detection of germ cell tumor cells in apheresis products using polymerase chain reaction1 · vol....

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: [email protected].

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.

Research. on August 6, 2020. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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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



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).



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.


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



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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1998;4:93-98. Clin Cancer Res Y Fan, L Einhorn, S Saxman, et al. polymerase chain reaction.Detection of germ cell tumor cells in apheresis products using

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