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T-cell blast crisis of chronic myelogenous leukemia manifesting as a large mediastinal tumor
HUMAN PATHOLOGY
Volume 33, No. 7 (July 2002)
T-CELL BLAST CRISIS OF CHRONIC MYELOGENOUS LEUKEMIA MANIFESTING AS A LARGE MEDIASTINAL TUMOR CHARLES C. YE, MD, CAROLINA ECHEVERRI, MD, JEANNE E. ANDERSON, MD, JANICE L. SMITH, PHD, ARMAND GLASSMAN, MD, MARGARET L. GULLEY, MD, DAVID CLAXTON, MD, AND FIONA E. CRAIG, MD We report an unusual case of T-cell blast crisis of chronic myelogenous leukemia (CML) with a clinical presentation more typical of de novo T-cell lymphoblastic lymphoma. The patient was a 32-yearold man who presented with acute superior vena cava syndrome 19 months after an initial diagnosis of CML and 5 months after allogeneic bone marrow transplantation. The tumor was composed of primitive lymphoid cells expressing CD2, CD3, CD4, CD5, CD7, CD8, and CD10. Although the clinical features were more typical of acute lymphoblastic leukemia/lymphoma, fluorescence in situ hybridization analysis showed the bcr-abl fusion gene within blastic tumor cells. This finding confirmed that the mass represented a blastic transformation of CML. We use the unusual features of the current case and the previous reports to suggest that the development of T-cell blast crisis of CML is dependent on the presence of both
marrow and extramedullary disease and a mechanism to evade apoptosis. HUM PATHOL 33:770-773. Copyright 2002, Elsevier Science (USA). All rights reserved. Key words: blast crisis, chronic myelogenous leukemia, fluorescence in situ hybridization, T-lymphoblast. Abbreviations: CML, chronic myelogenous leukemia; BMT, bone marrow transplant; RT-PCR, reverse transcriptase polymerase chain reaction; FNA, fine needle aspiration; FITC, fluorescein isothiocyanate; PE, phycoerythrin; FISH, fluorescence in situ hybridization; D-FISH, double bcr/abl fusion fluorescence in situ hybridization; S-FISH, single bcr/abl fusion fluorescence in situ hybridization; EBV, Epstein-Barr virus; Ig, immunoglobulin; ALL/L, acute lymphoblastic lymphoma leukemia; TCR, T-cell receptor; T-ALL/L, T-cell acute lymphoblastic lymphoma leukemia.
Chronic myelogenous leukemia (CML) is usually diagnosed in the chronic phase. After experiencing 3 to 5 years of stable disease, patients enter a terminal blast phase characterized by malaise, anorexia, bone pain, and weight loss. Approximately 70% of cases have a myeloid blast phenotype, and most of the remaining 30% have a primitive B-lymphocyte phenotype. Rare cases of blast crisis of CML with a primitive T-lymphocyte phenotype have been reported.1,2 In this report, we describe a patient who developed a mediastinal mass after undergoing HLA-matched, unrelated donor bone marrow transplant (BMT) for accelerated-phase CML. Biopsy of the mass showed a blastic process with an immature T-cell phenotype. We describe the studies performed to obtain a definitive diagnosis, review the literature on T-cell blast crisis of CML, and discuss the possible pathogenesis.
superior vena cava syndrome. Computed tomography scan of the neck and chest showed a large mediastinal tumor mass extending into the right supraclavicular fossa (Fig 1). There was an absence of circulating blasts and no leukoerythroblastosis. The patient underwent fine needle aspiration (FNA) and biopsy of the mass and repeat bone marrow aspiration. After a diagnosis of T-cell blast crisis of CML was established, the patient commenced treatment with conventional multidrug chemotherapy. Remission was not achieved, and he died of disease progression.
