anaplastic large cell lymphoma in leukemic phase: extraordinarily high white blood cell count

Case Report

Anaplastic large cell lymphoma in leukemic phase:Extraordinarily high white blood cell count

Jacqueline T. Nguyen,1 Michael R. Condron,1 Nghia D. Nguyen,1 Jitakshi De,1 L. Jeffrey Medeiros2 andAnthony Padula2

1Department of Pathology and Laboratory Medicine, University of Texas–Houston Medical School and 2Departmentof Hematopathology, University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA

Anaplastic large cell lymphoma (ALCL) is a distinct type ofT/null-cell non-Hodgkin lymphoma that commonly involvesnodal and extranodal sites. The World Health Organizationof lymphoid neoplasms recognizes two types: anaplasticlymphoma kinase (ALK) positive or ALK negative, theformer as a result of abnormalities involving the ALK geneat chromosome 2p23. Patients with ALCL rarely develop aleukemic phase of disease, either at the time of initial pre-sentation or during the clinical course. Described herein isa patient with ALK+ ALCL, small cell variant, associated withthe t(2;5)(p23;q35), who initially presented with leukemicinvolvement and an extraordinarily high leukocyte count of529 ¥ 109/L, which subsequently peaked at 587 ¥ 109/L.Despite chemotherapy the patient died 21/2 months afterdiagnosis. In the literature review 20 well-documentedcases are identified of ALCL in leukemic phase reportedpreviously, with a WBC ranging from 15 to 151 ¥ 109/L.Leukemic phase of ALCL occurs almost exclusively inpatients with ALK+ ALCL, most often associated with thesmall cell variant and the t(2;5)(p23;q35), similar to thepresent case. Patients with leukemic phase ALK+ ALCLappear to have a poorer prognosis than most patients withALK+ ALCL.

Key words: ALK positive, anaplastic large cell lymphoma, leu-kemic phase, small cell variant, t(2;5)(p23;q35)

Anaplastic large cell lymphoma (ALCL) is recognized in theWorld Health Organization (WHO) classification of lymphoidneoplasms.1,2 These neoplasms are strongly positive for

CD30, usually express T-cell and cytotoxic antigens, andcarry monoclonal T-cell receptor gene rearrangements. In theinitial version of the WHO classification, ALCL was describedas one entity in which anaplastic lymphoma kinase (ALK)+ orALK– subgroups were recognized. The former group is rec-ognized as a relatively homogeneous group with abnormali-ties involving the ALK gene at chromosome 2p23 resulting inALK overexpression. The ALK– group of neoplasms is moreheterogeneous and there has been no clear consensus for itsexistence.3 Savage et al., however, reported that patientswith ALK– ALCL have a prognosis intermediate betweenALK+ ALCL and other types of T-cell lymphomas,4 and thecurrent updated WHO classification now recognizes ALK+and ALK– ALCL as separate entities.1,2

ALK+ ALCL has a number of histological variants.1,3 In themost common or classical variant, the neoplastic cells arelarge and anaplastic with a cohesive growth pattern. Sinusoi-dal involvement is common. Other common variants of ALK+ALCL include the lymphohistiocytic and small cell variants,and there are a number of uncommon variants such as thesarcomatoid variant.1,3 Usually these neoplasms have asubset of neoplastic cells that have distinctive cytologicalfeatures known as hallmark cells. A typical hallmark cell hasa horseshoe-shaped nucleus associated with a perinuclearhof. Hallmark cells can be rare in the small cell variant.

Although a variety of B- and T-cell non-Hodgkin lympho-mas can present with or evolve into a leukemic phase,patients with ALCL in leukemic phase have been reportedonly rarely. In our review of the literature we have identifiedonly 20 cases of ALCL with a leukemic phase (Table 1).5–17

Typically, patients present with an elevated WBC count <100¥ 109/L.

In this report we describe a young man with ALK+ ALCLwho initially presented with an unusually high WBC count of529 ¥ 109/L, which subsequently peaked at 587 ¥ 109/L.Despite therapy the patient died 21/2 months after diagnosis.

