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Vol. 4, 93-98, January 1998 Clinical Cancer Research 93
Detection of Germ Cell Tumor Cells in Apheresis Products Using
Polymerase Chain Reaction1
Yi Fan, Lawrence Einhorn, Scott Saxman,
Barry Katz, Rafat Abonour, and
Kenneth Cornetta2
Divisions of Hematology/Oncology fY. F.. L. E., S. S., R. A.. K. C.]
and Biostatistics [B. K.], Department of Medicine, Indiana UniversitySchool of Medicine, Indianapolis, Indiana 46202-5121
ABSTRACT
The contamination of apheresis products with tumor
cells was evaluated in patients undergoing autologous pe-
ripheral blood stem cell transplantation for germ cell tu-mors. A blinded, retrospective analysis was performed on 63
apheresis products from 28 patients using the PCR and
primers for I� human chorionic gonadotropin (�-HCG). Of
the 20 patients with �-HCG-secreting tumors, 8 apheresisproducts from 7 patients were PCR positive. PCR was neg-
ative in the 8 patients whose tumors did not secrete f3-HCG.
Twenty-two apheresis products from patients with lym-
phoma and breast cancer were negative for �3-HCG expres-
sion. Evaluating the 20 patients with �-HCG-secreting tu-
mors, 100% of PCR-positive patients had elevated serum
�3-HCG at the time of apheresis compared to 46.2% of
PCR-negative patients (P = 0.04). A positive PCR was also
associated with a higher serum �-HCG at diagnosis (P =
0.03). Patients receiving a PCR-positive product had a
higher relapse rate (85.7 versus 61.5%) and were more likely
to have visceral metastasis (100 versus 61.5%), although the
numbers did not reach statistical significance (P = 0.35 and
0.11, respectively). The finding of �-HCG mRNA in aphere-sis products strongly suggests the presence of circulating
tumor cells in a significant number of germ cell patients
undergoing autobogous transplantation. This assay may be
useful in monitoring attempts at tumor cell depletion and in
developing improved prognostic models for assessing risk ofrelapse after transplantation.
Received 7/28/97; revised 10/13/97; accepted 10/23/97.
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with I 8 U.S.C. Section 1734 solely to
indicate this fact.
I This work was supported in part by American Cancer Society Grants
CRTG-97-042-EDT (to R. A.) and IRG-l6l (to S. S.) and a Center of
Excellence in Molecular Hematology Award (NIDDK, P50DK492I 8).R. A. is the recipient of a CAP. Award from the National Centers forResearch Resources (NIH MOl RR00750).
2 To whom requests for reprints should be addressed, at Bone MarrowTransplantation Program, Indiana University, 1044 West Walnut Street,
R4 202, Indianapolis, IN 46202-5121. Phone: (317) 274-0386; Fax:
(317) 274-0396; E-mail: ken_cornetta@iucc.iupui.edu.
INTRODUCTION
Although germ cell neoplasms are rare, they still rank as a
leading cause of cancer death in young adult men. Clinical
investigations in GCTs3 at Indiana University and elsewhere
during the 1970s and early 1980s saw the development of
well-tolerated effective cisplatin-combination chemotherapy for
disseminated germ cell cancer (1). Also, initial attempts at
developing predictive prognostic models allowed for the devel-
opment of clinical trials based on risk of failure (2-3). For
good-risk patients, trials sought to diminish therapy-rebated tox-
icities (4-7). For poor-risk patients, trials have sought treat-
ments that improve therapeutic results. Examples include dose
intensity, substitution of ifosfamide for bleomycin, additional
drugs, or initial autologous transplantation (8-1 1).
For those patients who relapse after initial therapy. con-
ventional-dose salvage chemotherapy results in durable com-
plete remissions for only 20-30% of patients (1). Patients who
relapse after salvage therapy or are platinum refractory are
incurable with conventional-dose chemotherapy. The use of
high-dose chemotherapy and autobogous BMT has been suc-
cessfub in some of these refractory patients (12-15). Certain
subsets of GCTs, in particular those refractory to platinum-
based chemotherapy (progression within 4 weeks of recent
platinum regimen) or nonseminomatous tumors arising in the
mediastinum, only rarely benefit from autologous BMT or other
salvage therapies ( 1 6, 17).
