Donor Cell Derived Acute Myeloid Leukemia AfterAllogeneic Cord Blood Transplantation in a Patient With

Adult T-Cell Lymphoma

Takuya Matsunaga,1* Kazuyuki Murase,1 Makoto Yoshida,1 Akihito Fujimi,1 Satoshi Iyama,1

Kageaki Kuribayashi,1 Tsutomu Sato,1 Katsuhisa Kogawa,2 Yasuo Hirayama,3

Sumio Sakamaki,3 Kyuhei Kohda,4 and Yoshiro Niitsu1

1 Fourth Department of Internal Medicine, Sapporo Medical University, School of Medicine, Japan2Department of Internal Medicine, Tokeidai Hospital, Japan

3Department of Internal Medicine, Higashi Sapporo Hospital, Japan4Department of Internal Medicine, Asahikawa Red Cross Hospital, Japan

We report a patient with adult T-cell lymphoma who developed acute myeloid leukemia

(AML) after allogeneic cord blood transplantation (CBT). Fluorescence in situ hybridiza-

tion (FISH) studies and molecular analysis using short tandem repeat (STR) sequences

proved the AML to be of donor origin. Although 25 cases of donor cell leukemia (DCL)

occurring after allogeneic bone marrow transplantation have previously been reported,

there have been no reports of DCL after CBT. This case is the first-reported DCL patient

after CBT. Am. J. Hematol. 79:294–298, 2005. ª 2005 Wiley-Liss, Inc.

Key words: donor cell derived leukemia; cord blood transplantation; adult T-cell

lymphoma

INTRODUCTION

Allogeneic bone marrow transplantation (BMT) isan effective therapy for hematological malignancies.Hematopoietic stem cells (HSC) are found not onlyin the bone marrow but also in the peripheral bloodand cord blood, and therefore HSC transplantationhas been recently carried out utilizing allogeneicperipheral-blood stem cells and cord blood stemcells [1,2]. In particular, cord blood stem-cell trans-plantation (CBT) has been successfully performednumerous times over the past decade and remainsan attractive option since donor coordination is notnecessary and the incidence and severity of acutegraft-versus-host disease are lower than those ofBMT or allogeneic peripheral-blood stem-cell trans-plantation (PBSCT) [2].However, there are still various causes of HSC

transplantation treatment failure after BMT for leu-kemia, the most frequent being the relapse of theunderlying host leukemia [3]. Furthermore, therehave been reports of donor cell leukemia (DCL) inrelapsed patients, raising a new type of ethical issue of

BMT and also providing new insights into themechanisms of leukemogenesis [4–11]. We reporthere a patient who developed DCL not after BMTbut after CBT.

CASE REPORT

A 57-year-old woman was admitted to the oto-laryngology department of our hospital for evalua-tion of left tonsillar swelling in May 1998. She wasdiagnosed with chronic-type adult T-cell lymphoma(ATL) by pathological examination of the resected

*Correspondence to: Takuya Matsunaga, M.D., Ph.D., 4th

Department of Internal Medicine, Sapporo Medical University,South-1 West-17, Chuo-ku, Sapporo, Japan.E-mail: matunaga@sapmed.ac.jp

Received for publication 6 September 2004; Accepted 30November 2004

Published online inWiley InterScience (www.interscience.wiley.com).DOI: 10.1002/ajh.20349

American Journal of Hematology 79:294–298 (2005)

ª 2005 Wiley-Liss, Inc.

tonsil. Regular follow-up without treatment wasplanned for the patient. She was referred andadmitted to our department in December 2001, forcervical, mediastinal, and abdominal lymphadeno-pathy. Southern-blot analysis of a neck lymph nodespecimen biopsied at the time of admission revealedmonoclonal integration of HTLV-1 proviral DNA,prompting a diagnosis of lymphoma type ATL.Laboratory tests showed a peripheral-blood flowercell (ATL cell) count of 2%, and elevations in theHTLV-1 viral titer (� 4,096), lactate dehydrogenase(LDH, 777 IU/L), and soluble IL-2 receptor (sIL2R,2,585 U/mL). From February 2002, she was treatedwith combination chemotherapy according to theLSG15 protocol [12], and achieved a completeresponse. The patient was discharged from our hos-pital in May 2002. Since lymphoma-type ATLpatients have a high incidence of relapse [12], allo-geneic hematopoietic stem-cell transplantation wasplanned for the patient. However, since neither anHLA-identical sibling nor an unrelated matcheddonor could be found for BMT or PBSCT, she wasreadmitted to our department to receive CBT in July2002. At the time of readmission, she had no lym-phadenopathy. Laboratory tests showed a flowercell count of 0%. Although LDH was normalized

and sIL2R was down to 630 U/mL, the HTLV-1viral titer remained elevated (� 8,192).In October 2002, the patient underwent the first CBT

using a cord unit from an HLA-mismatched (2 loci bygenotype), unrelated male donor (Fig. 1). The infusednucleated cell count was 2.65� 107 cells/kg (3.8� 104

