Donor cell leukemia following unrelated donor bone marrow transplantation for primary granulocytic sarcoma of the small intestine

Granulocytic sarcoma (GS) in the absence of bone marrow involvement at diagnosis is unusual. Treatment of primary (aleukemic) GS should follow intensive acute myeloid leukemia (AML)-style chemotherapy protocols, as patients otherwise appear destined to rapidly declare frank medullary disease. However, the role of hematopoietic stem cell transplantation (HSCT) is less clear. Here, we present an unusual case of isolated primary GS involving small bowel treated with chemotherapy consolidated by HSCT from an unrelated donor. Full donor chimerism was achieved and maintained, without relapse of the original disease. However, 28 months post-transplant our patient developed a distinct and rapidly progressive medullary AML, apparently derived from transplanted cells of donor origin. Donor cell leukemia (DCL) is a fascinating and increasingly recognized phenomenon, with at least 50 cases reported to date. Our case adds to this growing literature and highlights the ethical dilemmas concerning notification of unrelated donors in such cases, since evidence suggests against an increased risk of themselves developing leukemia. This also represents the first case of DCL reported after transplantation for primary GS. Granulocytic sarcoma (GS) in the absence of bone marrow involvement at diagnosis is unusual. Treatment of primary (aleukemic) GS should follow intensive acute myeloid leukemia (AML)-style chemotherapy protocols, as patients otherwise appear destined to rapidly declare frank medullary disease. However, the role of hematopoietic stem cell transplantation (HSCT) is less clear. Here we present an unusual case of isolated primary GS involving small bowel treated with chemotherapy consolidated by HSCT from an unrelated donor. Full donor chimerism was achieved and maintained, without relapse of the original disease. However, 28 months post-transplant our patient developed a distinct and rapidly progressive medullary AML, apparently derived from transplanted cells of donor origin. Donor cell leukemia (DCL) is a fascinating and increasingly recognized phenomenon, with at least 50 cases reported to date. Our case adds to this growing literature and highlights the ethical dilemmas concerning notification of unrelated donors in such cases, since there is little evidence that they are at increased risk of developing leukemia themselves. This also represents the first case of DCL reported after transplantation for primary GS. A 34-year-old woman presented with features of small bowel obstruction at 16 weeks gestation of first pregnancy. Emergency laparotomy was performed, with resection of the affected segment. Histology revealed tissue infiltration by myeloid blast cells staining positive for CD34, CD43, CD68, CD117, myeloperoxidase (MPO), leucocyte common antigen and lysozyme. Full blood count and bone marrow (BM) morphology were normal, with normal female karyotype in all BM metaphases examined. A diagnosis of primary granulocytic sarcoma was made. Following spontaneous pregnancy loss at 22 weeks gestation intensive chemotherapy with DAT (daunorubicin; cytarabine; 6-thioguanine) was commenced. Post-treatment CT scan showed only partial response, prompting further treatment with FLAG Ida (fludarabine; cytarabine; GCSF; idarubicin). Complete remission was achieved but complicated by persistent chemotherapy-induced aplasia. She subsequently underwent allogeneic bone marrow transplantation (BMT) from a 37-year-old male unrelated donor. Conditioning comprised cyclophosphamide, total body irradiation and alemtuzumab. Single antigen mismatch was present at HLA-DQB1 (and HLA-DRB4), i.e., 9/10 match by standard measures. Major ABO mismatch was present (recipient O+/donor A+). Both recipient and donor were seropositive for cytomegalovirus (CMV) IgG. Cell dose was 2.84 × 106 CD34+ cells/kg. Graft-versus-host disease (GVHD) prophylaxis comprised cyclosporin and methotrexate. GCSF was commenced day +14 and neutrophil engraftment occurred by day +25, but platelet engraftment was not observed. She suffered no GVHD and cyclosporin was discontinued 6 months post-transplantation. Serial BM examination and CT imaging confirmed complete morphological and radiological remission beyond 2 years post-transplant. Semiquantitative fragment analysis by polymerase chain reaction (PCR) amplification of short tandem repeats (STR) revealed continued 100% donor chimerism. Post-transplant course was complicated by CMV reactivation and bilateral CMV retinitis. An intraocular ganciclovir implant was inserted and systemic ganciclovir continued for total 90 days, achieving quiescence of the retinitis and sustained viral clearance. However, this resulted in secondary graft failure, compounding the already incomplete primary hematopoeitic engraftment. She remained transfusion-dependent and significantly neutropenic for over 2 years, showing only minor responses to regular GCSF and erythropoietin. Second transplant was not possible as the original donor had withdrawn from the registry and no other suitable