Article

Tumor load in patients with follicular lymphoma post stem cell transplantation may correlate with clinical course

Medical College of Wisconsin, Milwaukee, Wisconsin, United States
Bone Marrow Transplantation (Impact Factor: 3.57). 09/2003; 32(3):287-91. DOI: 10.1038/sj.bmt.1704130
Source: PubMed

ABSTRACT

The purpose of this study was to evaluate if the tumor load, as determined by a real-time quantitative PCR (RQ-PCR) assay, correlated with the clinical course of follicular lymphoma patients after stem cell transplantation (SCT). Cryopreserved bone marrow and/or peripheral blood samples obtained at different time intervals after SCT from 11 patients (seven allogeneic, T-cell depleted/four autologous) were tested for tumor load, as defined by t(14;18) positive cells/total cells, using RQ-PCR. None of the six patients who remained in remission had samples with a tumor load >0.01% after SCT, although fluctuating tumor loads of </=0.01% were observed in three of these patients. In contrast, four of the five patients (three allogeneic/two autologous) with relapsed/progressive disease had increasing tumor loads of >0.01% after SCT (0/6 vs 4/5, P<0.02, Fisher's exact). Our results suggest that RQ-PCR measurable tumor load >0.01% after SCT may correlate with relapsed/progressive disease. Prospective studies with greater numbers of cases are indicated to better determine the critical tumor load that predicts poor outcome after SCT with RQ-PCR.

Full-text

Available from: Carolyn A Keever-Taylor, Jun 26, 2014
Non-Hodgkin’s Lymphoma
Tumor load in patients with follicular lymphoma post stem cell
transplantation may correlate with clinical course
C-C Chang
1
, C Bredeson
2
, M Juckett
2
, B Logan
3
and CA Keever-Taylor
2
1
Department of Pathology, Medical College of Wisconsin, Milwaukee, WI, USA;
2
Department of Medicine, Medical College of
Wisconsin, Milwaukee, WI, USA; and
3
Division of Biostatistics, Medical College of Wisconsin, Milwaukee, WI, USA
Summary:
The purpose of this study was to evaluate if the tumor
load, as determined by a real-time quantitative PCR (RQ-
PCR) assay, correlated with the clinical course of
follicular lymphoma patients after stem cell transplanta-
tion (SCT). Cryopreserved bone marrow and/or periph-
eral blood samples obtained at different time intervals
after SCT from 11 patients (seven allogeneic, T-cell
depleted/four autologous) were tested for tumor load, as
defined by t(14;18) positive cells/total cells, using RQ-
PCR. None of the six patients who remained in remission
had samples with a tumor load 40.01% after SCT,
although fluctuating tumor loads of p0.01% were
observed in three of these patients. In contrast, four of
the five patients (three allogeneic/two autologous) with
relapsed/progressive disease had increasing tumor loads of
40.01% after SCT (0/6 vs 4/5, Po0.02, Fisher’s exact).
Our results suggest that RQ-PCR measurable tumor load
40.01% after SCT may correlate with relapsed/progres-
sive disease. Prospective studies with greater numbers of
cases are indicated to better determine the critical tumor
load that predicts poor outcome after SCT with RQ-PCR.
Bone Marrow Transplantation (2003) 32, 287–291.
doi:10.1038/sj.bmt.1704130
Keywords:
RQ-PCR; follicular lymphoma; SCT; relapse;
tumor load; quantitative PCR; clinical course
Stem cell transplantation (SCT), allogeneic or autologous,
holds promise for improving the survival of follicular
lymphoma (FL) patients.
1–5
FL is one of the most frequent
subtypes of malignant lymphoma in Western countries and
accounts for approximately 25% of all adult non-Hodg-
kin’s lymphoma.
6
However, despite the intensive condi-
tioning regimen and possible graft-versus-lymphoma (GvL)
effect of allogeneic SCT, some patients (16–38%) still have
relapse or progression of disease after allogeneic SCT.
1,2
The relapse rate is even higher in patients receiving
autologous SCT at about 58% at 8 years post-SCT.
3
Laboratory methods that can predict which patients
are at risk of clinical relapse/progression after SCT are
potentially valuable to improve the outcomes of SCT. The
best example is the application of quantitative RT-PCR to
measure BCR–ABL fusion transcripts as a marker of
tumor load in chronic myeloid leukemia (CML).
