Premonoclonal B-cell lymphocytosis: the CLL cell of origin

In this issue of Blood, Kolijn et al¹ demonstrate the findings of a longitudinal analysis of chronic lymphocytic leukemia (CLL) buffy coat genomic DNA.

Using next-generation sequencing, Kolijn et al sequenced the B-cell receptor (BCR) immunoglobulin heavy chain (IgH) repertoire for patients with CLL in blood samples collected up to 22 years before the diagnosis of CLL. Their findings include B-cell repertoire skewing and the dynamics of clonotypic evolution regardless of IgH mutational status or stereotypy. Mutational status is defined as unmuted CLL (U-CLL) if the IgHV sequence shows ≥98% homology (identical) to a reference germ line sequence and as mutated (M-CLL) if the IgH sequence shows <98% homology. Stereotypy is defined by the IgHV CDR3 sequence, and distinct subsets have been defined for CLL. The diagnostic BCR IgHV sequence could be detected in the background or as a dominate clonotype ≥2% in most samples tested. IgHV mutational status and stereotypy have clinical significance at the time of diagnosis in terms of indolent or aggressive disease, but there was no difference in the time from first appearance of the clonotype to the time of clinical diagnosis, whether U-CLL or M-CLL. The key finding is that these early clonotypic changes seem to prolong the already indolent preclinical stage of CLL. Also, the poor-prognosis stereotype subset 2 (CDR3 sequence associated with worse prognosis) and U-CLL clonotype were found 16 years before the diagnosis of CLL. These findings represent the earliest detection of a clonotypic precursor cell for CLL.

Some time ago, using routine flow cytometric immunophenotyping, we and others observed a precursor to CLL in healthy individuals without hematologic abnormalities.² It was defined as monoclonal B-cell lymphocytosis (MBL). Absolute B-cell lymphocyte count was used as a cutoff to define CLL as >5000 clonal B cells per μL of blood.³ Low-count MBL was further defined as MBL ≤500 cells per microliter or clone occupation ≥85% of the B-cell population and sometimes referred to as population-based MBL. High-count MBL, referred to as clinical MBL, exists when B-cell lymphocytosis is >500 B cells per μL but <5000 cells per microliter. Low-count MBL is usually stable and nonprogressive. However, the longitudinal study of MBL in familial CLL by Slager et al⁴ shows that low-count MBL may progress to high-count MBL.

Clinical high-count MBL is associated with increased infections and second primary tumors; Mayo data suggest that the risk of infection, even without hypogammaglobulinemia, is greater than the risk of progression to CLL,⁵ and a similar finding for the relationship between high-count MBL seems to exist for second primary tumors. Salamanca investigators showed that these clinical correlations were also present in patients with low-count MBL, accompanied by a decrease in overall survival.⁶ Given the fact that very few cases of low-count MBL progress to CLL, low-count MBL has been designated an age-related immune senescence.³ 

Given the recognition of MBL as the precursor state in CLL, a question arises as to what determines which MBL cases progress. A partial answer to this question is that some of the same prognostic markers of early CLL (Rai stage 0, Binet A) are found in high-count MBL and to a lesser extent in low-count MBL. The data would suggest that the pathway to the “evolution from low-count MBL to high-count MBL and subsequently [treatment naïve] CLL occurs in a stepwise fashion, with gradual acquisition of high-risk genetic abnormalities.”⁷ In particular, the studies by Kostopoulos et al⁷ have delineated some of these earliest cytogenetic changes in high-count MBL. Clonal evolution can occur in stable low count.

Now that the emergence of the clonotypic cell in CLL can be detected and followed through a pre-MBL stage to both low count and high count, what are the first steps in leukemogenesis and lymphomagenesis that precede the appearance of a clonotypic cell? And what is the anatomic location of this pre-MBL cell? Lymph node? Bone marrow? Peripheral blood? The laboratory of Wiestner in collaboration with the Chiorazzi laboratory studied the proliferation rate of a subpopulation of proliferating lymphocytes in CLL and found that the lymph node has the highest rate of proliferation, followed by cells circulating in blood, with bone marrow showing the lowest rate of proliferation.⁸ The presence of proliferation centers in CLL lymph nodes is also well known, and a nodal MBL correlate has been identified.

In terms of a pregerminal center or postgerminal center cell of origin, both suggest a lymph node as the site of origin but do not rule out either bone marrow or spleen as the initial source of this long-sought-after cell of origin in CLL. B-cell subsets have not been extensively examined, particularly in the setting of vaccination. However, the role of autonomous cell signaling and chronic immune stimulation in low-count MBL needs to be further investigated. Where do we go from here? CLL has a long evolutionary history in which early branching may start as an oligoclonal process (antigen stimulation) and include driver mutations. A long-term analysis of the B-cell repertoire in familial CLL might shed light on this process. Further clarification of the mechanisms of age-related immune senescence is also of interest.

In conclusion, a clonotypic cell or cluster predates the onset of either low-count or high-count MBL dating back to between 16 and 22 years. The multiclonal dynamics of pre-MBL may equal or surpass this process that has been observed during the course of CLL.⁹ Dissecting or separating this clonotypic cell from a polyclonal background presents a new challenge.