EBV negative Richter's syndrome from a coexistent clone after salvage ...

American Journal of Hematology 81:706–712 (2006)

EBV Negative Richter’s Syndrome from a Coexistent Clone after Salvage Treatment with Alemtuzumab in a CLL Patient Ann Janssens,1* Mario Berth,2 Pascale De Paepe,3 Bruno Verhasselt,2 Nadine Van Roy,4 Lucien Noens,1 Jan Philippe´,2 and Fritz Offner1 1

2

Department of Hematology, Ghent University Hospital, Ghent, Belgium Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, Belgium 3 Department of Pathology, Ghent University Hospital, Ghent, Belgium 4 Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium

Transformation of B cell chronic lymphocytic leukemia (B-CLL) to large cell lymphoma or Hodgkin’s disease is known as a Richter’s syndrome (RS). According to the literature, 1– 10% of B-CLL patients develop this high-grade lymphoid malignancy. The relationship between the immunosuppressive effect of nucleoside analogues (NA) and monoclonal antibodies and the development of large cell transformation still remains a controversial issue. We describe a CLL patient who developed a large B cell lymphoma 94 months after diagnosis and 3 months after the start of alemtuzumab. The CLL immunophenotype was retained by the transforming cells although a different light chain was expressed. Molecular analysis of the immunoglobulin heavy chain confirmed that the CLL and the RS had a different clonal origin. Subsequent molecular analyses of stored samples showed that the clone with transforming capacity already appeared two years before the clinical appearance of the RS. We hypothesize that alemtuzumab promoted the uncontrolled growth of the latest clone by eradicating the initial B-CLL clone efficiently, and by inducing a strong T cell depletion with consequent impairment of the immunosurveillance. We also ruled out that the RS was EBV driven. In conclusion, we report a case of EBV negative RS after alemtuzumab as salvage C 2006 Wiley-Liss, Inc. therapy. Am. J. Hematol. 81:706–712, 2006. V Key words: B cell chronic lymphocytic leukemia; Richter’s syndrome; alemtuzumab

INTRODUCTION

B cell chronic lymphocytic leukemia (B-CLL) is an indolent lymphoproliferative disorder with a progressive accumulation of small, morphologically mature B lymphocytes in the blood, bone marrow, and lymphoid tissues. Recently it has become evident that patients with mutated IgVH genes have a median survival of *25 years while patients with unmutated IgVH genes survive for only 10 years [1]. In both groups transformation to a higher grade lymphoma can occasionally occur, which leads to a shorter survival. Prolymphocytoid transformation is characterized by a progressive increase in the percentage of prolymphocytes in the blood and the bone marrow together with a worsening of cytopenia, increasing lymphadenopathy and organomegaly, and refractoC 2006 Wiley-Liss, Inc. V

riness to conventional treatment. It is thought to occur in 10–20% of patients [2]. Transformation or progression to a large cell lymphoma (LCL) or Hodgkin’s disease (HD) is known as Richter’s syndrome (RS). The most common clinical features of this syndrome are an abrupt change Contract grant sponsor: Fonds Wetenschappelijk Onderzoek (FWO) Vlaanderen; Contract grant number: G0050.01. *Correspondence to: Ann Janssens, University Hospital Gasthuisberg, Department of Hematology, Herestraat 49, 3000 Leuven, Belgium. E-mail: [email protected] Received for publication 24 January 2005; Accepted 12 May 2005 Published online 12 July 2006 in Wiley InterScience (www.interscience. wiley.com). DOI: 10.1002/ajh.20712