CD34+ cells/kg). Conditioning consisted of busulfan (4mg/kg for 2 days) and fludarabine (30 mg/m2 for 6days), and GVHD prophylaxis was facilitated bycyclosporine and short-course methotrexate. Neutro-phil engraftment was observed on day 28 post CBT.Donor–recipient chimerism was analyzed with XYchromosome FISH, and full-donor chimerism was con-firmed on day 35. However, the patient remained trans-fusion dependent throughout the post-transplantationperiod. There was no evidence of acute GVHD. Thepercentage of flower cells in peripheral blood, whichwas 1–2% just before CBT, became 0%after transplan-tation. The patient developed a recurrence of the cyto-megalovirus (CMV) viremia and was treated withganciclovir repeatedly. She developed a late graft failureon day 112 post CBT. A second CBT from an HLA-mismatched (1 locus by genotype), unrelated maledonor was undertaken in April 2003. The number ofcells infused was 2.38� 107 cells/kg (5.5� 104 CD34+

cells/kg). On this occasion, the conditioning regimen

Fig. 1. Clinical course. Peripheral-blood donor (black portions)–recipient (white portions) chimerism was analyzed withXY chromosome FISH. Abbreviations: Flu, fludarabine; Bu, busulfan; CyA, cyclosporine; MTX, methotrexate; PSL,prednisolone; ATG, anti-thymocyte globulin; mPSL, methylprednisolone; CMV, cytomegalovirus; flower cells, ATL cells;blasts, AML cells.

Case Report: Donor Cell Derived Leukemia After Cord Blood Transplantation 295

consisted of fludarabine (30 mg/m2 for 6 days), anti-thymocyte globulin (ATG) (15 mg/m2 for 4 days) andmethylprednisolone (mPSL) (1,000 mg/m2 for 4 days).Tacrolimus and mPSL were given as GVHDprophylaxis. Neutrophil engraftment was observed byday 24 post CBT. Full-donor chimerism was confirmedby XY chromosome FISH on day 28. However, sherequired transfusion support throughout the post-trans-plantation period. There was no evidence of acute orchronic GVHD. Although flower cells in peripheralblood were transiently increased, they disappearedafter tacrolimus and mPSL had tapered off. The patientdeveloped a recurrence of CMVviremia andwas treatedwith ganciclovir repeatedly. On day 256 after the secondCBT, blasts were detected in peripheral blood and sig-nificantly increased over the following week. We per-formed a bone marrow aspiration on day 263.Because the patient and her family rejected

chemotherapy for AML, she died due to AML onday 263 after the second CBT.

METHODS

Expression levels of surface markers on the leukemiccells were determined by flow cytometry. Abnormal kar-yotypes were classified according to the InternationalSystem for Human Cytogenetic Nomenclature [13].Slides for FISH were prepared following standard cyto-genetic procedures and stored at 37�C until use. The sexchromosome complement was determined by scoring1,000 interphase nuclei hybridized to the dual color

satellite X (DXZ1) and satellite III Y (DYZ3) probes(Vysis, Inc., Downers Grove, IL) according to the man-ufacturer’s protocols. DNA was extracted by DNAzolReagent (Invitrogen, Carlsbad, CA), and polymerasechain reaction (PCR) of short tandem repeat (STR)loci, D16S539, D7S820, D13S317, D5S818, CSF1PO,TPOX, TH01, and vWA, was performed with thePowerPlex 1.1 System (Promega, Madison, WI) [14].The PCR products were electrophoresed bypolyacrylamide gel, and the gel was scanned by aFMBIOII fluorescence scanner (Hitachi, Brisbane,CA). To determine the integration of the HTLV-I pro-virus genome in leukemic cells, 10 mg of DNA wassubjected to Southern-blot analysis using non-radioactive probes specific for total HTLV-1 genome aspreviously reported [15].

