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Radiomics involves high throughput extraction of mineable precise quantitative imaging features that serve as non-invasive prognostic or predictive biomarkers. High levels of hypoxia are associated with a poorer prognosis in prostate cancer and limit radiation therapy efficacy. Most patients with prostate cancer undergo magnetic resonance imaging (MRI) as a part of their diagnostics, and T2 imaging is the most utilised imaging method. The aim of this study was to determine whether hypoxia in prostate tumors could be identified using a radiomics model extracted from T2-weighted MR images.Eighty eight intermediate or high-risk prostate cancer patients were evaluated. Prior to radical prostatectomy, all patients received pimonidazole (PIMO). PIMO hypoxic scores were assigned in whole-mount sections from prostatectomy specimens by an experienced pathologist who was blinded to MRI. The region of interest used for radiomics analysis included the prostatic index tumor. Radiomics extraction yielded 165 features using a special evaluation version of RadiomiX [RadiomiX Research Toolbox version 20180831 (OncoRadiomics SA, Liège, Belgium)] for non-clinical use. Multivariable logistic regression with Elastic Net regularization was utilised using 10 times repeated 10-fold cross-validation to select the best model hyperparameters, optimizing for area under the receiver operating characteristic curve (AUC).The average (out of sample) performance based on the repeated cross validation using the ONESE model yielded an AUC of 0.60±0.2. Shape-based features were the most prominent in the model.The development of a radiomics hypoxia model using T2 weighted MR images, standard in the staging of prostate cancer, is possible.

DCE-MRI and digital histopathology

Validation of algorithm for assessing hypoxia levels in patient tumors

Construction of histology images of cell density and blood vessel density.

Pimonidazole immunoscore versus time after administration, DWI parameters and histology parameters

Tumor hypoxia levels range from mild to severe and have different biological and therapeutical consequences but are not easily assessable in patients. Here we present a method based on diagnostic dynamic contrast enhanced (DCE) MRI that reflects a continuous range of hypoxia levels in patients with tumors of cervical cancer. Hypoxia images were generated using an established approach based on pixel-wise combination of DCE-MRI parameters νe and Ktrans, representing oxygen consumption and supply, respectively. Using two tumor models, an algorithm to retrieve surrogate measures of hypoxia levels from the images was developed and validated by comparing the MRI-defined levels with hypoxia levels reflected in pimonidazole-stained histologic sections. An additional indicator of hypoxia levels in patient tumors was established on the basis of expression of nine hypoxia-responsive genes; a strong correlation was found between these indicator values and MRI-defined hypoxia levels in 63 patients. Chemoradiotherapy outcome of 74 patients was most strongly predicted by moderate hypoxia levels, whereas more severe or milder levels were less predictive. By combining gene expression profiles and MRI-defined hypoxia levels in cancer hallmark analysis, we identified a distribution of levels associated with each hallmark; oxidative phosphorylation and G2–M checkpoint were associated with moderate hypoxia, epithelial-to-mesenchymal transition, and inflammatory responses with significantly more severe levels. At the mildest levels, IFN response hallmarks together with HIF1A protein expression by IHC appeared significant. Thus, our method visualizes the distribution of hypoxia levels within patient tumors and has potential to distinguish levels of different prognostic and biological significance.Significance:These findings present an approach to image a continuous range of hypoxia levels in tumors and demonstrate the combination of imaging with molecular data to better understand the biology behind these different levels.

Development of the image integration algorithm.

Comparison of MRI with histology, construction of hypoxia images, and hypoxia levels in patients

The established role of hypoxia-induced signaling in prostate cancer growth, metastasis, and response to treatment suggests that a method to image hypoxia in tumors could aid treatment decisions. Here, we present consumption and supply-based hypoxia (CSH) imaging, an approach that integrates images related to oxygen consumption and supply into a single image. This integration algorithm was developed in patients with prostate cancer receiving hypoxia marker pimonidazole prior to prostatectomy. We exploited the intravoxel incoherent motion (IVIM) signal in diagnostic diffusion-weighted (DW) magnetic resonance (MR) images to generate separate images of the apparent diffusion coefficient (ADC) and fractional blood volume (fBV). ADC and fBV correlated with cell density (CD) and blood vessel density (BVD) in histology and whole-mount sections from 35 patients, thus linking ADC to oxygen consumption and fBV to oxygen supply. Pixel-wise plots of ADC versus fBV were utilized to predict the hypoxia status of each pixel in a tumor and to visualize the predicted value in a single image. The hypoxic fraction (HFDWI) of CSH images correlated strongly (R2 = 0.66; n = 41) with pimonidazole immunoscore (HSPimo); this relationship was validated in a second pimonidazole cohort (R2 = 0.54; n = 54). We observed good agreement between CSH images and pimonidazole staining in whole-mount sections. HFDWI correlated with tumor stage and lymph node status, consistent with findings for HSPimo. Moreover, CSH imaging could be applied on histologic CD and BVD images, demonstrating transferability to a histopathology assay. Thus, CSH represents a robust approach for hypoxia imaging in prostate cancer that could easily be translated into clinical practice.Significance: These findings present a novel imaging strategy that indirectly measures tumor hypoxia and has potential application in a wide variety of solid tumors and other imaging modalities.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/16/4774/F1.large.jpg. Cancer Res; 78(16); 4774–85. ©2018 AACR.