CASE REPORT The patient was diagnosed with chronic-phase CML in July 1998. Cytogenetic studies showed t(9;22), and reverse transcriptase polymerase chain reaction (RT-PCR) identified the bcr-abl p210 rearrangement. After cytoreduction with hydroxyurea and 8 months of treatment with ␣-interferon, the patient did not experience a cytogenetic remission and subsequently acquired trisomy 8. In September 1999, he underwent human leukocyte antigen–matched, gender-identical, unrelated donor BMT. Bone marrow aspirates performed on day 29 and day 100 after transplant were hypocellular and karyotypically normal but were persistently positive for bcr-abl p210 detected by RT-PCR. On day 154, he developed acute
From the Departments of Pathology and Hematology, University of Texas Health Science Center at San Antonio, San Antonio, TX, Laboratories for Genetics Services, Inc, Houston, TX, and M.D. Anderson Cancer Center, Houston, TX; Milton S. Hershey Medical Center, Hershey, PA; and the Department of Pediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada. Accepted, for publication April 22, 2002. Address correspondence and reprint requests to Fiona E. Craig, MD, Department of Pathology, University of Pittsburgh Medical Center, Presbyterian Hospital, Room C604, 200 Lothrop St, Pittsburgh, PA 15213. Copyright 2002, Elsevier Science (USA). All rights reserved. 0046-8177/02/3307-0015$35.00/0 doi:10.1053/hupa.2002.126190
MATERIALS AND METHODS FNA of the mediastinal tumor was performed, and the sample was processed using standard methods. Immunohistochemistry was performed on decalcified bone marrow biopsy sections using a routine streptavidin-biotin method.3 Flow cytometry was performed by dual-color analysis using a FACs Calibur flow cytometer (Becton, Dickinson and Company, San Jose, CA), SimulSET software (Becton, Dickinson), and fluorescein isothiocyanate (FITR)- and phycoerythrin (PE)-labeled antibodies (Becton, Dickinson).4 RT-PCR was used to amplify bcr-abl p210.5 Epstein-Barr (EBV) viral load was measured in plasma by competitive PCR as previously described6 using commercial reagents (ViralQuant, BioSource, Camarillo, CA). TCR␥ gene rearrangement was performed using multiplex primers directed at V1– 8, V9, V10 as well as V11:J1/2, JP and JP1/2 sequences.7 Immunoglobulin H gene rearrangement was assayed by PCR using FRIII:JHa primers and Hot-Start polymerase (AmpliTaq, Perkin-Elmer, Wellesley, MA).8 PCR assays were done on a Perkin-Elmer GeneAmp PCR System 9700 alongside a positive cell line control, at least two negative tissue controls, and a no-template blank. Products were sized on agarose or polyacrylamide gels. Fluorescence in situ hybridization (FISH) analysis was performed on paraffin-embedded sections mounted on silanecoated slides. The procedure was carried out according to a protocol adapted from that for HER-2/neu FISH analysis of breast tumor9 and the LSI bcr/abl probe (Vysis, Downers Grove, IL) detecting double bcr/abl gene fusion signal (D-FISH) was applied. FISH analysis was performed in a second laboratory on a fresh left supraclavicular lymph node biopsy sample and bone marrow aspirate. The target DNA was hybrid-
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CASE STUDIES
FIGURE 1. Computed tomography scan of the chest. Large mediastinal tumor mass (arrow) compressing the superior vena cava (arrowhead).
ized with an LSI probe (Vysis) detecting single bcr/abl gene fusion signal (S-FISH).10 RESULTS FNA smears and the core biopsy sample from the mediastinal tumor mass showed a monotonous blastic infiltrate (Fig 2). Flow cytometry showed expression of CD2, CD3, CD4, CD5, CD7, CD8, and CD10. The concurrent bone marrow aspirate was hypocellular, with a blast count of 0.2%. Sections from the corresponding iliac crest bone marrow biopsy sample showed focal areas of blastic infiltration that expressed CD3 by immunohistochemistry. There was no expression of CD20, myeloperoxidase, or EBV-latent membrane protein. RT-PCR of blood obtained at the time of the mediastinal biopsy was strongly positive for bcr-abl p210, suggesting persistent or relapsed leukemia. The band pattern was identical to that of a blood specimen drawn at the time of initial diagnosis 19 months earlier. EBV was not detectable in peripheral blood as measured by quantitative PCR. Clonal TCR␥ gene rearrangement was identified in the mediastinal tumor tissue and right bone marrow biopsy, with identical bands. Clonal IgH gene rearrangement also was identified in the mediastinal tumor tissue but not in the bone marrow, and the negative marrow result was interpreted as being caused by the paucity of neoplastic cells. D-FISH performed on paraffin sections from the mediastinal tumor using an LSI bcr/abl dual-color probe showed a gene fusion signal in 91 of 120 tumor cells (76%) (Fig 3). Conventional S-FISH analysis performed on frozen tissue showed the bcr-abl gene fusion signal in 194 of 200 interphase, aspirated cells (97%) from the supraclavicular biopsy and in 25 of 200 interphase/metaphase, aspirated marrow cells (12.5%). These values are higher than the background for these techniques; thus, they confirmed the morphological finding of a blastic infiltrate.
FIGURE 2. Histopathology of the mediastinal tumor specimen. The biopsy sample shows a focus composed of monotonous, small- to intermediate-sized lymphoid cells containing scant cytoplasm, round nuclei, and finely dispersed chromatin (Hematoxylin and eosin; original magnification x400).
mia (ALL/L), and blast crisis of CML. Subsequent bone marrow evaluation showed focal involvement by a blastic T-cell infiltrate, thus indicating the invasive nature of this process and confirming the T-lymphoid phenotype. Molecular diagnostic studies showed clonal T-cell receptor (TCR) and IgH rearrangement, thus indicating the presence of a clonal lymphoid proliferation. T-cell acute lymphoblastic lymphoma leukemia (T-ALL/L) is a disease that frequently affects young adults and is more predominant in male patients. Patients often present with bulky mediastinal disease related to involvement of the thymus. Although the clinical presentation of the patient described in this report is consistent with de novo ALL/L, identification of the bcr-abl p210 rearrangement by D- and S-FISH makes this diagnosis unlikely. In adult ALL, the bcr-abl rear-
DISCUSSION We report an unusual case of T-cell blast crisis of CML presenting as a rapidly growing mediastinal mass that appeared 5 months after allogeneic BMT. A biopsy sample was obtained. The initial differential diagnosis included normal thymic tissue, thymoma, posttransplantation lymphoproliferative disorder, de novo acute lymphoblastic lymphoma leuke-
FIGURE 3. FISH using a bcr-abl probe. (A) D-FISH analysis on paraffin-embedded mediastinal tumor tissue. Positive cells (arrows) contain 4 signals: a green signal indicates the normal chromosome 22; an orange signal, the normal chromosome 9; a yellow fusion signal, bcr-abl on the translocated chromosome 22. A second orange signal indicates the remaining abl gene on the translocated chromosome 9.
