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Background: Her2/neu is overexpressed in various human cancers of epithelial origin and is associated with increased metastatic potential and poor prognosis. Several attempts have been made using the extracellular domain of Her2/neu (ECD/Her2) as a prophylactic vaccine in mice with no success in tumor prevention.Methods: The extracellular domain of Her2/neu (ECD/Her2) was expressed in yeast P. pastoris, in a soluble highly mannosylated form. The immune response of the immunization with this recombinant ECD/Her2 was analyzed using immunoprecipitation and western blot analysis, proliferation and cytotoxicity assays as well as specific tumor growth assays.Results: Mannosylated ECD/Her2 elicited a humoral response with HER2/neu specific antibodies in vaccinated mice, which were able to reduce the proliferation rate of cancer cells in vitro. Moreover, it elicited a cellular response with Her2/neu-specific CTL capable of lysing tumor cells, in vitro. When immunized Balb/c and HHD mice were challenged with Her2/neu-overexpressing cells, tumor growth was inhibited.Conclusion: Here we report on the efficacy of the extracellular domain of human Her2/neu produced in yeast P. pastoris, which confers mannosylation of the protein, to act as a potent anti-tumor vaccine against Her2/neu overexpressing tumors. Specific cellular and humoral responses were observed as well as efficacy. [ABSTRACT FROM AUTHOR]

Inflammation is a key process in the pathophysiology of various diseases, with macrophages playing a central role in the inflammatory response. This study investigates the anti-inflammatory potential of a newly synthesized analog of oleuropein (OP), the major olive tree ( Olea europaea) metabolite. This derivative of OP, named Ole-Oxy, was designed by introducing an oxygen atom between the aromatic ring and the aliphatic chain of OP, to enhance interaction with proteins and improve bioactivity. Ole-Oxy demonstrated notable anti-inflammatory effects in vitro, particularly in phorbol 12-myristate 13-acetate-differentiated THP-1 macrophages, where it markedly reduced interleukin-6, tumor necrosis factor-α, and reactive oxygen species (ROS) levels, surpassing the effects of OP. In vivo, Ole-Oxy was evaluated in mouse models of acute skin and colon inflammation, showing significant efficacy in C57BL/6J mice, likely due to their Th1-biased immune response. Our results suggest that Ole-Oxy modulates inflammation through ROS scavenging and differential macrophage activation, underscoring the need for further research to fully elucidate its mechanism of action and optimize its pharmacokinetic properties for future therapeutic applications.

T-cell-mediated anti-tumoral responses may have significant clinical relevance as a biomarker for response to immunotherapy. The value of peripheral blood pre-existing tumor antigen-specific T cells (PreI + ) as a predictive immunotherapy biomarker in NSCLC patients was investigated, along with the frequency of various circulating immune cells. Fifty-two treatment-naïve, stage III/IV NSCLC patients, treated with front-line immune checkpoint inhibitors (ICI)-containing regimens were enrolled. PreI was calculated as the percentages of CD3 + IFNγ + cells after in vitro co-cultures of PBMCs with peptides against four different Tumor-Associated Antigens (TAA). Immunophenotyping of peripheral blood immune cells was performed using multicolor flow cytometry. PreI + T cells were detected in 44% of patients. Median overall survival (OS) was significantly higher in PreI + patients compared to PreI - patients (not reached vs. 321 days, respectively; p = 0.014). PreI + patients had significantly higher numbers of possible exhausted CD3 + CD8 + PD-1 + cells and lower percentages of immunosuppressive Tregs compared to PreI - patients. Additionally, patients with PreI + and low numbers of peripheral blood M-MDSCs had a significant survival advantage compared to the rest of the patients. Thus, combining pre-existing tumor antigen-specific immunity before initiation of ICI in NSCLC patients with selected immune-suppressive cells could identify patients who have a favorable clinical outcome when treated with ICI-containing regimens.

Using next-generation sequencing (NGS), we investigated DNA mutations in the plasma tumor cell-free circulating DNA (ctDNA) of 38 patients with inoperable squamous cell head neck cancer (SCHNC) before and after the completion of chemoradiotherapy (CRT). Baseline mutations of the TP53 were recorded in 10/38 (26.3%) and persisted in 4/10 patients after CRT. ΤP53 mutations were further detected post CRT in 7/38 additional patients with undetectable mutations at baseline (overall rate 44.7%). Furthermore, 4/38 patients exhibited baseline mutations of the EGFR , AR , FGFR3 , and FBXW3, and four new gene mutations were detected after CRT ( MTOR , EGFR3 , ALK , and SF3B1) . Τ4 stage was related with a significantly higher rate of mutations ( TP53 and overall). Mutations were observed in 8/30 (26.6%) responders (complete/partial response) vs. in 6/8 (75%) of the rest of the patients ( p = 0.03). Significant poorer LRFS was noted for patients with mutations detected before and after CRT ( p = 0.02). Patients who had detectable mutations either before or after CRT had significantly worse DMFS ( p = 0.04 overall, and p = 0.02 for TP53 mutations). It was concluded that assessment of mutations before and after the end of CRT is essential to characterize patients with a high risk of locoregional recurrence or metastatic progression.

