Daniel Zips

Exploratory prospective trial of hypoxia-specific PET imaging during radiochemotherapy in patients with locally advanced head-and-neck cancer

PURPOSE To explore in a prospective trial the prognostic value of hypoxia imaging before and during radiochemotherapy in patients with locally advanced head and neck cancer. PATIENTS AND METHODS Twenty-five patients with stage III/IV head and neck cancer were investigated with [(18)F]-fluoromisonidazole (FMISO) PET/CT at four time points during radiochemotherapy (baseline, 8-10 Gy, 18-20 Gy,50-60 Gy). FMISO PET/CT image parameters were extracted including maximum-tumour-to-background (TBR(max)) and thresholded volume at different TBR ratios. CT volume and baseline FDG-PET/CT image parameters were also included. Parameters at all time points were investigated for their prognostic value with the local-progression-free-survival endpoint (LPFS). Significance was evaluated with multivariate Cox (including clinical parameters) and Log-rank tests. RESULTS FMISO-image parameters were found to have a strong association with the LPFS endpoint, and were strongest at the week 1 and 2 time points (p = 0.023-0.048 and 0.042-0.061 respectively on multivariate Cox). Parameters measured at baseline were only significant on univariate analysis. None of the clinical parameters, and also FDG- or CT-delineated volumes, were significantly associated with LPFS. CONCLUSION This prospective, exploratory study demonstrated that FMISO-PET/CT imaging during the initial phase of treatment carries strong prognostic value. FMISO-PET/CT imaging at 1 or 2 weeks during treatment could be promising way to select patients that would benefit from hypoxia modification or dose-escalated treatment. A validation study is on-going.

Radiation oncology in the era of precision medicine

Technological advances and clinical research over the past few decades have given radiation oncologists the capability to personalize treatments for accurate delivery of radiation dose based on clinical parameters and anatomical information. Eradication of gross and microscopic tumours with preservation of health-related quality of life can be achieved in many patients. Two major strategies, acting synergistically, will enable further widening of the therapeutic window of radiation oncology in the era of precision medicine: technology-driven improvement of treatment conformity, including advanced image guidance and particle therapy, and novel biological concepts for personalized treatment, including biomarker-guided prescription, combined treatment modalities and adaptation of treatment during its course.

Glycolytic metabolism and tumour response to fractionated irradiation

BACKGROUND AND PURPOSE To study whether pre-therapeutic lactate or pyruvate predict for tumour response to fractionated irradiation and to identify possible coherencies between intermediates of glycolysis and expression levels of selected proteins. MATERIALS AND METHODS Concentrations of lactate, pyruvate, glucose and ATP were quantified via bioluminescence imaging in tumour xenografts derived from 10 human head and neck squamous cell carcinoma (HNSCC) lines. Tumours were irradiated with 30 fractions within 6 weeks. Expression levels of the selected proteins in tumours were measured at the mRNA and protein level. Tumour-infiltrating leucocytes were quantified after staining for CD45. RESULTS Lactate but not pyruvate concentrations were significantly correlated with tumour response to fractionated irradiation. Lactate concentrations in vivo did not reflect lactate production rates in vitro. Metabolite concentrations did not correlate with GLUT1, PFK-L or LDH-A at the transcriptional or protein level. CD45-positive cell infiltration was low in the majority of tumours and did not correlate with lactate concentration. CONCLUSIONS Our data support the hypothesis that the antioxidative capacity of lactate may contribute to radioresistance in malignant tumours. Non-invasive imaging of lactate to monitor radiation response and testing inhibitors of glycolysis to improve outcome after fractionated radiotherapy warrant further investigations.

Cancer stem cells and radiotherapy

Purpose: The present work summarises the history and current status of research into the importance of cancer stem cells for radiobiological research and for clinical radiation oncology. An effort is made to differentiate clonogenicity from stemness of cancer cells. Conclusion: In radiooncology, cancer stem cells have been an important research field for five decades. Quantitative transplantation assays with evaluation of the take dose 50% (TD50) remain the gold standard to verify the stemness of the selected cells. New technologies allow sorting of tumour cells according to their surface marker expression and thereby selecting subpopulations that are enriched in cancer stem cells (e.g., CD133, CD44, CD29). While development of surface-marker-based assays is a highly important step in cancer-stem-cell research, to date there are still problems to be solved, e.g., the specifity of markers, adequate animal models, and optimised in vitro assays. Of special concern for radiobiology is that clonogenic in vitro assays do not necessarily measure stemness of cancer cells. This hampers investigations into the important question of whether cancer stem cells are more radioresistant than non-stem cells. The most extensive of the limited data on this topic relate to glioma stem cells identified by the surface marker CD133. These do not provide firm evidence for difference of radiosensitivity between stem and non stem cells. In spite of many problems to be solved, the combination of stem cell markers with radiobiological assays bears considerable promise for advancing translational research in radiation oncology.

