Quantitative Imaging of Hypoxic CAIX-Positive Tumor Areas with Low Immune Cell Infiltration in Syngeneic Mouse Tumor Models.

Limited diffusion of oxygen in combination with increased oxygen consumption leads to chronic hypoxia in most solid malignancies. This scarcity of oxygen is known to induce radioresistance and leads to an immunosuppressive microenvironment. Carbonic anhydrase IX (CAIX) is an enzyme functioning as a catalyzer for acid export in hypoxic cells and is an endogenous biomarker for chronic hypoxia. The aim of this study is to develop a radiolabeled antibody that recognizes murine CAIX to visualize chronic hypoxia in syngeneic tumor models and to study the immune cell population in these hypoxic areas. An anti-mCAIX antibody (MSC3) was conjugated to diethylenetriaminepentaacetic acid (DTPA) and radiolabeled with indium-111 (111In). CAIX expression on murine tumor cells was determined using flow cytometry, and in vitro affinity of [111In]In-MSC3 was analyzed in a competitive binding assay. Ex vivo biodistribution studies were performed to determine in vivo radiotracer distribution. CAIX+ tumor fractions were determined by mCAIX microSPECT/CT, and the tumor microenvironment was analyzed using immunohistochemistry and autoradiography. We showed that [111In]In-MSC3 binds to CAIX-expressing (CAIX+) murine cells in vitro and accumulates in CAIX+ areas in vivo. We optimized the use of [111In]In-MSC3 for preclinical imaging such that it can be applied in syngeneic mouse models and showed that we can quantitatively distinguish between tumor models with varying CAIX+ fractions by ex vivo analyses and in vivo mCAIX microSPECT/CT. Analysis of the tumor microenvironment identified these CAIX+ areas as less infiltrated by immune cells. Together these data demonstrate that mCAIX microSPECT/CT is a sensitive technique to visualize hypoxic CAIX+ tumor areas that exhibit reduced infiltration of immune cells in syngeneic mouse models. In the future, this technique may enable visualization of CAIX expression before or during hypoxia-targeted or hypoxia-reducing treatments. Thereby, it will help optimize immuno- and radiotherapy efficacy in translationally relevant syngeneic mouse tumor models.

Novel VHH-Based Tracers with Variable Plasma Half-Lives for Imaging of CAIX-Expressing Hypoxic Tumor Cells.

Hypoxic areas are present in the majority of solid tumors, and hypoxia is associated with resistance to therapies and poor outcomes. A transmembrane protein that is upregulated by tumor cells that have adapted to hypoxic conditions is carbonic anhydrase IX (CAIX). Therefore, noninvasive imaging of CAIX could be of prognostic value, and it could steer treatment strategies. The aim of this study was to compare variants of CAIX-binding VHH B9, with and without a C-terminal albumin-binding domain with varying affinity (ABDlow and ABDhigh), for SPECT imaging of CAIX expression. The binding affinity and internalization of the various B9-variants were analyzed using SK-RC-52 cells. Biodistribution studies were performed in mice with subcutaneous SCCNij153 human head and neck cancer xenografts. Tracer uptake was determined by ex vivo radioactivity counting and visualized by SPECT/CT imaging. Furthermore, autoradiography images of tumor sections were spatially correlated with CAIX immunohistochemistry. B9-variants demonstrated a similar moderate affinity for CAIX in vitro. Maximal tumor uptake and acceptable tumor-to-blood ratios were found in the SCCNij153 model at 4 h post injection for [111In]In-DTPA-B9 (0.51 ± 0.08%ID/g and 8.1 ± 0.85, respectively), 24 h post injection for [111In]In-DTPA-B9-ABDlow (2.39 ± 0.44%ID/g and 3.66 ± 0.81, respectively) and at 72 h post injection for [111In]In-DTPA-B9-ABDhigh (8.7 ± 1.34%ID/g and 2.43 ± 0.15, respectively). An excess of unlabeled monoclonal anti-CAIX antibody efficiently inhibited tumor uptake of [111In]In-DTPA-B9, while only a partial reduction of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh uptake was found. Immunohistochemistry and autoradiography images showed colocalization of all B9-variants with CAIX expression; however, [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh also accumulated in non-CAIX expressing regions. Tumor uptake of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh, but not of [111In]In-DTPA-B9, could be visualized with SPECT/CT imaging. In conclusion, [111In]In-DTPA-B9 has a high affinity to CAIX and shows specific targeting to CAIX in head and neck cancer xenografts. The addition of ABD prolonged plasma half-life, increased tumor uptake, and enabled SPECT/CT imaging. This uptake was, however, partly CAIX- independent, precluding the ABD-tracers for use in hypoxia quantification in this tumor type.

