High-throughput Proteomics Identifies THEMIS2 as Independent Biomarker of Treatment-free Survival in Untreated CLL

<jats:p>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 &gt;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 <jats:italic toggle="yes">TP53</jats:italic> mutational status (hazard ratio, 2.49 [95% confidence interval, 1.62-3.84]; <jats:italic toggle="yes">P</jats:italic> &lt; 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.</jats:p>

section_content: The interval from diagnosis to the development of symptomatic disease necessitating treatment can vary considerably between patients with chronic lymphocytic leukemia (CLL). Whereas some patients may have stable disease for years, others experience rapid progression and require treatment within months following diagnosis. Heterogeneity in disease biology underpins this clinical variability. Indeed, the molecular landscape of CLL is characterized by numerous recurrently encountered aberrancies, rather than having a common single driver (1, 2). 

An important source of variability in CLL is the immunoglobulin heavy chain (IGH) variable (IGHV) mutational status. According to the burden of somatic hypermutation (SHM) present in the IGHV gene, CLL is classi ed as unmutated or mutated CLL (U-CLL or M-CLL), likely re ecting pre-and post-germinal center ancestry. M-CLL is associated with a considerably longer time-to-rst-treatment (TTFT), compared to U-CLL (3, 4). Other important recurrent molecular drivers are numerous cytogenetic anomalies, including trisomy 12, deletions of chromosome 13q14, 11q22 or 17p13, genomic complexity and single-gene lesions, most notably affecting the TP53 gene (5) (6) (7). 

Although characterization of some of these features is integrated in routine CLL care, it remains challenging to recognize CLL patients with unfavorable TTFT at an early disease stage, especially in the setting of M-CLL with intact TP53. In addition, the pathophysiological downstream effects of several wellknown molecular features of CLL are not completely clear. To address these issues, we performed largescale characterization of global intracellular protein expression by mass spectrometry in a cohort of previously untreated CLL patients, to identify downstream protein correlates of molecular features of CLL and nd putative biomarkers associated with inferior TTFT.
section_name: Introduction

section_name: Methods

section_content: Patient samples were obtained from the biobank for B-cell malignancies, coordinated by the Academic Medical Center in Amsterdam, the Netherlands. Patients provided written informed consent prior to enrollment and inclusion of samples in the biobank. Ethics approval was provided by the AUMC medical ethical and biobank committees (METC #2013_159), in accordance with the Declaration of Helsinki. Selection criteria for the mass spectrometry and validation cohorts included (1) an established diagnosis of CLL according to the criteria established by the international workshop on CLL (iwCLL) (ref. 8), (2) no indication for treatment at time of diagnosis and (3) Rai stage at diagnosis of 0, I or II. Cases were de ned as CLL patients with a TTFT of ≤ 24 months from sampling, whereas controls were de ned as CLL patients with a TTFT exceeding 24 months from sampling, corresponding to to previous de nitions of 'high-risk CLL' (9). For the mass spectrometry cohort, cases and controls were matched by sex and age at sampling (± 2 years).
section_name: Patient samples

section_content: Cryopreserved peripheral blood mononuclear cells (PBMCs) were thawed and 1*10 6 cells were aliquoted for targeted uorescence in situ hybridization (FISH). For all other downstream purposes, B cells were puri ed from PBMCs using untouched magnetic activated cell sorting (MACS) by negative selection (Human B Cell Isolation Kit II, Miltenyi Biotec, Bergisch Gladbach, Germany). 

