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Nucleophosmin (NPM1)–mutated acute myeloid leukemia (AML), which is recognized as a provisional entity in the World Health Organization 2008 classification of myeloid neoplasms, accounts for 30% of AML. We analyzed 1227 diagnostic and follow-up samples in 252 NPM1-mutated AML patients with 17 different NPM1 mutation–specific real-time quantitative polymerase chain reaction (RQ-PCR) assays. Paired diagnostic/relapse samples of 84 patients revealed stable NPM1 mutations in all cases, suggesting that they are pathogenetically early events and thus applicable for minimal residual disease detection. A total of 47 relapses were predictable because of an NPM1 mutation level (%NPM1/ABL1) increase of at least 1 log or in 15 cases because of NPM1 mutation levels not decreasing less than 3 log ranges. A high prognostic value of NPM1 levels was shown for 4 different intervals after therapy was initiated. Furthermore, thresholds of 0.1 and 0.01%NPM1/ABL1 during/after treatment discriminated between prognostic subgroups. Univariate analyses, including age, white blood cell count, blast count, CD34 positivity, FLT3 mutations status, FAB type, karyotype, NPM1 mutation type, and pretreatment NPM1 mutational level, showed that, besides NPM1 mutation level, only age and FLT3-LM mutation status were prognostically significant for EFS. Multivariate analysis, including age, FLT3-LM status, and NPM1 mutation level at different time points, demonstrated that NPM1 level was the most relevant prognostic factor during first-line treatment. Similar results were obtained in patients undergoing second-line chemotherapy or allogeneic stem cell transplantation.

Nucleophosmin(NPM1)–mutated acute myeloid leukemia (AML), which is recognized as a provisional entity in the World Health Organization 2008 classification of myeloid neoplasms, accounts for 30% of AML. We analyzed 1227 diagnostic and follow-up samples in 252 NPM1-mutated AML patients with 17 different NPM1mutation–specific real-time quantitative polymerase chain reaction (RQ-PCR) assays. Paired diagnostic/relapse samples of 84 patients revealed stable NPM1mutations in all cases, suggesting that they are pathogenetically early events and thus applicable for minimal residual disease detection. A total of 47 relapses were predictable because of an NPM1mutation level (%NPM1/ABL1) increase of at least 1 log or in 15 cases because of NPM1mutation levels not decreasing less than 3 log ranges. A high prognostic value of NPM1levels was shown for 4 different intervals after therapy was initiated. Furthermore, thresholds of 0.1 and 0.01%NPM1/ABL1during/after treatment discriminated between prognostic subgroups. Univariate analyses, including age, white blood cell count, blast count, CD34 positivity, FLT3mutations status, FAB type, karyotype, NPM1mutation type, and pretreatment NPM1mutational level, showed that, besides NPM1mutation level, only age and FLT3-LM mutation status were prognostically significant for EFS. Multivariate analysis, including age, FLT3-LM status, and NPM1mutation level at different time points, demonstrated that NPM1level was the most relevant prognostic factor during first-line treatment. Similar results were obtained in patients undergoing second-line chemotherapy or allogeneic stem cell transplantation.

Nucleophosmin (NPM1)exon-12 gene mutations are the hallmark of a large acute myelogenous leukemia (AML) subgroup with normal karyotype, but their prognostic value in this AML subset has not yet been determined. We screened 401 AML patients with normal karyotype treated within the German AML Cooperative Group Protocol 99 (AMLCG99) study for NPM1mutations. Results were related with partial tandem duplications within the MLLgene (MLL-PTD), Fms-like tyrosine kinase 3–length mutations (FLT3-LM), the tyrosine kinase domain of FLT3(FLT3-TKD), NRAS, KIT,and CEBPAmutations and with clinical characteristics and outcome. NPM1mutations were detected in 212 (52.9%) of 401 patients. Fourteen mutations, including 8 new variants, were identified. NPM1-mutated cases associated frequently with FLT3mutations but rarely with other mutations. The NPM1-mutated group had a higher complete remission (CR) rate (70.5% vs 54.7%, P= .003), a trend to a longer overall survival (OS; median 1012 vs 549 days, P= .076), and significantly longer event-free survival (EFS; median 428 vs 336 days; P= .012). The favorable impact of NPM1mutations on OS and EFS clearly emerged in the large group (264 [66.8%] of 395 cases) of normal-karyotype AML without FLT3-LM. This positive effect was lost in the presence of a concomitant FLT3-LM, since survival of the NPM1+/FLT3-LM+double positive was similar to NPM1–/FLT3-LM+cases. In conclusion, this study demonstrates that NPM1+/FLT3-LM–mutations are an independent predictor for a favorable outcome in AML with normal karyotype.

