Pierpaolo Pastina

Systemic inflammatory status at baseline predicts bevacizumab benefit in advanced non-small cell lung cancer patients.

Bevacizumab is a humanized anti-VEGF monoclonal antibody able to produce clinical benefit in advanced non-squamous non-small-cell lung cancer (NSCLC) patients when combined to chemotherapy. At present, while there is a rising attention to bevacizumab-related adverse events and costs, no clinical or biological markers have been identified and validated for baseline patient selection. Preclinical findings suggest an important role for myeloid-derived inflammatory cells, such as neutrophils and monocytes, in the development of VEGF-independent angiogenesis. We conducted a retrospective analysis to investigate the role of peripheral blood cells count and of an inflammatory index, the neutrophil-to-lymphocyte ratio (NLR), as predictors of clinical outcome in NSCLC patients treated with bevacizumab plus chemotherapy. One hundred twelve NSCLC patients treated with chemotherapy ± bevacizumab were retrospectively evaluated for the predictive value of clinical or laboratory parameters correlated with inflammatory status. Univariate analysis revealed that a high number of circulating neutrophils and monocytes as well as a high NLR were associated with shorter progression-free survival (PFS) and overall survival (OS) in bevacizumab-treated patients only. We have thus developed a model based on the absence or the presence of at least one of the above-mentioned inflammatory parameters. We found that the absence of all variables strongly correlated with longer PFS and OS (9.0 vs. 7.0 mo, HR: 0.39, p = 0.002; and 20.0 vs. 12.0 mo, HR: 0.29, p < 0.001 respectively) only in NSCLC patients treated with bevacizumab plus chemotherapy. Our results suggest that a baseline systemic inflammatory status is marker of resistance to bevacizumab treatment in NSCLC patients.

Immune-modulating effects of the newest cetuximab-based chemoimmunotherapy regimen in advanced colorectal cancer patients.

Cetuximab is a human–murine chimeric monoclonal antibody to the epidermal growth factor receptor, active for advanced colorectal cancer treatment in combination with chemotherapy. Cetuximab mainly acts by inhibiting epidermal growth factor receptor-mediated pathways in cancer cells; however, in the human host, its IgG1 backbone may offer additional antitumor activity that includes Fc&ggr;Rs-mediated antibody-dependent cell cytotoxicity, phagocytosis, cross priming, and tumor-specific T-cell–mediated immune response. These mechanisms are still under active investigation. At this purpose, we have performed an immunologic investigation in advanced colon cancer patients enrolled in an ongoing phase II trial aimed to test the toxicity and the biological and antitumor activity of a novel biochemotherapy regimen combining polychemotherapy with gemcitabine, irinotecan, levofolinic acid, and fluorouracil with cetuximab and with subcutaneous low-dose metronomic aldesleukin (GILFICet regimen). The peripheral blood mononuclear cells of the first 20 patients enrolled in the GILFICet trial were collected at baseline and after 6 treatment cycles and examined for immune-phenotype change by flow cytometry. Colon cancer-specific T-cell lines were also generated ex vivo from these samples and subsequently characterized for immune phenotype, functional activity, and antigen specificity. We found a treatment-related increase of circulating dendritic cells, natural killer cells, central memory T cells, and activated T cells with a T-helper 1 (Th1)-cytotoxic phenotype. In addition, the ex-vivo characterization of antigen-specific T cells derived from the treated patients revealed a significant increase in proliferating cytotoxic T-lymphocyte precursors specific for carcinoembryonic antigen and thymidylate synthase derivative epitope peptides. On these basis, we concluded that the GILFICet regimen exerts substantial immune-modulating activity that significantly affects tumor antigen-specific T-cell compartment with potential antitumor activity.

Gemcitabine, oxaliplatin, levofolinate, 5-fluorouracil, granulocyte-macrophage colony-stimulating factor, and interleukin-2 (GOLFIG) versus FOLFOX chemotherapy in metastatic colorectal cancer patients: the GOLFIG-2 multicentric open-label randomized phase III trial.