PINCH1 regulates Akt1 activation and enhances radioresistance by inhibiting PP1α

Tumor cell resistance to ionizing radiation and chemotherapy is a major obstacle in cancer therapy. One factor contributing to this is integrin-mediated adhesion to ECM. The adapter protein particularly interesting new cysteine-histidine-rich 1 (PINCH1) is recruited to integrin adhesion sites and promotes cell survival, but the mechanisms underlying this effect are not well understood. Here we have shown that PINCH1 is expressed at elevated levels in human tumors of diverse origins relative to normal tissue. Furthermore, PINCH1 promoted cell survival upon treatment with ionizing radiation in vitro and in vivo by perpetuating Akt1 phosphorylation and activity. Mechanistically, PINCH1 was found to directly bind to protein phosphatase 1alpha (PP1alpha) - an Akt1-regulating protein - and inhibit PP1alpha activity, resulting in increased Akt1 phosphorylation and enhanced radioresistance. Thus, our data suggest that targeting signaling molecules such as PINCH1 that function downstream of focal adhesions (the complexes that mediate tumor cell adhesion to ECM) may overcome radio- and chemoresistance, providing new therapeutic approaches for cancer.

Combination of EGFR/HER2 Tyrosine Kinase Inhibition by BIBW 2992 and BIBW 2669 with Irradiation in FaDu Human Squamous Cell Carcinoma

