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A phase I/II trial of rAd/p53 (SCH 58500) gene
replacement in recurrent ovarian cancer
Richard E Buller,
1
Ingo B Runnebaum,
2
Beth Y Karlan,
3
Jo Ann Horowitz,
4
Mark Shahin,
1
Thomas Buekers,
1
Stan Petrauskas,
4
Rolf Kreienberg,
5
Dennis Slamon,
3
and Mark Pegram
3
1
Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, The University of Iowa Hospitals
and Clinics, Iowa City, Iowa, USA;
2
Department of Obstetrics and Gynecology, University of Freiburg, Freiburg,
Germany;
3
University of California, Los Angeles, California, USA;
4
Schering-Plough Research Institute,
Kenilworth, New Jersey, USA; and
5
University of Ulm, Ulm, Germany.
Purpose: To determine the safety, gene transfer, host immune response, and pharmacokinetics of a replication - deficient adenovirus
encoding human, recombinant, wild-type p53 (SCH 58500) delivered into the peritoneal cavity (i.p.) alone and sequentially in
combination with platinum-based chemotherapy, of patients with recurrent ovarian, primary peritoneal, or fallopian tube cancer
containing aberrant or mutant p53. Methods: SCH 58500 was administered i.p. to three groups of patients with heavily pretreated
recurrent disease. Group 1 (n=17) received a single dose of SCH 58500 escalated from 7.510
10
to 7.510
12
particles. Group 2
(n=9) received two or three doses of SCH 58500 given alone for one cycle, and then with chemotherapy for two cycles. The SCH
58500 dose was further escalated to 2.510
13
particles/dose in group 2. A third group (n=15) received a 5-day regimen of SCH
58500 given at 7.510
13
particles/dose per day i.p. alone for cycle 1 and then with intravenous carboplatin / paclitaxel
chemotherapy for cycles 2 and 3. Results: No dose-limiting toxicity resulted from the delivery of 236/287 (82.2%) planned doses of
SCH 58500. Fever, hypotension abdominal complaints, nausea, and vomiting were the most common adverse events. Vector -
specific transgene expression in tumor was documented by RT-PCR in cells from both ascitic fluid and tissue biopsies. Despite
marked increases in serum adenoviral antibody titers, transgene expression was measurable in 17 of 20 samples obtained after two or
three cycles of SCH 58500. Vector was detectable in peritoneal fluid by 24 hours and persisted for as long as 7 days whereas none
was detected in urine or stool. There was poor correlation between CT scans and CA125 responses. CA125 responses, defined as a
greater than 50% decrement in serum CA125 from baseline, were documented in 8 of 16 women who completed three cycles of the
multidose regimen. Conclusion: CT scans are not a valid measure of response to i.p. SCH 58500 due to extensive adenoviral -induced
inflammatory changes. Intraperitoneal SCH 58500 is safe, well tolerated, and combined with platinum-based chemotherapy can be
associated with a significant reduction of serum CA125 in heavily pretreated patients with recurrent ovarian, primary peritoneal, or
fallopian tube cancer.
Cancer Gene Therapy (2002) 9, 553–566 doi:10.1038/sj.cgt.7700472
Keywords: p53; gene therapy; ovarian cancer; CA125
It is projected that 23,400 women will be diagnosed with
ovarian cancer and 13,900 will die from this disease
during the year 2001.
1
These statistics make ovarian cancer
the fifth leading cause of death among women in the United
States. Ovarian cancer offers several unique opportunities for
novel therapeutic intervention. First, despite the tendency to
present at advanced International Federation of Gynecology
and Obstetrics ( FIGO ) stage reflected by the observation
that nearly 73% of ovarian cancers are no longer confined to
the ovary at diagnosis,
2
this cancer often remains confined
within the abdominal cavity throughout its course.
3,4
Second, initial, complete clinical responses are the expected
norm following surgical cytoreduction and adjuvant sys-
temic chemotherapy. Unfortunately, recurrence, progression,
and death from disease is the eventual outcome for more than
75% of women diagnosed with epithelial ovarian cancer.
Finally, because both primary
5,6
and secondary
7,8
surgical
cytoreduction are cornerstones of the therapeutic approach to
this cancer, tissue samples are often available for molecular
genetic studies. Such studies have resulted in a better
understanding of some of the molecular changes associated
with ovarian cancer and how they may influence prognosis
or response to treatment.
Received April 4, 2002.
Address correspondence and reprint requests to: Dr Richard E Buller,
Department of Obstetrics and Gynecology, Division of Gynecologic
Oncology, 200 Hawkins Drive — #4630 JCP, Iowa City, IA 52242-
1009, USA. E - mail: richard - buller@uiowa.edu
Presented in part at the 7th International Conference on Gene Therapy
of Cancer, San Diego, CA, November 19 – 21, 1998 and the 30th Annual
Meeting of the Society of Gynecologic Oncologists, San Francisco, CA
March 20– 24, 1999.
Cancer Gene Therapy (2002) 9, 553 – 566
D2002 Nature Publishing Group All rights reserved 0929-1903/ 02 $25.00
www.nature.com / cgt
Mutation of the p53 tumor suppressor gene is one of the
most frequent molecular genetic changes in cancer.
9,10
Wild-
type p53 functions include roles in DNA repair following G1
cell cycle arrest, and directing irreparably damaged cells
toward apoptotic pathways, thus maintaining the integrity of
the genome.
11
Both in vitro and in vivo evidence suggests
that cells with altered p53 function may be less responsive to
certain chemotherapeutics than those that are able to express
wild-type p53.
12,13
p53 dysfunction frequently results from
mutations that can generate both missense and nonsense
inactivating mutations. Rare gain of function mutations has
also been described.
14
Nearly 70% of advanced stage ovarian
cancers contain p53 mutations and many of these mutations
render the cancers p53 null.
15 - 17
Overall, p53 null mutations
can be associated with extremely poor prognosis reflected, at
least in part, by early and distant metastasis.
4
These ob-
servations suggest that p53 mutation is of fundamental
importance in the progression of ovarian cancer.
Despite the association of distant metastasis with p53 null
mutation, most ovarian cancers usually remain confined to
the abdominal cavity throughout their course and provide a
unique opportunity for regional delivery of therapeutic
agents. Intraperitoneal delivery of chemotherapy can provide
a pharmacokinetic advantage over intravenous dosing by
maximizing delivery of drug directly to tumor and minimiz-
ing systemic side effects.
18
A seminal study by the Gyne-
cologic Oncology Group has demonstrated both response
and survival advantage to women with minimal residual
disease treated with intraperitoneal ( i.p. ) cisplatin after
primary cytoreductive surgery for ovarian cancer.
19
Thus,
ovarian cancer is a unique model for gene replacement
strategies.
20,21
Preclinical studies in several in vivo models have shown
that delivery of wild - type p53 to tumor cells can be
achieved.
22 - 31
Extension of these studies, particularly in
lung cancer, to phase I clinical trials has produced
encouraging results.
32 - 36
To date, gene transfer in these
systems has been accomplished with cationic lipids and a
variety of viral vectors including the retroviruses and
adenoviruses.
37
The use of an adenoviral vector, which has
been rendered replication deficient, offers several advantages
for therapeutic gene replacement strategies in cancer.