Nucleophosmin (NPM1) exon-12 gene mutations are the hallmark of a large acute myelogenous leukemia (AML) subgroup with normal karyotype, but their prognostic value in this AML subset has not yet been determined. We screened 401 AML patients with normal karyotype treated within the German AML Cooperative Group Protocol 99 (AMLCG99) study for NPM1 mutations. Results were related with partial tandem duplications within the MLL gene (MLL-PTD), Fms-like tyrosine kinase 3–length mutations (FLT3-LM), the tyrosine kinase domain of FLT3 (FLT3-TKD), NRAS, KIT, and CEBPA mutations and with clinical characteristics and outcome. NPM1 mutations were detected in 212 (52.9%) of 401 patients. Fourteen mutations, including 8 new variants, were identified. NPM1-mutated cases associated frequently with FLT3 mutations but rarely with other mutations. The NPM1-mutated group had a higher complete remission (CR) rate (70.5% vs 54.7%, P = .003), a trend to a longer overall survival (OS; median 1012 vs 549 days, P = .076), and significantly longer event-free survival (EFS; median 428 vs 336 days; P = .012). The favorable impact of NPM1 mutations on OS and EFS clearly emerged in the large group (264 [66.8%] of 395 cases) of normal-karyotype AML without FLT3-LM. This positive effect was lost in the presence of a concomitant FLT3-LM, since survival of the NPM1+/FLT3-LM+ double positive was similar to NPM1–/FLT3-LM+ cases. In conclusion, this study demonstrates that NPM1+/FLT3-LM– mutations are an independent predictor for a favorable outcome in AML with normal karyotype.

Schnittger: MLL Munich Leukemia Lab: Equity Ownership. Tschulik:MLL Munich Leukemia Lab: Employment. Wendland:MLL Munich Leukemia Lab: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Lab: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Abstract 438

In CML real time PCR for the detection of residual disease usually has been done by the detection of the BCR-ABL-fusion transcript. In addition some reports have shown the usefulness of WT1 as a target for follow up in CML blast crisis. The aim of this study was to evaluate whether WT1 in addition to BCR-ABL assessment in imatinib treated CML can give any new information. Expression of BCR-ABL as well as WT1 (ELN assay) was normalized to ABL and expressed as % to ABL. In total 268 bone marrow samples (spls) of 40 patients (pts) have been analysed. Sixteen spls were newly diagnosed untreated CML. 252 spls were analysed at several timepoint during treatment with imatinib starting with a “near to untreated” sample with a >50% BCR-ABL/ABL ratio. According to standard definitions 6 of the 40 pts showed no response, 16 minor (MR), and 18 major molecular response (MMR) as determined by BCR-ABL expression. Three of the MR and one of the MMR pts relapsed. The 14 pretreatment spls showed a median WT1 expression of 3.36 (range 0.176–14.9) whereas the median BCR-ABL level was 56.6 (range 11.4–149.2). Thus the BCR-ABL level was more than one log above the WT1 level and was more uniform within the cohort. This lower total expression and a background level of 0.04 (as estimated from 7 normal bone marrows) results in 2–3 log lower sensitivity of WT1 compared to BCR-ABL. The correlation of WT1 and BCR-ABL in the total cohort of 268 spls was low (r=0.259). Median WT1 expression in BCR-ABL negative spls (n=15) was 0.055 and thus correlates to that of normal spls. Regarding the total follow up in all 40 pts a good correlation of WT1 and BCR-ABL was found only in 9 cases, all of which had a WT1 expression >10 at the start of follow up. 7 pts had a WT1 expression between 1–7 with good correlation to BCR-ABL but restricted sensitivity. 24 pts had a WT1 expression of <1 and a BCR-ABL ratio >50 at start of follow up. In these pts there was no correlation of BCR-ABL and WT1 during follow up. In two of these 24 pts there was even a negative correlation with increasing WT1 levels during FU even though BCR-ABL remained undetectable. Cytogenetic investigation of these two pts revealed the development of Ph-negative aberrant clones (one with +8 and one with +11). This result raised the hypothesis that increasing WT1 expression in BCR-ABL negative CML during follow up may indicate the development of BCR-ABL negative clones. Therefore we measured the WT1 expression in a new cohort of 26 CML pts that developed Ph-clones during follow up. All these pts had low (<0.5) or undetectable BCR-ABL. Chromosomal aberrations in these cases were −7 (n=1), +8 (n=12), +11 (n=1), del(20q) (n=1), −Y (n=10)–X (n=1). A high WT1 expression (range 5–177) was detected in the two cases with +11 and −7 and in 10 of 12 cases with +8. A low WT1 expression (<0.5) was detected in the case with 20q- and in all 10 cases with–Y. An intermediate expression of 1.5 was detected in the case with +X. In summary a high WT1 expression was detected in 12/26 (46%) of all cases with new chromosomal aberrations in a Ph-negative clone and in 12/16 (75%) excluding the 10 cases with -Y. So far, the clinical relevance of Ph-negative clones is unclear. In rare cases the development of secondary MDS has been described. The cases that were followed here were all still in CR of CML with no signs of a secondary disease. However, high WT1 expression was detected in most cases with +8 but never with–Y, possibly indicating unequal biological relevance of these aberrations. The 10–100-times elevated WT1 expression in the +8 and +11 cases suggest that these aberrations may be of adverse impact. However, further clinical follow up is needed to show the outcome in these pts. In contrast, none of the cases with–Y revealed elevated WT1 expression suggestive for the expansion of healthy–Y clones that frequently can occur in a normal male bone marrow. 