t(8;21)(q22;q22) in Blast Phase of Chronic Myelogenous Leukemia

Hematopathology / t(8;21)(q22;q22) IN BLAST PHASE CML

t(8;21)(q22;q22) in Blast Phase of Chronic Myelogenous Leukemia C. Cameron Yin, MD, PhD, L. Jeffrey Medeiros, MD, Armand B. Glassman, MD, and Pei Lin, MD Key Words: Chronic myelogenous leukemia; Blast phase; Clonal evolution; t(9;22); t(8;21) DOI: 10.1309/H8JH6L094B9U3HGT

Abstract The blast phase of chronic myelogenous leukemia (CML) frequently is associated with cytogenetic evidence of clonal evolution, defined as chromosomal aberrations in addition to the t(9;22)(q34;q11.2). We identified the t(8;21)(q22;q22) and other cytogenetic abnormalities by conventional cytogenetics and fluorescence in situ hybridization in 2 patients with t(9;22)-positive CML at the time of blast phase. The t(8;21), which typically is associated with a distinct subtype of de novo acute myeloid leukemia (AML) carrying the aml1/eto fusion gene, was accompanied by increased bone marrow myeloblasts (33%) in case 1 and extramedullary myeloid sarcoma in case 2, suggesting its possible role in disease progression. In case 1, the leukemic cells in aspirate smears had salmon-colored cytoplasmic granules, and immunophenotypic studies showed that the blasts expressed CD19. These findings suggest that the pathologic features of blast phase CML with the t(8;21) resemble those of de novo AML with the t(8;21).

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder of pluripotent hematopoietic stem cells characterized by specific hematologic and chromosomal changes.1,2 The cytogenetic hallmark of CML, the Philadelphia chromosome (Ph), results from a reciprocal translocation involving the abl gene at chromosome 9q34 and the bcr gene at chromosome 22q11.2.3-5 The encoded chimeric protein, BCR/ABL, is a constitutively activated tyrosine kinase that has a central role in the pathogenesis of CML.6 Complex translocations involving chromosomes 9 and 22 with additional chromosomes also occur in a small subset of CML cases.7 The clinical course of patients with CML generally is characterized by an initial chronic phase, an ill-defined accelerated phase, and a terminal blast phase. The accelerated and blast phases are associated with disease progression, resistance to therapy, and a poor prognosis.8 During this evolution, the neoplastic cells usually acquire additional karyotypic abnormalities, most often trisomy 8, a second Ph, and isochromosome17q.9 We identified 2 patients with Ph-positive CML in blast phase with the t(8;21)(q22;q22). The t(8;21)(q22;q22) typically is associated with a distinct type of acute myeloid leukemia (AML) with characteristic morphologic features and a favorable clinical outcome as described in the World Health Organization classification.10 To our knowledge, the occurrence of this translocation in Ph-positive CML in blast phase has been reported previously only once, as a case report.11 We describe the clinicopathologic features of 2 additional cases.

Materials and Methods Case Selection The files of the Cytogenetics Laboratory, Department of Hematopathology, The University of Texas M.D. Anderson 836 836

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Hematopathology / ORIGINAL ARTICLE

