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Year : 2017  |  Volume : 60  |  Issue : 1  |  Page : 84-86
Biclonal chronic lymphocytic leukemia: A study of two cases and review of literature


1 Department of Pathology, Hematopathology Laboratory, Tata Memorial Centre, Mumbai, Maharashtra, India
2 Department of Cancer Cytogenetics, Tata Memorial Centre, Mumbai, Maharashtra, India

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Date of Web Publication14-Feb-2017
 

   Abstract 

Chronic lymphocytic leukemia (CLL) is a common, immunophenotypically well-defined mature B-cell neoplasm. Demonstration of more than 5000/μL CD5+ B-cell population with co-expression of CD23, weak expression of CD20, and one type of immunoglobin light chain (either kappa or lambda) is necessary for the diagnosis of CLL. However, CLL with two populations of B-cells expressing both kappa as well as lambda (biclonal) light chains are extremely rare and has not been reported from India. We report two cases of biclonal CLL presented with leukocytosis, typical morphological features, and distinct immunophenotype of CLL. These cases are also an example which suggests that careful attention to the morphology of the blood smear and the entire immunophenotype panel is a must and will aid the proper diagnosis as only light chain ratios can be misguiding.

Keywords: Biclonal chronic lymphocytic leukemia, chronic lymphocytic leukemia, immunophenotype

How to cite this article:
Ghodke KA, Patkar NV, Subramanian P G, Gujral S, Kadam PA, Tembhare PR. Biclonal chronic lymphocytic leukemia: A study of two cases and review of literature. Indian J Pathol Microbiol 2017;60:84-6

How to cite this URL:
Ghodke KA, Patkar NV, Subramanian P G, Gujral S, Kadam PA, Tembhare PR. Biclonal chronic lymphocytic leukemia: A study of two cases and review of literature. Indian J Pathol Microbiol [serial online] 2017 [cited 2017 May 26];60:84-6. Available from: http://www.ijpmonline.org/text.asp?2017/60/1/84/200019



   Introduction Top


Demonstration of clonality forms the basis for the diagnosis of chronic lymphoproliferative disorders (CLPDs). B-cell or T-cell clonal expansion for the diagnosis of is demonstrated by two methods, i.e., molecular assay or flow cytometric immunophenotyping (FCI). FCI is easy, widely available, and traditionally used for the confirmation of clonal proliferation of B-cells.[1] Normal and reactive B-cell populations show the expression of both kappa and lambda light immunoglobulin chains usually with ratio of 2–4:1.[1] In contrast to neoplasms of mature B cells usually represent a single clone of cells that express only one class of light chain (i.e., kappa or lambda) along with other immunophenotypic features.[2] However, rare cases of low-grade B-CLPD also have been reported have more than one clone of B-cells (i.e., biclonal) with two population of B-cells expressing same immunophenotype but different light chain expression (both kappa and lambda) and genetics features. In such cases, there is a possibility of miss the diagnosis considering these cells polyclonal and reactive. In this case study, we report two such cases of chronic lymphocytic leukemia (CLL) with biclonal B-cell population along with their clinicopathologic features. To the best of our knowledge, this is the first report of biclonal CLL from India.


   Materials and Methods Top


We evaluated clinical, and laboratory features of two cases of biclonal CLL presented in our center over a period of the last 2 years. The details of an individual case are given below. Complete blood cell count was done on Advia 212i. Peripheral blood smears (PBSs) were stained with Wright's stain. Peripheral blood (PB) specimens were further processed for FCI using a lyse-stain-wash technique and a comprehensive 8-color antibody panel for CLPD on Navios (Beckman Coulter, USA) and analyzed using kaluza-1.3 software (Beckman Coulter, USA). The CLPD panel included monoclonal antibodies against CD1d, CD2, sCD3, CD4, CD5, CD8, CD10, CD11c, CD16, CD19, CD20, CD22, CD23, CD38, CD43, CD45, CD56, CD81, CD200, Kappa, and lambda surface light chains. Data were collected and analyzed using a CD45–side scatter-based gating strategy [Figure 1].
Figure 1: Antigen expression pattern in the chronic lymphocytic leukemia cases. Dot plots of (a) case 1. (b) Case 2 with chronic lymphocytic leukemia cells (red dots) express CD19 and dim to negative CD20 with co-expression of CD5, co-expression of CD43 and CD200. Surface light chain immunostaining showed subset of atypical lymphoid cells is positive for kappa, and other subset is positive for lambda surface light chain

