we initially studied 90 patients ( age  21 years ) who had newly diagnosed all and were enrolled on the st . , we subsequently studied an additional 170 patients with all enrolled on the same treatment protocol , using publicly available snp data we had previously reported . jude children s research hospital , and informed consent was obtained from patients , their guardians , or both before enrollment . leukemia cells were isolated by applying a ficoll - hypaque gradient to bone marrow aspirates obtained at diagnosis ( median , 97% blast cells ) . normal leukocytes were isolated from peripheral blood samples obtained after the successful completion of remission induction therapy ( on days 4548 after the start of treatment ) . to assess gene copy number loss in msh2-l leukemia cells , dna was extracted from leukemia cells and normal peripheral blood leukocytes ( obtained when patients were in complete remission ) and genotyped for 600k snps using the affymetrix genechip human mapping 50k - hind-240 , 50k - xba-240 , 250k - sty and 250k nsp snp arrays . dna was restriction enzyme digested , pcr - amplified , purified , labeled , fragmented and hybridized to the arrays according to the manufacturer s instructions . snp array data were analyzed using dchip for chromosomal abnormalities . to improve the accuracy of copy number inference using dchip , we applied a normalization procedure that uses snps exclusively from regions shown to be diploid in the normal leukocytes and maps signals from those snps to a common target probability distribution . the snps with an estimated copy number lower than 1.40 were considered as evidence of deletions . we initially performed snp analysis on prkcz , because it is known to regulate msh2 stability . this led us to subsequently perform a broader pathway analysis , to interrogate additional genes upstream of prkcz ( figure 1c ) . for each snp , we evaluated the over - representation of deletions in msh2-l leukemia cells compared to msh - h leukemias , using fisher s exact test . the significance of each snp was then adjusted for multiple testing using 100,000 permutations . at each permutation , we recorded the smallest p - value among all the 122 snps based on randomly assigned msh2 status . the adjusted p - values were computed as the proportion of permutations whose smallest p - values were lower than or equal to the observed . the human t - lineage leukemia cell line ccrf - cem was obtained from the american type culture collection . the human pre - b leukemia cell lines 697 and nalm-6 were obtained from the german collection of microorganisms and cell cultures . cells were cultured in rpmi-1640 medium containing 2 mm glutamine and 10% fetal bovine serum at 37 c with 5% co2 . ccrf - cem cells were infected with mission lentiviral transduction particles ( sigma - aldrich ) produced from a library of sequence - verified shrnas targeting human pik3c2b , frap1/mtor , herc1 or prkcz transcripts . non - target shrna control particles ( shc002v ) were also purchased from sigma - aldrich . fisher s exact test was used to test the overrepresentation of losses among msh2 low patients versus msh2 positive patients . a linear regression model was used to test the correlation between the levels of msh2 mrna and protein . the expression levels of the probe sets were analyzed by applying a general linear model in which the effect of msh2 status ( positive vs. negative ) was adjusted for the all genetic subtypes . the cox proportional hazards model was used to identify independent prognostic effect of msh2 status . for patients who achieved complete remission , cumulative incidence of hematological relapse were constructed by the method of kalbfleisch and prentice , and compared with gray s test . we initially studied 90 patients ( age  21 years ) who had newly diagnosed all and were enrolled on the st . , we subsequently studied an additional 170 patients with all enrolled on the same treatment protocol , using publicly available snp data we had previously reported . jude children s research hospital , and informed consent was obtained from patients , their guardians , or both before enrollment . leukemia cells were isolated by applying a ficoll - hypaque gradient to bone marrow aspirates obtained at diagnosis ( median , 97% blast cells ) . normal leukocytes were isolated from peripheral blood samples obtained after the successful completion of remission induction therapy ( on days 4548 after the start of treatment ) . to assess gene copy number loss in msh2-l leukemia cells , dna was extracted from leukemia cells and normal peripheral blood leukocytes ( obtained when patients were in complete remission ) and genotyped for 600k snps using the affymetrix genechip human mapping 50k - hind-240 , 50k - xba-240 , 250k - sty and 250k nsp snp arrays . dna was restriction enzyme digested , pcr - amplified , purified , labeled , fragmented and hybridized to the arrays according to the manufacturer s instructions . snp array data were analyzed using dchip for chromosomal abnormalities . to improve the accuracy of copy number inference using dchip , we applied a normalization procedure that uses snps exclusively from regions shown to be diploid in the normal leukocytes and maps signals from those snps to a common target probability distribution . the snps with an estimated copy number lower than 1.40 were considered as evidence of deletions . we initially performed snp analysis on prkcz , because it is known to regulate msh2 stability . this led us to subsequently perform a broader pathway analysis , to interrogate additional genes upstream of prkcz ( figure 1c ) . , we evaluated the over - representation of deletions in msh2-l leukemia cells compared to msh - h leukemias , using fisher s exact test . the significance of each snp was then adjusted for multiple testing using 100,000 permutations . at each permutation , we recorded the smallest p - value among all the 122 snps based on randomly assigned msh2 status . the adjusted p - values were computed as the proportion of permutations whose smallest p - values were lower than or equal to the observed . the human t - lineage leukemia cell line ccrf - cem was obtained from the american type culture collection . the human pre - b leukemia cell lines cells were cultured in rpmi-1640 medium containing 2 mm glutamine and 10% fetal bovine serum at 37 c with 5% co2 . ccrf - cem cells were infected with mission lentiviral transduction particles ( sigma - aldrich ) produced from a library of sequence - verified shrnas targeting human pik3c2b , frap1/mtor , herc1 or prkcz transcripts . non - target shrna control particles ( shc002v ) were also purchased from sigma - aldrich . fisher s exact test was used to test the overrepresentation of losses among msh2 low patients versus msh2 positive patients . a linear regression model was used to test the correlation between the levels of msh2 mrna and protein . the expression levels of the probe sets were analyzed by applying a general linear model in which the effect of msh2 status ( positive vs. negative ) was adjusted for the all genetic subtypes . the cox proportional hazards model was used to identify independent prognostic effect of msh2 status . for patients who achieved complete remission , cumulative incidence of hematological relapse were constructed by the method of kalbfleisch and prentice , and compared with gray s test .