Ten-year clinical outcome, toxicity and compliance of dose-dense sequential adjuvant administration of cyclophosphamide & epirubicin followed by docetaxel in patients with early breast cancer: A hellenic cooperative oncology group observational study (HE 10/10) with concurrent investigation of significance of tumor infiltrating lymphocytes.

BACKGROUND: Dose-dense sequential (dds) chemotherapy has changed the clinical outcome of patients with early breast cancer (BC). To investigate the impact of dose intensity (DI) in the adjuvant setting of BC, this observational trial (HE 10/10) was conducted assessing the long-term survival outcome, safety and toxicity of a currently widely used chemotherapeutic regimen. In addition, the prognostic significance of tumor infiltrating lymphocytes (TILs) and infiltrating CD8+ lymphocytes were also evaluated in the same cohort. PATIENTS AND METHODS: Totally, 1054 patients were prospectively enrolled in the current study with 1024 patients being eligible, while adequate tissue was available for 596 of them. TILs, CD8+ lymphocytes in intratumoral areas in contact with malignant cells (iCD8), CD8+ lymphocytes in tumor stroma (sCD8) as well as the total number of CD8+ lymphocytes within the tumor area (total CD8) were assessed by immunohistochemistry. RESULTS: Within a median follow-up of 125.18 months, a total of 200 disease-free survival (DFS) events (19.5%) were reported. Importantly, the 10-year DFS and OS rates were 78.4% (95% CI 75.0-81.5) and 81.7% (95% CI 79.0-84.1), respectively. Interestingly, higher CD8+ T cells as well as TILs in the tumor microenvironment were associated with an improved long-term survival outcome. CONCLUSIONS: In conclusion, this study confirms the significance of dds adjuvant chemotherapeutic regimen in terms of long-term survival outcome, safety and toxicity as well as the prognostic significance of TILs and infiltrating CD8+ lymphocytes in BC patients with early-stage disease.

mRNA expression of specific HER ligands and their association with clinical outcome in patients with metastatic breast cancer treated with trastuzumab.

Prognostic and predictive biomarkers are being studied for the diagnosis and treatment of breast cancer. The present study retrospectively assessed the mRNA expression of HER family receptor ligands and of other potential prognostic biomarkers and their association with time to progression (TTP), survival and clinicopathological characteristics in patients with metastatic breast cancer (MBC) treated with trastuzumab. A total of 145 tumour tissue samples were analysed. mRNA expression analysis of the transcripts of interest was performed and the association of these markers with selected clinicopathological parameters was examined. HER2 status was centrally re-evaluated. Only 67.6% of patients were truly HER2-positive according to the central HER2 re-evaluation. Heparin binding epidermal growth factor (EGF)-like growth factor, transforming growth factor ß1 (TGFB1) and thyroid hormone receptor α (THRA) mRNA expression was higher in HER2-positive patients (P=0.026, P<0.001 and P<0.001). Insulin-like growth factor binding protein 4 was correlated with retinoic acid receptor α, TGFB1 and THRA (rho=0.45, rho=0.60 and rho=0.45). In HER2-positive patients, high neuregulin 1 and high betacellulin were unfavourable factors for TTP [hazard ratio (HR) = 1.78, P=0.040 and HR=2.00, P=0.043, respectively]. In patients with de novo MBC, high EGF expression was associated with a non-significant prolongation of TTP (HR=0.52, P=0.080) and significantly longer survival (HR=0.40, P=0.020). The present study examined clinical and biological implications of specific genes and it was concluded that their expression has an impact on the outcome of trastuzumab-treated patients with MBC.

Tumor Mutational Patterns and Infiltrating Lymphocyte Density in Young and Elderly Patients With Breast Cancer.

BACKGROUND/AIM: Age may pertain to different tumor genotype characteristics which may interfere with treatment efficacy and prognosis. We investigated the distribution and prognostic effect of mutations and tumor infiltrating lymphocyte (stromal TIL density) in young (≤35 years) and elderly (>65 years) early breast cancer patients. MATERIALS AND METHODS: Paraffin tumor genotypes of all clinical subtypes from 345 patients were examined. RESULTS: A total of 638 mutations were detected in 221 patients (64.1%). Compared to young, elderly patients presented with lower TIL density (p<0.001) but more TILs in TP53 mutated tumors (p=0.042). Mutation in one, rather than in 2 or more genes, conferred better outcome (DFS: HR=0.51, p=0.016; OS: HR=0.47, p=0.015) but the effect was age-independent. CONCLUSION: There are fewer TILs and different mutations patterns in tumors from elderly patients compared to young. Age and TIL-independent gene agnostic co-mutations affect patient outcome.