37
First,
in contrast to retroviruses, adenoviral vectors efficiently
transduce both dividing and quiescent cells. Second, they
can be produced in high titers with particle numbers
approaching the number of target cancer cells. Third, a
bystander effect has been observed to occur following dosing
with adenoviral vectors. Fourth, adenoviral vectors do not
integrate into the host genome minimizing concerns regard-
ing insertional mutagenesis. Taken together, these observa-
tions encouraged us to undertake a phase I/II trial of human
recombinant adenoviral p53 gene therapy with rAd / p53
(SCH 58500 ) in recurrent ovarian cancer. Preliminary results
have been presented in part.
38
The objectives of the study
were: ( a ) to determine safety and tolerability to SCH 58500
alone and in combination with chemotherapy, (b) to
determine the ability to transfer wild - type p53 sequences
into ovarian cancer cells in vivo, ( c ) to measure serum and
ascites antibody responses to this form of therapy along with
their influence on gene transfer, (d) to determine the
pharmacokinetics of SCH 58500 in ascites and serum, and
(e) to evaluate tumor response when multiple doses are
delivered to patients over a 3 - month period. Our findings
indicate that gene transfer of SCH 58500 can be accomplished
with minimal toxicity and that reduction in a surrogate
marker, CA125, suggests the potential for clinical activity.
Methods
SCH 58500
SCH 58500 is a novel antineoplastic agent consisting of a
recombinant adenoviral vector containing the cloned,
human, wild- type p53 tumor suppressor gene cDNA, which
is under the control of the human cytomegalovirus
immediate early promoter / enhancer element. SCH 58500
is derived from a type 5 adenovirus, a common serotype
belonging to subgroup C, which has been rendered
replication - defective through deletion of the viral genes
E1a, E1b, and protein IX.
39
Vector is produced using GMP
standards and has been tested for the presence of viral,
bacterial, and other contaminants.
Tumor p53 mutation status
For screening, a representative primary or recurrent tumor
sample from each patient who had signed informed consent
was analyzed for p53 mutation by immunohistochemistry
utilizing both Pab 1801 (diluted 1:40 ) and Pab 240 ( diluted
1:20) antibodies ( Pharmingen, San Diego, CA ). A positive
stain with either antibody was considered to reflect aberrant
tumor p53 protein and confirmed eligibility. Although this
finding does not always reflect a p53 mutation, most authors
consider immunopositive tumor to contain dysfunctional
p53.
16
Sections with < 10% of cells showing nuclear staining
were considered negative. Such individuals were excluded
from study entry unless a p53 DNA sequence abnormality
could be documented.
16
Antiadenovirus antibody assay
An ELISA was used to measure antiadenovirus antibodies
specific for adenoviral coat proteins ( antihexon antibodies )
in serum and ascites. Samples were assayed in parallel with
normal human serum and a ratio of sample titer versus
normal human serum titer was calculated. If this ratio was
less than 0.28 the sample was considered negative.
Patient eligibility and exclusion criteria
Only female patients at least 18 years of age previously
treated with surgery and chemotherapy for ovarian, fallopian
tube, or primary peritoneal carcinoma now presenting with
pathologically confirmed recurrence of disease were eligible.
An elevated CA125 was not required for entry. For those
individuals without malignant ascites at recurrence, we
required surgically documented i.p. disease accessible to
laparoscopic or percutaneous biopsy. A tumor p53 mutation
was required as described above. All treated individuals
functioned with a Karnofsky performance status of at least
60% and a minimum life expectancy of 3 months. Stand-
ardized clinical laboratory tests were within normal limits.
Cancer Gene Therapy
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RE Buller et al
554
Previous whole abdominal radiotherapy was not allowed.
Before the first treatment cycle a contrast study of the
abdomen demonstrated free flow of instilled agent. Either a
spiral CT with i.p. contrast, or i.p. Hypaque (Nycomed,
Princeton, NJ ) in 500 mL of normal saline followed in
30 minutes with a conventional flat plate x-ray was used to
determine adequate peritoneal distribution of the infuseate.
Three eligible, consented patients did not receive treatment
with SCH 58500 based on poor distribution of contrast.
Initially, only patients serologically positive for antiadeno-
virus type 5 antibody at screening were treated.
Patients not eligible for the study included those pregnant
or nursing, and those with presence of serious bacterial, viral,
fungal, or parasitic infection. Patients with evidence of
adenoviral infection, as determined by ELISA, at screening
were excluded and the chronic use of immunosuppressant
therapy or use of another investigational drug within
3 months of proposed treatment with SCH 58500 also
resulted in exclusion. Known human immunodeficiency
virus (HIV ) -positive individuals were also excluded. Short -
term bolus use of dexamethasone as an antiemetic, or as
premedication for paclitaxel, was allowed.
Registration
An institutional human subjects review board approved
informed consent was obtained before the performance of
any test or evaluation not considered standard of care for
patients with peritoneal carcinomatosis. The same consent
detailed the treatment with SCH 58500 and alternatives. No
patient received SCH 58500 without signing an informed
consent.
SCH 58500 delivery
SCH 58500 was infused over 20 minutes into the peritoneal
cavity via a Hickman ( Bard Systems, Salt Lake City, UT ),
Tenckhoff (CR Bard, Murray Hill, NJ ), or Porta Cath (SIMS
Deltec, St. Paul, MN ) catheter. In preliminary studies, all
catheters were shown to be compatible with SCH 58500. The
goals of the infusions were to use a constant volume for each
dose, with the volume large enough to generate adequate i.p.
distribution, while at the same time providing a tolerable
total volume. To achieve these goals, some variability of
infusion volumes was required. Patients with clinically
significant, preexisting ascites underwent drainage of the
ascites before dosing with SCH 58500. Patients in group
1 then received SCH 58500 in 1000 mL of 0.9% NaCl.
Group 2 and 3 patients received SCH 58500 in 250 mL, for
2 (Level 4), 3 (Level 5 ), and 5 ( Level 6 ) days. By the end of
five daily administrations ( i.e., level 6 ), a total infusion
volume of 1250 mL had been reached. Any additional ascites
that accumulated during the course of administration of
multiple doses was not removed except in one patient who
had a large volume of ascites with her recurrent disease.
Following this patient’s first dose in cycle 1, the day 2 dose
was delayed 24 hours to allow for ascites drainage. In the
absence of ascites, each dose of SCH 58500 was infused in
500 mL of 0.9% NaCl so that by the end of five daily
administrations ( i.e., level 6 ), a total infusion volume of
2500 mL had been reached. Patients were then rotated every
15 minutes for 2 hours into Trendelenberg, right lateral, left
lateral, and sitting positions.
Treatments
This sequential cohort, nonrandomized study, was conducted
in three groups of patients. Table 1 outlines the treatment
schema for i.p. SCH 58500. For group 1 patients, a single
treatment dose of SCH 58500 was escalated from 7.510
10
particles to 7.510
12
particles per dose in four steps. Three
patients were to be treated with SCH 58500 at each dose
level in this group. The decision to escalate or expand a dose
level was based on review of safety data for the patients
within the single - dose level cohort under study or after day 7
of the first cycle when multiple cycles were given. A single,
potential dose - limiting toxicity ( DLT; see Results ) promp-
ted us to expand level 2 from three to six patients. After
initial safety data were obtained, two additional antiadeno-
viral antibody negative individuals were allowed to enter at
level 1. Therefore, a total of 17 patients were treated in
group 1. Patients treated in this group were allowed to enter
the multiple - dose group ( see below ) if they continued to
meet all eligibility criteria.