7–9
In
patients with CML, rising or persistently high levels of
tumor load, BCR–ABL to ABL ratio of 40.02% or 4100
BCR–ABL transcripts/mg RNA, in two sequential speci-
mens more than 4 months following SCT are predictive of
overt clinical relapse.
7
Furthermore, treating CML patients
before there is overt clinical relapse of disease with donor
lymphocyte infusion (DLI) has improved the survival of
CML patients after SCT.
8
Unlike CML, studies to determine the clinical signifi-
cance of the tumor load in FL after SCT are very limited.
9
Hirt et al
10
have recently reported that relapses are
associated with increasing numbers of circulating t(14;18)-
positive cells, while continuous complete clinical remissions
are associated with stable t(14;18)-positive cell counts in
seven patients following autologous SCT. Mandigers et al
11
reported that levels of tumor load correlated with remission
after using DLI to treat the relapse of FL following
allogeneic SCT in one patient.
The main purpose of the current study was to evaluate
if the level of tumor load correlated with the clinical course
in 11 patients with FL after SCT (seven allogeneic, T-cell
depleted and four autologous). We measured the level of
tumor load, t(14;18)-positive cells/total mononuclear cells,
using a real-time quantitative PCR (RQ-PCR) assay.
Materials and methods
Patients and samples
We retrospectively studied 11 FL patients who underwent
T-cell-depleted allogeneic SCT or autologous SCT at the
Medical College of Wisconsin from February 1990 to
November 1999. The T-cell depletion was performed using
the protocol reported previously.
12
Allogeneic patients in
this study received grafts with a 2.270.7 log depletion of
T cells, resulting in T-cell doses/kg ranging from 4.15 10
4
to 3.6 10
5
. These patients were studied because cryopre-
served bone marrow (BM) and/or peripheral blood (PB)
mononuclear cells of these patients collected before SCT
Received 17 August 2002; accepted 7 March 2003
Correspondence: Dr C-C Chang, Department of Pathology, Medical
College of Wisconsin, 9200 W. Wisconsin Avenue, Milwaukee,
WI 53226, USA
Bone Marrow Transplantation (2003) 32, 287–291
& 2003 Nature Publishing Group All rights reserved 0268-3369/03 $25.00
www.nature.com/bmt
Page 1
were positive for t(14;18) using the RQ-PCR assay
described below. A total of 70 samples obtained at various
time intervals after SCT from these 11 patients was further
tested for tumor load using the RQ-PCR assay.
Demographic data and the clinical course of the 11
patients are summarized in Table 1. These patients received
SCT for relapsed/refractory FL. All patients except cases 4,
9, and 10 (Table 1) were conditioned with total body
irradiation, AraC, and Cytoxan. Case #4 was conditioned
with BEAM, Case #9 with etoposide, cisplatin, cyclopho-
sphamide, and carmustine and Case #10 with VP-16,
Cytoxan, cisplatin, and carmustine. The median length of
follow-up in these patients post-SCT was 76 months (85
months for survivors), ranging from 7 months to 152
months (45 to 152 months for survivors). The end point of
follow-up was either the date of the last visit or the date of
death up to December 2002. This study was approved by
the Institutional Review Board of Medical College of
Wisconsin.
RQ-PCR assay
This RQ-PCR assay was developed to detect and quantitate
the cells carrying t(14;18) at the major breakpoint region
(MBR). Briefly, total cellular DNA from the cell line,
SUDHL-6 containing t(14; 18)(q32;q21), normal human
placenta, and patients’ samples were prepared using Pure-
gene DNA Isolation Kit (Gentra Systems, Minneapolis,
MN, USA). The standard curve for quantitating the tumor
load, the ratio of t(14:18)+ cells/total cells, was established
by standardized samples, with the different ratios of
SUDHL-6 DNA/total DNA ( ¼ SUDHL-6 DNA+placen-
ta DNA). A total of four standardized samples with tumor
loads of 10% (one tumor cell in 10 total cell), 1% (one in
100), 0.1% (one in 1000), and 0.01% (one in 10 000),
respectively, was made for establishing a standard curve.
One additional standardized sample, with a tumor load of
0.0025% (one tumor cell in 40 000 total cells), was prepared
to test the sensitivity of the assay.
