Aberrant Dntt Expression, TdT-Aided Gene Length Mutations, and Prognosis Relevance in ATG/Alg Based Regimen Allo-HSCT in Acute Myeloid Leukemias

DNTT encodes the most template-independent DNA polymerases TdT. The canonical function of TdT is to boost the diversity of immunoglobulin and T cell receptors by incorporating non-templated nucleotides (NTN) to their variable regions via RAG1/2 mediated DNA breaks and non-homologous end joining (NHEJ) rearrangement process. This study aimed to investigate the relationship between aberrant DNTT expression and illegitimately TdT-aided microhomology-mediated replication-dependent recombination (MMRDR) with the mutagenesis of gene length mutations (LMs) in acute myeloid leukemia (AML), and their prognosis relevance. A cohort of 578 AML cases was enrolled. Fifty healthy donors for allogeneic hematopoietic stem cell transplantation (allo-HSCT), 393 B cell acute lymphoblastic leukemia (B-ALL) cases, 78 T-ALL cases, and 25 mixed-phenotype acute leukemia (MPAL) cases were used as control. Next-generation sequencing was performed for mutation analysis of 86 leukemia driver genes. RNA-seq was used to analyze the expression of DNTT and other non-homologous end joining (NHEJ) associated genes. Prognostic was investigated in a subset of 239 AML cases who underwent anti-thymocyte globulin (ATG) or anti-lymphocyte globulin (ALG) based regimen allo-HSCT. Based on sequence anatomy that considers the MMRDR mechanism and nucleotides characteristics of TdT mediated NTN incorporation, we formulate a classification algorithm for LMs and divide them into four subtypes (type I-IV). Type-I indicates pure duplicated/triplicated germline sequences with identifiable ≥2bp canonical triple microhomology (MH) sequences; type-II indicates pure duplicated/triplicated germline sequences without ≥2bp canonical MH sequences; type-III indicates any LMs with NTN insertions; type-IV indicates any other LMs, mainly deletions. FLT3-LMs has the highest overall incidence and occur across multiple lineage leukemias. We observed a significant FLT3-LMs subtypes distribution bias among acute leukemia subtypes (Figure 1A). Type-I FLT3 LMs are only observed in AML; there are mainly type-III FLT3 LMs in T-ALL and MPAL; type-II, III, IV FLT3 LMs are predominant in B-ALL. The overall DNTT expression was significantly lower in AML than in other leukemia subtypes and control groups (P < 0.001). This supports that FLT3-LMs subtypes distribution bias might be attributed to the difference in the overall DNTT expression among leukemia lineages. A total of 458 LMs events were observed in 295 cases (51.0%) within 25 genes (FLT3, NPM1, CEBPA, RUNX1, KIT, etc.) in our AML cohort. The incidence of type-II and type-III LMs, both of which the mutagenesis relies on TdT-aided MMRDR in theoretically speculate, were 31.2 % and 47.8 %, respectively. Type-I and type-II, which manifested as pure germline sequence duplications, account for 43.6% of the FLT3 LMs; type-III LMs, which with additional inserted NTN sequences, account for as high as 50.4% of the FLT3 LMs. We analyzed the G/C nucleotide contents adjacent to LMs junctions. A significantly high G/C bias was observed at +1 nucleotide position in type-II and type-III subsets (Figure 1B), suggesting that the TdT-aided MMRDR mechanism plays a role in the mutagenesis in these cases. We also observed a strong positive correlation between fragment length and G/C content of the inserted NTN sequences (P < 0.001) within the type-III subset of the 25 LMs genes (Figure 1C), suggesting a higher TdT activity mediates longer inserted sequences. DNTT expressions level of type-III LMs cases were significantly higher than that of type-I, II, IV LMs cases and cases without LMs in the total 25 LMs genes (Figure 1D) and the FLT3 LMs subset. Similar expression signatures of other NHEJ associated genes RAG2, XRCC4, and XRCC6 were also observed. For the survival analysis in the ATG/ALG based regimen allo-HSCT AML subset, we observed a significantly better overall survival (P = 0.024) in cases positive for type-III FLT3-LMs than that of type-I, II (Figure 1E). In this study, we proposed a subclassification algorithm for LMs (type I-IV) in AML. Both DNTT gene expression and sequence character suggesting that TdT-aided MMRDR plays a role in the mutagenesis of type-III and type-II LMs. We also observed AML cases with type-III FLT3 LMs benefit more from ATG/ALG based regimen allo-HSCT than cases with other FLT3 LMs types, which may be attributed to the aberrant lymphoid lineage antigen expression. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Mitochondrial Haplotype Analysis for Differentiation of Isolates of Phytophthora cinnamomi

