No abstract

BackgroundTo evaluate prevalence, radiological reporting and clinical management of pathologic vertebral body fractures (VBFs) of unknown origin in cancer patients receiving computed tomography (CT) examinations.MethodsWe investigated all CT examinations (over 1 year) of male and female patients with an underlying malignancy and an increased risk of osteoporosis (age 55–79 years) for the presence of VBFs. We evaluated midline sagittal CT-reformations of the spine for prevalence, fracture type, severity and location, the accuracy and style of radiological reporting, subsequent clinical management and documentation in hospital discharge letters.Results848 patients were investigated. We found 143 VBFs in 94 (11 %) patients. 6, 49, and 45 % were grade 1, grade 2, and grade 3 fractures, respectively, while 20, 66, and 14 % were wedge, biconcave and crush fractures, respectively. 32 (34 %) radiological reports correctly classified VBFs as fractures, 25 (27 %) reports recognized VBFs, but did not type them, and VBFs were not described in 37 (39 %) reports. In 3 (3 %) patients further clinical work-up of VBFs was performed, while only 8 (9 %) hospital discharge letters contained the information of the presence of pathologic VBFs of unknown origin.ConclusionsVBFs of unknown origin appear frequently in cancer patients, however, clinical management and documentation was found in only few cases. Moreover, especially in cancer patients consistent radiological reporting of VBFs seems important, as aetiology of VBFs could be from osteoporosis, disease progression or oncological therapy, however, reporting is still performed inconsistently.

407 CpG methylation is an epigenetic modification that is important for cellular development. The DNMT3A gene, located on chromosome 2p23.3, encodes for a DNA methyltransferase and plays a central role in de novo CpG methylation. Recently, DNMT3A has been reported to be mutated in 22% of AML and 8% of MDS (Ley et al., N Engl J Med, 2010; Walter et al., Leukemia, 2011). Further, DNMT3A mutations were observed to be associated with a short overall survival in both diseases, respectively. In order to determine the role of DNMT3A mutations in leukemia we investigated two different entities by next-generation sequencing: 145 AML patients and 83 cases harboring a T-cell acute lymphoblastic leukemia (T-ALL). We applied an amplicon based deep-sequencing assay (454 Life Sciences, Branford, CT) in combination with the 48.48 Access Array technology (Fluidigm, South San Francisco, CA). The peripheral blood or bone marrow samples were obtained from untreated patients. The AML cohort was restricted to cases with normal karyotype (CN-AML). 87/145 (60%) cases were specifically selected to be wild-type for NPM1, FLT3-ITD, CEBPA, and MLL-PTD, whereas 58/145 (40%) samples were mutated in NPM1 (n=33) or double-mutated in NPM1 and FLT3-ITD (n=25). In our cohort of AML cases without mutations in NPM1, FLT3-ITD, CEBPA, and MLL-PTD, we observed a DNMT3A mutation frequency of 17.2% (15/87 cases). The DNMT3A mutation rate in the NPM1 mutated/FLT3 wild-type cases (16/33, 48.5%, P=0.001) and NPM1/FLT3-ITD mutated cases (19/25, 76%, P<0.001) was significantly higher, confirming the association of DNMT3A mutations with NPM1 and FLT3-ITD mutations that had been reported previously (Ley et al.). Interestingly, also in the cohort of T-ALL we detected patients that carried a DNMT3A mutation (16/83, 19.3%), which is very similar to the mutation frequency in AML, and has not been described yet. To further address the biology of DNMT3A mutations in acute leukemias we combined the AML and T-ALL cohorts and identified in total 31 distinct missense mutations in 65 patients (49 AML, 16 T-ALL). Most frequently, amino acid R882 located in exon 23 was mutated (n=29 cases). In addition, we identified 7 frame-shift alterations, 5 nonsense and 2 splice-site mutations. Moreover, 9 of the 65 mutated cases had two independent mutations. Focusing on AML, only three (6.1%) of the 49 DNMT3A-mutated cases were observed to harbor two different mutations concomitantly. In contrast, in the cohort of T-ALL we detected two different mutations in 6/16 (37.5%, P=0.003) cases. Further, in the cohort of AML, no homozygous mutation was detected, however, in the T-ALL group, two cases harbored a homozygous mutation. Therefore, only 3/49 AML (6.1%) cases, but 8/16 T-ALL (50%) cases showed biallelic mutation status (P<0.001). With respect to overall survival, no association was seen in the complete cohort of CN-AML cases (n=145). After limiting this cohort to the cases without mutations in NPM1, FLT3-ITD, CEBPA and MLL-PTD (n=87), an inferior survival was observed for DNMT3A-mutated patients as compared to DNMT3A wild-type patients (n=15 vs. n=72; alive at 2 years: 27.9% vs. 56.6%; P=0.048). Remarkably, also in the cohort of T-ALL a worse survival for patients with DNMT3A mutations was seen which has not been reported thus far (n=13 vs. n=64; alive at 1 years: 28.6% vs. 80.9%; P=0.001). Subsequently, we were interested whether gain-of-function mutations of the DNMT3A gene were associated with trisomy 2 and acquired uniparental disomy (aUPDs) of the short arm of chromosome 2 where DNMT3A is located. As such, we investigated 9 cases harboring a trisomy 2 (AML n=4, MDS n=4, and CMML n=1) and one MDS patient harboring an aUPD 2p, as confirmed by SNP microarray analyses (SNP Array 6.0, Affymetrix, Santa Clara, CA). Not all, but 3/9 cases with trisomy 2 harbored a DNMT3A mutation (one AML, MDS, and CMML case each), suggesting that duplication of DNMT3A mutations can enhance the effect of the mutation. Moreover, the single case with aUPD 2p also showed a mutation, further suggesting that LOH leading to loss of the wild-type DNMT3A may be another mechanism of disease leading to progression of leukemia. In conclusion, we here report on a high mutation rate of DNMT3A in both AML and T-ALL and independently confirmed an inferior overall survival in these two entities, respectively. This indicates a significant role of DNMT3A alterations in myeloid as well as in lymphoid neoplasms. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Wild:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

