A diagnostic challenge of KIT p.V559D and BRAF p.G469A mutations in a paragastric mass.

A patient with gastrointestinal stroma tumor (GIST) and KIT p.V559D and BRAF p.G469A alterations was referred to our institutional molecular tumor board (MTB) to discuss therapeutic implications. The patient had been diagnosed with B-cell chronic lymphocytic leukemia (CLL) years prior to the MTB presentation. GIST had been diagnosed 1 month earlier. After structured clinical annotation of the molecular alterations and interdisciplinary discussion, we considered BRAF/KIT co-mutation unlikely in a treatment-naïve GIST. Discordant variant allele frequencies furthermore suggested a second malignancy. NGS of a CLL sample revealed the identical class 2 BRAF alteration, thus supporting admixture of CLL cells in the paragastric mass, leading to the detection of 2 alterations. Following the MTB recommendation, the patient received imatinib and had a radiographic response. Structured annotation and interdisciplinary discussion in specialized tumor boards facilitate the clinical management of complex molecular findings. Coexisting malignancies and clonal hematopoiesis warrant consideration in case of complex and uncommon molecular findings.

Liquid biopsy in head neck cancer: ready for clinical routine diagnostics?

PURPOSE OF REVIEW: The bodily fluids of patients with solid cancers representing a minimally-invasive source of clinically exploitable biomarkers have attracted an increasing amount of attention in recent years. In patients with head and neck squamous cell carcinoma (HNSCC), cell-free tumour DNA (ctDNA) belongs to the most promising liquid biomarkers for monitoring disease burden and identifying patients at high risk of recurrence. In this review, we highlight recent studies, evaluating the analytical validity and clinical utility of ctDNA as a dynamic biomarker in HNSCC, especially as it relates to risk stratification and contrasting human papilloma virus (HPV+ and HPV-) and carcinomas. RECENT FINDINGS: The clinical potential of minimal residual disease monitoring through viral ctDNA in identifying HPV+ oropharyngeal carcinoma patients at higher risk of recurrence has recently been demonstrated. Furthermore, accumulating evidence supports a potential diagnostic value of ctDNA dynamics in HPV-negative HNSCC. Altogether, recent data suggest that ctDNA analysis may be a valuable tool in guiding (de)escalation of surgical interventions as well as adaptation in radiotherapy dosage, both in the definitive and adjuvant settings. SUMMARY: Rigorous clinical trials with patient-relevant endpoints are critical in order to demonstrate that treatment decisions based on ctDNA dynamics result in better outcomes in HNSCC.

Tumor DNA-methylome derived epigenetic fingerprint identifies HPV-negative head and neck patients at risk for locoregional recurrence after postoperative radiochemotherapy.

Biomarkers with relevance for loco-regional therapy are needed in human papillomavirus negative aka HPV(-) head and neck squamous cell carcinoma (HNSCC). Based on the premise that DNA methylation pattern is highly conserved, we sought to develop a reliable and robust methylome-based classifier identifying HPV(-) HNSCC patients at risk for loco-regional recurrence (LR) and all-event progression after postoperative radiochemotherapy (PORT-C). The training cohort consisted of HPV-DNA negative HNSCC patients (n = 128) homogeneously treated with PORT-C in frame of the German Cancer Consortium-Radiation Oncology Group (DKTK-ROG) multicenter biomarker trial. DNA Methylation analysis was performed using Illumina 450 K and 850 K-EPIC microarray technology. The performance of the classifier was integrated with a series of biomarkers studied in the training set namely hypoxia-, 5-microRNA (5-miR), stem-cell gene-expression signatures and immunohistochemistry (IHC)-based immunological characterization of tumors (CD3/CD8/PD-L1/PD1). Validation occurred in an independent cohort of HPV(-) HNSCC patients, pooled from two German centers (n = 125). We identified a 38-methylation probe-based HPV(-) Independent Classifier of disease Recurrence (HICR) with high prognostic value for LR, distant metastasis and overall survival (P < 10-9 ). HICR remained significant after multivariate analysis adjusting for anatomical site, lymph node extracapsular extension (ECE) and size (T-stage). HICR high-risk tumors were enriched for younger patients with hypoxic tumors (15-gene signature) and elevated 5-miR score. After adjustment for hypoxia and 5-miR covariates, HICR maintained predicting all endpoints. HICR provides a novel mean for assessing the risk of LR in HPV(-) HNSCC patients treated with PORT-C and opens a new opportunity for biomarker-assisted stratification and therapy adaptation in these patients.

