Value of autologous stem cell transplantation with purged bone marrow as first-line therapy for follicular lymphoma with high tumor burden: A GOELAMS phase II study

Abstract

This prospective phase II study was undertaken to evaluate the efficacy and toxicity of early intensive therapy followed by purged autologous bone marrow transplantation (ABMT) in patients with follicular lymphoma with high tumor burden. All patients received the VCAP regimen (vindesine, cyclophosphamide, doxorubicin and prednisone) as conventional chemotherapy and DHAP as second-line therapy. Twenty-nine consecutive patients were included in the study. Twenty-seven patients were grafted, seven in first complete remission (CR) and 20 in first partial remission (PR). Preparative therapy consisted of cyclophosphamide and total body irradiation (TBI) in all the patients. With a median follow-up of 6 years, the actuarial overall survival is 64% and the actuarial event-free survival is 55%. Two treatment-related early deaths were observed. Eleven patients were informative for serial PCR analysis of minimal residual disease after ABMT: two relapsed, four remained disease-free with PCR positivity and five were disease-free with PCR negativity. These encouraging results lay the basis of future prospective randomized trials comparing autologous stem cell transplantation as front-line treatment with conventional chemotherapy for patients with bad prognostic factors.

Full text

Bone Marrow Transplantation (2000) 26, 971–977
2000 Macmillan Publishers Ltd All rights reserved 0268–3369/00 $15.00
www.nature.com/bmt
Value of autologous stem cell transplantation with purged bone
marrow as first-line therapy for follicular lymphoma with high tumor
burden: a GOELAMS phase II study
Ph Colombat
1
, P Cornillet
2
, E Deconinck
3
, JM Tourani
4
, M Gardembas
5
, M Delain
1
, JF Abgrall
7
,
C Kootz
1
and N Milpied
9
Departments of Hematology,
1
CHU Bretonneau, Tours;
2
CHU Reims, Ho
ˆpital Robert Debre
´, Reims;
3
CHU Besanc
¸on, Ho
ˆpital J
Minjoz, Besanc
¸on;
4
Ho
ˆpital Laennec, Paris;
5
CHU Angers, Angers;
6
CHU Brest, Ho
ˆpital Morvan, Brest; and
7
CHU Nantes, Ho
ˆtel
Dieu, Nantes, France
Summary:
This prospective phase II study was undertaken to
evaluate the efficacy and toxicity of early intensive ther-
apy followed by purged autologous bone marrow trans-
plantation (ABMT) in patients with follicular lym-
phoma with high tumor burden. All patients received
the VCAP regimen (vindesine, cyclophosphamide,
doxorubicin and prednisone) as conventional chemo-
therapy and DHAP as second-line therapy. Twenty-nine
consecutive patients were included in the study. Twenty-
seven patients were grafted, seven in first complete
remission (CR) and 20 in first partial remission (PR).
Preparative therapy consisted of cyclophosphamide and
total body irradiation (TBI) in all the patients. With a
median follow-up of 6 years, the actuarial overall sur-
vival is 64% and the actuarial event-free survival is
55%. Two treatment-related early deaths were
observed. Eleven patients were informative for serial
PCR analysis of minimal residual disease after ABMT:
two relapsed, four remained disease-free with PCR posi-
tivity and five were disease-free with PCR negativity.
These encouraging results lay the basis of future pro-
spective randomized trials comparing autologous stem
cell transplantation as front-line treatment with conven-
tional chemotherapy for patients with bad prognostic
factors. Bone Marrow Transplantation (2000) 26, 971–
977.
Keywords: autologous stem cell transplantation; chemo-
therapy; follicular lymphoma; minimal residual disease;
purging
Follicular lymphomas are B cell non-Hodgkin’s lym-
phomas (NHL) that are derived from follicle center cells.
They are classified as low-grade lymphomas and account
for approximately 15 to 30% of newly diagnosed NHL.
Although such lymphomas are sensitive to a variety of
chemotherapeutic agents or radiotherapy, several trials have
reported no plateau in the freedom from progression curve
Correspondence: Dr Ph Colombat, Department of Haematology, CHU
Bretonneau, 37044 Tours Cedex, France
Received 4 May 2000; accepted 16 July 2000
in advanced-stage low-grade lymphoma. Conventional
approaches have resulted in an average survival of 6–10
years in recent studies.
