The Immunoglobulin Gene Repertoire of Low-Count CLL-Like MBL Is Different from CLL: Diagnostic Considerations and Implications for Clinical Monitoring
Authors: Dagklis Antonis , Fazi Claudia , Sala Cinzia , Cantarelli Valeria , Scielzo Cristina , Massacane Roberto , Toniolo Daniela , Caligaris-Cappio Federico , Stamatopoulos Kostas , Ghia Paolo 
Published: October 2019

Abstract
The term Monoclonal B Lymphocytosis (MBL) defines the presence of Monoclonal B Lymphocytes in the blood of otherwise healthy individuals. Though phenotypically heterogeneous, most MBL cases resemble CLL cells (CD5+, CD20dim, CD79b dim, sIgdim). The interest in MBL increased after this entity was included in the revised NCI-WG/IWCLL guidelines for the diagnosis and management of CLL (Hallek et al, 2008) and defined as “the presence of fewer than 5×109/L B lymphocytes” in the peripheral blood. However, the concentration of MBL in the blood of any given individual is extremely variable accounting in some cases for the vast majority of circulating B cells while being a negligible portion of them in others. It is then plausible that molecular differences could exist between low- vs. high-count MBL, being the latter likely more advanced on the way to become CLL. In this context, it was recently reported that subjects with <5×109/L CLL-like MBL but with lymphocytosis will require treatment at a rate of 1.1% per year. The absolute B-cell count turned out to be the only independent prognostic factor associated with progressive lymphocytosis, as all MBL cases studied had immunoglobulin (IG) gene features and cytogenetic abnormalities similar to good prognosis CLL. In contrast, very little is known about low-count MBL cases accidentally found in the general population. By cytofluorograph analysis, we identified 89 CLL-like MBL in the blood of 1725 healthy individuals >18 years old (5.1%) and analyzed the IGHV-D-J rearrangements expressed by 51 of them, the majority being characterized by few clonal B cells (mean 6.9% of circulating B lymphocytes, with only 13 cases >10%).
CLL-like MBL cells showed a predominance of IGHV3 genes, followed by IGHV4 genes, resembling the normal repertoire. The most frequent IGHV gene in MBL was IGHV4- 59/61, rarely used in CLL. The MBL repertoire was also conspicuous for the lack of the IGHV1-69 gene (the predominant gene in unmutated CLL, ~25%) and the low frequency of the IGHV4-34 gene (2/51 sequences, 3.9%), the most frequent gene in mutated CLL (~12%). Following the 98% identity cut-off value, 36/51 sequences (70.5%) were defined as “mutated”, whereas the remainder had “unmutated” IGHV genes. Alignment of MBL HCDR3 sequences to a comprehensive panel of CLL HCDR3 sequences identified 2/51 (3.9%) MBL cases with a sequence similar to previously described CLL cases (“stereotyped receptors”). These results show that the IG gene repertoire in low-count MBL, accidentally found in the general population, does not show the typical CLL-related biases in terms of IGHV gene usage. This cannot be simply explained by the higher number of mutated cases among MBL, as unmutated cases account for almost a third of CLL-like MBL, indicating a molecular heterogeneity. In addition, HCDR3 stereotypy in MBL is significantly less frequent than in CLL (>25% of cases). Occasional MBL may indeed express a CLL “stereotyped receptor”, implying that the potential to evolve into a leukemia exists within MBL, though at low frequency, and may depend on precise selection mechanisms based on the molecular features of the B cell receptor.
Taken together, our results strongly suggest that the detection of MBL in an otherwise healthy subject is not always equivalent to a pre-leukemic state. Differential IG molecular features might provide a better tool to discriminate individuals at risk of progression than an arbitrary mathematical threshold. Detailed IG molecular analysis of individual MBL may help to identify those few cases that necessitate continuous clinical monitoring to anticipate disease progression and, on the other hand, to avoid the burden of lengthy and expensive follow-ups in the vast number of persons who are extremely unlikely to develop CLL, though carrying MBL in their blood.