Immunologic biomarkers in prostate cancer

Abstract

One major achievement in cancer therapy is to select patients who will most likely benefit from a specific treatment. Predictive biomarkers play an important role in this respect being already useful in management of breast cancer and melanoma. For example, HER-2/neu (HER-2) overexpression selects for breast cancer patients to be treated with trastuzumab, and BRAF_V600E mutations select for melanoma patients to be treated with vemurafenib. Identification of factors associated with T cell responsiveness to vaccination remains critical. Pre-existent immunity and circulating suppressor cells may regulate the levels of vaccine-specific T cell immunity after vaccination. The identification of immunologic endpoints to immunotherapy would thus considerably help guide the development of immunotherapy-based clinical trials. This commentary is based on a retrospective analysis we performed of data from prostate cancer patients vaccinated and boosted with the AE37 vaccine. The aim of these exploratory analyses was to identify factors useful in predicting which patients are more likely to respond to the treatment under study. The issue we are addressing here is to which extent common variables used pre- and/or following vaccinations with AE37 to assess the immune response status of the prostate cancer patients, may predict overall survival.

Keywords: :

• prostate cancer
• cancer vaccine
• biomarkers
• HER-2/neu
• HLA

Introduction

Prostate cancer (PCa) represents the second most common cause of cancer-related death among Caucasians. The protracted time-period from first diagnosis to death and the defined stages during disease progression, make PCa an appealing target for immunotherapies. Among the many concepts under investigation, augmenting immune responses to PCa has been proposed as a valid therapeutic approach, and there are a number of lines of evidence to support this. For example, studies of PCa samples show evidence of intratumoral infiltration by multiple leucocyte subtypes, including natural killer cells and T cells. These data suggest that both the innate and adaptive branches of the immune system are involved in spontaneous immune responses to PCa.¹ Many different immunotherapeutic strategies are being investigated in clinical trials of patients with PCa, with vaccines being most heavily exploited as single agents.²

Vaccines

Cancer vaccines represent an evolving type of immunotherapy that can be used to present single or multiple tumor antigens to the immune system, via a variety of delivery systems, in order to prime/boost an immune response. Over the last 2 decades, anticancer vaccines have generally yielded disappointing clinical results,³ however, increased understanding of the immune system, tumor immunology and vaccine technologies have allowed for the development of novel vaccine approaches that may be more efficacious.⁴ To this end, Sipuleucel-T became the first therapeutic cancer vaccine to receive Food and Drug Administration approval in the USA based on the prolongation of overall survival (OS) among men with metastatic castrate resistant PCa (MCRPCa), and the results from clinical trials with PROSTVAC-VF, a poxvirus based vaccine, have also been encouraging, thus providing proof-of- principle for vaccines as a therapeutic approach.^5,6 In a phase II study, PROSTVAC-VF was administered concurrently with ipilimumab in patients with MCRPCa who experienced a significantly longer OS vs those treated with PROSTVAC-VF alone.^7,8

HER-2 Expression in Prostate Cancer

HER-2 is a member of the family of oncogenes associated with tyrosine protein kinase activity. HER-2 oncoprotein is a 185-kDa transmembrane receptor (p185), with intracellular, transmembrane, and extracellular domains. The extracellular domain (ECD) can be detected in the serum of patients with PCa and its increase has been associated with metastatic hormone-independent disease stage.⁹ HER-2 has been proposed as a survival factor for PCa cells in the absence of androgens.^10-12 Overexpression of HER-2 promotes the activation of the androgen receptor (AR) and enhances the binding of AR to the promoters of androgen-regulated genes. In PCa patients, HER-2 overexpression is found mainly in those patients who have become resistant to hormonal ablation.¹³ Moreover, HER-2 expression in tissue samples of hormone-independent PCa patients has been associated with lower survival.^14-16 Therefore, HER-2 targeting has become an attractive treatment strategy for hormone-refractory PCa patients.^17-20 The efficacy of this strategy, however, has not been clinically proved to date.

The AE37 Vaccine

Work from our laboratory has shown that the HER-2(776–790) epitope serves as a compelling tumor antigen and that CD4⁺ T cells primed with the synthetic HER2(776–790) peptide help autologous cytotoxic T lymphocytes (CTL) for increased antitumor activity.^21-23 We have shown that HER2(776–790) chemically linked to a tetra-peptide from the invariant chain of MHC class II molecules (Ii-key/HER-2(776–790) hybrid peptide or AE37) induces more potent immunologic responses both in vitro and in vivo compared with the non-modified HER-2(776–790) peptide (AE36).^23-25 Recently, Holmes et al.²⁶conducted a phase I study of the AE37 hybrid peptide in HER-2/neu–positive breast cancer patients and showed strong immunologic responses to the vaccine.

