Johannes H.A.M. Kaanders

Activation of the PI3-K/AKT pathway and implications for radioresistance mechanisms in head and neck cancer

Activation of the phosphatidylinositol-3-kinase (PI3-K)/protein kinase B (AKT) pathway is associated with three major radioresistance mechanisms: intrinsic radioresistance; tumour-cell proliferation; and hypoxia. Monitoring and manipulation of this signal-transduction pathway can have important implications for the management of head and neck cancer, because activation of the PI3-K/AKT pathway is a frequent event in these tumours. PI3-K/AKT signalling regulates cellular processes, including proliferation, invasion, apoptosis, and the upregulation of hypoxia-related proteins. Activation of this pathway can be caused by stimulation of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR). In clinical trials, a strong and independent association has been noted between expression of activated AKT and treatment outcome. Therefore, the search for molecular predictors of sensitivity to EGFR-directed treatment should be extended to markers of PI3-K/AKT activation. Another strategy might be the direct targeting and inhibition of this pathway. Such inhibition will enhance the efficacy of radiotherapy, by antagonising radiation-induced cellular defense mechanisms, especially in tumours that have activated the PI3-K/AKT cascade. Thus, the activation status of this pathway might be a key element for the prediction of treatment response and for therapeutic targeting in head and neck cancer.

Tumor hypoxia at the micro-regional level: clinical relevance and predictive value of exogenous and endogenous hypoxic cell markers.

BACKGROUND AND PURPOSE Tumor oxygenation is recognized as an important determinant of the outcome of radiotherapy and possibly also of other treatment modalities in a number of tumor types and in particular in squamous cell carcinomas. The hypoxic status of various solid tumors has been related to a poor prognosis due to tumor progression towards a more malignant phenotype, with increased metastatic potential, and an increased resistance to treatment. It has been demonstrated in head and neck cancer that hypoxic radioresistance can be successfully counteracted by hypoxia modifying approaches. The microregional distribution and the level of tumor hypoxia depend on oxygen consumption and temporal and spatial variations in blood supply. It is unclear if severely hypoxic cells can resume clonogenicity when O(2) and nutrients become available again as a result of (treatment related) changes in the tumor microenvironment. Non-terminally differentiated hypoxic cells that are capable of proliferation are important for outcome because of their resistance to radiotherapy and possibly other cytotoxic treatments. Various exogenous and endogenous markers for hypoxia are currently available and can be studied in relation to each other, the tumor architecture and the tumor microenvironment. Use of nitroimidazole markers with immunohistochemical detection allows studying tumor cell hypoxia at the microscopic level. Co-registration with other microenvironmental parameters, such as vascular architecture (vascular density), blood perfusion, tumor cell proliferation and apoptosis, offers the possibility to obtain a comprehensive functional image of tumor patho-physiology and to study the effects of different modalities of cancer treatment. CONCLUSION A number of functional microregional parameters have emerged that are good candidates for future use as indicators of tumor aggressiveness and treatment response. The key question is whether these parameters can be used as tools for selection of treatment strategies for individual patients. This requires testing of these markers in prospective randomized clinical trials comparing standard treatment against experimental treatments targeting the relevant microregional constituent.

ARCON: a novel biology-based approach in radiotherapy

Two mechanisms of radiotherapy resistance which are of major importance in various tumour types are tumour-cell repopulation and hypoxia. ARCON (accelerated radiotherapy with carbogen and nicotinamide) is a new therapeutic strategy that combines radiation treatment modifications, with the aim of counteracting these resistance mechanisms. To limit clonogenic repopulation during therapy, the overall duration of the radiotherapy is reduced, generally by delivering several fractions per day. This accelerated radiotherapy is combined with inhalation of hyperoxic gas to decrease diffusion-limited hypoxia, and nicotinamide, a vasoactive agent, to decrease perfusion-limited hypoxia. Preclinical studies have been done to test the enhancing effects of these three components of ARCON, individually and in combination, in several experimentally induced tumours and normal tissues. In a mouse mammary carcinoma, the tumour-control rate obtained with ARCON was the same as that with conventional treatment, but with a radiation dose almost 50% lower. Phase 1 and 2 clinical trials have shown the feasibility and tolerability of ARCON, and have produced promising results in terms of tumour control. In particular in cancers of the head and neck and bladder, the local tumour-control rates are higher than in other studies, and phase 3 trials for these tumour types are underway. In conjunction with these trials, hypoxia markers detectable by immunohistochemistry are being tested for their potential use in predictive assays to select patients for ARCON and other hypoxia-modifying therapies.