Regulation of the Receptor Tyrosine Kinase AXL in Response to Therapy and Its Role in Therapy Resistance in Glioblastoma.

The receptor tyrosine kinase AXL (RTK-AXL) is implicated in therapy resistance and tumor progression in glioblastoma multiforme (GBM). Here, we investigated therapy-induced receptor modifications and how endogenous RTK-AXL expression and RTK-AXL inhibition contribute to therapy resistance in GBM. GBM cell lines U118MG and SF126 were exposed to temozolomide (TMZ) and radiation (RTX). Receptor modifications in response to therapy were investigated on protein and mRNA levels. TMZ-resistant and RTK-AXL overexpressing cell lines were exposed to increasing doses of TMZ and RTX, with and without RTK-AXL tyrosine kinase inhibitor (TKI). Colorimetric microtiter (MTT) assay and colony formation assay (CFA) were used to assess cell viability. Results showed that the RTK-AXL shedding product, C-terminal AXL (CT-AXL), rises in response to repeated TMZ doses and under hypoxia, acts as a surrogate marker for radio-resistance. Endogenous RTX-AXL overexpression leads to therapy resistance, whereas combination therapy of TZM and RTX with TKI R428 significantly increases therapeutic effects. This data proves the role of RTK-AXL in acquired and intrinsic therapy resistance. By demonstrating that therapy resistance may be overcome by combining AXL TKI with standard treatments, we have provided a rationale for future study designs investigating AXL TKIs in GBM.

Distinct immune evasion in APOBEC-enriched, HPV-negative HNSCC.

Immune checkpoint inhibition leads to response in some patients with head and neck squamous cell carcinoma (HNSCC). Robust biomarkers are lacking to date. We analyzed viral status, gene expression signatures, mutational load and mutational signatures in whole exome and RNA-sequencing data of the HNSCC TCGA dataset (n = 496) and a validation set (DKTK MASTER cohort, n = 10). Public single-cell gene expression data from 17 HPV-negative HNSCC were separately reanalyzed. APOBEC3-associated TCW motif mutations but not total single nucleotide variant burden were significantly associated with inflammation. This association was restricted to HPV-negative HNSCC samples. An APOBEC-enriched, HPV-negative subgroup was identified, that showed higher T-cell inflammation and immune checkpoint expression, as well as expression of APOBEC3 genes. Mutations in immune-evasion pathways were also enriched in these tumors. Analysis of single-cell sequencing data identified expression of APOBEC3B and 3C genes in malignant cells. We identified an APOBEC-enriched subgroup of HPV-negative HNSCC with a distinct immunogenic phenotype, potentially mediating response to immunotherapy.

Radiosurgery and fractionated stereotactic body radiotherapy for patients with lung oligometastases.

