ALBERT

Description: Introduction AML is an aggressive hematological neoplasm originating from hematopoietic precursor or stem cells and is rapidly fatal if untreated. Although rates of complete remission (CR) approach 80% in patients with favorable risk profile according to European Leukemia Net classification, CR rates drop dramatically within the intermediate or adverse prognostic groups, and most patients relapse in spite of CR. The origin of refractory or relapsed disease is - according to several studies - the bone marrow (BM) niche that protects the leukemia initiating cells (LIC) from cytotoxic drugs.The chemokine receptor CXCR4 is a key factor mediating the crosstalk between the BM niche and LICs. CXCR4 is a G-protein coupled chemokine receptor with Stromal cell-derived factor-1α (SDF-1α) as its single known chemokine-ligand, and high expression is associated with poor prognosis in AML. Targeting the CXCR4/SDF-1αaxis with small peptide inhibitors or monoclonal antibodies has shown promising results in preclinical and clinical studies, thus rendering it an attractive target for future therapeutic strategies (Peled & Tavor, Theranostics, 2014). In this observational study we analyzed whether the new CXCR4-targeted positron emission tomography (PET)-probe [68Ga]-Pentixafor (Philipp-Abbrederis et al, EMBO Mol Med, 2015; Wester et al, Theranostics, 2015) is applicable for molecular imaging of patients with AML with the goal to develop a CXCR4- targeted peptide-receptor-radiotherapy (PRRT). Methods Using myeloid malignancies (MDS, active AML) as an exemplary CXCR4-expressing cancer entity we imaged 10 patients with the CXCR4 specific PET-probe [68Ga]-Pentixafor by whole body PET/magnetic resonance imaging (PET/MR) after they signed informed consent for this observational assessment. Maximum standardized uptake values (SUVmax) of the involved BM areas were compared to BM from patients without BM malignancy assessed in a different study. CXCR4 surface expression of leukemic BM cells of an unselected cohort of 67 patients with myeloid malignancies was analyzed by flow cytometry. CXCR4 expression in 14 established AML cell lines was measured by flow cytometry and quantitative PCR. Results
 Out of 10 patients who were imaged with [68Ga]-Pentixafor-PET/MR, 4 - all of them with the diagnosis of an AML - showed visually positive PET signal of the BM. Those areas correlated well with disease infiltration as determined by MR imaging and the meanSUVmax was significantly higher as compared to visually CXCR4 negative patients (meanSUVmax 8.23 ± 5.23 vs. 2.26 ± 0.47; p=0.036). One of the 10 patients presenting with proven extramedullary relapse showed a CXCR4 positive PET signal within the lesion (SUVmax 5.12), whereas the cytological non-involved BM was PET negative (meanSUVmax 1.82). CXCR4 surface expression in established AML cell lines was variable and ranged from low to high, and correlated with mRNA levels. These data reflect the different expression values we found in the cohort of patients with myeloid malignancies. There was a trend to higher CXCR4 surface expression on blasts when AML patients were compared to MDS patients (median fluorescent intensity CXCR4/isotype 1.73 ± 0.25 vs. 0.96 ± 0.06; p=0.063) that became significant when blast frequency exceeds 30% (≤30% vs. 〉30% blasts: 1.07 ± 0.11 vs. 2.08 ± 0.38; p=0.006). In this patient cohort we did not observe any correlation between CXCR4 expression and factors such as diagnosis (secondary vs. de novo AML), disease status (refractory or relapse vs. no pretreatment), molecular subtypes or cytogenetic aberrations. Discussion Imaging of CXCR4 by PET/MR with [68Ga]-Pentixafor is feasible in patients with AML. Due to the higher uptake in leukemic BM compared to control BM and its low uptake in other organs (e.g. liver, brain and gut), CXCR4 could serve as an attractive new PRRT-target in selected patients with AML. Such PRRT approaches are of special interest in the setting of allogeneic BM transplantation as conventional myeloablative conditioning regimens - although they are associated with a better relapse free survival - show substantial rates of transplant-related mortality and are therefore not applicable in many AML patients due to their age. In particular, therapeutic targeting of CXCR4 by PRRT provides a unique opportunity of integrating a LIC/niche-directed treatment into the conditioning regimen. Such clinical studies are in development. Disclosures Wester: Scintomics GmbH: Other: General Manager. Keller:Roche: Consultancy, Honoraria; Pfizer: Consultancy.