21 pts with Ph-negative clones could be followed for 13–36 months. There was a good correlation of the WT1 expression level with the number of metaphases carrying the chromosomal aberration (r=0.729). In one case an increase of WT1 expression was measured 11 months before detection of the evolving +8 suggestive of a preexisting cytogenetically undetected clone. In conclusion,

It has previously been shown that imatinib uptake into chronic myeloid leukemia (CML) cells is dependent on human Organic Cation Transporter 1 (hOCT1; SLC22A1). In more recent work on clinical samples it was further shown that low hOCT1 expression of this influx transporter may be an important mechanism of imatinib resistance. To further evaluate this issue we have retrospectively quantified pretreatment hOCT1 mRNA expression in 92 CML patients (pts) that responded with major molecular remission within the first year of treatment and compared these results to 19 pts with primary resistance to imatinib. We found that all 19 resistant pts had low hOCT1 expression (median: 2.032 (expressed as %hOCT1/ABL); range 0.18–4.24). Although the median hOCT1 expression at diagnosis in the responders was higher (median 8.417) the range was very heterogeneous (0.45–188.2) with only 30% of all responders having a significantly higher expression than the resistant pts. As in vitro studies have shown that genetic variants of the SLC22A1 gene that codes for hOCT1 can have a negative effect on the transport of some substrates we hypothesized that not only certain hOTC1 expression levels but also different genetic variants within the SLC22A1 gene may be associated with different efficiencies of imatinib uptake. Using high resoluting melting and subsequent sequencing we have genotyped exons 1, 2, 5, 6, 7, 9, 10, and 11 in 109 responders as well as in 55 resistant pts, thus each 326 alleles were evaluated. We detected 12 different exonic polymorphisms. Two of these, a G38D and a Y404C were so far undescribed variants. Both nonsynonymous variants were detected in heterozygeous forms, the G38D in one responder and the Y404C variant in one resistant pt. All other variants were detected in frequencies similar to those that have already been described (R61C: 0.07, L160F: 0.76, P341L: 0.01, G401S: <0.01, M408V: 0.60, delM420: 0.19, G465R: 0.05, V519I: <0.01). In addition the silent variants S51S and V501V were detected with frequencies of 0.26% and 0.01% respectively. All variants in heterozygous as well as in homozygous form were distributed equally between responders and resistant patients. Thus we did not find any correlation between SLC22A1 genotype and imatinib response. In addition there was also no correlation of any of these polymorphisms to the high expressers. We found that those polymorphisms that have been described to severely affect hOTC1 functions in vitro were very rare (P341L and G401S with <0.01% each) or even never detected (P283L and R287L) in our cohort. Thus, although in vitro studies have shown that hOCT1 polymorphisms may severely affect function with respect to substrat specificity and transport efficiency of imatinib they do not seem to play a major role in response of CML patients to imatinib. In addition, we analyzed the three most frequent polymorphisms in exons 12, 21, and 26 in the multidrug resistence gene (MDR1) that codes for an efflux transporter implicated in imatinib efflux. In total 84 responders and 38 resistant patients were analyzed. We found that the exon12 nt1236t allele is more frequently observerd in resistant patients (p=0.045) whereas there was only a week association for the exon21 nt 2677t allele (p=0.121) and the exon26 nt3435t allele (p=0.139) to resistance. In conclusion, it seems to be unlikely that genetic variants of hOCT1 play a major role in imatinib resistance if at all, also the hOCT1 expression levels account for the response of only a few cases. It remains unclear whether hOCT1 plays a role in influx of imatinib or whether its function may be overwritten by other influx transporters like the very homologous and functionally redundant hOCT3 just in the vicinity of hOCT1. 3) The role of efflux transporters in imatinb resistance may be more important, however we detected only a weak association to certain polymorphisms in MDR1 to resistance in our cohort.