Correspondence: Anthony Padula, MD, Department of Hematopa-thology, Box 72, M. D. Anderson Cancer Center, 1515 HolcombeBoulevard, Houston, TX 77030, USA. Email: [email protected]

Received 19 November 2008. Accepted for publication 16 January2009.© 2009 The AuthorsJournal compilation © 2009 Japanese Society of Pathology

Pathology International 2009; 59: 345–353 doi:10.1111/j.1440-1827.2009.02376.x

Tab

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346 J. T. Nguyen et al.

© 2009 The AuthorsJournal compilation © 2009 Japanese Society of Pathology

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ALK+ ALCL in marked leukemic phase 347


Tab

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Con

tinue

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We discuss the clinicopathological, immunophenotypic, andcytogenetic and molecular features of this case, review theliterature, and discuss the differential diagnoses.

CLINICAL SUMMARY

A 26-year-old Hispanic man with no significant past medicalhistory presented with abdominal pain, fever, chills, and nightsweats. Physical examination and radiological imaging indi-cated cervical lymphadenopathy and multiple small mesen-teric and inguinal lymph nodes. The complete blood count atthe time of initial examination was: WBC 529 ¥ 109/L (normalrange, 4–11 ¥ 109/L), platelet count 174 ¥ 109/L (normalrange, 150–440 ¥ 109/L), and hematocrit 40.5% (normalrange, 41.5–50.44%). A manual differential count showed 5%neutrophils, 51% lymphocytes, and 25% atypical lympho-cytes. Bone marrow aspiration and biopsy were performedthat indicated numerous atypical lymphoid cells. Based onthe morphological findings and initial flow cytometry immu-nophenotypic data, a number of different diagnoses wereconsidered before the diagnosis of ALK+ ALCL was estab-lished. The patient was treated with intensive chemotherapycomposed of three cycles of Hyper-CVAD chemotherapy(fractionated cyclophosphamide, vincristine, doxorubicin,and dexamethasone with intrathecal methotrexate and cyt-arabine, alternating cycles every 3 weeks with high-dosemethotrexate and cytarabine).

The patient’s WBC count at the start of chemotherapy was587 ¥ 109/L, which decreased to 72.5 ¥ 109/L 2 weeks aftercycle 2, and reached a nadir of 32 ¥ 109/L after cycle 3. TheWBC increased to 147 ¥ 109/L, however, by the time cycle 4was to be administered. Consideration had been given tointensification with humanized monoclonal antibody to CD52(Alemtuzumab). In addition, the management plan includedevaluating the patient for potential hematopoietic stem celltransplantation, but as an undocumented resident in the USAhe was not eligible. Therefore, the patient decided to return tohis native country and within 1 week was admitted to a localhospital with visual complaints, headache, and obtundation.The patient died 2.5 months after initial presentation.

PATHOLOGICAL FINDINGS

Procedure

Hematology and histology

Peripheral blood and bone marrow aspirate smears were airdried and stained with Wright-Giemsa. A 400-cell differentialcount was performed on bone marrow aspirate smears. Thedecalcified bone marrow core biopsy and the aspirate clot

specimens were fixed in formalin and paraffin-embedded,and histological sections were stained with HE.

Immunohistochemistry

Immunohistochemistry was performed using formalin-fixed,paraffin-embedded tissue sections of the bone marrowaspirate clot specimen. The avidin–biotin peroxidasetechnique was used with either a Ventana Benchmarkautostainer (Tucson, AZ, USA) or a Dako autostainer(Santa Barbara, CA, USA) using antibodies specific forALK-1 (monoclonal), terminal deoxynucleotidyl transferase(TdT; polyclonal), Pax-5 (monoclonal), and CD117 (poly-clonal; Cell Marque, Hot Springs, AR, USA); epithelialmembrane antigen (EMA)/Mc5 (monoclonal, Ventana); andCD30/Ber-H2 (monoclonal) and LMP-1/CS.1-4 (monoclonal;Dako, Glostrup, Denmark). In situ hybridization for EBV-encoded RNA (EBER; PanPath Rembrandt, Amsterdam,The Netherlands) was also performed.