Recently, we have shown that HLA-A*02:01 and HLA-A*24:02 in de novo metastatic prostate cancer (MPCa) have an important role in disease progression. Since de novo MPCa represents a small group among patients diagnosed with prostate cancer (PCa), it was obvious to try to extend the validity of our results to larger cohorts of PCa patients. Herein, we analyzed patients irrespective of their disease status at diagnosis to include, besides patients with MPCa, those with localized PCa (LPCa). Our goal was to specify the prognostic value of HLA-A*02:01 and HLA-A*24:02 for overall survival (OS) prospectively and for early biochemical recurrence (BCR) and castrate resistance (CR) as additional clinical endpoints in a prospective/retrospective manner, to improve clinical decisions for patients covering all stages of PCa. On univariate analysis, HLA-A alleles were significantly associated as prognostic biomarkers with early BCR ( p = 0.028; HR = 1.822), OS ( p = 0.013; HR = 1.547) and showed a trend for CR ( p = 0.150; HR = 1.239). On multivariate analysis, HLA-A alleles proved to be independent prognosticators for early BCR ( p = 0.017; HR = 2.008), CR ( p = 0.005; HR = 1.615), and OS ( p = 0.002; HR = 2.063). Kaplan-Meier analyses revealed that patients belonging to the HLA-A*02:01 + HLA-A*24:02 - group progressed much faster to BCR and CR and had also shorter OS compared to HLA-A*24:02 + patients. Patients being HLA-A*02:01 - HLA-A*24:02 - exhibited varying clinical outcomes, pointing to the presence of additional HLA-A alleles with potential prognostic value. Our data underline the HLA-A alleles as valuable prognostic biomarkers for PCa that may assist with the appropriate treatment and follow-up schedule based on the risk for disease progression to avoid over-diagnosis and over-treatment.

Background/aim: The plasma levels of cell-free DNA (cfDNA) in cancer patients increase due to rapid cancer cell proliferation and death. Therefore, cfDNA can be used to study specific tumor-DNA features. In addition, the non-specific cfDNA concentration may be an important biomarker of cancer prognosis., Patients and Methods: We prospectively examined the predictive role of cfDNA levels and the kinetics in the outcome of chemo-radiotherapy (CRT) in a cohort of 47 patients with locally advanced squamous cell head-neck cancer (SCHNC) treated with definitive chemo-radiotherapy., Results: Increased cfDNA levels after therapy completion (after/before treatment ratio; A/B-ratio >1) were found in 26/47 patients (55.3%). Locally advanced T4-stage was significantly associated with higher cfDNA levels after CRT (3.3 ng/μl in T4-stage vs. 1.3 ng/μl in T1-3 stages, p=0.007). Patients who responded to CRT (partial/complete response) had significantly lower cfDNA levels before therapy (mean values 1.2 ng/μl vs. 2.7 ng/μl, p=0.03). A significantly worse locoregional progression-free survival in patients with an A/B-ratio >1 was documented (p=0.01; hazard ratio 3.5, 95%CI=1.2-9.7). This was also confirmed in multivariate analysis, where the A/B-ratio was an independent predictive variable of locoregional relapse (p=0.03, hazard ratio 3.9, 95%CI=1.2-13)., Conclusion: High post-CRT cfDNA levels could be an early biomarker for the immediate recruitment of patients with SCHNC in consolidation chemo-immunotherapy protocols., Competing Interests: There are no conflicts of interest to report in relation to this study., (Copyright 2023, International Institute of Anticancer Research.)

Prostate cancer (PCa) is one of the most common male cancers worldwide and one of the deadliest if unsuccessfully treated. Τhe need for reliable, easily accessible immune-related molecular biomarkers that could be combined with clinically defined criteria, including PSA and Gleason score, to accurately predict PCa patients' clinical outcomes is emerging. Herein, we describe for the first time a blood-identified immune-related gene signature comprising eight upregulated multi-functional genes associated with poor prognosis. Next-generation sequencing (NGS) analysis of PCa patients' peripheral blood samples revealed a more than three-fold upregulation of each of the eight genes as compared to samples originating from healthy donors. The construction of gene and protein interaction networks revealed different extents of the functional implications of these genes in the regulation of cell proliferation and immune responses. Analysis of the available data from The Cancer Genome Atlas (TCGA) regarding gene expression and survival of prostate adenocarcinoma (PRAD) and pan-cancer (PANCAN) patients revealed that intra-tumoral upregulation of this eight-gene signature (8-GS) was associated with poor 5-year progression-free intervals in PCa patients, even in those with high Gleason scores, and also with an unfavorable prognosis for cancer patients irrespective of the cancer type and even in the early stages. These observations suggest that further investigation of the 8-GS prospectively in randomized clinical trials, in which clinical benefit in terms of evaluating time to disease progression can be assessed, is warranted.

HER-2/ neu is the human epidermal growth factor receptor 2, which is associated with the progression of prostate cancer (PCa). HER-2/ neu -specific T cell immunity has been shown to predict immunologic and clinical responses in PCa patients treated with HER-2/ neu peptide vaccines. However, its prognostic role in PCa patients receiving conventional treatment is unknown, and this was addressed in this study. The densities of CD8+ T cells specific for the HER-2/ neu (780-788) peptide in the peripheral blood of PCa patients under standard treatments were correlated with TGF-β/IL-8 levels and clinical outcomes. We demonstrated that PCa patients with high frequencies of HER-2/ neu (780-788) -specific CD8+ T lymphocytes had better progression-free survival (PFS) as compared with PCa patients with low frequencies. Increased frequencies of HER-2/ neu (780-788) -specific CD8+ T lymphocytes were also associated with lower levels of TGF-β and IL-8. Our data provide the first evidence of the predictive role of HER-2/ neu -specific T cell immunity in PCa.