Description: Abstract 1328 Cks1 is an activator of the SCFSkp2 ubiquitin ligase complex that targets the cell cycle inhibitor p27Kip1 for degradation. Loss of Cks1 results in p27Kip1 accumulation and decreased proliferation. Cks1 expression is elevated in various B cell malignancies including Burkitt lymphoma and multiple myeloma. We have previously shown that loss of Cks1 results in elevated p27Kip1 levels and delayed tumor development in a mouse model of Myc-induced B cell lymphoma. Surprisingly, loss of Skp2 in the same mouse model also resulted in elevated p27Kip1 levels but exhibited no impact on tumor onset. Analysis of Cks1−/− murine embryonic fibroblasts (MEFs) unveiled a significant growth defect and comparison with double knock out Cks1−/−;p27Kip1−/− MEFs revealed a p27Kip1 independent defect at G1/S transition. These effects were associated with suppressed Cdk1 protein and Cdk2 activity in Cks1−/− cells. We hypothesized that Cks1 recruits further cell cycle regulators or facilitators of proliferation, which may explain the dramatic growth defect and the prolonged latency of lymphomagenesis upon loss of Cks1. Using Strep-Flag tagged Cks1 and tandem affinity purification followed by mass spectrometry we identified Geminin as a new Cks1 binding partner. Geminin is an inhibitor of rereplication and loss of Geminin leads to recurrent origin firing in S phase. The physical interaction of Cks1 and Geminin was present in cells of human and murine origin and was associated with Cdk2 binding to Geminin. Detailed analysis proved Cks1 dispensibel for cell cycle controlled Geminin degradation or phosphorylation. Surprisingly when mining public databases and RNA microarray data we found Geminin overexpressend in aggressive human lymphoma subtypes associated with high Ki-67 expression, and in murine Myc-induced lymphoma. Real time PCR revealed increased expression of Geminin during lymphomagenesis in Eμ-Myc mice, which correlated with protein expression. In conclusion, we established the physical Geminin-Cks1 protein interaction, present in human and murine cells, which could act in control of rereplication and genomic stability. Geminin is overexpressed in human and murine lymphoma revealing a potential role in lymphomagenesis. Disclosures: No relevant conflicts of interest to declare.

Description: Background: In AML patients (pts), pre-leukemic mutations in DNMT3A, TET2 and ASXL1 (DTA) were shown to persist in remission, which was not associated with survival (Jongen-Lavrencic et al. NEJM 2018). On the other hand Abelson et al. (Nature 2018) recently identified a specific pre-leukemic mutational spectrum in pts that eventually develop AML. NPM1 mutation (NPM1mut) identifies a WHO AML entity which accounts for about 30% of all AML. The absence of NPM1 transcripts following treatment defines complete molecular remission (CMR). We aimed to dissect the clonal hierarchy of co-mutations at diagnosis of NPM1mut AML and analyze the role of persistent mutations in this well-defined CMR setting. Methods: We investigated 136 pts with diagnosis of NPM1mut AML (from 2006 to 2016) who attained a CMR (absence of NPM1 transcripts, sensitivity 0.001%) after intensive treatment. The mean follow-up was 40 months (2-96). Next-generation sequencing of 39 genes associated with myeloid malignancies was performed for all pts in paired samples at diagnosis and at CMR. The median coverage was 5200x. FLT3-ITD was analyzed by gene scan. Results: At diagnosis, a total of 375 mutations were detected, with an average of 2.8/pt (range 1-6); at least one mutation other than NPM1 was present in 123/136 pts (90%). DTA-genes DNMT3A (39% of pts) and TET2 (15%) were among the most frequently mutated, while no ASXL1 mutation was observed. Mutated non-DTA genes included: FLT3-ITD (35%), IDH2 (27%), IDH1 (21%), NRAS (18%), FLT3-TKD (14%) and PTPN11 (13%). The mean variant allele frequency (VAF) was 36% (2.7-94), and was higher for DTA than non-DTA mutations (44% vs 34%, p