PCR was performed in a 20 ml volume containing 250 ng
DNA from each standardized sample or patient samples,
5.0 mm MgCl
2
, 200 mm of dATP, dCTP, dGTP, dTTP, and
dUTP, 1.0 mm of each primer (5
0
-agatggcaaatgaccagcaga-3
0
,
5
0
-acctgaggagacggtgacc-3
0
), 0.3 mm of 3
0
end fluorescein-
labeled (5
0
-ctgggtgggtctgtgttgaa-3
0
), and 0.2 mm of 5
0
end
LCRed 640-labeled (5
0
-caggccacgtaaagcaact-3
0
) probes, 1 U
of thermolabile uracil-N-glycosylase (Roche Diagnostics,
Indianapolis, IN, USA), and 10 U of Amplitaq Gold DNA
polymerase (Applied Biosystems, Forster City, CA, USA).
The PCR amplification and quantitation of PCR products
were performed on a LightCyclert (Roche Diagnostics).
After 10 min at 951C to activate Amplitaq Gold, the
following reaction was carried out: five cycles of denatura-
tion at 941C for 0 s, annealing at 671C for 5 s and extension
at 741C for 15 s, followed by 45 cycles of denaturation at
941C for 0 s, annealing at 631C for 5 s, and extension at
741C for 15 s. The tumor loads of each patient sample were
assessed using the standard curve established by the
standardized samples (Figure 1).
All the patients’ samples were tested in duplicate initially.
If both runs were negative, an additional two runs were
performed to reach the sensitivity of detecting one
neoplastic cell in 160K normal cells (0.0006%). The tumor
load was calculated as the average of two or four runs.
For each patient sample, the b-globin gene was amplified
separately using another RQ-PCR assay similar to the one
described above with primers (5
0
-ggctacgttcattgccagat-3
0
,
5
0
-gagaccattgtggcagtgat-3
0
),3
0
end fluorescein-labeled (5
0
-
tccttcttttcattcacagacagggatttt-3
0
), and 5
0
end LCRed 640-
labeled (5
0
-gagaccattgtggcagtgat-3
0
) probes targeting b-
globin gene. This was to ensure that all samples contained
similar amounts of DNA and were free of PCR inhibitors.
Results
The RQ-PCR assay showed the sensitivity of detecting a
single cell carrying t(14;18) in the background of 40 000
normal cells (ie a tumor load of 0.0025%). The linear
measuring range of the standard curve of quantitation
of the assay was between tumor loads of 0.01 and 10%
(Figure 1). For samples with a tumor load less than 0.01%,
the tumor load was estimated based on the standard curve
with the understanding that the measurement might not be
accurate. Additionally, an interassay (eight experiments
using standardized samples with tumor loads of 10, 1, 0.1,
and 0.01%) coefficient of variation (CV) of p10.6% and
an intraassay (six measurements of identical samples) CV
of p6.9% were achieved for reproducibility. The CV in-
creased significantly when the tumor load was below 0.01%.
Out of 11 patients, 10 achieved a complete clinical
remission after SCT as determined by physical, CT scan,
and/or bone marrow examination (Table 1). Six (two
autologous and four allogeneic) of these 10 patients
remained in complete clinical remission with a median
follow-up length of 132 months (ranging from 55 to 152
months) after SCT. None of these six patients received any
further treatment, including DLI or other immunomani-
pulation, after SCT. The remaining four patients (two
autologous and two allogeneic) had relapse at 5, 13, 14, and
36 months, respectively (cases #11, #9, #10, and #8,
respectively). Three relapsed patients ultimately died of
38
36
34
32
30
28
26
24
22
20
18
Threshhold cycle number
-3
-2.5
-2
-1.5 -1
-0.5
0 0.5
1
Log tumor load [%]
y = 25.35 - 4.15x
P < 0.0001 r
2
= 1.00
Figure 1 Standard curve for quantitation. The standard curve for
quantitation of tumor load is established by plotting the log of the tumor
load (in percentage) of each standardized sample vs the ‘threshold cycle
number (crossing point)’.
17
The results show that the log of the tumor load
(in percentage) is inversely correlated to the ‘threshold cycle number’, the
number of the PCR cycle at which the fluorescence intensity generated by
products (due to FRET) becomes greater than the background intensity.
17
Tumor load may correlate with clinical course
C-C Chang et al
288
Bone Marrow Transplantation
Page 2
progressive disease (cases #9, 10, and, 11) and one attained
complete clinical remission after DLI (case #8). Only one
allogeneic recipient did not achieve a complete remission
after SCT. She died of progressive disease 11 months after
SCT (case #7).