One potential cause of treatment failure after autobogous
transplantation for GCTs is the reinfusion of cancer cells mad-
vertently collected at the time of stem cell collection. Contam-
ination of bone marrow products has been shown to contribute
to disease relapse after autobogous transplantation for childhood
acute myeboid leukemia and adult chronic myeboid leukemia
using retroviral gene marking (18, 19). Whereas definitive ev-
idence of relapse arising from transplantation of lymphoma cells
will require similar marking studies, patients with detectable
lymphoma cells in stem cell products have a significantly higher
risk of relapse compared to individuals in which no detectable
lymphoma cells are present (20). In breast cancer, tumor cell
contamination of bone marrow and stem cell products is a
common finding (21-23), although the ability of reinfused
breast cancer cells to cause disease relapse has not been defi-
nitely shown (24).
In this study, we chose to evaluate apheresis products for
evidence of GCT contamination. Tumor cell detection used a
PCR-based method with primers specific for �3-HCG mRNA.
Patients with detectable tumor cells were compared to PCR-
negative patients with regard to serum �3-HCG levels at diag-
3 The abbreviations used are: GCT, germ cell tumor; BMT. bone mar-row transplantation; �-HCG, �3 human chorionic gonadotropin; AuSCT,
autobogous stem cell transplantation.
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I II I I I I
94 Germ Cell Tumor Cells in Apheresis Products
nosis, serum 3-HCG at apheresis, sites of disease, and evidence
of relapse after transplantation.
MATERIALS AND METHODS
Patient Samples. This study represents a retrospective
analysis of patients undergoing AuSCT. Apheresis products
were collected after a uniform mobilization protocol using daily
s.c. granulocyte colony-stimulating factor (10 p.g/kg/day) for 4
days before collection and daily during apheresis. Target cell
numbers were �6.5 X 108 mononuclear cells/kg for patients
with lymphoma and �5 X 108 MNC/kg for breast cancer
patients. Germ cell patients were scheduled for tandem trans-
plants, and � 10 x 108 mononuclear cells/kg were collected. For
GCT patients, one to four apheresis samples were required to
reach the target number of cells. Consent for use of the apheresis
product in research studies was obtained at the time of stem cell
collection.
Cell Isolation and PCR. RNA was isolated from l-ml
apheresis samples that had been stored in liquid nitrogen since
the day of collection. For analysis, cells were thawed in a 37#{176}C
water bath, washed once in 100% fetal bovine serum, and
resuspended in Iscove’s modified Dulbecco’s medium with 10%
fetal bovine serum. Cells were enumerated, and 1-5 X 106
viable cells were suspended in 1 ml of Tn Reagent (Molecular
Research Center, Inc., Cincinnati, OH). RNA was isolated and
resuspended in 12 p.1 of diethyl pyrocarbonate treated water.
