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advanced B cell chronic lymphocytic leukemia. L Sutton1, K ... hematopoietic stem cell collection could be obtained and to study the outcome of ...... chronic lymphocytic leukaemia and B-prolymphocytic leukemia. Br J Haematol 1997; 96: ...
Leukemia (1998) 12, 1699–1707  1998 Stockton Press All rights reserved 0887-6924/98 $12.00 http://www.stockton-press.co.uk/leu

Autologous hematopoietic stem cell transplantation as salvage treatment for advanced B cell chronic lymphocytic leukemia L Sutton1, K Maloum2, H Gonzalez1, H Zouabi1, N Azar3, C Boccaccio3, F Charlotte4, J-M Cosset5, J Gabarre1, V Leblond1, H Merle-Beral2 and J-L Binet1,2 Departments of 1Clinical Hematology, 2Biological Hematology, 3Hemobiology, and 4Pathology, Hoˆpital Pitie´-Salpeˆtrie`re; and 5Department of Radiotherapy, Institut Curie, Paris, France

Given the generally poor outcome of advanced B cell chronic lymphocytic leukemia, experimental approaches are warranted, especially for younger patients in whom classical treatments have failed. We therefore conducted a prospective singlecenter study, using polychemotherapy (ESHAP) to prepare patients for hematopoietic stem cell collection and autologous stem cell transplantation as consolidation therapy. Twenty patients entered the study. An adequate response to ESHAP was obtained in 13 patients, and sufficient stem cells for grafting were obtained in eight of the 12 patients who underwent the collection procedure. Six of these grafted patients are alive in complete clinical remission a median of 30 months after transplantation. It should be noted that we were only able to graft 40% of the patients enrolled in this study, either because a new remission could not be obtained or because not enough hematopoietic stem cells could be collected. This argues for stem cell collection as soon as a first remission is obtained, even if the autograft is done later in the course of the disease. Keywords: B-CLL; advanced disease; autologous transplantation; hematopoietic stem cell

Introduction Hematopoietic stem cell transplantation has been widely used in treating chronic lymphocytic leukemia (CLL), and is currently one of the only approaches yielding immunophenotypic and molecular remission.1–3 However, the use of stem cell transplantation as first-line treatment is debatable, as fludarabine and CHOP give 80% and 77% of responses, respectively, in previously untreated patients and a survival time tending towards 10 years in patients entering clinical remission.4–7 However, standard treatments are usually only palliative, and curative treatments for younger patients are clearly needed.8 The prognosis is worse when the disease relapses or does not respond to first-line therapies. Fludarabine only induces shortlived remission in such patients; in addition, the disease is rapidly life-threatening when it is resistant to this second-line therapy.4,8 Consequently, younger patients who relapse or are refractory after several lines of treatment need a salvage procedure. We conducted a prospective study aimed at inducing remission with the combination of etoposide, cisplatin, highdose cytarabine and high-dose methylprednisolone (ESHAP), as originally proposed by Velasquez et al,9 as it has given interesting results in relapsing and refractory lymphoma. The need for myeloablative treatment for consolidation was suggested by results in other lymphoproliferative disease.10–12 Autologous hematopoietic stem cell transplantation (ASCT) was thus planned for patients lacking an HLA-identical sibling and those over 50 years. Total body irradiation was used as a part of the myeloablative conditioning regimen, given the

Correspondence: L Sutton, Service d’He´matologie Clinique, Hoˆpital Pitie´-Salpeˆtrie`re, 47 boulevard de l’Hoˆpital, 75651 Paris cedex 13, France; Fax: 33 1 42 16 02 49 Received 8 June 1998; accepted 6 August 1998

known radiosensitivity of CLL lymphocytes.13 The aim of the study was to determine whether an adequate response for hematopoietic stem cell collection could be obtained and to study the outcome of autologous transplantation in patients with advanced CLL. Despite the limited number of patients, this study shows that it can be impossible to collect adequate stem cells for autografting in multitreated patients, and argues in favor of stem cell harvest immediately after the first remission is obtained.