BACKGROUND: Patients with oligometastatic disease can potentially be cured by using an ablative therapy for all active lesions. Stereotactic body radiotherapy (SBRT) is a non-invasive treatment option that lately proved to be as effective and safe as surgery in treating lung metastases (LM). However, it is not clear which patients benefit most and what are the most suitable fractionation regimens. The aim of this study was to analyze treatment outcomes after single fraction radiosurgery (SFRS) and fractionated SBRT (fSBRT) in patients with lung oligometastases and identify prognostic clinical features for better survival outcomes. METHODS: Fifty-two patients with 94 LM treated with SFRS or fSBRT between 2010 and 2016 were analyzed. The characteristics of primary tumor, LM, treatment, toxicity profiles and outcomes were assessed. Kaplan-Meier and Cox regression analyses were used for estimation of local control (LC), overall survival (OS) and progression-free survival. RESULTS: Ninety-four LM in 52 patients were treated using SFRS/fSBRT with a median of 2 lesions per patient (range: 1-5). The median planning target volume (PTV)-encompassing dose for SFRS was 24 Gy (range: 17-26) compared to 45 Gy (range: 20-60) in 2-12 fractions with fSBRT. The median follow-up time was 21 months (range: 3-68). LC rates at 1 and 2 years for SFSR vs. fSBRT were 89 and 83% vs. 75 and 59%, respectively (p = 0.026). LM treated with SFSR were significantly smaller (p = 0.001). The 1 and 2-year OS rates for all patients were 84 and 71%, respectively. In univariate analysis treatment with SFRS, an interval of ≥12 months between diagnosis of LM and treatment, non-colorectal cancer histology and BED < 100 Gy were significantly associated with better LC. However, none of these parameters remained significant in the multivariate Cox regression model. OS was significantly better in patients with negative lymph nodes (N0), Karnofsky performance status (KPS) > 70% and time to first metastasis ≥12 months. There was no grade 3 acute or late toxicity. CONCLUSIONS: Longer time to first metastasis, good KPS and N0 predicted better OS. Good LC and low toxicity rates were achieved after short SBRT schedules.

Novel prognostic clinical factors and biomarkers for outcome prediction in head and neck cancer: a systematic review.

Current algorithms for the clinical management of patients with squamous cell carcinoma of the head and neck (HNSCC) are based on a stage-dependent strategy where all patients at the same TNM stage receive the same treatment. Patient outcomes might be substantially improved by biomarker-guided treatment selection based on individual differences in the genetic and biological characteristics of tumours. Rapid technical advances enabling fast and affordable comprehensive molecular characterisation of tumours have led to increased knowledge of the molecular pathways involved in neoplastic transformation and disease progression in HNSCC. Despite notable successes in other tumour entities, the exploitation of molecular data for the improvement of tumour staging, prognosis, and individual treatment selection for patients with HNSCC has not yet become clinical routine. In this Review, we discuss and merge existing and new information on prognostic biomarkers for HNSCC, with the potential to improve clinical management of patients in the near future.

Morphomolecular analysis of the immune tumor microenvironment in human head and neck cancer.

Immunotherapy is effective in head and neck squamous cell carcinoma (HNSCC), but only a minority of patients responds to immune checkpoint blockade (ICB). To contribute to a better understanding of the underlying immune biology, we combined histomorphological evaluation and molecular analysis of the HNSCC immune microenvironment in the TCGA cohort. Analyzing digital HE-stained slides, a method for classification of tumor infiltrating lymphocytes (TILs) in the intra-epithelial compartment (ieTILs, present vs. absent) and the stromal compartment (strTILs, high vs. low) was established. We also analyzed the abundance of eight immune cell populations (estimated from RNAseq data) and PD-L1 mRNA expression. Status of ieTILs and status of strTILs were concordant for 61%, but discordant for 39% of tumors. In univariate survival analysis, ieTILs were a positive prognostic marker for DFS in the study cohort (HR = 0.66, p = 0.015) and in the HPV- subcohort (HR = 0.68, p = 0.04), but not in the HPV + subcohort. T cells were a positive prognostic marker for DFS in the study cohort (HR = 0.80, p = 0.03) and in the HPV + subcohort (HR = 0.20, p = 0.001), but not in the HPV- subcohort. In univariate survival analysis, PD-L1 mRNA expression was neither associated with DFS nor with OS. However, in bivariate and multivariate analyses including both PD-L1 mRNA levels and T cells, PD-L1 was a negative prognostic marker of DFS and OS, while T cells remained a positive prognostic marker. In conclusion, ieTILs and strTILs were non-redundant biomarkers in HNSCC and should be evaluated separately. The identified prognostic markers should be evaluated for predictivity in ICB-treated patients.

Basal subtype is predictive for response to cetuximab treatment in patient-derived xenografts of squamous cell head and neck cancer.

