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Dysfunctional T cells present in malignant lesions are characterized by a sustained and highly diverse expression of inhibitory receptors, also referred to as immune checkpoints. Yet, their relative functional significance in different cancer types remains incompletely understood. In this study, we provide a comprehensive characterization of the diversity and expression patterns of inhibitory receptors on tumor-infiltrating T cells from patients with non–small cell lung cancer. In spite of the large heterogeneity observed in the amount of PD-1, Tim-3, CTLA-4, LAG-3, and BTLA expressed on intratumoral CD8+ T cells from 32 patients, a clear correlation was established between increased expression of these inhibitory coreceptors and progression of the disease. Notably, the latter was accompanied by a progressively impaired capacity of T cells to respond to polyclonal activation. Coexpression of several inhibitory receptors was gradually acquired, with early PD-1 and late LAG-3/BTLA expression. PD-1 blockade was able to restore T-cell function only in a subset of patients. A high percentage of PD-1hi T cells was correlated with poor restoration of T-cell function upon PD-1 blockade. Of note, PD-1hi expression marked a particularly dysfunctional T-cell subset characterized by coexpression of multiple inhibitory receptors and thus may assist in identifying patients likely to respond to inhibitory receptor–specific antibodies. Overall, these data may provide a framework for future personalized T-cell–based therapies aiming at restoration of tumor-infiltrating lymphocyte effector functions. Cancer Immunol Res; 3(12); 1344–55. ©2015 AACR.

Supplementary Table 1. Clinical data of NSCLC patients. Supplementary Figure 1. Calculation of the iR score. Supp. Figure 2. Activation of PBMC from healthy donors by polyclonal stimulation.

T-cell Bispecific Antibodies (TCBs) elicit anti-tumor responses by cross-linking T-cells to tumor cells and mediate polyclonal T-cell expansion that is independent of T-cell receptor specificity. TCBs thus offer great promise for patients who lack antigen-specific T-cells or have non-inflamed tumors, which are parameters known to limit the response of checkpoint inhibitors. The current study deepens the understanding of TCB mode of action and elaborates on one of the adaptive resistance mechanisms following its treatment in vivo in humanized mice and syngeneic pre-clinical tumor models. Single-agent TCB treatment reduced tumor growth compared with controls and led to a 2-10-fold increase in tumor-infiltrating T-cells, regardless of the baseline tumor immune cell infiltration. TCB treatment strongly induced the secretion of CXCL10 and increased the frequency of intra-tumor CXCR3+ T-cells pointing to the potential role of the CXCL10-CXCR3 pathway as one of the mechanisms for T-cell recruitment to tumors upon TCB treatment. Tumor-infiltrating T-cells displayed a highly activated and proliferating phenotype, resulting in the generation of a highly inflamed tumor microenvironment. A molecular signature of TCB treatment was determined (CD8, PD-1, MIP-a, CXCL10, CXCL13) to identify parameters that most robustly characterize TCB activity. Parallel to T-cell activation, TCB treatment also led to a clear upregulation of PD-1 on T-cells and PD-L1 on tumor cells and T-cells. Combining TCB treatment with anti-PD-L1 blocking antibody improved anti-tumor efficacy compared to either agent given as monotherapy, increasing the frequency of intra-tumoral T-cells. Together, the data of the current study expand our knowledge of the molecular and cellular features associated with TCB activity and provide evidence that the PD-1/PD-L1 axis is one of the adaptive resistance mechanisms associated with TCB activity. This mechanism can be managed by the combination of TCB with anti-PD-L1 blocking antibody translating into more efficacious anti-tumor activity and prolonged control of the tumor outgrowth. The elucidation of additional resistance mechanisms beyond the PD-1/PD-L1 axis will constitute an important milestone for our understanding of factors determining tumor escape and deepening of TCB anti-tumor responses in both solid tumors and hematological disorders.

PD-L1/PD-1 blocking antibodies have demonstrated therapeutic efficacy across a range of human cancers. Extending this benefit to a greater number of patients, however, will require a better understanding of how these therapies instigate anticancer immunity. Although the PD-L1/PD-1 axis is typically associated with T cell function, we demonstrate here that dendritic cells (DCs) are an important target of PD-L1 blocking antibody. PD-L1 binds two receptors, PD-1 and B7.1 (CD80). PD-L1 is expressed much more abundantly than B7.1 on peripheral and tumor-associated DCs in patients with cancer. Blocking PD-L1 on DCs relieves B7.1 sequestration in cis by PD-L1, which allows the B7.1/CD28 interaction to enhance T cell priming. In line with this, in patients with renal cell carcinoma or non-small cell lung cancer treated with atezolizumab (PD-L1 blockade), a DC gene signature is strongly associated with improved overall survival. These data suggest that PD-L1 blockade reinvigorates DC function to generate potent anticancer T cell immunity.

Evidence from mouse chronic viral infection models suggests that CD8 + T cell subsets characterized by distinct expression levels of the receptor PD-1 diverge in their state of exhaustion and potential for reinvigoration by PD-1 blockade. However, it remains unknown whether T cells in human cancer adopt a similar spectrum of exhausted states based on PD-1 expression levels. We compared transcriptional, metabolic and functional signatures of intratumoral CD8 + T lymphocyte populations with high (PD-1 T ), intermediate (PD-1 N ) and no PD-1 expression (PD-1 - ) from non-small-cell lung cancer patients. PD-1 T T cells showed a markedly different transcriptional and metabolic profile from PD-1 N and PD-1 - lymphocytes, as well as an intrinsically high capacity for tumor recognition. Furthermore, while PD-1 T lymphocytes were impaired in classical effector cytokine production, they produced CXCL13, which mediates immune cell recruitment to tertiary lymphoid structures. Strikingly, the presence of PD-1 T cells was strongly predictive for both response and survival in a small cohort of non-small-cell lung cancer patients treated with PD-1 blockade. The characterization of a distinct state of tumor-reactive, PD-1-bright lymphocytes in human cancer, which only partially resembles that seen in chronic infection, provides potential avenues for therapeutic intervention.

A key question in cancer immunotherapy is the general immune status of the patient's tumor-microenvironment (TME) prior to therapy. A patient's underlying tumor immune-contexture may therefore a) guide the therapeutic intervention and b) help to identify potential resistance mechanisms to immunotherapies. The most commonly used classification of the patients' immune status is based on the CD8 tumor infiltrating lymphocytes (TIL) status referring to either “cold”- or “hot” tumors. Patients in early clinical development programs will mostly present with late metastatic disease and therefore the immune phenotypes from these lesions may more relevant to determine the actual tumor-immune contexture status than the currently used characterization based on the primary tumor type. In this study, we systematically analyzed the CD8 immune phenotype (CD8 IP) in 454 patients from various Phase 1 clinical trials using the commonly applied CD8 immnohistochemistry (IHC)-based classification into cold (CD8 deserts) and hot (CD8 excluded and inflamed) tumors and matched gene expression profiling. In total, we observed 116 inflamed, 95 excluded, and 243 desert phenotypes from 25 different indications. The main sources of tumor biopsy tissues were liver metastasis (N=151), lymph node (N=73) as well as lung (N=59). Overall, we identified a significant overrepresentation of the desert phenotype in liver metastasis (p=1e-05). In patients with metastatic disease, we compared the predominant CD8 IP in metastatic lesions to that of the primary tumor. The overall distribution for tumor types such as UBC, NSCLC or OvCa did not change, whereas PDAC, GC, and CRC revealed a significant change towards the desert phenotype for metastatic lesions. Beyond the predominant CD8 IP assessment via IHC, we characterized each CD8 subtype further using specific immune gene signatures. We could identify a reverse correlation of inflammatory and suppressive immune signatures along the CD8 phenotypes in metastatic lesions such as M2-like macrophages, MDSC, or Dendritic Cells. Further zooming into the desert phenotype, we were also able to show that liver metastasis have a more significant immunosuppressive tumor environment compared to other tumor biopsies. In summary, our data provide essential insights about the importance of the origin of the tissue biopsy to reduce impaired decision making based on the underlying tumor-immune contexture of cancer patients. Precision immune phenotyping beyond CD8 TIL infiltration is needed to identify specific resistance mechanisms to cancer immunotherapy and can be used for efficient patient enrichment and thus increase the probability of success for early clinical trials in immuno-oncology. Citation Format: Andreas Roller, Claudia Ferreira, Laura Jarassier, Astrid Heller, Gabriele Dietmann, Konstanty Korski, Bruno Gomes, Michael A. Cannarile. Precision immune phenotyping from primary tumor and metastatic lesions reveals novel insights into therapeutic intervention in cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2051.

Safety and efficacy of therapeutic antibodies are often dependent on their interaction with Fc receptors for IgG (FcγRs). The Göttingen minipig represents a valuable species for biomedical research but its use in preclinical studies with therapeutic antibodies is hampered by the lack of knowledge about the porcine FcγRs. Genome analysis and sequencing now enabled the localization of the previously described FcγRIIIa in the orthologous location to human FCGR3A. In addition, we identified nearby the gene coding for the hitherto undescribed putative porcine FcγRIIa. The 1′241 bp long FCGR2A cDNA translates to a 274aa transmembrane protein containing an extracellular region with high similarity to human and cattle FcγRIIa. Like in cattle, the intracellular part does not contain an immunoreceptor tyrosine-based activation motif (ITAM) as in human FcγRIIa. Flow cytometry of the whole blood and single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs) of Göttingen minipigs revealed the expression profile of all porcine FcγRs which is compared to human and mouse. The new FcγRIIa is mainly expressed on platelets making the minipig a good model to study IgG-mediated platelet activation and aggregation. In contrast to humans, minipig blood monocytes were found to express inhibitory FcγRIIb that could lead to the underestimation of FcγR-mediated effects of monocytes observed in minipig studies with therapeutic antibodies. Electronic supplementary material The online version of this article (10.1007/s00251-018-01099-1) contains supplementary material, which is available to authorized users.

We develop a mathematical model to examine the role of helper and cytotoxic T cells in an anti-tumour immune response. The model comprises three ordinary differential equations describing the dynamics of the tumour cells, the helper, and the cytotoxic T cells and implicitly accounts for immunosuppressive effects. The aim is to investigate how the anti-tumour immune response varies with the level of infiltrating helper and cytotoxic T cells. Through a combination of analytical studies and numerical simulations, our model exemplifies the three Es of immunoediting: elimination, equilibrium, and escape. Specifically, it reveals that the three Es of immunoediting depend highly on the infiltration rates of the helper and cytotoxic T cells. The model's results indicate that both the helper and cytotoxic T cells play a key role in tumour elimination. They also show that combination therapies that boost the immune system and block tumour-induced immunosuppression may have a synergistic effect in reducing tumour growth.

In patients with myelodysplastic syndromes (MDS), sole 20q deletion [del(20q)] is a recurrent favourable abnormality. We studied additional molecular and cytogenetic lesions and their prognostic impact in 305 MDS patients with del(20q) (229 males/76 females; 29-90 years). All patients were investigated by cytomorphology and chromosome banding analysis (CBA), subsets by fluorescence in situ hybridization, molecular mutation screening, and array comparative genomic hybridization (aCGH). By aCGH (n = 30), the minimal common deleted region (CDR) was flanked by PTPRT (20q13·11) and EYA2 (20q13·12). 210 (68·9%) patients had 'early MDS' without blast increase, 95 (31·1%) 'advanced' MDS with blast increase (5-19%). Additional chromosomal abnormalities (ACAs) were detected in 88/305 (28·9%) patients. Patients with advanced MDS more frequently had ACAs (P = 0·003) and had a higher mean number of ACAs (P = 0·020) and of molecular mutations (P = 0·060). Spliceosome mutations were frequent (U2AF1: n = 31/155; 20·0%; SRSF2: n = 31/159; 19·5%; SF3B1mut: n = 8/159; 5·0%). ASXL1mut (25/153; 16·3%) were associated with advanced MDS (P = 0·001). Presence of ≥3 ACAs (P = 0·003) and ASXL1mut (P = 0·002) were associated with worse 2-year survival. In conclusion, the cytogenetic subgroup of MDS with del(20q) has a good prognosis but may be further subclassified by additional cytogenetic and molecular lesions. U2AF1mut is overrepresented in MDS with del(20q), and ASXL1mut is prognostically adverse.