Mass spectrometry 1*10 6 sorted cells were washed three times with PBS and snap frozen in an ethanol and dry ice. Cell pellets were lysed in 1ml 50 mM Tris/HCl pH 8. 2, 0. 5% sodium deoxycholate (SDC) using a Bioruptor ultasonicator (Diagenode, Liège, Belgium). Protein concentrations were measured using the BCA assay (ThermoScienti c). Proteins were reduced with 5 mM DTT and cysteine residues were alkylated with 10 mM iodoacetamide. Protein was extracted by acetone precipitation at -20°C overnight. Samples were centrifuged at 8,000 g for 10 min at 4°C. After acetone removal, the pellet was allowed to dry and dissolved in 1ml 50mM Tris/HCl pH 8. 2, 0. 5% SDC. Proteins were digested with LysC (1:200 enzyme:protein ratio) for 4h at 37°C. Next, trypsin was added (1:100 enzyme:protein ratio) and the digestion proceeded overnight at 30°C. Digests were acidi ed with 50µl 10% formic acid and centrifuged at 8,000g for 10 min at 4°C to remove the precipitated SDC. The supernatant was transferred to a new centrifuge tube. The digests were puri ed with C18 solid phase extraction (Sep-Pak, Waters Corporation, Milford, MA, USA), lyophilized and stored at -20°C. 

Proteolytic peptides were labeled with TMT 11-plex labeling reagents (ThermoScienti c) for 10 separate TMT LC-MS runs allowing for peptide quantitation. Peptides were mixed at the 11-plex level and analyzed by nano ow LC-MS/MS. nLC-MS/MS was performed on EASY-nLC 1200 coupled to an Orbitrap Eclipse mass spectrometer (ThermoScienti c) operating in positive mode and equipped with a nanospray source. 

Peptides were separated on a ReproSil C18 reversed phase column (Dr Maisch GmbH; column dimensions 15 cm × 50 µm, packed in-house) using a linear gradient from 0 to 80% B (A = 0. 1% formic acid; B = 80% (v/v) acetonitrile, 0. 1% formic acid) in 70 min and at a constant ow rate of 200nl/min using a splitter. The column eluent was directly sprayed into the ESI source of the mass spectrometer. Field asymmetric waveform ion mobility spectrometry was used to separate peptides in the gas-phase (CV: 40, 50, 60 and 70). Mass spectra were acquired in continuum mode; fragmentation of peptides was performed in data-dependent mode using the multinotch SPS MS3 reporter ion-based quanti cation method.
section_name: Sample preparation

section_content: Mass spectrometry data were analyzed with Proteome Discoverer 2. 5 (ThermoScienti c). The Mascot search algorithm (version 2. 3. 2, MatrixScience) was used for searching against the Uniprot database (taxonomy: Homo sapiens, version 06-2021). The peptide tolerance was typically set to 10ppm and the fragment ion tolerance was set to 0. 6Da. The integration tolerance for reporter ion quantitation was set to 20ppm. A maximum number of 2 missed cleavages by trypsin were allowed and carbamidomethylated cysteine and oxidized methionine were set as xed and variable modi cations, respectively. Typical contaminants were omitted from the output tables. A pooled sample was used as an internal reference standard (IRS, TMT channel 126 in all runs) for inter-run normalization of peptide abundances. Raw reporter ion abundances were extracted from PD2. 5 and normalization of peptide abundances between the separate TMT-plexed LC-MS runs and further analysis was performed in R.
section_name: Raw data processing and quality control

section_content: Patient samples with ≥ 40% missingness or highly deviant values (> 2 SD from the mean) were excluded from downstream analysis (Supplemental Fig. 1 ). In addition, proteins with ≥ 50% missingness across all patients were excluded from downstream analysis. Protein abundance data were background corrected and scaled prior to analysis. For hierarchical clustering, missing values were imputed using the quantile regression imputation of left-censored data method using the imputeLCMD R package (10). To correct for batch effects, batch number was included as a covariate in all regression models and statistical tests. In addition, for data visualization, batch effects were reduced through the ComBat function in the R package sva (11). Data visualization was performed using the ggplot2 and pheatmap R packages (12, 13). 