In CML real time PCR for the detection of residual disease usually has been done by the detection of the BCR-ABL-fusion transcript. In addition some reports have shown the usefulness of WT1 as a target for follow up in CML blast crisis. The aim of this study was to evaluate whether WT1 in addition to BCR-ABL assessment in imatinib treated CML can give any new information. Expression of BCR-ABL as well as WT1 (ELN assay) was normalized to ABL and expressed as % to ABL. In total 268 bone marrow samples (spls) of 40 patients (pts) have been analysed. Sixteen spls were newly diagnosed untreated CML. 252 spls were analysed at several timepoint during treatment with imatinib starting with a “near to untreated” sample with a >50% BCR-ABL/ABL ratio. According to standard definitions 6 of the 40 pts showed no response, 16 minor (MR), and 18 major molecular response (MMR) as determined by BCR-ABL expression. Three of the MR and one of the MMR pts relapsed. The 14 pretreatment spls showed a median WT1 expression of 3.36 (range 0.176–14.9) whereas the median BCR-ABL level was 56.6 (range 11.4–149.2). Thus the BCR-ABL level was more than one log above the WT1 level and was more uniform within the cohort. This lower total expression and a background level of 0.04 (as estimated from 7 normal bone marrows) results in 2–3 log lower sensitivity of WT1 compared to BCR-ABL. The correlation of WT1 and BCR-ABL in the total cohort of 268 spls was low (r=0.259). Median WT1 expression in BCR-ABL negative spls (n=15) was 0.055 and thus correlates to that of normal spls. Regarding the total follow up in all 40 pts a good correlation of WT1 and BCR-ABL was found only in 9 cases, all of which had a WT1 expression >10 at the start of follow up. 7 pts had a WT1 expression between 1–7 with good correlation to BCR-ABL but restricted sensitivity. 24 pts had a WT1 expression of <1 and a BCR-ABL ratio >50 at start of follow up. In these pts there was no correlation of BCR-ABL and WT1 during follow up. In two of these 24 pts there was even a negative correlation with increasing WT1 levels during FU even though BCR-ABL remained undetectable. Cytogenetic investigation of these two pts revealed the development of Ph-negative aberrant clones (one with +8 and one with +11). This result raised the hypothesis that increasing WT1 expression in BCR-ABL negative CML during follow up may indicate the development of BCR-ABL negative clones. Therefore we measured the WT1 expression in a new cohort of 26 CML pts that developed Ph-clones during follow up. All these pts had low (<0.5) or undetectable BCR-ABL. Chromosomal aberrations in these cases were −7 (n=1), +8 (n=12), +11 (n=1), del(20q) (n=1), −Y (n=10)–X (n=1). A high WT1 expression (range 5–177) was detected in the two cases with +11 and −7 and in 10 of 12 cases with +8. A low WT1 expression (<0.5) was detected in the case with 20q- and in all 10 cases with–Y. An intermediate expression of 1.5 was detected in the case with +X. In summary a high WT1 expression was detected in 12/26 (46%) of all cases with new chromosomal aberrations in a Ph-negative clone and in 12/16 (75%) excluding the 10 cases with -Y. So far, the clinical relevance of Ph-negative clones is unclear. In rare cases the development of secondary MDS has been described. The cases that were followed here were all still in CR of CML with no signs of a secondary disease. However, high WT1 expression was detected in most cases with +8 but never with–Y, possibly indicating unequal biological relevance of these aberrations. The 10–100-times elevated WT1 expression in the +8 and +11 cases suggest that these aberrations may be of adverse impact. However, further clinical follow up is needed to show the outcome in these pts. In contrast, none of the cases with–Y revealed elevated WT1 expression suggestive for the expansion of healthy–Y clones that frequently can occur in a normal male bone marrow. 21 pts with Ph-negative clones could be followed for 13–36 months. There was a good correlation of the WT1 expression level with the number of metaphases carrying the chromosomal aberration (r=0.729). In one case an increase of WT1 expression was measured 11 months before detection of the evolving +8 suggestive of a preexisting cytogenetically undetected clone. In conclusion, BCR-ABL is superior to WT1 as marker for follow up in CML. Increasing WT1 levels in BCRABL negative follow up spls can indicate the development of Ph-negative clones under imatinib treatment. Future studies will show whether the WT1 level in Ph-clones may indicate the relevance of certain aberrations for outcome in these patients.

