prognosis patients with B-cell chronic lymphocytic leukemia ...

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1993 82: 1366-1376

Autologous and allogeneic bone marrow transplantation for poor prognosis patients with B-cell chronic lymphocytic leukemia SN Rabinowe, RJ Soiffer, JG Gribben, H Daley, AS Freedman, J Daley, K Pesek, D Neuberg, G Pinkus and PR Leavitt

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Autologous and Allogeneic Bone Marrow Transplantation for Poor Prognosis Patients With B-Cell Chronic Lymphocytic Leukemia By Susan N. Rabinowe, Robert J. Soiffer, John G. Gribben, Heather Daley, Arnold S. Freedman, John Daley, Kelly Pesek, Donna Neuberg, Geraldine Pinkus, Pearl R. Leavitt, Neil A . Spector, Michael L. Grossbard, Kenneth Anderson, Michael J. Robertson, Peter Mauch, Karen Chayt-Marcus, Jerome Ritz, and Lee M. Nadler Twenty patients with poor prognosis B-cell chronic lymphocytic leukemia (B-CLL) underwent uniform high-dose chemoradiotherapy followed by rescue with multiple monoclonal antibody-purged autologous bone marrow (BM) (12 patients) or T-cell-depleted allogeneic BM from HLA-identical siblings (8 patients) in a pilot study to assess the feasibility of BM transplantation (BMT) in this disease. All had poor prognosis disease by either staging, BM pattern, tumor doubling time criteria, or cytogenetics. All patients achieved remission criteria (defined as s2 cm adenopathy, absence of splenomegaly, 520% of the intertrabecular space involved on BM biopsy) before BMT. Despite the use of fludarabine, a median of three treatment regimens were required to achieve BMT eligibility. After BMT, all patients achieved complete hematologic engraftment. Toxicities

were not significantly different between autologous versus allogeneic BMT. Two toxic deaths were observed. Of 19 evaluable patients, 17 clinical complete clinical remissions (89%)were observed, with 2 patients (1 allogeneic and 1 autologous) exhibiting persistent BM disease. Complete clinical remissions were documented at the phenotypic and molecular level for the majority of patients in whom dual fluorescence for CD5 and CD20 (15 of 15; 100%)and lg gene rearrangements (11 of 14; 79%) were performed. Although long-term follow-up is needed to assess any potential impact on the disease-free and overall survival of these patients, this study shows the feasibility of using high-dose chemoradiotherapy and BMT in patients with poor prognosis B-CLL. 0 1993 by The American Society of Hematology.

A

advanced age, the majority ofthese younger patients will die from progressive B-CLL and its complications such that this young group of patients has a significantly shorter survival than their normal aged-matched counterparts.’ Regardless of age, several prognostic parameters, including clinical stage, lymphocyte doubling time, pattern of bone marrow (BM) infiltration, and cytogenetics, have been useful in predicting which patients will progress more rapidly, require therapy, and eventually succumb to their B-CLL.9-’9Patients in the younger age group with poor prognostic features have a median survival of less than 3 years.’ This patient subgroup might therefore merit a more aggressive treatment approach. To date, very few patients with CLL have undergone high-dose myeloablative therapy and BM transplantation (BMT). A recent report from a multicenter European study described the results of allogeneic (allo) BMT for I7 cases of young, predominantly resistant CLL patients.” These patients were treated with several different ablative regimens and methods of graft-versus-host disease (GVHD) prophylaxis. In contrast, there have been no reports of autologous (auto) BMT for CLL. The paucity ofpatients with CLL who have received BMT is due to several factors, including the older age of onset ofthis disease, its long natural history, the difficulty in achieving a minimal disease state before transplant, and concerns regarding potential transplant-related morbidity and mortality. In the present report, we present the findings of a pilot study to evaluate the role of allo-BMT and auto-BMT as consolidative therapy in B-CLL patients with poor prognostic features but who had chemosensitive disease at the time of BMT. Eligibility requirements were that patients had poor prognosis B-CLL based on the International Workshop on CLL staging (which integrates the Binet and Rai systems) B (II), C (111), and C (IV) as well as other characteristics.2’ Eight patients with HLA-matched sibling donors underwent allo-BMT with anti-T 12 (CD6)-depleted marrow,22whereas 12 patients without an HLA-compatible

DVANCED-STAGE chronic lymphocytic leukemia of B-cell origin (B-CLL) frequently affects individuals over 60 years of age. Therefore, the practical therapeutic approach has been to treat patients with single agents or relatively unaggressive combination chemotherapy to reduce the tumor burden, alleviate clinical symptoms, and decrease the need for transfusions as well as the frequency of infection. This palliative approach has shown that most BCLLs are responsive to conventional dose treatment and partial remissions are frequently achieved. However, few durable clinical complete remissions (CRs) are observed and few, if any, patients are cured. Although most responses tend to be short-lived, patients usually experience relatively prolonged survival. More recently, higher numbers of CRs have been reported using newer agents such as fludarabine.’-’ The long-term clinical impact of these new agents remains unknown, but it is unlikely that many patients will be cured.’ Up to 40% of patients with B-CLL are younger than 60 years of age at diagnosis.’ Whereas many patients in the older subgroup will ultimately die of unrelated causes due to

From the Division of Tumor Immunology and Biostatistics, Dana-Farber Cancer Institute: and the Joint Center of Radiation Therapy and Department ofPathology andmedicine, Harvard Medical School, Boston, MA. Submitted December 22, 1992; accepted April 16, 1993. Supported by Grant No. CA34183from the National Institutes of Health to L.M.N. Address reprint requests to Lee M. Nadler, MD, Division of Tiimor Immunology, Dana-Farber Cancer Institute, 44 Binney St, Boston, MA 021 IS. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1993 by The American Society of Hematology. 0006-4971/93/8204-0022$3.00/0

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Blood, VOI 8 2 , NO 4 (August 15). 1993:pp 1366-1376

From bloodjournal.hematologylibrary.org by guest on July 12, 2011. For personal use only. BONE MARROW TRANSPLANTATION FOR B-CLL

donor underwent auto-BMT using marrow purged with multiple anti-B-cell monoclonal antibodies ( M o A ~ s ) . ’All ~ patients were treated with the identical ablative regimen. After BMT, 17 of 19 (89%) evaluable patients achieved a clinical CR, 15 of 15 [ 100%) tested achieved a phenotypic CR, and 1 1 of 14 (79%) tested achieved a molecular CR, with acceptable morbidity and mortality. This study shows the feasibility of BMT as a therapeutic approach in suitable poor prognosis patients with B-CLL. MATERIALS AND METHODS

Selection ofpatients and treatment protocol. Patients with a histologic diagnosis of CLL were eligible for study if (1) they had BCLL documented by tumor cell reactivity with anti-B1 (CD20) MoAb; (2) they were less than or equal to 60 years of age; (3) their disease was International Workshop on CLL stage B (II), C (111), or C (IV);(4) there was absence ofcomorbid disease ofthe lungs, liver, and heart, and they had a Karnofsky score above 80%;and (5) they were able to attain a minimal disease state (12 cm nodes, no organomegaly, and 520% BM infiltration of the intertrabecular space as assessed by bilateral iliac crest biopsy). Although some patients had one or more poor prognostic features, includinga diffuse pattern on BM biopsy, abnormal BM cytogenetics, and/or a rapid tumor growth rate (defined as doubling of either lymphocyte count and/or lymphadenopathy within a 6- to 12-month period), these were not formal eligibility requirements for the study. Patients with T-CLL or prolymphocytic leukemia were not eligible. In addition, any circumstance that precluded the ability to deliver full-dose total body irradiation (TBI) was a reason for exclusion from the study. Patients with HLA-matched siblings underwent allo-BMT, whereas all other patients underwent auto-BMT. The preparative regimen was identical for all patients and consisted of cyclophosphamide (CTX) at 60 mg/kg of body weight infused on each of 2 consecutive days followed by 1,400 cGy TBI administered in fractionated doses (200 cGy twice daily on 3.5 consecutive days). Within 18 hours of the completion of radiotherapy, patients received either cryopreserved autologous BM that had been previously treated in vitro with multiple anti-B-cell MoAbs and rabbit complement or newly harvested BM from an HLA-identical sibling donor that had been treated in vitro with anti-TI2 (CD6) MoAb and complement. No other GVHD prophylaxis was given. Informed consent was obtained from all patients and the protocol was approved by the Human Protection Committee at Dana-Farber Cancer Institute. Collection,processing, and infusion of marrow. Within 4 weeks before admission for BMT, autologous BM was harvested from the iliac crests, treated in vitro as previously described for patients with non-Hodgkin’s lymphoma with anti-Bl (CD20), B5, and J5 (CDIO),and ~ryopreserved.’~ Anti-TI (CD5) was not used because of a previous report documenting an increased incidence of Epstein-Barr virus-associated lymphoproliferative syndrome after purging of the BM with anti-TI for patients undergoing autologous BMT for T-cell lymph~ma.’~ After completion of radiotherapy, the cryopreserved marrow cells were rapidly thawed and diluted in medium containing DNAase. The median number of cells infused was 2.87 X IO7/kg (range, 2.60 to 8.01) with 85% to 95% viability, as measured by trypan blue dye exclusion. For patients undergoing allo-BMT, BM was harvested from the iliac crests of normal HLAmatched sibling donors and incubated in vitro with anti-T12 (CD6) MoAb and rabbit complement, as previously described.’* The median number of allogeneic cells infused was 4.97 X 107/kg(range, 4.4 to 8.0). Supportive care. Patients were treated in reverse isolation