Targeted FISH FISH was carried out on xated PBMCs to determine structural and numerical abnormalities involving chromosome 11q22 (LSI ATM, limit of detection [LOD] 8% for hemizygosity), chromosome 12 (CEP12, LOD 3% for trisomy), chromosome 13q14/13q34 (LSI D13S319, LOD 7% for hemizygosity) or chromosome 17p13 (LSI p53, LOD 10% for hemizygosity) (all Vysis, Abbott, Abbott Park, IL, USA).
section_name: Protein abundance analysis

section_content: Genomic DNA was isolated from up to 10 6 sorted cells using the AllPrep DNA/RNA/miRNA Universal kit (Qiagen) and quanti ed using NanoDrop spectrophotometry (ThermoFisher). The leukemia-speci c IGH rearrangement was ampli ed using IGHV-leader primers in a multiplex PCR, followed by family-speci c ampli cation and Sanger sequencing using a BigDye Terminator v3. 1 Cycle Sequencing Kit (ThermoFisher). For selected, di cult-to-solve cases, IGH rearrangements were ampli ed using the IGHV EuroClonality leader primer set( 14 ), followed by NGS on a MiSeq using a v3 600 cycle kit (both Illumina, San Diego, CA, USA) and analysis using the ARResT/Interrogate immunopro ler (15). Nucleotide sequences were annotated using IMGT/V-QUEST(16). The IGHV mutational status was determined in accordance with the European Research Initiative on CLL (ERIC) recommendations (17, 18). TP53 mutations were detected by NGS as detailed previously (19, 20), with a variant allele frequency cut-off of 10%. IGLV3-21 R110 status was determined by Sanger sequencing of cDNA, as described previously (21).
section_name: IGHV, IGLV3-21 and TP53 mutational status pro ling

section_content: Total RNA was isolated from up to 10 6 sorted cells according to the manufacturer's protocol, using the AllPrep DNA/RNA/miRNA Universal kit (Qiagen) and quanti ed using NanoDrop spectrophotometry (ThermoFisher). cDNA was synthesized using SuperScript II reverse transcriptase (ThermoFisher). Realtime quantitative PCR (qPCR) of THEMIS2 transcript was performed in triplicate using a TaqMan gene expression assay (ThermoFisher, ID: Hs00982814_g1), universal mastermix (Applied Biosystems, Waltham, MA, USA) on a Quantstudio 3 (ThermoFisher). Replicates with a deviation of ≥ 0. 25 cycle from the mean were excluded from the analysis. Cycle threshold values were normalized against expression of ABL.
section_name: Gene expression analysis

section_content: For enzyme-linked immunosorbent assays (ELISA), up to 10 6 sorted cells were lysed on ice in 100µL RIPA buffer with 1% HALT phosphatase and protease inhibitor (both ThermoScienti c, Waltham, MA, USA). 

Protein concentration was quanti ed using a Pierce BCA protein assay kit (ThermoScienti c). ELISA was performed according to the manufacturer's protocol, using a Human Protein THEMIS2 ELISA kit (Abbexa Ltd, Cambridge, UK) with 500ng of total protein content input in duplicate wells. Average optical density was measured at 450nM and THEMIS2 concentration was interpolated using a standard curve generated from same-plate duplicate wells.
section_name: ELISA

section_content: Two publicly available, independently generated datasets were used for biomarker external validation. The rst dataset, generated by Herbst e. a., comprised bulk RNA sequencing and mass spectrometry data of 68 CLL patients (22). As not all patients in this cohort were treatment-free, TFS was reported as endpoint. Kaplan-Meier survival curves were generated using the online tool available at www. dietrichlab. de/CLL_Proteomics/. The second dataset, published by Knisbacher e. a., comprised bulk RNA sequencing, FFS and OS of 556 treatment-naive CLL patients. Clinical data and batch corrected transcript per million (TPM) data was downloaded from www. CLLmap. org.
section_name: Biomarker validation in external cohorts