The GOLFIG-2 phase III trial was designed to compare the immunobiological activity and antitumor efficacy of GOLFIG chemoimmunotherapy regimen with standard FOLFOX-4 chemotherapy in frontline treatment of metastatic colorectal cancer (mCRC) patients. This trial was conceived on the basis of previous evidence of antitumor and immunomodulating activity of the GOLFIG regimen in mCRC. GOLFIG-2 is a multicentric open/label phase III trial (EUDRACT: 2005-003458-81). Chemo-naive mCRC patients were randomized in a 1:1 ratio to receive biweekly standard FOLFOX-4 or GOLFIG [gemcitabine (1000 mg/m2, day 1); oxaliplatin (85 mg/m2, day 2); levofolinate (100 mg/m2, days 1–2), 5-fluorouracil (5-FU) (400 mg/m2 in bolus followed by 24 h infusion at 800 mg/m2,days 1–2), sc. GM-CSF (100 &mgr;g, days 3–7); sc. aldesleukin (0·5 MIU bi-daily, days 8–14 and 17–30)] treatments. The study underwent early termination because of poor recruitment in the control arm. After a median follow-up of 43.83 months, GOLFIG regimen showed superiority over FOLFOX in terms of progression-free survival [median 9·23 (95% confidence interval (CI), 6·9–11.5) vs. median 5.70 (95% CI, 3.38–8.02) months; hazard ratio (HR): 0.52 (95% CI, 0.35–0.77), P=0·002] and response rate [66.1% (95% CI, 0.41–0.73) vs. 37·0% (95% CI, 0.28–0.59), P=0.002], with a trend to longer survival [median 21.63 (95% CI, 18.09–25.18) vs. 14.57 mo (95% CI, 9.07–20.07); HR: 0·79 (95% CI, 0.52–1.21); P=0.28]. Patients in the experimental arm showed higher incidence of non-neutropenic fever (18.5%), autoimmunity signs (18.5%), an increase in the number of monocytes, eosinophils, CD4+ T lymphocytes, natural killer cells, and a decrease in immunoregulatory (CD3+CD4+CD25+FoxP3+) T cells. Taken together, these findings provide proof-of-principle that GOLFIG chemoimmunotherapy may represent a novel reliable option for first-line treatment of mCRC.

The combined EGFR protein expression analysis refines the prognostic value of the MGMT promoter methylation status in glioblastoma

BACKGROUND/AIMS To investigate the combined prognostic value of the EGFR expression level and the MGMT promoter methylation status in Glioblastoma (GB). METHODS We assessed the EGFR protein expression level by immune-histochemical (IHC) evaluation and the MGMT promoter methylation status by Polymerase Chain Reaction (PCR) in 169 patients affected by GB. We assessed the prognostic significance of combined MGMT methylation status and EGFR expression level in terms of Overall Survival (OS) with univariate and multivariate analysis, and validated this finding using an external data set of GB patient. RESULTS Clustering survival analysis for the methylation status of MGMT (methMGMT/unmethMGMT) and EGFR expression (High EGFR: H-EGFR; Low EGFR: L-EGFR), identified three different prognostic groups (p=0.001), as follows. Patients with unmethMGMT/H-EGFR had the shortest survival time (median OS: 5 months) and patients co-expressing methMGMT/L-EGFR had the best prognosis (median OS: 35 months), as compared to the other two sub-groups (methMGMT/H-EGFR; unmethMGMT/L-EGFR), which had respectively median OSs of 11 and 12 months. The combined MGMT methylation and EGFR amplification status analysis showed a similar prognostic impact in an independent series, which we used for validation (p=0.001). CONCLUSIONS The EGFR expression evaluation refines the prognostic value of MGMT methylation status in GBs.

