Ann Hematol (2003) 82:47–52DOI 10.1007/s00277-002-0569-x

C A S E R E P O R T

F. Kroschinsky · K. Friedrich · M. Hanel · B. Mohr ·T. Langer · M. Meinhardt · C. Thiede · M. Bornhauser ·G. Baretton · G. Ehninger

Extramedullary blast crisis of chronic myeloid leukemia after allogeneichematopoietic stem cell transplantation mimicking aggressive,translocation t(14;18)-positive B-cell lymphomaReceived: 21 February 2002 / Accepted: 8 October 2002 / Published online: 29 November 2002� Springer-Verlag 2002

Abstract We report the case of a 42-year-old male patientwho was diagnosed with a large tumor of the rightthoracic aperture 30 months after unrelated hematopoieticstem cell transplantation (HSCT) for accelerated phase ofPhiladelphia chromosome (Ph)-positive chronic myeloidleukemia (CML). Biopsy revealed an immature lymphoidneoplasia with blastic tumor cell morphology and immu-noreactivity for CD34, CD79a, CD43, and CD30 as wellas slight positivity for TdT and CD20. Bcr-Abl rear-rangement was found in interphase tumor cell nuclei byfluorescence in situ hybridization (FISH). Furthermore, atranslocation t(14;18)(q32;q21) was amplified by poly-merase chain reaction (PCR). Bone marrow (BM) exam-ination showed regular hematopoiesis including anegative FISH analysis for Bcr-Abl and complete donorchimerism. Nested PCR from peripheral blood (PB), butnot conventional PCR, was positive for the b3a2 Bcr-Abltranscript. Neither radiation nor intensive chemotherapywas capable of achieving a tumor remission, and thepatient died from progressive disease 6 months later.Postmortem examinations showed a shift of immunophe-notype with appearance of myeloperoxidase-positivetumor cells and loss of lymphoid antigens. In addition,there were characteristic cytogenetic findings of multiplePh chromosomes and a clonal loss of P53 tumorsuppressor gene. The latter was already deleted beforeHSCT. We conclude that lymphoid neoplasia occurring inour patient should be interpreted as an extramedullary,

very immature blast crisis of CML expressing lymphoiddifferentiation markers rather than a true de novo NHL.

Keywords Allogeneic hematopoietic stem celltransplantation · Chronic myeloid leukemia ·Extramedullary blast crisis · B-cell lymphoma

Introduction

Allogeneic hematopoietic stem cell transplantation(HSCT) remains the only curative treatment for chronicmyeloid leukemia (CML). If treated with conventionalcytoreductive therapy, the disease inevitably terminates ina final blast crisis (BC). BC blast cells predominantlyexpress myeloid markers; the immunophenotype is lym-phoid in only 15–20% [14, 17]. Leukemic infiltrates atsites other than bone marrow (BM), peripheral blood(PB), spleen, or liver are termed extramedullary disease(EMD), chloroma, or granulocytic sarcoma. In theliterature, the incidence of EMD during the course ofCML is reported to lie between 3 and 8% [13, 19, 20].CML is characterized by cytogenetic aberrations. Inaddition to the typical t(9;22) translocation, which fusesthe Abl protooncogene from chromosome 9 to thebreakpoint cluster region (Bcr) gene on chromosome 22[Bcr-Abl rearrangement, Philadelphia (Ph) chromosome],various chromosomal aberrations, such as trisomy 8 (+8),double Ph, trisomy 19 (+19), or abnormalities ofchromosome 17 are common findings in disease acceler-ation [7, 8].

We report the diagnostic and therapeutic difficulties ina 42-year-old CML patient who developed a Ph-positivelymphoid tumor of the right thoracic aperture 3 years afterunrelated HSCT. At the time of lymphoma presentation,the patient was in complete hematological remission andhad complete donor chimerism. Nested polymerase chainreaction (PCR) from peripheral blood was positive forBcr-Abl rearrangement, whereas conventional PCR re-mained negative. Interphase fluorescence in situ hybrid-

F. Kroschinsky ()) · M. Hanel · B. Mohr · T. Langer · C. Thiede ·M. Bornhauser · G. EhningerUniversit�tsklinikum Carl Gustav Carus,Medizinische Klinik und Poliklinik I, Fetscherstrasse 74,01307 Dresden, Germanye-mail: kroschinsky@mk1.med.tu-dresden.deTel.: +49-351-4588110Fax: +49-351-4588120

K. Friedrich · M. Meinhardt · G. BarettonUniversit�tsklinikum Carl Gustav Carus, Institut f�r Pathologie,Dresden, Germany

ization (FISH) analysis of BM also detected no Bcr-Abl-positive cells. Differential diagnosis between true de novomalignant non-Hodgkin’s lymphoma (NHL) and extra-medullary CML relapse became even more complicatedafter detection of a translocation t(14;18)(q32;q21) byPCR in the tumor cells. Translocation t(14;18) is a typicalaberration in peripheral B-cell neoplasias, found in 80%of follicular lymphomas and about 20% of diffuse largeB-cell lymphomas. In contrast, it is a rare finding inprecursor B-cell lymphomas/leukemias [3, 4].