section_content: To test for associations between molecular features and intracellular protein abundances, we used a twoway ANOVA, including protein abundance as the quantitative variable with the molecular feature and batch number as independent categorical variables. All P-values were adjusted for multiple testing, using the Benjamini-Hochberg method. Differences in mean expression of individual proteins and difference in gene expression levels were compared using unpaired t-tests. TTFT and TFS were de ned as the interval between the date of sampling and the initiation of rst-line or next line of therapy, respectively. Failure-free survival (FFS) was de ned as the interval between date of sampling and the initiation of the next line of therapy or death. Overall survival (OS) was de ned as the interval between date of sampling and death. Univariable survival analysis was performed using Kaplan-Meier estimation, evaluating statistical signi cance using a log-rank test. Multivariable survival analysis was performed using Cox proportional hazards modelling. Hazard ratios were evaluated for statistical signi cance using a Wald test with correction for multiple testing. All survival analysis was performed in R, using the survival package (23). Model goodness-of-t was compared using a likelihood ratio test.
section_name: Statistical analysis

section_name: Results

section_content: To characterize global protein expression of untreated, early-stage CLL, we measured the intracellular protein abundances of magnetically puri ed B-cells using mass spectrometry in a cohort of 40 CLL patients with TTFT ≤ 24 months after peripheral blood (PB) sampling (hereafter: cases) and 40 sex-and age-matched CLL patients with TTFT > 24 months after PB sampling (hereafter: controls) (Fig. 1A ). Cases or controls with ≥ 40% missing data or highly deviant protein expression values (> 2 SD from the mean), likely indicating technical artefacts, were excluded from the analysis (n = 5), resulting in a nal cohort of 75 patients (Supplemental Fig. 1, Supplemental Table 1 ). As expected, high-risk molecular features, such as unmutated IGHV, TP53 anomalies and IGLV3-21 R110 were enriched in the cases. Median TTFT in the cases was 7. 7 months, versus not reached (NR) in the controls (Fig. 1B ). 

In total, we quanti ed the expression of 3 268 proteins in the cohort. Principal component analysis and unsupervised clustering by global protein expression did not show segregation by outcome or molecular features (Supplemental Fig. 2A, B ).
section_name: Mass spectrometry analysis of the proteome in untreated CLL

section_content: To analyze the impact of well-known molecular characteristics of CLL on intracellular protein expression, we investigated differential protein expression (log 2 [fold change] ≥ 0. 5 or ≤-0. 5, adjusted P-value < 0. 05) associated with immunogenetic (IGHV mutational status and/or IGLV3-21 R110 light chain use), cytogenetic and genetic features (TP53 mutations). In our analysis, IGHV mutational status (32 proteins overexpressed in U-CLL, 7 proteins overexpressed in M-CLL) and trisomy 12 (32 proteins overexpressed in the presence of trisomy 12, 8 proteins overexpressed in the absence of trisomy 12) were most impactful on the proteome (Fig. 2A, Supplemental Tables 2 and 3 ). TP53 aberrancies (i. e., del(17p13) and/or TP53 mutations), IGLV3-21 R110, del(13q14) and del(11q22) were not associated with differential protein expression (Fig. 2A ). 

Comparing U-CLL to M-CLL, the highest fold change was observed for ZAP70, with a 4. 7-fold higher average abundance in U-CLL, compared to M-CLL (Fig. 2B, C ). High expression of ZAP70 is a well-known feature of U-CLL, thus serving as a validation of our dataset. In addition, abundance of the IG constant region mu and delta (IGHM and IGHD) and CD79B, part of the B cell receptor complex, was higher in U-CLL, in concordance with the higher surface IgM expression and BCR responsiveness observed in these patients (24, 25). In contrast to previous research(26), MARCKS was not differentially abundant between U-CLL and M-CLL patients in our cohort (Supplemental Fig. 3A ). Notably, 13/32 (41%) of proteins overexpressed in CLL with trisomy 12 were encoded by genes on chromosome 12, indicative of gene dosage effects (Fig. 2D ).
section_name: Downstream protein correlates of CLL molecular features

