Inhibition of the angiotensin II type 2 receptor AT2R is a novel therapeutic strategy for glioblastoma.

Glioblastoma (GBM) is an aggressive malignant primary brain tumor with limited therapeutic options. We show that the angiotensin II (AngII) type 2 receptor (AT2R) is a therapeutic target for GBM and that AngII, endogenously produced in GBM cells, promotes proliferation through AT2R. We repurposed EMA401, an AT2R antagonist originally developed as a peripherally restricted analgesic, for GBM and showed that it inhibits the proliferation of AT2R-expressing GBM spheroids and blocks their invasiveness and angiogenic capacity. The crystal structure of AT2R bound to EMA401 was determined and revealed the receptor to be in an active-like conformation with helix-VIII blocking G-protein or ß-arrestin recruitment. The architecture and interactions of EMA401 in AT2R differ drastically from complexes of AT2R with other relevant compounds. To enhance central nervous system (CNS) penetration of EMA401, we exploited the crystal structure to design an angiopep-2-tethered EMA401 derivative, A3E. A3E exhibited enhanced CNS penetration, leading to reduced tumor volume, inhibition of proliferation, and increased levels of apoptosis in an orthotopic xenograft model of GBM.

Aqueous Humour Ofloxacin Concentration after Topical Instillation in Patients with Dry Eye Disease.

Background and Objectives: A prospective, randomized clinical trial was conducted to evaluate the concentration of ofloxacin in the aqueous humour (AqH) of patients suffering from dry eye disease (DED) after topical instillation. Materials and Methods: Ninety-one (91) cataract patients scheduled for phacoemulsification were categorized into three groups according to DED severity. Group I (n = 17) was comprised of subjects without DED, patients in group II (n = 37) were evaluated as having non-severe DED, while group III (n = 37) consisted of patients suffering from severe DED. Preoperatively, patients received 4 drops of 0.3% of ofloxacin at 15 min intervals. One hour after the last instillation, aqueous samples were collected intraoperatively. Results: The median AqH concentration of ofloxacin in group I was 199.9 ng/mL (range 92.2−442.8 ng/mL), while in group II it was 530.5 ng/mL (range 283.7−1004.9 ng/mL), and 719.2 ng/mL (range 358.0−1512.4 ng/mL) in Group III, p < 0.001 (Kruskal-Wallis tests). Pairwise tests (two-tailed with Bonferroni corrections) between groups resulted in a p-value of 0.001 when group II was compared to group I and group III was compared to group I, and a p-value of 0.020 when group II was compared to group III. The severity of DED, across groups I, II, and III, and the levels of ofloxacin revealed a strong positive correlation (r = 0.639, p < 0.001). Conclusions: Ofloxacin concentration in the AqH after topical drop instillation may be affected by the degree of ocular surface inflammation in patients suffering from DED.

Correlation Between Bevacizumab Exposure and Survival in Patients with Metastatic Colorectal Cancer.

BACKGROUND: Bevacizumab treatment is subject to large interpatient variability in efficacy, which may partly be explained by differences in complex bevacizumab pharmacokinetic characteristics that influence bevacizumab exposure. Exposure-response relationships have been identified for other monoclonal antibodies. We aimed to identify possible exposure-survival relationships in bevacizumab-treated patients with metastatic colorectal cancer (mCRC). MATERIALS AND METHODS: Patients with mCRC who started first-line bevacizumab-based chemotherapy between July 2012 and July 2014, and from whom serial blood samples and survival were prospectively collected, were included. Follow-up was carried out until July 2018. Total bevacizumab trough concentrations were measured from cycle 2 to cycle 30 of treatment. The receiver operating characteristic (ROC) curve analysis and Cox analysis were used to identify the relationship between concentrations and overall survival (OS). In addition, OS was compared between different trough concentration groups. RESULTS: One hundred fifty-seven blood samples from 46 patients were evaluable for analyses. ROC analysis showed a clear separation in survival based on trough levels (area under the curve = 0.739, p = .009). Cox regression also showed a strong positive correlation between trough levels and survival (p = .0004). Three distinct groups of exposure were identified: low (median trough concentration [Ctm ] ≤41.9 mg/L); medium (Ctm 43-87.2 mg/L) with median OS of 12.8 and 36 months, respectively (p = .0003); and high (Ctm ≥7.9 mg/L), where the majority of patients were still alive 60 months after the initiation of treatment. CONCLUSION: This study shows that survival was proportional to the magnitude of exposure in patients with mCRC. Further clinical research should focus on clarifying these exposure-outcome relationships in order to optimize dosing. IMPLICATIONS FOR PRACTICE: Bevacizumab-based chemotherapy is standard first-line treatment in metastatic colorectal cancer. Moreover, bevacizumab presents complicated pharmacokinetics, and in many cases, clinical outcomes can be highly variable, with some patients responding remarkably well and others not. This study's results show that patients who experienced longer overall survival also had significantly higher exposure to bevacizumab. Therefore, bevacizumab trough concentrations could be used both as a predictive biomarker and as a tool for treatment monitoring and optimization. Finally, the development of validated, rapid, and sensitive assays for bevacizumab concentration measurements in combination with these results may lead to a therapeutic drug monitoring-guided approach in bevacizumab treatment with better clinical outcomes.

