Pedro A. Andrade Filho

Cetuximab-Activated Natural Killer and Dendritic Cells Collaborate to Trigger Tumor Antigen–Specific T-cell Immunity in Head and Neck Cancer Patients

Purpose: Tumor antigen–specific monoclonal antibodies (mAb) block oncogenic signaling and induce Fcγ receptor (FcγR)–mediated cytotoxicity. However, the role of CD8+ CTL and FcγR in initiating innate and adaptive immune responses in mAb-treated human patients with cancer is still emerging. Experimental Design: FcγRIIIa codon 158 polymorphism was correlated with survival in 107 cetuximab-treated patients with head and neck cancer (HNC). Flow cytometry was carried out to quantify EGF receptor (EGFR)–specific T cells in cetuximab-treated patients with HNC. The effect of cetuximab on natural killer (NK) cell, dendritic cell (DC), and T-cell activation was measured using IFN-γ release assays and flow cytometry. Results: FcγRIIIa polymorphism did not predict clinical outcome in cetuximab-treated patients with HNC; however, elevated circulating EGFR853–861–specific CD8+ T cells were found in cetuximab-treated patients with HNC (P < 0.005). Cetuximab promoted EGFR-specific cellular immunity through the interaction of EGFR+ tumor cells and FcγRIIIa on NK cells but not on the polymorphism per se. Cetuximab-activated NK cells induced IFN-γ–dependent expression of DC maturation markers, antigen processing machinery components such as TAP-1/2 and T-helper cell (TH1) chemokines through NKG2D/MICA binding. Cetuximab initiated adaptive immune responses via NK cell–induced DC maturation, which enhanced cross-presentation to CTL specific for EGFR as well as another tumor antigen, MAGE-3. Conclusion: Cetuximab-activated NK cells promote DC maturation and CD8+ T-cell priming, leading to tumor antigen spreading and TH1 cytokine release through “NK–DC cross-talk.” FcγRIIIa polymorphism did not predict clinical response to cetuximab but was necessary for NK–DC interaction and mAb-induced cross-presentation. EGFR-specific T cells in cetuximab-treated patients with HNC may contribute to clinical response. Clin Cancer Res; 19(7); 1858–72. ©2013 AACR.

Immunization with analog peptide in combination with CpG and montanide expands tumor antigen-specific CD8+ T cells in melanoma patients.

Analog peptides represent a promising tool to further optimize peptide-based vaccines in promoting the expansion of tumor antigen-specific cytotoxic T lymphocytes. Here, we report the results of a pilot trial designed to study the immunogenicity of the analog peptide NY-ESO-1 157-165V in combination with CpG 7909/PF3512676 and Montanide ISA 720 in patients with stage III/IV NY-ESO-1–expressing melanoma. Eight patients were immunized either with Montanide and CpG (arm 1, 3 patients); Montanide and peptide NY-ESO-1 157-165V (arm 2, 2 patients); or with Montanide, CpG, and peptide NY-ESO-1 157-165V (arm 3, 3 patients). Only the 3 patients immunized with Montanide, CpG, and peptide NY-ESO-1 157-165V in arm 3 developed a rapid increase of effector-memory NY-ESO-1–specific CD8+ T cells, detectable ex vivo. The majority of these cells exhibited an intermediate/late-stage differentiated phenotype (CD28−). Our study further demonstrated that our vaccine approach stimulated spontaneous tumor-reactive NY-ESO-1–specific CD8+ T cells in 2 patients with advanced disease, but failed to prime tumor-reactive NY-ESO-1–specific T cells in 1 patient with no spontaneously tumor-induced CD8+ T-cell responses to NY-ESO-1. Collectively, our data support the capability of the analog peptide NY-ESO-1 157-165V in combination with CpG and Montanide to promote the expansion of NY-ESO-1–specific CD8+ T cells in patients with advanced cancer. They also suggest that the presence of tumor-induced NY-ESO-1–specific T cells of well-defined clonotypes is critical for the expansion of tumor-reactive NY-ESO-1–specific CD8+ T cells after peptide-based vaccine strategies.