Radiotherapy and cGAS/STING signaling: Impact on MDSCs in the tumor microenvironment.

Myeloid derived suppressor cells (MDSCs) are a highly heterogeneous population of immature immune cells with immunosuppressive functions that are recruited to the tumor microenvironment (TME). MDSCs promote tumor growth and progression by inhibiting immune effector cell proliferation and function. MDSCs are affected by both novel anti-cancer therapies targeting the immune system to promote anti-tumor immunity, as well as by conventional treatments such as radiotherapy. Following radiotherapy, cytoplasmic double stranded DNA stimulates the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway, resulting in type I interferon production. Effectiveness of radiotherapy and cGAS/STING signaling are closely intertwined: activation of cGAS and STING is key to generate systemic anti-tumor immunity after irradiation. This review focuses on how radiotherapy and cGAS/STING signaling in MDSCs and/or tumor cells impact MDSC recruitment, expansion and function. The influence of conventional and ablative radiotherapy treatment schedules, inflammatory response following radiotherapy, and hypoxia are discussed as MDSC modulators.

Stereotactic ablative body radiotherapy (SABR) combined with immunotherapy (L19-IL2) versus standard of care in stage IV NSCLC patients, ImmunoSABR: a multicentre, randomised controlled open-label phase II trial.

BACKGROUND: About 50% of non-small cell lung cancer (NSCLC) patients have metastatic disease at initial diagnosis, which limits their treatment options and, consequently, the 5-year survival rate (15%). Immune checkpoint inhibitors (ICI), either alone or in combination with chemotherapy, have become standard of care (SOC) for most good performance status patients. However, most patients will not obtain long-term benefit and new treatment strategies are therefore needed. We previously demonstrated clinical safety of the tumour-selective immunocytokine L19-IL2, consisting of the anti-ED-B scFv L19 antibody coupled to IL2, combined with stereotactic ablative radiotherapy (SABR). METHODS: This investigator-initiated, multicentric, randomised controlled open-label phase II clinical trial will test the hypothesis that the combination of SABR and L19-IL2 increases progression free survival (PFS) in patients with limited metastatic NSCLC. One hundred twenty-six patients will be stratified according to their metastatic load (oligo-metastatic: ≤5 or poly-metastatic: 6 to 10) and randomised to the experimental-arm (E-arm) or the control-arm (C-arm). The C-arm will receive SOC, according to the local protocol. E-arm oligo-metastatic patients will receive SABR to all lesions followed by L19-IL2 therapy; radiotherapy for poly-metastatic patients consists of irradiation of one (symptomatic) to a maximum of 5 lesions (including ICI in both arms if this is the SOC). The accrual period will be 2.5-years, starting after the first centre is initiated and active. Primary endpoint is PFS at 1.5-years based on blinded radiological review, and secondary endpoints are overall survival, toxicity, quality of life and abscopal response. Associative biomarker studies, immune monitoring, CT-based radiomics, stool collection, iRECIST and tumour growth rate will be performed. DISCUSSION: The combination of SABR with or without ICI and the immunocytokine L19-IL2 will be tested as 1st, 2nd or 3rd line treatment in stage IV NSCLC patients in 14 centres located in 6 countries. This bimodal and trimodal treatment approach is based on the direct cytotoxic effect of radiotherapy, the tumour selective immunocytokine L19-IL2, the abscopal effect observed distant from the irradiated metastatic site(s) and the memory effect. The first results are expected end 2023. TRIAL REGISTRATION: ImmunoSABR Protocol Code: NL67629.068.18; EudraCT: 2018-002583-11; Clinicaltrials.gov: NCT03705403; ISRCTN ID: ISRCTN49817477; Date of registration: 03-April-2019.