Genetic mapping of pancreatic cancer by targeted next-generation sequencing in a cohort of patients managed with nab-paclitaxel-based chemotherapy or agents targeting the EGFR axis: a retrospective analysis of the Hellenic Cooperative Oncology Group (HeCOG).

Pancreatic cancer is one of the most fatal malignancies ranking fourth among the leading causes of cancer death with diagnosis at late stages carrying a dismal prognosis. The aim of our retrospective study was to describe the nature and the incidence of gene mutations and genomic instability in advanced pancreatic adenocarcinomas of a Greek patient population fully annotated with clinicopathological data. We used a targeted next-generation sequencing (NGS) panel encompassing genes commonly mutated in pancreatic tumours in a patient population managed with either nab-paclitaxel regimens or targeted compounds modulating the epidermal growth factor receptor (EGFR)/AKT/mTOR axis. We identified KRAS, TP53, SMAD4 and CDKN2A as being the most prevalent mutations in the study population with the exception of an intriguingly lower incidence regarding KRAS mutants. Homologous recombination gene mutations were found to be mutually exclusive with CDKN2A mutations. The coexistence of both KRAS and TP53 mutation seems to adversely affect the outcome of the patients whether treated with targeted therapy against EGFR/Akt/mTOR axis or cytotoxic drugs. The poor prognosis observed, correlated to late presentation, specific molecular mutations and to high mutational load warrant prospective validating studies and research into the mechanistic pathophysiology of pancreatic tumours for more effective therapeutic targeting.

Cyclin D1 differential activation and its prognostic impact in patients with advanced breast cancer treated with trastuzumab.

INTRODUCTION: We sought to determine the level of activation of the critical components of the cyclin D1-mediated pathway and to evaluate their prognostic significance across the different molecular subtypes of advanced breast cancer. PATIENTS AND METHODS: The study population comprised 219 female patients with advanced breast cancer who had been found to have human epidermal growth factor receptor 2 (HER2)-positive disease by local testing and were all treated with trastuzumab-based regimens. For all tumours, central testing for HER2 was performed, and cyclin D1 gene (CCND1) amplification, mRNA and protein expression were assessed by FISH, quantitative real-time-PCR and immunohistochemistry, respectively. Prognostic impact on clinical endpoints was evaluated with Cox regression analyses. RESULTS: After central testing, only 134 (61.2%) of 219 patients were confirmed to have HER2 gene amplification by FISH and/or 3+ HER2 protein expression by immunohistochemistry. After a median follow-up time of 136.0 months (95% CI 123.3 to 148.9), 105 (78.4%) HER2-positive patients and 76 (89.4%) HER2-negative patients had died, while 80% of the former and 87.1% of the latter had experienced a disease relapse. Patients with positive oestrogen receptor/progesterone receptor status presented with higher cyclin D1 mRNA expression. In the HER2-negative subgroup, patients with negative cyclin D1 protein expression were at higher risk of progression (HR= 1.66, 95%CI 1.01 to 2.72, Wald's p=0.045). Among de novo metastatic patients, the risk of progression was higher for patients with non-amplified CCND1 tumours (HR= 2.00, 95% CI 1.03 to 3.90, p=0.041). CONCLUSION: Aberrant activation of the cyclin D1-mediated pathway appears to reduce the risk of progression in HER2-negative tumours, but not in HER2-positive ones.

Tumor Infiltrating Lymphocytes Affect the Outcome of Patients with Operable Triple-Negative Breast Cancer in Combination with Mutated Amino Acid Classes.

BACKGROUND: Stromal tumor infiltrating lymphocytes (TILs) density is an outcome predictor in triple-negative breast cancer (TNBC). Herein we asked whether TILs are related to coding mutation load and to the chemical class of the resulting mutated amino acids, i.e., charged, polar, and hydrophobic mutations. METHODS: We examined paraffin tumors from TNBC patients who had been treated with adjuvant chemotherapy mostly within clinical trials (training cohort, N = 133; validation, N = 190) for phenotype concordance; TILs density; mutation load and types. RESULTS: Concordance of TNBC phenotypes was 42.1% upon local / central, and 72% upon central / central pathology assessment. TILs were not associated with mutation load, type and class of mutated amino acids. Polar and charged mutation patterns differed between TP53 and PIK3CA (p<0.001). Hydrophobic mutations predicted for early relapse in patients with high nodal burden and <50% TILs tumors (training: HR 3.03, 95%CI 1.11-8.29, p = 0.031; validation: HR 2.90, 95%CI 0.97-8.70, p = 0.057), especially if compared to patients with >50% TILs tumors (training p = 0.003; validation p = 0.015). CONCLUSIONS: TILs density is unrelated to mutation load in TNBC, which may be regarded as an unstable phenotype. If further validated, hydrophobic mutations along with TILs density may help identifying TNBC patients in higher risk for relapse.