Combined Epidermal Growth Factor Receptor and Beclin1 Autophagic Protein Expression Analysis Identifies Different Clinical Presentations, Responses to Chemo- and Radiotherapy, and Prognosis in Glioblastoma

Dysregulated EGFR in glioblastoma may inactivate the key autophagy protein Beclin1. Each of high EGFR and low Beclin1 protein expression, independently, has been associated with tumor progression and poor prognosis. High (H) compared to low (L) expression of EGFR and Beclin1 is here correlated with main clinical data in 117 patients after chemo- and radiotherapy. H-EGFR correlated with low Karnofsky performance and worse neurological performance status, higher incidence of synchronous multifocality, poor radiological evidence of response, shorter progression disease-free (PDFS), and overall survival (OS). H-Beclin1 cases showed better Karnofsky performance status, higher incidence of objective response, longer PDFS, and OS. A mutual strengthening effect emerges in correlative power of stratified L-EGFR and H-Beclin1 expression with incidence of radiological response after treatment, unifocal disease, and better prognosis, thus identifying an even longer OS group (30 months median OS compared to 18 months in L-EGFR, 15 months in H-Beclin1, and 11 months in all GBs) (P = 0.0001). Combined L-EGFR + H-Beclin1 expression may represent a biomarker in identifying relatively favorable clinical presentations and prognosis, thus envisaging possible EGFR/Beclin1-targeted therapies.

Immune-modulating effects of bevacizumab in metastatic non-small-cell lung cancer patients

The mPEBev is an anticancer regimen which combines a chemotherapy doublet, based on cisplatin and oral etoposide (mPE), with bevacizumab (mPEBev), a mAb targeting the vasculo-endothelial growth factor (VEGF). In previous studies, this regimen showed powerful anti-angiogenetic effects and significant antitumor activity in metastatic non-small-cell lung cancer (mNSCLC) patients. We also recorded the best benefit in patients exhibiting low-systemic inflammatory profile at baseline. On these bases, we hypothesized that mPEBev antitumor activity could be partially related to bevacizumab-associated immunological effects. For this reason, we performed an immunological monitoring in 59 out of 120 stage IIIb-IV NSCLC patients enrolled in the BEVA2007 phase II trial, who received fractioned cisplatin (30 mg/sqm days 1-3q21) and oral etoposide (50 mg, days 1-15q21) (mPE doublet) ±bevacizumab. In this group of patients, 12 received the mPE doublet alone and 47 the doublet in combination with bevacizumab (5 mg/kg on the day 3q21; mPEBev regimen). Blood cell counts, serum analysis, multiplex cytokine assay and immunocytofluorimetric analysis, performed on baseline and post-treatment on blood samples from these patients, revealed that bevacizumab addition to the doublet decreased levels of pro-angiogenic (VEGF, Angiostatin-1 and Follistatin) and inflammatory cytokines (interferon (IFN)γ, IL4 and IL17), improved in vivo and in vitro cytotoxic T-lymphocytes (CTL) response and promoted dendritic cell activation. These results suggest that the mPEBev regimen improve the micro-environmental conditions for an efficient antigen-specific CTL response, making it a feasible candidate regimen to be assessed in combination with immune-checkpoint inhibitors in NSCLC patients.

The effects of radiotherapy on the survival of patients with unresectable non-small cell lung cancer

ABSTRACT Introduction: Lung cancer represents the leading cause of cancer mortality across the worlds. At present, less than 30% of the patients can undergo curative surgery, while the majority of them (65%) are diagnosed with metastatic disease and directed to systemic treatments. In this context there is a subset of patients (25%) with locally advanced stage disease whose outcome might be improved by using combined strategies of treatment including chemotherapy, radiotherapy and surgery. Areas covered: Here we reviewed possible combination strategies aimed to improve the outcome of lung cancer patients, focusing on the role of radiotherapy both in the adjuvant and oligo-metastatic setting and in synergy with immunotherapy, and finally, we afforded the new challenges concerning the advanced RT and precision oncology. We carried out a focused analysis concerning the key clinical management weaknesses as well as the potential that current research holds. Expert commentary: We believe that the most promising clinical trials in this specific patient subset will build their rationale on the results of well-designed translational models aimed to test the combination of cytotoxic drugs, radiobiology, and immune-pharmacology. In this context, remarkable investigational fields are focused on the attempt to combine radiotherapy with chemo-immunological strategies and precision medicine protocols.