For group 2 patients ( n= 9 ), cytotoxic chemotherapy was
added in cycles 2 and 3 to allow differentiation between SCH
58500 side effects when it was given alone in cycle 1 and
those related to its combination with chemotherapy. The
dose of SCH 58500 was further escalated to 2.510
13
particles although single - day dosing was increased first to 2
and then to 3 days per treatment cycle. Six group 2 patients
received single- agent i.p. cisplatin at 100 mg/m
2
on day 1 of
cycles 2 and 3 for dose levels 4 and 5 only. A 30- minute
infusion of cisplatin was delivered in 1 liter of 0.9% NaCl
1 hour following the SCH 58500 infusion. The rest of the
multiple - cycle patients were treated with intravenous
chemotherapy. Paclitaxel at 175 mg/m
2
was infused over
3 hours immediately before SCH 58500 on day 1 whereas
carboplatin was infused over 30 minutes immediately after
SCH 58500 on day 3 of cycles 2 and 3 at dose levels 5 and 6.
The carboplatin dose was based on an area under the curve
Table 1 SCH 58500 treatment regimens
Dose
level
Particles
delivered
Treatment
days Chemotherapy Cycles*
Group 1 — single - dose SCH 58500
1 7.510
10
1 None 1
2 7.510
11
1 None 1
3 2.510
12
1 None 1
4 7.510
12
1 None 1
Group 2 — escalating - dose SCH 58500 plus chemotherapy
4 7.510
12
2 Cisplatin ( i.p. ) 3
5 2.510
13
3 Cisplatin ( i.p. ) 3
5 2.510
13
3 Carboplatin / Taxol ( i.v. ) 3
Group 3 — multiple - dose SCH 58500 plus conventional
chemotherapy
6 7.510
13
5 Carboplatin / Taxol ( i.v. ) 3
*Treatments were repeated at 28 - day intervals.
Cancer Gene Therapy
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RE Buller et al
555
(AUC) of 6 mg / mL min with the GFR based on the
Cockroff-Gault formula for creatinine clearance.
40
With
multiple- dose, multiple - cycle regimens, paclitaxel was
before the vector because of in vitro evidence that this agent
enhances transfection efficiency of SCH 58500.
23
Once safety was confirmed by interpatient escalation,
group 3 patients ( n= 15 ) were treated with intravenous
carboplatin and paclitaxel in combination with SCH 58500 at
7.510
13
particles, dose level 6. The number of doses of
SCH 58500 was escalated from 3 to 5 per cycle. For patients
in this group, either measurable or evaluable disease was
required. Measurable disease was defined as a bidirectionally
measurable lesion with clearly defined margins on physical
exam or x - ray, computed tomography (CT), or magnetic
resonance image analysis. Evaluable disease was defined as
an elevated CA125 tumor antigen level greater than two
times the institutional norm.
Tumor sampling. Twenty - four to 72 hours following single -
dose SCH 58500, or 24 hours after the last dose of SCH
58500 in each multiple dose of the study agent, the
peritoneal cavity was drained to obtain tumor cells. Patients
who did not have ascites with recurrence of their cancer, or
who had inadequate ascites following SCH 58500 dosing,
were separately consented to laparoscopy for cycles 1 and 3
to obtain tumor and normal tissue samples for the various
PCR studies. Pathologic, or cytologic, examination con-
firmed the presence of malignant cells in the samples of all
patients.
Toxicity. The study design with escalating doses of SCH
58500 was aimed to determine dose - limiting toxicities
utilizing standard WHO criteria. Any grade 4 ( G1; WHO )
toxicity, or a grade 3 (G3 ) toxicity lasting greater than
1 week, was to be considered dose limiting ( DLT), unless
the event was obviously related to another procedure ( e.g.,
anemia due to chronic test phlebotomy ).
Nausea, vomiting, and anorexia were excluded as dose -
limiting toxicities in patients receiving chemotherapy.
Patient monitoring. All single-dose patients were treated as
inpatients. A qualitative ELISA kit ( Cambridge Biotech,
Worcester, MA ) was used to confirm the absence of viral
shedding in urine and stool samples before dosing, during
treatment, and before hospital discharge. Vital signs were
obtained before and periodically following the administra-
tion of SCH 58500. Physical exams, performance status,
weight, and adverse event assessments were performed daily
whereas the patients were hospitalized and at prescribed
intervals following discharge: Day 7, 14, 21, and 2 months
after dosing, then every 3 months until death. Laboratory
data included serum and ascites sampling for pharmacoki-
netic studies, complete blood counts ( CBC ), fibrinogen,
fibrin split products, PT, PTT, serum C
3
,C
4
,CH
50
,
electrolytes including magnesium, blood glucose, and
CA125. Laboratory tests were performed at each visit,
except CA125, which was monthly. Baseline abdominal and
pelvic computed tomograms were obtained along with a
chest x-ray before dosing. Follow-up scans were obtained at
28 days and as clinically indicated for patients who received
multiple cycles of SCH 58500. Lesions were measured in
two perpendicular directions. Standard response definitions
were used, i.e., complete response ( CR ) required the
disappearance of all gross evidence of disease for at least
4 weeks; partial response ( PR ) required a reduction in lesion
size in excess of 50% lasting at least 4 weeks; progressive
disease was said to have occurred on the basis of a 25%
increase in lesion size; all other measurable disease cases
were considered to define stable disease ( SD ). As an
additional measure of response, changes in serum CA125
were evaluated. The 50% and 75% CA125 responses as
defined by Rustin et al
41,42
have been shown to correlate well
with conventional CT response measures.
Documentation of gene transfer and viral persistence. Total
RNA was extracted and cDNA prepared from ascites or
tissue biopsies. The QIAamp 96 Spin Blood Kit ( Qiagen,
Valencia, CA) was used to extract viral DNA from serum.
Polymerase chain reactions were carried out utilizing primers
specific for both the adenovirus and the p53 gene as well as
-actin or glyceraldehyde 3 - phosphate dehydrogenase
(G3PD) collectively referred to as housekeeping genes or
HKG. The MIMIC
2
(Clontech, Palo Alto, CA ) reverse
transcriptase technique allowed for semiquantitative com-
parisons of mRNA levels. Tripartite leader sequence -
specific primers permitted the resolution of SCH 58500
sequence from host p53 sequence ( See Results ).
In situ PCR. Five-micrometer sections of formalin - fixed,
paraffin-embedded tissue were placed on 1.2 - mm silane -
coated Perkin - Elmer ( Foster City, CA) in situ PCR glass
slides. Slides were baked 2 – 3 hours at 608C to reduce RNA
content. Slides were then treated sequentially with 0.02 N
HCl, Proteinase K, and acetic acid. Thirty - five PCR cycles
were carried out using dinitrophenyl - labeled primers
(DNP) specific for SCH 58500. Following incubation with
anti-DNP antibody conjugated to alkaline phosphatase,
visualization was achieved by adding nitro - blue tetrazo-
lium-5-bromo - 4 -chloro - 3 - indolyl phosphate as substrate
and counterstaining with Nuclear Fast Red. Negative staining
was pink, whereas positive staining was blue and nuclear.