Cetuximab is the single targeted therapy approved for the treatment of head and neck cancer (HNSCC). Predictive biomarkers have not been established and patient stratification based on molecular tumor profiles has not been possible. Since EGFR pathway activation is pronounced in basal subtype, we hypothesized this activation could be a predictive signature for an EGFR directed treatment. From our patient-derived xenograft platform of HNSCC, 28 models were subjected to Affymetrix gene expression studies on HG U133+ 2.0. Based on the expression of 821 genes, the subtype of each of the 28 models was determined by integrating gene expression profiles through centroid-clustering with previously published gene expression data by Keck et al. The models were treated in groups of 5-6 animals with docetaxel, cetuximab, everolimus, cis- or carboplatin and 5-fluorouracil. Response was evaluated by comparing tumor volume at treatment initiation and after 3 weeks of treatment (RTV). Tumors distributed over the 3 signature-defined subtypes: 5 mesenchymal/inflamed phenotype (MS), 15 basal type (BA), 8 classical type (CL). Cluster analysis revealed a strong correlation between response to cetuximab and the basal subtype. RTV MS 3.32 vs. BA 0.78 (MS vs. BA, unpaired t-test, p 0.0002). Cetuximab responders were distributed as following: 1/5 in MS, 5/8 in CL and 13/15 in the BA group. Activity of classical chemotherapies did not differ between the subtypes. In conclusion basal subtype was associated with response to EGFR directed therapy in head and neck squamous cell cancer patient-derived xenografts.

CCI-779 (Temsirolimus) exhibits increased anti-tumor activity in low EGFR expressing HNSCC cell lines and is effective in cells with acquired resistance to cisplatin or cetuximab.

BACKGROUND: The mammalian target of rapamycin (mTOR) signaling pathway plays a pivotal role in numerous cellular processes involving growth, proliferation and survival. The purpose of this study was to investigate the anti-tumoral effect of the mTOR inhibitor (mTORi) CCI-779 in HNSCC cell lines and its potency in cisplatin- and cetuximab-resistant cells. METHODS: A panel of 10 HNSCC cell lines with differences in TP53 mutational status and basal cisplatin sensitivity and two isogenic models of acquired resistance to cisplatin and cetuximab, respectively were studied. Cell survival after treatment with CCI-779, cisplatin and cetuximab alone or in combination was determined by MTT assays. Potential predictive biomarkers for tumor cell sensitivity to CCI-779 were evaluated. RESULTS: We observed considerable heterogeneity in sensitivity of HNSCC cell lines to CCI-779 monotherapy. Sensitivity was observed in TP53 mutated as well as wild-type cell lines. Total and p-EGFR expression levels but not the basal activity of the mTOR and MAPK signaling pathways were correlated with sensitivity to CCI-779. Resistant cells with increased EGFR activation could be sensitized by the combination of CCI-779 with cetuximab. Interestingly, cell lines with acquired resistance to cisplatin displayed a higher sensitivity to CCI-779 whereas cetuximab-resistant cells were less sensitive to the drug, but could be sensitized to CCI-779 by EGFR blockade. CONCLUSIONS: Activity of CCI-779 in HNSCC cells harboring TP53 mutations and displaying a phenotype of cisplatin resistance suggests its clinical potential even in patients with dismal outcome after current standard treatment. Cetuximab/mTORi combinations might be useful for treatment of tumors with high expression of EGFR/p-EGFR and/or acquired cetuximab resistance. This combinatorial treatment modality needs further evaluation in future translational and clinical studies.

Molecular subtyping of head and neck cancer - Clinical applicability and correlations with morphological characteristics.

AIM: We aimed to evaluate the applicability of a customized NanoString panel for molecular subtyping of recurrent or metastatic head and neck squamous cell carcinoma (R/M-HNSCC). Additionally, histological analyses were conducted, correlated with the molecular subtypes and tested for their prognostic value. MATERIAL AND METHODS: We conducted molecular subtyping of R/M-HNSCC according to the molecular subtypes defined by Keck et al. For molecular analyses a 231 gene customized NanoString panel (the most accurately subtype defining genes, based on previous analyses) was applied to tumor samples from R/M-HNSCC patients that were treated in the CeFCiD trial (AIO/IAG-KHT trial 1108). A total of 130 samples from 95 patients were available for sequencing, of which 80 samples from 67 patients passed quality controls and were included in histological analyses. H&E stained slides were evaluated regarding distinct morphological patterns (e.g. tumor budding, nuclear size, stroma content). RESULTS: Determination of molecular subtypes led to classification of tumor samples as basal (n = 46, 45 %), inflamed/mesenchymal (n = 31, 30 %) and classical (n = 26, 25 %). Expression levels of Amphiregulin (AREG) were significantly higher for the basal and classical subtypes compared to the mesenchymal subtype. While molecular subtypes did not have an impact on survival, high levels of tumor budding were associated with poor outcomes. No correlation was found between molecular subtypes and histological characteristics. CONCLUSIONS: Utilizing the 231-gene NanoString panel we were able to determine the molecular subtype of R/M-HNSCC samples by the use of FFPE material. The value to stratify for different treatment options remains to be explored in the future. The prognostic value of tumor budding was underscored in this clinically well annotated cohort.