Background Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms characterized by varying degrees of cytopenias and a predisposition to acute myeloid leukemia (AML). With conspicuous clinical and biological heterogeneity in MDS, an optimized choice of treatment based on accurate diagnosis and risk stratification in individual patients is central to the current therapeutic strategy. Diagnosis and prognostication in patients with myelodysplastic syndromes (MDS) may be improved by high-throughput mutation/copy number profiling. Methods A total of 944 patients with various MDS subtypes were screened for gene mutations and deletions in 104 known/putative genes relevant to MDS using targeted deep-sequencing and/or array-based genomic hybridization. Impact of genetic lesions on overall survival (OS) was investigated by univariate analysis and a conventional Cox regression, in which the Least Absolute Shrinkage and Selection Operator (lasso) was used for selecting variables. The linear predictor from the Cox regression was then used to assign the patients into discrete risk groups. Prognostic models were constructed in a training set (n=611) and confirmed using an independent validation cohort (n=175). Results After excluding sequencing/mapping errors and known or possible polymorphisms, a total of 2,764 single nucleotide variants (SNVs) and insertions/deletions (indels) were called in 96 genes as high-probability somatic changes. A total of 47 genes were considered as statistically significantly mutated (p10% of the cases. Less common mutations (2−10%) involved U2AF1, ZRSR2, STAG2, TP53, EZH2, CBL, JAK2, BCOR, IDH2, NRAS, MPL, NF1, ATM, IDH1, KRAS, PHF6, BRCC3, ETV6, and LAMB4. Intratumoral heterogeneity was evident in as many as 456 cases (48.3%), even though the small number of gene mutations available for evaluation was thought substantially to underestimate the real frequency. The number of observed intratumoral subpopulations tended to correlate with the number of detected mutations and therefore, advanced WHO subtypes and risk groups with poorer prognosis. Mean variant allele frequencies (VAFs) showed significant variations among major gene targets, suggesting the presence of clonogenic hierarchy among these common mutations during clonal evolution in MDS. The impact of these genetic lesions on clinical outcomes was initially investigated in 875 patients. In univariate analysis, 25 out of 48 genes tested significantly affected overall survival negatively (P A total of 14 genes, together with age, gender, white blood cell counts, hemoglobin, platelet counts, cytogenetic score in IPSS-R, were finally selected for the Cox regression in a proportional hazard model and based on the linear predictor of the regression model, we constructed a prognostic model (novel molecular model), in which patients were classified into 4 risk groups showing significantly different OS (“low”, “intermediate”, “high”, and “very high risk”) with 3-year survival of 95.2%, 69.3%, 32.8%, and 5.3%, respectively (P Conclusions Large-scale genetic and molecular profiling by cytogenetics, NGS and array-CGH not only provided novel insights into the pathogenesis and clonal evolution of MDS, but also helped to develop a powerful prognostic model based on gene mutations and other clinical variables that could be used for risk prediction. Molecular profiling of multiple target genes in MDS is feasible and provides an invaluable tool for improved diagnosis, biologic subclassification and especially prognostication for patients with MDS. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Bacher:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

We analyzed a large cohort of 1160 untreated CLL patients for novel genetic markers (SF3B1, NOTCH1, FBXW7, MYD88, XPO1) in the context of molecular, immunophenotypic and cytogenetic data. NOTCH1 mutations (mut) (12.3%), SF3B1mut (9.0%) and TP53mut (7.1%) were more frequent than XPO1mut (3.4%), FBXW7mut (2.5%) and MYD88mut (1.5%). SF3B1mut, NOTCH1mut, TP53mut and XPO1mut were highly correlated to unmutated, whereas MYD88mut were associated with mutated IGHV status. Associations of diverse cytogenetic aberrations and mutations emerged: (1) SF3B1mut with del(11q), (2) NOTCH1mut and FBXW7mut with trisomy 12 and nearly exclusiveness of SF3B1mut, (3) MYD88mut with del(13q) sole and low frequencies of SF3B1mut, NOTCH1mut and FBXW7mut. In patients with normal karyotype only SF3B1mut were frequent, whereas NOTCH1mut rarely occurred. An adverse prognostic impact on time to treatment (TTT) and overall survival (OS) was observed for SF3B1mut, NOTCH1mut and TP53 disruption. In multivariate analyses SF3B1mut, IGHV mutational status and del(11q) were the only independent genetic markers for TTT, whereas for OS SF3B1mut, IGHV mutational status and TP53 disruption presented with significant impact. Finally, our data suggest that analysis of gene mutations refines the risk stratification of cytogenetic prognostic subgroups and confirms data of a recently proposed model integrating molecular and cytogenetic data.

We studied the utility and clinical relevance of RUNX1 (runt-related transcription factor 1) mutations and their application as residual disease detection markers using next-generation deep-sequencing. Mutation screening was prospectively performed in 814 acute myeloid leukemia patients. At diagnosis, 211/814 (25.9%) patients harbored mutations with a median clone size of 39% (range: 2-96%). Furthermore, in 57 patients paired samples from diagnosis and relapse were analyzed. In 47/57 (82.5%) cases the same alterations detected at diagnosis were present at relapse, whereas in 1/57 (1.8%) cases the mutation from the diagnostic sample was no longer detectable. Discrepancies were observed in 9/57 (15.8%) cases, also including the occurrence of novel RUNX1 mutations not restricted to those regions affected at diagnosis. Moreover, in 103 patients the prognostic impact of residual levels of RUNX1 mutations during complete remission was studied. Separation of patients according to median residual mutation burden into 'good responders' and 'poor responders' (median: 3.61%; range: 0.03-48.0%) resulted in significant differences of both event-free (median 21.0 vs. 5.7 months, P

Abstract 1394 Background: Acute erythroid leukemia (AEL) is characterized by a predominant erythroid population and is comprising Aims: Molecular and cytogenetic characterization of AEL and identification of genes with prognostic impact. Patients and Methods: We studied an unselected cohort of 94 AEL patients including 32 female and 62 male cases; median age was 69.0 yrs (range: 21.3–88.3 yrs). Survival data was available in 73 cases; median survival was 15.9 months. First, chromosome banding analysis (n=94) was performed. In addition, all cases with normal karyotype (NK) were investigated by CGH arrays (n=32) (Human CGH 12×270K Whole-Genome Tiling Array, Roche NimbleGen, Madison, WI). Further, mutation screening for ASXL1 (n=87), CEBPA (n=94), DNMT3A (n=94), FLT3 (both internal tandem duplication (ITD) (n=93), and tyrosine-kinase domain (TKD) mutations (n=85)), IDH1 (n=93), IDH2 (n=65), NRAS (n=91), KRAS (n=93), MLL-PTD (n=79), NPM1 (n=94), RUNX1 (n=94), TP53 (n=94), and WT1 (n=90) was performed by 454 amplicon deep-sequencing (Roche, Branford, CT), Sanger sequencing or melting curve analyses. CGH array data analysis was performed using Nexus Copy Number 6.0 (BioDiscovery Inc, El Segundo, CA). Results: Cytogenetic data was available for all cases: 48 cases (51.1%) presented an intermediate-risk and 46 (48.9%) cases an unfavorable cytogenetic category according to the MRC Classification. By CGH array analysis 30/32 cases retained a NK, whereas in two cases small aberrations were detected: case 1: deletion of the CEBPA gene, case 2: duplication 11q13.3 to 11q25 including the ATM and MLL gene. Molecular mutations were detected in 85/94 patients (90.4%). 63.5% (54/85) of mutated patients carried one, whereas 36.5% (31/85) of cases harbored two (n=22) or more (n=9) mutations. In detail, TP53 was the most frequently mutated gene (41 cases, 43.6%). Other alterations were detected in NPM1 (15/94; 16.0%); DNMT3A (12/94; 12.8%); ASXL1 (8/87; 9.2%); MLL-PTD (7/79; 8.9%); RUNX1 (8/94; 8.5%); IDH1 (6/93; 6.5%); WT1 (5/90; 5.6%); IDH2 (3/65; 4.6%); NRAS (3/91; 3.3%); KRAS (3/93; 3.2%); FLT3-ITD (3/93, 3.2%), FLT3-TKD (3/85, 3.5%), and CEBPA (1/94). First, we were interested in any correlation with the respective karyotype and observed that NPM1, RUNX1, and WT1 mutations correlated with an intermediate-risk karyotype (NPM1: 15/48 vs 0/46, P Conclusions: (1) The frequency of cases with complex or other adverse karyotypes within the AEL cohort is very high (48.9%), (2) 93.7% of cases with NK also showed a NK using high-resolution CGH arrays. (3) Overall, a remarkably high mutation frequency of 90.4% was found. (4) NPM1 and RUNX1mut were exclusively detected in the cytogenetically intermediate-risk MRC, TP53 mut predominantly in the MRC adverse group and mainly in cases with complex karyotype. (5) In addition to chromosome banding analysis mutation screening of RUNX1 and NPM1 in cases with intermediate-risk karyotype should be considered for better prognostication. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Bacher:MLL Munich Leukemia Laboratory: Employment. Poetzinger:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Summary Acute myeloid leukaemia (AML) with CEBPA mutations is listed as a provisional entity in the current World Health Organization classification. A difference in clinical outcome between single- (sm) and double-mutated (dm) cases has been reported, whereupon CEBPAdm cases were shown to be associated with better overall survival (OS). The occurrence and prognostic impact of concomitant molecular mutations in addition to CEBPAdm has not been assessed until now with exception of GATA2 mutations. Here, we investigated a cohort of 95 AML CEBPAdm cases for concomitant mutations. TET2 was found to be most frequently mutated (34·0%) gene, followed by GATA2 (21·0%), WT1 (13·7%), DNMT3A (9·6%), ASXL1 (9·5%), NRAS (8·4%), KRAS (3·2%), IDH1/2 (6·3%), FLT3-internal tandem duplication (6·3%), FLT3-tyrosine kinase domain (2·1%), NPM1 (2·1%), and RUNX1 (1/94). Patients harbouring additional mutations in the TET2 gene showed significantly worse OS than TET2 wild-type cases (P = 0·035), whereas GATA2-mutated patients showed improved OS (P = 0·032). Serial analyses were performed for 39 CEBPAdm cases with concomitant mutations. Here, we observed that CEBPA mutations present the primary pathogenetic event in the majority of cases (76·9%). Further, a distinct gene expression profile (GEP) was confirmed for CEBPAdm versus CEBPAsm or CEBPA wild-type cases while no significant changes in GEP were observed related to additional mutations within the CEBPAdm AML.

The karyotype is so far the most important prognostic parameter in acute myeloid leukemia (AML). Molecular mutations have been analyzed to subdivide AML with normal karyotype into prognostic subsets. The aim of this study was to develop a prognostic model for the entire AML cohort solely based on molecular markers. One thousand patients with cytogenetic data were investigated for the following molecular alterations: PML-RARA, RUNX1-RUNX1T1, CBFB-MYH11, FLT3-ITD, and MLL-PTD, as well as mutations in NPM1, CEPBA, RUNX1, ASXL1, and TP53. Clinical data were available in 841 patients. Based on Cox regression and Kaplan-Meier analyses, 5 distinct prognostic subgroups were identified: (1) very favorable: PML-RARA rearrangement (n = 29) or CEPBA double mutations (n = 42; overall survival [OS] at 3 years: 82.9%); (2) favorable: RUNX1-RUNX1T1 (n = 35), CBFB-MYH11 (n = 31), or NPM1 mutation without FLT3-ITD (n = 186; OS at 3 years: 62.6%); (3) intermediate: none of the mutations leading to assignment into groups 1, 2, 4, or 5 (n = 235; OS at 3 years: 44.2%); (4) unfavorable: MLL-PTD and/or RUNX1 mutation and/or ASXL1 mutation (n = 203; OS at 3 years: 21.9%); and (5) very unfavorable: TP53 mutation (n = 80; OS at 3 years: 0%; P < .001). This comprehensive molecular characterization provides a more powerful model for prognostication than cytogenetics.

