|
Detailed
protocol
1.
Immunotherapy with Heat Shock Enhanced Autologous Tumour
Lysate, Alpha-Interferon and Granulocyte-Macrophage Colony
Stimulating Factor
in Patients with Malignant Mesothelioma:
A Phase II Study
2.
Study
Personnel
Professor Bruce Robinson phone 93462098
bwsrobin@cyllene.uwa.edu.au
Dr Alex
Powell phone 93462098
powelal@cygnus.uwa.edu.au
Dr
Michelle Murphy phone 93462098
murphym@cyllene.uwa.edu.au
Clinical
Professor Bill Musk phone 93464528
3.
Rationale
Malignant mesothelioma (MM) is a cancer of the pleural
surfaces and peritoneum. It is a relentlessly progressive
disease with no known curative treatment.1
Western Australia has one of the highest incidences of MM in
the world. The disease is characterized by debilitating
symptoms, such as shortness of breath and chest pain, and
shortened life expectancy (median survival 9 months).1
Currently, no form of treatment has a substantial impact on
quality of life or life expectancy. Whilst chemotherapy can
produce tumour response rates ranging from 10-48%,2
there is no evidence to support the notion that improved
response rates equate with improvement in quality of life or
increased life expectancy. Due to the relatively poor
results with standard anti-cancer therapies, clinical trials
are now underway evaluating new treatment strategies in MM.
Our
hypotheses are:
1.
A
vaccine manufactured out of the patient’s own tumour tissue
(‘autologous’) will stimulate a tumour-specific immune
response, which will have anti-tumour activity. This immune
response will be enhanced by the addition of three immune
adjuvants: heat shock proteins (HSPs),
granulocyte-macrophage colony stimulating factor (GM-CSF) &
alpha-interferon (aIFN).
2.
The
tumour-specific immune response will result in tumour
shrinkage and prolonged survival.
Autologous Tumour Vaccines
In
recent years, cancer vaccines have progressed from
pre-clinical to clinical studies and represent a new, novel,
safe and in some cases effective form of treatment for
patients with cancer. Vaccines can be manufactured from
whole autologous tumour cells, autologous tumour cell
lysates, allogeneic cell lines or cell membrane antigens
manufactured ex-vivo. Each of these methods can produce
immunogenic material.3 The addition of immune
adjuvants can boost the immune response to the vaccine.
We have
recently completed a clinical mesothelioma trial using an
autologous tumour lysate vaccine together with GM-CSF (80mg/d
for 4 days). The treatment was well tolerated with no
significant adverse effects. In the 14 patients who
completed the trial, 4 positive DTH skin tests
were seen.
No objective tumour responses were detected, though 3
patients have demonstrable stable disease. On an
intention-to-treat basis, the median survival was 10 months.
However, there were some cases of prolonged survival (of those
who completed the trial, the median survival was 25 months). We
believe that the anti-cancer immune response to the vaccine
could be improved by the induction of HSPs within the tumour
sample, and the addition of the immune adjuvant
aIFN.
Immune
Adjuvants: HSPs, GM-CSF and
aIFN
3.1.
HSPs
3.1.1
Theory
HSPs are
natural chaperones of intracellular antigenic peptides.4
HSP-peptide complexes have been shown to be highly immunogenic.5
When exposed to HSPs, macrophages and dendritic cells are
stimulated to secrete cytokines and to express antigen
presenting and co-stimulatory molecules.6 These
antigen presenting cells (APC) then take up the HSP-peptide
complex, and the peptide is re-presented by the class I MHC of
the APC.7 This has the potential to induce an
antigen-specific cytotoxic (CD8+) T cell response, though CD4+ T
lymphocytes and natural killer cells may also be involved in the
generalised immune response.5
Every cancer
expresses a unique group of antigens.5 Immunisation
with cancer-derived HSP-peptide complexes can elicit a specific immune response that is directed only against the
cancer from which the HSPs were obtained and not against
antigenically distinct tumours or normal tissues.4
Ideally this specific immune response would lead to the
recognition and destruction of tumour cells. Four classes of HSP
preparation (gp96, HSP70, HSP90 and calreticulin) have been used
successfully in anti-cancer vaccines.4,8
Relative to
normal tissues, some primary human tumour cell lysates are
enriched in the HSPs gp96 and HSP70.9 HSP production
in tumour cells can be further increased by exposure to a
stressor, such as heat, cold and radiation.10 The
metabolism of a cell slows significantly upon heating, with the
production of HSPs increasing rapidly upon return to normal
temperature (37°C).
Therefore, exposure of a tumour to heat stress, followed by a
subsequent rest period at 37°C
prior to cell lysis, will further increase the total HSP content
in the final cell lysate vaccine.11
3.1.2
Pre-clinical
studies of HSP tumour vaccines
Vaccination
with autologous tumour-derived HSP-peptide complexes has been
shown to result in both prophylactic and therapeutic antitumour
activity in multiple animal models.4,12 Preventive
immunisation against tumours using HSP vaccines in mice was
initially demonstrated by Srivastava et al.13 Tamura
et al5 later showed that treatment of mice with
pre-existing cancers with HSP preparations derived from
autologous cancer resulted in delayed progression of the primary
cancer, reduced metastatic load and prolonged life span. Tumour
types included melanoma, fibrosarcoma, colon, lung and spindle
cell carcinomas. The HSP vaccine could be derived from either
the primary or a metastatic lesion. However, treatment with HSP
preparations derived from cancers other than the autologous
cancer did not provide significant protection.