Purpose:To investigate the effect of the dual EGFR/HER2 (ErbB2) tyrosine kinase inhibitors BIBW 2992 and BIBW 2669 on proliferation and clonogenic cell survival of FaDu human squamous cell carcinoma in vitro, and on tumor growth after single-dose irradiation in nude mice.Material and Methods:Cell proliferation, cell-cycle distribution and clonogenic cell survival after irradiation were assayed with and without BIBW 2992 or BIBW 2669 (3, 30, and 300 nM) in vitro. Tumor volume and tumor growth delay (GDV2) were determined in tumors growing in NMRI (nu/nu) nude mice, treated with (a) BIBW 2992 (20 mg kg–1 body weight orally), BIBW 2669 (3–4 mg kg–1 body weight orally) or carrier until a final tumor diameter of 15 mm, or, (b) 3 days before a 20-Gy single-dose irradiation or, (c) after a 20-Gy single-dose irradiation until reaching the final tumor diameter.Results:BIBW 2992 and BIBW 2669 significantly increased the doubling time of FaDu cells in vitro. A marked dose-dependent antiproliferative effect with blockade of the cells in G0/G1-phase of the cell cycle was found. Incubation with BIBW 2669 or BIBW 2992 for 3 days marginally increased radiosensitivity of FaDu cells in vitro. For BIBW 2992, this effect was statistically significant (p = 0.006). Daily oral application of BIBW 2669 or BIBW 2992 in mice bearing unirradiated FaDu tumors showed a marked antiproliferative effect with a significant prolongation of tumor growth delay (p < 0.0001). After drug application for 3 days, followed by 20-Gy single-dose irradiation, a slight effect of both drugs on tumor growth delay was seen. For BIBW 2669, this effect was statistically significant (p = 0.007). However, this effect disappeared when tumor volumes were normalized to the time point of irradiation suggesting that both drugs showed no or only a slight radiosensitizing effect in vivo. Daily application of BIBW 2669 or BIBW 2992 after a single-dose irradiation showed a clear inhibition of tumor growth with a significantly longer tumor growth delay after drug treatment compared to control tumors (p < 0.002). Enhancement ratios were smaller for irradiated than for unirradiated tumors, suggesting an additive effect for combinations with radiotherapy. In all treatment arms, the effects of BIBW 2669 were not significantly different from BIBW 2992.Conclusion:BIBW 2669 and BIBW 2992 showed a clear antiproliferative effect in vitro, whereas radiosensitization was only marginal. The present data are the first to show an effect of combined irradiation and dual EGFR/ErbB2 inhibition on tumor growth delay in vivo. Further preclinical investigations using fractionated irradiation schedules and local tumor control as experimental endpoint are needed to evaluate a possible curative potential for the combination treatment.Ziel:In der vorliegenden Arbeit wurde die Wirkung der neuen dualen EGFR/HER2-Tyrosinkinaseinhibitoren BIBW 2992 und BIBW 2669 auf die Zellproliferation und das klonogene Zellüberleben in der humanen Plattenepithelkarzinomlinie FaDu in vitro sowie auf das Tumorwachstum und die Tumorwachstumsverzögerung nach Einzeldosisbestrahlung in vivo untersucht.Material und Methodik:Zellproliferation, Zellzyklusverteilung und klonogenes Zellüberleben nach Bestrahlung wurden mit und ohne BIBW 2992 oder BIBW 2669 (3, 30 und 300 nM) in vitro untersucht. In NMRI-(nu/nu-)Nacktmäusen wurden Tumorvolumen und Tumorwachstumsverzögerung (GDV2) nach a) alleiniger Applikation von BIBW 2992 (20 mg kg–1 KG oral), BIBW 2669 (3–4 mg kg–1 KG oral) oder Kontrollsubstanz bis zur Tumorendgröße von 15 mm, b) 3-tägiger Substanzapplikation und folgender 20-Gy-Einzeldosisbestrahlung und c) 20-Gy-Einzeldosisbestrahlung bis zur Tumorendgröße bestimmt.Ergebnisse:BIBW 2992 und BIBW 2669 führten zu einer signifikanten Verlängerung der Verdopplungszeit von FaDu-Zellen in vitro (Abbildung 1, Tabelle 1). Der ausgeprägte dosisabhängige antiproliferative Effekt ging mit einem G0/G1-Block einher (Abbildungen 1 und 2). Die Inkubation mit BIBW 2669 und BIBW 2992 führte zu einer geringen Erhöhung der Strahlenempfindlichkeit von FaDu-Zellen in vitro (Abbildung 3). Dieser Effekt war für BIBW 2992 statistisch signifikant (p = 0,006). Die tägliche orale Applikation von BIBW 2669 oder BIBW 2992 führte bei unbestrahlten Tumoren in vivo zu einem deutlichen proliferationshemmenden Effekt (Abbildung 4) mit signifikanter Verlängerung der Tumorwachstumsverzögerung (p < 0,0001; Abbildung 6, Tabelle 2). Eine 3-tägige Substanzapplikation und anschließende 20-Gy-Einzeldosisbestrahlung zeigten einen geringen Effekt auf die Tumorwachstumsverzögerung. Für BIBW 2669 war dieser Effekt signifikant (p = 0,007). Der Effekt verschwand, wenn die Tumorvolumina zum Zeitpunkt der Bestrahlung normiert wurden (Abbildung 5). Für beide Substanzen konnte somit kein oder nur ein geringer strahlensensitivierender Effekt in vivo nachgewiesen werden. Eine Einzeldosisbestrahlung mit 20 Gy und anschließender Substanzapplikation bis zur Tumorendgröße führte zusätzlich zum Effekt der Bestrahlung zu einem deutlichen proliferationshemmenden Effekt mit signifikanter Verlängerung der Tumorwachstumsverzögerung im Vergleich zu Kontrolltumoren (p < 0,002). Die Verstärkungsratios waren bei bestrahlten Tumoren geringer als bei unbestrahlten Tumoren, was auf einen additiven Effekt der Substanzen schließen lässt. In allen Behandlungsarmen konnte zwischen BIBW 2669 und BIBW 2992 kein signifikanter Unterschied gefunden werden.Schlussfolgerung:BIBW 2669 und BIBW 2992 zeigten einen deutlichen antiproliferativen Effekt in vitro bei nur geringer Strahlensensitivierung. Die vorliegenden Daten haben erstmals die Wirkung einer kombinierten Bestrahlung und dualen EGFR/ ErbB2-Inhibition auf die Verzögerung des Tumorwachstums in vivo gezeigt. Weitere präklinische Untersuchungen mit einem fraktionierten Bestrahlungsschema und lokaler Tumorkontrolle als Endpunkt sind nötig, um ein mögliches kuratives Potential von BIBW 2669 oder BIBW 2992 in Kombination mit Strahlentherapie zu untersuchen.

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)

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.