Poor outcome in hypoxic endometrial carcinoma is related to vascular density.

BACKGROUND: Identification of endometrial carcinoma (EC) patients at high risk of recurrence is lacking. In this study, the prognostic role of hypoxia and angiogenesis was investigated in EC patients. METHODS: Tumour slides from EC patients were stained by immunofluorescence for carbonic anhydrase IX (CAIX) as hypoxic marker and CD34 for assessment of microvessel density (MVD). CAIX expression was determined in epithelial tumour cells, with a cut-off of 1%. MVD was assessed according to the Weidner method. Correlations with disease-specific survival (DSS), disease-free survival (DFS) and distant disease-free survival (DDFS) were calculated using Kaplan-Meier curves and Cox regression analysis. RESULTS: Sixty-three (16.4%) of 385 ECs showed positive CAIX expression with high vascular density. These ECs had a reduced DSS compared to tumours with either hypoxia or high vascular density (log-rank p = 0.002). Multivariable analysis showed that hypoxic tumours with high vascular density had a reduced DSS (hazard ratio [HR] 3.71, p = 0.002), DDFS (HR 2.68, p = 0.009) and a trend for reduced DFS (HR 1.87, p = 0.054). CONCLUSIONS: This study has shown that adverse outcome in hypoxic ECs is seen in the presence of high vascular density, suggesting an important role of angiogenesis in the metastatic process of hypoxic EC. Differential adjuvant treatment might be indicated for these patients.

Quantitative Imaging of the Hypoxia-Related Marker CAIX in Head and Neck Squamous Cell Carcinoma Xenograft Models.

Tumor hypoxia plays a major role in radio- and chemotherapy resistance in solid tumors. Carbonic Anhydrase IX (CAIX) is an endogenous hypoxia-related protein, which is associated with poor patient outcome. The quantitative assessment of CAIX expression of tumors may steer cancer treatment by predicting therapy response or patient selection for antihypoxia or CAIX-targeted treatment. Recently, the single-photon emission computerized tomography (SPECT) tracer [111In]In-DTPA-girentuximab-F(ab')2 was developed and validated for targeting CAIX. The aim of this study was to optimize quantitative microSPECT/CT of CAIX expression in vivo in head and neck tumor models. Athymic mice with subcutaneous SCCNij153 and SCCNij202 head and neck squamous cell carcinoma xenografts were injected with [111In]In-DTPA-girentuximab-F(ab')2. First, the protein dose, timing, and image acquisition settings were optimized. Tracer uptake was determined by quantitative SPECT, ex vivo radioactivity counting, and by autoradiography of tumor sections. The same tumor sections were immunohistochemically stained for CAIX expression and hypoxia. Highest tumor-normal-tissue contrast was obtained at 24 h after injection of the tracer. A protein dose of 10 µg resulted in the highest tumor-to-muscle ratio at 24 h p.i. Ex vivo biodistribution studies showed a tumor uptake of 3.0 ± 0.6%ID/g and a tumor-to-muscle ratio of 8.7 ± 1.4 (SCCNij153). Quantitative analysis of the SPECT images enabled us to distinguish CAIX antigen blocked from nonblocked tumors, fractions positive for CAIX expression: 0.22 ± 0.02 versus 0.08 ± 0.01 ( p < 0.01). Immunohistochemical, autoradiographic, and microSPECT/CT analyses showed a distinct intratumoral spatial correlation between localization of the radiotracer and CAIX expression. Here, we demonstrate that [111In]In-DTPA-girentuximab-F(ab')2 specifically targets CAIX-expressing cells in head and neck cancer xenografts. SPECT imaging with indium-labeled girentuximab-F(ab')2 allows quantitative assessment of the fraction of CAIX positive tissue in head and neck cancer xenografts. These results indicate that [111In]In-DTPA-girentuximab-F(ab')2 is a promising tracer to image hypoxia-related CAIX expression.

Inter-observer variability in target delineation increases during adaptive treatment of head-and-neck and lung cancer.