Observations
from our own animal experiments have indicated a 40% survival
rate of mice vaccinated with tumour cell lysate enriched with
HSPs (through heat-shocking of live cells prior to lysis) and
subsequently challenged with viable tumour cells. Non-survivors
also displayed slower tumour growth kinetics (unpublished data).
C57Bl/6J
mice were vaccinated with placebo (saline) or autologous tumour
lysates (AE17sOVA lysate), some of which were prepared from
tumour cells that had been exposed to various stressors (UV,
radiation, heat). Mice were inoculated with live tumour on day
14. Mice that received the heat shocked lysate displayed slower
tumour growth (Fig A) and prolonged survival (Fig B).
3.1.3
Clinical
trials of HSP tumour vaccines
Janetzki et al14 performed a pilot study evaluating a
gp96-autologous tumour vaccine in patients with assorted
malignancies. A CD8+ restricted immune response was seen in 6
out of 12 patients. In four of these patients the response
intensified with successive vaccinations. Four of the patients
had stabilisation of disease for 3-7 months. Amato et al15
studied a similar vaccine in patients with renal cell carcinoma.
Of 16 patients, 1 had a complete response, 3 had partial
remissions, and 3 had prolonged stabilisation of disease (>52
weeks). Similar results were found in a larger follow-up trial
by the same authors16.
An
autologous tumour-derived HSP-gp96 vaccine (Oncophage) is
currently being studied in a number of phase I and II trials
involving >500 patients worldwide. In melanoma trials, responses
have been seen in 14 - 36% of patients.17,18 In a
group of melanoma patients who were disease-free post-surgery
and vaccinated with Oncophage, >75% of the group were
alive and disease-free 17 months later19. In renal
cell carcinoma, response rates of 10 % were seen, with disease
stabilisation in an additional 25%20. In 29 patients
with metastatic colorectal carcinoma who underwent surgery
followed by Oncophage, overall 2-year survival was 79%. A
significant, tumour-specific T cell immune response was seen in
60% of patients and was associated with a lower rate of
recurrence.21,22 A similar immune response has been
demonstrated in gastric carcinoma23,24, pancreatic
carcinoma25 and melanoma17,18.
Our vaccine
will differ somewhat from the above studies. As mentioned above,
4 different classes of HSPs have been used successfully in
vaccines. Rather than restrict the vaccine to a single class of
HSP, we are interested in the effect of exposure to multiple
HSPs. As such, we are not going to isolate and purify the
individual HSPs within our vaccine.
Importantly,
no autoimmune reactions or severe side effects have been
observed in any of the human trials of HSP vaccines to date.
3.2
GM-CSF
3.2.1
Preclinical
studies of adjuvant GM-CSF in tumour vaccines
GM-CSF
activates APCs, which in turn take up, process and present
tumour antigens in local draining lymph nodes. A classical
experiment demonstrating the importance of GM-CSF as an adjuvant
in tumour vaccination was published in 199326. In
this murine melanoma model tumour cells were genetically
engineered by viral transduction to secrete a variety of
cytokines. GM-CSF secreting tumours were the most strongly
immunogenic, resulting in both therapeutic and protective
immunity. These results have been confirmed in other preclinical
models.
Other tumour
vaccine studies have used soluble GM-CSF. In a B16 mouse
melanoma model, immunisation with tumour cells together with a
slow release preparation of GM-CSF resulted in a level of
immunity comparable to that of transduced tumour cells that
secreted GM-CSF27. Similar results were seen in a mouse lymphoma model, where mice
were vaccinated with a lymphoma-derived immunoglobulin idiotype
combined with 4 days of GM-CSF. This was associated with
significantly enhanced protective tumour immunity and prolonged
survival. Local (subcutaneous) administration of GM-CSF was more
effective than systemic (intraperitoneal) administration28.
Rhesus monkeys have been
vaccinated with an anti-idiotype antibody mimicking Lewis Y
antigen (which is expressed in adenocarcinoma cells) and given
two schedules of GM-CSF at 10
mg/kg
on day 1 only or the same dose for 4 days. Only the prolonged
schedule resulted in protective antibodies29.
3.2.2
Clinical
studies of adjuvant GM-CSF in tumour vaccines
A number of
clinical studies have used autologous tumour cells and GM-CSF as
immunotherapy for early stage or advanced cancer.
Simons et al30 treated patients
with prostate cancer with irradiated autologous tumour cells
genetically engineered to secrete GM-CSF.
This resulted in strong cell mediated and humoral
immunity to prostate cancer.
Leong et al31 treated patients with
metastatic melanoma with autologous tumour cells, recombinant
GM-CSF and BCG. This resulted in a 20% clinical response rate
with an extra 10% having stable disease.
Soiffer et al32 vaccinated melanoma
patients with irradiated autologous tumour cells engineered to
secrete GM-CSF. A potent antimelanoma immune response (both
cytotoxic T cells and antibodies) was associated with
destruction of both primary and metastatic lesions.
In patients with early stage colorectal cancer,
Samanci et al33 trialled a vaccine containing
rCEA (a colorectal tumour antigen) alone or with GM-CSF (80mg/d
for 4 days). The GM-CSF group developed strong humoral and cell
mediated immunity as opposed to the rCEA alone group. No
autoimmune reactions were seen.
Mellstedt34 et al treated colorectal
patients with a vaccine containing GA-73.3 antigen plus soluble
GM-CSF (75mg/d
for 4 days). Strong cell mediated responses were seen.
Side effects were minimal in all of these trials.
More recently, vaccination with irradiated autologous tumour
cells engineered to secrete GM-CSF was demonstrated to enhance
antitumour immunity in patients with metastatic non-small cell
lung cancer.35
Positive DTH responses were seen in 18 of 22 patients.