Ten p.1 of isolated RNA were added to 90 �il of DNAse I
solution (9 parts lOX DNAse I buffer:1 part 18 units/�il DNAse
I; Boehringer Mannheim, Indianapolis, IN). After a 15-mn
incubation at room temperature, the RNA was purified using a
High Pure filter tube (Boehringer Mannheim) and resuspended
in 20 p.1 of diethyl pyrocarbonate-treated water. The integrity
and quantity of RNA were verified by spectrophotometry and
gel electrophoresis. One p.g of RNA with 30 pmol of random
hexamer primers (Promega, Madison, WI) were heated to 72#{176}C
for 2 mm and then rapidly quenched on ice. To this mixture, SO
mM Tris-HC1, 30 mM KC1, 8 mr�i MgC12, 1 msi DTT (pH 8.5),
0.5 mM deoxynucleotide triphosphate, 0.75 unit of RNase in-
hibitor, and 300 units of avian myeloblastosis virus reverse
transcriptase were added and incubated at 42#{176}Cfor 1 h. After
cDNA generation, the reverse transcriptase was inactivated by
heating to 94#{176}Cfor 5 mm. Primary and secondary PCR included
35 cycles of 1 mm of denaturation at 94#{176}C, 1 mm of primer
annealing at 55#{176}C, and 1.5 mm of extension at 72#{176}Cafter an
initial denaturation for 5 mm at 94#{176}C.Ten p.1 of each cDNA
product were placed in a sterile 0.5-mi PCR tube in a final
volume of 100 p.1 with the following reaction conditions: 10 mr�i
Tris-HC1 (pH 8.3); 50 mM KC1; 2.0 mist MgC12; 0.2 mrvi each of
dATP, dCTP, dGTP, and dTTP; 500 pmol of each of the two
oligonucleotide primers specific for �3-HCG [gene products and
2.5 units of Taq polymerase (Perkin-Elmer Corp.)]. Two sets of
primer pairs specific for the majority of the �3-HCG gene cluster
(four of six) were designed. The first round primers were 5’-
TCGGGTCACGGCCTCCT-3’ (- 35 l/-335) and 5 ‘-AG-
GATCGGGGTGTCCGA-3’ (464/480), which amplified 831 bp
from cDNA and 1416 bp from genomic DNA (25, 26). The
nested PCR was initiated in this study by using a secondary pair
of primers, S’-ACATGGGCATCCAAGGAG-3’ (44/61) and
-� Intron t -� In(ron II �- �-
hCGSI (351-352nt) hCGS2 (234-.236nt) hCGAS2(-351-335) (44-61) (436-453) (464-480)
Fig. 1 PCR primers within the �3-HCG gene locus. The �3-HCG genelocus contains three exons (boxes) and two introns (lines). The 5’
untranslated portion of �3-HCG mRNA contains approximately 360nucleotides. The 3’ region codes for approximately 30 amino acids that
share no homology with other glycoprotein hormones. Nested PCR
primers used in sequential PCR amplification are shown as arrows. The
two antisense primers are within the unique 3’ region of the mRNA.
5 ‘-AGTCGGGATGGACTTGGA-3’ (436/453), which gave a
410-bp band in cDNA and a 644-bp band in genomic DNA.
PCR Laboratory Procedures. The laboratory adheres
to strict policies designed to decrease the likelihood of false
positive results due to contamination by previously amplified
DNA. To this end, samples were prepared in a dedicated PCR
hood (clean room) and amplified and electrophoresed in a
separate products room. Before initiating PCR work in either
room, personnel are instructed to always wash hands and don
gloves, to wear lab coats that have not come from other areas,
and to decontaminate all equipment using bleach or UV irradi-
ation. The PCR hood is decontaminated daily using bleach and
UV irradiation before and after each use. Nucleic acids, RNA,
and DNA from control or patient samples were stored separately
from PCR reagents, and amplified products, which were never
removed from the products room, were disposed of once results
were obtained.
Statistical Analysis. Fisher’ s exact tests were run to
compare clinical characteristics (elevated serum �3-HCG at
apheresis, visceral metastasis, and relapse) and the PCR results
among those patients with �3-HCG-secreting tumors. The ability
of serum �3-HCG levels at diagnosis or at the time of apheresis
to predict relapse was assessed with logistic regression (27)
using relapse as the dependent variable and the log of serum
�3-HCG as the independent variable. t tests were done to com-
pare serum �3-HCG levels for PCR-positive and PCR-negative
patients, and a log transformation was performed to correct for
unequal variances.
