In vitro and in vivo characterization of [64Cu][Cu(elesclomol)] as a novel theranostic agent for hypoxic solid tumors.

PURPOSE: Hypoxic tumors are associated with therapy resistance and poor cancer prognosis, but methods to detect and counter tumor hypoxia remain insufficient. Our purpose was to investigate 64Cu(II)-elesclomol ([64Cu][Cu(ES)]) as a novel theranostic agent for hypoxic tumors, by implementing an improved production method and assessing its therapeutic and diagnostic potential compared to the established Cu-64 radiopharmaceuticals [64Cu]CuCl2 and [diacetyl-bis(N4-methylthiosemicarbazone) [64Cu][Cu(ATSM)]. METHODS: Cu-64 was produced using a biomedical cyclotron at 12 MeV with the reaction 64Ni(p,n)64Cu, followed by synthesis of [64Cu]CuCl2, [64Cu][Cu(ATSM)], and [64Cu][Cu(ES)]. In vitro therapeutic effects were assessed in both normoxic and hypoxic cells (22Rv1 and PC3 prostate cancer cells, and U-87MG glioblastoma cells) using the clonogenic assay and analyzing cellular uptake and internalization. In vivo therapeutic effects were assessed in 22Rv1 xenografts in BALB/cAnN-Foxn1nu/nu/Rj mice receiving a single or multiple doses of radiopharmaceutical, before their feasibility to detect tumor hypoxia was assessed by positron emission tomography (PET) in 22Rv1 and U-87MG xenografts. RESULTS: In vitro and in vivo studies demonstrated that [64Cu][Cu(ES)] reduced cell survival and inhibited tumor growth more effectively than [64Cu][Cu(ATSM)] and [64Cu]CuCl2. Hypoxia increased the cellular uptake and internalization of [64Cu][Cu(ES)] and [64Cu][Cu(ATSM)]. [64Cu][Cu(ES)]-PET tumor hypoxia detection was feasible and also revealed an unexpected finding of uptake in the brain. CONCLUSION: To the best of our knowledge, this is the first time that ES is radiolabeled with [64Cu]CuCl2 to [64Cu][Cu(ES)]. We demonstrated superior therapeutic effects of [64Cu][Cu(ES)] compared to [64Cu][Cu(ATSM)] and [64Cu]CuCl2 and that [64Cu][Cu(ES)]-PET is feasible. [64Cu][Cu(ES)] is a promising theranostic agent for hypoxic solid tumors.

Next-generation sequencing reveals mitogenome diversity in plasma extracellular vesicles from colorectal cancer patients.

BACKGROUND: Recent reports have demonstrated that the entire mitochondrial genome can be secreted in extracellular vesicles (EVs), but the biological attributes of this cell-free mitochondrial DNA (mtDNA) remain insufficiently understood. We used next-generation sequencing to compare plasma EV-derived mtDNA to that of whole blood (WB), peripheral blood mononuclear cells (PBMCs), and formalin-fixed paraffin-embedded (FFPE) tumor tissue from eight rectal cancer patients and WB and fresh-frozen (FF) tumor tissue from eight colon cancer patients. METHODS: Total DNA was isolated before the mtDNA was enriched by PCR with either two primer sets generating two long products or multiple primer sets (for the FFPE tumors), prior to the sequencing. mtDNA diversity was assessed as the total variant number, level of heteroplasmy (mutant mtDNA copies mixed with wild-type copies), variant distribution within the protein-coding genes, and the predicted functional effect of the variants in the different sample types. Differences between groups were compared by paired Student's t-test or ANOVA with Dunnett's multiple comparison tests when comparing matched samples from patients. Mann-Whitney U test was used when comparing differences between the cancer types and patient groups. Pearson correlation analysis was performed. RESULTS: In both cancer types, EV mtDNA presented twice as many variants and had significantly more low-level heteroplasmy than WB mtDNA. The EV mtDNA variants were clustered in the coding regions, and the proportion of EV mtDNA variants that were missense mutations (i.e., estimated to moderately affect the mitochondrial protein function) was significantly higher than in WB and tumor tissues. Nonsense mutations (i.e., estimated to highly affect the mitochondrial protein function) were only observed in the tumor tissues and EVs. CONCLUSION: Taken together, plasma EV mtDNA in CRC patients exhibits a high degree of diversity. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01816607 . Registered 22 March 2013.