The aim of this study was to further evaluate the impact of minimal residual disease (MRD) in NPM1 mutated AML in comparison to other factors like FAB, cytogenetics, FLT3 mutations, NPM1 mutation type and age. In total 1002 samples of 219 NPM1 mutated (NPM1mut) patients (pts) were analysed at diagnosis, during, and after therapy. Pts were treated within different AML trials, and follow-up samples were referred to perform an NPM1 specific RQ-PCR for MRD. The cohort was comprised of 112 females and 107 males, median age was 58.8 years (range: 20–79 years). 207 had de novo AML (M0: n=5; M1: n=49; M2: n=55; M4=57; M5: n=28, M5: n=6; M7: n=1, nd: n=6), 4 s-AML and 5 t-AML. Cytogenetic data was available in 215 pts: 178 with normal (NK) and 37 with aberrant karyotypes (+4: n=4; +8: n=7; +21: n=2, two or more trisomies: n=4; -Y: n=4; del(7q): n=2; del(9q): n=3; del(20q): n=2; rare translocations: n=9). At diagnosis 87/219 pts (39.7%) had FLT3-ITDs in addition to the NPM1mut. FLT3-TKD status was available in 206 cases (14 mutated (6.7%) and 192 WT). The NPM1 mutation types were A (n=174), B (n=13), D (n=14), I: (n=4), L: (n=2), R: (n=4) and 8 with individual rare types. Univariate analysis for overall survival (OS) revealed unfavourable impact for age (p=0.049), and for FLT3-ITD (p=0.002), favourable impact for FLT3-TKD (p=0.046), and no impact for FAB, chromosomal aberrations or NPM1 mutation type. For MRD assessment for all 14 different NPM1 mutation types mRNA based RQ-PCR assays were established with sensitivities of 10,000–1,000,000. For each patient 2–17 samples (spls) were analyzed (median: 4) spanning a median follow up time of 252 days (range: 18–2347 days). Paired samples of diagnosis and relapse were available in 71 pts, in 8 pts also from second relapse. At relapse all cases had high NPM1 levels comparable to those at diagnosis. The FLT3-ITD status was mutated (+/+) at both time points in 25 pts and −/− in another 25 pts. 10 pts gained FLT3-ITD at relapse and 3 lost it. For 48 paired samples cytogenetics was available for both time points. A normal karyotype (NK) at both time points was detected in 36 pts, 7 cases showed a normal or aberrant karyotype (AK) at diagnosis and and AK at relapse (two of these gained additional aberrations at relapse), 2 different AK at both time points in were detected in 3 cases and a regression from AK to NK in 2 cases. These data show that NPM1 seems to be the primary genetic aberration in these cases and detection of NPM1 is more reliable to detect relapse than cytogenetics. To analyse the impact of NPM1 mutation levels on prognosis four different follow-up intervals were defined: interval 1: days 21–60 after start of therapy; interval 2: days 61–120; interval 3: days 121–365, 4: >365 days. First a set of 605 samples referred for analysis during first line treatment were analysed. Using Cox regression analysis a significant impact of MRD levels (as continuous variable) on EFS was detected for interval 2 (128 spls, p=0.008), interval 3 (214 spl; p<0.001), interval 4 (171 spls; p<0.001) but not for the early interval up to day 60 showing that early molecular response is not relevant for long time outcome. A multivariate analysis showed that MRD was the most significant prognostic parameter (p<0.001) (p-values for interval 3), followed by age (p=0.003), and pretreatment FLT3-ITD status (p=0.065). The same analysis was performed for a second set of 183 spls taken from 50 pts during salvage therapy after relapse. The most relevant interval for this group was between days 30–60 (26 spls; p=0.003). In a third set 87 spls from 28 pts after allogeneic bone marrow transplantation were analyzed. A prognostic impact of MRD could be shown for interval 2 (17 spls; p=0.005) and 3 (23 spls; p=0.006) (no samples from later intervals available). Of the total cohort 325 spls were analysed in parallel with RQ-PCR for NPM1 and genescan for the FLT3-ITD. A high correlation of both follow up markers was observed (r=0.807, p<0.001). Although the method for NPM1 detection is 3–4 log ranges more sensitive our data suggest parallel assessment for FLT3-ITD for high risk patients as many of them aquired FLT3-ITD as additional marker during progression. In conclusion, MRD is the most relevant prognostic marker in NPM1 mutated AML and it is a very useful tool to assess therapy response and to guide therapy.

