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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Departments of Hematology and Pathology and
the Central Hematology Laboratory, University Medical Center Nijmegen,
The Netherlands.
In follicular lymphoma, the t(14;18) status of the peripheral blood
and bone marrow analyzed by polymerase chain reaction (PCR) is assumed
to correlate with disease activity in patients with relapsed disease.
The clinical significance of quantitating circulating lymphoma cells by
real-time PCR is reported in patients on first-line treatment.
Thirty-four consecutive patients with previously untreated follicular
lymphoma and detectable t(14;18)-positive cells in pretreatment
peripheral blood samples were monitored. All patients were treated with
standard chemotherapy in combination with interferon alfa-2b. Before
and after induction therapy, blood samples were taken for quantitative
analysis of t(14;18). At presentation, a median of 262 t(14;18)-positive cells per 75 000 normal cells was found (range,
1-75 000). Patients with lower numbers of circulating tumor cells more
frequently had bulky disease (P = .02). Seventy-nine percent of the patients responded clinically to treatment. In 22 of 28 patients, including 4 patients in whom treatment had failed clinically,
the number of circulating t(14;18)-positive cells decreased to
undetectable or low levels after therapy. In the remaining responding
patients, circulating tumor cells persisted after therapy. These
quantitative data on circulating t(14;18)-positive cells call into
question the usefulness of molecular monitoring of the blood in a group
of patients with follicular lymphoma uniformly treated with a
noncurative first-line regimen. T(14;18)-positive cells decreased in
peripheral blood after treatment, irrespective of the clinical
response. Therefore, the significance of so-called molecular remission
should be reconsidered in follicular lymphoma.
(Blood. 2001;98:940-944) Follicular non-Hodgkin lymphoma (NHL) is
characterized by the chromosomal translocation
t(14;18)(q32;q21),1 leading to the overexpression of
bcl-2 with subsequent inhibition of apoptosis.2 The presence of t(14;18)-positive cells, analyzed by polymerase chain
reaction (PCR), is assumed to correlate with lymphoma activity. However, until now the clinical significance of the presence or absence
of t(14;18)-positive cells in bone marrow and peripheral blood has not
been settled.3-7 Quantitation of circulating
t(14;18)-positive cells, showing variation at diagnosis and over time,
may lead to a better understanding of the natural course of the disease and the effects of treatment.
Previously we observed an excellent correlation between numbers of
t(14;18)-positive cells in peripheral blood, analyzed by real-time PCR,
and the graft-versus-lymphoma effect of donor leukocyte infusion in a
patient with follicular NHL who had a relapse after allogeneic bone
marrow transplantation (BMT).8 Here, we present the
results of prospective monitoring of circulating t(14;18)-positive cells by real-time quantitative PCR before and during first-line treatment of patients with follicular lymphoma, consisting of standard
chemotherapy in combination with interferon alfa-2b and followed by
interferon alfa-2b maintenance therapy.9,10
Patients
Molecular analysis
Cells carrying t(14;18) with a bcl-2 gene breakpoint in the
MBR were quantified performing real-time PCR analysis using
Taq polymerase and an internal probe in the ABI/Prism 7700 sequence detector (Applied Biosystems, Foster City, CA).11
For this, 500 ng DNA of each patient sample, measured by optical
density at 260 nm, was amplified in duplicate in the presence of 300 nM MBR-2 (5'-TCC CTT TGA CCT TGT TTC TTG A-3') and JH-con
oligonucleotides,12 200 nM dual-labeled fluorogenic MBR
internal probe (5'-(FAM)-CAC AGA CCC ACC CAG AGC CC-(TAMRA)-3'), 250 µM dNTPs, 1.25 U AmpliTaq gold DNA polymerase, and 4 mM
MgCl2 in sample buffer A (Applied Biosystems) in a total
volume of 50 µL. Samples were heated for 10 minutes at 95°C and
amplified for 50 cycles of 15 minutes at 95°C and 60 minutes at
60°C. In parallel reactions, 500 ng patient DNA was amplified in
duplicate in the presence of 300 nM albumin forward (5'-TGA AAC ATA CGT
TCC CAA AGA GTT T-3') and reverse primer (CTC TCC TTC TCA GAA AGT GTG
CAT AT-3'), 200 nM dual-labeled fluorogenic albumin probe (5'-(JOE)-TGC
TGA AAC ATT CAC CTT CCA TGC AGA-(TAMRA)-3'), 250 µM dNTPs, 1.25 U
AmpliTaq gold DNA polymerase, and 4 mM MgCl2 in
sample buffer A in a total volume of 50 µL. Amplification conditions
for the albumin reaction were as mentioned above. Given that the
t(14;18) and albumin reactions had equivalent amplification
efficiencies (E = 0.91 ± 0.05 and
E = 0.93 ± 0.04, respectively),13 the
t(14;18) PCR signal, reflecting the total number of lymphoma cells, was
directly normalized to the albumin PCR signal, reflecting the total
number of cells used in the assay. For quantitation, normalized patient
PCR signals were compared to a single calibrator sample using the
comparative Ct-method. Briefly, for each patient sample, the t(14;18)
PCR Ct-value Statistical analysis Patient characteristics between groups were compared with the 2 test (level of significance: P < .05).
