Fabian Lohaus

We examined the prognostic value of tumour-infiltrating lymphocytes (TILs) in patients with squamous cell carcinoma of the head and neck (SCCHN) after surgery and postoperative cisplatin-based chemoradiotherapy. FFPE-tissue originating from the surgery of 161 patients treated in 8 DKTK partner sites was immunohistochemically stained for CD3 and CD8. Their expression was correlated with clinicopathological characteristics as well as overall survival (OS), local progression-free survival (LPFS) and distant metastases free-survival (DMFS), also in the context of the HPV16-DNA/p16 status. After a median follow-up of 48 months (range: 4100 months), OS at 4 years was 46.5% for the entire cohort. In multivariate analysis, high CD8 expression was confirmed as an independent prognostic parameter for OS (p = 0.002), LPFS (p = 0.004) and DMFS (p = 0.006), while CD3 expression lacked significance. In multivariate analysis HPV16 DNA positivity was associated with improved OS (p = 0.025) and LPFS (p = 0.013) and p16-positive patients showed improved DMFS (p = 0.008). Interestingly, high CD8 expression was a prognostic parameter for the clinical outcome in both HPV16 DNA-positive and HPV16 DNA-negative patients. Similar findings were observed in the multivariate analysis for the combined HPV16 DNA/p16 status. Altogether, CD8+ TILs constitute an independent prognostic marker in SCCHN patients treated with adjuvant chemoradiotherapy. These data indicate that CD8-positive TILs have antitumour activity and could be used for treatment stratification. Further validation of the prognostic value of CD8+ TILs as a biomarker and its role in the immune response in SCCHN patients after adjuvant chemoradiotherapy is warranted and will be performed in the prospective DKTK-ROG study.

Radiomics applies machine learning algorithms to quantitative imaging data to characterise the tumour phenotype and predict clinical outcome. For the development of radiomics risk models, a variety of different algorithms is available and it is not clear which one gives optimal results. Therefore, we assessed the performance of 11 machine learning algorithms combined with 12 feature selection methods by the concordance index (C-Index), to predict loco-regional tumour control (LRC) and overall survival for patients with head and neck squamous cell carcinoma. The considered algorithms are able to deal with continuous time-to-event survival data. Feature selection and model building were performed on a multicentre cohort (213 patients) and validated using an independent cohort (80 patients). We found several combinations of machine learning algorithms and feature selection methods which achieve similar results, e.g.C-Index = 0.71 and BT-COX: C-Index = 0.70 in combination with Spearman feature selection. Using the best performing models, patients were stratified into groups of low and high risk of recurrence. Significant differences in LRC were obtained between both groups on the validation cohort. Based on the presented analysis, we identified a subset of algorithms which should be considered in future radiomics studies to develop stable and clinically relevant predictive models for time-to-event endpoints.

Radiotherapy is one of the curative treatment options for localized prostate cancer (PCa). The curative potential of radiotherapy is mediated by irradiation-induced oxidative stress and DNA damage in tumor cells. However, PCa radiocurability can be impeded by tumor resistance mechanisms and normal tissue toxicity. Metabolic reprogramming is one of the major hallmarks of tumor progression and therapy resistance. Specific metabolic features of PCa might serve as therapeutic targets for tumor radiosensitization and as biomarkers for identifying the patients most likely to respond to radiotherapy. The study aimed to characterize a potential role of glutaminase (GLS)-driven glutamine catabolism as a prognostic biomarker and a therapeutic target for PCa radiosensitization. Methods: We analyzed primary cell cultures and radioresistant (RR) derivatives of the conventional PCa cell lines by gene expression and metabolic assays to identify the molecular traits associated with radiation resistance. Relative radiosensitivity of the cell lines and primary cell cultures were analyzed by 2-D and 3-D clonogenic analyses. Targeting of glutamine (Gln) metabolism was achieved by Gln starvation, gene knockdown, and chemical inhibition. Activation of the DNA damage response (DDR) and autophagy was assessed by gene expression, western blotting, and fluorescence microscopy. Reactive oxygen species (ROS) and the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) were analyzed by fluorescence and luminescence probes, respectively. Cancer stem cell (CSC) properties were investigated by sphere-forming assay, CSC marker analysis, and in vivo limiting dilution assays. Single circulating tumor cells (CTCs) isolated from the blood of PCa patients were analyzed by array comparative genome hybridization. Expression levels of the GLS1 and MYC gene in tumor tissues and amino acid concentrations in blood plasma were correlated to a progression-free survival in PCa patients. Results: Here, we found that radioresistant PCa cells and prostate CSCs have a high glutamine demand. GLS-driven catabolism of glutamine serves not only for energy production but also for the maintenance of the redox state. Consequently, glutamine depletion or inhibition of critical regulators of glutamine utilization, such as GLS and the transcription factor MYC results in PCa radiosensitization. On the contrary, we found that a combination of glutamine metabolism inhibitors with irradiation does not cause toxic effects on nonmalignant prostate cells. Glutamine catabolism contributes to the maintenance of CSCs through regulation of the alpha-ketoglutarate (α-KG)-dependent chromatin-modifying dioxygenase. The lack of glutamine results in the inhibition of CSCs with a high aldehyde dehydrogenase (ALDH) activity, decreases the frequency of the CSC populations in vivo and reduces tumor formation in xenograft mouse models. Moreover, this study shows that activation of the ATG5-mediated autophagy in response to a lack of glutamine is a tumor survival strategy to withstand radiation-mediated cell damage. In combination with autophagy inhibition, the blockade of glutamine metabolism might be a promising strategy for PCa radiosensitization. High blood levels of glutamine in PCa patients significantly correlate with a shorter prostate-specific antigen (PSA) doubling time. Furthermore, high expression of critical regulators of glutamine metabolism, GLS1 and MYC, is significantly associated with a decreased progression-free survival in PCa patients treated with radiotherapy. Conclusions: Our findings demonstrate that GLS-driven glutaminolysis is a prognostic biomarker and therapeutic target for PCa radiosensitization.

The current literature on stereotactic body radiotherapy (SBRT) for oligometastatic disease is characterized by small patient cohorts with heterogeneous primary tumors, metastases location and dose regimes. Hence, this study established a multi-institutional database of 700 patients treated with SBRT for pulmonary metastases to identify prognostic factors influencing survival and local control.All German radiotherapy departments were contacted and invited to participate in this analysis. A total number of 700 patients with medically inoperable lung metastases treated with SBRT in 20 centers between 1997 and 2014 were included in a database. Primary and metastatic tumor characteristics, treatment characteristics and follow-up data including survival, local control, distant metastases, and toxicity were evaluated. Lung metastases were treated with median PTV-encompassing single doses of 12.5Gy (range 3.0-33.0Gy) in a median number of 3 fractions (range 1-13).After a median follow-up time of 14.3 months, 2-year local control (LC) and overall survival (OS) were 81.2% and 54.4%, respectively. In multivariate analysis, OS was most significantly influenced by pretreatment performance status, maximum metastasis diameter, primary tumor histology, time interval between primary tumor diagnosis and SBRT treatment and number of metastases. For LC, independent prognostic factors were pretreatment performance status, biological effective dose (BED) at PTV isocenter (BEDISO) and single fraction (PTV-encompassing) dose in multivariate analysis. Radiation-induced pneumonitis grade 2 or higher was observed in 6.5% of patients. The only factor significantly influencing toxicity was BEDISO (p=0.006).SBRT for medically inoperable patients with pulmonary metastases achieved excellent local control and promising overall survival. Important prognostic factors were identified for selecting patients who might benefit most from this therapy approach.

To investigate the impact of HPV status in patients with locally advanced head and neck squamous cell carcinoma (HNSCC), who received surgery and cisplatin-based postoperative radiochemotherapy.For 221 patients with locally advanced squamous cell carcinoma of the hypopharynx, oropharynx or oral cavity treated at the 8 partner sites of the German Cancer Consortium, the impact of HPV DNA, p16 overexpression and p53 expression on outcome were retrospectively analysed. The primary endpoint was loco-regional tumour control; secondary endpoints were distant metastases and overall survival.In the total patient population, univariate analyses revealed a significant impact of HPV16 DNA positivity, p16 overexpression, p53 positivity and tumour site on loco-regional tumour control. Multivariate analysis stratified for tumour site showed that positive HPV 16 DNA status correlated with loco-regional tumour control in patients with oropharyngeal carcinoma (p=0.02) but not in the oral cavity carcinoma group. Multivariate evaluation of the secondary endpoints in the total population revealed a significant association of HPV16 DNA positivity with overall survival (p<0.01) but not with distant metastases.HPV16 DNA status appears to be a strong prognosticator of loco-regional tumour control after postoperative cisplatin-based radiochemotherapy of locally advanced oropharyngeal carcinoma and is now being explored in a prospective validation trial.

Objective To investigate the impact of the tumour volume, HPV status, cancer stem cell (CSC) marker expression and hypoxia gene signatures, as potential markers of radiobiological mechanisms of radioresistance, in a contemporary cohort of patients with locally advanced head and neck squamous cell carcinoma (HNSCC), who received primary radiochemotherapy (RCTx). Materials and Methods For 158 patients with locally advanced HNSCC of the oral cavity, oropharynx or hypopharynx who were treated at six DKTK partner sites, the impact of tumour volume, HPV DNA, p16 overexpression, p53 expression, CSC marker expression and hypoxia-associated gene signatures on outcome of primary RCTx was retrospectively analyzed. The primary endpoint of this study was loco-regional control (LRC). Results Univariate Cox regression revealed a significant impact of tumour volume, p16 overexpression, and SLC3A2 and CD44 protein expression on LRC. The tumour hypoxia classification showed a significant impact only for small tumours. In multivariate analyses an independent correlation of tumour volume, SLC3A2 expression, and the 15-gene hypoxia signature with LRC was identified (CD44 protein n/a because of no event in the CD44-negative group). Logistic modelling showed that inclusion of CD44 protein expression and p16 overexpression significantly improved the performance to predict LRC at 2 years compared to the model with tumour volume alone. Conclusions Tumour volume, HPV status, CSC marker expression and hypoxia gene signatures are potential prognostic biomarkers for patients with locally advanced HNSCC, who were treated by primary RCTx. The study also supports that the individual tumour volumes should generally be included in biomarker studies and that panels of biomarkers are superior to individual parameters.

Abstract Purpose: To investigate the impact of hypoxia-induced gene expression and cancer stem cell (CSC) marker expression on outcome of postoperative cisplatin-based radiochemotherapy (PORT-C) in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). Experimental Design: Expression of the CSC markers CD44, MET, and SLC3A2, and hypoxia gene signatures were analyzed in the resected primary tumors using RT-PCR and nanoString technology in a multicenter retrospective cohort of 195 patients. CD44 protein expression was further analyzed in tissue microarrays. Primary endpoint was locoregional tumor control. Results: Univariate analysis showed that hypoxia-induced gene expression was significantly associated with a high risk of locoregional recurrence using the 15-gene signature (P = 0.010) or the 26-gene signature (P = 0.002). In multivariate analyses, in patients with HPV16 DNA–negative but not with HPV16 DNA–positive tumors the effect of hypoxia-induced genes on locoregional control was apparent (15-gene signature: HR 4.54, P = 0.006; 26-gene signature: HR 10.27, P = 0.024). Furthermore, MET, SLC3A2, CD44, and CD44 protein showed an association with locoregional tumor control in multivariate analyses (MET: HR 3.71, P = 0.016; SLC3A2: HR 8.54, P = 0.037; CD44: HR 3.36, P = 0.054; CD44 protein n/a because of no event in the CD44-negative group) in the HPV16 DNA–negative subgroup. Conclusions: We have shown for the first time that high hypoxia-induced gene expression and high CSC marker expression levels correlate with tumor recurrence after PORT-C in patients with HPV16 DNA–negative HNSCC. After validation in a currently ongoing prospective trial, these parameters may help to further stratify patients for individualized treatment de-escalation or intensification strategies. Clin Cancer Res; 22(11); 2639–49. ©2016 AACR.

Background and purpose To evaluate whether local tumor control probability (TCP) in stereotactic body radiotherapy (SBRT) varies between lung metastases of different primary cancer sites and between primary non-small cell lung cancer (NSCLC) and secondary lung tumors. Materials and methods A retrospective multi-institutional (n = 22) database of 399 patients with stage I NSCLC and 397 patients with 525 lung metastases was analyzed. Irradiation doses were converted to biologically effective doses (BED). Logistic regression was used for local tumor control probability (TCP) modeling and the second-order bias corrected Akaike Information Criterion was used for model comparison. Results After median follow-up of 19 months and 16 months (n.s.), local tumor control was observed in 87.7% and 86.7% of the primary and secondary lung tumors (n.s.), respectively. A strong dose–response relationship was observed in the primary NSCLC and metastatic cohort but dose–response relationships were not significantly different: the TCD90 (dose to achieve 90% TCP; BED of maximum planning target volume dose) estimates were 176 Gy (151–223) and 160 Gy (123–237) (n.s.), respectively. The dose–response relationship was not influenced by the primary cancer site within the metastatic cohort. Conclusions Dose–response relationships for local tumor control in SBRT were not different between lung metastases of various primary cancer sites and between primary NSCLC and lung metastases.

We examined the prognostic role of PD-1+ and CD8+ tumor infiltrating lymphocytes (TILs), and PD-L1+ cells in patients with squamous cell carcinoma of the head and neck (SCCHN) treated with surgery and postoperative chemoradiotherapy (CRT). FFPE samples from 161 patients were immunohistochemically stained for PD-1, CD8 and PD-L1. The immune marker expression was correlated with clinicopathologic characteristics, and overall survival (OS), local progression-free survival (LPFS) and distant metastases free-survival (DMFS), also in the context of HPV16 DNA/p16 status. The median follow-up was 48 months (range: 4–100). The 2-year-OS was 84.1% for the entire cohort. High PD-1 and PD-L1 expression were more common in patients with positive HPV16 DNA (p < 0.001 and p = 0.008, respectively) and high infiltration by CD8+ TILs (p < 0.001 for both markers). High PD-L1 expression correlated with superior OS (p = 0.025), LPFS (p = 0.047) and DMFS (p = 0.048) in multivariable analysis, whereas no significance could be demonstrated for PD-1. Patients with CD8high/PD-L1high expression had favorable outcome (p < 0.001 for all endpoints) compared to other groups. We validated the superior OS data on CD8high/PD-L1high using the Cancer Genome Atlas TCGA dataset (n = 518; p = 0.032). High PD-L1 expression was a favorable prognostic marker in HPV16-negative but not HPV16-positive patients. In conclusion, HPV-positive tumors showed higher expression of immune markers. PD-L1 expression constitutes an independent prognostic marker in SCCHN patients post-adjuvant CRT. In conjunction with CD8 status, these data provide an important insight on the immune contexture of SCCHN and are directly relevant for future treatment stratification with PD-1/PD-L1 immune checkpoint inhibitors to complement CRT.

The aim of this analysis was to model the effect of local control (LC) on overall survival (OS) in patients treated with stereotactic body radiotherapy (SBRT) for liver or lung metastases from colorectal cancer.The analysis is based on pooled data from two retrospective SBRT databases for pulmonary and hepatic metastases from 27 centers from Germany and Switzerland. Only patients with metastases from colorectal cancer were considered to avoid histology as a confounding factor. An illness-death model was employed to model the relationship between LC and OS.Three hundred eighty-eight patients with 500 metastatic lesions (lung n = 209, liver n = 291) were included and analyzed. Median follow-up time for local recurrence assessment was 12.1 months. Ninety-nine patients with 112 lesions experienced local failure. Seventy-one of these patients died after local failure. Median survival time was 27.9 months in all patients and 25.4 months versus 30.6 months in patients with and without local failure after SBRT. The baseline risk of death after local failure exceeds the baseline risk of death without local failure at 10 months indicating better survival with LC.In CRC patients with lung or liver metastases, our findings suggest improved long-term OS by achieving metastatic disease control using SBRT in patients with a projected OS estimate of > 12 months.

Local ablative radiotherapy (aRT) of oligometastatic prostate cancer (PCa) is very promising and has become a focus of current clinical research.We hypothesize that aRT is safe and effective in gallium-68 prostate-specific membrane antigen targeted positron emission tomography (PSMA-PET)-staged oligometastatic PCa patients.A nonrandomized, prospective, investigator-initiated phase 2 trial recruited patients with oligometastatic PCa (five or fewer lymph node or osseous metastases) after local curative therapy, without significant comorbidity and androgen deprivation therapy (ADT), at two German centers from 2014 to 2018.All PSMA-PET-positive metastases were treated with aRT. No systemic therapy was initiated.The primary endpoint was treatment-related toxicity (grade ≥2) 24 mo after aRT. A one-sided single-sample test of proportions was planned to test whether the endpoint occurs in <15% of the patients. Key secondary endpoints were time to progression of prostate-specific antigen (PSA) and time to ADT, which were associated with potential prognostic factors by Cox regression.Of 72 patients, 63 received aRT (13% dropout rate). The median follow-up was 37.2 mo. No treatment-related grade ≥2 toxicity was observed 2 yr after treatment. The median time to PSA progression and time to ADT were 13.2 and 20.6 mo, respectively. Of the patients, 21.4% were free of PSA progression after 3 yr.It was observed that aRT is safe, and midterm PSA progression and ADT-free time were achieved in one of five patients. Randomized clinical trials are indicated to further evaluate the option of delaying ADT in selected patients.In this clinical trial, 63 patients with up to five metastases of prostate cancer without androgen deprivation therapy were included. We showed that local ablative radiotherapy is safe and that one in five patients had no recurrent prostate-specific antigen value after 3 yr. Local ablative radiotherapy might be an option to avoid systemic therapy in selected patients.

