A Comparison Between Radioimmunotherapy and Hyperthermic Intraperitoneal Chemotherapy for the Treatment of Peritoneal Carcinomatosis of Colonic Origin in Rats Background Cytoreductive surgery -LRB- CS -RRB- followed by heated intraperitoneal chemotherapy -LRB- HIPEC -RRB- is considered the standard of care for the treatment of patients with peritoneal carcinomatosis -LRB- PC -RRB- of colorectal cancer -LRB- CRC -RRB- . These surgical procedures result in a median survival of 2 years at the cost of considerable morbidity and mortality . In preclinical studies , radioimmunotherapy -LRB- RIT -RRB- improved survival after CS in a model of induced PC of colonic origin . In the present studies we aimed to compare the efficacy and toxicity of CS followed by adjuvant RIT in experimental PC to the standard of care , HIPEC . Peritoneal carcinomatosis -LRB- PC -RRB- of colorectal cancer -LRB- CRC -RRB- frequently is an end stage of colorectal cancer , occurring in 5 -- 50 % of the patients , either synchronous or metachronous .1 If untreated , patients suffering from PC have a median survival of only 6 months .2 Survival is significantly improved by radical surgical debulking procedures -LRB- cytoreduction -RRB- in combination with intraperitoneal chemotherapy , either in combination with normothermia or hyperthermia -LRB- HIPEC -RRB- .3 -- 7 The median survival after cytoreductive surgery and HIPEC is 13 -- 34 months ,4,8 and the 5-year survival rate is 19 -- 27 % at the cost of considerable morbidity and mortality rates of up to 23 % and 4 % , respectively .6,9 In the latest reported clinical trial on adjuvant RIT in the setting of colon cancer , Liersch et al. reported results of a Phase II trial with 131I-labeled anti-CEA antibody labetuzumab administered to patients after complete resection of colorectal liver metastases . This study , where RIT was applied in an adjuvant setting to complete resection , resulted in a promising 5-year survival rate of 51.5 % .10 Radioimmunotherapy using radiolabeled monoclonal antibodies directed against tumor-associated antigens may therefore be an attractive anticancer therapy in patients with small volume disease . We therefore have studied the application of RIT as adjuvant therapy following cytoreductive surgery -LRB- CS -RRB- in the setting of PC . In previous studies regarding PC of CRC in a rat model we showed that RIT could be an effective adjuvant treatment after CS . The efficacy of adjuvant RIT in combination with CS was investigated and compared with no treatment , CS only , and RIT only . The results of this study showed a significantly improved survival of animals treated with CS followed by RIT -LRB- median 88 days -RRB- compared with those treated with CS only -LRB- median 51 days -RRB- and RIT only -LRB- median 61.1 days -RRB- .11 Based on the encouraging results , showing the observed increase in survival that was achieved with low-dose RIT and concomitant low toxicity , we now aimed to compare the efficacy of this treatment to that of today 's standard of care , HIPEC ,12,13 in a preclinical setting . MATERIALS AND METHODS Animal Model of Peritoneal Carcinomatosis WAG/Rij rats -LRB- 10 -- 12 weeks old , body weight 240 -- 290 g , Harlan Horst , The Netherlands -RRB- were housed under nonsterile standard conditions -LRB- temperature , 20 -- 24 °C ; relative humidity , 50 -- 60 % ; 12-h light/dark cycle -RRB- in filter-topped cages -LRB- two rats per cage -RRB- , with free access to food -LRB- Ssniff , Bio Services Uden , The Netherlands -RRB- and water . Rats were accustomed to laboratory conditions for at least 1 week before experimental use . Peritoneal carcinomatosis was induced by intraperitoneal inoculation of 2.0 × 106 CC-531 colon cancer cells , as described previously .14 All experiments were approved by the local Animal Welfare Committee of the Radboud University Nijmegen and were carried out in accordance with the Dutch Animal Welfare Act of 1997 . Operative Procedure Prior to the laparotomy , all rats were given 10 mL of saline to prevent hypovolemia . Surgical procedures were performed under general anaesthesia using isoflurane 3 % , O2 and N2O 1:1 . Thirty minutes prior to and once daily until the third day postoperatively , rats were given buprenorphine -LRB- 5 μg , 0.1 mL/rat/day -RRB- for analgesia . During the operation , rats were placed on a warmed mattress to limit body heat loss . All rats underwent a midline laparotomy . After opening the abdomen the extent of intraperitoneal tumor growth was scored semiquantitatively , 0 -LRB- no macroscopic tumor -RRB- , 1 -LRB- little ; located at 1 -- 2 sites with a diameter of 1 -- 2 mm -RRB- , 2 -LRB- moderate ; located at 1 -- 2 sites and a diameter 2 -- 5 mm -RRB- , or 3 -LRB- abundant ; located at multiple sites and/or diameter > 5 mm -RRB- in all four quadrants of the abdomen . The sum of the tumor scores of all sites represented the peritoneal cancer index -LRB- PCI -RRB- .11 Subsequently , CS , including a routine omentectomy , was performed in all rats . Irresectable tumor deposits were cauterized using an electrocoagulation device . After completion of the surgical cytoreduction , the abdominal wall was closed in two layers using continuous Vicryl 3/0 sutures for the muscular component and iron wound clips for the skin in animal treated with CS only or CS + RIT . Monoclonal Antibody , Radiolabeling , and RIT The murine MG1 monocolonal antibody -LRB- MAb -RRB- , an anti-CC531 IgG2a monoclonal antibody that recognizes a 80 kDa cell surface antigen and localizes preferentially in tumors when injected in rats bearing CC-531 tumors ,15 was purchased from