Introduction: Inter-observer variability (IOV) in target volume delineation is a well-documented source of geometric uncertainty in radiotherapy. Such variability has not yet been explored in the context of adaptive re-delineation based on imaging data acquired during treatment. We compared IOV in the pre- and mid-treatment setting using expert primary gross tumour volume (GTV) and clinical target volume (CTV) delineations in locoregionally advanced head-and-neck squamous cell carcinoma (HNSCC) and (non-)small cell lung cancer [(N)SCLC]. Material and methods: Five and six observers participated in the HNSCC and (N)SCLC arm, respectively, and provided delineations for five cases each. Imaging data consisted of CT studies partly complemented by FDG-PET and was provided in two separate phases for pre- and mid-treatment. Global delineation compatibility was assessed with a volume overlap metric (the Generalised Conformity Index), while local extremes of IOV were identified through the standard deviation of surface distances from observer delineations to a median consensus delineation. Details of delineation procedures, in particular, GTV to CTV expansion and adaptation strategies, were collected through a questionnaire. Results: Volume overlap analysis revealed a worsening of IOV in all but one case per disease site, which failed to reach significance in this small sample (p-value range .063-.125). Changes in agreement were propagated from GTV to CTV delineations, but correlation could not be formally demonstrated. Surface distance based analysis identified longitudinal target extent as a pervasive source of disagreement for HNSCC. High variability in (N)SCLC was often associated with tumours abutting consolidated lung tissue or potentially invading the mediastinum. Adaptation practices were variable between observers with fewer than half stating that they consistently adapted pre-treatment delineations during treatment. Conclusion: IOV in target volume delineation increases during treatment, where a disparity in institutional adaptation practices adds to the conventional causes of IOV. Consensus guidelines are urgently needed.

Deep learning for lung cancer prognostication: A retrospective multi-cohort radiomics study.

BACKGROUND: Non-small-cell lung cancer (NSCLC) patients often demonstrate varying clinical courses and outcomes, even within the same tumor stage. This study explores deep learning applications in medical imaging allowing for the automated quantification of radiographic characteristics and potentially improving patient stratification. METHODS AND FINDINGS: We performed an integrative analysis on 7 independent datasets across 5 institutions totaling 1,194 NSCLC patients (age median = 68.3 years [range 32.5-93.3], survival median = 1.7 years [range 0.0-11.7]). Using external validation in computed tomography (CT) data, we identified prognostic signatures using a 3D convolutional neural network (CNN) for patients treated with radiotherapy (n = 771, age median = 68.0 years [range 32.5-93.3], survival median = 1.3 years [range 0.0-11.7]). We then employed a transfer learning approach to achieve the same for surgery patients (n = 391, age median = 69.1 years [range 37.2-88.0], survival median = 3.1 years [range 0.0-8.8]). We found that the CNN predictions were significantly associated with 2-year overall survival from the start of respective treatment for radiotherapy (area under the receiver operating characteristic curve [AUC] = 0.70 [95% CI 0.63-0.78], p < 0.001) and surgery (AUC = 0.71 [95% CI 0.60-0.82], p < 0.001) patients. The CNN was also able to significantly stratify patients into low and high mortality risk groups in both the radiotherapy (p < 0.001) and surgery (p = 0.03) datasets. Additionally, the CNN was found to significantly outperform random forest models built on clinical parameters-including age, sex, and tumor node metastasis stage-as well as demonstrate high robustness against test-retest (intraclass correlation coefficient = 0.91) and inter-reader (Spearman's rank-order correlation = 0.88) variations. To gain a better understanding of the characteristics captured by the CNN, we identified regions with the most contribution towards predictions and highlighted the importance of tumor-surrounding tissue in patient stratification. We also present preliminary findings on the biological basis of the captured phenotypes as being linked to cell cycle and transcriptional processes. Limitations include the retrospective nature of this study as well as the opaque black box nature of deep learning networks. CONCLUSIONS: Our results provide evidence that deep learning networks may be used for mortality risk stratification based on standard-of-care CT images from NSCLC patients. This evidence motivates future research into better deciphering the clinical and biological basis of deep learning networks as well as validation in prospective data.

Image-guided adaptive radiotherapy in patients with locally advanced non-small cell lung cancer: the art of PET.

