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Bone Marrow Transplantation (2011) 46, 1576–1578 & 2011 Macmillan Publishers Limited All rights reserved 0268-3369/11

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LETTER TO THE EDITOR

Clonally identical Hodgkin’s disease develops after allogeneic hematopoietic cell transplant for CLL Bone Marrow Transplantation (2011) 46, 1576–1578; doi:10.1038/bmt.2010.340; published online 24 January 2011 We present a patient with CLL who achieved remission following allogeneic BMT and donor lymphocyte infusion, but 1.5 years later developed clonally identical Hodgkin’s disease. In 2000, a 50-year-old woman was diagnosed with Rai Stage I CLL with diffuse lymphadenopathy and WBC of 21 000/mL. Translocation t(7;18) (q32;q11.2) was noted in 2 of 20 BM metaphases. When she experienced rapid lymph node progression 2 years later, she was treated with chlorambucil with no response. Partial response followed six cycles of fludarabine, cyclophosphamide and rituximab. In March 2003, she received a right breast lumpectomy and radiation for ductal carcinoma in situ. In 2004, she received six cycles of alemtuzumab (30 mcg subq tiw) with complete clinical response until CLL progressed with hepatosplenomegaly, fever, night sweats, 75 lb weight loss and hypercalcemia. BM showed 5% infiltration with small lymphocytes staining positive for CD5 and CD23 (Figure 1a–c). Heavy-chain IgG hypermutation analysis was unmutated, predicting poor outcome. She received salvage chemotherapy with dramatic clinical improvement and resolution of fever and night sweats, but splenomegaly and diffuse lymphadenopathy persisted. Five years after diagnosis, she underwent reduced intensity conditioning with TLI and anti-thymoglobulin, followed by allogeneic hematopoietic cell transplant (alloHCT) from an unrelated 21-year-old female 10/10 HLAidentical donor.1 Four doses of rituximab (anti-CD20) followed at 56–77 days after HCT on a GVHD prevention protocol. By 90 days after transplant, she achieved 100% donor CD3 T-cell chimerism (blue; Figure 1g) and CD19 B-cell chimerism. She obtained molecular remission with no detectable CLL by allele-specific quantitative PCR assay (VH IgG assay (red)). Her CD56 NK cell and CD15 myeloid chimerism remained mixed, with a maximum donor contribution of 90% (black) and 76%, respectively. CLL was redetected by allele-specific quantitative PCR 180 days post-HCT. New hypermetabolic lymph nodes were detected by PET-CT scan (Figure 1h). Figure 1g shows CLL expansion as donor CD3 T cell and CD56 NK-cell chimerism fell to 30% off immune-suppressive medications. Unmanipulated donor lymphocyte infusion dosed 3  107 CD3 cells/kg at 270 days post-transplant converted her to full donor T- and NK-cell chimerism. By 90 days after donor lymphocyte infusion, she reachieved CR by allele-