Combined RBE and OER optimization in proton therapy with FLUKA based on EF5-PET.

INTRODUCTION: Tumor hypoxia is associated with poor treatment outcome. Hypoxic regions are more radioresistant than well-oxygenated regions, as quantified by the oxygen enhancement ratio (OER). In optimization of proton therapy, including OER in addition to the relative biological effectiveness (RBE) could therefore be used to adapt to patient-specific radioresistance governed by intrinsic radiosensitivity and hypoxia. METHODS: A combined RBE and OER weighted dose (ROWD) calculation method was implemented in a FLUKA Monte Carlo (MC) based treatment planning tool. The method is based on the linear quadratic model, with α and ß parameters as a function of the OER, and therefore a function of the linear energy transfer (LET) and partial oxygen pressure (pO2 ). Proton therapy plans for two head and neck cancer (HNC) patients were optimized with pO2 estimated from [18 F]-EF5 positron emission tomography (PET) images. For the ROWD calculations, an RBE of 1.1 (RBE1.1,OER ) and two variable RBE models, Rørvik (ROR) and McNamara (MCN), were used, alongside a reference plan without incorporation of OER (RBE1.1 ). RESULTS: For the HNC patients, treatment plans in line with the prescription dose and with acceptable target ROWD could be generated with the established tool. The physical dose was the main factor modulated in the ROWD. The impact of incorporating OER during optimization of HNC patients was demonstrated by the substantial difference found between ROWD and physical dose in the hypoxic tumor region. The largest physical dose differences between the ROWD optimized plans and the reference plan was 12.2 Gy. CONCLUSION: The FLUKA MC based tool was able to optimize proton treatment plans taking the tumor pO2 distribution from hypoxia PET images into account. Independent of RBE-model, both elevated LET and physical dose were found in the hypoxic regions, which shows the potential to increase the tumor control compared to a conventional optimization approach.

Energy Metabolism Is Altered in Radioresistant Rectal Cancer.

Resistance to neoadjuvant chemoradiation therapy is a significant clinical challenge in the management of rectal cancer. There is an unmet need to identify the underlying mechanisms of treatment resistance to enable the development of biomarkers predictive of response and novel treatment strategies to improve therapeutic response. In this study, an in vitro model of inherently radioresistant rectal cancer was identified and characterized to identify mechanisms underlying radioresistance in rectal cancer. Transcriptomic and functional analysis demonstrated significant alterations in multiple molecular pathways, including the cell cycle, DNA repair efficiency and upregulation of oxidative phosphorylation-related genes in radioresistant SW837 rectal cancer cells. Real-time metabolic profiling demonstrated decreased reliance on glycolysis and enhanced mitochondrial spare respiratory capacity in radioresistant SW837 cells when compared to radiosensitive HCT116 cells. Metabolomic profiling of pre-treatment serum samples from rectal cancer patients (n = 52) identified 16 metabolites significantly associated with subsequent pathological response to neoadjuvant chemoradiation therapy. Thirteen of these metabolites were also significantly associated with overall survival. This study demonstrates, for the first time, a role for metabolic reprograming in the radioresistance of rectal cancer in vitro and highlights a potential role for altered metabolites as novel circulating predictive markers of treatment response in rectal cancer patients.

MRI-based automatic segmentation of rectal cancer using 2D U-Net on two independent cohorts.