Head and neck cancer (HNC) is a term collectively used to describe a heterogeneous group of tumors that arise in the oral cavity, larynx, nasopharynx, oropharynx, and hypopharynx, and represents the sixth most common type of malignancy worldwide. Despite advances in multimodality treatment, the disease has a recurrence rate of around 50%, and the prognosis of metastatic patients remains poor. HNCs are characterized by a high degree of genomic instability, which involves a vicious circle of accumulating DNA damage, defective DNA damage repair (DDR), and replication stress. Nonetheless, the damage that is induced on tumor cells by chemo and radiotherapy relies on defective DDR processes for a successful response to treatment, and may play an important role in the development of novel and more effective therapies. This review summarizes the current knowledge on the genes and proteins that appear to be deregulated in DDR pathways, their implication in HNC pathogenesis, and the rationale behind targeting these genes and pathways for the development of new therapies. We give particular emphasis on the therapeutic targets that have shown promising results at the pre-clinical stage and on those that have so far been associated with a therapeutic advantage in the clinical setting.

The cancer immunoediting theory describes the dual ability of endogenous antitumor immunity to inhibit or promote progressing cancers. Tumor-specific neoantigens arising from somatic mutations serve as targets for the endogenous T-cell-mediated antitumor immunity and therefore possess a crucial role for tumor development. Additionally, targeting these molecules is conceptually appealing because neoantigens are not expressed in healthy tissue and therefore confer less toxicity and greater specificity when used in therapeutic interventions. Moreover, intratumor neo-antigenic heterogeneity is believed to play a pivotal role in the activation of adaptive immunity and in the efficacy of immunotherapies that are based on immune checkpoint inhibition. In this respect, mutual interactions between tumor cells and immune lymphocytes regulate the levels of antitumor immunity, but also shape tumor heterogeneity through the selective outgrowth of tumor subclones. Therefore, the exploration of the mechanistic pathways and the identification of the genomic aberrations underlying the clonal evolution of tumors is considered mandatory for improving the clinical outcomes of therapies, as it will assist in the selection of the appropriate therapeutic decisions so as to delay, avoid, or overcome resistance through the identification of the most effective therapeutic strategies., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

Acquired immune resistance (AIR) describes a situation in which cancer patients who initially responded clinically to immunotherapies, after a certain period of time, progress with their disease. Considering that AIR represents a feedback response of the tumor against the immune attack generated during the course of immunotherapies, it is conceivable that AIR may also occur before treatment initiation as a mechanism to escape endogenous adaptive antitumor immunity (EAAI). In the present study, we assessed the EAAI in paraffin-embedded breast primary tumor tissue samples and drew correlations with the clinical outcomes. In particular, we analyzed densities of CD8+ cells as elements mediating antitumor cytotoxicity, and of CD163+ and FoxP3+ cells as suppressor elements. We found a direct correlation between the densities of CD8+ cells and of CD163+ and/or FoxP3+ cells in the vast majority of patients' tumors. Importantly, the vast majority of patients whose tumors were overpopulated by CD8+ cells developed AIR, which was characterized by high intratumoral CD163+ and/or FoxP3+ cell densities and reduced overall survival (OS). We also showed that AIR depends on the levels of CD8+ cell-ratios in the tumor center to the invasive margin. Our data suggest that tumors develop AIR only when under a robust endogenous immune pressure.

Radiotherapy for localized prostate cancer has increased the cure and survival rates of patients. Besides its local tumoricidal effects, ionizing radiation has been linked to mechanisms leading to systemic immune activation, a phenomenon called the abscopal effect. In this study, we performed gene expression analysis on peripheral blood from prostate cancer patients obtained post- radiotherapy and showed that 6 genes, including CCR7 , FCGR2B , BTLA , CD6 , CD3D , and CD3E , were down-regulated by a range of 1.5-2.5-fold as compared to pre-radiotherapy samples. The expression of the signature consisting of these six genes was also significantly lower post- vs. pre-radiotherapy. These genes are involved in various tumor-promoting immune pathways and their down-regulation post-radiotherapy could be considered beneficial for patients. This is supported by the fact that low mRNA expression levels for the 6-gene signature in the prostate tumor tissue was linked to better survival. Importantly, we report that this 6-gene signature strongly correlated with a favorable prognosis regardless of poor standard clinicopathological parameters (i.e., Gleason score ≥ 8 and T3 (including T3a and T3b). Our pioneering data open the possibility that the 6-gene signature identified herein may have a predictive value, but this requires further long-term studies.

Head and neck cancers (HNCs) comprise a heterogeneous group of tumors that extend from the oral cavity to the upper gastrointestinal tract. The principal etiologic factors for oral tumors include tobacco smoking and alcohol consumption, while human papillomavirus (HPV) infections have been accused of a high incidence of pharyngeal tumors. Accordingly, HPV detection has been extensively used to categorize carcinomas of the head and neck. The diverse nature of HNC highlights the necessity for novel, sensitive, and precise biomarkers for the prompt diagnosis of the disease, its successful monitoring, and the timely prognosis of patient clinical outcomes. In this context, the identification of certain microRNAs (miRNAs) and/or the detection of alterations in their expression patterns, in a variety of somatic fluids and tissues, could serve as valuable biomarkers for precision oncology. In the present review, we summarize some of the most frequently studied miRNAs (including miR-21, -375, -99, -34a, -200, -31, -125a/b, -196a/b, -9, -181a, -155, -146a, -23a, -16, -29, and let-7), their role as biomarkers, and their implication in HNC pathogenesis. Moreover, we designate the potential of given miRNAs and miRNA signatures as novel diagnostic and prognostic tools for successful patient stratification. Finally, we discuss the currently ongoing clinical trials that aim to identify the diagnostic, prognostic, or therapeutic utility of miRNAs in HNC.

