Detailed protocol

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 carcinoma^23,24, pancreatic carcinoma²⁵ and melanoma^17,18.

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

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

9.1             Power calculations

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.

9.2             Data analysis

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 survival^53,54. 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. 

9.3             Statistical Analysis

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