Patients and methods

Patient eligibility B-CLL was diagnosed on the basis of classical criteria, using blood cell counts, cytology, immunophenotyping and bone marrow cell morphology. Patients with B-CLL were eligible for the study if they had received at least two different chemotherapy regimens and were refractory to or had relapsed after the last treatment. Binet’s staging and new cytologic, phenotypic and bone marrow biopsy studies were performed at enrolment. Tumor burden was assessed by physical examination and by chest and abdominal-pelvic computed tomography. From September 1992 to January 1997, all consecutive patients aged up to 66 years, and those under 50 lacking an HLA-identical sibling, were offered the protocol. Informed consent was obtained from all the patients. The treatment protocol was reviewed by the scientific committee of the French Cooperative Group on Chronic Lymphocytic Leukemia and approved by the local Institutional Review Board.

Treatment plan Patients were first treated with ESHAP chemotherapy every 4 weeks, combining intravenous infusions of methylprednisolone (500 mg/day) for 5 days, etoposide (40 mg/m2/day) and cisplatin (25 mg/m2/day as a 24-h continuous infusion) for 4 days, and cytarabine (2 g/m2 at the end of the last infusion of cisplatin).9 The response to this treatment was studied after the first three cycles and patients were moved towards hematopoietic stem cell (HSC) collection procedures if they qualified. Additional cycles of ESHAP could be performed to improve the response before collection, and also after collection pending ASCT. Trimethoprim/sulphamethoxazole (160 mg/800 mg per day, three times a week) was administered from the start of the first ESHAP cycle through the ASCT procedure as prophylaxis against Pneumocystis carinii infection.

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Cell collection and processing Patients were eligible for HSC collection when they fulfilled the criteria of partial response (PR) and in addition had less than 4 × 109/1 peripheral blood lymphocytes (PBLy), irrespective of the PBLy phenotype and the bone marrow pattern of infiltration (see below).14 Bone marrow (BM) and/or peripheral blood stem cells (PBSC) were used as appropriate. BM was harvested from the iliac crests and treated in vitro with anti-CD19 and anti-CD20 monoclonal antibodies plus rabbit complement.15 PBSC were mobilized with cyclophosphamide (60 mg/kg/day intravenously on 2 consecutive days) plus filgrastim (5 ␮g/kg/day) subcutaneously until neutrophil recovery and completion of harvesting; or with filgrastim plus molgramostim (5 ␮g/kg/day each); or with filgrastim alone (10 ␮g/kg/day subcutaneously for 6 days), with collection on days 5, 6 and 7.16 Mononuclear cells (MNC) were analyzed for CD19-positive and CD5-CD19 double-positive cells, CD34-positive cells and granulocyte– macrophage colony-forming units (CFU-GM) in the PBSC or post-purge BM, and the cells were then cryopreserved.15,16 CD19-positive cells were also evaluated in pre-purge BM. Patients were eligible for ASCT when CFU-GM and/or CD34positive cell counts reached 5 × 104/kg and 1 × 106/kg, respectively, in BM, or 15 × 104/kg and 2 × 106/kg, respectively, in PBSC, in a single sample, as currently recommended in our institution.

109 lymphocytes per liter of blood, and normal BM (except for a few nodules: CRnod). Phenotypic remission was defined when less than 5% of PBLy were CD5-CD19 double-positive in which case a molecular analysis of immunoglobulin gene rearrangement was performed to detect minimal residual disease.1 The persistence of remission was assessed every 6 months or more frequently if clinically indicated. Hematologic recovery after ASCT was based on daily blood counts, and defined as a neutrophil count of 0.5 × 109/1 on 2 consecutive days and a platelet count of 50 × 109/1 without transfusion. The number of packed red cell and platelet transfusions was recorded.17