Description: Background: Genome instability is a hallmark of cancer. Mutations in DNA repair pathway genes are frequent in a number of solid tumors. Defects in DNA repair or damage response can weaken response to conventional chemotherapy and are frequently regarded as poor prognostic markers. However, a high tumor mutation burden (TMB, number of somatic mutations per mega base) was recently found to correlate with better response to immune checkpoint inhibitors e.g. in colon cancer. Patients with defects in the DNA mismatch repair (MMR) pathway in solid tumors are among the cases with the highest TMB. Hematological malignancies are generally expected on the lower end of the TMB spectrum. We used whole genome sequencing (WGS) for 3256 patients with hematological malignancies (lymphatic and myeloid) to determine factors of genetic instability across all entities. Aim: To determine the number of known mutations in genes from the DNA repair pathway To estimate TMB using WGS and identify cases with high TMB in hematologic malignancies Methods: We investigated a cohort of 3256 patients with hematological malignancies, who were analyzed according to WHO diagnostic gold standards for routine purposes (incl. 584 acute myeloid leukemia [AML] and 635 myelodysplastic syndromes [MDS] samples). We performed amplification-free library preparation and sequencing on HiseqX and NovaSeq 6000 with a median coverage of 106x. Mapping and variant calling was performed with standard pipelines via BaseSpace (all Illumina, San Diego, CA). A pool of gender-matched genomic DNA (Promega, Madison, WI) was used for a tumor-unmatched normal variant calling. (a) In detail we evaluated 180 genes involved in DNA repair. We filtered on (likely) pathogenic variants from ClinVar and for TP53 on protein-truncating variants and (likely, possibly) pathogenic variants from the IARC database. (b) For TBM calculation we determined protein-altering changes and then subtracted all gnomAD listed variants in order to eliminate most germline variants. Results: We found 479 of 3256 (15%) patients with at least one pathogenic variant according to current database annotations in DNA repair or damage response genes. Most pathogenic variants were found in TP53 (330/3256; 10%) and ATM (25/3256, 1%), however, this is probably the effect of the already available systematic database annotation for both genes. For routine diagnostic purposes TP53 mutation status had been analyzed for 1184 patients with Sanger sequencing (7%) or amplicon next-generation sequencing (93%). A 98% and 99% concordance of the pathogenic and non-pathogenic TP53 status was found in comparison to WGS. Mutations in genes from the DNA double-strand break repair (and/or homologous DNA pairing and strand exchange) pathway were found in 93 patients (3%). Pathogenic and potentially germline MMR gene mutations were found in only 3 patients (0.1%, 2 MLH1, 1 MSH6), which equals the expected frequency in the Western population (0.05-0.3%). Next, we calculated TMB. The average was 2.4 [range: 0.4-39.2]. Only samples above the 95th percentile were defined as "TMBhi" (TMB ≥5). TMB was lowest in chronic myeloid leukemia (CML) and essential thrombocythemia (ET) (〈 2) and no ET or CML patient was found among the TMBhi. We then focused on MDS, which is our largest subcohort: 56 of 635 (9%) patients were in TMBhi. Furthermore, among MDS patients, a significantly higher TMB was found in MDS-EB-2 (average 3.3 vs. 2.3 for non EB-2, p

Description: Background: Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare disorder, occurs almost exclusively in smoking women and is characterized by a chronic polyclonal lymphocytosis with circulating binucleated lymphocytes, clonal cytogenetic abnormalities involving chromosome 3, and chromosomal instability. Outcome of PPBL patients is mostly benign, but subsequent malignancies (non-Hodgkin´s lymphomas and solid tumors) were described. Potential molecular factors leading to their development are yet unclear. Aims: Detailed molecular genetic characterization of PPBL by whole genome sequencing (WGS) and RNA sequencing (RNAseq) in comparison to the well-characterized lymphoid malignancy CLL. Patient cohorts and methods: The total cohort comprised 27 PPBL (3 male, 24 female) and 250 CLL cases (163 male, 87 female). WGS