Spatial heterogeneity of tumor cells and the tissue microenvironment in oral squamous cell carcinoma.

PURPOSE: This study describes the morphologic and phenotypic spatial heterogeneity of tumor cells and the tissue microenvironment (TME), focusing on immune infiltration in OSCCs. STUDY DESIGN: Patients with OSCCs and planned surgical tumor resection were eligible for the study. Two biopsies each from the tumor center and the tumor rim were obtained. Immunohistochemical characterization of tumor and immune cells was performed using a panel of immunohistochemical markers. RESULTS: Thirty-six biopsies were obtained from the 9 patients. All patients showed an individual marker expression profile with ITH. Within the same biopsy, the CPS and TPS scores showed relevant variations in PD-L1 expression. Comparisons between the tumor center and rim revealed significant differences in the up/downregulation of p53. Marker expression of patients with recurrences clustered similarly, with the higher expression of FoxP3, IDO, CD4, CD68, and CD163 at the tumor rim. CONCLUSION: OSCCs were found to exhibit relevant ITH involving both tumor cells and TME, suggesting that biomarker analysis of multiple tumor regions may be helpful for clinical decision making and tumor characterization. The analysis of multiple spots within a biopsy is recommended for a reliable determination of PD-L1 expression and other biomarkers, impacting current clinical assessments.

Tumour mutational burden and survival with molecularly matched therapy.

BACKGROUND: The impact of tumour mutational burden (TMB) on outcome with molecularly matched therapy is unknown. Higher TMB could predict resistance to molecularly matched therapy through co-occurring driver mutations. METHODS: One hundred and four patients with advanced cancers underwent molecular profiling in the DKTK-MASTER program. Fifty-five patients received systemic therapy excluding immunotherapy. Patients with molecularly matched (n = 35) or non-molecularly informed therapy (n = 20) were analysed for TMB and survival. Results were validated in an independent cohort of patients receiving molecularly matched (n = 68) or non-molecularly informed therapy (n = 40). Co-occurring driver mutations and TMB were analysed in the exploratory cohort and The Cancer Genome Atlas (TCGA) datasets. RESULTS: Patients were stratified by the median TMB of 1.67 mutations per Megabase (mut/Mb) of 35 patients receiving molecularly matched therapy into TMB-high or TMB-low groups. Median overall survival (4 months [95% CI, 3.3-7.6] versus 12.8 months [95% CI, 10-not reached], p < 0.001) and progression-free survival (1.8 months [95% CI, 1.1-3.7] versus 7.9 months [95% CI, 2.8-17.0], p = 0.003) were significantly shorter in the TMB-high group compared to the TMB-low group. In the validation cohort, shorter OS and PFS were identified in the TMB-high group (TMB cut-off of 4 mut/Mb) treated with molecularly matched therapy. No differences were observed in patients receiving non-molecularly informed systemic therapy. A significant correlation between co-occurring driver mutations and TMB (n = 104, r = 0.78 [95% CI, 0.68-0.85], p < 0.001) was found in the exploratory cohort as well as the majority (24/33) of TCGA studies. CONCLUSION: A high TMB was associated with unfavourable outcome in patients receiving molecularly matched therapy, indicating untargeted resistance pathways. Therefore, TMB should be further investigated as a predictive biomarker in precision oncology programs.

Immune-related pan-cancer gene expression signatures of patient survival revealed by NanoString-based analyses.

