Abstract: Half the patients with acute myeloid leukemia (AML) who achieve complete remission (CR), ultimately relapse. Residual treatment-surviving leukemia is considered responsible for the outgrowth of AML. In many retrospective studies, detection of minimal residual disease (MRD) has been shown to enable identification of these poor-outcome patients by showing its independent prognostic impact. Most studies focus on molecular markers or analyze data in retrospect. This study establishes the value of immunophenotypically assessed MRD in the context of a multicenter clinical trial in adult AML with sample collection and analysis performed in a few specialized centers. Patients and Methods In adults (younger than age 60 years) with AML enrolled onto the Dutch-Belgian Hemato-Oncology Cooperative Group/Swiss Group for Clinical Cancer Research Acute Myeloid Leukemia 42A study, MRD was evaluated in bone marrow samples in CR (164 after induction cycle 1, 183 after cycle 2, 124 after consolidation therapy). Results After all courses of therapy, low MRD values distinguished patients with relatively favorable outcome from those with high relapse rate and adverse relapse-free and overall survival. In the whole patient group and in the subgroup with intermediate-risk cytogenetics, MRD was an independent prognostic factor. Multivariate analysis after cycle 2, when decisions about consolidation treatment have to be made, confirmed that high MRD values ( 〉 0.1% of WBC) were associated with a higher risk of relapse after adjustment for consolidation treatment time-dependent covariate risk score and early or later CR. Conclusion In future treatment studies, risk stratification should be based not only on risk estimation assessed at diagnosis but also on MRD as a therapy-dependent prognostic factor.

Abstract: To evaluate the prognostic value of ecotropic viral integration 1 gene (EVI1) overexpression in acute myeloid leukemia (AML) with MLL gene rearrangements. Patients and Methods We identified 286 patients with AML with t(11q23) enrolled onto German-Austrian Acute Myeloid Leukemia Study Group and Dutch-Belgian-Swiss Hemato-Oncology Cooperative Group prospective treatment trials. Material was available from 177 AML patients for EVI1 expression analysis. Results We divided 286 MLL-rearranged AMLs into three subgroups: t(9;11)(p22;q23) (44.8%), t(6;11)(q27;q23) (14.7%), and t(v;11q23) (40.5%). EVI1 overexpression (EVI1 + ) was found in 45.8% of all patients with t(11q23), with t(6;11) showing the highest frequency (83.9%), followed by t(9;11) at 40.0%, and t(v;11q23) at 34.8%. Concurrent gene mutations were rare or absent in all three subgroups. Within all t(11q23) AMLs, EVI1 + was the sole prognostic factor, predicting for inferior overall survival (OS; hazard ratio [HR], 2.06; P = .003), relapse-free survival (HR, 2.28; P = .002), and event-free survival (HR, 1.79; P = .009). EVI1 + AMLs with t(11q23) in first complete remission (CR) had a significantly better outcome after allogeneic transplantation compared with other consolidation therapies (5-year OS, 54.7% v 0%; Mantel-Byar, P = .0006). EVI1 − t(9;11) AMLs had lower WBC counts, more commonly FAB M5 morphology, and frequently had additional trisomy 8 (39.6%; P 〈 .001). Among t(9;11) AMLs, EVI1 + again was the sole independent adverse prognostic factor for survival. Conclusion Deregulated EVI1 expression defines poor prognostic subsets among AML with t(11q23) and AML with t(9;11)(p22;q23). Patients with EVI1 + MLL-rearranged AML seem to benefit from allogeneic transplantation in first CR.

