Non-Contrast-Enhanced Multiparametric MRI of the Hypoxic Tumor Microenvironment Allows Molecular Subtyping of Breast Cancer: A Pilot Study.

Tumor neoangiogenesis is an important hallmark of cancer progression, triggered by alternating selective pressures from the hypoxic tumor microenvironment. Non-invasive, non-contrast-enhanced multiparametric MRI combining blood-oxygen-level-dependent (BOLD) MRI, which depicts blood oxygen saturation, and intravoxel-incoherent-motion (IVIM) MRI, which captures intravascular and extravascular diffusion, can provide insights into tumor oxygenation and neovascularization simultaneously. Our objective was to identify imaging markers that can predict hypoxia-induced angiogenesis and to validate our findings using multiplexed immunohistochemical analyses. We present an in vivo study involving 36 female athymic nude mice inoculated with luminal A, Her2+, and triple-negative breast cancer cells. We used a high-field 9.4-tesla MRI system for imaging and subsequently analyzed the tumors using multiplex immunohistochemistry for CD-31, PDGFR-ß, and Hif1-α. We found that the hyperoxic-BOLD-MRI-derived parameter ΔR2* discriminated luminal A from Her2+ and triple-negative breast cancers, while the IVIM-derived parameter fIVIM discriminated luminal A and Her2+ from triple-negative breast cancers. A comprehensive analysis using principal-component analysis of both multiparametric MRI- and mpIHC-derived data highlighted the differences between triple-negative and luminal A breast cancers. We conclude that multiparametric MRI combining hyperoxic BOLD MRI and IVIM MRI, without the need for contrast agents, offers promising non-invasive markers for evaluating hypoxia-induced angiogenesis.

Hyperoxic BOLD-MRI-Based Characterization of Breast Cancer Molecular Subtypes Is Independent of the Supplied Amount of Oxygen: A Preclinical Study.

Hyperoxic BOLD-MRI targeting tumor hypoxia may provide imaging biomarkers that represent breast cancer molecular subtypes without the use of injected contrast agents. However, the diagnostic performance of hyperoxic BOLD-MRI using different levels of oxygen remains unclear. We hypothesized that molecular subtype characterization with hyperoxic BOLD-MRI is feasible independently of the amount of oxygen. Twenty-three nude mice that were inoculated into the flank with luminal A (n = 9), Her2+ (n = 5), and triple-negative (n = 9) human breast cancer cells were imaged using a 9.4 T Bruker BioSpin system. During BOLD-MRI, anesthesia was supplemented with four different levels of oxygen (normoxic: 21%; hyperoxic: 41%, 71%, 100%). The change in the spin-spin relaxation rate in relation to the normoxic state, ΔR2*, dependent on the amount of erythrocyte-bound oxygen, was calculated using in-house MATLAB code. ΔR2* was significantly different between luminal A and Her2+ as well as between luminal A and triple-negative breast cancer, reflective of the less aggressive luminal A breast cancer's ability to better deliver oxygen-rich hemoglobin to its tissue. Differences in ΔR2* between subtypes were independent of the amount of oxygen, with robust distinction already achieved with 41% oxygen. In conclusion, hyperoxic BOLD-MRI may be used as a biomarker for luminal A breast cancer identification without the use of exogenous contrast agents.

Detecting Phylogenetic Signal and Adaptation in Papionin Cranial Shape by Decomposing Variation at Different Spatial Scales.

Phylogenetic reconstruction based on morphometric data is hampered by homoplasies. For example, many similarities in cranial form between primate taxa more strongly reflect ecological similarities rather than phylogenetic relatedness. However, the way in which the different cranial bones constitute cranial form is, if at all, of less functional relevance and thus largely hidden from selection. We propose that these "constructional details" are better indicators of phylogenetic history than any large-scale shape feature or raw form variable. Within a geometric morphometric context, we show how to analyze the relative extent of bones independently of differences in overall shape. We also show how to decompose total shape variation into small-scale and large-scale shape variation. We apply both methods to the midsagittal cranial morphology of papionin monkeys, which are well known for the discrepancy between morphological similarities and phylogenetic relationships. We study phylogenetic signal and functional adaptation using a molecular phylogeny and contextual data on feeding ecology and locomotor behavior. As expected, total cranial shape, bone outline shape, and large-scale shape features were only weakly associated with phylogenetic distance. But the relative bone contributions and small-scale shape features were both highly correlated with phylogenetic distances. By contrast, the association with ecological and behavioral variables was strongest for the outline shape and large-scale shape features. Studies of morphological adaptation and phylogenetic history thus profit from a decomposition of shape variation into different spatial scales. [Adaptation; canalization; cranial shape; geometric morphometrics; papionini; partial warps; phylogeny.].

