© 2000 Nature America, Inc. 0929-1903/00/$15.00/⫹0
www.nature.com/cgt
Biological activity and safety of adenoviral vector-expressed
wild-type p53 after intratumoral injection in melanoma and
breast cancer patients with p53-overexpressing tumors
Reinhard Dummer,1 Jonas Bergh,2 Ylva Karlsson,2 Jo Ann Horowitz,3 Nanno H. Mulder,4
Daan Ten Bokkel Huinink,5 Günter Burg,1 Günther Hofbauer,1 and Susanne Osanto5
1
Department of Dermatology, University Hospital of Zürich, Zürich, Switzerland; 2Department of Oncology,
University Hospital of Uppsala, Uppsala, Sweden; 3Oncology, Clinical Research, Schering-Plough Research
Institute, Kenilworth, New Jersey 07033; 4Department of Internal Medicine, University Hospital Groningen,
Groningen, The Netherlands; and 5Department of Clinical Oncology, Leids Universitair Medisch Centrum,
Leiden, The Netherlands.
p53 mutations are common genetic alterations in human cancer. Gene transfer of a wild-type (wt) p53 gene reverses the loss of
normal p53 function in vitro and in vivo. A phase I dose escalation study of single intratumoral (i.t.) injection of a
replication-defective adenoviral expression vector containing wt p53 was carried out in patients with metastatic melanoma or breast
cancer with increased p53 protein immunoreactivity in pretreatment tumor biopsies. The biological activity of the injected wt p53
was assayed by reverse transcriptase-polymerase chain reaction in tumor tissue. A total of six (five melanoma and one breast
adenocarcinoma) patients were treated at dose levels dependent upon tumor size/dose escalation sequence. Five of six patients
became positive for the transfer of wt p53 into tumor tissue 2 days after injection of the vector. Of the four patients assayed, all
developed anti-adenoviral antibodies. Adverse reactions associated with i.t. injection were mild, with no obvious correlation
between the incidence, severity, or relationship of the events and drug dose. p53 gene therapy by i.t. injection of a
replication-defective adenoviral expression vector is safe, feasible, and biologically effective (with respect to transduction frequency)
in patients with either metastatic melanoma or breast cancer. Cancer Gene Therapy (2000) 7, 1069 –1076
Key words: Melanoma; breast cancer; p53; adenoviral vector; gene therapy; phase I trial.
M
utations of the tumor suppressor gene p53 are
detected in ⬃50% of colon and non-small cell lung
cancers (NSCLCs)1–3 and in 20 –25% of breast cancers,4
with missense mutations within the DNA binding domain being the most common gene alteration observed.5
The resultant defective protein accumulates intracellularly at high concentrations,6 where the loss of a functional p53 protein impairs both cell cycle control and
DNA damage repair mechanisms.7 Mice with a homozygous deletion of p53 have a greatly enhanced susceptibility to early cancer development.8 In humans, a familial germline mutation in p53 (Li-Fraumeni syndrome)
resulting in increased breast cancer, sarcomas, and other
neoplasms has been described.9,10 Also, mutations affecting p53 may curtail the apoptosis induced by several
cytotoxic agents or radiation.11,12
Structural alterations in the p53 gene of tumors in
NSCLC are associated with a worse prognosis.13–15 A
Received December 17, 1999; accepted April 15, 2000.
Address correspondence and reprint requests to Dr. Reinhard Dummer,
Department of Dermatology, University Hospital of Zürich, Gloriastr. 31,
CH-8091 Zürich, Switzerland. E-mail address: [email protected]
Cancer Gene Therapy, Vol 7, No 7, 2000: pp 1069 –1076
recent study reported induction of apoptosis and tumor
regression in patients with advanced NSCLC after injection of a retroviral vector containing wild-type (wt) p53
into tumors.16 However, although vector sequences were
detected in posttreatment biopsies by DNA polymerase
chain reaction (PCR) or in situ hybridization, there was
no demonstration of transgene expression. Intratumoral
(i.t.) injection of a replication-deficient adenoviral vector containing wt p5317 demonstrated effective local
disease control, as well as vector-specific wt p53 RNA
expression, in four of six NSCLC patients.