Infiltrates of T cells associated with tumour necrosis were
demonstrated in metastatic lesions in 3 of 6 patients. 2
patients rendered disease-free by surgery remained disease-free
at >40 months and 5 patients showed stable disease. Toxicity was
limited to grade 1-2 local skin reactions.
Collectively, these pre-clinical and clinical data strongly
support the use of GM-CSF as an adjuvant for cancer vaccines. We
have chosen to use soluble GM-CSF as opposed to virally
transduced cells engineered to secrete GM-CSF because: i) it is
easier, ii) the amount of GM-CSF delivered is predictable, and
iii) it has been used effectively in other trials with minimal
side effects.
3.3
aIFN
3.3.1
Preclinical
studies of adjuvant
aIFN
in tumour vaccines
aIFN
is known to have immune modulating activity including the
augmentation of T cell responses to cancer. It can also have a
direct inhibitory effect on tumour growth. In murine tumour
models,
aIFN
has been demonstrated to result in a significant delay in onset
of clinical symptoms and death compared to placebo. Some tumour
inhibitory effect has also been demonstrated.36 In
combination with cisplatinum it has resulted in tumour
regression in human mesothelioma xenografts in nude mice.37
An aIFN-producing
tumour cell vaccine +/- lung radiation was studied in a murine
model of metastatic renal cell carcinoma.38 The
growth rate of the
aIFN-secreting
tumour cells was significantly reduced in vitro and in
vivo. The host anti-tumour response to subcutaneous
vaccination with
aIFN-secreting
tumour cells was systemic, with a significant reduction in lung
metastases. The effect was enhanced by lung irradiation.
Santodonato et al39 demonstrated that
vaccination with irradiated,
aIFN-producing
tumour cells inhibited the growth of primary and metastatic
tumours, whereas control (ie non-aIFN
producing) cells were ineffective. Both CD4+ and CD8+ T
lymphocytes were involved in the anti-tumour response.
3.3.2
Clinical
studies of adjuvant
aIFN
in tumour vaccines
In patients
with gastrointestinal and lung malignancies, Itoh et al40
evaluated a vaccine containing carcinoembryonic antigen in
combination with adjuvant
aIFN
(1 million units twice weekly). Positive DTH responses were seen
in 2 out of 10 patients, who remained stable for 6 and 9 months.
No toxicity was observed. Anton et al41 used a
weekly autologous tumour cell vaccine combined with
aIFN
(1 million units) and IL2 in 208 patients undergoing surgery for
renal cell carcinoma. Toxicity was mild. Compared with
historical controls, there was an improvement in disease-free
survival (21 months).
Like GM-CSF,
pre-clinical and clinical data demonstrates that
aIFN
is an effective adjuvant for tumour vaccines. We have chosen a
low dose (250,000 units) of
aIFN
to exploit its immunomodulatory effects as opposed to its
cytotoxic effects. The low dose will mainly act locally and
therefore have minimal systemic side effects.
3.4 Potential adverse events
3.4.1 Procurement of Tumour Tissue for Vaccine manufacture
-
Video
assisted thoracoscopy:
risks associated with a general anaesthetic, the surgery
(pain, bleeding), the intercostal catheter in the postoperative
period (pain, infection, bleeding); potential for tumour to
track along the thoracoscopy site – this can be prevented by
postoperative irradiation to the area.
-
Resection/
Biopsy of a Subcutaneous Tumour Deposit:
reaction to the local anaesthetic, postoperative pain, and wound
infection.
3.4.2 The Vaccine
·
local
cutaneous skin reaction/ discomfort at the injection site
·
local
infection (rare)
·
HSPs: no
significant side effects, in particular no autoimmune reactions,
have been reported in HSP vaccine trials to date.
·
systemic
side effects of the cytokines: malaise, fever, fatigue, bone
pain, myalgia and headache (uncommon at the low doses we plan to
use)
·
GM-CSF:
increase in white cell count or eosinophil count (uncommon). In
our recently completed mesothelioma trial, GM-CSF (80mg/d
for 4 days/ fortnight) was well tolerated with no significant
side effects.
·
aIFN:
adverse effects are dose related and unlikely at the very low
dose to be used. Rare, but serious, side effects include:
depression, liver, heart, bone marrow and lung toxicity. These
are more common at high dose and in those with preexisting
disease of these organs.
·
theoretical
risk of reintroduction of live tumour cells: the methods we will
use to inactivate tumour cells have been well validated in other
published clinical trials, with no reports of tumour growth at
injection sites. In our experience, 34 patients have been
treated with no evidence of tumour growth at vaccination sites.
Every possible precaution will be taken to ensure cells are
non-viable prior to reintroduction.
3.4.3 Safety of the GM-CSF /
aIFN
Combination
The
combination of GM-CSF and
aIFN
has not previously been used in malignant mesothelioma. However,
this combination has been studied in human trials of malignant
melanoma, renal cell carcinoma (RCC), chronic myelogenous
leukaemia (CML) and chronic hepatitis B (HBV) and hepatitis C
(HCV) viral infection. In these trials, much higher doses of
a
IFN were used (³1
million units (MU) vs 250,000 units in our trial). A high dose
is more likely to have a systemic effect, and therefore side
effects, than a low dose that would mainly act locally. In some
trials the cytokines were given sequentially, rather than
concurrently. Many of the melanoma and renal cell carcinoma
trials included interleukin2 (IL2). IL2 is known to commonly
cause side effects. When a patient experiences an adverse event
whilst receiving a combination of IL2, GM-CSF and
aIFN,
it is more likely to be due to the IL2, though this can be hard
to prove conclusively. We will not be using IL2 in this
study. Lastly, some of the studies included chemotherapy and the
toxicity reported may be due to this, rather than the
aIFN/
GM-CSF combination.