Disruption of the anaphase-promoting complex confers resistance to TTK inhibitors in triple-negative breast cancer.

TTK protein kinase (TTK), also known as Monopolar spindle 1 (MPS1), is a key regulator of the spindle assembly checkpoint (SAC), which functions to maintain genomic integrity. TTK has emerged as a promising therapeutic target in human cancers, including triple-negative breast cancer (TNBC). Several TTK inhibitors (TTKis) are being evaluated in clinical trials, and an understanding of the mechanisms mediating TTKi sensitivity and resistance could inform the successful development of this class of agents. We evaluated the cellular effects of the potent clinical TTKi CFI-402257 in TNBC models. CFI-402257 induced apoptosis and potentiated aneuploidy in TNBC lines by accelerating progression through mitosis and inducing mitotic segregation errors. We used genome-wide CRISPR/Cas9 screens in multiple TNBC cell lines to identify mechanisms of resistance to CFI-402257. Our functional genomic screens identified members of the anaphase-promoting complex/cyclosome (APC/C) complex, which promotes mitotic progression following inactivation of the SAC. Several screen candidates were validated to confer resistance to CFI-402257 and other TTKis using CRISPR/Cas9 and siRNA methods. These findings extend the observation that impairment of the APC/C enables cells to tolerate genomic instability caused by SAC inactivation, and support the notion that a measure of APC/C function could predict the response to TTK inhibition. Indeed, an APC/C gene expression signature is significantly associated with CFI-402257 response in breast and lung adenocarcinoma cell line panels. This expression signature, along with somatic alterations in genes involved in mitotic progression, represent potential biomarkers that could be evaluated in ongoing clinical trials of CFI-402257 or other TTKis.

Symptomatic suspected gluten exposure is common among patients with coeliac disease on a gluten-free diet.

BACKGROUND: A gluten-free diet is the only recommended treatment for coeliac disease. AIM: To determine the prevalence and characteristics of reactions to gluten among persons with coeliac disease on a gluten-free diet. METHODS: Adults with biopsy proven, newly diagnosed coeliac disease were prospectively enrolled. A survey related to diet adherence and reactions to gluten was completed at study entry and 6 months. The Coeliac Symptom Index, Coeliac Diet Assessment Tool (CDAT) and Gluten-Free Eating Assessment Tool (GF-EAT) were used to measure coeliac disease symptoms and gluten-free diet adherence. RESULTS: Of the 105 participants, 91% reported gluten exposure <1 per month and median CDAT score was 9 (IQR 8-11), consistent with adequate adherence. A suspected symptomatic reaction to gluten was reported by 66%. Gluten consumption was unsuspected until a reaction occurred (63%) or resulted from problems ordering in a restaurant (29%). The amount of gluten consumed ranged from cross-contact (30%) to a major ingredient (10%). Median time to symptom onset was 1 h (range 10 min to 48 h), and median symptom duration was 24 h (range 1 h to 8 days). Common symptoms included abdominal pain (80%), diarrhoea (52%), fatigue (33%), headache (30%) and irritability (29%). CONCLUSIONS: Reactions to suspected gluten exposure are common among patients with coeliac disease on a gluten-free diet. Eating at restaurants and other peoples' homes remain a risk for unintentional gluten exposure. When following individuals with coeliac disease, clinicians should include questions regarding reactions to gluten as part of their assessment of gluten-free diet adherence.

Living gluten-free: adherence, knowledge, lifestyle adaptations and feelings towards a gluten-free diet.

BACKGROUND: A gluten-free diet (GFD) requires tremendous dedication, involving substantive changes to diet and lifestyle that may have a significant impact upon quality of life. The present study aimed io assess dietary adherence, knowledge of a GFD, and the emotional and lifestyle impact of a GFD. METHODS: Community dwelling adults following a GFD completed a questionnaire with items related to reasons for avoiding gluten, diagnostic testing, GFD adherence, knowledge and sources of information about a GFD, the Work and Social Adjustment Scale, and the effect of a GFD diet on lifestyle, feelings and behaviours. RESULTS: Strict GFD adherence among the 222 coeliac disease (CD) patients was 56%. Non-CD individuals (n = 38) were more likely to intentionally ingest gluten (odds ratio = 3.7; 95% confidence interval = 1.4-9.4). The adverse impact of a GFD was modest but most pronounced in the social domain. Eating shifted from the public to the domestic sphere and there were feelings of social isolation. Affective responses reflected resilience because acceptance and relief were experienced more commonly than anxiety or anger. Non-CD respondents were less knowledgeable and less likely to consult health professionals. They experienced less anger and depression and greater pleasure in eating than CD respondents. CONCLUSIONS: The findings obtained in the present suggest there is good potential for positive adaptation to the demands of a GFD; nevertheless, there is a measurable degree of social impairment that merits further study. The GFD may be a viable treatment option for conditions other than CD; however, education strategies regarding the need for diagnostic testing to exclude CD are required.