Statistics. Differences in toxicities between SCH 58500 and
SCH 58500 plus chemotherapy cycles were evaluated with
Fisher’s Exact test, 2-tailed. Mean CA125 changes were
analyzed by ANOVA, or ttests as appropriate. A Pvalue of
<.05 was required for significance.
Results
Patient selection and characteristics
One hundred and fifty - five patients signed informed consent
and entered into screening at three sites. Overexpression of
p53 protein by immunohistochemistry was demonstrated for
79 of the 155 ( 51% ) cancers tested. The Iowa site carried out
p53 gene sequencing on 25 of 28 p53 immunonegative
cancers screened at that institution. An additional 8 patients
(7 with p53 null mutations ) met the p53 eligibility criteria in
this fashion. So that 57% ( 88 /155 ) of patients screened were
eligible for entry. Overall, 36 patients were dosed with SCH
58500. Five patients were treated on both the single - dose
arm of the study and the later multiple - dose program.
Therefore, 41% ( 36 / 88 ) of the eligible patients representing
Cancer Gene Therapy
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RE Buller et al
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23% (36/155 ) of the patients screened were actually treated
with SCH 58500. The mean age of the individuals dosed was
60 years (range: 39–76). Demographic and disease - related
parameters for this cohort of heavily pretreated individuals
with recurrent peritoneal carcinomatosis are summarized in
Table 2. Most individuals had recurrent ovarian cancer. The
mean interval from primary diagnosis to dosing with SCH
58500 was 778 days ( range: 115–2360 days). The mean
platinum -free interval to dosing with SCH 58500 was
263 days (range: 37–711 days). Nine of 36 patients had a
platinum -free interval of more than one year. The mean
number of prior chemotherapy regimens was 2.8 with 22%
of individuals receiving four or more prior regimens and
33% receiving just one prior regimen. All individuals had
previously received platinum - based chemotherapy, and all
but two had prior treatment with a taxane. Three patients had
recurrent disease evaluable only on the basis of laparoscopic
biopsy. Fourteen patients could be considered to have
small-volume disease, arbitrarily defined as less than or
equal to 2 cm.
Toxicity
Two hundred and thirty- six different signs or symptoms
were recorded as adverse events. These varied from single
patient WHO grade 1 (G1 ) events such as increased earwax
and nonspecific breast complaints to a G4 transient ischemic
attack. Because adenoviral particles delivered in this study
are more than a log higher than in any previously reported
gene therapy trial, great attention was paid to complete
reporting of all potential adverse events. From a practical
standpoint, we have chosen to present all serious G3 or G4
events, but only the G1 and grade 2 (G2) events that
occurred in three or more treated individuals. This of course
underreports the total number of minor adverse events. Each
treatment - related adverse event is recorded as the highest -
grade toxicity experienced out of all treatment cycles
received by that patient as explained in the legend to
Table 3. The events are listed in this table on the basis of
occurrence in either the single - dose or multiple - dose
groups. To show that there was no cumulative toxicity, we
have listed the five patients who were treated with multiple -
dose SCH 58500 after completion of the single -dose portion
of the study separately. The most common adverse events in
the single - dose group included fever ( 47% ), nausea ( 41% ),
edema (41% ), abdominal complaints ( 41% ), and anemia
(29%). Seven patients experienced 5 different adverse
events whereas only 1 patient had no adverse events at all.
Eight G3 or G4 adverse events were reported in four patients.
These included anemia ( 2:G3; 1:G4 ), abdominal complaints
(1:G3), dehydration ( 1:G3 ), pain ( 1:G3 ), tachycardia
(1:G3), and vomiting ( 1:G3 ). There was no unusual toxicity
in the two serum antiadenoviral antibody negative patients
dosed at level 1.
Fever was also the most common adverse event experi-
enced by 100% of the multiple - dose patients. This sign
developed within 2 to 4 hours of dosing. The highest reported
temperature was 40.58C. Four cycles were accompanied by
G3 febrile responses ( >408C ) among two different patients.
After fever was noted in the initial dosing cohorts, patients
were generally given prophylactic acetaminophen. The
subsequent febrile responses were attenuated, but this may
also have been due to the steroid premedication given before
chemotherapy for cycles 2 and 3. Figure 1 demonstrates this
observation graphically for a patient treated at dose level 5. In
the multidose cohort, the next most frequent signs and
symptoms related to SCH 58500 included hypotension
(89%), a variety of abdominal complaints ( 79% ), hyper-
tension (68% ), nausea ( 63% ), tachycardia ( 58% ), vomiting
(58%), and fatigue (53%) — often in the same patient and
cycle as the hypertension was noted. The blood pressure
changes prevalent in this group were not seen at all in the
single-dose group, but they were generally considered mild
because only one G3 toxicity occurred. All of these most
common adverse events, except hypertension, also occurred
in 100% of the single - dose patients who reenrolled in the
multiple - dose regimen. However, there was no progression
of toxicity grade in those re - treated relative to those initially
treated at the same dose of SCH 58500.
Forty-seven G3 or G4 toxicities were reported in
13 patients who received multiple - dose SCH 58500. Many
of the new WHO G3 toxicities were probably related to
chemotherapy because they usually appeared in cycles 2
and 3. The patient with congestive heart failure also
developed a G4 neutropenia with concomitant thrombocy-
topenia in cycle 3. A few new low - grade adverse events
were reported when chemotherapy was combined with SCH
Table 2 Study cohort demographics
Primary diagnosis
Ovarian cancer 30
Peritoneal cancer 5
Fallopian tube cancer 1
Prior chemotherapy
Mean number of regimens 2.8 range [ 1 – 8 ]
Mean treatment cycle 13 range [ 5 – 31 ]
Mean drugscycles 24.4 range [ 8 – 78 ]
SCH 58500 treatment
Single dose 17
Multiple dose 24
Both 5
Interval: ( days )
Diagnosis to SCH 58500 777.7 range [ 115 – 2360 ]
median = 630
Platinum - free to SCH
58500 first dose*
263 range [ 37 – 711 ]
median = 261
Disease status
Elevated CA125 33
CT measurable lesionsy
>2 cm 22
2cm 5
Normal CA125 and CT scan 3
*The platinum - free interval for nine patients was 365 days.
yAll CT - measurable disease was accompanied by an elevated
CA125.
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557
58500. These included lower extremity myalgias, myoclo-
nus, ileus, gastritis with hematemesis, hyperactive bowel
sounds, pulmonary hypertension, peripheral neuropathy,
oliguria, mucositis, port site cellulitis, agitation, generalized
weakness, and cachexia. Only three multiple - dose patients
had 3 adverse events whereas 11 reported 10 adverse
events. Overall, G3 toxicities accompanied approximately
one-third of the treatment cycles. Antiemetics generally
alleviated the gastrointestinal symptoms and were used
prophylactically at the investigator’s discretion. As the total
amount of SCH 58500 delivered was increased, there was a
trend toward more G3 adverse events: the number of adverse
events went from 0.5 to 1.6 to 2.9 per patient as the treatment
was advanced from single dose to level 4 / 5 and then to
level 6. The addition of chemotherapy produced additional
nausea and vomiting ( P= .03, Fisher’s exact test, 2 - tailed ).
