High-Throughput Proteomics Identifies THEMIS2 As Biomarker of Inferior Treatment-Free Survival in Chronic Lymphocytic Leukemia

Background Notwithstanding advances in the prognostication of chronic lymphocytic leukemia (CLL), it remains challenging to distinguish between patients with favorable and unfavorable treatment-free survival (TFS). In addition, the downstream protein correlates of well-known genomic features of CLL are not always clear. To address this, we performed large-scale, unbiased characterization of global intracellular protein expression in a cohort of previously untreated CLL patients.

Materials and methods We selected 40 CLL cases (patients in need of treatment within 24 months of sampling) and 40 age- and sex-matched CLL controls (patients without need for treatment for at least 24 months since sampling) from the Dutch natural history CLL cohort. In this cohort, we characterized global intracellular protein expression of magnetically purified B cells using tandem-mass-tagging, followed by high-performance liquid chromatography and triple tandem mass-spectrometry. In addition, recurrent cytogenetic aberrations (del13q, del11q, del17p and trisomy 12), IGHV mutational status and TP53 mutations were characterized using targeted fluorescence in situ hybridization (FISH), Sanger sequencing and next-generation sequencing, respectively.

Results Expression of 3268 proteins was quantified in the cohort, allowing for a comprehensive characterization of the CLL proteome. CLL with unmutated IGHV (U-CLL) was associated with significant overexpression of 32 proteins, including ZAP70 and proteins involved in B-cell receptor signaling, such as CD79b and immunoglobulin constant regions mu and delta (adjusted P-value <0.05). CLL with mutated IGHV (M-CLL) was associated with higher intracellular expression of 7 proteins, including both subunits of ferritin (FTH1 and FTL, adjusted P-value <0.05). Trisomy 12 was associated with increased intracellular expression of 32 proteins, 13 (40%) of which are encoded by genes on chromosome 12, and decreased expression of 8 proteins. Many of these hits are concordant with the results of independently performed mass-spectrometry screens. Interestingly, the intracellular concentration of THEMIS2 was significantly higher in cases, compared to controls. THEMIS2 is an protein that is constitutively bound to Grb2, Lyn and PLCγ2 and lowers the threshold for B-cell receptor activation by low-avidity antigens. THEMIS2 protein levels were higher in both U-CLL cases (mean fold change 2, P-value <0.0001) and M-CLL cases (mean fold change 1.7, P-value <0.01), compared to controls. Concordantly, Cox proportional hazards modelling demonstrated that higher concentration of THEMIS2 was significantly associated with shorter TTFT, independent of IGHV mutational status and TP53 aberrations (standardized HR 2.79 [95%CI 1.71-4.54], P<0.0001). ROC analysis indicated a good discriminatory capacity of elevated THEMIS2 concentration, with an AUC of 0.83. THEMIS2 protein quantification by ELISA in a cohort of 40 patients confirmed that higher expression of THEMIS2 was associated with inferior TTFT (median TTFT for patients with THEMIS2 levels above versus below the mean 11 months versus 66 months, P=0.04). Gene expression analysis by qPCR confirmed higher expression of THEMIS2 in cases, compared to controls (mean fold change of 3, P<0.01). Analysis of a publicly available, independently generated mass-spectrometry dataset of 68 CLL patients confirmed the association of elevated intracellular THEMIS2 expression and TFS (median TFS 3 months versus not reached, P<0.0001).

Conclusions We present a comprehensive characterization of the proteome of untreated CLL. Of note, we identify elevated gene and protein expression of THEMIS2 as putative biomarker of inferior TTFT, independent of IGHV mutational status and TP53 aberrations.

Westerweel:Novartis: Research Funding. Kater:Janssen, LAVA: Patents & Royalties: Pending; Abbvie, Astra Zeneca, Janssen: Other: Speakers fee; Astra Zeneca, BMS, Roche/Gennetech, Janssen, Abbvie, LAVA: Membership on an entity's Board of Directors or advisory committees; Abbvie, Astra Zeneca, BMS, Janssen, Roche/Genentech: Research Funding; Amsterdam UMC, University of Amsterdam: Current Employment. Langerak:Genentech: Research Funding; Gilead: Speakers Bureau; Janssen: Speakers Bureau; Roche: Research Funding. Levin:AbbVie, Roche and Janssen: Other: Travel expenses.