Frequency of additional clonal populations detected by high sensitivity flow cytometry in patients with hairy cell leukemia
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- Roshal, M. & Cherian, S. J Hematopathol (2012) 5: 123. doi:10.1007/s12308-012-0137-9
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Samples from 115 patients with hairy cell leukemia (HCL) were analyzed by high sensitivity flow cytometry for evidence of an additional B cell or plasma cell clone. We found that 10.4% of HCL patients harbored an additional clonal population in either peripheral blood or bone marrow. Among the patients with additional clones, 58% had the second population present at the time of HCL diagnosis and 42% developed the second clone in a subsequent sample. Half of the clonal populations identified represented true disease entities rather than a monoclonal B lymphocytosis (MBL) or monoclonal gammopathy of undetermined significance (MGUS). The frequency of MBL and MGUS was similar to that reported in the general population, but the frequency of B cell-derived malignancies appears increased in the HCL cohort. Compared to patients without additional clones, the patients with second clones were older (median age 66 vs. 55 years old). Patients over age 60 were at higher risk for a second clone (18.2% vs. 5.6%, OR 3.7, P < 0.05). Among the patients with a second clone, 75% had second B cell clone, 42% had a plasma cell clone, and 17% had both. We have also identified a clear case of bi-clonal HCL; a finding not previously reported using contemporary diagnostic criteria. We conclude that second clonal expansions are relatively common in patients with HCL, in particular in older patients, and may represent clinically significant neoplasms.
KeywordsHairy cell leukemiaFlow cytometryB cell lymphomaPlasma cell neoplasm
Hairy cell leukemia (HCL) is a rare blood-, bone marrow-, and spleen-based lymphoproliferative disorder of mature B cells. The neoplastic population can be readily identified immunophenotypically by the expression of CD11c, CD25, and CD103 [1, 2]. Detection of the expression of these antigens on an expanded, clonal B cell population is diagnostic for this disorder. Additionally, the IL3 receptor (CD123) has been proposed as a useful marker for HCL . The origin of the neoplastic cell in hairy cell leukemia is not entirely clear, but the near universal absence of expression of the activation marker CD38, germinal center marker Bcl-6, and germinal center-derived memory cell marker CD27  argue against a germinal center derivation of these cells, despite a not-infrequent expression of CD10 [5, 6].
Patients with HCL are at increased risk for subsequent non-hematopoietic and hematopoietic neoplasms [7–12]. Reasons for the increase are not entirely clear. Immunosuppressive therapy for treatment of HCL, immunosuppressive effect of HCL itself, and/or intrinsic cancer susceptibility of patients with HCL may all play a role.
Aside from the aforementioned epidemiologic studies, reports of second hematologic malignancies in patients with HCL are mostly contained in case reports and series containing very few HCL patients [13–16]. Thus, the true incidence of second clonal expansions in HCL is not established. The largest study to date contained 13 HCL and HCL variant (HCL-v) cases from both unselected patient samples and selected patient samples referred to the reference flow cytometry laboratory for confirmation of a second clone. That study demonstrated a second clonal expansion in 4 out the 13 patients. This finding suggested a possible increased incidence of second clones in HCL, although the study design precluded a definite conclusion . We build on these observations in a much larger study specifically focused on an unselected cohort of HCL patients.
In a retrospective analysis, we have identified 115 individual patients with HCL that had definitive immunophenotypic findings characteristic of HCL (positivity for CD25, CD11c, and CD103 on a clonal mature B cell population) over a 5-year period in a single large academic institution. The frequency of additional clonal hematopoietic proliferations and their immunophenotypic and clinical characteristics are described.
In addition, we detected an unequivocal case of bi-clonal HCL that was confirmed by molecular studies. This, to our knowledge, is the second report of such a case and the first that uses modern diagnostic criteria of HCL. A prior case report of a single patient was published in 1986, and it is not entirely clear whether the patient’s diagnosis in that report would meet the modern diagnostic criteria for HCL as it was based on a relatively limited immunophenotyping that would not distinguish HCL from HCL-v . Finally, we took advantage of the large number of cases to further characterize additional immunophenotypic characteristics of HCL.