Flow cytometry immunophenotyping

Immunophenotypic analysis of peripheral blood and bonemarrow aspirate samples was performed using a whole bloodlysis method followed by three-color flow cytometry on aFACSort analyzer (Becton Dickinson, San Jose, CA, USA)using the CD45 versus side-scatter gating technique. Mono-clonal antibodies, conjugated to fluorescein isothiocyanate orphycoerythrin, were used that were specific for CD3 (Leu-4,clone SK7), CD4 (Leu-3a, clone SK3), CD5 (clone 17F12),CD7 (clone 4H9), CD8 (Leu-2a, clone SK1), CD10 (cloneHI10a), CD11c (clone S-HCL-3), CD13 (clone L138), CD14(clone MFP9), CD16 (Leu-11b, clone GO22), CD19 (Leu-12,clone 4G7), CD20 (clone L27), CD22 (clone S-HCL-1), CD23(clone EBVCS-5), CD25 (clone 2A3), CD33 (clone P67.6),CD34 (clone My10), CD45 (anti–Hle-1, clone 2D1), CD52(clone CF1D12), CD56 (clone MY31), human leukocyteantigen (HLA)-DR (clone L243), and surface immunoglobulinkappa (clone TB28-2) and lambda (clone 1-155-2) lightchains. All antibodies were supplied by Becton Dickinsonexcept CD52, which was supplied by Caltag Laboratories(Invitrogen, Carlsbad, CA, USA).

Cytogenetics and molecular analysis

Conventional cytogenetic analysis was performed on thebone marrow aspirate material. Three metaphases wereanalyzed from one 24 h unstimulated culture, and anadditional 17 metaphases were examined from a 72 hlipopolysaccharide-stimulated culture.

Fluorescence in situ hybridization (FISH) was performedon bone marrow aspirate smears using the LSI ALK dualcolor breakapart DNA probe (Vysis, Downers Grove, IL,USA) which hybridizes to the 2p23 locus according to themanufacturer’s instructions.



Molecular analysis for T-cell receptor gamma chain generearrangements was performed using a peripheral bloodspecimen and a polymerase chain reaction (PCR)-basedmethod utilizing primers specific for the variable (V) andjoining (J) regions of the T-cell receptor gamma chain gene.The amplified products were analyzed by capillary electro-phoresis.

Molecular analysis for immunoglobulin heavy chain immu-noglobulin gene rearrangements was performed using aperipheral blood specimen and a PCR-based method usingprimers specific for the frameworks 1, 2, and 3 of the immu-noglobulin heavy chain gene V regions and primers specificfor the J region. The amplified products were analyzed bycapillary electrophoresis.

RESULTS

Morphological findings

The peripheral blood smear (Fig. 1) showed normocytic nor-mochromic anemia with marked leukocytosis and lymphocy-tosis. The white blood cell count was 529 ¥ 109/L with 51%lymphocytes. Most of the lymphocytes were atypical, small tomedium in size, with irregular nuclear contours. A smallernumber, <5%, were larger with vacuolated and basophiliccytoplasm. Rare cells with an irregularly shaped nucleusresembling cloverleaf and horseshoe shapes were observed.

The bone marrow aspirate smears showed numerousatypical lymphocytes admixed with maturating bone marrowelements. The bone marrow aspirate clot specimen (Fig. 2)was hypercellular (90%) and also showed a predominantpopulation of atypical lymphoid cells, representing approxi-mately 60% of all cells. The bone marrow biopsy specimenwas of very poor quality, being extensively fragmented withaspiration artifact and almost no hematopoietic bone marrow.

Immunophenotype

Flow cytometry immunophenotyping of peripheral blood andbone marrow aspirate specimens showed an abnormal popu-lation of cells positive for CD4, CD5, CD13, CD25, CD45,CD52, and HLA-DR, and negative for CD3, CD7, CD8,CD10, CD11C, CD14, CD16, CD19, CD20, CD22, CD23,CD33, CD34, CD56, and immunoglobulin kappa and lambdalight chains.

