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Blood First Edition
prepublished
online
March
27, 2003;
DOI 10.1182/blood-2002-08-2476
Long term remission in mantle cell lymphoma following highdose sequential chemotherapy and in vivo rituximab-purged
stem cell autografting (R-HDS regimen)
Short Title: In vivo rituximab-purging for mantle cell lymphoma
Alessandro M. Gianni*^, Michele Magni*, Maurizio Martelli°, Massimo Di Nicola*,
Carmelo Carlo-Stella*^, Silvana Pilotti*^^, Alessandro Rambaldi°°, Sergio Cortelazzo°°, KatiaPatti**, Guido Parvis*°,
Fabio Benedetti^^, Saveria Capria°, Paolo Corradini°°*, Corrado Tarella***, and Tiziano Barbui°°
*Division of Medical Oncology C - Donazione Cristina Gandini, Istituto Nazionale Tumori, Milan, Italy; ^Chair of
Medical Oncology, University of Milan, Milan, Italy; *^^Experimental Molecular Pathology Unit, Department of
Pathology, Istituto Nazionale Tumori, Milan, Italy; °Department of Cellular Biotechnology and Hematology,
University La Sapienza, Rome; °°Division of Hematology, Ospedali Riuniti, Bergamo; **Division of Hematology,
Ospedale Cervello, Palermo; *°Division of Internal Medicine and Hematology, Ospedale San Luigi, Orbassano, Turin;
^^Institute of Hematology, University of Verona, Verona; ***Department of Hematology, University of Turin, Turin;
and °°*Division of Hematology, Istituto Nazionale Tumori, and Chair of Hematology I, University of Milan.
Supported in part by grant from AIRC and Ministero della Sanità, Italy, ICS 030.1/RF 96.278
Corrisponding Author:
Alessandro M. Gianni, MD,
Division of Medical Oncology C,
Istituto Nazionale Tumori,
via Venezian 1, 20133 Milan, Italy
Phone +390223902532
Fax +390223902678
e-mail: [email protected]
Word Counts
Abstract: 240 words
Text: 4332 words
1
Copyright (c) 2003 American Society of Hematology
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Abstract
Mantle-cell lymphoma (MCL) is rarely cured with standard-dose chemotherapy. From January 1997 and February
2000, 28 previously untreated and younger than 61 years patients with advanced-stage MCL were treated at nine Italian
hematological departments with 3 cycles of standard-dose debulking chemotherapy, followed by a high-dose rituximabsupplemented sequence (R-HDS) including intravenous administration of high-dose cyclophosphamide, high-dose
cytarabine, high-dose melphalan, and high-dose mitoxantrone plus melphalan. Study end points included toxicity,
clinical and molecular response rates, long-term event-free survival (EFS) and overall survival (OS) rates, as well as the
ability to harvest tumor-free peripheral blood stem cells. Optimal amounts of polymerase chain reaction-negative (PCRnegative) CD34+ cells were collected from all 20 informative patients. One patient died of toxicity. All 27 patients
assessable for response achieved a complete response (CR), of which 24 remain in continuous complete remission
(CCR) after a median follow-up of 35 months. Three patients had transient evidence of PCR-detectable disease in the
bone marrow. The OS and EFS rates at 54 months were 89% and 79%, respectively. These results compare with the
42% OS rate and the 18% EFS rate observed in 35 age-matched historical controls treated with standard-dose
chemotherapy at the participating centers. The use of rituximab in combination with high-dose chemotherapy represents
a very effective in vivo purging method. The R-HDS regimen can be safely applied in a multicenter hematology setting,
and leads to long-term EFS and OS in the majority of patients with an otherwise incurable disease.
[email protected]
2
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Introduction
Mantle cell lymphoma is a CD5+, CD20+ malignancy, characterized by a t(11:14) translocation causing overexpression
of cyclin D1 and cell cycle dysregulation1 2. It is now recognized by the Revised European-American Classification of
Lymphoid Neoplasms (REAL) and the World Health Organization (WHO) classification systems for non-Hodgkin’s
lymphomas (NHL)3 4as a distinct clinical entity, which comprises around 5% of all cases of NHLs. Patients with MCL
typically present with disseminated disease which frequently includes bone marrow, blood, and splenic involvement 5.
Complete response to standard chemotherapy regimens is poor (in the order of 50% or less)6 7, with progression
typically occurring within just one year after diagnosis, and an overall survival of only three years6 7.
Patients with MCL are clearly in need of an alternative treatment to conventional chemotherapy, and therefore highdose chemotherapy with autologous hematopoietic stem cell transplantation has been considered for these patients.
