Radiochemotherapy with the apoptosis stimulator xevinapant demonstrated superiority over radiochemotherapy in a randomized phase 2 trial in head and neck squamous cell carcinoma (HNSCC) but the subsequent phase 3 trial failed. Here, we compared the radiosensitization through xevinapant with two other emerging approaches, the inhibition of ATR and PARP through tuvusertib and olaparib, in a panel of four radioresistant HPV-negative HNSCC cell lines (HSC4, UT-SCC-60A, SAS, SAT). Without irradiation the analyses of proliferation and colony formation showed varying sensitivities of the cell lines towards the different agents and their combinations. When combined with radiation in colony formation assays, we observed moderate radiosensitization through xevinapant in individual cell lines but not in general. Both tuvusertib and olaparib induced stronger radiosensitization than xevinapant and combining both agents resulted in especially profound radiosensitization in three out of the four cell lines tested, whereas their combination with xevinapant or the combination of xevinapant with cisplatin was less effective. Assessment of cell death induction via annexin V/DAPI staining failed to generally predict cytotoxicity or radiosensitization of these approaches. Overall, our data are in line with the recent failure of the phase 3 TrilynX trial and suggest further investigation of ATR and PARP inhibition for the curative treatment of HPV-negative HNSCC.

ABSTRACT The incidence rate of human papillomavirus-driven oropharyngeal cancer (HPV-OPC) is increasing in countries with high human development index. HPV cell-free DNA (cfDNA) isolated from 3 to 4 mL blood plasma has been successfully used for therapy surveillance. A highly discussed application of HPV-cfDNA is early detection of HPV-OPC. This requires sensitive and specific cfDNA detection as cfDNA levels can be very low. To study the predictive power of pre-diagnostic HPV-cfDNA, archived samples from epidemiological cohorts with limited plasma volume are an important source. To establish a cfDNA detection workflow for low plasma volumes, we compared cfDNA purification methods [MagNA Pure 96 (MP96) and QIAamp ccfDNA/RNA] and digital PCR systems (Biorad QX200 and QIAGEN QIAcuity One). Final assay validation included 65 low-volume plasma samples from oropharyngeal cancer (OPC) patients with defined HPV status stored for 2–9 years. MP96 yielded a 28% higher cfDNA isolation efficiency in comparison to QIAamp. Both digital PCR systems showed comparable analytical sensitivity (6–17 copies for HPV16 and HPV33), but QIAcuity detected both types in the same assay. In the validation set, the assay had 80% sensitivity ( n = 28/35) for HPV16 and HPV33 and a specificity of 97% ( n = 29/30). In samples with ≥750 µL plasma, the sensitivity was 85% ( n = 17/20), while in samples with <750 µL plasma, it was 73% ( n = 11/15). Despite the expected drop in sensitivity with decreased plasma volume, the assay is sensitive and highly specific even in low-volume samples and thus suited for studies exploring HPV-cfDNA as an early HPV-OPC detection marker in low-volume archival material. IMPORTANCE HPV-OPC has a favorable prognosis compared to HPV-negative OPC. However, the majority of tumors is diagnosed after regional spread, thus making intensive treatment necessary. This can cause lasting morbidity with a large impact on quality of life. One potential method to decrease treatment-related morbidity is early detection of the cancer. HPV cfDNA has been successfully used for therapy surveillance and has also been detected in pre-diagnostic samples of HPV-OPC patients. These pre-diagnostic samples are only commonly available from biobanks, which usually only have small volumes of blood plasma available. Hence, we have developed a workflow optimized for small-volume archival samples. With this method, a high sensitivity can be achieved despite sample limitations, making it suitable to conduct further large-scale biobank studies of HPV-cfDNA as a prognostic biomarker for HPV-OPC.