RESULTS

On day 263 post second CBT, peripheral-bloodWBCcount was 31,700/mL, and blast count was 93% (Fig. 1).The blasts were positive for CD11b (98.0%), CD33(98.2%), and CD34 (43.2%) and had a karyotypicabnormality of 46,XY,add(19)(p13). The examinationof a bone marrow smear disclosed a blast count of 60%,and the blasts were positive for myeloperoxidase andchloroacetate esterase staining. Based on these findings,AML-M2 (according to the French-American-Britishclassification). As shown in Fig. 2, 98.3% of peripheralblood cells were donor-derived male cells containing theXY chromosomes. Molecular analysis of STR sequence

Fig. 2. FISH performed on the peripheral blood with probes specific for chromosome X and Y: (A) patient; (B) femalecontrol; (red) fluorescence of X chromosome; (green) fluorescence of Y chromosome. [Color figure can be viewed in theonline issue, which is available at www.interscience.wiley.com.]

296 Case Report: Matsunaga et al.

of peripheral-blood leukemic cells showed a profilecompletely matching the second donor (Fig. 3). Mono-clonal integration of the HTLV-I provirus genome wasnot detected in leukemic cells (data not shown).

DISCUSSION

To our knowledge, although 25 cases of DCLoccurring after BMT have previously been reported[4–11], there have been no reports of DCL occurringafter either PBSCT or CBT.

In these 25 DCL patients, several mechanisms havebeen offered to explain how DCL arose. Niederwieseret al. [4] reported on a donor cell derived AMLpatient who had been administered AML blasts con-taminated donor bone marrow cells. Because the cordblood from the second donor in our case did notcontain any AML blasts, this mechanism is unlikelyto explain the DCL in our patient. In addition to thismechanism, Cooley et al. [6] proposed the followingthree mechanisms for the development of DCL afterBMT: (i) the conditioning regimen for BMT, espe-cially total body irradiation (TBI), may damage therecipient tumor cells and cause the release of viral ornonviral oncogenic materials, which are then trans-ferred to donor cells; (ii) the residual host cellsundergo fusion with donor cells, and the oncogenicmaterials in the host cells are transferred to donorcells; (iii) immunosuppressive agents such as steroidsand cyclosporine used after BMT impair immunesurveillance and allow the development of DCL.Our patient had not been treated with TBI, and the

patient’s DCL cells contained no detectable monoclo-nal integration of the HTLV-1 proviral DNA. FISHanalysis of the sex chromosomes and PCR analysis ofSTR exclude the possibility of cell fusion in thispatient. The patient experienced repeated risingCMV antigenemia, which suggested that the host-immune surveillance was severely suppressed byATL itself and repetitive CBT. However, this alonecannot explain why DCL occurred early after thesecond CBT.Bielorai et al. [11] reported a patient who developed

donor-derived AML after BMT, and 9 years later thedonor developed B-cell lymphoma. Cytogeneticanalysis of this donor’s lymphoma cells revealedmultiple chromosomal aberrations. The donor washeterozygous for the Ashkenazi mutation of Bloom’ssyndrome, suggesting that this donor-type leukemiacould have resulted from genomic instability in thedonor cells. This possibility cannot be excluded in ourpatient.Yamamoto et al. [16] reported that in vitro, the

ATL-derived factor (ADF) produced by HTLV-1infected ATL cells and lymphocytes stimulate theproliferation of blasts obtained from AML patients.Imamura et al. [17] reported that ATL patients andHTLV-1 carriers have an increased incidence of sec-ondary malignancies such as adenocarcinoma of thethyroid or lung and squamous cell carcinoma of thelarynx, lip, or lung; they speculated that there existsan association between ATL cells and premalignantcells, mediated through the activation of ras onco-genes by ADF. They also speculated that subsequentsuppression of host-immune defense mechanismsin ATL permits the emergence of the secondary

Fig. 3. STR locus marker gels. D16S539, D7S820,D13S317, D5S818, CSF1PO, TPOX, TH01, and vWA areSTR loci: lane 1, recipient profile, pre-transplant; lane 2, 1st

donor profile; lane 3, 2nd donor profile; lane 4, recipientprofile, post-1st transplant; lane 5, recipient profile, post-2nd transplant.

Case Report: Donor Cell Derived Leukemia After Cord Blood Transplantation 297

neoplasm. In our patient, it remains possible that theADF produced by the ATL cells, which transientlyincreased after the second CBT, stimulated the leuke-mogenesis of the cord blood stem cells. Demonstrat-ing leukemogenesis of cord blood stem cells by ADFrequires future studies.The second CBT donor was did not have congeni-

tal AML and is healthy so far.In conclusion, we report on a patient with ATL

who developed DCL after CBT. Further studies areneeded to clarify the mechanism of the pathogenesisof DCL after CBT and to establish measures to pre-vent the development of DCL.

ACKNOWLEDGMENT

The authors thank Mr. Kevin Litton (Bachelor ofArts in English) for editorial assistance.

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