Cancer Center, Houston, were searched for cases of t(8;21)(q22;q22) during the period April 1998 to September 2003. Of the 44 cases identified, 2 had the t(8;21)(q22;q22) and the t(9;22)(q34;q11.2). In both cases, the patients had a well-established diagnosis of t(9;22)-positive CML, confirmed by review of medical records, peripheral blood and bone marrow morphologic findings, and cytogenetic analysis. Conventional Cytogenetics and Fluorescence In Situ Hybridization Conventional G-band karyotyping was performed on metaphase cells from bone marrow aspirate samples cultured for 24 or 48 hours using previously described methods.12 Metaphase cells were obtained by using a methanol acetic acid fixation method. Cell suspensions were dropped on airdried, clean slides. Fluorescence in situ hybridization (FISH) was performed on interphase nuclei of matched samples. Assessment for the t(8;21)(q22;q22) and t(9;22)(q34;q11.2) was performed using commercial probes, the LSI aml1/eto dualcolor, dual-fusion translocation probe and the LSI BCR/ABL ES dual color translocation probe (Vysis, Downers Grove, IL), designed for their respective purposes. Slides was treated with 2× sodium saline citrate for 30 minutes at 37°C, dehydrated in 70%, 85%, and 100% ethanol solutions for 2 minutes each, and immediately transferred serially into cold 70%, 85%, 100% ethanol solutions for 2 minutes each and air dried. Probes were denatured and hybridized according to the manufacturer’s instructions. Slides were examined using a fluorescent microscope (Carl Zeiss, Thornwood, NY) with appropriate filters. Reference values (within the background range) in the Cytogenetics Laboratory are less than 1.5% positive for the t(9;22) and less than 0.5% for the t(8;21). For retrospective FISH performed on formalin-fixed, paraffin-embedded tissue sections, the slides were first deparaffinized overnight in a 60°C oven, washed with 2× sodium saline citrate solution, and pretreated with protease. The subsequent hybridization procedure was performed similarly to that described in the preceding paragraph. Real-Time Quantitative RT-PCR Assay A real-time quantitative reverse transcription–polymerase chain reaction (RT-PCR) assay was performed for the detection of bcr/abl fusion transcripts. RNA was extracted from bone marrow samples using Trizol reagent (Gibco-BRL, Gaithersburg, MD) according to the manufacturer’s instructions. Reverse transcription was performed on 1 µg of total RNA using random hexamers and superscript II reverse transcriptase (Gibco-BRL) as described previously.13,14 The resulting complementary DNA was subjected to PCR to amplify bcr/abl fusion transcripts in an ABI PRISM 7700 Sequence Detector

(Perkin Elmer/Applied Biosystems, Foster City, CA) using primers and conditions as described previously.13,14 Flow Cytometric Immunophenotyping Three- or 4-color flow cytometric analysis was performed on peripheral blood or bone marrow aspirate specimens collected in EDTA. After incubation of cells with a panel of monoclonal antibodies for 10 minutes at 4°C, the RBCs were lysed with NH4Cl for 10 minutes, followed by 2 washing steps using phosphate-buffered saline solution. The cells then were resuspended and fixed with 1% paraformaldehyde. The panel of monoclonal antibodies included those specific for CD45 (peridinin-chlorophyll-αprotein conjugated), CD2 (fluorescein isothiocyanate [FITC]), CD3 (allophycocyanin [APC]), CD7 (FITC or phycoerythrin [PE]), CD10 (FITC), CD13 (PE), CD14 (APC), CD19 (FITC or APC), CD33 (FITC or PE), CD34 (FITC), CD64 (PE), and CD117 (FITC or PE). All antibodies were from BD Biosciences (San Jose, CA), and analysis was performed using a FACScan or FACSCaliber cytometer (BD Biosciences). For each antibody, negative staining levels were set by comparison with an isotypematched control. Immunohistochemical Analysis Immunohistochemical studies were performed using formalin-fixed, paraffin-embedded tissue sections and monoclonal antibodies specific for CD3 (dilution 1:80), CD20 (dilution 1:700), CD43 (dilution 1:20), CD45 (LCA, dilution 1:300), and myeloperoxidase (dilution 1:1,000) (DAKO, Carpinteria, CA), as previously described.15

Results Report of Cases Case 1 A 54-year-old woman had a history of easy bruising for several months before seeking medical care in April 2001. She denied systemic B-type symptoms such as fever, weight loss, or night sweats. The physical examination showed no lymphadenopathy or organomegaly. A CBC count revealed a WBC count of 19,500/µL (19.5 × 109/L; reference range, 4,000-11,000/µL [4.0-11.0 × 109/L]) with a differential count of 79% neutrophils (0.79), 2% bands (0.02), 10% lymphocytes (0.10), 6% monocytes (0.06), 2% basophils (0.02), and 1% eosinophils (0.01). The hemoglobin level was 15.1 g/dL (151 g/L; reference range, 14.0-18.0 g/dL [140-180 g/L]), and the platelet count was 367 × 103/µL (367 × 109/L; reference range, 140-440 × 103/µL [140-440 × 109/L]). The Am J Clin Pathol 2004;121:836-842