1367 rooms. All patients received oral prophylactic antibiotics, with either ciprofloxacin or trimethoprim-sulfamethoxazole when chemotherapy was begun. These were discontinued when intravenous antibiotics were instituted for fever. In addition, all patients received prophylactic acyclovir ( 5 mg/kg intravenously [IV] or 400 mg orally every 8 hours) for herpes simplex infections. Cytomegalovirus-negative or leuco-filtered blood products were used in all patients regardless of prior exposure to cytomegalovirus. All blood products were irradiated (2,000 cGy) to prevent transfusion-related GVHD. Clinical evaluation. Before admission for BMT, all patients were evaluated by physical examination, blood-chemistry profile, complete blood count (CBC), differential, serum protein immunoelectrophoresis, chest x-ray, abdominal-pelvic computer tomography (CT) scanning, as well as bilateral BM aspirates and biopsies. Chest CT scans were performed in patients with evidence of abnormalities on chest x-ray evaluation. Splenomegaly was defined by CT scan evaluation: mild, 12to 14 cm; moderate, greater than 14to 17 cm; massive, greater than 17 cm. Clinical CR was defined as the absence of any detectable disease. The criteria for achievement of clinical CR were identical after BMT, with all evaluations performed within 3 months of BM infusion. Follow-up restaging for assessment of the durability of remissions was performed every 3 to 6 months thereafter or more frequently if clinically indicated. Pathologic review. BM aspirates were reviewed at the DanaFarber Cancer Institute and the Hematopathology Department at Brigham and Women’s Hospital (Boston, MA). BM biopsies, lymph node biopsies (when available), and all splenectomy specimens were reviewed by the Hematopathology Department at Brigham and Women’s Hospital. BM biopsies were examined for cellularity, lymphocyte percentages of the overall cellularity, and intertrabecular space and pattern of BM infiltration. Five different patterns on biopsy were reported: ( I ) normal, with no evidence of lymphoproliferation in the BM; (2) nodular, with nodules of small mature lymphocytes lacking clear germinal centers; (3) interstitial, with replacement of normal hematopoietic tissue by small mature lymphocytes infiltrating between fat without distortion of the marrow architecture; (4) mixed, with both nodular and interstitial involvement;and (5) diffuse, with effacement of the marrow architecture by small mature 1ymph0cytes.l~ Hematologic engraftment. Neutrophil engraRment was defined as the achievement of an absolute neutrophil count (ANC) of greater than or equal to 500/pL on 2 consecutive days, with the time to neutrophil engraftment calculated using day 0 as the day of BM infusion and measuring to the first of 2 consecutive days. Platelet engraftment was defined as the first of 2 consecutive days with platelets greater than or equal to 20,00O/pL, and followed by 7 days without platelet transfusion. Dual fluorescence analysis. To further assess remission status immediatelybefore and after BMT, dual fluorescenceof BM mononuclear cells for CD5 and CD20 was performed for the majority of patients. Cells were analyzed at the time of initial evaluation, immediately before BMT, and at varying intervals between 3 and 6 months after BMT. Immunophenotypic analysiswas performed on an EPICS ELITE flow cytometer (Coulter Cytometry, Hialeah, FL). Negative isotypic controls as well as single color (fluorescein isothiocyanate [FITC] and phycoerythrin [PE]) stained samples were analyzed to insure that the flow cytometer was adjusted for maximum sensitivity as well as proper fluorescence compensation. For dual-labeled samples, the degree of reactivity was performed on 1 X IO4 scatter gated lymphocytesand percent of coexpression was determined by using a Quad-stat statistics program (Coulter Electronics, Hialeah, FL). The presence of more than 10% of the total

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RABINOWE ET AL

lymphocyte population coexpressing CD5 and CD20 was considered positive for residual disease. Ig gene rearrangements. Assessment of Ig gene rearrangements were performed on BM and peripheral blood (PB) samples for the majority of patients. High molecular weight DNA was extracted from cells using standard protocols and 10 pg of individual DNA samples digested using the restriction enzymes EcoRI, BumHI, and Hind111 (New England Biolabs, Beverley, MA). The digested samples were analyzed by electrophoresis in 0.8% agarose gels and Southern blotted onto Nylon membranes (BioRad, Richmond, CA). Hybridization of the Southern blots was performed using oligolabeled probes to the JH region of the Ig heavy chain region. After hybridization, the blots were exposed for 3 days at -70°C. On dilution assays, the detection of Ig gene rearrangements was sensitive to the 1% level. Statistical methods. Descriptive statistics based on ranks (such as the median, minimum, and maximum) are used to present time to engraftment, disease-free survival, and overall survival. Survival variables are assessed by the method of Kaplan and Meier, with confidence intervals calculated using Greenwood's formula. RESULTS

Patient characteristics. Eight patients (7 males and I female) with B-CLL underwent allo-BMT and 12 patients (10 males and 2 females) underwent auto-BMT between January 1990 and August 1992. The characteristics of these patients are shown in Table 1. The median age ofthe cohort that underwent allo-BMT was 40 years (range, 31 to 54),

whereas that of the group that underwent auto-BMT was 45 years (range, 27 to 54). Five patients had a positive family history for CLL. Ofthe entire group, 13 patients had International Working Group stage B (11), 3 had stage C (Ill), 3 had stage C (IV), and 1 had Stage B (I) with a history of Richter's transformation. At the time of initial evaluation for BMT, the majority of patients had a diffuse BM pattern on b i o p ~ y ,whereas '~ the remainder exhibited a variety of patterns (3, mixed; 1, interstitial; 1, nodular; and 1, histologically normal) (Table I ) . Of 18 patients evaluated, 5 had cytogenetic abnormalities and I3 had normal analyses. Ten of the 20 patients had evidence for a rapid tumor growth rate, with doubling of either lymphocyte count and/or lymphadenopathy within a 6- to 12-month period. Before BMT, 7 patients were inevaluable for tumor doubling time because of extensive disease at presentation requiring immediate therapy and were also considered to have rapid tumor growth. In addition, 2 patients (nos. 10 and 20) had 20% to 50%prolymphocytes on peripheral blood smear, which was ~ * * patients ~ assessed to be a poor prognostic f e a t ~ r e . ~Eight had a history of massive splenomegaly (patient no. 18 had a postsplenectomy status), whereas 4 had moderate splenomegaly and 6 had mild splenomegaly as documented on CT scan evaluation (Table I). Treatment before B M T for B-CLL. The therapy received before BMT is shown in Table 2. Nine patients had