RESULTS
PCR Amplification of �-HCG mRNA Using NestedPrimers. Using PCR for detection of �3-HCG expression is
complicated by two factors: (a) 3-HCG is a glycoprotein hor-
mone with extensive homology with three other hormones,
luteinizing hormone, follicule-stimulating hormone, and thy-
roid-stimulating hormone. Differences in the 3’ portion of the
f3-HCG gene permit the design of specific PCR primers (25, 26),
but the relatively small introns within the genomic sequence
generate nested PCR products that vary by only 234-236 bp
from the mRNA product (Fig. 1); and (b) a second factor to
consider is the potential competition for primers between
genomic and mRNA sequences. �3-HCG is encoded within a
multigene cluster composed of six homologous sequences, each
containing three exons and two introns (26). Because we antic-
ipate that very few cells in the peripheral blood express �3-HCG
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A J32M � � � �
�-HCG2#{176} � � -, �
�-.- � .4-, B�HCG 644 bp�enomlc DNA. � . .4-S., �3-HCG 410
cDNA
Clinical Cancer Research 95
lOl8bp-
506 bp -
304 bp �
- 4
0
Z(�o
00
C)I- C)C) �.�o � E E�C) C�JC) tu tuC)�xmcncotn
B
1� � � .�- 132M 344 bp
Fig. 2 Reverse transcription-PCR of apheresis products using nested
primers for �3-HCG. A, comparison of signals obtained from apheresissamples with and without DNAse I treatment. Lane 1, Marker, molec-
ular weight marker; Lane 2, H,O control, PCR reaction run with H2Osubstituting for RNA; Lane 3, BeWo, f3-HCG-secreting BeWo cell line
RNA; Lane 4, Sample w/o DNase I, positive apheresis sample after PCR
amplification without DNAse I treatment; Lane 5, Sample wi DNase I,apheresis product shown in Lane 4 treated with DNase I before reverse
transcriptase and PCR; Lane 6, BeWo genomic DNA, PCR of genomicDNA from BeWo cell line. B, 32M, PCR for �32-microglobulin as anRNA control.
mRNA, whereas all blood cells will contain six copies of the
genomic sequence, primer competition may decrease the sensi-
tivity of detecting small numbers of 3-HCG-expressing cells.
Therefore, we chose to DNAse treat isolated RNA before re-
verse transcriptase treatment to decrease the potential for primer
competition. The utility of this approach is shown in Fig. 2. PCR
of cDNA from the �3-HCG-expressing BeWo cell line (Ameri-
can Type Culture Collection CCL 98) generates a 4l0-bp band
corresponding to �3-HCG mRNA (Fig. 2, Lane 3). In contrast,
RNA prepared from apheresis products demonstrates a predom-
inant band of 644 bp corresponding to the genomic DNA band
(Fig. 3, Lane 4). The 644-bp band indicates inadvertent con-
tamination of the RNA preparation with genomic DNA. Pre-
treating isolated RNA with DNAse I before reverse transcription
eliminates or greatly diminishes the genomic DNA band while
maintaining or increasing the intensity of the corresponding
mRNA band (Fig. 2, Lane 5). DNAse I treatment maintained
sensitivity of �-HCG detection, because as little as one 3-HCG-
expressing BeWo cell was detected among 106 3-HCG-nonex-
pressing K562 cells (Fig. 3).
PCR Analysis of Apheresis Products from Non-GCTPatients. The frequency of �3-HCG-expressing cells in the
peripheral blood of cancer patients undergoing apheresis is
unknown. To begin to address this question, RNA was prepared
from 1 1 apheresis products from 5 men undergoing AuSCT for
lymphoma and 1 1 apheresis products obtained from 7 women
undergoing AuSCT for breast cancer. All patients were mobi-
lized in an identical fashion to those GCT patients analyzed in
106 io� 104103102 101 iOO o
Fig. 3 Detection of �3-HCG mRNA sensitivity after DNase I treatment.
Various concentrations of �3-HCG-expressing BeWo cells were mixedwith a constant number of nonexpressing K562 cells. RNA was isolated
from each mixture, treated with DNase I for 15 mm, and then subjectedto sequential reverse transcription-PCR using nested primers for �3-HCG
(�3-HCG 2#{176})or single amplification using �32-microgbobulin (�32M) prim-
ers. The top row indicates the number of BeWo cells mixed with 106
K562 cells.
this study (granubocyte colony-stimulating factor, 10 �i.g1kg/day
for 4 days). No detectable �3-HCG mRNA was found in the 22
samples tested (Table 1).
PCR Analysis of Apheresis Products in GCT Patients.To evaluate �3-HCG expression in mobilized peripheral blood of
patients undergoing tandem AuSCT for relapsed or refractory
GCT, we retrospectively analyzed 63 apheresis products from
28 consecutive patients. The PCR analysis was blinded to the
serologic �3-HCG and clinical status of transplanted patients.