Radiation therapy (RT) is an essential component in the therapeutic treatment of patients with localized prostate cancer (LPCa). Besides its local effects, ionizing radiation has been linked to mechanisms leading to systemic immune activation. The present study explored the effect of RT on the T‑cell receptor variable β (TCR Vβ) chain repertoire of peripheral blood T cells in patients with LPCa. High‑throughput TCR Vβ sequencing was performed on 20 blood samples collected from patients with LPCa at baseline and 3 months post‑RT. The diversity index was altered, as were TCR Vβ clonal evenness and convergence before and post‑RT; however, these findings were not significant. Notably, marked changes in the frequencies among the top 10 TCR Vβ clonotypes were detected and some patients developed new clonotypes of high abundance. These data provided initial evidence that RT in patients with LPCa may induce systemic immune changes, which could be exploited by future therapies for improved clinical results.

Radiotherapy (RT) is a therapeutic modality that aims to eliminate malignant cells through the induction of DNA damage in the irradiated tumor site. In addition to its cytotoxic properties, RT also induces mechanisms that result in the promotion of antitumor immunity both locally within the irradiation field but also at distant tumor lesions, a phenomenon that is known as the "abscopal" effect. Because the immune system is capable of sensing the effects of RT, several treatment protocols have been assessing the synergistic role of radiotherapy combined with immunotherapy, collectively referred to as radioimmunotherapy. Herein, we discuss mechanistic insights underlying RT-based immunomodulation, which also enhance our understanding of how RT regulates antitumor T-cell-mediated immunity. Such knowledge is essential for the discovery of predictive biomarkers and for the improvement of clinical trials investigating the efficacy of radio-immunotherapeutic modalities in cancer patients.

The 4th Summer School in Immuno-Oncology was held from July 1st-July 3rd as a web meeting. Many eminent researchers and leading oncologists from Europe and the USA working on basic, translational and clinical cancer research participated, presented, and discussed the most recent advances in cancer immunology and immunotherapy. Besides sharing the newest information in the field of cancer immunology and immunotherapy, the meeting also focused on the actual translation of new knowledge acquired in the lab to the clinical setting; particular emphasis was given to the mode of action of novel therapeutic modalities and to biomarkers helpful for treatment decision-making, as well as to means that may improve cancer immunotherapeutic protocols used for the treatment of a variety of malignancies. The main topics presented by the speakers included: (1) mechanisms of tumor immune evasion and resistance; (2) host-tumor interactions and means to regulate antitumor immunity; (3) exploitation of new biomarkers and tumor or immune signatures able to potentially guide therapeutic interventions; (4) emerging therapeutic modalities for cancer treatment and specific immunotherapeutics for thoracic, genito-urinary, gastrointestinal, skin and breast cancers; and (5) innovative treatment options and alternatives to minimize the toxic adverse events of cancer immunotherapy., (© 2021 The Author(s). Published by BRI.)

Cancer evolution is a complex process influenced by genetic factors and extracellular stimuli that trigger signaling pathways to coordinate the continuous and dynamic interaction between tumor cells and the elements of the immune system. For over 20 years now, the immune mechanisms controlling cancer progression have been the focus of intensive research. It is well established that the immune system conveys protective antitumor immunity by destroying immunogenic tumor variants, but also facilitates tumor progression by shaping tumor immunogenicity in a process called "immunoediting". It is also clear that immune-guided tumor editing is associated with tumor evasion from immune surveillance and therefore reinforcing the endogenous antitumor immunity is a desired goal in the context of cancer therapies. The tumor microenvironment (TME) is a complex network which consists of various cell types and factors having important roles regarding tumor development and progression. Tumor infiltrating lymphocytes (TILs) and other tumor infiltrating immune cells (TIICs) are key to our understanding of tumor immune surveillance based on tumor immunogenicity, whereby the densities and location of TILs and TIICs in the tumor regions, as well as their functional programs (comprising the "immunoscore") have a prominent role for prognosis and prediction for several cancers. The presence of tertiary lymphoid structures (TLS) in the TME or in peritumoral areas has an influence on the locally produced antitumor immune response, and therefore also has a significant prognostic impact. The cross-talk between elements of the immune system with tumor cells in the TME is greatly influenced by hypoxia, the gut and/or the local microbiota, and several metabolic elements, which, in a dynamic interplay, have a crucial role for tumor cell heterogeneity and reprogramming of immune cells along their activation and differentiation pathways. Taking into consideration the recent clinical success with the application immunotherapies for the treatment of several cancer types, increasing endeavors have been made to gain better insights into the mechanisms underlying phenotypic and metabolic profiles in the context of tumor progression and immunotherapy. In this review we will address (i) the role of TILs, TIICs and TLS in breast cancer (BCa); (ii) the different metabolic-based pathways used by immune and breast cancer cells; and (iii) implications for immunotherapy-based strategies in BCa., (Copyright © 2019. Published by Elsevier Ltd.)