While some pilot studies have given encouraging results8
9 10
, long-term follow-up has generally revealed a poor
11
clinical outcome, with no evidence for long-term remission . On the assumption that reinfusion of lymphomacontaminated stem cell harvests might contribute to relapse, attempts have been made to improve remission duration by
purging the stem cell graft of tumor cells both in vitro
12 13
or in vivo14. However, immunologic purging failed to
eradicate PCR-detectable disease in the vast majority of patients, with little evidence of improvement over patients
reinfused with non-purged cells 12
13 14
. In conclusion, available evidence supports the need for more effective purging
methods, as well as for better induction therapy to enhance the complete response rate before autografting tumor-free
hematopoietic stem cells.
In a previous study from our group, six consecutive patients with MCL at diagnosis have been treated with multiple
cycles of high-dose chemotherapy, given in combination with the monoclonal antibody rituximab15. All six patients
achieved a complete response (including a PCR-negative bone marrow status) and yielded PCR-negative peripheral
blood stem cell harvests, thus providing the proof of principle that in vivo purging is feasible, and that the chemoimmunotherapy strategy adopted is effective against this highly resistant lymphoma type. This initial series has been
subsequently implemented with 2 more patients from our Unit, and 20 additional patients from 8 other Italian centers
treated with the same protocol. In the present multicenter study we report results after 54 months follow-up of a
retrospective analysis of the latter 28 patients, who underwent high-dose chemo-immunotherapy and in vivo purged
peripheral blood stem cell autografting for advanced-stage MCL at diagnosis. In contrast with historical controls, a
significant number of patients remain disease free and in molecular remission after completing the program. This
suggests that high-dose chemo-immunotherapy may contribute to improved outcome in selected (i.e. younger than 61
years, and clinically fit) patients with mantle cell lymphoma.
Patients and Methods
Selection of patients
Between January 1997 and February 2000, 28 patients with MCL at diagnosis treated at 9 Italian Hematological
Departments were included in this high-dose sequential chemotherapy study. Eligibility criteria included: written
informed consent; age 60 years or younger; absence of severe organ dysfunction not due to tumor; and no previous viral
infections (hepatitis B or C, or human immunodeficiency virus). All pathological materials were reviewed by a single
pathologist at the Milan Cancer Institute using a combination of morphologic, immunophenotypic (CD20+, CD5+,
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CD23-), cytogenetic [t(11;14)(q13;q32)], and molecular (bcl-1 rearrangement) criteria. Diffuse histology, nodular
histology, and blastic variant was present in 75%, 7%, and 18% of the cases, respectively.
For controls, we reviewed the medical records of all adult patients with MCL who were seen between May 1985 and
December 1999 at the same 9 Centers participating in the present study. Patients were eligible for inclusion in the
control group if they had: a pathological confirmed diagnosis of MCL; age 60 years or younger; and had completed a
full course (at least six cycles) of CHOP or CHOP-like chemotherapy. Two patients, who had received high-dose
myeloablative chemotherapy as second line chemotherapy, were excluded. A total of 35 adult patients met the latter
criteria, and served as historical controls. Diffuse histology was present in 86% of the cases, and blastic variant in 14%,
respectively.
Treatment schedule
The treatment program, including its schema, has been described in detail previously15. As outlined in Table 1, after an
initial standard-dose phase consisting of 2 to 3 cycles of either doxorubicin- or cisplatin-containing chemotherapy, all
patients were assigned to receive a rituximab-supplemented 4-step high-dose sequence (R-HDS) including intravenous
administration of: high-dose cyclophosphamide (7g/m2), high-dose cytarabine (2 g/m2 every 12 hours for 6 consecutive
days), high-dose melphalan (180 mg/m2), and high-dose mitoxantrone plus melphalan (60 and 180 mg/m2,
respectively). Rituximab was infused intravenously for a total of 6 doses, as previously indicated (twice after
cyclophosphamide, twice after cytarabine, and twice after the final myeloablative step).15 Because of medical decision
or patient refusal, yet in the absence of compelling medical contraindications, 4 patients did not complete the tandem
myeloablative transplantation program. Among the latter, 3 patients (no. 11, 13, and 15) received 1 cycle of
mitoxantrone plus melphalan, and 1 patient (no. 20) received 1 cycle of melphalan only.