Toxicity in Human Malignancy Trials using combined GM-CSF/aIFN
1.
Malignant Melanoma:
·
de Gast et
al42 trialled 5 days of oral temozolomide followed by
12 days of a combination of IL2 (4 MU/m2/d),
aIFN
(5 MU/d) and GM-CSF (2.5mg/kg/d)
in 74 patients. All patients experienced flu-like symptoms,
fatigue and anorexia. Two patients withdrew due to malaise. 58
patients had transient abnormalities of liver function tests.
·
Groenewegen
et al43 studied dacarbazine, GM-CSF (2.5
mg/kg/d
for days 2-12), IL2 (1.8 MU/d for days 8-18) and
aIFN
(6 MU/d for days 15-20) in 32 patients. Therapy was well
tolerated without significant toxicity. One patient had a skin
rash attributed to GM-CSF. Mild flu-like symptoms occurred due
to aIFN.
·
Vaughan et
al44 combined cisplatin, dacarbazine, tamoxifen and
IL2 (9 MU/m2/d) with intermittent
aIFN
(5 MU/m2 on days 6-10 and 17-21) and varying doses of
GM-CSF in 19 patients. Constitutional symptoms and lymphopaenia
were the main side effects.
These
studies all included IL2 & used much higher doses of
aIFN
than we are planning to use.
·
Schachter et
al45 used
aIFN
(3 MU/d on days 1,3,5) followed by chemotherapy then GM-CSF in
40 patients with acceptable toxicity. The
aIFN
and GM-CSF were not concurrent in this trial.
2.
Melanoma and RCC
·
De Gast et
al46 This study was designed to determine maximally
tolerated dose (MTD) and dose-limiting toxicity (DLT) of
combined GM-CSF, IL2 and
aIFN.
Therapy was given for 12 consecutive days every 3 weeks. MTD was
2.5 mg/kg/d
of GM-CSF, 4 MU/m2/d of IL2 and 5 MU/d of
aIFN.
DLT was grade 4 fever, chills with hypotension, grade 3 fatigue/
malaise and fluid retention. There was one episode of angioedema
that was attributed to GM-CSF. One patient died from a
cerebrovascular accident during the study. This was thought to
be possibly treatment related, though in an ongoing phase 2
study (70 patients) by the same authors there have been no
cerebrovascular accidents. The cytokine doses we are planning to
use are well below the MTDs in this study. When not using IL2,
the MTD of the other cytokines may actually be higher.
3.
Renal Cell carcinoma
·
Ryan et al47
trialled chemoimmunotherapy in a poor prognosis group of
patients with metastatic RCC. Patients received daily
cis-retinoic acid combined with GM-CSF (125
mg/d)
for 2 weeks, followed by IL2 (11 MU/d for 4 days/wk) and
aIFN
(10 MU/d for 2 days/wk) for 4 weeks. Dose reductions of IL2 and
aIFN
were required for dehydration, respiratory difficulty, fatigue,
fever, myalgia and mental status changes. Eosinophilia occurred
in most patients and mild leucocytosis not requiring dose
reduction in one patient. Other side effects included fever,
fatigue and anorexia. Mucositis, chelitis and dermatitis were
due to cis-retinoic acid. There was one possible therapy-related
death from a cardiopulmonary arrest. This patient had had
previous cardiovascular toxicity (tachycardia and hypotension)
and a dose reduction had been planned. This study used very high
doses of cytokines combined with chemotherapy in unwell
patients.
·
Lummen et al48
studied
a
IFN (10 MU) plus GM-CSF (15-300
mg)
3 times per week in 21 patients with advanced RCC. 24% of
patients dropped out due to grade 3 toxicities associated with
high dose GM-CSF and
aIFN.
Doses of 120-150mg
GM-CSF 3 times per week were tolerated.
·
Westermann
et al49 studied varying combinations of GM-CSF (5
mg/kg
3x/wk), IL2 (4 MU/m2 5x/wk) and
aIFN
(5 MU/m2 3x/wk) in metastatic RCC. There was no
severe organ toxicity, though there was one case of marked
eosinophilia. Fatigue and flu-like symptoms were more common
when the three cytokines were combined.
4. CML
·
Cortes et al50
used GM-CSF (30-60
mg/m2
/d) in patients with CML already taking
aIFN
(8 MU 2x/wk to 11 MU/d). The combination was well tolerated,
with less myelosuppression than
aIFN
alone. These patients had already been stabilised on
aIFN,
so there may have been selection bias for patients who would
tolerate the combination.
4.
Objectives
To assess
the response of a vaccine containing heat shock enhanced
autologous tumour lysate with adjuvant GM-CSF and αIFN on
the outcome of patients with malignant mesothelioma.
The primary
outcomes will be:
1.
Stimulation of a tumour specific immune response
2.
Disease response
3.
Time taken to disease progression.
4.
Overall survival
5.
Study Plan & Schedule of Assessments
5.1
Methods of collecting data
The stimulation of a tumour specific immune response will be
measured by way of a delayed type hypersensitivity skin test
(DTH) using non-viable autologous tumour tissue. The material is
rendered non-viable by the same way as the autologous tumour
lysate material (see laboratory protocol). The tests will be
read as either positive or negative, a positive test being an
erythematous, raised skin reaction measuring > 5mm 48 hours
after its administration. Western blots will also be performed
on patients’ serum to determine the presence of protein bands
that will indicate antibody formation. Again the test will be
either positive or negative.