Targeting Hypoxia, HIF-1, and Tumor Glucose Metabolism to Improve Radiotherapy Efficacy

Radiotherapy, an important treatment modality in oncology, kills cells through induction of oxidative stress. However, malignant tumors vary in their response to irradiation as a consequence of resistance mechanisms taking place at the molecular level. It is important to understand these mechanisms of radioresistance, as counteracting them may improve the efficacy of radiotherapy. In this review, we describe how the hypoxia-inducible factor 1 (HIF-1) pathway has a profound effect on the response to radiotherapy. The main focus will be on HIF-1–controlled protection of the vasculature postirradiation and on HIF-1 regulation of glycolysis and the pentose phosphate pathway. This aberrant cellular metabolism increases the antioxidant capacity of tumors, thereby countering the oxidative stress caused by irradiation. From the results of translational studies and the first clinical phase I/II trials, it can be concluded that targeting HIF-1 and tumor glucose metabolism at several levels reduces the antioxidant capacity of tumors, affects the tumor microenvironment, and sensitizes various solid tumors to irradiation. Clin Cancer Res; 18(20); 5585–94. ©2012 AACR.

Method for quantitative mapping of dynamic MRI contrast agent uptake in human tumors.

A method is presented for the acquisition and analysis of dynamic contrast‐enhanced (DCE) MRI data, focused on the characterization of tumors in humans. Gadolinium (Gd) contrast was administered by bolus injection, and its effect was monitored in time by fast T1‐weighted MRI. A simple algorithm was developed for automatic extraction of the arterial input function (AIF) from the DCE‐MRI data. This AIF was used in the pixelwise pharmacokinetic determination of physiological vascular parameters in normal and tumor tissue. Maps were reconstructed to show the spatial distribution of parameter values. To test the reproducibility of the method 11 patients with different types of tumors were measured twice, and the rate of contrast agent uptake in the tumor was calculated. The results show that normalizing the DCE‐MRI data using individual coregistered AIFs, instead of one common AIF for all patients, substantially reduces the variation between successive measurements. It is concluded that the proposed method enables the reproducible assessment of contrast agent uptake rates. J. Magn. Reson. Imaging 2001;14:457–463.

18F-FLT PET Does Not Discriminate Between Reactive and Metastatic Lymph Nodes in Primary Head and Neck Cancer Patients

Repopulation of clonogenic tumor cells is inversely correlated with radiation treatment outcome in head and neck squamous cell carcinomas. A functional imaging tool to assess the proliferative activity of tumors could improve patient selection for treatment modifications and could be used for evaluation of early treatment response. The PET tracer 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) can image tumor cell proliferation before and during radiotherapy, and it may provide biologic tumor information useful in radiotherapy planning. In the present study, the value of 18F-FLT PET in determining the lymph node status in squamous cell carcinoma of the head and neck was assessed, with pathology as the gold standard. Methods: Ten patients with newly diagnosed stage II–IV squamous cell carcinoma of the head and neck underwent 18F-FLT PET before surgical tumor resection with lymph node dissection. Emission 18F-FLT PET and CT images of the head and neck were recorded and fused, and standardized uptake values (SUVs) were calculated. From all 18 18F-FLT PET-positive lymph node levels and from 8 18F-FLT PET-negative controls, paraffin-embedded lymph node sections were stained and analyzed for the endogenous proliferation marker Ki-67 and for the preoperatively administered proliferation marker iododeoxyuridine. The sensitivity, specificity, positive predictive value, and negative predictive value were calculated for 18F-FLT PET. Results: Primary tumor sites were oral cavity (n = 7), larynx (n = 2), and maxillary sinus (n = 1). Nine of the 10 patients examined had 18F-FLT PET-positive lymph nodes (SUVmean: median, 1.2; range, 0.8–2.9), but only 3 of these patients had histologically proven metastases. All metastatic lymph nodes showed Ki-67 and iododeoxyuridine staining in tumor cells. In the remaining 7 patients, there was abundant Ki-67 and iododeoxyuridine staining of B-lymphocytes in germinal centers in PET-positive lymph nodes, explaining the high rate of false-positive findings. The sensitivity, specificity, positive predictive value, and negative predictive value of 18F-FLT PET were 100%, 16.7%, 37.5%, and 100%, respectively. Conclusion: In head and neck cancer patients, 18F-FLT PET showed uptake in metastatic as well as in nonmetastatic reactive lymph nodes, the latter due to reactive B-lymphocyte proliferation. Because of the low specificity, 18F-FLT PET is not suitable for assessment of pretreatment lymph node status. This observation may also negatively influence the utility of 18F-FLT PET for early treatment response evaluation of small metastatic nodes.