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IGVH mutation study was performed on the genomic RNA extracted from the PB samples on the Qiagen QIAcube. cDNA was synthesized from the RNA by reverse transcriptase. The DNA was amplified by PCR using forward “leader” primer sets for Vh1 to Vh6 regions of the IGVH gene and a common reverse primer. A negative control and no template control were put up with each PCR. The amplified products were subjected to gel electrophoresis. Bidirectional sequencing of the monoclonal amplicons was performed on ABI3500 genetic analyzer. Comparative sequence analysis of the tumor with the germline IGVH sequences was performed on NCBI and IMGT reference databases. A difference of >2% from the germline sequence was used as a cutoff to assign mutated status.

For cytogenetic evaluation, fluorescence in situ hybridization studies were performed on interphase cells from PB with panel of probes consisting of LSI D13S319 (13q14.3)/LSI 13q34, LSI ATM (11q22.3)/CEP11, LSI TP53 (17p13.1)/CEP17, CEP 12, LSI 6q21/SE6, and LSI break apart IgH translocation probe (Vysis Abbott Molecular, Delkenheim, Germany and Kreatech Diagnostics, The Netherlands) according to the manufacturer's protocol and a total of 200 cells were analyzed in each specimen.


   Results Top


The details of clinical presentation and laboratory findings were included in [Table 1]. In both cases because of leukocytosis with atypical lymphocytosis on PBS, FC IPT was asked.
Table 1: Clinical details and laboratory findings of two cases

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Independent immunophenotypic analysis in both cases revealed abnormal lymphoid cells which are small in size by forward light scatter and express B-cell markers (CD19, CD20, and CD22) with co-expression of CD5 and CD23, co-expression of CD43 and CD200 but negative for CD3, CD4, CD7, CD8, CD10, CD11c, CD38, CD81, CD16, CD56, and sTCRgd. On evaluation of surface light chains, subset of atypical lymphoid cells is positive for kappa, and other subset is positive for lambda surface light chain. This biclonality is specific because of applied gating was sequential and negative expression of isotype control.

The immunoglobulin surface light chain staining was also repeated using different company antibody [Figure 1]. IGVH mutation study data was available in the second case only, and it revealed unmutated IGVH sequence. Cytogenetic analysis revealed evidence of heterozygous 13q deletion (75% of cells) using LSI 13q14 and LSI 13q ter probe in the first case while there was no evidence of trisomy 12, TP53 deletion, ATM (11q) deletion, MYB (6q) deletion, and IgH translocation. The second case did not reveal any cytogenetic abnormality.

Both the patients kept under observation with no active treatment. In the recent clinical follow-up, there was no change in disease status in both the patients after 18 months.


   Discussion Top


We are describing two unique cases of CLL that had the typical morphologic and immunophenotypic features of CLL except for the biclonal pattern of immunoglobulin light chain expression. CLL is a common mature B-cell lymphoproliferative neoplasm of elderly with B-cell proliferation expressing characteristic immunophenotype features, i.e. intermediate to weak CD19, weak to negative CD20, weak CD22 and CD79a, and co-expression of CD5 and CD23 with weak monoclonal (either kappa or lambda) surface immunoglobulin light chain expression.[3] CLL with biclonal B-cell proliferation is rare. The reported incidence of biclonal CLL among all CLL cases varies from 0.7% to 3.4%.[4],[5] We diagnosed two cases of biclonal CLL in 194 cases of CLL over a period of 18 months with the incidence of 1.03%. Gonzalez-Campos et al.[4] found one biclonal CLL by FC IPT among 130 (0.7%) CLL patients. Sanchez et al.[6] reported biclonal CLL by FC IPT in 12/353 (3.4%) typical CLL and in 4/29 (13.8%) atypical CLL patients. The largest study by Kern et al.[5] identified 76 patients (1.4%) with biclonal CLL by FC IPT.