was performed for all patients: 150bp paired-end reads where generated on Illumina HiseqX and NovaSeq 6000 machines (Illumina, San Diego, CA). A mixture genomic DNA from multiple anonymous donors was used as normal controls. To remove potential germline variants, each variant was queried against the gnomAD database, variants with global population frequencies 〉1% where excluded. Final analysis was performed only on protein-altering and splice-site variants. For further analysis, a virtual panel of 355 lymphoid genes was selected. All reported p-values are two-sided and were considered significant at p 5 CPM) in at least 66% of the samples. Genes with FDR (false discovery rate) 〈 0.05 and an absolute logFC 〉 1.5 were considered differentially expressed (DE). Results: Median age was 46 years for PPBL patients (range: 23-67 years) and 67 years for CLL patients (range: 39-94 years). Mean number of mutations per patient was 18 for PPBL and 20 for CLL. For both entities, the majority of mutations were missense mutations (88% in PPBL vs. 81% in CLL), followed by splice-site mutations (7% vs. 10%), other mutation types were only rarely detected. In PPBL, 42 genes were found to be mutated at a frequency of 〉15%, including ATM (22%), CREBBP (19%), NCOR2 (19%), AHNAK2 (15%), JAK3 (15%), NOTCH2 (15%) and TRAF1 (15%), all of which have been associated with a variety of cancers. Moreover, ATM, NOTCH2 and TRAF1 mutations were described before to be associated with lymphomas. In PPBL patients, mutations in TRAF1 and ATM as well as mutations in TRAF1 and NOTCH2 were found to be mutually exclusive. For CLL patients, 29 genes showed a mutation frequency of 〉15%, comprising ATM (26%), KMT2D (23%), NOTCH1 (23%), LRP1B (19%), TP53 (16%) and CREBBP (15%). Comparison of the mutation frequencies between the two entities revealed several genes with significant differences: whereas mutations in CKAP5 (11% vs. 2%, p=0.022), DNMT3A (11% vs. 3%, p=0.033), MAP2 (19% vs. 4%, p=0.009), ROBO1 (15% vs. 4%, p=0.046) and TRAF1 (15% vs. 2%, p=0.006) were found to be more frequent in PPBL cases compared to CLL cases, KMT2D (4% vs. 23%, p=0.014), TDRD6 (0% vs. 14%, p=0.032) and TP53 (4% vs. 16%, p=0.048) mutations were more abundantly detected in CLL patients. Moreover, NOTCH1 was mutated more frequently in CLL cases (7% vs. 23%, p=0.082), whereas mutated NOTCH2 (known to be frequently mutated in splenic marginal zone lymphoma), was more abundant in PPBL patients (15% vs. 6%, p=0.116), although both correlations were not statistically significant. Gene expression analyses by RNAseq revealed 337 genes to be differentially expressed between the entities. 207 genes were upregulated in PPBL, including PTPRK, CXCR1, BCL11B, CEPBA, CCR4 and MYC, whereas 130 genes were found to be upregulated in CLL cases, comprising ID3, BCL2, FGF2 and FLT1. Conclusions: 1) WGS analysis identifies high frequencies of cancer/lymphoma-associated gene mutations in PPBL, including mutated ATM, NOTCH2 and TRAF1. 2) Five genes showed a higher mutation frequency compared to CLL including TRAF1,DNMT3A, CKAP5 and MAP2. 3) Lymphoma associated genes (BCL11B and MYC) were overexpressed in PPBL vs CLL. 4) Taken together our results question PPBL as a benign entity and identify molecular markers that might contribute to development of subsequent malignancies. Disclosures Stengel: MLL Munich Leukemia Laboratory: Employment. Höllein:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Walter:MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Description: The WHO recognizes tMN as a distinct disease entity. The majority of tMN arise after treatment of solid tumors with cytotoxic chemotherapy or radiation therapy. In many tMN patients, clonal hematopoiesis was demonstrated to be already present at diagnosis of the solid tumor and later gave rise to the tMN. Cases of tMN following successful treatment of AML are rare and poorly characterized. We therefore set out to study the genetic profile of these patients and evaluated if AML and subsequent tMN are two genetically distinct diseases or share a common ancestral clone. We selected cases of AML sent to our laboratory