Accelerated Radiotherapy With Carbogen and Nicotinamide for Laryngeal Cancer: Results of a Phase III Randomized Trial

PURPOSE To report the results from a randomized trial comparing accelerated radiotherapy (AR) with accelerated radiotherapy plus carbogen inhalation and nicotinamide (ARCON) in laryngeal cancer. PATIENTS AND METHODS Patients with cT2-4 squamous cell laryngeal cancer were randomly assigned to AR (68 Gy within 36 to 38 days) or ARCON. To limit the risk of laryngeal necrosis, ARCON patients received 64 Gy on the laryngeal cartilage. The primary end point was local control. Secondary end points were regional control, larynx preservation, toxicity, disease-free survival, and overall survival. In a translational side study, the hypoxia marker pimonidazole was used to assess the oxygenation status in tumor biopsies. RESULTS From April 2001 to February 2008, 345 patients were accrued. After a median follow-up of 44 months, local tumor control rate at 5 years was 78% for AR versus 79% for ARCON (P = .80), with larynx preservation rates of 84% and 87%, respectively (P = .48). The 5-year regional control was significantly better with ARCON (93%) compared with AR (86%, P = .04). The improvement in regional control was specifically observed in patients with hypoxic tumors and not in patients with well-oxygenated tumors (100% v 55%, respectively; P = .01). AR and ARCON produced equal levels of toxicity. CONCLUSION Despite lack of benefit in local tumor control for advanced laryngeal cancers, a significant gain in regional control rate, with equal levels of toxicity, was observed in favor of ARCON. The poor regional control of patients with hypoxic tumors is specifically countered by ARCON treatment.

Dynamics of tumor hypoxia measured with bioreductive hypoxic cell markers.

Abstract Ljungkvist, A. S. E., Bussink, J., Kaanders, J. H. A. M. and van der Kogel, A. J. Dynamics of Tumor Hypoxia Measured with Bioreductive Hypoxic Cell Markers. Radiat. Res. 167, 127–145 (2007). Hypoxic cells are common in tumors and contribute to malignant progression, distant metastasis and resistance to radiotherapy. It is well known that tumors are heterogeneous with respect to the levels and duration of hypoxia. Several strategies, including high-oxygen-content gas breathing, radiosensitizers and hypoxic cytotoxins, have been developed to overcome hypoxia-mediated radioresistance. However, with these strategies, an increased tumor control rate is often accompanied by more severe side effects. Consequently, development of assays for prediction of tumor response and early monitoring of treatment responses could reduce both over- and undertreatment, thereby avoiding unnecessary side effects. The purpose of this review is to discuss different assays for measurement of hypoxia that can be used to detect changes in oxygen tension. The main focus is on exogenous bioreductive hypoxia markers (2-nitroimidazoles) such as pimonidazole, CCI-103F, EF5 and F-misonidazole. These are specifically reduced and bind to macromolecules in viable hypoxic cells. A number of these bioreductive drugs are approved for clinical use and can be detected with methods ranging from noninvasive PET imaging (low resolution) to microscopic imaging of tumor sections (high resolution). If the latter are stained for multiple markers, hypoxia can be analyzed in relation to different microenvironmental parameters such as vasculature, proliferation and endogenous hypoxia-related markers, for instance HIF1α and CA-IX. In addition, temporal and spatial changes in hypoxia can be analyzed by consecutive injection of two different hypoxia markers. Therefore, bioreductive exogenous hypoxia markers are promising as tools for development of predictive assays or as tools for early treatment monitoring and validation of potential endogenous hypoxia markers.