None of the 63 products tested had detectable �3-HCG mRNA
after the first round of amplification. Eight samples from seven
patients had detectable 3-HCG mRNA after the second round of
amplification (Table 1). The PCR findings were then compared
to serologic �3-HCG data, because approximately one-third of
GCTs do not secrete �3-HCG. Among the 28 patients evaluated,
20 had an elevated serum �3-HCG at some time during their
course, and all 7 patients with a positive PCR were among those
patients with �3-HCG-secreting tumors (Table 1).
At the time of apheresis, elevated serum �3-HCG was noted
in 100% of PCR-positive patients compared to 46.2% of patients
whose PCR was negative (Table 2). This difference was statis-
tically significant when compared by Fisher’s exact test (P =
0.04). As shown in Fig. 4, a positive PCR was associated with
a slightly higher serum �3-HCG at the time of apheresis com-
pared with PCR-negative values (mean ± SD, 818 ± 1772
versus 1 17 ± 232, respectively). Using t test analysis and a log
transformation to correct for unequal variances, the serum
�3-HCG at apheresis was slightly higher for PCR-positive pa-
tients (P = 0.09). Interestingly, a positive PCR was also asso-
ciated with a higher serum �3-HCG at diagnosis (mean,
169,986 ± 207,712 versus 27,886 ± 77,038, respectively), a
difference that was statistically significant (P = 0.03). Of those
patients with detectable �3-HCG by PCR, 100% had a history of
visceral metastasis (pulmonary or hepatic), compared to 61.5%
of patients whose PCR was negative (Table 2), but the differ-
ence did not reach statistical significance (P = 0.1 1). A greater
number of patients with a positive PCR relapsed after transplan-
tation (85.7 versus 61.5%), but the numbers were not statisti-
cally significant (P = 0.35).
Research. on August 6, 2020. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Table 2 Clinical characteristics of PCR+ and PCR- patients among
those with �3-HCG-secreting tumors (number with characteristic/total
number of patients)
100000
10000
1000
100
10
$
$
$
A.
1�0
Sci,
B.
100000
10000
1,C.,I-�E iooo
U)100
10
PCR+ pc’R.
*
t
PCR+ PCR-
Fig. 4 Comparison of serum �-HCG and PCR assay for �-HCG
mRNA. The serum �3-HCG values at the time of apheresis (A) and at
diagnosis (B) are illustrated for patients with and without detectable�3-HCG mRNA by PCR.
96 Germ Cell Tumor Cells in Apheresis Products
Table I Evaluation of apheresis products for the presence of �3-HCG mRNA using nested PCR primers
Elevated serum No. of No. of Apheresis with
�3-HCG’� Sex patients apheresis tested �3-HCG by PCRPatients with
3-HCG by PCR
Lymphoma
Breast cancer
Germ cell tumorGerm cell tumorGerm cell tumor
NT Male 7 1 1 0
Negative Female 7 1 1 0
All patients Male 28 63 8
Negative Male 8 16 0Positive Male 20 47 8
0
0
7
07
(I Elevated seru m �3-HCG refers to patients with elevated �3-HCG at any time during their disease. NT. not tested.
Elevated
�3-HCG at BMT
Visceral
metastasis Relapse
PCR+ 7/7 7/7 6/7
PCR- 6/13 8/13 8/13
The ability of serum �3-HCG to predict relapse, irrespective
of PCR results, was analyzed with logistic regression using
relapse as the dependent variable and log of �3-HCG at diagnosis
or log of �3-HCG at BMT as the independent variable. In both
cases, the odds ratio was not significant (P = 0. 14 and 0.069,
respectively).
DISCUSSION
In this study, OCT patients undergoing autobogous trans-
plantation were found to have circulating tumor cells at the time
of apheresis, as measured by PCR for �3-HCG mRNA. The PCR
assay seemed specific for �-HCG-secreting tumors, and no
detectable �3-HCG mRNA was noted in apheresis samples from
patients with breast cancer, bymphoma, and GCTs that did not
secrete �3-HCG. Positive detection of �3-HCG mRNA was asso-
ciated with poor-risk factors at diagnosis, specifically a mark-
edly elevated serum �3-HCG. All seven patients with detectable
�3-HCG had a history of pulmonary or liver metastasis, and six
of the seven patients relapsed after transplantation.