Progression-free survival was measured from the start of induction
therapy with CVP and interferon alfa until the time of disease
progression or until the end of the observation period in patients
without progressive disease. Ending the protocol treatment because of
other causes than progressive disease for example, intolerable
toxicity or death from diseases other than malignant lymphomawas
censored. If a patient had had an initial wait-and-see policy,
progression-free survival was still measured from the date induction
therapy was started. Survival curves were calculated according to the
Kaplan-Meier method; the log-rank test was used for analyzing
differences between curves.
Clinical characteristics Table 1 summarizes the clinical characteristics of the 34 patients with circulating t(14;18)-positive cells at presentation. Seventy-nine percent of the patients responded to induction therapy with 8 CVP courses combined with interferon alfa-2b. After a median follow-up of 44 months (range, 8-69 months), the median progression-free survival time of patients was 23 months. Median overall survival has not yet been reached.
Quantitative t(14;18) at diagnosis In blood samples taken before the start of treatment, the median number of t(14;18)-positive cells was 262 (range, 1-75 000) per 75 000 cells. Table 2 shows the clinical characteristics of patients with high numbers ( 262 per 75 000
cells) and low numbers (up to 262 per 75 000 cells) of
t(14;18)-positive cells. In patients with the higher numbers of
t(14;18)-positive cells, stage IV disease (mostly due to bone marrow
involvement) was more frequent than in those with the lower numbers
(P = .01). On the other hand, bulky disease was more often
present in patients with the lower numbers of circulating
t(14;18)-positive cells (P = .02). The highest numbers of
t(14;18)-positive cells at presentation were found in the 2 patients
with overt leukemic NHL, with lymphocyte counts of 15 and
22 × 109/L respectively (patients 1 and 2).
Progression-free survival curves of patients with higher and lower
numbers of circulating t(14;18)-positive cells before therapy are shown
in Figure 1. In the patients with lower
numbers, we excluded those with up to 10 circulating t(14;18)-positive
cells per 75 000 cells because this number approximates the maximum number of t(14;18)-positive cells we and others17 found in
the blood of healthy persons through real-time PCR analysis.
Progression-free survival did not differ between these groups
(P = .26). Even if the patients with pretreatment 1 to 10 circulating t(14;18)-positive cells per 75 000 cells were included in
the group of patients with lower numbers, progression-free survival of
this group did not differ from that of the group with higher numbers
(P = .23) (data not shown).
Sequential quantitative t(14;18) monitoring In 28 patients, t(14;18)-positive cells could be quantitated in paired blood samples, taken before and after induction treatment with CVP and interferon alfa-2b (Table 3). In all but one patient, the number of circulating t(14;18)-positive cells dropped, irrespective of the clinical response. In 13 patients, t(14;18) could not be detected after treatment. Twelve of them had a clinical response, but one showed progressive disease. In 9 patients, numbers of t(14;18) dropped to low levels of 1 to 10 per 75 000 cells, yet treatment had failed in 3 of these. One of them (patient 5) was the only patient who, in retrospect, did not have low-grade lymphoma but had discordant NHL, with a small cell component in lymph node and bone marrow and a large cell component in the femur. After induction therapy, therapy-resistant progressive disease of the large cell type was found in lymph node and femur. Finally, in 4 responding patients, the numbers decreased but remained positive at levels of 12, 79, 177, and 361 per 75 000 cells, respectively. In one patient, a decrease to 11 per 75 000 cells was found though treatment failed. In most patients, circulating t(14;18)-positive cells decreased by one or more logs after induction therapy. Analysis of absolute leukocyte and lymphocyte counts in the blood before and after induction showed that the decrease in t(14;18) did not result from leukopenia or lymphopenia per se. As induction treatment led to a decrease in the median number of t(14;18)-positive cells from 262 to 1 per 75 000 cells, it led to a decrease in the median numbers of leukocytes from 7.5 to 4.0 × 109/L and to a decrease of lymphocytes from 1.3 to 0.7 × 109/L.