Case report

Pretreatment and phase of transplantation

A 38-year-old male patient was diagnosed with chronicphase of Ph-positive CML in January 1996. After initialtreatment with hydroxyurea and interferon-alpha, diseaseacceleration with increased leukocyte count in peripheralblood and 10% myeloperoxidase (MPO)-positive blastcells in hypercellular bone marrow occurred in November1997. At that time, cytogenetic analysis revealed anunbalanced translocation t(17;18)(q11.2;p11.3), resultingin partial monosomies 17p and 18p, respectively. As aconsequence of this additional aberration, one P53 allelewas deleted. Both variants of the major Bcr-Abl rear-rangement (b2a2 and b3a2) could be amplified by reversetranscriptase (RT-) PCR.

Allogeneic HSCT from a HLA-matched unrelatedmale donor was performed in May 1998. Preparativeregimen consisted of intravenous busulfan 4�3.3 mg/kg(total dose 13.2 mg/kg) and cyclophosphamide 4�50 mg/kg (200 mg/kg), accompanied by antithymocyte globulin(Pasteur M�rieux, Lyon, France) 4�2.5 mg/kg (10 mg/kg). Tacrolimus adjusted to plasma levels and methotrex-ate 10 mg/m2 on days +1, +3, and +6 were given asprophylaxis for graft-versus-host disease (GVHD). Thebone marrow graft contained 0.6�108/kg mononucleatedcells (1.24�106/kg CD34-positive cells). Engraftmentwith neutrophils exceeding 0.5�109/l and stable plateletcount above 50�109/l was delayed until day +35;nevertheless, the clinical course after transplantationproceeded without major complications. Sequential quan-titative determination of donor-recipient chimerism wasperformed using a PCR assay with multiplex amplifica-tion of several short tandem repeats (STR). This showed acomplete donor chimerism on day +28. On day +50,grade III GVHD of the skin developed with a maculo-papular rash and prednisolone 2 mg/kg was started. Afterinitial response to steroid treatment, mild erythemapersisted until day +225. Following the resolving of theskin reaction, immunosuppression could be tapered andstopped on day +300. On day +95, complete cytogeneticand molecular remission was determined by negativenested RT-PCR for Bcr-Abl rearrangement from periph-eral blood and bone marrow as well as FISH examinationof bone marrow.

Diagnosis of relapse and salvage treatment

From August 2000 on, the patient experienced increasingpain in the right scapula and shoulder, spreading to hisright arm. This pain was associated with hyperesthesia indermatome T2 and a slight motoric weakness of the righthand. Under the diagnosis of a vertebral disorder, he wastreated with nonsteroidal antiphlogistics and physicaltherapy. Conventional X-ray and spinal magnetic reso-nance imaging (MRI) did not demonstrate any relevantaffection. However, computed tomography (CT) of thechest, performed in November 2000, showed a largetumor of the right thoracic aperture (90 mm in largestdiameter) with infiltration and destruction of the first rib.Fine needle biopsy revealed blastic tumor cell morphol-ogy with immunoreactivity for CD34, CD79a, CD43, andCD30, and a slight positivity for TdT and CD20 (Fig. 1 A,B, C). FISH evaluation (LSI Bcr/Abl dual color translo-cation probe; VYSIS) showed Bcr-Abl rearrangement ininterphase tumor cell nuclei with a high number of cellscontaining two or more fusion signals for the Bcr-Ablhybrid gene. Surprisingly, PCR analysis of the tumor cellsrevealed a translocation t(14;18)(q32;q21) (Fig. 2). Un-fortunately, the amount of biopsy tissue was not sufficientto perform Bcl-2 immunohistochemistry. No furthertumor manifestations were found by clinical examinationand abdomen CT scan. Bone marrow analysis showedregular hematopoiesis with complete donor chimerism aswell as a negative FISH analysis for Bcr-Abl in interphasenuclei. However, one of 49 analyzed BM metaphases wassuspicious for a t(9;22). In addition, nested PCR, but notconventional PCR, from peripheral blood was positive forthe b3a2 transcript.