18F-FLT PET/CT for Early Response Monitoring and Dose Escalation in Oropharyngeal Tumors

Accelerated tumor cell proliferation is an important mechanism adversely affecting therapeutic outcome in head and neck cancer. 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) is a PET tracer to noninvasively image tumor cell proliferation. The aims of this study were to monitor early tumor response based on repetitive 18F-FLT PET/CT scans and to identify subvolumes with high proliferative activity eligible for dose escalation. Methods: Ten patients with oropharyngeal tumors underwent an 18F-FLT PET/CT scan before and twice during radiotherapy. The primary tumor and metastatic lymph nodes (gross tumor volume, or GTV) were delineated on CT (GTVCT) and after segmentation of the PET signal using the 50% isocontour of the maximum signal intensity or an adaptive threshold based on the signal-to-background ratio (GTVSBR). GTVs were calculated, and similarity between GTVCT and GTVSBR was assessed. Within GTVSBR, the maximum and mean standardized uptake value (SUVmax and SUVmean, respectively) was calculated. Within GTVCT, tumor subvolumes with high proliferative activity based on the 80% isocontour (GTV80%) were identified for radiotherapy planning with dose escalation. Results: The GTVCT decreased significantly in the fourth week but not in the initial phase of treatment. SUVmax and SUVmean decreased significantly as early as 1 wk after therapy initiation and even further before the fourth week of treatment. For the primary tumor, the average (±SD) SUVmean of the GTVSBR was 4.7 ± 1.6, 2.0 ± 0.9, and 1.3 ± 0.2 for the consecutive scans (P < 0.0001). The similarity between GTVCT and GTVSBR decreased during treatment, indicating an enlargement of GTVSBR outside GTVCT caused by the increasing difficulty of segmenting tracer uptake in the tumor from the background and by proliferative activity in the nearby tonsillar tissue. GTV80% was successfully identified in all primary tumors and metastatic lymph nodes, and dose escalation based on the GTV80% was demonstrated to be technically feasible. Conclusion: 18F-FLT is a promising PET tracer for imaging tumor cell proliferation in head and neck carcinomas. Signal changes in 18F-FLT PET precede volumetric tumor response and are therefore suitable for early response assessment. Definition of tumor subvolumes with high proliferative activity and dose escalation to these regions are technically feasible.

CD44 Expression Predicts Local Recurrence after Radiotherapy in Larynx Cancer

Purpose: To find molecular markers from expression profiling data to predict recurrence of laryngeal cancer after radiotherapy. Experimental Design: We generated gene expression data on pre-treatment biopsies from 52 larynx cancer patients. Patients developing a local recurrence were matched for T-stage, subsite, treatment, gender and age with non-recurrence patients. Candidate genes were then tested by immunohistochemistry on tumor material from a second series of 76 patients. Both series comprised early stage cancer treated with radiotherapy alone. Finally, gene expression data of eight larynx cancer cell lines with known radiosensitivity were analyzed. Results: Nineteen patients with a local recurrence were matched with 33 controls. Gene sets for hypoxia, proliferation and intrinsic radiosensitivity did not correlate with recurrence, whereas expression of the putative stem cell marker CD44 did. In a supervised analysis, probes for all three splice variants of CD44 on the array appeared in the top 10 most significantly correlated with local recurrence. Immunohistochemical analysis of CD44 expression on the independent validation series confirmed CD44s predictive potential. In 8 larynx cancer cell lines, CD44 gene expression did not correlate with intrinsic radiosensitivity although it did correlate significantly with plating efficiency, consistent with a relationship with stem cell content. Conclusions: CD44 was the only biological factor tested which significantly correlated with response to radiotherapy in early stage larynx cancer patients, both at the mRNA and protein levels. Further studies are needed to confirm this and to assess how general these findings are for other head and neck tumor stages and sites. Clin Cancer Res; 16(21); 5329–38. ©2010 AACR.