Using this RQ-PCR assay, none of the six patients who
remained in complete clinical remission had any samples
reaching a tumor load of 0.01% after SCT (Figure 2).
Three (cases #1, #2, and #6) of the six patients in complete
clinical remission showed fluctuating tumor loads of
o0.01%, while the other three patients had no detectable
tumor load after SCT (Figure 2). In contrast, four of the
five patients who had relapsed/progressive disease had one
or more samples showing tumor load 40.01% (ranging
from 0.012 to 5.79%, Figure 3) (0/6 vs 4/5, Po0.02,
Fisher’s exact). Furthermore, clinical relapse/progression
was accompanied by a tumor load of 40.1% in four
patients (cases #7, #8, #9, and #10, Figure 3). In two
patients (cases #9 and #10), CT scan/bone marrow/physical
examination failed to show any signs of relapsed/progres-
sive disease at the time of tumor load 40.01%. The time
interval between the samples showing tumor load 40.01%
and clinical relapse in these two patients were 56 and 359
days, respectively (Figure 3). In one patient, clinical relapse
was associated with at least 10-fold higher level of tumor
load than the initial sample showing tumor load 40.01%
(case #9, Figure 3).
None of the seven patients who received allogeneic SCT
developed acute GVHD of grade III or higher (Table 1).
Five patients had extensive chronic GVHD, one had
limited chronic GVHD, and one had no chronic GVHD.
There was, however, no apparent difference in the degree of
acute or chronic GVHD between patients with relapsed/
progressive disease (two of three acute GVHD and two of
three chronic GVHD) and those without (three of four
acute GVHD and four of four chronic GVHD).
Discussion
Our results suggest that the tumor load measured by the
RQ-PCR assay correlates with clinical course of patients
with follicular lymphoma after SCT. The patients with
tumor loads constantly below 0.01% had enduring remis-
sion of disease, while those with tumor load of 40.01%
were at risk of relapse/progression. Furthermore, clinical
relapse/progression after SCT was associated with a tumor
load 40.1%. Our results in patients with autologous SCT
agree with those reported by Hirt et al.
10
Using a different
quantitative PCR assay, they observed that patients with
continuous complete remission had circulating t(14;18)-
positive cells at the levels of o0.01%. In one of their two
patients, the relapse was accompanied by a logarithmic
increase of t(14;18)-positive cells over a tumor load of
Table 1 Demographic data and clinical course of the patients studied
Case # Age at
Dx/Gender
Stage
at Dx
Therapy prior
to SCT
Status of
disease
at SCT
Age
at
SCT
SCT
type
GVHD
(acute/chronic)
Best
response
to SCT
Current status
post-SCT
Cause of
death
Remained remission
1 43/F IV CHOP-Bleox5 PR1 44 TD, RM II/extensive CR Alive* at 125 mos NA
2 44/M IV COPA 8 Relapse 2 48 AU NA CR CR
#
at 148 mos NA
Chlor+predx6
3 56/M IV CHOP 6 Relapse 1 58 TD, RM II/extensive CR CR at 76 mos NA
DHAPx2
4 49/M I CHOP 7 PR2 53 AU NA CR CR at 55 mos NA
CHOPx6
ICE 2
5 27/F IV CVP 1 Relapse 1 27 TD, RM None/extensive CR CR at 152 mos NA
CHOPx6
6 41/M IV MBACOD 7 Relapse 2 43 TD, RM I/limited CR CR at 139 mos NA
ProMACE-MOPP
4 MBCOD 3
VP16+Mito-xantronex5
Local XRTx2
Relapsed/progressed
7 60/F IV CHOP 6 PR2 62 TD, RM None/none Progressive Dead at 11 mos Progressive
Hux5 Disease Disease
8 45/F IV CHOP 6 +INF Relapse 1 49 TD, RM II/extensive CR Relapsed at 36 mos NA
Alive at 51 mos
CR post-DLI
9 47/M IV CHOP 6 PR1 48 AU NA CR Relapsed at 13 mos Progressive
CVPx1 Dead at 72 mos Disease
10 40/M IV ProMACE-MOPP 7 Relapse 2 51 AU NA CR Relapsed at 14 mos Progressive
Local XRT 2 Dead at 52 mos Disease
11 46/F III CHOP 6 PR1 46 TD, RM II/extensive CR Relapsed at 5 mos Progressive
CDEP 2 Dead at 7 mos Disease
AU: autologous SCT, Chlor: chlorambucil, CR: complete remission, Dx: diagnosis, mos: months, GVHD: graft-versus-host-disease.