Deficiency of activated STAT1 in head and neck cancer cells mediates TAP1-dependent escape from cytotoxic T lymphocytes

Squamous cell carcinoma of the head and neck (SCCHN) cells can escape recognition by tumor antigen (TA)-specific cytotoxic T lymphocytes (CTL) by downregulation of antigen processing machinery (APM) components, such as the transporter associated with antigen processing (TAP)-1/2 heterodimer. APM component upregulation by interferon gamma (IFN-γ) restores SCCHN cell recognition and susceptibility to lysis by CTL, but the mechanism underlying TAP1/2 downregulation in SCCHN cells is not known. Because IFN-γ activates signal transducer and activator of transcription (STAT)-1, we investigated phosphorylated (p)-STAT1 as a mediator of low basal TAP1/2 expression in SCCHN cells. SCCHN cells were found to express basal total STAT1 but low to undetectable levels of activated STAT1. The association of increased pSTAT1 levels and APM components likely reflects a cause–effect relationship, since STAT1 knockdown significantly reduced both IFN-γ-mediated APM component expression and TA-specific CTL recognition of IFN-γ-treated SCCHN cells. On the other hand, since oncogenic pSTAT3 is overexpressed in SCCHN cells and was found to heterodimerize with pSTAT1, we also tested whether pSTAT3 and pSTAT1:pSTAT3 heterodimers inhibited IFN-γ-induced STAT1 activation and APM component expression. First, STAT3 activation or depletion did not affect basal or IFN-γ-induced expression of pSTAT1 and APM components or recognition of SCCHN cells by TA-specific CTL. Second, pSTAT1:pSTAT3 heterodimers did not interfere with IFN-γ-induced STAT1 binding to the TAP1 promoter or APM protein expression. These findings demonstrate that APM component downregulation is regulated primarily by an IFN-γ-pSTAT1-mediated signaling pathway, independent of oncogenic STAT3 overexpression in SCCHN cells.

Oropharyngeal complications of suspension laryngoscopy: a prospective study.

Objective: This study was designed to evaluate the oropharyngeal complications of suspension laryngoscopy (SL).

SHP2 Is Overexpressed and Inhibits pSTAT1-Mediated APM Component Expression, T-cell Attracting Chemokine Secretion, and CTL Recognition in Head and Neck Cancer Cells

Purpose: Human leukocyte antigen (HLA) class I antigen processing machinery (APM) component downregulation permits escape of malignant cells from recognition by cytotoxic T lymphocytes (CTL) and correlates with poor prognosis in patients with head and neck cancer (HNC). Activated STAT1 (pSTAT1) is necessary for APM component expression in HNC cells. We investigated whether an overexpressed phosphatase was responsible for basal suppression of pSTAT1 and subsequent APM component-mediated immune escape in HNC cells. Experimental Design: Immunohistochemical staining and reverse transcription PCR of paired HNC tumors was performed for the phosphatases src homology domain-containing phosphatase (SHP)–1 and SHP2. Depletion of phosphatase activity in HNC and STAT1−/− tumor cells was achieved by siRNA knockdown. HLA class I–restricted, tumor antigen-specific CTL were used in IFN-γ ELISPOT assays against HNC cells. Chemokine secretion was measured after SHP2 depletion in HNC cells. Results: SHP2, but not SHP1, was significantly upregulated in HNC tissues. In HNC cells, SHP2 depletion significantly upregulated expression of pSTAT1 and HLA class I APM components. Overexpression of SHP2 in nonmalignant keratinocytes inhibited IFN-γ–mediated STAT1 phosphorylation, and SHP2 depletion in STAT1−/− tumor cells did not significantly induce IFN-γ–mediated APM component expression, verifying STAT1 dependence of SHP2 activity. SHP2 depletion induced recognition of HNC cells by HLA class I–restricted CTL and secretion of inflammatory, T-cell attracting chemokines, RANTES and IP10. Conclusion: These findings suggest for the first time an important role for SHP2 in APM-mediated escape of HNC cells from CTL recognition. Targeting SHP2 could enhance T-cell–based cancer immunotherapy. Clin Cancer Res; 19(4); 798–808. ©2012 AACR.

