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Breast cancer is a unique tumor disease in terms of the stringent requirement of predictive biomarker assessments. As recommended by current international guidelines, the established markers consist of estrogen receptor (ER), progesterone receptor, human epidermal growth factor and Ki67, and are primarily analyzed by immunohistochemistry. However, new diagnostic methods based on microarray or next-generation sequencing on DNA and mRNA level are gaining ground. These analyses require fresh-frozen tumor tissue that is generally not available from tumors <10 mm in diameter, comprising almost 25% of all resected breast cancer at our department. We here present a simple and standardized method to generate material from small tumors without risking the histopathological examination. Furthermore, we show that the quality of this material is sufficient for subsequent analysis on mRNA, DNA, and epigenetic level. We were also able to use this method for isolation and expansion of cancer stem cells from the majority of tumors. Consequently, researches can be provided with clinically relevant material for translational studies. In conclusion, this method opens up a new possibility for usage of valuable fresh tumor material for research purposes, biobanking, and next-generation sequencing.

Aims: The aim of this study was to investigate in primary breast cancer the congruency of routine clinical predictive biomarker evaluations, including ER, PR and Ki67, obtained using immunocytochemistry (ICC) and immunohistochemistry (IHC)., Methods and Results: Clinicopathological data were collected on all women diagnosed with primary breast cancer at Karolinska University Hospital in 2011. A total of 346 patients were included in a retrospective paired comparison of predictive biomarker evaluations on direct smear ICC and IHC. This showed a low congruency between findings with the two methods, especially evident for Ki67 (κ = 0.35-0.42). By suggested adjustments to ICC cut-offs, we managed to improve the inter-rater agreement of Ki67 classification slightly to κ = 0.46., Conclusions: Our findings suggest that routine clinical ICC and IHC evaluations of predictive biomarkers produce discordant results. Consequently, basing therapeutic decisions on cytology with cut-offs defined for IHC induces a risk that patients will receive suboptimal therapy. However, our analysis shows that local adjustments to biomarker cut-off levels may improve congruency and increase the probability of correct classifications., (© 2013 John Wiley & Sons Ltd.)

Microvascular assessment would benefit from co-registration of blood flow and hemoglobin oxygenation dynamics during stimulus response tests. We used a fiber-optic probe for simultaneous recording of white light diffuse reflectance (DRS; 475-850 nm) and laser Doppler flowmetry (LDF; 780 nm) spectra at two source-detector distances (0.4 and 1.2 mm). An inverse Monte Carlo algorithm, based on a multiparameter three-layer adaptive skin model, was used for analyzing DRS data. LDF spectra were conventionally processed for perfusion. The system was evaluated on volar forearm recordings of 33 healthy subjects during a 5-min systolic occlusion protocol. The calibration scheme and the optimal adaptive skin model fitted DRS spectra at both distances within 10%. During occlusion, perfusion decreased within 5 s while oxygenation decreased slowly (mean time constant 61 s; dissociation of oxygen from hemoglobin). After occlusion release, perfusion and oxygenation increased within 3 s (inflow of oxygenized blood). The increased perfusion was due to increased blood tissue fraction and speed. The supranormal hemoglobin oxygenation indicates a blood flow in excess of metabolic demands. In conclusion, by integrating DRS and LDF in a fiber-optic probe, a powerful tool for assessment of blood flow and oxygenation in the same microvascular bed has been presented.

The tissue fraction of red blood cells (RBCs) and their oxygenation and speed-resolved perfusion are estimated in absolute units by combining diffuse reflectance spectroscopy (DRS) and laser Doppler flowmetry (LDF). The DRS spectra (450 to 850 nm) are assessed at two source-detector separations (0.4 and 1.2 mm), allowing for a relative calibration routine, whereas LDF spectra are assessed at 1.2 mm in the same fiber-optic probe. Data are analyzed using nonlinear optimization in an inverse Monte Carlo technique by applying an adaptive multilayered tissue model based on geometrical, scattering, and absorbing properties, as well as RBC flow-speed information. Simulations of 250 tissue-like models including up to 2000 individual blood vessels were used to evaluate the method. The absolute root mean square (RMS) deviation between estimated and true oxygenation was 4.1 percentage units, whereas the relative RMS deviations for the RBC tissue fraction and perfusion were 19% and 23%, respectively. Examples of in vivo measurements on forearm and foot during common provocations are presented. The method offers several advantages such as simultaneous quantification of RBC tissue fraction and oxygenation and perfusion from the same, predictable, sampling volume. The perfusion estimate is speed resolved, absolute (% RBC×mm/s), and more accurate due to the combination with DRS.