There was no trend for adverse events to worsen in a given
individual as the number of doses delivered was increased.
Likewise, there was no evidence of cumulative toxicity as
patients progressed from the single - dose arm to treatment
with multiple doses and multiple cycles.
One G4 toxicity occurred in a patient who became anemic
in cycle 2. This complication along with the other G3
toxicities due to anemia occurred in individuals who were
anemic at the start of the study and has been attributed to the
volume of blood drawn for the multiple laboratory studies,
Table 3 Treatment - related adverse events*
Single - dose SCH 58500
(N=17)
Multiple - dose SCH 58500
(N=19)
Single - and multiple - dose
SCH 58500 ( N=5)
Adverse event G1 G2 G3 G4 G1 G2 G3 G4 G1 G2 G3 G4
Abdominal complaintsy4210 44702120
Anxiety 0 0 0 0 2 0 0 0 2 0 0 0
Anemia 1 1 2 1 0 3 1 0 0 0 1 1
Anorexia 1 0 0 0 5 0 1 0 2 0 0 0
Asthenia 0 0 0 0 2 4 2 0 2 0 0 0
Bradycardia 0 0 0 0 0 0 1 0 0 0 0 0
Chills 1 0 0 0 7 3 0 0 0 0 0 0
Cellulitis ( Port) 1 0 0 0 3 1 0 0 0 1 0 0
CHF 0000 00100000
Dehydration 0 0 1 0 2 0 0 0 0 0 1 0
Diaphoresis 2 0 0 0 2 0 0 0 0 0 0 0
Diarrhea 0 1 0 0 7 0 1 0 1 0 1 0
Dizziness 1 0 0 0 3 2 0 0 2 0 0 0
Dyspnea 1 0 0 0 3 1 0 0 0 0 0 0
Edema 6 1 0 0 0 1 1 0 1 0 0 0
Fatigue 2200 33402300
Fever 3 5 0 0 1 16 2 0 2 3 0 0
Gastritis 0 0 0 0 0 0 1 0 0 0 0 0
Headache 0 0 0 0 8 1 0 0 1 0 0 0
Hypertensionz0000103001100
Hypotensionx0000113204100
Lethargy 0 0 0 0 0 1 1 0 0 0 0 0
Loss of Consciousness 0 0 0 0 0 0 1 0 0 0 0 0
Malaise 1000 34202000
Nausea 7 0 0 0 2 3 7 0 1 1 3 0
Neutropenia 0 0 0 0 0 0 0 1 0 0 0 0
Pain{1210 42100200
Peritonitis 0 0 0 0 0 0 2 0 0 0 0 0
Tachycardia 1 0 1 0 11 0 0 0 5 0 0 0
Thrombocytopenia 0 0 0 0 0 0 0 1 0 0 0 0
TIA 0000 00010000
Vomiting 4 1 1 0 2 3 6 0 2 2 1 0
*All WHO G3 or G4 toxicities and any toxicity, including G1 and G2 reported by three or more patients. Values are number of patients with a
given event in each group. Only the worst toxicity level is reported for each patient, i.e., a G2 fever in cycle 1, G1 in cycle 2, and G3 in cycle 3
appears as a single entry, G3.
yIncludes abdominal enlargement, bloating, contractions, cramping discomfort, distention, fullness, pain, pressure, or tenderness.
zHypertension: G1=asymptomatic transient increase by greater than 20 mmHg or to >150/ 100 if previously within normal limits; no treatment
required. G2= recurrent or persistent increase by greater than 20 mmHg or to >150 / 100 if previously within normal limits; no treatment required.
G3 = requires therapy. G4 = hypertensive crisis.
xHypotension: G1 = 20 mmHg decrease in SBP or DBP requiring no therapy ( including transient orthostatic hypotension ). G2 = 20 mmHg
decrease in SBP or DBP requiring fluid replacement or other therapy but not hospitalization. G3 = requires therapy and hospitalization. G4 = life -
threatening.
{
Includes pain, back pain, breast pain, chest pain, substernal chest pain, or flank pain.
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anemia of chronic disease, and anemia secondary to chemo-
therapy treatments. There was no evidence of hemolysis in
any patients. One individual with liver metastasis and
progressive disease following single - dose SCH 58500 at
level 2 developed a potential DLT reflected by an increase in
alkaline phosphatase from 57 U / L at baseline to 742 U /L 28
days following dosing. This was accompanied by an increase
inAST to 106 U/ Land ALT to111U/L. She refused a follow-
up CT scan and died 51 days after dosing. The family
declined a request for an autopsy. Because of this adverse
event, three additional patients were treated at this dose level
before moving on to level 3. Therefore, although one cannot
rule out SCH 58500 as a cause of this potential DLT, the
investigator felt that the clinical course of this patient was
quite consistent with progression of disease as the proximal
cause of these events. Supporting this conclusion was the
additional observation that no other patient, at any dose,
developed evidence of G3 or G4 hepatic toxicity. Five
individuals ( 14% ) developed potentially worrisome small
bowel obstructions between 2 and 8 months after initial
dosing. These events occurred both on the single - dose
(n=2) and multiple - dose ( n= 3 ) arms. Only one episode
was attributed to SCH 58500, rather than to disease
progression and / or underlying adhesions. All five resolved
with conservative nonsurgical management. Two i.p.
catheter-related infections also complicated treatment and
led to patient removal before completion of the anticipated
number of cycles. Both were associated with abdominal
Hickman ( Bard Systems, Salt Lake City, UT) catheters. One
of these individuals developed vancomycin - resistant enter-
ococcal peritonitis. She was found to be a nasal carrier of this
organism. Another individual developed a sterile pelvic
abscess. Both patients received only five doses of SCH
58500 alone before withdrawal from the multiple - dose arm.
Overall, 82.2% of the planned doses of SCH 58500 were
delivered. In addition to the catheter problems outlined
above, failure to complete the planned number of cycles of
chemotherapy plus SCH 58500 resulted from disease
progression ( two patients ), side effects ( one patient ), and
a withdrawn consent ( two patients ).
Pharmacokinetics. SCH 58500–specific PCR was carried
out on serum samples of all patients during cycle 1. Samples
were obtained pretreatment at 15, 30 minutes, 1, 2, 4, 6, 12,
24, 36, 48, and 72 hours; and days 7, 14, 21, and 28 following
administration of SCH 58500. Detectable serum levels of
SCH 58500 were found in seven patients. In four of these, the
levels were detectable but not quantifiable. Only one patient
had a quantifiable level after 24 hours. There was no vector
shedding in either urine or stool of any patient as determined
by ELISA assay. One patient underwent a therapeutic
thoracentesis 72 hours after dosing with SCH 58500 on the
single-dose arm. The pleural fluid was positive for vector by
ELISA. Patient peritoneal fluid analysis consistently dem-
onstrated the presence of viral DNA for 24 hours. For a
subset of three patients, viral DNA was detected on day 6 for
one patient and day 7 for two. ELISA positive peritoneal
fluid was noted for periods in excess of 1 year following the
last dose of SCH 58500. However, we were unable to culture
live virus or demonstrate infectivity by the FACS assay
43
from the prolonged ELISA positive fluid.