1,2
For patients with poor prognostic
features, ie the high-risk group in the International Prognos-
tic Index (IPI), high tumor burden, patients with primary
refractory disease or partial remission after front-line
therapy, the median survival is significantly shorter.
Attention has recently been focused on the use of very
intensive therapy supported by autologous stem cell trans-
plantation (ASCT) for patients with intermediate and high-
grade histology treated in relapse
3
or in first-line therapy.
4–
5
There is considerably less experience with transplantation
for relapsing patients with low-grade NHL: the results in
heterogeneous groups suggest that this approach can
achieve 25 to 50% long-term survival, according to the
series.
6–14
Very few findings have been published about the value
of ASCT as first-line therapy for poor-risk patients with
low-grade NHL.
15–18
It has been demonstrated using mol-
ecular biology techniques that minimal residual disease
may be present in the bone marrow and peripheral blood
of patients with follicular lymphoma who are otherwise
considered to be in complete remission (CR).
19
As reported
by Gribben et al,
20
bone marrow purging apparently has an
impact on disease-free survival (DFS) of patients grafted
for low-grade NHL. Patients with negative autografts after
purging for t(14;18) using polymerase chain reaction (PCR)
had significantly better disease-free survival compared with
those transplanted with PCR-positive bone marrow.
We present the long-term results of a phase II pilot study
of 29 patients with follicular NHL with bad prognostic fac-
tors according to the Groupe d’Etude des Lymphomes Fol-
liculaires (GELF) criteria
21
who received myeloablative
therapy and purged bone marrow stem cells as first-line
therapy. Minimal residual disease (MRD) was evaluated in
12 patients who were PCR-positive on diagnosis: (1) to
evaluate the possibility of achieving PCR negativity after
conventional chemotherapy; (2) to evaluate the capacity of
BM purging to obtain PCR-negative transplants; (3) to
evaluate the clinical relevance of PCR analysis after auto-
grafting.

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et al
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Materials and methods
Selection of patients and treatment protocol
Patients were eligible for entrance into this phase II trial if
they were aged 60 years or less, had previously untreated
follicular lymphoma according to Working Formulation
(WF) criteria including: follicular small cleaved cell (WF-
B), follicular mixed small cleaved and large cell (WF-C)
and follicular large cell lymphoma (WF-D). Patients were
required to have a high tumor burden according to the
GELF classification,
21
ie including one of the following cri-
teria: nodal or extranodal disease of more than 7 cm, pres-
ence of B symptoms, considerable splenomegaly, pleural
effusion or ascites, complications such as compression,
increased LDH or
␤
2
microglobulin levels. Informed con-
sent was obtained from all patients.
All patients received the VCAP regimen as first-line ther-
apy combining vindesine 3 mg/m
2
on day 1, cyclophos-
phamide 1500 mg/m
2
on day 2, doxorubicin 80 mg/m
2
on
day 2 and prednisolone 50 mg/m
2
/day on days 1 to 5. After
three cycles every 3 weeks, patients in complete remission
(CR) or in partial remission (PR) went on to bone marrow
harvesting. Patients with minimal response received two to
three courses of DHAP combining cisplatine 100 mg/m
2
on
day 1, cytarabine 4 g/m
2
on day 2 and dexamethasone
40 mg/m
2
on days 1 to 4 as second-line therapy. If a partial
or a complete response were obtained after DHAP, bone
marrow was harvested. After BM harvesting and before the
conditioning regimen, patients received one or two courses
of IMVP16, which combines ifosfamide 1.5 g/m
2
on days
1 to 3, VP16 100 mg/m
2
on days 1 and 3 and methotrexate
30 mg/m
2
on days 1 and 10.
Preparative therapy consisted of cyclophosphamide
(60 mg/kg body weight) infused on each of 2 consecutive
days of radiotherapy. Total body irradiation (TBI) was
administered in fractionated doses (200 cGy) twice daily on
3 consecutive days in all patients.
BM collection, purging and cryopreservation procedures
BM collection was performed in patients in CR or PR after
VCAP or DHAP regimen. Three types of immunological
purging to reduce MRD in the transplants were performed
according to the center:immunological purging with com-
plement in 20 patients, immunological purging with immu-
nobeads in five patients and CD34 positive cell selection
in two patients.