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serum β2-microglobulin level was elevated to 2.7 µg/mL (230 nmol/L) (reference range, 0.6-2.0 µg/mL [51-170 nmol/L]), and the lactate dehydrogenase was 568 U/L (reference range, 313–618 U/L). Bone marrow biopsy and cytogenetic analysis confirmed the diagnosis of CML in chronic phase. Therapy with imatinib mesylate (Gleevec) was started, and complete remission was achieved 3 months later with a normal CBC count, normal bone marrow cellularity, and a diploid karyotype shown by conventional cytogenetics and FISH analyses. However, a b3a2 bcr/abl fusion transcript coding for the 210-kd BCR/ABL protein was detected by RT-PCR. The patient remained in complete clinical remission until April 2002, when blast-phase CML developed. Laboratory tests revealed a WBC count of 15,100/µL (15.1 × 109/L), with 24% circulating blasts, and left-shifted granulocytes. Also noted were mild anemia with a hemoglobin of 13.9 g/dL (139 g/L) and mild thrombocytopenia with a platelet count of 134 × 103/µL (134 × 109/L). Bone marrow aspirate and biopsy confirmed the diagnosis of CML in blast phase. Cytogenetic analysis revealed a variant Ph chromosome and additional changes including the t(8;21). The patient was treated by stem cell transplantation with a matched unrelated donor in June 2003. She remains free of disease at the time of writing. Case 2 A 47-year-old man noted a painful mass in his left arm in July 2000. He denied systemic B-type symptoms, and physical examination revealed no lymphadenopathy or organomegaly. His initial laboratory data showed a WBC

❚Image 1❚ (Case 1) Bone marrow biopsy specimen at time of initial diagnosis showing hypercellular bone marrow with leftshifted granulopoiesis and increased micromegakaryocytes (H&E, ×200).

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count of 37,600/µL (37.6 × 109/L) with a moderate left shift in granulocyte maturation, 2% blasts (0.02), and 4% basophilia (0.04 basophils). Mild anemia with a hemoglobin of 11.1 g/dL (111 g/L) and thrombocytosis with a platelet count of 545 × 103/µL (545 × 109/L) were present. The serum lactate dehydrogenase level was elevated to 3,416 U/L. Bone marrow aspirate and biopsy showed features of CML in chronic phase with 1% blasts. However, a biopsy of the left arm mass revealed myeloid sarcoma. Cytogenetic analysis of the bone marrow aspirate specimen identified the Ph. Based on the presence of extramedullary myeloid sarcoma, the patient was considered clinically to be in blast phase, despite the low blast count in the bone marrow. The patient was treated with troxacitabine, and complete clinical and hematologic remission were achieved 1 month later, with complete resolution of the left arm mass. However, the bone marrow remained Ph-positive by conventional cytogenetic analysis. The therapy was changed to imatinib mesylate, interferon α, and cytarabine in June 2001, but conventional cytogenetic analysis of bone marrow aspirate material continued to show the Ph. In April 2002, a recurrent mass developed on the patient’s shoulder, and a second mass developed in an axillary lymph node 2 months later, which were treated with triapine and local radiation. Conventional cytogenetic analysis of bone marrow and shoulder mass biopsy specimens obtained at this time (19 months after initial diagnosis) showed the Ph, the t(8;21), and additional chromosomal abnormalities. In November 2002, neutropenic fever, hyperbilirubinemia, and radiation pneumonitis developed, in addition to extensive leukemic infiltration of the skeletal muscles in the shoulder and left upper arm. He died shortly thereafter. Morphologic and Immunophenotypic Findings Aspirate smears and histologic sections of the bone marrow biopsy specimens from cases 1 and 2 at the time of initial examination were similar. Both specimens had characteristic features of CML in chronic phase with marked trilineage hyperplasia, left-shifted granulopoiesis, increased micromegakaryocytes, basophilia, and eosinophilia ❚Image 1❚. In case 1, the disease progressed to blast phase 12 months after the initial diagnosis of CML. At this time, 33% blasts were present in bone marrow aspirate smears, and the aspirate clot section showed 90% cellularity ❚Image 2A❚ . The blasts were of medium to large size with a moderate amount of cytoplasm and salmon-colored cytoplasmic granules ❚Image 2B❚. Auer rods were not identified. Most blasts were positive for myeloperoxidase shown by cytochemical analysis. Flow cytometry immunophenotyping studies showed that the blasts were positive for CD13, CD33, CD34, and HLA-DR and were negative for CD3, CD10, and terminal deoxynucleotidyl transferase, © American Society for Clinical Pathology