BACKGROUND: Tumor delineation is time- and labor-intensive and prone to inter- and intraobserver variations. Magnetic resonance imaging (MRI) provides good soft tissue contrast, and functional MRI captures tissue properties that may be valuable for tumor delineation. We explored MRI-based automatic segmentation of rectal cancer using a deep learning (DL) approach. We first investigated potential improvements when including both anatomical T2-weighted (T2w) MRI and diffusion-weighted MR images (DWI). Secondly, we investigated generalizability by including a second, independent cohort. MATERIAL AND METHODS: Two cohorts of rectal cancer patients (C1 and C2) from different hospitals with 109 and 83 patients, respectively, were subject to 1.5 T MRI at baseline. T2w images were acquired for both cohorts and DWI (b-value of 500 s/mm2) for patients in C1. Tumors were manually delineated by three radiologists (two in C1, one in C2). A 2D U-Net was trained on T2w and T2w + DWI. Optimal parameters for image pre-processing and training were identified on C1 using five-fold cross-validation and patient Dice similarity coefficient (DSCp) as performance measure. The optimized models were evaluated on a C1 hold-out test set and the generalizability was investigated using C2. RESULTS: For cohort C1, the T2w model resulted in a median DSCp of 0.77 on the test set. Inclusion of DWI did not further improve the performance (DSCp 0.76). The T2w-based model trained on C1 and applied to C2 achieved a DSCp of 0.59. CONCLUSION: T2w MR-based DL models demonstrated high performance for automatic tumor segmentation, at the same level as published data on interobserver variation. DWI did not improve results further. Using DL models on unseen cohorts requires caution, and one cannot expect the same performance.

Development of an automated production process of [64 Cu][Cu (ATSM)] for positron emission tomography imaging and theranostic applications.

Cyclotron-produced copper-64 radioisotope tracers offer the possibility to perform both diagnostic investigation by positron emission tomography (PET) and radiotherapy by a theranostic approach with bifunctional chelators. The versatile chemical properties of copper add to the importance of this isotope in medicinal investigation. [64 Cu][Cu (ATSM)] has shown to be a viable candidate for imaging of tumor hypoxia; a critical tumor microenvironment characteristic that typically signifies tumor progression and resistance to chemo-radiotherapy. Various production and radiosynthesis methods of [64 Cu][Cu (ATSM)] exist in labs, but usually involved non-standardized equipment with varying production qualities and may not be easily implemented in wider hospital settings. [64 Cu][Cu (ATSM)] was synthesized on a modified GE TRACERlab FXN automated synthesis module. End-of-synthesis (EOS) molar activity of [64 Cu][Cu (ATSM)] was 2.2-5.5 Ci/µmol (HPLC), 2.2-2.6 Ci/µmol (ATSM-titration), and 3.0-4.4 Ci/µmol (ICP-MS). Radiochemical purity was determined to be >99% based on radio-HPLC. The final product maintained radiochemical purity after 20 h. We demonstrated a simple and feasible process development and quality control protocols for automated cyclotron production and synthesis of [64 Cu][Cu (ATSM)] based on commercially distributed standardized synthesis modules suitable for PET imaging and theranostic studies.

The circulating soluble form of the CD40 costimulatory immune checkpoint receptor and liver metastasis risk in rectal cancer.

BACKGROUND: In colorectal cancer, the inflamed tumour microenvironment with its angiogenic activities is immune- tolerant and incites progression to liver metastasis. We hypothesised that angiogenic and inflammatory factors in serum samples from patients with non-metastatic rectal cancer could inform on liver metastasis risk. METHODS: We measured 84 angiogenic and inflammatory markers in serum sampled at the time of diagnosis within the population-based cohort of 122 stage I-III patients. In a stepwise manner, the statistically strongest proteins associated with time to development of liver metastasis were analysed in the corresponding serum samples from 273 stage II-III rectal cancer patients in three independent cohorts. RESULTS: We identified the soluble form of the costimulatory immune checkpoint receptor cluster of differentiation molecule 40 (sCD40) as a marker of liver metastasis risk across all patient cohorts-the higher the sCD40 level, the shorter time to liver metastasis. In patients receiving neoadjuvant treatment, the sCD40 value remained an independent variable associated with progression to liver metastasis along with the local treatment response. Of note, serum sCD40 was not associated with progression to lung metastasis. CONCLUSIONS: Circulating sCD40 is a marker of liver metastasis risk in rectal cancer and may be developed for use in clinical practice.

Sex disparities in vitamin D status and the impact on systemic inflammation and survival in rectal cancer.