Immunological purging technique with complement: BM
cells were treated with three monoclonal antibodies
(MoAb): CD19/B64, CD20/B B6, CD10/B E3)
(Innotherapy, Besanc
¸on, France) and newborn rabbit
complement (CRTS Besanc
¸on, France).
The MNC obtained after Ficoll centrifugation were
washed twice and adjusted with 4% human albumin to a
concentration of 2 ×10
7
cells/ml. A mixture of CD19 and
CD20 (10
␮
g of each for 1 ×10
7
cells) and CD10 (5
␮
g
for 1 ×10
7
cells) was added and incubated for 30 min at
+4°C, with gentle tilting. Then three cycles were completed
with rabbit complement.
For each cycle, 1 vol complement was added to 2 vol
cells and after incubation for 30 min at 37°C, the mixture
was centrifuged, the supernatant discarded and the cells
resuspended in 4%. human albumin.
Immunomagnetic purging technique: The procedure was
completed using the Max SEP TM system (Baxter Immun-
otherapy Division, Germany) and five Moab (anti CD19,
CD20, CD22, CD23, CD37). After Ficoll, the MNC were
incubated with 1 mg of each MoAb per 1 ×10
8
total
nucleated cells for 30 min at +4°C. Excess MoAb was
removed by washing cells twice in cold buffer. Immuno-
magnetic sheep anti-mouse beads were then added to obtain
a ratio of two beads per cell and incubated for 30 min at
+4°C. Magnetic separation was performed on Max SEP,
and the negative MNC collected were remixed once more
with beads.
CD34-positive selection of BM cells: Selection was perfor-
med according to the manufacturer’s instructions (Ceprate
SC Stem Cell Concentration System, Cellpro, Bothell, WA,
USA). Washed BM cells were incubated with 20
␮
g/ml of
biotinylated anti-CD34 monoclonal antibody. The treated
cells were washed with PBS (phosphate-buffered saline
without Ca
++
or Mg
++
(Cell Pro, Bothell, WA, USA)) to
remove unbound antibodies and processed through the avi-
din-coated column of the Ceprate SC apparatus.
The cryopreserved marrow cells were rapidly thawed and
reinfused within 48 h of the completion of chemotherapy.
Evaluation
All patients were evaluated before treatment by physical
examination, blood chemistry profile, complete blood
count, chest X-ray, thoraco-abdominal CT scan and bone
marrow biopsy. Follow-up restaging was performed after
VCAP and, if necessary, DHAP chemotherapy, every 6
months after transplantation for 2 years post ABMT and
yearly thereafter. CR was defined as the disappearance of
all measurable and evaluable disease. PR was defined as
⬎50% reduction in measurable lesions without the appear-
ance of new lesions or by persistence of BM infiltration.
PCR amplification of Bcl-2/IgH translocation
Blood and bone marrow cells were obtained from patients
and healthy donors after informed consent. Mononuclear
cells were separated by Ficoll–Hypaque density centrifug-
ation (Seromed, Biochrom KG, Berlin, Germany).
DNA was obtained after proteinase K digestion (Merck,
Darmstadt, Germany), phenol-chloroform extraction and
ethanol precipitation or by the salting out procedure as pre-
viously described.
The major breakpoint region of bcl-2 (MBR) was sub-
jected to nested PCR. The primers used to amplify the bcl2-
JH translocation were those previously described by
Gribben et al.
20
The first amplification was performed with
250 ng DNA in a total volume of 50
␮
l containing
150
␮
mol/l dNTPs (Amersham Pharmacia Biotech, Saclay,
France), I ×Taq buffer (10 mmol/l Tris-HCl pH 8.3,
50 mmol/l KCl, 1 mmol/l MgCl
2
), 125 ng of each primer

ABMT in follicular lymphoma
Ph Colombat
et al
973
and 1 U of Taq Polymerase (Life Technologies, Cergy-Pon-
toise, France). The second amplification was performed on
1
␮
l of the first PCR product in the same reaction mixture
but for dNTPs (100
␮
mol/l) and the internal primer (50 ng).