Immunohistochemistry performed on the bone marrowaspirate clot specimen showed that the cells were positive forALK-1, CD30, and EMA. The CD30 pattern of expressionwas membranous and paranuclear (Fig. 3). The pattern ofALK-1 was nuclear and cytoplasmic (Fig. 4). The neoplasticcells were negative for CD117, TdT, PAX5, and EBV latent

membrane protein type 1. In situ hybridization for EBER wasnegative.

Chromosome and molecular studies

Conventional cytogenetics of the bone marrow aspirate speci-men (Fig. 5) showed eight normal and 12 abnormal meta-phases with the following karyotype: 46,XY,t(2;5)(p23;q35),add(18)(q21). FISH showed disruption of the ALK gene on2p23.

Molecular studies of a peripheral blood specimen showeda monoclonal T-cell receptor gamma chain gene rearrange-ment and no evidence of monoclonal immunoglobulin heavychain gene rearrangement.

DISCUSSION

Leukemic involvement is highly unusual in patients withALCL. Including the present case, we have identified a totalof 21 cases of ALCL in leukemic phase reported in the litera-ture (Table 1). Almost all of these cases, 20 of 21 (95.2%),were ALK+ ALCL. Eighteen of the 20 (95%) ALK+ ALCL weredescribed as or designated as the small cell variant andtherefore we agree with Onciu et al. that this variant seems tobe particularly prone to entering the blood in large numbers.12

Although the explanation for why small cell variant of ALCLcommonly presents with a leukemic phase is unknown, cyto-genetic abnormalities in addition to the t(2;5) translocationmay provide a clue. Twenty cases reported have availablecytogenetic data. Most cases of leukemic phase ALK+ ALCLhave been associated with the t(2;5)(p23;q35): 17 of 20(85%). Two neoplasms reported had three-way translo-cations: t(2;5;13)(p23;q35;q14) and t(2;5;3)(p23;q35;p21).Of the three cases without the t(2;5), one case involvedt(2;19)(p23;p13.1),11 one case involved poorly describedabnormalities of chromosome 2, 5, and 20,12 and one case ofALK– ALCL involved a complex karyotype with abnormalitiesof chromosomes 1, 2, 5, 7, 8, and 15.15 Because the t(2;5)occurs in approximately 80% of all ALK+ ALCL, it does notappear that the t(2;5) is preferentially associated with theleukemic phase of disease.

Clinically, the age range of affected patients is wide, from7 months to 63 years, but 15 of 21 patients (71.4%) wereyounger than 30 years of age. There is a male predominance(61.9%). These findings are in keeping with the tendency ofALK+ ALCL to affect younger male patients. Most patientshave B-type symptoms, with fever most common, reported in18 of 21 patients (85.7%). Weight loss and night sweats areless common, reported in fewer than one-third of patients.Patients with leukemic phase ALK+ ALCL typically havewidespread involvement of many organ systems at the time



of diagnosis, as shown by physical examination and radio-logical studies. The degree of leukocytosis is variable, but isusually under 100 ¥ 109/L in most patients. Four patientshave had WBC counts >100 ¥ 109/L: 106, 118, 149.9, and151 ¥ 109/L. The present case was highly unusual, involvinga peak WBC of 587 ¥ 109/L, almost fourfold higher than in anyother case reported.

Patients with ALK+ ALCL are usually younger and have amore favorable clinical course than patients with other typesof T-cell lymphoma when treated with appropriate multi-agentchemotherapy. This is true despite the fact that patients withALK+ ALCL commonly present with advanced disease and Bsymptoms.1,3 Patients with leukemic phase ALK+ ALCL,however, appear to be a poor prognostic subset. At least 13patients with leukemic phase ALK+ ALCL have died ofdisease, usually within a few months, and additional patientshad relapsed by the time their case was reported. Thissuggestion has been made previously by others,12 and thepresent case supports this interpretation.