section_content: Three other mass spectrometry screens of the CLL proteome have recently been published (22, 26, 27). To investigate the validity and reproducibility of mass spectrometry screens in CLL, we compared our ndings to the DEPs reported in these screens. Indeed, in spite of the differences in study design and methodology, a large number of DEPs found in our screen were concordantly reported in one or multiple other screens. Speci cally, 28/39 (72%) DEPs associated with IGHV mutational status (Fig. 3A ) and 34/40 (85%) DEPs associated with trisomy 12 (Fig. 3B ) were found both in our screen, and in one or more screens from the literature, in all cases sharing directionality. Regarding IGHV mutational status, four proteins were consistently identi ed as differentially expressed in all four screens: ZAP70 and Lamin type A/C, overexpressed in U-CLL, and FTH1 and FTL, both overexpressed in M-CLL.
section_name: Cross-validation with publicly available mass spectrometry screens in CLL cohort

section_content: When comparing the DEPs between cases and controls, 5 proteins were signi cantly upregulated in the cases, whereas 3 proteins were signi cantly downregulated (Fig. 4A ). Inevitably, many of these DEPs were correlated to IGHV mutational status, as U-CLL was exclusively present among the cases. To identify which of these proteins may aid prognostication beyond IGHV and TP53 mutational status, we performed Cox PH modelling, estimating the hazard of progression to therapy as a function of protein expression, while correcting for IGHV mutational status and TP53 aberrancies. Out of all 8 proteins, only elevated protein expression of THEMIS2 was independently associated with TTFT (HR 2. 98, [95%CI 1. 87-4. 73], P < 0. 001) (Fig. 4B ). Indeed, average THEMIS2 protein levels were elevated in both U-CLL and M-CLL cases, compared to controls, which were all M-CLL (Fig. 4C ), and was unrelated to the presence of IGLV3-21 R110 (Supplemental Fig. 3B ). High intracellular protein levels of THEMIS2 were associated with shorter TTFT (relative THEMIS2 expression > mean, median TTFT 10. 7 months [95%CI 6. 4-24. 6] vs < mean, median TTFT NR [95%CI 41. 4-NR], P < 0. 001) and failure-free survival (FFS) (relative THEMIS2 expression > mean, median FFS 9. 3 months [95%CI 6. 6-NR] vs < mean, median FFS 44. 2 [95%CI 14. 43-NR], P = 0. 0019) (Fig. 4D, supplemental Fig. 3C ). An increase in THEMIS2 protein abundance was inversely correlated to TTFT (Spearman's r= -0. 58, P < 0. 001) (Fig. 4E ). Receiver operating characteristic (ROC) analysis demonstrated good discriminatory capacity of relative THEMIS2 protein levels, distinguishing cases from controls with an AUC of 0. 83 (Fig. 4F ).
section_name: Biomarker identi cation in untreated CLL

section_content: The International Prognostic Score for Early-stage CLL (IPS-E) index is a prediction model that estimates TTFT in early-stage CLL based on the IGHV mutational status, an absolute lymphocyte count (ALC) > 15*10 9 /L and lymph node palpability(28). Addition of THEMIS2 as predictor variable in a Cox PH model including IGHV mutational status, ALC and Rai stage at diagnosis, as surrogate for lymph node involvement, signi cantly improved model goodness-of-t (likelihood ratio test, P < 0. 0001) (Supplemental table 4 ). 

