Leukemia (2002) 16, 1454–1459  2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu Simultaneous diagnosis of hairy cell and chronic lymphocytic leukemia/small lymphocytic : a frequent association? E Gine´1, F Bosch1, N Villamor2, M Rozman2, D Colomer2,ALo´pez-Guillermo1, E Campo2 andE Montserrat 1

1Institute of and , Department of Hematology, Hospital Cli´nic, Institut d’Investigacions Biome`diques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; and 2Hematopathology Unit, Hospital Clı´nic, Institut d’Investigacions, Biome`diques August Pi i Sunyer (IDIBAPS), Barcelona, Spain

The association of hairy cell leukemia (HCL) with other neo- phomas are the most frequently hematological malignancies plasms, mainly non-Hodgkin’s , is well known. reported.10,15,16 The association of HCL andchronic lympho- However, the simultaneous diagnosis of HCL and chronic lym- phocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is cytic leukemia/small lymphocytic lymphoma (CLL/SLL) is con- rare, with only few cases of such an association having been sidered to be exceedingly rare. Thus, in an extensive com- reported. We describe three patients with a well-characterized puter-assistedsearch of the literature, only one case of HCL in whom a CLL/SLL population was detected. Of note, transformation of CLL/SLL into HCL,17 one case of HCL diag- these cases represent a significant proportion (11.5%; 95% CI: nosedyears after the diagnosisof CLL/SLL, 18 andsome cases 0% to 24%) of the total number of HCL cases diagnosed in our of lymphoproliferative disorders with intermediate features institution during the same period of time. All three patients 19–21 were treated with deoxycoformycin. They achieved a complete between HCL andCLL/SLL have been reported. More- response of the HCL, whereas the CLL/SLL population per- over, in the four largest studies assessing the incidence of sisted in all cases. The immunoglobulin gene rearrangement secondneoplasms in patients with HCL, includinga total of analysis, in two informative cases, suggested that the HCL and 1433 patients, no case of CLL/SLL has been described.12–15 CLL/SLL populations arose from different clones. This Since 1991, all the cases of HCL diagnosed in our center study indicates that the association of HCL and CLL/SLL might were systematically screened for the presence of additional be much more frequent than previously recognized. Therefore, a large panel of monoclonal antibodies, including those neces- B cell populations using an extensive panel of monoclonal sary to detect CLL/SLL, should be employed when studying antibodies. Using this approach, three patients with the simul- patients with HCL. taneous existence of a HCL and CLL/SLL were identified. The Leukemia (2002) 16, 1454–1459. doi:10.1038/sj.leu.2402553 aim of the present study is to describe the clinical, morpho- Keywords: hairy cell leukemia; chronic lymphocytic leukemia; logic, immunophenotypic andmolecular features of these small lymphocytic lymphoma; immunoglobulin gene rearrangement cases andto comment on the association between HCL and CLL/SLL.