Leukaphereses were performed during growth-factor expanded mobilization of progenitor cells occurring after
administration of both high-dose cyclophosphamide (median 1 procedure, range 1-3), and high-dose cytarabine (median
1, range 1-3). On the first recovery day after chemotherapy when the CD34+ cell count reached at least 0.02 x 109/L, the
patients received 1 dose of rituximab; 24 hours later, they underwent one or more once-daily leukaphereses until the
target number of CD34+ cells was collected. Mononuclear cells were harvested with use of a novel automated
leukapheresis system (Auto PBSC Spectra; COBE, Lakewood, CO), as previously described.16
To hasten hematopoietic recovery and to reduce hematologic toxicity, each patient received 3 progenitor/stem cell
reinfusions, both after the severely myelotoxic, yet non-myeloablative course of high-dose cytarabine (reinfusion 1, see
Table 1), and after the 2 subsequent myeloablative courses of melphalan and mitoxantrone plus melphalan (reinfusion 2
and 3, respectively). The minimum target dose of CD34+ cells per kg of body weight to be reinfused after each of the 3
autografts were 2 x 106, 3 x 106, and 5 x 106, respectively. The timing and number of CD34+ cell collections, as well as
the choice of the different products to be reinfused, were guided by real-time assessment of circulating progenitor
counts,17 and by results of overnight PCR analysis. Briefly, if the first harvest during the post-cyclophosphamide
recovery phase was both quantitatively adequate (i.e. yielded
10 x 106 CD34+ cells/kg) and PCR-negative, the
harvested cells were used for all 3 subsequent reinfusions, and the additional leukaphereses performed after cytarabine
administration were kept as back-up. If the CD34+ cells harvested after cyclophosphamide were either less than 10 x
106/kg, or PCR-positive, or PCR-unknown (probe unavailable), the post-cyclophosphamide harvested cells were kept as
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back-up, and the post-cytarabine leukapheresed cells were used for reinfusion 2 and 3, irrespective of PCR-status
(negative, positive or unknown). Thus, the goal was to reinfuse, whenever possible, optimal amounts of PCR-negative
cells harvested after cyclophosphamide treatment.
The supportive care given during the entire course of R-HDS therapy (including administration of hematopoietic
growth factors) was described in detail previously,15 with additional measures prompted by the common occurrence of
herpes simplex virus and/or cytomegalovirus (CMV) reactivation. Briefly, starting after the high-dose
cyclophosphamide treatment, all patients received acyclovir or a derivative as prophylaxis, and were monitored weekly
for the presence of CMV pp65-positive white blood cells for a period of 3 months after the final autograft. Patients
becoming positive for CMV pp65 antigen received a course of gancyclovir, with or without foscarnet, as treatment.
Patients with hypogammaglobulinemia received intravenous gammaglobulins.
Patient evaluation and response criteria
Before treatment, all patients were evaluated by physical examination, blood chemistry profile, blood cell counts, chest
x-ray, total body computed tomographic scan, bone marrow aspirate and biopsy, and immunophenotypic analysis of
bone marrow (BM) and peripheral blood (PB) cells. When a molecular probe was available, molecular analysis of
minimal residual disease (MRD) in bone marrow and peripheral blood cells was performed. Additional examinations,
like gallium scan, positron emission tomography, nuclear magnetic resonance or bipedal lymphography were performed
as needed to determine the extent of the disease. Whenever possible, the patients were reevaluated before the start of
each of the four high-dose chemotherapy cycles, and one month after completion of the final high-dose chemotherapy
treatment. Follow-up restaging was scheduled every 6 months after transplantation or as clinically indicated for the first
2 years, and yearly thereafter.
Complete response (CR) and partial response (PR) were defined according to the recommendations of the International
Workshop.18 Molecular complete response was defined as the absence of neoplastic cells within peripheral blood and/or
bone marrow mononuclear cells as demonstrated by semi-nested PCR assay to identify bcl-1/IgH translocations or
clonal IgH rearrangements, respectively (see below). Toxicity was evaluated according to the WHO criteria.
Molecular monitoring of MRD
Molecular monitoring of MRD was accomplished by assessing DNA samples from peripheral blood, bone marrow, and
leukapheresis products with use of either semi-nested PCR amplification of the bcl-1/IgH translocation or semi-nested
amplification of clonal rearrangement of IgH genes, essentially as described by Corradini et al. 14 and Andersen et al. 12
The limit of detection of MRD was reproducible at the level of 105 for both rearrangements. The progenitor cells to be
autografted were processed, cryopreserved, thawed, and reinfused as described previously.19
Statistical analysis
Overall survival (OS) was measured from diagnosis until death. Event was defined as progression, relapse of disease,
death in remission or toxic death. Event-free survival (EFS) was calculated from the day of starting chemotherapy to
date of event, or to date of last known alive and disease-free. OS and EFS curves were calculated according to the
Kaplan and Meier method.20 The log-rank test was used to compare event-free and overall survival duration between
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patient groups. To account for the longer follow-up of the control patients, and to prevent later events in the control
group to negatively influence curve comparisons, all control patients were censored at 54 months. Thus, the 2 additional
relapses and the 8 additional deaths that did occur in the control group at later time points, were not counted in the
event-free and survival analysis, respectively.