BACKGROUND: We reported previously that rectal cancer patients given curative-intent chemotherapy, radiation, and surgery for non-metastatic disease had enhanced risk of metastatic progression and death if circulating levels of 25-hydroxyvitamin D [25(OH) D] were low. Here we investigated whether the association between the vitamin D status and prognosis pertains to the general, unselected population of rectal cancer patients. METHODS: Serum 25(OH) D at the time of diagnosis was assessed in 129 patients, enrolled 2013-2017 and representing the entire range of rectal cancer stages, and analyzed with respect to season, sex, systemic inflammation, and survival. RESULTS: In the population-based cohort residing at latitude 60°N, 25(OH) D varied according to season in men only, who were overrepresented among the vitamin D-deficient (< 50 nmol/L) patients. Consistent with our previous findings, the individuals presenting with T4 disease had significantly reduced 25(OH) D levels. Low vitamin D was associated with systemic inflammation, albeit with distinct modes of presentation. While men with low vitamin D showed circulating markers typical for the systemic inflammatory response (e.g., elevated erythrocyte sedimentation rate), the corresponding female patients had elevated serum levels of interleukin-6 and the chemokine (C-X-C motif) ligand 7. Despite disparities in vitamin D status and the potential effects on disease attributes, significantly shortened cancer-specific survival was observed in vitamin D-deficient patients irrespective of sex. CONCLUSION: This unselected rectal cancer cohort confirmed the interconnection of low vitamin D, more advanced disease presentation, and poor survival, and further suggested it may be conditional on disparate modes of adverse systemic inflammation in men and women. TRIAL REGISTRATION: ClinicalTrials.gov NCT01816607 ; registration date: 22 March 2013.

Immunogenic cell death by neoadjuvant oxaliplatin and radiation protects against metastatic failure in high-risk rectal cancer.

OBJECTIVE: High rates of systemic failure in locally advanced rectal cancer call for a rational use of conventional therapies to foster tumor-defeating immunity. METHODS: We analyzed the high-mobility group box-1 (HMGB1) protein, a measure of immunogenic cell death (ICD), in plasma sampled from 50 patients at the time of diagnosis and following 4 weeks of induction chemotherapy and 5 weeks of sequential chemoradiotherapy, both neoadjuvant modalities containing oxaliplatin. The patients had the residual tumor resected and were followed for long-term outcome. RESULTS: Patients who met the main study end point-freedom from distant recurrence-showed a significant rise in HMGB1 during the induction chemotherapy and consolidation over the chemoradiotherapy. The higher the ICD increase, the lower was the metastatic failure risk (hazard ratio 0.26, 95% confidence interval 0.11-0.62, P = 0.002). However, patients who received the full-planned oxaliplatin dose of the chemoradiotherapy regimen had poorer metastasis-free survival (P = 0.020) than those who had the oxaliplatin dose reduced to avert breach of the radiation delivery, which is critical to maintain efficient tumor cell kill and in the present case, probably also protected the ongoing radiation-dependent ICD response from systemic oxaliplatin toxicity. CONCLUSION: The findings indicated that full-dose induction oxaliplatin followed by an adapted oxaliplatin dose that was compliant with full-intensity radiation caused induction and maintenance of ICD and as a result, durable disease-free outcome for a patient population prone to metastatic progression.

High level of circulating vitamin D during neoadjuvant therapy may lower risk of metastatic progression in high-risk rectal cancer.

BACKGROUND: Following curative-intent neoadjuvant therapy in locally advanced rectal cancer, metastatic progression is still dominant. We investigated if patients' circulating 25-hydroxyvitamin D [25(OH)D] levels were associated with outcome. METHODS: Serum 25(OH)D concentration was assessed by liquid chromatography-mass spectrometry in samples collected from 84 patients at baseline, completion of the neoadjuvant therapy, and treatment evaluation before surgery, and analyzed with respect to season, disease presentation, and treatment effects. RESULTS: In the cohort of patients residing at latitude 58-62°N, baseline 25(OH)D differed significantly over the seasons, with highest measures (mean of 71.2 ± 5.6 nmol/L) in summer and lowest (48.7 ± 4.5 nmol/L) in spring, and changed over the three-month neoadjuvant period till response evaluation solely owing to season. The patient subgroup with slightly reduced performance status, anemia, and T4 disease that did not respond to the neoadjuvant therapy (ypT4 cases), had significantly lower baseline 25(OH)D (below 50 nmol/L) than T4 cases with response (ypT0-3) and T2-3 cases (above 60 nmol/L). Compared to the T4 patients with levels above 50 nmol/L, regarded as sufficient for a healthy bone status, those presenting levels below had significantly heightened risk of disease progression (mainly metastasis) and death, with hazard ratio of 3 and 17, respectively, on adjustment for age, sex, body mass index, and season. CONCLUSION: Rectal cancer T4 cases had high risk of metastatic progression and death if circulating 25(OH)D levels were insufficient but obtained short-term and long-term outcome to neoadjuvant treatment no worse than patients with T2-3 disease when 25(OH)D was sufficient. TRIAL REGISTRATION: ClinicalTrials.gov NCT00278694 ; registration date: 16 January 2006, retrospective to enrollment of the first 10 patients of the current report.