The primers used were as follows: 5′-CAG CCT TGA AAC
ATT GAT GG-3′and 5′-TAT GGT GGT TTG ACC TTT
AG-3′as bcl2 primers, 5′-ACC TGA GGA GAC GGT
GAC C-3′and 5′-ACC AGG GTC CCT TGG CCC CA as
JH consensus primers, for the first and nested PCR, respect-
ively. The amplification mixture was incubated for 7 min
for denaturation at 94°C. The DNA template was amplified
for 35 cycles at 94°C for 45 s then at 60°C for the first or
58°C for the nested PCR for 45 s and at 72°C for 45 s and
finally incubated for a final elongation step for 7 min at
72°C in a thermal cycler 2400 (Perkin Elmer, Applied Bio-
system, Courtaboeuf, France). Ten
␮
l of the second ampli-
fication products were electrophoresed through a 6% acry-
malide gel (BioProbe, Paris, France) stained with ethidium
bromide and visualized by UV light. If necessary, the speci-
ficity of the PCR products was verified by hybridization
with a bcl-2 oligoprobe (5′-GCC TGT TTC AAC GAC CC-
3′) or sequencing.
The absence of amplication of the bcl2/JH hybrid DNA
fragment was ratified if the amplification of a 553 bp frag-
ment of the
␤
-actin gene confirmed the characteristics of
the amplified DNA sample. A negative result was estab-
lished only if the 10
5
dilution in normal DNA of the control
cell line was positive. Positive results were validated only
if the negative controls (healthy donor and tube without
DNA) were unamplified.
Statistical methods
An event was defined as progression, relapse of disease or
death in remission. Event-free survival (EFS) was calcu-
lated from the date of the beginning of treatment until the
occurrence of the event using the Kaplan and Meier
methods.
22
Overall survival time was defined as the time
from inclusion until death from any cause or until last
follow-up evaluation for patients who were still alive.
Results
Patient characteristics
Twenty-nine patients (median age 42 years; range 28–60)
with previously untreated high tumor burden follicular lym-
phomas were included in this study between December
1991 and January 1994. At diagnosis, nine of the 29
patients had follicular small cleaved cell (FSC), 18 had fol-
licular mixed small cleaved and large cell lymphoma (FM)
and two had follicular large cell lymphoma (FLC).
Patient characteristics are summarized in Table 1. All but
one were stage III–IV. Sixteen patients had an abdominal
mass greater than 10 cm.
Response to induction therapy
After the three courses of VCAP, six patients were in CR,
20 patients in PR, two had stable disease and one patient
Bone Marrow Transplantation
Table 1 Patient characteristics
Sex
Male 15
Female 14
Histology
Follicular small cleaved 9
Mixed 18
Large cell 2
Stage
II 1
III 4
IV 24
B symptoms
Yes 4
No 25
ECOG
05
118
⭓26
Abdominal mass ⬎10 cm
Yes 16
No 13
Splenomegaly ⬎15 cm
Yes 5
No 24
LDH level
Normal 19
Increased 9
Not done 1
IPI index
Low 1
Low intermediate 18
High intermediate 8
High 3
had progressed. Three patients received two to three
courses of the DHAP regimen and 15 patients received one
to three courses of IMVP16. Twenty-seven patients were
grafted, seven in CR and 20 in PR. Two patients were not
grafted because of refusal (one patient) or progressive dis-
ease (one patient). These results suggest that this type of
first-line chemotherapy can induce sufficient tumor
response to permit autologous stem cell transplantation in
newly diagnosed FL with high tumor burden.
In vitro treatment of bone marrow
Three different techniques of bone marrow purging were
used to reduce the number of tumor cells as far as possible,
as previously described. The number of mononuclear cells
(MNC) obtained ranged from 1.3 to 5.6 ×10
8
/kg. After the
in vitro treatment, a median of 0.18 ×10
8
/kg (range 0.02–
0.42) of MNC and a median of 1.25 ×10
4
/kg (range 0.11–
7.11) of CFU-GM were obtained.
Clinical toxicity and hematologic recovery after
autologous bone marrow transplantation
Of the 27 patients who were transplanted, there were two
treatment-related early deaths from septicemia (n=1) and
non-documented interstitial pneumonitis (n=1). Eighteen
patients received G-CSF after transplantation.
The median time to engraftment was 19 days for neutro-
phil recovery ⬎0.5 ×10
9
/l (range 10–⬎90) and 25 days for

ABMT in follicular lymphoma
Ph Colombat
et al
974
Bone Marrow Transplantation
platelet recovery ⬎30 ×10
9
/l (range 14–⬎90). Only one
patient had not engrafted 2 months after reinfusion of
treated marrow and an untreated back-up marrow was
used successfully.