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rangement appears to be confined to cases with a B-cell or, rarely, mixed-lineage phenotype.11 Although pediatric T ALL/L may be Ph1-positive, the majority of cases have a p190 breakpoint.11 Furthermore, and more importantly, the patient had a history of CML involving the bcr-abl p210 breakpoint. T-cell blast crisis of CML is a rare disorder; only 25 cases have been previously reported. Patients vary in age from 13 to 67 years, and the male-to-female ratio is 1.5:1. The interval between the diagnosis of CML and the onset of blast crisis ranges from 5 months to 6 years. Many patients present with hepatosplenomegaly and lymphadenopathy.2,12-22 Lymph node biopsy was performed in 13 of the reported cases, and all samples showed diffuse blastic infiltration and the presence of the Ph1-chromosome and/or bcr-abl translocation using conventional karyotyping and Southern blot analysis, respectively.12-14,16,17,19-22 In 9 cases, the blast count in bone marrow was less than 30% at the time the lymph node blast crisis was diagnosed.12-14,16,19,22 One of the best documented cases was published by Sun et al1 who reported a 63-year-old female patient who developed progressive back pain 5 years after an initial diagnosis of CML. Magnetic resonance imaging of the spine showed lytic lesions and a soft tissue mass at T11. The extramedullary mass was biopsied, and the sample showed a T-cell blastic infiltrate that was positive by cytogenetics for the Ph1-chromosome. Concurrent posterior iliac crest bone marrow examination showed only 1% blasts.1 Sun et al1 observed that in T-lymphoblastic transformation of CML, extramedullary blast crisis often precedes overt blastemia and overt blastic infiltration in the bone marrow. The presence of a predominant extramedullary component is documented in at least 9 of the 25 previously reported cases as well as in the current case.12-14,16,19,21,22 Fermand et al18 reported a case of a 19-year-old female patient who developed T-cell blast crisis 4 years after CML was diagnosed. Her clinical manifestations included rapid onset of coma, hepatosplenomegaly, lymphadenopathy, and a large mediastinal mass involving the thymic region. Unfortunately, this mediastinal mass was not biopsied.18 Allogeneic BMT was performed in only 1 of the 25 reported cases, in which a 37-year-old female patient developed nodal T-cell blast crisis in her supraclavicular regions approximately 5 months after undergoing allogeneic BMT for CML. Chest x-ray showed a large pleural effusion but no mediastinal lymphadenopathy.21 Although the latter case resembles the current case most closely, the mediastinal involvement in the current report raised the possibility of de novo T-ALL/L. Clues to the pathogenesis of T-cell blastic transformation of CML lie in the physiology of normal T-lymphocyte maturation and the lack of involvement of T-lymphocytes in CML. During normal T-lymphocyte maturation, prothymocytes migrate from the bone marrow to the thymic cortex. There, the fate of the cell (activation, anergy, or apoptosis) is determined by T-cell receptor– generated intracellular signaling pathways.23 Many cells die by apoptosis within the cortex.23 Those that survive migrate to the medulla and are released into peripheral blood.23 The requirement for a thymic pathway for T-cell maturation may explain the absence of the Ph-chromosome in T-lymphocytes in patients with CML despite involvement of hematopoietic stem cells.24 Most patients with CML are adults who have a relatively inactive thymus because of age-related involution. Immature abnormal T-cells may reach the thymus, but they die by apoptosis within the cortex. In this case, the patient presented with prominent mediastinal disease consistent with thymic involvement. This suggests that immature cells containing the bcr-abl translocation escaped from the bone marrow and migrated to the thymus where they proliferated and evaded apoptosis. An-