Novel Immunogenic HLA-A*0201-restricted Epidermal Growth Factor Receptor-specific T-cell Epitope in Head and Neck Cancer Patients

Therapeutic targeting of the epidermal growth factor receptor (EGFR), which is highly overexpressed and correlated with poor prognosis in colorectal and head and neck squamous cell carcinoma (SCCHN), has shown clinical efficacy using the blocking mAbs, cetuximab or panitumumab, but only in 10% to 20% of patients. Clinical responsiveness is correlated with certain Fcγ receptor genotypes, suggesting immune activity may contribute to therapeutic efficacy. In addition, cetuximab-resistant tumor cells exhibit ubiquitination and degradation of EGFR, which would increase its processing as a tumor antigen for cytotoxic T lymphocyte (CTL) lysis. Thus, T cell-based immunotherapy might enhance the antitumor efficacy of EGFR-specific mAbs, but CTL epitopes are poorly defined. To permit combinatorial EGFR-targeted immunotherapy, we identified a novel immunogenic wild-type sequence peptide, EGFR853−861 and modified its anchor sequence to enhance HLA-A*0201 binding and stimulation of cross-reactive anti-wild–type EGFR853−861-specific CTL. Cross-reactivity was also observed with HER2861−869. EGFR853−861-specific CTL recognition of SCCHN cells was increased by incubation of tumor cells with cetuximab, which led to EGFR degradation. In addition, EGFR853−861-specific CTLs were elevated in the circulation of SCCHN patients as compared with healthy control peripheral blood mononuclear cells. Thus, a novel, immunogenic EGFR-encoded CTL epitope may be incorporated into vaccines and would be useful for combinatorial immunotherapy with EGFR-specific mAbs in cancer patients.

Quantitative Expression and Immunogenicity of MAGE-3 and -6 in Upper Aerodigestive Tract Cancer

The MAGE antigens are frequently expressed cancer vaccine targets. However, quantitative analysis of MAGE expression in upper aerodigestive tract (UADT) tumor cells and its association with T‐cell recognition has not been performed, hindering the selection of appropriate candidates for MAGE‐specific immunotherapy. Using quantitative RT‐PCR (QRT‐PCR), we evaluated the expression of MAGE‐3/6 in 65 UADT cancers, 48 normal samples from tumor matched sites and 7 HLA‐A*0201+ squamous cell carcinoma of the head and neck (SCCHN) cell lines. Expression results were confirmed using Western blot. HLA‐A*0201:MAGE‐3‐ (271–279) specific cytotoxic T lymphocytes (MAGE‐CTL) from SCCHN patients and healthy donors showed that MAGE‐3/6 expression was highly associated with CTL recognition in vitro. On the basis of the MAGE‐3/6 expression, we could identify 31 (47%) of the 65 UADT tumors, which appeared to express MAGE‐3/6 at levels that correlated with efficient CTL recognition. To confirm that the level of MAGE‐3 expression was responsible for CTL recognition, 2 MAGE‐3/6 mRNAhigh SCCHN cell lines, PCI‐13 and PCI‐30, were subjected to MAGE‐3/6‐specific knockdown. RNAi‐transfected cells showed that MAGE expression and MAGE‐CTL recognition were significantly reduced. Furthermore, treatment of cells expressing low MAGE‐3/6 mRNA with a demethylating agent, 5‐aza‐2′‐deoxycytidine (DAC), increased the expression of MAGE‐3/6 and CTL recognition. Thus, using QRT‐PCR UADT cancers frequently express MAGE‐3/6 at levels sufficient for CTL recognition, supporting the use of a QRT‐PCR‐based assay for the selection of candidates likely to respond to MAGE‐3/6 immunotherapy. Demethylating agents could increase the number of patients amenable for targeting epigenetically modified tumor antigens in vaccine trials.