The immune system plays a central role in the onset and progression of cancer. A better understanding of transcriptional changes in immune cell-related genes associated with cancer progression, and their significance in disease prognosis, is therefore needed. NanoString-based targeted gene expression profiling has advantages for deployment in a clinical setting over RNA-seq technologies. We analysed NanoString PanCancer Immune Profiling panel gene expression data encompassing 770 genes, and overall survival data, from multiple previous studies covering 10 different cancer types, including solid and blood malignancies, across 515 patients. This analysis revealed an immune gene signature comprising 39 genes that were upregulated in those patients with shorter overall survival; of these 39 genes, three (MAGEC2, SSX1 and ULBP2) were common to both solid and blood malignancies. Most of the genes identified have previously been reported as relevant in one or more cancer types. Using Cibersort, we investigated immune cell levels within individual cancer types and across groups of cancers, as well as in shorter and longer overall survival groups. Patients with shorter survival had a higher proportion of M2 macrophages and γδ T cells. Patients with longer overall survival had a higher proportion of CD8+ T cells, CD4+ T memory cells, NK cells and, unexpectedly, T regulatory cells. Using a transcriptomics platform with certain advantages for deployment in a clinical setting, our multi-cancer meta-analysis of immune gene expression and overall survival data has identified a specific transcriptional profile associated with poor overall survival.

DNA-Methylome-Based Tumor Hypoxia Classifier Identifies HPV-Negative Head and Neck Cancer Patients at Risk for Locoregional Recurrence after Primary Radiochemotherapy.

PURPOSE: 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. EXPERIMENTAL DESIGN: 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). RESULTS: 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. CONCLUSIONS: 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.

A simple multicolor flow cytometry protocol for detection and molecular characterization of circulating tumor cells in epithelial cancers.

Circulating tumor cells (CTCs) might not only serve as prognostic marker but could also be useful for monitoring treatment efficacy. A multicolor flow cytometry protocol for their detection and molecular characterization in peripheral blood was developed which consisted of erythrocyte lysis followed by staining of cells with fluorochrome-labeled antibodies against CD45 and the epithelial markers EpCam and cytokeratin 7/8. For reducing the number of events acquired by flow cytometry, an electronic threshold for the fluorescent signals from the epithelial markers was applied. After establishment of the protocol by using spiking experiments, its suitability to determine the absolute number of CTCs as well as their expression of epidermal growth factor receptor (EGFR) and its phosphorylated form (phospho-EGFR) in blood samples from patients with squamous cell carcinoma of the head and neck (SCCHN) was validated. Spiking experiments demonstrated an excellent recovery (mean 85%) and a linear performance (R(2) = 0.98) of the protocol. Sensitivity and specificity were comparable to our former protocol using immunomagnetic CTC pre-enrichment. The analysis of 33 SCCHN patient samples revealed the presence of CTCs in 33.3% of cases with a mean ± SD of 1.5 ± 0.5 CTCs per 3.75 ml blood. EGFR was expressed in 100% and phospho-EGFR in 36.4% of the CTC+ cases. We have established a simple and sensitive multicolor flow cytometry protocol for detection of CTCs in patients with epithelial cancers including SCCHN which will allow their detailed molecular characterization.

Circulating tumour cells escape from EpCAM-based detection due to epithelial-to-mesenchymal transition.