Table 1 . Characteristics of Patients Undergoing BMT for B-CLL

Patient No. AIIO-BMT 1 2 3 4 5 6 7 8 Auto-BMT 9 10 11 12 13 14 15 16 17 18 19 20

Age W S e x

Date of Dx

48/F 51/M 34/M 31/M 36/M 41/M 39/M 54/M

1/88 9/85 2/88 1/90 4/88 6/90 8/86 4/85

40/M 45/M 35/M 27/F 46/F 54/M 54/M 32/M 45/M 50/M 51/M 45/M

7/88 3/89 3/88 10188 5/89 1/82 419 1 7/89 219 1 4/90 10/89 5/91

Family History

International Workshop Stage

BM Pattern

+ -

+ +

Rapid Tumor Growth

Trisomy 12 Normal Normal Normal t(11;14) Normal Normal ND

+

-

BM

Cytogenetics

D M D' D D I D D D D NI D

Normal Trisomy 12 Normal Normal ND Trisomy 12 Normal Normal Normal Normal t(11;22) Normal

Splenomegaly Mild Mild Mild Mild Massive Moderate Massive Moderate

+ +t

+ + + -

+ + + + + +t

Massive Mild Moderate Massive Normal Mild Massive Moderate Massive Massivet Normal Massive

Abbreviations: D, diffuse; N, nodular; I, interstitial; M, mixed; NI normal; ND, not done; mild, spleen 12 to 14 cm on CT scan; moderate, spleen >14 to 17 cm on CT scan; massive, spleen > 17 cm on CT scan. * Dry BM aspirate. t Twenty percent to 50% prolymphocytes on peripheral smear. t Status postsplenectomy.


BONE MARROW TRANSPLANTATION FOR B-CLL Table 2. Treatment Before B M T for B-CLL

Patient No

AIIO-BMT 1

Therapy Pre-BMT Evaluation

2

None CHL/P

3 4

None None

5 6

CHL/P None

7

CVP (3) M-BACOD (5)

8

CHL/P

Auto-BMT 9

None

10 11

None None

12

None

13 14

CHOP (2) PROMACE-MOPP (8) CHL

15 16

CHL/P None

17

None

18

Splenectomy PROMACE-MOPP (4) MOPP (2) CHL/P COP-BLAM (4) None

19 20

Therapy to Achieve

Total No. of Regimens

Minimal Disease

Pre-BMT

CHOP (6) CHOP (6) FLU (7) FLU (6) CHOP (6) FLU (3) CVP (12) CHOP (7) CEP (1) FLU (3) Local XRT CHOP (2) 6 0 mg/kg CTX FLU (5) ESAP (3) FLU (3) CHOP (6) CVP (2) ESAP (1 ) CHOP (6); 60 mg/kg CTX (1 ) CHOP (6) CHOP (6); 6 0 mg/kg CTX (1) FLU (7) CHOP (6) 6 0 mg/kg CTX (1) FLU (7) FLU (7) CHOP (6) FLU (3) FLU (6) CHOP (4) FLU (6) CHOP (4) FLU (6) 6 0 mg/kg CTX (1) FLU (6) 60 mg/kg CTX (2)

IntervalFrom Dx to BMT Splenectomy

(mo)

31 68 40 18 42 17

+

6

64

5

85

2

18

1 3

12 32

+

3

25

3

25

3

115

+

2 2

12 34

-

3

16

4

26

None

2

34

FLU (4) CHOP (6) ESAP (1) CVP (3)

4

16

~~

~~

~~

Abbreviations: CHL/P, chlorambucil/prednisone; CVP, cyclophosphamide, vincristine, prednisone; M-BACOD, methotrexate, bleomycin, adriamycin. cyclophosphamide, oncovin. dexamethasone; PROMACE-MOPP. prednisone, methotrexate, adriamycin, cyclophosphamide, etoposide, nitrogen mustard, vincristine. procarbazine; COP-BLAM, cyclophosphamide, vincristine, prednisone, bleomycin, doxorubicin, procarbazine; ESAP, etoposide, methylprednisolone, cytosine arabinoside, cis platinum; FLU, fludarabine; CEP, cyclophosphamide, etoposide, cis platinum; XRT, radiotherapy.

received chemotherapy before the initial BMT evaluation. With the exception of 1 patient (no. 19), a variety of induction regimens were subsequently required to achieve the minimal disease criteria requisite for BMT, inl:luding CVP

(cyclophosphamide, vincristine, prednisone) (n = 3), CHOP (cyclophosphamide, adriamycin, vincristine, prednisone) (n = 14), fludarabine (n = 1 9 , CEP (cyclophosphamide, etoposide, cis platinum) (n = l), 60 mg/kg CTX (n =

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1370

6), and ESAP (etoposide, methylprednisolone, cytosine arabinoside, cis platinum) (n = 3) (Table 2). Patients received between one and six treatment regimens before BMT (median of 3). In addition, 6 patients underwent splenectomy before BMT because of persistent splenic enlargement, with 5 of 6 having evidence of persistent disease on pathologic examination (Table 2). The median interval from the time of diagnosis to BMT was 28 months (range, 12 to 115 months) (Table 2). Diseuse status ut BMT. Despite aggressive attempts to induce CR, only 3 patients were in clinical CR before BMT (patients no. 3, 15, and 19),whereas 1 patient achieved a CR in the BM but still had residual adenopathy (patient no. 6) (Table 3). Although having achieved the minimal disease state requisite for BMT, 8 patients continued to have evidence of both lymph node and BM involvement, whereas 8 patients had residual BM involvement alone. The degree of residual BM involvement as assessed by percentage of intertrabecular space infiltrated varied from less than 5% (6 pa-

Table 3. Disease Status at B M T

Status at BMT

Site at BMT

1

MD

LN BM

2 3 4

MD CR MD

BM

5

MD

6 7

MD MD

8

MD

LN BM

MD MD

BM BM LN BM BM BM LN BM

(% IT) (pattern)

LN BM LN BM LN LN

BM

11 12 13 14

MD MD MD MD

15 16 17

CR MD MD

18 19 20

MD CR MD

Allo-BMT 1 2 3 4 5 6 7 8 Auto-BMT 9 10 11 12 13 14 15 16 17 18 19 20

7/90 5/91 6/91 7/91 10/91 10/91 12/91 5/92

CTX/TBI CTX/TBI CTX/TBI

1 /90 3/90 10/90 11/90 6/91 8/91 4/92 4/92 5/92 6/92 8/92 8/92

CTX/TBI

15 20 26 24 29 26 14 21

34 21 15 24 20 26 13 20

91 26 31 30 35 35 60 27

CTX/TBI CTX/TBl CTX/TBI CTX/TBI CTX/TBI CTX/TBl CTX/TBl CTX/TBl CTX/TBl CTX/TBI CTX / TBI CTX/TBI

22 19 18 18 27 30 19 26 25 20 21 21

29 16 29 >61 69 61 20 23 47 47 19 20

26 27 30 70 122 47 41 25 34 24 29 31

CTX/TBI

CTX/TBI CTX/TBl CTX/TBl

Abbreviation: PLT. platelets

AIIO-BMT

Auto-BMT 9 10

for B-CLL Conditioning PLT Regimen ANC (220X lo3/ Duration of Date of (CTX 120 mg/kg. (2500/ pL X 7 Hospitalization Patient No. BMT 1,400cGy TBI) pL X 2 d) d) (d)

BM Bx Status at Harvest

Disease Patient No.

