green I  and the DNA fragments were detected with a laser scanning imager . The bands representing LH DNAs were then excised and crushed in a small quantity of water, before an aliquot of each extract was re-amplified by PCR. The PCR products were subcloned into the pCR4TOPO  plasmid vector, and the nucleotide sequences were confirmed. All PCR amplifications and elongation reactions with LH probes were performed with Accuprime Taq polymerase together with a primer-template hybridisation-enhancing reagent (Invitrogen).The LH-MSA, a modified heteroduplex technique, was used to analyse the EGFR gene mutations. Briefly, two genomic DNA fragments spanning the mutation hotspots in exons 19 and 21 were amplified by PCR with the primers e19F and e19R (for exon 19) or e21F and e21R (for exon 21) . At the χ2 test was used to identify differences in the gefitinib responses between wild-type and mutant EGFR genes. The influence of each factor on the response to gefitinib was examined by logistic regression analysis. The Kaplan–Meier method was used to estimate the probability of survival, and differences in survival were analysed by the log-rank and Wilcoxon tests. Differences at P<0.05 were considered significant. All analyses were performed using StatView or Fisher's software.The A feasibility study was carried out to determine whether cytology specimens obtained by transbronchial abrasion were applicable for analysis of the EGFR gene status. A total of 52 patients who were diagnosed cytologically as having class V NSCLC by transbronchial abrasion were entered into this study. A Pinpoint Slide DNA Isolation System was used to extract DNA from cancer cells alone on glass slides , and then=11) and a wild-type EGFR group (n=14), and compared their EGFR statuses and clinical outcomes. Among the 11 patients with EGFR mutations, 10 showed tumour regression after gefitinib treatment and one showed no cancer progression over 1 year. Only two of 14 patients with the wild-type EGFR gene showed gefitinib-induced tumour regression. The response rate of patients with EGFR mutations was significantly higher than that of patients with the wild-type EGFR gene . Logistic regression analysis revealed that EGFR mutations were the only significant factor contributing to gefitinib sensitivity . This is a very high percentage compared with previous studies in which the EGFR gene status was clarified in about 30% of patients using biopsy or resected tumour specimens . In geneGefitinib is not currently a first-line anticancer drug, and is usually used after previous treatments with several conventional chemotherapeutic reagents. It is probable that the preceding chemotherapy may modify the sensitivity to gefitinib, as acquired cross-resistance of cancer cells to multiple anticancer drugs is a commonly encountered clinical phenomenon. Therefore, we consider that it is critical to evaluate the efficacy of anticancer drugs, including gefitinib, just before their use. The LH-MSA used in the present study requires only a small number of cancer cells, which may be sampled using common clinical procedures, such as collection of sputum, pleural effusion or peripheral blood. Our present findings suggest that the majority of patients could be tested in this manner for the presence of EGFR mutations, thus allowing selection of patients who would be likely to benefit from gefitinib treatment.Our results confirmed that specific missense and deletion mutations in the tyrosine kinase domain of the EGFR gene are associated with the response to gefitinib. However, some of our patients without EGFR mutations also responded to gefitinib, suggesting that the clinical benefits of the drug cannot be explained only by the presence of EGFR mutations. Previous studies have demonstrated that the EGFR gene copy number is significantly associated with the response to gefitinib, and that gefitinib-treated patients showing EGFR gene amplification or high polysomy have significantly better responses, a longer time to progression and longer survival than patients with no or low EGFR genomic gain (


During unexpected infectious disease outbreaks, public health agencies must make effective use of limited resources. Vaccine distribution may be delayed and staggered through time, as underscored by the 2009 H1N1 influenza pandemic. Using a mathematical model parametrized with data from the 2009 H1N1 pandemic, we found that optimal allocations of vaccine among people in different age groups and people with high-risk conditions depends on the schedule of vaccine availability relative to the progress of the epidemic. For the projected schedule of H1N1 vaccine availability, the optimal strategy to reduce influenza-related deaths is to initial target high-risk people, followed by school-aged children (5–17) and then young adults (18–44). The optimal strategy to minimize hospitalizations, however, is to target ages 5–44 throughout the vaccination campaign, with only a tiny amount of vaccine used on high-risk people. We find that optimizing at each vaccine release time independently does not give the overall optimal strategy. In this manuscript, we derive policy recommendations for 2009 H1N1 vaccine allocation using a mathematical model. In addition, our optimization procedures, which consider staggered releases over the entire epidemic altogether, are applicable to other outbreaks where not all supplies are available initially. Vaccines are critical to controlling the spread of influenza. The development and distribution of effective vaccines for new pandemic strains may take several months, and not all the vaccine doses