Clinical characteristics, pimonidazole immunoscore and DWI parameters versus clinicopathological markers

PDF file - 25K, Figure showing IFP and v0 versus tumor size for TS-415 and U-25 tumors

MOV file - 305K, Video showing the outward movement of the peritumoral high-signal-intensity rim of a human cervical carcinoma

MOV file - 305K, Video showing the outward movement of the peritumoral high-signal-intensity rim of a TS-415 cervical carcinoma xenograft

SummaryA phase II trial, investigating whether the antidiabetic drug metformin could reduce hypoxia in cervical cancer, used imaging to preselect patients and test the biological hypothesis behind the drug effect. This trial design would be of importance for the implementation of targeted treatment in the clinic.See related article by Han et al., p. 5263

MRI, applying dynamic contrast-enhanced (DCE) and diffusion-weighted (DW) sequences, and 18F-fluorodeoxyglucose (18F-FDG) PET/CT provide information about tumor aggressiveness that is unexploited in treatment of locally advanced cervical cancer (LACC). We investigated the potential of a multimodal combination of imaging parameters for classifying patients according to their risk of recurrence.Eighty-two LACC patients with diagnostic MRI and FDG-PET/CT, treated with chemoradiotherapy, were collected. Thirty-eight patients with MRI only were included for validation of MRI results. Endpoints were survival (disease-free, cancer-specific, overall) and tumor control (local, locoregional, distant). KHF showed a stronger association with outcome than the single modality parameters. This association was confirmed in the validation cohort. Low HF identified low-risk patients with 95% precision. Based on the 50th SUV-percentile (SUVMultimodal combination of MR- and FDG-PET/CT-images improves classification of LACC patients compared to single modality images and clinical factors.

Tumor hypoxia levels range from mild to severe and have different biological and therapeutical consequences but are not easily assessable in patients. Here we present a method based on diagnostic dynamic contrast enhanced (DCE) MRI that reflects a continuous range of hypoxia levels in patients with tumors of cervical cancer. Hypoxia images were generated using an established approach based on pixel-wise combination of DCE-MRI parameters νe and Ktrans, representing oxygen consumption and supply, respectively. Using two tumor models, an algorithm to retrieve surrogate measures of hypoxia levels from the images was developed and validated by comparing the MRI-defined levels with hypoxia levels reflected in pimonidazole-stained histologic sections. An additional indicator of hypoxia levels in patient tumors was established on the basis of expression of nine hypoxia-responsive genes; a strong correlation was found between these indicator values and MRI-defined hypoxia levels in 63 patients. Chemoradiotherapy outcome of 74 patients was most strongly predicted by moderate hypoxia levels, whereas more severe or milder levels were less predictive. By combining gene expression profiles and MRI-defined hypoxia levels in cancer hallmark analysis, we identified a distribution of levels associated with each hallmark; oxidative phosphorylation and G2–M checkpoint were associated with moderate hypoxia, epithelial-to-mesenchymal transition, and inflammatory responses with significantly more severe levels. At the mildest levels, IFN response hallmarks together with HIF1A protein expression by IHC appeared significant. Thus, our method visualizes the distribution of hypoxia levels within patient tumors and has potential to distinguish levels of different prognostic and biological significance. Significance: These findings present an approach to image a continuous range of hypoxia levels in tumors and demonstrate the combination of imaging with molecular data to better understand the biology behind these different levels.

Tumours exhibit abnormal interstitial structures and vasculature function often leading to impaired and heterogeneous drug delivery. The disproportionate spatial accumulation of a drug in the interstitium is determined by several microenvironmental properties (blood vessel distribution and permeability, gradients in the interstitial fluid pressure). Predictions of tumour perfusion are key determinants of drug delivery and responsiveness to therapy. Pharmacokinetic models allow for the quantification of tracer perfusion based on contrast enhancement measured with non-invasive imaging techniques. An advanced cross-voxel exchange model (CVXM) was recently developed to provide a comprehensive description of tracer extravasation as well as advection and diffusion based on cross-voxel tracer kinetics (Sinno et al 2021). Transport parameters were derived from DCE-MRI of twenty TS-415 human cervical carcinoma xenografts by using CVXM. Tracer velocity flows were measured at the tumour periphery (mean 1.78–5.82 μm.s−1) pushing the contrast outward towards normal tissue. These elevated velocity measures and extravasation rates explain the heterogeneous distribution of tracer across the tumour and its accumulation at the periphery. Significant values for diffusivity were deduced across the tumours (mean 152–499 μm2.s−1). CVXM resulted in generally smaller values for the extravasation parameter K e x t (mean 0.01–0.04 min−1) and extravascular extracellular volume fraction v e (mean 0.05–0.17) compared to the standard Tofts parameters, suggesting that Toft model underestimates the effects of inter-voxel exchange. The ratio of Tofts’ extravasation parameters over CVXM’s was significantly positively correlated to the cross-voxel diffusivity (P < 0.0001) and velocity (P = 0.0005). Tofts’ increased v e measurements were explained using Sinno et al (2021)’s theoretical work. Finally, a scan time of 15 min renders informative estimations of the transport parameters. However, a duration as low as 7.5 min is acceptable to recognize the spatial variation of transport parameters. The results demonstrate the potential of utilizing CVXM for determining metrics characterizing the exchange of tracer between the vasculature and the tumour tissue. Like for many earlier models, additional work is strongly recommended, in terms of validation, to develop more confidence in the results, motivating future laboratory work in this regard.