It has previously been shown that imatinib uptake into chronic myeloid leukemia (CML) cells is dependent on human Organic Cation Transporter 1 (hOCT1; SLC22A1). In more recent work on clinical samples it was further shown that low hOCT1 expression of this influx transporter may be an important mechanism of imatinib resistance. To further evaluate this issue we have retrospectively quantified pretreatment hOCT1 mRNA expression in 92 CML patients (pts) that responded with major molecular remission within the first year of treatment and compared these results to 19 pts with primary resistance to imatinib. We found that all 19 resistant pts had low hOCT1 expression (median: 2.032 (expressed as %hOCT1/ABL); range 0.18–4.24). Although the median hOCT1 expression at diagnosis in the responders was higher (median 8.417) the range was very heterogeneous (0.45–188.2) with only 30% of all responders having a significantly higher expression than the resistant pts. As in vitro studies have shown that genetic variants of the SLC22A1 gene that codes for hOCT1 can have a negative effect on the transport of some substrates we hypothesized that not only certain hOTC1 expression levels but also different genetic variants within the SLC22A1 gene may be associated with different efficiencies of imatinib uptake. Using high resoluting melting and subsequent sequencing we have genotyped exons 1, 2, 5, 6, 7, 9, 10, and 11 in 109 responders as well as in 55 resistant pts, thus each 326 alleles were evaluated. We detected 12 different exonic polymorphisms. Two of these, a G38D and a Y404C were so far undescribed variants. Both nonsynonymous variants were detected in heterozygeous forms, the G38D in one responder and the Y404C variant in one resistant pt. All other variants were detected in frequencies similar to those that have already been described (R61C: 0.07, L160F: 0.76, P341L: 0.01, G401S: <0.01, M408V: 0.60, delM420: 0.19, G465R: 0.05, V519I: <0.01). In addition the silent variants S51S and V501V were detected with frequencies of 0.26% and 0.01% respectively. All variants in heterozygous as well as in homozygous form were distributed equally between responders and resistant patients. Thus we did not find any correlation between SLC22A1 genotype and imatinib response. In addition there was also no correlation of any of these polymorphisms to the high expressers. We found that those polymorphisms that have been described to severely affect hOTC1 functions in vitro were very rare (P341L and G401S with <0.01% each) or even never detected (P283L and R287L) in our cohort. Thus, although in vitro studies have shown that hOCT1 polymorphisms may severely affect function with respect to substrat specificity and transport efficiency of imatinib they do not seem to play a major role in response of CML patients to imatinib. In addition, we analyzed the three most frequent polymorphisms in exons 12, 21, and 26 in the multidrug resistence gene (MDR1) that codes for an efflux transporter implicated in imatinib efflux. In total 84 responders and 38 resistant patients were analyzed. We found that the exon12 nt1236t allele is more frequently observerd in resistant patients (p=0.045) whereas there was only a week association for the exon21 nt 2677t allele (p=0.121) and the exon26 nt3435t allele (p=0.139) to resistance. In conclusion,