T-cell bispecific antibodies (TCBs) are a novel therapeutic tool designed to selectively recruit T-cells to tumor cells and simultaneously activate them. However, it is currently unknown whether the dysfunctional state of T-cells, embedded into the tumor microenvironment, imprints on the therapeutic activity of TCBs. We performed a comprehensive analysis of activation and effector functions of tumor-infiltrating T-cells (TILs) in different tumor types, upon stimulation by a TCB targeting folate receptor 1 and CD3 (FolR1-TCB). We observed a considerable heterogeneity in T-cell activation, cytokine production and tumor cell killing upon exposure to FolR1-TCB among different FolR1-expressing tumors. Of note, tumors presenting with a high frequency of PD-1hi TILs displayed significantly impaired tumor cell killing and T-cell function. Further characterization of additional T-cell inhibitory receptors revealed that PD-1hi TILs defined a T-cell subset with particularly high levels of multiple inhibitory receptors compared with PD-1int and PD-1neg T-cells. PD-1 blockade could restore cytokine secretion but not cytotoxicity of TILs in a subset of patients with scarce PD-1hi expressing cells; in contrast, patients with abundance of PD-1hi expressing T-cells did not benefit from PD-1 blockade. Our data highlight that FolR1-TCB is a promising novel immunotherapeutic treatment option which is capable of activating intratumoral T-cells in different carcinomas. However, its therapeutic efficacy may be substantially hampered by a pre-existing dysfunctional state of T-cells, reflected by abundance of intratumoral PD-1hi T-cells. These findings present a rationale for combinatorial approaches of TCBs with other therapeutic strategies targeting T-cell dysfunction.

Dysfunctional T cells present in malignant lesions are characterized by a sustained and highly diverse expression of inhibitory receptors, also referred to as immune checkpoints. Yet, their relative functional significance in different cancer types remains incompletely understood. In this study, we provide a comprehensive characterization of the diversity and expression patterns of inhibitory receptors on tumor-infiltrating T cells from patients with non–small cell lung cancer. In spite of the large heterogeneity observed in the amount of PD-1, Tim-3, CTLA-4, LAG-3, and BTLA expressed on intratumoral CD8+ T cells from 32 patients, a clear correlation was established between increased expression of these inhibitory coreceptors and progression of the disease. Notably, the latter was accompanied by a progressively impaired capacity of T cells to respond to polyclonal activation. Coexpression of several inhibitory receptors was gradually acquired, with early PD-1 and late LAG-3/BTLA expression. PD-1 blockade was able to restore T-cell function only in a subset of patients. A high percentage of PD-1hi T cells was correlated with poor restoration of T-cell function upon PD-1 blockade. Of note, PD-1hi expression marked a particularly dysfunctional T-cell subset characterized by coexpression of multiple inhibitory receptors and thus may assist in identifying patients likely to respond to inhibitory receptor–specific antibodies. Overall, these data may provide a framework for future personalized T-cell–based therapies aiming at restoration of tumor-infiltrating lymphocyte effector functions. Cancer Immunol Res; 3(12); 1344–55. ©2015 AACR.

Recent advances in cancer immunotherapies with PD-1/PD-L1 pathway blockade have transformed the way that cancer is being treated, leading to durable responses and prolonged overall survival. The general thinking is that PD-1/PD-L1 blockade reinvigorates tumor-infiltrating PD-1+ T cells with exhausted phenotypes. However, a mechanistic understanding on why only a subset of patients (10-30%) responds to checkpoint inhibition remains largely unknown, as does the exact immune mechanism of PD-1/PD-L1 blockade. Here, we discovered that PD-L1 blockade mediates anti-tumor immunity via dendritic cells. In human blood, in vitro and ex vivo dendritic cells (DCs) express both PD-1 and PD-L1, and an activation signal via TLR agonists triggers downregulation of PD-1 to empower the T cell stimulatory capability of DCs. In contrast, tolerogenic DCs remain PD-1 positive, correlating to T cell-unresponsiveness. Anti-PD-L1 Ab directly induces maturation of DCs and renders them capable of stimulating T cell proliferation. Similarly, anti-PD-L1 Ab treatment in tumor-bearing mice induces massive infiltration and activation of CD11c+ DCs in the spleen and draining lymph node, indicating that PD-1 is a negative regulator in DCs. Furthermore, ablation of DCs prior to anti-PD-L1 Ab treatment in an established MC38 tumor model in CD11c-DTR mice suggests a crucial role of DCs in mediating response to PD-L1 treatment. In support of the preclinical evidence that DCs are the primary target of anti-PD-L1 Ab, we analyzed RNA-seq data from tumor biopsies at baseline in patients with renal cell carcinoma prior to treatment with atezolizumab and found that the abundance of genes related to cross-presenting DC subsets (such as XCR1) correlates with a survival advantage in response to atezolizumab (HR=0.13, median OS is 16.2 months in patients with DC gene score 50%). In conclusion, we discovered abundance of tumor-infiltrating DCs as a novel biomarker that can predict the clinical response of PD-L1 blockade. We postulate that checkpoint inhibition directly on tumor-infiltrating DCs likely contributes to amplification of Ag-specific priming of tumor-specific T cells. Citation Format: Maud Mayoux, Marieke F. Fransen, Andreas Roller, Ines Matos, Vesna Pulko, Vaios Karanikas, Pablo Umana, Christian Klein, Ferry A. Ossendorp, Wei Xu. Dendritic cells dictate the responsiveness of PD-L1 blockade in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3658. doi:10.1158/1538-7445.AM2017-3658

The role of neuronal noncoding RNAs in energy control of the body is not fully understood. The arcuate nucleus (ARC) of the hypothalamus comprises neurons regulating food intake and body weight. Here we show that Dicer-dependent loss of microRNAs in these neurons of adult (DicerCKO) mice causes chronic overactivation of the signaling pathways involving phosphatidylinositol-3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) and an imbalance in the levels of neuropeptides, resulting in severe hyperphagic obesity. Similarly, the activation of PI3K-Akt-mTOR pathway due to Pten deletion in the adult forebrain leads to comparable weight increase. Conversely, the mTORC1 inhibitor rapamycin normalizes obesity in mice with an inactivated Dicer1 or Pten gene. Importantly, the continuous delivery of oligonucleotides mimicking microRNAs, which are predicted to target PI3K-Akt-mTOR pathway components, to the hypothalamus attenuates adiposity in DicerCKO mice. Furthermore, loss of miR-103 causes strong upregulation of the PI3K-Akt-mTOR pathway in vitro and its application into the ARC of the Dicer-deficient mice both reverses upregulation of Pik3cg, the mRNA encoding the catalytic subunit p110γ of the PI3K complex, and attenuates the hyperphagic obesity. Our data demonstrate in vivo the crucial role of neuronal microRNAs in the control of energy homeostasis.

The clinical impact of aberrant CEBPA promoter methylation (PM) in AML is controversially discussed. The aim of this study was to clarify the significance of aberrant CEBPA PM with regard to clinical features in a cohort of 623 cytogenetically normal (CN) de novo AML. 555 cases had wild-type CEBPA, 68 cases harbored CEBPA mutations. The distal promoter was methylated in 238/623 cases (38.2%), the core promoter in 8 of 326 cases (2.5%), whereas proximal PM was never detected. CEBPA PM and CEBPA mutations were mutually exclusive. CEBPA distal PM positive cases were characterized by reduced CEBPA mRNA expression levels and elevated white blood cell counts. CEBPA distal PM was less frequent in patients with mutations in FLT3, NPM1 and TET2 and more frequent in cases with RUNX1 and IDH2R140 mutations. Overall, no association of methylation to prognosis was seen. However CEBPA distal PM was associated with inferior outcome in cases with low FLT3-ITD ratio or TET2 mutations. A distinct gene expression profile of CEBPA distal PM positive cases compared to CEBPA mutated and CEBPA distal PM negative cases was observed. In conclusion, the presence of aberrant CEBPA PM is associated with distinct biological features but impact on outcome is weak.

Acute myeloid leukaemia (AML) with CEBPA mutations is listed as a provisional entity in the current World Health Organization classification. A difference in clinical outcome between single- (sm) and double-mutated (dm) cases has been reported, whereupon CEBPAdm cases were shown to be associated with better overall survival (OS). The occurrence and prognostic impact of concomitant molecular mutations in addition to CEBPAdm has not been assessed until now with exception of GATA2 mutations. Here, we investigated a cohort of 95 AML CEBPAdm cases for concomitant mutations. TET2 was found to be most frequently mutated (34·0%) gene, followed by GATA2 (21·0%), WT1 (13·7%), DNMT3A (9·6%), ASXL1 (9·5%), NRAS (8·4%), KRAS (3·2%), IDH1/2 (6·3%), FLT3-internal tandem duplication (6·3%), FLT3-tyrosine kinase domain (2·1%), NPM1 (2·1%), and RUNX1 (1/94). Patients harbouring additional mutations in the TET2 gene showed significantly worse OS than TET2 wild-type cases (P = 0·035), whereas GATA2-mutated patients showed improved OS (P = 0·032). Serial analyses were performed for 39 CEBPAdm cases with concomitant mutations. Here, we observed that CEBPA mutations present the primary pathogenetic event in the majority of cases (76·9%). Further, a distinct gene expression profile (GEP) was confirmed for CEBPAdm versus CEBPAsm or CEBPA wild-type cases while no significant changes in GEP were observed related to additional mutations within the CEBPAdm AML.

Part of the sequencing of þ 12 cohorts was supported by Roche Applied Science (Penzberg, Germany).

The thermal properties influencing seam performance of polymer modified bituminous roofing membranes are investigated using thermogravimetry and differential scanning calorimetry. Ten different commercial roofing materials are included: three atactic polypropylene (APP) modified bitumens and seven styrene-butadiene-styrene copolymer (SBS) modified bitumens. The results show that the difference in specific heat among different SBS or APP products was small. However, the specific heat of APP products was approximately 0.2 J g−1 K−1 higher than that of SBS products. It may indicate that for APP products a higher amount of energy is required for welding. Overheating of the membranes above 200°C during heat welding may cause decomposition in the polymer modified bitumen, since the degradation of pyrolysable organics begins at ∼200°C.

Currently there are multiple approaches for measuring gene expression from human tissue samples on the market. These methods range from measuring the expression of a few genes using quantitative RT-PCR to measuring every gene in a sample by whole transcriptome RNA sequencing (RNA-Seq). Although many techniques and products for measuring gene expression are available, the highly degraded RNA isolated from formalin-fixed, paraffin embedded (FFPE) tumor tissues still poses a challenge for many of them. FFPE tumor samples are an abundant source of biomarker information and gene expression analysis in these samples has the potential to identify new signatures, biomarkers, or diagnostics that could predict patient response to treatment. Accurately measuring gene expression in a high throughput manner from this sample type has long been a struggle in the field. To determine the ability of RNA-Seq to accurately measure gene expression from FFPE-derived RNA, we utilized a large collection of matched fresh frozen (FF) and FFPE samples from 12 different tumor indications. By comparing the results to the matching FF sample, we were able to determine the accuracy and sensitivity of each platform when using degraded FFPE-derived RNA. The FFPE samples in the collection had a wide range of RNA quality scores (RIN score: 0-6.8, DV200:0-78) representing what is typically isolated from clinical trial samples. We further compared whole transcriptome RNA-Seq with a hybrid capture method, RNA Access, to determine the accuracy and feasibility of using this technology on degraded RNA. RNA Access selects for protein coding transcripts by hybridization, whereas whole transcriptome RNA-seq removes ribosomal RNA, but analyzes everything else i.e. the global transcriptome. All samples were sequenced to an average of 50 or 25 million paired-end reads for whole transcriptome RNA-Seq and RNA Access, respectively and 100 ng of RNA was used as input in both assays. We conclude that both methods can be used for analyzing FFPE tumor samples. A similar dynamic range was observed for both methods and both show similar correlation between FF and FFPE samples within the method (0.76 vs 0.74). The overall concordance between methods was 0.69 and 0.63 for FF and FFPE, respectively. When looking specifically at the detection of 90 low expressing immune related genes, both assays were able to detect >94% with an inter-platform correlation of 0.66 and 0.56 in FF and FFPE, respectively. Whole transcriptome RNA-seq provides the maximum amount of information from clinical samples including anti-sense and non-coding RNA detection while RNA Access can be applied more cost-efficiently when one is mostly interested in the protein coding transcriptome. Citation Format: Erica B. Schleifman, Anna Kiialainen, Andreas Roller, Sabine Bader, Maipelo Motlhabi, Priti Hegde, Ian McCaffery, Garret Hampton, Michael Cannarile, Craig Cummings, Olivia Spleiss, Eric Peters. Whole transcriptome and exome targeted RNA sequencing for FFPE tumor samples from clinical trials. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3631.