With a worldwide annual incidence of 1.8 million cases, lung cancer is the most diagnosed form of cancer in men and the third most diagnosed form of cancer in women. Histologically, 80-85% of all lung cancers can be categorized as non-small cell lung cancer (NSCLC). For patients with locally advanced NSCLC, standard of care is fractionated radiotherapy combined with chemotherapy. With the aim of improving clinical outcome of patients with locally advanced NSCLC, combined and intensified treatment approaches are increasingly being used. However, given the heterogeneity of this patient group with respect to tumor biology and subsequent treatment response, a personalized treatment approach is required to optimize therapeutic effect and minimize treatment induced toxicity. Medical imaging, in particular positron emission tomography (PET), before and during the course radiotherapy is increasingly being used to personalize radiotherapy. In this setting, PET imaging can be used to improve delineation of target volumes, employ molecularly-guided dose painting strategies, early response monitoring, prediction and monitoring of treatment-related toxicity. The concept of PET image-guided adaptive radiotherapy (IGART) is an interesting approach to personalize radiotherapy for patients with locally advanced NSCLC, which might ultimately contribute to improved clinical outcomes and reductions in frequency of treatment-related adverse events in this patient group. In this review, we provide a comprehensive overview of available clinical data supporting the use of PET imaging for IGART in patients with locally advanced NSCLC.

Tumor Delineation and Quantitative Assessment of Glucose Metabolic Rate within Histologic Subtypes of Non-Small Cell Lung Cancer by Using Dynamic 18F Fluorodeoxyglucose PET.

Purpose To assess whether dynamic fluorine 18 (18F) fluorodeoxyglucose (FDG) positron emission tomography (PET) has added value over static 18F-FDG PET for tumor delineation in non-small cell lung cancer (NSCLC) radiation therapy planning by using pathology volumes as the reference standard and to compare pharmacokinetic rate constants of 18F-FDG metabolism, including regional variation, between NSCLC histologic subtypes. Materials and Methods The study was approved by the institutional review board. Patients gave written informed consent. In this prospective observational study, 1-hour dynamic 18F-FDG PET/computed tomographic examinations were performed in 35 patients (36 resectable NSCLCs) between 2009 and 2014. Static and parametric images of glucose metabolic rate were obtained to determine lesion volumes by using three delineation strategies. Pathology volume was calculated from three orthogonal dimensions (n = 32). Whole tumor and regional rate constants and blood volume fraction (VB) were computed by using compartment modeling. Results Pathology volumes were larger than PET volumes (median difference, 8.7-25.2 cm3; Wilcoxon signed rank test, P < .001). Static fuzzy locally adaptive Bayesian (FLAB) volumes corresponded best with pathology volumes (intraclass correlation coefficient, 0.72; P < .001). Bland-Altman analyses showed the highest precision and accuracy for static FLAB volumes. Glucose metabolic rate and 18F-FDG phosphorylation rate were higher in squamous cell carcinoma (SCC) than in adenocarcinoma (AC), whereas VB was lower (Mann-Whitney U test or t test, P = .003, P = .036, and P = .019, respectively). Glucose metabolic rate, 18F-FDG phosphorylation rate, and VB were less heterogeneous in AC than in SCC (Friedman analysis of variance). Conclusion Parametric images are not superior to static images for NSCLC delineation. FLAB-based segmentation on static 18F-FDG PET images is in best agreement with pathology volume and could be useful for NSCLC autocontouring. Differences in glycolytic rate and VB between SCC and AC are relevant for research in targeting agents and radiation therapy dose escalation. © RSNA, 2016 Online supplemental material is available for this article.

The potential of hyperpolarized 13C magnetic resonance spectroscopy to monitor the effect of combretastatin based vascular disrupting agents.