Table 4. Type of BMT and Hematologic Engraftment After B M T

BM LN BM BM BM

Abbreviations: MD, minimal disease; LN, lymph nodes; M, mixed; N, nodular; IT. intertrabecular space; CR, complete clinical remission (defined by CBC, differential, blood-chemistry profile, serum protein, immunoelectrophoresis,physical examination, x-rays, CT scans, bilateral BM aspirates and biopsies).

tients), to 5% to 10%(9 patients), to 10%to 20% (1 patient). The predominant pattern of BM involvement was nodular (10 patients), but 6 patients exhibited a mixed pattern (Table 3). Ten patients were hypogammaglobulinemic, whereas 3 patients had monoclonal serum proteins before BMT (patient no. 13, IgM 564 mg/dL; patient no. 18, IgG 2,360 mg/dL; patient no. 20, IgM 666 mg/dL). Type of BMT and hematologic engraftment. Eight patients underwent allo-BMT with anti-T12-depleted BM from normal HLA-identical siblings and 12 patients underwent auto-BMT (Table 4).All patients achieved neutrophil engraftment. After auto-BMT, 3 patients (nos. 14, 17, and 20) received recombinant human granulocyte colony-stimulating factor (rhG-CSF) on day 29 (patient no. 14) and day 20 (patients no. I7 and 20), respectively, with achievement of an ANC 2 500/pL on days 30,25, and 2 1, respectively. For the remaining I7 patients who did not receive rhG-CSF, the median time to an ANC 2500/pL was 22.5 days (range, 14 to 29) for 8 patients after allo-BMT and 20 days (range, 18 to 27) for 9 patients after auto-BMT (Table 4). All patients achieved platelet engraftment. The median time to platelet recovery (220,00O/pL) was 20.5 days (range, 13 to 34) after allo-BMT and 29 days after auto-BMT (range, 16 to 69). Patients were discharged from the hospital at a median of 33 days (range, 26 to 9 1) after allo-BMT and 3 1 days (range, 24 to 122) after auto-BMT (Table 4). Acute and chronic toxicity post-BMT. Eighteen patients developed fever (2101°F) (6 after allo-BMT; all 12 after auto-BMT) and all patients experienced mucositis in association with neutropenia (Table 5). Other acute infectious toxicities included Hickman line infections with coagulasewhich led to septic thrombophlenegative Stuphylococcu~,~ bitis in 1 patient and bacteremia in another, one transient culture negative pneumonitis, and 1 life-threatening case of


BONE MARROW TRANSPLANTATION FOR B-CLL

Table 5. Acute Toxicitv Post-BMT for B-CLL Infectious Noninfectious Type of BMT

No of Patients

Fever

Line

Allo-BMT Auto-BMT

8 12

6 12

1

Bacteremia/ Sepsis

1 1

3

Toxic Deaths

Other'

CHF

Pericarditis

GVHD

VOD

DAHS

0

0

1

2

1

0

3 0

0 0

0 1

Abbreviations: VOD, venocclusive disease; DAHS, diffuse alveolar hemorrhage syndrome; CHF, congestive heart failure. CNS toxoplasmosis (transient pneumonitis)

central nervous system (CNS) toxoplasmosis (Table 5). Acute noninfectious complications included one congestive heart failure and one transient pericarditis, as well as GVHD (2 with grade I and 1 with grade IV) (Table 5). Venocclusive disease was not observed. One acute in-hospital death (day 62; patient no. 12) occurred after auto-BMT secondary to diffuse alveolar hemorrhage syndrome. There was no significant increase in toxicities observed for those patients who had received fludarabine before BMT. Late infectious complications (after discharge) included one Staph epidermidis bacteremia from a Hickman line infection, one bacterial sinusitis, and three Herpes zoster (Table 6). Of interest, all 3 patients who developed bacterial sinusitis had undergone splenectomy before BMT. Late noninfectious complications included three cases of hemolyticuremic syndrome and one chronic GVHD (Table 6). The patient with grade IV chronic GVHD of the skin and gastrointestinal tract (patient no. 1) required immunosuppressive therapy and ultimately succumbed to pneumocystis pneumonia. No significant differences in toxicities were observed between those patients who underwent allo-BMT versus those who underwent auto-BMT. Treatment outcome. Nineteen patients survived for greater than 3 months and were evaluable for response to BMT. Sixteen patients were documented to achieve clinical CR within the first 3 months after BMT and remain in unmaintained CR at last restaging (6 for allo-BMT; 10 for auto-BMT) (median follow-up, 1 1.7 months for allo-BMT, 5.0 months for auto-BMT; range, 2 to 3 1 months) (Table 7). Two patients (nos. 8 and 14) had evidence of persistent BM infiltration on BM biopsy after allo- and auto-BMT, respectively. Both patients remain alive and well untreated 15 months (no. 14) and 6 months (no. 8), respectively, postBMT and continue to have the BM as their only site of disease. One patient (no. 1) was not restaged until 12

months post-BMT, when she was found to be in clinical CR. Patient no. 12 was never restaged to assess whether a clinical CR had been achieved. Of the 5 patients with known cytogenetic abnormalities before BMT, 4 evaluated after BMT reverted to normal. Ten of 16 patients who were documented to be in clinical CR 2 3 months post-BMT have been restaged at various intervals after BMT and continue in clinical CR (Table 7). Of the 10 patients who were hypogammaglobulinemic before BMT, 5 of 7 who were evaluable and in clinical CR were examined at 2 6 months after BMT and found to have a normal serum protein electrophoresis (SPEP). Patient no. 13 has reverted to a normal SPEP greater than 1 year post-BMT (from a monoclonal serum IgM), whereas patient no. 18 continues to show a monoclonal serum protein (2,210 mg/dL) within the first 3 months post-BMT. Follow-up is short for this patient population and, therefore, the long-term impact of achievement of clinical CR after BMT for B-CLL is unknown. The predicted disease-free survival of the combined patient group who have undergone allo-BMT and auto-BMT is shown in Fig 1. At 12 months, the predicted disease-free survival is 82% (95% CI, 64% to 100%). Assessment of CR by phenotypic and molecular biologic analysis. In an attempt to assess more accurately the remission status of these patients, samples were assessed for the presence of CLL cells by phenotypic analysis and for the presence of detectable Ig gene rearrangements by restriction fragment analysis and Southern blotting. At the time of initial evaluation, the CLL cells of all 20 patients were documented to coexpress CD5 and CD20 by dual-color immunofluorescence of BM, PB, or lymph node samples. Similarly, all 20 patients had evidence of monoclonal Ig gene rearrangements at the time of initial evaluation. At the time of BM harvest, all 20 patients had persistent evidence ofdetectable Ig gene rearrangements, including the 3 patients who

Table 6. Late Toxicity Post-BMT for B-CLL Infectious

Toxic Deaths Noninfectious

Type of BMT

No. of Patients

Line

AIIO-BMT Auto-BMT

8 11

0 1

Abbreviation: HUS, hemolytic uremic syndrome. Chronic-skin, GI tract.

Bacteremia1 Sepsis

Bacterial Sinusitis

Herpes Zoster

0

1

2

I*

1

1

1

2

1

0

2

0

GVHD

HUS

Pneumocystis Pneumonia


RABINOWE ET AL

Table 7. Treatment Outcome After BMT for B-CLL Status at Last Staging

Status r 3 mo Post-BMT

Cytogenetic Response

Months Post BMT

Clinical Response

NA Complete Complete Complete Complete Complete Complete PD

NA Normal NA Normal Normal Normal Normal Normal

12 18 11 12 11 12 12 6

Complete Complete Complete Complete Complete Complete Complete PD

Complete Complete Complete NE (died 62 d) Complete PD Complete Complete Complete Complete Complete Complete

Normal Normal NA

31 27 25

Complete Complete Complete

NA Normal Normal Normal NA Normal Normal NA

13 15 6

Complete* PD Complete Too Early Too Early Too Early Too Early Too Early

Clinical

Patient No.

Response

AIIO-BMT 1 2 3 4 5 6 7 8 Auto-BMT 9 10 11 12 13 14 15 16 17 18 19 20

Complete response by clinical staging: CBC, differential, blood-chemistry profile, serum protein immunoelectrophoresis, physical examination, x-rays, CT scans, bilateral BM aspirates and biopsies. Abbreviations: NA, not assessed; PD, persistent disease; NE, not evaluable. * Refused BM

had achieved a clinical CR. However, only 4 of I3 patients assessed had evidence of detectable cells expressing both CD5 and CD20 (data not shown). The results of both dual-color immunofluorescence and Southern blot analysis of BM samples obtained at intervals after allogeneic BMT are shown in Fig 2. All samples analyzed were obtained when the patients were assessed to be in clinical CR. As can be seen, 5 of these 6 patients had detectable residual CLL cells within 6 months of receiving the

d

8

0.4 . 0.2

'

L 0

I

I

I

I

I

I

5

10

15

20

25

30

Months Fig 1. Predicted disease-free survival (DFS) of 20 patients undergoing auto-BMT or allo-BMT for B-CLL.