Antibodies for Research Applications BV -LRB- Gouda , The Netherlands -RRB- . Labeling of the antibody with 177Lu was carried out as previously described .11 In brief , the MAb was conjugated with 2 - -LRB- 4-isothiocyanatobenzyl -RRB- - diethylenetriaminepenta-acetic acid -LRB- ITC-DTPA -RRB- -LRB- Macrocyclics , Dallas , TX -RRB- and subsequently labeled with 177Lutetium -LRB- IDB Holland , Baarle Nassau The Netherlands -RRB- and purified by gel filtration on a PD10 column -LRB- Amersham , Pharmacia Biotech , Maarsen , The Netherlands -RRB- . The purified 177Lu-MG1 preparation was diluted in PBS with 0.5 % BSA for injection , the specific activity of the administered 177Lu-MG1 preparation was 0.4 MBq/μg . The labeling procedure using 177Lu was performed under strict metal-free conditions . RIT -LRB- 185 μg MG1 / rat , radiolabeled with 74 MBq 177Lu in 3.0 mL -RRB- was intraperitoneally injected immediately after surgery , as this was determined to be the most optimal time for adjuvant administration .16 Mitomycin-C Mitomycin-C -LRB- MMC -RRB- was obtained from Nycomed Christiaens BV -LRB- Breda , The Netherlands -RRB- as a powder in glass vial -LRB- 40 mg/vial -RRB- . Immediately before use , MMC was dissolved in 0.9 % sodium chloride to the appropriate concentrations . HIPEC Procedure Following CS , while the abdomen was still exposed , two multiperforated catheters -LRB- Argyle , Sherwood Medical , Ireland -RRB- were inserted laterally through the abdominal wall and subsequently fixed in the abdominal cavity . The inflow drain was placed in the right paracolic gutter , the outflow drain in the left subdiaphragmatic space . The intraperitoneal temperature was monitored with an intra-abdominal thermometer -LRB- PTFE Insulated thermocouple , VWR International , Amsterdam , The Netherlands -RRB- , at the site with generally the highest tumor load -LRB- omentum -RRB- . In addition , a thermometer was placed inside the rectum . After placement of the catheters , the abdominal wall was closed using a continuous suture -LRB- Ethilon 3.0 , Johnson & Johnson , Ethicon -RRB- -LRB- Fig. 1 -RRB- . During the HIPEC procedure , rats were removed from the warmed mattress to prevent general hyperthermia . The perfusion system was filled with 250 mL saline , containing 4 mg MMC -LRB- Mitomycin-C Kyowa , Christiaens -RRB- . The perfusate was heated in a tube coil using a thermostatically regulated water bath set to a temperature of 48 °C and infused into the peritoneal cavity by a roller pump -LRB- Ismatec IPS-8 , Ismatec SA , Glattbrugg , Switzerland -RRB- for the duration of 60 minutes at 10 mL/min . Abdominal inflow temperature was set at 44 °C . In order to achieve a uniform heat distribution , gentle massage of the abdomen was applied throughout the duration of the HIPEC procedure . After completion of the perfusion , the abdominal cavity was flushed with warmed -LRB- 37 °C -RRB- saline for a period of 10 minutes . The abdomen was opened again to remove the catheters . Subsequently , the abdomen was closed as described previously . FIG. 1 . HIPEC Perfusion System ; MMC Mitomycin C Kyowa 16 mg/L perfusate . Adapted from Ref . 19 . Reproduced with permission . Intraperitoneal Distribution of MMC and Dose Determination Prior to the therapy experiment with HIPEC , we investigated the intraperitoneal distribution of the perfusion fluid using a methylene blue stained perfusate . The perfusate was administered in the same fashion as in the therapeutic experiment . After completion , the abdominal cavity was inspected for the presence of blue dye in all quadrants on both parietal as well as visceral peritoneum of the intra-abdominal organs and the diaphragm . Subsequently , a study to determine the dose of MMC that resulted in acceptable toxicity was performed in nine animals -LRB- three animals per group -RRB- . Animals underwent a laparotomy including an omentectomy and complete bowel inspection followed by heated perfusion of the abdominal cavity with MMC at 4 mg/L or 16 mg/L . Control rats underwent laparotomy and an omentectomy only . Body weight and physical condition were monitored during 6 days following the procedure to assess treatment-related toxicity . Treatment Efficacy Seven days after intraperitoneal tumor induction with 2.0 × 106 CC-531 tumor cells , 45 rats , 15 per treatment group , were randomly assigned to undergo either CS only , CS + RIT or CS + HIPEC . The operative procedures and application of the adjuvant therapies were performed as described previously . Toxicity of the treatment was determined clinically and by weighing the rats . Body weight was expressed as relative body weight compared with the body weight on the day of surgery . Survival was scored , and at autopsy the extent of tumor growth was determined . Follow-Up The primary endpoint was 16-week survival . As part of monitoring the physical condition during the immediate postoperative period , the general condition was monitored and the body weight was measured daily during the first 2 weeks . When the humane endpoint -LRB- HEP -RRB- was reached -LRB- signs of massive hemorrhagic ascites , physical inactivity or signs of intra-abdominal tumor growth with invalidating consequences -RRB- , rats were killed by O2/CO2-administration and immediately dissected . The HEP was determined by an experienced biotechnician who was blinded to the therapeutic regimen . At the time of the HEP rats were generally lethargic , showing signs of advanced PC as the presence of ascites . At dissection , the intraperitoneal tumor growth was scored as described previously . At 16 weeks postoperatively , the study was terminated