Introduction Patients and methods Hairy cell leukemia (HCL) is an infrequent chronic lympho- proliferative disorder (2% of all adult ), initially Patients and treatment described by Bouroncle et al,1 which is characterizedby the infiltration of the andthe by mature lym- Between 1991 and1997, 26 consecutive patients were diag- phocytes with typical cytoplasmic projections. , nosed with HCL in our institution according to standard clini- , peripheral involvement andopportunistic cal, morphological andphenotypic criteria. 22 During the same infections are common clinical manifestations of HCL. In most periodof time, 270 patients were diagnosedwith CLL/SLL. 23,24 instances the prognosis of patients with HCL is good, with an In all cases the immunophenotype of the neoplasic cells was overall survival of around85% at 8 years from diagnosis. 2–4 studied by cytofluorometry as described below. Phenotypic Hairy cells strongly express CD20, CD22 andmonoclonal andmolecular analysis from peripheral bloodandbone mar- surface immunoglobulins, together with the distinctive row samples were performed at diagnosis and during the expression of CD25, CD11c, FMC7 andCD103. 5, course of the disease. Most patients with HCL were treated structural abnormalities involving the pericentromeric regions with deoxycoformycin (DCF) at a dose of 4 mg/m2 every 2 of chromosomes 5 and2, 1q42 abnormalities, and14q alter- weeks, until maximal response or for a minimum of six cycles ations are frequently foundin patients with HCL, 5 but there is in case of treatment failure. Response was evaluatedaccord- no recurrent chromosomal aberration typical of this disease. ing to conventional criteria.3,25 Cyclin D1 gene overexpression, in absence of t(11;14), is foundin the majority of cases. 6 Although the normal counter- part of the hairy cells has not yet been identified, this disease Immunophenotypic analysis is considered to derive from clonal post- B cells, basedon the assessment of the immunoglobulin gene Peripheral bloodor bone marrow samples were usedto assess rearrangements7 andthe analysis of somatic hypermutation of the membrane phenotype of . Briefly, 5 × 105 VH genes.8 cells were incubated(15 min) with the appropriate amounts The occurrence of secondneoplasms in patients with HCL of monoclonal antibodies using double or triple color combi- is a well-known fact.9–15 Among them, non-Hodgkin’s lym- nations, followedby redcell lyses using FACS lysing solution. Stainedsamples were acquiredin a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA, Correspondence: F Bosch, Department of Hematology, Hospital Clı´nic Villarroel no. 170, 08036-Barcelona, Spain; Fax: 34-93- USA), andanalyzedusing the Paint-a Gate Pro software 2275428 (Becton Dickinson). The reactivity against antigens (at Received4 September 2001; accepted27 February 2002 least CD3, CD4, CD5, CD8), B cell antigens (at least CD19, HCL and CLL E Gine´ et al 1455 CD22, CD20, kappa andlambdalight chain), andother anti- Patient 1 gens (at least CD11c, CD23, CD25, CD103) was analyzed. When required, immunohistochemical analysis was perfor- A 59-year-oldman was referredto our center with a palpable medin bone marrow biopsies using CD79a andCD5 anti- splenomegaly andretroperitoneal lymph nodes.The WBC bodies in order to ascertain CLL/SLL infiltrates. count was 9.53 × 109/l (60% lymphocytes, the great majority with a hairy cell appearance). The bone marrow aspirate showedthe presence of 54% hairy cells. A bone marrow Immunoglobulin gene rearrangement biopsy revealedinterstitial infiltration by two distinctlympho- cyte subtypes: (1) medium-sized lymphocytes, with a round High molecular weight DNA was extractedfrom mononuclear nuclear profile andfine chromatin, widelyseparatedfrom cells isolated by Ficoll gradient according to standard pro- each other by their cytoplasm, (2) small lymphocytes, with cedures.26 Consensus primers for the framework 3 (FR3) por- roundnucleus andmature chromatin; there was a marked tion of the variable region andof the 3 Ј portion of the joining increase in reticulin fibers andthe small infiltrates region of the immunoglobulin heavy chain gene, basedon were positive for CD79a andCD5 (Figure 1). The immuno- 26 phenotype of peripheral bloodshowed40% of lymphocytes previously publishedstudies, were usedto amplify the + + + CDR3 region. The reaction mixture of PCR contained30 with a HCL phenotype (CD19 ,CD25, CD11c , andlambda chain restriction), whereas 12% of lymphocytes hada CLL/SLL pmols of each primer, 200 mM of each dNTPs, 2.5 mM MgCl2 + + + +/− and1.25 U of High ExpandFidelity(Roche Diagnostics, phenotype (CD19 ,CD5,CD23 ,CD22 , andlambdachain Mannheim, Germany) and1 ␮g of template. The reaction con- restriction) (Figure 2-1). sistedof 40 cycles of a denaturationstep at 94 °C for 1 min The patient was treatedwith DCF. After treatment, a com- (10 min first cycle), an annealing step at 55°C for 2 min and plete response was documented. However, both hairy cell an extension step for 1 min at 72°C (10 min last cycle). Ampli- andCLL/SLL immunophenotypic markers were observedin fiedDNA was electrophoresedon an acrylamide-bis(19:1) the peripheral blood. Six years after diagnosis the patient gel 5% and was stained using ethidium bromide and vis- presentedwith clinical manifestations relatedto the CLL/SLL, × 9 ualizedunderUV. (20 10 /l), anda palpable splenomegaly. The presence of both B cell lymphocytic popu- lations was again confirmedby morphologic andimmuno- phenotyping methods (53% of CLL/SLL lymphocytes and 2% Literature search of hairy cells in the peripheral blood). After completing treat- ment with 2-chlorodeoxyadenosine, the hematological para- A literature search of the periodJanuary 1966 to July 2001 meters normalizedandthe immunophenotypic analysis was performedusing the Pub MedService, National Library detected only the presence of the CLL/SLL population. The of Medicine – National Center for Biotechnology information PCR analysis performedat that time showeda single heavy (www.ncbi.nlm.nih.gov/) USA. Key words consulted were: chain immunoglobulin gene (JH) band. hairy cell leukemia andchronic lymphocytic leukemia, hairy cell leukemia, hairy cell leukemia andtreatment. Patient 2