BMT. After 6 months, no evidence of these residual CLL cells could be detected by dual-color immunofluorescence. Of interest, 4 patients had complete resolution of detectable CLL cells at further follow-up despite the fact that they received no additional therapy. One patient (no. 7) who remains in clinical CR at 12 months post-BMT has persistent residual CLL cells, as assessed by the presence of detectable Ig gene rearrangements. Five patients had no evidence of residual CLL cells by phenotypic or molecular biologic assessment of their most recent BM samples. The results of dual-color immunofluorescence and Southern blot analysis of BM samples obtained at intervals after auto-BMT are shown in Fig 3 . Again, all samples analyzed were obtained when the patients were in clinical CR. Six of these patients (nos. 15 through 20) have been observed for only a very short period and in 3 cases residual CLL cells were detected within 3 months after BMT. In 1 of these cases (no. 1 3 , subsequent analysis documented a return to germline configuration. In the 3 cases in whom longer-term follow-up samples are available for analysis, no residual lymphoma cells were detected by either phenotypic or molecular biologic analysis. The results of dual-color immunofluorescence analysis for CD5 and CD20 of samples obtained from patient no. 9, who underwent auto-BMT, are shown in Fig 4. As can be seen, at the time of initial evaluation (Fig 4A) the majority of cells are CLL cells and coexpress CD5 and CD20. At the time of BM harvest (Fig 4B), a


BONE MARROW TRANSPLANTATION FOR B-CLL

la Gene Rearranae.

CD5 I CD20

CD51CD20

€

1

Fig 2. Assessment of patients for residual disease after alloBMT by dual fluorescence for CD5 and CD20 and Southern blot analysis of BM samples. Those patients who had evidence of CLL cells are representedas ( ). Those samples analyzed that showed no evidence of residual CLL cells are represented as ( ).

+

-

clinical CRs were achieved for those 15 patients who were treated with CHOP induction therapy. Of only 3 patients who attained a clinical CR before BMT, 2 received fludarabine. The CR rates for CHOP and fludarabine in this patient population are certainly lower than has been reported p r e v i o ~ s I y . ' - ~These , ~ ~ , ~lower ~ response rates are likely due both to the poor prognostic features of our patient cohort as well as our aggressive staging, which included CT scan evaluations. In addition, higher response rates might have been achieved if fludarabine had been used more frequently as first-line therapy. Both the time to engraftment and toxicity observed in this study appeared to be comparable to those previously reported for auto-BMT and allo-BMT in patients with other hematologic malignancies treated at our i n s t i t u t i ~ n . * ~ . ~ ~ However, 1 patient who underwent auto-BMT developed CNS toxoplasmosis and survived. CNS toxoplasmosis has not been observed in nearly 500 patients undergoing autoBMT for other hematologic malignancies at Dana-Farber Cancer Institute. Whether the immunosuppressive effects of fludarabine contributed to this event is unknown but certainly is of concern. The majority of complications after allo-BMT were comparable to those seen after auto-BMT. The similar toxicities were due to the fact that only 1 ofthe 8

small but distinct residual population of CLL cells are observed. By 24 months after auto-BMT (Fig 4C), CLL cells coexpressing CD5 and CD20 are no longer detectable. DISCUSSION

CLL is one of the few hematologic malignancies' for which high-dose myeloablative therapy and BMT has played a limited therapeutic role. We report the results of a pilot study in which patients with aggressive poor prognosis B-CLL underwent either allo-BMT or auto-BMT to examine the safety and efficacy of this treatment approach. Twenty patients with poor prognosis B-CLL were treated to a minimal disease state using conventional chemotherapy. Only 3 attained a clinical CR, whereas the remainder attained a good partial remission before BMT. After BMT. all patients fully engrafted and transplant-related mortality was relatively low (10%).After BMT, of the 19 patients evaluable for response, 17 (89%) achieved a clinical CR. Although follow-up is too short to evaluate the impact of BMT on long-term disease-free survival (median follow-up, 1 1.7 months), assessment for residual B-CLL cells in the BM by CD5/CD20 dual-color flow cytometry as well as Ig gene rearrangements shows that most patients evaluated have achieved both phenotypic (100%)and molecular CRs (79%) with this approach. Although B-CLL is traditionally considered to be sensitive to conventional therapy, this was not our experience in the subgroup of poor prognosis patients treated on this study. Most patients were initially treated with CHOP, which usually resulted in cytoreduction of nodal disease but did not result in dramatic marrow response^.^^.^^ In fact, no

CD5 I CD20 la Gene Rearranae.

CD5 I CD20

CD5 I CD20 lg Gene Rearrange.

CD5 I CD20

Fig 3. Assessment of patients for residual disease after autoBMT by dual fluorescence for CD5 and CD20 and Southem blot analysis of B M samples. Those patients who had evidence of CLL cells are represented as ( ). Those samples analyzed that showed no evidence of residual CLL cells are represented as ( -).

+


RABINOWE ET AL

Fig 4. Dual fluorescence (CD5 and CD20) of B M mononuclearcells for patient no. 9 (A) at initial evaluation, (B) at BM harvest, and (C) at 24 months after auto-BMT.

patients who underwent allo-BMT developed clinically significant GVHD (grade 4). We conclude that these toxicities are acceptable and should not preclude further use of this approach for suitable patients with B-CLL. Because it will require several more years of follow-up and significantly larger numbers of patients transplanted before any statement of long-term efficacy can be made, we attempted to determine whether both phenotypic and molecular remissions were achieved in the marrow after alloBMT and auto-BMT. Recently, Robertson et a15have examined the response rates of patients with progressive or symptomatic CLL treated with fludarabine and prednisone. Of note, CT scans were not routinely performed to determine remission status. Response was defined by clinical criteria as well as assessment of residual disease by dual-color flow cytometry for CDS/CD20-positive cells and Ig gene rearrangements. Although most patients in clinical CR (89%) had no residual disease detected by flow cytometry, only 5 1% of those with a nodular marrow CR and 19% of the partial responders had phenotypic remissions. A small subgroup of patients in clinical CR were also examined for Ig gene rearrangements, with a return to the germline configuration observed in the majority (5 of 7 CRs and 2 of 8 nodular CRs). In addition, all patients who reverted to a germline DNA pattern after treatment remained free of disease. Of the 19 evaluable patients post-BMT in this study, 89% attained a clinical CR. Of the 15 patients in clinical CR who were evaluated for phenotypic remission, all lacked evidence of residual disease at follow-up 3 to 24 months post-BMT. Of the 14 patients in clinical CR who were evaluated for molecular remission, 79% had returned to the germline configuration from 3 to 24 months post-BMT. It is important to note that of the 9 patients who underwent auto-BMT and were assessed for phenotypic and/or molecu-

lar CR, only 4 have been observed for more than a 3-month period (range, 6 to 24 months). Nevertheless, these results are encouraging and suggest that both allo-BMT and autoBMT for B-CLL can result in phenotypic and molecular CRs. Clearly, much longer follow-up will be essential to determine whether the attainment of such a CR will lead to prolonged disease-free survival. To date, our results extend the encouraging results of the previously reported European multi-center study.20Transplant-related toxicity was greater in their study, with 5 toxic deaths observed (29%)and all 15 evaluable patients developing GVHD (5 cases being chronic). Fourteen patients received non-T-cell-depleted BM, which probably explains the high incidence of GVHD. Nevertheless, 9 patients remained disease free with a median follow-up of 25.6 months. Our study differed in several respects. Only patients who achieved a minimal disease state after a variety of chemotherapy regimens were eligible for BMT and T-cell depletion of the donor BM was the only method of GVHD prophylaxis. In addition, auto-BMT was performed if an HLA-identical donor was not available. Finally, all patients in this study received a uniform myeloablative regimen. In addition, we have documented both phenotypic and molecular CRs in the majority of patients after both allo-BMT and auto-BMT. Long-term follow-up will be essential to determine whether the CRs that we have observed after BMT for BCLL will translate into long-term disease-free survival and overall survival. Our observation that several patients in whom BMs initially positive for Ig gene rearrangements subsequently returned to the germline configuration suggests that CLL cells may die slowly over time and that there may be a potential role for immune modulation after BMT.Z9It seems clear that CRs can be achieved after both

From bloodjournal.hematologylibrary.org by guest on July 12, 2011. For personal use only. BONE MARROW TRANSPLANTATION FOR B-CLL

allo-BMT and auto-BMT for B-CLL and thus far we have observed no significant differences in either toxicity or treatment outcome between these two approaches. Because we are a tertiary referral center, and therefore evaluate a relatively selected subset of patients with B-CLL, it is difficult to assess how applicable this technique will be to the entire population of patients with poor prognosis disease. However, our experience of patients who were referred to this institution for consideration of this approach has been that virtually every patient who presents with International Workshop stage I1 could be rendered into a protocol eligible minimal disease state and proceed to transplant. In contrast, of those patients with International Workshop stage I11 and IV, less than one-third of patients responded to conventional dose therapy to achieve a protocol eligible minimal disease state and proceed to transplant. Because the nonresponders probably represent the worst prognostic group, it is difficult to determine whether BMT will have any impact on overall survival. Although the present study shows that allo-BMT and auto-BMT are feasible treatment options for selected younger patients with CLL, future studies will be required to delineate the role of BMT in the management of this disease.