Validation of THEMIS2 as putative biomarker for short TTFT THEMIS2, which functions downstream of the B cell receptor (BCR), increases BCR sensitivity to lowavidity antigens and increases BCR activation in the absence of antigen(29). To con rm the relationship between THEMIS2 and TTFT, we measured THEMIS2 gene expression by qPCR in cases and controls. Average normalized expression of THEMIS2 mRNA was 2. 9-fold higher in cases, compared to controls (P = 0. 005) (Fig. 5A ). Furthermore, direct quanti cation of THEMIS2 by ELISA in a cohort of 40 patients (21 patients from the mass spectrometry screen and 19 new patients, Supplemental Table 5 ) demonstrated that higher intracellular concentrations of THEMIS2 were associated with shorter TTFT (THEMIS2 concentration > mean, median TTFT 11 months [95%CI 7-NR], versus < mean, 66 months [95%CI 50-NR], P = 0. 04) (Fig. 5B ). Finally, we evaluated the prognostic utility of THEMIS2 in two publicly available, independently generated datasets. The rst, generated by Herbst et al., encompasses bulk RNA sequencing and mass spectrometry data of 68 CLL patients (22). As not all patients in this cohort were treatment-naïve, treatment-free survival (TFS) rather than TTFT was reported. In this cohort, elevated expression of THEMIS2 was associated with inferior TFS, both on the mRNA level (high expression, median TFS approximately 10 months, versus low expression, median TFS NR, P = 0. 007) (Fig. 5C ) and on the protein level (high abundance, median TFS approximately 4 months, versus low abundance, median TFS NR, P < 0. 0001) (Fig. 5D ) (22). In the second dataset, assembled by Knisbacher et al., bulk RNA sequencing and clinical data was available for 556 treatment-naïve CLL patients (30). In these patients, high gene expression of THEMIS2 was associated with signi cantly shorter failure free survival (FFS) and overall survival (OS) (median FFS, THEMIS2 expression > mean 38. 2 months [95%CI 33. 6-49] vs < mean, 81. 8 months [95%CI 65. 1-109], P < 0. 001 and median OS, THEMIS2 expression > mean 154 months [95%CI 139-NR] vs 191 months [95%CI 157-NR], P = 0. 009) (Fig. 5E, F ) (30). In a Cox PH model, including IGHV mutational status and presence or absence of TP53 aberrations and IGLV3-21 R110 as covariables, THEMIS2 gene expression signi cantly predicted FFS, but not OS (Supplemental Tables 6 and 7 ).
section_name: Additive value of THEMIS2 to the IPS-E index

section_content: In this study we have characterized the global intracellular proteome of 75 early-stage, treatment-naïve CLL patients. We describe the downstream effects on protein expression of recurrent molecular features of CLL and use the unique case-control design of our early-stage cohort to identify THEMIS2 as novel, independent biomarker associated with TTFT. THEMIS2, alternatively known as ICB-1, is a newly described member of the Themis family of proteins. 

Whereas THEMIS1 plays a nonredundant role in positive selection of CD8 + CD4 + T cells in the thymus, THEMIS2 is mainly expressed in monocytes, follicular B cells and B1 B cells (29, 31). The latter observation is noteworthy, as human B1-like B cells have been proposed to be the cell of origin in CLL (32). Although THEMIS2 is not essential for B cell biology, it increases B cell receptor (BCR) sensitivity to lowavidity, but not high-avidity antigens by constitutive binding to Grb2, Lyn and PLCγ2 (29). Crucially, THEMIS2 may increase constitutive BCR activation in the absence of antigen (29). Whether the relationship between THEMIS2 and survival is purely associative, or whether THEMIS2 may play a role in the pathophysiology of CLL, remains to be determined. 

The association between THEMIS2 and outcome was present at both mRNA and protein level, as demonstrated by qPCR, mass spectrometry and ELISA. External validation was performed by analysis of two independently generated datasets, published by Herbst et al. and Knisbacher et al., which demonstrated an inverse association between THEMIS2 gene and protein expression and TFS, FFS and OS (22, 30). Of note, the association between THEMIS2 and TTFT or FFS was independent of IGHV mutational status and TP53 aberrancies. Moreover, incorporation of THEMIS2 protein levels in a model containing all components of the IPS-E index(28), a validated prognostic model in the context of earlystage CLL, improved model performance. As such, high intracellular THEMIS2 concentration, as measured by clinically applicable techniques such as ELISA, could be putatively positioned as biomarker of poor prognosis in early-stage CLL, complementary to IGHV and TP53 mutational pro ling. De ning the optimal measurement modality and threshold value for THEMIS2-based prognostication requires additional work in a larger cohort of patients. 