In vivo quantification and pharmacokinetic studies of cotinine in mice after smoke exposure by LC-MS/MS.

A sensitive analytical method was developed and validated for the quantification of cotinine in mouse plasma after exposure to smoke of 0.5, 1.0, and 1.5 commercially available cigarettes, using liquid chromatography tandem mass spectrometry. The method was validated over a linear concentration range of 0.075-20.0 ng/mL with the R2 value being higher than 0.99. Both the precision (coefficient of variation; %) and accuracy (relative error; %) were within acceptable criteria of <15%. The lower limit of quantification (LLOQ) for cotinine was 0.075 ng/mL with sufficient specificity, accuracy, and precision. Following exposure to 0.5, 1.0, and 1.5 cigarette smoke, it was observed that the AUC and the Cmax increased linearly as the doses increased. The pharmacokinetics of cotinine was found linear for the range of 0.5-1.5 commercial cigarette smoke. The quantification of the concentration of cotinine in mouse plasma after smoke exposure will facilitate future behavioral and toxicological experiments in animals and may prove useful in predicting cotinine levels in humans during smoking.

Pharmacogenetics in Model-Based Optimization of Bevacizumab Therapy for Metastatic Colorectal Cancer.

Vascular endothelial growth factor A (VEGF-A) and intercellular adhesion molecule 1 (ICAM-1) are significant regulators of angiogenesis, an important biological process involved in carcinogenesis. Bevacizumab, an anti-VEGF monoclonal antibody (MAB), is approved for the treatment of metastatic Colorectal cancer (mCRC), however clinical outcomes are highly variable. In the present study, we developed a pharmacokinetic (PK), a simplified quasi-steady state (QSS) and a pharmacokinetic/pharmacodynamic (PK/PD) model to identify potential sources of variability. A total of 46 mCRC patients, who received bevacizumab in combination with chemotherapy were studied. VEGF-A (rs2010963, rs1570360, rs699947) and ICAM-1 (rs5498, rs1799969) genes' polymorphisms, age, gender, weight, and dosing scheme were investigated as possible co-variates of the model's parameters. Polymorphisms, trough, and peak levels of bevacizumab, and free VEGF-A were determined in whole blood and serum. Data were analyzed using nonlinear mixed-effects modeling. The two-compartment PK model showed that clearance (CL) was significantly lower in patients with mutant ICAM-1 rs1799969 (p < 0.0001), inter-compartmental clearance (Q) was significantly higher with mutant VEGF-A rs1570360 (p < 0.0001), and lower in patients with mutant VEGF-A rs699947 (p < 0.0001). The binding QSS model also showed that mutant ICAM-1 rs1799969 was associated with a lower CL (p = 0.0177). Mutant VEGF-A rs699947 was associated with a lower free VEGF-A levels, prior to the next dose (p = 0.000445). The above results were confirmed by the PK/PD model. Findings of the present study indicated that variants of the genes regulating angiogenesis might affect PK and PD characteristics of bevacizumab, possibly influencing the clinical outcomes.

Development and validation of analytical methodologies for the quantification of PCK3145 and PEG-PCK3145 in mice.