for diagnostic work-up from 2005 to 2018 who subsequently developed a tMN. To exclude events of AML relapse from the cohort, known AML driver mutations such as in NPM1 or AML defining rearrangements present at diagnosis (Dx) of AML had to be absent at Dx of tMN. Based on sample availability, whole exome sequencing (WES) was carried out for 25 pts at the time points of Dx of AML and Dx of tMN as well as on matched remission samples that were available for 19 pts. Enrichment based library preparation was performed using the xGen Exome Research Panel and sequenced (2x151bp) on a NovaSeq instrument. Data was processed with BaseSpace using the BWA Enrichment app with BWA for Alignment (against hg19) and GATK for variant calling with default parameters. Data was subsequently loaded into BaseSpace Variant Interpreter to filter and prioritize variants of interest. Only passed protein changing variants were considered with an ExAC population frequency of less than 1% for further analysis. The cohort included 13 females and 12 males, aged 28 to 77 years (median: 60 yrs). Median time to Dx of tMN following Dx of AML was 42 months (range: 8-96 months). Thirteen pts had developed tMDS and 12 pts tAML. While WES identified numerous recurrently mutated genes, we focused our further analyses on 28 genes associated with myeloid neoplasms and detected a total of 97 mutations (1-8 mutations per patient, median: 4). Following genes were mutated in 〉15% of pts: NPM1 (14/25 of pts), DNMT3A (9/25), TET2 (9/25), SRSF2 (7/25), CEBPA (6/25), RUNX1 (6/25), ASXL1, FLT3, IDH2 and TP53 (4/25 of pts, each). We did not identify a mutation pattern distinguishing between pts who developed a tAML or tMDS, however, pts with tAML had a significantly higher total number of mutations than those with tMDS (median of 5 vs 3 mutations, p= 0.022). When comparing matched initial AML and subsequent tMN samples for each patient, a total of 43 mutations were detected at Dx of AML only, while 25 mutations were exclusive to the tMN sample. Interestingly, 18/25 (72%) of pts harbored mutations present at both time points (Figure 1). Moreover, in 8/22 of pts with available material, mutations in DNMT3A (n=6), IDH1, SRSF2 and TET2 (n=1, each) persisted during remission. Of the 7 pts without shared mutations between time point of AML and tMN, 2 were initially positive for the PML-RARA rearrangement, and 1 patient showed a CBFB-MYH11 rearrangement at Dx of AML. Next, we compared gene mutation data with cytogenetic information available for 21/25 pts at both time points. Nine patients with a normal karyotype (NK) at Dx of AML developed chromosome aberrations at Dx of tMN. In this group, 8/9 pts harbored mutations in epigenetic modifiers (DNMT3A, TET2 and SRSF2) present both at AML and tMN. Similarly, 2/3 patients with NK AML and NK tMN shared mutations in IDH2, SRSF2 and ASXL1 at both time points. Eight pts showed either independent cytogenetic aberrations between AML and tMN or harbored chromosome aberrations at Dx of AML that were absent at Dx of tMN. Even in this group of cytogenetically independent clones, 3/8 pts harbored the same mutations at both time points. Our study provides novel insights into the pathogenesis of tMN. In the majority of analyzed cases, we detected mutations present in the AML and tMN clones of the same patient. Considering the absence of known AML driver gene mutations such as in NPM1 in the tMN sample, we speculate the presence of a common ancestral clone with mutations mostly affecting epigenetic modifiers which have previously been linked to clonal hematopoiesis. Unlike the full-blown leukemia, this clone is refractory to chemotherapy and later gives rise to the tMN. The absence of shared mutations between AML and tMN in patients with PML-RARA or CBFB-MYH11 rearrangement is in line with thought that these entities are true de novo AML that do not evolve from a clonal hematopoiesis. Disclosures Hartmann: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Höllein:MLL Munich Leukemia Laboratory: Employment. Vetro:MLL Munich Leukemia Laboratory: Employment. Stengel: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.