Systemic immune response induced by oxaliplatin-based neoadjuvant therapy favours survival without metastatic progression in high-risk rectal cancer.

BACKGROUND: Systemic failure remains a challenge in rectal cancer. We investigated the possible systemic anti-tumour immune activity invoked within oxaliplatin-based neoadjuvant therapy. METHODS: In two high-risk patient cohorts, we assessed the circulating levels of the fms-like tyrosine kinase 3 ligand (Flt3L), a factor reflecting both therapy-induced myelosuppression and activation of tumour antigen-presenting dendritic cells, at baseline and following induction chemotherapy and sequential chemoradiotherapy, both modalities containing oxaliplatin. The primary end point was progression-free survival (PFS). RESULTS: In both cohorts, the median Flt3L level was significantly higher at completion of each sequential modality than at baseline. The 5-year PFS (most events being metastatic progression) was 68% and 71% in the two cohorts consisting of 33% and 52% T4 cases. In the principal cohort, a high Flt3L level following the induction chemotherapy was associated with low risk for a PFS event (HR: 0.15; P < 0.01). These patients also had available dose scheduling and toxicity data, revealing that oxaliplatin dose reduction during chemoradiotherapy, undertaken to maintain compliance to the radiotherapy protocol, was associated with advantageous PFS (HR: 0.47; P = 0.046). CONCLUSION: In high-risk rectal cancer, oxaliplatin-containing neoadjuvant therapy may promote an immune response that favours survival without metastatic progression.

Dynamic multi-echo DCE- and DSC-MRI in rectal cancer: Low primary tumor Ktrans and ΔR2* peak are significantly associated with lymph node metastasis.

PURPOSE: To implement a dynamic contrast-based multi-echo MRI sequence in assessment of rectal cancer and evaluate associations between histopathologic data and the acquired dynamic contrast-enhanced (DCE) and dynamic susceptibility contrast (DSC) -MRI parameters. MATERIALS AND METHODS: This pilot study reports results from 17 patients with resectable rectal cancer. Dynamic contrast-based multi-echo MRI (1.5T) was acquired using a three-dimensional multi-shot EPI sequence, yielding both DCE- and DSC-data following a single injection of contrast agent. The Institutional Review Board approved the study and all patients provided written informed consent. Quantitative analysis was performed by pharmacokinetic modeling on DCE data and tracer kinetic modeling on DSC data. Mann-Whitney U-test and receiver operating characteristics curve statistics was used to evaluate associations between histopathologic data and the acquired DCE- and DSC-MRI parameters. RESULTS: For patients with histologically confirmed nodal metastasis, the primary tumor demonstrated a significantly lower Ktrans and peak change in R2*, R2*-peakenh , than patients without nodal metastasis, showing a P-value of 0.010 and 0.005 for reader 1, and 0.043 and 0.019 for reader 2, respectively. CONCLUSION: This study shows the feasibility of acquiring DCE- and DSC-MRI in rectal cancer by dynamic multi-echo MRI. A significant association was found between both Ktrans and R2*-peakenh in the primary tumor and histological nodal status of the surgical specimen, which may improve stratification of patients to intensified multimodal treatment. LEVEL OF EVIDENCE: 4 Technical Efficacy: Stage 2 J. MAGN. RESON. IMAGING 2017;46:194-206.

Diffusion-weighted magnetic resonance imaging of rectal cancer: tumour volume and perfusion fraction predict chemoradiotherapy response and survival.