Despite clear differences in clinical presentation and outcome, squamous cell carcinomas of the head and neck (SCCHN) arising from human papilloma virus (HPV) infection or heavy tobacco/alcohol consumption are treated equally. Next-generation sequencing is expected to reveal novel targets for more individualised treatment.Tumour specimens from 208 patients with locally advanced squamous cell carcinoma of the hypopharynx, oropharynx or oral cavity, all uniformly treated with adjuvant cisplatin-based chemoradiation, were included. A customised panel covering 211 exons from 45 genes frequently altered in SCCHN was used for detection of non-synonymous point and frameshift mutations. Mutations were correlated with HPV status and treatment outcome.Mutational profiles and HPV status were successfully established for 179 cases. HPV- tumours showed an increased frequency of alterations in tumour suppressor genes compared to HPV+ cases (TP53 67% versus 4%, CDKN2A 18% versus 0%). Conversely, HPV+ carcinomas were enriched for activating mutations in driver genes compared to HPV- cases (PIK3CA 30% versus 12%, KRAS 6% versus 1%, and NRAS 4% versus 0%). Hotspot TP53 missense mutations in HPV- carcinomas correlated with an increased risk of locoregional recurrence (hazard ratio [HR] 4.3, 95% confidence interval [CI] 1.5-12.1, P=0.006) and death (HR 2.2, 95% CI 1.1-4.4, P=0.021). In HPV+ SCCHN, driver gene mutations were associated per trend with a higher risk of death (HR 3.9, 95% CI 0.7-21.1, P=0.11).Distinct mutation profiles in HPV- and HPV+ SCCHN identify subgroups with poor outcome after adjuvant chemoradiation. Mutant p53 and the phosphoinositide 3-kinase pathway were identified as potential druggable targets for subgroup-specific treatment optimisation.

BackgroundRadical local treatment of pulmonary metastases is practiced with increasing frequency due to acknowledgment and better understanding of oligo-metastatic disease. This study aimed to develop a nomogram predicting overall survival (OS) after stereotactic body radiotherapy (SBRT) for pulmonary metastases.Patients and methodsA multi-institutional database of 670 patients treated with SBRT for pulmonary metastases was used as training cohort. Cox regression analysis with bidirectional variable elimination was performed to identify factors to be included into the nomogram model to predict 2-year OS. The calibration rate of the nomogram was assessed by plotting the actual Kaplan–Meier 2-year OS against the nomogram predicted survival. The nomogram was externally validated using two separate monocentric databases of 145 and 92 patients treated with SBRT for pulmonary metastases.ResultsThe median follow up of the trainings cohort was 14.3 months, the 2-year and 5-year OS was 52.6% and 23.7%, respectively. Karnofsky performance index, type of the primary tumor, control of the primary tumor, maximum diameter of the largest treated metastasis and number of metastases (1 versus >1) were significant prognostic factors in the Cox model (all p < 0.05). The calculated concordance-index for the nomogram was 0.73 (concordance indexes of all prognostic factors between 0.54 and 0.6). Based on the nomogram the training cohort was divided into 4 groups and 2-year OS ranged between 24.2% and 76.1% (predicted OS between 30.2% and 78.4%). The nomogram discriminated between risk groups in the two validation cohorts (concordance index 0.68 and 0.67).ConclusionsA nomogram for prediction of OS after SBRT for pulmonary metastases was generated and externally validated. This tool might be helpful for interdisciplinary discussion and evaluation of local and systemic treatment options in the oligo-metastatic setting.Key messageA nomogram for prediction of overall survival after stereotactic body radiotherapy (SBRT) for pulmonary metastases was developed and externally validated. This tool might be helpful for interdisciplinary discussion and evaluation of local and systemic treatment options in the oligo-metastatic setting.

Abstract Purpose: The heavy chain of the CD98 protein (CD98hc) is encoded by the SLC3A2 gene. Together with the light subunit LAT1, CD98hc constitutes a heterodimeric transmembrane amino acid transporter. High SLC3A2 mRNA expression levels are associated with poor prognosis in patients with head and neck squamous cell carcinoma (HNSCC) treated with radiochemotherapy. Little is known regarding the CD98hc protein–mediated molecular mechanisms of tumor radioresistance. Experimental Design: CD98hc protein expression levels were correlated with corresponding tumor control dose 50 (TCD50) in HNSCC xenograft models. Expression levels of CD98hc and LAT1 in HNSCC cells were modulated by siRNA or CRISPR/Cas9 gene editing. HNSCC cell phenotypes were characterized by transcription profiling, plasma membrane proteomics, metabolic analysis, and signaling pathway activation. Expression levels of CD98hc and LAT1 proteins were examined by IHC analysis of tumor tissues from patients with locally advanced HNSCC treated with primary radiochemotherapy (RCTx). Primary endpoint was locoregional tumor control (LRC). Results: High expression levels of CD98hc resulted in an increase in mTOR pathway activation, amino acid metabolism, and DNA repair as well as downregulation of oxidative stress and autophagy. High expression levels of CD98hc and LAT1 proteins were significantly correlated and associated with an increase in radioresistance in HNSCC in vitro and in vivo models. High expression of both proteins identified a poor prognosis subgroup in patients with locally advanced HNSCC after RCTx. Conclusions: We found that CD98hc-associated signaling mechanisms play a central role in the regulation of HNSCC radioresistance and may be a promising target for tumor radiosensitization.

Classical robust optimization considers uncertainties in patient setup and particle range. However, anatomical changes occurring during the treatment are neglected. Our aim was to compare classical robust optimization (cRO) with anatomical robust optimization (aRO), to quantify the influence of anatomical variations during the treatment course, and to assess the need of adaptation.Planning CT and weekly control CTs (cCTs) from 20 head and neck patients were analysed. Three intensity-modulated proton therapy (IMPT) plans were compared: conventional PTV-based plan; cRO, using solely the planning CT, and aRO, including additionally the first 2 cCTs in the optimization. Weekly and total cumulative doses, considering anatomical variations during the treatment, were calculated and compared with the nominal plans.Nominal plans fulfilled clinical specifications for target coverage (D98% ≥95% of prescribed dose). The PTV-based and cRO approaches were not sufficient to account for anatomical changes during the treatment in 10 and 5 patients, respectively, resulting in the need of plan adaptation. With the aRO approach, in all except one patient the target coverage was conserved, and no adaptations were necessary.In 25% of the investigated cases, classical robust optimization is not sufficient to account for anatomical changes during the treatment. Adding additional information of random anatomical variations in the optimization improves plan robustness.

Combining stereotactic radiosurgery (SRS) and active systemic therapies (STs) achieved favourable survival outcomes in patients with melanoma brain metastases (MBMs) in retrospective analyses. However, several aspects of this treatment strategy remain poorly understood. We report on the overall survival (OS) of patients with MBM treated with a combination of radiotherapy (RT) and ST as well as the impact of the v-Raf murine sarcoma viral oncogene homolog B (BRAF)-V600 mutation (BRAFmut) status, types of RT and ST and their sequence.Data of 208 patients treated with SRS or whole brain radiation therapy (WBRT) and either immunotherapy (IT) or targeted therapy (TT) within a 6-week interval to RT were analysed retrospectively. OS was calculated from RT to death or last follow-up. Univariate and multivariate Cox proportional hazard analyses were performed to determine prognostic features associated with OS.The median follow-up was 7.3 months. 139 patients received IT, 67 received TT and 2 received IT and TT within 6 weeks to RT (WBRT 45%; SRS 55%). One-year Kaplan-Meier OS rates were 69%, 65%, 33% and 18% (P < .001) for SRS with IT, SRS with TT, WBRT with IT and WBRT with TT, respectively. Patients with a BRAFmut receiving IT combined with RT experienced higher OS rates (88%, 65%, 50% and 18%). TT following RT or started before and continued thereafter was associated with improved median OS compared with TT solely before RT (12.2 [95% confidence interval {CI} 9.3-15.1]; 9.8 [95% CI 6.9-12.6] versus 5.1 [95% CI 2.7-7.5]; P = .03).SRS and IT achieved the highest OS rates. A BRAFmut appears to be a favourable prognostic factor for OS. For the combination of RT and TT, the sequence appears to be crucial. Combinations of WBRT and ST achieved unprecedentedly high OS rates and warrant further studies.

Radiotherapy and surgery are curative treatment options for localized prostate cancer (PCa) with a 5-year survival rate of nearly 100%. Once PCa cells spread into distant organs, such as bone, the overall survival rate of patients drops dramatically. The metastatic cascade and organotropism of PCa cells are regulated by different cellular subtypes, organ microenvironment, and their interactions. This cross-talk leads to pre-metastatic niche formation that releases chemo-attractive factors enforcing the formation of distant metastasis. Biological characteristics of PCa metastasis impacting on metastatic sites, burden, and latency is of clinical relevance. Therefore, the implementation of modern hybrid imaging technologies into clinical routine increased the sensitivity to detect metastases at earlier stages. This enlarged the number of PCa patients diagnosed with a limited number of metastases, summarized as oligometastatic disease. These patients can be treated with androgen deprivation in combination with local-ablative radiotherapy or radiopharmaceuticals directed to metastatic sites. Unfortunately, the number of patients with disease recurrence is high due to the enormous heterogeneity within the oligometastatic patient population and the lack of available biomarkers with predictive potential for metastasis-directed radiotherapy. Another, so far unmet clinical need is the diagnosis of minimal residual disease before onset of clinical manifestation and/or early relapse after initial therapy. Here, monitoring of circulating and disseminating tumor cells in PCa patients during the course of radiotherapy may give us novel insight into how metastatic spread is influenced by radiotherapy and vice versa. In summary, this review critically compares current clinical concepts for metastatic PCa patients and discuss the implementation of recent preclinical findings improving our understanding of metastatic dissemination and radiotherapy resistance into standard of care.

Tumor cells frequently overexpress heat shock protein 70 (Hsp70) and present it on their cell surface, where it can be recognized by pre‐activated NK cells. In our retrospective study the expression of Hsp70 was determined in relation to tumor‐infiltrating CD56 + NK cells in formalin‐fixed paraffin embedded (FFPE) tumor specimens of patients with SCCHN ( N = 145) as potential indicators for survival and disease recurrence. All patients received radical surgery and postoperative cisplatin‐based radiochemotherapy (RCT). In general, Hsp70 expression was stronger, but with variable intensities, in tumor compared to normal tissues. Patients with high Hsp70 expressing tumors (scores 3–4) showed significantly decreased overall survival (OS; p = 0.008), local progression‐free survival (LPFS; p = 0.034) and distant metastases‐free survival (DMFS; p = 0.044), compared to those with low Hsp70 expression (scores 0–2), which remained significant after adjustment for relevant prognostic variables. The adverse prognostic value of a high Hsp70 expression for OS was also observed in patient cohorts with p16‐ ( p = 0.001), p53‐ ( p = 0.0003) and HPV16 DNA‐negative ( p = 0.001) tumors. The absence or low numbers of tumor‐infiltrating CD56 + NK cells also correlated with significantly decreased OS ( p = 0.0001), LPFS ( p = 0.0009) and DMFS ( p = 0.0001). A high Hsp70 expression and low numbers of tumor‐infiltrating NK cells have the highest negative predictive value ( p = 0.00004). In summary, a strong Hsp70 expression and low numbers of tumor‐infiltrating NK cells correlate with unfavorable outcome following surgery and RCT in patients with SCCHN, and thus serve as negative prognostic markers.

Abstract Purpose: The aim of this study was to identify and independently validate a novel gene signature predicting locoregional tumor control (LRC) for treatment individualization of patients with locally advanced HPV-negative head and neck squamous cell carcinomas (HNSCC) who are treated with postoperative radio(chemo)therapy (PORT-C). Experimental Design: Gene expression analyses were performed using NanoString technology on a multicenter training cohort of 130 patients and an independent validation cohort of 121 patients. The analyzed gene set was composed of genes with a previously reported association with radio(chemo)sensitivity or resistance to radio(chemo)therapy. Gene selection and model building were performed comparing several machine-learning algorithms. Results: We identified a 7-gene signature consisting of the three individual genes HILPDA, CD24, TCF3, and one metagene combining the highly correlated genes SERPINE1, INHBA, P4HA2, and ACTN1. The 7-gene signature was used, in combination with clinical parameters, to fit a multivariable Cox model to the training data (concordance index, ci = 0.82), which was successfully validated (ci = 0.71). The signature showed improved performance compared with clinical parameters alone (ci = 0.66) and with a previously published model including hypoxia-associated genes and cancer stem cell markers (ci = 0.65). It was used to stratify patients into groups with low and high risk of recurrence, leading to significant differences in LRC in training and validation (P &amp;lt; 0.001). Conclusions: We have identified and validated the first hypothesis-based gene signature for HPV-negative HNSCC treated by PORT-C including genes related to several radiobiological aspects. A prospective validation is planned in an ongoing prospective clinical trial before potential application in clinical trials for patient stratification. Clin Cancer Res; 24(6); 1364–74. ©2018 AACR.

<h3>Purpose</h3> Many technological and methodical advances have made stereotactic body radiotherapy (SBRT) more accurate and more efficient during the last years. This study aims to investigate whether experience in SBRT and technological innovations also translated into improved local control (LC) and overall survival (OS). <h3>Methods and Materials</h3> A database of 700 patients treated with SBRT for lung metastases in 20 German centers between 1997 and 2014 was used for analysis. It was the aim of this study to investigate the impact of fluorodeoxyglucose positron-emission tomography (FDG-PET) staging, biopsy confirmation, image guidance, immobilization, and dose calculation algorithm, as well as the influence of SBRT experience, on LC and OS. <h3>Results</h3> Median follow-up time was 14.3 months (range, 0-131.9 months), with 2-year LC and OS of 81.2% (95% confidence interval [CI] 75.8%-85.7%) and 54.4% (95% CI 50.2%-59.0%), respectively. In multivariate analysis, all treatment technologies except FDG-PET staging did not significantly influence outcome. Patients who received pre-SBRT FDG-PET staging showed superior 1- and 2-year OS of 82.7% (95% CI 77.4%-88.6%) and 64.8% (95% CI 57.5%-73.3%), compared with patients without FDG-PET staging resulting in 1- and 2-year OS rates of 72.8% (95% CI 67.4%-78.8%) and 52.6% (95% CI 46.0%-60.4%), respectively (<i>P</i>=.012). Experience with SBRT was identified as the main prognostic factor for LC: institutions with higher SBRT experience (patients treated with SBRT within the last 2 years of the inclusion period) showed superior LC compared with less-experienced centers (<i>P</i>≤.001). Experience with SBRT within the last 2 years was independent from known prognostic factors for LC. <h3>Conclusion</h3> Investigated technological and methodical advancements other than FDG-PET staging before SBRT did not significantly improve outcome in SBRT for pulmonary metastases. In contrast, LC was superior with increasing SBRT experience of the individual center.

Background Tumour mutational burden (TMB) estimated from whole exome sequencing or comprehensive gene panels has previously been established as predictive factor of response to immune checkpoint inhibitors (ICIs). Its predictive value for the efficacy of concurrent chemoradiation (cCRTX), a potential combination partner of ICI, remains unknown. Methods The accuracy of TMB estimation by an in-house 327-gene panel was established in the Cancer Genome Atlas (TCGA) head and neck squamous cell carcinoma (HNSCC) data set. Interference of TMB with outcome after cCRTX was determined in a multicentre cohort of patients with locally advanced HNSCC uniformly treated with cCRTX. Targeted next-generation sequencing was successfully applied in 101 formalin-fixed, paraffin-embedded pretreatment tumour samples. In a subset of cases (n = 40), tumour RNA was used for immune-related gene expression profiling by the nanoString platform. TMB was correlated with TP53 genotype, human papilloma virus (HPV) status, immune expression signatures and survival parameters. Results were validated in the TCGA HNSCC cohort. Results A high accuracy of TMB estimation by the 327-gene panel was established. High TMB was significantly associated with an increased prevalence of TP53 mutations and immune gene expression patterns unrelated to T cell–inflamed gene expression profiles. Kaplan-Meier analysis revealed significantly reduced overall survival in the patient group with high TMB (hazard ratio for death: 1.79, 95% confidence interval: 1.02–3.14; P = 0.042) which remained significant after correcting for confounding factors in the multivariate model. The prognostic value of TMB was confirmed in the TCGA HNSCC cohort. Conclusion High TMB identifies HNSCC patients with poor outcome after cCRTX who might preferentially benefit from CRTX-ICI combinations.

Renal cell carcinoma (RCC) is traditionally considered to be radioresistant. Radiotherapy response rates are believed to improve with hypofractionated, high dose stereotactic body radiotherapy (SBRT). However, limited data exist regarding the role of SBRT in the treatment of pulmonary metastases.The working group "Stereotactic Radiotherapy" of the German Society of Radiation Oncology analyzed its multi-institutional database of more than 700 patients who received SBRT for pulmonary metastases. Treatment was performed at 10 centers between 2001 and 2016. Patients with metastatic RCC were included in the study. Tumor characteristics, treatment details, and follow-up data including survival, local control (LC), distant metastases, and toxicity were evaluated.A total of 46 RCC patients treated with SBRT for 67 lung metastases were identified, who received a median total biologically effective dose (BEDiso) at planning target volume (PTV) isocenter of 117.0 Gy (range, 48.0-189.0 Gy). A median fractional dose of 20.8 Gy at isocenter (range, 6.0-37.9 Gy) was administered in a median number of 3 fractions (1-8 fractions). After a median follow-up time of 28.3 months for all patients, 1- and 3-year LC rates were 98.1% and 91.9%, with corresponding 1- and 3-year overall survival (OS) of 84.3% and 43.8%, respectively. Pulmonary metastases treated with BEDiso ≥130 Gy showed a trend for superior LC (P=0.054). OS was significantly improved in both uni- and multivariate analysis for patients with higher Karnofsky performance scale, lower maximum pulmonary metastasis diameter and lack of post-SBRT systemic therapy due to progression (P=0.014; P=0.049; P=0.006). Only mild acute and late toxicity was reported.SBRT for pulmonary metastases from RCC was associated with low treatment-associated toxicity, promising survival, and excellent LC, especially in those patients receiving a BEDiso ≥130 Gy.