Therapeutic cancer vaccines have been at the forefront of cancer immunotherapy for more than 20 years, with promising results in phase I and-in some cases-phase II clinical trials, but with failures in large phase III studies. After dozens of clinical studies, only Dendreon's dendritic cell vaccine Sipuleucel-T has succeeded in receiving US FDA approval for the treatment of metastatic castrate-resistant prostate cancer. Although scientists working on cancer immunotherapy feel that this is an essential breakthrough for the field, they still expect that new vaccine regimens will yield better clinical benefits compared to the four months prolonged median overall survival (OS) Sipuleucel-T demonstrated in the IMPACT phase III clinical trial. Clinical development of cancer vaccines has been unsuccessful due to failures either in randomized phase II or-even worse-phase III trials. Thus, rigorous re-evaluation of these trials is urgently required in order to redefine aspects and optimize the benefits offered by therapeutic cancer vaccines. The scope of this review is to provide to the reader our thoughts on the key challenges in maximizing the therapeutic potentials of cancer vaccines, with a special focus on issues that touch upon clinical trial design., Competing Interests: The authors declare no conflict of interest.

The prognostic value of human leukocyte antigen (HLA) class I molecules in prostate cancer (PCa) remains unclear. Herein, we investigated the prognostic relevance of the most frequently expressed HLA-A alleles in Greece (A*02:01 and HLA-A*24:02) in de novo metastatic hormone-sensitive PCa (mPCa), which is a rare and aggressive disease characterized by a rapid progression to castration-resistance (CR) and poor overall survival (OS), contributing to almost 50% of PCa-related deaths. We identified 56 patients who had either progressed to CR (these patients were retrospectively analyzed for the time to the progression of CR and prospectively for OS) or had at least three months' follow-up postdiagnosis without CR progression and, thus, were prospectively analyzed for both CR and OS. Patients expressing HLA-A*02:01 showed poor clinical outcomes vs. HLA-A*02:01 - negative patients. HLA-A*24:02 - positive patients progressed slower to CR and had increased OS. Homozygous HLA-A*02:01 patients progressed severely to CR, with very short OS. Multivariate analyses ascribed to both HLA alleles significant prognostic values for the time to progression (TTP) to CR and OS. The presence of HLA-A*02:01 and HLA-A*24:02 alleles in de novo mPCa patients are significantly and independently associated with unfavorable or favorable clinical outcomes, respectively, suggesting their possible prognostic relevance for treatment decision-making in the context of precision medicine.

Purpose: AE37 and GP2 are HER2 derived peptide vaccines. AE37 primarily elicits a CD4+ response while GP2 elicits a CD8+ response against the HER2 antigen. These peptides were tested in a large randomized trial to assess their ability to prevent recurrence in HER2 expressing breast cancer patients. The primary analyses found no difference in 5-year overall disease-free survival (DFS) but possible benefit in subgroups. Here, we present the final landmark analysis., Methods: In this 4-arm, prospective, randomized, single-blinded, multi-center phase II trial, disease-free node positive and high-risk node negative breast cancer patients enrolled after standard of care therapy. Six monthly inoculations of vaccine (VG) vs. control (CG) were given as the primary vaccine series with 4 boosters at 6-month intervals. Demographic, safety, immunologic, and DFS data were evaluated., Results: 456 patients were enrolled; 154 patients in the VG and 147 in CG for AE37, 89 patients in the VG and 91 in CG for GP2. The AE37 arm had no difference in DFS as compared to CG, but pre-specified exploratory subgroup analyses showed a trend towards benefit in advanced stage (p = 0.132, HR 0.573 CI 0.275-1.193), HER2 under-expression (p = 0.181, HR 0.756 CI 0.499-1.145), and triple-negative breast cancer (p = 0.266, HR 0.443 CI 0.114-1.717). In patients with both HER2 under-expression and advanced stage, there was significant benefit in the VG (p = 0.039, HR 0.375 CI 0.142-0.988) as compared to CG. The GP2 arm had no significant difference in DFS as compared to CG, but on subgroup analysis, HER2 positive patients had no recurrences with a trend toward improved DFS (p = 0.052) in VG as compared to CG., Conclusions: This phase II trial reveals that AE37 and GP2 are safe and possibly associated with improved clinical outcomes of DFS in certain subgroups of breast cancer patients. With these findings, further evaluations are warranted of AE37 and GP2 vaccines given in combination and/or separately for specific subsets of breast cancer patients based on their disease biology.

Tumors and their surrounding area represent spatially organized "ecosystems", where tumor cells and the immune contextures of the different compartments are in a dynamic interplay, with potential clinical impact. Here, we aimed to investigate the prognostic significance of peritumoral tertiary lymphoid structures (TLS) either alone or jointly with the intratumoral densities and spatial distribution of CD8 + and CD163 + cells in breast cancer (BCa) patients. TLS were identified peritumorally, within the area distancing up to 5 mm from the infiltrative tumor border, counted and further characterized as adjacent or distal, in formalin-fixed, paraffin-embedded tumor tissue samples from a cohort of 167 patients, with histologically confirmed invasive ductal BCa. TLS and tumor-infiltrating immune cells were determined by H&E and immunohistochemistry. Clinical follow-up was available for 112 of these patients. Patients with peritumoral TLS exhibited worse disease-free survival (DFS) and overall survival (OS) as compared to patients lacking TLS. Moreover, the density of peritumoral TLS was found to be crucial for prognosis, since patients with abundant TLS exhibited the worst DFS and OS. By combining the density of adjacent TLS (aTLS) with our recently published intratumoral signatures based on the differential distribution of CD8 + and CD163 + in the tumor center and invasive margin, we created two improved immune signatures with superior prognostic strength and higher patient population coverage. Our observations strengthen the notion for the fundamental role of the dynamic interplay between the immune cells within the tumor microenvironment (center/invasive margin) and the tumor surrounding area (peritumoral TLS) on the clinical outcome of BCa patients.