Recently, several mass spectrometry screens have been published in the context of CLL. Beckmann et al. compared the proteome and phosphoproteome in a small series of three U-CLL and three M-CLL patients, using isobaric tags for relative and absolute quanti cation (iTRAQ)-based mass spectrometry(26). Meier-Abt et al. used data-independent acquisition (DIA)-mass spectrometry to characterize the proteome of 117 CLL patients (27). Lastly, Herbst et al., using in-depth high-resolution isoelectric (HiRIEF)-mass spectrometry, characterized protein expression in a cohort of 68 CLL patients (22). Comparing the result of these contemporaneous, independently performed mass spectrometry screens allows for an assessment of the validity and reproducibility of proteome pro ling in CLL. 

There was high concordance between all four reported screens. Similarly to Meier-Abt et al. and Herbst et al., in our screen, the molecular features with the highest impact on the proteome were IGHV mutational status and trisomy 12(refs. 22,27). The proteomic effects of other features, speci cally IGLV3-21 R110 light chain use, del(13q14), del(11q22) and TP53 aberrancies, were negligible. Importantly, 72% of DEPs associated with IGHV mutational status and 85% of DEPs associated with trisomy 12 identi ed in our analysis were reported in at least one of the three screens from the literature, underscoring the robust nature of these mass spectrometry screens from a Bayesian perspective. Differential expression of four proteins was consistently associated with IGHV mutational status across all four screens: ZAP70 and Lamin type A/C, with high expression in U-CLL, and the heavy and light chain of ferritin, with high expression in M-CLL. Although the association between ZAP70 expression and IGHV mutational status has been widely established, to our knowledge, the role of these other proteins has not been studied in the context of CLL. Lamin A/C belongs to the lamin family of proteins, components of the nuclear envelope. Lamin B1, a similar perinuclear protein, has been implicated in the regulation of SHM by binding to the immunoglobulin domains: LMNB1 expression is decreased in germinal centers, induction of SHM lowers Lamin B1 expression and suppression of Lamin B1 grossly increases SHM rates (33). Of note, in contrast to Lamin type A/C, LMNB1 expression was upregulated in M-CLL, and low LMNB1 expression was associated with inferior survival (33). Whether LMNA is similarly involved in the molecular machinery of SHM warrants further investigation. The heavy and light chains of ferritin, FTH1 and FTL, form polymers of 24 subunits to comprise ferritin, which is responsible for the intracellular sequestration of iron. Free intracellular iron is cytotoxic, as it catalyzes the formation of free radicals in the Fenton reaction. As such, the high expression of ferritin may, speculatively, render M-CLL cells differentially susceptible to ferroptosis. Of note, a recent study correlated the expression of eight ferroptosis-related genes to the clinical outcome of CLL patients, and to the predicted in vitro susceptibility of CLL cells to udarabine, cyclophosphamide and ibrutinib, further indicating that ferroptosis may play an important role in the context of CLL (34). 

Our study has some limitations. We con ned the molecular characterization of our cohort to those features commonly determined in clinical practice. As such, we were unable to study the proteomic effects of other recurrent mutations, such as lesions affecting NOTCH1 and SF3B1, although in other screens, their effects on global protein expression were found to be minimal (22, 27). In addition, as biobank participation terminated upon treatment initiation, the effects of protein expression on treatment e cacy and OS could not be evaluated in this CLL biobank cohort. Finally, our cohort was powered and matched to evaluate differences using a TTFT threshold of 24 months. Consequently, proteins identi ed as DEPs at this threshold might not have the same discriminatory capacity at shorter (i. e., 6 to 12 months) or longer (i. e., 36 or 48 months) TTFT intervals. That said, our survival analyses demonstrate that the discriminatory capacity of THEMIS2 persists across the entire length of follow-up time in our cohort. 