Case Report: Richter’s Syndrome after Alemtuzumab

in patient’s condition (fever, sweats, and weight loss), a rapid and often asymmetric enlargement of lymph nodes, extra nodal involvement, a rapidly increasing lactate dehydrogenase (LDH), and possibly a monoclonal gammopathy. According to the literature, this type of transformation occurs in 1– 10% of CLL patients [3]. The presence of acute lymphoblastic leukemia (ALL) [4] or multiple myeloma (MM) [5] in patients with pre-existing B-CLL has also been described in case reports. The development of RS, but also ALL or MM may represent either a clonal progression of the B-CLL or a de novo development of an independent lymphoid malignancy [4–6]. The long term immunosuppression accompanying B-CLL may be a mechanism allowing transformed clones to progress. This immunodeficiency status specific for CLL may be aggravated by the given treatment [4]. Controversy still remains about the relationship between the immunosuppressive effects of nucleoside analogues (NA) and the generation of large cell lymphoma. The monoclonal antibody alemtuzumab (Campath-1H), a humanized antiCD52 antibody approved for use in CLL patients who have failed purine analogue treatment, induces an even more profound and prolonged depletion of CD4 and CD8 subpopulations [7,8]. There are no reports so far that suggest a relation between treatment with alemtuzumab and RS, outside the context of EBV-transformed clones.

CASE REPORT Diagnosis

A 64-year-old woman was referred to our department in 1996 because of lymphocytosis. The white blood cell count was 18.000 with 78% lymphocytes. Morphologically these lymphocytes were small with clumped chromatin. No excess of prolymphocytes or pleomorphic cells was seen. Flow cytometry revealed a CLL score of 5/5 (CD5, CD19, and CD23 positive; CD79b, sIg kappa dim, and FMC7 negative), confirming the diagnosis of a B-CLL. Taking into account the absence of peripheral lymphadenopathy, spleno- and hepatomegaly, anemia or thrombocytopenia the patient was staged as RAI 0 or Binet A. Prognostic Factors

CD38 expression was seen in 89% of the B-lymphocytes. IgVH gene mutation analysis showed 94.6% homology to the nearest germ line sequence with use of the VH2-70 family. 11q22.3 deletion was found as high risk genetic aberration in 88% of the cells with fluorescence in situ hybridization (FISH).

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ZAP70 expression evaluated with flow cytometry and RT-PCR was found positive. Treatment History

Because of the short lymphocyte doubling time and the appearance of multiple enlarged lymph nodes, treatment with chlorambucil was started in 1998, 2000, and 2001. Each time a partial remission was obtained. Six years after diagnosis an autoimmune hemolytic anemia (AIHA) occurred. Corticosteroids could hardly control this process. At the beginning of 2004, lymphocytosis exceeded 100,000 and thrombocytopenia was noted. Because of the preceding AIHA, fludarabine was not an option and therapy with alemtuzumab was started. After 4 weeks severe neutropenia developed and treatment was withheld. Thrombocytopenia recovered and no hemolysis was seen. Twelve weeks after the start of alemtuzumab, the patient became febrile and a rapidly increasing LDH (2000 U/L) was observed along with recurrent thrombocytopenia and anemia. No enlargement of lymph nodes, increasing organomegaly or extra nodal involvement was noticed. Morphology at Transformation

The white blood cell count rose to 3000/ml 3 months after the start of alemtuzumab. Twentyfive per cent of the cells were still recognized as small lymphocytes, another 25% was designated as morphologically abnormal. These lymphocytes were large sized, had vesicular nuclei, sometimes with visible nucleoli, and an abundant basophilic cytoplasm, which was frequently vacuolated (see Fig. 1). The bone marrow smears revealed 31% of these lymphoma cells and 13% small lymphocytes. The trephine biopsy was highly cellular. The marrow showed a diffuse infiltration by large blasts, which replaced most of the normal hemopoietic and fat cells (see Fig. 2). Flow Cytometry at Transformation

Four-color immunophenotypic analysis (FACSCalibur, BD Biosciences) was performed on bone marrow and peripheral blood. Two monotypic Bcell populations could be identified and easily discerned on light-scatter properties: small cells (CLL cells) and large cells. Both populations were positive for CD19, CD5, CD20 (dim), CD22 (dim), CD79b (dim), CD23, CD52 and negative for CD10 and FMC7. A dim expression of the surface immunoglobulin was observed on both populations, but there was a lambda light chain restriction in the large cell American Journal of Hematology DOI 10.1002/ajh

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Case Report: Janssens et al.