A

B

❚Image 2❚ (Case 1) A, Bone marrow clot specimen at time of blast crisis showing hypercellular bone marrow with sheets of immature myeloid cells (H&E, ×200). B, Bone marrow aspirate smear showing large blasts with basophilic cytoplasm and salmon-colored granules (Wright-Giemsa, ×1,000).

supporting myeloid lineage. The blasts also expressed the B-cell antigen CD19. In case 2, the histologic sections of the left arm mass biopsy specimen demonstrated small fragments of skeletal muscle extensively infiltrated by a hematopoietic neoplasm. Some cells were immature and blastoid, but other cells had more mature nuclei resembling myelocytes and metamyelocytes. Rare bands and mature neutrophils also were scattered throughout the infiltrate ❚Image 3❚. Immunostains revealed that the neoplastic cells were positive for myeloperoxidase and CD43 and negative for CD3, CD20, and CD45, supporting myeloid lineage. Fine-needle aspiration performed on subsequent (19 months later) masses arising in the shoulder and axillary lymph node demonstrated similar cytologic and immunohistochemical findings. Both specimens showed myeloid cells with many early and intermediate forms. Concurrent bone marrow aspirate and biopsy specimens showed 30% cellularity with 1% blasts. Cytogenetic Findings Conventional cytogenetic analysis of bone marrow aspirate material obtained at the time of initial examination revealed the t(9;22;19;20)(q34;q11;p13.1;q22) in case 1 and the t(9;22)(q34;q11.2) in case 2. FISH analysis detected bcr/abl nuclear fusion signals in more than 90% of the interphases in both cases. During the course of the disease, in both cases conventional cytogenetics detected additional chromosomal abnormalities that included the t(8;21)(q22;q22) ❚Table 1❚ These abnormalities were detected in the bone marrow (cases 1 and 2) and the shoulder and axillary masses (case 2) 12 to 19 months after the initial diagnosis of CML.

For case 2, we retrospectively performed FISH studies for the t(9;22) and t(8;21) on histologic sections of the left arm mass biopsy specimen and for the t(8;21) on the initial bone marrow biopsy specimen. Fusion signals were identified in the corresponding specimens, demonstrating the presence of both the t(9;22) and t(8;21) in the left arm mass specimen ❚Image 4A❚ and ❚Image 4B❚ , and the t(8;21) in the initial bone marrow biopsy specimen.

❚Image 3❚ (Case 2) Histologic sections of the left arm mass biopsy specimen show increased immature myeloid cells. The infiltrating cells have a moderate amount of eosinophilic cytoplasm (H&E, ×500).

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❚Table 1❚ Cytogenetic Results and Corresponding Morphologic Features for Two Cases of CML With t(8;21)(q22;q22) Case No.