Clinical outcome (Figure 1)
Nineteen patients remain alive with a median follow-up of
6 years. As noted earlier, there were two treatment-related
early deaths and one patient with progression after induc-
tion therapy who died 14 months after beginning treatment.
Nine patients relapsed after ABMT and seven of them have
subsequently died (between 13 and 67 months after
diagnosis).
The patient who refused ABMT relapsed 36 months after
initial therapy and is still alive.
The actuarial overall survival (OS) is 64% (95% confi-
dence interval =53–74%; and the actuarial event-free sur-
vival 55% (95% confidence interval =43–66%). At this
time medians for OS and EFS have not yet been reached.
PCR analysis (Figure 2)
Blood and bone marrow samples were available at the time
of initial diagnosis in 23/29 patients. Eleven were negative
for MBR. Twelve patients were therefore available for
sequential PCR analysis.
After induction chemotherapy, only one patient was
negative for the t(14;18) in blood and BM. After in vitro
treatment, two BM samples became PCR-negative and nine
of 11 remained PCR-positive. The technique used for BM
purging for the two patients who became negative was
immunological purging with complement.
Twelve patients were informative for serial analysis of
minimal residual disease after ABMT. One year after
ABMT, six were PCR-positive and five PCR-negative. Of
the six patients who were PCR-positive, one relapsed and
five remained in CR, two with subsequent negativization
of the PCR. Of the five patients who were PCR-negative
one year after ABMT, 1 relapsed after becoming PCR-
positive again and four remained disease-free, three without
PCR-detectable disease.
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
847260483624120
Months
Probability
Overall survival
Event-free survival
Figure 1 Overall survival and event-free survival of follicular lymphoma
patients who received high-dose therapy with autologous purged stem cells
as first line therapy. Median follow up, 56 months.
Discussion
In this report, we present the results of 29 patients entered
in a phase II study testing the value of purged bone marrow
transplantation after high-dose conventional chemotherapy
in newly diagnosed follicular lymphoma with the longest
follow-up yet published. We found an overall survival of
64% and an event-free survival of 53% at 8 years. This
preliminary trial shows that the majority of patients can
achieve partial remission after conventional chemotherapy
and could be grafted.
Very few studies have reported the results of autologous
stem cell transplantation as first-line therapy of follicular
lymphomas.
15–18
Ten patients autografted for consolidation of early
remission were published by Fouillard et al.
15
Marrows
were purged in vitro by mafosfamide at individually
adjusted levels. Eight patients remained in unmaintained
CR 15 to 43 months after ABMT at the time of publication.
Haas et al
17
reported 28 patients grafted with unpurged per-
ipheral blood stem cells and conditioned by total body
irradiation and cyclophosphamide. With a median follow-
up of 15 months, 27 out of the 28 patients grafted in first
remission were alive without relapse. Corradini et al
18
reported 21 follicular lymphomas (eight with transformed
histology) who were grafted after conventional chemo-
therapy, peripheral blood stem cell harvesting (or BM
harvesting if PBSC collection failed) and a conditioning
regimen combining mitoxantrone and high-dose melphalan;
16 of the 21 patients with follicular lymphoma were auto-
grafted and evaluable for clinical results: 14 patients (80%)
were alive in continuous CR at a median follow-up of 19
months. In the largest series published by Freedman et al,
16
83 patients with previously untreated follicular lymphoma
received CHOP induction; for patients in PR or CR, bone
marrow was harvested and BM cells were purged with three
monoclonal antibodies and complement. With a median fol-
low-up of 45 months, disease-free survival and overall sur-
vival were estimated to be 63% and 89% at 3 years.
Our results appear relatively comparable whilst all the
patients in our study had poor prognostic factors according
to the GELF criteria
21
and were probably a subgroup of
patients with a worse prognosis than the patients included
in other studies. The relapse rate that we observed might
have been related to the fact that most patients had a high
tumor burden and only seven were in CR at the time of
BMT. A combination of conditioning regimens using only
pharmacological drugs and involving field radiotherapy
could perhaps improve these results.