products revealed prominent peaks of 72, 80 (FR3A), and 247 (FR2) base pairs long in the diagnostic sample. Follow-up after 6 years showed the same peaks, with the addition of minor peaks of 130 (FR3A) and 295 (FR2) base pairs long. The sample taken at transformation showed significant increase of the intensity of these latter two peaks, while the products detected in the diagnostic sample were still present, although declining in intensity (see Fig. 5). Cytogenetics at Transformation

Fig. 1. Buffy coat film (May-Gru¨newald-Giemsa, x500). Large sized cells with vesicular nuclei, sometimes with visible nucleoli and abundant basophilic cytoplasm that is frequently vacuolated among small lymphocytes. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

FISH on t (12), 13q, 11q, and 17p confirmed the presence of the 11q22.3 deletion in 90% of the cells as solitary aberration just before the start of treatment with alemtuzumab. Genetic analysis was also performed on the transforming bone marrow. Trisomy 12, 13q14.3, p53 deletion and 8q24 (myc) rearrangement were all absent. The 11q22.3-23.1 deletion had disappeared. Conventional chromosome banding analysis showed the existence of complex alterations in all the mitoses (46XX, der(16)t(1;16) (q25;q13), add(21)(p11.2)). EBV Status

EBV in-situ hybridization, searching for EBER RNA, was done on the bone marrow biopsy specimen. On peripheral blood a PCR against EBNA-2 was performed. Both tests were negative, which enabled us to rule out that the lymphoproliferation was EBV driven. DISCUSSION

Fig. 2. Bone marrow trephine biopsy (Hematoxylin and eosin, x100). Diffuse infiltration by large blasts replacing the normal hemopoietic and fat cells. [Color figure can be viewed in the online issue, which is available at www. interscience.wiley.com.]

population and a kappa light chain restriction in the small cell population (see Fig. 3). The expression of CD38 was dim in the small cell population and strong in the large cell population (see Fig. 4). Immunoglobulin Heavy-Chain Rearrangement Studies

Peripheral blood DNA was purified from cryopreserved cells. Capillary gel electrophoresis of immunoglobulin heavy-chain rearrangement (IgH) PCR American Journal of Hematology DOI 10.1002/ajh

This case reports on the transformation of a BCLL into a diffuse large B-cell lymphoma 94 months after diagnosis and 3 months after starting treatment with alemtuzumab. Flow cytometric examination of the lymphoma cells showed a typical CLL profile despite the large cell characteristics of the B cells in transformation. However, the abnormal cell population expressed lambda immunoglobulin light chain while the initial and remaining smaller B cells were still positive for kappa immunoglobulin light chain. Kroft et al. compared the immunophenotypes of RS and the previous or coexistent CLL and concluded that the LCL mostly retained a CLL phenotype with only minor modulations in antigen expression [9]. The different light chain phenotype of the CLL and subsequent RS does not necessarily indicate bi-clonality. Clonality might be more convincingly documented by molecular analysis of immunoglobulin gene rearrangement [4,6,10]. Subsequent clonality analyses in this case showed that the RS had


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Fig. 3. Scattergrams without gating showing the CD19+/lambda+ lymphocytes being the large lymphocytes. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 4. Scattergrams with gating on CD19 cells showing the CD38+dim cells being the small lymphocytes. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

evolved from a clone that was not seen at diagnosis and emerged after 6 years. It seemed that the initial CLL clone was effectively suppressed by the monoclonal therapy while the evolving clone could suddenly multiply unchecked. According to the literature approximately two thirds of all cases of RS evolve from the B-CLL clone while one third develops as an independent de novo lymphoid malignancy [4,6,10]. RS is a relatively rare event in our practice. We found in our CLL database of 310 patients only five cases with transformation to a LCL and one case with transformation to Hodgkin disease. Transformation was observed in two cases during or at the end of fludarabine treatment (2/42), and in this case 3 months after starting alemtuzumab (1/7). Although these three patients had no signs or symptoms of RS at the start of NA or alemtuzumab, we can not exclude for certain begin-