Alcohol dependence is a heterogeneous disorder where several signalling systems play important roles. Recent studies implicate that the gut-brain hormone ghrelin, an orexigenic peptide, is a potential mediator of alcohol related behaviours. Ghrelin increases whereas a ghrelin receptor (GHS-R1A) antagonist decreases alcohol consumption as well as operant self-administration of alcohol in rodents that have consumed alcohol for twelve weeks. In the present study we aimed at investigating the effect of acute and repeated treatment with the GHS-R1A antagonist JMV2959 on alcohol intake in a group of rats following voluntarily alcohol consumption for two, five and eight months. After approximately ten months of voluntary alcohol consumption the expression of the GHS-R1A gene (Ghsr) as well as the degree of methylation of a CpG island found in Ghsr was examined in reward related brain areas. In a separate group of rats, we examined the effect of the JMV2959 on alcohol relapse using the alcohol deprivation paradigm. Acute JMV2959 treatment was found to decrease alcohol intake and the effect was more pronounced after five, compared to two months of alcohol exposure. In addition, repeated JMV2959 treatment decreased alcohol intake without inducing tolerance or rebound increase in alcohol intake after the treatment. The GHS-R1A antagonist prevented the alcohol deprivation effect in rats. There was a significant down-regulation of the Ghsr expression in the ventral tegmental area (VTA) in high- compared to low-alcohol consuming rats after approximately ten months of voluntary alcohol consumption. Further analysis revealed a negative correlation between Ghsr expression in the VTA and alcohol intake. No differences in methylation degree were found between high- compared to low-alcohol consuming rats. These findings support previous studies showing that the ghrelin signalling system may constitute a potential target for development of novel treatment strategies for alcohol dependence.

Development of alcohol use disorders largely depends on the effects of alcohol on the brain reward systems. Emerging evidence indicate that common mechanisms regulate food and alcohol intake and raise the possibility that endocrine signals from the gut may play an important role for alcohol consumption, alcohol-induced reward and the motivation to consume alcohol. Glucagon-like peptide 1 (GLP-1), a gastrointestinal peptide regulating food intake and glucose homeostasis, has recently been shown to target central brain areas involved in reward and motivation, including the ventral tegmental area and nucleus accumbens. Herein we investigated the effects of the GLP-1 receptor agonist, Exendin-4 (Ex4), on various measures of alcohol-induced reward as well as on alcohol intake and alcohol seeking behavior in rodents. Treatment with Ex4, at a dose with no effect per se, attenuated alcohol-induced locomotor stimulation and accumbal dopamine release in mice. Furthermore, conditioned place preference for alcohol was abolished by both acute and chronic treatment with Ex4 in mice. Finally we found that Ex4 treatment decreased alcohol intake, using the intermittent access 20% alcohol two-bottle-choice model, as well as alcohol seeking behavior, using the progressive ratio test in the operant self-administration model, in rats. These novel findings indicate that GLP-1 signaling attenuates the reinforcing properties of alcohol implying that the physiological role of GLP-1 extends beyond glucose homeostasis and food intake regulation. Collectively these findings implicate that the GLP-1 receptor may be a potential target for the development of novel treatment strategies for alcohol use disorders., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: New medications for alcohol use disorder (AUD) are needed. Long-term alcohol consumption leads to a dysregulated dopamine system. A novel approach to normalize these dysregulations might be treatment with "monoamine stabilizers," a novel class of compounds characterized by the ability to either suppress, stimulate, or not influence dopamine activity depending on the prevailing dopaminergic tone., Methods: The effects of the monoamine stabilizer (-)-OSU6162 (OSU6162) on voluntary ethanol intake and ethanol withdrawal symptoms were evaluated in rats voluntarily consuming ethanol for at least 3 months before testing. Furthermore, effects of OSU6162 on ethanol seeking behavior were evaluated with the progressive ratio and cue-induced reinstatement paradigms. Finally, the interaction of OSU6162 with ethanol on dopamine output and metabolism was studied with microdialysis., Results: The OSU6162 attenuated several ethanol-mediated behaviors, including voluntary ethanol consumption, ethanol withdrawal symptoms, operant ethanol self-administration under progressive ratio schedule, and cue-induced reinstatement of ethanol seeking in rats that had voluntarily consumed ethanol for at least 3 months before treatment. In addition, OSU6162 blunted ethanol-induced dopamine output in nucleus accumbens of ethanol-naïve rats., Conclusions: These results highlight the ability of OSU6162 to stabilize dopamine activity depending on the prevailing dopaminergic tone and indicate that OSU6162 might decrease ethanol intake by attenuating the acute rewarding properties of ethanol. In addition, OSU6162 might have potential to prevent relapse triggered by alcohol craving, alcohol related cues, and or an urge to relieve abstinence symptoms. The present study is to our knowledge the first indicating that OSU6162 might serve as a novel medication for AUD., (Copyright © 2012 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