Although Phytophthora cinnamomi is heterothallic, there are few instances of successful crossing in laboratory experiments, and analysis of field populations indicates a clonally reproducing population. In the absence of sexual recombination, the ability to monitor mitochondrial haplotypes may provide an additional tool for identification of clonal isolates and analysis of population structure. To determine mitochondrial haplotypes for this species, seven mitochondrial loci spanning a total of 6,961 bp were sequenced for 62 isolates representing a geographically diverse collection of isolates with A1 and A2 mating type. Three of the regions were primarily intergenic regions between trnG and rns, rns and nad3, and nad6 and cox1, while the remaining loci spanned cox2, nad9, rps10, and secY coding regions and some of the flanking spacer regions. In total, 45 mitochondrial haplotypes were identified (75% of the total isolates examined) with differences due to single-nucleotide polymorphisms (SNPs, totaling 152 bp) and length mutations (17 indels >2 bp representing a total of 910 bp in length). SNPs were the predominate mutation in the four coding regions and their flanking intergenic regions, while both SNPs and length mutations were observed in the three primarily intergenic regions. Some of the length mutations in these regions were due to addition or loss of unique sequences while others were due to variable numbers of subrepeats (in the trnG-rns region, there were 3 to 12 copies of a 24-bp subrepeat sequence that differentiated 17 haplotypes). Network analysis of the haplotypes identified eight primary clades, with the most divergent clade representing primarily A1 isolates collected from Papua New Guinea. The isolate grouping in the network corresponded to mating type and previously published isozyme classifications, with three exceptions: a haplotype representing an A1 mating type (H29) was placed well within the A2 mating type haplotype grouping, one haplotype (H26) had isolates with two isozyme classifications, and one isozyme group was represented on separate network clades, suggesting that recombination has occurred in the past. Among the 62 isolates examined, several examples were identified of isolates recovered from different geographic regions having the same mitochondrial haplotype, suggesting movement of isolates via plant material. Analysis of the data set to determine whether fewer loci could be sequenced to classify haplotypes indicated that the trnG-rns and rns-nad6 loci would classify 87% of the haplotypes identified in this study, while additional sequencing of the nad9 or secY loci would further differentiate the remaining six haplotypes. Based on conservation of gene order in Phytophthora spp., the trnG-rns locus should be useful for mitochondrial haplotype classification in other species, as should the cox2, nad9, rps10, and secY loci. However, the rns-nad3 and nad6-cox1 loci span regions that can have a different gene order in some Phytophthora spp.

Specific Molecular Mutations with Prognostic Relevance in AML with Normal Karyotype Are Also Associated with Outcome in AML with Aberrant Karyotype: A Study on 1981 Cases,