ning transformation at that time point. The other three cases were only pretreated with chlorambucil (3/117). All patients had received corticosteroids during their disease. An additive or synergistic immunosuppressive effect can therefore not be excluded. We reviewed the available literature to find a relationship between treatment with NA and the development of RS (Table I). With these data we are not able to show a statistically significant difference in RS incidence between patients treated respectively with or without NA. Nevertheless, most papers report more patients with RS when purine analogue treatment has been given (range 3.4 vs. 18%). Some case reports describe the occurrence of LCL, HD [18], and even MM [5] shortly after the introduction of fludarabine in B-CLL. Some of these lymphoproliferations were clearly EBV-mediated [18]. The appearance of a LCL during or soon after initiation American Journal of Hematology DOI 10.1002/ajh

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Case Report: Janssens et al.

Fig. 5. IgVH rearrangement study (FR2). Black – 2 years after diagnosis, one peak of 247 bp. Red – 6 years after diagnosis, addition of a second peak of 295 bp. Blue – at transformation, initial peak declining, latest clone increasing; IgVH rearrangement study (FR3A). Black – 2 years after diagnosis, prominent peaks of 72 and 80 bp. Red – 6 years after diagnosis, addition of a peak of 130 bp. Blue – at transformation, initial peak declining, latest clone increasing. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] TABLE I. Relationship between Treatment with NA and the Development of RS Incidence of RS

NA as salvage therapy Robertson et al., 1993 Mauro et al., 1999 Cheson et al., 1999 Tabuteau et al., 2000 Robak et al., 2003 NA as initial therapy Keating et al., 1998 Morrison et al., 1999 Leporrier et al., 2001

Incidence RS according to treatment (%)

Total number

Percentage (%)

Without NA

With NA

Refs.

46/1374 22/1011 23/595 23/298 10/1487

3.30 2.20 3.90 7.70 0.60

2.40 No data No data 5.40 No difference

3.40 No data 3.90 18 No difference

3 11 12 13 14

13/174 39/544 2/939

7.50 7.20 0.20

No data 5.75 No difference

7.50 8.50 No difference

15 16 17

of treatment may indeed imply a causal relationship between the treatment and the disease progression. Monoclonal antibodies such as rituximab and alemtuzumab are both active in CLL and are avidly incorporated in treatment protocols. An increase in opportunistic infections is a well known consequence of the drug induced immunosuppression. We questioned the literature for a relation between treatment with monoclonal antibodies and the ocAmerican Journal of Hematology DOI 10.1002/ajh

currence of a transforming lymphoma. Cohen et al. published two cases of CLL, pretreated with fludarabine and developing transformed, large clonal masses two months after the start of treatment with rituximab [19]. Byrd et al. also reported two deaths caused by RS in patients treated concurrently with fludarabine and rituximab as part of the CALGB 9712 trial [20]. The efficacy, safety, and clinical benefit of alemtuzumab in refractory and relapsing