Tumor sampling
Following cycle 1, 22 patients had ascites sampled, 5 had a
laparoscopic biopsy, and 11 had both. Only ascitic fluid was
sampled after cycle 2. Following cycle 3, 8 patients had
ascitic fluid sampled, 5 underwent laparoscopic biopsy, and
1 had both procedures.
Determination of gene transfer
The unique tripartite leader sequence incorporated into the
recombinant p53 gene sequence allowed us to differentiate
35.0
36.0
37.0
38.0
39.0
40.0
41.0
952
1122
2000
800
1140
1240
2000
800
1145
1230
2100
900
1921
2021
651
2000
1200
1845
2000
600
1600
1000
1844
1945
2030
245
1445
1535
1655
1740
1455
1555
1624
1709
T
E
M
P
Cycle 2
Cycle 1
Time
Cycle 3
Figure 1 Typical febrile response to SCH 58500 over time with
multiple doses and cycles. "indicates dose of i.p. SCH 58500
delivered. SCH 58500 toxicity by treatment cycle.
Figure 2 MIMIC
2
PCR assessment of gene transfer. The numbers
correspond to lane numbers in a 3% agarose gel. Total RNA was
extracted from a tumor biopsy obtained laparoscopically from a
patient 72 hours after administration of a single dose of 2.510
12
particles of SCH 58500. For this sample, the effect of serial dilution of
- actin message template cDNA prepared from tissue RNA and
added to a MIMIC
2
PCR reaction is reflected by the decreasing
intensity of the upper band in lanes 2 – 5 of the agarose gel. A
precisely calculated amount ( 500 molecules ) of - actin MIMIC
2
has
been spiked into the PCR reaction and results in the generation of the
lower band in the same lanes. Lanes 11 (500 molecules of -actin
MIMIC
2
) and 12 ( 100,000 molecules of - actin MIMIC
2
) have been
used for quantitative calculations. Similarly 500 molecules of the p53
MIMIC
2
have been spiked into the PCR reactions run in lanes 6 – 8
from serial dilutions of the template cDNA. In these lanes the MIMIC
2
product is the lower band and corresponds to the single band in lane 1
( 500 molecules of p53 MIMIC
2
without template cDNA ). The upper
band in lanes 6 – 8 represents p53 product containing the tripartite
leader. In the absence of transfection, as in lanes 9 and 10, no upper
band is seen because the wild - type p53 sequence does not contain
sequence that will bind the leader sequence specific primers.
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RE Buller et al
559
mRNA expression due to transduced p53 gene from any host
wild-type p53 mRNA co -isolated from contaminating
normal cells. Figure 2 shows a gel containing both sample
and MIMIC
2
PCR reaction that demonstrates this principle.
The equivalence of band intensities in lane 7 at a 1:4 dilution
of template cDNA allows for the calculation of the number
of molecules of p53 mRNA isolated from the sample
normalized for the sample - actin message content. In this
case 1.6 molecules of p53 transgene mRNA per 1000
molecules of - actin message were detected. Similar studies
were carried out using mRNA isolated from cells separated
from ascites or from biopsies obtained at the indicated times
and cycles for all patients treated. Figure 3 summarizes these
results. Transgene expression was seen at doses as low as
7.510
10
particles and consistently at or above 7.510
11
particles per dose. Three samples were negative for - actin
and were excluded from this analysis. In two cases samples
thawed during shipment. In another patient, a tumor biopsy
obtained at day 3 was negative; however, her ascites was
positive at day 7. Overall, transgene expression at the RNA
level occurred in 3 of 5, 4 of 4, 3 of 3, 8 of 11, 9 of 11, and 25
of 28 samples analyzed for SCH 58500 doses of 7.510
10
,
7.510
11
,2.510
12
,7.510
12
,2.510
13
, and 7.510
13
particles per dose, respectively. The most significant
observation from this analysis is that transgene expression
was detectable in 17 of 20 ( 85%) samples following multiple
dosing with SCH 58500.
Demonstration of vector-encoded DNA in tumor target
cells
The RT-PCR transgene expression data presented above
were generated from ascitic fluid cell pellets or tissue
biopsies. Such samples may contain normal cells as well as
tumor cells. Thus, whereas we have clearly demonstrated
transgene expression in our biopsy and ascitic fluid samples,
we have not demonstrated the presence of either agent or
transgene product from within tumor cells. To achieve this
goal, in situ PCR was carried out on sequential tissue
samples from a single patient. The primers used were
specific for SCH 58500. Figure 4A shows a sample obtained
before dosing with SCH 58500. The pink stain indicates the
absence of viral DNA. This contrasts with the blue nuclear
stain of the sample shown in Figure 4B obtained after three
cycles of SCH 58500. A negative control is shown in
Figure 4C wherein Taq polymerase was omitted from the
reaction. These results clearly demonstrate the presence of
viral DNA within tumor cells. Finally, Figure 4D shows a
hematoxylin and eosin stained section corresponding to the
tissue sample in panels B and C. In this figure, apoptotic
bodies and dying tumor cells are readily differentiated from
healthy tumor cells deeper within the biopsy.
Antiadenoviral antibody response
Baseline serum antiadenoviral antibody titers ranged from
1:160 to 1:16,000 before the first dose of SCH 58500. A 2 -
fold rise in titer could be seen by day 3 following i.p. SCH
58500. Increases in titer on day 28 ranged from 2 - to 1600 -
fold over screening values. For patients enrolled in the
multiple - dose regimens, or those re - treated with SCH
58500, a transient decrease in antibody titer on the order of
2- to 4-fold was sometimes seen. Twelve- to fifty- fold
increases over the baseline titer were observed for up to
11 months following a single dose of SCH 58500. With
multiple dosing, continued increases in titer were measured
to as high as 1:2,560,000. An immune response was
documented in one of the two individuals treated at level 1
who entered with negative titers. There was no apparent
correlation between change in antibody titer and alterations
in CA125 levels ( see below ). Likewise, there was no
correlation between the dose of SCH 58500 and the mean
change in antibody titer or the mean change in antibody titer
with transgene expression ( data not shown ).
Measures of response
Table 4 compares conventional CT response determinations
to the change in CA125 measured from study entry to study
Figure 3 p53 gene transfer following multidose i.p. delivery of SCH
58500. MIMIC
2
PCR reactions were carried out as described in the
legend to Figure 1. Measurable levels of mRNA are plotted in the
graphs according to cycle and dose of SCH 58500. Seven additional
samples were RT - PCR positive, but at expression below levels that
could be quantitated. Only 9 of 62 samples expressing - actin were
negative for p53 transgene expression.
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RE Buller et al
560
exit. Three tumors, all in group 1, were CA125 negative
( < 35 U / dL ) at the time of enrollment. Although all three
had biopsy-proven recurrence of disease, none had CT-
measurable disease either. Six other individuals with
elevated CA125 levels did not have CT- measurable disease
at study entry. Two individuals without CT- measurable
disease were treated in both group 1 and group 2. There were
no CR or PRs documented by CT scan. On the contrary, the
best CT responses were four cases of SD, three from group 1
and one from group 2. The most striking feature of the follow
up CT scans was the frequency that disease progression was
called on the basis of the development of new lesions —
documented in 18 treatment regimens. For nine of these
cases, apparent disease progression was accompanied by at
least a 26% decrease in CA125 from baseline. In several of
these cases, apparent CT progression was found at laparo-
scopy to represent a pocket of inflammatory cells. Five of
nine patients treated in groups 2 and 3 with purported CT
disease progression demonstrated at least a 50% CA125
response. In contrast, for six of nine group 1 patients, the
development of new CT lesions was accompanied by at least
a 25% increase in CA125 disease. Together these observa-
tions are consistent with the hypothesis that the new CT
lesions often occurred due to SCH 58500 – induced inflam-
matory changes rather than disease progression. This
conclusion prompted us to carry out a more detailed analysis
of the response to SCH 58500 on the basis of the associated
CA125 change from baseline.