Although the exact histologic diagnosis of the tumor(CML or NHL) remained unclear, radiation therapy wasinitiated because of increasing pain and neurologicalsymptoms. Initial clinical response included reduction ofpain and improvement of neurological function. However,radiation had to be stopped at a dose of 20 Gy because ofprogressive disease. Progression was confirmed by CTscan, which now showed additional enlarged hilar andmediastinal lymph nodes. Because the tumor showedfeatures of precursor B-cell neoplasm with Bcr-Ablrearrangement, we decided to start intensive inductionchemotherapy including high-dose methotrexate, ifosfa-mide, VM-26, cytarabine, and dexamethasone accompa-nied by intrathecal prophylaxis with methotrexate andcytarabine. Chemotherapy led to a significant improve-ment of clinical symptoms. Partial tumor response (PR)was confirmed by CT scan and by negative Bcr-Abl(nested) PCR from peripheral blood. Unfortunately, thepatient presented with progressive disease again 3 weekslater. Second-line chemotherapy consisting of mitox-antrone, fludarabine, cytarabine, and G-CSF (MitoFLAG)[10] was initiated, but was not able to induce a response.Progressive tumor growth during aplasia led to paralysisof the right arm. Obstruction of the left superiorpulmonary lobe bronchus was associated with increasingdyspnea and extensive need for morphine. Bone marrow

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examination, which was performed because of prolongedcytopenia, showed reduction of hematopoiesis. Moreover,FISH analysis revealed a subpopulation of Bcr-Abl-positive cells containing two to five fusion signals per cell(Fig. 3). Donor chimerism continued to be complete atthis time. As a final therapeutic approach, the patient wastreated with tyrosine kinase inhibitor STI571 (Glivec,Novartis, Basel, Switzerland), which was stopped after21 days due to further progression. He died 4 days aftertherapy was terminated.

Postmortem examination

Postmortem examination showed a large, partly necrotictumor mass located at the right thoracic aperture with amaximum diameter of 9.5 cm. It had led to destruction ofthe second and third ribs and occlusion of the bronchus ofthe right upper lobe. Dissemination to the pleura,infiltration of the apex of the lung, the right atrium ofthe heart, and the mediastinum with stenosis of the uppervena cava were also noted. Surprisingly, autopsy revealedsystemic tumor spread to the mesenteric nodules, thespleen, the liver, the meninges, and an extensive infiltra-tion of bone marrow. All these infiltrates had not beendetected intra vitam.

Fig. 2A, B Detection of translocation t(14; 18) in a fine needlebiopsy specimen of chest wall tumor by PCR. A Electrophoresis(agar gel); B Southern blot after PCR with primers for Mbr-B and

H9 (1 DNA marker, 2 positive control for mbr-B, 3 negative controlfor mbr-B, 4, 5 patient’s DNA)

Fig. 1A–F Biopsy of chest wall tumor revealed blastic cellmorphology (A, H&E, �60) with strong immunoreactivity againstCD34 (B, �60) and a slight positivity for CD20 (C, �60). Tumorcells showed increased polymorphism in postmortem examination

(D, H&E, �60) and were positive for CD34 (E, �60) again. Incontrast to initial diagnosis, single cells showed a positiveimmunostaining for MPO (F, �60)

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Histological evaluation showed leukemic infiltrates ofmedium-sized cells with immature morphology and avariable amount of large polymorphic blasts. Most of thecells expressed CD34, CD79a, and CD43, while a fewshowed positive immunostaining for MPO (Fig. 1 C, D,E). In contrast to the initial biopsy, there was noexpression of lymphoid antigens such as CD20, CD30,and TdT. Results of immunohistochemistry of differenttumor sites are summarized in Table 1.

FISH examination of interphase nuclei from bonemarrow and chest wall tumor cells revealed two or morefusion signals for the Bcr-Abl hybrid gene in more than80%, frequently accompanied by a loss of a single Bcrsignal. Furthermore, analysis of the P53 gene wasperformed on interphase nuclei, using a centromere-specific probe for chromosome 17 (LSI P53, CEP17;VYSIS). A mosaic was detected with 30% of tumor cellsexhibiting loss of both P53 alleles and 70% of cellscontaining one or two signals. The initial finding of apositive PCR for t(14;18) was not confirmed by post-mortem examination of the chest wall tumor or the bonemarrow.

Discussion

Coexpression of precursor cell antigens (CD34 and TdT)and B-cell markers (CD79a, CD20, CD30) combined withthe finding of a t(14;18) were indicative for the devel-opment of a de novo malignant lymphoma in the reported

patient. On the other hand, clinical history as well asdetection of Bcr-Abl rearrangement in the tumor cells andthe peripheral blood led to the assumption of anextramedullary CML blast crisis.