ACKNOWLEDGMENT

The authors thank Kerstin Hildebrandt for assistance in preparation of the manuscript as well as Mary Whelan and Andrea Freeman for their expert assistance in data management.

REFERENCES

I. Grever MR, Kopecky KJ, Coltman CA, Files JC, Greenberg BR, Hutton JJ, Talley R, Von Hoff D. Balcerak S P Fludarabine monophosphate: A potentially useful agent in chronic lymphocytic leukemia. Nouv Rev Fr Hematol 30:457, 1988 2. Keating MJ, Kantarjian H, Talpaz M, Redman J, Koller C, Barlogie B, Velasquez W, Plunkett W, Freireich EJ, McCredie KB: Fludarabine: A new agent with major activity against chronic lymphocytic leukemia. Blood 74: 19, I989 3. Keating MJ, Kantarjian H, OBrien S, Koller C, Talpaz M, Schachner J, Childs CC, Freireich EJ, McCredie KB: Fludarabine: A new agent with marked cytoreductive activity in untreated chronic lymphocytic leukemia. J Clin Oncol9:44, 1991 4. Keating MJ: Fludarabine phosphate in the treatment of chronic lymphocytic leukemia. Semin Oncol 17:49, 1990 5. Robertson LE, Huh YO, Butler JJ, Pugh WC, C. H-G, Stass S, Kantarjian H, Keating MJ: Response assessment in chronic lymphocytic leukemia after fludarabine plus prednisone: Clinical, pathologic, immunophenotypic, and molecular analysis. Blood 80:29, 1992 6. Piro LD, Carrera CJ, Beutler E, Carson DA: 2-Chlorodeoxyadenosine: An effective new agent for the treatment ofchronic lymphocytic leukemia. Blood 72: 1069, 1988 7. Saven A, Carrera CJ, Carson DA, Beutler E, Piro L D 2Chlorodeoxyadenosine treatment of refractory chronic lymphocytic leukemia. Leuk Lymphoma 5:133, 1991 8. Lee JS, Dixon DO, Kantarjian HM, Keating MJ, Talpaz M: Prognosis of chronic lymphocytic leukemia: A multivariate regression analysis of 325 untreated patients. Blood 69:929. 1987

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9. Montserrat E, Gomis F, Vallespi T, Rios A, Romero A, Soler J, Alcala A, Morey M, Ferran C, Dib-Mediavilla J, Flores A, Woessner S , Batelle J, Gonzalez-Aza C , Rovira M, Reverter J-C, Rozman C Presenting features and prognosis of chronic lymphocytic leukemia in younger adults. Blood 78: 1545, 1991 IO. Rai KR, Sawitsky A, Cronkite EP, Chanana AD, Levy RN, Pasternack BS: Clinical staging of chronic lymphocytic leukemia. Blood 46:219, 1975 11. Binet JL, Auquier A, Dighiero G, Chastang C, Piguet H, Goasguen J, Vaugier G, Potron G, Colona P, Oberling F, Thomas M, Tchernia G, Jacquillat C, Boivin P, Lesty C, Duault T, Monconduit M, Belabes S, Gremy F A new prognostic classification of chronic lymphocytic leukemia derived from multivariate survival analysis. Cancer 48:198. 1981 12. Montseratt E, Sanchez-Bison0 J, Vinolas N, Rozman C: Lymphocyte doubling time in chronic lymphocytic leukaemia: Analysis of its prognostic significance. Br J Haematol62567, 1986 13. Vinolas N, Reverter JS, Urbano-Ispizua A, et al: Lymphocyte doubling time in chronic lymphocytic leukemia: An update of its prognostic significance. Blood Cells 12:457, 1987 14. Rozman C, Montserrat E, Roriguez-Fernandez JM, Ayats R, Vallespi T, Parody R, Rios A, Prados D, Morey M, Gomis F, Alcala A, Gutierrez M, Maldonado J, Gonzalez C, Giralt M, HemandezNieto L, Cabrera A, Fernandez-Ranada JM: Bone marrow histologic pattern-The best single prognostic parameter in chronic lymphocytic leukemia: A multivariate survival analysis of 329 cases. Blood 64:642, 1984 15. Rozman C, Montserrat E: Bone marrow biopsy in chronic lymphocytic leukemia. Nouv Rev Fr Hematol 30:369, 1988 16. Pangalis GA, Roussou PA, Kittas C, Kokkinou S, Fessas P: B-chronic lymphocytic leukemias. Prognostic implication of bone marrow histology in 120patients. Experience from a single hematology unit. Cancer 59:767, 1987 17. Pangalis GA, Boussiotis VA, Kittas C: B-chronic lymphocytic leukemia. Disease progression in 150 untreated stage A and B patients as predicted by bone marrow pattern. Nouv Rev Fr Hemato1 30:373, 1988 18. Juliusson G, Oscier DG, Fitchett M, Ross FM, Stockdill G. Mackie MJ, Parker AC, Castoldi GL, Cuneo A, Knuutila S, Elonen E, Gahrton G: Prognostic subgroups in B-cell CLL defined by specific chromosomal abnormalities. N Engl J Med 323:720, 1990 19. Juliusson G, Gahrton G: Chromosome aberrations in B-cell chronic lymphocytic leukemia. Pathogenetic and clinical implications. Cancer Genet Cytogenet 45: 143, 1990 20. Michallet M, Corront B, Hollard D, Gratwohl A, Milpied N, Dauriac C, Brunet S, Sober J, Jouet JP, Sperou E, Bourdeau H, Arcese W, Witz F, Moine A, Zwaan FE: Allogeneic bone marrow transplantation in chronic lymphocytic leukemia: I7 cases. Report from the EBMTG. Bone Marrow Transplant 7:275, 1991 2 1. Binet JL, Catovsky D, Chandra P, Dighiero G, Montserrat E, Rai KR, Sawitsky A: Chronic lymphocytic leukemia: Proposals for a revised prognostic staging system. Br J Haematol48:365, 1981 22. Soiffer RJ, Murray C, Mauch P, Anderson KC, Freedman AS, Rabinowe SN, Takvorian T, Robertson MJ, Spector N, Gonin R, Miller KB, Rudders RA, Freeman A, Blake K, Coral F, Nadler LM, Ritz J: Prevention of graft-versus-host disease by selective depletion of CD6-positive T lymphocytes from donor bone marrow. J Clin Oncol 10:119l, 1992 23. Freedman AS, Takvorian T, Anderson KC, Mauch P, Rabinowe SN, Blake K, Yeap B, Soiffer R, Coral F, Heflin L, Ritz J, Nadler LM: Autologous bone marrow transplantation in B-cell non-Hodgkin’s lymphoma: Very low treatment-related mortality in 100 patients in sensitive relapse. J Clin Oncol 8:784, 1990


24. Anderson KC, Soiffer R, DeLage R, Takvorian T, Freedman AS, Rabinowe SN, Nadler LM, Dear K, Heflin L, Mauch P, Ritz J: T-cell-depleted autologous bone marrow transplantation therapy: Analysis of immune deficiency and late complications. Blood 76:235, 1990 25. Melo JV, Catovsky D, Galton DAG The relationship between chronic lymphocytic leukaemia and prolymphocytic leukaemia. I. Clinical and laboratory features of 300 patients and characterization of an intermediate group. Br J Haematol 63:377, 1986 26. Melo JV, Catovsky D, Gregory WM, Galton DAG. The relationship between chronic lymphocytic leukaemia and prolymphocytic leukaemia. IV. Analysis of survival and prognostic features. Br J Haematol65:23, 1987