Our results appear better in terms of EFS but comparable
in terms of OS if we compare them with the best results
of conventional chemotherapy yet published. Solal-Celigny
et al
21
recently reported the final analysis of a randomized
study where 123 patients with the same inclusion criteria
were treated with a combination of cyclophosphamide,
doxorubicin, teniposide and prednisone (CHVP) and inter-
feron; with a 6 year median follow-up, the median pro-
gression-free survival was 2.9 years and the 5 year overall
survival was 70.5%.
The timing of autologous stem cell transplantation as
first-line or second-line therapy of high-risk follicular lym-

ABMT in follicular lymphoma
Ph Colombat
et al
975
Years post grafting 1 2 3 4 5 6
Patient Diagnosis BM Harvest After BMT
Purging
Pre Post
CR+
CR+
CR+
CR+
CR+
CR+
CR+
CR+
CR+
CR+
1
2
3
4
5
6
7
8
9
10
11
12
Figure 2 Clinical outcome according to sequential PCR analysis: 쐌, represents positive PCR result; 앩, represents negative PCR result; 쎲, indicates relapse.
phoma remains questionable. We recently reported the
results for patients with high tumor burden grafted as first-
line therapy or in relapse.
23
Apostolidis et al
24
evaluated
the survival pattern of patients with FC following recur-
rence after myeloablative therapy and showed that survival
after recurrence was similar to that of a historical control
group who received conventional therapy as initial
treatment.
Caution about possible benefits is also demanded by the
toxicity of the treatment. Two toxic deaths occurred in our
study, but no case of myelodysplasia has occurred to date.
These early and late toxicities have to be taken into account
in the therapeutic approaches to indolent lymphoma.
A major issue when using autologous BM or PBSC is
whether reinfused tumor cells may contribute to relapse.
No randomized study comparing purged vs unpurged BMT
in the treatment of FL has been published to date. The Eur-
opean Marrow and Blood Transplant Registry (EBMTR)
25
has reported that the 5 year OS of patients who received
purged (monoclonal antibody or cyclophosphamide) mar-
row was higher than that of those receiving untreated BM
(54% vs 48%) with no difference in PFS (44% in both
arms).
The Dana Farber Cancer Institute has examined the
impact of the presence of residual neoplastic cells in the
autograft upon DFS.
13,20
In the more recent publication,
13
patients whose BM was negative by PCR for bcl2/IgH
rearrangement after purging experienced longer freedom
from recurrence than those whose BM remained PCR-
positive: the 8 year FFR was 83% for PCR
−
and 14% for
PCR
+
patients.
Although the purging of harvested stem cells appears
important, it appears that the type of stem cells (PBSC/BM)
and the purging technique could play a role. McQuaker et
al
26
and Voso et al
27
showed the lack of t(14;18) PCR-
positive cells in highly purified CD34
+
peripheral blood
stem cells. Although negative selection of BM stem cells
Bone Marrow Transplantation
with monoclonal antibodies and complement or immuno-
magnetic beads seems unable to eradicate the t(14;18)-
bearing clone in most patients,
28,29
the technique of nega-
tive purging appears important. Gribben et al
30
showed that
the addition of a fourth monoclonal antibody increases the
efficiency of complement-mediated lysis and that immuno-
magnetic bead depletion is superior to complement-
mediated purging.
Some studies
31,32
have also evaluated the prognostic
value of molecular monitoring of minimal residual disease
after PB or BM stem cell transplantation as first- or second-
line therapy. All these studies confirmed the prognostic
value of bcl2 PCR negativity post transplantation. These
results were recently confirmed by Lopez-Guillermo et al
33
who showed that patients treated with conventional chemo-
therapy achieved a molecular response during the first year
of treatment and sustained it, had better failure-free survival
than those who either reverted back to positive PCR or who
never achieved a molecular response. In our trial, we con-
firmed the prognostic value of PCR negativity post trans-
plantation and also the possibility of very long complete
remission in some patients who remain PCR-positive, as
previously described in stage I–II localized FL treated by
radiotherapy alone.
34
In conclusion, this phase II trial appears encouraging and
large phase III trials are therefore needed to show the
superiority of purged or unpurged autologous stem cell
transplantation in comparison with conventional chemo-
therapy in the treatment of advanced stage of FL with
poor prognosis.
Acknowledgements
The authors thank Marie-Claude Chamard for expert technical
assistance.

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Ph Colombat
et al
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