PCK3145 is an anti-metastatic synthetic peptide against prostate cancer. The objective of the study is to develop and validate novel and sensitive methods for the determination of PCK3145 and Pegylated PCK3145 in mouse plasma. An LC-MS/MS method was developed and validated for the determination of PCK3145 giving high sensitivity and linearity in the range of 0.125-4.0 µg/mL. PCK3145 characterised by short half-life, therefore, it was conjugated with the poly ethylene glycol (PEG). However, LC-MS/MS has been more difficult to apply for the quantitative analysis of PEGylated peptides due to the large size. A UHPLC-UV method was developed and validated for the determination of PEG-PCK3145, with linearity of 0.05-2.0 mg/mL. In order to further improve the sensitivity for the detection of PEG-PCK3145, an indirect ELISA method was used. It was found that this method was capable of detecting PCK3145 through the quantification of PEG with excellent sensitivity found at 0.132 ng/mL. The in vitro proteolytic stability of PCK3145 and PEG-PCK3145 in mouse plasma and whole blood was studied by LC-MS/MS and UHPLC, respectively. The LC-MS/MS and ELISA methods can be applied for monitoring levels of PCK3145 in mouse plasma for in vivo pharmacokinetic and bioavailability animal studies.

VEGF-A and ICAM-1 Gene Polymorphisms as Predictors of Clinical Outcome to First-Line Bevacizumab-Based Treatment in Metastatic Colorectal Cancer.

Bevacizumab is used to treat metastatic colorectal cancer (mCRC). However, there are still no available predictors of clinical outcomes. We investigated selected single nucleotide polymorphisms (SNPs) in the genes involved in VEGF-dependent and -independent angiogenesis pathways and other major intracellular signaling pathways involved in the pathogenesis of mCRC as an attempt to find predictors of clinical outcome. Forty-six patients treated with first-line bevacizumab-based chemotherapy were included in this study with a 5 year follow up. Genomic DNA was isolated from whole blood for the analysis of VEGF-A (rs2010963, 1570360, rs699947), ICAM-1 (rs5498, rs1799969) SNPs and from tumor tissue for the detection of genomic variants in KRAS, NRAS, BRAF genes. PCR and next generation sequencing were used for the analysis. The endpoints of the study were progression-free survival (PFS) and overall survival (OS). The VEGF-A rs699947 A/A allele was associated with increased PFS (p = 0.006) and OS (p = 0.043). The ICAM-1 rs1799969 G/A allele was associated with prolonged OS (p = 0.036). Finally, BRAF wild type was associated with increased OS (p = 0.027). We identified VEGF-A and ICAM-1 variants in angiogenesis and other major intracellular signaling pathways, such as BRAF, that can predict clinical outcome upon bevacizumab administration. These identified biomarkers could be used to select patients with mCRC who may achieve long-term responses and benefit from bevacizumab-based therapies.

Characterization and Biological Evaluation of Propolis from Poland.

In this study, we assessed the therapeutic potential of propolis from Poland and performed chemical analysis by GC-MS, as well as determined its botanical origin. Chemical constituents typical for bud exudates of Populusnigra (section Aigeiros) were determined, however, glycerol esters of phenolic acids, as well as unusually high amounts of p-coumaric and ferulic acid and their benzyl esters, were also detected. These constituents are characteristic for buds of Populustremula (section Leuce). We also evaluated the antiproliferative effect of propolis extracts against nine human cancer cell lines. Additionally, promising antibacterial activity of the dichloromethane extract (Minimal Inhibitory Concentration MIC values of 0.95-1.24 mg/mL), as well as a moderate antifungal activity (MIC values of 1.25-1.40 mg/mL), was noticed. Propolis from Poland appeared as a rich source of antibacterial and antiproliferative compounds and this confirmed that it is a valuable natural product with the potential to improve human health.

Development and validation of a UHPLC-UV method for the determination of a prostate secretory protein 94-derived synthetic peptide (PCK3145) in human plasma and assessment of its stability in human plasma.