Only one patient (patient 33) showed an increase after induction therapy from 1 to 406 circulating t(14;18)-positive cells per 75 000 cells. This patient had stage IVB bulky follicular NHL with bone marrow localization and achieved partial remission after induction therapy. In the original lymph node biopsy of this patient, t(14;18) could not be demonstrated. The course of t(14;18) in the peripheral blood in this patient remains unexplained.
This is the first report on prospective quantitative monitoring of t(14;18)-positive cells in the blood of patients with follicular lymphoma receiving first-line treatment. In this multicenter study, we used peripheral blood as a source for t(14;18) analysis; serial bone marrow analyses are less attractive because of patient inconvenience and sampling error, and serial lymph node sampling is not feasible. Thus far, only qualitative t(14;18) studies in follicular lymphoma have been reported, and the clinical significance of results has been conflicting. Several authors claimed that the risk for relapse after autologous stem cell transplantation and after standard chemotherapy correlated with the presence of t(14;18) in bone marrow or peripheral blood at the time of treatment or shortly thereafter.5-7 However, others found persistently t(14;18)-positive blood samples in patients with long-lasting remission.3,4 These patients have been followed up predominantly through clinical examination, making statements on real remission status impossible. The median number of circulating t(14;18)-positive cells in this study was 262 per 75 000 cells (3.5 per 1000 cells) before the start of therapy, with only 6 patients having low numbers (1 to 10 per 75 000). Patients with stage IV disease and bone marrow involvement were more likely to have higher numbers of t(14;18)-positive cells than those with less advanced stages. This finding can be considered to reflect the systemic dissemination of stage IV disease. In contrast, patients with bulky disease were more likely to have lower numbers of circulating t(14;18)-positive cells than those without bulky disease. This finding may be explained by an increased expression of adhesion receptors, such as L-selectin, in bulky lymphoma, resulting not only in the formation of a large contiguous tumor mass but also in preferential homing of lymphoma cells in lymph nodes rather than in bone marrow and peripheral blood.18 Our finding that the progression-free survival rates of patients with higher numbers of circulating t(14;18)-positive cells before treatment did not differ from those of patients with lower numbers suggests that the lymphoma load in peripheral blood at presentation does not influence prognosis. First-line treatment appears to result in an easy clearing of lymphoma cells from the blood, irrespective of their numbers. In contrast, this therapy may not always affect other compartments of disease, such as lymph nodes or bone marrow, from which progressive disease may originate later in the course of the disease. Earlier we observed a clear correlation between numbers of t(14;18)-positive cells in blood and bone marrow, and the post-BMT course of a patient with a follicular NHL.8 In this patient, t(14;18)-positive cells persisted in the blood after allogeneic BMT and increased before clinical relapse. After subsequent donor leukocyte infusion, t(14;18)-positive cells in blood and bone marrow decreased gradually while the patient responded clinically. A similar course was seen in another patient with follicular lymphoma treated with allogeneic BMT, in whom clinical findings correlated well with molecular analysis of t(14;18)-positive cells, quantitated by a competitive PCR method.19 These findings suggested a contribution of monitoring t(14;18)-positive cells in predicting response and relapse in patients with follicular lymphoma, as also assumed by Hirt and Dölken,20 quantitating circulating t(14;18)-positive cells in patients with follicular lymphoma treated with autologous BMT. Real-time quantitative t(14;18) monitoring in serial blood samples in this previously untreated patient group showed a sharp decrease in t(14;18)-positive cells after conventional therapy with CVP and interferon alfa, regardless of the clinical response. This decrease in t(14;18) cannot be explained by a parallel lowering of circulating white blood cells. In most patients who responded to induction therapy, we found a considerable decrease or even disappearance of circulating t(14;18)-positive cells. When molecular remission in patients with follicular lymphoma may be indicative of cure or at least prolonged remission, the disappearance of t(14;18)-positive cells after noncurative treatment with CVP and interferon alfa cannot be expected. Remarkably, even in the 5 nonresponders, we observed a similar decrease in t(14;18). Increasing the sensitivity of this real-time PCR analysis of t(14;18) will not contribute to understanding the