Hu: hydroxyurea, NA: not applicable, PR: partial remission, RM: related matched, TD: T-cell depleted, XRT: radiation therapy. *Developed post-
transplant lymphoproliferative disorder (PTLD) 60 months post-SCT. The PTLD lesion showed evidence of EBV infection and was negative for t(14;18)
using the same PCR assay. #Received allogeneic SCT at 48 months after autologous SCT secondary to MDS.
Tumor load may correlate with clinical course
C-C Chang et al
289
Bone Marrow Transplantation
Page 3
0.01%. There are no data available in the literature to be
compared with our findings for patients with allogeneic
SCT.
The correlation between the levels of tumor load and
clinical course in FL recipients of T-cell-replete SCT
requires further investigation, as all of the allogeneic
recipients in our study received T-cell-depleted SCT. T-
cell-depleted SCT may have the disadvantage of weaker
GVL effect associated with the desired less-severe GVHD.
However, there were no apparent differences in the degree
of acute or chronic GVHD between patients with relapsed/
progressive disease and those with stable remission in this
small series of patients.
Our findings further indicate that long-term monitoring
of tumor load with RQ-PCR may be necessary for FL
patients receiving SCT, since tumor load may gradually
increase following a transient decrease after SCT (Figure 2).
One patient (case #8) demonstrated an increase in tumor
load associated with clinical relapse 1081 days after SCT.
This observation is in agreement with van Besien et al
2
who
reported that, although uncommon, disease relapse may
occur 2 years or more after allogeneic SCT. Additionally,
consistent with previous studies, we showed that some
patients may have fluctuating low levels (o0.01%) of
tumor loads after SCT without clinical relapse.
3,10,13–16
These patients, however, may require close follow-up
to evaluate if the tumor load is stable, decreasing, or
increasing over time. Further studies are needed to
determine the optimal length and interval of monitoring
tumor load.
In the current study, we identified two patients from
whom samples with tumor load 40.01% were obtained up
to 359 days prior to clinical relapse. In these two patients,
there was no evidence of clinical relapse by either physical,
bone marrow, or CT scan examination at the time the
samples were obtained. However, gallium scan, which may
be more sensitive than CT scan, was not performed in these
patients. Our findings agree with the observation by
Mandigers et al.
11
They observed a patient whose tumor
load remained over 0.01% after allogeneic SCT, even
though he was in complete clinical remission. This patient
Un-Detected
0.0006
0.001
0.010
0.100
1.000
10.000
Tumor load (%)
-100 -10
100 200
300
400 500
600 1600
2600
3600 4000
Days post BMT
Case #1
Case #2
Case #3
Case #4
Case #5
Case #6
Figure 2 Levels of tumor load over time in patients who remained in remission. Specimens with no detectable tumor loads are shown as the data points
below tumor loads of 0.0006% (sensitivity limit of the assay with same samples run in quadruplicate). The data points below this level (0.0006%) shouldbe
regarded as the same although they are shown differently for illustration purpose. The case number designated is identical to that of Table 1.
10.000
1.000
0.100
0.010
0.001
0.0006
Un-Detected
Tumor load (%)
0
200
400
600
800
1000
1200
Days post BMT
Case #7
Case #8
Case #9
Case #10
Case #11
Figure 3 Levels of tumor load over time in patients who relapsed/
progressed. Specimens with no detectable tumor loads are shown as the
data points below tumor loads of 0.0006% (sensitivity limit of the assay
with same samples run in quadruplicate). The arrow indicates the
occurrence of clinical relapse. Case #11 had a pair of samples obtained
on the same date before SCT, one with very low level of tumor load and the
other with undetected tumor load. The case number designated is identical
to that of Table 1.
Tumor load may correlate with clinical course
C-C Chang et al
290
Bone Marrow Transplantation
Page 4
eventually had clinical relapse 2 years later. Furthermore, it
is important to note that the tumor load increased at least
one log when overt clinical relapse occurred in one of our
patients and the case reported by Mandigers.