A spectroscopic probe with multiple detecting fibers was used for quantifying absorption and scattering in liquid optical phantoms. The phantoms were mixtures of Intralipid and red and blue food dyes. Intensity calibration for the detecting fibers was undertaken using either a microsphere suspension (absolute calibration) or a uniform detector illumination (relative calibration between detectors). Two different scattering phase functions were used in an inverse Monte Carlo algorithm. Data were evaluated for residual spectra (systematic deviations and magnitude) and accuracy in estimation of scattering and absorption. Spectral fitting was improved by allowing for a 10% intensity relaxation in the optimization algorithm. For a multi-detector setup, non-systematic residual spectrum was only found using the more complex Gegenbauer-kernel phase function. However, the choice of phase function did not influence the accuracy in the estimation of absorption and scattering. Similar estimation accuracy as in the multi-detector setup was also obtained using either two relative calibrated detectors or one absolute calibrated detector at a fiber separation of 0.46 mm.

Model based data analysis of diffuse reflectance spectroscopy data enables the estimation of optical and structural tissue parameters. The aim of this study was to present an inverse Monte Carlo method based on spectra from two source-detector distances (0.4 and 1.2 mm), using a multilayered tissue model. The tissue model variables include geometrical properties, light scattering properties, tissue chromophores such as melanin and hemoglobin, oxygen saturation and average vessel diameter. The method utilizes a small set of presimulated Monte Carlo data for combinations of different levels of epidermal thickness and tissue scattering. The path length distributions in the different layers are stored and the effect of the other parameters is added in the post-processing. The accuracy of the method was evaluated using Monte Carlo simulations of tissue-like models containing discrete blood vessels, evaluating blood tissue fraction and oxygenation. It was also compared to a homogeneous model. The multilayer model performed better than the homogeneous model and all tissue parameters significantly improved spectral fitting. Recorded in vivo spectra were fitted well at both distances, which we previously found was not possible with a homogeneous model. No absolute intensity calibration is needed and the algorithm is fast enough for real-time processing.

Laser Doppler flowmetry (LDF) can be used for assessing the microcirculatory perfusion. However, conventional LDF (cLDF) gives only a relative perfusion estimate for an unknown measurement volume, with no information about the blood flow speed distribution. To overcome these limitations, a model-based analysis method for quantitative LDF (qLDF) is proposed. The method uses inverse Monte Carlo technique with an adaptive three-layer skin model. By analyzing the optimal model where measured and simulated LDF spectra detected at two different source-detector separations match, the absolute microcirculatory perfusion for a specified speed region in a predefined volume is determined. qLDF displayed errors<12% when evaluated using simulations of physiologically relevant variations in the layer structure, in the optical properties of static tissue, and in blood absorption. Inhomogeneous models containing small blood vessels, hair, and sweat glands displayed errors<5%. Evaluation models containing single larger blood vessels displayed significant errors but could be dismissed by residual analysis. In vivo measurements using local heat provocation displayed a higher perfusion increase with qLDF than cLDF, due to nonlinear effects in the latter. The qLDF showed that the perfusion increase occurred due to an increased amount of red blood cells with a speed>1 mm∕s.