In prostate cancer, disease progression after primary treatment and subsequent androgen deprivation therapy is common. Intensification of systemic treatment is the standard of care. Recently, 68Ga prostate-specific membrane antigen positron emission tomography (PSMA-PET) imaging was introduced to identify oligometastatic prostate cancer patients. In this retrospective, exploratory study, we report on the efficacy of PSMA-PET-guided local ablative radiotherapy (aRT) in 15 oligometastatic castration-resistant prostate cancer (CRPC) patients, selected from our prospective institutional database and treated between 2013 and 2016. After multidisciplinary discussion, aRT was delivered with two different schedules. Androgen deprivation therapy remained unchanged. Prostate-specific antigen (PSA) response and time to PSA progression were analysed. For comparison, individual time to PSA progression without aRT was estimated by individual PSA doubling time (PSADT). PSA response was observed in 11 patients (73%). Mean time to PSA progression or last follow-up was 17.9 mo, as opposed to 2.9 mo estimated from the PSADT without aRT (p < 0.001). A relevant subset of CRPC patients had a PSA response with aRT to PET-positive lead metastases. A prospective trial is in preparation. In selected patients with prostate-specific antigen (PSA) increase during androgen deprivation, metastases were detected with prostate-specific membrane antigen positron emission tomography imaging. Fifteen patients with three or fewer metastases were treated with high-dose radiotherapy. Subsequently, PSA values dropped in 11 patients and in six patients no PSA progression was detected for >12 mo.

Introduction This multicenter study aims to analyze outcome as well as early versus late patterns of recurrence following pulmonary stereotactic body radiotherapy (SBRT) for patients with oligometastatic non–small-cell lung cancer (NSCLC). Materials and Methods This analysis included 301 patients with oligometastatic NSCLC treated with SBRT for 336 lung metastases. Although treatment of the primary tumor consisted of surgical resection, radiochemotherapy, and/or systemic therapy, pulmonary oligometastases were treated with SBRT. Results The median follow-up time was 16.1 months, resulting in 2-year overall survival (OS), local control (LC), and distant control (DC) of 62.2%, 82.0%, and 45.2%, respectively. Multivariate analysis identified age (P = .019) and histologic subtype (P = .028), as well as number of metastatic organs (P < .001) as independent prognostic factors for OS. LC was superior for patients with favorable histologic subtype (P = .046) and SBRT with a higher biological effective dose at isocenter (P = .037), whereas DC was inferior for patients with metastases in multiple organs (P < .001) and female gender (P = .027). Early (within 24 months) local or distant progression was observed in 15.3% and 36.5% of the patients. After 24 months, the risk of late local failure was low, with 3- and 4-year local failure rates of only 4.0%, and 7.6%. In contrast, patients remained at a high risk of distant progression with 3- and 4-year failure rates of 13.3% and 24.1%, respectively, with no plateau observed. Conclusion SBRT for pulmonary oligometastatic NSCLC resulted in favorable LC and promising OS. The dominant failure pattern is distant with a continuously high risk of disease progression for many years.

Abstract For treatment individualisation of patients with locally advanced head and neck squamous cell carcinoma (HNSCC) treated with primary radiochemotherapy, we explored the capabilities of different deep learning approaches for predicting loco-regional tumour control (LRC) from treatment-planning computed tomography images. Based on multicentre cohorts for exploration (206 patients) and independent validation (85 patients), multiple deep learning strategies including training of 3D- and 2D-convolutional neural networks (CNN) from scratch, transfer learning and extraction of deep autoencoder features were assessed and compared to a clinical model. Analyses were based on Cox proportional hazards regression and model performances were assessed by the concordance index (C-index) and the model’s ability to stratify patients based on predicted hazards of LRC. Among all models, an ensemble of 3D-CNNs achieved the best performance (C-index 0.31) with a significant association to LRC on the independent validation cohort. It performed better than the clinical model including the tumour volume (C-index 0.39). Significant differences in LRC were observed between patient groups at low or high risk of tumour recurrence as predicted by the model ( $$p=0.001$$ <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow> <mml:mi>p</mml:mi> <mml:mo>=</mml:mo> <mml:mn>0.001</mml:mn> </mml:mrow> </mml:math> ). This 3D-CNN ensemble will be further evaluated in a currently ongoing prospective validation study once follow-up is complete.

Abstract Ablative radiotherapy is a highly efficient treatment modality for patients with metastatic prostate cancer (PCa). However, a subset of patients does not respond. Currently, this subgroup with bad prognosis cannot be identified before disease progression. We hypothesize that markers indicative of radioresistance, stemness and/or bone tropism may have a prognostic potential to identify patients profiting from metastases‐directed radiotherapy. Therefore, circulating tumor cells (CTCs) were analyzed in patients with metastatic PCa (n = 24) during radiotherapy with CellSearch, multicolor flow cytometry and imaging cytometry. Analysis of copy‐number alteration indicates a polyclonal CTC population that changes after radiotherapy. CTCs were found in 8 out of 24 patients (33.3%) and were associated with a shorter time to biochemical progression after radiotherapy. Whereas the total CTC count dropped after radiotherapy, a chemokine receptor CXCR4‐expressing subpopulation representing 28.6% of the total CTC population remained stable up to 3 months. At once, we observed higher chemokine CCL2 plasma concentrations and proinflammatory monocytes. Additional functional analyses demonstrated key roles of CXCR4 and CCL2 for cellular radiosensitivity, tumorigenicity and stem‐like potential in vitro and in vivo. Moreover, a high CXCR4 and CCL2 expression was found in bone metastasis biopsies of PCa patients. In summary, panCK + CXCR4 + CTCs may have a prognostic potential in patients with metastatic PCa treated with metastasis‐directed radiotherapy.

Background Stereotactic body radiotherapy (SBRT) for oligometastatic disease is characterized by an excellent safety profile; however, experiences are mostly based on treatment of one single metastasis. It was the aim of this study to evaluate safety and efficacy of SBRT for multiple pulmonary metastases. Patients and methods This study is based on a retrospective database of the DEGRO stereotactic working group, consisting of 637 patients with 858 treatments. Cox regression and logistic regression were used to analyze the association between the number of SBRT treatments or the number and the timing of repeat SBRT courses with overall survival (OS) and the risk of early death. Results Out of 637 patients, 145 patients were treated for multiple pulmonary metastases; 88 patients received all SBRT treatments within one month whereas 57 patients were treated with repeat SBRT separated by at least one month. Median OS for the total patient population was 23.5 months and OS was not significantly influenced by the overall number of SBRT treatments or the number and timing of repeat SBRT courses. The risk of early death within 3 and 6 months was not increased in patients treated with multiple SBRT treatments, and no grade 4 or grade 5 toxicity was observed in these patients. Conclusions In appropriately selected patients, synchronous SBRT for multiple pulmonary oligometastases and repeat SBRT may have a comparable safety and efficacy profile compared to SBRT for one single oligometastasis.

68Gallium prostate specific membrane antigen (PSMA) ligand positron emission tomography (PET) is an increasingly used imaging modality in prostate cancer, especially in cases of tumor recurrence after curative intended therapy. Owed to the novelty of the PSMA-targeting tracers, clinical evidence on the value of PSMA-PET is moderate but rapidly increasing. State of the art imaging is pivotal for radiotherapy treatment planning as it may affect dose prescription, target delineation and use of concomitant therapy.This review summarizes the evidence on PSMA-PET imaging from a radiation oncologist's point of view. Additionally a short survey containing twelve examples of patients and 6 additional questions was performed in seven mayor academic centers with experience in PSMA ligand imaging and the findings are reported here.

Abstract Background Metastasis directed treatment (MDT) is increasingly performed with the attempt to improve outcome in non-small cell lung cancer (NSCLC) patients receiving targeted- or immunotherapy (TT/IT). This study aimed to assess the safety and efficacy of metastasis directed stereotactic radiotherapy (SRT) concurrent to TT/IT in NSCLC patients. Methods A retrospective multicenter cohort of stage IV NSCLC patients treated with TT/IT and concurrent (≤ 30 days) MDT was established. 56% and 44% of patients were treated for oligoprogressive disease (OPD) or polyprogressive disease (PPD) under TT/IT, polyprogressive respectively. Survival was analyzed using Kaplan–Meier and log rank testing. Toxicity was scored using CTCAE v4.03 criteria. Predictive factors for overall survival (OS), progression free survival (PFS) and time to therapy switch (TTS) were analyzed with uni- and multivariate analysis. Results MDT of 192 lesions in 108 patients was performed between 07/2009 and 05/2018. Concurrent TT/IT consisted of EGFR/ALK-inhibitors (60%), immune checkpoint inhibitors (31%), VEGF-antibodies (8%) and PARP-inhibitors (1%). 2y-OS was 51% for OPD and 25% for PPD. After 1 year, 58% of OPD and 39% of PPD patients remained on the same TT/IT. Second progression after MDT was oligometastatic (≤ 5 lesions) in 59% of patients. Severe acute and late toxicity was observed in 5.5% and 1.9% of patients. In multivariate analysis, OS was influenced by the clinical metastatic status (p = 0.002, HR 2.03, 95% CI 1.30–3.17). PFS was better in patients receiving their first line of systemic treatment (p = 0.033, HR 1.7, 95% CI 1.05–2.77) and with only one metastases-affected organ (p = 0.023, HR 2.04, 95% CI 1.10–3.79). TTS was 6 months longer in patients with one metastases-affected organ (p = 0.031, HR 2.53, 95% CI 1.09–5.89). Death was never therapy-related. Conclusions Metastases-directed SRT in NSCLC patients can be safely performed concurrent to TT/IT with a low risk of severe toxicity. To find the ideal sequence of the available multidisciplinary treatment options for NSCLC and determine what patients will benefit most, a further evaluated in a broader context within prospective clinical trials is needed continuation of TT/IT beyond progression combined with MDT for progressive lesions appears promising but requires prospective evaluation. Trial registration : retrospectively registered

To validate the impact of HPV status, cancer stem cell (CSC) marker expression and tumour hypoxia status in patients with locally advanced head and neck squamous cell carcinoma (HNSCC), who received postoperative radiotherapy. The results of the exploration cohort have previously been reported by the German Cancer Consortium Radiation Oncology Group (DKTK-ROG; Lohaus et al., 2014; Linge et al., 2016).For 152 patients with locally advanced HNSCC the impact of HPV16 DNA status, CSC marker expression and hypoxia-associated gene signatures on outcome of postoperative radiotherapy were retrospectively analysed. Out of them, 40 patients received postoperative radiochemotherapy. Cox models presented in a previous study were validated using the concordance index as a performance measure. The primary endpoint of this study was loco-regional control. Results were compared to those previously reported by DKTK-ROG.Loco-regional control, freedom from distant metastases and overall survival were inferior to the previously reported cohort. Despite of this, the prognostic value of the combination of HPV infection status, CSC marker expression (SLC3A2) and tumour hypoxia status could be validated in univariate analyses using an independent validation cohort. For multivariate models, the concordance index was between 0.58 and 0.69 in validation, indicating a good prognostic performance of the models. The inclusion of CD44 and the 15-gene hypoxia signature moderately improved the performance compared to a baseline model without CSC markers or hypoxia classifiers.The HPV status, CSC marker expression of CD44 and SLC3A2 as well as hypoxia status are potential prognostic biomarkers for patients with locally advanced HNSCC treated by postoperative radiotherapy.

Proton beam therapy is promising for the treatment of head and neck cancer (HNC), but it is sensitive to uncertainties in patient positioning and particle range. Studies have shown that the planning target volume (PTV) concept may not be sufficient to ensure robustness of the target coverage. A few planning studies have considered irradiation of unilateral HNC targets with protons, but they have only taken into account the dose on the nominal plan, without considering anatomy changes occurring during the treatment course.Four pencil beam scanning (PBS) proton therapy plans were calculated for 8 HNC patients with unilateral target volumes: single-field (SFO) and multi-field optimized (MFO) plans, either using the PTV concept or clinical target volume (CTV)-based robust optimization. The dose was recalculated on computed tomography (CT) scans acquired during the treatment course. Doses to target volumes and organs at risk (OARs) were compared for the nominal plans, cumulative doses considering anatomical changes, and additional setup and range errors in each fraction. If required, the treatment plan was adapted, and the dose was compared with the non-adapted plan.All nominal plans fulfilled the clinical specifications for target coverage, but significantly higher doses on the ipsilateral parotid gland were found for both SFO approaches. MFO PTV-based plans had the lowest robustness against range and setup errors. During the treatment course, the influence of the anatomical variation on the dose has shown to be patient specific, mostly independent of the chosen planning approach. Nine plans in four patients required adaptation, which led to a significant improvement of the target coverage and a slight reduction in the OAR dose in comparison to the cumulative dose without adaptation.The use of robust MFO optimization is recommended for ensuring plan robustness and reduced doses in the ipsilateral parotid gland. Anatomical changes occurring during the treatment course might degrade the target coverage and increase the dose in the OARs, independent of the chosen planning approach. For some patients, a plan adaptation may be required.

Introduction Preclinical and clinical data suggest that the chemokine pathway governed by SDF-1 and CXCR4 contributes to a resistant phenotype. This retrospective biomarker study aims to explore the specific prognostic value of SDF-1 and CXCR4 expression in locally advanced head and neck squamous cell carcinomas (HNSCC) treated with primary radiochemotherapy (RT-CT). Material and methods Biopsies from 141 HNSCC tumours of the oral cavity, oropharynx and hypopharynx were evaluated for SDF-1 and CXCR4 expression by immunofluorescence. SDF-1 and CXCR4 expression was correlated with clinico-pathological characteristics and outcome after RT-CT. Results Patients with tumours exhibiting overexpression of intracellular SDF-1 and CXCR4 have a higher risk for loco-regional relapse and a worse overall survival after RT-CT (multivariate analysis, hazard ratio 2.33, CI [1.18–4.62], p = 0.02 and hazard ratio 2.02, CI [1.13–3.59], p = 0.02, respectively). Similar results were observed when only the subgroup of HPV DNA negative patients were analysed (hazard ratio 2.23 and 2.16, p = 0.02 and p = 0.01, respectively). Conclusions Our data support the importance of SDF-1 and CXCR4 expression for loco-regional control and overall survival in HNSCC after primary radiochemotherapy. Prospective multivariate validation and further studies into CXCR4 inhibition to overcome radiation resistance are warranted.

ObjectiveTo independently validate the impact of tumour volume, p16 status, cancer stem cell (CSC) marker expression and hypoxia-associated gene signatures as potential prognostic biomarkers for patients with locally advanced head and neck squamous cell carcinoma (HNSCC), who underwent primary radiotherapy or radiochemotherapy (RCTx). These markers have previously been reported in a study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG) (Linge et al., 2016).Materials and methodsIn this retrospective monocentric study, 92 patients with locally advanced HNSCC were included. Univariable and multivariable logistic regressions and Cox models presented in the study of the DKTK-ROG were validated using the area under the curve (AUC) and the concordance index (ci), respectively. The primary endpoint of this study was loco-regional tumour control (LRC) after primary RCTx.ResultsAlthough both cohorts significantly differed in the proportion of the tumour subsites, the parameters tumour volume, p16 status and N stage could be validated regarding LRC and overall survival (OS) using multivariable Cox regression (LRC ci: 0.59, OS ci: 0.63). These models were slightly improved by combination with the putative CSC marker CD44 (LRC ci: 0.61, OS ci: 0.69). The logistic regression model for 2-year LRC based on tumour volume, p16 status and CD44 protein was validated with an AUC of 0.64. The patient stratification based on hypoxia-associated gene signatures status was similar to the original study but without significant differences in LRC and OS.ConclusionsIn this validation study, the inclusion of the putative CSC marker CD44 slightly improved the prognostic performance of the baseline parameters tumour volume, p16 status and N stage. No improvement was observed when including expressions of the hypoxia-associated gene signatures. Prospective validation on a larger cohort is warranted to assess the clinical relevance of these markers.

Imaging features for radiomic analyses are commonly calculated from the entire gross tumour volume (GTV entire). However, tumours are biologically complex and the consideration of different tumour regions in radiomic models may lead to an improved outcome prediction. Therefore, we investigated the prognostic value of radiomic analyses based on different tumour sub-volumes using computed tomography imaging of patients with locally advanced head and neck squamous cell carcinoma. The GTV entire&nbsp;was cropped by different margins to define the rim and the corresponding core sub-volumes of the tumour. Subsequently, the best performing tumour rim sub-volume was extended into surrounding tissue with different margins. Radiomic risk models were developed and validated using a retrospective cohort consisting of 291 patients in one of the six Partner Sites of the German Cancer Consortium Radiation Oncology Group treated between 2005 and 2013. The validation concordance index (C-index) averaged over all applied learning algorithms and feature selection methods using the GTVentire achieved a moderate prognostic performance for loco-regional tumour control (C-index: 0.61 ± 0.04 (mean ± std)). The models based on the 5 mm tumour rim and on the 3 mm extended rim sub-volume showed higher median performances (C-index: 0.65 ± 0.02 and 0.64 ± 0.05, respectively), while models based on the corresponding tumour core volumes performed less (C-index: 0.59 ± 0.01). The difference in C-index between the 5 mm tumour rim and the corresponding core volume showed a statistical trend (p = 0.10). After additional prospective validation, the consideration of tumour sub-volumes may be a promising way to improve prognostic radiomic risk models.