Background Gene signatures measured in a biopsy have been proposed as hypoxia biomarkers in prostate cancer. We assessed a previously developed signature, and aimed to determine its relationship to hypoxia and its heterogeneity within the dominant (index) lesion of prostate cancer. Methods The 32-gene signature was assessed from gene expression data of 141 biopsies from the index lesion of 94 patients treated with prostatectomy. A gene score calculated from the expression levels was applied in the analyses. Hypoxic fraction from pimonidazole immunostained whole-mount and biopsy sections was used as reference standard for hypoxia. Results The gene score was correlated with pimonidazole-defined hypoxic fraction in whole-mount sections, and the two parameters showed almost equal association with clinical markers of tumour aggressiveness. Based on the gene score, incorrect classification according to hypoxic fraction in whole-mount sections was seen in one third of the patients. The incorrect classifications were apparently not due to intra-tumour heterogeneity, since the score had low heterogeneity compared to pimonidazole-defined hypoxic fraction in biopsies. The score showed prognostic significance in uni-and multivariate analysis in independent cohorts. Conclusions Our signature from the index lesion reflects tumour hypoxia and predicts prognosis in prostate cancer, independent of intra-tumour heterogeneity in pimonidazole-defined hypoxia.

Simple Summary Hypoxia is a common feature of solid tumors and associated with poor outcome in most cancer types and treatment modalities, including radiotherapy, chemotherapy, surgery and, most likely, immunotherapy. Emerging strategies, such as proton therapy and combination therapies with radiation and hypoxia targeted drugs, provide new opportunities to overcome the hypoxia barrier and improve therapeutic outcome. Hypoxia is heterogeneously distributed both between and within tumors and shows large variations across patients not only in prevalence, but importantly, also in level. To best exploit the emerging strategies, a better understanding of how individual hypoxia levels from mild to severe affect tumor biology is vital. Here, we discuss our current knowledge on this topic and how we should proceed to gain more insight into the field. Abstract Hypoxia arises in tumor regions with insufficient oxygen supply and is a major barrier in cancer treatment. The distribution of hypoxia levels is highly heterogeneous, ranging from mild, almost non-hypoxic, to severe and anoxic levels. The individual hypoxia levels induce a variety of biological responses that impair the treatment effect. A stronger focus on hypoxia levels rather than the absence or presence of hypoxia in our investigations will help development of improved strategies to treat patients with hypoxic tumors. Current knowledge on how hypoxia levels are sensed by cancer cells and mediate cellular responses that promote treatment resistance is comprehensive. Recently, it has become evident that hypoxia also has an important, more unexplored role in the interaction between cancer cells, stroma and immune cells, influencing the composition and structure of the tumor microenvironment. Establishment of how such processes depend on the hypoxia level requires more advanced tumor models and methodology. In this review, we describe promising model systems and tools for investigations of hypoxia levels in tumors. We further present current knowledge and emerging research on cellular responses to individual levels, and discuss their impact in novel therapeutic approaches to overcome the hypoxia barrier.

The purpose of the present study is to investigate if consumption and supply hypoxia (CSH) MR-imaging can depict breast cancer hypoxia, using the CSH-method initially developed for prostate cancer. Furthermore, to develop a generalized pan-cancer application of the CSH-method that doesn’t require a hypoxia reference standard for training the CSH-parameters. In a cohort of 69 breast cancer patients, we generated, based on the principles of intravoxel incoherent motion modelling, images reflecting cellular density (apparent diffusion coefficient; ADC) and vascular density (perfusion fraction; fp). Combinations of the information in these images were compared to a molecular hypoxia score made from gene expression data, aiming to identify a way to apply the CSH-methodology in breast cancer. Attempts to adapt previously proposed models for prostate cancer included direct transfers and model parameter rescaling. A novel approach, based on rescaling ADC and fp data to give more nuanced response in the relevant physiologic range, was also introduced. The new CSH-method was validated in a prostate cancer cohort with known hypoxia status. The proposed CSH-method gave estimates of hypoxia that was strongly correlated to the molecular hypoxia score in breast cancer, and hypoxia as measured in pathology slices stained with pimonidazole in prostate cancer. The generalized approach to CSH-imaging depicted hypoxia in both breast and prostate cancers and requires no model training. It is easy to implement using readily available technology and encourages further investigation of CSH-imaging in other cancer entities and in other settings, with the goal being to overcome hypoxia-induced resistance to treatment.

Background Emerging biomarkers from medical imaging or molecular characterization of tumour biopsies open up for combining the two and exploiting their synergy in treatment planning of cancer patients. We generated a paired data set of imaging- and gene-based hypoxia biomarkers in cervical cancer, appraised the influence of intratumour heterogeneity in patient classification, and investigated the benefit of combining the methodologies in prediction of chemoradiotherapy failure. Methods Hypoxic fraction from dynamic contrast enhanced (DCE)-MR images and an expression signature of six hypoxia-responsive genes were assessed as imaging- and gene-based biomarker, respectively in 118 patients. Findings Dichotomous biomarker cutoff to yield similar hypoxia status by imaging and genes was defined in 41 patients, and the association was validated in the remaining 77 patients. The two biomarkers classified 75% of 118 patients with the same hypoxia status, and inconsistent classification was not related to imaging-defined intratumour heterogeneity in hypoxia. Gene-based hypoxia was independent on tumour cell fraction in the biopsies and showed minor heterogeneity across multiple samples in 9 tumours. Combining imaging- and gene-based classification gave a significantly better prediction of PFS than one biomarker alone. A combined dichotomous biomarker optimized in 77 patients showed a large separation in PFS between more and less hypoxic tumours, and separated the remaining 41 patients with different PFS. The combined biomarker showed prognostic value together with tumour stage in multivariate analysis. Interpretation Combining imaging- and gene-based biomarkers may enable more precise and informative assessment of hypoxia-related chemoradiotherapy resistance in cervical cancer. Funding Norwegian Cancer Society, South-Eastern Norway Regional Health Authority, and Norwegian Research Council.