BackgroundHead and neck squamous cell carcinoma (HNSCC) negative for Human Papillomavirus (HPV) has remained a difficult to treat entity, whereas tumors positive for HPV are characterized by radiosensitivity and favorable patient outcome. On the cellular level, radiosensitivity is largely governed by the tumor cells` ability to repair radiation-induced DNA double-strand breaks (DSBs), but no biomarker is established that could guide clinical decision making. Therefore, we tested the impact of the expression levels of ATM, the central kinase of the DNA damage response as well as DNA-PKcs and Ku80, two major factors in the main DSB repair pathway non-homologous end joining (NHEJ).MethodsA tissue microarray of a single center HNSCC cohort was stained for ATM, DNA-PKcs and Ku80 and the expression scored based on staining intensity and the percentages of tumor cells stained. Scores were correlated with clinicopathological parameters and survival.ResultsSamples from 427 HNSCC patients yielded interpretable stainings and were scored following an established algorithm. The majority of tumors showed strong expression of both NHEJ factors, whereas the expression of ATM varied more. The expression scores of ATM and DNA-PKcs were not associated with patient survival. For HPV-negative HNSCC, the minority of tumors without strong Ku80 expression trended towards superior survival when treatment included radiotherapy. Focusing stronger on staining intensity to define the subgroup with lowest and therefore potentially insufficient expression levels in the HPV-negative subgroup, we observed significantly better overall survival for patients treated with radiotherapy but not with surgery alone.ConclusionsOur data suggest that HPV-negative HNSCC with particularly low Ku80 expression represent a highly radiosensitive subpopulation. Confirmation in independent cohorts is required.

Numerous natural substances have anti-cancer properties. Especially indigenous people use aqueous plant extracts for tea or ointments including Dioscorea sansibarensis Pax to treat various diseases. The aim of this study was to evaluate the cytotoxic and radiosensitizing potential of aqueous extracts from Dioscorea sansibarensis Pax collected from Kenya in a panel of HPV-negative and -positive head and neck squamous cell carcinoma (HNSCC) cells grown in three-dimensional laminin-rich extracellular matrix (3D lrECM). The results show cytotoxicity, radiosensitization and increased levels of residual double strand breaks (DBS) by Dioscorea sansibarensis Pax extracts in HPV-negative and -positive HNSCC models in a concentration- and cell model-dependent manner. Application of ROS scavengers indicated an association between ROS-induced DSB and radiosensitization through Dioscorea sansibarensis Pax pretreatment. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based characterization of Dioscorea sansibarensis Pax identified the main components of the extract including camptothecin. Overall, Dioscorea sansibarensis Pax aqueous extracts alone and in combination with X-ray irradiation showed effective anticancer properties, which are worthy of further mechanistic investigation.

Introduction Sinonasal squamous cell carcinoma (SNSCC) is a rare, heterogeneous, and difficult-to-treat cancer with limited understanding of its tumor biology. A multimodal treatment approach involving surgery and radio(chemo)therapy is often necessary, but tumor relapse is common. Patient-derived tumor tissue slices of SNSCC have the potential to personalize targeted (radio-)therapy for these patients. Therefore, we compare individual radiosensitivity and the radiosensitizing capacity of dual targeting PARP and the intra-S/G2 checkpoint through Wee1 inhibition in different specimens.

Introduction Recently, a combination of the pro-apoptotic SMAC mimetic xevinapant and radiochemotherapy (RCT) was the first study arm ever to show superiority over RCT alone in HNSCC. The proportion of HPV-pos. patients in this randomized phase 2 trial was only 8% and there are very limited preclinical data on radiosensitization of HPV-pos. HNSCC. Since further clinical trials with xevinapant are currently being initiated that also include HPV-pos. HNSCC we are performing thorough preclinical analyses on the radiosensitization of HPV-pos. HNSCC cells through xevinapant alone, as well as the combination with another promising substance, the ATR-inhibitor tuvusertib.

Introduction Whilst the worldwide incidence of HPV-driven oropharyngeal cancer (HPV-OPC) is still increasing, early diagnosis is hampered by the lack of detectable precursor lesions. Serum antibodies against HPV16 early proteins are detectable several years before diagnosis, and cell-free HPV DNA in liquid biopsies is emerging as additional pre-diagnostic marker. PHORECAST aims defining the positive predictive value of these markers in the Hamburg City Health Study (HCHS), a single center, prospective epidemiologic cohort study that started enrolling 45,000 participants (45-74 years) in 2016.

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.