Motion mitigation is of crucial importance in particle therapy (PT) of patients with abdominal tumors to ensure high-precision irradiation. Magnetic resonance imaging (MRI) is an excellent modality for target volume delineation and motion estimation of mobile soft-tissue tumors. Thus, the aims of this study were to develop an MRI- and PT-compatible abdominal compression device, to investigate its effect on pancreas motion reduction, and to evaluate patient tolerability and acceptance.In a prospective clinical study, 16 patients with abdominal tumors received an individualized polyethylene-based abdominal corset. Pancreas motion was analyzed using time- and phase resolved MRI scans (orthogonal 2D-cine and 4D MRI) with and without compression by the corset. The pancreas was manually segmented in each MRI data set and the population-averaged center-of-mass motion in inferior-superior (IS), anterior-posterior (AP) and left-right (LR) directions was determined. A questionnaire was developed to investigate the level of patient acceptance of the corset, which the patients completed after acquisition of the planning computed tomography (CT) and MRI scans.The corset was found to reduce pancreas motion predominantly in IS direction by on average 47 % - 51 % as found in the 2D-cine and 4D MRI data, respectively, while motion in the AP and LR direction was not significantly reduced. Most patients reported no discomfort when wearing the corset.An MRI- and PT-compatible individualized abdominal corset was presented, which substantially reduced breathing-induced pancreas motion and can be safely applied with no additional discomfort for the patients. The corset has been successfully integrated into our in-house clinical workflow for PT of tumors of the upper abdomen.

Tumor hypoxia is a paradigmatic negative prognosticator of treatment resistance in head and neck squamous cell carcinoma (HNSCC). The lack of robust and reliable hypoxia classifiers limits the adaptation of stratified therapies. We hypothesized that the tumor DNA methylation landscape might indicate epigenetic reprogramming induced by chronic intratumoral hypoxia.A DNA-methylome-based tumor hypoxia classifier (Hypoxia-M) was trained in the TCGA (The Cancer Genome Atlas)-HNSCC cohort based on matched assignments using gene expression-based signatures of hypoxia (Hypoxia-GES). Hypoxia-M was validated in a multicenter DKTK-ROG trial consisting of human papillomavirus (HPV)-negative patients with HNSCC treated with primary radiochemotherapy (RCHT).Although hypoxia-GES failed to stratify patients in the DKTK-ROG, Hypoxia-M was independently prognostic for local recurrence (HR, 4.3; P = 0.001) and overall survival (HR, 2.34; P = 0.03) but not distant metastasis after RCHT in both cohorts. Hypoxia-M status was inversely associated with CD8 T-cell infiltration in both cohorts. Hypoxia-M was further prognostic in the TCGA-PanCancer cohort (HR, 1.83; P = 0.04), underscoring the breadth of this classifier for predicting tumor hypoxia status.Our findings highlight an unexplored avenue for DNA methylation-based classifiers as biomarkers of tumoral hypoxia for identifying high-risk features in patients with HNSCC tumors. See related commentary by Heft Neal and Brenner, p. 2954.

<h3>Purpose</h3> Most radiobiological models for prediction of tumor control probability (TCP) do not account for the fact that many events could remain unobserved because of censoring. We therefore evaluated a set of TCP models that take into account this censoring. <h3>Methods and Materials</h3> We applied 2 fundamental Bayesian cure rate models to a sample of 770 pulmonary metastasis treated with stereotactic body radiation therapy at German, Austrian, and Swiss institutions: (<i>1</i>) the model developed by Chen, Ibrahim and Sinha (the CIS99 model); and (<i>2</i>) a mixture model similar to the classic model of Berkson and Gage (the BG model). In the CIS99 model the number of clonogens surviving the radiation treatment follows a Poisson distribution, whereas in the BG model only 1 dominant recurrence-competent tissue mass may remain. The dose delivered to the isocenter, tumor size and location, sex, age, and pretreatment chemotherapy were used as covariates for regression. <h3>Results</h3> Mean follow-up time was 15.5 months (range: 0.1-125). Tumor recurrence occurred in 11.6% of the metastases. Delivered dose, female sex, peripheral tumor location and having received no chemotherapy before RT were associated with higher TCP in all models. Parameter estimates of the CIS99 were consistent with the classical Cox proportional hazards model. The dose required to achieve 90% tumor control after 15.5 months was 146 (range: 114-188) Gy₁₀ in the CIS99 and 133 (range: 101-164) Gy₁₀ in the BG model; however, the BG model predicted lower tumor control at long (<mml:math><mml:mrow><mml:mi>≳</mml:mi><mml:mn>20</mml:mn></mml:mrow></mml:math> months) follow-up times and gave a suboptimal fit to the data compared to the CIS99 model. <h3>Conclusions</h3> Biologically motivated cure rate models allow adding the time component into TCP modeling without being restricted to the follow-up period which is the case for the Cox model. In practice, application of such models to the clinical setting could allow for adaption of treatment doses depending on whether local control should be achieved in the short or longer term.

To compare six HPV detection methods in pre-treatment FFPE tumour samples from patients with locally advanced head and neck squamous cell carcinoma (HNSCC) who received postoperative (N = 175) or primary (N = 90) radiochemotherapy.HPV analyses included detection of (i) HPV16 E6/E7 RNA, (ii) HPV16 DNA (PCR-based arrays, A-PCR), (iii) HPV DNA (GP5+/GP6+ qPCR, (GP-PCR)), (iv) p16 (immunohistochemistry, p16 IHC), (v) combining p16 IHC and the A-PCR result and (vi) combining p16 IHC and the GP-PCR result. Differences between HPV positive and negative subgroups were evaluated for the primary endpoint loco-regional control (LRC) using Cox regression.Correlation between the HPV detection methods was high (chi-squared test, p < 0.001). While p16 IHC analysis resulted in several false positive classifications, A-PCR, GP-PCR and the combination of p16 IHC and A-PCR or GP-PCR led to results comparable to RNA analysis. In both cohorts, Cox regression analyses revealed significantly prolonged LRC for patients with HPV positive tumours irrespective of the detection method.The most stringent classification was obtained by detection of HPV16 RNA, or combining p16 IHC with A-PCR or GP-PCR. This approach revealed the lowest rate of recurrence in patients with tumours classified as HPV positive and therefore appears most suited for patient stratification in HPV-based clinical studies.

Progression of prostate-specific antigen (PSA) values after curative treatment of prostate cancer patients is common. Prostate-specific membrane antigen (PSMA-) PET imaging can identify patients with metachronous oligometastatic disease even at low PSA levels. Metastases-directed local ablative radiotherapy (aRT) has been shown to be a safe treatment option. In this prospective clinical trial, we evaluated local control and the pattern of tumor progression. Between 2014 and 2018, 63 patients received aRT of 89 metastases (MET) (68 lymph node (LN-)MET and 21 bony (OSS-)MET) with one of two radiation treatment schedules: 50 Gy in 2 Gy fractions in 34 MET or 30 Gy in 10 Gy fractions in 55 MET. The mean gross tumor volume and planning target volume were 2.2 and 14.9 mL, respectively. The median follow-up time was 40.7 months. Local progression occurred in seven MET, resulting in a local control rate of 93.5% after three years. Neither treatment schedule, target volume, nor type of lesion was associated with local progression. Regional progression in the proximity to the LN-MET was observed in 19 of 47 patients with at least one LN-MET (actuarial 59.3% free of regional progression after 3 years). In 33 patients (52%), a distant progression was reported. The median time to first tumor-related clinical event was 16.6 months, and 22.2% of patients had no tumor-related clinical event after three years. A total of 14 patients (22%) had another aRT. In conclusion, local ablative radiotherapy in patients with PSMA-PET staged oligometastatic prostate cancer may achieve local control, but regional or distant progression is common. Further studies are warranted, e.g., to define the optimal target volume coverage in LN-MET and OSS-MET.

Patients with locally advanced head and neck squamous cell carcinoma (HNSCC) may benefit from personalised treatment, requiring biomarkers that characterize the tumour and predict treatment response. We integrate pre-treatment CT radiomics and whole-transcriptome data from a multicentre retrospective cohort of 206 patients with locally advanced HNSCC treated with primary radiochemotherapy to classify tumour molecular subtypes based on radiomics, develop surrogate radiomics signatures for gene-based signatures related to different biological tumour characteristics and evaluate the potential of combining radiomics features with full-transcriptome data for the prediction of loco-regional control (LRC). Using end-to-end machine-learning, we developed and validated a model to classify tumours of the atypical subtype (AUC [95% confidence interval] 0.69 [0.53-0.83]) based on CT imaging, observed that CT-based radiomics models have limited value as surrogates for six selected gene signatures (AUC < 0.60), and showed that combining a radiomics signature with a transcriptomics signature consisting of two metagenes representing the hedgehog pathway and E2F transcriptional targets improves the prognostic value for LRC compared to both individual sources (validation C-index [95% confidence interval], combined: 0.63 [0.55-0.73] vs radiomics: 0.60 [0.50-0.71] and transcriptomics: 0.59 [0.49-0.69]). These results underline the potential of multi-omics analyses to generate reliable biomarkers for future application in personalized oncology.

The aim of this study was to evaluate interobserver agreement (IOA) on target volume definition for pancreatic cancer (PACA) within the Radiosurgery and Stereotactic Radiotherapy Working Group of the German Society of Radiation Oncology (DEGRO) and to identify the influence of imaging modalities on the definition of the target volumes.Two cases of locally advanced PACA and one local recurrence were selected from a large SBRT database. Delineation was based on either a planning 4D CT with or without (w/wo) IV contrast, w/wo PET/CT, and w/wo diagnostic MRI. Novel compared to other studies, a combination of four metrics was used to integrate several aspects of target volume segmentation: the Dice coefficient (DSC), the Hausdorff distance (HD), the probabilistic distance (PBD), and the volumetric similarity (VS).For all three GTVs, the median DSC was 0.75 (range 0.17-0.95), the median HD 15 (range 3.22-67.11) mm, the median PBD 0.33 (range 0.06-4.86), and the median VS was 0.88 (range 0.31-1). For ITVs and PTVs the results were similar. When comparing the imaging modalities for delineation, the best agreement for the GTV was achieved using PET/CT, and for the ITV and PTV using 4D PET/CT, in treatment position with abdominal compression.Overall, there was good GTV agreement (DSC). Combined metrics appeared to allow a more valid detection of interobserver variation. For SBRT, either 4D PET/CT or 3D PET/CT in treatment position with abdominal compression leads to better agreement and should be considered as a very useful imaging modality for the definition of treatment volumes in pancreatic SBRT. Contouring does not appear to be the weakest link in the treatment planning chain of SBRT for PACA.

To assess the robustness of prognostic biomarkers and molecular tumour subtypes developed for patients with head and neck squamous cell carcinoma (HNSCC) on cell-line derived HNSCC xenograft models, and to develop a novel biomarker signature by combining xenograft and patient datasets.Mice bearing xenografts (n = 59) of ten HNSCC cell lines and a retrospective, multicentre patient cohort (n = 242) of the German Cancer Consortium-Radiation Oncology Group (DKTK-ROG) were included. All patients received postoperative radiochemotherapy (PORT-C). Gene expression analysis was conducted using GeneChip Human Transcriptome Arrays. Xenografts were stratified based on their molecular subtypes and previously established gene classifiers. The dose to control 50 % of tumours (TCD50) was compared between these groups. Using differential gene expression analyses combining xenograft and patient data, a gene signature was developed to define risk groups for the primary endpoint loco-regional control (LRC).Tumours of mesenchymal subtype were characterized by a higher TCD50 (xenografts, p < 0.001) and lower LRC (patients, p < 0.001) compared to the other subtypes. Similar to previously published patient data, hypoxia- and radioresistance-related gene signatures were associated with high TCD50 values. A 2-gene signature (FN1, SERPINE1) was developed that was prognostic for TCD50 (xenografts, p < 0.001) and for patient outcome in independent validation (LRC: p = 0.007).Genetic prognosticators of outcome for patients after PORT-C and subcutaneous xenografts after primary clinically relevant irradiation show similarity. The identified robust 2-gene signature may help to guide patient stratification, after prospective validation. Thus, xenografts remain a valuable resource for translational research towards the development of individualized radiotherapy.

<h2>Abstract</h2><h3>Introduction</h3> Outcome after postoperative radiochemotherapy (RT-CT) for patients with advanced head and neck squamous cell carcinomas (HNSCC) remains unsatisfactory, especially among those with HPV negative tumours. Therefore, new biomarkers are needed to further define subgroups for individualised therapeutic approaches. Preclinical and first clinical observations showed that the chemokine receptor CXCR4 and its ligand SDF-1 (CXCL12) play an important role in tumour cell proliferation, survival, cancer progression, metastasis and treatment resistance. However, the data on the prognostic value of SDF-1/CXCR4 expression for HNSCC are conflicting. The aim of our hypothesis-generating study was to retrospectively explore the prognostic potential of SDF-1/CXCR4 in a well-defined cohort of HNSCC patients collected within the multicenter biomarker study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG). <h3>Material and methods</h3> Patients with stage III and IVA HNSCC of the oral cavity, oropharynx and hypopharynx were treated with resection and adjuvant radiotherapy (RT) with ≥60Gy and concurrent cisplatin-based chemotherapy (CT). Tissue micro-arrays (TMAs) from a total of 221 patients were generated from surgical specimens, 201 evaluated for the SDF-1 and CXCR4 expression by immunofluorescence and correlated with clinico-pathological and outcome data. <h3>Results</h3> In univariate and multivariate analyses intracellular SDF-1 expression was associated with lower loco-regional control (LRC) in the entire patient group as well as in the HPV16 DNA negative subgroup. CXCR4 expression showed a trend for lower LRC in the univariate analysis which was not confirmed in the multivariate analysis. Neither for SDF-1 nor CXCR4 expression associations with distant metastasis free or overall survival were found. <h3>Conclusions</h3> Our exploratory data support the hypothesis that overexpression of intracellular SDF-1 is an independent negative prognostic biomarker for LRC after postoperative RT-CT in high-risk HNSCC. Prospective validation is warranted and further exploration of SDF-1/CXCR4 as a potential therapeutic target to overcome treatment resistance in HNSCC appears promising.

The increasing use of targeted therapy (TT) has resulted in prolonged disease control and survival in many metastatic cancers. In parallel, stereotactic radiotherapy (SRT) is increasingly performed in patients receiving TT to obtain a durable control of resistant metastases, and thereby to prolong the time to disseminated disease progression and switch of systemic therapy. The aims of this study were to analyze the safety and efficacy of SRT combined with TT in metastatic cancer patients and to assess the influence of continuous vs. interrupted TT during metastasis-directed SRT. The data of 454 SRTs in 158 patients from the international multicenter database (TOaSTT) on metastatic cancer patients treated with SRT and concurrent TT (within 30 days) were analyzed using Kaplan-Meier and log rank testing. Toxicity was defined by the CTCAE v4.03 criteria. The median FU was 19.9 mo (range 1-102 mo); 1y OS, PFS and LC were 59%, 24% and 84%, respectively. Median TTS was 25.5 mo (95% CI 11-40). TT was started before SRT in 77% of patients. TT was interrupted during SRT in 44% of patients, with a median interruption of 7 (range 1-42) days. There was no significant difference in OS or PFS whether TT was temporarily interrupted during SRT or not. Any-grade acute and late SRT-related toxicity occurred in 63 (40%) and 52 (33%) patients, respectively. The highest toxicity rates were observed for the combination of SRT and EGFRi or BRAF/MEKi, and any-grade toxicity was significantly increased when EGFRi (p = 0.016) or BRAF/MEKi (p = 0.009) were continued during SRT. Severe (≥grade 3) acute and late SRT-related toxicity were observed in 5 (3%) and 7 (4%) patients, respectively, most frequently in patients treated with EGFRi or BRAF/MEKi and in the intracranial cohort. There was no significant difference in severe toxicity whether TT was interrupted before and after SRT or not. In conclusion, SRT and continuous vs. interrupted TT in metastatic cancer patients did not influence OS or PFS. Overall, severe toxicity of combined treatment was rare; a potentially increased toxicity after SRT and continuous treatment with EGFR inhibitors or BRAF(±MEK) inhibitors requires further evaluation.

PurposeTo develop and validate a CT-based radiomics signature for the prognosis of loco-regional tumour control (LRC) in patients with locally advanced head and neck squamous cell carcinoma (HNSCC) treated by primary radiochemotherapy (RCTx) based on retrospective data from 6 partner sites of the German Cancer Consortium - Radiation Oncology Group (DKTK-ROG).Material and methodsPre-treatment CT images of 318 patients with locally advanced HNSCC were collected. Four-hundred forty-six features were extracted from each primary tumour volume and then filtered through stability analysis and clustering. First, a baseline signature was developed from demographic and tumour-associated clinical parameters. This signature was then supplemented by CT imaging features. A final signature was derived using repeated 3-fold cross-validation on the discovery cohort. Performance in external validation was assessed by the concordance index (C-Index). Furthermore, calibration and patient stratification in groups with low and high risk for loco-regional recurrence were analysed.ResultsFor the clinical baseline signature, only the primary tumour volume was selected. The final signature combined the tumour volume with two independent radiomics features. It achieved moderately good discriminatory performance (C-Index [95% confidence interval]: 0.66 [0.55–0.75]) on the validation cohort along with significant patient stratification (p = 0.005) and good calibration.ConclusionWe identified and validated a clinical-radiomics signature for LRC of locally advanced HNSCC using a multi-centric retrospective dataset. Prospective validation will be performed on the primary cohort of the HNprädBio trial of the DKTK-ROG once follow-up is completed.