Results
Patient characteristics
The characteristics of the 28 R-HDS patients and the 35 controls, are listed in Table 2. The presence of bcl-1/IgH or
clonal IgH rearrangement was detected in 11 and 9, respectively, of the 28 patients, while no molecular probe was
available for the remaining 8 patients (29%), and for all the controls. As expected for a disease which occurs
predominantly in males,21 the female sex was under-represented in both arms. While the age of the study population
was younger (median, 48.5 years) than the typical mantle cell patient population (median slightly above 60 years), it
was superimposable to the age of the historical control group (median, 48.8 years). The majority of patients had stage
IV disease by virtue of morphologic bone marrow involvement, with other extranodal sites in approximately half
patients. The most common extranodal site of disease was the gastrointestinal tract. The presence of molecular and/or
immunophenotypic markers for Cyclin D1 disregulation was documented in 89% of the R-HDS patients, and in 68% of
the controls, respectively. Molecular lymphoma involvement in both bone marrow and peripheral blood was detected
by PCR in all 20 R-HDS patients for whom a molecular probe was available. According to the International Prognostic
Index (IPI),22 36% of patients had 1 risk factor (none had zero factors), 50% had 2 or 3 factors, and 14% had 4 or 5
factors. The corresponding figures in the control group were 17%, 77%, and 6%, respectively. Thus, even if
stratification by IPI is of limited value in MCL, 64% of the R-HDS patients, and 77% of the controls had an
unfavorable presentation, with 2 or more risk factors present. 23 None of the differences in the characteristics of the
patients was statistically significant (Fisher’s exact test).
CD34+ cell harvesting and PCR assessment of leukapheresis products
Figure 1 shows the yield (left panel), and the PCR analysis (right panel) of the CD34+ cells harvested after
cyclophosphamide and cytarabine, respectively. Quantitatively, no significant differences were observed in the total
number of CD34+ cells harvested from the 28 patients after the two treatments, thus confirming the surprising lack of
impairment that a prior and close cyclophosphamide infusion had on post-cytarabine mobilization.15
24
A molecular
assessment on leukapheresed cells was carried out in 17 patients following cyclophosphamide, and in 19 patients
following cytarabine, respectively. Following cyclophosphamide, 4 patients with clonal IgH rearrangement, and 3 with
bcl-1/IgH rearrangement yielded PCR-negative harvests, for an overall 42% of negative cases. Conversely, all 19 postcytarabine leukaphereses analyzed (or 100%) were lymphoma-free as documented by IgH (9 cases) or bcl-1/IgH
rearrangement analysis (10 cases). As already pointed out, a reproducible sensitivity level of 10-5 for both probes was
observed.
Detection of MRD in bone marrow samples
Figure 2 shows the results of PCR analysis for the 20 patients in whom a probe for IgH (9 patients) or a bcl-1/IgH
rearrangement (11 patients) was available for evaluation. The analysis was carried out on bone marrow (BM) samples
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obtained at diagnosis, after standard-dose chemotherapy, after cyclophosphamide, after cytarabine, after the second
autografting (day 0), and at the indicated follow-up times, respectively. Following the final autografting, all but 1
patient (no. 18, filled circle) achieved a molecular remission. At a median observation time for molecular analysis of 21
months (range, 4 to 47), all the 20 informative patients were in clinical and molecular remission. A transient PCRpositivity was documented in 3 patients (no. 3, 5, and 18, respectively).
Toxicity
The high response rate seen with R-HDS was not accompanied by unacceptable toxicity. As expected with high-dose
chemotherapy, all patients had grade 4 hematological adverse events, but in general the safety profile of this regimen
indicates that R-HDS is a feasible treatment for younger patients with MCL. One toxic death was seen, but this was a
cardiac event which occurred in a patients with prior history of tachyarrhythmias, and may not have been related to
study treatment. Reported adverse events of grade 3 or higher are shown in Table 3. Of note, 6 patients (21%),
experienced reactivation of CMV. This otherwise rare event in the autologous stem cell transplantation setting occurred
within the interval between cytarabine infusion and 3 months after the final autograft. In all instances CMV reactivation
promptly responded to anti-CMV therapy (gancyclovir, with or without foscarnet). No cases of secondary
myelodysplasia or acute myelogenous leukemia have been reported to date.