Enhanced Susceptibility of Irradiated Tumor Vessels to Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibition

Previous experiments with PTK787/ZK222584, a specific inhibitor of vascular endothelial growth factor receptor (VEGFR) tyrosine kinases, using irradiated human FaDu squamous cell carcinoma in nude mice, suggested that radiation-damaged tumor vessels are more sensitive to VEGFR inhibition. To test this hypothesis, the tumor transplantation site (i.e., the right hind leg of nude mice) was irradiated 10 days before transplantation of FaDu to induce radiation damage in the host tissue. FaDu tumors vascularized by radiation-damaged blood vessels appeared later, grew at a slower rate, and showed more necrosis and a smaller vessel area per central tumor section than controls. PTK787/ZK222584 at a daily dose of 50 mg/kg body weight had no impact on growth of control tumors. In contrast, tumors vascularized by radiation-damaged vessels responded to PTK787/ZK222584 with longer latency and slower growth rate than controls, and a trend toward further increase in necrosis, indicating that irradiated tumor vessels are more susceptible to VEGFR inhibition than unirradiated vessels. Although not proving causality, expression analysis of VEGF and VEGFR2 shows that enhanced sensitivity of irradiated vessels to a specific inhibitor of VEGFR tyrosine kinases correlates with increased expression of the molecular target.

A strategy for multimodal deformable image registration to integrate PET/MR into radiotherapy treatment planning

Abstract Background. Combined positron emission tomography (PET)/magnetic resonance imaging (MRI) is highly promising for biologically individualized radiotherapy (RT). Hence, the purpose of this work was to develop an accurate and robust registration strategy to integrate combined PET/MR data into RT treatment planning. Material and methods. Eight patient datasets consisting of an FDG PET/computed tomography (CT) and a subsequently acquired PET/MR of the head and neck (HN) region were available. Registration strategies were developed based on CT and MR data only, whereas the PET components were fused with the resulting deformation field. Following a rigid registration, deformable registration was performed with a transform parametrized by B-splines. Three different optimization metrics were investigated: global mutual information (GMI), GMI combined with a bending energy penalty (BEP) for regularization (GMI+ BEP) and localized mutual information with BEP (LMI+ BEP). Different quantitative registration quality measures were developed, including volumetric overlap and mean distance measures for structures segmented on CT and MR as well as anatomical landmark distances. Moreover, the local registration quality in the tumor region was assessed by the normalized cross correlation (NCC) of the two PET datasets. Results. LMI+ BEP yielded the most robust and accurate registration results. For GMI, GMI+ BEP and LMI+ BEP, mean landmark distances (standard deviations) were 23.9 mm (15.5 mm), 4.8 mm (4.0 mm) and 3.0 mm (1.0 mm), and mean NCC values (standard deviations) were 0.29 (0.29), 0.84 (0.14) and 0.88 (0.06), respectively. Conclusion. Accurate and robust multimodal deformable image registration of CT and MR in the HN region can be performed using a B-spline parametrized transform and LMI+ BEP as optimization metric. With this strategy, biologically individualized RT based on combined PET/MRI in terms of dose painting is possible.

Identification of Patient Benefit From Proton Therapy for Advanced Head and Neck Cancer Patients Based on Individual and Subgroup Normal Tissue Complication Probability Analysis

PURPOSE The purpose of this study was to determine, by treatment plan comparison along with normal tissue complication probability (NTCP) modeling, whether a subpopulation of patients with head and neck squamous cell carcinoma (HNSCC) could be identified that would gain substantial benefit from proton therapy in terms of NTCP. METHODS AND MATERIALS For 45 HNSCC patients, intensity modulated radiation therapy (IMRT) was compared to intensity modulated proton therapy (IMPT). Physical dose distributions were evaluated as well as the resulting NTCP values, using modern models for acute mucositis, xerostomia, aspiration, dysphagia, laryngeal edema, and trismus. Patient subgroups were defined based on primary tumor location. RESULTS Generally, IMPT reduced the NTCP values while keeping similar target coverage for all patients. Subgroup analyses revealed a higher individual reduction of swallowing-related side effects by IMPT for patients with tumors in the upper head and neck area, whereas the risk reduction of acute mucositis was more pronounced in patients with tumors in the larynx region. More patients with tumors in the upper head and neck area had a reduction in NTCP of more than 10%. CONCLUSIONS Subgrouping can help to identify patients who may benefit more than others from the use of IMPT and, thus, can be a useful tool for a preselection of patients in the clinic where there are limited PT resources. Because the individual benefit differs within a subgroup, the relative merits should additionally be evaluated by individual treatment plan comparisons.