colorectal cancer ( crc ) is a common malignancy and a leading cause of cancer death worldwide , and the incidence of crc is increasing rapidly in eastern countries including korea as a result of recent changes in diet and lifestyle . the primary treatment of crc is a complete resection in stage i - iii patients followed by adjuvant chemotherapy in stage iii and high - risk stage ii patients . in the considerable number of patients with distant metastases ( i.e. , stage iv disease ) at the time of diagnosis , complete resection of colorectal liver metastases can provide a chance for long - term survival in approximately 20% of these patients . oxaliplatin plus fluoropyrimidine combination chemotherapy is widely used in both the adjuvant and palliative setting to improve survival of crc patients . with increasing incorporation of liver metastasectomy in the treatment strategy , sinusoidal obstruction syndrome ( sos ) in the liver is a long - term toxicity of oxaliplatin reported in patients who underwent hepatic resection after oxaliplatin - based chemotherapy [ 6 - 10 ] . sos , previously known as hepatic veno - occlusive disease , was most commonly reported to occur after bone marrow transplantation for hematologic malignancies . sos has been reported in 19%-79% of crc patients who underwent hepatic resection after oxaliplatin - based chemotherapy . toxic effect of oxaliplatin on sinusoidal endothelial cells ( sec ) causes disruption of the sinusoidal wall , subsequently causing congestive obstruction with impairment of sinusoidal blood flow . as a result , diffuse sinusoidal injury leads to portal hypertension , hepatomegaly , and hyperbilirubinemia with severe complications such as ascites and variceal bleeding in rare cases . in addition , development of sos may be associated with increased morbidity and mortality following hepatic resection in patients treated with preoperative oxaliplatin - based regimens . prediction of sos development is important in selection of proper candidates for hepatic resection and preoperative management of patients at risk of sos . increase in spleen size , elevated aspartate aminotransferase to platelet ratio index , and hyaluronic acid levels have been reported as reliable indicators of sos . however , there is no reliable biomarker for prediction of sos before oxaliplatin treatment that could help in the decision of oxaliplatin use . in addition , association of the susceptibility to sos with antitumor efficacy in the adjuvant setting has not been studied . many recent efforts have been made to clarify the pathogenesis of sos at the molecular level and to use this as a molecular marker of sos . this model also suggested that additional mechanisms such as an increase in expression of matrix metalloproteinase-9 ( mmp-9 ) ( and to a lesser extent mmp-2 ) , reduced synthesis in nitric oxide , and oxidative stress contribute to sec injury . vascular endothelial growth factor ( vegf ) is known to regulate mmp-9 activation by inducing its expression , and the degree of the increase in vegf serum level parallels the clinical severity of sos . therefore , vegf blockade may attenuate sinusoidal injury by down - regulating mmp-9 production . in the liver , nitric oxide is produced by the nitric oxide synthase 3 ( nos3 ) expressed in the sec . decreased activity of glutathione s - transferase ( gst ) leads to a decrease of adduct formation between glutathione and platinum , consequently attenuating a defense mechanism against oxaliplatin . in this study , we have chosen several genes related to the pathogenesis of sos ( vegfa , mmp9 , nos3 , and gstp1 ) , as shown in the previous model , and studied the association between their genetic polymorphisms and sos following oxaliplatin treatment using spleen size as a surrogate of sos . crc patients receiving adjuvant 5-fluorouracil , leucovorin , and oxaliplatin ( folfox ) were selected in order to eliminate the potential bias caused by the extent of liver metastasis . we also analyzed the association of sos with disease - free survival ( dfs ) following adjuvant folfox . this retrospective analysis included stage iii or high - risk stage ii crc patients who received adjuvant folfox chemotherapy after complete resection of crc from september 2005 to december 2009 at seoul national university hospital ( snuh ) and participated in pharmacogenomics study of chemotherapy among cancer patients . other eligibility criteria were age over 18 years , adenocarcinoma histology , complete resection of primary tumor with negative margin , adequate organ function , completion of at least six cycles of the planned 12 cycles of chemotherapy , and computed tomography ( ct ) images obtained before and after chemotherapy adequate for measurement of spleen size . for adequate measurement , contrast - enhanced ct images were obtained with at least 5 mm slice thickness or less . patients with underlying severe liver disease such as active viral hepatitis , severe steatohepatitis , or liver cirrhosis before chemotherapy were excluded . each cycle of folfox-4 consisted of oxaliplatin ( 85 mg / m ) on day 1 and leucovorin ( 200 mg / m ) and a bolus of 5-fluorouracil ( 5-fu ; 400 mg / m ) followed by a 22-hour infusion of 5-fu ( 600 mg / m ) on days 1 and 2 , which was repeated every 2 weeks . modified folfox-6 consisted of oxaliplatin ( 85 mg / m ) , leucovorin ( 400 mg / m ) , and a bolus of 5-fu ( 400 mg / m ) followed by a 46-hour infusion of 5-fu ( 2,400 mg / m ) repeated every 2 weeks . the study protocol was reviewed and approved by the institutional review board of snuh and was conducted in accordance with the declaration of helsinki . genomic dna was extracted from peripheral blood mononuclear cells using a qiaamp dna kit ( qiagen , valencia , ca ) , and each polymorphism was determined using a pyrosequencing method ( pyromark q96 i d , qiagen ) . primer sequence and biological effect of each polymorphism ten polymorphisms in 4 potentially sos - related genes were analyzed : 2578c / a , 1154g / a , 634g / c , and 936c / t in vegfa ; 1562c / t , 836a / g , and 2003g / a in mmp9 ; 786t / c and 894g / t in nos3 , and ile105val in gstp1 . spleen size was measured by loading the ct images into a 3d workstation ( rapidia ver . the outline of the spleen on each axial image of ct scans was traced using an electronic free - curve in the software , cross - sectional areas were calculated , and then the sum of the areas was multiplied by slice thickness for calculation of spleen volume . spleen sizes measured from ct images obtained before chemotherapy and after completion of chemotherapy were compared . association between change in spleen size and each genetic polymorphism was analyzed using student s t test . the relationship between splenomegaly and each polymorphism was also analyzed using pearson s chi - square or fisher s exact test . splenomegaly was functionally defined as a  50% increase in spleen size after oxaliplatin - based chemotherapy . multivariate logistic regression analysis was performed to evaluate the effect of other clinicopathologic factors on splenomegaly . in this analysis , the backward stepwise regression model including only variables with a p - value < 0.10 in univariate analysis was used . survival functions for dfs were estimated using the kaplan - meier method , and differences between groups were tested using a log - rank test . this retrospective analysis included stage iii or high - risk stage ii crc patients who received adjuvant folfox chemotherapy after complete resection of crc from september 2005 to december 2009 at seoul national university hospital ( snuh ) and participated in pharmacogenomics study of chemotherapy among cancer patients . other eligibility criteria were age over 18 years , adenocarcinoma histology , complete resection of primary tumor with negative margin , adequate organ function , completion of at least six cycles of the planned 12 cycles of chemotherapy , and computed tomography ( ct ) images obtained before and after chemotherapy adequate for measurement of spleen size . for adequate measurement , contrast - enhanced ct images were obtained with at least 5 mm slice thickness or less . patients with underlying severe liver disease such as active viral hepatitis , severe steatohepatitis , or liver cirrhosis before chemotherapy were excluded . each cycle of folfox-4 consisted of oxaliplatin ( 85 mg / m ) on day 1 and leucovorin ( 200 mg / m ) and a bolus of 5-fluorouracil ( 5-fu ; 400 mg / m ) followed by a 22-hour infusion of 5-fu ( 600 mg / m ) on days 1 and 2 , which was repeated every 2 weeks . modified folfox-6 consisted of oxaliplatin ( 85 mg / m ) , leucovorin ( 400 mg / m ) , and a bolus of 5-fu ( 400 mg / m ) followed by a 46-hour infusion of 5-fu ( 2,400 mg / m ) repeated every 2 weeks . the study protocol was reviewed and approved by the institutional review board of snuh and was conducted in accordance with the declaration of helsinki . genomic dna was extracted from peripheral blood mononuclear cells using a qiaamp dna kit ( qiagen , valencia , ca ) , and each polymorphism was determined using a pyrosequencing method ( pyromark q96 i d , qiagen ) . primer sequence and biological effect of each polymorphism are shown in supplementary table 1 . ten polymorphisms in 4 potentially sos - related genes were analyzed : 2578c / a , 1154g / a , 634g / c , and 936c / t in vegfa ; 1562c / t , 836a / g , and 2003g / a in mmp9 ; 786t / c and 894g / t in nos3 , and ile105val in gstp1 . spleen size was measured by loading the ct images into a 3d workstation ( rapidia ver . the outline of the spleen on each axial image of ct scans was traced using an electronic free - curve in the software , cross - sectional areas were calculated , and then the sum of the areas was multiplied by slice thickness for calculation of spleen volume . spleen sizes measured from ct images obtained before chemotherapy and after completion of chemotherapy were compared . association between change in spleen size and each genetic polymorphism was analyzed using student s t test . the relationship between splenomegaly and each polymorphism splenomegaly was functionally defined as a  50% increase in spleen size after oxaliplatin - based chemotherapy . multivariate logistic regression analysis was performed to evaluate the effect of other clinicopathologic factors on splenomegaly . in this analysis , the backward stepwise regression model including only variables with a p - value < 0.10 in