Serum CA125 levels were measured immediately before
dosing with SCH 58500 and following each treatment cycle.
Two of the three patients with baseline CA125s < 35 U / dL
more than doubled their CA125 during study. CA125 could
thus be considered a valid response parameter for all but a
single patient. In addition, it is clear that the inflammatory
response initiated by SCH 58500 did not uniformly give rise
to an increase in CA125 by itself. The percent change in
CA125 was then calculated for each individual for each
treatment cycle, and overall at 28 days after study
Figure 4 Analysis of tumor biopsies after i.p. SCH 58500. In situ PCR measurement of viral DNA. Panel A: Biopsy from a patient before SCH
58500 ( 400 ). Panel B: Biopsy from a patient after three cycles of SCH 58500 ( 200 ). Panel C: 5-m section from the same sample as (4-B)
but a negative control based on omission of Taq polymerase from the PCR reaction ( 40 ). Panel D: An H and E section from the same sample
(200 ).
Cancer Gene Therapy
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RE Buller et al
561
completion. Comparison of CA125 levels following treat-
ment with SCH 58500 alone at 2.510
11
particles / dose to
treatment at 2.510
12
particles / dose demonstrated a mean
increase in serum CA125 of 94% versus a mean decrease
of 8.5% at the higher dose ( P= .07, 2 - tailed, unequal
variances). Thus, SCH 58500 alone at higher doses provides
a favorable change in CA125 not seen at lower doses of
vector. Figure 5 summarizes the CA125 response data.
Dosing with SCH 58500 alone resulted in a mean decrease in
CA125 of 33.6% for the 16 of 41 women whose CA125
levels declined during the 28 days following dosing. These
declines ranged from 4% to 77%. One additional patient’s
CA125 was unchanged at 46 U / mL. In contrast, with the
addition of chemotherapy for cycle 2, 15 of 18 women
demonstrated a mean decrement of 47.7% in their pre – cycle
2 CA125 levels. This result indicates enhanced CA125
response over treatment with SCH 58500 alone. For cycle 3,
11 of 16 of treated women showed a mean decline in CA125
levels of 36.6% compared to cycle 2 day 28 CA125. Thus, a
continued response was seen in excess of that seen with SCH
58500 alone. Overall, 2 of 14 women demonstrated a 50% or
greater decline in CA125 following a single dose of SCH
58500. For the 16 women who completed all three multiple -
dose cycles, 8 registered a CA125 decline 54% from study
entry. Among all responders, the average decline in CA125
was 60.1% (P=.06 vs SCH 58500 alone ). Favorable
changes in CA125 levels were independent of the time
interval from initial diagnosis to SCH 58500 dosing.
Likewise, the number of prior treatment cycles and regimens
did not preclude a CA125 response and there was no
apparent relationship between transgene expression and
CA125 response.
Discussion
Because p53 tumor suppressor gene dysfunction is seen in
50–60% of all human malignancies,
9,10
this gene has
become a leading candidate for clinical studies involving
gene transfer technology for the treatment of cancer.
Preclinical studies utilizing a variety of cell lines have
shown efficient transduction, cell cycle arrest, apoptosis, and
enhanced cell death following treatment with adenoviral
constructs containing wild - type p53 gene sequence alone
and in combination with cytotoxic chemotherapy.
22 - 31
Although some evidence suggests that this effect may not
be solely dependent on the presence of mutant p53, others
have found greater efficacy when the endogenous p53 is
mutant.
31,44
Results from in vitro xenograft models of
several malignancies, including ovarian cancer, suggest
0
10
20
30
40
50
60
70
80
C1 C2 C3 Overall
Treatment Cycle
% Decline in CA125
Figure 5 CA125 responses following treatment with SCH 58500.
In cycle 1 ( C1: 5) all patients received SCH 58500 alone. Cycles 2
and 3 ( C2, C3: &) includes all patients who received chemotherapy
in addition to SCH 58500. The mean decline in serum CA125 was
calculated for responders only. Overall ( E) is the average percent
decline at the end of the study for individuals who responded relative
to their screening CA125 level. In each case, CA125 is measured
over a 28 - day interval or at the beginning of the subsequent
treatment cycle. The error bars are 95% confidence limits of the
mean.
Table 4 Relationship of CT - based response to CA125 response
CA125 change
Treatment regimen Best response Cases >+ 25% + 24% to 25% 26% to 74% > 75%
Single - dose SCH 58500 SD 3 – 1 2 –
PD 3 3 – – –
PD
n
96 1 1 1
NM 2 1 1 – –
Multiple - dose SCH 58500 SD 1 – 1 – –
PD 9 3 4 1 1
PD
n
92 – 3 4
NM 5 2 1 – 2
SD = stable disease; PD = progressive disease by enlargement of response lesion; PD
n
= progressive disease by virtue of new lesions; NM = no
measurable disease.
Cancer Gene Therapy
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RE Buller et al
562
promise for the strategy of p53 gene replacement as a novel
cancer therapeutic approach.
25,30,31,45
There is no apparent
effect of wild -type p53 overexpression on normal tissue
such as fibroblasts.
46
Of particular relevance is the
preclinical observation that the effects of p53 gene replace-
ment are synergistic with both cisplatin and paclitaxel, the
two mainstays of ovarian cancer chemotherapy.
22,23,30
Most
clinical data to date with p53 gene replacement are limited to
intratumoral injections,
32 - 36
in contrast to the body cavity
exposure of the large surface area of the peritoneal cavity
exposed to SCH 58500 in the present study. Finally, although
carcinogenesis clearly involves multiple gene defects, data
support a therapeutic approach that corrects only a single,
critical gene defect.
47,48
Intraperitoneal therapy of ovarian cancer was initially
reported in 1955 by Weisberger et al.
49
In the past 5–10 years
encouraging results from the i.p. delivery of a variety of
chemotherapeutics and biologics have been reported both for
primary therapy and for small -volume recurrent or persistent
disease.
19,50 - 55
These studies have suggested the importance
of treating small - volume disease and have established safety
and symptom data to which one can then compare results of
i.p. gene therapy. Indeed, encouraged by these data, phase I
trials of the herpes simplex thymidine kinase / ganciclovir
system,
56 - 58
and adenoviral E1a gene therapy,
59
have been
initiated for recurrent ovarian cancer. Early results of phase
I/II retroviral and adenoviral BRCA1 i.p. gene replacement
have also been published.
60,61
Several potential limiting factors associated with i.p. drug
delivery of gene therapy per se have been identified. For
example, the uniformity of drug distribution is always of
concern. For the present study, all patients were required to
have widespread i.p. distribution verified by a pretreatment
radiologic study before initial dosing. The fraction of the
cancer cells that needs to be transduced in order for a clinical
effect to be measured is unknown. It is clear that not all
tumor target cells will be transduced, especially with a single
administration of vector because the depth of penetration
into tumor appears limited.