Response and survival
After the final transplantation, all 27 study patients evaluable for tumor response were in clinical complete remission
(100% CR rate). The one remaining patient died of toxicity without evidence of disease (NED) on day +15 post
transplantation, i.e. too early for response evaluation. For 12 patients it was possible to accurately measure tumor
shrinkage, and to asses response at various time points during the treatment, thus helping define the contribution of the
various steps to the final overall outcome. As shown in Figure 3, all phases were followed by a measurable reduction in
tumor size. Despite a dramatic initial tumor shrinkage (half patients experienced a 50% tumor reduction following
standard-dose induction therapy), a NED status was achieved late in the course of the treatment schedule. In fact, the
number of patients who became NED was none after standard-dose chemotherapy, none after high-dose
cyclophosphamide, 6 (or 50%) after high-dose cytarabine, and 6 after the final autografting. Of the 35 patients in the
historical control group treated with CHOP or CHOP-like chemotherapy, only 13 (or 37%) achieved a CR, and 11 a
partial response (PR), for an overall response rate (RR) of 69%. As shown in Table 2, in 57% of the patients lymphoma
cells were detected in the peripheral blood by immophenotypic analysis, with absolute counts of CD20+,CD5+,CD23cells ranging from 10 to as high as 41,000/µL. Of note, during the hematologic recovery following the standard-dose
induction chemotherapy cycles, these patients showed a marked increase in the number of circulating lymphoma cells at
a time when solid tumor masses were shrinking. This chemotherapy-elicited leukemization, already reported by Jacquy
et al.25, was no longer evident after subsequent high-dose chemotherapy cycles.
Of the 28 study patients, there was 1 acute in-hospital treatment-related death from cardiac arrhythmia, and 3 relapses at
9, 13 and 31 months, respectively. Of the 3 patients who relapsed, 2 relapsed in sites of prior disease, and 1 in the
central nervous system only. Two of the relapsed patients have died because of disease progression 14 and 3 months
following relapse, respectively. Twenty-four patients remain in continuous complete remission (CCR) with a median
follow-up for the entire study of 35 months (range, 21 to 54 months). The Kaplan-Meier estimate of the percentage of
patients alive or event-free at 54 months was 89% (95% confidence intervals [CI], 78% to 100%), and 79% (95% [CI],
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58% to 100%), respectively (Figure 4). Of the 35 historical control patients, 11 never obtained a CR and progressed
shortly after completion of CHOP or CHOP-like chemotherapy, 8 relapsed, and 16 remain in CCR with a median
follow-up of 51 months (range, 16 to 54 months). For these patients the 54-month OS rate was 42% (95% [CI], 24% to
59%), and the 54-month EFS was 18% (95% [CI], 5% to 31%), respectively (Figure 4). When the results of the 28
study-group patients were compared with the 35 historical controls, there was a significant advantage for the high-dose
treated patients in terms of both EFS (P<0.0001) and OS (P=0.027). Since patients without molecular, cytogenetic or
immunohistochemical evidence of Cyclin D1 disregulation might have a better prognosis, a subset analysis was carried
out, limited to patients (23 in the R-HDS group, and 19 in the control group) with Cyclin D1 overexpression, and/or
bcl1 rearrangement. The results were hardly distinguishable from the results in the whole group, with a 54-month EFS
rate of 87% vs 26% (P=0.034), and an OS rate of 87% vs 18% (P<0.0001) in favor of the R-HDS arm.
Discussion
Because of its invariably poor outcome, MCL has become a preferred target for new treatment approaches. These
include the use of new drugs, aggressive leukemialike regimens
26
chemo-radioimmunotherapy
10
, and high-dose myeloablative chemotherapy or
followed by either autologous or allogeneic stem cell transplantation, as reviewed by
27
Sweetenham . The main goals of our study were: (i) to confirm the original observation15 that the combination of highdose chemotherapy and rituximab is an efficient in vivo purging strategy in MCL; (ii) to assess in a multicenter setting
the feasibility, toxicity and long-term efficacy of a high-dose sequential treatment which included a tandem
transplantation, as well as the use of rituximab (R-HDS), as induction treatment in a larger series of consecutive patients
with newly diagnosed, advanced-stage MCL.
