Plasma Proteomics Differentiates between Patients with B-Cell Lymphomas
Authors: Babačić Haris , Chowdhury Nashif Mahruf , Berglund Mattias , Hashemi Jamileh , Collin Jeremia , Pettersson Emma , Ly Ann-Marie , Nikkarinen Anna , Enblad Gunilla , Molin Daniel , Glimelius Ingrid , Pernemalm Maria , Hellström Mats 
Published: December 2024

Introduction
Techniques for assessing the blood plasma proteome with high precision and at great depth are rapidly developing and have demonstrated utility in carrying diagnostic and prognostic information for patients with cancer, including hematological malignancies. However, it is not known whether the plasma proteome can be useful in distinguishing the more closely related cancer entities, such as different B-cell lymphomas (BCLs). Performing affinity-based plasma proteomics analyses in a population-based cohort of BCLs, we aimed at discovering plasma proteome differences between BCL subtypes and identifying potential biomarkers that can aid differential diagnosis.
Material and Methods
We analyzed 592 BCLs (221 diffuse large BCL (DLBCL), 94 follicular lymphoma (FL), 123 Hodgkin lymphoma (HL), 91 mantle cell lymphoma (MCL), and 63 primary CNS lymphoma (PCNSL)) from the U-CAN biobank (www.u-can.uu.se). Plasma samples collected at diagnosis were analyzed using the Olink Explore 1536 platform, which provided relative quantification of 1463 unique proteins. The plasma proteomes between a given group and all the remaining groups were compared with a two-sided t test and further adjusted for age and sex in multivariable linear limma models. To identify panels of plasma proteins that can differentiate between the different subtypes of BCLs, we trained two types of machine learning (ML) models based on the random forest (RF) algorithm and logistic regression with regularization (LRR). The entire dataset was proportionally partitioned into a training (70%) and testing (30%) dataset. Both model types were trained in one thousand iterations, with cross-validation, on a non-filtered dataset and implementing different filtering approaches based on varying cut-offs of mean log2-difference (log2-diff) of differentially altered proteins (DAPs) and 0.1% false discovery rate (FDR). Finally, the best-performing model from the iterations of the two ML methods on the training data was selected and tested on the testing dataset for performance. Both balanced accuracy and area under the curve (AUC) were considered as main outcomes of performance.
Results
Comparing the plasma proteomes between BCL subtypes showed many DAPs in each subtype compared to the rest of the cohort at 5% FDR. PCNSL patients had the largest number of DAPs, followed by HL, MCL, DLBCL, and FL. However, most of these alterations were of smaller log2-diff between the subgroups. Less than ten proteins per group had a log2-diff &gt; 1 in a subgroup compared to other subtypes, apart from MCL patients, who had 64 DAPs with log2-diff &gt; 1. The findings remained consistent in the multivariable analyses, where the log2-diff between subgroups was adjusted for age and sex. Yet, each subgroup had more DAPs that were uniquely altered in that subgroup and in no other group, regardless of the log2-FC, with most DAPs observed again in the MCL, followed by DLBCL, HL, FL, and PCNSL. This was reflected in the ML models, where combining smaller differences in protein levels into multivariate models showed reliable performance in differentiating the BCLs. Filtering improved the model's accuracy, and the derived best-performing LRR model showed moderate to high accuracy in differentiating the BCLs on testing data. The LRR model had the highest accuracy in classifying MCL, with AUC of 91%, followed by HL (90%), PCNSL (89%), DLBCL (85%), and FL (80%), the latter being repeatedly misclassified in the ML iterations. Although the model's sensitivity was variable, being highest for HL and lowest for FL, the specificity was very high (&gt;93%) for excluding FL (94%), HL (96%), MCL (98%), and particularly PCNSL (99%), with the negative predictive value of the model for CNS involvement being 98%.
Conclusions
Plasma proteomics can differentiate between distinct types of BCLs with a moderate to high accuracy, between 80% and 91%. The models showed the highest accuracy in classifying MCL, likely due to the highest number of unique DAPs and proteins with large log2-diff observed in this subtype On average, the models showed better specificity, which is highly relevant for DLBCL, where a blood biomarker can serve as a quick diagnostic tool for initial exclusion of CNS involvement in a patient, with very high predictive value.
This suggests that plasma proteomics could assist in the differential diagnosis of B-cell lymphomas and potentially for CNS-involvement.