Radiochemotherapy (RCT) has been shown to induce changes in immune cell homeostasis which might affect antitumor immune responses. In the present study, we aimed to compare the composition and kinetics of major lymphocyte subsets in the periphery of patients with non-locoregional recurrent (n = 23) and locoregional recurrent (n = 9) squamous cell carcinoma of the head and neck (SCCHN) upon primary RCT.EDTA-blood of non-locoregional recurrent SCCHN patients was collected before (t0), after application of 20-30 Gy (t1), in the follow-up period 3 (t2) and 6 months (t3) after RCT. In patients with locoregional recurrence blood samples were taken at t0, t1, t2 and at the time of recurrence (t5). EDTA-blood of age-related, healthy volunteers (n = 22) served as a control (Ctrl). Major lymphocyte subpopulations were phenotyped by multiparameter flow cytometry.Patients with non-recurrent SCCHN had significantly lower proportions of CD19+ B cells compared to healthy individuals before start of any therapy (t0) that dropped further until 3 months after RCT (t2), but reached initial levels 6 months after RCT (t3). The proportion of CD3+ T and CD3+/CD4+ T helper cells continuously decreased between t0 and t3, whereas that of CD8+ cytotoxic T cells and CD3+/CD56+ NK-like T cells (NKT) gradually increased in the same period of time in non-recurrent patients. The percentage of CD4+/CD25+/FoxP3+ regulatory T cells (Tregs) decreased directly after RCT, but increased above initial levels in the follow-up period 3 (t2) and 6 (t3) months after RCT. Patients with locoregional recurrence showed similar trends with respect to B, T cells and Tregs between t0 and t5. CD4+ T helper cells remained stably low between t0 and t5 in patients with locoregional recurrence compared to Ctrl. NKT/NK cell subsets (CD56+/CD69+, CD3-/CD56+, CD3-/CD94+, CD3-/NKG2D+, CD3-/NKp30+, CD3-/NKp46+) increased continuously up to 6 months after RCT (t0-t3) in patients without locoregional recurrence, whereas in patients with locoregional recurrence, these subsets remained stably low until time of recurrence (t5).Monitoring the kinetics of lymphocyte subpopulations especially activatory NK cells before and after RCT might provide a clue with respect to the development of an early locoregional recurrence in patients with SCCHN. However, studies with larger patient cohorts are needed.Observational Study on Biomarkers in Head and Neck Cancer (HNprädBio), NCT02059668. Registered on 11 February 2014, https://clinicaltrials.gov/ct2/show/NCT02059668 .

To assess the relation of the previously reported classification of molecular subtypes to the outcome of patients with HNSCC treated with postoperative radio(chemo)therapy (PORT-C), and to assess the association of these subtypes with gene expressions reflecting known mechanisms of radioresistance.Gene expression analyses were performed using the GeneChip Human Transcriptome Array 2.0 on a multicentre retrospective patient cohort (N = 128) of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG) with locally advanced HNSCC treated with PORT-C. Tumours were assigned to four molecular subtypes, and correlation analyses between subtypes and clinical risk factors were performed. In addition, the classifications of eight genes or gene signatures related to mechanisms of radioresistance, which have previously shown an association with outcome of patients with HNSCC, were compared between the molecular subtypes. The endpoints loco-regional control (LRC) and overall survival (OS) were evaluated by log-rank tests and Cox regression.Tumours were classified into the four subtypes basal (19.5%), mesenchymal (18.8%), atypical (15.6%) and classical (14.1%). The remaining tumours could not be classified (32.0%). Tumours of the mesenchymal subtype showed a lower LRC compared to the other subtypes (p = 0.012). These tumours were associated with increased epithelial-mesenchymal transition (EMT) and overexpression of a gene signature enriched in DNA repair genes. The majority of the eight considered gene classifiers were significantly associated to LRC or OS in the whole cohort.Molecular subtypes, previously identified on HNSCC patients treated with primary radio(chemo)therapy or surgery, were related to LRC for patients treated with PORT-C, where mesenchymal tumours presented with worse prognosis. After prospective validation, subtype-based patient stratification, potentially in combination with other molecular classifiers, may be considered in future interventional studies in the context of personalised radiotherapy and may guide the development of combined treatment approaches.

To develop prognostic biomarker signatures for patients with locally advanced head and neck squamous cell carcinoma (HNSCC) treated by primary radiochemotherapy (RCTx) based on previously published molecular analyses of the retrospective biomarker study of the German Cancer Consortium - Radiation Oncology Group (DKTK-ROG).In previous studies on the retrospective DKTK-ROG HNSCC cohort treated with primary RCTx, the following clinical parameters and biomarkers were evaluated and found to be significantly associated with loco-regional tumour control (LRC) or overall survival (OS): tumour volume, p16 status, expression of cancer stem cell markers CD44 and SLC3A2, expressions of hypoxia-associated gene signatures, tumour mutational burden (TMB), single nucleotide polymorphisms (SNPs) in the ERCC2 gene (rs1799793, rs13181) and ERCC5 gene (rs17655) as well as the expression of CXCR4, SDF-1 and CD8. These biomarkers were combined in multivariable modelling using Cox-regression with backward variable selection.A baseline signature containing the widely accepted parameters tumour volume, p16 status, cancer stem cell marker expression (CD44), and hypoxia-associated gene expression has been defined, representing the main hypothesis of the study. Furthermore, the baseline signature was extended by additional prognostic biomarkers and a data-driven signature without any pre-hypothesis was generated for both endpoints. In these signatures, the SNPs rs1799793 and rs17655 as well as CXCR4, SDF-1 and SLC3A2 expression were additionally included. The signatures showed significant patient stratifications for LRC and OS.Three biomarker signatures were defined for patients with locally advanced HNSCC treated with primary RCTx for the endpoints LRC and OS. These signatures will be validated in the prospective HNprädBio study of the DKTK-ROG that recently completed recruitment, before potential application in an interventional trial.

The aim of this study was to develop and validate a novel gene signature from full-transcriptome data using machine-learning approaches to predict loco-regional control (LRC) of patients with human papilloma virus (HPV)-negative locally advanced head and neck squamous cell carcinoma (HNSCC), who received postoperative radio(chemo)therapy (PORT-C).Gene expression analysis was performed using Affymetrix GeneChip Human Transcriptome Array 2.0 on a multicentre retrospective training cohort of 128 patients and an independent validation cohort of 114 patients from the German Cancer Consortium - Radiation Oncology Group (DKTK-ROG). Genes were filtered based on differential gene expression analyses and Cox regression. The identified gene signature was combined with clinical parameters and with previously identified genes related to stem cells and hypoxia. Technical validation was performed using nanoString technology.We identified a 6-gene signature consisting of four individual genes CAV1, GPX8, IGLV3-25, TGFBI, and one metagene combining the highly correlated genes INHBA and SERPINE1. This signature was prognostic for LRC on the training data (ci = 0.84) and in validation (ci = 0.63) with a significant patient stratification into two risk groups (p = 0.005). Combining the 6-gene signature with the clinical parameters T stage and tumour localisation as well as the cancer stem cell marker CD44 and the 15-gene hypoxia-associated signature improved the validation performance (ci = 0.69, p = 0.001).We have developed and validated a novel prognostic 6-gene signature for LRC of HNSCC patients with HPV-negative tumours treated by PORT-C. After successful prospective validation the signature can be part of clinical trials on the individualization of radiotherapy.

Rating both a lung segmentation algorithm and a deformable image registration (DIR) algorithm for subsequent lung computed tomography (CT) images by different evaluation techniques. Furthermore, investigating the relative performance and the correlation of the different evaluation techniques to address their potential value in a clinical setting.Two to seven subsequent CT images (69 in total) of 15 lung cancer patients were acquired prior, during, and after radiochemotherapy. Automated lung segmentations were compared to manually adapted contours. DIR between the first and all following CT images was performed with a fast algorithm specialized for lung tissue registration, requiring the lung segmentation as input. DIR results were evaluated based on landmark distances, lung contour metrics, and vector field inconsistencies in different subvolumes defined by eroding the lung contour. Correlations between the results from the three methods were evaluated.Automated lung contour segmentation was satisfactory in 18 cases (26%), failed in 6 cases (9%), and required manual correction in 45 cases (66%). Initial and corrected contours had large overlap but showed strong local deviations. Landmark-based DIR evaluation revealed high accuracy compared to CT resolution with an average error of 2.9 mm. Contour metrics of deformed contours were largely satisfactory. The median vector length of inconsistency vector fields was 0.9 mm in the lung volume and slightly smaller for the eroded volumes. There was no clear correlation between the three evaluation approaches.Automatic lung segmentation remains challenging but can assist the manual delineation process. Proven by three techniques, the inspected DIR algorithm delivers reliable results for the lung CT data sets acquired at different time points. Clinical application of DIR demands a fast DIR evaluation to identify unacceptable results, for instance, by combining different automated DIR evaluation methods.