Eradication of PCR-detectable disease in stem cell products is very difficult to obtain in MCL. The recent finding that
rituximab and combination chemotherapy28 transiently cleared peripheral blood (PB) and the bone marrow (BM) of
detectable tumor cells in 36% of 25 informative patients, has been obtained from the analysis of small samples of BM
and/or PB, and might not predict the ability to obtain negative harvests. In fact, it has been shown that the number of
progenitor cells harvested is directly proportional to lymphoma contamination of mobilized stem cells29 . In the few
published instances in which the molecular analysis was performed on preparative harvests, both ex vivo 12 and in vivo 14
, the purging methods used have generally failed: a 12% success rate has been reported for both. In contrast, the results
in Figure 1 show that all 20 informative patients treated with R-HDS yielded very large amounts of CD34+ cells,
devoid of mantle cell contamination. Of note, this very high eradication rate was obtained in patients starting with an
overt leukemic PB pattern in 57% of the cases, and showing (as observed previously by Jacquy et al. 25), a massive
lymphoma cell mobilization following the initial three courses of conventional chemotherapy. These results confirm
and extend our original observation15 that the combination of high-dose chemotherapy and rituximab represents a very
effective in vivo purging method in this otherwise hard to purge disease. The role of a successful purging in autologous
stem cell transplantation has yet to be established. However, the observation that eradication of PCR-detectable
lymphoma cells from BM harvests was associated with a markedly reduced relapse rate30 , makes the harvest of
uncontaminated products a worthwhile and appealing objective. This is particularly relevant if purging can be achieved
with a simple and highly reproducible in vivo method like the one described here.
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One patient with a prior history of cardiac arrhythmia died suddenly on day 14 after mitoxantrone and melphalan,
following an otherwise uneventful recovery. In all remaining patients, acute toxicity attributable to the treatment was
limited, and mainly consisted of severe but short-term pancytopenia after each high-dose course (see Table 2). The only
acute toxicity that could not be anticipated from our previous experience with similar HDS regimens, was CMV
reactivation, which occurred in 21% of the patients here and very infrequently in the past. This suggests a higher degree
of immunosuppression of the present intensified schedule, in which cytarabine, rituximab and a tandem transplant were
added. Early diagnosis through pp65-positive white blood cell monitoring, and timely treatment with antiviral drugs,
prevented the appearance of signs and symptoms of overt CMV infection in all patients. After a median follow-up of 35
months, no secondary malignancies or myelodysplasias have been observed in this previously untreated study
population. The manageable toxicity of the program, at least in this relatively young patient population, was best
attested by its applicability in this multicenter setting. In fact, 24 of the 28 study patients could complete the program as
scheduled, while 4 patients received only one of the 2 programmed myeloablative courses because of patient refusal or
clinical decision.
Of the 27 patients eligible for response assessment, a 100% CR rate was observed and was durable in 24 patients, with a
projected EFS rate at 4-5 years of 79%. In addition, we observed molecular remission in the BM of all 20 patients for
which a molecular probe was available. Seventeen of these 20 patients have remained
in molecular remission
throughout the period of molecular follow-up, while 3 have shown a transient PCR-positivity (see Figure 2).
An accurate appraisal of the clinical efficacy of this approach is difficult in the absence of a randomized control group.