other clue to the pathogenesis of T-cell blast crisis of CML is the presence in our case of clonal IgH gene rearrangement in addition to rearrangement of the T-cell receptor (TCR) gamma gene. Coexisting IgH and TCR receptor rearrangements have been described in up to 30% of T-ALL/L cases.25 Promiscuous IgH rearrangement also has been reported in normal mouse thymocytes and has been assumed to be an event that would normally direct cells toward apoptosis.26 Cases of T-ALL/L, and our case of T-cell blast crisis of CML, may represent immortalization of these abnormally rearranged T-cells by a malignant transformation that rescues them from this fatal pathway. In summary, we present a case of T-cell blast crisis of CML with a clinical presentation resembling that of T-ALL/L. The differential diagnosis was resolved using interphase FISH analysis showing bcr-abl rearrangement. The presentation and aberrant IgH gene rearrangement suggest a pathogenesis for T-cell blast crisis of CML that is similar to that of de novo ALL/L. Inhibition of apoptosis may play a role in the development of both diseases. The rarity of T-cell blast crisis of CML may relate to the apparent requirement for both a bone marrow (stem cell bcr-abl rearrangement) and extramedullary (thymic maturation) component of disease and a mechanism to evade apoptosis. REFERENCES 1. Sun T, Susin M, Koduru P, et al: Extramedullary blast crisis in chronic myelogenous leukemia: Demonstration of T-cell lineage and Philadelphia chromosome in a paraspinal tumor. Cancer 68:605-610, 1991 2. Dorfman DM, Longtine JA, Fox EA, et al: T-cell blast crisis in chronic myelogenous leukemia: Immunophenotypic and molecular biologic findings. Am J Clin Pathol 107:168-176, 1997 3. Elias JM, Margiotta M, Gaborc O: Sensitivity and detection efficiency of the peroxidase antiperoxidase (PAP), avidin-biotin peroxidase complex (ABC) and peroxidase-labeled avidin-biotin (LAB) methods. Am J Clin Pathol 92:6267, 1989 4. Stelzer GT, Marti G, Hurley A, et al: US-Canadian consensus recommendations on immunophenotypic analysis of hematologic neoplasia by flow cytometry: Standardization and validation of laboratory procedures. Cytometry 30:214-230, 1997 5. Radich JP, Gehly G, Gooley E, et al: Polymerase chain reaction detection of the BCR-ABL fusion transcript after allogenic marrow transplantation for chronic myeloid leukemia: Results and implications in 346 patients. Blood 85:2632-2638, 1995 6. Fan H, Gulley ML: EBV viral load by competitive PCR, in Killeen A (ed): Molecular Pathology Protocols. Humana Press, Totowa, NJ, 2001 7. Krafft AE, Taubenberger JK, Sheng ZM, et al: Enhanced sensitivity with a novel TCR gamma PCR assay for clonality studies in 569 formalin-fixed, paraffin-embedded (FFPE) cases. Molecular Diagnosis 4:119-133, 1999 8. Gill JI, Gulley ML: Immunoglobulin and T-cell receptor gene rearrangement. Hematol Oncol Clin North Am 8:751-770, 1994 9. Masood S, Bui MM, Yung JF, et al: Her-2/neu SpectrumOrangeTM and CEP 17 SpectrumGreen TM dual color deoxyribonucleic acid probe kit. Ann Clin Lab Sci 28:215-223, 1998 10. DeWald GW, Wyatt WA, Juneau AL, et al: Highly sensitive fluorescence in situ hybridization method to detect double BCR/ABL fusion and monitor response to therapy in chronic myeloid leukemia. Blood 91:3357-3365, 1998 11. Crist W, Carroll A, Shuster J, et al: Philadelphia chromosome positive childhood acute lymphoblastic leukemia: Clinical and cytogenetic characteristic and treatment outcome: A Pediatric Oncology Group study. Blood 76:489494, 1990 12. Palutke M, Eisenberg L, Nathan L: Ph1-positive T-lymphoblastic transformation of chronic granulocytic leukemia in a lymph node. Lancet 2:1053. 13. Jacobs P, Greaves M: Ph1-positive T lymphoblastic transformation. Leuk Res 8:737-739, 1984 14. Vannier JP, Bizet M, Bastard A, et al: Simultaneous occurrence of a T-cell lymphoma and a chronic myelogenous leukemia with an unusual phenotype. Leuk Res 8:647-657, 1984 15. Cervantes F, Anegon I, Rozman C, et al: Early T-cell features in blast crisis of Ph1-positive chronic myelogenous leukaemia. Scand J Haematol 35:71-76, 1985 16. Allouche M, Bourinbaiar A, Goergoulias V, et al: T cell lineage involvement in lymphoid blast crisis of chronic myelogenous leukemia. Blood 66:11551161, 1985 17. Falini BM, Tabilio A, Pelicci PG, et al: T-cell receptor -chain gene rearrangement in a case of Ph1-positive chronic myelogenous leukaemia blast crisis. Br J Haematol 62:776-779, 1986
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18. Fermand JP, Sigaux F, Tsapis A, et al: T-cell derived blast crisis in chronic myelogenous leukemia. Leukemia 1:210-212, 1987 19. Yasukawa M, Iwamsa K, Kawamura S, et al: Phenotypic and genotypic analysis of chronic myelogenous leukemia with T-lymphoblastic and megakaryoblastic mixed crisis. Br J Haematol 66:331-336, 1987 20. Giannone L, Whitlock JA, Kinney MC, et al: Use of the BCR probe to demonstrate extramedullary recurrence of CGL with a T cell lymphoid phenotype following bone marrow transplantation. Bone Marrow Transplant 3:631635, 1988 21. Kuriyama K, Gale RP, Tomonaga M, et al: CML-T1: A cell line derived from T-lymphocyte acute phase of chronic myelogenous leukemia. Blood. 74:1381-1387, 1989 22. Advani SH, Malhotra H, Kadam PR, et al: T-lymphoid blast crisis in chronic myelogenous leukemia. Am J Hematol 36:86-92, 1991