Results A 49-year-oldman was referredto our center because of pan- cytopenia foundin a routine analysis. The physical examin- Three of the 26 patients with HCL (11.5%; 95% CI: 0% to ation revealedpalpable splenomegaly at 5 cm below the cos- 24%) showeda concomitant B cell population consistent with tal margin. The hemoglobin level was 97 g/l, the WBC count B cell CLL/SLL. The main clinical, immunophenotypic and 1.7 × 109/l (89% lymphocytes) andthe count 69 × molecular characteristics of these patients are detailed in 109/l. A sternal bone marrow aspirate was hypercellular with Table 1 andbriefly describedbelow. 41% hairy cells. A bone marrow biopsy revealeda diffuse

Table 1 Biological characteristics of the three patients with coexistence of HCL andCLL/SLL at diagnosis

Patient Age/sex Cytogenetics (metaphases) Immunophenotype PCR analysis

HCL CLL/SLL

1 59/M 46,XY [20] PB (40%) PB (12%) Monoclonal CD19+, CD25+, CD11c+, ␭ CD19+, CD5+, CD23+, 1 band CD20+/−, ␭ 2 49/M 46,XY [20] PB (6%) PB (12%) Monoclonal CD19+, CD25+, CD103+, CD19+, CD5+, CD23+, 2 bands CD11c+ CD20+/− 3 48/M t(1;19) (p35; q40) [2] BM (1%) PB (80%) Monoclonal 46,XY [58] CD19+, CD103+, CD25+, CD19+, CD5+, CD23+, 2 bands CD11c+ CD20+/−

PB, peripheral blood; BM, bone marrow.

Leukemia HCL and CLL E Gine´ et al 1456 Patient 3

A 48-year-oldman previously diagnosedwith HCL at another center, where he was treatedwith DCF, was referredto us 1 year later for evaluation. At that time the WBC count was 51 × 109/l, with 75% of lymphocytes with typical features of CLL/SLL. Peripheral bloodandbone marrow immunopheno- typic analysis was also characteristic of CLL/SLL with a lambda clonal light chain restriction. Moreover, in 1% of bone marrow lymphocytes a typical HCL phenotype was found (Figure 2-3). The patient achieveda clinical response after treatment with fludarabine combined with andmitoxantrone. However, the immunophenotypic and molecular studies demonstrated the persistence of both B cell populations. The patient underwent an autologous peripheral stem cell transplantation, and1 year later neither CLL/SLL nor HCL are detectable.