Radiotherapy prolongs the survival of advanced non-small-cell lung cancer patients undergone to an immune-modulating treatment with dose-fractioned cisplatin and metronomic etoposide and bevacizumab (mPEBev)

Radiotherapy (RT), together with a direct cytolytic effect on tumor tissue, also elicits systemic immunological events, which sometimes result in the regression of distant metastases (abscopal effect). We have shown the safety and anti-tumor activity of a novel metronomic chemotherapy (mCH) regimen with dose-fractioned cisplatin, oral etoposide and bevacizumab, a mAb against the vasculo-endothelial-growth-factor (mPEBev regimen), in metastatic non-small-cell-lung cancer (mNSCLC). This regimen, designed on the results of translational studies, showed immune-modulating effects that could trigger and empower the immunological effects associated with tumor irradiation. In order to assess this, we carried out a retrospective analysis in a subset of 69 consecutive patients who received the mPEBev regimen within the BEVA2007 trial. Forty-five of these patients, also received palliative RT of one or more metastatic sites. Statistical analysis (a Log-rank test) revealed a much longer median survival in the group of patients who received RT [mCH vs mCH + RT: 12.1 +/-2.5 (95%CI 3.35-8.6) vs 22.12 +/-4.3 (95%CI 11.9-26.087) months; P=0.015] with no difference in progression-free survival. In particular, their survival correlated with the mPEBev regimen ability to induce the percentage of activated dendritic cells (DCs) (CD3-CD11b+CD15-CD83+CD80+) [Fold to baseline value (FBV) ≤1 vs >1: 4+/-5.389 (95%CI,0- 14.56) vs 56+/-23.05 (95%CI,10.8-101.2) months; P:0.049)] and central-memory- T-cells (CD3+CD8+CD45RA-CCR7+) [FBV ≤ 1 vs >1: 8+/-5.96 (95%CI,0-19.68) vs 31+/-12.3 (95%CI,6.94-55.1) months; P:0.045]. These results suggest that tumor irradiation may prolong the survival of NSCLC patients undergone mPEBev regimen presumably by eliciting an immune-mediated effect and provide the rationale for further perspective clinical studies.

Anti-cancer activity of dose-fractioned mPE +/− bevacizumab regimen is paralleled by immune-modulation in advanced squamous NSLC patients

Background Results from the BEVA2007 trial, suggest that the metronomic chemotherapy regimen with dose-fractioned cisplatin and oral etoposide (mPE) +/- bevacizumab, a monoclonal antibody to the vascular endothelial growth factor (VEGF), shows anti-angiogenic and immunological effects and is a safe and active treatment for metastatic non-small cell lung cancer (mNSCLC) patients. We carried out a retrospective analysis aimed to evaluate the antitumor effects of this treatment in a subset of patients with squamous histology. Methods Retrospective analysis was carried out in a subset of 31 patients with squamous histology enrolled in the study between September 2007 and September 2015. All of the patients received chemotherapy with cisplatin (30 mg/sqm, days 1-3q21) and oral etoposide (50 mg, days 1-15q21) (mPE) and 14 of them also received bevacizumab 5 mg/kg on the day 3q21 (mPEBev regimen). Results This treatment showed a disease control rate of 71% with a mean progression free survival (PFS) and overall survival (OS) of 13.6 and 17 months respectively. After 4 treatment courses, 6 patients showing a remarkable tumor shrinkage, underwent to radical surgery, attaining a significant advantage in term of survival (P=0.048). Kaplan-Meier and log-rank test identified the longest survival in patients presenting low baseline levels in neutrophil-to-lymphocyte ratio (NLR) (P=0.05), interleukin (IL) 17A (P=0.036), regulatory-T-cells (Tregs) (P=0.020), and activated CD83+ dendritic cells (DCs) (P=0.03). Conclusions These results suggest that the mPE +/- bevacizumab regimen is feasible and should be tested in comparative trials in advanced squamous-NSCLC (sqNSCLC). Moreover, its immune-biological effects strongly suggest the investigation in sequential combinations with immune check-point inhibitors.