specific quantitative PCR and PET-CT scan (Figures 1g and i). She concurrently developed skin and liver GVHD, which was effectively treated with 1 mg/kg prednisone. The patient was asymptomatic 1.5 years after transplant when surveillance PET-CT scan showed widespread lymphadenopathy, despite there being no detectable CLL using allele-specific quantitative PCR assay, BM biopsy or flow cytometry. Surprisingly, cervical lymph node biopsy showed classical Hodgkin’s disease (HD) with Hodgkin Reed–Sternberg-like cells expressing CD30, CD15 and Pax5 (Figures 1d–f). Hodgkin Reed–Sternberglike cells were in a histiocyte-rich, lymphocyte-depleted background and EBV was not detected by in situ hybridization. Cytogenetic studies were not obtained. The absence of CLL cells was confirmed by immunostaining and by flow cytometry for CD5 and CD23. However, the original CLL allele-specific VH IgH sequence amplified from the lymph node sample (784 658 clonal IgH/mg DNA) and from laser captured Hodgkin Reed–Sternberg-like cells, strongly supporting a clonal relationship between the original CLL and the newly detected HD. Despite chemotherapy treatment with adriamycin, VCR and dacarbazine, the patient continued to progress. Her PETCT scan showed hypermetabolic activity in cervical, mediastinal, retroperitoneal and pelvic lymph nodes with skeletal lesions. She expired on 4 November 2008. Although Hodgkin’s disease coexisting with CLL cells has been previously reported,2–5 this is the first report of Hodgkin’s disease being clonally related to CLL following remission of CLL after HCT. We offer several explanations for the clonal relationship between this patient’s CLL B cells and Hodgkin Reed–Sternberg-like cells. We favor the explanation that a cancer stem cell may exist that gives rise to CLL and later to Hodgkin Reed–Sternberg cells sharing the same VH IgG sequence following remission of CLL. This cell would be long-lived, persisting through the 1.5 years of molecular remission. Alternatively, the cell that gives rise to both cancers may be a ‘normal’ memory B cell that bears the same heavy chain immunoglobulin gene rearrangement shared by CLL and HD. We hypothesize that this cell may share a transcriptome with normal memory B cells and long-term hematopoietic stem cells.6 Another possibility is that a non-cancer stem cell CLL clone could have given rise to HD. This would imply that CLL cells persisted for 1.5 years below the technical limit of detection of our molecular assay. The absence of EBV involvement2,7 makes this less likely, but conceivably other inciting factors could have triggered this transformation. Improvements in our understanding of normal B-cell development and cancer stem cells in lymphoid

Letter to the Editor

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Quantitative assessment of CLL and donor chimerism over time DLI 3e7/kg

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malignancies can lead to therapeutic advances to directly target the malignant cell of origin or target them in combination with HCT.8

Conflict of interest The authors declare no conflict of interest. Bone Marrow Transplantation

Letter to the Editor

1578 Figure 1

Histopathology, quantitative assessment of CLL and donor chimerism, and PET-CT images post-HCT/post-DLI. Histopathology of CLL and Hodgkin’s disease. (a–f) Formalin-fixed, paraffin-embedded samples of the left posterior iliac crest BM biopsy and left cervical lymph node excision were stained with hematoxylin and eosin according to standard methods. (a–c) BM biopsy showing involvement by CLL. (a) Hematoxylin and eosinstain (H&E), (b) anti-CD5 (Leica, Wetzlar, Germany), (c) anti-CD23 (Leica). (d–f) Anterior cervical lymph node biopsy with Hodgkin’s cells. (d) H&E with close-up inset, (e) anti-CD30 (Dako, Carpinteria, CA, USA), (f) anti-CD15. Slides were developed with horseradish peroxidase-labeled secondary Ab and diaminobenzidine chromogen using the EnVision þ method.9 Images were acquired with an Olympus BX51 microscope (Olympus, Center Valley, PA, USA). Images were captured using a DP70 digital camera and software (Olympus) and processed using Adobe Illustrator software (Adobe Systems, San Jose, CA, USA). (g) Quantitative Assessment of CLL and Donor Chimerism following allo-HCT Assessment of minimal residual disease (MRD) in the peripheral blood was performed by quantitative PCR using ASO-Q-PCR, largely as previously described.10 VH-region consensus probes and rearranged-IgH allele-specific primers, including a CDR3-region primer, were used. The method employs absolute quantification, as CLL is reported as copy number per microgram (on the y axis with corresponding values trended in red). GAPDH is used as an endogenous control gene to measure the input quantity of amplifiable DNA and ASO-IgH plasmid standards. The sensitivity for each assay is p10 copies/mg. Specificity is demonstrated using several negative controls including normal tonsillar DNA as a polyclonal control. TaqMan primers and probes were designed using PrimerExpress from Applied Biosystems, Foster City, CA, USA (version 3.0). MRD assays were performed on the ABI 7700 or 7900 (Applied Biosystems). Molecular remission was defined as a result p30 copies/mg WBC–DNA (1.8  104 WBCs). Percentage of donor chimerism is displayed on the y axis (right), with T-cell chimerism (CD3) trended in blue and NK-cell chimerism (CD56) trended in black. Days post-HCT is displayed on the x axis. Rituxan and donor lymphocyte infusion were administered at the time points indicated above. Unmanipulated donor lymphocyte infusion was dosed at 3  107 CD3 cells per recipient wt kg. CsA and prednisone taper and GVHD course are shown. Lymph node biopsy on post-HCT day þ 574 makes the diagnosis of classic Hodgkin’s disease. PET-CT images post-HCT/post-DLI. (h) Day þ 270/16; (i) day þ 374/ þ 88; (j) day þ 545/ þ 259. The [18F]FDG-PET scans were performed at Stanford University Medical Center on a Discovery LS [18F]FDG-PET/CT scanner (GE Medical Systems, Waukesha, WI, USA). Patients were injected with 10–15 mCi of [18F]FDG and a standard 45- to 60-min tracer uptake period was used.