and the remaining rats were euthanized and dissected . In case of absence of macroscopic tumor , all relevant organs , including the greater momentum , the mesentery , and the diaphragm were removed for histopathological staining to determine tumor presence microscopically . Sections were stained using hematoxylin & eosin -LRB- H&E -RRB- and/or immunohistochemical staining using the murine MG1 antibody in combination with a horse-anti-mouse IgG antibody , HRP conjugated -LRB- Vector Laboratories Inc. , Burlingame , CA , USA -RRB- . Statistical Analysis Statistical analysis was performed using SPSS -LRB- Chicago , IL -RRB- software and Graphpad Prism -LRB- Graphpad Software Inc. , San Diego CA -RRB- for analysis . Comparison of dichotomous values was done using chi-square or Fisher 's Exact test . Nonparametric testing was performed using two-way ANOVA testing . Survival curves were analyzed using Kaplan -- Meier curves and compared by means of the log-rank test . Posttesting using Bonferroni was applied to correct for multiple groups . All tests were two-sided ; the level of statistical significance was set at a P value of < .05 . RESULTS Intraperitoneal Distribution of HIPEC and Dose Determination The intraperitoneal distribution of the perfusate administered according to the previously described procedure , showed a distribution pattern amongst all quadrants , including the diaphragm bilaterally and at the mesenterial root -LRB- Fig. 2 -RRB- . FIG. 2 . Intraperitoneal distribution of methylene blue stained perfusate . The applied dose of 16 mg MMC/L resulted in a maximum mean weight loss of 13.7 ± 2.9 % at 4 days postoperatively . In addition , the first 3 days following the heated perfusion , animals were lethargic and suffered from diarrhea from day 2 until day 4 postoperatively . In contrast , the maximum weight loss in the 4 mg/L was 8.3 ± 2.9 % at day 3 and 7.5 ± 2.3 % at day 3 in the control group -LRB- Fig. 3 -RRB- . None of the animals died during the immediate postoperative period . Based on these observations , HIPEC , when administered at a dose of 16 mg/L for the duration of 60 minutes at the given temperature , was considered to be the maximal tolerable dose to be used for the HIPEC procedure . FIG. 3 . The relative body weight of Wag/Rij rats after exploratory laparotomy -LRB- control -RRB- and heated intraperitoneal chemotherapy -LRB- HIPEC -RRB- given immediately postoperatively in different doses . Data represent means ± standard error of the mean -LRB- SEM -RRB- . Operative Procedure Preoperative body weight did not differ between groups , P = .52 -LRB- Table 1 -RRB- At laparotomy , tumor nodules were present in the omentum , liver hilum , the mesentery , and gonadal fatpads -LRB- 1 -- 3 mm diameter -RRB- . Median PCI score at time of surgery was 5 -LRB- range 4 -- 8 -RRB- and was similar in all experimental groups . After surgical cytoreduction , residual disease remained in situ in 7 rats after cauterization and was equally distributed among the groups -LRB- P = .84 -RRB- . The surgical procedures without adjuvant therapy took 20 -- 30 minutes per animal . TABLE 1 . Treatment group characteristicsPCIMedian -LRB- range -RRB- CSCS + HIPECCS + RITPreoperative body weight -LRB- g -RRB- 266 -LRB- 251 -- 287 -RRB- 264 -LRB- 245 -- 285 -RRB- 262 -LRB- 244 -- 276 -RRB- Tumor score per site Greater omentum2 -LRB- 2 -- 3 -RRB- 2 -LRB- 1 -- 2 -RRB- 2 -LRB- 1 -- 2 -RRB- Liver hilum1 -LRB- 0 -- 1 -RRB- 1 -LRB- 0 -- 1 -RRB- 1 -LRB- 0 -- 1 -RRB- Perisplenic0 -LRB- 0 -- 1 -RRB- 0 -LRB- 0 -RRB- 0 -LRB- 0 -RRB- Mesentery1 -LRB- 0 -- 2 -RRB- 1 -LRB- 0 -- 2 -RRB- 1 -LRB- 0 -- 2 -RRB- Gonadal fatpads0 -LRB- 0 -- 2 -RRB- 0 -LRB- 0 -- 2 -RRB- 1 -LRB- 0 -- 2 -RRB- Diaphragm0 -LRB- 0 -- 1 -RRB- 0 -LRB- 1 -RRB- 0 -LRB- 0 -- 1 -RRB- Parietal peritoneum1 -LRB- 0 -- 1 -RRB- 1 -LRB- 0 -- 1 -RRB- 1 -LRB- 0 -- 1 -RRB- Total5 -LRB- 4 -- 8 -RRB- 5 -LRB- 4 -- 6 -RRB- 5 -LRB- 4 -- 8 -RRB- Resection macroscopically complete Yes121313 No322Treatment group characteristics -LRB- peritoneal cancer index ; PCI -RRB- found during laparotomy before the administration of the adjuvant therapy.CS , cytoreductive surgery ; HIPEC , heated intraperitoneal chemotherapy ; RIT , radioimmunotherapy . PCI is expressed as median and range . There was no intraoperative mortality . However , one rat in the CS + HIPEC and one rat in the CS + RIT group were euthanized after 2 and 9 days , respectively . The animal in the CS + HIPEC group showed massive weight loss as a result of bowel necrosis and subsequent perforation , the cause of death of the animal in the CS + RIT group remained unclear . The median intra-abdominal temperature during the HIPEC procedure , measured at the site where the greater omentum was removed , was 41.0 °C -LRB- range 40.4 -- 41.6 °C -RRB- . In contrast , the median rectal temperature was 34.6 °C -LRB- range 34.1 -- 34.8 °C -RRB- -LRB- Fig. 4 -RRB- . FIG. 4 . The recorded intra-abdominal and rectal temperature during the HIPEC procedure . Data represent means ± standard error of the mean -LRB- SEM -RRB- . CS and CS + RIT were well tolerated , whereas animals in the CS + HIPEC groups showed signs of physical discomfort ; animals of the latter group were generally lethargic and showed pilo erection two days following the procedure . In addition , these animals all suffered from diarrhea up to 4 days after the HIPEC procedure . The relative body weight after of the various treatment groups is depicted in Fig. 5 . Maximum body weight loss after CS or CS + RIT was similar -LRB- 7.3 ± 2.6 % vs 9.3 ± 1.8 % 4 days postoperatively , P > .05 -RRB- . Rats that received adjuvant HIPEC had