Adjusting breast cancer patient prognosis with non-HER2-gene patterns on chromosome 17.

BACKGROUND: HER2 and TOP2A gene status are assessed for diagnostic and research purposes in breast cancer with fluorescence in situ hybridization (FISH). However, FISH probes do not target only the annotated gene, while chromosome 17 (chr17) is among the most unstable chromosomes in breast cancer. Here we asked whether the status of specifically targeted genes on chr17 might help in refining prognosis of early high-risk breast cancer patients. METHODS: Copy numbers (CN) for 14 genes on chr17, 4 of which were within and 10 outside the core HER2 amplicon (HER2- and non-HER2-genes, respectively) were assessed with qPCR in 485 paraffin-embedded tumor tissue samples from breast cancer patients treated with adjuvant chemotherapy in the frame of two randomized phase III trials. PRINCIPAL FINDINGS: HER2-genes CN strongly correlated to each other (Spearman's rho >0.6) and were concordant with FISH HER2 status (Kappa 0.6697 for ERBB2 CN). TOP2A CN were not concordant with TOP2A FISH status (Kappa 0.1154). CN hierarchical clustering revealed distinct patterns of gains, losses and complex alterations in HER2- and non-HER2-genes associated with IHC4 breast cancer subtypes. Upon multivariate analysis, non-HER2-gene gains independently predicted for shorter disease-free survival (DFS) and overall survival (OS) in patients with triple-negative cancer, as compared to luminal and HER2-positive tumors (interaction p = 0.007 for DFS and p = 0.011 for OS). Similarly, non-HER2-gene gains were associated with worse prognosis in patients who had undergone breast-conserving surgery as compared to modified radical mastectomy (p = 0.004 for both DFS and OS). Non-HER2-gene losses were unfavorable prognosticators in patients with 1-3 metastatic nodes, as compared to those with 4 or more nodes (p = 0.017 for DFS and p = 0.001 for OS). CONCLUSIONS: TOP2A FISH and qPCR may not identify the same pathology on chr17q. Non-HER2 chr17 CN patterns may further predict outcome in breast cancer patients with known favorable and unfavorable prognosis.

Cavitating lung lesion as a manifestation of inflammatory tumor (pseudotumor) of the lung: A case report and literature review.

PATIENT: Female, 60 FINAL DIAGNOSIS: Inflammatory pseudotumor of the lung Symptoms: Cough dry • fever MEDICATION: - Clinical Procedure: - Specialty: - OBJECTIVE: Rare disease. BACKGROUND: Inflammatory pseudotumor of the lung involves a benign, non-neoplastic lung lesion of unknown etiology. CASE REPORT: We present a case of a 60-year-old female smoker who had been under intermittent immunosuppressive medication for discoid lupus, who was admitted to hospital with fever of 39.5°C of 10-day duration, not responding to an oral cephalosporin. Chest CT examination showed a cavitating opacity in the upper zone of the left lung. It was not feasible to establish a diagnosis based on clinical and laboratory testing nor based on CT scanning and bronchoscopy. Thus, the patient underwent left thoracotomy and sphenoid resection of the lesion, which was sent for biopsy. The histopathologic features aided by immunohistochemical staining proved the lesion to be an inflammatory pseudotumor of the lung. CONCLUSIONS: The case is reported because of the extremely rare radiologic presentation of the development of a lung pseudotumor emerging as a cavitated lesion, which relapsed during the follow-up period while the patient was still under immunosuppressive medication.

Development of a capillary electrophoresis-mass spectrometry method for the determination of rivastigmine in human plasma--optimization of the limits of detection and quantitation.

A capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) method was developed for the analysis of the acetylcholinesterase inhibitor rivastigmine. Several electrophoretic and ESI-MS parameters were investigated in order to improve sensitivity. These parameters were categorized in three areas: (i) background electrolyte (BGE) parameters, (ii) sheath liquid parameters, and (iii) spray chamber parameters. The optimized results were obtained by using 40-mM ammonium acetate at pH 9 as BGE, a sheath liquid of 1% acetic acid in water:MeOH (50:50 v/v) at a flow rate of 10 µL/min, and a drying gas flow rate that was set at 6 L/min and at a temperature of 200°C. These parameters provided limit of detection and limit of quantitation of 2.8 ng/mL and 8.4 ng/mL, respectively. The optimal CZE-ESI-MS conditions were applied to a plasma sample obtained from an Alzheimer's disease patient following rivastigmine patch administration, and the mean (±standard deviation) plasma concentration was estimated to be 14.6 (±1.7) ng/mL. Several sample preparation procedures were examined, and solid-phase extraction using a C18 cartridge proved to be the most effective procedure, since higher sensitivity and recovery were obtained. In addition, precision was evaluated based on migration time and peak area in plasma, and the relative standard deviations were in the range of 0.10-0.16% and 0.62-9.0%, respectively.