This article compares the expression and applicability of biomarkers, from single genes and gene signatures, identified in patients with locally advanced head and neck squamous cell carcinoma using the GeneChip Human Transcriptome Array 2.0, nCounter, and real-time PCR analyses. Two multicenter, retrospective cohorts of patients with head and neck squamous cell carcinoma from the German Cancer Consortium Radiation Oncology Group who received postoperative radiochemotherapy or primary radiochemotherapy were considered. Real-time PCR was performed for a limited number of 38 genes of the cohort who received postoperative radiochemotherapy only. Correlations between the methods were evaluated by the Spearman rank correlation coefficient. Patients were stratified based on the expression of putative cancer stem cell markers, hypoxia-associated gene signatures, and a previously developed seven-gene signature. Locoregional tumor control was compared between these patient subgroups using log-rank tests. Gene expressions obtained from nCounter analyses were moderately correlated to GeneChip analyses (median ρ = approximately 0.68). A higher correlation was obtained between nCounter analyses and real-time PCR (median ρ = 0.84). Significant associations with locoregional tumor control were observed for most of the considered biomarkers evaluated by GeneChip and nCounter analyses. In general, all applied biomarkers (single genes and gene signatures) classified approximately 70% to 85% of the patients similarly. Overall, gene signatures seem to be more robust and had a better transferability among different measurement methods. This article compares the expression and applicability of biomarkers, from single genes and gene signatures, identified in patients with locally advanced head and neck squamous cell carcinoma using the GeneChip Human Transcriptome Array 2.0, nCounter, and real-time PCR analyses. Two multicenter, retrospective cohorts of patients with head and neck squamous cell carcinoma from the German Cancer Consortium Radiation Oncology Group who received postoperative radiochemotherapy or primary radiochemotherapy were considered. Real-time PCR was performed for a limited number of 38 genes of the cohort who received postoperative radiochemotherapy only. Correlations between the methods were evaluated by the Spearman rank correlation coefficient. Patients were stratified based on the expression of putative cancer stem cell markers, hypoxia-associated gene signatures, and a previously developed seven-gene signature. Locoregional tumor control was compared between these patient subgroups using log-rank tests. Gene expressions obtained from nCounter analyses were moderately correlated to GeneChip analyses (median ρ = approximately 0.68). A higher correlation was obtained between nCounter analyses and real-time PCR (median ρ = 0.84). Significant associations with locoregional tumor control were observed for most of the considered biomarkers evaluated by GeneChip and nCounter analyses. In general, all applied biomarkers (single genes and gene signatures) classified approximately 70% to 85% of the patients similarly. Overall, gene signatures seem to be more robust and had a better transferability among different measurement methods. Patients with locally advanced head and neck squamous cell carcinomas (HNSCC) have a heterogeneous response to radiochemotherapy (RCT), thus leading to an overall survival of only approximately 50%.1Baumann M. Krause M. Overgaard J. Debus J. Bentzen S.M. Daartz J. 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Krause M. Neumann K. Endris V. Sak A. Stuschke M. Balermpas P. Rödel C. Avlar M. Grosu A.L. Abdollahi A. Debus J. Belka C. Pigorsch S. Combs S.E. Mönnich D. Zips D. Baumann M. Targeted next-generation sequencing of locally advanced squamous cell carcinomas of the head and neck reveals druggable targets for improving adjuvant chemoradiation.Eur J Cancer. 2016; 57: 78-86Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 17Balermpas P. Rödel F. Rödel C. Krause M. Linge A. Lohaus F. Baumann M. Tinhofer I. Budach V. Gkika E. Stuschke M. Avlar M. Grosu A.-L. Abdollahi A. Debus J. Bayer C. Stangl S. Belka C. Pigorsch S. Multhoff G. Combs S.E. Mönnich D. Zips D. Fokas E. CD8+ tumour-infiltrating lymphocytes in relation to HPV status and clinical outcome in patients with head and neck cancer after postoperative chemoradiotherapy: a multicentre study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG): CD8+.Int J Cancer. 2016; 138: 171-181Crossref PubMed Scopus (148) Google Scholar, 18Lohaus F. Linge A. Tinhofer I. Budach V. Gkika E. Stuschke M. Balermpas P. Rödel C. Avlar M. Grosu A.L. Abdollahi A. Debus J. Bayer C. Belka C. Pigorsch S. Combs S.E. Mönnich D. Zips D. Von Neubeck C. Baretton G.B. Löck S. Thames H.D. Krause M. Baumann M. HPV16 DNA status is a strong prognosticator of loco-regional control after postoperative radiochemotherapy of locally advanced oropharyngeal carcinoma: results from a multicentre explorative study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG).Radiother Oncol. 2014; 113: 317-323Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 19Wintergerst L. Selmansberger M. Maihoefer C. Schüttrumpf L. Walch A. Wilke C. Pitea A. Woischke C. Baumeister P. Kirchner T. Belka C. Ganswindt U. Zitzelsberger H. Unger K. Hess J. A prognostic mRNA expression signature of four 16q24.3 genes in radio(chemo)therapy-treated head and neck squamous cell carcinoma (HNSCC).Mol Oncol. 2018; 12: 2085-2101Crossref PubMed Scopus (16) Google Scholar, 20Chen J. Fu G. Chen Y. Zhu G. Wang Z. Gene-expression signature predicts survival benefit from postoperative chemoradiotherapy in head and neck squamous cell carcinoma.Oncol Lett. 2018; 16: 2565-2578PubMed Google Scholar However, molecular methods are varying from array-based approaches to single-gene assays, thus impeding the comparability, transferability, and reproducibility of the results. Common approaches include chip-based whole transcriptome arrays (GeneChip analyses), customized nanoString (NanoString Technologies, Seattle, WA) gene panels (nCounter analyses), and real-time PCR analyses. To date, several studies have found a high correlation between the expressions obtained by GeneChip and nCounter analyses.21Brant R. Sharpe A. Liptrot T. Dry J.R. Harrington E.A. Barrett J.C. Whalley N. Womack C. Smith P. Hodgson D.R. Clinically viable gene expression assays with potential for predicting benefit from MEK inhibitors.Clin Cancer Res. 2017; 23: 1471-1480Crossref PubMed Scopus (20) Google Scholar, 22Ho T.H. Serie D.J. Parasramka M. Cheville J.C. Bot B.M. Tan W. Wang L. Joseph R.W. Hilton T. Leibovich B.C. Parker A.S. Eckel-Passow J.E. Differential gene expression profiling of matched primary renal cell carcinoma and metastases reveals upregulation of extracellular matrix genes.Ann Oncol. 2017; 28: 604-610Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 23Altenbuchinger M. Schwarzfischer P. Rehberg T. Reinders J. Kohler C.W. Gronwald W. Richter J. Szczepanowski M. Masqué-Soler N. Klapper W. Oefner P.J. Spang R. Molecular signatures that can be transferred across different omics platforms.Bioinformatics. 2017; 33: i333-i340Crossref PubMed Scopus (12) Google Scholar, 24Hess A.-K. Müer A. Mairinger F.D. Weichert W. Stenzinger A. Hummel M. Budach V. Tinhofer I. MiR-200b and miR-155 as predictive biomarkers for the efficacy of chemoradiation in locally advanced head and neck squamous cell carcinoma.Eur J Cancer. 2017; 77: 3-12Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar An even higher correlation is stated between the gene expression measurements obtained with nCounter and real-time PCR measurements.25Geiss G.K. Bumgarner R.E. Birditt B. Dahl T. Dowidar N. Dunaway D.L. Fell H.P. Ferree S. George R.D. Grogan T. James J.J. Maysuria M. Mitton J.D. Oliveri P. Osborn J.L. Peng T. Ratcliffe A.L. Webster P.J. Davidson E.H. Hood L. Dimitrov K. Direct multiplexed measurement of gene expression with color-coded probe pairs.Nat Biotechnol. 2008; 26: 317-325Crossref PubMed Scopus (1554) Google Scholar,26Reis P.P. Waldron L. Goswami R.S. Xu W. Xuan Y. Perez-Ordonez B. Gullane P. Irish J. Jurisica I. Kamel-Reid S. mRNA transcript quantification in archival samples using multiplexed, color-coded probes.BMC Biotechnol. 2011; 11: 46Crossref PubMed Scopus (215) Google Scholar However, most of these studies were based on a limited number of patients or samples and were performed using versions of Affymetrix (Thermo Fisher Scientific, Santa Clara, CA) arrays considered outdated today.27Veldman-Jones M.H. Lai Z. Wappett M. Harbron C.G. Barrett J.C. Harrington E.A. Thress K.S. Reproducible, quantitative, and flexible molecular subtyping of clinical DLBCL samples using the NanoString nCounter system.Clin Cancer Res. 2015; 21: 2367-2378Crossref PubMed Scopus (56) Google Scholar Therefore, the aim of this study was to compare gene expression data obtained by the three approaches, GeneChip analyses, nCounter analyses, and real-time PCR, that are available to a large number of patients and that were also based on the same RNA as starting material. We report on the two retrospective HNSCC cohorts of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG), including 327 patients in total who were diagnosed with locally advanced disease and received postoperative or primary RCT. For these cohorts, the impact of the putative cancer stem cell (CSC) markers CD44, MET, and SLC3A2, hypoxia-associated gene panels, and a seven-gene signature (postoperative RCT only) have previously been investigated and reported using nCounter gene expression data.9Linge A. Lohaus F. Löck S. Nowak A. Gudziol V. Valentini C. von Neubeck C. Jütz M. Tinhofer I. Budach V. Sak A. Stuschke M. Balermpas P. Rödel C. Grosu A.-L. Abdollahi A. Debus J. Ganswindt U. Belka C. Pigorsch S. Combs S.E. Mönnich D. Zips D. Buchholz F. Aust D.E. Baretton G.B. Thames H.D. Dubrovska A. Alsner J. Overgaard J. Krause M. Baumann M. HPV status, cancer stem cell marker expression, hypoxia gene signatures and tumour volume identify good prognosis subgroups in patients with HNSCC after primary radiochemotherapy: a multicentre retrospective study of the German Cancer Consortium Radiation.Radiother Oncol. 2016; 121: 364-373Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar,12Schmidt S. Linge A. Zwanenburg A. Leger S. Lohaus F. Krenn C. Appold S. Gudziol V. Nowak A. Von Neubeck C. Tinhofer I. Budach V. Sak A. Stuschke M. Balermpas P. Rodel C. Bunea H. Grosu A.L. Abdollahi A. Debus J. Ganswindt U. Belka C. Pigorsch S. Combs S.E. Monnich D. Zips D. Baretton G.B. Buchholz F. Baumann M. Krause M. Lock S. Development and validation of a gene signature for patients with head and neck carcinomas treated by postoperative radio(chemo)therapy.Clin Cancer Res. 2018; 24: 1364-1374Crossref PubMed Scopus (37) Google Scholar,15Linge A. Löck S. Gudziol V. Nowak A. Lohaus F. von Neubeck C. Jütz M. Abdollahi A. Debus J. Tinhofer I. Budach V. Sak A. Stuschke M. Balermpas P. Rödel C. Avlar M. Grosu A.-L. Bayer C. Belka C. Pigorsch S. Combs S.E. Welz S. Zips D. Buchholz F. Aust D.E. Baretton G.B. Thames H.D. Dubrovska A. Alsner J. Overgaard J. Baumann M. Krause M. DKTK-ROGLow cancer stem cell marker expression and low hypoxia identify good prognosis subgroups in HPV(-) HNSCC after postoperative radiochemotherapy: a multicenter study of the DKTK-ROG.Clin Cancer Res. 2016; 22: 2639-2649Crossref PubMed Scopus (96) Google Scholar,18Lohaus F. Linge A. Tinhofer I. Budach V. Gkika E. Stuschke M. Balermpas P. Rödel C. Avlar M. Grosu A.L. Abdollahi A. Debus J. Bayer C. Belka C. Pigorsch S. Combs S.E. Mönnich D. Zips D. Von Neubeck C. Baretton G.B. Löck S. Thames H.D. Krause M. Baumann M. HPV16 DNA status is a strong prognosticator of loco-regional control after postoperative radiochemotherapy of locally advanced oropharyngeal carcinoma: results from a multicentre explorative study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG).Radiother Oncol. 2014; 113: 317-323Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar Here, the reproducibility of these results is tested using GeneChip analyses as well as real-time PCR gene expression data, which is important for conducting clinical trials, including multicenter biomarker trials, and may also play a role in meta-analyses of biomarker data. For this study, patients of the two retrospective HNSCC cohorts of the DKTK-ROG were considered. All patients were diagnosed with locally advanced disease and were treated with either postoperative RCT15Linge A. Löck S. Gudziol V. Nowak A. Lohaus F. von Neubeck C. Jütz M. Abdollahi A. Debus J. Tinhofer I. Budach V. Sak A. Stuschke M. Balermpas P. Rödel C. Avlar M. Grosu A.-L. Bayer C. Belka C. Pigorsch S. Combs S.E. Welz S. Zips D. Buchholz F. Aust D.E. Baretton G.B. Thames H.D. Dubrovska A. Alsner J. Overgaard J. Baumann M. Krause M. DKTK-ROGLow cancer stem cell marker expression and low hypoxia identify good prognosis subgroups in HPV(-) HNSCC after postoperative radiochemotherapy: a multicenter study of the DKTK-ROG.Clin Cancer Res. 2016; 22: 2639-2649Crossref PubMed Scopus (96) Google Scholar,18Lohaus F. Linge A. Tinhofer I. Budach V. Gkika E. Stuschke M. Balermpas P. Rödel C. Avlar M. Grosu A.L. Abdollahi A. Debus J. Bayer C. Belka C. Pigorsch S. Combs S.E. Mönnich D. Zips D. Von Neubeck C. Baretton G.B. Löck S. Thames H.D. Krause M. Baumann M. HPV16 DNA status is a strong prognosticator of loco-regional control after postoperative radiochemotherapy of locally advanced oropharyngeal carcinoma: results from a multicentre explorative study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG).Radiother Oncol. 2014; 113: 317-323Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar,28Linge A. Schötz U. Löck S. Lohaus F. von Neubeck C. Gudziol V. Nowak A. Tinhofer I. Budach V. Sak A. Stuschke M. Balermpas P. Rödel C. Bunea H. Grosu A.-L. Abdollahi A. Debus J. Ganswindt U. Lauber K. Pigorsch S. Combs S.E. Mönnich D. Zips D. Baretton G.B. Buchholz F. Krause M. Belka C. Baumann M. Comparison of detection methods for HPV status as a prognostic marker for loco-regional control after radiochemotherapy in patients with HNSCC.Radiother Oncol. 2018; 127: 27-35Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar or primary R-CT9Linge A. Lohaus F. Löck S. Nowak A. Gudziol V. Valentini C. von Neubeck C. Jütz M. Tinhofer I. Budach V. Sak A. Stuschke M. Balermpas P. Rödel C. Grosu A.-L. Abdollahi A. Debus J. Ganswindt U. Belka C. Pigorsch S. Combs S.E. Mönnich D. Zips D. Buchholz F. Aust D.E. Baretton G.B. Thames H.D. Dubrovska A. Alsner J. Overgaard J. Krause M. Baumann M. HPV status, cancer stem cell marker expression, hypoxia gene signatures and tumour volume identify good prognosis subgroups in patients with HNSCC after primary radiochemotherapy: a multicentre retrospective study of the German Cancer Consortium Radiation.Radiother Oncol. 2016; 121: 364-373Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar,28Linge A. Schötz U. Löck S. Lohaus F. von Neubeck C. Gudziol V. Nowak A. Tinhofer I. Budach V. Sak A. Stuschke M. Balermpas P. Rödel C. Bunea H. Grosu A.-L. Abdollahi A. Debus J. Ganswindt U. Lauber K. Pigorsch S. Combs S.E. Mönnich D. Zips D. Baretton G.B. Buchholz F. Krause M. Belka C. Baumann M. Comparison of detection methods for HPV status as a prognostic marker for loco-regional control after radiochemotherapy in patients with HNSCC.Radiother Oncol. 2018; 127: 27-35Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar at one of the eight DKTK-ROG partner sites. The retrospective postoperative RCT cohort of the DKTK-ROG included 221 patients and was previously described.15Linge A. Löck S. Gudziol V. Nowak A. Lohaus F. von Neubeck C. Jütz M. Abdollahi A. Debus J. Tinhofer I. Budach V. Sak A. Stuschke M. Balermpas P. Rödel C. Avlar M. Grosu A.-L. Bayer C. Belka C. Pigorsch S. Combs S.E. Welz S. Zips D. Buchholz F. Aust D.E. Baretton G.B. Thames H.D. Dubrovska A. Alsner J. Overgaard J. Baumann M. Krause M. DKTK-ROGLow cancer stem cell marker expression and low hypoxia identify good prognosis subgroups in HPV(-) HNSCC after postoperative radiochemotherapy: a multicenter study of the DKTK-ROG.Clin Cancer Res. 2016; 22: 2639-2649Crossref PubMed Scopus (96) Google Scholar,18Lohaus F. Linge A. Tinhofer I. Budach V. Gkika E. Stuschke M. Balermpas P. Rödel C. Avlar M. Grosu A.L. Abdollahi A. Debus J. Bayer C. Belka C. Pigorsch S. Combs S.E. Mönnich D. Zips D. Von Neubeck C. Baretton G.B. Löck S. Thames H.D. Krause M. Baumann M. HPV16 DNA status is a strong prognosticator of loco-regional control after postoperative radiochemotherapy of locally advanced oropharyngeal carcinoma: results from a multicentre explorative study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG).Radiother Oncol. 2014; 113: 317-323Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar,28Linge A. Schötz U. Löck S. Lohaus F. von Neubeck C. Gudziol V. Nowak A. Tinhofer I. Budach V. Sak A. Stuschke M. Balermpas P. Rödel C. Bunea H. Grosu A.-L. Abdollahi A. Debus J. Ganswindt U. Lauber K. Pigorsch S. Combs S.E. Mönnich D. Zips D. Baretton G.B. Buchholz F. Krause M. Belka C. Baumann M. Comparison of detection methods for HPV status as a prognostic marker for loco-regional control after radiochemotherapy in patients with HNSCC.Radiother Oncol. 2018; 127: 27-35Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar Briefly, patients were treated between 2004 and 2012 with curatively intended cisplatinum-based postoperative RCT with a median dose of 64.0 Gy (range, 56.0 to 68.0 Gy) according to standard protocols. Each patient presented with a tumor stage pT4 and/or >3 positive lymph nodes and/or positive microscopic resection margins and/or extracapsular spread. The primary RCT cohort originally consisted of 158 patients and was previously described.9Linge A. Lohaus F. Löck S. Nowak A. Gudziol V. Valentini C. von Neubeck C. Jütz M. Tinhofer I. Budach V. Sak A. Stuschke M. Balermpas P. Rödel C. Grosu A.-L. Abdollahi A. Debus J. Ganswindt U. Belka C. Pigorsch S. Combs S.E. Mönnich D. Zips D. Buchholz F. Aust D.E. Baretton G.B. Thames H.D. Dubrovska A. Alsner J. Overgaard J. Krause M. Baumann M. HPV status, cancer stem cell marker expression, hypoxia gene signatures and tumour volume identify good prognosis subgroups in patients with HNSCC after primary radiochemotherapy: a multicentre retrospective study of the German Cancer Consortium Radiation.Radiother Oncol. 2016; 121: 364-373Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar,28Linge A. Schötz U. Löck S. Lohaus F. von Neubeck C. Gudziol V. Nowak A. Tinhofer I. Budach V. Sak A. Stuschke M. Balermpas P. Rödel C. Bunea H. Grosu A.-L. Abdollahi A. Debus J. Ganswindt U. Lauber K. Pigorsch S. Combs S.E. Mönnich D. Zips D. Baretton G.B. Buchholz F. Krause M. Belka C. Baumann M. Comparison of detection methods for HPV status as a prognostic marker for loco-regional control after radiochemotherapy in patients with HNSCC.Radiother Oncol. 2018; 127: 27-35Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar Patients received primary RCT (based on cisplatinum or mitomycin-C) with a median dose of 72.0 Gy (range, 68.4 to 74.0 Gy) between 2005 and 2011. Some of the samples of the original patient cohorts had to be omitted from the analysis because of insufficient tumor material or a too low RNA yield. The frequency of RNA assay failure was 1.2% for nCounter, 1.8% for GeneChip, and 4.6% for real-time PCR analyses. Results of both GeneChip and nCounter analyses were available for 191 patients in the postoperative RCT cohort and 136 patients in the primary RCT cohort. For 187 patients in the postoperative RCT cohort, an additional analysis with real-time PCR was performed. Both cohorts are summarized in Table 1. Expression and outcome data are available in Supplemental Table S1.Table 1Patient and Tumor Characteristics for the Postoperative and Primary RCT CohortsCharacteristicsPostoperative RCT cohortPrimary RCT cohortFollow-up, median (95% CI), months56.7 (11.5–94.5)54.7 (10.9–81.1)Age, median (range), years57.0 (24.0–75.2)59.0 (39.2–81.9)Dose, Gy64.0 (56.0–68.4)72.0 (68.4–74.0)Sex, n (%) Male152 (76.6)112 (82.4) Female39 (30.4)24 (17.6)Localization Oropharynx113 (59.2)70 (51.5) Oral cavity53 (27.7)23 (16.9) Hypopharynx25 (13.1)43 (31.6)UICC stage 1 27 (3.7) 330 (15.7)12 (8.8) 4154 (80.6)124 (91.8)HPV16 status Negative125 (65.4)119 (87.5) Positive65 (34.0)16 (11.8) Unknown1 (0.5)1 (0.7)Locoregional recurrences28 (14.7)54 (39.7)Distant metastases36 (18.9)24 (17.7)Deaths61 (31.9)70 (51.5)Data are presented as n (%) of patients unless otherwise indicated. Compared with previous studies of these cohorts,9Linge A. Lohaus F. Löck S. Nowak A. Gudziol V. Valentini C. von Neubeck C. Jütz M. Tinhofer I. Budach V. Sak A. Stuschke M. Balermpas P. Rödel C. Grosu A.-L. Abdollahi A. Debus J. Ganswindt U. Belka C. Pigorsch S. Combs S.E. Mönnich D. Zips D. Buchholz F. Aust D.E. Baretton G.B. Thames H.D. Dubrovska A. Alsner J. Overgaard J. Krause M. Baumann M. HPV status, cancer stem cell marker expression, hypoxia gene signatures and tumour volume identify good prognosis subgroups in patients with HNSCC after primary radiochemotherapy: a multicentre retrospective study of the German Cancer Consortium Radiation.Radiother Oncol. 2016; 121: 364-373Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar,12Schmidt S. Linge A. Zwanenburg A. Leger S. Lohaus F. Krenn C. Appold S. Gudziol V. Nowak A. Von Neubeck C. Tinhofer I. Budach V. Sak A. Stuschke M. Balermpas P. Rodel C. Bunea H. Grosu A.L. Abdollahi A. Debus J. Ganswindt U. Belka C. Pigorsch S. Combs S.E. Monnich D. Zips D. Baretton G.B. Buchholz F. Baumann M. Krause M. Lock S. Development and validation of a gene signature for patients with head and neck carcinomas treated by postoperative radio(chemo)therapy.Clin Cancer Res. 2018; 24: 1364-1374Crossref PubMed Scopus (37) Google Scholar,15Linge A. Löck S. Gudziol V. Nowak A. Lohaus F. von Neubeck C. Jütz M. Abdollahi A. Debus J. Tinhofer I. Budach V. Sak A. Stuschke M. Balermpas P. Rödel C. Avlar M. Grosu A.-L. Bayer C. Belka C. Pigorsch S. Combs S.E. Welz S. Zips D. Buchholz F. Aust D.E. Baretton G.B. Thames H.D. Dubrovska A. Alsner J. Overgaard J. Baumann M. Krause M. DKTK-ROGLow cancer stem cell marker expression and low hypoxia identify good prognosis subgroups in HPV(-) HNSCC after postoperative radiochemotherapy: a multicenter study of the DKTK-ROG.Clin Cancer Res. 2016; 22: 2639-2649Crossref PubMed Scopus (96) Google Scholar,18Lohaus F. Linge A. Tinhofer I. Budach V. Gkika E. Stuschke M. Balermpas P. Rödel C. Avlar M. Grosu A.L. Abdollahi A. Debus J. Bayer C. Belka C. Pigorsch S. Combs S.E. Mönnich D. Zips D. Von Neubeck C. Baretton G.B. Löck S. Thames H.D. Krause M. Baumann M. HPV16 DNA status is a strong prognosticator of loco-regional control after postoperative radiochemother

Abstract Background Melanoma patients frequently develop brain metastases. The most widely used score to predict survival is the molGPA based on a mixed treatment of stereotactic radiotherapy (SRT) and whole brain radiotherapy (WBRT). In addition, systemic therapy was not considered. We therefore aimed to evaluate the performance of the molGPA score in patients homogeneously treated with SRT and concurrent targeted therapy or immunotherapy (TT/IT). Methods This retrospective analysis is based on an international multicenter database (TOaSTT) of melanoma patients treated with TT/IT and concurrent (≤30 days) SRT for brain metastases between May 2011 and May 2018. Overall survival (OS) was studied using Kaplan-Meier survival curves and log-rank testing. Uni- and multivariate analysis was performed to analyze prognostic factors for OS. Results One hundred ten patients were analyzed. 61, 31 and 8% were treated with IT, TT and with a simultaneous combination, respectively. A median of two brain metastases were treated per patient. After a median follow-up of 8 months, median OS was 8.4 months (0–40 months). The molGPA score was not associated with OS. Instead, cumulative brain metastases volume, timing of metastases (syn- vs. metachronous) and systemic therapy with concurrent IT vs. TT influenced OS significantly. Based on these parameters, the VTS score (volume-timing-systemic therapy) was established that stratified patients into three groups with a median OS of 5.1, 18.9 and 34.5 months, respectively ( p = 0.001 and 0.03). Conclusion The molGPA score was not useful for this cohort of melanoma patients undergoing local therapy for brain metastases taking into account systemic TT/IT. For these patients, we propose a prognostic VTS score, which needs to be validated prospectively.