BACKGROUND: Targeting tumor vasculature with vascular disrupting agents (VDAs) results in substantial cell death that precede tumor shrinkage. Here, we investigate the potential of hyperpolarized magnetic resonance spectroscopy (HPMRS) to monitor early metabolic changes associated with VDA treatment. METHODS: Mice bearing C3H mammary carcinomas were treated with the VDAs combretastatin-A4-phosphate (CA4P) or the analog OXi4503, and HPMRS was performed following [1-13C]pyruvate administration. Similarly, treated mice were positron emission tomography (PET) scanned following administration of the glucose analog FDG. Finally, metabolic imaging parameters were compared to tumor regrowth delay and measures of vascular damage, derived from dynamic contrast-agent enhanced magnetic resonance imaging (DCE-MRI) and histology. RESULTS: VDA-treatment impaired tumor perfusion (histology and DCE-MRI), reduced FDG uptake, increased necrosis, and slowed tumor growth. HPMRS, revealed that the [1-13C]pyruvate-to-[1-13C]lactate conversion remained unaltered, whereas [1-13C]lactate-to-[13C]bicarbonate (originating from respiratory CO2) ratios increased significantly following treatment. CONCLUSIONS: DCE-MRI and FDG-PET revealed loss of vessel functionality, impaired glucose delivery and reduced metabolic activity prior to cell death. [1-13C]lactate-to-[13C]bicarbonate ratios increased significantly during treatment, indicating a decline in respiratory activity driven by the onset of hypoxia. HPMRS is promising for early detection of metabolic stress inflicted by VDAs, which cannot easily be inferred based on blood flow measurements.

The mechanical microenvironment in cancer: How physics affects tumours.

The tumour microenvironment contributes greatly to the response of tumour cells. It consists of chemical gradients, for example of oxygen and nutrients. However, a physical environment is also present. Apart from chemical input, cells also receive physical signals. Tumours display unique mechanical properties: they are a lot stiffer than normal tissue. This may be either a cause or a consequence of cancer, but literature suggests it has a major impact on tumour cells as will be described in this review. The mechanical microenvironment may cause malignant transformation, possibly through activation of oncogenic pathways and inhibition of tumour suppressor genes. In addition, the mechanical microenvironment may promote tumour progression by influencing processes such as epithelial-to-mesenchymal transition, enhancing cell survival through autophagy, but also affects sensitivity of tumour cells to therapeutics. Furthermore, multiple intracellular signalling pathways prove sensitive to the mechanical properties of the microenvironment. It appears the increased stiffness is unlikely to be caused by increased stiffness of the tumour cells themselves. However, there are indications that tumours display a higher cell density, making them more rigid. In addition, increased matrix deposition in the tumour, as well as increased interstitial fluid pressure may account for the increased stiffness of tumours. Overall, tumour mechanics are significantly different from normal tissue. Therefore, this feature should be further explored for use in cancer prevention, detection and treatment.

Therapeutic targeting of autophagy in cancer. Part I: molecular pathways controlling autophagy.

Autophagy is a process in which cells can generate energy and building materials, by degradation of redundant and/or damaged organelles and proteins. Especially during conditions of stress, autophagy helps to maintain homeostasis. In addition, autophagy has been shown to influence malignant transformation and cancer progression. The precise molecular events in autophagy are complex and the core autophagic machinery described to date consists of nearly thirty proteins. Apart from these factors that execute the process of autophagy, several signalling pathways are involved in converting internal and external stimuli into an autophagic response. In this review we provide an overview of the signalling pathways that influence autophagy, particularly in cancer cells. We will illustrate that interference with multiple of these signalling pathways can have significant effects on cancer cell survival.

Therapeutic targeting of autophagy in cancer. Part II: pharmacological modulation of treatment-induced autophagy.

Autophagy, the catabolic pathway in which cells recycle organelles and other parts of their own cytoplasm, is increasingly recognised as an important cytoprotective mechanism in cancer cells. Several cancer treatments stimulate the autophagic process and when autophagy is inhibited, cancer cells show an enhanced response to multiple treatments. These findings have nourished the theory that autophagy provides cancer cells with a survival advantage during stressful conditions, including exposure to therapeutics. Therefore, interference with the autophagic response can potentially enhance the efficacy of cancer therapy. In this review we examine two approaches to modulate autophagy as complementary cancer treatment: inhibition and induction. Inhibition of autophagy during cancer treatment eliminates its cytoprotective effects. Conversely, induction of autophagy combined with conventional cancer therapy exerts severe cytoplasmic degradation that can ultimately lead to cell death. We will discuss how autophagy can be therapeutically manipulated in cancer cells and how interactions between the conventional cancer therapies and autophagy modulation influence treatment outcome.

PERK/eIF2α signaling protects therapy resistant hypoxic cells through induction of glutathione synthesis and protection against ROS.

