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Tumor immunologic microenvironment is strongly involved in tumor progression and the presence of tumor infiltrating lymphocytes (TIL) with different phenotypes has been demonstrated to be of prognostic relevance in different malignancies. We investigated whether TIL infiltration of tumor tissues could also predict the outcome of prostate cancer patients. To this end, we carried out a retrospective analysis correlating the outcome of locally advanced prostate cancer patients undergone salvage radiotherapy upon relapse after radical surgery with the infiltration by different TIL populations. Twenty-two patients with resectable prostate cancer, with a mean age of 67 (+/−3.93) years, who received salvage radiotherapy with a mean of 69.66 (+/− 3.178) Gy in 8 weeks, between June 1999 and January 2009 and with a median follow up of 123 (+/− 55.82) months, were enrolled in this study. We evaluated, by immunohistochemistry, the intratumoral (t) and peripheral stroma (p) infiltration by CD45, CD3, CD4, CD8, CCR7, FoxP3 or PD-1-positive cells on tumor samples taken at the diagnosis (d) and relapse times (R). We correlated these variables with patients' biochemical progression free survival (bPFS), post-radiotherapy progression free survival (PFS), and overall survival (OS). Substantial changes in the rate of TIL subsets were found between the first and the second biopsy with progressive increase in CD4, CCR7, FoxP3, PD-1+ cells. Our analysis revealed that higher CD8p,R+ and lower PD-1R+ TIL scores correlated to a longer bPFS. Higher CD8p,R+ and CCR7t,R+ TIL scores and lower CD45p,R+ and FoxP3p,R+ TIL scores correlated to a prolonged PFS and OS. These results suggest that the immunological microenvironment of primary tumor is strictly correlated with patient outcome and provide the rationale for immunological treatment of prostate cancer.

Even though the crucial role played by inflammation in cancer development and progression was first hypothesized by Rudolf Virchow at the beginning of the nineteenth century, only recently inflammation has been recognized as a hallmark of cancer. At present, the biology underlying the humoral and cellular immune-suppressive cancer-associated inflammatory microenvironment is an active area of preclinical and clinical investigation.1, 2 Indeed, the possibility to modulate the inflammatory/immune microenvironment, by either antagonizing the tumor-associated immune-suppression or by enhancing the pre-existing anti-cancer immune response in tumor tissues, is a promising therapeutic option for cancer patients. In this context, the presence of infiltrating lymphocytes with specific immune-phenotypes within the tumor or in the surrounding stroma has predicted long-lasting responses and improved patients' survival. Indeed, in a series of homogenously treated metastatic colorectal cancer (mCC) patients, we observed longest survival when high CCR7+ or FoxP3+ (Treg) lymphocytes infiltration occurred within tumor tissues. This latter finding provided a 'paradigm shift', since for the first time associated the density of an immune-suppressive population with a better outcome in mCC patients. This finding may be now explained by the recently disclosed 'regulatory' rather than 'suppressive' nature of Tregs. In this vision, in fact, these cells may work as specific repressor of the IL-17/Th17-driven inflammation, produced by alteration in the gut 'microbiota', and this event, in turn, may account for the survival benefit.3, 4, 5 Accordingly, we reported that a high baseline inflammatory status negatively affects patients' prognosis and impairs the response to standard and immune-modulatory anti-cancer treatments in different malignancies including colorectal cancer, multiple myeloma and non-small cell lung cancer (NSCLC).6, 7, 8, 9 In our latest report, recently published in Cell Death Discovery,10 we added a new piece to the puzzle (Figure 1), by investigating the immune-modulatory properties of bevacizumab, a monoclonal antibody (mAb) that acts by sequestering VEGFA, in NSCLC patients enrolled in mPEBev (BEVA2007) trial; our findings provide novel evidence of a functional link between angiogenesis and the immune/inflammatory response.6, 11, 12 Our work has been designed taking into account that the biological relevance of VEGFA is not limited to endothelial cells, and consequently to angiogenesis. Indeed, this molecule, produced and released by tumor cells, platelets and inflammatory cells, such as neutrophils and monocytes, in the cancer-associated inflammatory microenvironment, attracts myeloid-derived suppressor cells and tumor-associated macrophages, and impairs the immune-system by counteracting the activity of dendritic cells (DCs) and specific T-cell subsets.13 In the BEVA2007 clinical study, we evaluated the efficacy and safety of the mPE (cisplatinum+metronomic oral etoposide)+bevacizumab schedule as frontline therapy in a series of NSCLC patients. We found that this regimen is safe and highly active. Furthermore, according to the immune-modulatory role of VEGFA, we observed that patients who presented a low inflammatory status at baseline, as indicated by a low neutrophil-to-lymphocyte ratio (NLR), experienced a 20 months median overall survival with an impressive 29% hazard ratio.6 In our recently published paper, we added novel information demonstrating that the addition of bevacizumab to chemotherapy in NSCLC patients decreases systemic inflammatory status and promotes anticancerimmune-modulating effects. Specifically, we observed a significant reduction in the levels of proangiogenic factors (VEGFA, Angiopoietin 2 and Follistatin) and inflammatory cytokines (IFNγ, IL4 and IL17A) in the serum of these patients and a progressive decline in NLR. Additionally, we found that both mPE and mPE+bevacizumab (mPEBev) regimens induced a significant increase of central memory T lymphocytes in peripheral blood, while only patients who underwent mPEBev treatment presented a significant rise in the percentage of mature DCs. The latter finding is suggestive of improved antigen presentation consequent to bevacizumab-dependent VEGFA deprivation. These data were also in line with a functional ex vivo study performed on antigen-specific cytotoxic T lymphocyte (CTL) lines generated in vitro from peripheral blood mononuclear cells at baseline and after mPE or mPEBev treatment. In particular, antigen-specific CTL proliferation and Th1 polarization was more pronounced in T-cell lines derived from patients who received the mPEBev regimen as compared with T cells lines derived from patients who received chemotherapy alone. On these bases, we hypothesized that the anti-tumor activity of bevacizumab may at least in part rely on the modulation of the immune/inflammatory response.