We analyzed the mutational hotspot region of SRSF2 (Pro95) in 275 cases with chronic myelomonocytic leukemia (CMML). In addition, ASXL1, CBL, EZH2, JAK2V617F, KRAS, NRAS, RUNX1, and TET2 mutations were investigated in subcohorts. Mutations in SRSF2 (SRSF2mut) were detected in 47% (129 of 275) of all cases. In detail, 120 cases had a missense mutation at Pro95, leading to a change to Pro95His, Pro95Leu, Pro95Arg, Pro95Ala, or Pro95Thr. In 9 cases, 3 new in/del mutations were observed: 7 cases with a 24-bp deletion, 1 case with a 3-bp duplication, and 1 case with a 24-bp duplication. In silico analyses predicted a damaging character for the protein structure of SRSF2 for all mutations. SRSF2mut was correlated with higher age, less pronounced anemia, and normal karyotype. SRSF2mut and EZH2mut were mutually exclusive, but SRSF2mut was associated with TET2mut. In the total cohort, no effect of SRSF2mut on survival was observed. However, in the RUNX1mut subcohort, SRSF2 Pro95His had a favorable effect on overall survival. This comprehensive mutation analysis found that 93% of all patients with CMML carried at least 1 somatic mutation in 9 recurrently mutated genes. In conclusion, these data show the importance of SRSF2mut as new diagnostic marker in CMML.

CEBPA (CCAAT/enhancer-binding protein alpha) is a member of the C/EBP family of bZIP transcription factors encoding two different translational protein isoforms. The CEBPA transcription factor is involved in cell cycle arrest, repression of self-renewal and myeloid differentiation during normal hematopoiesis. In acute myeloid leukemia (AML), mutations in CEBPA result in a cellular differentiation block.1 They occur in around 8% of normal karyotype AML (CN-AML). Approximately half of the patients harbor two CEPBA mutations associated to a favorable prognosis. CEBPA function can also be affected by promoter methylation2 or alterations in other oncogenes, for example through the t(8;21)(q22;q22)/RUNX1–RUNX1T1, which suppresses CEBPA mRNA expression. The t(8;21)(q22;q22) translocation replaces the C terminus, including the transactivation domain (TAD) of RUNX1, with RUNX1T1.3 RUNX1–RUNX1T1 in t(8;21) blocks CEBPA-dependent activation of its own (CEBPA) promoter and thereby inhibits autoregulation.4 Other leukemic fusion proteins involving core-binding factor (CBF) family members, for example t(3;21)(q26;q22)/RUNX1–EVI1 or inv(16)(p13q22)/CBFB–MYH11, did not suppress CEBPA mRNA, indicating a RUNX1–RUNX1T1-specific effect on CEBPA transcriptional control.1 Intragenic RUNX1 mutations confer an adverse prognosis in AML. Previously, we identified RUNX1 mutations in 32.7% of CN-AML or with non-complex chromosomal imbalances.5 RUNX1 mutations are absent in CBF–AML and acute promyelocytic leukemia.6 They are inversely correlated with CEBPA mutations.5, 6 To clarify whether intragenic RUNX1 mutations such as RUNX1–RUNXT1 fusions also result in CEBPA mRNA downregulation, we investigated 359 AML patients consisting of two independent cohorts: cohort 1 with 209 AML cases (109 males/100 females; median age 65.4 years; 19.7–88.1 years) from different cytogenetic subgroups (normal karyotype (n=93), t(8;21)(q22;q22)/RUNX1–RUNX1T1 (n=16), t(15;17)(q22;q12)/PML-RARA (n=15), sole +8 (n=12), sole +13 (n=10), complex karyotypes (n=10) and other rare noncomplex genetic abnormalities (n=53)). Cohort 2 comprised 150 normal karyotype patients selected according to RUNX1 mutation status (92 males/58 females; median age, 69.7 years; 18.3–88.1 years; 81/150 (54.0%) RUNX1 mutated) with survival data in 124 cases. Bone marrow and/or peripheral blood samples were sent to the MLL Munich Leukemia Laboratory in 2005–2011. All patients gave their written informed consent to genetic analysis and scientific studies. Chromosome-banding analysis was performed in all cases, when needed, combined with fluorescent in situ hybridization. RUNX1 mutations were analyzed by Sanger sequencing or an amplicon-based high-throughput deep-sequencing assay (454 Life Sciences, Branford, CT, USA). CEBPA (mRNA) expression was quantified in cohort 1 by gene expression microarray profiling (Affymetrix HG-U133 Plus 2.0 microarrays; Santa Clara, CA, USA). The gene expression raw data were processed according to the manufacturer's recommendations. Detection calls, that is present, marginal, or absent expression, were determined by default parameters. For measurement of CEBPA expression in cohort 2, a quantitative real-time reverse transcriptase PCR (RT-PCR) assay was established (Taqman, Life Technologies, Carlsbad, CA, USA; CEBPA TaqMan Gene Expression Assay: HS00269972_S1). mRNA expression of CEBPA was normalized against expression of ABL1; ratios were given as %CEBPA/ABL1. First, we investigated 209 AML cases from different cytogenetic subgroups using gene expression microarray profiling (Table 1a). The RUNX1 mutation status was analyzed in 178 cases (RUNX1–RUNX1T1 or PML-RARA-mutated cases had been excluded), in 41/178 (23%) of patients RUNX1 was mutated. The median CEBPA expression intensity value in all patients was 670 (range 48–5244). RUNX1-mutated cases showed a lower CEBPA expression than RUNX1 wild-type cases (n=41 vs 137, mean±s.d. 429±395 vs 998±717; P148 CEBPA expression (using the median CEBPA expression as threshold in the cohort), we observed no association with OS and EFS (median OS was 16.8 and 18.8 months, OS at 3 years was 25.6 and 24.3%, P=0.507; median EFS was 11.0 and 9.2 months, OS at 3 years was 19.1 and 15.0% P=0.541). Table 1b Investigation of CEBPA expression as determined by quantitative real-time PCR in cohort 2 of 150 patients with normal karyotype AML In addition, we investigated whether the localization of the RUNX1 mutations had any impact on CEBPA expression. However, no significant difference was detected between CEBPA expression levels, when RUNX1 mutations were located either outside or within the DNA-binding domain (RUNT) (n=46 cases), or behind the RUNT and within the TAD (n=35) (mean±s.d. 161±107 vs 148±87; P=NS), respectively. Also when comparing missense mutations (n=19) against in-frame, frameshift and nonsense alterations (n=62), no significant difference was detectable (mean±s.d. 177±114 vs 148±93; P=NS). In contrast, separating cases in single-mutated (n=60) or double-mutated/homozygous (n=21) RUNX1 mutations, we observed a non-significant trend towards a lower CEBPA expression in cases with double-mutated/homozygous mutations (mean±s.d. 126±89 vs 165±100; P=0.116) (Figure 1c). In murine experiments, RUNX1 gene deletion was reducing CEBPA mRNA in lineage-negative marrow cells in granulocyte–monocyte progenitors or common myeloid progenitors.7 Here, we demonstrated a negative effect of RUNX1 mutations on CEBPA expression levels in AML patients, similar to the RUNX1–RUNX1T1 fusion, which have previously been reported.4 By gene expression profiling, downregulation of different hematological transcription regulators such as CEBPA or ETV6 had been described by Silva et al.8 in AML FAB M0, which is closely associated with RUNX1 mutations. RUNX1 mutation localization seems to have no impact on CEBPA expression. In summary, downregulation of CEBPA expression may contribute to leukemogenesis in RUNX1-mutated AML.

We investigated the robustness of amplicon deep sequencing to study its utility in routine clinical applications offering patient-specific individualized assays for molecular disease characterization and monitoring. Amplicons were designed targeting RUNX1, CEBPA, CBL, NRAS, KRAS, DNMT3A, EZH2, and TP53 using different PCR amplification strategies and Roche GS FLX Titanium and Illumina MiSeq sequencing platforms. Thirty-three patients with leukemia were selected as an exemplary cohort representing heterogeneous cancer specimens. Both standard two-primer amplification and four-primer microfluidics PCRs yielded highly linear characteristics in detecting molecular alterations in series of dilution experiments. By fitting a linear mixed-effects model to the logarithmized data, a slope β of −1.000 (95% CI, ±0.046) was obtained for two-primer assays and of −0.998 (95% CI, ±0.105) was obtained for four-primer assays, which represented a near-perfect decrease of the mutation load. Furthermore, data are presented on technical precision, limit of detection, and occurrence of small subclones in TP53- and RUNX1-mutated patients to identify clonal disease progression and residual disease. We demonstrate that, depending on the local sequence context for each amplicon, the limit of detection of the assay cannot be lower than a range of 0.25% to 3.5%. In conclusion, amplicon deep sequencing enabled the assessment of mutations in a highly robust manner and across a broad range of relative frequencies of mutations. This assay detects residual disease or identifies mutations with predictive relevance to direct treatment strategies.

DNA hypermethylation has long been implicated in the pathogenesis of myelodysplastic syndromes (MDS) and also highlighted by the frequent efficacy of demethylating agents to this disease. Meanwhile, recent genetic studies in MDS have revealed high frequency of somatic mutations involving epigenetic regulators, suggesting a causative link between gene mutations and epigenetic alterations in MDS. The accumulation of genetic and epigenetic alterations promotes tumorigenesis, hypomethylating agents such as Azacitidine exert their therapeutic effect through inhibition of DNA methylation. However, the relationship between patterns of epigenetic phenotypes and mutations, as well as their impact on therapy, has not been clarified. To address this issue, we performed genome-wide DNA methylation profiling (Infinium 450K) in combination with targeted-deep sequencing of 104 genes for somatic mutations in 291 patients with MDS. Beta-mixture quantile normalization was performed for correcting probe design bias in Illumina Infinium 450k DNA methylation data. Of the >480,000 probes on the methylation chip, we selected probes using the following steps: (i) probes annotated with "Promotor_Associated" or "Promoter_Associated_Cell_type_specific; (ii) probes designed in "Island", "N_Shore" or "S_Shore"; (iii) removing probes designed on the X and Y chormosomes; (iv) removing probes with >10% of missing value. Consensus clustering was performed utilizing the hierarchical clustering based on Ward and Pearson correlation algorithms with 1000 iterations on the top 0.5% (2,000) of probes showing high variation by median absolute deviation across the dataset using Bioconductor package Consensus cluster plus. The number of cluster was determined by relative change in area under cumulative distribution function curve by consensus clustering. Unsupervised clustering analysis of DNA methylation revealed 3 subtypes of MDS, M1-M3, showing discrete methylation profiles with characteristic gene mutations and cytogenetics. The M1 subtype (n=121) showed a high frequency of SF3B1 mutations, exhibiting the best clinical outcome, whereas the M2 subtype (n=106), characterized by frequent ASXL1, TP53 mutations and high-risk cytogenetics, showed the shortest overall survival with the hazard ratios of 3.4 (95% CI:1.9-6.0) and 2.2 (95% CI:1.2-4.0) compared to M1 and M3, respectively. Finally, the M3 subtype (n=64) was highly enriched (70% of cases) for biallelic alterations of TET2 and showed the highest level of CpG island methylation and showed an intermediate survival. In the current cohort, we had 47 patients who were treated with demethylating agents, including 11 responders and 36 non-responders. When DNA methylation status at diagnosis was evaluated in terms of response to demethylating agents, we identified 54 differentiated methylated genes showing >20% difference in mean methylation levels between responders and non-responders (q < 0.1). Twenty-five genes more methylated in responders were enriched in functional pathways such as chemokine receptor and genes with EGF-like domain, whereas 29 less methylated gene in responders were in the gene set related to regulation of cell proliferation. Genetic alterations were also assessed how they affected treatment responses. In responders, TET2 mutated patients tended to more frequently respond (45% vs 34%), whereas patients with IDH1/2 and DNMT3A mutations were less frequently altered (0% vs 14%, 9% vs 14%) in responders, compared in non-responders. In conclusion, our combined genetic and methylation analysis unmasked previously unrecognized associations between gene mutations and DNA methylation, suggesting a causative link in between. We identified correlations between genetic/epigenetic profiles and the response to demethylating agents, which however, needs further investigation to clarify the mechanism of and predict response to demethylation agents in MDS. Disclosures Alpermann: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kiyoi:Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.k.: Research Funding; Pfizer Inc.: Research Funding; Takeda Pharmaceutical Co.,Ltd.: Research Funding; MSD K.K.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Alexion Pharmaceuticals.: Research Funding; Teijin Ltd.: Research Funding; Zenyaku Kogyo Company,Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Patents & Royalties, Research Funding; Nippon Shinyaku Co.,Ltd.: Research Funding; Japan Blood Products Organization.: Research Funding; Eisai Co.,Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Kyowa-Hakko Kirin Co.,Ltd.: Consultancy, Research Funding; Fujifilm Corporation.: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Bristol-Myers Squibb.: Research Funding; Chugai Pharmaceutical Co.,LTD.: Research Funding; Mochida Pharmaceutical Co.,Ltd.: Research Funding. Kobayashi:Gilead Sciences: Research Funding. Naoe:Toyama Chemical CO., LTD.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Pfizer Inc.: Research Funding; Astellas Pharma Inc.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Celgene K.K.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Miyazaki:Chugai: Honoraria, Research Funding; Shin-bio: Honoraria; Sumitomo Dainippon: Honoraria; Celgene Japan: Honoraria; Kyowa-Kirin: Honoraria, Research Funding.