PCK3145 is a synthetic peptide, derived from the Prostate Secreted Protein 94 (PSP94), with promising in vitro and animal in vivo results in prostate cancer. The aim of the present study was to develop and validate a fast and robust ultra-high-performance liquid chromatography with ultraviolet detection for the determination of PCK3145 in human plasma which would be suitable for the assessment of PCK3145 stability to proteolytic degradation. Following protein precipitation, chromatographic separation was carried out on an Aeris Peptide C18 column with mobile phase consisting of acetonitrile-water at a flow-rate of 0.50 mL/min. The calibration curve was linear over the range 0.50-20.00 µg/mL. Intra- and inter-day percentage relative standard deviation and relative error were ≤10%. The limit of detection and the lower limit of quantification were 0.15 and 0.50 µg/mL, respectively. Recovery of PCK3145 from human plasma was ≥96%. The peptide presented high stability in whole blood and in human plasma (>98% intact peptide after 24 h incubation at 37°C in human plasma), which represents a distinctive advantage in the therapeutic use of the compound. This is the first validated UHPLC method for the determination of PCK3145 reported, and it was successfully applied in the study of the proteolytic stability of PCK3145 in human plasma ex vivo. Copyright © 2016 John Wiley & Sons, Ltd.

Liposomal forms of anticancer agents beyond anthracyclines: present and future perspectives.

Liposomes are widely used as delivery systems of cytotoxic drugs. The encapsulation into liposomes improves pharmacological properties and as a result therapeutic index and outcomes. To date, liposomal vincristine and cytarabine are approved and marketed for intravenous and intrathecal administration, respectively. The main goal of this review is to examine the clinical use and pharmacological properties, as well as the safety of liposomal forms of less widely used liposomal forms of anticancer agents compared to their conventional forms and to present data regarding clinical development of other liposomal agents. Liposomal forms of cytarabine and vincristine are less widely used and unknown compared to liposomal anthracyclines, because they are approved only for specific indications and only in the United States.

Machine learning-guided covariate selection for time-to-event models developed from a small sample of real-world patients receiving bevacizumab treatment.

Therapeutic outcomes in patients with metastatic colorectal cancer (mCRC) receiving bevacizumab treatment are highly variable, and a reliable predictive factor is not available. Progression-free survival (PFS) and overall survival (OS) were recorded from an observational, prospective study after 5 years of follow-up, including 46 patients with mCRC receiving bevacizumab treatment. Three vascular endothelial growth factor (VEGF)-A and two intercellular adhesion molecule-1 genes polymorphisms, age, gender, weight, dosing scheme, and co-treatments were collected. Given the relatively small number of events (37 [80%] for the PFS and 26 [57%] for the OS), to study the effect of these covariates on PFS and OS, a covariate analysis was performed using statistical and supervised machine learning techniques, including Cox regression, penalized Cox regression techniques (least absolute shrinkage and selection operator [LASSO], ridge regression, and elastic net), survival trees, and survival forest. The predictive performance of each method was evaluated in bootstrapped samples, using prediction error curves and the area under the curve of the receiver operating characteristic. The LASSO penalized Cox-regression model showed the best overall performance. Nonlinear mixed effects (NLME) models were developed, and a conventional stepwise covariate search was performed. Then, covariates identified as important by the LASSO model were included in the base NLME models developed for PFS and OS, resulting in improved models as compared to those obtained with the stepwise covariate search. It was shown that having gene polymorphisms in VEGFA (rs699947 and rs1570360) and ICAM1 (rs1799969) are associated with a favorable clinical outcome in patients with mCRC receiving bevacizumab treatment.

Effectiveness and Tolerability of Natural Herbal Formulations in the Prevention of Radiation-Induced Skin Toxicity in Patients Undergoing Radiotherapy.

The aim of this study is to investigate the preventive role of 3 herbal formulation products on reducing the incidence of radiation-induced dermatitis in patients undergoing radiotherapy for either breast or head and neck cancer. A total of 59 patients participated in the study. The novel herbal products, a combination of beeswax, olive oil, Calendula and Hypericum oils and Aloe gel, were daily and regularly being used by the patients during radiotherapy and 2 weeks after treatment end. Acute skin toxicity was scored weekly during radiotherapy and after treatment for a further 4-week follow-up period. Demographic data were analyzed by descriptive statistics. Statistical analyses of the study objectives were based on an intent-to-treat principle. Most of the patients presented with grade I (RTOG/EORTC) toxicity in the first weeks of radiotherapy, progressed to grade II but reverted to grade I toxicity up until the study end. A total of 94.9% of the patients had Dermatology Life Quality Index up to 1, and 66.1% remained in this scale. The application of the novel natural product combinations proved to be statistically significantly effective in reducing the intensity of radiation dermatitis, positively affecting the quality of life of the patients.

Pharmacogenomics, Pharmacokinetics and Circulating Proteins as Biomarkers for Bevacizumab Treatment Optimization in Patients with Cancer: A Review.