donors were identified. On day +809 she received recombinant human stem cell factor (Stemgen®: 20 μg/kg for 5 days) as a further attempt to expand and support hematopoiesis. By day +833 total white cell count had risen to 63.2 × 109/l, comprising mostly circulating myeloblasts. BM aspirate revealed ∼40% myeloid blasts with CD34+/CD13+/CD33+/CD14+/CD117+/MPO+ phenotype on flow cytometry, indicating a diagnosis of AML. However, G-banded cytogenetics revealed abnormal male karyotype (45,XY) with monosomy 7 in all 30 metaphases examined. Fluorescence in situ hybridization revealed monosomy 7 in nine of 100 interphase cells; all cells possessed Y chromosome (i.e., donor-type; Fig. 1). PCR amplification of five fully informative STR markers (D3S1358, THO-1, D7S820, TPOX, CSFIPO) confirmed continued 100% donor chimerism (Fig. 2), reproduced on serial peripheral blood analysis including an additional two STR markers (D10S2325, D5S818). These findings confirmed the AML to have derived from cells of donor origin. Re-induction chemotherapy was commenced but was complicated by neutropenic sepsis and fungal pneumonia to which she eventually succumbed. Diagnostic material from bone marrow specimens at day +833 following hematopoietic stem cell transplant. Panel A: Light microscopy showing presence of myeloblasts in bone marrow, confirming diagnosis of acute myeloid leukemia (×100 magnification). Panel B: G-banded karyogram displaying 45,XY (male i.e., donor karyotype) with monosomy 7 (present in 30 of 30 metaphases examined). Panel C: Fluorescence in situ hybridization revealing monosomy 7 within male (donor) cells in 15 of 200 interphase nuclei inspected; the remaining cells displayed 2 copies of chromosome 7, but all 200 cells displayed hybridization signals with the CEP Y probe, consistent with full donor chimerism and consequently suggesting donor origin of the acute myeloid leukemia. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Results of short tandem repeat chimerism analysis by polymerase chain reaction at time of diagnosis of acute myeloid leukemia (D+833 post-hematopoietic stem cell transplant). Lane A: Patient (recipient) pretransplant bone marrow specimen. Lane B: Donor fibroblast specimen. Lane C: Patient (recipient) bone marrow specimen at diagnosis of acute myeloid leukemia post hematopoietic stem cell transplant (day +833). Lane C shows identical allele peaks corresponding with donor fibroblast alleles in Lane 2, confirming 100% donor chimerism at time of leukemia “relapse.” [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] This case displays two unusual and remarkable features: (1) The initial occurrence of granulocytic sarcoma within the small intestine without BM involvement and (2) subsequent development of a distinct donor cell leukemia following allogeneic BMT from an unrelated donor. GS describes an extramedullary tumor composed of myeloblasts and other immature cells of myeloid lineage. GS usually occurs in the context of AML, but can rarely arise as an isolated mass in the absence of BM disease (primary GS). The gastrointestinal tract is an uncommon but well-recognized site of involvement, and 20 cases of small intestinal GS have been reviewed by Kohl and Aoun [1]. Most patients with primary GS appear destined to develop frank AML. In one retrospective series frank AML developed in all patients receiving tumor resection ± local irradiation alone, at median 3–6 months. Onset of AML was significantly delayed in patients treated with systemic chemotherapy (median 12 months), with 19% remaining leukemia-free for >24 months [2]. Intensive AML-type chemotherapy is therefore the standard for primary GS, even in the absence of BM disease. A retrospective review of 51 GS patients receiving HSCT reported 5-year disease-free and overall survival rates of 36 and 47%, respectively. Consequently the authors concluded that first-line transplantation in GS is a valid therapeutic option [3]. However, most of these patients had concurrent or preexisiting BM disease, and the role of HSCT in primary GS remains unclear. Nevertheless, successful use of HSCT and a clinically significant graft-versus-disease effect have been described in various case reports [4]. Only two cases of primary small bowel GS (without BM involvement) treated with HSCT have been reported previously. Both underwent resection of the original tumor followed by AML-style chemotherapy, consolidated with myeloablative HSCT (from sibling and unrelated donor respectively). Both remained disease-free at 4 years [5, 6]. We here present a third case of primary small bowel GS treated with myeloablative HSCT. Complete donor chimerism was sustained, with no relapse of the original disease beyond 2 years. However, our patient's post-transplant course was complicated by the rare development of de novo AML in cells of donor origin. By definition, leukemia relapses following allogeneic HSCT involve recurrence of the original clone, and while this can display phenotypic differences from the original disease (e.g., through clonal evolution or emergence of a resistant subclone) true DCL remains a rare and difficult-to-diagnose