The presence of immune infiltrates in the tumor microenvironment has been documented in many types of cancer. Moreover, the preexistent or endogenous immunity which consists of interactions between intratumoral lymphocytes and tumor cells is mostly relevant for the successful application of various anticancer therapies, including standard chemotherapy, immune checkpoint inhibition-based immunotherapy and targeted therapies. The immunoscore defines densities of intratumoral immune infiltrates which determine poor or favorable prognosis depending on their quantity and quality in the tumor compartments. Results from large clinical studies have demonstrated an association between high densities of cytotoxic and memory TILs in the tumor compartments with improved prognosis. Importantly, we have demonstrated that differential combined densities of immune infiltrates jointly analyzed in the tumor center (TC) and the invasive margin (IM) have a significant prognostic value in breast cancer patients with poor clinicopathological parameters.

The concept of a dual functional programme of the immune system to destroy malignant cells but also to edit their immunogenic profile, considerably improved our understanding of the process of tumor evolution in the context of a continuum of interactions between tumor cells and immune lymphocytes. Such an endogenous antitumor immunity throughout the period of cancer development established the concept of cancer immunomodulation which is practically based on a process of selection of more clonal tumors which are manageable by the immune system and constitute the equilibrium phase of immunoediting. The duration of this phase is very important, because the immune system keeps the tumor in a dormant state via cell interactions which establish a balanced state of tumor immunosurveillance versus tumor immune evasion. Depending on the quality and quantity of antitumor immune reactivity and the effectiveness of resistance mechanisms employed by the tumor cells to counteract this immune attack, the equilibrium phase may have shorter or longer duration. Notwithstanding its natural course, the equilibrium phase should be considered as a part of tumor evolutionary process guided by genetic as well as epigenetic changes which in turn activate endogenous cellular immunity to certain levels capable of controlling tumor growth rates and maintain tumor dormancy.

Breast cancer (BCa) is a heterogeneous disease with different histological, prognostic and clinical aspects. Therefore, the need for identification of novel biomarkers for diagnosis, prognosis and monitoring of disease, as well as treatment outcome prediction remains at the forefront of research. The search for circulating elements, obtainable by simple peripheral blood withdrawal, which may serve as possible biomarkers, constitutes still a challenge. In the present study, we have evaluated the expression of 6 circulating miRNAs, (miR-16, miR-21, miR-23α, miR-146α, miR-155 and miR-181α), in operable BCa patients, with non-metastatic, invasive ductal carcinoma, not receiving neoadjuvant chemotherapy. These miRNAs, known to be involved in both tumor cell progression and immune pathways regulation, were analyzed in relation to circulating cytokines, tumor immune-cell infiltration and established prognostic clinicopathological characteristics. We have identified three different clusters, with overall low (C1), moderate (C2) or high (C3) expression levels of these six circulating miRNAs, which define three distinct groups of non-metastatic BCa patients characterized by different clinicopathological and immune-related characteristics, with possibly different clinical outcomes. Our data provide the proof-of-principle to support the notion that, up- or down-regulation of the same circulating miRNA may reflect different prognosis in BCa. Nonetheless, the prognostic and/or predictive potential of these three "signatures" needs to be further evaluated in larger cohorts of BCa patients with an, at least, 5-year clinical follow-up.

This is the 2nd Symposium of a series organized annually. It aims to integrate tumor immunology basic research with results from most recent clinical trials based on the use of anti-cancer agents targeting immune system components.

Melanoma, like most solid tumors, is highly heterogeneous in terms of invasive, proliferative, and tumor-initiating potential. This heterogeneity is the outcome of differential gene expression resulting from conditions in the tumor microenvironment and the selective pressure of the immune system. To investigate possible signatures combining immune-related gene expression and lymphocyte infiltration, we established a preclinical model using B16.F1-derived clones, in the context of melanoma aggressiveness. Combinatorial analyses revealed that tumors concomitantly expressing low levels of Tnf-a, Pd-1, Il-10, Il-1ra, Ccl5, Ido, high Il-9, and with low infiltration by CD45 + , CD3 + , CD4 + and CD8 + cells and a high CD4 + :CD8 + T cell ratio exhibited the most aggressive growth characteristics. Overall, these results support the notion that the intratumoral immunologic network molds aggressive melanoma phenotypes.

Background: Tumor immune cell infiltrates are essential in hindering cancer progression and may complement the TNM classification. CD8+ and CD163+ cells have prognostic impact in breast cancer but their spatial heterogeneity has not been extensively explored in this type of cancer. Here, their potential as prognostic biomarkers was evaluated, depending on their combined densities in the tumor center (TC) and the tumor invasive margin (IM)., Methods: CD8+ and CD163+ cells were quantified by immunohistochemistry of formalin-fixed, paraffin-embedded (FFPE) tumor tissue samples from a cohort totaling 162 patients with histologically-confirmed primary invasive non-metastatic ductal breast cancer diagnosed between 2000 and 2015. Clinical follow-up (median 6.9 years) was available for 97 of these patients., Results: Differential densities of CD8+ and CD163+ cells in the combined TC and IM compartments (i.e., high(H)/low(L), respectively for CD8+ cells and the reverse L/H combination for CD163+ cells) were found to have significant prognostic value for survival, and allowed better patient stratification than TNM stage, tumor size, lymph node invasion and histological grade. The combined evaluation of CD8+ and CD163+ cell densities jointly in TC and IM further improves prediction of clinical outcomes based on disease-free and overall survival. Patients having the favorable immune signatures had favorable clinical outcomes despite poor clinicopathological parameters., Conclusions: Given the important roles of CD8+ and CD163+ cells in regulating opposing immune circuits, adding an assessment of their differential densities to the prognostic biomarker armamentarium in breast cancer would be valuable. Larger validation studies are necessary to confirm these findings., Trial Registrations: Study code: IRB-ID 6079/448/10-6-13 Date of approval: 10/06/2013 Retrospective study (2000-2010) First patient prospectively enrolled 14/2/2014.