univariate analysis was used . survival functions for dfs were estimated using the kaplan - meier method , and differences between groups were tested using a log - rank test . of a total of 124 patients included in this study , 73 patients ( 58.9% ) were male ( table 1 ) . patients received median 12 cycles ( range , 6 to 12 cycles ) of adjuvant folfox ; 108 patients ( 87.1% ) completed all 12 cycles of adjuvant folfox . the median cumulative dose of oxaliplatin was 935 mg / m ( range , 459 to 1,020 mg / m ) . increase in spleen size was observed in 109 patients ( 87.9% ) after completion of chemotherapy compared with baseline size before chemotherapy . the median change in spleen size was 31% ( range , 42% to 168% ) . the mean increase in spleen size was higher in patients receiving a full dose of oxaliplatin during 12 cycles ( n=54 ) compared with patients receiving a reduced dose of oxaliplatin ( n=70 ) ( 50.740.4% vs. 30.234.4% , p=0.003 ) . ten genetic polymorphisms within four genes were analyzed ( vegfa , mmp9 , nos3 , and gstp1 ) . when the relationship between these genetic polymorphisms and change in spleen size or splenomegaly was analyzed in univariate analysis , no genetic polymorphism was associated with change in spleen size or splenomegaly ( p - value , not significant ) ( table 2 ) . in univariate analysis for development of splenomegaly , only the cumulative dose of oxaliplatin ( full vs. reduced dose ) was significantly associated with development of splenomegaly . no significant associations were found between splenomegaly and other clinical factors ( age [ > 65 years vs.  65 years ] , lowest platelet count during chemotherapy [ < 75,000/mm vs.  75,000/mm ] and the presence of chronic liver disease ) . in multivariate logistic regression analysis performed using variables with p - values < 0.10 in univariate analysis , the cumulative dose of oxaliplatin and the lowest platelet count during the chemotherapy period were associated with splenomegaly ( table 3 ) . during adjuvant folfox chemotherapy and the follow - up period after completion of chemotherapy , patients with splenomegaly had lower values of mean platelet count compared to patients without splenomegaly . this difference in mean platelet count was the most prominent from 3 months to 6 months after initiation of folfox ( p < 0.05 , by student s t test ) , and the difference was gradually reduced after completion of chemotherapy ( fig . 1 ) . in addition , patients with splenomegaly had more severe thrombocytopenia compared to those without splenomegaly during the chemotherapy period ( mean lowest platelet count , 85,00028,000/mm vs. 115 , 00042,000/mm ; p < 0.001 ) . we also analyzed the association of splenomegaly , a surrogate marker of sos , with treatment outcome of adjuvant folfox chemotherapy . the 3-year dfs rate was 89.1% ( 95% confidence interval [ ci ] , 79.9% to 98.3% ) in patients who developed splenomegaly and 85.7% ( 95% ci , 77.7% to 93.7% ) in patients without splenomegaly ( p=0.42 by log - rank test ) ( fig . of a total of 124 patients included in this study , 73 patients ( 58.9% ) were male ( table 1 ) . patients received median 12 cycles ( range , 6 to 12 cycles ) of adjuvant folfox ; 108 patients ( 87.1% ) completed all 12 cycles of adjuvant folfox . the median cumulative dose of oxaliplatin was 935 mg / m ( range , 459 to 1,020 mg / m ) . increase in spleen size was observed in 109 patients ( 87.9% ) after completion of chemotherapy compared with baseline size before chemotherapy . the median change in spleen size was 31% ( range , 42% to 168% ) . the mean increase in spleen size was higher in patients receiving a full dose of oxaliplatin during 12 cycles ( n=54 ) compared with patients receiving a reduced dose of oxaliplatin ( n=70 ) ( 50.740.4% vs. 30.234.4% , p=0.003 ) . ten genetic polymorphisms within four genes were analyzed ( vegfa , mmp9 , nos3 , and gstp1 ) . when the relationship between these genetic polymorphisms and change in spleen size or splenomegaly was analyzed in univariate analysis , no genetic polymorphism was associated with change in spleen size or splenomegaly ( p - value , not significant ) ( table 2 ) . in univariate analysis for development of splenomegaly , only the cumulative dose of oxaliplatin ( full vs. reduced dose ) was significantly associated with development of splenomegaly . no significant associations were found between splenomegaly and other clinical factors ( age [ > 65 years vs.  65 years ] , lowest platelet count during chemotherapy [ < 75,000/mm vs.  75,000/mm ] and the presence of chronic liver disease ) . in multivariate logistic regression analysis performed using variables with p - values < 0.10 in univariate analysis , the cumulative dose of oxaliplatin and the lowest platelet count during the chemotherapy period were associated with splenomegaly ( table 3 ) . during adjuvant folfox chemotherapy and the follow - up period after completion of chemotherapy , patients with splenomegaly had lower values of mean platelet count compared to patients without splenomegaly . this difference in mean platelet count was the most prominent from 3 months to 6 months after initiation of folfox ( p < 0.05 , by student s t test ) , and the difference was