Kern et al.[5] reported mutated IGHV status with similar frequencies in biclonal CLL and monoclonal CLL (61.1% vs. 60.9%). In addition, they also found two different B-cell receptor rearrangements more frequently in immunophenotypically biclonal disease than in monoclonal CLL (37.1% vs. 2.7%; P < 0.001).[5] Sanchez et al.[6] also found two different B-cell clones in 92.8% CLL patients with biclonal immunophenotype. It is known that normal and malignant B-cells could show double productive IGVH rearrangements; however, only one rearrangement will be translated to protein and expressed on the cell surface due to allelic exclusion. Therefore, biclonal CLL may reflect lack of allelic exclusion.[7] Thus, the absence of two different B-cell receptor rearrangements might be found in biclonal CLL.

In the literature, most frequent cytogenetic abnormality in biclonal CLL found was del 13q at 62.2%, followed by trisomy 12 (15.9%).[5] In the present study, we have also found del 13q in single patient.

Clinically, biclonal CLL are significant as they require early initiation of treatment as compared to the monoclonal CLL.[5],[6] In addition, these patients also had a slightly higher incidence of splenomegaly and deaths.[6] In some of these cases, loss of biclonal nature and its conversion to monoclonal disease in a course and follow-up investigations have been identified.[5]


   Conclusion Top


These are unique cases which highlight biclonality in CLL that actually reflects two different subpopulations as detected by FC IPT with different genetic background and thus the light chain ratios can be misguiding. These cases are also an example which suggests that careful attention to the morphology of the blood smear and the entire immunophenotype panel is a must and will aid the proper diagnosis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
Craig FE. Flow cytometric evaluation of B-cell lymphoid neoplasms. Clin Lab Med 2007;27:487-512, vi.  Back to cited text no. 1
    
2.
Craig FE, Foon KA. Flow cytometric immunophenotyping for hematologic neoplasms. Blood 2008;111:3941-67.  Back to cited text no. 2
    
3.
Swerdlow S, Campo E, Harris N, Jaffe, E.S., Pileri, S.A., Stein, H., et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC; 2008.  Back to cited text no. 3
    
4.
González-Campos J, Ríos-Herranz E, De Blas-Orlando JM, Martín-Noya A, Parody-Ruiz-Berdejo R, Rodríguez-Fernández JM. Chronic lymphocytic leukemia with two cellular populations: A biphenotypic or biclonal disease. Ann Hematol 1997;74:243-6.  Back to cited text no. 4
    
5.
Kern W, Bacher U, Schnittger S, Dicker F, Alpermann T, Haferlach T, et al. Flow cytometric identification of 76 patients with biclonal disease among 5523 patients with chronic lymphocytic leukaemia (B-CLL) and its genetic characterization. Br J Haematol 2014;164:565-9.  Back to cited text no. 5
    
6.
Sanchez ML, Almeida J, Gonzalez D, Gonzalez M, Garcia-Marcos MA, Balanzategui A, et al. Incidence and clinicobiologic characteristics of leukemic B-cell chronic lymphoproliferative disorders with more than one B-cell clone. Blood 2003;102:2994-3002.  Back to cited text no. 6
    
7.
European Research Initiative on CLL, ERIC. Available from: http://www.ericll.org/index.php. [Last accessed on 2007 Jan 21].  Back to cited text no. 7
    

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Correspondence Address:
Prashant R Tembhare
Department of Pathology, Hematopathology Laboratory, 7th Floor, Annex Building, Tata Memorial Centre, Dr E Borges Road, Parel, Mumbai - 400 012, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.200019

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