Taken together, our results suggest the tumor load
measured by the RQ-PCR assay may predict the clinical
course of FL patients receiving SCT. The current study is,
however, limited by its retrospective nature without
planned sampling of patients at regular intervals after
SCT. Prospective studies with a larger sample size and
regular sampling are indicated to evaluate if a critical level
of tumor load, beyond which clinical relapse is inevitable,
can be determined. If so, pre-emptively treating patients at
this critical level, but without overt clinical relapse, can be
considered. This approach may allow treatment at a stage
when tumor burden is relatively low, resulting in improved
outcome. This may be particularly important for FL
patients undergoing allografts, as lower dose DLI may be
used at this stage to generate an efficacious GVL effect
while avoiding severe GVHD, as shown in CML patients.
8
Acknowledgements
We thank Dr. Carl G Becker, Professor and Chairman,
Department of Pathology, Medical College of Wisconsin, for
his critical review of this manuscript.
References
1 Juckett M, Rowlings P, Hessner M et al. T cell-depleted
allogeneic bone marrow transplantation for high-risk non-
Hodgkin’s lymphoma: clinical and molecular follow-up. Bone
Marrow Transplant 1998; 21: 893–899.
2 van Besien K, Sobocinski KA, Rowlings PA et al. Allogeneic
bone marrow transplantation for low-grade lymphoma. Blood
1998; 92: 1832–1836.
3 Freedman AS, Neuberg D, Mauch P et al. Long-term follow-
up of autologous bone marrow transplantation in patients with
relapsed follicular lymphoma. Blood 1999; 94: 3325–3333.
4 Friedberg JW, Freedman AS. High-dose therapy and stem cell
transplantation in follicular lymphoma. Ann Hematol 1999; 78:
203–211.
5 Mounier N, Socie G, Gisselbrecht C. High-dose therapy for
indolent lymphoma. Crit Rev Oncol Hematol 2002; 41: 225–239.
6 Armitage JO, Weisenburger DD. New approach to classifying
non-Hodgkin’s lymphomas: clinical features of the major
histologic subtypes. Non-Hodgkin’s Lymphoma Classification
Project. J Clin Oncol 1998; 16: 2780–2795.
7 Lin F, van Rhee F, Goldman JM et al. Kinetics of increasing
BCR–ABL transcript numbers in chronic myeloid leukemia
patients who relapse after bone marrow transplantation. Blood
1996; 87: 4473–4478.
8 Dazzi F, Szydlo RM, Goldman JM. Donor lymphocyte
infusions for relapse of chronic myeloid leukemia after
allogeneic stem cell transplant: where we now stand. Exp
Hematol 1999; 27: 1477–1486.
9 Bagg A. Commentary: minimal residual disease: how low do
we go? Mol Diagn 2001; 6: 155–160.
10 Hirt C, Dolken G. Quantitative detection of t(14; 18)-positive
cells in patients with follicular lymphoma before and after
autologous bone marrow transplantation. Bone Marrow
Transplant 2000; 25: 419–426.
11 Mandigers CM, Meijerink JP, Raemaekers JM et al. Graft-
versus-lymphoma effect of donor leucocyte infusion shown by
real-time quantitative PCR analysis of t(14; 18). Lancet 1998;
352: 1522–1523.
12 Keever-Taylor CA, Craig A, Molter M et al. Complement-
mediated T-cell depletion of bone marrow: comparison of
T10B9.1A-31 and Muromonab-Orthoclone OKT3. Cyto-
therapy 2001; 3: 467–481.
13 Corradini P, Astolfi M, Cherasco C et al. Molecular
monitoring of minimal residual disease in follicular and mantle
cell non-Hodgkin’s lymphomas treated with high-dose che-
motherapy and peripheral blood progenitor cell autografting.
Blood 1997; 89: 724–731.
14 Meijerink JP, Goverde GJ, Smetsers TF et al. Quantitation of
follicular non-Hodgkin’s lymphoma cells carrying t(14; 18) in a
patient before and after allogeneic bone marrow transplanta-
tion. Ann Oncol 1994; 5: 43–45.
15 Masuda R, Teshima T, Ishimaru F et al. Allogeneic peri-
pheral blood stem cell transplantation for the treatment
of refractory follicular lymphoma. Intern Med 1998; 37:
1050–1054.