In summary, we provide a comprehensive characterization of the global proteome of early-stage CLL. We identify high expression of THEMIS2 as a biomarker of short TTFT, independent of IGHV mutational status or TP53 aberrations. B) Dot-and-whisker plot, representing the hazard ratio per protein in a Cox proportional hazards model, estimating time to rst treatment with IGHV and TP53 mutation status as covariables. Dots represent the point estimate, whereas the whiskers extend to the upper and lower limit of the 95% con dence interval. Statistical signi cance was evaluated using a Wald test. C) Dotplot demonstrating the normalized relative intracellular abundance of THEMIS2, strati ed by case/control identity and IGHV mutational status. Fold-change values were normalized to the average abundance of M-CLL controls. Statistical signi cance was evaluated using a t-test.
section_name: Discussion

section_name: Declarations Figures

section_name: Acknowledgements

section_content: The authors would like to thank all patients, their families, and investigators involved in the biobank for B cell malignancies. In addition, we would like to thank Abt-Meier et al., Herbst et al. and Knischbacher et al. for making their datasets publicly available as interactive web modules. This project was partly funded by the 2021 Albert Schweitzer Hospital Stipend for Research.
section_name: Acknowledgements

section_name: Data availability statement

section_content: The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository, with the dataset identi er PXD036918 (35). The scripts used for the bioinformatic analyses have been uploaded to https://github. com/paul10011993/CLLproteome.
section_name: Data availability statement

section_name: Authorship contributions

section_content: PJH, MDL and AWL contributed to study design. PJH performed the experimental procedures. PMK contributed to data analysis and interpretation. JAAD and WD performed the mass spectrometry and contributed to data analysis. JMND and APK were involved in the coordination of the biobank for B cell malignancies. MR performed the TP53 sequencing. HJB performed the IG light chain sequencing. KJG performed the targeted FISH. JLJCA, LvdS, PJMV and PEW contributed to data interpretation. PJH, PMK, MDL and AWL wrote the manuscript. All authors critically read and approved the nal version of the manuscript. 

Competing Interest Statement has received research funding from Roche/Genentech. APK has received personal fees from AbbVie, LAVA, Genmab, Janssen, AstraZeneca, Roche/Genentech, and Bristol Myers Squibb; and research funding from AbbVie, Janssen, AstraZeneca, Roche/Genentech, and Bristol Myers Squibb. M-DL has received personal fees from AbbVie, Janssen, and Roche; and research funding from AbbVie, Janssen, AstraZeneca, and Roche/Genentech. AWL has received research funding via an unrestricted grant from Roche-Genentech and speaker-fees from Janssen. The remaining authors declare no competing nancial interests. 

B) Kaplan-Meier survival plot, estimating the proportion of treatment-free patients over time, strati ed by above or below average intracellular levels of THEMIS2 protein, quanti ed by ELISA. Asterisks indicate right-censoring. Statistical signi cance was evaluated using a log-rank test. 

C, D) Kaplan-Meier survival plots, estimating the proportion of treatment-free patients over time, strati ed by above or below average THEMIS2 gene expression (C) or THEMIS2 protein expression (D). Statistical signi cance was evaluated using a log-rank test. These gures were generated using an external validation cohort, through the online application kindly provided by Herbst et al, available at https://www. dietrichlab. de/CLL_Proteomics/ E, F) Kaplan-Meier survival plots, estimating the proportion of failure-free (C) or alive (D) patients over time, strati ed by above or below average THEMIS2 gene expression. Statistical signi cance was evaluated using a log-rank test. These gures were generated using an external validation cohort published by Knisbacher
section_name: Authorship contributions