Background and purpose Oesophageal mobility relative to bony anatomy is a major source of geometrical uncertainty in proton radiotherapy of oesophageal carcinoma. To mitigate this uncertainty we investigated the use of implanted fiducial markers for direct target verification in terms of safety, visibility, and stability. Materials and methods A total of 19 helical gold markers were endoscopically implanted in ten patients. Their placement at the proximal and distal tumour borders was compared to tumour demarcations derived from [18F]Fluorodeoxyglucose positron emission tomography, their visibility quantified via the contrast-to-noise ratio on daily orthogonal X-ray imaging, and their mobility relative to bony anatomy analysed by means of retrospective triangulation. Results Marker implantation proceeded without complications, but the distal tumour border could not be reached in two patients. Marker locations corresponded reasonably well with metabolic tumour edges (mean: 5.4 mm more distally). Marker visibility was limited but mostly sufficient (mean contrast-to-noise ratio: 1.5), and sixteen markers (84%) remained in situ until the end of treatment. Overall, marker excursions from their planned position were larger than 5(10) mm in 59(17)% of all analysed fractions. On one occasion severe target displacement was only identified via markers and was corrected before treatment delivery. Conclusion Implanted helical gold fiducial markers are a safe and reliable method of providing target-centric positioning verification in proton beam therapy of oesophageal carcinoma.

Classical robust optimization (cRO) in intensity-modulated proton therapy (IMPT) considers isocenter position and particle range uncertainties; anatomical robust optimization (aRO) aims to consider additional non-rigid positioning variations. This work compares the influence of different uncertainty sources on the robustness of cRO and aRO IMPT plans for head and neck squamous cell carcinoma (HNSCC).Two IMPT plans were optimized for 20 HNSCC patients who received weekly control CTs (cCT): cRO, using solely the planning CT, and aRO, including 2 additional cCTs. The robustness of the plans in terms of clinical target volume (CTV) coverage and organ at risk (OAR) sparing was analyzed considering stepwise the influence of (1) non-rigid anatomical variations given by the weekly cCT, (2) with fraction-wise added rigid random setup errors and (3) additional systematic proton range uncertainties.cRO plans presented significantly higher nominal CTV coverage but are outperformed by aRO plans when considering non-rigid anatomical variations only, as cRO and aRO plans presented a median target coverage (D98%) decrease for the low-risk/high-risk CTV of 1.8/1.1 percentage points (pp) and -0.2 pp/-0.3 pp, respectively. Setup and range uncertainties had larger influence on cRO CTV coverage, but led to similar OAR dose changes in both plans. Considering all error sources, 10/2 cRO/aRO patients missed the CTV coverage and a limited number exceeded some OAR constraints in both plans.Non-rigid anatomical variations are mainly responsible for critical target coverage loss of cRO plans, whereas the aRO approach was robust against such variations. Both plans provide similar robustness of OAR parameters.The influence of different uncertainty sources was quantified for robust IMPT HNSCC plans.

Abstract Identifying patients with locally advanced head and neck carcinoma on high risk of recurrence after definitive concurrent radiochemotherapy is of key importance for the selection for consolidation therapy and for individualized treatment intensification. In this multicenter study we analyzed recurrence-associated single-nucleotide polymorphisms (SNPs) in DNA repair genes in tumor DNA from 132 patients with locally advanced head and neck carcinoma (LadHnSCC). Patients were treated with definitive radiotherapy and simultaneous cisplatin-based chemotherapy at six partner sites of the German Cancer Consortium (DKTK) Radiation Oncology Group from 2005 to 2011. For validation, a group of 20 patients was available. Score selection method using proportional hazard analysis and leave-one-out cross-validation were performed to identify markers associated with outcome. The SNPs rs1799793 and rs13181 were associated with survival and the same SNPs and in addition rs17655 with freedom from loco-regional relapse (ffLRR) in the trainings datasets from all patients. The homozygote major rs1799793 genotype at the ERCC2 gene was associated with better (Hazard ratio (HR): 0.418 (0.234–0.744), p = 0.003) and the homozygote minor rs13181 genotype at ERCC2 with worse survival (HR: 2.074, 95% CI (1.177–3.658), p = 0.017) in comparison to the other genotypes. At the ffLRR endpoint, rs1799793 and rs13181 had comparable prognostic value. The rs1799793 and rs13181 genotypes passed the leave-one-out cross-validation procedure and associated with survival and ffLRR in patients with LadHnSCC treated with definitive radiochemotherapy. While findings were confirmed in a small validation dataset, further validation is underway within a prospective biomarker study of the DKTK.

(1) Background: Patients with locally advanced head and neck squamous cell carcinoma (HNSCC) who are biologically at high risk for the development of loco−regional recurrences after postoperative radiotherapy (PORT) but at intermediate risk according to clinical risk factors may benefit from additional concurrent chemotherapy. In this matched-pair study, we aimed to identify a corresponding predictive gene signature. (2) Methods: Gene expression analysis was performed on a multicenter retrospective cohort of 221 patients that were treated with postoperative radiochemotherapy (PORT-C) and 283 patients who were treated with PORT alone. Propensity score analysis was used to identify matched patient pairs from both cohorts. From differential gene expression analysis and Cox regression, a predictive gene signature was identified. (3) Results: 108 matched patient pairs were selected. We identified a 2-metagene signature that stratified patients into risk groups in both cohorts. The comparison of the high-risk patients between the two types of treatment showed higher loco−regional control (LRC) after treatment with PORT-C (p < 0.001), which was confirmed by a significant interaction term in Cox regression (p = 0.027), i.e., the 2-metagene signature was indicative for the type of treatment. (4) Conclusion: We have identified a novel gene signature that may be helpful to identify patients with high-risk HNSCC amongst those at intermediate clinical risk treated with PORT, who may benefit from additional concurrent chemotherapy.

In their recent editorial Kimple and Harari (1) reviewed the current knowledge of the importance of HPV status of head and neck squamous cell carcinoma (HNSCC) for outcome of radiotherapy. The editorial summarized, among others, a retrospective multicenter study by the German Cancer Consortium Radiation Oncology Group (DKTK-ROG) which demonstrated significant prognostic value of the HPV status in patients with locally advanced HNSCC on the outcome of postoperative radiochemotherapy (PORT-C) (2). The overall aim of the DKTK-ROG head and neck program is to identify and validate biomarkers including biological and clinical parameters as well as imaging data for patient stratification in terms of individualization of radiotherapy by multimodal treatment. The study design includes retrospective explorative analyses in patients who received PORT-C or primary radiochemotherapy (RCT) for biomarker identification. The most promising biomarkers will then be validated in a prospective validation cohort, which is currently recruiting at all eight DKTK-ROG partner sites. Based on these results, interventional studies are under preparation. Biomarker analyses are being performed at all eight DKTK partner sites and are addressing different topics such as HPV status, hypoxia, cancer stem cells, tumor infiltrating lymphocytes, tumor volume, targeted next generation sequencing, transcriptomics and methylome analyses.

6006 Background: The genetic landscape of SCCHN is currently being unravelled, but the role of distinct mutations for treatment outcome remains largely unknown. We compared mutational patterns of HPV+ and HPV- tumors with outcome after uniform chemoradiation. Methods: Archival tumor specimens from 208 patients with carcinomas of the hypopharynx, oropharynx or oral cavity, all uniformly treated with surgery and adjuvant cisplatin-based radiochemotherapy, were included in this study. An in-house gene panel for semiconductor-based next-generation ultra-deep sequencing, covering 211 exons from 45 genes frequently altered in SCCHN was used for mutational analysis. Genetic alterations were correlated with HPV status and patient outcome. Results: Mutational profiles were successfully established for 185 SCCHN cases. Interestingly, HPV+ carcinomas were significantly enriched for activating mutations in driver genes (PIK3CA 27%, KRAS 8%, NRAS 4%, HRAS 2%) compared to HPV- cases (P = 0.002). Conversely, HPV- tumors showed an increased frequency of loss-of-function alterations in tumor suppressor genes (TP53 67%, CDKN2A 30%, PTEN 4%, SMAD4 3%) compared to HPV+ cases (P < 0.001). After a median follow-up of 55 months, alterations in tumor suppressor genes significantly increased the risk of death (HR 2.9, 95% CI 1.5-5.8, P = 0.001), locoregional recurrence (HR 5.4, 95% CI 1.6-18.1, P = 0.006) and distant metastasis (HR 2.3, 95% CI 1.0-5.1, P = 0.04). The occurrence of activating driver gene mutations did not influence outcome in the total cohort of patients; however, they were associated per trend with increased risk of locoregional recurrence and death (HR 3.7, 95% CI 0.7-20.6, P = 0.12) in HPV+ oropharyngeal carcinomas. Conclusions: Overall, loss-of-function tumor suppressor gene mutations negatively interfere with efficacy of adjuvant cisplatin-based chemoradiation, whereas activating driver gene mutations define poor risk specifically in HPV-driven SCCHN. These genes and their signalling pathways might represent therapeutic targets for improving cure rates of SCCHN.

An amendment to this paper has been published and can be accessed via the original article.

SBRT for early stage NSCLC has been characterized in many studies and pooled analyses resulting in clear recommendations regarding patient selection, planning, delivery, dose and quality assurance. This report of the German working group stereotactic radiotherapy pools SBRT patient data of German centers and aims to describe local control, overall survival and toxicity for the subgroups of different primary tumors. All radiotherapy departments in Germany were contacted and invited to participate in this study. Six hundred fifty-one patients with 868 lung metastases treated with SBRT were included in this database. Biggest subgroups were NSCLC (n = 223), colorectal cancer (n = 196), renal cell cancer (n = 58) and breast cancer (n = 54). Two hundred sixty-four were treated as single fraction radiosurgery, 313 in a three fraction approach, 291 lesions with different fractionation. The PTV encompassing isodose ranged from 33.5 Gy BED to 177 Gy BED. For the entire patient cohort a local control rate after 1 year of 90.8% was achieved. A total of 5.9% of the patients experienced grade 3 toxicities. Pneumonitis grade 1 was found in 14.9%, grade 2 in 4.6% and grade 3 in 0.8%. One patient with grade 5 toxicity was reported. Kaplan-Meier estimations of local control and overall survival are currently in preparation. In addition multivariate analysis are planned in order to identify the relevant factors for therapy success depending on primary tumor histology. Despite a very heterogeneous practice throughout Germany excellent results regarding local control and toxicity could be achieved. The ongoing investigations will hopefully be the basis for practical guidelines for the individual subgroups.

Die Leber ist einer der Hauptmetastasierungsorte gastrointestinaler, aber auch anderer Malignome. Aus diesem Grunde spielt die Therapie von Lebermetastasen eine zentrale Rolle in der onkologischen Behandlung. In der vorliegenden Arbeit werden die aktuellen Therapieoptionen und multimodalen Therapiekonzepte für die Therapie von Lebermetastasen dargestellt. Nach Analyse aktueller Studienergebnisse sowie Empfehlungen nationaler und internationaler Expertengruppen werden die verschiedenen Therapieoptionen für Lebermetastasen im Allgemeinen zusammengefasst und anschließend für die jeweilige Tumorentität dargestellt. Lebermetastasen werden in 3 große Gruppen unterteilt, die sich hinsichtlich Therapie und Prognose unterscheiden: die kolorektalen Lebermetastasen (CRLM), die neuroendokrinen Lebermetastasen und die nichtkolorektalen, nichtneuroendokrinen Lebermetastasen, zu welchen Lebermetastasen aller übrigen Malignome gezählt werden. CRLM sind dabei aufgrund der höchsten Inzidenz von zentralem Interesse. Die Wahl der Therapie richtet sich nach der onkologischen Gesamtsituation und den funktionellen Aspekten, insbesondere der Leberfunktion, und den Komorbiditäten der Patienten. Die chirurgische Therapie stellt die beste kurative Option dar. Aus diesem Grunde sollte für jeden Patienten eine chirurgische Resektion geprüft werden. Trotz höherer Lokalrezidivraten stellen interventionelle Ablationsverfahren bei kleinen und in der Anzahl limitierten Lebermetastasen eine gute Alternative zur lokalen Kontrolle dar. Systemtherapien sowie Bestrahlungsoptionen finden in der Palliation sowie im Rahmen multimodaler Therapiekonzepte mit dem Ziel einer Kuration Anwendung.

&lt;p&gt;Univariate analyses of both hypoxia gene signatures using nanoString or RT-PCR analyses. HR = hazard ratio; 95% CI = 95 percent confidence interval.&lt;/p&gt;

&lt;p&gt;Multivariate analyses of hypoxia-gene signatures and additional prognostic factors assessed for all patients. HR = hazard ratio; 95% CI = 95 percent confidence interval; ECE = extracapsular extension.&lt;/p&gt;

&lt;p&gt;Combined multivariate analyses of HPV16 DNA, cancer stem cell markers, hypoxia and additional prognostic factors for all patients (endpoint loco-regional control). HR = hazard ratio; 95% CI = 95 percent confidence interval; ECE = extracapsular extension.&lt;/p&gt;

&lt;p&gt;Univariate analyses of CSC markers. HR = hazard ratio; 95% CI = 95 percent confidence interval.&lt;/p&gt;

&lt;p&gt;A. Correlation of hypoxia gene-signatures and HPV16 DNA. B. Correlation of CSC marker expression and HPV16 DNA.&lt;/p&gt;

&lt;p&gt;A. Patient characteristics. B. Hypoxia gene signatures.&lt;/p&gt;

&lt;p&gt;Univariate analyses of both hypoxia gene signatures using nanoString or RT-PCR analyses. HR = hazard ratio; 95% CI = 95 percent confidence interval.&lt;/p&gt;

&lt;p&gt;A. Correlation of CSC marker expressions. B. Multivariate analyses of stem cell markers and additional prognostic factors for all patients. HR = hazard ratio; 95% CI = 95 percent confidence interval ; ECE = extracapsular extension. C. Correlation of CSC marker expression and HPV16 DNA.&lt;/p&gt;

&lt;p&gt;Multivariate analyses of hypoxia-gene signatures and additional prognostic factors assessed for all patients. HR = hazard ratio; 95% CI = 95 percent confidence interval; ECE = extracapsular extension.&lt;/p&gt;

&lt;p&gt;Univariate analyses of CSC markers. HR = hazard ratio; 95% CI = 95 percent confidence interval.&lt;/p&gt;

&lt;p&gt;Combined multivariate analyses of HPV16 DNA, cancer stem cell markers, hypoxia and additional prognostic factors for all patients (endpoint loco-regional control). HR = hazard ratio; 95% CI = 95 percent confidence interval; ECE = extracapsular extension.&lt;/p&gt;

&lt;p&gt;Correlation of tumor hypoxia using nanoString vs PCR.&lt;/p&gt;

&lt;p&gt;Table S1 Analyzed genes of the hypothesis-driven gene set. Table S2 Programs and packages used for evaluation. Figure S1 Results of repeated 3-fold cross validation for LRC. Figure S2 Mean oob ci depending of the signature size for LRC. Figure S3 Importance score for genes associated with LRC. Table S3 Increasing robustness by including highly correlated genes. Figure S4 Patient stratification by the 7-gene signature for LRC. Figure S5 Patient stratification by the 7-gene signature and clinical parameters for LRC. Table S4 Multivariable Cox regression of OS. Figure S6 Patient stratification by the 7-gene signature and clinical parameters for OS. Table S5 Multivariable Cox regression of DM. Figure S7 Patient stratification by the 7-gene signature and clinical parameters for DM. Table S6 Means and standard deviations of the 7-gene signature gene expressions. Table S7 Comparison of gene expressions between patient groups. Table S8 Hyper-parameters for feature selection algorithms and predictive models. Table S9 Patient characteristics of all patients. Figure S8 Identification of the final gene signature.&lt;/p&gt;