16 Gribben JG, Neuberg D, Freedman AS et al. Detection by
polymerase chain reaction of residual cells with the bcl-2
translocation is associated with increased risk of relapse after
autologous bone marrow transplantation for B-cell lymphoma.
Blood 1993; 81: 3449–3457.
17 Wittwer CT, Herrmann MG, Moss AA et al. Continuous
fluorescence monitoring of rapid cycle DNA amplification.
Biotechniques 1997; 22: 130–131, 134–138.
Tumor load may correlate with clinical course
C-C Chang et al
291
Bone Marrow Transplantation
Page 5
  • Source
    • "The resistance of cancer stem cells (CSCs) to conventional chemotherapy and radiotherapy has been attributed to cellular mechanisms such as multidrug resistance, quiescence, enhanced DNA repair abilities , and anti-apoptotic mechanisms [7]. Dysregulation of key signaling pathways such as the Wnt, Hedgehog, Notch, and TGF-β/BMP pathways plays an important role in modulating tumorigenesis as well as in stem cell function [8] . These studies suggest that identification and targeting cancer stem cells are key aspects to optimizing cancer therapy. "
    [Show abstract] [Hide abstract] ABSTRACT: Recent data suggest that tumor persistence and recurrence could be caused by the presence of cancer stem cells (CSCs). Aldehyde dehydrogenase 1 (ALDH1) has been implicated in cancer pathogenesis and used as a CSC marker. We previously reported that cervical carcinoma contains a small subpopulation of cells expressing ALDH1 [1]. In this study, we used small interfering RNA to suppress ALDH1 expression and introduced an ALDH1 reporting vector into HeLa cells followed by various in vitro assays. We showed that knockdown of ALDH1 expression reduced the cell migration ability of HeLa cells, whereas augmented expression of ALDH1 increased cell migration. However, there was no difference in the cellular proliferation, apoptosis, cell cycle, and invasion. These results indicate that ALDH1 is directly involved in HeLa migration.
    Full-text · Article · Apr 2015 · Tumor Biology
  • Source
    [Show abstract] [Hide abstract] ABSTRACT: In order to increase the prognostic significance of polymerase chain reaction (PCR) data it has been suggested that quantitative PCR can be used to measure tumor burden. However, this option has not yet been definitely supported or refuted in patients with follicular lymphoma (FL). We decided to evaluate whether knowledge of the quantitative level of minimal residual disease and its variations can be of use in the management of FL patients. We used qualitative and competitive PCR to study 11 patients with refractory or relapsed FL harboring the t(14;18) translocation who underwent autologous (nine patients) or allogeneic (two patients) stem cell transplantation (SCT). Competitive PCR was performed with a multiple competitor carrying specific sequences including Bcl2/IgH MBR and mcr, and the beta-globin gene. After a median post-SCT follow-up of 44 months (range, 12-62), overall survival was 91% and disease-free survival 82%. The quantitative PCR data showed that: 1) effective chemotherapy before SCT substantially (1-2 log) reduced the tumor burden in the bone marrow (BM); 2) the increase in rearranged DNA detected in BM was associated with disease progression and relapse; 3) a PCR-negative autograft seemed to lead to lasting molecular remission even when it was performed in patients with a low level of BM infiltration before transplant; and 4) allo-SCT made and maintained the BM PCR negative even in the presence of a greater tumor burden before SCT. Six of the nine patients having CR after SCT (four auto and two allo) are in continuous molecular remission. In FL patients qualitative and quantitative PCR may provide data that can be helpful for the prognostic evaluation of tumor progression and the early detection of impending relapse by highlighting biological features such as the quality of the infused material, the tumor burden at transplant, and the behavior of tumor cells after transplant.