Bevacizumab is a monoclonal antibody that targets VEGF-A and inhibits tumor angiogenesis. Bevacizumab is approved for the treatment of various cancer, including metastatic colorectal cancer (mCRC), ovarian cancer, lung cancer, and others. Thus, it is widely used in oncology, but contrary to other therapeutic classes, there is still a lack of validating predictive factors for treatment outcomes with these agents. In recent years, the research for factors predictive of anti-VEGF treatments and especially bevacizumab response has been one of the most competitive translational research fields. Herein, we review and present the available literature of the clinical use of biomarkers, pharmacogenomics (PG), and therapeutic drug monitoring (TDM) approaches that can be used for the optimization of bevacizumab use in the era of precision medicine.

Development of a validated LC-MS/MS method for the in vitro and in vivo quantitation of sunitinib in glioblastoma cells and cancer patients.

Sunitinib is a multi-targeted tyrosine kinase inhibitor approved for the treatment of renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumor and is currently being investigated against other forms of malignant tumors. Recently great interest has emerged for the application of sunitinib to glioblastoma treatment. In order to have a method with broad applicability it will be of importance to have access to a method that could be applied both in human plasma and cell uptake studies. No method has been reported thus far for the estimation of sunitinib uptake in glioma cells. We therefore set out to develop a method that could be applied for quantifying sunitinib in human plasma and in cell uptake studies. The method was validated and accredited according to ISO 17025:2005 guideline in human plasma and successfully applied to cancer patient plasma. Also, the method was effectively recruited to establish a protocol for the evaluation of sunitinib accumulation into M095K glioma cells. This method could significantly contribute to developmental phases in repurposing this drug in different cancer types.

Development and validation of simple step protein precipitation UHPLC-MS/MS methods for quantitation of temozolomide in cancer patient plasma samples.

Temozolomide (TEMODAL™) (TMZ) is an antineoplastic agent that is primarily used for the treatment of glioblastoma and anaplastic gliomas, two aggressive forms of brain cancer. Due to the poor prognosis of brain tumour patients, there is an increasing body of research into improving the stability and delivery of TMZ past the blood brain barrier using carrier molecules. These require accurate determination of TMZ levels for biodistribution and pharmacokinetic evaluation. Unfortunately, current methodologies for the determination of TMZ in human plasma suffer from low reproducibility, recovery, sensitivity or cost ineffective procedures associated with extensive sample cleaning. To surpass these disadvantages, we developed two bioanalytical methods with high sensitivity and excellent recovery for the determination of TMZ in human plasma at minimum cost. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used and both methods were validated under US Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) guidelines. The two methods had minor differences in the sample pre-treatment and each method was developed and applied in separate laboratories. Theophylline was selected as internal standard (IS). Calibration curves were linear over the range of 10-500 ng/mL with extraction recovery ranging from 77.3 to 97.3% while all validation parameters met the acceptance criteria and proved the methods' reliability. The validated methods were successfully applied to plasma samples donated from cancer patient following treatment with temozolomide.

Intraoperative avidination for radionuclide therapy: a prospective new development to accelerate radiotherapy in breast cancer.

PURPOSE: In a continuous effort to seek for anticancer treatments with minimal side effects, we aim at proving the feasibility of the Intraoperative Avidination for Radionuclide Therapy, a new procedure for partial breast irradiation. EXPERIMENTAL DESIGN: To assess doses of 90Y-DOTA-biotin to target (i.e., breast tumor bed) and nontarget organs, we did simulation studies with 111In-DOTA-biotin in 10 candidates for conservative breast surgery. Immediately after quadrantectomy, patients were injected with 100-mg avidin in the tumor bed. On the following day, patients were given 111In-DOTA-biotin (approximately 111 MBq) i.v. after appropriate chase of biotinylated albumin (20 mg) to remove circulating avidin. Biokinetic studies were done by measuring radioactivity in scheduled blood samples, 48-h urine collection, and through scintigraphic images. The medical internal radiation dose formalism (OLINDA code) enabled dosimetry assessment in target and nontarget organs. RESULTS: Images showed early and long-lasting radioactive biotin uptake in the operated breast. Rapid blood clearance (<1% at 12 h) and urine excretion (>75% at 24 h) were observed. Absorbed doses, expressed as mean+/-SD in Gy/GBq, were as low as 0.15+/-0.05 in lungs, 0.10+/-0.02 in heart, 0.06+/-0.02 in red marrow, 1.30+/-0.50 in kidneys, 1.50+/-0.30 in urinary bladder, and 0.06+/-0.02 in total body, whereas in the targeted area, they increased to 5.5+/-1.1 Gy/GBq (50% ISOROI) and 4.8+/-1.0 Gy/GBq (30% ISOROI). CONCLUSION: Our preliminary results suggest that Intraoperative Avidination for Radionuclide Therapy is a simple and feasible procedure that may improve breast cancer patients' postsurgical management by shortening radiotherapy duration.