phenomenon. Fialkow reported the first case of DCL in 1971, in a 16-year-old female patient with acute lymphoblastic leukemia who relapsed 62 days after BMT from her HLA-identical brother. Relapse cytogenetics revealed only male (XY) metaphases, suggesting that leukemic transformation had arisen in engrafted donor cells [7]. Overall, around 50 cases of DCL have been reported in the literature [8]. However, this is the first report of DCL occurring after transplantation for primary GS. A survey by the European Group for Blood and Marrow Transplantation identified 14 cases of suspected/proven DCL from a total 10,489 transplants, estimating incidence at 124 cases per 100,000 transplants [9]. Elsewhere incidence as high as 5% has been proposed [10]. Until recently, unequivocal confirmation of donor origin was hampered by limited sensitivity of diagnostic techniques, with most early cases relying solely on demonstration of sex-mismatchby conventional cytogenetics. However, assumptions from this approach alone can be unreliable and consequently the validity of some early DCL reports has been questioned. More recent molecular chimerism techniques based on PCR analysis of STR markers have extended ability to diagnose DCL to sex-matched transplant pairs, and enabled confirmation of donor origin with greater confidence through enhanced sensitivity in detecting presence of a minor recipient clone. While this could still be misleading (e.g., loss of an informative STR locus with a total or partial chromosomal deletion) reliability can be recovered by employing additional STR markers from a number of different chromosomal loci. In our case we repeatedly confirmed 100% donor hematopoiesis using seven different STR markers (distributed across six chromosomes), sufficiently negating any chance ofinformative allele loss and strongly supporting donor derivation of the leukemic clone. Increasing awareness of DCL as an entity has stimulated considerable interest, since the leukemic transformation of previously healthy donor hematopoietic stem cells when transferred into a new host environment provides a useful (and unique) in vivo model for investigating mechanisms of human leukemogenesis. The simplest explanation for DCL would be the inadvertent transmission of occult malignancy present within the donor at time of donation. Indeed, several examples of such have been reported in the literature [11-14], in which identical disease subsequently developed within the donor and was retrospectively detected within archived donor BM material. Conversely, Frey et al. recently reported the transmission and engraftment of a clone bearing trisomy 8, initially masquerading as possible DCL, from a donor later identified to display constitutive trisomy 8 mosaicism [15]. However, simple transfer of frank malignancy cannot explain the majority of DCL cases since no other donors were reported to themselves develop leukemia. Moreover, many had been subsequently recalled for clinical assessment and BM examination, with no evidence of the culpable disease clone detected. Therefore, additional donor-, recipient-, and transplant-related factors must be involved in triggering and/or supporting the frank malignant transformation. Several putative mechanisms have been proposed, including: pre-leukemic potential of donor HSCs; transfection of latent viruses or other oncogenic material released during conditioning; hybridization of transplanted cells with residual malignant host cells; increased replicative stress and accelerated telomere attrition of donor HSCs; residual effects of prior chemo-/radiotherapy (including “radiation-induced bystander effects”); defective BM microenvironment (either innate or consequent on prior therapy); and impaired immune tumor surveillance in the immunocompromised transplant recipient. However, the paucity of reported cases and absence of prospective series preclude identification of any unifying mechanisms or risk factors, although a multifactorial, multiple hit pathogenesis appears likely. The entity of DCL raises important ethical questions regarding the duty towards donor notification in these cases. Most registries have a policy of informing donors of serious developments that might have impact on their health, yet from the available evidence it seems doubtful that donors are at increased risk of developing leukemia. Consequently, most commentators have concluded that the anxiety resulting from such communication cannot currently be justified. This issue is particularly complex in the context of DCL following cord blood (CB) transplantation, after which DCL might be disproportionately common (owing to the observed high prevalence of potentially pre-leukemic clones detectable in CB samples) [16]. Furthermore, in some cases donors may have withdrawn from the registry (as in our case) and contact details may not have been maintained. This question remains unanswered, but the growing awareness of this important transplant complication should enable prospective collation and detailed examination of future cases, and ultimately provide clarification of this difficult ethical dilemma.