BACKGROUND: In locally advanced rectal cancer (LARC), responses to preoperative treatment are highly heterogeneous and more accurate diagnostics are likely to enable more individualised treatment approaches with improved responses. We investigated the potential of diffusion-weighted magnetic resonance imaging (DW MRI), with quantification of the apparent diffusion coefficient (ADC) and perfusion fraction (F), as well as volumetry from T2-weighted (T2W) MRI, for prediction of therapeutic outcome. MATERIAL AND METHODS: In 27 LARC patients receiving neoadjuvant chemotherapy (NACT) before chemoradiotherapy (CRT), T2W- and DW MRI were obtained before and after NACT. Tumour volumes were delineated in T2W MRI and ADCs and Fs were estimated from DW MRI using a simplified approach to the intravoxel incoherent motion (IVIM) model. Mean tumour values and histogram analysis of whole-tumour heterogeneity were correlated with histopathologic tumour regression grade (TRG) and 5-year progression-free survival (PFS). RESULTS: At baseline, high tumour F predicted good tumour response (TRG1-2) (AUC = 0.79, p = 0.01), with a sensitivity of 69% and a specificity of 100%. The combination of F and tumour volume (Fpre/Vpre) gave the highest prediction of poor tumour response (AUC = 0.93, p < 0.001) with a sensitivity of 88% and a specificity of 91%, and also predicted PFS (p < 0.01). Baseline tumour ADC was not significantly related to therapeutic outcome, whereas a positive change in ADC from baseline to after NACT, ΔADC, significantly predicted good tumour response (AUC = 0.83, p < 0.01, 83% sensitivity, 73% specificity), but not PFS. CONCLUSIONS: The MRI parameter F/V at baseline was a remarkably strong predictor of both histopathologic tumour response and 5-year PFS in patients with LARC.

Pro-survival responses to the dual inhibition of anti-apoptotic Bcl-2 family proteins and mTOR-mediated signaling in hypoxic colorectal carcinoma cells.

BACKGROUND: The use of targeted agents to impel dual inhibition of anti-apoptotic mechanisms and mTOR-mediated pro-survival signaling in colorectal carcinoma (CRC) cell lines with KRAS or BRAF mutation has been shown to induce apoptosis, a timely result given CRC entities harboring such mutations are in need of new therapies. Since CRC comprises heterogeneous tumors with predominant hypoxic components, we investigated effects of an inhibitor of anti-apoptotic Bcl-2 family proteins (ABT-737) in combination with an mTOR inhibitor (AZD8055)-collectively referred to as combo-Rx, in hypoxic CRC cell lines. METHODS: Cell viability measures, expression of proteins implicated in apoptosis and MAPK/PI3K-AKT/mTOR pathway signaling, and profiling of composite kinase activities were undertaken in a panel of 14 cell lines. RESULTS: In hypoxic conditions, combo-Rx suppressed viability of 13 of the cell lines, albeit ABT-737 did not significantly potentiate the inhibitory effect of single-agent AZD8055 in six of the models. Hypoxic KRAS/PIK3CA-mutant HCT-116 and HCT-15 cell lines (both with low endogenous expression of the anti-apoptotic Mcl-1 protein and showing augmented inhibition of viability following the addition of ABT-737 to AZD8055) responded to combo-Rx by induction of apoptosis but with the simultaneous strong Mcl-1 up-regulation and activation of MAPK/PI3K-conducted signaling. In contrast, in hypoxic KRAS-mutant LoVo (devoid of PIK3CA mutation), BRAF/PIK3CA-mutant RKO, and wild-type Colo320DM cell lines (all with high endogenous Mcl-1 expression and being resistant to the additional effect of ABT-737 to AZD8055), combo-Rx did not elicit apoptotic or pro-survival responses. CONCLUSIONS: The concurrent inhibition of anti-apoptotic proteins and mTOR-mediated signaling in hypoxic KRAS/PIK3CA-mutant CRC cell lines resulted in pro-survival responses in parallel with the intended anti-proliferative effects, a finding that should be of note if considering combinatory targeting of multiple pathways in this CRC entity.

Sulfamate inhibitor S4 influences carbonic anhydrase IX ectodomain shedding in colorectal carcinoma cells.