Time

Site

Conventional Cytogenetic Findings

Morphologic Findings

1

Initial 12 mo

BM BM

CML-CP CML-BP

Initial

BM* Arm† BM Shoulder, axilla

46,XX,t(9;22;19;20)(q34;q11;p13.1;q22)[20] 48,XX,+8,t(8;21)(q22;q22),t(9;22;19;10)(q34;q11;p13.1;q22), +der(22)t(9;22;19;10)[20] 46,XY,t(9;22)(q34;q11.2)[20] Not done 47,XY,+8,t(8;21)(q22;q22),del(9)(q13q32),t(9;22)(q34;q11.2)[2] 47,XY,+8,t(8;21)(q22;q22),del(9)(q13q32),t(9;22)(q34;q11.2)[20]

2

19 mo

CML-CP EMS CML-CP EMS

BM, bone marrow; BP, blast phase; CML, chronic myelogenous leukemia; CP, chronic phase; EMS, extramedullary myeloid sarcoma. * The t(8;21) was identified retrospectively in a bone marrow aspirate specimen by FISH. † The t(8;21) and t(9;22) were identified retrospectively on formalin-fixed, paraffin-embedded sections by FISH.

Discussion Secondary chromosomal aberrations that occur as part of clonal evolution are demonstrable in 60% to 80% of cases of CML in accelerated and blast phases.16,17 The secondary changes usually are complex, with trisomy 8, an extra Ph, and isochromosome 17q most common, occurring in 34%, 30%, and 20% of cases, respectively.9,17 Rare cases involve translocations and inversions that typically are associated with myelodysplastic syndromes or de novo AML, such as inv(3)(q21q26), t(3;21)(q26;q22), and inv(16)(p13q22).9,18,19 We describe 2 cases in which the t(8;21)(q22;q22) was detected by conventional cytogenetic analysis after the initial diagnosis and treatment for Ph-positive CML. The t(8;21)(q22;q22) is found in 10% to 15% of de novo AML

cases, designated as one type of AML with recurrent cytogenetic abnormalities in the World Health Organization classification and representing a subset of AML with maturation in the French-American-British classification (FAB M2).10,20 This translocation disrupts the aml1 (recently renamed runx1) gene on chromosome 21q22 and the eto gene on chromosome 8q22, resulting in the aml1/eto fusion gene on the derivative chromosome 8.21 AML with t(8;21) occurs predominantly in young patients with a propensity to involve extramedullary sites and is associated with a good prognosis.21,22 Characteristic morphologic features include the presence of large blasts with abundant basophilic cytoplasm, salmon-colored granules, long and slender Auer rods, a variable degree of dysplasia, and eosinophilia or basophilia. The blasts in AML with t(8;21) frequently

B

A

❚Image 4❚ (Case 2) Fluorescence in situ hybridization performed on histologic sections of paraffin-embedded tissue from the left arm mass biopsy specimen (×630). A, t(9;22)(q34;q11.2). B, t(8;21)(q22;q22). In a Ph+ cell, the hybridization produces 1 fused yellow signal (5’bcr/3’abl), 1 larger orange signal (native abl), 1 smaller orange signal (residual arginosuccinic synthetase gene), and 1 green signal (native bcr). In a cell with t(8;21), the hybridization produces 2 fused yellow signals (aml1/eto), 1 orange signal (eto), and 1 green signal (aml1). The normal control for each translocation produces 2 green and 2 orange signals. Arrows indicate cells with fusion signals.