The interaction between caveolin-1 and Rho-GTPases promotes metastasis by controlling the expression of alpha5-integrin and the activation of Src, Ras and Erk.

Proteins containing a caveolin-binding domain (CBD), such as the Rho-GTPases, can interact with caveolin-1 (Cav1) through its caveolin scaffold domain. Rho-GTPases are important regulators of p130(Cas), which is crucial for both normal cell migration and Src kinase-mediated metastasis of cancer cells. However, although Rho-GTPases (particularly RhoC) and Cav1 have been linked to cancer progression and metastasis, the underlying molecular mechanisms are largely unknown. To investigate the function of Cav1-Rho-GTPase interaction in metastasis, we disrupted Cav1-Rho-GTPase binding in melanoma and mammary epithelial tumor cells by overexpressing CBD, and examined the loss-of-function of RhoC in metastatic cancer cells. Cancer cells overexpressing CBD or lacking RhoC had reduced p130(Cas) phosphorylation and Rac1 activation, resulting in an inhibition of migration and invasion in vitro. The activity of Src and the activation of its downstream targets FAK, Pyk2, Ras and extracellular signal-regulated kinase (Erk)1/2 were also impaired. A reduction in α5-integrin expression, which is required for binding to fibronectin and thus cell migration and survival, was observed in CBD-expressing cells and cells lacking RhoC. As a result of these defects, CBD-expressing melanoma cells had a reduced ability to metastasize in recipient mice, and impaired extravasation and survival in secondary sites in chicken embryos. Our data indicate that interaction between Cav1 and Rho-GTPases (most likely RhoC but not RhoA) promotes metastasis by stimulating α5-integrin expression and regulating the Src-dependent activation of p130(Cas)/Rac1, FAK/Pyk2 and Ras/Erk1/2 signaling cascades.

The Cancer Research UK experience of pre-clinical toxicology studies to support early clinical trials with novel cancer therapies.

Pre-clinical toxicology studies in rodents and Phase I clinical trial data are summarised for 14 novel anticancer therapies. With only one exception, an antifolate antimetabolite, rodent toxicology predicted a safe Phase I trial starting dose and the majority of the dose limiting toxicities, in particular haematological toxicity. For targeted agents with well-defined pharmacodynamic markers, illustrated in the current study by 3 anti-endocrine drugs and one resistance modifier, the definition of a maximum tolerated dose can be avoided. Together with earlier data, the current study confirms that pre-clinical toxicology studies in a non-rodent species are not routinely needed for the safe conduct of early clinical trials with new cancer chemotherapies.

Pulmonary availability of isotretinoin in rats after inhalation of a powder aerosol.

Repeated oral administration of chemopreventive retinoids such as isotretinoin over extended periods of time is associated with intolerable systemic toxicity. Here isotretinoin was formulated as a powder aerosol, and its delivery to the lungs of rats was studied with the aim to explore the possibility of minimizing adverse effects associated with its oral administration. Rats received isotretinoin orally (0.5, 1 or 10 mg kg(-1)) or by inhalation (theoretical dose approximately 1 or approximately 10 mg kg(-1)) in a nose-only inhalation chamber. Isotretinoin was quantitated by high-pressure liquid chromatography in plasma and lung tissue. The ratios of mean area of concentration-vs-time curve (AUC) values in the lungs over mean AUCs in the plasma for isotretinoin following single or repeated aerosol exposure surpassed those determined for the oral route by factors of between two (single low-dose) and five (single high-dose). Similarly, the equivalent ratios for the maximal peak concentrations in lungs and plasma obtained after aerosol exposure consistently exceeded those seen after oral administration, suggesting that lungs were exposed to higher isotretinoin concentrations after aerosol inhalation than after oral administration of similar doses. Repeated high doses of isotretinoin by inhalation resulted in moderate loss of body weight, but microscopic investigation of ten tissues including lung and oesophagus did not detect any significant aerosol-induced damage. The results suggest that administration of isotretinoin via powder aerosol inhalation is probably superior to its application via the oral route in terms of achieving efficacious drug concentrations in the lungs.