Tumor hypoxia levels range from mild to severe and have different biological and therapeutical consequences, but are not easily assessable in patients. We present a method based on diagnostic dynamic contrast enhanced (DCE) magnetic resonance imaging (MRI) that visualizes a continuous range of hypoxia levels in tumors of cervical cancer patients. Hypoxia images were generated using an established approach based on pixel-wise combination of the DCE-MRI parametersνeandKtrans, reflecting oxygen consumption and supply, respectively. An algorithm to retrieve hypoxia levels from the images was developed and validated in 28 xenograft tumors, by comparing the MRI-defined levels with hypoxia levels derived from pimonidazole stained histological sections. We further established an indicator of hypoxia levels in patient tumors based on expression of nine hypoxia responsive genes. A strong correlation was found between these indicator values and the MRI-defined hypoxia levels in 63 patients. Chemoradiotherapy outcome of 74 patients was most strongly predicted by moderate hypoxia levels, whereas more severe or milder levels were less predictive. By combining gene expression profiles and MRI-defined hypoxia levels in cancer hallmark analysis, we identified a distribution of levels associated with each hallmark; oxidative phosphorylation and G2/M checkpoint were associated with moderate hypoxia, and epithelial-to-mesenchymal transition and inflammatory responses with significantly more severe levels. At the mildest levels, interferon response hallmarks, together with stabilization of HIF1A protein by immunohistochemistry, appearred significant. Thus, our method visualizes the distribution of hypoxia levels within patient tumors and has potential to distinguish levels of different prognostic and biological significance.

The established role of hypoxia-induced signaling in prostate cancer growth, metastasis, and response to treatment suggests that a method to image hypoxia in tumors could aid treatment decisions. Here, we present consumption and supply-based hypoxia (CSH) imaging, an approach that integrates images related to oxygen consumption and supply into a single image. This integration algorithm was developed in patients with prostate cancer receiving hypoxia marker pimonidazole prior to prostatectomy. We exploited the intravoxel incoherent motion (IVIM) signal in diagnostic diffusion-weighted (DW) magnetic resonance (MR) images to generate separate images of the apparent diffusion coefficient (ADC) and fractional blood volume (fBV). ADC and fBV correlated with cell density (CD) and blood vessel density (BVD) in histology and whole-mount sections from 35 patients, thus linking ADC to oxygen consumption and fBV to oxygen supply. Pixel-wise plots of ADC versus fBV were utilized to predict the hypoxia status of each pixel in a tumor and to visualize the predicted value in a single image. The hypoxic fraction (HFDWI) of CSH images correlated strongly (R2 = 0.66; n = 41) with pimonidazole immunoscore (HSPimo); this relationship was validated in a second pimonidazole cohort (R2 = 0.54; n = 54). We observed good agreement between CSH images and pimonidazole staining in whole-mount sections. HFDWI correlated with tumor stage and lymph node status, consistent with findings for HSPimo. Moreover, CSH imaging could be applied on histologic CD and BVD images, demonstrating transferability to a histopathology assay. Thus, CSH represents a robust approach for hypoxia imaging in prostate cancer that could easily be translated into clinical practice. Significance: These findings present a novel imaging strategy that indirectly measures tumor hypoxia and has potential application in a wide variety of solid tumors and other imaging modalities. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/16/4774/F1.large.jpg. Cancer Res; 78(16); 4774–85. ©2018 AACR.

Background and purpose: Several investigators have indicated that dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has the potential to provide biomarkers for personalized treatment of cervical carcinoma. However, some clinical studies have suggested that treatment failure is associated with low tumor signal enhancement, whereas others have reported associations between high signal enhancement and poor outcome. The purpose of this investigation was to clear up these conflicting reports and to provide a method for identifying biomarkers that easily can be implemented in routine DCE-MRI diagnostics. Methods: The study involved 85 patients (FIGO stage IB through IVA) treated with concurrent chemoradiotherapy. Low-enhancing tumor volume (LETV) and low-enhancing tumor fraction (LETF), defined as the volume and fractional volume of low-enhancing voxels, respectively, were calculated from signal intensities recorded within 1 min after contrast administration by using two methods reported to give conflicting conclusions. Results: Multivariate analysis involving tumor volume, lymph node status, FIGO stage, and LETV or LETF revealed that LETV and LETF provided independent prognostic information on treatment outcome, independent of the method of calculation. Conclusion: Low signal enhancement is associated with poor prognosis in cervical carcinoma, and biomarkers predicting poor outcome can be provided by short-term DCE-MRI without advanced image