Numerous studies have revealed a variety of pathways involved in the development of melanoma, however, the molecular and genetic divergence of underlying mechanisms remain vague. In a mouse model, we studied the expression pattern of insulin-like growth factor 2 mRNA-binding protein 1 (Igf2bp1) and target genes microphthalmia-associated transcription factor (Mitf), v-myc avian myelocytomatosis viral oncogene homolog (Myc), B-cell lymphoma 2 (Bcl2), prothymosin alpha (Ptma) and melan-A (Mlana) in relation to tumor-growth characteristics. The in vivo expression of the aforementioned genes was assessed by quantitative Real Time-Polymerase Chain Reaction (RT-PCR) in tumors established by B16-F1-derived clones. Gene expression was correlated with tumor growth characteristics. Simultaneous expression of elevated levels of Myc, Igf2bp1, Ptma and Mitf characterizes tumors with a more aggressive phenotype. Our findings introduce a tumor-specific molecular signature possibly associated with melanoma heterogeneity. The concomitant overexpression of key molecules such as IGF2BP1, PTMA, MYC and MITF could serve as prognostic or predictive marker., (Copyright© 2017 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Purpose: Breast cancer is a leading cause of cancer deaths in women, but despite steady improvements in therapies, treatment is still suboptimal. Immunotherapy holds promise as a more effective therapy for breast cancer; supporting this, our prior study showed that patients possessing HER2-reactive CD8+ T cells in blood experience survival superior to patients without these cells. Here, we define a composite set of biomarkers that identify patients with T cell responses to tumour antigens., Methods: We assessed T cell responses following in vitro stimulation with the HER2, MUC1 and SUR tumour-associated antigens (TAA) by flow cytometry and intracellular cytokine staining in 50 breast cancer patients. We also measured HLA type, serum cytokines, tumour-infiltrating leukocytes and blood leukocyte populations., Results: We found few correlations between TAA-reactive T cells and HLA type, serum cytokines and tumour-infiltrating leukocytes, whereas blood leukocyte phenotypes broadly correlated with TAA responses. This showed monocytes, natural killer cells, dendritic cells and T cells to be inversely associated with both CD4+ and CD8+ T cells reactive to tumour antigens. Moreover, combining multiple parameters improved the accuracy in predicting patients with TAA-responsive T cells., Conclusion: This study therefore defines composite immune profiles that identify patients responding to TAAs which may allow better personalisation of cancer therapies.

Background: Cancer vaccines aim at eliciting not only an immune response against specific tumor antigens, but also at enhancing a preexisting immunity against the tumor. In this context, we recently reported on the levels of preexisting immunity in prostate cancer patients vaccinated with the HER-2 hybrid peptide (AE37), during a phase I clinical trial. The purpose of the current study was to correlate between preexisting immunity to the native HER-2 peptide, AE36, and expression of HLA-A2 and -A24 molecules with the clinical outcome. Additionally, we investigated the ability of the AE37 vaccine to induce an antitumor immune response against other tumor associated antigens, not integrated in the vaccine formulation, with respect to the clinical response., Methods: We analyzed prostate cancer patients who were vaccinated with the AE37 vaccine [Ii-Key-HER-2 /neu (776-790) hybrid peptide vaccine (AE37), which is a MHC class II long peptide vaccine encompassing MHC class I epitopes, during a phase I clinical trial. Preexisting immunity to the native HER-2 /neu (776-790) (AE36) peptide was assessed by IFNγ response or dermal reaction at the inoculation site. Antigen specificity against other tumor antigens was defined using multimer analysis. Progression free survival (PFS) was considered as the patients' clinical outcome. Two-tailed Wilcoxon signed rank test at 95 % confidence interval was used for statistical evaluation at different time points and Kaplan-Meier curves with log-rank (Mantel-Cox) test were used for the evaluation of PFS., Results: Preexisting immunity to AE36, irrespectively of HLA expression, was correlated with longer PFS. Specific CD8 + T cell immunity against E75 and PSA 146-151 (HLA-A2 restricted), as well as, PSA 153-161 (HLA-A24 restricted) was detected at relatively high frequencies which were further enhanced during vaccinations. Specific immunity against PSA 153-161 correlated with longer PFS in HLA-A24 + patients. However, HLA-A2 + patients with high preexisting or vaccine-induced immunity to E75, showed a trend for shorter PFS., Conclusions: Our data cast doubt on whether preexisting immunity or epitope spreading specific for HLA-class I-restricted peptides can actually predict a favorable clinical outcome. They also impose that preexisting immunity to long vaccine peptides, encompassing both HLA class II and I epitopes should be considered as an important prerequisite for the improvement of future immunotherapeutic protocols. Protocol ID Code: Generex-06-07 National Organization for Medicines (EOF) ID Code: IS-107-01-06 NEC Study Code: EED107/1/06 EudraCT Number: 2006-003299-37 Date of registration: 07/06/2006 Date of enrolment of the first participant to the trial: Nov 1st, 2007.