The aim of this study was to further evaluate the impact of minimal residual disease (MRD) in NPM1 mutated AML in comparison to other factors like FAB, cytogenetics, FLT3 mutations, NPM1 mutation type and age. In total 1002 samples of 219 NPM1 mutated (NPM1mut) patients (pts) were analysed at diagnosis, during, and after therapy. Pts were treated within different AML trials, and follow-up samples were referred to perform an NPM1 specific RQ-PCR for MRD. The cohort was comprised of 112 females and 107 males, median age was 58.8 years (range: 20–79 years). 207 had de novo AML (M0: n=5; M1: n=49; M2: n=55; M4=57; M5: n=28, M5: n=6; M7: n=1, nd: n=6), 4 s-AML and 5 t-AML. Cytogenetic data was available in 215 pts: 178 with normal (NK) and 37 with aberrant karyotypes (+4: n=4; +8: n=7; +21: n=2, two or more trisomies: n=4; -Y: n=4; del(7q): n=2; del(9q): n=3; del(20q): n=2; rare translocations: n=9). At diagnosis 87/219 pts (39.7%) had FLT3-ITDs in addition to the NPM1mut. FLT3-TKD status was available in 206 cases (14 mutated (6.7%) and 192 WT). The NPM1 mutation types were A (n=174), B (n=13), D (n=14), I: (n=4), L: (n=2), R: (n=4) and 8 with individual rare types. Univariate analysis for overall survival (OS) revealed unfavourable impact for age (p=0.049), and for FLT3-ITD (p=0.002), favourable impact for FLT3-TKD (p=0.046), and no impact for FAB, chromosomal aberrations or NPM1 mutation type. For MRD assessment for all 14 different NPM1 mutation types mRNA based RQ-PCR assays were established with sensitivities of 10,000–1,000,000. For each patient 2–17 samples (spls) were analyzed (median: 4) spanning a median follow up time of 252 days (range: 18–2347 days). Paired samples of diagnosis and relapse were available in 71 pts, in 8 pts also from second relapse. At relapse all cases had high NPM1 levels comparable to those at diagnosis. The FLT3-ITD status was mutated (+/+) at both time points in 25 pts and −/− in another 25 pts. 10 pts gained FLT3-ITD at relapse and 3 lost it. For 48 paired samples cytogenetics was available for both time points. A normal karyotype (NK) at both time points was detected in 36 pts, 7 cases showed a normal or aberrant karyotype (AK) at diagnosis and and AK at relapse (two of these gained additional aberrations at relapse), 2 different AK at both time points in were detected in 3 cases and a regression from AK to NK in 2 cases. These data show that NPM1 seems to be the primary genetic aberration in these cases and detection of NPM1 is more reliable to detect relapse than cytogenetics. To analyse the impact of NPM1 mutation levels on prognosis four different follow-up intervals were defined: interval 1: days 21–60 after start of therapy; interval 2: days 61–120; interval 3: days 121–365, 4: >365 days. First a set of 605 samples referred for analysis during first line treatment were analysed. Using Cox regression analysis a significant impact of MRD levels (as continuous variable) on EFS was detected for interval 2 (128 spls, p=0.008), interval 3 (214 spl; p<0.001), interval 4 (171 spls; p<0.001) but not for the early interval up to day 60 showing that early molecular response is not relevant for long time outcome. A multivariate analysis showed that MRD was the most significant prognostic parameter (p<0.001) (p-values for interval 3), followed by age (p=0.003), and pretreatment FLT3-ITD status (p=0.065). The same analysis was performed for a second set of 183 spls taken from 50 pts during salvage therapy after relapse. The most relevant interval for this group was between days 30–60 (26 spls; p=0.003). In a third set 87 spls from 28 pts after allogeneic bone marrow transplantation were analyzed. A prognostic impact of MRD could be shown for interval 2 (17 spls; p=0.005) and 3 (23 spls; p=0.006) (no samples from later intervals available). Of the total cohort 325 spls were analysed in parallel with RQ-PCR for NPM1 and genescan for the FLT3-ITD. A high correlation of both follow up markers was observed (r=0.807, p<0.001). Although the method for NPM1 detection is 3–4 log ranges more sensitive our data suggest parallel assessment for FLT3-ITD for high risk patients as many of them aquired FLT3-ITD as additional marker during progression. In conclusion, MRD is the most relevant prognostic marker in NPM1 mutated AML and it is a very useful tool to assess therapy response and to guide therapy.