Tumour response will be assessed by serial CT scan measurements.
The now standard RECIST51 criteria will be used to
measure tumour response (Appendix 2).
Time to progression will be measured as the time from the first
vaccination to tumour progression as assessed by radiological
means (see above) or the development of a new lesion.
5.1
Study Plan
   
5.2
Schedule of assessments
|
Visit |
Time
(weeks) |
Surgery |
Vaccine |
FVC |
DTH &
WB |
CT Scan
Chest |
FBP
U&Es
LFTs |
|
Initial |
-2 |
|
|
|
|
|
Ö |
|
2 |
0 |
Ö |
|
|
|
|
|
|
3 |
2 |
|
Ö |
Ö |
Ö |
Ö |
Ö |
|
4 |
4 |
|
Ö |
Ö |
|
|
|
|
5 |
6 |
|
Ö |
Ö |
Ö |
|
Ö |
|
6 |
8 |
|
Ö |
Ö |
|
|
|
|
8 |
10 |
|
Ö |
Ö |
Ö |
|
Ö |
|
10 |
12 |
|
Ö |
Ö |
|
|
|
|
14 * |
14 |
|
|
Ö |
Ö |
Ö |
Ö |
Notes:
FVC: forced
vital capacity
DTH:
delayed-type hypersensitivity skin test
WB: western blot
FBP: full blood profile; LFTs: liver function tests; U&Es: urea
and electrolytes
* At week 14
a decision will be made as to whether a patient continues with
fortnightly vaccinations or has follow up at monthly intervals.
5.3
Clinical
Protocol
Eligible
patients will consent to a video assisted thoracoscopy (VAT) or
subcutaneous tumour deposit resection or will be having a
surgical pleurectomy as a therapeutic, non-experimental
procedure. The surgeon will be asked to provide us with at least
5 cc of tumour tissue that will be placed in a sterile jar and
retrieved by one of our staff and transported immediately to the
laboratory. Therapy will begin within 2 weeks. The vaccines will
be administered as a sub-cutaneous injection over the deltoid
once a fortnight for 12 weeks. A dose of 0.25 x 106 U
aIFN
and 80ug of GM-CSF will be administered as a daily subcutaneous
injection
at the vaccine site
for 5 days beginning on the day of the vaccine. CT
scans of the thorax will be obtained at baseline (ie in the
immediate post surgical period), and at the end of the 12 week
vaccination period. In those patients with measurable disease
tumour thickness will be measured by RECIST criteria. Lung
function, as measured by forced vital capacity (FVC), will be
tested at fortnightly intervals. DTH testing will be performed
at baseline, followed by 3 further tests at monthly intervals.
Patients will return to clinic 2 days after each DTH skin test
to have the result read and recorded. FBP and U&Es will be
performed at baseline and monthly for 3 months to monitor for
any potential haematological and renal toxicity or electrolyte
disturbance.
Those
patients assessed as having responding or stable disease (by
RECIST criteria) at the end of the vaccination period will be
offered further vaccinations at fortnightly intervals until the
vaccine supply runs out, or there is clear evidence of disease
progression. CT scans will be performed at 2 monthly intervals,
or monthly if lung function has deteriorated by more than 25%,
during this prolonged vaccination phase.
Patients
with progressive disease will be followed up monthly until time
of death. A history, physical examination and details of
subsequent therapies will be obtained. All patients will also be
referred back to their original treating doctor for ongoing
follow-up.
5.4
Laboratory
Protocol
10 mls of
serum will be collected from the patient prior to surgery.
Approximately 10 grams of fresh tumour tissue will be collected
in a sterile container from theatre and transported directly to
the laboratory (Bone Bank, Hollywood Private Hospital). This is
the only TGA approved area in the vicinity that is suitable for
handling transplantable human tissue. Specimens will be given a
unique identification code and processed immediately. No other
tissue culture work will be done at the same time or using the
same equipment.
A sufficient
amount of tissue will be processed to obtain a final cell count
of 1-2 x 108 cells, in order for the patient to
receive the equivalent of 1-2 x 107 lysed tumour
cells per vaccination. A separate sample of tumour will also be
set aside for analysis of baseline expression of heat shock
protein 70 (HSP70) by ELISA.
Manufacturing the vaccine:
Working in a
laminar flow hood, the tumour tissue will be coarsely diced up
using a sterile scalpel and petri dish, and then transferred to
a 50ml centrifuge tube (Falcon). Hepes buffered culture medium
(Sigma, Invitrogen) and 5-10mls of autologous serum will be
added. The specimen will be transferred to a preheated incubator
(Sanyo MIR 153 incubator) and heat-shocked at 43°C
for 1 hour. The incubator temperature will then be rapidly
reduced to 37°C
and maintained for 3 hours to allow the cells to recover. The
supernatant (media + sera) will be removed from the tissue
sample and discarded. The sample will then be rinsed in sterile
saline to remove any residual media, prior to being frozen in
liquid nitrogen overnight.
Wei et al52
demonstrated the induction of HSP in fresh human tumours by
heat exposure. Tumours were surgically resected and
enzymatically digested with collagenase and DNase, prior to
being placed in a water bath at 42°C
for 30-60 minutes. The samples were then incubated at 37°C
for 2 hours. Western blot analysis revealed an increased
expression of heat shock proteins in the heat-treated tumour
cells.