Abstract: Background Acute myeloid leukemia (AML) is caused by cooperating oncogenic driver mutations that induce uncontrolled proliferation in combination with maturation arrest in myeloid precursor cells. The majority of oncogenic drivers are somatically acquired. Recurrent genetic lesions are used for molecular classification and prognosis of AML. In addition, expression levels of selected genes contribute to AML subclassification (CD34, KIT) and prognostication (e.g. EVI1). Actionable aberrations and pathways are molecular targets for personalized pharmacotherapy. Structures, incidences, and mutual associations of recurrent genetic aberrations have been elucidated by large whole genome/exome sequencing efforts. However, 10-20% of AML appear to be caused by hitherto unrecognized or individual driver lesions. Current AML diagnostics are poorly standardized and comprise a combination of morphology, flow cytometry, cytogenetics, targeted gene amplification with electrophoresis, sequence analysis, and qRT-PCR. We here test the hypothesis that whole transcriptome sequencing (RNAseq) without parallel germ-line sequencing could potentially be used as a single and cost-efficient platform for AML diagnosis and prognostication. Methods Poly(A)+ RNA was isolated from 100 cryopreserved AML samples with a blast count of 10-99% (median: 75%). These samples were obtained from 97 patients (2 diagnosis-relapse pairs, 1 de novo AML with subsequent tAML). RNA was sequenced at a depth of 59x106 paired-end reads per sample with median read length of 126 bp through a ISO17025-accredited Illumina HiSeq 2500 pipeline. After alignment against the GRCh38 reference genome, single nucleotide variants (SNV) and small indels were called by VarScan with a threshold of 20% aberrant reads. Variants were filtered for known polymorphisms occurring at 〉 5% in defined ethnic subpopulations of the 1000 Genomes and Genome of the Netherlands Projects. Internal tandem duplications (ITD) were identified by frequency and distribution of soft clipped reads. Detection of fusion genes was performed on raw reads using STAR-Fusion and FusionCatcher. Geneexpression levels were measured relative to HBMS as a housekeeping gene by counting the number of reads aligned to gene exons normalized to the sum of exon lengths. HAMLET was developed and implemented as an integrated RNAseq pipeline to measure relevant recurrent AML-associated aberrations, i.e. SNV and small indels in recurrently mutated genes, the FLT3-ITD, AML-associated gene fusions, and EVI1 overexpression. Results A total of 221 SNV/indels (15 homozygous) were detected at expected or higher frequencies in NPM1 (33%), FLT3 (ITD+TKD: 37%), DNMT3A (30%), TET2 (27%), IDH1 (14%), IDH2 (14%), RUNX1 (23%), CEBPA (4%), KIT (7%), WT1 (8%), ASXL1 (11%), and TP53 (3%). Sensitivities to detect SNV/small indels and FLT3-ITD were 96% and 94%, respectively, with no apparent relation to the blast count. Fusion genes relevant to the WHO AML classification were found with a sensitivity of 100% when compared to metaphase cytogenetics and FISH. Specifically, 10 cases of CBFB-MYH11, 3 RUNX1-RUNX1T1, 2 PML-RARA, 1 FUS-ERG, 1 KMT2A-MLLT3, and 1 DEK-NUP214 fusion events were correctly detected. In addition, 3 variant KMT2A translocations with MLLT1, MLLT4, and MLLT6 as fusion partners were correctly called. The specificity of HAMLET for recurrent SNV, indels, and fusion genes was 100%. Finally, RNAseq quantitatively detected variability in gene expression as exemplified for EVI1 overexpression and confirmed by qRT-PCR (r=0.85). Conclusions In conclusion, RNAseq analysis of AML samples without concomitant germ-line sequencing detects molecular information with relevance for classification, prognosis, and targeted therapy with 96% overall sensitivity and 100% specificity. HAMLET is currently optimized to further improve the sensitivity to detect SNV/small indel and ITD events. With a cost price of €980,- per case and full accreditation for diagnostic application of the RNAseq raw data, RNAseq facilitates comprehensive and cost-effective AML diagnostics in a single assay. Additional predicted benefits of this RNAseq approach to be explored include identification of non-recurrent individual drivers, neoantigens, and minor histocompatibility antigens, a potential to replace conventional HLA typing, and as a potential alternative for flow cytometry. Disclosures Janssen: GenomeScan BV: Employment.

