The HPV and p63 Status in Penile Cancer Are Linked with the Infiltration and Therapeutic Availability of Neutrophils.

Squamous penile cancer displays a rare human papillomavirus (HPV)-associated tumor entity. Investigations on the molecular pathogenesis of HPV-driven penile cancer are impaired by the rareness of clinical specimens and, in particular, are missing relevant cell culture models. Here, we identified in HPV-positive penile cancer cell lines that HPV16 oncoproteins control TP63 expression by modulating critical regulators, while integration into the TP63 open reading frame facilitates oncogene expression. The resulting feed-forward loop leads to elevated p63 levels that in turn enhance the release of the neutrophil-recruiting chemokine CXCL8. Remarkably, elevated CXCL8 amounts lead to the increased surface exposition of the Fc receptor of human IgA antibodies, FcαRI, on neutrophils and correlated with a higher susceptibility to antibody-dependent neutrophil-mediated cytotoxicity (ADCC) using an EGFR-specific IgA2 antibody. IHC staining of tissue microarrays proved that elevated expression of p63 together with neutrophil infiltration were significantly more frequent in HPV-positive penile cancer displaying a higher tumor grade. In summary, we identified a promising marker profile of patients with penile cancer at higher risk for worse prognosis. However, these patients may benefit from immunotherapeutic approaches efficiently engaging neutrophils for tumor cell killing.

Monitoring kinetics reveals critical parameters of IgA-dependent granulocyte-mediated anti-tumor cell cytotoxicity.

Human IgA antibodies effectively engage myeloid cells for the FcαRI-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells. Established methods to investigate ADCC are the 51chromium and Calcein release assays. Their critical limitations are the end-point measurement, the unspecific release of the probes, the requirement of target cells in suspension and thus do not reflect physiologic conditions of adherently growing cells. Here we report the label-free real-time monitoring of granulocyte-mediated ADCC using an impedance-based method. We investigated the efficacy of an engineered epidermal growth factor receptor (EGFR)-directed IgA2 antibody to engage neutrophils for ADCC against a panel of adherently growing EGFR-expressing cancer cell lines majorly head and neck squamous cell carcinoma (HNSCC). The impedance assay allowed the documentation of the IgA-neutrophil-and FcαRI-signaling dependent ADCC of adherently growing target cells. While at a short-term it provided comparable results to release assays, in the long run real time monitoring also revealed cell-line specific kinetics and long-term efficacy. Although short-term results may depend on EGFR expression, long-term efficacy did not correlate with the surface level of EGFR nor of the myeloid checkpoint CD47 pointing to additional critical parameters to predict the treatment efficacy. Real-time monitoring of neutrophil-mediated ADCC allowed documenting effector cell activity and exhaustion. Along with excess expression of Mac-1 ligands, which may explain the target cell resistance, this eventually leads to tumor cell outgrowth at later time points. In conclusion, the impedance assay provides valuable information on the kinetics, effector cell performance, efficacy and critical parameters of IgA-dependent granulocyte-mediated cytotoxicity and is expected to become an important tool in its evaluation.