GP2 is a HER2-derived, HLA-A2+ restricted peptide. Phase I studies showed GP2 administered with GM-CSF to be safe and immunogenic. Here we report the primary analysis of a prospective, randomized, multicenter phase II adjuvant trial conducted to determine the vaccine's efficacy. The trial enrolled HLA-A2+, clinically disease-free, node-positive and high-risk node-negative breast cancer patients with tumors expressing HER2 (immunohistochemistry[IHC] 1+-3+). Patients were randomized to GP2+GM-CSF versus GM-CSF alone. Disease-free survival (DFS) was analyzed in intention-to-treat (ITT) and per-treatment cohorts; pre-specified subgroup analyses were performed for patients with IHC 3+ or FISH+ disease. The trial enrolled 180 patients; 89 received GP2+GM-CSF and 91 received GM-CSF alone. The groups were well-matched for clinicopathologic characteristics. Toxicities have been minimal. The Kaplan-Meier estimated 5-year DFS rate in the ITT analyses was 88% (95% CI:78-94%) in vaccinated vs. 81% (95% CI:69-89%) (P = 0.43) in control patients after a 34 month median follow-up. In the per-treatment analysis, the estimated 5-year DFS rates were 94% (95% CI:83-98%) and 85% (73-92%) (P = 0.17). In IHC 3+/FISH+ patients, the estimated 5-year DFS rate was 94% (82-98%) in vaccinated patients (n = 51) vs. 89% (71-96%) in control patients (n = 50), (P = 0.86) in the ITT analyses and 100% vs. 89% (71-96%) in vaccinated vs. control patients in the per-treatment analyses (P = 0.08). While the overall ITT analysis did not demonstrate benefit to vaccination, this trial confirmed that the GP2 vaccine is safe and suggests that vaccination may have clinical activity, particularly in patients with HER2 overexpression who received the full vaccine series (ie per-treatment group).

Background: AE37 is the Ii-Key hybrid of the MHC class II peptide, AE36 (HER2 aa:776-790). Phase I studies showed AE37 administered with granulocyte macrophage colony-stimulating factor (GM-CSF) to be safe and highly immunogenic. A prospective, randomized, multicenter phase II adjuvant trial was conducted to evaluate the vaccine's efficacy., Methods: Clinically disease-free node-positive and high-risk node-negative breast cancer patients with tumors expressing any degree of HER2 [immunohistochemistry (IHC) 1-3+] were enrolled. Patients were randomized to AE37 + GM-CSF versus GM-CSF alone. Toxicity was monitored. Clinical recurrences were documented and disease-free survival (DFS) analyzed., Results: The trial enrolled 298 patients; 153 received AE37 + GM-CSF and 145 received GM-CSF alone. The groups were well matched for clinicopathologic characteristics. Toxicities have been minimal. At the time of the primary analysis, the recurrence rate in the vaccinated group was 12.4% versus 13.8% in the control group [relative risk reduction 12%, HR 0.885, 95% confidence interval (CI) 0.472-1.659, P = 0.70]. The Kaplan-Meier estimated 5-year DFS rate was 80.8% in vaccinated versus 79.5% in control patients. In planned subset analyses of patients with IHC 1+/2+ HER2-expressing tumors, 5-year DFS was 77.2% in vaccinated patients (n = 76) versus 65.7% in control patients (n = 78) (P = 0.21). In patients with triple-negative breast cancer (HER2 IHC 1+/2+ and hormone receptor negative) DFS was 77.7% in vaccinated patients (n = 25) versus 49.0% in control patients (n = 25) (P = 0.12)., Conclusion: The overall intention-to-treat analysis demonstrates no benefit to vaccination. However, the results confirm that the vaccine is safe and suggest that vaccination may have clinical benefit in patients with low HER2-expressing tumors, specifically TNBC. Further evaluation in a randomized trial enrolling TNBC patients is warranted., (© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Realizing the basis for generating long-lasting clinical responses in cancer patients after therapeutic vaccinations provides the means to further ameliorate clinical efficacy. Peptide cancer vaccines stimulating CD4(+) T helper cells are often promising for inducing immunological memory and persistent CD8(+) cytotoxic T cell responses. Recent reports from our clinical trial with the AE37 vaccine, which is a HER2 hybrid polypeptide, documented its efficacy to induce CD4(+) T cell immunity, which was associated with clinical improvements preferentially among HLA-DRB1*11(+) prostate cancer patients. Here, we performed in-depth investigation of the CD4(+) T cell response against the AE37 vaccine. We used the DR11/AE37 tetramer in combination with multicolor flow cytometry to identify and characterize AE37-specific CD4(+) T cells regarding memory and Tregs phenotype in HLA-DRB1*11(+) vaccinated patients. To verify vaccine-specific immunological memory in vivo, we also assessed AE37-specific CD4(+) T cells in defined CD4(+) memory subsets by cell sorting. Finally, vaccine-induced AE37-specific CD4(+) T cells were assessed regarding their functional profile. AE37-specific memory CD4(+) T cells could be detected in peptide-stimulated cultures from prostate cancer patients following vaccination even 4 y post-vaccination. The vast majority of vaccine-induced AE37-specific CD4(+) T cells exhibited a multifunctional, mostly Th1 cytokine signature, with the potential of granzyme B production. In contrast, we found relatively low frequencies of Tregs among AE37-specific CD4(+) T cells. This is the first report on the identification of vaccine-induced HER2-specific multifunctional long-lasting CD4(+) T cells in vaccinated prostate cancer patients.