DEFINICIÓN DE TÉRMINOS

Having demonstrated that multiple antigen nucleofection and subsequent ELISpot could be done with a protocol that minimized background responses, we wanted to apply the assay to prostate cancer patients. To explore this, we undertook a study titled, ‘immune responses in adenocarcinoma of the prostate (IRAP). The aim of this study was to apply the assay in a clinical setting, allowing us to further refine the assay in preparation for the ADUP clinical trial. It would also give us an opportunity to catalogue immune responses in prostate cancer patients to help us interpret the results from the ADUP trial. By sampling patients with various disease grades and stages, the data would also help hypothesis formation for future studies. After suitable ethical and research and development approval, prostate patients were recruited from prostate clinic and from the ward.

It was reassuring to find that patient recruitment to the study was straightforward. Only 5 patients (12%) declined to participate demonstrating the acceptability of the tests to this patient population. There were no difficulties encountered with the retrieval of blood samples from theatres and outpatients and their transfer to the laboratories in the School for Cancer Sciences. The cellular yields from the samples were satisfactory for the completion of the assays with cells to spare. It was noted that

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the yields from the theatre patients were slightly higher, and that could reflect their relative dehydration (as the patient has been starved for a general anaesthetic) or that they were a younger cohort of patients. In the forthcoming ADUP trial, the patients will have their primary sample taken in theatre and I expect the cellular yields to also be high.

The assay performed well in this patient population with consistently low background readings and consistent spot counts. We found evidence of immune responses to prostate tumour antigens in both patients with prostate cancer, and patients without prostate cancer. The levels of response were much less in the non-cancer patients but within the cancer patient cohort we found a wide variety of responses. In patients with low volume early stage disease the responses were brisk, as were the responses in patients on active surveillance. These findings are consistent with immuno-editing theory but, in the active surveillance patients, these findings could be attributed to the fact the patients are undergoing regular prostate biopsies. In those patients with advanced disease, the immune responses were far less evident. This could be due to exhaustion of the T cell populations targeting our panel of prostate specific tumour antigens and this may be contributing to the progression of the disease. It may be because of progressive loss of MHC expression in these tumours, their increasing heterogeneity or mechanisms within the progressive tumour microenvironment that there is suppression of this T cell population. It is possible that these patients have always had poor responses, and that has contributed to their disease progression. It was interesting to observe a trend towards an increase in immune responses from low risk to intermediate risk patients before the drop in immune responses seen in high risk patients. Initially, this increase could be due to increase in disease volume without a major shift in cellular morphology driving greater immune responses. As

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the disease progresses and the cellular morphology becomes more poorly differentiated the immune responses coincidently drop away. There will usually be greater disease bulk in these patients making it difficult to identify why the immune responses decrease. It could be morphology, tumour volume or both that is driving the exhaustion of the T cell response. It could also be that failure in the immune response facilitates the progression of prostate cancer in these patients.

This raises interesting considerations for the timing of immune–related therapies in prostate cancer. Clinical trials using the autologous dendritic cell based vaccine therapy, Sipuleucel T were undertaken on patients with advanced disease. If our observations are a genuine reflection of the patterns of immune responses in prostate cancer patients then these patients might be expected to benefit least from this type of treatment. If it were practical, it would be interesting to see how efficacious this treatment would be in patients with earlier staged disease. The ADUP trial will be recruiting patients at an earlier stage in their disease pathway than those in the Sipuleucel T trials and the disease bulk will be much less. The immune stimulating element of the treatment should benefit from this and it will be interesting to monitor the immune responses in these patients as their treatment progresses.

In the study, we were fortunate enough to get sequential samples on 10 patients. In those patients in whom there were no treatment modifications, the immune responses appear to be gradually declining. Those patients in whom a direct insult to the prostate is offered, there appears to be an upward trend in the immune responses. The most remarkable change in immune responses is seen in the patient who underwent HIFU. This observation is consistent with observations made in the literature relating to this treatment in the prostate and other organs. Given the coagulative necrosis caused by the HIFU, and the rim of thermally stressed but surviving tissues, there would be an

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expectation to see immune responses mounted to antigens released within the treatment environment. It would be worth further study to see if this is a consistent feature for this treatment and if similar responses are seen in other thermotherapeutic modalities such as cryotherapy. If this is a consistent observation, it would be worth investigating whether neoadjuvant use of immune stimulants alongside thermotherapies can be used to enhance their efficacy, or even see them used in advanced disease to try and offer local disease control whilst delivering a systemic immune benefit.

If the assay were used serially in a patient it would be interesting to see if it could have a role in predicting outcome, particularly in those patients who would ordinarily select active surveillance. We have seen the gradual decline over time in patients on stable treatment, and this kind of assay could serve as an indicator for contemplating a treatment change or escalation. The limitations with the assay, would be in the cost and logisitics involved in delivering it in mainstream healthcare and, consequently, it is likely to remain a research tool.

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In this thesis I have demonstrated the modified virus AdGMNR will infect prostate cancer cells and the NTR and GMCSF genes are expressed. Those cancer cells expressing NTR and subsequently exposed to CB1954 will die with neighbouring cells also dying, due to the bystander effect. The GMCSF produced by infected prostate cancer cells will induce morphological changes in PBMCs consistent differentiation into immature dendritic cells.

I have adapted an ELISpot assay that gave high background readings. Modifying the assay has minimized those background readings. The platform allows us to test for responses to a panel of 5 prostate specific cancer antigens. I expect this to increase the sensitivity of the assay.

I have used the assay on a number of prostate cancer patients at various disease stages and have been able to measure immune responses in a satisfactory way. The observed immune responses show consistencies with immune editing theory, and generates some good background data against which we can compare the results from the forthcoming ADUP trial as well as helping to formulate possible hypotheses to be tested in future studies.