Thymidylate synthase (TS) is a tumor-associated enzyme critical for DNA replication and main 5′-fluorouracil (5′-FU) target. TSPP/VAC1 is a multi-arm trial phase-Ib trial program aimed to investigate the toxicity and biomodulatory activity of a poly-epitope-peptide vaccine to TS (TSPP) in cancer patients (pts). Here, we present the results of the TSPP/VAC1/arm C trial aimed to evaluate TSPP in combination with chemo-immunotherapy in pretreated metastatic colo-rectal cancer (mCRC) pts. Twenty-nine pts, 14 males and 15 females, received poly-chemotherapy with gemcitabine [GEM; 1,000mg/sqm, day-1], oxaliplatin [OX; 80mg/sqm, day-2], levofolinate [100mg/sqm, days 1–2], bolus/infusional 5′-FU [400mg/800mg/sqm, days 1–2], sargramostim [50μg, days 3–7/q30], and interleukin-2 [sc. 0.5 MIU twice a day, days 8–14/18–30] [GOLFIG-regimen]. Seventeen pts received sc. TSPP injections at escalating dosage [3 pts, 100µg (DL-1); 3 pts, 200µg (DL-2) and 11pts, 300µg (DL-3)] one week after each chemotherapy cycle (concomitant module), while 10 out 12 pts received TSPP (300µg) after 12 GOLFIG courses [dose level (DL)-0] (sequential module). TSPP MTD was not achieved. Adverse events consisted in swelling/erythema at injection sites (17 cases), G1–2 haematological (16 cases) and gastro-enteric events (12), fever, rhinitis, conjunctivitis, and poly-arthralgia and rise in auto-antibodies [ANA, ENA, c-ANCA, p-ANCA in the DL1–3 pts]. Both treatment-modules showed immunomodulating and antitumor activity (disease-control-rate, DL1–3 and DL0 were 70.6% and 83.3%, respectively) with a better survival recorded in the second group [median OS DL1–3vs. DL0 = 8vs. 16mo, p = 0.049]. The promising long-term survival produced by the sequential treatment module deserves further phase II evaluation.

Thymidylate synthase (TS) poly-epitope peptide (TSPP) is a 27-mer peptide vaccine containing the amino acidic sequences of three epitopes with HLA-A2.1-binding motifs of TS, an enzyme overexpressed in cancer cells, which plays a crucial role in DNA repair and replication. Based on the results of preclinical studies, we designed a phase Ib trial (TSPP/VAC1) to investigate, in a dose escalation setting, the safety and the biological activity of TSPP vaccination alone (arm A) or in combination with GM-CSF and IL-2 (arm B) in cancer patients. Twenty-one pretreated metastatic cancer patients, with a good performance status (ECOG≤1) and no severe organ failure or immunological disease, were enrolled in the study (12 in arm A, nine in arm B) between April 2011 and January 2012, with a median follow-up of 28months. TSPP resulted safe, and its maximal tolerated dose was not achieved. No grade 4 toxicity was observed. The most common adverse events were grade 2 dermatological reactions to the vaccine injection, cough, rhinitis, fever, poly-arthralgia, gastro-enteric symptoms and, to a lesser extent, moderate hypertension and hypothyroidism. We detected a significant rise in auto-antibodies and TS-epitope-specific CTL precursors. Furthermore, TSPP showed antitumor activity in this group of pretreated patients; indeed, we recorded one partial response and seven disease stabilizations (SD) in arm A, and three SD in arm B. Taken together, our findings provide the framework for the evaluation of the TSPP anti-tumor activity in further disease-oriented clinical trials.