Background: Activation of the extrinsic apoptotic pathway by TRAIL is dependent on clustering of death receptors (DR) on the surface of cells. However, current TRAIL-based strategies have proven ineffective in clustering death receptors and failed to demonstrate robust therapeutic activity in clinical trials. More potent DR agonist therapies could help to overcome insufficient pathway activation and resistance to TRAIL activation. RG7386 is a novel bispecific FAP-DR5 antibody, binding with high affinity to fibroblast activation protein (FAP) and with low affinity to DR5. FAP is expressed at high prevalence on cancer associated fibroblasts (CAFs) in various tumor types as well as on tumors of mesenchymal origin, such as sarcomas. Avidity-driven binding of the bispecific antibody induces hyperclustering of DR5, which leads to potent induction of extrinsic apoptosis pathway signaling and tumor cell death. Biomarkers will be crucial in predicting sensitivity to DR5 activation and apoptosis induction and for selection of patients most likely to benefit from treatment with RG7386. Aim: The aim of the study was to explore the efficacy of RG7386 in vitro and in vivo. CRC and PDAC xenograft models expressing FAP on tumor stroma as well as sarcoma models were used to explore in vivo efficacy. Molecular profiling of sensitive and resistant tumors was also performed to identify response prediction markers. Results: RG7386 demonstrated additive efficacy in vitro with clinically relevant combinations (e.g. irinotecan, paclitaxel) in a variety of CRC and PDAC cell lines. In a xenograft model, where CRC cells (DLD-1) were co-injected with fibroblasts, RG7386 showed strong anti-tumor efficacy in combination with irinotecan. Remarkably, in a patient-derived CRC xenograft model (Co5896), the efficacy of RG7386 in combination with irinotecan induced complete tumor remission in all mice (n = 10/10). Furthermore, the combination of RG7386 with doxorubicin generated complete remissions in FAP+ sarcoma patient and desmoplastic melanoma cell line derived xenograft models such as Sarc4605 and LOX-IMVI. Finally, extensive molecular profiling of sensitive and resistance models in vitro revealed a distinct response prediction signature of DR5 sensitivity. Conclusion: RG7386 is a novel bispecific antibody inducing avidity-driven DR5 crosslinking by binding to FAP. This induces potent apoptosis of tumor cells, making it an attractive therapeutic approach for treatment of FAP+ solid tumors. Encouraging data indicate the high potential of RG7386 to treat FAP positive sarcomas. A comprehensive biomarker program will be employed in the early clinical development of RG7386 to enable selection of patients likely to benefit and to corroborate the mode of action, anti-tumor activity and potential response prediction markers. Citation Format: Thomas Friess, Stefanie Lechner, Esther Abraham, Ann-Marie Broeske, Sabine Bader, Andreas Roller, Meher Majety, Katharina Wartha, Suzana Vega-Harring, Hadassah Sade, Oliver Krieter, Peter Bruenker. Induction of avidity-driven hyperclustering of DR5 by a new FAP-DR5 bispecific antibody (RG7386) leads to strong anti-tumor efficacy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 952. doi:10.1158/1538-7445.AM2015-952