The incidence of melanoma has increased rapidly
worldwide.18 The prognosis for patients with thin primary tumors is excellent after adequate surgical removal, but a significant proportion of these patients still
develop distant metastases that ultimately lead to
death.19 The incidence of mutant p53 expression in
primary melanoma is low;20,21 a much higher rate of p53
alterations is found for advanced lesions.22 As melanoma is characterized by a resistance to cytotoxic drugs,
the alteration of p53 mutant phenotypes might provide a
reasonable addition to the present repertoire of therapeutic interventions. In addition, most melanomas ex1069
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DUMMER, BERGH, KARLSSON, ET AL: PHASE I TRIAL USING A P53-EXPRESSING ADENOVIRAL VECTOR
press the human Coxsackie and adenovirus receptors,
which facilitate adenoviral entry into these tumor cells.23
Breast cancer, which accounts for 18% of all cancer
deaths in women, is the leading cause of death for
women between the ages of 40 and 55. For patients with
breast cancer, local control, in addition to control of
distant metastases, is one of the common treatment
approaches undertaken. The mutant p53 genotype has
been identified in 20 –30% of all patients with breast
cancer24 and has prognostic implications.25 Introduction
of the wt p53 phenotype in human breast cancer cell
lines has lead to suppression of tumor cell growth in cell
lines.26,27 After the delivery of normal p53 via liposomes,
tumor regressions and decreased metastatic spread occurred in xenografts of a p53 mutant human breast
cancer in athymic mice.28 Regression of xenografts
derived from three breast carcinoma lines has also been
reported in severe combined immunodeficient and athymic mice after the injection of the wt p53 adenoviral
construct (SCH 58500) used in the present study.29
A phase I study was performed to examine the ability
to alter the p53 phenotype in cutaneous tumors from
patients with melanoma or breast cancer after the
injection of a wt p53 contained in a replication-defective
adenovirus vector. This vector has been successfully used
in NSCLC patients to achieve local control of the
tumor.17
contraception for at least 6 months after study treatment;
uncontrolled serious infections; seropositivity for the HIV;
prior local antitumoral therapy directed against cutaneous or
subcutaneous lesions; systemic antitumoral therapy within 28
days of study medication; or the receipt of immunosuppressive
or corticosteroid treatment within the last 3 months before
entry into the study. Acute adenoviral infection, as determined
by enzyme-linked immunosorbent assay (ELISA) screening,
was ruled out within 24 hours of therapy. All patients gave
written informed consent.
Study design
This was a multicenter, open-label, single-dose, phase I dose
escalation study. One patient was treated at each dose level,
and dose escalation was continued until two consecutive dose
levels confirmed biological activity of the treatment, as determined by the presence of wt p53 using reverse transcriptase
(RT)-PCR in at least two patients at the same dose level, or
until any significant dose-limiting toxicity was encountered.
Each patient was followed weekly for 28 days, with day 1 being
the date of treatment. Upon completion of the observation
period, all patients were followed at the study centers at
regular intervals. The consent form and the protocol were
approved by the ethics committee of the participating center,
the National Health Authorities (Switzerland), the Ministry of
Environment (The Netherlands), and the Medical Products
Agency (Sweden). The study was performed according to the
Declaration of Helsinki and according to the principles of
Good Clinical Practice. All biosafety precautions and procedures required by the Institutional Biosafety Committee and
local regulatory authorities were observed.
PATIENTS AND METHODS
Patients
All patients enrolled in the study either had histologically
confirmed cutaneous or subcutaneous recurrence of metastatic
melanoma at least 5 cm from the primary scar (five patients) or
breast adenocarcinoma (one patient) for which curative treatment alternatives did not exist, with evidence of enhanced p53
protein levels in the tumor tissue. Evidence of enhanced p53
protein levels was determined by immunohistochemical (IHC)
detection of strong nuclear p53 immunoreactivity by monoclonal antibodies (mAbs) Pab 1801 and/or Pab 240 (PharMingen,
San Diego, Calif) in at least 10% of the tumor cells. A positive
reaction served as a surrogate marker of mutant p53 status.6
Currently, it is known that IHC gives false-positive rates of up
to 30% and false-negative rates of up to 10%, and that
sequencing for p53 status is the more accurate method.30,31
However, when this study was planned, these findings were not
generally accepted. Because the primary objective of this study
was transgene expression and safety, and immunohistochemistry is a rapid test and was readily available at all study sites,
patients were enrolled based on IHC rather than the more
stringent technology of gene sequencing. Staining of paraffinembedded tissue was performed using a streptavidin-biotin
detection procedure, and reactivity was visualized by reaction
with 3,3⬘ diaminobenzidine. Sections were scored as negative if
⬍10% of tumor cells stained positive with the aforementioned
mAbs.