Background and purpose Locoregional treatment failure and poor survival rates are associated with extensive hypoxia in the primary tumor in advanced cervical carcinoma. The potential of gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in assessing the hypoxic fraction, radioresponsiveness, and metastatic propensity of cervical carcinomas was investigated in this preclinical study. Materials and methods CK-160 and TS-415 cervical carcinoma xenografts were used as tumor models. DCE-MRI was carried out at 1.5 T, and parametric images of K trans and v e were produced by pharmacokinetic analysis of the DCE-MRI series. Pimonidazole was used as a hypoxia marker. Tumor radioresponsiveness was determined by irradiating tumors with five fractions of 4Gy in 48h and measuring cell survival in vitro. Metastatic propensity was determined by examining host mice for tumor growth in lymph nodes. Results Low values of K trans were associated with extensive hypoxia and radiation resistance in tumors of both lines and with high incidence of metastases in CK-160 tumors. Associations between v e and hypoxia, radioresponsiveness, or metastatic propensity were not found in any of the tumor lines. Conclusion K trans is a potentially useful biomarker of tumor hypoxia, radiation resistance, and metastatic growth in advanced cervical carcinoma.

Background. A high fraction of stroma in malignant tissues is associated with tumor progression, metastasis, and poor prognosis. Possible correlations between the stromal and physiologic microenvironments of tumors and the potential of dynamic contrast-enhanced (DCE) and diffusion-weighted (DW) magnetic resonance imaging (MRI) in quantification of the stromal microenvironment were investigated in this study. Material and methods. CK-160 cervical carcinoma xenografts were used as preclinical tumor model. A total of 43 tumors were included in the study, and of these tumors, 17 were used to search for correlations between the stromal and physiologic microenvironments, 11 were subjected to DCE-MRI, and 15 were subjected to DW-MRI. DCE-MRI and DW-MRI were carried out at 1.5 T with a clinical MR scanner and a slotted tube resonator transceiver coil constructed for mice. Fraction of connective tissue (CTFCol) and fraction of hypoxic tissue (HFPim) were determined by immunohistochemistry. A Millar SPC 320 catheter was used to measure tumor interstitial fluid pressure (IFP). Results. CTFCol showed a positive correlation to IFP and an inverse correlation to HFPim. The apparent diffusion coefficient assessed by DW-MRI was inversely correlated to CTFCol, whereas no correlation was found between DCE-MRI-derived parameters and CTFCol. Conclusion. DW-MRI is a potentially useful method for characterizing the stromal microenvironment of tumors.

Gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has been suggested to be a useful non-invasive method for providing biomarkers for personalized cancer treatment. In this preclinical study, we investigated whether Gd-DTPA-based DCE-MRI may have the potential to differentiate between poorly and highly metastatic tumors.CK-160 cervical carcinoma and V-27 melanoma xenografts were used as tumor models. Fifty-six tumors were imaged, and parametric images of K(trans) (the volume transfer constant of Gd-DTPA) and v(e) (the fractional distribution volume of Gd-DTPA) were produced by pharmacokinetic analysis of the DCE-MRI series. The host mice were examined for lymph node metastases immediately after the DCE-MRI.Highly metastatic tumors showed lower values for median K(trans) than poorly metastatic tumors (p = 0.00033, CK-160; p0.00001, V-27). Median v(e) was lower for highly than for poorly metastatic V-27 tumors (p = 0.047), but did not differ significantly between metastatic and non-metastatic CK-160 tumors (p0.05).This study supports the clinical attempts to establish DCE-MRI as a method for providing biomarkers for tumor aggressiveness and suggests that tumors showing low K(trans) and low ve values may have high probability of lymphogenous metastatic dissemination.

Background and purpose Extensive hypoxia and high interstitial fluid pressure (IFP) in the primary tumor may cause resistance to radiation treatment and promote metastatic spread. The potential of gadomelitol-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in assessing the extent of hypoxia and the level of interstitial hypertension in tumors was investigated in this preclinical study. Materials and methods Twenty-three A-07 tumors were subjected to DCE-MRI and subsequent measurement of IFP and fraction of pimonidazole-positive hypoxic tissue (HF Pim ). Parametric tumor images of K trans , v e , and V b Tofts (Tofts model) and of K i and V b Patlak (Patlak model) were produced by pharmacokinetic analyses of the DCE-MRI series. Results There was no correlation between IFP and HF Pim in the tumors. K trans and K i decreased significantly with increasing HF Pim , whereas V b Tofts and V b Patlak increased significantly with increasing IFP. Conclusion Information on both the extent of hypoxia and the level of interstitial hypertension in A-07 tumors can be derived from a single DCE-MRI series by using gadomelitol as contrast agent.

Background and purpose High tumor interstitial fluid pressure (IFP) is associated with poor outcome in locally advanced carcinoma of the uterine cervix. We have recently developed a noninvasive assay of the IFP of tumors, and in this assay, the outward interstitial fluid flow velocity at the tumor surface ( v 0 ) is measured by Gd-DTPA-based DCE-MRI and used as a parameter for IFP. Here, we investigated the independent prognostic significance of v 0 in cervical cancer patients given cisplatin-based concurrent chemoradiotherapy with curative intent. Patients The study involved 62 evaluable patients from a cohort of 74 consecutive patients (Stage IB through IIIB) with a median follow-up of 5.5years. Results The actuarial disease-free survival (DFS) and overall survival (OS) at 5years were 67% and 76%, respectively. Significant associations were found between v 0 dichotomized about the median value and DFS and OS, both in the total patient cohort and a subcohort of 40 Stage IIB patients. Multivariate analysis involving stage, tumor volume, lymph node status, and v 0 revealed that only v 0 provided independent prognostic information about DFS and OS. Conclusion This investigation demonstrates a strong, independent prognostic impact of the pretreatment peritumoral fluid flow velocity in cervical cancer.