Carbonic anhydrase IX (CAIX) is a pivotal pH regulator under hypoxia, which by its tumor-specific expression represents an attractive target for cancer therapy. Here, we report on effects of the sulfamate CAIX inhibitor S4 (4-(3'-(3″,5″-dimethylphenyl)ureido)phenyl sulfamate) in colorectal carcinoma cell lines. S4 was administered under experimental hypoxia or normoxia to HT29, KM20L2 and HCT116 cells. Effects on survival, proliferation, pH, lactate extrusion and CAIX protein expression were evaluated. S4 treatment resulted in attenuated hypoxia-induced extracellular acidification and reduced clonogenic survival under hypoxia in HT29 cells. The pH effects were present only in a [Formula: see text]-free buffer system and were accompanied by decreased lactate extrusion. The main finding of this work was that S4 treatment caused alterations in CAIX ectodomain shedding. This merits further investigation to understand how sulfamates influence CAIX activity and how such drugs may be of use in cancer treatment.

Early increase in circulating carbonic anhydrase IX during neoadjuvant treatment predicts favourable outcome in locally advanced rectal cancer.

BACKGROUND: Locally advanced rectal cancer (LARC) comprises heterogeneous tumours with predominant hypoxic components. The hypoxia-inducible metabolic shift causes microenvironmental acidification generated by carbonic anhydrase IX (CAIX) and facilitates metastatic progression, the dominant cause of failure in LARC. METHODS: Using a commercially available immunoassay, circulating CAIX was assessed in prospectively archived serial serum samples collected during combined-modality neoadjuvant treatment of LARC patients and correlated to histologic tumour response and progression-free survival (PFS). RESULTS: Patients who from their individual baseline level displayed serum CAIX increase above a threshold of 224 pg/ml (with 96 % specificity and 39 % sensitivity) after completion of short-course neoadjuvant chemotherapy (NACT) prior to long-course chemoradiotherapy and definitive surgery had significantly better 5-year PFS (94 %) than patients with below-threshold post-NACT versus baseline alteration (PFS rate of 56 %; p < 0.01). This particular CAIX parameter, ΔNACT, was significantly correlated with histologic ypT0-2 and ypN0 outcome (p < 0.01) and remained an independent PFS predictor in multivariate analysis wherein it was entered as continuous variable (p = 0.04). CONCLUSIONS: Our results indicate that low ΔNACT, i.e., a weak increase in serum CAIX level following initial neoadjuvant treatment (in this case two cycles of the Nordic FLOX regimen), might be used as risk-adapted stratification to postoperative therapy or other modes of intensification of the combined-modality protocol in LARC. TRIAL REGISTRATION: ClinicalTrials.gov NCT00278694.

Interim 18F-FDG-PET based response-adaptive dose escalation of proton therapy for head and neck cancer: a treatment planning feasibility study.

BACKGROUND: Image-driven dose escalation to tumor subvolumes has been proposed to improve treatment outcome in head and neck cancer (HNC). We used 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) acquired at baseline and into treatment (interim) to identify biologic target volumes (BTVs). We assessed the feasibility of interim dose escalation to the BTV with proton therapy by simulating the effects to organs at risk (OARs). METHODS: We used the semiautomated just-enough-interaction (JEI) method to identify BTVs in 18F-FDG-PET images from nine HNC patients. Between baseline and interim FDG-PET, patients received photon radiotherapy. BTV was identified assuming that high standardized uptake value (SUV) at interim reflected tumor radioresistance. Using Eclipse (Varian Medical Systems), we simulated a 10% (6.8 Gy(RBE1.1)) and 20% (13.6 Gy(RBE1.1)) dose escalation to the BTV with protons and compared results with proton plans without dose escalation. RESULTS: At interim 18F-FDG-PET, radiotherapy resulted in reduced SUV compared to baseline. However, spatial overlap between high-SUV regions at baseline and interim allowed for BTV identification. Proton therapy planning demonstrated that dose escalation to the BTV was feasible, and except for some 20% dose escalation plans, OAR doses did not significantly increase. CONCLUSION: Our in silico analysis demonstrated the potential for interim 18F-FDG-PET response-adaptive dose escalation to the BTV with proton therapy. This approach may give more efficient treatment to HNC with radioresistant tumor subvolumes without increasing normal tissue toxicity. Studies in larger cohorts are required to determine the full potential for interim 18F-FDG-PET-guided dose escalation of proton therapy in HNC.