Moreover, as for other similar intensive regimens, it cannot be safely applied to elderly patients (over 60 years or so),
who are not candidates for this approach. However, the present results are very different from the results invariably
obtained after standard-dose anthracycline-based chemotherapy, with a CR rate less than 50%6 , and a median survival
of only 3 years31 . More recent studies using rituximab in combination with CHOP chemotherapy failed to achieve an
improved CR rate or to prolong the time to progression28. This finding, at variance with aggressive B-cell lymphoma in
the elderly,32 indicates that the addition of rituximab to CHOP is unable to significantly improve the clinical outcome of
this disease. In the present study we have compared the results of R-HDS with a historical control population consisting
of 35 adult patients younger than 61 years who had received a full course of anthracycline-based chemotherapy at the
same Centers participating in the present study. Allowing for the limitations of studies using historical controls, the RHDS regimen was associated with higher CR (100% vs 37%), EFS (79% vs 18%), and OS rates (89% vs 42%),
respectively. These results did not change when only patients with Cyclin D1 expressions and/or bcl1 rearrangement
were compared. Our figures also compare favorably with the results from studies that used high-dose chemotherapy as
first-line treatment in MCL. In general, the best results were reported for studies on limited numbers of patients and
short follow-up,8 33 or on patients selected for having achieved a CR following standard-dose induction chemotherapy34
. Most series analogous to our in terms of long-term follow-up duration (median over 2 years) and without patient
selection according to initial complete response, have shown progression-free survival rates ranging from 40% to 55%,
with overall survival rates of 50% to 88%8 11
35 36
. Our results are very similar to those reported by Khoury et al. 10 for
25 chemotherapy-naive patients who received an aggressive multiagent acute lymphoblastic leukemia-like induction
therapy (Hyper-CVAD and high-dose methotrexate/cytarabine), followed by stem-cell transplantation. After a median
follow-up of 25 months, the 3-year EFS rate of the latter study was 72%, with a 3-year OS rate of 92%. More recently
the same group reported encouraging results on 36 previously untreated MCL patients younger than 66 years, treated
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with rituximab-supplemented Hyper-CVAD, and without stem-cell transplantation if the patients had achieved a CR
with 6 courses of the treatment. The CR rate was 92%, and after a median follow-up of 14 months, the 2-year EFS was
80%, and the 2-year OS rate 87%37 . If confirmed after a longer follow-up, these results could suggest that, when
supplemented with rituximab, an intensive leukemia-like treatment also might be curative in MCL. However, its
applicability, efficacy and toxicity as compared with stem-cell supported approaches would require ad hoc prospective
studies. Today, the reinfusion of hematopoietic progenitors is simply a convenient and effective type of supportive
therapy, increasingly being used also after non-myeloablative treatments (like high-dose cytarabine, as used here). The
absence of autografting should not represent per se a critical issue in decision making.
In conclusion, our regimen achieved complete and durable responses in a very high proportion of patients, thus
confirming in a relatively large population of patients that rituximab-supplemented high-dose chemotherapy has a role
in the initial management of MCL in a relatively young patient population (up to 60 years). We are presently
implementing a phase II multicenter trial to prospectively substantiate these findings, while the precise role of this highdose approach awaits direct comparison with novel, competing strategies26 37.
Acknowledgments
We thank staff at the various INT Units for expert patient care; Marco Milanesi and Paolo Longoni for technical
assistance; Dr Attilio Gabbas, for referring patients with MCL; Dr. Alessandra Cuttica; and Dr Enrica Gamba from
Roche Spa, Milan, Italy.
10
DAY OF CYCLE
CYCLE 1
CYCLE 3*
CYCLE 4*
Cytarabine (2 g/m2 Q12H)
G-CSF (5 &g/kg die)
Rituximab (375 mg/m2)
Reinfusion 1 (32 x 10e6 CD34+/kg)
Apheresis 2
x x x x x x
14
21
28
35
Start Cycle 2
Start Cycle 3
x
x x x x x x x x x x x x
x
¬
Melphalan (180 mg/m2)
G-CSF (5 &g/kg die)
Reinfusion 2 (33 x 10e6 CD34+/kg)
x
Start Cycle 4
x x x x x x x x x x x x
Mitoxantrone (60 mg/m2)
Melphalan (180 mg/m2)
G-CSF (5 &g/kg die)
Rituximab (375 mg/m2)
Reinfusion 3 (35 x 10e6 CD34+/kg)
x
x
x x x x x x x x x x x x
x
x
° If quantitatively adequate (i.e. 310 x 10e6 /kg) and PCR-negative, the post-cyclophosphamide leukapheresed cells (Apheresis 1) were used for all
three subsequent reinfusions. Alternatively, 2 x 10e6 /kg of them were reinfused after cytarabine, while the post-cytarabine harvested cells (Apheresis
2) were used for Reinfusions 2 and 3, respectively.
* Cycles 2, 3 and 4 were started as soon as both hematological and non-hematological toxicities from prior cycle had disappeared (usually on day 21
after Cycle 1 and 2, and on day 28 after Cycle 3).
