Unclassifiable Isolated Monoclonal Lymphocytosis: Comprehensive Description of a Retrospective Cohort

According to the World Health Organization (WHO) classification, the nosology of B-cell neoplasms integrates clinical, morphological, phenotypic, and genetic data. In this retrospective analysis, we identified 18 patients with isolated neoplastic lymphocytosis that could not be accurately classified within the WHO classification. Most of them were asymptomatic at the time of diagnosis and the evolution was relatively indolent, as only five patients required treatment after a median follow-up of 48 months. The neoplastic B-cells expressed CD5 in most cases, but the Royal Marsden Hospital score was strictly below 3. Trisomy 12 was the most frequent cytogenetic abnormality. High-throughput sequencing highlighted mutations found in both chronic lymphocytic leukemia (CLL) and marginal zone lymphoma (MZL). Similarly, the immunoglobulin heavy chain variable region repertoire was distinct from those reported in CLL or MZL. However, as treatment choice is dependent on the correct classification of the lymphoproliferative disorder, a histological diagnosis should be performed in case patients need to be treated.


Introduction
As stated in the fourth edition of the World Health Organization (WHO) classification of hematological malignancies, "classification is the language of medicine" [1]. The classification of lymphoid neoplasms integrates clinical, morphological, phenotypic, and genetic data to associate a malignant cell to its supposed normal counterpart. An ideal classification system should minimize overlap between categories, and every patient should be assigned to one (and only one) category after extensive characterization of the disease [1]. Given the huge variability in the outcome of lymphoid neoplasms and their clinical management, the precise refinement of their classification is ongoing work that is of utmost importance. The accuracy of classification is even more important with the use of targeted therapies, as their authorization is restricted to specific nosological entities with a given molecular alteration [2].
Lymphocytosis (defined as an absolute lymphocyte count above 5 G/L) is a frequent clinical presentation of B-cell neoplasms. Lymphocytosis can represent the dissemination in the blood of an overt lymphoma, or can be the only detectable manifestation of the neoplasm. In the latter case, accurate diagnosis relies exclusively on the characterization of the circulating lymphocytes, based on cytology, immunophenotyping, cytogenetics, and/or molecular biology. For instance, the diagnosis of chronic lymphocytic leukemia (CLL) requires a Royal Marsden Hospital (RMH) score [3,4] of three or more. The diagnosis of mantle cell lymphoma (MCL) relies on the demonstration of the t(11;14)(q13;q32) translocation leading to cyclin D1 overexpression [1]. Some patients present an isolated monoclonal lymphocytosis that does not fulfill the diagnostic criteria for any lymphoid neoplasm and thus cannot be accurately classified within the WHO classification. In order to describe the clinical and biological characteristics of these patients, we performed a retrospective single-center study of 18 patients with unclassifiable isolated absolute lymphocytosis.

Clinical and Biological Characterization of the Cohort
The study included 18 cases (10 women and 8 men; Table 1), consulting for absolute lymphocytosis above 5 G/L without clinically detectable enlarged lymph node, splenomegaly, or hepatomegaly. This was confirmed by imaging (i.e., ultrasound, radiography, and/or scanner) in 12 patients. The median age at diagnosis was 79 years (range: 55-92 years). With the exception of one patient who had a moderately altered performance status (Eastern Cooperative Oncology Group (ECOG) score: 1), all patients were asymptomatic. The median lymphocytosis was 9.55 G/L (range: 5.30-25.69 G/L).
The cytological analysis of blood smears was performed for 17 patients, and the three types of lymphocytes (CLL-like, atypical, and normal lymphocytes) were found in all cases. Thirteen patients (76%) had a majority of CLL-like lymphocytes, whereas the remaining four (24%) had a majority of atypical lymphocytes (Table 1).
Two patients had a bone marrow biopsy, which showed bone marrow involvement in both cases by an unclassifiable small B-cell lymphoid malignancy.