Background Antiangiogenic treatment may change the tumor microenvironment and hence influence the effect of conventional therapies. The potential of diffusion weighted magnetic resonance imaging (DW-MRI) and dynamic contrast enhanced MRI (DCE-MRI) in assessing microenvironmental effects of sunitinib treatment was investigated in this preclinical study. Methods Sunitinib-treated and untreated A-07 tumors were subjected to DW-MRI and DCE-MRI, and parametric images of ADC and Ktrans were produced. Microvascular density, hypoxic fraction, and necrotic fraction were assessed from immunohistochemical preparations, and tumor interstitial fluid pressure (IFP) was assessed with probe measurement. Results Sunitinib-treated tumors showed reduced microvascular density, increased hypoxic fraction, increased necrotic fraction, increased ADC, and reduced Ktrans, but did not differ from untreated tumors in growth rate and IFP. Conclusions Sunitinib treatment affected the tumor microenvironment without affecting tumor size. DW-MRI and DCE-MRI were sensitive to the sunitinib-induced changes in the tumor microenvironment.

Background Cancer patients with primary tumors showing extensive hypoxia and highly elevated interstitial fluid pressure (IFP) have poor prognosis. The potential of diffusion-weighted magnetic resonance imaging (DW-MRI) in assessing the hypoxic fraction, IFP, and metastatic propensity of tumors was investigated in this study. Methods A-07 and R-18 melanoma xenografts were used as general models of human cancer. DW-MRI was performed at 1.5 T, and maps of the apparent diffusion coefficient (ADC) were produced with in-house-made software developed in Matlab. Pimonidazole was used as a hypoxia marker. Tumor cell density and hypoxic fraction were assessed by quantitative analysis of histological sections. IFP was measured with a Millar catheter. Metastatic propensity was determined by examining tumor-bearing mice for pulmonary micrometastases post mortem. Results ADC decreased with increasing tumor cell density, independent of whether the A-07 and R-18 data were analyzed separately or together. In the A-07 line, ADC decreased with increasing hypoxic fraction and increasing IFP and was lower in metastatic than in nonmetastatic tumors, and in the R-18 line, ADC decreased with increasing hypoxic fraction. There was a strong inverse correlation between ADC and hypoxic fraction as well as between ADC and IFP across the two tumor lines, primarily because low ADC as well as high hypoxic fraction and high IFP were associated with high cell density. Conclusion Low ADC is a potentially useful biomarker of poor prognosis in cancer, since low ADC is mainly a consequence of high cell density, and high cell density may lead to increased hypoxia and interstitial hypertension and, therefore, increased microenvironment-associated metastasis.

Poor disease-free and overall survival rates in locally advanced cervical cancer are associated with a tumor micro-environment characterized by extensive hypoxia, interstitial hypertension, and high lactate concentrations. The potential of gadolinium diethylenetriamine pentaacetic acid-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in assessing the microenvironment and microenvironment-associated aggressiveness of cervical carcinomas was investigated in this preclinical study. CK-160 and TS-415 cervical carcinoma xenografts were used as tumor models. DCE-MRI was carried out at 1.5 T, and parametric images of K (trans) and v e were produced by pharmacokinetic analysis of the DCE-MRI series. Pimonidazole was used as a marker of hypoxia. A Millar catheter was used to measure tumor interstitial fluid pressure (IFP). The concentrations of glucose, adenosine triphosphate (ATP), and lactate were measured by induced metabolic bioluminescence imaging. High incidence of lymph node metastases was associated with high hypoxic fraction and high lactate concentration in CK-160 tumors and with high IFP and high lactate concentration in TS-415 tumors. Low K (trans) was associated with high hypoxic fraction, low glucose concentration, and high lactate concentration in tumors of both lines and with high incidence of metastases in CK-160 tumors. Associations between v e and microenvironmental parameters or metastatic propensity were not detected in any of the tumor lines. Taken together, this preclinical study suggests that K (trans) is a potentially useful biomarker for poor outcome of treatment in advanced cervical carcinoma. The possibility that K (trans) may be used to identify patients with cervical cancer who are likely to benefit from particularly aggressive treatment merits thorough clinical investigations.

Cancer patients showing highly elevated interstitial fluid pressure (IFP) in the primary tumor may benefit from particularly aggressive treatment. There is some evidence that gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) may be a useful non-invasive method for providing information on the IFP of tumors. The purpose of this preclinical study was to investigate whether any association between DCE-MRI-derived parametric images and tumor IFP can be strengthened by using MR contrast agents with higher molecular weights than that of Gd-DTPA.A-07 human melanoma xenografts were used as preclinical models of human cancer. Three contrast agents were compared: Gd-DTPA (0.55 kDa), P846 (3.5 kDa), and gadomelitol (6.5 kDa). A total of 46 tumors were subjected to DCE-MRI and subsequent measurement of IFP. Parametric images of K(trans) (the volume transfer constant of the contrast agent) and v(e) (the fractional distribution volume of the contrast agent) were produced by pharmacokinetic analysis of the DCE-MRI series.Significant inverse correlations were found between median K(trans) and IFP for Gd-DTPA (p = 0.0076; R(2) = 0.46; n = 14) and P846 (p = 0.0042; R(2) = 0.45; n = 16), whereas there was no correlation between median K(trans) and IFP for gadomelitol (p0.05; n = 16). Significant correlation between median v(e) and IFP was not found for any of the contrast agents (p0.05 for Gd-DTPA, P846, and gadomelitol).K(trans) images, but not v(e) images, derived by pharmacokinetic analysis of DCE-MRI data for low-molecular-weight contrast agents may provide information on the IFP of tumors. Any association between K(trans) and IFP cannot be expected to be improved by using contrast agents with higher molecular weights than those of Gd-DTPA and P846.