Background The gene of the Epidermal growth factor receptor ( EGFR ) is one of the most frequently altered genes in glioblastoma (GBM), with deletions of exons 2–7 (EGFRvIII) being amongst the most common genomic mutations. EGFRvIII is heterogeneously expressed in GBM. We already showed that EGFRvIII expression has an impact on chemosensitivity, replication stress, and the DNA damage response. Wee1 kinase is a major regulator of the DNA damage induced G2 checkpoint. It is highly expressed in GBM and its overexpression is associated with poor prognosis. Since Wee1 inhibition can lead to radiosensitization of EGFRvIII-negative (EGFRvIII−) GBM cells, we asked, if Wee1 inhibition is sufficient to radiosensitize also EGFRvIII-positive (EGFRvIII+) GBM cells. Methods We used the clinically relevant Wee1 inhibitor adavosertib and two pairs of isogenetic GBM cell lines with and without endogenous EGFRvIII expression exhibiting different TP53 status. Moreover, human GBM samples displaying heterogenous EGFRvIII expression were analyzed. Expression of Wee1 was assessed by Western blot and respectively immunohistochemistry. The impact of Wee1 inhibition in combination with irradiation on cell cycle and cell survival was analyzed by flow cytometry and colony formation assay. Results Analysis of GBM cells and patient samples revealed a higher expression of Wee1 in EGFRvIII+ cells compared to their EGFRvIII− counterparts. Downregulation of EGFRvIII expression by siRNA resulted in a strong decrease in Wee1 expression. Wee1 inhibition efficiently abrogated radiation-induced G2-arrest and caused radiosensitization, without obvious differences between EGFRvIII− and EGFRvIII+ GBM cells. Conclusion We conclude that the inhibition of Wee1 is an effective targeting approach for the radiosensitization of both EGFRvIII− and EGFRvIII+ GBM cells and may therefore represent a promising new therapeutic option to increase response to radiotherapy.

Recent advances in understanding the tumor’s biology in line with a constantly growing number of innovative technologies have prompted characterization of patients’ individual malignancies and may display a prerequisite to treat cancer at its patient individual tumor vulnerability. In recent decades, radiation- induced signaling and tumor promoting local events for radiation sensitization were explored in detail, resulting the development of novel molecular targets. A multitude of pharmacological, genetic, and immunological principles, including small molecule- and antibody-based targeted strategies, have been developed that are suitable for combined concepts with radiation (RT) or chemoradiation therapy (CRT). Despite a plethora of promising experimental and preclinical findings, however, so far, only a very limited number of clinical trials have demonstrated a better outcome and/or patient benefit when RT or CRT are combined with targeted agents. The current review aims to summarize recent progress in molecular therapies targeting oncogenic drivers, DNA damage and cell cycle response, apoptosis signaling pathways, cell adhesion molecules, hypoxia, and the tumor microenvironment to impact therapy refractoriness and to boost radiation response. In addition, we will discuss recent advances in nanotechnology, e.g., RNA technologies and protein-degrading proteolysis-targeting chimeras (PROTACs) that may open new and innovative ways to benefit from molecular-targeted therapy approaches with improved efficacy.

Preclinical research rarely predict outcomes of clinical research. One main reason for translational failure is the incapacity of cell lines/animal models to reliably predict the complex and diverse tumor behavior in humans. This underlines the necessity of innovative tumor models. Tumor tissue slice cultivation is a patient-derived model preserving tumor cells and tumor microenvironment. Tumor biopsies were cut in 400 μm sections. The slices were cultivated on cell culture inserts at air-medium interface. Tumor-individual radiosensitivity-assays were performed and associated to clinical characteristics. We succeeded to cultivate over 50 HNSCC with the aim to understand specific radiobiology of HPV-positive oropharyngeal cancer (OPSCC): HPV-positive OPSCC tissues (n=14) showed a ATM-dependent defect in DNA damage repair (4.9 vs. 1.2 DNA double strand break foci per nucleus after 3 Gy; p <0.0001). The HPV-positive OPSCC samples derived from patients with smoking of 10 or more pack years (n=7) showed less intrinsic radiosensitivity than these without or little smoking exposure (p=0,0105). Analyse potential radiosensitizer: we tested dual targeting of PARP and the intra-S/G2 checkpoint through Wee1 inhibition for its radiosensitizing capacity. The combination appeared to be highly effective in HPV-negative locally advanced HNSCC (UICC: Stadium 4). Analyse radiosensitivity of rare tumors: intrinsic radiosensitivity of sinonasal squamous cell carcinoma is similiar to other HNSCC in the Hamburger tumor tissue slice cohort (n=6). Tumor tissue slices cultivation is an effective tool to examine individual radiosensitivity in HNSCC patients. In future, it could be used in clinical trials to stratify therapeutic responders from nonresponders and personalize treatment.

