Squamous cell carcinoma of the head and neck (HNSCC) consist of two distinct biological entities. While the numbers of classical, tobacco-induced HNSCC are declining, tumors caused by human papillomavirus (HPV) infection are increasing in many countries. HPV-positive HNSCC mostly arise in the oropharynx and are characterized by an enhanced sensitivity towards radiotherapy and a favorable prognosis. To identify molecular differences between both entities on the protein level, we conducted a mass spectrometric comparison of eight HPV-positive and nine HPV-negative oropharyngeal tumors (OPSCC). Overall, we identified 2051 proteins, of which 31 were found to be differentially expressed. Seventeen of these can be assorted to three functional groups, namely DNA replication, nuclear architecture and cytoskeleton regulation, with the differences in the last group potentially reflecting an enhanced migratory and invasive capacity. Furthermore, a number of identified proteins have been described to directly impact on DNA double-strand break repair or radiation sensitivity (e.g., SLC3A2, cortactin, RBBP4, Numa1), offering explanations for the differential prognosis. The unequal expression of three proteins (SLC3A2, MCM2 and lamin B1) was confirmed by immunohistochemical staining using a tissue microarray containing 205 OPSCC samples. The expression levels of SLC3A2 and lamin B1 were found be of prognostic relevance in patients with HPV-positive and HPV-negative OPSCC, respectively.

The receptor tyrosine kinase Axl is described to promote migration, metastasis and resistance against molecular targeting, radiotherapy, and chemotherapy in various tumor entities, including head and neck squamous cell carcinoma (HNSCC). Since clinical data on Axl and its ligand Gas6 in HNSCC are sparse, we assessed the association of Axl and Gas6 expression with patient survival in a single center retrospective cohort in a tissue microarray format. Expression was evaluated manually using an established algorithm and correlated with clinicopathological parameters and patient survival. A number of 362 samples yielded interpretable staining, which did not correlate with T- and N-stage. Protein expression levels were not associated with the survival of patients with p16-positive oropharyngeal SCC. In HPV-negative tumors, Axl expression did not impact patients treated with primary or adjuvant radio(chemo)therapy, but was significantly associated with inferior overall and recurrence-free survival in patients treated with surgery alone. Gas6 was a positive predictor of survival in patients whose treatment included radiotherapy. Associations remained significant in multivariable analysis. Our data question a meaningful contribution of the Axl/Gas6 pathway to radio-resistance in HNSCC and instead suggest that strong Axl expression identifies tumors requiring adjuvant radio(chemo)therapy after surgery.

Introduction HNSCC negative for Human Papillomavirus (HPV) remain a difficult to treat entity and the morbidity of current multimodal treatment is high. Radiotherapy in combination with molecular targeting could represent suitable, less toxic treatment options especially for cisplatin ineligible patients. Therefore, we tested dual targeting of PARP and the intra-S/G2 checkpoint through Wee1 inhibition for its radiosensitizing capacity in radioresistant HPV-negative HNSCC cells. Material and Methods Three radioresistant HPV-negative cell lines (HSC4, SAS, UT-SCC-60a) were treated with olaparib, adavosertib and ionizing irradiation. The impact on cell cycle, G2 arrest and replication stress was assessed through flow cytometry after DAPI, phospho-histone H3 and γH2AX staining. Long term cell survival after treatment was determined through colony formation assay and DNA double-strand break (DSB) levels were assessed through quantification of nuclear 53BP1 foci in cell lines and patient-derived HPV± tumor slice cultures. Results Wee1 and dual targeting induced replication stress but failed to effectively inhibit radiation-induced G2 cell cycle arrest. Single as well as combined inhibition increased radiation sensitivity and residual DSB levels, with the largest effects induced through dual targeting. Dual targeting also enhanced residual DSB levels in patient-derived slice cultures (4/6) from HPV-negative but not HPV+ HNSCC. Conclusion We conclude that the combined inhibition of PARP and Wee1 results in enhanced residual DNA damage levels after irradiation and effectively sensitizes radioresistant HPV-negative HNSCC cells. Ex vivo tumor cultures may predict the response of individual patients with HPV-negative HNSCC to this dual targeting approach. Deutsche Krebshilfe (Antrag 70113259; SK, KR, TR) & BMBF (Antrag 02NUK032; MK, KR, TR) Article published online: 12 May 2023 Georg Thieme Verlag Rüdigerstraße 14, 70469 Stuttgart, Germany

Einleitung Die aktuellen multimodalen Therapien für HPV-negative HNSCC sind bei nach wie vor nicht-optimalen Heilungsraten mit hoher Morbidität verbunden. Die Kombination der Strahlentherapie mit molekularem Targeting könnte, insbesondere für Cisplatin-ungeeignete Patienten, wirksame Alternativen ermöglichen. In diesem Kontext wurde hier die duale Inhibition von PARP und Wee1 auf ihre radiosensibilisierende Wirkung getestet. Material und Methoden Behandlung dreier strahlenresistenter HPV-negativer Zelllinien (HSC4, SAS, UT-SCC-60a) mittels Olaparib, Adavosertib und Bestrahlung. Analyse des Zellzyklus, G2-Arrest und Replikationsstress mittels Durchflusszytometrie. Analyse des langfristigen Zellüberlebens mittels Kolonieassay und Analyse der Reparatur strahleninduzierter DNA-Schäden über Quantifizierung nukleärer 53BP1-Foci in Zelllinien und Patienten-abgeleiteten Tumorschnittkulturen. Resultate Inhibition von Wee1 und duales Targeting induzieren Replikationsstress, können aber den strahleninduzierten G2-Zellzyklusarrest nicht wirksam inhibieren. Einzelne sowie kombinierte Inhibition erhöht die Strahlenempfindlichkeit und die Anzahl nicht-reparierter, residualer DNA-Doppelstrangbrüche, den toxischsten strahleninduzierten Läsionen. Duales Targeting zeigte hier die höchste Wirksamkeit und erhöhte die Anzahl residualer Doppelstrangbrüche ebenfalls in Patienten-abgeleiteten Schnittkulturen (4/6) HPV-negativer, aber nicht HPV-positiver Tumoren. Schlussfolgerungen Kombinierte PARP und Wee1 Inhibition führt zu wirksamer Radiosensibilisierung strahlenresistenter, HPV-negativer HNSCC Zellen aufgrund verminderter DNA-Reparatur. Ex vivo-Tumorschnittkulturen könnten das Ansprechen individueller HPV-negativer Tumoren vorhersagen und personalisierte Behandlungskonzepte ermöglichen. Deutsche Krebshilfe (Antrag 70113259; SK, KR, TR) & BMBF (Antrag 02NUK032; MK, KR, TR) Article published online: 12 May 2023 Georg Thieme Verlag Rüdigerstraße 14, 70469 Stuttgart, Germany