The high cure rate of GCTs challenges us to intensify
treatment for patients destined to fail standard therapies without
increasing treatment-related toxicity for those patients destined
to be cured. A variety of prognostic factors have been identified,
and classification systems have successfully used clinical and
serologic parameters to identify those patients at high risk of
treatment failure ( I , 2, 28). Although these systems are accurate
at predicting those patients who are destined to do well (4-7), a
relatively large number of curable patients are included in
high-risk groups. The next level of prognostication will likely
come from combinations of currently available and newly de-
veboped laboratory-based predictors of outcome. PCR has been
used to identify cancer cells in apheresis products of patients
with hematobogic malignancies and breast cancer (20, 23). It has
also been used to predict disease relapse after abbogeneic BMT
for patients with chronic myeloid leukemia and acute lymphoid
leukemia (29-3 1 ). Interestingly. detecting minimal residual dis-
ease does not necessarily predict disease relapse after standard-
dose chemotherapy or BMT, but a quantitative increase in
PCR-detectable disease has correlated with disease recurrence
(29, 32, 33).
In this study, we chose to evaluate patients for the presence
of �3-HCG mRNA in peripheral blood cells as a marker of
circulating tumor cells. Using PCR, �3-HCG mRNA was iden-
tified in eight apheresis products from seven patients. Data to
support our hypothesis that detection of �3-HCG mRNA in
peripheral blood represents circulating GCT cells include: (a) no
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Clinical Cancer Research 97
5. Loehrer, P., Johnson, D., Elson, P., Einhorn, L., and Trump, D.Importance of bleomycin in favorable-prognosis disseminated germ cell
detectable �3-HCG mRNA in 22 apheresis products from breast
and lymphoma patients; (b) no detectable �3-HCG mRNA in 16
apheresis products from GCT patients whose tumors did not
secrete �3-HCG; (c) 100% of PCR-positive patients had elevated
serologic �3-HCG levels at the time of apheresis; and (d) all
seven PCR-positive patients had a history of metastatic disease
to the lung or liver, indicating prior hematogenous spread of
tumor cells.
The precise role of �3-HCG mRNA PCR in determining
prognosis after AuSCT requires further evaluation. Our finding
that six of seven patients with a positive PCR relapsed suggests
that this may be more accurate than serum �3-HCG at the time of
transplantation in predicting outcome. Because serum �3-HCG at
diagnosis, a known poor prognostic factor, was associated with
a positive PCR, a multivariate analysis on a larger group of
patients will be required to determine if PCR will serve as an
independent variable in predicting response to autologous trans-
plantation. Of note, the one patient with a positive PCR who has
not relapsed received three cycles of oral VP-l6 (50 mg/m2
daily for 21 days of a 28-day cycle) after transplantation (34).
The utility of using PCR to identify candidates for posttrans-
plantation chemotherapy deserves further study.
The ability to detect circulating GCT cells may have other
applications in GCT management. PCR performed at diagnosis
may identify patients with advanced disease destined to fail
standard chemotherapy. PCR may also identify patients cur-
rently treated with surgery alone who are destined to relapse and
may benefit from adjuvant chemotherapy. In the setting of
autologous transplantation, PCR may identify those patients
who could benefit from stem cell manipulations such as tumor
cell purging or CD34 selection.
The finding of �3-HCG mRNA in apheresis products
strongly suggests the presence of circulating tumor cells in germ
cell patients undergoing autobogous transplantation. Whether
these tumor cells have the capacity to contribute to disease
relapse remains to be determined. Nevertheless, PCR technol-
ogy may prove a useful prognostic tool in predicting response to
transplantation and identifying those patients requiring addi-
tional therapy after AuSCT.
ACKNOWLEDGMENTS
We thank the Indiana University Stem Cell Laboratory, pheresis
nurses, and BMT staff for excellent technical and clinical care.
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