Gene Mutations
Eight patients were analyzed by the high-throughput DNA sequencing of a large panel of genes recurrently mutated in hematological malignancies (Table S1). Between zero and four mutations per patient were identified ( Table 2). Most of these were described in the COSMIC database [6], and all single nucleotide variation was predicted as deleterious and/or disease causing by SIFT and/or Mutation Taster algorithms [7,8]. TP53 was the most frequently mutated gene (3/8 cases, with 17p deletion in 2 of them). Two patients harbored the same mutation of MYD88 (p.S219C). MYD88 mutations areslightly more frequent in marginal zone lymphoma (MZL) than CLL, but one of these two patients also harbored an SF3B1 mutation, which is far more frequent in CLL (Table S2). The remaining patients harbored mutations found with similar frequencies in both diseases.

IGH Gene Repertoire
Productive clonal IGH rearrangements could be obtained for nine cases (Table S3). Based on the percentage of IGH variable region (IGHV) gene identity to the germline sequences, 7/9 cases could be classified as "significantly mutated" (identity <97%), whereas the two remaining cases were "borderline/minimally mutated" (97-99.9% identity). The majority of cases (6/9) used IGHV3 subgroup genes while the remaining used IGHV1 subgroup genes. Among the latter, IGHV1-2 was identified in two cases-a gene which has been found to be over represented in splenic marginal zone lymphoma (SMZL) [9], although significantly less mutated than in the present study. In contrast, neither of the two predominant genes that contribute to the CLL repertoire [10]-IGHV1-69 (mostly non-mutated) and IGHV4-34 (mostly mutated)-were identified in the present cohort.

Clinical Outcome
All patients were initially monitored with a wait and watch strategy. After a median follow-up of 48 months, the lymphocyte count doubled in 12/18 patients (67%, median doubling time: 16 months, range: 5-46 months). Eight (44%) patients developed a clinically detectable adenopathy and/or organomegaly (median time to develop clinically detectable adenopathy and/or organomegaly: 22 months; range: 8-48 months). Four patients were treated with chemotherapy or immunochemotherapy, and one underwent splenectomy because of a symptomatic increase of spleen volume associated with anemia (Table 1 and Figure 1a). For these five patients, the median time to first treatment was 23 months. Among them, two patients presented both a chromosome 17p deletion and a TP53 mutation. Two patients achieved complete remission with a rituximab-chlorambucil regimen, and one achieved partial remission after attenuated R-CHOP (rituximab + cyclophosphamide + doxorubicin + vincristine + prednisone). One patient died a few weeks after initiation of chlorambucil. The splenectomized patient achieved a partial response and a diagnosis of MZL was made on the spleen histology. With a median follow-up of 48 months, the overall survival rate was 83% (Figure 1b). Of note, histological transformation into diffuse large B-cell lymphoma was observed in one patient after two lines of treatment. This patient harbored a chromosome 17p deletion associated with a TP53 mutation, both known as poor prognosis markers in CLL [11,12].