2268 Since the introduction of imatinib mesylate for treating patients with chronic myeloid leukemia (CML) data has shown that a small proportion of patients in chronic phase experience refractoriness to kinase inhibitor treatment due to mutations in the ABL1 domain. A variety of molecular methods provide a clinically feasible workflow to detect these mutations but have both strengths and weaknesses: e.g. direct sequencing of nested PCR assays amplifying the ABL1 part of BCR-ABL1 fusion transcript has a low diagnostic detection sensitivity of 10%; and more sensitive methods such as allele-specific PCRs are only available for known mutations. Here, we investigated the utility of ultra-deep next-generation sequencing (NGS) to detect and monitor the composition of ABL1 kinase mutations at diagnosis and during inhibitor treatment. We included 62 samples from 13 CML patients, diagnosed between 10/2005 - 07/2009. The chimeric BCR-ABL1 fusion transcript was amplified from cDNA. Subsequently, 2 overlapping amplicons were designed to amplify a 740 bp stretch of the ABL1 kinase domain to be processed by 454 Titanium amplicon chemistry protocol (454 Life Sciences, Branford, CT). In median, 454 sequencing data was generated for each patient across 4 time points (range 3–11) with a median time span of 11 months (range 5–35) from state of first diagnosis to the most recent investigation. For each investigation a median of 2234 reads specific for the ABL1 kinase domain was obtained. In this selected cohort, in 12/13 patients, previous Sanger sequencing had detected a spectrum of 10 different kinase domain mutations during course of inhibitor treatment. In all 12 patients deep-sequencing enabled the quantitative detection of these mutations with 100% concordance. In 1/13 cases carrying a t(2;3)(q31,q27), neither NGS nor routine methods detected any mutation over four distinct time points, although the %BCR-ABL1/ABL1 was persistently high (ratios of 108, 84, 80, and 108, respectively). According to European LeukemiaNet guidelines (Baccarani et al., JCO 2009), kinase domain mutation screening is only recommended if BCR-ABL1 transcript levels are increasing at consecutive time points. We therefore investigated at which point in time NGS would allow to detect resistant clones subsequent to inhibitor therapy even though alternative testings had not been performed. In 11/12 cases resistant clones were already early detectable after start of treatment with tyrosine kinase inhibitors, with a median time span of 3.9 months from first diagnosis (range 1.1–19.5). We here demonstrate in 6 patients, treated with Imatinib or Dasatinib that even at transcript levels of BCR-ABL1/ABL1, ranging in our cohort from 1.1%-13%, mutations were detectable (range of mutations: 4%-97% of sequencing reads). These points to a very fast selection process of resistance-defining clones. More importantly, NGS was able to quantitate in 4 patients an increasing mutational burden of resistant clones while the %BCR-ABL1/ABL1 stayed consistently low. For example, one patient harbored the L387M mutation, detected with 54% sequencing reads harboring the mutation (1.1 %BCR-ABL1/ABL1). After 8 months, the L387M mutation was detectable in 97% NGS reads, while the %BCR-ABL1/ABL1 was 3.7. Moreover, it was possible to monitor in detail the molecular composition in 3 patients harboring concomitantly more than one mutation. NGS demonstrated that one of these mutations or even a novel clone was dominant in a subsequent time point, in particular after changing the inhibitor. For example, one patient harbored concomitantly the mutations L284V (30%), Y253H (17%), and T315I (4%). The L284V and Y253H mutations disappeared during Dasatinib treatment, and T315I was thereafter persistent -within 38 days- with a high mutation burden of 98%. Importantly, 454 NGS never detected more than 3 resistant clones concomitantly per patient and thus in no case indicated the existence of a variety of coexisting low-level mutations. However, in all 12 patients at the last time point of our investigation there was only one resistance mutation detectable (in 6/12 cases: T315I). In conclusion, as investigated in this work, NGS is technically feasible for high-throughput serial monitoring of CML patients and, moreover, provides an accurate and highly-sensitive quantitative assessment of mutations leading to therapy resistance but also allows uncovering novel mutations. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Wild: MLL Munich Leukemia Laboratory: Employment. Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann: MLL Munich Leukemia Laboratory: Employment.