a maximum body weight loss of 12.3 ± 1.7 % , which was significantly higher than that after CS alone -LRB- P < .001 -RRB- or CS + RIT -LRB- P < .001 -RRB- . Rats generally gained weight from the fifth postoperative day onward . In the HIPEC group , however , postoperative mean body weight remained significantly lower than that of the animals in the CS group , until 5 weeks postoperatively . FIG. 5 . The relative body weight of Wag/Rij rats with small peritoneal CC-531 tumors in the first 14 days after cytoreductive surgery -LRB- CS -RRB- only , CS + radioimmunotherapy given immediately postoperatively -LRB- RIT -RRB- or heated intraperitoneal chemotherapy -LRB- HIPEC -RRB- given immediately postoperatively . Data represent means ± standard error of the mean -LRB- SEM -RRB- . Treatment Efficacy During the experiment , 29 animals were euthanized because of massive amounts of ascites that resulted from intraperitoneal tumor growth . The mean amount of ascites at the humane endpoint was 31 ± 22.6 mL , 26.5 ± 23.8 mL , and 26.4 ± 22.6 mL in the CS , RIT , and HIPEC groups , respectively -LRB- P = .82 -RRB- . At the time of death , mean PCI in the CS , CS + HIPEC , and CS + RIT groups was 18 -LRB- range 9 -- 22 -RRB- , 12 -LRB- range 5 -- 15 -RRB- , and 18 -LRB- range 16 -- 19 -RRB- , respectively , with significant differences between the CS + HIPEC and both other treatment groups -LRB- P < .001 for both comparisons -RRB- . The survival curves of the various treatment groups are depicted in Fig. 6 . Median survival of the rats that were treated with CS only was 57 days -LRB- range 36 -- 112 -RRB- . Adjuvant HIPEC resulted in a median survival of 76 days -LRB- range 33 -- 112 -RRB- , P = .17 , when compared with CS only . Median survival of the rats that were treated with CS followed by the adjuvant administration of RIT was improved to a median survival of 97 days -LRB- range 49 -- 112 -RRB- , P < .001 compared with CS only . When compared with CS followed by adjuvant HIPEC , the adjuvant administration of RIT to surgery did not result in an improved survival -LRB- P = .33 -RRB- . FIG. 6 . Kaplan -- Meier survival curves for Wag/Rij rats with small peritoneal CC-531 tumors after cytoreductive surgery -LRB- CS -RRB- , CS + RIT -LRB- RIT -RRB- or CS + HIPEC -LRB- HIPEC -RRB- . At the endpoint of the study , 16 weeks after CS , 14 animals -LRB- two animals in the CS group , five animals in the CS + HIPEC group , and seven animals in the CS + RIT group -RRB- were still alive , without any physical signs of intraperitoneal tumor growth . Of these 14 animals that were still alive 16 weeks after surgery , one animal in the CS + HIPEC group and three animals in the CS + RIT group showed macroscopic evidence of tumor at dissection . In the remaining 10 animals -LRB- two in the CS alone group , four in the HIPEC group , and four in the RIT group -RRB- not even microscopic tumor presence was found . DISCUSSION In the present study , adjuvant radioimmunotherapy after cytoreductive surgery for peritoneal carcinomatosis of colorectal origin in rats significantly improved survival , whereas HIPEC did not . In addition , the application of HIPEC was associated with considerably more toxicity as compared to RIT . The treatment of peritoneal carcinomatosis was studied with CC-531 syngeneic tumors that grew intraperitoneally in Wag/Rij rats . This model is highly reproducible , and the growth and distribution pattern throughout the abdominal cavity are similar to the human entity of PC .14 Cytoreduction performed at 7 days after tumor reduction resulted in minimal residual disease -LRB- < 1 mm -RRB- . In this setting , both HIPEC and RIT result in maximum therapeutic efficacy .12,13,17 The MG1 MAb preferentially localizes in the CC-531 tumors ,11 with only minor localization in thymus , lymph node , salivary gland tissue , and skin .15177 Lu was selected as the radionuclide for RIT of minimal residual disease because of its high tumor uptake and adequate physical properties including a medium-energy β-emission with a maximum penetration range in tissue of 2.5 mm . In our previous studies we have used the combination of 177Lu-MG1 radionuclide-antibody . These studies demonstrated the combination to be highly effective for the improvement of survival in the model of PC as described previously .11,16 Moreover , in previous experiments we have shown that radioimmunotherapy with a radiolabeled irrelevant antibody is less effective by far compared with therapy with a radiolabeled specific antibody .18 HIPEC has been studied in only a few preclinical studies .19 -- 22 These studies showed that its use was associated with a decreased tumor load compared with control groups .19 However , in these studies , HIPEC was associated with a considerable toxicity , indicated by lethargy , marked body weight loss , and bacterial translocation .23 These results on toxicity are in corroboration with the results of our study and mimic the clinical effects of HIPEC . Clinical studies with postoperative intraperitoneal chemotherapy are associated with a high mortality and morbidity .12 Of 16 published reports on the use of HIPEC in the clinical setting , 13 reports described the administration of MMC .12 In vitro , MMC has also shown to inhibit growth of CC531 cells in a concentration a thousand-fold lower than the concentration used in the present experiment .24 The applied dose of 16 mg/L MMC in our study is within the range of doses applied in clinical practice -LRB- 5 -- 20 mg/L25 -RRB- and is higher than described in other preclinical studies -LRB- 2.25 mg/L19 and 4 mg/L22 -RRB- . In addition , in the present