Background In patients with myelodysplastic syndromes (MDS), a 20q deletion [del(20q)] is considered to define a cytogenetic subgroup and, if present as a sole cytogenetic aberration, is associated with a favorable prognosis according to the revised IPSS (IPSS-R). However, associated genetic lesions and their prognostic impact in MDS with del(20q) remain to be clarified. Study Design We studied 305 MDS patients with del(20q) for associated molecular and cytogenetic markers and clinical outcomes. All patients (229 males/76 females; median age, 74 yrs) were investigated by cytomorphology (CM) and chromosome banding analysis (CBA). Additionally, subsets were characterized by fluorescence in situ hybridization (FISH), molecular genetics including high-throughput sequencing (NGS), and array comparative genomic hybridization (aCGH). Results A total of 210 (68.9%) patients had “early” MDS with blasts below 5%, and 95 (31.1%) “advanced” MDS i.e. RAEB-1/-2. When only patients with a sole del(20q) (n=217) were considered, 161 (74.2%) had early MDS, 56 (25.8%) had advanced MDS. This confirmed the preponderance of good-risk MDS subtypes in patients with del(20q). Additional chromosomal aberrations (ACAs), e.g. -Y, del(5q), -7/del(7q), and +8, were detected in 88/305 (28.9%) patients. Cytogenetic complexity was higher in advanced MDS patients as they showed a higher frequency of ACAs than early MDS (39/95; 41.1% vs. 49/210; 23.3%; P=0.003) and a higher mean number of ACAs (mean±SD, 1.3±2.7 vs. 0.6±1.5; P=0.020). The minimal commonly deleted region (CDR) was determined using aCGH (n=30 investigated) and was located from position 41,067,253 to 45,700,000 on 20q13.11-q13.12 (4.6 Mb in size). The flanking genes of the minimal CDR were PTPRT (receptor-type tyrosine-protein phosphatase T) on 20q13.11 and EYA2 (Eyes Absent Homolog 2) on 20q13.12. A total of 159 patients (104 early MDS, 55 advanced MDS) were investigated for additional molecular mutations, 82 patients (51.6%) had at least one mutation. The mean number of molecular mutations per patient was higher in advanced MDS as compared to early MDS (0.9±0.9 vs. 0.6±0.7; P=0.060). In detail, spliceosome mutations were frequent (U2AF1: n=28/140 investigated; 20.0%; SRSF2: n=29/159; 18.2%; SF3B1: n=8/159; 5.0%) and were mutually exclusive and equally distributed in early und advanced MDS. ASXL1mut which were found in 25/153 cases (16.3%) investigated in the total cohort showed a significantly lower frequency in early MDS as compared to advanced MDS (9/100; 9.0%; vs. 16/53; 30.2%; P=0.001). Lower mutation frequencies we observed for RUNX1 (14/157; 8.9%) and MLL-PTD (2/134; 1.5%). The 2-year overall survival (2-yr OS) was better in patients with early MDS as compared to advanced MDS (92.3% vs. 73.7%; P=n.s.). Patients with a sole del(20q) showed similar 2-yr OS to patients with del(20q) and occurrence of ACAs (87.1% vs 81.1%, P=n.s.). Patients with ASXL1mut had significantly worse 2-yr OS as compared to wild-type ASXL1 (45.5% vs 87.9%; P=0.002). The SRSF2 and U2AF1 mutation status did not significantly impact survival. Conclusion The cytogenetic subgroup of MDS with del(20q) has a good prognosis but may be further subclassified by additional cytogenetic and molecular lesions. U2AF1 mutations were overrepresented in MDS with del(20q) as compared to published frequencies in overall MDS. ASXL1 mutations were relevant for disease progression. Therefore, analyses of molecular mutations may contribute to prognostication and to therapeutic decisions in patients with MDS and del(20q). Disclosures: Bacher: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Krauth:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Background Gains and losses of chromosomal material are frequent in AML and MDS and usually lead to loss or gain of a single copy of a whole chromosome, a chromosome arm or small stretches of the chromosome that may be microscopically invisible. More rarely, amplifications of chromosomal regions (defined as the presence of more than 6 copies of a region per cell) are observed. These supernumerary copies are located either extrachromosomally as small acentric chromosomal structures - so called double-minutes (dmin) - or intrachromosomally as large contiguous stretches of amplified DNA, so called homogeneously staining regions (HSR). Aims Characterize AML and MDS cases with gene amplifications with respect to size, affected genes and accompanying chromosomal abnormalities as well as TP53 status. Patients and Methods 84 AML and 31 MDS cases with cytogenetically visible amplifications were selected for this study. All cases were analyzed by array CGH, chromosome banding analysis, sequencing for TP53 mutations as well as FISH for TP53 deletions. Results The cohort comprised 55 (47.8%) males and 60 (52.2%) females with a median age of 72.0 years (range 38.0 - 90.3 years). A complex karyotype (≥4 aberrations) was present in 92/115 (80.0%) cases (AML=65/84 (77.4%); MDS=27/31 (87.1%)). In total, 385 amplified regions were identified by array CGH. In more detail: 3q26 (AML: n=6; MDS: n=3), 8q24 (AML: n=15; MDS: n=1), 11q21-25 (AML: n=42; MDS: n=13), 13q12 (AML: n=3; MDS: n=1), 13q31 (AML: n=3; MDS: n=2), 19p13 (AML: n=2; MDS: n=4), and 21q21-q22 (AML: n=24; MDS: n=5). The median number of amplified regions was 3 (range 1-18). In 14/115 (12.2%) cases, the amplification was located in dmins (AML: n=11; MDS: n=3) and in 101/115 (87.8%) patients in HSR (AML: n=73; MDS: n=28). In 40 of the latter 101 cases (39.6%) (AML: n=24; MDS: n=16) the amplification was located on a ring chromosome (rc). In patients with complex karyotypes we detected a significantly higher number of amplified regions as compared to non-complex karyotypes (3.5 vs. 2.8; p=0.015). No association between the complexity of the karyotype and the structural type of the amplification (dmin vs rc) was observed. Cases with non-complex karyotypes frequently harbored a 5q deletion (6/23; 26.1%) or chromosome 8 abnormalities (3/23; 13.0%). Within the subgroup of non-complex karyotypes del(5q) cases showed a tendency to a higher number of amplified regions (3.6 vs. 1.9; p=0.140). Further, amplifications of 11q genes were more frequent in complex karyotypes (54.4% vs. 21.7%; p=0.005), whereas 8q amplifications were more frequent in non-complex karyotypes (43.5% vs. 4.4%; p Conclusions 1. MLL is the most frequently amplified gene in AML and MDS. 2. Patients with complex karyotypes or TP53mut harbored more amplified regions compared to patients with non-complex karyotypes and TP53wt. 3. Amplifications on 11q were more frequent in complex karyotype whereas gene amplifications on 8q were predominantly observed in non-complex karyotypes. 4. EVI1 and ERG gene amplifications lead to a higher expression of the respective genes. Disclosures: Roller: MLL Munich Leukemia Laboratory: Employment. Weber:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller: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. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Introduction In acute myeloid leukemia (AML), the karyotype and molecular mutation profile are the strongest determinants for prognosis and biological subclassification. Yet, diagnostic analyses rely on chromosome banding technique and sequencing of a constantly growing number of genes. Aims In an era of novel high-throughput sequencing assays becoming viable options for diagnostic implementation we aimed to evaluate whether the application of targeted exome sequencing can reliably identify copy number states and molecular mutations in a single-step procedure. Patients and Methods The pilot cohort included four AML cases with a complex karyotype with known chromosomal alterations as detected by chromosome banding analysis, 24-color FISH and array CGH (12x270K microarrays, NimbleGen, Madison, WI). The size of the aberrant clone was determined by suitable probes using interphase-FISH on bone marrow smears. For sequencing analysis genomic DNA was extracted from mononuclear cells and 50 ng were processed using the TruSight Rapid Capture kit (Illumina, San Diego, CA). Sequencing was performed on a MiSeq instrument using the 2x150 bp paired-end read chemistry targeting a subset of the human exome (2,761 genes; 37,366 exons). This exome enrichment library contained >50,000 probes (7.75 Mb) focusing on disease-causing variants in specific inherited conditions (Illumina). Data analysis was performed applying default settings of the on-board MiSeq Reporter Software version 2.2.29 using the Burrows-Wheeler Aligner to align the reads against the hg19 reference genome. Further processing to delineate copy number states was performed using the ExomeCNV package. Results Each patient was analyzed in a single MiSeq run and in median 22,022,240 (range 19,233,134 - 23,507,016) reads were generated. The median coverage per target region was in the range of 74-186 reads. Coverage uniformity was assessed according to the manufacturer's recommendations. Over 98% of bases were covered at 0.12X mean coverage for each sample. Next, two data analysis pipelines were triggered, i.e. copy number states and mutation analysis. With respect to copy number alterations (CNA), in total 65 CNA were detected by chromosome banding analysis/array CGH. Of these, 21 were gains, 44 were losses. The size of the deletions ranged between 378,377 and 141,048,720 bp (median 10,731,680 bp), the size of the gains ranged between 281,608 and 46,404,876 bp (median 4,947,125 bp), respectively. In total, 63/65 (96.9%) copy number alterations were correctly identified by targeted exome sequencing. The NGS assay was able to detect copy number alterations that were present in only 23% of cells as determined by interphase-FISH. In detail, one of the deletions was homozygous with a larger deletion on the long arm of chromosome 17 (size: 1,070,162 bp) and a small intragenic deletion within the NF1 gene. This homozygous deletion was detected by array-CGH and by exome sequencing. Interestingly, the higher resolution of the exome sequencing assay in this area enabled the exact localization (exons 37 to 58) and size determination (78,415 bp) of the deletion. Overall, only 2 gains escaped detection. These were two small gained regions on a highly rearranged chr. 19. Secondly, with respect to mutation analysis, the same assay detected 19, 20, 21 and 28 mutations in the four analyzed patients. This pipeline took only putative variants into account that were not present in the control sample, were having a coverage ≥30 reads with a mutation load ≥10%, and had a confirmed COSMIC mutation entry (v66). 12/2,761 (0.4%) genes harbored mutations in at least 2/4 patients. This included genes known to be involved in leukemogenesis. TP53 mutations were detected in all four cases and all were confirmed by Sanger sequencing. Conclusions A targeted exome sequencing assay allowed to robustly assess copy number states in AML at diagnosis at a resolution greater than current conventional array CGH analyses. Moreover, exome sequencing read data also can be used to delineate mutation profiles. Thus, this workflow enabled to call gene mutations and copy number states in a single assay and is a promising option for a routine diagnostics assay in the future. The gene panel has to be further optimized by adding genes known to be mutated in hematological malignancies. More data is necessary to precisely determine the detection limit and to optimize software tools for a routine use. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Kuznia:MLL Munich Leukemia Laboratory: Employment. Zenger: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. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Introduction In 2012, whole-genome, whole-exome and global transcriptome sequencing studies unraveled the molecular complexity of Burkitt lymphoma (BL). Amongst a set of up to 70 recurrently mutated genes (Love C. et al., Nat. Genet. 2012), essential affected oncogenic pathways were discovered including mutations in MYC and the transcription factor TCF3 (Schmitz R. et al., Nature 2012), as well as inactivating mutations of ID3 (Richter J. et al., Nat. Genet. 2012). Aims (1) To validate the mutation frequency and mutational landscape of selected candidate target genes (ID3, MYC, TCF3, and TP53) in an independent cohort of 60 adult patients with Burkitt Lymphoma/Leukemia. (2) To investigate the clonal composition and associations with BCL2 rearrangements using a quantitative next-generation sequencing assay. Methods The patient cohort included 33 cases with Burkitt Leukemia (33/60, 55.0%) and 27 cases with Burkitt Lymphoma (27/60, 45.0%). 36 (60.0%) of these were single-hit lymphomas only harboring MYC-rearrangements, whereas 24 (40.0%) cases were multiple-hit lymphomas harboring in addition at least one of the following gene rearrangements: BCL2, BCL6, and/or CCND1. The median age was 60.8 years (range 5.5 - 82.7). All cases were comprehensively characterized by cytomorphology, cytogenetics and FISH, including evaluation for MYC-, BCL2-, BCL6-, and CCND1-rearrangements. Four candidate genes were sequenced using a quantitative deep-sequencing NGS assay (median coverage of 633 reads per mutation). The lower limit of detection of mutations was set at 2%. Amplicon sequencing libraries were prepared using genomic DNA extracted from mononuclear cells. The following regions were investigated: ID3 (exons 1-2), MYC (exons 2-3), TCF3 (exons 18), and TP53 (exons 4-11), respectively. Results In total, 112 mutations were detected in 39/60 (56.5%) patients. 9/112 (8.0%) were detected with a clone size of Conclusion In this independent validation study we can confirm the high frequency of mutations in ID3, MYC, TCF3 and TP53 in adult BL. As our understanding of the genetic landscape is further increased novel therapeutic approaches seem possible in this disease. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Kienast:MLL Munich Leukemia Laboratory: Employment. Denzel:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Alpermann: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. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Background In CLL, the TP53 gene may be inactivated by deletion and/or mutations. Most cases with 17p deletion also carry TP53 mutations on the second allele. However, in a subset of cases only one allele seems to be disrupted by either mutation or deletion. It is still a matter of debate whether monoallelic TP53 abnormalities have the same poor prognostic effect as biallelic alterations. Further, a small subset of patients with TP53 deletions harboring mutated IGHV genes were described to exhibit a slowly progressive disease without treatment indication for years. Aims In this study, we addressed the following questions: 1. Frequency of TP53 alterations: mutation and deletion. 2. Characterization of the TP53 altered subsets with respect to IGHV mutation status, other molecular mutations and cytogenetics. 3. Impact on survival. Patients and Methods 3,988 CLL patients were analyzed by DNA sequencing for TP53 mutations and by FISH for TP53 deletion status as well as for del(13q), del(11q) and +12. IGHV mutation status was determined in 3,505 patients. Further, SF3B1 (n=1,245), MYD88 (n=1,026), XPO1 (n=1,025), NOTCH1 (n=973), and FBXW7 (n=962) were analyzed by DNA sequencing. Results 488/3,988 (12.2%) harbored a TP53 mutation (TP53mut) and 308/3,988 (7.7%) patients showed a TP53 deletion (TP53del) by FISH. 268 cases (6.7%) showed both a TP53del and a TP53mut, while 220 cases (5.5%) harbored a TP53mut only and 40 (1.0%) a TP53del only. 20.5% of TP53mut cases harbored more than one TP53mut. The frequency of TP53mut and TP53del increased significantly with age (≤40 yrs: 2.4%/2.4%; 41-50 yrs: 7.5%/4.0%; 51-60 yrs: 12.4%/6.8%; 61-70 yrs: 12.1%/8.1%; 71-80 yrs: 13.4%/9.1%; >80 yrs: 16.0%/9.9%; p=0.006 and p=0.013, respectively). In the entire cohort, 1,428/3,505 (40.7%) cases showed an unmutated and 2,077/3,505 (59.3%) a mutated IGHV status. The lowest frequency of IGHV unmutated was observed in cases without TP53 alteration (1,148/3,094; 37.1%) and the highest in patients with both TP53mut and TP53del (156/201; 77.6%). The frequency was in between in patients with TP53mut sole (106/176; 60.2%) and TP53del sole (18/34; 52.9%). Patients with both TP53mut and TP53del as well as patients with TP53del sole had a significantly shorter overall survival (OS) compared to patients with TP53mut sole or patients without TP53 alteration (OS at 5 yrs: 40.2% vs. 36.4% vs. 68.8% vs 85.4%; p60 yrs (HR: 3.3), del(11q) (HR: 2.1), del(13q) sole (HR: 0.6), SF3B1mut (HR: 2.5) (for all p60 yrs (HR: 2.6, p Conclusions 1. TP53 alterations were observed in 13.2% of CLL patients, 6.7% showed both a deletion and a mutation, while 1% showed a deletion only and 5.5% a mutation only. 2. Both TP53 mutations and TP53 deletions are associated with an unmutated IGHV status. 3. TP53 deletions had the most adverse impact on survival, TP53 mutations had a significant impact on OS only if the mutation load was ≥20%. A small subset of patients with TP53 deletion sole and a mutated IGHV status seems to have a favorable outcome. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Worseg:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Background MYC translocations are observed in a variety of mature B-cell neoplasms and are also found in ALL. A clear assignment to an entity is not always possible in terms of immunophenotyping and morphology alone. So far, neither cytogenetics nor molecular genetics have been used to classify this group of diseases. Aims Analyze cytogenetic aberrations and molecular mutations in a large cohort of patients with B-cell neoplasms and MYC rearrangements to evaluate their respective value for classification. Patients and Methods 155 patients with B-cell neoplasms harboring a MYC translocation were included in this study. Chromosome banding analysis, FISH verifying the MYC rearrangement and mutation screening of the genes ID3, MYC, TP53 and SF3B1 (only in CLL) was performed. Results The cohort comprised 103 (66.5%) males and 52 (33.5%) females with a median age of 66.2 yrs (range: 5.5-87.1 yrs). Cytomorphologic and flow cytometric findings were heterogeneous with patients presenting as ALL (n=33, 21.3%), Burkitt lymphoma (n=29, 18.7%), CLL (n=29, 18.7%), and other mature B-cell neoplasms including follicular lymphoma, mantle cell lymphoma and PLL (n=64, 41.3%). MYC translocations occurred either as a single translocation event (“single hit”) or in addition to one, two or three other translocations involving BCL2, BCL6 and/or CCND1 (“double hit”, “triple hit” and “quadruple hit”, respectively (combined: “multiple hit”). Accordingly, the cohort was subdivided into 98/155 (63.2%) patients with single hit and 57/155 (36.8%) patients with multiple hit lymphoma (double hit: 39 (25.2%), triple hit: 16 (10.3%) and quadruple hit: 2 (1.3%)). Results of mutation analysis are depicted in the figure. TP53 mutations were identified in 57/155 (36.8%) patients and were significantly more frequent in Burkitt lymphoma compared to all other entities (18/29 (62.1%) vs. 39/126 (31.0%), p=0.003). MYC mutations were identified with a frequency of 48/155 (31.0%), without significant differences between entities. However, in patients with CLL we observed a slightly lower frequency than in all other entities (17.2% vs 34.1%, p=0.117). ID3 mutations were found in 20/155 (12.9%) cases and were significantly more frequent in Burkitt lymphoma compared to all other entities (10/29 (34.5%) vs. 10/126 (7.9%), p=0.001). Remarkably, in the subgroup of patients presenting as CLL no ID3 mutation was detected (0/29). Single hit lymphoma showed higher mutation frequencies of TP53 and ID3 compared to multiple hit lymphoma (TP53mut: 45.9% vs. 21.1%, p=0.002, ID3mut: 19.4% vs. 1.8%, p=0.001). In contrast, in single hit lymphoma the number of cytogenetic abnormalities observed in addition to MYC-translocation (ACA) was lower than in multiple hit lymphoma (mean: 3.5 vs. 8.5, p CLL patients were additionally analyzed for mutations in SF3B1. Interestingly, this subgroup showed a higher frequency of SF3B1 mutations compared to published data (Cazzola et al. Blood 2013,121:260-9) (11/29; 37.9% vs. 5-17%). Conclusions 1. Mutations in ID3 and TP53 were most frequently observed in cases presenting as Burkitt lymphoma and especially associated with single hit lymphoma. 