Elevated interstitial fluid pressure (IFP) in tumors can cause metastatic dissemination and treatment resistance, but its study poses a challenge because of a paucity of noninvasive imaging strategies. In this study, we address this issue by reporting the development of a noninvasive tool to assess tumor IFP and interstitial hypertension-induced lymph node metastasis. Using mouse xenograft models of several types of human cancer, we used gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA) as a contrast agent for dynamic contrast-enhanced MRI (DCE-MRI). Immediately after Gd-DTPA administration, a high-signal-intensity rim was observed in the tumor periphery, which moved outward with time. Assuming the velocity of Gd-DTPA to be equal to the fluid flow velocity, we used a simple model of peritumoral interstitial fluid flow to calculate the fluid flow velocity at the tumor surface (v0) based on the rim movement. Significant positive correlations were found between v0 and IFP in all tumor xenografts. Moreover, the primary tumors of metastasis-positive mice displayed higher IFP and v0 than the primary tumors of metastasis-negative mice. Findings were confirmed in cervical cancer patients with pelvic lymph node metastases, where we found v0 to be higher compared with patients without lymph node involvement (P < 0.00001). Together, these findings establish that Gd-DTPA-based DCE-MRI can noninvasively visualize tumor IFP, and they reveal the potential for v0 determined by this method to serve as a novel general biomarker of tumor aggressiveness. Cancer Res; 72(19); 4899–908. ©2012 AACR.

The prognosis is particularly poor for patients with advanced squamous cell carcinoma of the uterine cervix when the primary tumor has developed severe physiological abnormalities. The impact of the physiological microenvironment of the primary tumor on lymph node metastasis was investigated in this preclinical study.Xenografted tumors of two human cervical carcinoma lines (CK-160 and TS-415) transplanted into BALB/c nu/nu mice were included in the study. The fraction of radiobiologically hypoxic cells (HF(Rad)), interstitial fluid pressure (IFP), and extracellular pH (pH(e)) were measured in 22 CK-160 tumors and 16 TS-415 tumors and related to the metastatic status of the host mice.In CK-160, HF(Rad) was significantly higher in the metastatic than in the nonmetastatic tumors, whereas the metastatic and nonmetastatic tumors did not differ significantly in IFP or pH(e). In TS-415, IFP was significantly higher in the tumors that metastasized than in those that did not metastasize, whereas the tumors of the metastasis-positive and metastasis-negative mice did not differ significantly in HF(Rad) or pH(e).Lymph node metastasis is associated with abnormalities in the physiological microenvironment of the primary tumor in cervical carcinoma xenografts, and tumor line-specific mechanisms are probably involved.

The purpose of this preclinical study was to investigate the effect of two antiangiogenic agents, properdistatin and sunitinib, on the morphology and function of tumor vasculature. Properdistatin is a novel peptide derived from the thrombospondin-1 domain of the plasma protein properdin. Properdistatin treatment has been shown to inhibit angiogenesis and tumor growth in breast cancer xenografts. Sunitinib is a tyrosine kinase inhibitor that interacts with several receptors including vascular endothelial growth factor receptors 1-3, and platelet-derived growth factor receptors α-β. Sunitinib treatment has been shown to inhibit angiogenesis and tumor growth in a number of tumor models, and has been approved clinically for several indications. A-07 human melanoma xenografts grown in dorsal window chambers or as intradermal flank tumors were used as preclinical tumor model. In window chamber tumors, morphologic parameters of tumor vascular networks were assessed from high-resolution transillumination images, and blood supply time and plasma velocities were assessed from first-pass imaging movies recorded after a bolus of 155kDa tetramethylrhodamine isothiocyanate-labeled dextran had been administered intravenously. In flank tumors, parametric images of ADC and Ktrans were produced from diffusion weighted magnetic resonance imaging (DW-MRI) and dynamic contrast-enhanced MRI (DCE-MRI). Tumor hypoxia and necrosis were assessed from immunohistochemical preparations of the imaged tissue, and tumor interstitial fluid pressure (IFP) was assessed with probe measurement. Properdistatin treatment reduced the density of small-diameter vessels, reduced blood supply time, and increased plasma velocities, whereas sunitinib treatment did not alter blood supply time, reduced overall vessel density, induced tumor hypoxia and necrosis, and did not alter tumor IFP. Sunitinib-treated flank tumors showed reduced Ktrans values corresponding to a decrease in vessel density, and increased ADC values corresponding to induction of tumor necrosis. In conclusion, properdistatin treatment improved vascular function, whereas sunitinib treatment did not improve vascular function but induced tumor hypoxia and necrosis in A-07 tumors. This data demonstrates that different antiangiogenic agents may affect vascular function fundamentally different in the same tumor model. DW-MRI and DCE-MRI were sensitive to the sunitinib-induced changes in the tumor microenvironment, demonstrating that these MR-techniques may be used to identify tumors where antiangiogenic treatment does not improve vascular function. Citation Format: Jon-Vidar Gaustad, Trude G. Simonsen, Tord Hompland, Lise Mari K. Andersen, Einar K. Rofstad. Vascular function affected fundamentally different by different antiangiogenic agents. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A03.