Figure 2 Bone marrow clot specimen (original magnification ¥500)showing sheets of lymphoma cells, mostly small–intermediate, withscattered large cells having irregular nuclei and occasional promi-nent nucleoli, admixed with normal hematopoietic elements. A mitoticfigure is observed.

Figure 3 Bone marrow clot specimen (original magnification ¥500).Immunohistochemical staining for CD30 is strongly positive in thesubset of large neoplastic cells (as is usually the case in this variant)in a membranous and paranuclear dot-like pattern.

Figure 4 Bone marrow clot specimen (original magnification ¥500).Immunohistochemical staining for anaplastic lymphoma kinase high-lights the neoplastic cells in a nuclear and cytoplasmic pattern. Thelarger cells show a stronger staining pattern, as is usually the case inthis variant.

Figure 1 (a) Peripheral blood smear(original magnification ¥1000) showingsmall–intermediate sized lymphomacells with irregular nuclei and a giant cellwith hallmark-like features. (b) Periph-eral blood smear (original magnification¥1000) showing smaller lymphoma cellsand occasional large cells, one with aprominent mitosis.

a b



Microscopically, most of the neoplastic cells of ALK+ ALCLin a peripheral blood smear often have ‘flower-like’ or irregu-lar nuclear shapes and do not resemble the common appear-ance of ALK+ ALCL cells in tissue biopsy specimens. Rarelarge cells with horseshoe shaped nuclei, however, cansometimes be identified. The presence of these large cells isa clue to the correct diagnosis, although the predominantsmall cell features and the rarity of leukemic presentationcreate a diagnostic challenge.

Other entities that are part of the morphological differentialdiagnosis include adult T-cell leukemia/lymphoma, T lympho-blastic leukemia/lymphoma, Sezary syndrome, and T-cellprolymphocytic leukemia. The circulating neoplastic cells ofadult T-cell leukemia/lymphoma are well known for theirflower-like nuclear appearance, and patients often presentsimilarly to the present patient, with a high WBC count andwidespread disease. Adult T-cell leukemia/lymphoma alsocan express CD30 in a subset of cases. This possibilitywas excluded in the current case by serological testing forhuman T-cell lymphotropic virus (HTLV)-1 and HTLV-2, whichwas negative. In addition, cases of adult T-cell leukemia/lymphoma do not express cytotoxic markers, the pattern andintensity of CD30 expression is usually variable and lessintense, and these neoplasms lack ALK expression andt(2;5)(p23;q35). The neoplastic cells of T-lymphoblasticleukemia/lymphoma typically have more blastic chromatinand they have an immature T-cell immunophenotype, includ-ing expression of TdT. T-lymphoblastic leukemia/lymphomais negative for CD30 and ALK. Sezary syndrome is charac-terized by the presence of larger Sezary cells and smallerLutzner type cells that can appear multilobate or flower-like inthe peripheral blood smear. The present patient did not haveerythroderma or skin lesions to support the diagnosis of

Sezary syndrome. The neoplastic cells of Sezary syndromerarely express CD30 or cytotoxic antigens and are negativefor ALK. Lastly, T-cell prolymphocytic leukemia was initiallyconsidered as a potential diagnosis. Patients with T-cell pro-lymphocytic leukemia (T-PLL) often present with clinicallyaggressive disease, a high WBC count, and extensive bonemarrow disease, as seen in the present patient. AlthoughT-cell prolymphocytes usually have a distinct central nucleo-lus, a subset of cases designated as small cell variant ofT-PLL can have Sezary cell-like features. Unlike the presentpatient, T-cell prolymphocytic leukemia usually affects olderpatients and the neoplastic cells do not express CD30 orALK.