Additional inclusion criteria were as follows: Karnofsky
performance status of ⱖ70; age of 18 –75 years; absence of
clinically relevant hematological, hepatic, or renal insufficiency; and a life expectancy of at least 3 months. Exclusion
criteria included the following: pregnant or lactating women;
fertile males or females not practicing medically acceptable
Study endpoints
The primary objectives of the study were to determine the
safety, feasibility, and biological activity of a single i.t. injection
of SCH 58500, as determined by RT-PCR detection of vectorspecific wt p53 RNA sequences in posttreatment tumor biopsies. The secondary objective of the study was to assess any
clinical evidence of the antitumoral efficacy of i.t. SCH 58500
injection in patients with either metastatic melanoma or breast
cancer.
Staging procedures
At baseline and on day 28, all patients underwent staging of all
sites of disease and determination of tumor dimensions for all
sites of measurable disease, defined as any lesion with a
perpendicular dimension of ⱖ0.5 cm ⫻ ⱖ0.5 cm, as assessed by
physical examination and/or radiographic imaging scan. Patients underwent computed tomography scans of the chest and
abdomen/pelvis and bone scans at baseline and at day 28 if
needed to evaluate all sites of disease for staging purposes.
Treatment
SCH 58500 (rAd/p53) is a replication-defective recombinant
adenovirus type 5-containing wt p53 cDNA under the control
of the human cytomegalovirus immediate early gene promoter.27,32,33 SCH 58500 was manufactured under current good
manufacturing practice compliance in validated plant facilities
under strict environmental monitoring and control conditions
by Schering-Plow Werthenstein Chemie AG (Schachen, Switzerland)17 and was supplied in vial strengths of 1 ⫻ 108
plaque-forming units (PFU)/mL and 1 ⫻ 109 PFU/mL (7.5 ⫻
1010 particles/mL and 7.5 ⫻ 1011 particles/mL). All patients
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DUMMER, BERGH, KARLSSON, ET AL: PHASE I TRIAL USING A P53-EXPRESSING ADENOVIRAL VECTOR
were treated once with a calculated total dose of SCH 58500
(based on tumor size) divided and administered as four
injections of equal volume, with one injection given per tumor
quadrant within the tumor at its perimeter. The absolute dose
levels used per patient were: 2 ⫻ 107 PFU (0.2 mL), 3 ⫻ 107
PFU (0.3 mL), 5 ⫻ 107 PFU (0.5 mL), 3 ⫻ 108 PFU (0.3 mL),
4 ⫻ 108 PFU (0.4 mL), and 5 ⫻ 108 PFU (5 mL). The
maximum diameters of the tumors given these injections were:
1.3 cm, 2.5 cm, 4.5 cm, 2.1 cm, 3.6 cm, and 4.7 cm, respectively.
After treatment, all patients were hospitalized in single rooms
in an isolation unit for at least 72 hours or until adenovirus
shedding by the patients became negative. All patients underwent a 5- to 8-mm punch biopsy of the treated tumor lesion
either 24 or 48 hours after injection of SCH 58500 to confirm
transgene expression.
The World Health Organization grading system was used to
grade the severity of acute and subacute adverse events. For
adverse events not covered by the World Health Organization
grading system, the following definitions were used: mild
(awareness of sign, symptom, or event, but easily tolerated),
moderate (discomfort enough to cause interference with usual
activity; may warrant intervention), severe (incapacitating, with
inability to perform usual activities or significantly effected
clinical status; warranting intervention), and life-threatening
(immediate risk of death).