2. CLL patients with MYC translocation showed a specific mutation pattern with no ID3 mutations, a low frequency of MYC mutations, but a very high frequency of SF3B1 mutations. Further, MYC translocated CLL is characterized by a low frequency of additional chromosome abnormalities and presents predominantly as single hit lymphoma. 3. Thus far, B-cell neoplasms are mainly classified based on morphological criteria and the immunophenotype. Our data suggest that the cytogenetic and molecular genetic profile might help to establish an improved classification system. The differentiation between single hit lymphoma and multiple hit lymphoma as well as the separation of the category of CLL with MYC-rearrangements is anticipated to be clinically relevant and should be further studied. Disclosures: Denzel: MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kienast: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. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Background FLT3-ITD has been established as an adverse prognostic factor in acute myeloid leukemia (AML). However, it has been repeatedly shown that not the presence of FLT3-ITD by itself confers the unfavorable effect, but the excess load of the FLT3-ITD in comparison to the FLT3 wild-type (wt) allele, which occurs by loss of heterozygosity (LOH) due to somatic isodisomy of chromosome 13q. Aim As now analysis of FLT3-ITD in many countries is restricted by patent claims we aimed at developing a novel assay for LOH detection on 13q as a substitute for FLT3-ITD to identify patients (pts) with a higher risk for shorter survival. Patients and Methods 1) First an intensively treated AML cohort with normal karyotype (NK) (n=453) was selected in which the FLT3-ITD/wt ratio was assessed by a standard procedure (fragment length analysis by gene scan). 2) In a second step, a subcohort (cohort 2) of 233/453 pts was analyzed for LOH in 13q by fragment analysis of polymorphic single tandem repeat (STR) markers flanking the FLT3 gene as previously described (Whitman et al., JCO 2001). 3) A further subset of 91 pts from cohort 2 was subjected to a proof-of-principle study of applying next generation amplicon sequencing (NGS) for detection of LOH using a combination of Access Arrays (Fluidigm, South San Francisco, CA) and the 250 bp paired-end read chemistry on a MiSeq instrument (Illumina, San Diego, CA). In total, 41 amplicons were designed covering 67 different known SNPs (single nucleotide polymorphisms), with 43 SNPs flanking the FLT3 gene in the telomeric and the centromeric direction (genomic position: chr13:26,755,356-38,231,018), 22 SNPs being located in FLT3 intronic regions and one SNP each in exons 2 and 6. The achieved median coverage per SNP was 6,143 reads (range 58 - 30,311). Results 1) In the control cohort of 453 NK-AML (FLT3-ITD+: n=162, 35.8%) the adverse impact of high FLT3-ITD burden was reproduced. According to FLT3-ITD/FLT3wt a ratio of 1 (LOH) was significantly shorter as compared to all others (EFS: 3.6 vs 14.4 months, p1: n=83, FLT3-ITD+ load ≤1: n=105, FLT3wt: n=45). The median mutation load was 0.80 (range: 0.03-181.7). In all patients at least one informative STR marker was identified: 1: n=6 pts, 2: n=23 pts, 3: n=60 pts, 4: n=77 pts, 5: n=67 pts). STR allele ratios were similar for FLT3wt and FLT3-ITD with burden of ≤1. A threshold of a STR aIlelic ratio ≥70% allows identification of 81/83 samples with LOH (positive detection: 97.6%). Median EFS was 4.7 months in the 13q LOH group compared to 14.4 months in all others (p=0.001), clearly demonstrating that STR analysis using five 13q-specific markers can be used as a powerful tool to generate prognostic data. 3) As the gene scan procedure was time consuming, we next tested whether an NGS-based high-throughput SNP analysis can detect the LOH cases with the same precision. In total, 110 different SNPs were detected in this cohort (43 more than the anticipated 67, as many pts had additional rare SNPs). Complete homozygous SNP constellations were regarded as not informative. A median of 67 SNPs/pts (range: 44-80) were detected with a median informative allele constellation of 51/pts (range: 27-70). The allelic SNP load was calculated from the mean allelic load of all informative SNPs and then correlated to the FLT3-ITD load as calculated by gene scan. Across the total subset of 91 pts a very good correlation was detected (Spearman, R=0.807, p Conclusions 1) Not the presence of FLT3-ITD per se but a high FLT3-ITD load due to LOH of 13q has prognostically adverse impact. 2) Assessment of LOH can be done either by STR analysis or even more accessible by quantification of SNP allelic ratios using NGS amplicon deep-sequencing with a positive detection rate of 97.9%. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Weber:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Introduction Massively parallel next-generation sequencing (NGS) data have changed the landscape of molecular mutations in chronic lymphocytic leukemia (CLL). The number of molecular markers continues to constantly increase. As such, physicians and laboratories face a great unmet yet challenging need to test panels of genes at a high level of sensitivity. Aim To develop an assay that is easily adoptable to adjust gene targets and amplicons according to current state-of-the-art evidence regarding the published landscape of mutations in CLL. Methods We developed a sensitive deep-sequencing assay for routine diagnostics. In total, 13 genes with relevance in CLL providing in part adverse prognostic information were chosen: ATM, BIRC3, BRAF (V600), FBXW7, KLHL6, KRAS, NOTCH1 (PEST domain), NRAS, MYD88, POT1, SF3B1 (HEAT repeats), TP53, and XPO1. Targets of interest comprised either complete coding gene regions or hotspots. In summary, 323 amplicons were designed with a median length of 204 bp (range 150-240 bp), representing a total target sequence of 39.36 kb. The sequencing library was constructed starting off 2.2 μg genomic DNA per patient using a single-plex microdroplet-based assay (RainDance, Lexington, MA). Sequencing data was generated using the MiSeq instrument (Illumina, San Diego, CA) loading up to 10 patients per run. The total turn-around time of the assay was less than 5 days. As a proof-of-principle cohort, 18 clinically well-annotated CLL patients were analyzed during the evaluation phase. The median age was 78 years (range: 52 – 87 years). Results Using the 500 cycles sequencing by-synthesis chemistry, in median 7,262 millions of paired-end reads were generated per run. This resulted in a median coverage per gene of 7,476 reads (range: 5,595 - 10,337). (1) In this cohort of 18 cases, a total of 71 mutation analyses had already been previously performed for eight of the 13 genes using either capillary Sanger sequencing or alternative amplicon deep-sequencing assays (454 LifeSciences or Illumina MiSeq). In detail, in these 8 genes these 71 assays detected 56 known polymorphisms or mutations in ATM (n=8), BIRC3 (n=6), FBXW7 (n=4), MYD88 (n=4), NOTCH1 (n=10), SF3B1 (n=5), TP53 (n=14), and XPO1 (n=4) and 28 analyses revealed a wild-type status. When comparing these results with data obtained using the 13-gene NGS panel, in all 84/84 (100%) parallel assessments concordant results were obtained underlining the robustness of this assay. (2) Overall and extending the previous results, the comprehensive 13-gene NGS panel then detected in 18/18 patients a total of 46 mutations in 10 of the 13 genes with a range of 1-5 mutations per case (median: 2). The mutation types comprised 22 missense, 4 nonsense, 16 frame-shifts, 3 insertions and 1 splice-site alterations. In median, the coverage per variant was 10,390-fold, thus enabling a sensitive detection of mutations at a lower limit of detection set at 3%. The mutation burden ranged from 3.0% to 62.0%. 18/46 (39.13%) mutations were detected with a clone size Conclusion We demonstrated that microdroplet-based sample preparation enabled to robustly target 13 genes for next-generation sequencing in a routine diagnostics environment. This included also larger gene targets such as ATM, being represented by 119 amplicons. Thus, this approach provides the potential to screen for prognostically relevant mutations in all CLL patients in a fast and comprehensive way providing actionable information suitable to guide therapy. Disclosures: Kohlmann MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Albuquerque:MLL Munich Leukemia Laboratory: Employment. Kuznia:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Background In MDS, cytogenetic aberrations play an important role for classification and prognostication. The original IPSS and the revised IPSS classifiers have clearly demonstrated the prognostic impact of distinct cytogenetic abnormalities. The vast majority of chromosome aberrations in MDS are gains or losses of chromosomal material while balanced rearrangements are rare. However, more than 50% of MDS and even more in low risk MDS harbor a normal karyotype. Chromosome banding analysis can only detect gains and losses of more than 10 Mb size due to its limited resolution and is dependent on proliferation of the MDS clone in vitro to obtain metaphases. Array CGH has a considerably higher resolution and does not rely on proliferating cells. Aims In this study we addressed the question whether MDS with normal karyotype harbor cytogenetically cryptic gains and losses. Patients and Methods 520 MDS patients with normal karyotype were analyzed by array CGH (Human CGH 12x270K Whole-Genome Tiling Array, Roche NimbleGen, Madison, WI). For all patients cytomorphology and chromosome banding analysis had been performed in our laboratory. The cohort comprised the following MDS subtypes: RA (n=22), RARS (n=43), RARS-T (n=27), RCMD (n=124), RCMD-RS (n=111), RAEB-1 (n=104), and RAEB-2 (n=89). Median age was 72.2 years (range: 8.9-90.1 years). Subsequently, recurrently deleted regions detected by array CGH were validated using interphase-FISH. Results In 52/520 (10.0%) patients copy number changes were identified by array CGH. Only eight cases (1.5%) harbored large copy number alterations >10 Mb in size, as such generally detectable by chromosome banding analysis. These copy number alterations were confirmed by interphase-FISH. They were missed by chromosome banding analysis due to small clone size (n=2), insufficient in vitro proliferation (n=3) or poor chromosome morphology (n=3). In the other 44 patients with submicroscopic copy number alterations 18 gains and 32 losses were detected. The sizes ranged from 193,879 bp to 1,690,880 bp (median: 960,176 bp) in gained regions and 135,309 bp to 3,468,165 bp (median: 850,803 bp) in lost regions. Recurrently deleted regions as confirmed by interphase-FISH encompassed the genes TET2 (4q24; n=9), DNMT3A (2p23; n=3), ETV6 (12p13; n=2), NF1 (17q11; n=2), RUNX1 (21q22; n=2), and STAG2 (Xq25, deleted in 2 female patients). No recurrent submicroscopic gain was detected. In addition, we performed survival analysis and compared the outcome of patients with normal karyotype also proven by array CGH (n=462) to patients with aberrant karyotype as demonstrated by array CGH (n=52). No differences in overall survival were observed. However, overall survival in 35 patients harboring deletions detected solely by array CGH was significantly shorter compared to all others (median OS: 62.1 vs 42.4 months, p=0.023). Conclusions 1. Array CGH detected copy number changes in 10.0% of MDS patients with cytogenetically normal karyotype as investigated by the gold standard method, i.e. chromosome banding analysis. 2. Most of these alterations were submicroscopic deletions encompassing the genes TET2, ETV6, DNMT3A, NF1, RUNX1, and STAG2. 3. Interphase-FISH for these loci can reliably pick up these alterations and is an option to be easily performed in routine diagnostics in MDS with normal karyotype. 4. Patients harboring deletions detected solely by array-CGH showed worse prognosis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Raitner:MLL Munich Leukemia Laboratory: Employment. Holzwarth:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Introduction Molecular mutation analyses are performed in myeloid malignancies either in a stepwise procedure, i.e. one target gene after each other or are not performed at all, e.g. in low-risk MDS. A comprehensive pan-myeloid panel to simultaneously target mutations in 26 genes allows a comprehensive analysis with the perspective to detect disease defining mutations in the majority of patients. Aims To test the utility of a pan-myeloid panel in routine diagnostics. Methods We developed sensitive next-generation deep-sequencing (NGS) assays comprising in total 26 genes: ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3 (TKD), GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PHF6, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1, and WT1. With the exception of RUNX1, which was sequenced on the 454 Life Sciences NGS platform (Branford, CT), all remainder genes were studied using a combination of a microdroplet-based assay (RainDance, Lexington, MA) and the MiSeq sequencing instrument (Illumina, San Diego, CA). The assay's turn-around time was less than 6 days, loading up to eight patients per sequencing run. In summary, 389 amplicons were designed with a median length of 206 bp (range 150-240 bp), representing a total target sequence of 78.15 kb. The sequencing library was constructed starting off 2.2 μg genomic DNA per patient, purified from isolated mononuclear cells. Using the 500 cycles sequencing-by-synthesis chemistry in median 7.644 millions of paired-end reads were generated per run. This resulted in a median coverage per gene of 7,626 reads (range 174-12,256). The lower limit of detection was set at a cut-off of 3%. Results Thus far, 191 prospectively collected cases have been analyzed during routine operations. In all cases the assay was successfully performed. Mutations (range 0-7) have been found in 119/191 (62.3%) cases. The major disease categories were as follows: MDS (n=76), suspected MDS (n=28), MDS/MPN (n=10), reactive bone marrow conditions (n=46), AML (n=8), CML (n=3), other conditions (n=20). We first were interested to address the utility of the panel in MDS when the analysis was restricted to the five prognostically relevant predictors of poor overall survival according to Bejar et al. (N Engl J Med. 2011;364:2496-506), i.e. ASXL1, ETV6, EZH2, RUNX1, and TP53. In detail, 69 cases with MDS were studied and in 42.0% (29/69) of cases mutations had been detected in these five genes while 40 patients showed no mutation. Interestingly, upon extending the analysis to the remainder 21 genes, at least one more mutation was discovered in 72.5% (29/40) of these cases, thereby extending the number of cases with at least one mutation to 84.1% (58/69) of patients. Of note, in 65.5% (19/29) of these latter cases, spliceosome mutations occurred in a mutually exclusive manner (SRSF2, SF3B1, U2AF1), thus also detecting mutations conferring a favorable clinical outcome, i.e. SF3B1 alterations. We next studied in more detail 28 cases with suspected MDS according to cytomorphology, i.e. cases with dysplastic features not sufficient to diagnose MDS. When again in a first step the five predictors of poor overall survival according to Bejar et al. were analyzed, mutations in ASXL1, ETV6, EZH2, RUNX1, and TP53 were observed in 25.0% of cases (7/28). In the group of 75.0% (21/28) of samples with no mutations according to Bejar et al., 28.6% (6/21) of cases harbored a mutation in the group of the 21 remainder genes analyzed simultaneously in the gene panel assay. Thus, in total the number of cases with at least one mutation increased to 46.4% (13/28) of patients. Of note, 6 of the 13 suspected MDS cases with mutations had a normal karyotype. In summary, with respect to correlations between these two cohorts, we observed that morphologically confirmed MDS cases (n=69) showed a higher number of mutated genes compared to “suspected MDS” cases (n=28) (1.88 vs 0.71; p Conclusion A pan-myeloid screening assay using NGS allows to address 26 relevant gene mutations in myeloid malignancies with diagnostic or prognostic impact. This approach is scalable and adoptable to accommodate the inclusion of novel gene targets according to the latest evidence from the literature. Importantly, given the broad spectrum of mutations in myeloid diseases covered by such a panel, mutations can be identified in the majority of patients and enable to support a more comprehensive classification in these complex diseases. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Kuznia:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Albuquerque:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Abstract 2873 Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Recurrent activating mutations of NOTCH1 have been reported, including the relevance of NOTCH1 mutations as independent negative prognostic marker (Rossi et al., Blood 2012). Aims: 1. Determine the frequency and prognostic impact of NOTCH1 mutations (NOTCH1 mut) in a large unselected cohort of adult CLL patients. 2. Evaluate the range of mutational burden by amplicon deep-sequencing. Patients and Methods: We investigated 538 patients (189 female, 349 male; median age: 66.1 years, range: 37.8 – 90.5 years) with untreated CLL by FISH and for NOTCH1 mut (Transcript ID ENST00000277541) using next-generation amplicon deep-sequencing (454 Life Sciences, Branford, CT). Additionally, TP53 (n=45 mut/523 screened, 8.6%) and IGHV mutation status were analyzed. IGHV status was unmutated in 39.2% (209/533) and mutated in 60.8% (324/533) cases. Since NOTCH1 mut in CLL are known to be located predominantly within the C-terminal PEST domain (Rossi et al., Blood 2012) we sequenced exons 33–34 (covering codons 2029 to 2556), represented by 7 distinct PCR reactions with a median amplicon length of 345 bp. In median, 608 bidirectional reads (range 140–2,117) were generated per amplicon, thereby allowing a sensitive detection of variants, i.e. at a cut-off value of 5% ∼30 independent reads were sequenced. Results: All patients were investigated by FISH: del(17p) (30/538, 5.6%), del(11q) (57/538, 10.6%), +12 (103/538, 19.1%), del(6q) (8/538, 1.5%), normal karyotype according to FISH (NK) (111/538, 20.6%) and del(13q) as sole abnormality (229/538, 42.6%). In total, 81 NOTCH1 mut were observed in 71/538 (13.2%) patients. The vast majority of mutations (98.8%) were found to be heterozygous, only 1/81 mutation (1.2%) was homozygous. We identified 23 point mutations (6 missense and 17 nonsense; 28.4%) and 58 frame-shift alterations (57 deletions and 1 indel; 71.6%). The most frequently occurring mutation was as previously described p.Pro2514ArgfsX4 (c.7541_7542delCT), which was identified in 51/81 (62.7%) variants. The median mutational burden as assessed by deep-sequencing read counts was 28% of sequence reads carrying the mutation (range: 2% - 69%). Of note, in 54/81 (66.7%) variations the detected mutation load was ≤20% and therefore would be below the detection level of Sanger sequencing. In detail, a mutational burden ≤20% was observed in 32/81 (39.5%) variations and ≤10% in 22/81 (27.2%) mutations. 10/71 (14.1%) NOTCH1 mut patients carried 2 mutations. In 9/10 patients a different mutational load between the 2 NOTCH1 mut was detected, indicating the presence of 2 independent clones or clonal evolution with acquisition of a second mutation in the initially NOTCH1 single mutated clone. Mutations mainly clustered in the C-terminal part, i.e. codons 2,385 to 2,555 of exon 34 where 72/81 (88.8%) alterations were located. Confirming published data, statistical analyses revealed NOTCH1 mut being associated with unmutated (unmut) IGHV status (unmut vs mut: 59/209, 28.2% vs 11/324, 3.4%; P Conclusion: 1. We present the first deep-sequencing study of NOTCH1 mutations in a large unselected CLL cohort and report an overall frequency of 13.2%. 2. The mutational burden of 66.7% of NOTCH1 mutations in CLL patients was ≤20%. 3. NOTCH1 mutations are an adverse prognostic parameter associated with shorter TTT and represent yet another novel important biomarker in CLL. Disclosures: Weissmann: MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment.