Hypoxia is a common feature of tumors and an important contributor to malignancy and treatment resistance. The ability of tumor cells to survive hypoxic stress is mediated in part by hypoxia-inducible factor (HIF)-dependent transcriptional responses. More severe hypoxia activates endoplasmatic reticulum stress responses, including the double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α)-dependent arm of the unfolded protein response (UPR). Although several studies implicate important roles for HIF and UPR in adaption to hypoxia, their importance for hypoxic cells responsible for therapy resistance in tumors is unknown. By using isogenic models, we find that HIF and eIF2α signaling contribute to the survival of hypoxic cells in vitro and in vivo. However, the eIF2α-dependent arm of the UPR is uniquely required for the survival of a subset of hypoxic cells that determine tumor radioresistance. We demonstrate that eIF2α signaling induces uptake of cysteine, glutathione synthesis, and protection against reactive oxygen species produced during periods of cycling hypoxia. Together these data imply that eIF2α signaling is a critical contributor to the tolerance of therapy-resistant cells that arise as a consequence of transient changes in oxygenation in solid tumors and thus a therapeutic target in curative treatments for solid cancers.

The unfolded protein response as a target for cancer therapy.

Various physiological and pathological conditions generate an accumulation of misfolded proteins in the endoplasmic reticulum (ER). This results in ER stress followed by a cellular response to cope with this stress and restore homeostasis: the unfolded protein response (UPR). Overall, the UPR leads to general translational arrest and the induction of specific factors to ensure cell survival or to mediate cell death if the stress is too severe. In multiple cancers, components of the UPR are overexpressed, indicating increased dependence on the UPR. In addition, the UPR can confer resistance to anti-cancer treatment. Therefore, modification of the UPR should be explored for its anti-cancer properties. This review discusses factors associated with the UPR that represent potential therapeutic targets.

Epidermal growth factor receptor imaging in human head and neck cancer xenografts.

Molecular imaging of specific biomarkers can have prognostic, predictive or monitoring value in head and neck squamous cell carcinoma (HNSCC). The epidermal growth factor receptor (EGFR) is involved in various radiation resistance mechanisms as it steers the pathways related to DNA damage repair, proliferation, hypoxia and apoptosis. Radiolabeled labeled F(ab')2 fragments of the EGFR antibody cetuximab can be applied for non-invasive imaging of this receptor. Preclinical studies have shown that radioresistant tumors had a higher tracer uptake after irradiation, probably due to upregulation of membranous EGFR, thereby increasing target availability possibly as a compensation mechanism. Tumors with increased EGFR availability were also more responsive to the EGFR inhibitor cetuximab. Potentially, radionuclide imaging of the EGFR can be applied for monitoring treatment regimens in clinical practice.

Semiautomatic methods for segmentation of the proliferative tumour volume on sequential FLT PET/CT images in head and neck carcinomas and their relation to clinical outcome.

PURPOSE: Radiotherapy of head and neck cancer induces changes in tumour cell proliferation during treatment, which can be depicted by the PET tracer (18)F-fluorothymidine (FLT). In this study, three advanced semiautomatic PET segmentation methods for delineation of the proliferative tumour volume (PV) before and during (chemo)radiotherapy were compared and related to clinical outcome. METHODS: The study group comprised 46 patients with 48 squamous cell carcinomas of the head and neck, treated with accelerated (chemo)radiotherapy, who underwent FLT PET/CT prior to treatment and in the 2nd and 4th week of therapy. Primary gross tumour volumes were visually delineated on CT images (GTV CT). PVs were visually determined on all PET scans (PV VIS). The following semiautomatic segmentation methods were applied to sequential PET scans: background-subtracted relative-threshold level (PV RTL), a gradient-based method using the watershed transform algorithm and hierarchical clustering analysis (PV W&C), and a fuzzy locally adaptive Bayesian algorithm (PV FLAB). RESULTS: Pretreatment PV VIS correlated best with PV FLAB and GTV CT. Correlations with PV RTL and PV W&C were weaker although statistically significant. During treatment, the PV VIS, PV W&C and PV FLAB significant decreased over time with the steepest decline over time for PV FLAB. Among these advanced segmentation methods, PV FLAB was the most robust in segmenting volumes in the third scan (67 % of tumours as compared to 40 % for PV W&C and 27 % for PV RTL). A decrease in PV FLAB above the median between the pretreatment scan and the scan obtained in the 4th week was associated with better disease-free survival (4 years 90 % versus 53 %). CONCLUSION: In patients with head and neck cancer, FLAB proved to be the best performing method for segmentation of the PV on repeat FLT PET/CT scans during (chemo)radiotherapy. This may potentially facilitate radiation dose adaptation to changing PV.