Detection of wt p53 gene transfer
Expression of vector-specific wt p53 RNA was assessed in
posttreatment biopsies by means of RT-PCR according to
previously published methods.32 In brief, total cellular RNA
was isolated from homogenized tumor biopsies and reverse
transcribed into cDNA. PCR was performed for 28 cycles on
all samples. Samples from two patients with negative results
after 28 cycles were also run for 45 cycles to increase sensitivity.
The vector-encoded p53 cDNA was amplified using specific
primers, one within the Ad2 tripartite leader (at the 5⬘ end of
the mRNA), and the second within the p53 coding region.
Parallel PCRs using primers specific for human ␤-actin served
as controls for the RNA extraction as well as for cDNA
synthesis. Semiquantitation was carried out for vector-encoded
p53 cDNA as well as for the human ␤-actin through the use of
DNA “mimics” specific for each region. The mimic carried
sequences at its ends, which allowed it to be amplified by the
same primers used to amplify the target sequence. Addition of
a known concentration of mimic to a given reaction enabled
the determination of the relative amount of each cDNA
present. Quantification of the amplified DNA bands was
performed on a Molecular Dynamics FluorImager (Molecular
Dynamics, Sunnyvale, Calif).
Virology studies
Adenovirus shedding and excretion was assessed in a patient’s
urine and stool by means of a commercially available ELISA
kit (Adenoclone; Cambridge Biotech, Worcester, Mass). In
addition, serum, nasal, urine, and stool samples were assayed
to detect the presence of adenovirus using a fluorescenceactivated cell sorter (FACS) assay and a PCR assay (if
necessary). Briefly, samples from patients recovered at pretreatment and at 2 and 3 days postinjection were incubated
with human embryonic kidney 293 cells for 48 hours. The cells
were fixed and reacted with an anti-adenovirus type 5 antibody
(Ab) followed by FACS sorting. Samples deemed to be
positive for infectious activity were to be reassayed after
incubation with 293 cells for 24 – 48 hours followed by PCR
assay with paired primers specific for SCH 58500. An ELISA
Cancer Gene Therapy, Vol 7, No 7, 2000
1071
system was established to detect serum Abs against SCH
58500, which also detects cross-reacting Abs against wt adenoviruses. In brief, microtiter plates were coated overnight with
SCH 58500 and subsequently washed and blocked with bovine
serum albumin. After initially diluting serum samples 1/40 in
phosphate-buffered saline/1% bovine serum albumin/0.05%
Tween, a further set of seven serial 2-fold dilutions of serum
samples was incubated overnight in the microtiter plates. A
positive control and pooled normal sera were also assayed on
each plate. After washing, anti-SCH 58500 Abs were detected
by a reaction with biotin-labeled protein A/G (Jackson Immunoresearch Labs, West Grove, Penn) followed by streptavidinlabeled horseradish peroxidase, and were visualized by reaction with tetramethyl benzidine. Samples were considered
positive for the presence of anti-SCH 58500 Abs if the ratio of
the mean optical densities (ODs) of the sample dilutions
versus the mean ODs of the normal human serum dilutions
was above the threshold value of 0.28. Also, patients were
considered positive for the development of anti-SCH 58500
Abs if the posttreatment sample was positive and there was at
least a 2-fold increase in mean OD compared with pretreatment values.
Anti-adenovirus serum neutralizing Ab assays were performed using the Saos-2 antiproliferative assay, which measures the proliferation of the human osteosarcoma Saos-2 line
in vitro. The assay measures the neutralization of adenovirus by
anti-adenovirus serum Abs by comparing the proliferation of
cells incubated with adenovirus and serum with the proliferation of unexposed cells. The following criteria were used to
assess the reactivity of samples: a value of 0 –10% of control
was considered a high positive for the presence of serum
neutralizing Abs; 10% to ⬍75% of control was considered
positive; 75% to ⬍85% of control was considered low positive;
and ⬎85% of control was considered negative for the presence
of serum neutralizing Abs.
RESULTS
Wt p53 gene transfer
A total of six patients were enrolled in the study between
February 18, 1997 and June 30, 1997. The baseline
characteristics of these patients are shown in Table 1. All
patients had detectable levels of p53 protein in their
tumors as determined by positive reactivity with mAbs
Pab 1801 or Pab 240 (not detectable in normal tissue by
ICH methods).