Objective: To compare the microcirculatory velocity distribution in type 2 diabetic patients and nondiabetic control subjects at baseline and after local heating., Research Design and Methods: The skin blood flow response to local heating (44 degrees C for 20 min) was assessed in 28 diabetic patients and 29 control subjects using a new velocity-resolved quantitative laser Doppler flowmetry technique (qLDF). The qLDF estimates erythrocyte (RBC) perfusion (velocity x concentration), in a physiologically relevant unit (grams RBC per 100 g tissue x millimeters per second) in a fixed output volume, separated into three velocity regions: v <1 mm/s, v 1-10 mm/s, and v >10 mm/s., Results: The increased blood flow occurs in vessels with a velocity >1 mm/s. A significantly lower response in qLDF total perfusion was found in diabetic patients than in control subjects after heat provocation because of less high-velocity blood flow (v >10 mm/s). The RBC concentration in diabetic patients increased sevenfold for v between 1 and 10 mm/s, and 15-fold for v >10 mm/s, whereas no significant increase was found for v <1 mm/s. The mean velocity increased from 0.94 to 7.3 mm/s in diabetic patients and from 0.83 to 9.7 mm/s in control subjects., Conclusions: The perfusion increase occurs in larger shunting vessels and not as an increase in capillary flow. Baseline diabetic patient data indicated a redistribution of flow to higher velocity regions, associated with longer duration of diabetes. A lower perfusion was associated with a higher BMI and a lower toe-to-brachial systolic blood pressure ratio.

Background: Breast cancer is uncommon in young women and correlates with a less favourable prognosis; still it is the most frequent cancer in women under 40, accounting for 30-40% of all incident female cancer. The aim of this study was to study prognosis in young women, quantifying how much stage at diagnosis and management on the one hand, and tumour biology on the other; each contribute to the worse prognosis seen in this age group., Methodology/principal Findings: In a registry based cohort of women aged 20-69 (n = 22 017) with a primary diagnosis of invasive breast cancer (1992-2005), women aged 20-34 (n = 471), 35-39 (n = 858) and 40-49 (n = 4789) were compared with women aged 50-69 years (n = 15 899). The cumulative 5-year relative survival ratio and the relative excess mortality (RER) were calculated. The cumulative 5-year relative survival ratio was lowest in women aged 20-34. The RER was 2.84 for women aged 20-34 and decreased with increasing age (RER 1.76 and 1.17 for women aged 35-39 and 40-49, respectively). The excess risk was, however, present only in disease stages I and II. For women aged 20-34 with stage I disease RER was 4.63, and 6.70 in the subgroup with tumour size 1-10 mm. The absolute difference in stage I between the youngest and the reference groups amounted to nearly 8%, with a 90% 5-year survival in women aged 20-34. In stages IIa and IIb, the relative excess risk was not as dramatic, but the absolute differences approached 15%. The youngest women with small tumours generally received more aggressive treatment than women in older age groups., Conclusions: After correction for stage, tumour characteristics and treatment, age remained an independent risk factor for breast cancer death in women <35 years of age. The excess risk for young women was only seen in early stages of disease and was most pronounced in women with small tumours. Young women affected by breast cancer have a high risk of dying compared to their middle-aged counterparts even if diagnosed early and receiving an intense treatment.

An electrode with adjacent optical fibers for measurements during navigation and radio frequency lesioning in the brain is modeled for Monte Carlo simulations of light transport in brain tissue. Relative reflected light intensity at 780 nm, I780, from this electrode and probes with identical fiber configuration are simulated using the intensity from native white matter as reference. Models are made of homogeneous native and coagulated gray, thalamus, and white matter as well as blood. Dual layer models, including models with a layer of cerebrospinal fluid between the fibers and the brain tissue, are also made. Simulated I780 was 0.16 for gray matter, 0.67 for coagulate gray matter, 0.36 for thalamus, 0.39 for coagulated thalamus, unity for white matter, 0.70 for coagulated white matter, and 0.24 for blood. Thalamic matter is also found to reflect more light than gray matter and less than white matter in clinical studies. In conclusion, the reflected light intensity can be used to differentiate between gray and white matter during navigation. Furthermore, coagulation of light gray tissue, such as the thalamus, might be difficult to detect using I780, but coagulation in darker gray tissue should result in a rapid increase of I780.

A new method for estimating the measurement depth and volume in laser Doppler flowmetry (LDF) is presented. The method is based on Monte Carlo simulations of light propagation in tissue. The contribution from each individual Doppler shift is calculated and thereby multiple Doppler shifts are handled correctly. Different LDF setups for both probe based (0.0, 0.25, 0.5, and 1.2 mm source-detector separation) and imaging systems (0.5 and 2.0 mm beam diameter) are considered, at the wavelengths 543 nm, 633 nm, and 780 nm. Non-linear speckle pattern effects are accounted for in the imaging system setups. The effects of tissue optical properties, blood concentration, and blood oxygen saturation are evaluated using both homogeneous tissue models and a layered skin model. The results show that the effect on the measurement depth of changing tissue properties is comparable to the effect of changing the system setup, e.g. source-detector separation and wavelength. Skin pigmentation was found to have a negligible effect on the measurement depth. Examples of measurement depths are (values are given for a probe based system with 0.25 mm source-detector separation and an imaging system with a 0.5 mm beam diameter, respectively, both operating at 780 nm): muscle - 0.55/0.79 mm; liver - 0.40/0.53 mm; gray matter - 0.48/0.68 mm; white matter - 0.20/0.20 mm; index finger pulp - 0.41/0.53 mm; forearm skin - 0.53/0.56 mm; heat provoked forearm skin - 0.66/0.67 mm.