course of t(14;18) because this PCR is already capable of detecting t(14;18)-positive cells in healthy persons. A longer follow-up period with more consecutive samples for t(14;18) analysis will give information on variation in circulating t(14;18)-positive cells during maintenance therapy and off maintenance therapy. It cannot be excluded that interferon alfa plays a role in the decrease of t(14;18)-positive cells after induction therapy. Therefore, we quantitated circulating t(14;18)-positive cells in another group of previously untreated patients with stages III and IV follicular lymphoma, treated now with 8 CVP courses without interferon alfa-2b according to a European protocol (EORTC 20963/HOVON 35). In this study 19 patients had pretreatment circulating t(14;18)-positive cells with a bcl-2 gene breakpoint in MBR (median, 23 per 75 000 cells; range, 1-5027 cells). After induction therapy, again irrespective of clinical response, a sharp decrease in circulating t(14;18)-positive cells to (nearly) undetectable levels was observed in all 12 patients whose blood was serially tested (data not shown). From these data, we conclude that it is unlikely that treatment with interferon alfa caused the decrease in circulating t(14;18)-positive cells in the patient group presented here. Our observation that circulating t(14;18)-positive cells decreased or disappeared after induction therapy in most patients, regardless of clinical response, should prompt a reappraisal of so-called molecular remissions. At least after this conventional first-line treatment, monitoring t(14;18)-positive cells in the peripheral blood does not correlate with lymphoma activity. Whether correlations are more reliable in pretreated patients with more advanced disease, as suggested in studies of high-dose therapy and stem cell transplantation,5,7,8,19 must be analyzed in future prospective monitoring studies.
We thank the participating centers of the South-East Cancer Centers Cooperative Group for their collaboration. Furthermore, we thank Lian Roovers and Sandra Seeger (Data Center, Department of Hematology, University Medical Center Nijmegen) for excellent data management. We also thank Han van Krieken (Department of Pathology, University Medical Center Nijmegen) for assistance in reviewing original lymph node biopsy samples and for critically reading the manuscript. Finally, we thank Schering-Plough for technical support in collecting blood samples.
A complete list of the members of the South-East Netherlands Comprehensive Cancer Centers Cooperative Group is given at the end of this article in the Appendix.
Submitted December 1, 2000; accepted March 30, 2001.
Supported by an unrestricted grant from Schering-Plough (J.R.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Caroline M. P. W. Mandigers, Department of Hematology, University Medical Center Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands; e-mail: c.mandigers{at}hemat.azn.nl.
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Apostolidis J, Gupta RK, Grenzelias D, et al.
High-dose therapy with autologous bone marrow support as consolidation of remission in follicular lymphoma: long-term clinical and molecular follow-up.
J Clin Oncol.
2000;18:527-536 8. Mandigers CMPW, Meijerink JPP, Raemaekers JMM, Schattenberg AVMB, Mensink EJBM. Graft-versus-lymphoma effect of donor leucocyte infusion shown by real-time quantitative PCR analysis of t(14;18). Lancet. 1998;352:1522-1523[CrossRef][Medline] [Order article via Infotrieve].
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J Clin Oncol.
1998;16:41-47 11. Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology. 1993;11:1026-1030[CrossRef][Medline] [Order article via Infotrieve]. 12. Meijerink JPP, Smetsers TFCM, Raemaekers JMM, Bogman MJJT, De Witte T, Mensink EJBM. Quantitation of follicular non-Hodgkin's lymphoma cells carrying t(14;18) by competitive polymerase chain reaction. Br J Haematol. 1993;84:250-256[Medline] [Order article via Infotrieve].
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A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR.
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2001;3:55-61 14. Limpens J, De Jong D, Van Krieken JHJM, et al. BCL-2/JH rearrangements in benign lymphoid tissues with follicular hyperplasia. Oncogene. 1991;6:2271-2276[Medline] [Order article via Infotrieve].
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2000;95:1900-1910 19. Meijerink JPP, Goverde GJ, Smetsers TFCM, et al. Quantitation of follicular non-Hodgkin's lymphoma cells carrying t(14;18) in a patient before and after allogeneic bone marrow transplantation. Ann Oncol. 1994;5:S43-S45[Abstract]. 20. Hirt C, Dölken G. Quantitative detection of t(14;18)-positive cells in patients with follicular lymphoma before and after autologous bone marrow transplantation for follicular lymphoma. Bone Marrow Transplant. 2000;25:419-426[CrossRef][Medline] [Order article via Infotrieve].