study HIPEC was applied as an adjuvant treatment to cytoreduction , whereas Pelz et al. 19 applied HIPEC as monotherapy with subsequent killing of the animals after only 10 days . The intraperitoneal distribution of MMC during the perfusion was studied before the start of the actual experiment and showed equal distribution among all quadrants . The effect of the perfusion technique on distribution differences and their associated differences in survival have never been studied clinically . Glehen and colleagues performed a large clinical study in 506 patients that were treated with both the open and closed abdomen perfusion technique . The authors reported no differences in survival between both perfusion techniques .6 This observation was confirmed by the study of Sugarbaker and colleagues .26 RIT with 74 MBq of the 177Lu-MG1 radionuclide-antibody in 3 mL has previously been shown to be an effective treatment for experimentally induced PC of colonic origin when administered intraperitoneally .11,16 The biodistribution of intraperitoneally injected 111In-labeled MG1 was studied by Koppe et al. and showed a preferential uptake of the radiolabeled antibody in the tumor .11 The extraperitoneal temperature of 48 °C necessary to obtain inflow temperatures of 44 °C would not have had a negative influence on the cytotoxicity of MMC , since Ahrar et al. 27 showed that only temperatures exceeding 60 °C decreased its cytotoxic effect . On the other hand , one has to bear in mind the fact that the additive effect of hyperthermia in HIPEC in the clinical setting has not yet been proven in a randomized trial . Elias et al. and Glehen et al. both reported the use of early postoperative chemotherapy -LRB- EPIC -RRB- , without hyperthermia , ranging from day 1 to day 5 after surgery , and HIPEC . Elias et al. found no significant difference in survival . Similarly , in the multicenter study in 506 patients of whom 53.3 and 24.3 % underwent HIPEC and EPIC alone , no significant difference was found in survival between the two treatment groups .6 The duration of the heated perfusion , 60 min . in this experiment , is in concordance with the recent consensus statement regarding Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy in the Management of Peritoneal Surface Malignancies of Colonic Origin , stating that the perfusion should last 60 -- 120 minutes .13 These data on the dose of MMC , the used perfusion time and temperature , together with our results that showed antitumor effect in the model of induced PC from CC531 cells -LRB- significantly lower PCI at HEP in favor of CS + HIPEC -RRB- , we can conclude that the HIPEC model used in our study was able to induce regression of PC of colonic origin . In the latest reported clinical trial on adjuvant RIT in the setting of colon cancer , Liersch et al. reported results of a Phase II trial with 131I-labeled anti-CEA antibody labetuzumab administered to patients after complete resection of colorectal liver metastases . This study , where RIT was applied in an adjuvant setting to complete resection , resulted in a promising 5-year survival rate of 51.5 % .10 The results of this study is in accordance with the conclusion of a recent review on the use of RIT to treat colon cancer .17 In this review , the authors state that the time may have come for clinical trials in which RIT is added to standard regimens to establish the place of this treatment modality as an adjuvant treatment after CS . To our knowledge , the present study is the first study comparing the use of RIT and HIPEC in an adjuvant setting to CS for the treatment of PC in colorectal cancer . Our preclinical studies indicate that the application of RIT immediately following CS can improve survival in rats with PC of CRC . Moreover , from the present study we conclude that the use of adjuvant RIT is an effective treatment with low toxicity . When compared with today 's standard of care , HIPEC , RIT was at least as effective . RIT consisted of an activity dose of 74 MBq of 177Lu-labeled MG1per rat , resulting in only minor toxicity , whereas the theoretical MTD of 177Lu-labeled antibodies in 250 g rats could be approximately 150 MBq .11 There are , however , some challenges to the clinical applications of adjuvant RIT . For example , after cytoreductive surgery , patients are transferred to the intensive care unit . Optimal patient care has to be balanced with radiation safety issues for the medical staff . Previously , we reported on the optimal time interval between RIT and CS .16 In that study , we showed that RIT should be administered as soon as possible after CS , with a window of opportunity for RIT administration up to 4 days after surgery . It can therefore be envisioned that for radiation safety reasons the therapy should be given not before discharge of the patient from ICU and removal of the abdominal drains . Our study therefore justifies the consideration of intraperitoneal radioimmunotherapy after cytoreductive surgery in case of peritoneal carcinomatosis of colorectal cancer . In clinical studies , this approach should be compared with HIPEC . CONCLUSION This study showed that RIT adjuvant to CS significantly improved survival compared to CS alone in a rat model of PC of CRC , whereas the contemporary gold standard , HIPEC , did not cause a significant improvement in survival . This improvement of survival was associated with a decreased level of treatment-related toxicity compared with HIPEC . Adjuvant radioimmunotherapy might therefore be an alternative adjuvant treatment after cytoreductive surgery of PC of colorectal origin in a clinical trial setting .