Systematic analysis of 18F-FDG PET and metabolism, proliferation and hypoxia markers for classification of head and neck tumors.

BACKGROUND: Quantification of molecular cell processes is important for prognostication and treatment individualization of head and neck cancer (HNC). However, individual tumor comparison can show discord in upregulation similarities when analyzing multiple biological mechanisms. Elaborate tumor characterization, integrating multiple pathways reflecting intrinsic and microenvironmental properties, may be beneficial to group most uniform tumors for treatment modification schemes. The goal of this study was to systematically analyze if immunohistochemical (IHC) assessment of molecular markers, involved in treatment resistance, and 18F-FDG PET parameters could accurately distinguish separate HNC tumors. METHODS: Several imaging parameters and texture features for 18F-FDG small-animal PET and immunohistochemical markers related to metabolism, hypoxia, proliferation and tumor blood perfusion were assessed within groups of BALB/c nu/nu mice xenografted with 14 human HNC models. Classification methods were used to predict tumor line based on sets of parameters. RESULTS: We found that 18F-FDG PET could not differentiate between the tumor lines. On the contrary, combined IHC parameters could accurately allocate individual tumors to the correct model. From 9 analyzed IHC parameters, a cluster of 6 random parameters already classified 70.3% correctly. Combining all PET/IHC characteristics resulted in the highest tumor line classification accuracy (81.0%; cross validation 82.0%), which was just 2.2% higher (p = 5.2×10-32) than the performance of the IHC parameter/feature based model. CONCLUSIONS: With a select set of IHC markers representing cellular processes of metabolism, proliferation, hypoxia and perfusion, one can reliably distinguish between HNC tumor lines. Addition of 18F-FDG PET improves classification accuracy of IHC to a significant yet minor degree. These results may form a basis for development of tumor characterization models for treatment allocation purposes.

Effect of hypoxia on the expression of αB-crystallin in head and neck squamous cell carcinoma.

BACKGROUND: The presence of hypoxia in head and neck squamous cell carcinoma (HNSCC) is associated with therapeutic resistance and increased risk of metastasis formation. αB-crystallin (HspB5) is a small heat shock protein, which is also associated with metastasis formation in HNSCC. In this study, we investigated whether αB-crystallin protein expression is increased in hypoxic areas of HNSCC biopsies and analyzed whether hypoxia induces αB-crystallin expression in vitro and in this way may confer hypoxic cell survival. METHODS: In 38 HNSCC biopsies, the overlap between immunohistochemically stained αB-crystallin and pimonidazole-adducts (hypoxiamarker) was determined. Moreover, expression levels of αB-crystallin were analyzed in HNSCC cell lines under hypoxia and reoxygenation conditions and after exposure to reactive oxygen species (ROS) and the ROS scavenger N-acetylcysteine (NAC). siRNA-mediated knockdown was used to determine the influence of αB-crystallin on cell survival under hypoxic conditions. RESULTS: In all biopsies αB-crystallin was more abundantly present in hypoxic areas than in normoxic areas. Remarkably, hypoxia decreased αB-crystallin mRNA expression in the HNSCC cell lines. Only after reoxygenation, a condition that stimulates ROS formation, αB-crystallin expression was increased. αB-crystallin mRNA levels were also increased by extracellular ROS, and NAC abolished the reoxygenation-induced αB-crystallin upregulation. Moreover, it was found that decreased αB-crystallin levels reduced cell survival under hypoxic conditions. CONCLUSIONS: We provide the first evidence that hypoxia stimulates upregulation of αB-crystallin in HNSCC. This upregulation was not caused by the low oxygen pressure, but more likely by ROS formation. The higher expression of αB-crystallin may lead to prolonged survival of these cells under hypoxic conditions.