23. Conroy LA, Alexander DR: The role of intracellular signalling pathways regulating thymocyte and leukemic T-cell apoptosis. Leukemia 10:14221435, 1996 24. Takahashi N, Miura I, Saitoh K, et al: Lineage involvement of stem cells bearing the Philadelphia chromosome in chronic myeloid leukemia in the chronic phase as shown by a combination of fluorescence-activated cell sorting and fluorescence in situ hybridization. Blood 92:4758-4763, 1998 25. Medeiros LJ, Carr J: Overview of the molecular methods in the diagnosis of malignant lymphomas. Arch Pathol Lab Med 123:1189-1207, 1999 26. Szczepanski T, Pongers-Willemse MJ, Langerak AW, et al: Ig heavy chain gene rearrangements in T-cell acute lymphoblastic leukemia exhibit predominant DH6-19 and DH7-27 gene usage, can result in complete V-D-J rearrangements, and are rare in T-cell receptor ␣ lineage. Blood 93:40794085, 1999
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T-CELL BLAST CRISIS OF CHRONIC MYELOGENOUS LEUKEMIA MANIFESTING AS A LARGE MEDIASTINAL TUMOR CHARLES C. YE, MD, CAROLINA ECHEVERRI, MD, JEANNE E. ANDERSON, MD, JANICE L. SMITH, PHD, ARMAND GLASSMAN, MD, MARGARET L. GULLEY, MD, DAVID CLAXTON, MD, AND FIONA E. CRAIG, MD We report an unusual case of T-cell blast crisis of chronic myelogenous leukemia (CML) with a clinical presentation more typical of de novo T-cell lymphoblastic lymphoma. The patient was a 32-yearold man who presented with acute superior vena cava syndrome 19 months after an initial diagnosis of CML and 5 months after allogeneic bone marrow transplantation. The tumor was composed of primitive lymphoid cells expressing CD2, CD3, CD4, CD5, CD7, CD8, and CD10. Although the clinical features were more typical of acute lymphoblastic leukemia/lymphoma, fluorescence in situ hybridization analysis showed the bcr-abl fusion gene within blastic tumor cells. This finding confirmed that the mass represented a blastic transformation of CML. We use the unusual features of the current case and the previous reports to suggest that the development of T-cell blast crisis of CML is dependent on the presence of both
marrow and extramedullary disease and a mechanism to evade apoptosis. HUM PATHOL 33:770-773. Copyright 2002, Elsevier Science (USA). All rights reserved. Key words: blast crisis, chronic myelogenous leukemia, fluorescence in situ hybridization, T-lymphoblast. Abbreviations: CML, chronic myelogenous leukemia; BMT, bone marrow transplant; RT-PCR, reverse transcriptase polymerase chain reaction; FNA, fine needle aspiration; FITC, fluorescein isothiocyanate; PE, phycoerythrin; FISH, fluorescence in situ hybridization; D-FISH, double bcr/abl fusion fluorescence in situ hybridization; S-FISH, single bcr/abl fusion fluorescence in situ hybridization; EBV, Epstein-Barr virus; Ig, immunoglobulin; ALL/L, acute lymphoblastic lymphoma leukemia; TCR, T-cell receptor; T-ALL/L, T-cell acute lymphoblastic lymphoma leukemia.
Chronic myelogenous leukemia (CML) is usually diagnosed in the chronic phase. After experiencing 3 to 5 years of stable disease, patients enter a terminal blast phase characterized by malaise, anorexia, bone pain, and weight loss. Approximately 70% of cases have a myeloid blast phenotype, and most of the remaining 30% have a primitive B-lymphocyte phenotype. Rare cases of blast crisis of CML with a primitive T-lymphocyte phenotype have been reported.1,2 In this report, we describe a patient who developed a mediastinal mass after undergoing HLA-matched, unrelated donor bone marrow transplant (BMT) for accelerated-phase CML. Biopsy of the mass showed a blastic process with an immature T-cell phenotype. We describe the studies performed to obtain a definitive diagnosis, review the literature on T-cell blast crisis of CML, and discuss the possible pathogenesis.
superior vena cava syndrome. Computed tomography scan of the neck and chest showed a large mediastinal tumor mass extending into the right supraclavicular fossa (Fig 1). There was an absence of circulating blasts and no leukoerythroblastosis. The patient underwent fine needle aspiration (FNA) and biopsy of the mass and repeat bone marrow aspiration. After a diagnosis of T-cell blast crisis of CML was established, the patient commenced treatment with conventional multidrug chemotherapy. Remission was not achieved, and he died of disease progression.