Histopathologic studies BM aspirates and posterior iliac crest biopsies were reviewed jointly at the Hematobiology and Hematopathology Departments of Hoˆpital Pitie´-Salpeˆtrie`re. BM biopsies were examined for cellularity, lymphocyte percentages and the intertrabecular pattern of BM infiltration, which was noted as normal (no evidence of lymphoproliferation), nodular (nodules of small mature lymphocytes lacking clear germinal centers), interstitial (replacement of normal hematopoietic tissue by small mature lymphocytes infiltrating through fat, with no distortion of the marrow architecture), mixed (both nodular and interstitial involvement), or diffuse (disruption of marrow architecture by small mature lymphocytes).18,19

Preparatory regimen and HSC infusion Immunophenotyping Fractionated total body irradiation (TBI), (3.3 Gy every 24 h for 3 days), was first administered at a mean dose rate below 0.25 Gy/min, using a linear accelerator; the total dose to the lungs was limited to 8 to 9 Gy. TBI was followed by cyclophosphamide (60 mg/kg/day for 2 days). After 2 days the cryopreserved HSC were rapidly thawed and infused via a central venous line. Filgrastim (5 ␮g/kg/day) was administered from the day after graft reinfusion until neutrophil recovery. Patients were treated in a laminar-flow protected environment until granulocyte recovery. They received oral prophylactic antibiotics for gut decontamination. All blood products were filtered and irradiated. Trimethoprim/sulfamethoxazole was interrupted at the outset of the conditioning regimen and reintroduced 1 month after ASCT for 6 months.

Response evaluation Responses were scored according to the NCI Working Group for CLL criteria, which defined complete remission (CR), partial response (PR), stable disease (SD) and progressive disease (PD), with the addition of the nodular remission (CRnod) group based on the presence of residual lymphoid nodules in BM, as described by Robertson et al.14,17,18 Patients were monitored at the Hematology Department of Hoˆpital Pitie´-Salpeˆtrie`re and the disease was staged after three cycles of ESHAP, at HSC collection, at ASCT, and within 6 months after the graft, by physical examination, chest and abdominal-pelvic computed tomography, complete blood counts and peripheral blood lymphocyte (PBLy) counts, PBLy immunophenotyping, and bone marrow aspiration and biopsy. We thus assessed clinical complete remission (CR) as the absence of detectable disease, together with more than 110 g/l hemoglobin, 1011 platelets, 1.5 × 109 neutrophils and fewer than 4 ×

Immunophenotyping was performed on peripheral blood, BM harvested before and after purging, and PBSC, by flow cytometry with single or dual-color staining on a FACStar device (Becton Dickinson, San Jose, CA, USA). The following monoclonal antibodies were always used: CD19 (B4), CD20 (B1), CD10 (J5), and CD23 (B6) from Coultronics (Margency, France); and CD5 (OK23), anti-kappa, anti-lambda and antiIgM from Dako (Trappes, France).20,21 Residual disease was defined by coexpression of CD5 on CD19-positive B lymphocytes.22 When more than 5% of the total lymphocyte population coexpressed CD19 and CD5, residual disease was diagnosed.1 Purging efficiency was evaluated by the percentage of CD19-positive cells before and after in vitro treatment. The percentage of contaminating CD5-CD19 double-positive cells and the quantity of CD34-positive cells among MNC were calculated before freezing.

Immunoglobulin gene rearrangements Complementary determining region 3 (CDR3) PCR amplification was first performed by using consensus framework 3 (FW3) and JH regions as 5⬘ and 3⬘ primers, respectively, as previously described.22 PCR products from a normal polyclonal population of B lymphocytes should appear as a smear on the ethidium bromide-stained acrylamide gel (defined as CDR3 molecular remission). Amplified DNA from cells containing an abnormal B cell population formed a single tight band in the size range of 100–130 base pairs. This method is capable of detecting tumor cell proportion exceeding 1%.22 In addition, each patient’s CDR3 sequence was determined and a CDR3 clone-specific probe was synthesized. Using the patient’s previously determined leukemic cell immunoglob-

ASCT in advanced B-CLL L Sutton et al

ulin VH family as 5⬘ primer and the clone-specific CDR3 probe in 3⬘, specific PCR amplification was performed (in case of a CDR3 molecular remission) and allowed the detection of about one clonal cell in 105 cells.23,24 A negative result defined clone-specific molecular remission.