Our protocol
differs slightly from this for the following reasons. We are not
going to isolate and purify the different HSPs within the sample
because: i) we are interested in the effect of exposure to HSPs
in general, rather than an individual class of HSPs; ii)
isolating the HSPs is time consuming, costly and
difficult; and iii) we will be reintroducing the processed
tumour lysate back into patients, rather than just analysing the
HSPs in the laboratory. It is important that the samples remain
sterile and are exposed to as few foreign substances and
materials as possible. Mechanical rather than enzymatic
digestion of tumour tissue will be used because residual small
amounts of collagenase in the final sample can cause false
positive DTH skin test results. This would make it difficult to
determine if a positive result was due to an immune response to
the tumour lysate or the contaminating enzyme. Lastly, we will
be using an incubator rather than a water bath because it can be
programmed to adjust the temperature quickly and accurately.
To determine
that the proposed heat-shock procedure is effective in inducing
heat shock proteins, pre-heat shock and post-heat shock tumour
samples will be analysed using a human HSP70 ELISA kit. This
detects the level of HSP70, the main inducible stress protein,
in cell lysate. As HSP70 is present at basal levels in human
tissues, small amounts of the protein should be detectable by
ELISA in the pre-heat shock tumour lysate, and this amount
should increase after the heat shock procedure.
The
following day the tissue will be thawed and mechanically
digested using a syringe plunger and a 40
mm
nylon cell strainer. The cells will then be suspended in saline
to a final volume of 5mls and freeze-thawed an additional five
cycles by freezing in -195°C
liquid nitrogen and warming to 45°C.
This will cause cell lysis. Finally, the cell suspension will be
irradiated with 20,000Gy (QEII radiation department) to ensure
cell non-viability. This will be confirmed using the trypan blue
method. This is the method used by other clinical cancer vaccine
trials to ensure cell non-viability.
We have
performed preliminary experiments using human tumour tissue
obtained fresh from the operating theatre and shown that
following two freeze-thaw cycles no viable cells can be
detected. If during the conduct of this trial viable cells are
detected, the freeze-thaw process will be repeated until no
viable cells are present.
A 0.5ml
aliquot of the 5ml tumour lysate sample will be taken and made
up to 5mls with normal saline. This will be divided into 10 x
0.5ml aliquots for DTH skin testing. The remaining 4.5mls of
tumour lysate will be divided into 9 x 0.5ml aliquots (6
vaccinations, 1 western blot, 1 HSP70 ELISA and 1 spare sample).
Vials will be labelled with a unique identification code and
stored in a designated cryogenic storage unit located within the
Bone Bank.
If
sufficient tumour tissue is available we will process the
samples in duplicate. This will enable the vaccine dose to be
increased in those patients who have not developed a positive
DTH skin test after receiving the initial two doses of vaccine.
Alternatively, those patients who have developed an immune and
radiological response to the vaccine would be able to undergo
further vaccinations.
This
protocol will abide by the Australian Code of Good Manufacturing
Practice for Medicinal Products (16 August 2002), which covers
investigational medicinal products. A copy of this document will
be held in the laboratory, along with a standard operating
procedure manual that we have prepared especially for this trial.
6.
Inclusion
Criteria
·
Males and females with histologically or cytologically confirmed
MM, with disease that is: 1. Surgically resectable via
pleurectomy, or
2.
Suitable for debulking surgery, or
3.
Chest wall masses which can be resected under local anaesthetic,
or
4.
Diffuse pleural disease which is amenable to video assisted
thoracoscopy
·
18 years of age and over
·
ECOG 0 – 2
·
Life expectancy of > 3 months
·
Bilirubin, AST and/or ALT < 2.5 times the upper limit of normal
level.
·
Serum creatinine < 200 umol/L
·
Neutrophil count > 1 x 109, platelet count > 50 x 109
·
Geographically accessible for treatment and follow up.
·
Ability to sign informed consent.
·
Women of child bearing potential must either practice abstinence
from intercourse through the duration of the study or take
appropriate contraceptive measures (including sterilization of
partner)
·
Men whose partners are of child bearing potential must practice
abstinence from intercourse for the duration of the trial, be
surgically sterile, or ensure their partner is taking adequate
contraceptive measures.
7.
Exclusion Criteria
·
Patients who are immunosuppressed or on oral steroids
(aerosolised steroids acceptable).
·
Patients on current investigational treatments.
·
Patients who have received chemotherapy within the last 4 weeks
·
Patients who have received previous immunotherapy
·
Serious uncontrolled disease (including serious psychological
disorders) likely to interfere with the study and/or likely to
cause death during the study duration
·
Previous participation in the trial
·
Active infections requiring antibiotic or anti-viral therapy
·
Non-compensated heart failure
·
Myocardial infarction during the last 6 months
·
Severe non-compensated hypertension
·
Severe non-compensated diabetes
·
Clinical signs of cerebral dysfunction
·
Women during the lactation period, pregnancy or of childbearing
potential not using a reliable contraceptive method
·
Severe psychiatric disease
·
Known HIV or active chronic hepatitis B or C infection
8.
Prohibited Drugs and Interventions
·
Patients must not take steroids (inhaled acceptable) during the
duration of the study, but steroids are allowed during the
follow up phase.
·
Radiotherapy must not be given to sites of measurable disease.
·
Concurrent chemotherapy.
9.
Study Plan and Analysis
Untreated
malignant mesothelioma can be assumed to have a tumour response
rate of 0%. Assuming that chemotherapy achieves a response
(i.e. tumour shrinkage) in 30% of patients, we would aim to
observe a similar response rate with the study vaccine.
In our previous trial,
4% of patients with malignant mesothelioma had a positive
DTH and western blot prior to therapy. Therefore, if an
extra 30% of patients develop positive DTH skin tests or
positive western blots during the study, this would be
considered a significant immune response to the vaccine.