Two forced detection (FD) variance reduction Monte Carlo algorithms for image simulations of tissue-embedded objects with matched refractive index are presented. The principle of the algorithms is to force a fraction of the photon weight to the detector at each and every scattering event. The fractional weight is given by the probability for the photon to reach the detector without further interactions. Two imaging setups are applied to a tissue model including blood vessels, where the FD algorithms produce identical results as traditional brute force simulations, while being accelerated with two orders of magnitude. Extending the methods to include refraction mismatches is discussed.

A method for determining a two-parametric Gegenbauer-kernel phase function that accurately describes the diffuse reflectance from a polydispersive scattering media at small source-detector separations (0.23 to 1.2 mm), is presented. The method involves spectral collimated transmission measurements, spatially resolved spectral diffuse reflectance (SRDR) measurements, and inverse Monte Carlo technique. Both absolute calibration (using a monodispersive polystyrene microsphere suspension) and relative calibration (eliminating differences between fibers) of SRDR spectra yielded comparable results. When applied to water dilutions of milk, simulated and measured spectra deviated less than 6.5% and 2.5% for the absolute and relative calibration case, respectively, even for the closest fiber separation. Corresponding values for milk including ink as an absorber, were 13.4% and 7.3%.

Introduction: Some patients with breast cancer develop local recurrence after breast-conservation surgery despite postoperative radiotherapy, whereas others remain free of local recurrence even in the absence of radiotherapy. As clinical parameters are insufficient for identifying these two groups of patients, we investigated whether gene expression profiling would add further information., Methods: We performed gene expression analysis (oligonucleotide arrays, 26,824 reporters) on 143 patients with lymph node-negative disease and tumor-free margins. A support vector machine was employed to build classifiers using leave-one-out cross-validation., Results: Within the estrogen receptor-positive (ER+) subgroup, the gene expression profile clearly distinguished patients with local recurrence after radiotherapy (n = 20) from those without local recurrence (n = 80 with or without radiotherapy). The receiver operating characteristic (ROC) area was 0.91, and 5,237 of 26,824 reporters had a P value of less than 0.001 (false discovery rate = 0.005). This gene expression profile provides substantially added value to conventional clinical markers (for example, age, histological grade, and tumor size) in predicting local recurrence despite radiotherapy. Within the ER- subgroup, a weaker, but still significant, signal was found (ROC area = 0.74). The ROC area for distinguishing patients who develop local recurrence from those who remain local recurrence-free in the absence of radiotherapy was 0.66 (combined ER+/ER-)., Conclusion: A highly distinct gene expression profile for patients developing local recurrence after breast-conservation surgery despite radiotherapy has been identified. If verified in further studies, this profile might be a most important tool in the decision making for surgery and adjuvant therapy.

An optical microvascular skin model, valid at 780 nm, was developed. The model consisted of six layers with individual optical properties and variable thicknesses and blood concentrations at three different blood flow velocities. Monte Carlo simulations were used to evaluate the impact of various model parameters on the traditional laser Doppler flowmetry (LDF) measures. A set of reference Doppler power spectra was generated by simulating 7000 configurations, varying the thickness and blood concentrations. Simulated spectra, at two different source detector separations, were compared with in vivo recorded spectra, using a nonlinear search algorithm for minimizing the deviation between simulated and measured spectra. The model was validated by inspecting the thickness and blood concentrations that generated the best fit. These four parameters followed a priori expectations for the measurement situations, and the simulated spectra agreed well with the measured spectra for both detector separations. Average estimated dermal blood concentration was 0.08% at rest and 0.63% during heat provocation (44 degrees C) on the volar side of the forearm and 1.2% at rest on the finger pulp. The model is crucial for developing a technique for velocity-resolved absolute LDF measurements with known sampling volume and can also be useful for other bio-optical modalities.