Statistical methods Survival data were updated on January 1998. Kaplan–Meier overall survival and relapse-free survival curves were calculated from the dates of inclusion and ASCT, respectively.25 The end-point taken for relapse-free survival was the date of clinical relapse. Results

Patients From September 1992 to January 1997, 20 consecutive patients with repeatedly relapsing or refractory CLL were enroled in this prospective single-center trial. There were 15 male and five female patients. All the patients had CD5-positive B-CLL diagnosed on morphologic and phenotypic grounds, except for UPN 4, whose leukemic cells were CD5negative at diagnosis. The median time from diagnosis to inclusion in the trial was 4.5 years (range 0.7 to 9 years). At inclusion the median age was 55 years (range 38 to 66 years). Bone marrow morphology showed a diffuse pattern of involvement in 11 patients, mixed in four, interstitial in one and nodular in four; 11 patients were in Binet’s stage B and nine in Binet’s stage C, with a high tumor burden. All the patients had received at least two different regimens; all had received a purine analog (mean 5.6 cycles, range 2–14) and 18 had received a doxorubicin-containing regimen (mean 7.2 cycles, range 2–12) (Table 1).

Response to ESHAP (Table 2) Complete clinical responses (CR) and partial responses (PR) were obtained in 10 and three patients, respectively, after a median of four cycles of ESHAP (range 3 to 6). However, in the 10 patients who entered complete remission after ESHAP, the malignant clone remained detectable in both PCR methods. One (UPN 6) of these 13 patients died of bleeding while in CR, 5 months after entering the study. Among the five patients who did not respond to ESHAP (UPN 3, 5, 9, 12 and 15), four died of disease progression (associated with an infection in one) at 3, 4, 12 and 16 months, and one patient (UPN 12) is alive at 40 months after 4-Gy hemibody radiotherapy followed by fludarabine (Famp) plus cyclophosphamide (CPM).26,27 The last two patients (UPN 2 and UPN 8) were not assessable for the response because one died in aplasia of intra-alveolar hemorrhage after the second cycle of ESHAP and the other died of sepsis, at 2 months and 1 month, respectively. Four patients had severe infections: UPN 7, who experienced severe and unusual cytopenia after each cycle of ESHAP, had Listeria monocytogenes meningitis occurring after the fourth cycle of ESHAP; it was cured by antibiotics but delayed the next two cycles; HSC collection and ASCT were delayed too at 14 and 18 months from inclusion, respectively. UPN 8, who responded to the first cycle of ESHAP, died at 1 month of Candida septicemia, in aplasia after the second

cycle. UPN 9 died of disseminated herpes virus type 1 infection and disease progression at 4 months. UPN 10 had M. tuberculosis meningitis that resolved on specific antibiotic therapy; HSC collection was possible but further treatments had to be delayed and she died of disease progression at 12 months. The hematologic toxicity of chemotherapy was difficult to distinguish from cytopenia related to the disease in resistant patients, but we observed a gradual recovery of blood counts over successive cycles in responding patients (data not shown).