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express the B-cell antigen CD19, in addition to myeloid markers such as CD13, CD33, and myeloperoxidase.23,24 The blast phase of CML in our patients showed features similar to AML with t(8;21). In case 1, the blasts were large with basophilic cytoplasm and salmon-colored granules. They also expressed CD19. In case 2, the blasts in the extramedullary myeloid sarcoma studied in H&E-stained tissue sections had abundant eosinophilic cytoplasm. However, salmon-colored granules are not detectable in tissue sections. These findings suggest that Ph-positive CML in blast phase with the t(8;21)(q22;q22) as an additional abnormality recapitulates the features of de novo AML carrying the same translocation. Other authors have observed similar findings, but involving other translocations, in blast-phase CML. Mohamed and colleagues19 reported 1 case of CML in which the inv(16)(p13q22) occurred at the time of blast phase. At this time, the leukemia had morphologic and immunophenotypic features of acute myelomonocytic leukemia with eosinophilia (FAB M4Eo). Nishii and colleagues25 reported a case of CML with t(11;17)(q23;q21) involving the MLL gene that expressed myeloid and B-lymphoid antigens. It seems that CML in blast phase with secondary cytogenetic abnormalities identical to those commonly associated with de novo AML also shares similar morphologic and/or immunophenotypic features with de novo AML. Hematologic responses to imatinib mesylate typically occur within 2 weeks after initiation of therapy, and complete hematologic responses often are achieved by 4 weeks.26 The rate of cytogenetic remission is related to pretreatment laboratory values. Patients showing complete cytogenetic responses tend to have lower WBC and platelet counts and fewer than 5% bone marrow blasts.27 At initial diagnosis, case 1 had CML in chronic phase with an isolated variant Ph, and clinical and cytogenetic remission occurred 3 months after treatment, although bcr/abl fusion transcripts were detected by real-time RT-PCR analysis. This remission lasted for 9 months until the disease progressed to blast phase, which correlated with the detection of secondary cytogenetic abnormalities. In contrast, at initial diagnosis, case 2 had myeloid sarcoma involving the left arm and a higher WBC count. Although clinical remission was obtained 1 month after treatment with troxacitabine, complete cytogenetic remission never occurred, and a recurrent extramedullary myeloid sarcoma erupted 19 months later, at which time secondary cytogenetic abnormalities were detected. Secondary genetic changes in CML may provide alternative survival or proliferation signals bypassing the bcr/abl pathway. aml1 contains a region of sequence homology to the Drosophila runt gene and the mouse polyomavirus enhancer binding protein pebp2 α gene.21 eto has a sequence with zinc finger motifs and proline-rich domains, suggestive

of its role as a transcription factor.21 Thus, the t(8;21) is a fusion between 2 probable transcription factors. The production of AML1/ETO fusion protein alters transcriptional regulation of normal aml1 and eto target genes. This may have a role in progression of CML by binding to DNA promoter and/or enhancer domains, thus increasing the transcription and expression of certain genes involved in cell growth. Although transgenic mice studies have shown that the t(8;21) is insufficient to generate a leukemic phenotype,28 the synergistic effect of bcr/abl and aml1/eto might provide an additional growth advantage necessary for neoplastic transformation. Interestingly, despite evidence of the t(8;21)(q22;q22) and other abnormalities, the bone marrow specimen from case 2 at the time of recurrent extramedullary disease showed normal cellularity without increased blasts. The discordant findings between the bone marrow and the shoulder mass suggest that the development of leukemia is determined by the microenvironment as well as cytogenetic alterations in the transformed cells. The risk for extramedullary disease may not be reduced even when tumor proliferation in the bone marrow is controlled by therapy. Furthermore, although there was no morphologic and conventional cytogenetic evidence of transformation in the bone marrow in case 2 at the time of initial examination, retrospective FISH analysis of the bone marrow aspirate specimen showed the presence of the t(8;21). This suggests that a subclone of cells with clonal evolution (under the detection threshold by conventional cytogenetics) already was present at that time and may have had a role in the development of extramedullary lesions. In summary, we describe the clinical, morphologic, immunophenotypic, and cytogenetic features of 2 cases of Phpositive CML in which the t(8;21)(q22;q22) developed along with other abnormalities at the time of blast crisis. This translocation was accompanied by morphologic and immunophenotypic features similar to de novo AML with the t(8;21). From the Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston. Address reprint requests to Dr Lin: Dept of Hematopathology, Box 72, UT M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030. Acknowledgments: We thank Kimberly Hayes and the staff of the Cytogenetics Laboratory for their contribution to the cytogenetic analysis.

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