Background and purpose Studies of intradermal melanoma xenografts have suggested that dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) may be a useful method for assessing the extent of hypoxia in tumors. Because the microvascular network of tumors is influenced significantly by the site of growth, we challenged this possibility in the present work by studying relationships between DCE-MRI-derived parameters and hypoxia in intramuscular melanoma xenografts. Materials and methods Intramuscular R-18, U-25, and V-27 tumors were subjected to DCE-MRI and measurement of the fraction of radiobiologically hypoxic cells (HF Rad ). Parametric images of K trans and v e were produced by pharmacokinetic analysis, and K trans and v e were related to HF Rad in individual tumors. Results K trans decreased with increasing HF Rad . The correlations between K trans and HF Rad were similar for the three tumor lines and were highly significant ( P v e and HF Rad . However, v e decreased significantly with increasing cell survival after single dose irradiation. Conclusion Intramuscular melanoma xenografts show similar inverse correlations between K trans and HF Rad as intradermal tumors, which support the current clinical attempts to establish DCE-MRI as a method for detecting hypoxia and defining therapeutic targets in tumors.

The fibrillar collagen network in tumor and normal tissues is different due to remodeling of the extracellular matrix during the malignant process. Collagen type I fibers have the crystalline and noncentrosymmetric properties required for generating the second-harmonic signal. The content and structure of collagen were studied by imaging the second-harmonic generation (SHG) signal in frozen sections from three tumor tissues, osteosarcoma, breast carcinoma, and melanoma, and were compared with corresponding normal tissues, bone/femur, breast, and dermis/skin. The collagen density was measured as the percentage of pixels containing SHG signal in tissue images, and material parameters such as the second-order nonlinear optical susceptibility given by the d22 coefficient and an empirical anisotropy parameter were used to characterize the collagen structure. Generally, normal tissues had much more collagen than tumor tissues. In tumor tissues, a cap of collagen was seen at the periphery, and further into the tumors, the distribution of collagen was sparse and heterogeneous. The difference in structure was reflected in the two times higher d22 coefficient and lower anisotropy values in normal tissues compared with tumor tissues. Together, the differences in the collagen content, distribution, and structure show that collagen signature is a promising diagnostic marker.

Characteristic changes in the organization of fibrillar collagen can potentially serve as an early diagnostic marker in various pathological processes. Tissue types containing collagen I can be probed by pulsed high-intensity laser radiation, thereby generating second harmonic light that provides information about the composition and structure at a microscopic level. A technique was developed to determine the essential second harmonic generation (SHG) parameters in a laser scanning microscope setup. A rat-tail tendon frozen section was rotated in the xy-plane with the pulsed laser light propagating along the z-axis. By analyzing the generated second harmonic light in the forward direction with parallel and crossed polarizer relative to the polarization of the excitation laser beam, the second-order nonlinear optical susceptibilities of the collagen fiber were determined. Systematic variations in SHG response between ordered and less ordered structures were recorded and evaluated. A 500 microm-thick z-cut lithiumniobate (LiNbO(3)) was used as reference. The method was applied on frozen sections of malignant melanoma and normal skin tissue. Significant differences were found in the values of d(22), indicating that this parameter has a potential role in differentiating between normal and pathological processes.

Background High interstitial fluid pressure (IFP) in the primary tumor is associated with poor disease-free survival in locally advanced cervical carcinoma. A noninvasive assay is needed to identify cervical cancer patients with highly elevated tumor IFP because these patients may benefit from particularly aggressive treatment. It has been suggested that dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA) as contrast agent may provide useful information on the IFP of cervical carcinomas. In this preclinical study, we investigated whether DCE-MRI with contrast agents with higher molecular weights (MW) than Gd-DTPA would be superior to Gd-DTPA-based DCE-MRI. Methods CK-160 human cervical carcinoma xenografts were subjected to DCE-MRI with Gd-DTPA (MW of 0.55 kDa) or gadomelitol (MW of 6.5 kDa) as contrast agent before tumor IFP was measured invasively with a Millar SPC 320 catheter. The DCE-MRI was carried out at a spatial resolution of 0.23 × 0.23 × 2.0 mm3 and a time resolution of 14 s by using a 1.5-T whole-body scanner and a slotted tube resonator transceiver coil constructed for mice. Parametric images were derived from the DCE-MRI recordings by using the Tofts iso-directional transport model and the Patlak uni-directional transport model. Results When gadomelitol was used as contrast agent, significant positive correlations were found between the parameters of both pharmacokinetic models and tumor IFP. On the other hand, significant correlations between DCE-MRI-derived parameters and IFP could not be detected with Gd-DTPA as contrast agent. Conclusion Gadomelitol is a superior contrast agent to Gd-DTPA in DCE-MRI of the IFP of CK-160 cervical carcinoma xenografts. Clinical studies attempting to develop DCE-MRI-based assays of the IFP of cervical carcinomas should involve contrast agents with higher MW than Gd-DTPA.