O O O B O O O O O O O O B I O O O O O O B I O O O O O B I I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O B I I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O B I O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O B O O B I O O O O O O O O O O O

radioimmunotherapy
cytoreductive surgery
heated intraperitoneal chemotherapy
peritoneal carcinomatosis
colon cancer
adjuvant


Iron and thrombosis Although essential for cell physiology , an increase or depletion of body iron has harmful effects on health . Apart from iron deficiency anemia and iron overload-related organ tissue damage , there are increasing evidences that body iron status is implicated in atherosclerotic cardiovascular diseases . The hypothesis formulated in 1981 that iron depletion may protect against cardiovascular events is intriguing and has generated a significant debate in the last two decades . Indeed , to study this phenomenon , several investigators have tried to design appropriate experimental and clinical studies and to identify useful biochemical and genetic markers of iron status . The results of the literature on the effect of iron deficiency and overload on vascular health are critically reviewed in this study from a pathogenic and clinical point of view . Introduction Iron is an essential nutrient for living cells because of its role as a cofactor for enzymes in the mitochondrial respiration chain , in the DNA synthesis , and being the central molecule for binding and transport of oxygen by hemoglobin and myoglobin . While the lack of iron leads to growth arrest and anemia , an increased accumulation of this metal is associated with toxic radical formation and progressive tissue damage . It is interesting to note that both iron deficiency and excess have been associated with an increased risk of developing thromboembolic events -LSB- 1 -- 7 -RSB- . This review will analyze , from a clinical and pathogenic point of view , the existing literature data on the relationship between iron and arterial and venous thrombosis . Iron deficiency and thrombosis There are several reports in the literature on thrombotic complications in iron-deficient children and adults -LSB- 8 -- 26 -RSB- . Secondary thrombocytosis has been implicated in many cases . Indeed , iron deficiency is a cause of reactive thrombocytosis , usually mild -LSB- 27 -RSB- . For instance , within a study group of children with iron deficiency , reactive thrombocytosis was found in up to one-third of them -LSB- 28 -RSB- . Nagai et al. -LSB- 29 -RSB- reported a case of severe iron deficiency with marked thrombocytosis -LRB- 1,020 × 109/l -RRB- that was complicated by central retinal vein occlusion . By contrast , Kinoshita et al. -LSB- 26 -RSB- described two cases of cerebral venous sinus thrombosis associated with iron deficiency and normal platelet count . Hartfield et al. -LSB- 13 -RSB- reported six children with iron deficiency who developed an ischemic stroke or venous thrombosis . Four of them had a concomitant thrombocytosis . In a prospective case -- control study , Stolz et al. -LSB- 30 -RSB- found that severe anemia , along with thrombophilia and hypercholesterolemia , were independent risk factors for cerebral venous thrombosis . The mechanisms causing reactive thrombocytosis in iron deficiency anemia are not completely understood . Iron is an important regulator of thrombopoiesis -LSB- 31 , 32 -RSB- . Whereas normal iron levels are required to prevent thrombocytosis by inhibiting thrombopoiesis , a minimum amount of iron is required to maintain platelet production . Thus , while thrombocytosis is usually associated with a mild iron deficiency and is the result of a lack of inhibition of thrombopoiesis , a severe defect of this metal may be accompanied by thrombocytopenia . However , studies on thrombopoietic cytokines failed to show any effect on reactive thrombocytosis in iron deficiency -LSB- 27 -RSB- . For instance , Akan et al. -LSB- 33 -RSB- assayed the serum levels of thrombopoietin , erythropoietin , leukemia inhibitor factor , IL-6 , and IL-11 in patients with iron-deficient anemia with or without elevated platelet count . Only erythropoietin level was elevated , correlated with thrombocytosis , and decreased with iron replacement . The other cytokines remained unchanged after therapy , suggesting that they probably do not play any significant role in iron deficiency-associated reactive thrombocytosis . Recently , Bilic and Bilic reported that the amino acid sequence homology of thrombopoietin and erythropoietin may explain the thrombocytosis in children with iron deficiency anemia -LSB- 34 -RSB- . By contrast , two other reports suggested that the relationship between iron deficiency and reactive thrombocytosis is more complex than a mere consequence of a crossreactivity between erythropoietin and thrombopoietin -LSB- 35 , 36 -RSB- . In addition to the increased thrombotic risk associated with high platelet count , other authors have suggested that the decrease in antioxidant defense in iron deficiency anemia may cause increased oxidant stress , which in turn may result in a tendency toward platelet aggregation -LSB- 37 -RSB- . Thus , the abnormal platelet count and function observed in iron deficiency anemia could act synergistically to promote thrombus formation , especially in the setting of an underlying atherosclerotic disease -LSB- 23 -RSB- . However , as not all cases of iron-related thrombotic events occur in patients with concomitant high platelet count -LSB- 13 , 26 -RSB- , other pathogenic mechanisms have been proposed in these last years . Thus , iron deficiency may contribute to a hypercoagulable state by affecting blood flow patterns within the vessels because of reduced deformability and increased viscosity of microcytic red blood cells -LSB- 13 -RSB- . Furthermore , anemic hypoxia secondary to iron deficiency could precipitate situations of increased metabolic stress -LRB- i.e. , infections -RRB- in particularly vulnerable areas of the brain supplied by end arteries , such as the basal ganglia , thalamus , and hypothalamus -LSB- 38 -RSB- . This phenomenon could explain the association between iron-deficient anemia and reversible focal deficits and stroke found by some authors -LSB- 1 , 13 , 39 , 40 -RSB- . Iron overload and thrombosis Accumulation of iron in excess of physiologic requirements has been implicated in the development of several chronic illnesses , including cardiovascular diseases -LSB- 7 -RSB- . As previously reported , iron is a prooxidant cofactor associated with an increased production of hydroxyl radical in cardiovascular tissues and increased progression of atherosclerosis in experimental models -LSB- 41 -- 44 -RSB- . Indeed , in an experimental mouse thrombosis model , a moderate iron overload markedly accelerated thrombus formation , impaired vasoreactivity , and enhanced the production of reactive oxygen species and systemic markers of oxidative stress -LSB- 41 -RSB- . It is interesting to note that the administration of dl-cysteine , a reactive oxygen species scavenger , completely abrogated the iron load-induced thrombus formation thus corroborating the hypothesis that iron accelerates thrombosis through a prooxidant mechanism . Similarly , laboratory investigations have demonstrated iron-dependent generation of reactive oxygen species in endothelial cell cultures and increased aortic atherosclerosis in the apolipoprotein E-deficient mice and cholesterol-fed rabbits with increased iron intake -LSB- 42 -- 44 -RSB- . The research in this field has focused on the evaluation of the impact of iron depletion and iron overload on cardiovascular outcomes . Sullivan first postulated in 1981 the `` iron hypothesis '' by which the chronic iron depletion has a protective effect against ischemic heart disease and may account for the reduced risk of cardiovascular events in menstruating women -LSB- 45 -- 47 -RSB- . Basing on these observations , some investigators have studied the effect of serial blood donations on the coronary heart disease risk , but their results were inconsistent -LSB- 48 -- 53 -RSB- . However , an important support to the hypothesis of a potential link between blood donation and reduced cardiovascular risk came from a recent study conducted by Zheng et al. -LSB- 54 -RSB- who found that high-frequency blood donors had decreased serum ferritin levels , a marker of body iron stores ; decreased serum 3-nitrotyrosine levels , a marker of oxidative stress ; and greater flow-mediated dilation in the brachial artery , a marker of vascular function . It is interesting to note that in a clinical study , the iron chelation with deferoxamine improved endothelial function in patients with coronary artery disease -LSB- 55 -RSB- . By contrast , in a multicenter , randomized controlled trial -LRB- the iron -LSB- Fe -RSB- and Atherosclerosis Study -LSB- FeAST -RSB- -RRB- on 1,277 patients with symptomatic peripheral arterial disease , the reduction of body iron stores by phlebotomy did not significantly decrease all-cause mortality or death plus nonfatal myocardial infarction and stroke -LSB- 56 -RSB- . Similarly , in a prospective analysis of the second National Health and Nutrition Examination Study -LRB- NHANES II -RRB- , Sempos et al. -LSB- 57 -RSB- observed either no association -LRB- in Caucasian men -RRB- or a possible nonsignificant increased risk -LRB- in Caucasian women -RRB- of cardiovascular or coronary heart disease death among individuals with low ferritin concentrations . On the other hand , the association between biochemical markers of body iron load and the risk of developing cardiovascular disease have been investigated by several studies -LSB- 7 , 58 -- 72 -RSB- . In the Kuopio Ischemic Heart Disease Risk Factor Study -LRB- KIHD -RRB- , serum ferritin levels were found to be one of the strongest risk factors for acute myocardial infarction among Finnish men -LSB- 61 -RSB- . Similar findings were reported from a Canadian study -LSB- 65 -RSB- that observed an increased risk of myocardial infarction among subjects in the highest serum iron category and from the Bruneck study -LSB- 64 -RSB- in which a positive association between serum ferritin levels and ultrasound measures of progression of carotid atherosclerosis over a 5-year follow-up period was found . A relationship between serum ferritin levels and carotid atherosclerosis was also identified by a recent study conducted by Wolff et al. -LSB- 71 -RSB- . Haidari et al. -LSB- 60 -RSB- observed a significant correlation between serum ferritin levels and risk of coronary heart disease in male Iranian patients . However , a number of epidemiological studies did not find an association between iron status and coronary artery disease -LSB- 61 , 69 , 70 , 72 -RSB- . For instance , Bozzini et al. -LSB- 61 -RSB- found that the mean serum ferritin concentrations were slightly higher in coronary heart disease patients than in controls , but this difference disappeared after adjusting for sex and C-reactive protein . The 1996 discovery of HFE gene mutations responsible for most cases of hereditary hemochromatosis has led to the use of genetic markers of iron overload , which are not influenced by external factors such as inflammation , in epidemiologic studies . Thus , several authors have investigated in recent years the relationship between C282Y and H63D mutations in the HFE gene and the risk of cardiovascular diseases -LSB- 73 -- 88 -RSB- . Three prospective population-based studies have reported an association between heterozygotes and vascular events -LSB- 75 -- 77 -RSB- . The first study was from a subgroup of the original Finnish KIHD cohort -LSB- 75 -RSB- . Of 68 individuals , 8 -LRB- 11.8 % -RRB- were diagnosed with acute myocardial infarction , and of 1,150 noncoronary heart disease participants , 77 -LRB- 6.7 % -RRB- were carriers of C282Y . The crude relative risk of myocardial infarction was 2.0 -LRB- 95 % CI = 0.9 -- 4.1 -RRB- and the adjusted relative risk was 2.3 -LRB- 95 % CI = 1.1 -- 4.8 -RRB- . In a cohort of 12,239 Dutch postmenopausal women , the C282Y carrier status was assessed among 531 women who died of cardiovascular disease and 555 randomly selected women who did not die of cardiovascular disease -LSB- 76 -RSB- . This study reported a relative risk of 1.6 -LRB- 95 % CI = 1.1 -- 2.4 -RRB- for total cardiovascular death . Finally , in the United States Atherosclerosis Risk in Communities -LRB- ARIC -RRB- study -LSB- 77 -RSB- , a C282Y carrier frequency of 9.9 % among 243 coronary heart disease cases and 6.1 % among 535 controls was reported . The crude relative risk of coronary heart disease associated with C282Y carrier status was 1.6 -LRB- 95 % CI = 0.9 -- 3.0 -RRB- and was 2.7 -LRB- 95 % CI = 1.2 -- 6.0 -RRB- after being controlled for other risk factors . To support of these findings , Gaenzer et al. -LSB- 78 -RSB- found an association between increased iron stores and impaired endothelial function -LRB- measured as endothelium-dependent dilation and intima-media thickness -RRB- in patients homozygous for C282Y mutation . Iron-depletion therapy normalized the endothelial function in such patients thus reducing the increased risk of cardiovascular events . However , the majority of the studies disagreed with these results -LSB- 61 , 79 -- 89 -RSB- . Indeed , Bozzini et al. -LSB- 61 -RSB- found a similar rate of carriers of C282Y mutation among patients with coronary atherosclerotic disease and controls . In a case -- control study on 1,098 subjects , Rossi et al. -LSB- 80 -RSB- found that C282Y mutation was not a predictor of asymptomatic carotid atherosclerosis . Franco et al. -LSB- 81 -RSB- reported that the HFE genes were not associated with coronary or peripheral atherosclerosis in patients aged less than 50 years . Similarly , in the West of Scotland Coronary Prevention Study -LRB- WOSCOPS -RRB- , Gunn et al. -LSB- 86 -RSB- found that the presence of a C282Y mutation in the HFE gene did not predict the occurrence of coronary events over a mean follow-up of 4.9 years . It is interesting to note that Yunker et al. -LSB- 89 -RSB- analyzed the relationship between biochemical and genetic markers of iron overload and carotid intima-media thickness and brachial flow-mediated vasodilation by high-resolution ultrasound in 907 males , but neither ferritin nor hemochromatosis genotype were related to brachial endothelial function and carotid atherosclerosis . In addition , a recent large study from Denmark found no increased risk of coronary heart disease among carriers of the C282Y mutation or individuals who had compound heterozygosity for the C282Y and H63D mutations -LSB- 79 -RSB- . Other investigation have focused on the association between genetic markers of iron overload and idiopathic dilated cardiomyopathy and stroke , but their results were conflicting -LSB- 90 -- 94 -RSB- . Finally , some authors have suggested that HFE C282Y could interact with other predisposing factors for venous thromboembolism , such as factor V Leiden , thus exacerbating their prothrombotic effect -LSB- 95 -RSB- . Table 1 summarizes the most important studies on the association between genetic markers of iron overload and cardiovascular diseases . Table 1Summary of the most important studies on the association between HFE gene mutations -LRB- C282Y and H63D -RRB- and the risk of cardiovascular diseasesAuthors -LSB- reference -RSB- Study designPopulationResultsTuomainen et al. -LSB- 75 -RSB- Prospective1 ,150 individualsC282Y heterozygosity is associated with a 2.3 RR for AMI compared with noncarriersRoest et al. -LSB- 76 -RSB- Prospective12 ,239 postmenopausal womenC282Y heterozygosity is associated with a 1.6 RR for TCD compared with noncarriersRasmussen et al. -LSB- 77 -RSB- Prospective243 CHD cases and 535 controlsC282Y heterozygosity is associated with a 2.7 RR for CHT compared with noncarriersGaenzer et al. -LSB- 78 -RSB- Case -- control41 C282Y/C282Y cases and 51 controlsC282Y homozygosity is associated with impaired endothelial functionBozzini et al. -LSB- 61 -RSB- Case -- control546 CHD cases and 303 controlsC282Y mutation is not associated with CHDRossi et al. -LSB- 80 -RSB- Case -- control1 ,098 subjectsC282Y mutation is not a risk factor for asymptomatic carotid atherosclerosisFranco et al. -LSB- 81 -RSB- Case -- control256 CHD cases and 272 controlsC282Y and H63D mutations are not associated with CHDEllervik et al. -LSB- 79 -RSB- Prospective9 ,178 individualsC282Y and H63D mutations are not associated with CHDCase -- control2 ,441 CHD and 1,113 AMI cases vs 8,080 controlsC282Y and H63D mutations are not associated with CHDGunn et al. -LSB- 86 -RSB- Case -- control482 CHD cases and 1,104 controlsC282Y mutation is not associated with CHDCampbell et al. -LSB- 88 -RSB- Case -- control924 AMI cases and 1,029 controlsC282Y mutation is not associated with CHDYunker et al. -LSB- 89 -RSB- Case -- control907 individualsHFE genotype is not related to brachial endothelial function and carotid atherosclerosisAMI : acute myocardial infarction , CHD : coronary heart disease , RR : relative risk , TCD : total cardiovascular death Conclusions It is interesting to note that although with different pathogenic mechanisms , both iron deficiency and overload have been associated with an increased thrombotic risk in experimental and clinical studies . However , several aspects need to be still elucidated in this field . In particular , large prospective controlled trials are needed to elucidate the role of genetic markers of iron stores and the impact of long-term iron depletion on morbidity and mortality from cardiovascular events .

B O B O O O O O O O O O O O O B O O O O O O O O B O B O B O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O B O B O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O