Abstract 3517 Background and Aim: The karyotype and molecular mutations are well established prognostic parameters in AML. However, the impact on outcome of molecular mutations has been evaluated mainly in the subset of AML with normal karyotype (NK). The aim of this study was 1. to determine the frequency of NPM1 mutations (NPM1 mut), partial tandem duplications within the MLL gene (MLL -PTD), length mutations within the FLT3 gene (FLT3 -ITD) and CEPBA mutations (CEPBA mut) in distinct cytogenetic subgroups and 2. to evaluate the prognostic impact of these mutations in relation to chromosome abnormalities. Patients and Methods: 1981 patients with AML and evaluable cytogenetics were included. Mutation data was available in the majority of cases: NPM1: 1646, CEPBA: 1324, FLT3 -ITD: 1726 and MLL -PTD: 1656. Based on the karyotype the cohort was subdivided according to revised MRC criteria (Grimwade et al. Blood 2010) and in addition into distinct cytogenetic subgroups. Results: According to cytogenetics 170 cases were assigned to the favorable MRC class (MRCF), 1414 to the intermediate MRC (MRCI) and 397 to the unfavorable MRC subset (MRCU). The frequency of NPM1 mut, CEPBA mut, MLL -PTD and FLT3 -ITD differed significantly between MRC classes and distinct cytogenetic groups. In the MRCI subset we evaluated overall survival (OS) between patients with normal or aberrant karyotype (AK) within the respective mutation groups. Within NPM1 mut, CEPBA mut, MLL -PTD+ and FLT3 -ITD+ patients no significant differences in OS were observed between patients with NK or AK. When separating CEPBA mut into biallelic (n=52) and monoallelic cases (n=40) also no difference in OS was observed between pats with NK or AK. Next we tested the prognostic impact of the respective molecular mutations within MRCI including 966 cases with NK and 448 cases with AK. OS was significantly longer in patients with NPM1 mut or CEPBA mut (median OS (mOS) 49.6 months (mo) vs 18.6 mo, p=0.003; not reached (n.r.) vs 21.1 mo, p=0.016) and significantly worse for patients with MLL -PTD or FLT3 -ITD (mOS 10.8 vs 23.0 mo, p=0.039; 13.8 vs 24.9 mo, p=0.003). Analyzing biallelic and monoallelic CEPA mut separately revealed that only biallelic CEPBA mut was associated with a longer OS (p=0.006). Restricting the analysis to MRCI patients with aberrant karyotype revealed a longer OS for patients with NPM1 mut/FLT3 -ITD-, and a shorter OS for patients with FLT3 -ITD (mOS n.r. vs 18.0 mo, p=0.033; 5.7 vs 23.0 mo, p=0.015). A trend towards better OS was observed for biallelic CEBPA mut. Conclusions: 1. The frequency of molecular mutations varies significantly between distinct cytogenetic subsets. They are particularly common in AK within MRCI. NPM1 mut, CEBPA mut and MLL -PTD were not observed in MRCF, in AML with 11q23/MLL -rearrangements, or in 3q26/EVI1 -rearrangements. Their frequency was below 5% in AML with complex karyotype. 2. The outcome of NPM1 mut, CEPBA mut and MLL -PTD+ cases was not different in AML with normal or aberrant karyotype within MRCI. 3. This data suggest to extend mutation screening for NPM1 mut, CEPBA mut, MLL -PTD and FLT3 -ITD to all AML with intermediate risk cytogenetics, as they are significantly associated with outcome not only in AML with normal karyotype but also in AML with cytogenetic abnormalities assigned to MRCI. This consequently will lead to better characterization of a reasonable number of cases from MRCI with important implication on treatment. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Clinical Phenotype of Myeloproliferative Neoplasms with Activating CBL-mutations Resembles Chronic Myelomonocytic Leukemia.

Abstract 3895 Poster Board III-831 A genome-wide single nucleotide polymorphism (SNP) screen led to the identification of 11q aUPD in patients diagnosed with various subtypes of myeloproliferative neoplasms (MPN), e.g. chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML) and myelofibrosis (MF) (Grand et al., Blood 2009;113:6182). Further molecular analyses revealed acquired activating point and length mutations in CBL exons 8 and 9 in 10% of CMML, 8% of aCML and 6% of MF cases. Most variants were missense substitutions in the RING or linker domains that abrogated CBL ubiquitin ligase activity and conferred a proliferative advantage to 32D cells overexpressing FLT3. In this study, 160 patients with BCR-ABL and JAK2 V617F negative MPNs were screened for CBL mutations by PCR and direct sequencing. Eighteen known (Y371H, L380P [2x], C381R, C381Y [2x], C384Y, C396Y, H398P, H398Q, W408C, P417H, F418L, R420Q [5x]) and four new (F378L, G397V, I423N, V430M) missense mutations affecting fourteen residues were identified in 20 patients. Two patients harbored two different mutations. The clinical phenotype could be characterized more precisely in 17 patients. Median age was 68 years (range 59–85) with a slight female predominance (f, n=10; m, n=7). Striking hematological features were leukocytosis (14/17; 82%; median 29,000/μl, range 4,500-141,000) with continuously left-shifted granulopoiesis (blasts, promyelocytes, myelocytes, metamyelocytes) in 85% and elevated monocytes (median 2,500/μl, range 630-10,656) >1,000/μL in 88% (15/17) of patients. Eosinophilia (>1,500/μL) was rare (3/17, 18%). Anemia (normal values: f, Hb <12g/dL; m, Hb <14g/dL) was present in all 17 patients (f, median 10g/dL, range 8.7-11.8; m, median 11.2g/dL, range 8.6-12.9). Platelets did not exceed 300,000/μL in any patient while 11/17 (65%) patients presented with thrombocytopenia (median 125,000/μL, range 18,000-271,000). Splenomegaly was present in 11/17 patients (65%) and LDH was elevated (median 304U/L, range 189-729) in 9/17 patients (52%). Bone marrow histology and immunohistochemistry were available from 12 patients. Relevant features were hypercellularity, marked granulopoiesis and microlobulated megakaryocytes without clusters in 11/12 patients (92%), respectively. Increased fibres were seen in 8/12 (67%) patients of whom one showed severe fibrosis. Clinical follow-up was available from 17 patients. Thirteen patients (76%) have died because of progression to secondary acute myeloid leukemia/blast phase (n=7), cytopenia-related complications (n=2) or for unknown reasons (n=4) after a median of 23 months (range 3-60) following diagnosis. In conclusion, point mutations of CBL exons 8 and 9 are present in approximately 6-12% of BCR-ABL and JAK2 V617F negative MPNs. They are associated with a distinct clinical and hematological phenotype presenting with myeloproliferative features allowing diagnosis of a proliferative subtype of CMML rather than aCML or MF in the majority of cases. Patients with left-shifted leukocytosis, monocytosis, anemia and lack of thrombocytosis who are negative for BCR-ABL and point or length mutations of JAK2 should be routinely screened for CBL mutations. Disclosures: No relevant conflicts of interest to declare.