RABINOWE ET AL

27. French Cooperative Group on Chronic Lymphocytic Leukaemia: Long-term results of the CHOP regimen in stage C chronic lymphocytic leukaemia. Br J Haematol 73:334, 1989 28. French Cooperative Group on Chronic Lymphocytic Leukaemia: Effectiveness of “CHOP” regimen in advanced untreated chronic lymphocytic leukaemia. Lancet 1: 1346, 1986 29. Grossbard ML, Gribben JG, Freedman AS, Rabinowe SN, Eliseo L, Epstein CL, Blattler WA, Nadler LM: Adjuvant therapy with anti-blocked ricin (anti-B4-BR) following autologous bone marrow transplantation (ABMT) for B-cell non-Hodgkin’s Iymphoma (NHL): Phase 1/11 trials. Blood 80:158a, 1992 (abstr, SUPPI 1)


CORRESPONDENCE

FUNCTIONAL IRON DEFICIENCY To the Editor: Brugnara et all have recently shown that the well-established effect of recombinant human erythropoietin in chronic renal failre'-^ also occurs in normal subjects. Therapeutic erythropoietin can stimulate erythropoiesis to the extent that the demand for iron by the proliferating marrow exceeds the rate at which it can be released from intracellularpools. The result of this is that the supply of iron to the developing erythron is limited. This functional iron deficiency" occurs even when there is ample iron in the stores, as reflected by the serum femtin concentration. Functional iron deficiency has been associated with reduced transfemn saturation, but this parameter may be comprised by many factors5 It is most closely reflected by the production of hypochromic red blood cells. The emergence of these cells in the circulation may be uniquely quantified by the Technicon H* System (Miles Diagnostics Inc, Tarrytown, NY). Although it is not clear from the study of Brugnara et a l l what proportion of the cells were hypochromic, we have found that transferrin saturation is markedly and consistently reduced when more than 20% of the cells in the circulation are hypo~hromic.~ Because this condition is the result of a failure of the rate of delivery of iron through the plasma transfemn pool to the proliferating erythroblast,the remedy should concentrate on this area. It is pointless to address the level of iron stores when these are already adequate. We have shown that iron dextran, which is rapidly metabolized to release iron to the transferrin pool> will allow sufficient iron to reach the proliferating erythroblast to abrogate production of hypochromic red blood cells.' The occurrence of functional iron deficiency in patients treated with recombinant human erythropoietin is an important limiting factor in the effectiveness of this expensivetherapy. We suggest that it is more widespreadthan measurements of serum ferritin or mean corpuscular volume might indicate and that its presence should be sought by the quantitation of hypochromia whenever there is an inadequate response to recombinant human erythropoietin.*

Blood, VOI 82,NO 4 (August 15),1993:pp 1377-1380

I. CAVILL Department of Haematology University of Wales College of Medicine Heath Park Cardix Wales I. C. MACDOUGALL Renal Unit St Bartholomews Hospital London, UK REFERENCES

1. Brugnara C, Chambers LA, Malynn E, Goldberg MA, Kruskall MS: Red blood cell regeneration induced by subcutaneous recombinant erythropoietin: Iron-deficient erythropoiesisin iron-replete subjects. Blood 81:956, 1993 2. Eschbach JW, Downing MR, Egrie JC, Browne JK, Adamson TW: USA multicenter clinical trial with recombinant human erythropoietin. Contrib Nephrol 76: 160, 1989 3. Macdougall IC, Hutton RD, Cavill I, Coles GA, Williams JD: Poor response to treatment of renal anaemia with erythropoietin corrected by iron given intravenously. Br Med J 299:157, 1989 4. Van Wyck DB, Stivelman JC, Ruiz J, Kirlin LF, Katz MA, Ogden DA: Iron status studies in patients receiving erythropoietin for dialysis-associatedanemia. Kidney Int 35:7 12, 1989 5. Macdougall IC, Cavill I, Hulme B, Bain B, McGregor E, McKay P, Sanders E, Coles GA, Williams J D Detection of functional iron deficiency during erythropoietin treatment: A new approach. Br Med J 304:225, 1992 6. Kanakakorn K, Cavill I, Jacobs A: The metabolism of intravenously administered iron-dextran. Br J Haematol25:637, 1973 7. Cavill 1, Macdougall IC: Erythropoiesis and iron supply in patients treated with erythropoietin. Erythropoiesis 350, I992 8. Macdougall IC, Berner CM, Wild BJ, Cavill I, Baker LRI, Raine AEG Measurement of red cell hypochromia is a useful indicator of iron status in renal patients. J Am Soc Nephrol3:427, 1992

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PLATELET FUNCTION AND THROMBOXANE SYNTHESIS IN MYELOPROLIFERATIVE DISORDERS To the Editor: We have read with interest the paper of Landolfi et al,’ “Increased Thromboxane Biosynthesis in Patients With Polycythemia Vera: Evidence for Aspirin-Suppressible Platelet Activation In Vivo.” In a recent publication’ on 1 1 patients with chronic myeloproliferative disorders (CMPD), 7 with essential thrombocytosis and 4 with polycytnemia vera, we have reached a similar conclusion (with respect to enhanced thromboxane generation) in our patients. Yet, the mechanism of this abnormality seems to be different from that suggested by Landolfi et al and, consequently, might have important clinical implications. It seems unlikely that in CMPD patients there is a “biochemical selective alteration in cycloxygenase/thromboxane synthetase pathway” as stated by Lan-

Blood, VOI 82,NO 4 (August 15), 1993: pp 1377-1380

dolfi et al, but rather an increase in platelet aggregation inducer (possibly thrombin) that stimulates platelet thromboxane A, (TXA,) synthesis. Pertinent is our observation’ on normal TXB, generation (corrected to a normal platelet concentration: 2.5 X lo8/ mL) in platelet-rich plasma of our patients after stimulation by ADP, collagen, or epinephrine (described also by Landolfi et al), despite a reduced extent of platelet aggregation by these inducers.’ However, this finding could not explain a pronounced increase in serum TXB2 generation (measured in suboptimal conditions, 22”C, and corrected to a normal platelet concentration) 2.9 to 7.1 times higher than that of controls’ and the presence of a potent agonist, possibly thrombin, stimulating platelet TXBz generation was indicated? The latter was also supported by the reported elevated plasma fibrinopeptideA level,3a marker of thrombin genera-

1377


CORRESPONDENCE

ti or^,^ in CMPD patient^.^ Moreover, other investigators have observed even a defective signal transduction through the TXA, receptor in a CMPD patient,’ contrary to the conclusion of Landolfi et al. We agree with Landolfi et a1 that increased TXA, synthesis in CMPD patients may represent an enhanced platelet activation for two reasons. We have obtained a markedly elevated plasma ,8thromboglobulin level (corrected to a normal platelet concentration), a marker of platelet activation,6 in our CMPD patients. In addition, picotamide (a thromboxane synthetase/receptor antagonist) was beneficial in the management of thromboembolic complications and in the reduction of elevated plasma fibrinopeptide A level in CMPD patient^.^ Finally, and in accordance with Landolfi et al, aspirin might be effective in the suppression of enhanced TXA, biosynthesis, but it was ineffective in the management of thrombotic complications (arterial and venous) present in 8 of our 1 1 CMPD patients. It was therefore substituted with success by heparin-coumadin, compatible with our above-mentioned mechanism. MIRIAM ZAHAVI JOSEPH ZAHAVI Ichilov Hospital Tel Aviv. Israel

REFERENCES 1. Landolfi R, Ciabattoni G, Patrignani P, Castellana MAL, Pogliani E, Bizzi B, Patrono C: Increased thromboxane biosynthesis in patients with polycythemia vera: Evidence for aspinn-suppressible platelet activation in vivo. Blood 80: 1965, 1992

2. Zahavi J, Zahavi M, Firsteter E, et al: An abnormal pattern of multiple platelet function abnormalities and increased thromboxane generation in patients with thrombocytosis and thrombotic complications. Eur J Haematol47:326, 199 1