Maturation pathways of dendritic cells determine TAP1 and TAP2 levels and cross-presenting function.

Ability to cross-present exogenous antigens in the human leukocyte antigen class I pathway is key to the antigen presenting function of mature tumor cell-loaded dendritic cells (DC). Conditions of DC maturation have been shown to be important for DCs ability to produce proinflammatory cytokines and induce T cell effector functions. However, it remains unknown if the different pathways of maturation are associated with modulation of the ability of mature DCs to cross-present tumor antigens (TA). Here, we compare DC matured with 3 clinically relevant cytokine combinations including interleukin (IL)-1β, tumor necrosis factor-α, IL-6 (termed DC-0), DC-0 cells incubated with prostaglandin-2 (termed DC-0+prostaglandin-2), or DC treated with interferon-γ, interferon-α, tumor necrosis factor-α, Poly I:C, and IL1-β (termed DC-1). We found that these DC vary in their ability to cross-present TA to cytotoxic T lymphocytes (CTL), with the DC-1 cytokine combination being significantly more effective than the other 2. TA cross presentation and CTL priming were strongly correlated with level of expression of the antigen processing machinery components, TAP1 and TAP2, indicating that these components could be used as biomarkers to standardize DC preparations for optimal function. However, the up-regulation of TAP1/TAP2 was not sufficient to explain the enhanced cross-presentation ability of DC-1 cells, as the use of IFN-γ alone to up-regulate TAP1/TAP2 did not generate DC as effective at cross-presentation as the full DC-1 maturation cytokine combination. These data indicate for the first time that the pathways of DC maturation modulate antigen processing machinery component expression to different extents and that differently matured DC vary in the ability to cross-present TA to human leukocyte antigen class I-restricted CTL.

Clinicopathologic features as stronger prognostic factors than histology or grade in risk stratification of primary parotid malignancies

The aim of this study, using a retrospective chart review as the primary study design, was to determine the relative contribution of clinicopathologic risk factors versus low‐ and high‐risk grade histologic groups to assist management of primary parotid cancers.

CD8+ T cell recognition of polymorphic wild-type sequence p5365-73 peptides in squamous cell carcinoma of the head and neck

The TP53 tumor suppressor gene contains a well-studied polymorphism that encodes either proline (P) or arginine (R) at codon 72, and over half of the world’s population is homozygous for R at this codon. The wild-type sequence (wt) p53 peptide, p5365–73, has been identified as a CD8+ T cell-defined tumor antigen for use in broadly applicable cancer vaccines. However, depending on the TP53 codon 72 polymorphism of the recipient, the induced responses to the peptides incorporating R (p5372R) or P (p5372P) can be “self” or “non-self.” Thus, we sought to determine which wt p5365–73 peptide should be used in wt p53-based cancer vaccines. Despite similar predicted HLA-A2-binding affinities, the p5372P peptide was more efficient than the p5372R peptide in HLA-A2 stabilization assays. In vitro stimulation (IVS) of CD8+ T cells obtained from healthy HLA-A2+ donors with these two peptides led to the generation of CD8+ T cell effectors in one-third of the samples tested, at a frequency similar to the responsiveness to other wt p53 peptides. Interestingly, regardless of their p53 codon 72 genotype, CD8+ T cells stimulated with either p5372P or p5372R peptide were cross-reactive against T2 cells pulsed with either peptide, as well as HLA-A2+ head and neck cancer (HNC) cell lines presenting p5372P and/or p5372R peptides for T cell recognition. Therefore, the cross-reactivity of CD8+ T cells for the polymorphic wt p5365–73 peptides, irrespective of their p53 codon 72 polymorphism, suggests that employing either peptide in wt p53-based vaccines can result in efficient targeting of this epitope.