Materials and methods
Cases selection criteria
This study was approved by the institutional review board at the University of Washington. All immunophenotyping studies were performed at the University of Washington flow cytometry laboratory in the course of routine clinical work. Cases were selected based on the flow cytometric detection of HCL between January 2004 and December 2009. In order to qualify for the study, HCL populations needed to demonstrate monoclonal expression of light chains with bright or moderate co-expression of CD11c, CD25, and CD103. Whenever indicated, clinical and morphologic correlation was performed to confirm the diagnosis. Cases that lacked expression of any of the antigens classically associated with HCL were excluded from further analysis to avoid contamination of the data set with HCL-v and other B cell lymphoproliferative disorders. The cases with a second clone were re-reviewed by two hematopathologists (SC and MR) in order to assure uniform flow cytometric detection of bi-clonal populations and second B cell or plasma cell clones. Only cases where both reviewers agreed about the unequivocal presence of the second clonal population were included. Clinical and pathologic data for cases with a second clone were then reviewed to determine if the second clone met the 2008 WHO criteria  for a true distinct disease entity (B cell lymphoma or plasma cell myeloma) as opposed to monoclonal B lymphocytosis (MBL) or monoclonal gammopathy of undetermined significance (MGUS).
Cell sorting was used to separate kappa- and lambda-restricted hairy cell populations in the bi-clonal HCL case. Cells were processed as described under “immunophenotyping”. Kappa- and lambda-restricted populations were individually sorted using flow cytometric sorting on a FACS-ARIA instrument from BD. Cell sorting was performed using the same staining and fixation method as for flow cytometric analysis using kappa-FITC, lambda-PE, and CD19-ECD.
Molecular analysis on the individually sorted kappa- and lambda-expressing populations as well as unsorted peripheral blood from the patient with bi-clonal HCL was performed as described below. DNA was prepared using the Puregene kit (Gentra Systems) according to the method of the manufacturer. The DNA segment of interest was amplified by PCR using Biomed-2 framework one to three fluorescently labeled primer sets . The products were then size-fractionated by capillary electrophoresis using Applied Biosystems 3130 Sequence Analyzer. Negative and positive controls were included in each assay.
Characteristics of the study group and HCL immunophenotype
Demographic and immunophenotypic characteristics of patients with HCL
Without additional clones
With additional clones
Immunophenotypic and clinical characterization of second clonal expansions
Twelve of the 115 cases showed flow cytometric evidence of a second clonal population. Compared to patients with HCL alone, the group with a second clone was significantly older [median age 66 (47–85) vs. 55 (27–95), p < 0.05]. Overall, patients 60 years and older demonstrated a significantly increased risk for a second clone [18.2% vs. 5.6%, odds ratio 3.7 (1.04–13.2), p < 0.05]. No significant differences were found in sex or immunophenotype (specifically the likelihood of expression of CD10 or CD123) when comparing HCL cases with and without a second clonal population. These finding are summarized in Table 2.
Immunophenotype and clinical characterization of additional clones in HCL patients
HCL light chain/clone size by FC
Additional B cell clone
Immunophenotype of additional population/% of WBC
Second clone detected at presentation
CD19 (absent), CD45 (absent), CD56 (subset), and cytoplasmic kappa light chain restriction with normal expression of CD38/3.0%
CD19 (absent), monoclonal kappa cytoplasmic light chain restriction with variable CD45, low CD20, and absent CD56/0.3%
MZL/LPL, MBL, possible MGUS
(1) CD25 and kappa light chain restriction with normal expression of CD45, CD19, and CD20 without CD5, CD10, CD11c, CD103, or FMC-7 (MZL/LPL)/10%; (2) CD5, CD20 (low), CD23, and absent to low level lambda light chain expression with normal expression of CD45 and CD19 without FMC7 or CD10 (MBL)/0.3%; (3) CD19 (variable, subset absent), CD45 (subset decreased), CD56 (subset), and monoclonal kappa cytoplasmic light chain restriction with normal expression of CD38 and CD138 (MGUS)/0.65%
CD45, CD19, and CD20, co-expression of CD5, CD38, low FMC7, and low CD25 with dim kappa light chain restriction without CD10, CD103, or CD23 (FISH demonstrated trisomy 12)/18.5%
CD5, CD20 (low), CD23 (low to absent), and kappa surface light chain restriction (low) and normal expression of CD45 and CD19 without FMC7/0.7%
CD5, CD20, and absent to low kappa surface light chain restriction with normal expression of CD45 and CD19 without significant CD10 or CD38/5.3%
CD5, CD20 (low), CD23, and bi-clonal surface light chain restriction (low kappa predominant) and normal expression of CD45 and CD19 without FMC7 or CD10/0.5%
Second clone detected at follow-up
MM/plasma cell leukemia
CD19 (absent), CD38 (low), CD45 (variable low), CD56, and monoclonal kappa cytoplasmic light chain restriction/20%
Mantle cell lymphoma