An inhibitor of poly (ADP-ribose) synthetase activity reduces contractile dysfunction and preserves high energy phosphate levels during reperfusion of the ischaemic rat heart.

The cardioprotective properties of inhibition of poly (ADP-ribose) synthetase (PARS) were investigated in the isolated perfused heart of the rat. Hearts were perfused in the Langendorff mode and subjected to 23 min total global ischaemia and reperfused for 60 min. Left ventricular function was assessed by means of an intra-ventricular balloon. High energy phosphates were measured by 31P-NMR spectroscopy. Intracellular levels of NAD were measured by capillary electrophoresis of perchloric acid extracts of hearts at the end of reperfusion. Reperfusion in the presence of the PARS inhibitor 1,5 didroxyisoquinoline (ISO, 100 microM) attenuated the mechanical dysfunction observed following 1 h of reperfusion; 27+/-13 and 65+/-8% recovery of preischaemic rate pressure product for control and 100 microM ISO, respectively. This cardioprotection was accompanied by a preservation of intracellular high-energy phosphates during reperfusion; 38+/-2 vs 58+/-4% (P<0.05) of preischaemic levels of phosphocreatine (PCr) for control and 100 microM ISO respectively and 23+/-1 vs 31+/-3% (P < 0.05) of preischaemic levels of ATP for control and 100 microM ISO respectively. Cellular levels of NAD were higher in ISO treated hearts at the end of reperfusion; 2.56+/-0.45 vs 4.76+/-1.12 micromoles g(-1) dry weight (P<0.05) for control and ISO treated. These results demonstrate that the cardioprotection afforded by inhibition of PARS activity with ISO is accompanied by a preservation of high-energy phosphates and cellular NAD levels and suggest that the mechanism responsible for this cardioprotection may involve prevention of intracellular ATP depletion.

Photodynamically generated bovine serum albumin radicals: evidence for damage transfer and oxidation at cysteine and tryptophan residues.

Porphyrin-sensitized photoxidation of bovine serum albumin (BSA) results in oxidation of the protein at (at least) two different, specific sites: the Cys-34 residue giving rise to a thiyl radical (RS.); and one or both of the tryptophan residues (Trp-134 and Trp-214) resulting in the formation of tertiary carbon-centred radicals and disruption of the tryptophan ring system. In the case of porphyrins such as hematoporphyrin, which bind at specific sites on BSA, these species appear to arise via long-range transfer of damage within the protein structure, as the binding site is some distance from the ultimate site of radical formation. This transfer of damage is shown to depend on a number of factors including the conformation of the protein, the presence of blocking groups and pH. Alteration of the protein conformation results in radical formation at additional (or alternative) sites, as does blocking of the preferred loci of radical formation. The formation of these thiyl and tryptophan-derived radicals does not lead to significant aggregation or fragmentation of the protein, though it does result in a dramatic enhancement in the susceptibility of the oxidised protein to proteolytic degradation by a range of proteases. The generation of protein-derived radicals also results in an enhancement of photobleaching of the porphyrin, suggesting that protein radical generation is linked to porphyrin photooxidation.

Protein hydroperoxides and carbonyl groups generated by porphyrin-induced photo-oxidation of bovine serum albumin.

Porphyrin-sensitized photo-oxidation of bovine serum albumin results in oxidation at specific sites to produce protein radical species: at the Cys-34 residue (to give a thiyl radical) and at one or both tryptophan residues (Trp-134 and Trp-214) to give tertiary carbon-centered radicals and cause disruption of the indole ring system. This study shows that these photo-oxidation processes also consume oxygen and give rise to hydrogen peroxide, protein hydroperoxides, and carbonyl functions. The yield of hydrogen peroxide, protein hydroperoxides, and carbonyl functions is shown to be dependent on illumination time, the nature of the sensitizer, and the concentration of oxygen; the yield of hydroperoxides can also be markedly diminished by the presence of a spin trap which reacts with the initial protein radicals. The mechanism of formation of the protein hydroperoxides is suggested to be primarily through type I processes (i.e., independent of singlet oxygen), while type II (singlet oxygen) mechanisms may play a significant role in protein carbonyl formation. Reaction of the protein hydroperoxide species with metal ion complexes is shown to produce further protein-derived radicals which are predominantly present on amino acid side chains.

Use of isotopically labelled spin-traps to determine definitively the presence or absence of non-radical addition artefacts in EPR spin-trapping systems.