The AE37 Vaccine in Prostate Cancer

In a phase I trial, we have immunized 29 prostate cancer patients with AE37 plus granulocyte/macrophage-colony stimulating factor (GM-CSF) as for an adjuvant. The vaccine was given monthly for 6 mo and was well-tolerated with minimal toxicity. AE37-induced strong immunological responses in vivo (delayed type hypersensitivity; DTH) and in vitro (IFNγ production) could be measured in the majority of patients.²⁷ Long-term immunity to AE37 was still detectable 6 mo post vaccinations and could be considerably prolonged for an additional period of 36 mo after one single booster AE37 injection.²⁸

Biomarkers Predicting Immunologic and Clinical Responses to AE37

Despite advances in the field of cancer immunotherapy, therapeutic cancer vaccines have yet to fulfil their promised potential to become standard treatment options for patients with cancer. Considering the time of translation of vaccination-induced immune responses into clinical efficacy, clinical responses in vaccinated patients may appear at much later time points after treatment initiation.²⁹ Thus, it becomes mandatory to identify immunological biomarkers as intermediate endpoints, which will be useful to determine clinical benefit shortly after immunotherapy. Such immunological endpoints will be essential to demonstrate the development of vaccine-induced immune responses and their clinical relevance. Although new paradigms for the development of immunotherapies are being discussed,⁴ few studies have directly addressed the link between tumor burden and pre-existent or induced immunological parameters with treatment benefit from the immunotherapy being tested.³⁰ The results from our aforementioned trial²⁷ support the general consensus that cancer vaccines are more likely to demonstrate immunologic benefit in patients with lower tumor burdens and with less aggressive disease. Indeed, we have demonstrated enhanced AE37-induced immunity in prostate cancer patients at the earlier stages of disease (II-III vs IV) and with low HER-2/neu expression (HER 1+, 2+ vs HER 3+). A mechanistic explanation for this observation is likely to include the physiological changes related to disease progress, which may impact on the quantity and/or quality of the induced immune response.

By extending the time frame of clinical monitoring of our vaccinated patients we could identify immunologic endpoints to AE37 immunotherapy which may help guide the development of our AE37-based vaccination studies (Table 1, manuscript in preparation). Such a development should then consider the optimization of the booster injections schedule and the combination of AE37 vaccine with modalities aiming at reducing tumor-induced suppression. At a median follow-up of 73 mo from first diagnosis (range: 17–161 mo) and of 60 mo from enrolment (range: 12–65 mo), we found that the AE37 vaccine had a significant clinical efficacy in the group of patients who exhibited decreased pre-existing (i.e., before vaccinations) TGFβ plasma levels. During this time frame, we could also demonstrate that strong DTH reactions post vaccinations were correlated with decreased pre-existing TGFβ levels and also showed a significantly prolonged OS. Also in vitro immune responses to the AE37 vaccine indicated by elevated IFNγ production were associated with improved OS.

Table 1. Biomarkers associated with improved OS in AE37-vaccinated prostate cancer patients

	Immunologic biomarkers
Patient stratification*	IFNγ	DTH^§	TGFβ^§	Tregs	IDO
• Staging (II+III vs IV)	NS/T	S	NS/T	NS	ND
• Hormone sensitivity (CS vs CR)	NS/T	NS/T	NS/T	NS	ND
• HER-2/neu expression levels (HER 1^+, 2^+ vs HER 3^+)	S	NS/T	NS/T	NS	ND
HLA-alleles as biomarkers
• HLA-DR11^+ vs HLA-DR11^-	S	NS/T	NS	NS	NS
• HLA-A24^+ vs HLA-A24^-	NS/T	NS	NS/T	NS/T	NS/T
• HLA-A24^+/DR11^+ vs rest**	S	NS/T	NS/T	NS/T	NS
• HLA-A02^- vs HLA-A02^+	NS	NS	NS	S	NS/T

*Comparisons were made between the indicated groups for each parameter or HLA biomarkers. **Includes A24⁺/DR11^-, A24^-/DR11⁺ and A24^-/DR11^- patients. S, indicates statistically significant differences (P ≤ 0.05); NS, indicates non statistically significant differences; NS/T, indicates trends in favor of the prognostic significance of the respective biomarkers; ND, not determined; ^§Patients with increased DTH or decreased TGFβ levels exhibited significantly increased OS.

From the same analyses, we have made associations between HLA alleles with immunologic and clinical responses (Table 1, manuscript in preparation). The majority of patients expressing HLA-A24 and or HLA-DR11 demonstrated increased in vitro and in vivo immunity to vaccination with AE37, whereas the HLA-A24 positive ones had additionally endogenous pre-existing immunity to PSA. More important, there was a strong trend for prolonged OS in patients expressing either HLA-A24 or HLA-DR11 (as opposed to those carrying neither of these alleles) which became almost significant among patients being positive for both alleles. In this particular group of patients, the prognostic significance of regulatory T cells (Tregs) (i.e., elevated numbers) was poor. In line with previous reports,^31,32 we found an inverse association between OS and HLA-A2: the expression of this allele seemed to negatively influence both, immunological responses to the vaccine, mostly due to elevated frequencies of circulating Tregs, as well as clinical outcome and was accompanied with significant increases in the frequencies of Tregs. Nevertheless, the associations between clinical outcome and the above biomarkers which have emerged from our studies need to be validated prospectively in large phase II/III clinical studies to establish their clinical utility.

Conclusions

We consider the results presented here (albeit obtained from trials with a limited number of patients) as providing further support for the increasing body of data implicating immunologic parameters from peripheral blood in a patient’s response to cancer immunotherapy, which in our case was peptide-based vaccinations utilizing a HER-2 hybrid peptide.

Although the mechanistic aspects of this remain to be clarified, the findings seem sufficient to incorporate the HLA and immunological biomarkers identified here into larger scale clinical trials of peptide-based vaccinations, but also other types of immunotherapies, to validate prospectively the working hypothesis that such biomarkers are correlated with OS. A greater understanding of biomarkers will permit a more personalized approach to cancer immunotherapies resulting in maximal benefit, minimizing toxicities and designing/conducting clinical trials tailored to patients most likely to experience optimized clinical responses.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

10.4161/hv.28032