3. Rafanelli D, Grossi A, Vannuchi AM, et a]: The effect ofpicotamide on platelet function in patients with myeloproliferative disorders. Thromb Haemost 63525, 1990 4. Nossel HL, Yudelman I, Canfield RE, et al: Measurement of fibrinopeptide A in human blood. J Lab Clin Invest 54:43, 1974 5. Ushikubi F, Ishikushi T, Nasumiya S, et al: Analysis of the defective signal transduction mechanism through platelet thromboxane A, receptor in a patient with polycythemia vera. Thromb Haemost 67:144, 1992 6. Zahavi J, Kakkar VV: P-Thromboglobulin-A specific marker of in vivo platelet release reaction. Thromb Haemost 44:23, 1980

RESPONSE We are grateful to Drs M. Zahavi and J. Zahavi for their comments. The mechanisms underlying enhanced thromboxane biosynthesis in polycythemia vera’ and essential thrombocythemia’ remain elusive. As we reported, it seems likely that thromboxane is produced in response to in vivo stimuli and that the platelet cycloxygenase/Tx-synthase pathway is involved in their transduction. Unfortunately, the findings of Drs Zahavi do not provide clues for the identification of these stimuli mainly because triggers to platelet activation operating in vivo are unlikely to be detected by ex vivo capacity measurements. As for the role of aspirin in preventing thrombotic complications in this setting, this requires a randomized clinical trial of adequate sample size. One such trial has just started (GISP: Gruppo Italian0 di Studio della Policitemia) with the aim of assessing the long-term efficacy and safety of low-dose aspirin (40 mg/d) in patients with polycythemia vera.

RAFFAELE LANDOLFI Catholic University School of Medicine Rome, Italy CARLO PATRON0 University of Chieti “G. Dhnniinzio” School of Medicine Chieti, Italy REFERENCES 1. Landolfi R, Ciabattoni G, Patrignani P, Castellana MAL,

Pogliani E, Bizzi B, Patrono C : Increased thromboxane biosynthesis in patients with polycythemia vera: Evidence for aspirin-suppressible platelet activation in vivo. Blood 80: 1965, 1992 2. Landolfi R, Rocca B, Ciabattoni G, Cortelazzo S, Barbui T, Tartaglione R, Patrono C: Increased thromboxane biosynthesis in essential thrombocythemia. Clin Res 41 :164A, 1993


CORRESPONDENCE

REMISSION AFTER ERYTHROPOIETIN ADMINISTRATION FOR ERYTHROLEUKEMIA-A To the Editor: Erythropoietin (EPO) has proved useful for the recovery of anemia under the condition of renal insufficiency or myeloma. In vitro colony assay has shown that EPO stimulates the growth and differentiation of burst-forming unit-erythroid and colony-forming uniterythroid colonies of bone marrow progenitor cells.' It has been noted that marrow erythroblasts in erythroleukemia were decreased

CASE STUDY

after transfusion, suggesting that a humoral factor such as EPO in serum may contribute to the regulation of erythroleukemia cells.' However, clinicians have withheld application of EPO in erythroleukemia because this agent may stimulate the proliferation of leukemia cells; on the other hand, it has not yet been shown that EPO is capable in vitro to force differentiation of erythroleukemia cells, as all-trans retinoic acid has been observed to do for promyelocytic leukemia cells.' We present here a case of erythroleukemia who


CORRESPONDENCE

Fig 1. May-Giemsa stain of erythroleukemia bone marrow cells before (A) and after (B) in vitro culture with EPO.

achieved complete remission after it was confirmed in vitro that EPO had brought about differentiation of leukemia cells. A 17-year-old male was admitted to our hospital for anemia. His red blood cell (RBC) count was 266 X IO"/pL hemoglobin (Hb) 9.5 g/dL: reticulocyte count 2.7%: white blood cell (WBC) count 1.700/ pL with 42% neutrophils. 54% lymphocytes, and 4%. monocytes: and platelet count 7.5 x IOJ/pL. Nuclear RBCs were found in 36 of I00 RBCs. The bone marrow aspirate disclosed an erythroid dominant marrow with a nuclear cell count of 206.0OO/pL. with 68% erythroid cells. The percentage ofblastsamongnonerythroid mononuclear cells was 37.8%. The erythroid cells showed a high nuclear/ cytoplasmic ratio and were positive for Periodic acid Schiff stain. A diagnosis of erythroleukemia (M6 in the French-American-British classification) was made and he was subsequently treated with. several chemotherapies. including daunomycin. 6-mercaptopurine.

€PO 12,000 unltslday

1

7 8 9 10 11 Months after Admission

12

Fig 2. The clinical course after EPO administration. (0)Hb concentration; (0)erythroleukemic cells in the bone marrow; ( 0 )nonerythroid blast cells in the bone marrow.

prednisolone. and behenoyl cytosine arabinoside. etc: however, these were not effective. We then investigated the potential ofdifferentiation therapy. The patient's bone marrow aspirates were subjected to Ficoll-Hypaque discontinuous centrifugation and mononuclear cells were obtained. The cells were incubated in 10%fetal calfserum supplemented with Iscove's modified Dulbecco's Medium for 7 days and cultured for another 7 days in the presence of EPO." After EPO addition. the total cell number (0.5 X IO"/mL) started to increase. reached a plateau (3.5 X I Oh/mL)at day 5. and thereafter rapidly decreased. It was noteworthy that the leukemic blasts in culture had almost disappeared and that the erythroblasts and mature RBCs increased by day 7. as shown in the photomicrograph (Fig I ) . These in vitro studies showed that differentiation can he stimulated in erythroleukemia cells by EPO. As the patient was refractory to conventional chemotherapies. we explained his situation and the results of the in vitro study to his family and obtained informed consent to use EPO. EPO was administered subcutaneously at a dose of 12.000 U/d. The patient's clinical course is shown in Fig 2. After 6 days of EPO administration, the Hb value started to increase and the nuclear RBCs had disappeared in the peripheral blood at day 14. Bone marrow aspirate showed a remarkable decrease in megaloblasts and an increase in polychromatic and orthochromatic erythroblasts. By 2 weeks of administration of EPO, the leukemia had gone into remission without any other cytocydal drugs. We decided to continue daily administration of EPO hecause it appeared to be the decisive factor in the patient's general improvement as well as his increase in the Hb. WBC. and platelet count. No donor for a bone marrow transplant could be found with matching HLA. However, 3 months after his first treatment with EPO. the patient developed pancytopenia and showed an increase of unclassified blasts both in peripheral blood and bone marrow. Conventional chemotherapies including cytosine arabinoside and other drugs were ineffective. These blasts were also refractory to EPO addition in vitro. The patient died from cerebral hemorrhage. Erythroleukemia is still one ofthe leukemias with a poor prognosis, because it has a high tendency to deteriorate into acute nonlymphocytic leukemia. Our patient's clinical course strongly suggests that EPO has potential for differentiation therapy of erythroleukemia. As far as we known. this is the first reported case in which EPO


CORRESPONDENCE

ETSU MIYAZAKI YUTAKA KOHGO MICHIAKI HIRAYAMA JOHJI KAWANlSHl JUNJI KAT0 SUM10 SAKAMAKI YOSHIRO NIITSU Department of Internal Medicine, Section 4 , Sapporo Medical Universiiy, School of Medicine Sapporo, Japan

has stimulated differentiation of erythroleukemia cells in vitro as well as substantively improving a patient’s condition. Further clinical studies of this agent seem to be warranted; however, users must take the potential for tumor growth stimulation into account and interrupt the treatment promptly if rapid progression occurs. ACKNOWLEDGMENT

We thank G r i n Brewery Co (Tokyo, Japan) for providing recombinant EPO for the management of this case. REFERENCES 1. Gregory CJ, Eaves A C Three stages oferythropoietic progeni-

tor cell differentiation distinguished by a number of physical and biologic properties. Blood 5 1527, 1978 2. Gabuzda TG, Shute HE, Erslev AJ: Regulation oferythropoiesis in erythroleukemia. Arch Intern Med 123:60, 1969

3. Breitman TR, Selonick SE, Colines SJ: Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc Natl Acad Sci USA 77:2936, 1980 4. Wada H, Suda T, Miura Y, Kajii E, Ikemoto S, Yawata Y: Expression of major blood group antigens on human erythroid cells in a two phase liquid culture system. Blood 75:505. 1990