Abstract 403 Background: According to WHO classification Mixed Phenotype Acute Leukemia, T/myeloid, NOS (MPAL-TM) is defined by the immunophenotype with expression of both myeloid and T-lymphatic antigens. Only limited data is available on genetic aberrations. Aims: Molecular and cytogenetic characterization of MPAL-TM in comparison to AML and T-ALL. Methods: We studied 18 patients with MPAL-TM (7 female, 11 male; median age 61.6 yrs, range 19.6–89.3). Five patients had complex karyotypes (≥3 aberrations), 4 had other chromosomal abnormalities and 8 had a normal karyotype (n=1 no data available). Survival data was available in 16 cases (median survival 19.0 months). Two cases were selected for whole-exome sequencing (NimbleGen SeqCap EZ Human Exome, Roche NimbleGen, Madison, WI; HiSeq 2000, Illumina, San Diego, CA), mutation data was subsequently validated in the 16 remainder cases. The detailed genetic characterization included also a comprehensive targeted mutation screening of 32 genes with relevance in myeloid malignancies. Using microdroplet-based library construction (RainDance, Lexington, MA) next-generation amplicon sequencing data was generated for ASXL1, BCOR, BCORL1, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3, IDH1, IDH2, JAK2, KDM6A, KIT, KRAS, NOTCH1, NPM1, NRAS, PHF6, PRPF40B, PTPN11, RUNX1, SF1, SF3A1, SF3B1, SRSF2, TET2, TP53, U2AF1, U2AF2, ZRSR2 (MiSeq instrument; Illumina, San Diego, CA). MLL-PTD was analyzed by quantitative real time PCR. Results: In both cases selected for whole-exome sequencing mutations in DNMT3A were detected. When extending DNMT3A analysis to the complete cohort, mutations were detected in 10/18 cases (55.6%). This represents the largest mutation frequency reported thus far both for DNMT3A itself and for any gene mutation in MPAL-TM. DNMT3A mutations were associated with a more mature T-lymphatic immunophenotype with CD2 expressed more often (8/10 cases) as compared to cases without DNMT3A mutations (2/8 cases, p=0.054). The majority of DNMT3A mutations were missense mutations (n=9) with 3 mutations affecting the hotspot codon Arg882 in the methyltransferase domain, followed by 2 splicesite mutations, 1 nonsense, and 1 frame-shift mutation. Notably, a biallelic mutation status occurred at high frequency in these cases (7/10). In detail, except for three cases with a mutation in Arg882, all other mutated cases showed either two distinct mutations (n=3) or one homozygous mutation (n=4). This contrasts AML which is known to virtually lack biallelic DNMT3A mutations (3/49 cases (6.1%), Grossmann et al., ASH 2011) and suggests a closer association of MPAL-TM to T-ALL in which half of cases show a biallelic DNMT3A mutation status (8/16 cases (50.0%), Grossmann et al., ASH 2011). Contrasting data for both AML and T-ALL where DNMT3A mutations are related to an adverse prognosis, DNMT3A mutations in MPAL-TM were associated with a longer overall survival (12.0 vs. 3.1 months), although this difference was not significant. Moreover, the broad targeted mutation screening resulted in the detection of mutations in the following genes: TP53 (n=3), IDH1/2 (n=3), BCOR (n=3), NOTCH1 (n=3), NRAS (n=3), BRAF (n=2), PHF6 (n=2), ETV6 (n=2), RUNX1 (n=1), FLT3 (n=1), TET2 (n=1), ASXL1 (n=1), ZRSR2 (n=1), and SF1 (n=1). The median number of mutations per patient was 2 (range 0–7), only one patient had no mutation. TP53 mutations were observed only in cases with complex karyotypes. Interestingly, the genetic mutations encountered were predominantly limited either to genes known to play a role in epigenetic processes (DNMT3A, TET2, IDH1/2, ASXL1; n=11) or to transcription factors (TP53, RUNX1, ETV6; n=6). Furthermore, these two groups of mutations occurred virtually mutually exclusive, except for one case harboring both a mutation in DNMT3A and ETV6. There were only two remaining cases not carrying aberrations in these two gene categories. Conclusions: 1. A remarkably high mutation frequency for DNMT3A (55.6%) was observed in MPAL-TM. 2. In MPAL-TM DNMT3A mutations were most frequently biallelic and lacked an adverse prognostic impact. 3. Other molecular mutations in MPAL-TM were limited to genes with epigenetic function or to transcription factors. 4. Cytogenetic abnormalities in MPAL-TM were neither typical for AML nor for T-ALL. 5. Analysis of DNMT3A mutations should be considered in the diagnostic work-up of patients with MPAL-TM in the future. Disclosures: Kern: MLL Munich Leukemia Laboratory: Equity Ownership. Grossmann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Schoeck:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment.

In chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant BCR-ABL mutants. We used an ultra-deep sequencing (UDS) approach to resolve qualitatively and quantitatively the complexity of mutated populations surviving TKIs and to investigate their clonal structure and evolution over time in relation to therapeutic intervention. To this purpose, we performed a longitudinal analysis of 106 samples from 33 patients who had received sequential treatment with multiple TKIs and had experienced sequential relapses accompanied by selection of 1 or more TKI-resistant mutations. We found that conventional Sanger sequencing had misclassified or underestimated BCR-ABL mutation status in 55% of the samples, where mutations with 1% to 15% abundance were detected. A complex clonal texture was uncovered by clonal analysis of samples harboring multiple mutations and up to 13 different mutated populations were identified. The landscape of these mutated populations was found to be highly dynamic. The high degree of complexity uncovered by UDS indicates that conventional Sanger sequencing might be an inadequate tool to assess BCR-ABL kinase domain mutation status, which currently represents an important component of the therapeutic decision algorithms. Further evaluation of the clinical usefulness of UDS-based approaches is warranted.