    Full-text · Article · Jan 2005 · Tumori
  • Source
    [Show abstract] [Hide abstract] ABSTRACT: Die für das follikuläre Lymphom charakteristische Translokation t(14;18) verlagert das Onkogen bcl-2 auf Chromosom 18 in den Schwerketten-Immunglobulin-Genlokus auf Chromosom 14. Daraus resultiert eine bcl-2-Überexpression mit Hemmung der Apoptose während der B-Zell-Entwicklung. Im Rahmen dieser Arbeit konnte gezeigt werden, dass die quantitative „real-time“ PCR (RQ-PCR) mit Hilfe der LightCycler®-Technologie genaue und reproduzierbare Messungen der t(14;18)-positiven Zellen im Knochenmark (KM) und im peripheren Blut (PB) möglich macht. Dabei wurde die t(14;18)-spezifische MBR („major breakpoint region“)-DNA amplifiziert und die Menge der Translokation-positiven Zellen quantifiziert. Die Methode konnte eine Tumorzelle in 105 normalen mononukleären Zellen nachweisen. Wir prüften den Zusammenhang zwischen molekularbiologisch nachweisbaren t(14;18)-positiven Zellen mit klinischen Parametern wie Knochenmarkinfiltration oder Remissionsstatus und der Behandlung von Patienten mit follikulärem Lymphom. Hierfür wurden 52 Patienten innerhalb von dreieinhalb Jahren zu verschiedenen Zeitpunkten KM- und PB-Proben entnommen und daraus gewonnene DNA im LightCycler® auf die Translokation untersucht. Der Vergleich zwischen KM und PB zeigte ähnliche Werte t(14;18)-positiver Zellen, so dass zukünftig PB-Proben für das molekulare Monitoring ausreichen. Die Menge der t(14;18)-positiven Zellen korrelierte signifikant mit der Krankheitsphase, da wir im Gegensatz zu Patienten mit Erstdiagnose in Patienten mit Rezidiv zehnmal höhere Mengen an t(14;18)-positiven Zellen nachweisen konnten. Bei Erstdiagnose-Patienten im Stadium IV erhielten wir höhere Werte als bei solchen in den Stadien II und III. Proben aus histopathologisch infiltriertem Knochenmark enthielten mehr t(14;18)-positive Zellen als solche aus morphologisch unauffälligem Mark. Darüber hinaus gestattete die quantitative PCR die serielle molekulare Untersuchung einer „minimal residual disease“ (MRD) in Patienten, die eine konventionelle oder dosiserhöhte zytotoxische Chemotherapie in Kombination mit dem monoklonalen Anti-CD20-Antikörper Rituximab (R) erhielten. Im Rahmen dieser Studie wurde erstmals die Wirksamkeit des Antikörpers in Kombination mit Chemotherapie als Erstlinientherapie bei Patienten mit follikulärem Lymphom auf molekularer Ebene untersucht. Im Hinblick auf das „in vivo-Purging“-Potential verschiedener Chemotherapeutika erwies sich die Kombination R/CHOP wirkungsvoller als die Kombination R/FM. Nach Hochdosistherapie mit R/BEAM sanken die t(14;18)-Werte sogar unter die Nachweisgrenze. Diese Arbeit zeigt, dass die RQ-PCR mit dem LightCycler® eine zuverlässige und sensitive Methode zur Quantifizierung t(14;18)-positiver Zellen im KM und PB von Patienten mit follikulärem Lymphom darstellt. Sie erlaubt die Untersuchung des therapeutischen Ansprechens sowie die Evaluation neuer Therapiekonzepte auf molekularer Ebene. Objectives: Quantitative real-time PCR (qPCR) is a suitable method to measure residual disease in hematological malignancies. Our objective was to assess a LightCycler®-based qPCR for t(14;18)(q32;q21)(IgH/bcl-2)-positive cells quantification in the context of clinical and morphopathological characteristics of patients with follicular lymphoma treated with Rituximab (R) in combination with conventional or high-dose chemotherapy. Methods: 270 bone marrow (BM) and peripheral blood (PB) samples collected from 52 patients with follicular lymphoma at diagnosis or at relapse before or sequentially during therapy were examined by qPCR and nested-PCR. Results: A greater amount of t(14;18) positive cells was observed in BM in comparison with PB in 76% of paired samples. Presence and number of t(14;18)-positive cells in BM and PB correlated with lymphoma activity. Significantly higher numbers of lymphoma cells were found in non-remission patients compared to patients in clinical remission. While non-remission, ten-fold higher numbers were measured at relapse than at diagnosis. During remission, significantly higher levels were found in partial compared to complete remission. During first-line therapy, R/CHOP had higher in vivo purging ability than R/FM. After R/HAM or R/BEAM, the level of t(14;18)-positive cells dropped under the detection limit in 80% of patients. Conclusions: LightCycler® qPCR is a reliable method for quantitative molecular monitoring of t(14;18) positive cells in BM and PB of patients with follicular lymphoma. It reflects clinical characteristics of patients and allows assessment of response to different treatment regimens on a molecular level.
    Full-text · Article ·
Show more