Discussion
We report herein a comprehensive description of a cohort of 18 patients with monoclonal lymphocytosis that cannot be accurately classified according to current guidelines [1]. For these patients, the diagnostic procedure was limited to the analysis of circulating lymphocytes or bone marrow because at the time of diagnosis enlarged lymph node, splenomegaly, or hepatomegaly were not detected. This is not a common situation (less than 1/100 CLL diagnoses in our center).
An important question is to what extent these patients can be included within an existing class of patients described by the WHO. Cytological examination of blood smears found in all patients both CLL-like and MZL-like atypical lymphocytes [13]. Immunological features were also shared between CLL (CD5 + ) and MZL (RMH score < 3, CD20 high , CD43 − ) [3,4,13,14]. Most cases harbored a chromosome 12 trisomy-a cytogenetic feature found in both CLL and MZL but at lower frequencies [11,15,16]. As we found other cytogenetic abnormalities associated with CLL (chromosome 13q deletion and chromosome 17p deletion) [11], but not those associated with MZL (chromosome 3 and 18 trisomies, or chromosome 7q deletion) [15][16][17], the cytogenetic data were more evocative of CLL. High-throughput sequencing did not further resolve the classification of these cases, as most patients had a molecular profile compatible with both CLL and MZL. Note that we did not find NOTCH1 mutations, which have been associated with chromosome 12 trisomy in CLL [18]. Interestingly, in two of the eight patients for whom a molecular profile was available, we found an MYD88 p.S219C mutation, which is rarely found in other B-cell neoplasms [12,[19][20][21][22][23]. Furthermore, the IGHV repertoire, which was mostly mutated, seemed distinct from those reported in CLL [10], MZL [9], or in the recently described clonal B-cell lymphocytosis of marginal zone origin (CBL-MZ) [24]. Finally, the diagnosis of lymphoplasmacytic lymphoma/Waldenström macroglobulinemia seems unlikely, as an MYD88 mutation was found in only 2/8 patients, and the classical p.L265P mutation was never observed [23].
CBL-MZ was defined as clonal B-cell lymphocytosis with an RMH score < 3 by Xochelli et al. in a population that included patients with absolute lymphocytosis [24]. Most of these patients had negative or weak CD5 staining and had cytogenetic features consistent with MZL, which contrasts with the cohort reported in this paper. These differences might be explained by the exclusion criteria of the present study, in particular the exclusion of lymphocytosis with cytoplasmic projections that were diagnosed as splenic diffuse red pulp small B-cell lymphoma or SMZL according to the WHO classification [1]. However, the only patient in our study with spleen histology was finally diagnosed with an SMZL. An alternative diagnosis is atypical CLL, which is not recognized as an entity in the WHO classification. Atypical CLL refers to clonal absolute lymphocytosis with atypical cytological [25] or immunological features (RMH score < 4) [26,27]. However, the lack of a widely accepted definition has compromised its recognition as a true entity. Interestingly, increased prevalence of chromosome 12 trisomy has been constantly reported in atypical CLL as compared with classical CLL [27], as it was the case in our cohort. Atypical CLL with chromosome 12 trisomy has been associated with t (14;19) [IGH/BCL3] translocation [5]-a genetic abnormality found in one case of the cohort.
Overall, we report a series of cases displaying intermediate features between CLL and MZL which cannot currently be accurately classified. It is possible that this cohort includes some cases of circulating MZL, expressing CD5 and without classical cytogenetical features diagnosed in early phase, without enlarged lymph node or organomegaly. For example, the patient who underwent a splenectomy received an MZL diagnosis. Only two patients underwent a bone marrow trephine biopsy, which did not allow us to correctly classify the lymphoproliferative disease. However, we cannot exclude the possibility that a bone marrow trephine biopsy would have resulted in a diagnosis for some of these patients. Another limitation of this study is that imaging was not performed for all patients, thus the organomegaly cannot be completely excluded in these cases.
This study highlights limitations of the WHO classification. It provides evidence that refining this classification system is of utmost importance. Larger studies are necessary to precisely know the features of these cases and classify them correctly, especially if a histological diagnosis is not possible. The correct classification of these cases has potential consequences for therapeutic management. Consider the three patients with a TP53 alteration. The first-line of treatment should be a B-cell receptor inhibitor if considered as CLL [28], whereas there is no first-line indication for these drugs if these pathologies are considered as MZL [2]. Consequently, a histological diagnosis based on bone marrow trephine biopsy must be realized at diagnosis. If treatment is needed, this will allow for the most suitable therapeutic option to be chosen in accordance with the lymphoproliferative disorder involved. However, sometimes this is not sufficient to classify the disease. If lymph node enlargement appears during follow-up and the lymph node is easily accessible, it seems reasonable to complete the diagnostic work-up with the histological analysis of a biopsy. Of note, in our series, the five patients who needed treatment developed a lymph node enlargement or an organomegaly before the treatment began.