After allogeneic HSCT, most patients become RT-PCR negative for Bcr-Abl gene. In general, qualitativePCR is not able to predict relapse in an individual patient.However, the reappearance of Bcr-Abl transcripts issuspicious for disease recurrence [5, 11]. The majority ofpatients with recurrent CML after HSCT show features ofchronic phase disease, whereas relapse into BC afterHSCT is a rare event (3%) [6]. In a retrospective analysisof the European Group for Blood and Marrow Trans-plantation (EBMT) only 6 of 2753 patients (0.22%) afterallogeneic HSCT for CML or myelodysplasia developedgranulocytic sarcoma [2]. Unfortunately, the study pro-vides no information about the incidence of lymphoiddifferentiated EMD. Ichinohasama et al. reviewed theliterature for association of CML and malignant lympho-ma and found 22 cases of combined Ph-positive CML andlymphoid neoplasia [12]. Interestingly there were onlythree cases with B-cell phenotype. Two patients devel-oped Ph-negative large cell lymphoma (B-cell and null-cell phenotype) independent from underlying Ph-positiveCML. Ph-positive malignant lymphoma was found in 12cases without history of CML or acute lymphoblasticleukemia. Recently, Beedassy et al. reported an extra-medullary CD20 and TdT-positive blast crisis in a patientwith CML in complete cytogenetic and molecular remis-sion on interferon-alpha therapy [1].

Fig. 3A, B FISH analysis forBcr-Abl hybrid gene of bonemarrow interphase nuclei (lastintra vitam biopsy in final stageof disease). The cell shows fiveBcr-Abl fusion signals, oneisolated Abl copy, and a loss ofBcr gene, indicating progressivegenomic instability (red signalsAbl gene, green signals Bcrgene; A Bcr-Abl, B Abl)

Table 1 Results of immunohistochemistry at different sites of disease

Antigen Intra vitam biopsyof chest wall tumor

Postmortem examination

Tumor Lymph nodes Bone marrow

CD34 + + + Single cells +CD79a + – + +CD43 + + + +CD30 + – – –TdT (+) – – –CD20 (+) – – –MPO – Single cells + Single cells + Single cells +CD68 + (reactive cells) + (reactive cells) + (reactive cells) + (reactive cells)

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Comparison of tumor cells from initial biopsy andfrom postmortem examination revealed a change insurface antigen expression. Whereas tumor biopsy wasnegative for MPO, there were MPO-reactive cells in allpostmortem specimens; in contrast to this, expression ofTdT, CD20, and CD30 were lost. We interpret this shift inantigen expression as a further biological evolution of theaggressive neoplasia.

Even more surprising was the detection of a translo-cation t(14;18) in the tumor cells. As mentioned, t(14;18)is a common feature of peripheral B-cell lymphomas.This chromosomal aberration leads to overexpression ofBcl-2 oncogene, the protein product of which has beenshown to prolong cellular survival by inhibiting apoptosis.To date stem cell involvement in t(14;18)-positiveneoplasms is subject to controversy. In sorted bonemarrow cells of patients with t(14;18)-positive B-cellNHL, Yarkoni et al. [22] could demonstrate that trans-location is present also in erythroid, myeloid, andmegakaryocytic lineages and in progenitor stem cells. Incontrast to this, Voso et al. [21] were not able to detect theaberration in CD34-positive progenitor cells or CD34/CD19-positive B-cell progenitors. Other reports discuss arole of the Bcl-2 oncogene in the molecular genesis ofacute and chronic leukemias [8, 15, 16] and the occur-rence of Bcl-2/IgH rearrangement in healthy individuals[18, 23]. The biological meaning of t(14;18) in our patientremains unclear, in particular because no Bcr-Abl andBcl-2 genes could be detected in the same tumor cells.The existence of a t(14;18)-positive subpopulation oftumor cells which was eliminated by intensive cytotoxictreatment might be a possible explanation. However, wesuggest that final diagnostic and therapeutic decisionshave to be based on clinical, morphological, and molec-ular means and the result of a single assay, especially ahighly sensitive method like PCR, should be interpretedwith caution.

Finally, we conclude that the lymphoid neoplasia inour case was not a true de novo NHL, but should beinterpreted as an extramedullary, very immature lym-phoid blast crisis. This conclusion is supported by theshift of immunophenotype with the appearance of MPO-expressing tumor cells as well as the characteristiccytogenetic findings of multiple Ph chromosomes andthe clonal reappearance of P53 deletion. The aggressivenature of the disease with progression after radiation andchemotherapy fits the common experience in BC patients.The effect of signal transduction inhibitor (STI) 571cannot be assessed in this patient due to the short periodof treatment. Furthermore, progressive Bcr-Abl amplifi-cation in genetically complex cancers seems to beassociated with resistance to STI treatment [9], makinga potential success of this treatment in very advanceddiseases unlikely.

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