The flow cytometry immunophenotypic findings in thepresent case also caused some initial confusion because theneoplastic cells expressed CD13, a myeloid-associatedmarker, which led to the diagnosis of acute biphenotypicleukemia of mixed T-cell and myeloid lineage being consid-ered. Recent flow cytometry immunophenotyping of ALCL,however, has shown that ALK+ ALCL commonly expressesCD13.18,19 This is now a recognized pitfall in establishing thediagnosis of ALCL by flow cytometry immunophenotyping.

In summary, we report a patient with ALK+ ALCL whopresented with an extraordinarily high WBC of 529 ¥ 109/Lthat subsequently peaked at 587 ¥ 109/L before therapy.Leukemic phase ALCL is uncommon, with only 19 cases ofALK+ ALCL reported in the literature. The present case wasunusual in that the degree of leukocytosis far exceeded pre-viously reported cases. Patients who present with leukemicphase ALCL are a diagnostic challenge, particularly initially,because morphological and flow cytometry immunopheno-typing can suggest other possible diagnoses. Consideringthe possibility of leukemic phase of ALCL, and performing anappropriate workup including immunohistochemical stains(e.g. CD30, ALK) and cytogenetic studies readily leads to thecorrect diagnosis.

REFERENCES

1 Delsol G, Jaffe ES, Falini B et al. Anaplastic large cell lym-phoma (ALCL), ALK-positive. In: Swerdlow SH, Campo E,Harris NL et al., eds. WHO Classification of Tumours of Hae-matopoietic and Lymphoid Tissues, 4th edn. Lyon: InternationalAgency for Research on Cancer, 2008; 312–16.

2 Mason DY, Campo E, Harris NL et al. Anaplastic large celllymphoma, ALK-negative. In: Swerdlow SH, Campo E, HarrisNL et al., eds. WHO Classification of Tumours of Haematopoi-etic and Lymphoid Tissues, 4th edn. Lyon: International Agencyfor Research on Cancer, 2008; 317–19.

3 Medeiros LJ, Elenitoba-Johnson KS. Anaplastic large cell lym-phoma. Am J Clin Pathol 2007; 127: 707–22.

4 Savage K, Harris NL, Vose JM et al. International PeripheralT-cell Lymphoma Project. Blood 2008; 111: 5496–504.

5 Anderson MM, Ross CW, Singleton TP, Sheldon S, Schnitzer B.Ki-1 anaplastic large cell lymphoma with a prominent leukemicphase. Hum Pathol 1996; 27: 1093–5.

Figure 5 Karyotype showing the t(2;5) translocation with additionalchromatin material on the long arm of chromosome 18.



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7 Bayle C, Charpentier A, Duchayne E et al. Leukaemic presen-tation of small cell variant anaplastic large cell lymphoma:Report of four cases. Br J Haematol 1999; 104: 680–88.

8 Grewal JS, Smith LB, Winegarden JD et al. K-positive anaplas-tic large cell lymphoma with a leukemic phase and multi-organinvolvement: A report of three cases and a review of the litera-ture. Ann Hematol 2007; 86: 499–508.

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10 Kong SY, Cho HJ, Suk JH et al. A novel complext(2;5;13)(p23;q35;q14) in small cell variant type anaplastic largecell lymphoma with peripheral involvement. Cancer Genet Cyto-genet 2004; 154: 183–5.

11 Meech SJ, McGavran L, Odom LF et al. Unusual childhoodextramedullary hematologic malignancy with natural killer cellproperties that contains tropomyosin 4-anaplastic lymphomakinase gene fusion. Blood 2001; 98: 1209–16.

12 Onciu M, Behm FG, Raimondi SC et al. ALK-positive anaplasticlarge cell lymphoma with leukemic peripheral blood involvementis a clinicopathologic entity with an unfavorable prognosis.Report of three cases and review of the literature. Am J ClinPathol 2003; 120: 617–25.

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16 Takahashi D, Nagatoshi Y, Nagayama J et al. Anaplastic largecell lymphoma in leukemic presentation: A case report and areview of the literature. J Pediatr Hematol Oncol 2008; 30:696–700.

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anaplastic large cell lymphoma in leukemic phase: extraordinarily high white blood cell count

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