may be available at the same time. In addition, prior to and throughout the vaccination period, a substantial fraction of the population may become infected and thereby naturally immunized to re-infection. These dynamics change the transmission pattern and need to be considered in order to optimize vaccine allocation. Such changing dynamics are also the case for the 2009 H1N1 influenza pandemic. The first 2009 H1N1 vaccines were distributed in the US in early October mainly to health care personnel and young children  Public health agencies often consider projected impacts on deaths and hospitalizations when determining intervention policy  Our analysis indicates that both deaths and hospitalizations can be minimized by prioritizing the vaccination of school-aged children (5–17), young adults (18–44), and people at high-risk of severe infections. However, the temporal order of prioritization differs between these two outcome measures when staggered vaccination schedules are considered. Deaths can be minimized by prioritizing high-risk people first, followed by schoolchildren and young adults. In contrast, hospitalizations are optimally averted by first prioritizing schoolchildren and young adults, followed by high-risk people. In contrast to both optimal strategies, the CDC recommends prioritizing high-risk people and people aged 6 months through 24 years  To minimize deaths, the optimal policy is for the first two batches of vaccine to be allocated to high-risk people, followed by schoolchildren (ages 5–17) and then young adults (ages 18–44) . To minimize hospitalizations, the first five batches should be allocated to schoolchildren and young adults, with a small amount of vaccine also allocated to high-risk people in the fifth batch . The optimal allocation is sensitive to the scheduling of release times. Consequently, evaluation of each batch separately does not generate the allocation strategy that is optimal overall . For example, when only two total batches are available (45 million doses on 15 October and 20 million on 22 October), deaths can be minimized by allocating the first batch to schoolchildren and the second batch to adults aged 18–44 . In contrast, when 3 total batches are available (45 million doses on 15 October and 20 million on each of 22 October and 29 October), the first two batches are optimally split between schoolchildren and high-risk people and the third batch is used exclusively on high-risk people. The projected reductions in infections, deaths, and hospitalizations are substantial for both sets of optimized vaccination strategies . The strategy that minimizes hospitalizations is predicted to reduce the total attack rate more effectively than the strategy that minimizes deaths . Comparing the number of deaths and hospitalizations, the strategy that minimizes deaths results in 8000 fewer deaths (a 23% reduction) and 44,000 more hospitalizations (a 9% increase) than the strategy that minimizes hospitalizations. Since the strategy that minimizes deaths also greatly reduces hospitalizations, it may be preferred by decision makers. The staggered vaccine delivery schedule that we considered had a total of 125 million doses in five batches starting on 15 October. To explore the effect of the delay in vaccine delivery, we also calculated the optimal allocations when all 125 million doses were delivered in one batch, either on 15 October or before the outbreak began . The epidemiological impact of the initial vaccination delay is substantial: the staggered release results in 23% more deaths and 26% more hospitalizations, respectively, compared to optimal allocation of the complete distribution in October. By contrast, the optimal distribution of 125 million doses before the outbreak begins prevented the outbreak entirely . Vaccination not only protects those who are vaccinated, but also reduces transmission to others in the population. Schoolchildren, and secondarily young adults, are responsible for most of the influenza transmission  We evaluated the optimal age-specific and risk-specific allocation of 2009 H1N1 influenza vaccine doses based on the schedule of vaccine availability projected by the CDC. In general, our results suggest that low-risk children between 6 months and 4 years of age and adults 65 and older should receive the lowest priority to minimize both hospitalizations and deaths. These groups are the least responsible for transmission, and are estimated to have relatively low case mortality ratios for 2009 H1N1  Despite the current stage of the 2009 H1N1 outbreak, the vaccination of those most responsible for transmission remains important. For diseases that are more transmissible than the current 2009 H1N1 influenza, and particularly if vaccination is not available early on during the outbreak, control of the outbreak via the reduction of transmission becomes very difficult, increasing the importance of vaccinating high-risk people  model of Medlock & Galvani  We assumed that the mean latent period was 2 days  The transmission model was parametrized with a mean of 22.5% of the population defined as at high risk for influenza complications, due to other, preexisting medical conditions, with the proportion at high risk varying among age groups Text S1: Detailed Methods Funding was provided in part by NIH grant 1U01GM087719-01.  JM was supported in part by start-up funding from the Clemson University Department of Mathematical Sciences.  The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. All of the authors have no competing financial, personal, or professional interests that have influenced this paper.