HSC collection and ASCT BM was harvested once from seven patients and twice from two patients. Seven BM graft were purged (Table 3). The amount of CD34 cells or CFU-GM was too small for grafting in four cases (UPN 11, 16, 17 and 19). Further PBSC collections were only successful in UPN 11. UPN 17 underwent a total of six collections (two BM, four PBSC), all of which were unsuccessful. Three patients (UPN 7, 10 and 20) had PBSC collection alone, which was successful in UPN 7 and UPN 20. UPN 16 and UPN 17 could not be grafted and were later treated with the CPM/Famp combination when their disease progressed.27 The percentage of contaminating CD5-CD19 positive cells in the graft, the number of CD34 cells recovered, and CFU-GM growth are shown in Table 3. In total, sufficient stem cells for autografting were only obtained in eight of the 12 patients who underwent the procedure. We were able to graft these eight patients, after a median of two more cycles of ESHAP (range 1–3). The median time from inclusion in the trial to the ASCT procedure was 8 months (range 5.5–18 months), only one patient being grafted after more than 9 months (Table 4). Four patients (UPN 1, 4, 13 and 14) were grafted with purged BM; three patients (UPN 7, 11 and 20) received unselected PBSC; and UPN 18 received both purged and unpurged BM. We used the same conditioning regimen for the eight patients, and G-CSF (5 ␮g/kg) was routinely added on day 1 after ASCT. The median time required for peripheral blood neutrophil recovery was 15 days (range 11– 28 days). Platelet recovery took 40 days (range 18–60 days), with the exception of UPN 4, who did not recover before relapsing. Except for UPN 4, the patients received an average of 6.6 packed red cell transfusions (range 2–13) and eight platelet transfusions from single donors (range 6–16). None had life-threatening complications after ASCT.

Outcome of ASCT All eight grafted patients were in CR after ESHAP chemotherapy, ie before the graft. However, only three had a normal BM morphology, one had a phenotypic remission (⬍5% CD5CD19 double-positive blood lymphocytes) and none had CDR3 molecular remission (Tables 2 and 5). Within 6 months after ASCT, BM remission was obtained in all the patients (one nodular CR), and all the patients but one (UPN 18) entered phenotypic remission in blood (Table 4). Furthermore, three patients (UPN 7, 13 and 14) entered CDR3 molecular remission. UPN 7 was also in clone-specific molecular remission at 6 months, but not at 24 months. Another patient (UPN 1) later entered both CDR3 and clone-specific molecular remissions, which persisted 42 months after ASCT. Two patients (UPN 4 and 11) never entered CDR3 molecular remission and had a clinical relapse at 8 and 19 months,

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Table 1

Patient’s status at inclusion in the study

Patient (age years)

Time from dg to inclusion (years)

UPN 1 (58) UPN 2 (62)

0.7

UPN 3 (66)

9

UPN 4 (55)

8

UPN 5 (45)

6

UPN 6 (54)

6

UPN 7 (56)

6

UPN 8 (59)

4.5

UPN 9 (55)

5

UPN 10 (46)

7

UPN 11 (56)

7.5

UPN 12 (66)

4

UPN (54) UPN (57) UPN (45) UPN (48) UPN (51)

13

8

14

4.5

15

1

16

3

17

4.5

UPN 18 (63) UPN 19 (50) UPN 20 (38)

8.5

2

1 1

Treatments before inclusion (No of cycles) Famp (3) CAP (2) CAP (6) mCAP (5) Famp (3) Clb (6 y) CHOP (6) Famp (6) clb pdn (12) COP (11) Clb (5y) Famp (6) clb pdn (12) CHOP (6) Clb (3 y) COP (3) Famp (3) Clb (2 y) Famp (12) CAP (3) CHOP (12) MIME (5) Famp (2) Clb (2 y) COP (6) CAP (3) Famp (12) 2cda (2) Clb (3 y) CHOP (8) Famp (3) 2cda (1) Clb (4 y) Famp (7) Clb. pdn (5) 2cda (2) Clb (6 y) Clb pdn (4) CHOP (3) Famp (6) CHOP (6) Clb pdn (4) Famp (6) CHOP (12) Famp (6) CHOP (12) Famp (6) CHOP (6) Famp (3) CHOP (8) Famp (6) CAP (6) mCAP (6) Famp (6) 2 cda (3) CAP (6) CHOP (6) Famp (4) Famp (3) CHOP (4) CHOP* (3)

Binet’s stage

BM involvement (% of Ly)

B

nodular (31) diffuse (NE)

C C

diffuse (NE)

B

mixed (74)