All patients were treated by a single i.t. injection of
SCH 58500 (range of 2 ⫻ 107 to 5 ⫻ 108 PFU per
patient). In all patients, sufficient amounts of RNA were
recovered from posttreatment biopsies for analysis of
transgene expression by RT-PCR. In five of six patients,
expression of vector-specific wt p53 RNA sequences
could be demonstrated in a non-dose-dependent manner in relation to SCH 58500 dose levels at 2 days
postinjection (Table 2). In one metastatic melanoma
patient treated with 3 ⫻ 107 PFU, wt p53 could not be
detected even after 45 cycles of amplification. In occasional samples, there was a failure to detect ␤-actin (and
p53); this was usually a result of small tissue samples that
were procured for analysis.
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DUMMER, BERGH, KARLSSON, ET AL: PHASE I TRIAL USING A P53-EXPRESSING ADENOVIRAL VECTOR
Table 1. Patient Characteristics
Characteristic
Gender
Age (mean, range)
Race
Histology
Melanoma
Adenocarcinoma, breast
Site of current disease at enrollment*
Skin
Lung
Lymph nodes (unspecified)
Hilar lymph nodes
Abdomen soft tissue
Pelvic lymph nodes
Karnofsky performance status
Screening
100%
80%
70%
Study end (day 28)
100%
80%
70%
⬍70%
Previous therapy*
Biological
Chemotherapy
Hormonal
Immunotherapy
Radiation
Surgical
3 female, 3 male
53.3 years, 37–68 years
6 white
Number of patients
5
1
p53 expression. The patient who received 3 ⫻ 108 PFU
was found to have a mild reactive mononuclear infiltrate
between tumor cells at 1 day postinjection. The patient
who received 3 ⫻ 107 PFU was found to have a mild
lymphocytic-histiocytic perivascular infiltrate with eosinophils as well as infiltrate around the nerves; swelling of
the endothelial cells was also present. This reaction was
reported as an adverse event (injection site inflammation).
Clinical response and patient follow-up
6
3
2
1
1
1
3
2
1
3
1
1
1
2
5
1
1
3
6
*The total number of patients for all categories combined will
be ⬎100% of all patients, because patients had ⬎1 disease site
or previous therapy.
Histopathology
In the patient given 5 ⫻ 108 PFU, tumor necrosis and a
dense cellular infiltration were noted 1 day after i.t.
injection. Also, in the patient given 2 ⫻ 107 PFU, the
tumor cells were noted to be moderately infiltrated by
small round cells at 1 day postinjection. Both of these
patients were found to have the greatest vector-specific
Clinical results in relation to SCH 58500 dose levels and
wt p53 RT-PCR status are summarized in Table 2. One
patient treated with 2 ⫻ 107 PFU had stable disease at
day 29 and was treated thereafter with chemotherapy
(dacarbazine, cisplatin, interferon-␣, interleukin-2). One
patient treated with 3 ⫻ 107 PFU had stable disease at
day 28, with two of three measured tumors unchanged in
size and one of three measured tumors having a slight
increase in size. One patient treated with 5 ⫻ 107 PFU
was hospitalized on day 25 and died on day 31 (30 days
after receiving study drug) due to disease progression.
The death was considered unrelated to the study drug,
and the patient had discontinued from the study due to
a general worsening of his medical condition. One
patient treated with 3 ⫻ 108 PFU had stable disease at
day 27, but her condition deteriorated over the next 4
weeks. One patient treated with 4 ⫻ 108 PFU had
disease progression by day 29; multiple tumors, all 0.5
cm in diameter, appeared that surrounded the four
tumors which were present at screening. One patient
treated with 5 ⫻ 108 PFU had stable disease at day 29.
Toxicity
In general, the toxicity of the i.t. injection of SCH 58500
was mild. The most common events reported by patients
were edema, fever, fatigue, and tachycardia (Table 3).
There were no apparent correlations between the frequencies, severities, or relationships of the adverse
events reported by these patients and the dose of SCH
58500 given.