EPR spin-trapping, although a powerful, sensitive technique for the study of free radicals, can be susceptible to artefacts; one of the most intractable to determine has been the non-radical addition of a substrate to a spin-trap followed by oxidation of the product to an EPR-detectable nitroxide. This work details how differentially isotopically labelled spin-traps (either nitroso or nitrone) may be used to determine the presence (or absence) of such artefacts, and provide a semi-quantitative measure of the extent of their contribution to the total EPR spectra in spin-trapping reactions. Artefactual 'ene' addition of the nitroso spin-trap 3,5-dibromo-4-nitroso-benzenesulphonic acid (DBNBS) to tryptophan followed by oxidation to EPR-detectable products has been confirmed, as has its nucleophilic addition to the thiol of glutathione to give non-EPR detectable products. The nitrone α-phenyl-N-tert-butylnitrone (PBN) exhibited no such reactivity.

A study of photochemically-generated protein radical spin adducts on bovine serum albumin: the detection of genuine spin-trapping and artefactual, non-radical addition in the same molecule.

Photo-oxidation of bovine serum albumin (BSA) by porphyrins produces protein-centred radicals that can be spin trapped by 3, 5-dibromo-4-nitrosobenzenesulphonic acid (DBNBS) and 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO). In the case of DMPO, a thiyl radical from the Cys-34 residue is trapped, whereas with DBNBS signals from both this thiyl and tertiary carbon-centred species are observed. However, specific chemical modification of the Cys-34 residue, in combination with dual-isotope spin-trapping techniques, shows that the signal assigned to the Cys-34 thiyl adduct with DBNBS is a nitroxide artefact resulting from sequential (non-radical) nucleophilic addition and oxidation. In contrast, both the Cys-34 thiyl DMPO adduct and the tertiary carbon-centred DBNBS adducts result from genuine spintrapping. This study shows that such artefacts can be detected-even with anisotropic EPR spectra-through the use of appropriately substituted spin-traps, and that nitroso spin-traps need to be employed with great care in systems containing free thiol groups.

Using spoken attributions to classify abusive families.

Despite claims that beliefs held by abusive parents are important indicators of family functioning, few studies have explored the relationship between patterns of beliefs and severity of abuse. This study applies findings from marital research that demonstrate that maladaptive attributional patterns predict the level of distress experienced in adult relationships. It examines spoken attributions produced by 18 families during diagnostic therapy sessions following serious abuse of a child. Attributions were identified from transcripts and coded using a standard system. Patterns of attributions, defined on the basis of previous work, successfully predicted classification of families by therapists as Good, Uncertain, and Poor, in terms of prognosis for rehabilitation. Using this classification to test hypotheses based on attributional style, group differences were found. In families rated Good, parents were more likely to attribute more control to self than child for negative outcomes. They were also more likely to nominate themselves as causing negative events. Case accounts of families from each category are presented to illustrate how attributional analysis can contribute to an understanding of the individual nature of child abuse.

Locally Optimal Run-Length Compression Applied to CT Images.

Run-length data compression techniques are described that preserve image content. After decompression, images are restored to their original state without loss in image gray scale or resolution. The first technique introduces terminology and illustrates a simple method for efficient picture archiving. It demonstrates the principle of run-length techniques. A second more general approach encodes picture information in a manner that adapts to local variation in pixel standard deviation. Among several options of compression formats, the one that delivers the best local compression is selected. Results of our compression techniques are given for several hundred computed tomography (CT) pictures with comparison to image entropy measures. A general solution to the optimal run-length compression of digital data is outlined. Routine application of the locally optimal method is also described.

Alternative types of duodenal ulcer induced in mice by partial X irradiation of the thorax.

The present study extends our earlier observations [A. Michalowski, Br. J. Radiol. 54, 713 (1981); A. Michalowski and J. Burgin, in Progress in Radio-Oncology II, pp. 105-110] on gastrointestinal pathology in thorax-irradiated female CFLP mice. It shows that exposure of the lower mediastinum to single doses of 14-30 Gy X rays results in the formation of the proximal duodenal ulcer accompanied frequently by erosion of the antral gastric mucosa. X irradiation of the lateral thoracic fields is responsible for single ulcers in the proximity of duodenal papilla, often associated with a circumscribed area of degeneration of the fundic mucosa of the stomach. In view of the small amount of radiation received by the subdiaphragmatic parts of the alimentary tract, these gastro-duodenal lesions represent abscopal effects of thoracic irradiation.