Low frequency of RAS and absence of FLT3-ITD gene mutations in patients with Myelodysplastic Syndromes in India: AIIMS experience

e22231 Background: Chromosomal abnormalities and molecular detection has potential importance for diagnosis and prognosis of MDS, although the mechanisms underlying the development of MDS and their progressive evolution to AML are still largely unknown. Since, no studies have been reported from India on the prevalence of N-RAS, K- RAS point mutation in codon 12 and FLT3-ITD mutations in patients with MDS, we undertook this study. Methods: DNA and RNA were extracted from bone marrow /peripheral blood. Using RT-PCR the patients were screened for length mutations in FLT3 gene. PCR-RFLP and nested PCR-RFLP were used for the detection of point mutation in codon 12 of N-RAS and K-RAS. Results: A total of 53 patients (median age 39 yrs, range 9–78yrs; M: F 2:1; median TLC-3.9×109/l, range 0.8–116 ×109/l Median platelet count- 87 ×109/l, range 1–349 ×109/l, Median hemoglobin -6.8 g/dl, range 2.7- 16.1 g/dl, were studied. One out of 53 patients (2%) was found positive for N-RAS and four patients were positive for K-RAS (8%) mutation. FLT3-ITD mutation was studied in 47 patients; all the patients were found negative. The mean observation of all the patients was 30 months and the median overall survival was 28 months. Nine patients died during follow up. The presence of N-RAS codon 12 mutation was associated with the poor survival. FLT3-ITD mutation was not observed in any of our cases, which is in contrast to 3% reported from the West. Conclusions: Thus, it appears that the RAS and FLT3 mutations are uncommon in MDS patients in India. No significant financial relationships to disclose.

Gene expression of MLH1 related to microsatellite instability, immuno-histochemistry, and promoter hypermethylation in colorectal cancer

3601 Background: The subgroup of highly microsatellite instable (MSI-H) colorectal cancers (CRC), is well described with DNA microsatellite and immuno-histochemistry (IHC) analyses. MSI-H CRCs are most often caused by hypermethylation inactivation of MLH1 or to a lesser extent by hereditary mutations in this or other mismatch repair genes. Microsatellite analysis and IHC are qualitative rather than quantitative methods. Reports showing that MMR deficient CRC has a favorable prognosis and that these cancers may respond less to chemotherapy may be more informative if based on a quantitative method instead. The aim of the present study was to correlate the expression of MLH1 to microsatellite analysis, promoter hypermethylation, and IHC. Methods: Sections of paraffin-embedded tumors from 206 unselected consecutive patients with CRC were microdissected and RNA was isolated and reverse-transcribed to cDNA. The relative gene expression of MLH1 was measured with real-time PCR. DNA from tumor and blood was examined with five standard microsatellite markers to find length mutations indicating instability. Tumor DNA was further analyzed with a methylation specific PCR in the C region of the MLH1 promoter. Protein expression of MLH1 was analyzed with IHC. Results: For all quantifiable tumors the median (and lower and upper quartiles) of the relative MLH1 gene expression was 0.90 (0.68, 1.42). For MSI-H tumors, defined as presense of instability in at least two markers or lack of protein expression with IHC, the median was 0.36 (0.26, 0.56). For microsatellite stable cancers the median was 0.96 (0.73, 1.44), p<0.0001 (Wilcoxon test). Median and quartiles of MLH1 expression in hypermethylated and non-methylated tumors was 0.31 (0.26, 0.45) and 0.96 (0.73, 1.43) respectively (p<0.0001). Relative MLH1 expression in MSI-H tumors range up to 0.64. Fourteen % of MSS tumors are in this range, too. Conclusions: In this large unselcted group of CRC patients we found a significant relation between low gene expression, MSI-H, MLH1-hypermethylation and negative IHC. We further identify a subgroup of MSS patients with low gene expression of MLH1 and suggest this quantitative marker in studies of prognosis and treatment effect. [Table: see text]