C

diffuse (90)

C

diffuse (88)

C

diffuse (80)

C

mixed (NE)

C

diffuse (100)

C

diffuse (99)

B

mixed (58)

B

diffuse (88)

B

interst (20) diffuse (76) diffuse (91) nodular (39) nodular (33)

B B C B B B B

nodular (10) diffuse (80) mixed (NE)

BM, bone marrow; CAP, cyclophosphamide-doxorubicin (50 mg/m2) – prednisone; 2cda, 2-chloro-deoxy-adenosine; CHOP, cyclophosphamide-doxorubicin (25 mg/m2) vincristine-prednisone; CHOP*, CHOP with doxorubicin (50 mg/m2); clb, chlorambucil; COP, CHOP without doxorubicin; dg, diagnosis; Famp, fludarabin; interst, interstitial; Ly, lymphocytes; mCAP, miniCAP (doxorubicine 25 mg/m2); MIME, guanylhydrazone dichlorohydrate-ifosfamide-methotrexate-etoposide; pdn, prednisone; NE, not evaluated.

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Response to ESHAP treatment

Patient

No. cycles to response

Clin status

BM involvement (% of Ly)

Follow-up from inclusion (⬎, alive) (months)

Clinical status at follow-up (cause of death)

UPN 1

3

CR

⬎63

CR

UPN 2 UPN 3 UPN 4

2 2 4

NE prog CR

2 3 18

NE (intra-alv Hrg) prog (dis) Rel (dis)

UPN 5

9

prog

16

prog (dis)

UPN 6

5

CR

5

CR (Hrg)

UPN 7

6

CR

UPN 8 UPN 9 UPN 10

2 4 3

NE prog PR

UPN 11

4

CR

UPN 12

5

prog

UPN 13

3

CR

UPN 14

3

CR

UPN 15

4

prog

UPN 16

5

PR

UPN 17

4

PR

UPN 18

3

CR

UPN 19

5

CR

UPN 20

5

CR

none (8) ND ND nodular (11) diffuse (100) nodular (NE) none (37) ND ND diffuse (81) nodular (12) diffuse (NE) nodular (15) nodular (20) diffuse (92) nodular (16) mixed (NE) none (4) nodular (10) nodular (40)

⬎53

CR

1 4 12

NE (sepsis) prog (sepsis) prog (dis)

⬎43

Rel

⬎40

prog

⬎37

CR

⬎31

CR

12

prog (dis)

⬎30

prog

⬎27

prog

⬎24

CR

⬎17

CR

⬎10

Rel

clin, clinical; CR, clinical complete remission; dis, disease; Hrg, hemorrhage; intra-alv Hrg, intra-alveolar hemorrhage; ND, not done; PR, partial remission; prog, progression; Rel, relapse.

respectively, after the blood phenotype had again become positive. UPN 4 died at 11 months from progression, and UPN 11, who entered a second CR with the Famp and CPM combination, was alive 35 months after ASCT.27 It is too early to assess the molecular status of UPN 20. The median follow-up of the 11 survivors in 36 months after inclusion in the trial. The Kaplan–Meier (KM) overall survival rate is 53% ± 22% (95% CI), 36 months after inclusion in the study (Figure 1). The KM relapse-free survival rate is 69% ± 36%, 30 months after ASCT (Figure 2). Discussion The lack of curative therapy for CLL, coupled with the potentially indolent course of the disease and the advanced age of many patients make symptom relief and prolongation of survival reasonable therapeutic goals in most patients requiring therapy. However, experimental approaches aimed at achieving a cure are warranted for younger patients with poor-risk factors.28,29 Moreover, patients in failure or relapse after firstline therapy have a very short survival time, with a median of 29 or 9 months according to whether or not they respond to a second treatment with fludarabine.4 Here we discuss the