B-CLL were published, respectively, by Keating et al. and Ferrajoli et al. [7,21]. Some deaths were attributed to disease progression but no allusion was made to RS. Faderl et al. reported on the safety and efficacy of alemtuzumab plus rituximab in patients with relapsed or refractory lymphoid malignancies. With a median follow up of 6.5 months no cases with RS have been seen [22]. At the end of 2003, four cases of EBV-positive lymphoproliferative disorders were published after alemtuzumab treatment. Ghobrial et al. described an EBV-related transformation in a CD4+ T-CLL pretreated with fludarabine [23]. O’Brien et al. reported on the use of alemtuzumab for residual disease in patients with B-CLL after chemotherapy with at least fludarabine and cyclophosphamide. 3/41 patients developed an EBV-positive LCL [24]. Recently, the long-term follow-up of alemtuzumab as first line therapy in B-CLL was published by Karlsson et al. in abstract form. The incidence of RS was 13% (5/38). These cases were not associated with EBV. The incidence of RS in the total control population was only 5% (n ¼ 310) [25]. Large B cell lymphomas are also reported in severe congenital immunodeficiencies, in persons with acquired immunodeficiency syndrome and in transplant recipients receiving immunosuppressive drugs. HIV-infected individuals have a high risk of developing NHL. About 4% of the people with AIDS have a NHL at AIDS diagnosis and at least the same proportion develop non-Hodgkin lymphoma (NHL) during the next few years [26]. However, after the introduction of the highly active antiretroviral therapies (HAART), the incidence of NHL in patients with sufficient immunologic response has decreased significantly [27,28]. The incidence of post-transplant lymphoproliferative disorders (PTLD) in solid-organ transplantation ranges from 3% for renal or liver transplants to 10% for lung transplants [29]. The PTLD incidence for allogeneic and also non myeloablative allogeneic stem cell transplantation (NMST) approaches 3% [30]. Although alemtuzumab is frequently incorporated in the conditioning regimen of NMST to achieve in vivo T-cell depletion, the PTLD incidence is much lower than the 24% reported in the setting of a T-cell-depleted mismatched bone marrow transplant [29]. This means that the incidence of PTLD in solid organ transplants and allogenic bone marrow transplantation varies with the degree of immunosuppression. Thus it is reasonable to suppose that purine analogues and alemtuzumab contribute to the development of RS in some CLL patients through the profound and prolonged T-cell depletion. Fludarabine, given as first line or salvage treatment, can

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reduce CD4 and CD8 cells to less than 200/ml during the treatment period and this suppression can persist for more than one year [15]. Alemtuzumab preferentially depletes malignant lymphocytes but normal B and T cells also bear CD52 and are also profoundly reduced. The Swedish group did a longterm follow-up of lymphocyte subsets after subcutaneous alemtuzumab treatment as primary therapy for CLL and found CD4 and CD8 cells as low as 50/ml during treatment. It took these cells *6 months to recover to 200/ml [8]. CLL patients treated with alemtuzumab after failing fludarabine treatment showed an even more pronounced CD4 depletion. At week 4 CD4 levels of 2/ml were measured. At week 12 the T helper cells were already increasing to 84/ml [7]. This profound and early occurring T cell depletion could be responsible for the development of RS soon after the start of and during treatment with NA and alemtuzumab, whereas the prolonged immunosuppression can explain the development of RS in CLL patients later on even when complete remission is sustained [4]. Some authors suggest using NA up front because the immunosuppression inherent to CLL is at that time not that high, and additional clones occurring during the natural history of CLL have not yet developed and therefore can not be selected by the given treatments [31]. However, the incidence rates of RS in patients treated first line with fludarabine or alemtuzumab seem to be at least equally high as the incidence of transformation found in patients treated with these agents as salvage regimen. In conclusion, this case report describes the development of an EBV negative LCL shortly after the start of alemtuzumab. The origin of this transformation was in a clone, which was different from the original B-CLL clone and appeared already two years earlier. Careful follow up of larger cohorts of patients treated with alemtuzumab will show if the development of RS after this treatment remains an exceptional event. REFERENCES 1. Oscier D, Gardiner A, Mould S, et al. Multivariate analysis of prognostic factors in CLL: Clinical stage, IGVH gene mutational status and loss or mutations of the p53 gene are independent prognostic factors. Blood 2002;100:1177–1184. 2. D’Onofrio G, Zini G, Bain B. Lymphoproliferative disorders. In: D’Onofrio G, Zini G, Bain B, editors. Morphology of the Blood. Oxford: Butterworth Heinemann; 1998. pp 257–315. 3. Robertson L, Pugh W, O’ Brien S, et al. Richter syndrome: A report on 39 patients. J Clin Oncol 1993;11:1985–1989. 4. Gilles F, O’Brien S, Keating M. Chronic lymphocytic leukaemia in (Richter’s) transformation. Semin Oncol 1998;25:117–125. 5. Patriarca F, Gaidano G, Capello D, Zaja F, Fanin R, Baccarani M. Occurrence of multiple myeloma after fludarabine treatment of a chronic lymphocytic leukaemia: Evidence of a biclo-

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