Discussion

The association of HCL andCLL/SLL is consideredto be rare. To challenge this concept we report three patients with HCL in whom a population of lymphocytes with the classical immunophenotypic features of CLL/SLL was detected. These cases representeda noteworthy percentage (11.5%), albeit with extremely wide 95% CI (0% to 24%), of the total number of HCL cases diagnosed in our institution during the same per- iodof time. Although the patients reportedpresentedwith clinical andmorphological characteristics of HCL, the concur- rent diagnosis was made after an accurate morphological and immunophenotypical analysis of the B lymphocyte population in peripheral bloodor bone marrow. Furthermore, after receiving treatment for the HCL, two of the patients developed clinical manifestations relatedto the progression of the CLL/SLL population, whereas the HCL remained, in all cases, silent. The coexistence of two different lymphoproliferative dis- Figure 1 (a) CD79a immunostaining of the bone marrow biopsy orders in the same patient is a well-known situation, most of corresponding to patient 1. Hairy cells (H) were bigger in size and the cases being transformations from a low-grade into high- showeddiffusecytoplasmic projections comparedto the CLL/SLL cells grade lymphoma. In this regard, HCL can also evolve into × (C) ( 500). (b) Bone marrow aspirate of patient 2 showing an infil- aggressive lymphoma, mimicking the so-calledRichter’s syn- tration composedof cells with morphologic characteristics of CLL/SLL 10,27 (C), andcells presenting typical cytoplasmic projections of the HCL drome in CLL/SLL. The association of HCL with other (H) (hematoxylin & eosin, ×500). lymphoproliferative disorders is unusual. Thus, only a few cases of HCL associated with Hodgkin’s disease,11,12 multiple myeloma15 or CLL/SLL17,18 have been described. In a com- puter-assistedliterature search, we have identifiedonly two reports describing the association of CLL/SLL and HCL.18,19 In these cases the HCL population was detected several years infiltration by cells typical of HCL anda moderateincrease in after the diagnosis and treatment of the CLL/SLL. Conversely, reticulin fibers. Peripheral bloodimmunophenotypic analysis in our patients both populations were detected at the same detected 5% cells with a characteristic HCL phenotype, and time. It is worth emphasizing the significant prevalence of this in 12% a CLL/SLL phenotype (Figure 2-2). The bone marrow association in our series of HCL (11%). This couldbe due,in cytogenetic study was normal in the 20 metaphases analyzed. part, to the large panel of monoclonal antibodies used for the The PCR analysis of the heavy chain immunoglobulin gene diagnosis of chronic lymphoproliferative disorders in our insti-

(JH) pattern in peripheral bloodlymphocytes showedtwo dif- tution, andto the refinement andaccuracy of the phenotype ferentiatedclonal bands.The patient was treatedwith six techniques. On the other hand, a referral center bias cannot cycles of DCF. After completing such treatment, the cytolog- be excluded. Nevertheless, the coexistence of HCL and ical andimmunophenotypic analysis disclosedonly the CLL/SLL seems to be a unique phenomenom since, among CLL/SLL population, andthe molecular analysis showeda sin- 270 patients diagnosed with CLL/SLL during the same period gle monoclonal band. One year later, coincidentally with the of time in whom the same panel of monoclonal antibodies immunophenotypic detection of minimal HCL population, was used, none was found to have HCL. In any case, the high two monoclonal bands appeared in the VH analysis of the prevalence foundin this series shouldbe verifiedin a larger bone marrow sample (Figure 3). panel of HCLs. In an attempt to explain the coexistence of HCL and

Leukemia HCL and CLL E Gine´ et al 1457

Figure 2 Immunophenotype of peripheral blood(lines 1 and2) or bone marrow (line 3) of the three patients. Hairy cells are representedin red, CLL/SLL cells in blue and normal B lymphocytes in green. Two different abnormal B cell populations were detected in each patient corresponding to CLL/SLL cells and hairy cells. Only in patient 2 was a residual normal B cell population detected.

CLL/SLL, some hypotheses can be formulated. Patients with mutations of the immunoglobulin variable region genes,8 a CLL/SLL have an impairedimmune surveillance that might hallmark of a germinal center origin.31,32 Conversely, about contribute to the development of second neoplasms.28,29 Like- 50% of CLL/SLL derive from post-germinal center CD5+ wise, a trendfor the developmentof secondmalignancies has CD27+ memory B cells,33 whereas the remaining CLL/SLL been described in HCL.12,15,16 Basedon phenotypic andmol- originate in CD5+ pre-germinal cells.33 Of note, CLL/SLL cells ecular studies, it is now well established that HCL derives can be differentiated under a variety of stimuli and, upon cul- from germinal or post-germinal center cells.30 Thus, hairy cells ture treatedwith phorbol ester (TPA), can acquire the pheno- have the phenotype of activatednormal B cells (CD22, CD25 type of HCL activatedcells. 34 In this setting, the presence of andCD40 ligand).Moreover, ‘hairy cell restrictedantigens’, the same immunoglobulin gene rearrangement in two differ- such as CD11c andCD103, have been associatedwith the ent lymphoproliferative disorders, although not definitive, activation of certain lymphoidandnon-lymphoidcell types. 25 argues strongly in favor of a common precursor origin. One From the molecular standpoint, hairy cells have somatic of the two previously reportedcases associating CLL/SLL and

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