results of ESHAP, the difficulty of collecting HSC in this series, and the good results of autografting in general. We used ESHAP to induce remission in such patients.9 Our results are comparable to those obtained with fludarabine by Keating et al,4 as we obtained a 65% response rate among our 20 heavily pretreated patients. Four of the five nonresponders died of disease progression within 16 months, confirming the poor outcome of refractory CLL.6 In addition, another three patients (15%) died of toxicity (one in response and two before evaluation). All but one of the 20 patients had previously been treated with fludarabine and four (20%) had life-threatening opportunistic infections. We gave all our patients trimethoprim/sulfamethoxazole prophylaxis against Pneumocystis carinii and observed no cases of this infection. This marked toxicity was partly offset by the fact that these patients had highly advanced disease and were heavily pretreated. Forthcoming strategies using other drugs in combination with fludarabine, or/and monoclonal antibodies against B cells (eg Campath-1H or anti-CD20) should increase the response rate in advanced B-CLL relative to ESHAP.8,30–32 HSC collection failed in four patients. This failure is probably due to the cumulative marrow toxicity of previous treatments. The difficulty of collecting HSC after more than two lines of treatment illustrates the need to perform the collection

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Table 3

Patient

UPN UPN UPN UPN UPN

HSC collection procedure when response to ESHAP

Type of HSCC

Mobilization procedure

Ex vivo B cell depletion

BM BM PBSC PBSC BM PBSC BM BM BM PBSC BM BM PBSC PBSC PBSC BM BM BM PBSC PBSC

— — CPM + G.CSF CPM + G.CSF — G.CSF — — — G.CSF — — CPM + G.CSF G.CSF + GM.CSF G.CSF —

+ +

1 4 7 10 11

UPN 13 UPN 14 UPN 16 UPN 17

UPN 18 UPN 19 UPN 20

% Frozen CD5 Frozen CFU-GM CD19 cells ×104/kg 0 0 6 65 2 30 0 0 12 4 4 35 25 22 30 0 9 ND ND 1

+ + + +

+

— CPM + G.CSF CPM + G.CSF

Frozen CD34 ×106/kg

27.5 14 19.5 0.3 4.6 45 11 14.7 3.5 1.6 2.5 0.9 2.8 4.2 1.25 2 1.5 1.4 3 28.5

1.3 1 2.25 0.8 ND 1.5 ND 1.5 ND 0.4 0.7 0.25 1 1.3 0.4 1.8 1.8 ND 0.9 5.6

CFU-GM, colony-forming unit granulocyte–macrophage; CPM, cyclophosphamide (120 mg/kg); G-CSF, granulocyte colony-stimulatingfactor; GM-CSF, granulocyte–macrophage colony-stimulating-factor; HSCC, hematopoı¨etic stem cell collection; MNC, mononuclear cells; PBSC, peripheral blood stem cells.

Table 4

Patient

UPN UPN UPN UPN UPN UPN UPN UPN

1 4 7 11 13 14 18 20a

Follow-up post-ASCT

Time from No. ESHAP from inclusion to HSCC to ASCT ASCT (months) 7 7 18 8 5.5 7 8 9

3 2 1 2 2 2 3 1

Status at 6 months

BM involvement at 6 months (%)

PB Ly at 6 months

CR CR CR CR CR CR CR CR

none (4) none (NE) none (22) none (7) none (8) nodular (NE) none (10) none (NE)

0.7 1.2 1.8 1.9 1.4 0.85 0.9 2.4

% db pos Ly Relapse Last follow-up CD5 CD19 (months) from ASCT (CD19 CD23) (⬎, alive) 3 (4) 2 (1) 2 (3) 4 (3) 1 (1) 3 (3) 7 (4) 2.5 (2)

no 8 no 19 no no no no

⬎55 11 ⬎35 ⬎35 ⬎31 ⬎28 ⬎16 ⬎2

a Evaluation at 2 months. ASCT, autologous stem cell transplantation; PB Ly, peripheral blood lymphocyte; db pos Ly, double positive lymphocyte.