62
Furthermore, there is concern
that the accumulation of adhesions and the host immune
response may prevent effective gene transfer with repetitive
dosing of a viral vector. In the present study, multiple
laparoscopies on the same patient provided the opportunity
to demonstrate that individual inflammatory response was
highly variable and that peritoneal distribution can clearly
change over time.
The present study was designed to determine the safety of
the SCH 58500 adenoviral vector delivered into the
peritoneal cavity of women with refractory ovarian cancer.
No maximum tolerated dose ( MTD ) was established as the
protocol- defined DLT was not met. The doses delivered
ranged from 7.510
10
to 7.510
13
particles per i.p. infusion.
The highest dose tested was limited by practical consid-
erations including the i.p. delivery volume for multiple - day
dosing regimens. Tolerance to SCH 58500 was excellent
with manageable toxicity. Aside from fever, the toxicity
profile, even with multiple cycles was similar to that reported
for i.p. chemotherapy in general.
19,50,51
Overall, 82.2% of
the planned doses were delivered and this included 219 of
270 (81%) doses on the multiple - dose / multiple - cycle
regimens. By way of comparison, 84% of the planned i.p.
chemotherapy was delivered in a similarly sized study by
Morgan et al
51
whereas 76.8% of the planned i.p. cisplatin
doses were delivered in the large cooperative group study
reported by Alberts et al.
19
Progression of disease was the
most common reason for incomplete dosing rather than side
effects in the present study.
Vector-specific gene transfer and mRNA expression of
SCH 58500 was seen at doses as low as 7.510
10
particles /
single dose and was frequently detected in patients that
received 7.510
11
particles / dose. It seemed desirable to
increase the dose level and number of doses to a maximum
based on the theoretical tumor burden within the peritoneal
cavity and the need to maximize exposure of tumor cells to
SCH 58500. Preclinical modeling indicated that multiple
fractionated doses of SCH 58500 had greater efficacy than a
single bolus injection.
22
Early concerns that the presence of serum neutralizing
antibodies to the adenovirus might limit its effectiveness,
particularly with repetitive exposure are not borne out by our
results.
63
Preclinical work with immunized rodents treated
with intratumoral injection of an adenoviral vector express-
ing IL-12 demonstrated minimal reduction in transfer
efficiency.
25
Despite the generation of increased antiadeno-
viral antibody titers to SCH 58500 in all treated patients, we
were also able to demonstrate transgene expression after
multiple cycles of dosing. There was no obvious enhanced
transgene expression in the two individuals who were treated
at level 1 because of no demonstrable adenoviral immunity.
Not all patients underwent sampling with each cycle of
treatment, due to the invasive nature of laparoscopy.
Nonetheless, our data clearly show the presence of transgene
expression in RNA isolated from both ascitic fluid and tumor
biopsies. The alternative explanation of persistent, stable
expression of SCH 58500 over time is inconsistent with in
vitro and in vivo preclinical observations.
For a single case, in situ PCR data confirmed gene
transfer in tumor cells obtained at laparoscopic biopsy. It is
not possible to determine the percent of tumor cells
transduced because of variability in the size of the biopsies
obtained and the variation in the depth of SCH 58500
penetration. For example, in the case of a 3-mm biopsy
with 1 mm of penetration and 100% transduction to the
level of penetration, one might infer 33% transduction
efficiency. However, because the size of the lesion is
unknown, the true transduction efficiency cannot be
calculated. Similarly, a smaller (2 mm) biopsy from the
same site would provide a different estimate of transduction
efficiency. This important parameter cannot be estimated
nearly as well in human clinical trials as it can be in cell
culture, or in orthotopic animal models with smaller and
more uniform lesions. Further in situ PCR studies are
ongoing and will be the subject of a separate report ( S Wen
et al, in preparation ).
Several investigators have postulated that adenoviral
transfection efficiency is determined by the presence of
coxsackie viral receptor ( CAR ) on the surface of epithelial
cells.
64,65
We did not have sufficient samples to test this
hypothesis as an explanation for the failure to achieve
transfection in all samples collected or the differential
Cancer Gene Therapy
Ovarian cancer gene therapy
RE Buller et al
563
expression of transgene, which varied between barely
detectable to 89,000 copies / per copy of - actin. Variations
in CAR receptor levels, however, may explain differences in
transfection efficiency between ovarian cancer cell lines
transduced with SCH 58500 in vitro.
66
The inclusion of a subset of patients who received
multiple courses of SCH 58500, both alone and in
combination with chemotherapy, provided the opportunity
not only to compare cumulative toxicity but also to gain
preliminary data relevant to clinical response. As we have
demonstrated, the combination of SCH 58500 with conven-
tional chemotherapy for ovarian cancer added little to the
toxicity of SCH 58500 alone. The frequent appearance of
new CT- measurable lesions during the course of treatment
with SCH 58500 accompanied by concomitant dramatic
decreases in CA125 suggests that for gene replacement
studies utilizing adenoviral vectors, CT scans are not a valid
means to assess response. Also supporting this conclusion is
the observation of mixed clinical responses observed in the
same individual with objective responses of some lesions
accompanied by the simultaneous development of new
lesions in the same individual. Fortunately, other studies
have demonstrated that CA125 responses to ovarian cancer
treatment correlate very well with CT responses when CT is
a valid measure of response.
41,42
Because CA125 responses
also correlate well with overall survival,
67 - 70
they should not
be dismissed out of hand. Indeed, because inflammatory
changes in the peritoneal cavity may effect modest elevations
of CA125 independent of ovarian cancer,
71 - 74
the interpre-
tation of the overall responses in this study solely on the
basis of CA125 response in the face of extensive
inflammation, may actually serve to underestimate true
response rates. The number of CA125 responders and the
degree of response observed in groups 2 and 3 is remarkable
based on the heavily pretreated nature of these patients.
These data suggest that SCH 58500 has no negative impact
on clinical outcome expected from standard chemotherapy
treatments. Finally, it should also be noted that our multiple-
dose cohort contained bulky tumor deposits, not the most
optimal group to study i.p. regimens of any type.
75
We
conclude that SCH 58500 is safe, well tolerated, and in
combination with platinum - based chemotherapy provides
response data to justify its further clinical testing for efficacy
in the newly initiated phase III trial for front - line treatment
of minimal residual ovarian cancer after primary surgical
cytoreduction.
Acknowledgments
The following individuals contributed significantly to the
development, monitoring, and / or execution of this trial:
from Iowa — Barrie Anderson MD, Joel Sorosky, MD, Anil
Sood MD, Teresa Benda, RN, Karen Powliss, RN, Linda
Sanders, BS, Melanie Hatterman, BS; from UCLA —
Natalie Uhorne, Lenore Gordon, Lisa Yanemoto, Malgarzata
Beryt; from SPRI — Michelle Kerin, Mary Ann Fritz, PhD,
L Nielsen PhD, Shu FenWen, PhD; from Ulm — Dres T
Hawighorst, S Regele, K Maidel, Ms T Kohler, Dres E
Stickeler, T Einzmann, and Ms L Walz.
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