CD45, CD5, CD19, CD20, low CD38, and lambda light chain restriction without CD10 or CD23 (FISH demonstrated a CCND1 translocation)/25.3%
CD5, CD20 (low), CD23, and kappa surface light chain restriction (low) and normal expression of CD45 and CD19 without FMC7 or CD10/60%
Initially thought to represent a plasma cell neoplasm not further classified; subsequent LN biopsy showed DLBCL with plasmacytic differentiation
Abnormal monoclonal kappa cytoplasmic light chain restriction with normal expression of CD19, CD45, and CD38 without CD56/5.4%. This was initially thought to represent a PCN; however, a subsequent lymph node biopsy demonstrated both an abnormal B cell population with CD20 (variably decreased), CD38 (variably increased), and kappa light chain restriction with normal expression of CD45 and CD19 without CD5 or CD10/6.2% and an abnormal plasma cell population expressing CD45 (increased) and cytoplasmic kappa light chain restriction with normal expression of CD19 and CD38 without CD56/16.7%. The findings in the lymph node were felt to represent DLBCL with plasmacytic differentiation
CD5, CD20 (low), CD23, FMC7, and absent surface light chain expression with normal expression of CD45 and CD19 without CD10/0.02%
Bi-clonal hairy cell leukemia
To our knowledge, this investigation is the first large systematic study analyzing data from unselected cases of HCL that explores the frequency of bi-clonal HCL proliferations or the coexistence of a second B cell or plasma cell clone in patients with HCL by flow cytometry. We documented that more than one in ten patients with HCL harbor one or more additional clonal B cell or plasma cell populations. Moreover, nearly one in five older patients with HCL harbors an additional detectable clone. The frequency of incidental second clones representing MBL or MGUS (5.2% and 1.7%, respectively) was similar in this cohort of patients with HCL as compared to that previously reported in the literature for healthy adults [22–26]. However, half of the second clones detected (6 of 12) represented true disease entities corresponding to an overall frequency of 5.2% of patients in this cohort. Although it is difficult to find comparable data for the general population, for non-Hodgkin’s lymphoma, the reported lifetime risk is 2.13% and the age-adjusted incidence is 19.8 per 100,000 per year, while for myeloma, the reported lifetime risk is 0.65% and the age-adjusted incidence is 5.7 per 100,000 per year as determined by data from the SEER database (calculations based on cases from 2004 to 2008 from 17 SEER geographic regions, see http://seer.cancer.gov/statfacts/html/nhl.html#prevalence, http://seer.cancer.gov/statfacts/html/mulmy.html#risk). These data suggest that the frequency of B cell lymphoma and/or myeloma may be higher in patients with HCL than in the general population.
More than half (7 of 12) of second clonal populations were detected at presentation and the remaining five at follow up for HCL. From this limited sample, it appears that HCL itself or a predisposition to developing a neoplasm of B cell or plasma cell origin, rather than HCL treatment, may be a primary factor in the development of second clone. This conclusion is supported by prior studies demonstrating lack of association of the presence of a second neoplasm in HCL and a specific therapeutic regimen [7, 12]. However, it is interesting to note that while the majority of second clones detected at presentation were clinically insignificant, the majority detected at follow-up were clinically significant. Although it is tempting to speculate that HCL or HCL treatment may contribute to the progression of indolent clonal expansions to true neoplasms, follow-up longitudinal studies would be needed to test this hypothesis.
The clonal relationship between the HCL clone and additional clone is not entirely clear on the basis of the data available for review. Because this study was retrospective, we were unable to analyze the populations by molecular methods. The clones were clearly immunophenotypically distinct and there was no significant association between the light chain restriction in HCL and additional clonal population. Whether the two clones shared a more distant precursor is a question that requires further study. In light of recent data suggesting that CLL may represent a stem cell disorder rather than a neoplasm originating in a disordered mature B cell  it would be interesting to investigate whether a similar phenomenon may be occurring in HCL.
Our study may not be entirely reflective of a true frequency of second clones in HCL. Observation bias, particularly in patients presenting with symptomatic neoplasms other than HCL and in whom HCL may have been an incidental diagnosis may certainly cause the study to overstate the true prevalence of second clones in HCL. Additionally, as only blood or bone marrow was evaluated in all but one case in this study, we may have missed nodal or extranodal lymphomas. Finally, longer follow-up times may reveal a greater number of cases with a second clone. Nonetheless, our experience is likely to be typical for samples seen in pathology laboratories performing flow cytometric and morphologic evaluation in patients with HCL.