in first remission and either to await disease progression before autografting, or to consolidate the good results of fludarabine by immediate autografting. In our study only eight patients were able to undergo ASCT. As the median age of our patients was high, we chose to reduce the fractionated TBI dose to 10 Gy in every case, principally to avoid pulmonary complications. All eight grafted patients were in clinical CR 6 months after ASCT. Two relapsed and the other six remain in CR a median of 29.5 months (range 2–55 months) after ASCT. Four are in CDR3 molecular remission and two entered clonespecific molecular remission (Table 5). The two relapsing patients never entered molecular remission. This suggests that UPN 18 is likely to relapse, as his PBLy phenotype remains abnormal at 13 months. Other teams have reported larger series of autografted patients with similar good results. Rabinowe et al2 obtained clinical CR at 3 months in 83% of 12 patients, which persisted for between 6 and 31 months in five cases. In Provan’s series of 21 patients,3 80% of the grafted patients remained in PCR-negative remission after between 7 months and more than 5 years; the absence of molecular remission at

any time after ASCT regularly led to relapse. Ten of the 11 patients reported by Khouri et al1 were in clinical CR 1 month after ASCT. Only three remained in CR between 4 and 29 months; three died of Richter’s syndrome and two from toxicity; three were still alive but in relapse or partial response at 17, 15 and 2 months. We also discovered a case of Richter’s syndrome on a post-mortem analysis of lymph nodes in the patient who relapsed 8 months after ASCT. It is important to note that no deaths were attributed to the autograft procedure itself. This good tolerability confirms previous reports and contrasts with the high rate of transplant-related mortality in allografting.33 A prospective study is required to determine the value of BM purging in vitro. In our series, the outcome of the patients seemed to be related more to the quality of the response before the graft than to the type of reinjected HSC. In the series of Provan et al,3 all BM grafts were purged in vitro using a panel of anti-B monoclonal antibodies and rabbit complement, but the post-autograft remission status did not depend on the efficiency of the purge, as assessed by immunoglobulin

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Figure 1

Kaplan–Meier estimate of overall survival in the 20 patients, from inclusion in the trial.

Figure 2

Kaplan–Meier estimate of relapse-free survival in the eight autografted patients, from the date of transplantation.

gene rearrangement PCR analysis. The study reported by Khouri et al1 does not support the use of BM purging either. The good results reported by Provan et al,3 which are confirmed in our series, raise the possibility that both teams selected good-prognosis patients for ASCT, whose outcome would have been favorable anyway. Effectively, in our study, seven of the eight autografted patients remained in Binet’s stage B at inclusion, as opposed to only four of the 12 non-grafted patients. However, irrespective of Binet’s status, all four patients who remained alive after responding to ESHAP but failing HSC collection had further disease progression, compared to only two of the eight grafted patients. Furthermore, the median follow-up of our autografted patients is more than 29 months, whereas heavily treated refractory patients usually die within a year.4 Comparable survival was reported by Rabi-

nowe2 and Provan,3 but their patients seemed to have a shorter disease history before the graft. Anyway, ASCT is the only strategy (except for allogeneic marrow transplantation) giving a high rate of molecular remission in CLL. This is the only prospective study of ASCT in 20 consecutive patients with CLL based on an intention-to-treat analysis. It stresses the difficulty of HSC collection and ASCT in such heavily pretreated patients, as only 40% underwent both procedures. As only stem cell grafting can yield molecular remissions, HSC collection and ASCT should perhaps be done earlier in the disease. In our opinion, patients under 65 years who have a first response to conventional treatment should undergo stem cell collection and should be randomized to ASCT or classical maintenance therapy in a prospective trial. In case of relapse or refractory disease after a first-line treat-

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Table 5

Peripheral blood biological remission study post-ASCT

ment, HSC collection, rapidly followed by ASCT should be proposed next to second-line chemotherapy. Such a trial could also assess the potential benefit of BM purging or nonleukemic PBSC selection.

6 7

Acknowledgements We thank David Young for checking the English and Florence Laffere for typing the manuscript. 8

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