Quantitative MRI-based radiomics analysis identifies blood flow feature associated to overall survival for rectal cancer patients.

Radiomics objectively quantifies image information through numerical metrics known as features. In this study, we investigated the stability of magnetic resonance imaging (MRI)-based radiomics features in rectal cancer using both anatomical MRI and quantitative MRI (qMRI), when different methods to define the tumor volume were used. Second, we evaluated the prognostic value of stable features associated to 5-year progression-free survival (PFS) and overall survival (OS). On a 1.5 T MRI scanner, 81 patients underwent diagnostic MRI, an extended diffusion-weighted sequence with calculation of the apparent diffusion coefficient (ADC) and a multiecho dynamic contrast sequence generating both dynamic contrast-enhanced and dynamic susceptibility contrast (DSC) MR, allowing quantification of Ktrans, blood flow (BF) and area under the DSC curve (AUC). Radiomic features were extracted from T2w images and from ADC, Ktrans, BF and AUC maps. Tumor volumes were defined with three methods; machine learning, deep learning and manual delineations. The interclass correlation coefficient (ICC) assessed the stability of features. Internal validation was performed on 1000 bootstrap resamples in terms of discrimination, calibration and decisional benefit. For each combination of image and volume definition, 94 features were extracted. Features from qMRI contained higher prognostic potential than features from anatomical MRI. When stable features (> 90% ICC) were compared with clinical parameters, qMRI features demonstrated the best prognostic potential. A feature extracted from the DSC MRI parameter BF was associated with both PFS (p = 0.004) and OS (p = 0.004). In summary, stable qMRI-based radiomics features was identified, in particular, a feature based on BF from DSC MRI was associated with both PFS and OS.

Imaging the tumour microenvironment in rectal cancer: Decline in tumour blood flow during radiotherapy predicts good outcome.

Background and purpose: Measuring rectal tumour response to radiation is pivotal to restaging patients and for possibly stratification to a watch-and-wait strategy. Recognizing the importance of the tumour microenvironment, we investigated a less explored quantitative imaging marker assessing tumour blood flow (BF) for its potential to predict overall survival (OS). Materials and methods: 24 rectal cancer patients given curative-intent neoadjuvant radiotherapy underwent a multi-echo dynamic magnetic resonance imaging (MRI) sequence with gadolinium contrast for quantification of tumour BF before either 25x2 Gy (n = 18) with concomitant chemotherapy or 5x5 Gy (n = 6). CD34 staining of excised tumour tissue was performed and baseline blood samples were analysed for lactate dehydrogenase (LDH) and angiopoietin-2 (ANGPT-2). Tumour volumes were measured before and after treatment. After subsequent surgery, ypTN scoring assessed tumour response. Cox regression for 5-year OS analysis and t-test for group comparisons were performed. Results: The change in tumour BF (ΔBF) during neoadjuvant radiotherapy was a significant marker of OS, whereas tumour stage and volume were not related to OS. All patients with >20 % decline in BF were long-term survivors. Separating cases in two groups based on ΔBF revealed that patients with increase or a low decrease had higher baseline LDH (p = 0.032) and ANGPT-2 (p = 0.028) levels. Conclusion: MRI-assessed tumour ΔBF during neoadjuvant treatment is a significant predictor of OS in rectal cancer patients, making ΔBF a potential quantitative imaging biomarker for treatment stratification. Blood LDH and ANGPT-2 indicate that non-responding tumours may have a hypoxic microenvironment resistant to radiotherapy.

Potential of Deep Learning in Quantitative Magnetic Resonance Imaging for Personalized Radiotherapy.

Quantitative magnetic resonance imaging (qMRI) has been shown to provide many potential advantages for personalized adaptive radiotherapy (RT). Deep learning models have proven to increase efficiency, robustness and speed for different qMRI tasks. Therefore, this article discusses the current state-of-the-art and potential future opportunities as well as challenges related to the use of deep learning in qMRI for target contouring, quantitative parameter estimation and also the generation of synthetic computerized tomography (CT) data based on MRI in personalized RT.