Abstract: BCR-ABL1 and BCR-ABL1-like acute lymphoblastic leukemia (ALL) are two major pre-B cell acute leukemia subtypes characterized by genetic alterations affecting lymphoid-specific transcription factors. Studies examining the chain of genetic events necessary to develop leukemia established that the BCR-ABL1 fusion gene and kinase-activating BCR-ABL1-like lesions are initiating events, however, insufficient for leukemia development. Secondary genetic events targeting B cell development genes are therefore an essential requirement for overt ALL. A recent study (Papaemmanuil et al, Nat. Genet., 2014) revealed that illegitimate RAG-mediated recombination is the predominant mutational mechanism establishing these secondary genetic events in ETV6-RUNX1 ALL. Of note, ETV6-RUNX1ALL is mainly restricted to pediatric cases and it remains unanswered whether this mutational process also plays a prominent role in adult ALL pathogenesis. We carried out a detailed genomic characterization to determine whether aberrant RAG activity is also a prominent mutational driver in certain adult B cell ALL (B-ALL) subtypes. Diagnostic material of 53 unselected B-ALL cases and matched remission specimens were characterized using DNA mapping arrays to discern copy number alterations (CNAs). We observed multiple BCR-ABL1/BCR-ABL1-like patients with abundant genetic lesions and selected 5 cases for targeted sequencing of CNA boundaries to determine whether these lesions were driven by RAG-mediated recombination. Whole genome sequencing (WGS) for a single BCR-ABL1-like patient was used to asses this mutational mechanism genome-wide. In total 64 structural variants (SVs) could be analyzed at base-pair level. De novo motif detection on breakpoint sequences revealed the prominence of the heptamer CACAGTG (E-value=5.68x10-91), a constituent of the recombination signal sequence (RSS), present in 121 out of 128 breakpoints (94.5%). RSS detection revealed that 58 out of 64 SVs (90.6%) had a cryptic RSS (cRSS) on one or both sides of the lesion. Incorporation of non-templated sequences was observed for 54 out of the 64 (84.4%) SVs. Superimposition of breakpoints on chromatin marks revealed a strong enrichment for active promoters and enhancers (p 〈 2.2x10-16). WGS data revealed cRSS motifs and incorporation of non-templated sequences for 23 out of 26 SVs (88.5%). Integrative analysis of all 6 cases confirmed 125 unique SV breakpoints strongly enriched for the active chromatin marks H3K4me3 and H3K27ac. STAT5 binding, a postulated regulator of V(D)J recombination, is similarly enriched at the breakpoints. Promiscuous binding of RAG1 and RAG2 was previously noted in human thymocytes and murine pre-B cells (Teng et al, Cell, 2015). Strikingly, the breakpoints are frequently bound by RAG2 in human thymocytes. In total 66 out of 125 breakpoints could be translated to the murine genome and revealed a strong enrichment of RAG1 and RAG2 binding at homologous positions in murine pre-B cells. Exhaustive mutation detection revealed complex somatic mutations within cRSS motifs, which are rare V(D)J recombination products introduced by erroneous cleavage and error-prone repair (open-and-shut joints). Strikingly, 4 out of 6 BCR-ABL1/BCR-ABL1-like cases had mutations in the BTLA promoter-situated cRSS, frequently in combination with a RAG-mediated deletion of the other allele (Figure 1). Genomic screening in 142 B-ALL patients confirmed 8 additional cases with BTLA promoter mutations, predominantly (6 out of 8) belonging to the BCR-ABL1/BCR-ABL1-like subgroups. We provide strong evidence that aberrant RAG activity plays a pivotal role in the development of BCR-ABL1/BCR-ABL1-like adult ALL. We demonstrate that breakpoints are strongly enriched for RAG binding implying a predisposition for illegitimate V(D)J recombination. Importantly, we report on a novel mutational mechanism introducing mutations in cRSS motifs through open-and-shut joints, frequently resulting in the biallelic inactivation of BTLA. Proliferation and V(D)J recombination during pre-B cell development is orchestrated by the interplay of IL7R and pre-BCR signalling. Strikingly, most kinase-activating lesions constitutively activate these signalling cascades and could enact, in concert with BTLA inactivation, constant proliferation, pro-survival and V(D)J recombination-initiating signals with disastrous consequences. Disclosures No relevant conflicts of interest to declare.