CASE REPORT The patient was diagnosed with chronic-phase CML in July 1998. Cytogenetic studies showed t(9;22), and reverse transcriptase polymerase chain reaction (RT-PCR) identified the bcr-abl p210 rearrangement. After cytoreduction with hydroxyurea and 8 months of treatment with ␣-interferon, the patient did not experience a cytogenetic remission and subsequently acquired trisomy 8. In September 1999, he underwent human leukocyte antigen–matched, gender-identical, unrelated donor BMT. Bone marrow aspirates performed on day 29 and day 100 after transplant were hypocellular and karyotypically normal but were persistently positive for bcr-abl p210 detected by RT-PCR. On day 154, he developed acute
From the Departments of Pathology and Hematology, University of Texas Health Science Center at San Antonio, San Antonio, TX, Laboratories for Genetics Services, Inc, Houston, TX, and M.D. Anderson Cancer Center, Houston, TX; Milton S. Hershey Medical Center, Hershey, PA; and the Department of Pediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada. Accepted, for publication April 22, 2002. Address correspondence and reprint requests to Fiona E. Craig, MD, Department of Pathology, University of Pittsburgh Medical Center, Presbyterian Hospital, Room C604, 200 Lothrop St, Pittsburgh, PA 15213. Copyright 2002, Elsevier Science (USA). All rights reserved. 0046-8177/02/3307-0015$35.00/0 doi:10.1053/hupa.2002.126190
MATERIALS AND METHODS FNA of the mediastinal tumor was performed, and the sample was processed using standard methods. Immunohistochemistry was performed on decalcified bone marrow biopsy sections using a routine streptavidin-biotin method.3 Flow cytometry was performed by dual-color analysis using a FACs Calibur flow cytometer (Becton, Dickinson and Company, San Jose, CA), SimulSET software (Becton, Dickinson), and fluorescein isothiocyanate (FITR)- and phycoerythrin (PE)-labeled antibodies (Becton, Dickinson).4 RT-PCR was used to amplify bcr-abl p210.5 Epstein-Barr (EBV) viral load was measured in plasma by competitive PCR as previously described6 using commercial reagents (ViralQuant, BioSource, Camarillo, CA). TCR␥ gene rearrangement was performed using multiplex primers directed at V1– 8, V9, V10 as well as V11:J1/2, JP and JP1/2 sequences.7 Immunoglobulin H gene rearrangement was assayed by PCR using FRIII:JHa primers and Hot-Start polymerase (AmpliTaq, Perkin-Elmer, Wellesley, MA).8 PCR assays were done on a Perkin-Elmer GeneAmp PCR System 9700 alongside a positive cell line control, at least two negative tissue controls, and a no-template blank. Products were sized on agarose or polyacrylamide gels. Fluorescence in situ hybridization (FISH) analysis was performed on paraffin-embedded sections mounted on silanecoated slides. The procedure was carried out according to a protocol adapted from that for HER-2/neu FISH analysis of breast tumor9 and the LSI bcr/abl probe (Vysis, Downers Grove, IL) detecting double bcr/abl gene fusion signal (D-FISH) was applied. FISH analysis was performed in a second laboratory on a fresh left supraclavicular lymph node biopsy sample and bone marrow aspirate. The target DNA was hybrid-
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FIGURE 1. Computed tomography scan of the chest. Large mediastinal tumor mass (arrow) compressing the superior vena cava (arrowhead).
ized with an LSI probe (Vysis) detecting single bcr/abl gene fusion signal (S-FISH).10 RESULTS FNA smears and the core biopsy sample from the mediastinal tumor mass showed a monotonous blastic infiltrate (Fig 2). Flow cytometry showed expression of CD2, CD3, CD4, CD5, CD7, CD8, and CD10. The concurrent bone marrow aspirate was hypocellular, with a blast count of 0.2%. Sections from the corresponding iliac crest bone marrow biopsy sample showed focal areas of blastic infiltration that expressed CD3 by immunohistochemistry. There was no expression of CD20, myeloperoxidase, or EBV-latent membrane protein. RT-PCR of blood obtained at the time of the mediastinal biopsy was strongly positive for bcr-abl p210, suggesting persistent or relapsed leukemia. The band pattern was identical to that of a blood specimen drawn at the time of initial diagnosis 19 months earlier. EBV was not detectable in peripheral blood as measured by quantitative PCR. Clonal TCR␥ gene rearrangement was identified in the mediastinal tumor tissue and right bone marrow biopsy, with identical bands. Clonal IgH gene rearrangement also was identified in the mediastinal tumor tissue but not in the bone marrow, and the negative marrow result was interpreted as being caused by the paucity of neoplastic cells. D-FISH performed on paraffin sections from the mediastinal tumor using an LSI bcr/abl dual-color probe showed a gene fusion signal in 91 of 120 tumor cells (76%) (Fig 3). Conventional S-FISH analysis performed on frozen tissue showed the bcr-abl gene fusion signal in 194 of 200 interphase, aspirated cells (97%) from the supraclavicular biopsy and in 25 of 200 interphase/metaphase, aspirated marrow cells (12.5%). These values are higher than the background for these techniques; thus, they confirmed the morphological finding of a blastic infiltrate.