During the course of the study, there were no changes
in laboratory grade from common toxicity criteria grade
Table 2. SCH 58500 Gene Transfer Results and Clinical Disease Status by SCH 58500 Dose Level
Patient diagnosis and dose (PFU)
2
3
5
3
4
5
⫻
⫻
⫻
⫻
⫻
⫻
107
107
107
108
108
108
PFU
PFU
PFU
PFU
PFU
PFU
melanoma
melanoma
melanoma
melanoma
breast adenocarcinoma
melanoma
Vector-specific p53
expression*
p53 molecules/1000
actin molecules
Day 28 clinical
status†
Positive
Negative
Positive
Positive
Positive
Positive
39
ND‡
ND§
0.5¶
2
46
SD
SD
PD
SD
PD
SD
*Positive, p53 expression detected; negative, p53 expression not detected.
†SD, stable disease; PD, progressive disease.
‡ND, not determined.
§SCH 58500 was detected after 45 cycles but could not be quantified.
¶In this patient, SCH 58500 expression was also found in normal tissue at 0.2 p53 molecules/100 actin molecules.
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DUMMER, BERGH, KARLSSON, ET AL: PHASE I TRIAL USING A P53-EXPRESSING ADENOVIRAL VECTOR
1073
Table 3. Adverse Events in Patients Receiving SCH 58500
Patient SCH 58500 dose level and
diagnosis
Adverse events
Type and duration (days)
Severity
Relationship
2 ⫻ 107 PFU melanoma
Coughing (13)
Edema (⬍1)
Erythema (⬍1)
Wheezing (19)
Mild
Mild
Mild
Mild
Possible
Definite
Definite
Possible
3 ⫻ 107 PFU melanoma
Edema (⬍1)
Injection site inflammation (9)
Mild
Mild
Possible
Possible
5 ⫻ 107 PFU melanoma
Condition aggravated (ongoing)*
Fatigue (ongoing)*
Fever (16)
Tachycardia (⬍1)
Severe
Severe
Mild
Mild
Unrelated
Unrelated
Unrelated
Unrelated
3 ⫻ 108 PFU melanoma
Edema (ongoing)†
Fever (5)
Malaise (5)
Tachycardia (ongoing)‡
Mild
Severe
Moderate
Mild
Possible
Probable
Possible
Possible
4 ⫻ 108 PFU breast adenocarcinoma
Erysipelas (ongoing)†
Mild
Unrelated
5 ⫻ 108 PFU melanoma
Fatigue (10)
Fever (⬍1)
Mild
Mild
Possible
Possible
*Ongoing adverse event was still present at the lat visit (day 31).
†Ongoing adverse event was still present at the last visit (day 29).
‡Ongoing adverse event was still present at the follow-up visit (day 66).
0, 1, or 2 at baseline to grade 3 or 4 in any patient. In
addition, there were few clinically relevant changes in
vital signs during the course of the study. Both reports of
tachycardia were considered to be mild in severity. One
patient (receiving 5 ⫻ 107 PFU) had an increase in heart
rate from 80 beats per minute (bpm) at day 23 to 120
bpm at day 31, with no follow-up due to the patient’s
death on day 31. The second patient (receiving 3 ⫻ 108
PFU) had an increase in heart rate from 89 bpm before
injection to 112 bpm at 4 hours postinjection, with a
continued increased rate (116 bpm) up to day 66. There
were no changes noted in electrocardiogram tracings
from pre- to posttreatment for any patient.
Virology studies
In all patients, adenovirus shedding was assessed on a
daily basis in urine and stool before treatment and for 48
hours posttreatment. All patients were confirmed to be
negative for adenovirus shedding before treatment, and
none were positive after treatment using ELISA. Serum,
urine, stool, and nasal swab samples from patients
receiving 3 ⫻ 107 and 5 ⫻ 108 PFU were also examined
before treatment and for 48 hours after treatment with
the FACS assay and found to be negative for adenovirus
shedding.