To detect a
34% response to therapy with a power of 90% and an
a
level of significance of 5%, we would need 49 patients.
This calculation is for a single measurement in time. Our study
will have repeated measurements taken over time; this will
increase our overall power.
Statistical
advice was obtained from the Biostatistical Consulting Group,
School of Population Health, University of WA.
The
development of a tumour-specific immune response to the
vaccine will be assessed by DTH skin testing and western blot
analysis. A review of published clinical cancer vaccine trials
reveals that evaluation of tumour-specific T cell responses by
DTH testing is the simplest and most informative approach. The
advantages of DTH testing are its convenience and specificity
(an autologous tumour cell lysate is used in the test). There is
also clinical data to suggest that the clone of T cells that
infiltrate DTH lesions are of the same type that infiltrate
metastatic deposits of tumour53. Some studies have
suggested that a response on DTH testing is correlated with
patient survival. Two trials in stage III melanoma patients
demonstrated that a DTH response to a polyvalent melanoma
vaccine correlated with overall survival53,54. McCune
et al demonstrated a similar association in patients with renal
cell carcinoma55.
A
large prospective study by Harris et al evaluating the effect of
an autologous tumour cell vaccine on the risk of tumour
recurrence in colorectal cancer demonstrated a positive
correlation between DTH response and reduced risk of recurrent
tumour56.
In untreated
patients with mesothelioma, the tumour would be expected to
progress and therefore increase in size on CT over time. If the
vaccine is effective in eliciting an anti-tumour response, the
abnormalities on CT may instead be stable or regress. To
determine disease response, a CT scan of the chest will
be obtained after surgery (i.e. pre-vaccination baseline) and at
the end of the vaccination period. In patients with measurable
disease, tumour thickness will be measured and disease response
determined by RECIST criteria.
On the baseline (pre-vaccination) CT scan, the sum
of the longest diameter for all target lesions will be
calculated
(see diagram, Appendix 2). This is referred to as
the baseline sum longest diameter (BSLD), and will be used as
the reference by which to characterise the objective tumour
response. A complete response is the disappearance of all
target lesions. A partial response is defined as at least
a 30% decrease in the sum of the longest diameter of target
lesions, relative to the BSLD. Progressive disease is
defined as at least a 20% increase in the sum of the longest
diameter of target lesions, relative to the BSLD. Stable
disease is defined as neither sufficient shrinkage to
qualify for partial response nor sufficient increase to qualify
for progressive disease.
Lastly, lung
function (Forced Vital Capacity) will be recorded on a
fortnightly basis during the vaccination period.
At the conclusion of the trial, data will be analysed with the
assistance of a statistician from the Biostatistical Consulting
Group, School of Population Health, University of WA. At
baseline descriptive analysis will be undertaken. To analyse
continuous data utilizing all the data measured at various time
points, a generalised linear mixed model will be used with a
compound symmetry variance-covariance structure. For binary
outcome measures, a generalised estimating equation model will
be used. In specific detail:
-
DTH
skin tests:
these will be performed at 4 time points during the trial
and will be reported as positive or negative. In order to
investigate changes over time a generalised estimating
equation method will be used for analysis to incorporate the
data collected at every time point.
-
Western blot analysis:
these will be used to assess immune response to the tumour
vaccine, not to quantitate a HSP response (see question 3 &
4). The western blot results will be reported as a
descriptive analysis.
-
Tumour measurement:
CT scans of the chest will be performed at baseline and at
the end of the vaccine period, and disease response will be
measured by RECIST criteria (described below). Polytomous
logistic regression with 3 outcome measures (partial
response, stable disease, progressive disease) will be used
to analyse the results.
-
Adverse events:
will be
reported as a descriptive analysis
-
Lung
function:
will be performed at each clinic visit. A generalised linear
mixed model with a compound symmetry variance-covariance
structure will be used to analyse the results for the
average change in lung function over time.
-
Survival:
A Kaplan Meier curve will be given. To investigate factors
related to survival, a proportional hazard regression model
will be used.
-
Time
to disease progression:
will be analysed in a similar way to survival.
9.4
Withdrawals
If a patient
withdraws due to a protocol violation, lack of interest, or
toxicity they will be included in the final analysis. They will
be invited to partake in the follow up process but if not their
progress will be followed by way of contact with their general
practitioner.
9.5
Analysis Population
All eligible
patients who enter the trial will have their results analysed,
including patients who withdraw. Assessment of disease response,
time taken to disease progression and overall survival will be
on an intention-to-treat basis. DTH responses and western blot
results will be analysed in all patients in whom they were
performed, irrespective of the number of vaccines received
9.6 Interim Analysis
An interim
analysis of data will be performed at 12 months, or when 25
patients have been recruited to the study, whichever is sooner.
An independent person who is not directly involved in the study
will undertake this analysis. The results of the interim
analysis will be discussed only with the Chief Investigator
(Professor Bruce Robinson), who will then decide to continue or
cease the study (eg. if the vaccine has proved itself effective,
or if there are unexpected safety issues).
If the study continues, then
appropriate adjustment to the p-values will be made due to the
interim analysis.
10. Safety:
Reporting of Adverse Events
10.1
Definition
of Adverse Events
·
adverse drug
reactions
·
illnesses
with onset during the study
·
exacerbation
of pre-existing illness, including the disease under study
·
abnormal
objective test findings (abnormal U&Es, FBP) or any test that
results in a change in study drug dosage or in discontinuation
of the drug/vaccine.
10.2
Investigator's Responsibility to Report Adverse Events
For all
adverse events, we will pursue and obtain information adequate
both to determine the outcome of the adverse event and to assess
whether it meets the criteria for classification as a serious
adverse event requiring immediate notification to CDTC/RIEC. For
all adverse events, sufficient information will be obtained by
us to determine the causality of the adverse event (i.e. study
drug or other illness).