Patients
We selected patients referred to our tertiary care center between 1999 and 2015 who had absolute lymphocytosis (above 5 G/L) due to the presence of monoclonal B cells, but without a clinically detectable enlarged lymph node, splenomegaly, or hepatomegaly. We excluded patients with definitive diagnosis according to the WHO classification [1], such as those with CLL (RMH score ≥ 3) [3], those with MCL (detection of the t(11;14)(q13;q32) translocation) [1], as well as those with cytological, phenotypical, and/or cytogenetical features characteristic of a specific lymphoid neoplasm (e.g., hairy cell leukemia, SMZL, prolymphocytic leukemia). Overall, 18 patients who fulfilled these criteria were identified.

Morphological Analysis
Peripheral blood smears were stained with May-Grünwald-Giemsa and lymphocyte cytology was independently evaluated by two experimented cytologists (MD and LB). Lymphoid cells were classified as CLL-like lymphocytes (i.e., small lymphocytes with a round, clumped nucleus and scanty cytoplasm), atypical lymphocytes (i.e., larger cells with an irregular nucleus and chromatin condensed in small irregular clumps, with more abundant and pale cytoplasm) or cytologically normal lymphocytes.

Cytogenetic Analysis
Conventional cytogenetic studies were performed on peripheral blood samples after 48-72 h expansion in RPMI 1640 medium supplemented with 12% fetal calf serum, L-glutamine, penicillin, and streptomycin. Cell stimulation was induced using either Phorbol12-myristate 13 acetate (TPA 0.1 µg/mL) or CpG-oligonucleotides added to interleukin 2. Chromosome analyses were carried out on RHG and GBG-banded metaphases as previously described and evaluated according to the International System for Human Cytogenomic Nomenclature recommendations [31,32].
Interphase FISH experiments were performed on chromosome spreads as previously reported using the XL CLL probe kit (XL DLEU/LAMP/12 cen and XL ATM/TP53) and the XL CCND1 break Apart Probe (Metasystems, Altlussheim, Germany) [31].

High-Throughput Sequencing
Libraries were prepared for a custom panel (provided in Table S1) with the Haloplex target enrichment system (Agilent, Santa Clara, CA, USA), according to the supplier's instructions, and sequenced using a NextSeq 500 (Illumina, San Diego, CA, USA). Raw data from sequencing were analyzed in parallel by two commercial pipelines: Surecall (Agilent, Santa Clara, CA, USA) and Nextgene (SoftGenetics, State College, PA, USA) and manually reviewed. Single-nucleotide polymorphisms with a minor allele frequency above 1% were filtered out.

PCR Amplification and Sequence Analysis of Immunoglobulin Gene Rearrangements
Amplification of IGH gene rearrangements was performed on genomic DNA according to the Biomed2 protocols [33]. Clonal PCR products were purified and directly sequenced on both strands. Sequences were then analyzed using the international ImMunoGeneTics information system (IMGT) database [34] and the IMGT/V-QUEST tool (http://www.imgt.org) [35].

Definitions of Treatment Responses
Treatment response was assessed according to the 2008 guidelines from the International Workshop on Chronic Lymphocytic Leukemia [36]. Treatment-free survival was defined as the interval between diagnosis and first treatment. Overall, survival was defined as the interval between diagnosis and death.

Statistical Analysis
Survival curves were drawn according to the Kaplan-Meier method using GraphPad PRISM 6 software (GraphPad Software, La Jolla, CA, USA). The index date was the date of diagnosis.

Conclusions
This work describes a group of patients who expressed intermediate features between recognized WHO entities. Even if this situation is rare, and must occur especially when the evolution seems indolent and explorations are limited, it highlights a limitation of the WHO classification. However, as treatment choice becomes increasingly dependent on classification, a correct diagnosis must be sought in case patients need treatment. Studies of larger cohorts are needed to precisely determine the features of these cases and classify them correctly, especially when a histological diagnosis is not possible.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/11/10/1495/s1, Table S1: Composition of the panel of genes analyzed by high throughput sequencing, Table S2: Prevalence of gene mutations in MZL and CLL, according to published data, Table S3: Sequence analysis of immunoglobulin gene rearrangements for 9 patients.

Conflicts of Interest:
The authors declare no conflicts of interest.