FIGURE 2. Histopathology of the mediastinal tumor specimen. The biopsy sample shows a focus composed of monotonous, small- to intermediate-sized lymphoid cells containing scant cytoplasm, round nuclei, and finely dispersed chromatin (Hematoxylin and eosin; original magnification x400).
mia (ALL/L), and blast crisis of CML. Subsequent bone marrow evaluation showed focal involvement by a blastic T-cell infiltrate, thus indicating the invasive nature of this process and confirming the T-lymphoid phenotype. Molecular diagnostic studies showed clonal T-cell receptor (TCR) and IgH rearrangement, thus indicating the presence of a clonal lymphoid proliferation. T-cell acute lymphoblastic lymphoma leukemia (T-ALL/L) is a disease that frequently affects young adults and is more predominant in male patients. Patients often present with bulky mediastinal disease related to involvement of the thymus. Although the clinical presentation of the patient described in this report is consistent with de novo ALL/L, identification of the bcr-abl p210 rearrangement by D- and S-FISH makes this diagnosis unlikely. In adult ALL, the bcr-abl rear-
DISCUSSION We report an unusual case of T-cell blast crisis of CML presenting as a rapidly growing mediastinal mass that appeared 5 months after allogeneic BMT. A biopsy sample was obtained. The initial differential diagnosis included normal thymic tissue, thymoma, posttransplantation lymphoproliferative disorder, de novo acute lymphoblastic lymphoma leuke-
FIGURE 3. FISH using a bcr-abl probe. (A) D-FISH analysis on paraffin-embedded mediastinal tumor tissue. Positive cells (arrows) contain 4 signals: a green signal indicates the normal chromosome 22; an orange signal, the normal chromosome 9; a yellow fusion signal, bcr-abl on the translocated chromosome 22. A second orange signal indicates the remaining abl gene on the translocated chromosome 9.
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HUMAN PATHOLOGY
Volume 33, No. 7 (July 2002)
rangement appears to be confined to cases with a B-cell or, rarely, mixed-lineage phenotype.11 Although pediatric T ALL/L may be Ph1-positive, the majority of cases have a p190 breakpoint.11 Furthermore, and more importantly, the patient had a history of CML involving the bcr-abl p210 breakpoint. T-cell blast crisis of CML is a rare disorder; only 25 cases have been previously reported. Patients vary in age from 13 to 67 years, and the male-to-female ratio is 1.5:1. The interval between the diagnosis of CML and the onset of blast crisis ranges from 5 months to 6 years. Many patients present with hepatosplenomegaly and lymphadenopathy.2,12-22 Lymph node biopsy was performed in 13 of the reported cases, and all samples showed diffuse blastic infiltration and the presence of the Ph1-chromosome and/or bcr-abl translocation using conventional karyotyping and Southern blot analysis, respectively.12-14,16,17,19-22 In 9 cases, the blast count in bone marrow was less than 30% at the time the lymph node blast crisis was diagnosed.12-14,16,19,22 One of the best documented cases was published by Sun et al1 who reported a 63-year-old female patient who developed progressive back pain 5 years after an initial diagnosis of CML. Magnetic resonance imaging of the spine showed lytic lesions and a soft tissue mass at T11. The extramedullary mass was biopsied, and the sample showed a T-cell blastic infiltrate that was positive by cytogenetics for the Ph1-chromosome. Concurrent posterior iliac crest bone marrow examination showed only 1% blasts.1 Sun et al1 observed that in T-lymphoblastic transformation of CML, extramedullary blast crisis often precedes overt blastemia and overt blastic infiltration in the bone marrow. The presence of a predominant extramedullary component is documented in at least 9 of the 25 previously reported cases as well as in the current case.12-14,16,19,21,22 Fermand et al18 reported a case of a 19-year-old female patient who developed T-cell blast crisis 4 years after CML was diagnosed. Her clinical manifestations included rapid onset of coma, hepatosplenomegaly, lymphadenopathy, and a large mediastinal mass involving the thymic region. Unfortunately, this mediastinal mass was not biopsied.18 Allogeneic BMT was performed in only 1 of the 25 reported cases, in which a 37-year-old female patient developed nodal T-cell blast crisis in her supraclavicular regions approximately 5 months after undergoing allogeneic BMT for CML. Chest x-ray showed a large pleural effusion but no mediastinal lymphadenopathy.21 Although the latter case resembles the current case most closely, the mediastinal involvement in the current report raised the possibility of de novo T-ALL/L. Clues to the pathogenesis of T-cell blastic transformation of CML lie in the physiology of normal T-lymphocyte maturation and the lack of involvement of T-lymphocytes in CML. During normal T-lymphocyte maturation, prothymocytes migrate from the bone marrow to the thymic cortex. There, the fate of the cell (activation, anergy, or apoptosis) is determined by T-cell receptor– generated intracellular signaling pathways.23 Many cells die by apoptosis within the cortex.23 Those that survive migrate to the medulla and are released into peripheral blood.23 The requirement for a thymic pathway for T-cell maturation may explain the absence of the Ph-chromosome in T-lymphocytes in patients with CML despite involvement of hematopoietic stem cells.24 Most patients with CML are adults who have a relatively inactive thymus because of age-related involution. Immature abnormal T-cells may reach the thymus, but they die by apoptosis within the cortex. In this case, the patient presented with prominent mediastinal disease consistent with thymic involvement. This suggests that immature cells containing the bcr-abl translocation escaped from the bone marrow and migrated to the thymus where they proliferated and evaded apoptosis. An-