The following colleagues participated in this study by the Interzol (South-East Netherlands Cancer Centers Cooperative Group) (in order of number of patients included): Bron and Erdkamp, Maasland Ziekenhuis Sittard; Derleyn and Koch, Elkerliek Ziekenhuis Helmond; Raemaekers and Mandigers, Universitair Medisch Centrum St Radboud Nijmegen; Roelofs and Werter, St Elisabeth Ziekenhuis Venray, Dullemond, Twee Steden Ziekenhuis Tilburg; Liem and Wils, St Laurentius Ziekenhuis Roermond; Mol, Rijnstate Ziekenhuis Arnhem; Van Reisen, Ziekenhuis Zeeuws Vlaanderen Terneuzen; Smals and Smeets, St Anna Ziekenhuis Geldrop; Schade, St Anna Ziekenhuis Oss; Fickers, Atrium Heerlen; Burghouts and Croles, Bosch Medicentrum `s-Hertogenbosch; Oosten and Van Turnhout, Canisius Wilhelmina Ziekenhuis Nijmegen; Loosveld, De Baronie Breda; Vlasveld, Diaconessenhuis Eindhoven; Beudeker and Creemers, Ziekenhuis Walcheren Vlissingen; Wittebol, Eemland Ziekenhuis Amersfoort; Van der Heul, St Elisabeth Ziekenhuis Tilburg; Janssen, St Franciscus Ziekenhuis Roosendaal; Van Nierop, St Jansdal Harderwijk; Keuning, St Joseph Ziekenhuis Veldhoven; Stouthard, Merwede Ziekenhuis Dordrecht; Jonkers, Rijnland Ziekenhuis Leiderdorp.
© 2001 by The American Society of Hematology.
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  M. J. Bishton, R. J. Hicks, D. A. Westerman, M. H. Prince, M. Wolf, and J. F. Seymour A prospective study of the separate predictive capabilities of 18[F]-FDG-PET and molecular response in patients with relapsed indolent non-Hodgkin's lymphoma following treatment with iodine-131-rituximab radio-immunotherapy Haematologica, May 1, 2008; 93(5): 789 - 790. [Full Text] [PDF]
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A. Koster, H. A. Tromp, J. M.M. Raemaekers, G. F. Borm, K. Hebeda, M. A. MacKenzie, and J. H.J.M. van Krieken The prognostic significance of the intra-follicular tumor cell proliferative rate in follicular lymphoma Haematologica, February 1, 2007; 92(2): 184 - 190. [Abstract] [Full Text] [PDF]
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 A. Koster, J. H.J.M. van Krieken, M. A. MacKenzie, M. Schraders, G. F. Borm, J. A.W.M. van der Laak, W. Leenders, K. Hebeda, and J. M.M. Raemaekers Increased Vascularization Predicts Favorable Outcome in Follicular Lymphoma Clin. Cancer Res., January 1, 2005; 11(1): 154 - 161. [Abstract] [Full Text] [PDF]
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 M. Ghielmini, S.-F. H. Schmitz, S. B. Cogliatti, G. Pichert, J. Hummerjohann, U. Waltzer, M. F. Fey, D. C. Betticher, G. Martinelli, F. Peccatori, et al. Prolonged treatment with rituximab in patients with follicular lymphoma significantly increases event-free survival and response duration compared with the standard weekly x 4 schedule Blood, June 15, 2004; 103(12): 4416 - 4423. [Abstract] [Full Text] [PDF]
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E. Fernandez-Ruiz, M. Cabrerizo, M. Ortega, C. Blas, P. Llamas, M. Santos-Roncero, S. Nieto, A. Acevedo, G. Perez, C. Nicolas, et al. High Molecular Response Rate and Clinical Correlation in Patients with Follicular Lymphoma Treated with Cyclophosphamide-Vincristine-Prednisone plus Interferon {alpha} 2b Clin. Cancer Res., July 1, 2003; 9(7): 2497 - 2503. [Abstract] [Full Text] [PDF]
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A. Rambaldi, M. Lazzari, C. Manzoni, E. Carlotti, L. Arcaini, M. Baccarani, T. Barbui, C. Bernasconi, G. Dastoli, G. Fuga, et al. Monitoring of minimal residual disease after CHOP and rituximab in previously untreated patients with follicular lymphoma Blood, February 1, 2002; 99(3): 856 - 862. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
20°C and stored in the participating center until transport on dry
ice to our laboratory for DNA isolation and subsequent
molecular analysis.
CtS). All
patient samples are quantified against normalized Ct values of a
calibrator sample (
XSample = XCal × 2
(
Cts
indicates
end of observation period for individual patients.