Acknowledgements We thank our patient and her family. We also thank Peter Maxim, PhD, from the Stanford University Department of Radiation Oncology for helping us acquire the PET-CT images, and Matt Inlay, PhD, from Irving Weissman’s laboratory at Stanford University for reviewing the manuscript. This research was supported by Leukemia and Lymphoma Society Translational Research Grant 6204-06 and NCI P01 CA049605.

D Tseng1, CD Jones2, M Anderson2, R Warnke2, JL Zehnder2 and DB Miklos3 1 Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA, USA; 2 Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA and 3 Department of Medicine, Division of Bone Marrow Transplantation, Stanford University School of Medicine, Stanford, CA, USA E-mail: [email protected]

References 1 Lowsky R, Takahashi T, Liu YP, Dejbakhsh-Jones S, Grumet FC, Shizuru JA et al. Protective conditioning for acute graftversus-host disease. N Engl J Med 2005; 353: 1321–1331. 2 Momose H, Jaffe ES, Shin SS, Chen YY, Weiss LM. Chronic lymphocytic leukemia/small lymphocytic lymphoma with Reed-Sternberg-like cells and possible transformation to Hodgkin’s disease. Mediation by Epstein-Barr virus. Am J Surg Pathol 1992; 16: 859–867.

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3 Williams J, Schned A, Cotelingam JD, Jaffe ES. Chronic lymphocytic leukemia with coexistent Hodgkin’s disease. Implications for the origin of the Reed-Sternberg cell. Am J Surg Pathol 1991; 15: 33–42. 4 Kanzler H, Kuppers R, Helmes S, Wacker HH, Chott A, Hansmann ML et al. Hodgkin and Reed-Sternberg-like cells in B-cell chronic lymphocytic leukemia represent the outgrowth of single germinal-center B-cell-derived clones: potential precursors of Hodgkin and Reed-Sternberg cells in Hodgkin’s disease. Blood 2000; 95: 1023–1031. 5 Ohno T, Smir BN, Weisenburger DD, Gascoyne RD, Hinrichs SD, Chan WC. Origin of the Hodgkin/Reed-Sternberg cells in chronic lymphocytic leukemia with ‘Hodgkin’s transformation’. Blood 1998; 91: 1757–1761. 6 Luckey CJ, Bhattacharya D, Goldrath AW, Weissman IL, Benoist C, Mathis D. Memory T and memory B cells share a transcriptional program of self-renewal with long-term hematopoietic stem cells. Proc Natl Acad Sci USA 2006; 103: 3304–3309. 7 Tsang WY, Chan JK, Ng CS. Epstein-Barr virus and Reed-Sternberg-like cells in chronic lymphocytic leukemia. Am J Surg Pathol 1993; 17: 853–854. 8 Park CY, Tseng D, Weissman IL. Cancer stem cell-directed therapies: recent data from the laboratory and clinic. Mol Ther 2009; 17: 219–230. 9 Paules RS, Levedakou EN, Wilson SJ, Innes CL, Rhodes N, Tlsty TD et al. Defective G2 checkpoint function in cells from individuals with familial cancer syndromes. Cancer Res 1995; 55: 1763–1773. 10 Ladetto M, Donovan JW, Harig S, Trojan A, Poor C, Schlossnan R et al. Real-time polymerase chain reaction of immunoglobulin rearrangements for quantitative evaluation of minimal residual disease in multiple myeloma. Biol Blood Marrow Transplant 2000; 6: 241–253.