Development and validation of analytical methodology for the quantification of aldehydes in e-cigarette aerosols using UHPLC-UV.

Aldehydes are produced in e-cigarette aerosols, as a result of the thermal decomposition of vegetable glycerin, propylene glycerol and flavorings in the atomizer. These aldehydes were collected with derivatization into 2,4-dinitrophenylhydrazine using impinger trapping. A new methodology for simultaneous quantitative analysis of aldehydes in base liquids was developed and validated. Chromatographic separation was carried out on a Jupiter Proteo 90A column, with the mobile phase consisting of 0.1% formic acid in water and acetonitrile, at a flow rate of 0.4 mL/min. Linearity was demonstrated over the range of concentrations 0.025-10 µg/mL, with correlation of determination ≥ 0.999. Intra- and inter-day % relative standard deviation and relative error were ≤ 10%. The lower limit of detection and quantification were 0.008 and 0.025 µg/mL, respectively. This method was further used for the quantification of aldehydes emitted by different base liquids. It has been shown that vegetable glycerin produces the highest percentage of aldehydes after thermal decomposition compared to propylene glycerol. We propose that it can be used for future e-liquids emissions studies.

Development of a novel conjugatable sunitinib analogue validated through in vitro and in vivo preclinical settings.

Sunitinib is an oral FDA/EMEA approved multi-targeted tyrosine kinase inhibitor. It possesses anti-angiogenic and antitumor activity against a variety of advanced solid tumors. However, its chemical core does not allow a potential linkage to tumor-homing elements that could eventually enhance its potency. Therefore, a novel linkable sunitinib derivative, designated SB1, was rationally designed and synthesized. The pharmaceutical profile of SB1 was explored both in vitro and in vivo. Mass spectrometry and NMR spectroscopy were utilized for characterization, while MTT assays and LC-MS/MS validated protocols were used to explore its antiproliferative effect and stability, respectively. Cytotoxicity evaluation in three glioma cells showed that SB1 preserved the antiproliferative effect of sunitinib. SB1 was stable in vitro after 24 h incubation in mouse plasma, while both agents exhibited bioequivalent pharmacokinetic characteristics after i.v. administration in Balb/c mice. To evaluate the levels of SB1 in mouse plasma, a novel analytical method was developed and validated in accordance to the US FDA and the EU EMA guidelines. We formulated a novel linkable sunitinib analog exhibiting similar antiproliferative and apoptotic properties with native sunitinib in glioma cell lines. Both SB1 and native sunitinib showed identical in vitro stability in mouse plasma and pharmacokinetics after i.v. administration in Balb/c mice.

Neoadjuvant therapy in locally advanced breast cancer: 99mTc-MIBI mammoscintigraphy is not a reliable technique to predict therapy response.

Mammoscintigraphy (MMS) has been indicated as a useful tool in predicting response to therapy in cancer. However, contrasting results have been reported in the literature for breast cancer patients. The aim of this study was to explore the role of MMS in locally advanced breast cancer (LABC) patients. Fifty-one patients affected by LABC and scheduled for neoadjuvant therapy were enrolled. Breast tumor status was evaluated at baseline, during therapy and at the completion of therapy by radiological techniques and by MMS. Pre-therapy (MMS1) and post-therapy MIBI (2-methoxyisobutilysonitrile) images (MMS2-3) were analyzed. MMS1 was performed in all pts, 41 carried out MMS2 and 27 had MMS3. Tumor uptake and washout in MMS1 did not show any correlation with the therapy response. The absence of any association between tumor uptake and washout with respect to therapy response suggests that MMS is not a reliable technique to predict therapy response in LABC.