Präklinische Forschung korreliert selten mit Ergebnissen klinischer Forschung. Ein Grund ist die Unfähigkeit von Zelllinien/Tiermodellen, das komplexe Tumorverhalten beim Menschen vorherzusagen. Dies unterstreicht die Notwendigkeit innovativer Tumormodelle. Tumorschnittkulturen basieren auf der Kultivierung frischer von Patienten stammenden Proben und enthalten Tumorzellen in ihrem originalen Tumormikromillieu. Es wurden tumorindividuelle Radiosensitivitäts-Assays an über 50 KHT durchgeführt mit Ergebnissen zur folgenden Themen: – Strahlenbiologie von HPV-positivem Oropharynxkarzinom (OPSCC): HPV-positive OPSCC (n=14) zeigen einen ATM-abhängigen Defekt in der DNA-Schadensreparatur (4,9 vs. 1,2 DNA-Doppelstrangbruche/Kern nach 3 Gy; p<0,0001). Die Proben, die von Patienten stammten, die 10 oder mehr „pack years“ geraucht hatten (n = 7), zeigten eine geringere intrinsische Strahlenempfindlichkeit als solche ohne/mit geringer Raucherexposition (p = 0,0105). – Analyse potenzieller „Radiosensitizer“: Die radiosensibilisierende Wirkung durch duale Inhibition von PARP und des Intra-S/G2-Checkpoint durch Wee1-Hemmung wurde bei verschiedenen KHT untersucht. Diese Kombination zeigte sich insbesondere bei HPV-negativem, lokal fortgeschrittenem HNSCC (UICC: Stadium 4) hochwirksam. – Analyse der Strahlenempfindlichkeit seltener Tumoren: Die intrinsische Strahlenempfindlichkeit sinonasaler Plattenepithelkarzinoms ist vergleichbar mit anderen KHT der Hamburger Kohorte (n=6). Die Kultivierung von Tumorgewebeschnitten ist ein effektives Instrument zur Untersuchung individueller Strahlenempfindlichkeit bei Patienten mit KHT. Das Tumormodell könnte zukünftig z.B. im Rahmen klinischer Studien therapeutische Responder von Non-Respondern unterscheiden und Behandlungen personalisieren

Einleitung Trotz zuletzt steigender Inzidenz von HPV-assoziierten Oropharynxkarzinomen (HPV-OPC), ist die Früherkennung weiterhin herausfordernd. Antikörper gegen frühe (early, E) HPV16-Proteine sind teils Jahre vor Diagnose nachweisbar. Unsere Proof-of-Concept-Studie identifizierte drei asymptomatische HPV-OPC-Patienten im Stadium I von insg. elf Teilnehmern mit hohem HPV-OPC-Risiko i. R. der Hamburg City Health Study (HCHS) bis Juni 2022.Patienten und

BACKGROUND:We have recently shown a frequent upregulation of Src-family kinases (SFK) in head and neck squamous cell carcinoma (HNSCC). Here we tested, if SFK targeting is effective especially in HNSCC cells with upregulated SFK signaling.METHODS:The impact of SFK inhibitors SU6656, PP2 and dasatinib on three HNSCC cell lines with different SFK activity levels was analyzed using proliferation and colony formation assays, Western blot and functional kinomics.RESULTS:Proliferation was blocked by all inhibitors in a micro-molar range. With respect to cell kill, dasatinib was most effective, while SU6656 showed moderate and PP2 minor effects. Cellular signaling was affected differently, with PP2 having no effect on SFK signaling while dasatinib probably has non-SFK specific effects. Only SU6656 showed clear SFK specific effects on signaling.CONCLUSION:The results demonstrate potential benefit of SFK inhibition in HNSCC but they also highlight challenges due to non-specificities of the different drugs.

Objectives:In head and neck squamous cell carcinoma (HNSCC), tumors 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. Materials 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 & gamma;H2AX 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 & PLUSMN; 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 from HPV-negative but not HPV+ HNSCC (5/7 vs. 1/6). 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 slice cultures may predict the response of individual patients with HPV-negative HNSCC to this dual targeting approach.