Abstract: The monitoring of response in chronic myeloid leukemia ( CML ) is of great importance to identify patients failing their treatment in order to adjust TKI choice and thereby prevent progression to advanced stage disease. Cytogenetic monitoring has a lower sensitivity, is expensive, and requires invasive bone marrow sampling. Nevertheless, chronic myeloid leukemia guidelines continue to recommend performing routine cytogenetic response assessments, even when adequate molecular diagnostics are available. Methods In a population‐based registry of newly diagnosed CML patients in the Netherlands, all simultaneous cytogenetic and molecular assessments performed at 3, 6, and 12 months were identified and response of these matched assessments was classified according to European Leukemia Net ( ELN ) recommendations. The impact of discrepant cytogenetic and molecular response classifications and course of patients with additional chromosomal abnormalities were evaluated. Results The overall agreement of 200 matched assessments was 78%. In case of discordant responses, response at 24 months was consistently better predicted by the molecular outcome. Cytogenetic response assessments provided relevant additional clinical information only in some cases of molecular “warning.” The development of additional cytogenetic abnormalities was always accompanied with molecular failure. Conclusion We conclude that it is safe to omit routine cytogenetics for response assessment during treatment and to only use molecular monitoring, in order to prevent ambiguous classifications, reduce costs, and reduce the need for invasive bone marrow sampling. Cytogenetic re‐assessment should still be performed when molecular response is suboptimal.

Abstract: Abstract 760 Currently, the most important prognostic factors for acute myeloid leukemia (AML) include molecular aberrancies and karyotype of the leukemic blasts. Although these factors have showed to be of utmost importance in upfront risk stratification in current treatment schedules, the treatment outcome of patients within as such defined risk groups is still quite heterogeneous. Therefore, there is an unmet need for therapy-dependent prognostic factors which can be implemented into risk-adapted treatment strategies. Minimal residual disease (MRD) frequency is such a parameter. MRD cells are considered responsible for the outgrowth of AML after treatment, leading to a relapse in 30–40% of the patients in complete remission (CR). In this study, we are the first to report prospective multicenter data on the prognostic impact of MRD frequency in adult AML. In our retrospective study (N.Feller et al. Leukemia 2004), we explored which cut-off points for percentage of MRD cells would define MRD positive (levels above cut-off, MRD+) patients with a relatively poor prognosis, from MRD negative (levels below cut-off, MRD-) patients who showed a longer overall and relapse-free survival (OS and RFS). In search for the most optimal cut-off level which can be used for clinical purpose in risk stratification-directed therapy, we used these cut-offs to evaluate the prognostic value in the current prospective setting. Diagnosis and follow-up samples were collected of 462 patients treated uniformly according to the HOVON/SAKK42a protocol (www.hovon.nl) and MRD frequency was assessed blindly without knowledge of clinical course. MRD detection was accomplished by immunophenotyping by flow cytometry (FCM) through aberrant expression of markers on AML blasts. Together with the expression of normal immature cell markers and/or myeloid lineage markers, this offers a leukemia associated phenotype (LAP). Each LAP was individually designed for each patient in diagnosis bone marrow (BM) or peripheral blood. Subsequently, BM samples obtained during follow-up were analysed for the presence of LAP-positive cells. MRD frequency was expressed as a percentage of leukocytes. The median MRD frequencies of patients in clinical CR after first induction cycle (n=164), second induction cycle (n=182) and consolidation (n=121) were 0.040%, 0.022% and 0.020%, respectively. The cut-off levels for MRD frequency as defined retrospectively were all significant in the identification of patients with adverse (MRD+) and favourable (MRD-) OS and RFS, respectively. After the first cycle, the most significant cut-off was 0.8%, leading to 17 MRD+ patients who showed a median RFS of only 8.6 months, while 147 MRD- patients had a median RFS of 〉 47 months (p=0.003,A). The relative risk of relapse (RR) was 2.9 (95% c.i. 1.4–6.0, p=0.004). After the second induction cycle, a cut-off level of 0.06% was most significant. Above this cut-off, 49 patients showed a median RFS of 7 months, while 133 MRD- patients showed a RFS of more than 47 months (p 〈 0.00001, fig B). The RR was 3.2 (95% c.i. 2.0–5.0, p 〈 0.00001). After consolidation therapy, 11 MRD+ patients with extremely poor prognosis were identified (median RFS 7.3 months vs. 〉 47 months for 110 MRD- patients, p 〈 0.00001, fig C), with a RR of 10.6 (95% c.i. 4.9–22.8, p 〈 0.00001). Multivariate analysis was performed with conventional prognostic factors for AML: cytogenetic risk groups and time to achieve CR. After every cycle of therapy, MRD frequency was an independent prognostic factor for RFS after all cycles (1st cycle: p=0.010, 2nd cycle and consolidation p 〈 0.00001) and for OS after 1st (p=0.023) and 2nd induction cycle (p=0.010). In this prospective multicenter study, already after first induction cycle, MRD detection by FCM was an independent significant factor in the identification of poor prognostic patients. In future treatment studies, risk stratification, e.g. for allogeneic stem cell transplantation, should not only be based on risk estimation determined at diagnosis, but also on MRD frequency as a therapy-dependent prognostic factor. This work was supported by Netherlands Cancer Foundation KWF. Disclosures: No relevant conflicts of interest to declare.