The ELISA anti-adenovirus Abs assay showed that of
four patients tested, all had previous exposure to natural
adenovirus infections, as indicated by Ab titers of ⱖ1:
320 at the screening/preinjection visits (Table 4). Three
of the four patients developed increased Ab titers that
met the protocol criteria for a response to SCH 58500
Cancer Gene Therapy, Vol 7, No 7, 2000
(Table 4). The anti-adenovirus neutralizing Ab assay
(Saos-2) indicated that patients developed neutralizing
Abs toward SCH 58500 (Table 5). Of these patients, only
the patient receiving 3 ⫻ 108 PFU was positive for the
presence of neutralizing Abs at screening. This patient
also had the highest preinjection background level of
anti-SCH 58500 Abs as detected by the ELISA method.
DISCUSSION
Most of the common human cancers are often associated with mutations of the tumor suppressor gene p53,
underscoring its central role in the prevention of malignant transformation and tumor progression. The expression of so-called wt p53 within tumor cells, which should
in theory restore the normal regulation of growth control to transformed cells, has become a goal for cancer
gene therapy. Suppression of tumor cell growth using
the p53 adenoviral construct described in this study has
been found in p53 mutant or null cell lines derived from
tumors from a variety of tissues,27 and tumor suppression has been demonstrated in several xenograft experiments in vivo.29
Recent reports have indicated the successful transfer
of wt p53 cDNA as suggested by increased apoptosis,
using a retroviral vector, into the tumors of patients with
lung cancer, although transgene expression was not
actually detected.16 In the present study, i.t. expression
of vector-specific wt p53 RNA, as detected as by RTPCR, was used as the primary endpoint. Immunohistochemistry is inferior to sequencing, as the latter has been
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DUMMER, BERGH, KARLSSON, ET AL: PHASE I TRIAL USING A P53-EXPRESSING ADENOVIRAL VECTOR
Table 4. Anti-SCH 58500 Ab ELISA Results by SCH 58500 Dose Level
Dose (PFU)
Visit
Ratio OD
to NHS*
Ratio OD
Pre/Post
Ab
presence†
Ab
development‡
Ab
titer
2 ⫻ 107
Screen
Day 3
Day 14
Day 22
Day 28
0.80
0.84
1.65
1.74
1.55
NA§
1.1
2.1
2.3
2.1
Positive
Positive
Positive
Positive
Positive
NA
Negative
Positive
Positive
Positive
640
1280
⬎5120
⬎5120
⬎5120
3 ⫻ 107
Before
Day 3
Day 7
Day 14
Day 21
Day 28
0.43
0.33
1.05
2.38
1.33
1.34
NA
0.8
2.5
5.5
6.8
6.9
Positive
Positive
Positive
Positive
Positive
Positive
NA
Negative
Positive
Positive
Positive
Positive
320
160
640
⬎5120
⬎5120
⬎5120
3 ⫻ 108
Screen
Before
Day 3
Day 8
Day 14
Day 21
Day 28
1.25
0.95
0.94
2.06
1.68
1.67
1.65
NA
NA
0.9
2.0
1.6
1.6
1.6
Positive
Positive
Positive
Positive
Positive
Positive
Positive
NA
NA
Negative
Positive
Positive
Positive
Positive
1280
2560
1280
⬎5120
⬎5120
⬎5120
⬎5120
4 ⫻ 108
Screen
Before
Day 3
Day 7
Day 14
Day 21
Day 28
1.45
1.17
1.09
1.14
1.30
1.11
1.20
NA
NA
0.9
1.0
1.1
1.0
1.1
Positive
Positive
Positive
Positive
Positive
Positive
Positive
NA
NA
Negative
Negative
Negative
Negative
Negative
2560
5120
2560
2560
⬎5120
⬎5120
⬎5120
*NHS, normal human serum
†Samples are positive for the Abs capable of binding to SCH 58500 if the ratio of the OD from the sample to the OD from NHS is
⬎0.28.
‡Samples are positive for the development of Abs capable of binding to SCH 58500 if, in the addition to the condition listed in the
second footnote, there is at leat a 2-fold increase for the predose sample to the postdose sample.
§NA, not applicable.
shown to yield better prognostic information in patients
with breast cancer.30 An adenoviral expression vector
system was chosen because of its established safety in
clinical trials and its organotropism,34 – 40 and because of
its capability to effect wt p53 gene transfer in vitro and in
preclinical animal models.27,32 Unlike retroviruses, integration into the cell genome is not an obligate part of the
adenoviral life cycle; rather, adenovirus DNA functions
in the extrachromosomal portion of the cell’s nucleus.41
Thus, in comparison with retroviral vectors for gene
delivery, this process has the therapeutic advantage of
reducing the risk of insertional mutagenesis and reducing the chance that viral DNA will be active in cell
progeny after cell division.