Abstract LB-229: Treatment of advanced cancer patients with a newest poly-epitope peptide vaccine to the thymidylate synthase(TSPP): a phase Ib (TSPP/VAC1) trial

Background TSPP is a poly-epitope peptide vaccine to the thymidylate syntase (TS) an enzyme commonly over-expressed in replicating cancer cells and recognized as a major target of fluoropyrimidines. TSPP showed immunological and anticancer activity in preclinical models, thus in the present study we investigated its safety and immunobiological activity in advanced cancer patients. This study was designed to identify TSPP Maximum Tolerated Dose (MTD) and Optimal Biological Dose (OBD) when used alone (arm A) or in combination with GM-CSF and IL-2 according to the IG-1 regimen (arm B). Patients and methods TSPP/VAC-1 (Eudract 2009-016897-33) is a monocentric phase Ib trial including 21 pretreated advanced cancer patients (12 in arm A and 9 in arm B) enrolled between April and October 2011 (12 with colorectal, 1 with breast, 1 with gastric, 1 with biliary tract and 6 with non small cell lung cancer). All patients presented a good performance status (ECOG ≤ 1). Each patient received every 3 weeks a sc administration of TSPP diluted in motanide ISA720 (1:1) at the dosage of 100 µg (6 patients), 200 µg (6 patients), and 300 µg (9 patients). Patients enrolled in arm B also received daily sc GM-CSF (100 µg) from day 1 to 5 and sc IL-2 (0.5 MIU) bid from day 6 to 15. Results No life-threatening adverse events were recorded; 1 case of grade 3 hypothyroidism (arm B), 4 cases of poly-articular arthropaty (arm A), and 4 cases of conjuntivitis (arm A) were observed. In arm B, cytokine-related flu-like syndrome, nausea, vomiting, and dyarrhea were commonly reported. An IFN-gamma ELISPOT assay revealed an increase in TS specific CTL precursors in 5 patients in arm A and 2 in arm B. It was also observed an increase in terminal effector memory and central memory T cells in the peripheral blood in both arms. Patients enrolled in arm A showed a significant decline in peripheral immune-regulatory (CD4+CD25hi+FoxP3+) T cells (baseline vs 3 vaccinations (B vs III): 3.8 vs 1.7 %; p= 0.041) and inhibitory myeloid cells (CD11c+CD15+) [Baseline (Bs) vs III vaccinations (Vc): 5.09 vs 1.02%; p= 0.05] associated to an increase in anti-proteinase III (Bs vs III Vs:0.48 vs 1.38; p= 0.04) and anti-myeloperoxidase auto-antibodies (Bs vs III Vc: 0.76 vs 1.50; p= 0.02) and reduced level of inflammatory markers (CRP, ESR, and LDH). A quality of life analysis did not reveal significant treatment-related changes in arm A, while a detrimental effect was observed in arm B. It was recorded 1 partial response (arm A);11 disease stabilitazions (8/12 patients arm A, and 3/9 arm B) and 8 disease progressions (3/12 patients arm A and 5/9 arm B). MTD was not reached, while OBD was defined as a TSPP dosage of 300 µg. Conclusion Our results suggest that the TSPP vaccine is safe and immunologically active. On these bases we believe that TSPP deserves to be evaluated in further phase II trials in cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-229. doi:1538-7445.AM2012-LB-229