11
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CYCLE 2*
Cyclophosphamide (7 g/m2)
G-CSF (5 &g/kg die)
Rituximab (375 mg/m2)
Apheresis 1°
Table 1 - HDS Treatment Schema
0
7
x
x x x x x x x x x x x x
x
x
¬
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Parameter
Total No
Sex
male
female
Table 2 - Patient Characteristics
Patients Controls
n (%)
n (%)
28
35
24 (86)
4 (14)
26 (74)
9 (26)
48.5
23-65
48.8
28-60
Molecular rearrangement
bcl1
IgH
Probe not available
11
9
8
ND
ND
35
Cyclin D1 expression
Positive
Negative
Not done
18
3
7
19
9
7
Cyclin D1 and bcl1 status
One or both markers available
Cyclin D1 and/or bcl1 positive
26
23 (89)
28
19 (68)
CD20+/CD5+/CD23- immunophenotype
28 (100) 35 (100)
Ann Arbor Stage III-IV
28 (100) 35 (100)
Age (years), median
range
B symptoms
11(39)
14 (40)
Mass > 10 cm
8 (29)
7 (20)
LDH (abnormal)
10 (36)
9 (26)
10 (36)
14 (50)
4 (14)
6 (17)
27 (77)
2 (6)
IPI
0-1
2-3
4-5
Sites of involvement
No. of nodal sites (median, inclusive of spleen)
Extra-nodal sites (exclusive of BM)
BM involvement (histology)
BM involvement (PCR)
Lymphoma cells in PB (immunophenotype)
Lymphoma cells in PB (PCR)
ND: not done
* 20 out of 20 informative patients
12
5.5
4.4
13 (46) 17 (48)
26 (93) 22 (62)
20 (100*)
ND
16 (57)
ND
20 (100*)
ND
Table 3 - Main Toxicities by Course
Cyclophosphamide
Cytarabine
Overall
4
4
100
100
100
4
8
11
21
21
64
7
17
21
Grade 5
Cardiac
Grade 4
Hematological
100
100
Mucositis
Grade 3
FUO/doc. infection
11
14
Hepatic
4
Neurologic
8
CMV reactivation
13
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Mitoxantrone and
Melphalan
Melphalan
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Figure Legends
Figure 1 – CD34+ cell harvesting and PCR assessment of leukapheresis products. Yield (left panel), and PCR
analysis (right panel) of the CD34+ cells harvested after cyclophosphamide and cytarabine. Each patient underwent the
minimum number of procedures sufficient to complete the therapeutic program. Boxes extend from the 25th to the 75th
percentile, with the horizontal line indicating the median. Whiskers extend from the largest to smallest values.
Figure 2 - Detection of MRD in bone marrow samples. Results of PCR analysis for the 20 patients in whom a probe
for IgH (9 patients) or a bcl-1/IgH rearrangement (11 patients) were available. The analysis was carried out on bone
marrow samples obtained at diagnosis, after standard-dose chemotherapy, after cyclophosphamide, after cytarabine,
after the second autografting (day 0), and at the indicated follow-up times. Black circle: PCR-positive; White circle:
PCR-negative.
Figure 3 - Tumor size reduction after each phase of R-HDS program. Patients were reevaluated before the start of
each of the four high-dose chemotherapy cycles. All phases were followed by a measurable tumor size reduction with
CR in 50% of patients after HD-Ara-C and 50% after the final myeloablative phase(s).
Figure 4 – Overall survival (OS) and event free survival (EFS) of patients treated with R-HDS vs conventional
chemotherapy. The Kaplan-Meier estimate of the percentage of patients alive or event-free at 54 months was 89%
(95% confidence intervals [CI], 78% to 100%), and 79% (95% [CI], 58% to 100%), respectively. For the 35 historical
control patients, 54-month OS rate was 42% (95% [CI], 24% to 59%), and the 54-month EFS was 18% (95% [CI], 5%
to 31%), respectively. There was a significant advantage for the R-HDS treated patients in terms of both EFS
(P<0.0001) and OS (P=0.027).
14
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Figure 1
100
90
80
70
60
50
40
30
20
10
0
100
80
60
40
20
0
15
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Figure 2
UP N
28
27
26
25
24
23
22
18
16
15
12
11
9
8
7
6
5
4
3
2
0
10
20
30
40
MONTHS FROMAUTOGRAFTING
(DAY 0)
16
50
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Figure 3
17
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Figure 4
100
100
80
80
60
R-HDS
60
CONTROLS
40
40
20
20
0
0
0
12
24
36
48
60
0
12
MONTHS FROM BEGINNING
18
24
36
48
60
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Prepublished online March 27, 2003;
doi:10.1182/blood-2002-08-2476
Long-term remission in mantle-cell lymphoma following high-dose
sequential chemotherapy and in vivo rituximab-purged stem cell
autografting (R-HDS regimen)
Alessandro M Gianni, Michele Magni, Maurizio Martelli, Massimo Di Nicola, Carmelo Carlo-Stella,
Silvana Pilotti, Alessandro Rambaldi, Sergio Cortelazzo, Katia Patti, Guido Parvis, Fabio Benedetti,
Saveria Capria, Paolo Corradini, Corrado Tarella and Tiziano Barbui
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