Description: Background: Resistance to apoptosis is one of the hallmarks in hematological neoplasms, most typically via dysregulation of the intrinsic mitochondrial pathway. The most important antiapoptotic/pro-survival proteins in this pathway are BCL2, BCL2L1 and MCL1. Molecules targeting each of these proteins are in various stages of preclinical and clinical development. The BCL2 inhibitor venetoclax is the first FDA approved drug in this setting and was shown to be highly effective in CLL and some B-cell lymphoma subtypes. Kumar et al. (Blood 2017) reported that in multiple myeloma response to venetoclax correlated with higher BCL2, BCL2:BCL2L1 and BCL2:MCL1 mRNA expression ratios. Aim: To determine the gene expression levels of BCL2, BCL2L1 and MCL1 across various hematological neoplasms in order to identify best candidates for pro-survival protein inhibitors. Patients and Methods: 2239 patients with hematological neoplasms (Table1) were evaluated by whole transcriptome sequencing (WTS) and 64 cases without hematological neoplasms were analysed as controls. Total RNA was sequenced on the NovaSeq 6000 with a median of 50 million reads per sample. The obtained estimated gene counts were normalized and the resulting log2 counts per million (L2CPM) were used as a proxy of gene expression. Results: The median BCL2 expression (exp) was significantly higher in lymphoid malignancies (lyM) compared to controls and myeloid malignancies (myM) (L2CPM: 5.8 vs 3.4 vs 3.4, p

Description: Background: The diagnosis and risk stratification of multiple myeloma (MM) is based on clinical and cytogenetic tests. Magnetic CD138 enrichment followed by interphase FISH is the gold standard to identify prognostic translocations and copy number alterations. Gene expression (GEP) studies show that MM might consist of various subgroups with distinct clinical outcomes (Zhan et al., Blood 2006). Whole genome sequencing (WGS) and targeted sequencing studies have further shed light onto recurrent mutations in MM and clinical implications are being derived (Lohr et al., Cancer Cell 2014). We set up a single workflow to analyze MM by WGS and RNA-Seq to evaluate whether this diagnostic workup is superior to conventional diagnostic testing. Methods: The cohort comprised 211 patients (pts) diagnosed with MM at our institution from 2011 to 2017. For all pts FISH and WGS was performed on CD138 enriched cells. For WGS 150bp paired-end sequences where generated on Illumina HiseqX and NovaSeq 6000 (Illumina, San Diego, CA). A mixture genomic DNA from multiple anonymous donors was used as normal controls. To remove potential germline variants, each variant was queried against the gnomAD database, variants with global population frequencies 〉1% where excluded. 47 genes recurrently mutated in MM were selected for evaluation (Kortüm et. al., Blood 2016). Copy number alterations (CNAs) were called using GATK4 and structural variations (SVs) were called using MANTA accounting for missing matched-normal samples. For transcriptome analysis total RNA was sequenced and the resulting estimated gene counts were pre-processed and normalized, applying trimmed mean of M-values normalization method. Results: WGS allowed us to detect 98/102 (96%) translocations that had previously been identified by FISH. Specifically we confirmed 24/24 of t(4;14), 6/7 of t(6;14), 11/12 of t(8;14), 51/53 of t(11;14) and 6/6 of t(14;16) cases by WGS. Moreover, by conventional FISH 12 pts had an IGH (n=4) or MYC (n=8) translocation with an unknown partner chromosome. We identified all 12 translocations by WGS. By WGS we also identified 679/740 (92 %) copy number alterations (CNA) detected by FISH. In detail these were 100/103 del(13q), 17/21 del(17p), 10/10 del(1p) and 79/87 +1q. Concordance rates for trisomies 3, 5, 9, 11, 15 and 19 were 80/91, 75/87, 92/97, 87/97, 53/55 and 86/92, respectively. Zhan et al. defined 7 MM subgroups (CD-1, CD-2, HY, MF, MS, LB, PR) based on GEP. 4 groups (CD-1/CD-2, MF, MS) were genetically defined by recurrent translocations and 1 by hyperploidy (HY). They used 700 probes to separate the groups of which 400 transcripts were recovered in our RNA-Seq analysis (GEPSeq). Supervised clustering grouped all 211 pts at the following frequencies: CD-1 (5%), CD-2 (25%), HY (30%), MF (5%), MS (11%), LB (14%), (PR 10%). 