10.3 Definition of Serious Adverse Events
A serious
adverse event is any adverse drug experience occurring at
any dose that:
1. results
in death;
2. is life
threatening;
3. results
in inpatient hospitalisation or prolongation of existing
hospitalisation;
4. results
in a persistent or significant disability/incapacity; or
5. results
in congenital anomaly/birth defect.
If timely
treatment saves the patient from death, from illness of
life-threatening severity or from hospitalisation, the reaction
is still classified as serious.
A life-threatening
adverse event is one that actually places
the patient/subject at immediate risk of death. A patient's
reaction is not classified as life-threatening simply because
more severe manifestations of the same adverse reaction can be
fatal, eg mild airway obstruction is not life threatening but
severe airway obstruction is.
Hospitalisation
includes any
inpatient admission, any transfer within a hospital for the
purpose of treatment (for example, transfer of a patient from a
psychiatric ward to a general medical ward) and prolongation of
admission. Prolongation of hospitalisation is defined as any
extension of an inpatient hospitalisation beyond the stay
anticipated/required in relation to the original reason for the
initial admission, as determined by the investigator or treating
physician. The following events, however, are not classified as
serious adverse events:
a)
Admission for treatment of a pre-existing condition not
associated with the development of a new adverse event or with
a worsening of the pre-existing condition.
b) Social
admission (e.g. subject has no place to sleep) or respite care
(e.g. caregiver relief)
c)
Administrative admission (e.g. for yearly physical exam)
d)
Protocol-specified admission during a clinical trial (e.g. for a
procedure required by the study protocol)
e) Optional
admission not associated with a precipitating clinical adverse
event
f) Hospice
admission, nursing home admission, admission to custodial care,
admission to a clinical research unit, admission to a
rehabilitation facility, attendance at
outpatient/same-day/ambulatory care /accident and emergency
facility unless another criterion for severity has been
satisfied
Disability
is defined
as is a substantial disruption of a person’s ability to conduct
normal life functions.
10.3
Investigator's Responsibility to Follow-up and Characterise
Adverse Events
This will be
in accordance with the CDTC guidelines
10.5 Adverse Events Reported through the Sponsor
This is not
a sponsored trial.
10.6
Collection
of Adverse Event and Safety Information (Appendix 1)
The
instruments to be used for collection of adverse event data are
outlined in Appendix 1.
11. Drug
Storage and Handling
GM-CSF and
αIFN vials to be stored under refrigeration within a secure
place in the University Department of Medicine. The vaccines are
stored and coded within a secure cylinder within the Hollywood
Bone Bank.
12.
Compliance with Good Clinical Practice, Ethical Considerations &
Informed Consent
This study
will be conducted in compliance with the conditions stipulated
by the Clinical Drug Trials Committee and Research Institute
Ethics Committee, informed consent regulations and NH&MRC
Guidelines. In addition, all local regulatory requirements will
be adhered to, in particular those which afford greater
protection to the safety of the trial participants.
This study
will be conducted according to the current revision of the
Declaration of Helsinki (Revised South Africa 1996) and with
local laws and regulations relevant to the use of new
therapeutic agents in Australia.
All
amendments to the trial will be placed before the Research
Institute Ethics Committee and the Clinical Drug Trials
Committee for approval.
Any
information that may affect either committee's decision to
continue approval for the trial will be forwarded to the
committees without delay.
An annual
report will be issued, in accordance with the guidelines
provided by the Research Institute Ethics Committee
13.
Informed Consent
The investigator, or a person designated by the investigator,
will explain the benefits and risks of participation in the
study to each subject, the subject’s legally acceptable
representative or impartial witness. Written informed consent
will be obtained prior to the subject entering the study. Each
subject’s original consent form, signed and dated by the subject
or by the subject’s legally acceptable representative, and by
the person who conducted the informed consent discussion, will
be retained by the investigator.
14.
Patient Information Sheet (attached)
15.
Patient Consent Documents (attached)
-
clinical
trial consent form
-
consent for
blood/tissue taking and cell line/tissue banking for clinical
research
16. Record
retention
Records will
be retained for 15 years in a locked facility, in accordance
with NH&MRC guidelines. Access to the records will be limited to
the study personnel listed at the beginning and authorised
representatives of drug regulatory bodies.
17.
Withdrawal criteria
Participants
will be withdrawn from the study under the following conditions:
·
Patient
request
·
If, in the
opinion of the treating clinician, the patient's interests are
best served by withdrawing from the trial
·
Patient
non-compliance
·
Circumstances which, in the opinion of the investigator will
prevent the patient from completing the trial treatments and
procedures
18.
Emergency Procedures
In the event
of anaphylactic shock we have a supply of adrenalin in the
treatment room. If patients are acutely unwell due to their
disease or toxicity from therapy they will be admitted to SCGH
under the care of either the chief investigator or associate
investigators.
19.
Liability and Indemnity
Please note
this is not a sponsored trial. SCGH and the University of WA
will provide indemnity.
20. Study
Monitoring and Auditing/Documentation of Study Findings
This will be
conducted by Miss Judy Innes-Rowe ph 93464520
21. Study
Dates
June 2003 –
June 2006
23.
References (attached)
24.
Investigator Brochure (attached)
25.
Investigator Protocol Agreement
Please
note: this is not a sponsored trial. However we will abide by
the following:
Consent forms
Patient information
sheet
For more
information contact:
jolghazi@cyllene.uwa.edu.au
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