Nucleoplasmin (NPM) mutations have recently been described as a new kind of mutation in AML. In the presented work we have analyzed 977 AML cases from different prognostic subgroups. Fifteen different mutations (type A:n=245 (78.3%), B:n=21 (6.7%), D: n=28 (11.4%), I:n=3 (1%), J:n=4 (1.3%), K:n=2, G,H, L, M, N, O, P, Q, R: n=1, each (0.3%) were detected in 313 cases. No mutation was found in inv(16) (n=89), t(8;21) (n=98), t(15;17) (n=17), t(11q23)/MLL (n=8), inv(3) (n=8), other reciprocal translocations (n=3), and complex aberrant karyotypes (n=59). In the normal karyotype group the incidence was 299/603 (49.6%) and in all others 13/90 (14.4%). In the latter cohort cases with NPM+ were: − Y (1 case), +4 alone (1 case), +4 and +8 (2 cases), +4 with multiple trisomies (1 case),+8 (1 case), multiple other trisomies (1 case), del(5q) (1 case), − 7 alone (2 cases), del(9q) (2 cases), unbalanced translocation (1 case). Data for evaluation of prognosis were available in 401 AML patients with normal karyotype treated within the AMLCG99 study. Results were calculated in relation to MLL-PTD, FLT3-LM, FLT3-TKD, NRAS, KIT, and CEBPA mutations and correlated to further clinical characteristics. NPM-mutated cases were frequently associated with FLT3 mutations but rarely with other mutations. The NPM-mutated group had a higher CR rate (70.5% vs. 54.7%, p=0.003), a trend to longer OS (median 1012 vs. 549 days, p=0.0762), and significantly longer EFS (median 428 vs. 336 days; p=0.0121). There was a clear favourable impact of NPM+/FLT3-LM- on OS and EFS (264/395 cases; 66.8%). This positive effect was lost in the presence of a concomitant FLT3-LM, since survival of the NPM+/FLT3-LM+ double positive pts. was similar to NPM-/FLT3-LM+ cases. We further analysed stability for the NPM mutation at diagnosis and relapse and found stability in 14/15 paired samples. One case with AML M5 lost the NPM mutation at relapse. This case had also a shift from normal karyotype to 46,XX,del(7)(q22),der(7)t(7;21)(p11;q?) and a complete shift of immunophenotype indicating a therapy-related AML. In addition, we have analyzed the applicability of NPM mutations as targets for minimal residual disease (MRD) detection. In total, 82 samples of 22 selected cases were analyzed by use of quantitative real time PCR for the three most common mutation types. Fifteen of these cases had a type A, 3 a type B, and 4 a type D mutation. Samples at diagnosis had an NPM/ABL ratio of 267.6 (range: 54.9 to 3174.7) while each 10 NPM negative cases had a median ratio of 0.03 (assay for A), 0.0001 (assay for B), and 0.002 (assay for D), showing that these assays were nearly specific for the respective mutation. Using limited dilution series of NPM+ in NPM- cDNA a median sensitivity of 1:100.000 could be shown. In both analyzed cases with relapse these were early detectable by increasing NPM levels. Thus, NPM mutations can be used for highly sensitive PCR-based MRD detection in AML, especially if other markers are lacking, i.e. in normal karyotype. In conclusion, our data showa nearly exclusive association of NPM+ to the normal karyotype,a good prognosis of NPM+/FLT3-LM- in normal karyotype.an intermediate prognosis of NPM+/FLT3-LM+ in normal karyotypeNPM as a new marker for PCR-based MRD-detection.

Nucleoplasmin (NPM) mutations have recently been described as a new kind of mutation in AML. In the presented work we have analyzed 977 AML cases from different prognostic subgroups. Fifteen different mutations (type A:n=245 (78.3%), B:n=21 (6.7%), D: n=28 (11.4%), I:n=3 (1%), J:n=4 (1.3%), K:n=2, G,H, L, M, N, O, P, Q, R: n=1, each (0.3%) were detected in 313 cases. No mutation was found in inv(16) (n=89), t(8;21) (n=98), t(15;17) (n=17), t(11q23)/MLL (n=8), inv(3) (n=8), other reciprocal translocations (n=3), and complex aberrant karyotypes (n=59). In the normal karyotype group the incidence was 299/603 (49.6%) and in all others 13/90 (14.4%). In the latter cohort cases with NPM+ were: − Y (1 case), +4 alone (1 case), +4 and +8 (2 cases), +4 with multiple trisomies (1 case),+8 (1 case), multiple other trisomies (1 case), del(5q) (1 case), − 7 alone (2 cases), del(9q) (2 cases), unbalanced translocation (1 case).