other clue to the pathogenesis of T-cell blast crisis of CML is the presence in our case of clonal IgH gene rearrangement in addition to rearrangement of the T-cell receptor (TCR) gamma gene. Coexisting IgH and TCR receptor rearrangements have been described in up to 30% of T-ALL/L cases.25 Promiscuous IgH rearrangement also has been reported in normal mouse thymocytes and has been assumed to be an event that would normally direct cells toward apoptosis.26 Cases of T-ALL/L, and our case of T-cell blast crisis of CML, may represent immortalization of these abnormally rearranged T-cells by a malignant transformation that rescues them from this fatal pathway. In summary, we present a case of T-cell blast crisis of CML with a clinical presentation resembling that of T-ALL/L. The differential diagnosis was resolved using interphase FISH analysis showing bcr-abl rearrangement. The presentation and aberrant IgH gene rearrangement suggest a pathogenesis for T-cell blast crisis of CML that is similar to that of de novo ALL/L. Inhibition of apoptosis may play a role in the development of both diseases. The rarity of T-cell blast crisis of CML may relate to the apparent requirement for both a bone marrow (stem cell bcr-abl rearrangement) and extramedullary (thymic maturation) component of disease and a mechanism to evade apoptosis. REFERENCES 1. Sun T, Susin M, Koduru P, et al: Extramedullary blast crisis in chronic myelogenous leukemia: Demonstration of T-cell lineage and Philadelphia chromosome in a paraspinal tumor. Cancer 68:605-610, 1991 2. Dorfman DM, Longtine JA, Fox EA, et al: T-cell blast crisis in chronic myelogenous leukemia: Immunophenotypic and molecular biologic findings. Am J Clin Pathol 107:168-176, 1997 3. Elias JM, Margiotta M, Gaborc O: Sensitivity and detection efficiency of the peroxidase antiperoxidase (PAP), avidin-biotin peroxidase complex (ABC) and peroxidase-labeled avidin-biotin (LAB) methods. Am J Clin Pathol 92:6267, 1989 4. Stelzer GT, Marti G, Hurley A, et al: US-Canadian consensus recommendations on immunophenotypic analysis of hematologic neoplasia by flow cytometry: Standardization and validation of laboratory procedures. Cytometry 30:214-230, 1997 5. Radich JP, Gehly G, Gooley E, et al: Polymerase chain reaction detection of the BCR-ABL fusion transcript after allogenic marrow transplantation for chronic myeloid leukemia: Results and implications in 346 patients. Blood 85:2632-2638, 1995 6. Fan H, Gulley ML: EBV viral load by competitive PCR, in Killeen A (ed): Molecular Pathology Protocols. Humana Press, Totowa, NJ, 2001 7. Krafft AE, Taubenberger JK, Sheng ZM, et al: Enhanced sensitivity with a novel TCR gamma PCR assay for clonality studies in 569 formalin-fixed, paraffin-embedded (FFPE) cases. Molecular Diagnosis 4:119-133, 1999 8. Gill JI, Gulley ML: Immunoglobulin and T-cell receptor gene rearrangement. Hematol Oncol Clin North Am 8:751-770, 1994 9. Masood S, Bui MM, Yung JF, et al: Her-2/neu SpectrumOrangeTM and CEP 17 SpectrumGreen TM dual color deoxyribonucleic acid probe kit. Ann Clin Lab Sci 28:215-223, 1998 10. DeWald GW, Wyatt WA, Juneau AL, et al: Highly sensitive fluorescence in situ hybridization method to detect double BCR/ABL fusion and monitor response to therapy in chronic myeloid leukemia. Blood 91:3357-3365, 1998 11. Crist W, Carroll A, Shuster J, et al: Philadelphia chromosome positive childhood acute lymphoblastic leukemia: Clinical and cytogenetic characteristic and treatment outcome: A Pediatric Oncology Group study. Blood 76:489494, 1990 12. Palutke M, Eisenberg L, Nathan L: Ph1-positive T-lymphoblastic transformation of chronic granulocytic leukemia in a lymph node. Lancet 2:1053. 13. Jacobs P, Greaves M: Ph1-positive T lymphoblastic transformation. Leuk Res 8:737-739, 1984 14. Vannier JP, Bizet M, Bastard A, et al: Simultaneous occurrence of a T-cell lymphoma and a chronic myelogenous leukemia with an unusual phenotype. Leuk Res 8:647-657, 1984 15. Cervantes F, Anegon I, Rozman C, et al: Early T-cell features in blast crisis of Ph1-positive chronic myelogenous leukaemia. Scand J Haematol 35:71-76, 1985 16. Allouche M, Bourinbaiar A, Goergoulias V, et al: T cell lineage involvement in lymphoid blast crisis of chronic myelogenous leukemia. Blood 66:11551161, 1985 17. Falini BM, Tabilio A, Pelicci PG, et al: T-cell receptor -chain gene rearrangement in a case of Ph1-positive chronic myelogenous leukaemia blast crisis. Br J Haematol 62:776-779, 1986
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