BACKGROUND: Circulating tumour cells (CTCs) have shown prognostic relevance in metastatic breast, prostate, colon and pancreatic cancer. For further development of CTCs as a biomarker, we compared the performance of different protocols for CTC detection in murine breast cancer xenograft models (MDA-MB-231, MDA-MB-468 and KPL-4). Blood samples were taken from tumour bearing animals (20 to 200 mm2) and analysed for CTCs using 1. an epithelial marker based enrichment method (AdnaTest), 2. an antibody independent technique, targeting human gene transcripts (qualitative PCR), and 3. an antibody-independent approach, targeting human DNA-sequences (quantitative PCR). Further, gene expression changes associated with epithelial-to-mesenchymal transition (EMT) were determined with an EMT-specific PCR assay. METHODS: We used the commercially available Adna Test, RT-PCR on human housekeeping genes and a PCR on AluJ sequences to detect CTCs in xenografts models. Phenotypic changes in CTCs were tested with the commercially available "Human Epithelial to Mesenchymal Transition RT-Profiler PCR Array". RESULTS: Although the AdnaTest detects as few as 1 tumour cell in 1 ml of mouse blood spiking experiments, no CTCs were detectable with this approach in vivo despite visible metastasis formation. The presence of CTCs could, however, be demonstrated by PCR targeting human transcripts or DNA-sequences - without epithelial pre-enrichment. The failure of CTC detection by the AdnaTest resulted from downregulation of EpCAM, whereas mesenchymal markers like Twist and EGFR were upregulated on CTCs. Such a change in the expression profile during metastatic spread of tumour cells has already been reported and was linked to a biological program termed epithelial-mesenchymal transition (EMT). CONCLUSIONS: The use of EpCAM-based enrichment techniques leads to the failure to detect CTC populations that have undergone EMT. Our findings may explain clinical results where low CTC numbers have been reported even in patients with late metastatic cancers. These results are a starting point for the identification of new markers for detection or capture of CTCs, including the mesenchymal-like subpopulations.

Multiparametric Phenotyping of Circulating Tumor Cells for Analysis of Therapeutic Targets, Oncogenic Signaling Pathways and DNA Repair Markers.

Detection of circulating tumor cells (CTCs) has been established as an independent prognostic marker in solid cancer. Multiparametric phenotyping of CTCs could expand the area of application for this liquid biomarker. We evaluated the Amnis® brand ImageStream®X MkII (ISX) (Luminex, Austin, TX, USA) imaging flow cytometer for its suitability for protein expression analysis and monitoring of treatment effects in CTCs. This was carried out using blood samples from patients with head and neck squamous cell carcinoma (n = 16) and breast cancer (n = 8). A protocol for negative enrichment and staining of CTCs was established, allowing quantitative analysis of the therapeutic targets PD-L1 and phosphorylated EGFR (phospho-EGFR), and the treatment response marker γH2AX as an indicator of radiation-induced DNA damage. Spiking experiments revealed a sensitivity of 73% and a specificity of 100% at a cut-off value of ≥3 CTCs, and thus confirmed the suitability of the ISX-based protocol to detect phospho-EGFR and γH2AX foci in CTCs. Analysis of PD-L1/-L2 in both spiked and patient blood samples further showed that assessment of heterogeneity in protein expression within the CTC population was possible. Further validation of the diagnostic potential of this ISX protocol for multiparametric CTC analysis in larger clinical cohorts is warranted.

Predictors for Adherence to Treatment Strategies in Elderly HNSCC Patients.

Finding a cure may be less important than ensuring the quality of life in elderly patients with head and neck squamous cell carcinoma (HNSCC). The aim of this study was to determine predictors for adherence. Clinical and pathological data from patients ≥70 years with HNSCC (initial diagnoses 2004-2018) were investigated retrospectively. Evaluated clinical predictors included biological age (Charlson Comorbidity Index; CCI), patient health (Karnofsky Performance Status; KPS) and therapy data. A total of 1125 patients were included. The median age was 75 years, 33.1% reached CCI ≥ 6, and 53.7% reached KPS ≤ 70%. In total, 968 patients were adherent, whereas 157 were nonadherent. Nonadherent patients were significantly more often smokers (p = 0.003), frequent drinkers (p = 0.001), had a worse health status (p ≤ 0.001) and a lower biological age (p = 0.003), an advanced T classification and lymph node involvement or UICC stage (each p ≤ 0.001). Approximately 88.0% of the included patients received a curative treatment recommendation. A total of 6.9% discontinued the therapy, and 7.0% refused the therapy. With the increasing complexity of a recommended therapy, adherence decreased. The 5-year overall survival was significantly higher in adherent patients (45.1% versus 19.2%). In contrast to the chronological patient age, biological age is a significant predictor for adherence. The evaluated predictors for nonadherence need to be verified prospectively.

Biomarker signatures for primary radiochemotherapy of locally advanced HNSCC - Hypothesis generation on a multicentre cohort of the DKTK-ROG.

PURPOSE: 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). MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.