Abstract: Introduction: Somatic TP53 mutations and deletions of 17p, to which TP53 is mapped, (TP53mut) occur in 8-10% of de novo Acute myeloid leukemia (AML) and in up to 37-46% of patients (pts) with adverse-risk cytogenetics and treatment-related myeloid neoplasms and confer a poor prognosis. In addition to its well-characterized function as a tumor suppressor, emerging evidence implicates mutant TP53 in activating genes involved in immune response and inflammation such as chemokines, cytokines and extracellular matrix modulators. An analysis of The Cancer Genome Atlas (TCGA) transcriptomic data showed that TP53 mutations, in 30 diverse cancer types, correlated with increased leukocyte infiltration into tumors with higher proportions of PD-L1-expressing CD8+ T cells and increased expression of T-cell effector genes and interferon (IFN)-γ-related genes. We recently characterized tumor microenvironmental (TME) immune gene sets that capture elements of both type I- and IFN-γ-driven biology and stratify AML into immune-infiltrated and immune-depleted subtypes. Our immune classifier predicted survival in patients receiving cytarabine-based induction and immunotherapy with flotetuzumab (FLZ), an investigational CD123×CD3 bispecific DART® molecule. We hypothesized that TP53-mutated AML represents immune-infiltrated AML that would be particularly responsive to FLZ. Methods: Fifteen TP53mut AML pts have been treated with FLZ on clinical trial CP-MGD006-01 (NCT#02152956). Disease status was assessed by modified International Working Group (IWG) criteria. Specifically, overall response rate (ORR), collectively complete response, defined as & lt;5% bone marrow (BM) blasts (CR, CRh, CRi or morphologic leukemia-free state [MLFS]), and partial response (PR), defined as & gt;50% decrease or decrease to 5-25% BM blasts. Microenvironmental RNAs were profiled using the PanCancer IO 360™ gene expression panel on the nCounter® platform. Baseline formalin-fixed paraffin embedded BM samples were evaluated for PD-L1, FoxP3, CD8 and CD3 expression by immunohistochemistry (IHC). Slides were stained using a Leica BondRx autostainer. Fluorescence was imaged using a Polaris Vectra 3 and analyzed using inForm software. A density-based clustering algorithm developed and run in QuPath was used to quantify T-cell 'hotspots". Results: Baseline (BL) BM samples for immune gene expression profiling were available in 13 pts with TP53mut (median age 61yrs [range 27-81]; 46.7% [7] pts female); among these, 77% (10/13) had high or intermediate immune infiltration in the TME compared with pts with 33% (10/30) TP53-WT AML (pt characteristics in the TP53-WT AML cohort were balanced) (Fig. 1A). IHC analysis confirmed high CD8+ T-cell, regulatory T cell (Treg) and PD-L1+ cell infiltration in TP53mut BL BM samples (Fig. 1B). ORR was 60% (9/15), with 47% (7/15) achieving complete response. In the TP53mut subgroup, the reduction of BM blasts relative to baseline averaged 51.2% (Fig. 1C). Time on treatment and time to death and/or censoring are summarized in Fig. 1D, including three pts who proceeded to receive allogeneic hematopoietic stem cell transplantation (HSCT). In pts who achieved a complete remission (CR, CRi), median OS was 10.3 months. Furthermore, the tumor inflammation signature (TIS), inflammatory chemokine, Treg and IFN-γ gene expression scores were significantly higher at baseline in pts with complete remission compared with non-responders (Fig. 1E), highlighting the association between response to T-cell engagers and a T cell-infiltrated TME. Conclusion: TP53 mutated AML is associated with immune infiltration in the TME and FLZ immunotherapy demonstrated activity in pts with TP53 alterations. This suggests that FLZ immunotherapy may alleviate the negative prognostic immunological impact of TP53 mutation. Figure 1 Disclosures Lai: Abbvie: Consultancy; Agios: Consultancy; Macrogenics: Consultancy; Astellas: Speakers Bureau; Jazz: Speakers Bureau. Church:NanoString Technologies, Inc.: Current Employment. Advani:Novartis: Consultancy, Other: advisory board; Abbvie: Research Funding; Pfizer: Honoraria, Research Funding; Kite: Other: Advisory board/ honoraria; Amgen: Consultancy, Other: steering committee/ honoraria, Research Funding; Seattle Genetics: Other: Advisory board/ honoraria, Research Funding; Immunogen: Research Funding; Glycomimetics: Consultancy, Other: Steering committee/ honoraria, Research Funding; Macrogenics: Research Funding; OBI: Research Funding; Takeda: Research Funding. Wieduwilt:Macrogeneics: Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Shire: Research Funding; Merck: Research Funding; Leadiant: Research Funding; Amgen: Research Funding. Arellano:Hanmi: Research Funding; Cephalon Oncology: Research Funding; Gilead Sciences, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees. Uy:Pfizer: Consultancy; Agios: Consultancy; Genentech: Consultancy; Jazz Pharmaceuticals: Consultancy; Daiichi Sankyo: Consultancy; Astellas Pharma: Honoraria. Ravandi:Macrogenics: Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Orsenix: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Xencor: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria. Foster:Bellicum Pharmaceuticals: Research Funding; Daiichi Sankyo: Consultancy; Macrogenics: Consultancy, Research Funding. Stiff:Atara: Research Funding; Delta-Fly: Research Funding; Kite, a Gilead Company: Research Funding; Amgen: Research Funding; Unum: Research Funding; Gamida Cell: Research Funding; Macrogenics: Research Funding. Emadi:NewLink Genetics: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; KinaRx: Other: co-founder and scientific advisor; Jazz Pharmaceuticals: Research Funding. Walter:Aptevo Therapeutics: Research Funding. Tran:MacroGenics: Current Employment. Kaminker:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Muth:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Guo:Macrogenics: Current Employment. Gojo:Genentech: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Amphivena: Research Funding; Amgen: Research Funding; Merck: Research Funding. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees. Davidson-Moncada:Macrogenics: Current Employment. Rutella:MacroGenics, Inc.: Research Funding; NanoString Technologies, Inc.: Research Funding; Kura Oncology: Research Funding.

Abstract: It remains challenging in chronic lymphocytic leukemia (CLL) to distinguish between patients with favorable and unfavorable time-to-first treatment (TTFT). Additionally, the downstream protein correlates of well-known molecular features of CLL are not always clear. To address this, we selected 40 CLL patients with TTFT ≤24 months and compared their B cell intracellular protein expression with 40 age- and sex-matched CLL patients with TTFT 〉 24 months using mass spectrometry. In total, 3268 proteins were quantified in the cohort. Immunoglobulin heavy-chain variable (IGHV) mutational status and trisomy 12 were most impactful on the CLL proteome. Comparing cases to controls, 5 proteins were significantly upregulated, whereas 3 proteins were significantly downregulated. Of these, only THEMIS2, a signaling protein acting downstream of the B cell receptor, was significantly associated with TTFT, independently of IGHV and TP53 mutational status (hazard ratio, 2.49 [95% confidence interval, 1.62-3.84]; P 〈 0.001). This association was validated on the mRNA and protein level by quantitative polymerase chain reaction and ELISA, respectively. Analysis of 2 independently generated RNA sequencing and mass spectrometry datasets confirmed the association between THEMIS2 expression and clinical outcome. In conclusion, we present a comprehensive characterization of the proteome of untreated CLL and identify THEMIS2 expression as a putative biomarker of TTFT.