In the present study, i.t. transgene expression of wt
p53 could be detected in tumor samples taken 48 hours
posttreatment in five (four melanoma and one breast
cancer) of six patients given SCH 58500, although it was
only possible to quantify the level relative to ␤-actin in
four patients due to the small amounts of tumor tissue
recovered. Of the patients with quantifiable levels of
vector-specific p53 sequences, the two patients with the
most pronounced wt p53 expression also had protocol-
Table 5. Anti-Adenovirus Neutralizing Ab Assay (Saos-2)
Results by Dose Level
Dose (PFU)
2 ⫻ 107
3 ⫻ 107
5 ⫻ 107
3 ⫻ 108
Visit
% of Control
Result*
Screen
Day 22
Day 28
Before
Day 3
Day 7
Day 14
Day 21
Day 28
Before
Day 3
Day 7
Day 14
Day 21
Day 28
Screen
Day 3
Day 8
Day 14
171
⬍0.3
⬍0.3
115
180
⬍0.3
⬍0.3
⬍0.3
⬍0.3
121
142
9.1
⬍0.9
⬍1
⬍1
51
69
⬍0.4
⬍0.3
Negative
High positive
High positive
Negative
Negative
High positive
High positive
High positive
High positive
Negative
Negative
High positive
High positive
High positive
High positive
Positive
Positive
High positive
High positive
*Result criteria: 0 to ⬍10, high positive; 1 to ⬍75, positive; 75
to ⬍85, low positive; ⬎85, negative.
Cancer Gene Therapy, Vol 7, No 7, 2000
DUMMER, BERGH, KARLSSON, ET AL: PHASE I TRIAL USING A P53-EXPRESSING ADENOVIRAL VECTOR
defined stable disease at day 28. However, the RT-PCR
data need to be interpreted with caution, as patients did
not exhibit a correlation of disease state with wt p53
level.
The introduction of vector-specific p53 adenoviral
constructs into the patients could also be inferred from
the increase in anti-adenovirus titers in three of four
patients assayed as well as from an increase in antiadenovirus neutralizing Abs in all four patients tested.
The induction of anti-SCH 58500 neutralizing Abs
would have had no effect on the expression of wt p53
introduced by the adenoviral vector because the levels of
these Abs were low initially and did not increase (days
7– 8 for three of the four patients assayed) until after the
samples for RT-PCR were taken (day 2). Earlier studies
have indicated that recombinant adenovirus injected
directly into the tumor is highly efficient for immunizing
patients against the transgene despite anti-adenovirus
responses.42
Adenovirus shedding, which is a potential safety concern if genetic technology is to be used as a standard
treatment, did not occur with any of the doses employed
in this study. The use of a replication-deficient adenoviral vector also makes transmission of genetically engineered adenoviruses to other individuals very unlikely.
In the present study, patients exhibited mild, generally
unrelated side effects after exposure to SCH 58500, and
no clinically significant toxicity was observed.
In conclusion, i.t. transgene expression of wt p53, as
determined by adenovirus-mediated gene transfer, was
shown to be safe and feasible in patients with advanced
metastatic melanoma and breast cancer. This proof of
concept study will lay the groundwork to support the
development of p53 therapy in oncology.
ACKNOWLEDGMENTS
We thank the patients for their participation in the trial and
the affiliates of Schering-Plow for their support and laboratory
investigations. The following individuals were of major importance for completion of the study at the center in Uppsala:
Associate Professor Hans Nordgren, for histopathological
evaluation and immunohistochemistry; Professor Jonas
Blomberg, for viral antigen testing; Research Nurses Carina
Andersson and Rita Grönberg; and Research Assistant Gunilla Känf. In the Leiden center, we thank J. H. J. M. Van
Krieken, A. C. M. Kroes, and Jan Ouwerkerk. In the Zürich
center, we thank Dr. C. Ruef for the surveillance of biosafety
aspects, and the nurses and technicians involved.
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