gradually reduced after completion of chemotherapy ( fig . , patients with splenomegaly had more severe thrombocytopenia compared to those without splenomegaly during the chemotherapy period ( mean lowest platelet count , 85,00028,000/mm vs. 115 , 00042,000/mm ; p < 0.001 ) . we also analyzed the association of splenomegaly , a surrogate marker of sos , with treatment outcome of adjuvant folfox chemotherapy . the 3-year dfs rate was 89.1% ( 95% confidence interval [ ci ] , 79.9% to 98.3% ) in patients who developed splenomegaly and 85.7% ( 95% ci , 77.7% to 93.7% ) in patients without splenomegaly ( p=0.42 by log - rank test ) ( fig . in the current study , splenomegaly , a surrogate of sos , was frequently observed after adjuvant folfox chemotherapy in crc patients . an increase in spleen size compared with baseline size before starting oxaliplatin - based chemotherapy was observed in 109 patients ( 87.9% ) , with a median increase in spleen size of 31% . although the direct comparison was difficult due to the difference in the cumulative dose of oxaliplatin and treatment duration every study , this change in spleen size is comparable to that reported in another study . the cumulative dose of oxaliplatin and the lowest platelet count during chemotherapy were clinical factors associated with splenomegaly . patients with splenomegaly showed more severe thrombocytopenia than patients without splenomegaly during or after oxaliplatin - based chemotherapy . this is in line with a previous study reporting on the relationship between splenomegaly and thrombocytopenia , which suggested splenic sequestration induced by portal hypertension as a possible mechanism of thrombocytopenia in patients with sos . thrombocytopenia related to sos is common , but usually not severe . in this study , thrombocytopenia less than 50,000 mm was only observed in five cases and there were no significant bleeding events . thrombocytopenia due to oxaliplatin - induced sos can be prolonged until 2 - 3 years after completion of oxaliplatin treatment . oxaliplatin - induced sos is infrequently presented with ascites , jaundice , and hepatomegaly suggestive of portal hypertension . in our study , none of the patients showed clinically apparent symptoms and signs of portal hypertension except splenomegaly . a number of studies have been conducted for discovery of molecular predictive biomarkers of sos based on its pathogenesis . rubbia - brandt et al . , who examined gene expression profiles in livers with oxaliplatin - induced sos and matched normal controls , found 913 differentially expressed genes . results of pathway analysis showed significant upregulation of expression in six pathways : acute phase response , coagulation system , hepatic fibrosis / hepatic stellate cell activation , and oxidative stress . in addition , angiogenic and hypoxic factors including vegfc and hypoxia - inducible factor 1-alpha ( hif1a ) were upregulated . a similar study conducted by a french group also confirmed the upregulation of genes involved in angiogenesis and coagulation in oxaliplatin - induced sinusoidal injuries . glutathione forms an adduct with platinum by gst , which leads to detoxification of oxaliplatin . in another study evaluating the role of gst polymorphism as a risk factor for sos , gstm1-null genotype we have analyzed the association between splenomegaly and genetic polymorphisms in four sos - related genes . we hypothesized that these genes might be relevant to the pathogenesis of sos , as suggested in an animal model . in this model , upregulation of mmp-9 and subsequent decrease of nitric oxide contributed to sec injury , as well as glutathione depletion followed by production of reactive oxygen species . further studies are needed to determine the role of other genes or polymorphisms as a risk factor of sos . some studies have suggested a negative impact of oxaliplatin - related sos on long term outcomes with early recurrence and decreased overall survival in patients with colorectal liver metastases . in addition , more severe grade of sos was correlated with lower histopathological tumor regression . in our study , splenomegaly , used as a biomarker of sos , showed no association with survival outcome . a possible explanation is that , unlike previous studies , our study was conducted in patients in the adjuvant setting without distant metastasis . in addition , the frequency of hepatic recurrence was not significantly different according to the development of splenomegaly ( 2.1% [ 1/47 ] in patients with splenomegaly vs. 5.2% [ 4/77 ] in patients without splenomegaly , p=0.65 ) . the current study has some limitations including the retrospective nature of the study and no histopathological confirmation of sos . however , the homogeneity of the study population including only patients in the adjuvant setting is the strength of the study . in summary , we show that splenomegaly occurred in 87.9% of crc patients receiving adjuvant folfox treatment and it was also associated with more severe thrombocytopenia . as we found no association between the genetic polymorphisms analyzed herein and development of splenomegaly , future studies investigating other biomarker candidates are warranted for prediction of sos .