&lt;p&gt;Supplementary discussion; Supplementary methods; Supplementary tables S1-S8; Supplementary figures S1-S8.&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; The aim of this study was to identify and independently validate a novel gene signature predicting locoregional tumor control (LRC) for treatment individualization of patients with locally advanced HPV-negative head and neck squamous cell carcinomas (HNSCC) who are treated with postoperative radio(chemo)therapy (PORT-C).&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; Gene expression analyses were performed using NanoString technology on a multicenter training cohort of 130 patients and an independent validation cohort of 121 patients. The analyzed gene set was composed of genes with a previously reported association with radio(chemo)sensitivity or resistance to radio(chemo)therapy. Gene selection and model building were performed comparing several machine-learning algorithms.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; We identified a 7-gene signature consisting of the three individual genes &lt;i&gt;HILPDA, CD24, TCF3&lt;/i&gt;, and one metagene combining the highly correlated genes &lt;i&gt;SERPINE1, INHBA, P4HA2&lt;/i&gt;, and &lt;i&gt;ACTN1&lt;/i&gt;. The 7-gene signature was used, in combination with clinical parameters, to fit a multivariable Cox model to the training data (concordance index, ci = 0.82), which was successfully validated (ci = 0.71). The signature showed improved performance compared with clinical parameters alone (ci = 0.66) and with a previously published model including hypoxia-associated genes and cancer stem cell markers (ci = 0.65). It was used to stratify patients into groups with low and high risk of recurrence, leading to significant differences in LRC in training and validation (&lt;i&gt;P&lt;/i&gt; &lt; 0.001).&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; We have identified and validated the first hypothesis-based gene signature for HPV-negative HNSCC treated by PORT-C including genes related to several radiobiological aspects. A prospective validation is planned in an ongoing prospective clinical trial before potential application in clinical trials for patient stratification. &lt;i&gt;Clin Cancer Res; 24(6); 1364–74. ©2018 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;The heavy chain of the CD98 protein (CD98hc) is encoded by the &lt;i&gt;SLC3A2&lt;/i&gt; gene. Together with the light subunit LAT1, CD98hc constitutes a heterodimeric transmembrane amino acid transporter. High &lt;i&gt;SLC3A2&lt;/i&gt; mRNA expression levels are associated with poor prognosis in patients with head and neck squamous cell carcinoma (HNSCC) treated with radiochemotherapy. Little is known regarding the CD98hc protein–mediated molecular mechanisms of tumor radioresistance.&lt;/p&gt;Experimental Design:&lt;p&gt;CD98hc protein expression levels were correlated with corresponding tumor control dose 50 (TCD&lt;sub&gt;50&lt;/sub&gt;) in HNSCC xenograft models. Expression levels of CD98hc and LAT1 in HNSCC cells were modulated by siRNA or CRISPR/Cas9 gene editing. HNSCC cell phenotypes were characterized by transcription profiling, plasma membrane proteomics, metabolic analysis, and signaling pathway activation. Expression levels of CD98hc and LAT1 proteins were examined by IHC analysis of tumor tissues from patients with locally advanced HNSCC treated with primary radiochemotherapy (RCTx). Primary endpoint was locoregional tumor control (LRC).&lt;/p&gt;Results:&lt;p&gt;High expression levels of CD98hc resulted in an increase in mTOR pathway activation, amino acid metabolism, and DNA repair as well as downregulation of oxidative stress and autophagy. High expression levels of CD98hc and LAT1 proteins were significantly correlated and associated with an increase in radioresistance in HNSCC &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt; models. High expression of both proteins identified a poor prognosis subgroup in patients with locally advanced HNSCC after RCTx.&lt;/p&gt;Conclusions:&lt;p&gt;We found that CD98hc-associated signaling mechanisms play a central role in the regulation of HNSCC radioresistance and may be a promising target for tumor radiosensitization.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; To investigate the impact of hypoxia-induced gene expression and cancer stem cell (CSC) marker expression on outcome of postoperative cisplatin-based radiochemotherapy (PORT-C) in patients with locally advanced head and neck squamous cell carcinoma (HNSCC).&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; Expression of the CSC markers &lt;i&gt;CD44, MET&lt;/i&gt;, and &lt;i&gt;SLC3A2&lt;/i&gt;, and hypoxia gene signatures were analyzed in the resected primary tumors using RT-PCR and nanoString technology in a multicenter retrospective cohort of 195 patients. CD44 protein expression was further analyzed in tissue microarrays. Primary endpoint was locoregional tumor control.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; Univariate analysis showed that hypoxia-induced gene expression was significantly associated with a high risk of locoregional recurrence using the 15-gene signature (&lt;i&gt;P&lt;/i&gt; = 0.010) or the 26-gene signature (&lt;i&gt;P&lt;/i&gt; = 0.002). In multivariate analyses, in patients with HPV16 DNA–negative but not with HPV16 DNA–positive tumors the effect of hypoxia-induced genes on locoregional control was apparent (15-gene signature: HR 4.54, &lt;i&gt;P&lt;/i&gt; = 0.006; 26-gene signature: HR 10.27, &lt;i&gt;P&lt;/i&gt; = 0.024). Furthermore, &lt;i&gt;MET, SLC3A2, CD44&lt;/i&gt;, and CD44 protein showed an association with locoregional tumor control in multivariate analyses (&lt;i&gt;MET&lt;/i&gt;: HR 3.71, &lt;i&gt;P&lt;/i&gt; = 0.016; &lt;i&gt;SLC3A2&lt;/i&gt;: HR 8.54, &lt;i&gt;P&lt;/i&gt; = 0.037; &lt;i&gt;CD44&lt;/i&gt;: HR 3.36, &lt;i&gt;P&lt;/i&gt; = 0.054; CD44 protein n/a because of no event in the CD44-negative group) in the HPV16 DNA–negative subgroup.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; We have shown for the first time that high hypoxia-induced gene expression and high CSC marker expression levels correlate with tumor recurrence after PORT-C in patients with HPV16 DNA–negative HNSCC. After validation in a currently ongoing prospective trial, these parameters may help to further stratify patients for individualized treatment de-escalation or intensification strategies. &lt;i&gt;Clin Cancer Res; 22(11); 2639–49. ©2016 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; The aim of this study was to identify and independently validate a novel gene signature predicting locoregional tumor control (LRC) for treatment individualization of patients with locally advanced HPV-negative head and neck squamous cell carcinomas (HNSCC) who are treated with postoperative radio(chemo)therapy (PORT-C).&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; Gene expression analyses were performed using NanoString technology on a multicenter training cohort of 130 patients and an independent validation cohort of 121 patients. The analyzed gene set was composed of genes with a previously reported association with radio(chemo)sensitivity or resistance to radio(chemo)therapy. Gene selection and model building were performed comparing several machine-learning algorithms.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; We identified a 7-gene signature consisting of the three individual genes &lt;i&gt;HILPDA, CD24, TCF3&lt;/i&gt;, and one metagene combining the highly correlated genes &lt;i&gt;SERPINE1, INHBA, P4HA2&lt;/i&gt;, and &lt;i&gt;ACTN1&lt;/i&gt;. The 7-gene signature was used, in combination with clinical parameters, to fit a multivariable Cox model to the training data (concordance index, ci = 0.82), which was successfully validated (ci = 0.71). The signature showed improved performance compared with clinical parameters alone (ci = 0.66) and with a previously published model including hypoxia-associated genes and cancer stem cell markers (ci = 0.65). It was used to stratify patients into groups with low and high risk of recurrence, leading to significant differences in LRC in training and validation (&lt;i&gt;P&lt;/i&gt; &lt; 0.001).&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; We have identified and validated the first hypothesis-based gene signature for HPV-negative HNSCC treated by PORT-C including genes related to several radiobiological aspects. A prospective validation is planned in an ongoing prospective clinical trial before potential application in clinical trials for patient stratification. &lt;i&gt;Clin Cancer Res; 24(6); 1364–74. ©2018 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;The heavy chain of the CD98 protein (CD98hc) is encoded by the &lt;i&gt;SLC3A2&lt;/i&gt; gene. Together with the light subunit LAT1, CD98hc constitutes a heterodimeric transmembrane amino acid transporter. High &lt;i&gt;SLC3A2&lt;/i&gt; mRNA expression levels are associated with poor prognosis in patients with head and neck squamous cell carcinoma (HNSCC) treated with radiochemotherapy. Little is known regarding the CD98hc protein–mediated molecular mechanisms of tumor radioresistance.&lt;/p&gt;Experimental Design:&lt;p&gt;CD98hc protein expression levels were correlated with corresponding tumor control dose 50 (TCD&lt;sub&gt;50&lt;/sub&gt;) in HNSCC xenograft models. Expression levels of CD98hc and LAT1 in HNSCC cells were modulated by siRNA or CRISPR/Cas9 gene editing. HNSCC cell phenotypes were characterized by transcription profiling, plasma membrane proteomics, metabolic analysis, and signaling pathway activation. Expression levels of CD98hc and LAT1 proteins were examined by IHC analysis of tumor tissues from patients with locally advanced HNSCC treated with primary radiochemotherapy (RCTx). Primary endpoint was locoregional tumor control (LRC).&lt;/p&gt;Results:&lt;p&gt;High expression levels of CD98hc resulted in an increase in mTOR pathway activation, amino acid metabolism, and DNA repair as well as downregulation of oxidative stress and autophagy. High expression levels of CD98hc and LAT1 proteins were significantly correlated and associated with an increase in radioresistance in HNSCC &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt; models. High expression of both proteins identified a poor prognosis subgroup in patients with locally advanced HNSCC after RCTx.&lt;/p&gt;Conclusions:&lt;p&gt;We found that CD98hc-associated signaling mechanisms play a central role in the regulation of HNSCC radioresistance and may be a promising target for tumor radiosensitization.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; To investigate the impact of hypoxia-induced gene expression and cancer stem cell (CSC) marker expression on outcome of postoperative cisplatin-based radiochemotherapy (PORT-C) in patients with locally advanced head and neck squamous cell carcinoma (HNSCC).&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; Expression of the CSC markers &lt;i&gt;CD44, MET&lt;/i&gt;, and &lt;i&gt;SLC3A2&lt;/i&gt;, and hypoxia gene signatures were analyzed in the resected primary tumors using RT-PCR and nanoString technology in a multicenter retrospective cohort of 195 patients. CD44 protein expression was further analyzed in tissue microarrays. Primary endpoint was locoregional tumor control.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; Univariate analysis showed that hypoxia-induced gene expression was significantly associated with a high risk of locoregional recurrence using the 15-gene signature (&lt;i&gt;P&lt;/i&gt; = 0.010) or the 26-gene signature (&lt;i&gt;P&lt;/i&gt; = 0.002). In multivariate analyses, in patients with HPV16 DNA–negative but not with HPV16 DNA–positive tumors the effect of hypoxia-induced genes on locoregional control was apparent (15-gene signature: HR 4.54, &lt;i&gt;P&lt;/i&gt; = 0.006; 26-gene signature: HR 10.27, &lt;i&gt;P&lt;/i&gt; = 0.024). Furthermore, &lt;i&gt;MET, SLC3A2, CD44&lt;/i&gt;, and CD44 protein showed an association with locoregional tumor control in multivariate analyses (&lt;i&gt;MET&lt;/i&gt;: HR 3.71, &lt;i&gt;P&lt;/i&gt; = 0.016; &lt;i&gt;SLC3A2&lt;/i&gt;: HR 8.54, &lt;i&gt;P&lt;/i&gt; = 0.037; &lt;i&gt;CD44&lt;/i&gt;: HR 3.36, &lt;i&gt;P&lt;/i&gt; = 0.054; CD44 protein n/a because of no event in the CD44-negative group) in the HPV16 DNA–negative subgroup.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; We have shown for the first time that high hypoxia-induced gene expression and high CSC marker expression levels correlate with tumor recurrence after PORT-C in patients with HPV16 DNA–negative HNSCC. After validation in a currently ongoing prospective trial, these parameters may help to further stratify patients for individualized treatment de-escalation or intensification strategies. &lt;i&gt;Clin Cancer Res; 22(11); 2639–49. ©2016 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Table S1 Analyzed genes of the hypothesis-driven gene set. Table S2 Programs and packages used for evaluation. Figure S1 Results of repeated 3-fold cross validation for LRC. Figure S2 Mean oob ci depending of the signature size for LRC. Figure S3 Importance score for genes associated with LRC. Table S3 Increasing robustness by including highly correlated genes. Figure S4 Patient stratification by the 7-gene signature for LRC. Figure S5 Patient stratification by the 7-gene signature and clinical parameters for LRC. Table S4 Multivariable Cox regression of OS. Figure S6 Patient stratification by the 7-gene signature and clinical parameters for OS. Table S5 Multivariable Cox regression of DM. Figure S7 Patient stratification by the 7-gene signature and clinical parameters for DM. Table S6 Means and standard deviations of the 7-gene signature gene expressions. Table S7 Comparison of gene expressions between patient groups. Table S8 Hyper-parameters for feature selection algorithms and predictive models. Table S9 Patient characteristics of all patients. Figure S8 Identification of the final gene signature.&lt;/p&gt;

&lt;p&gt;A. Correlation of hypoxia gene-signatures and HPV16 DNA. B. Correlation of CSC marker expression and HPV16 DNA.&lt;/p&gt;

&lt;p&gt;Correlation of tumor hypoxia using nanoString vs PCR.&lt;/p&gt;

&lt;p&gt;A. Correlation of CSC marker expressions. B. Multivariate analyses of stem cell markers and additional prognostic factors for all patients. HR = hazard ratio; 95% CI = 95 percent confidence interval ; ECE = extracapsular extension. C. Correlation of CSC marker expression and HPV16 DNA.&lt;/p&gt;

&lt;p&gt;A. Patient characteristics. B. Hypoxia gene signatures.&lt;/p&gt;

&lt;p&gt;Supplementary discussion; Supplementary methods; Supplementary tables S1-S8; Supplementary figures S1-S8.&lt;/p&gt;

&lt;p&gt;Statistical Output&lt;/p&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;Tumor hypoxia is a paradigmatic negative prognosticator of treatment resistance in head and neck squamous cell carcinoma (HNSCC). The lack of robust and reliable hypoxia classifiers limits the adaptation of stratified therapies. We hypothesized that the tumor DNA methylation landscape might indicate epigenetic reprogramming induced by chronic intratumoral hypoxia.&lt;/p&gt;Experimental Design:&lt;p&gt;A DNA-methylome–based tumor hypoxia classifier (Hypoxia-M) was trained in the TCGA (The Cancer Genome Atlas)-HNSCC cohort based on matched assignments using gene expression–based signatures of hypoxia (Hypoxia-GES). Hypoxia-M was validated in a multicenter DKTK-ROG trial consisting of human papillomavirus (HPV)–negative patients with HNSCC treated with primary radiochemotherapy (RCHT).&lt;/p&gt;Results:&lt;p&gt;Although hypoxia-GES failed to stratify patients in the DKTK-ROG, Hypoxia-M was independently prognostic for local recurrence (HR, 4.3; &lt;i&gt;P&lt;/i&gt; = 0.001) and overall survival (HR, 2.34; &lt;i&gt;P&lt;/i&gt; = 0.03) but not distant metastasis after RCHT in both cohorts. Hypoxia-M status was inversely associated with CD8 T-cell infiltration in both cohorts. Hypoxia-M was further prognostic in the TCGA-PanCancer cohort (HR, 1.83; &lt;i&gt;P&lt;/i&gt; = 0.04), underscoring the breadth of this classifier for predicting tumor hypoxia status.&lt;/p&gt;Conclusions:&lt;p&gt;Our findings highlight an unexplored avenue for DNA methylation–based classifiers as biomarkers of tumoral hypoxia for identifying high-risk features in patients with HNSCC tumors.&lt;/p&gt;&lt;p&gt;&lt;i&gt;&lt;a href="https://aacrjournals.org/clincancerres/article/doi/10.1158/1078-0432.CCR-23-1132" target="_blank"&gt;See related commentary by Heft Neal and Brenner, p. 2954&lt;/a&gt;&lt;/i&gt;&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Supplementary Table S3. Distribution of clinical and biomarker-related characteristics between Methylation Hypoxia High vs Low in the training cohort (TCGA-HNSCC)&lt;/p&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;Tumor hypoxia is a paradigmatic negative prognosticator of treatment resistance in head and neck squamous cell carcinoma (HNSCC). The lack of robust and reliable hypoxia classifiers limits the adaptation of stratified therapies. We hypothesized that the tumor DNA methylation landscape might indicate epigenetic reprogramming induced by chronic intratumoral hypoxia.&lt;/p&gt;Experimental Design:&lt;p&gt;A DNA-methylome–based tumor hypoxia classifier (Hypoxia-M) was trained in the TCGA (The Cancer Genome Atlas)-HNSCC cohort based on matched assignments using gene expression–based signatures of hypoxia (Hypoxia-GES). Hypoxia-M was validated in a multicenter DKTK-ROG trial consisting of human papillomavirus (HPV)–negative patients with HNSCC treated with primary radiochemotherapy (RCHT).&lt;/p&gt;Results:&lt;p&gt;Although hypoxia-GES failed to stratify patients in the DKTK-ROG, Hypoxia-M was independently prognostic for local recurrence (HR, 4.3; &lt;i&gt;P&lt;/i&gt; = 0.001) and overall survival (HR, 2.34; &lt;i&gt;P&lt;/i&gt; = 0.03) but not distant metastasis after RCHT in both cohorts. Hypoxia-M status was inversely associated with CD8 T-cell infiltration in both cohorts. Hypoxia-M was further prognostic in the TCGA-PanCancer cohort (HR, 1.83; &lt;i&gt;P&lt;/i&gt; = 0.04), underscoring the breadth of this classifier for predicting tumor hypoxia status.&lt;/p&gt;Conclusions:&lt;p&gt;Our findings highlight an unexplored avenue for DNA methylation–based classifiers as biomarkers of tumoral hypoxia for identifying high-risk features in patients with HNSCC tumors.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Supplementary Methods, Results, Table of Contents&lt;/p&gt;

&lt;p&gt;Supplementary Figure S2. (Top) Hypoxia-M V2 is prognostic in the DKTK-ROG cohort. Patients predicted to have higher tumor hypoxia by Hypoxia-M V2 had higher rates of death (p=0.0097), local recurrence (p=0.0037), distant metastasis (p=0.027) and disease progression (p=0.00096). (Bottom) Hypoxia-M V2 remains an independent prognosticator of OS, LR and disease progression after adjusting for age, smoking status, GTV and anatomical site. Additionally, there is a trend towards higher rates of distant metastasis (HR=2.33, p=0.13)&lt;/p&gt;

&lt;p&gt;Supplementary Table S1. Multivariate Analysis for the Hypoxia-M and Brooks signature.&lt;/p&gt;

&lt;p&gt;Supplementary Figure S3. (Top) Hypoxia-M V2 is associated with decreased OS in the TCGApancancer cohort (p&lt;0.002) (Bottom) Hypoxia-M V2 is an independent prognosticator of OS (HR=1.81, p&lt;0.03) after adjusting for demographic and clinical factors.&lt;/p&gt;

&lt;p&gt;Supplementary Table S4. Correlations with CIBERSORT immune signature expressions in the TCGA-HNSCC cohort&lt;/p&gt;

&lt;p&gt;Supplementary Table S6. Contingency Table of Hypoxia-M and CD8-immunohistochemistry (IHC) staining for CD8-T cells&lt;/p&gt;

&lt;p&gt;Supplementary Figure 1. (Top) Venn Diagram showing the intersect between Hypoxia-M V2 significant DMPs (n=3397) and Hypoxia-M V1 significant DMPs (n=5129) 2551 (66%) of significant DMPs identified based on the 85th percentile of standard deviation are also included in the selection criteria of Hypoxia-M V1. (middle) Hypoxia-M V2 is prognostic in the TCGA HNSCC cohort (p=0.041) and the prognostic effect remained independent on multivariate analysis. (bottom) Multivariate Analysis revealed that patients predicted to have high Hypoxia by Hypoxia-M V2 had worsened OS outcomes (HR=1.59, p&lt;0.026). Additionally, increasing age and pN2 nodal status were negatively prognostic of OS (p&lt;0.05)&lt;/p&gt;