INTRODUCTION Chronic Lymphocytic Leukemia (CLL) is the most frequent leukemia in adults and is typically preceded by a pre-malignant condition called Monoclonal B cell Lymphocytosis (MBL), which is detected in up to 10% of otherwise healthy individuals. Monoclonal gammopathy of undetermined significance (MGUS) is also a common pre-neoplastic condition involving healthy asymptomatic individuals. MGUS can be concomitantly present along with an MBL/CLL diagnosis and can be detected before, after or simultaneously to the leukemia diagnosis. Using sensitive multi-proteomics methods, this study aims to address the question of whether clonal CLL B cells can release monoclonal immunoglobulin (Ig) proteins into the serum, thus being directly responsible for co-existing MGUS.

METHODS We included 361 patients diagnosed with either CLL (N=263) or MBL (N=98) and characterized the expressed clonotypic Ig heavy and light chains (HC and LC, in 358 and 207 cases, respectively). As a first step, RNA was isolated from peripheral blood monoclonal B cells of patients whose samples were cryopreserved in the dedicated Dana-Farber Cancer Institute (DFCI) biobank. Using synthesized cDNA, PCR amplification was performed with specific Ig gene primers, followed by bidirectional Sanger sequencing. In parallel, sera from the same patients were analyzed with two different proteomic techniques: (i) Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry (MS) to achieve a precise quantification of the LC's mass-to-charge ratio (m/z), allowing the comparison to the Ig molecular mass results calculated from the Sanger sequences; and, (ii) high resolution quantitative proteomic sequencing using an EvoSep One Liquid Chromatography system coupled to a timsTOF Pro 2 mass spectrometer (Bruker) to identify the most abundant serum Ig and compare them to the expressed Ig HC and LC.

RESULTS In a cohort of 3127 patients with CLL/MBL followed up at DFCI with serum protein electrophoresis (SPEP) results, 658 (21%) cases presented with a serum monoclonal component (MC, either IgM, IgG or IgA or free Kappa/Lambda). For this study, we selected 361 cases, with a median age of 66 years and a male prevalence (62.6%; 226/361), of whom 195 (173 CLL, 22 MBL) showed a positive result after serum protein electrophoresis (SPEP) tests (Range 0.07-3.18 g/dL; 57 cases non-quantifiable). The remaining 166 cases, presenting no detectable MC, included 90 CLL and 76 MBL.

Initially, we compared data from the Ig LC expressed by the leukemic cells, as obtained by Sanger sequencing and flow cytometry, with the SPEP and MALDI-TOF MS data for the LC associated with the serum MC. The kappa or lambda expression by immunophenotyping matched the isotype of the sequenced LC in 88.3% (76/86) of the cases. More specifically, the serum Ig LC mass measured by MALDI-TOF MS matched the calculated mass from the sequences of the expressed clonotypic Ig LC in 61.2% (52/85, 36 kappa+ cases; 16 lambda+ cases) of the MBL/CLL cases.

Furthermore, using the high-resolution quantitative MS technique, which allows for the exact identification of the variable domain of the Ig HC and/or LC, we showed identity between the serum Ig protein sequences and the Ig expressed by the CLL & MBL cells in 78.6% (136/173) and 50% (11/22) of the cases, respectively. Interestingly, MS analysis in the 166 cases (90 CLL and 76 MBL) without an apparent serum MC on SPEP or below the detection limit of SPEP, led to similar findings in 64% (58/90) of CLL and 30% (23/76) of MBL cases, showing a match for the Ig HC and/or LC domain. In the remaining cases, MS was not able to identify a discrete abundant protein amongst the entire pool of Ig proteins.

CONCLUSIONS With this innovative approach, we provide, for the first time, robust evidence that in the majority (up to 3/4) of CLL/MBL cases, it is possible to detect the expressed monoclonal Ig circulating in the serum. This evidence supports the notion that the clonal B cells may be responsible for the production and release of monoclonal Ig to the levels that can be detected as paraproteins in routine clinical practice by SPEP. These results suggest the need for future work that will lead to a better characterization of the immunological nature of the leukemic cells underlying Ig production, potentially highlighting an alternative path of B cell maturation and transformation leading to an MGUS presentation, an aspect so far poorly investigated in CLL.

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2025
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