56/62 (90%) of pts that were allocated to CD-1/CD-2 had the characteristic translocation (t(11;14) or t(6;14)), while 23/24 (92%) of pts and 5/10 (50%) pts in GEPSeq group MS and MF had the respective t(4;14) or t(14;16). GEP allocates pts with hyperdiploidy to group HY and 51/63 (81%) pts in HY had a hyperdiploid karyotype by FISH. We specifically queried the WGS data for patients with discrepant FISH and GEPSeq results: WGS identified hyperdiploidy in 10/12 patients that were allocated to HY and could not be assigned by FISH due to insufficient material for complete testing, resulting in a 97% final concordance of GEPSeq and karyotype. One patient in group MS without FISH data for t(4;14) could be confirmed by WGS as harbouring the translocation (concordance 100%). Interestingly in 5/5 patients that were allocated to the MF group by GEPSeq 2 had an IGH-MYC or IGH-MYCN rearrangement respectively and 2 had another IGH rearrangement involving chromosome 8q. By WGS the most frequently mutated (mut) genes were KRAS (26%), NRAS (23 %), TP53 (8%), BRAF (4%) and ATM (2%), which is in line with published data (Lohr 2014). NRASmut was significantly associated with GEPSeq groups CD-2 and HY (p=0.001) and ATMmut with MF, MS and PR (p=0.047). Conclusion: RNA-Seq and WGS prove highly valuable in differentiating genetically distinct MM subgroups. The simultaneous analysis of gene mutations might have future implications for study design and selecting treatment options. A single workflow based on WGS and RNA-Seq provides a comprehensive genetic analysis in MM, is feasible and might substitute conventional diagnostic testing in the near future. Disclosures Höllein: MLL Munich Leukemia Laboratory: Employment. Twardziok:MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Hernández:MLL Munich Leukemia Laboratory: Employment. Meggendorfer: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.

Description: Key Points The Myc oncoprotein targets central regulators of the SUMOylation machinery, resulting in a hyper-SUMOylation state in Myc-induced lymphoma. Targeting SUMOylation by genetic or pharmacologic means represents a novel therapeutic option for lymphomas with MYC involvement.

Description: Background: Multi-parameter flow cytometry (MFC) data is a diagnostic keystone for hematologic malignancies. Data analysis and interpretation requires resources and expertise, full automation is desired. Combination of automated gating strategies and machine learning on MFC data proved effective in detecting AML cell populations vs normal (Aghaeepour et al., 2013). Aim of our approach was to create tools capable of comprehensively analyzing and classifying MFC data. We developed a proof of principle framework to process MFC data generated from samples with suspected B-cell lymphomas. Methods: MFC data was obtained from routine diagnostics of 16,384 patients as follows: normal controls (n=8,493), chronic lymphocytic leukemia (CLL, n=3,412), CLL with increased prolymphocytes (CLL/PL, n=603), follicular lymphoma (FL, n=219), hairy cell leukemia (HCL, n=193), lymphoplasmacytic lymphoma (LPL, n=629), mantle cell lymphoma (MCL, n=269), marginal zone lymphoma (MZL, n=979), monoclonal B-cell lymphocytosis (MBL, n=1,480). Two 9-color combinations of monoclonal antibodies were applied to analyze surface expression of the following antigens besides scatter signals: FMC7, CD10, IgM, CD79b, CD20, CD23, CD19, CD5, CD45, Kappa, Lambda, CD38, CD25, CD11c, CD103, CD22. For dimensionality reduction we used an unsupervised clustering approach (Kohonen et al., 1990). We first generated a consensus self-organizing map (CS) of 100 nodes including all MFC events of multiple preselected cases (5 samples/cohort). For a test case the fluorescent profile of a cell was assigned to its nearest node in the CS (upsampling). We used the resulting distribution for classification by a sequential neural network (NN). Computationally intensive processing steps were run using Amazon Web Services. Mean accuracies were calculated counting individual cases across the entire dataset. All accuracies are described for 10 CS generations and 10-fold cross validation in the classification process. Approaches of data processing were sequentially developed aiming at maximization of classification accuracy. Results: First, we assessed the performance in a binary classification, i.e. the merged group of CLL and MBL (n=4,877) vs. normal (n=8,493) achieving 97% accuracy. Secondly, we applied our approach to the classification of all 9 classes with an accuracy of 74%. Visual inspection of tSNE (van der Maaten et al., 2008) transformed upsampling information revealed good separability between CLL and normal cases but substantial overlap between other lymphoma subtypes (Figure). The following approaches were applied to improve classification: 1) Automated pregating of lymphocytes (CD45/SSC) by DBSCAN analysis (Ester et al., 1996) improved classification. Applying this to both CS generation and upsampling the accuracy of the 9-class problem increased to 78% (p