Separation of acetylcholinesterase inhibitors using polymeric surfactants in polyelectrolyte multilayer coatings.

The main objective of this study is the use of polymeric surfactants in polyelectrolyte multilayer (PEM) coatings for the separation of the pharmaceutical substances acetylcholinesterase inhibitors (AChEIs). AChEIs are used for the treatment of Alzheimer's Disease and Myasthenia Gravis. In the open-tubular capillary electrochromatography (OT-CEC) mode, the PEM coating is evaluated using nine AChEIs. Optimal conditions are established by altering several experimental parameters such as the pH of the background electrolyte (BGE), the anionic polymer for the PEM coating, the concentration of NaCl, which is used as an additive in the polymer deposition solutions, the number of bilayers, the deposition time, and the concentration of the polymeric surfactant. 25 mM NaH(2)PO(4).H(2)Ο and 25 mM Na(2)HPO(4) at pH 7 is used as BGE. Two bilayers of poly(diallyl dimethyl ammonium chloride) and poly(sodium N-undecanoyl L-leucinate) provide a baseline separation of all nine analytes in less than 4.5 min. Run-to-run reproducibility studies are also performed, and the relative standard deviation values of the migration times of the nine-analyte peaks are less than 2%. In addition, day-to-day, week-to-week and capillary-to-capillary reproducibilities are evaluated, and the relative standard deviation values of the electroosmotic flow are less than 2%. Finally, using the PEM coating approach, we were able to perform more than 150 runs in the same column. Neither the addition of the polymeric surfactant to the mobile phase, nor the reconstruction of the coating was necessary.

Chiral separation of Huperzine A using CE - method validation and application in pharmaceutical formulations.

The aim of this work is the development, validation and application of an MEKC method for the chiral separation of Huperzine A. Huperzine A is an important compound that is used to treat Alzheimer's disease. However, only the (-)-form of this compound is biologically active and behaves as a potential acetylcholinesterase inhibitor. Therefore, the separation of the (-)-form from the (+)-form is of greatest importance. Optimal conditions, regarding resolution and analysis time, were established by altering several experimental parameters, such as temperature, field strength, pH, type and concentration of BGE and chiral selector. A major problem that had to be solved in this study was the low intensity and efficiency of the peaks. More parameters were examined, such as the addition of modifiers, to optimize further the separation, and particularly the efficiency. Baseline enantioseparation was achieved by using a BGE of 50 mM acetate (pH 5.0), supplemented with 0.2% w/v poly(sodium N-undecanoyl-ll-alanyl-valinate) and 10% v/v tert-butanol. Finally, the optimum conditions were applied to a pharmaceutical formulation, to demonstrate the ability of the method to control the purity of the (-)-Huperzine A in pharmaceutical formulations.

Simultaneous determination of nine acetylcholinesterase inhibitors using micellar electrokinetic chromatography.

MEKC was used for the separation of nine acetylcholinesterase inhibitors (AChEIs). AChEIs are an important group of drug compounds that are used medicinally to treat Alzheimer's disease and Myasthenia Gravis. At the time of the experiment, this is the first time that nine AChEIs are used simultaneously in a study. Several chromatographic parameters, such as buffer concentration, pH, surfactants and their concentration, background electrolyte composition, etc., were evaluated to optimize the separation. The optimum separation of the nine AChEIs was achieved in less than 15 min by using 12.5 mM Na(2)HPO(4), 12.5 mM Na(2)B(4)O(7) and 20 mM SDS at pH 10, an applied voltage of 30 kV and a temperature of 25 °C. The reproducibility of the method was also evaluated by computing the RSDs of the migration times and the areas of the nine analyte-peaks, and the migration time and the area of the peak that corresponds to rivastigmine added in the blood sample. The RSD values of the migration times and the peak areas were less than 2% and 6%, respectively, in most cases. The limits of detection and quantification were 0.5 µg/mL and 1.7 µg/mL, respectively. The MEKC method developed was applied to a real blood sample that was obtained from a patient who was not under any of this medication. The sample was spiked with rivastigmine in order to establish the ability of the method to separate the drug from other components that might exist in the blood sample.

Analysis of polyphenols using capillary zone electrophoresis--determination of the most effective wine sample pre-treatment method.

A simple CZE method has been developed for the simultaneous determination of eight polyphenolic compounds. The influence of several experimental conditions such as buffer (concentration and pH) and temperature were studied. Optimum separation was achieved in less than 25 min by using a BGE of 50 mM Na(2)B(4)O(7) and 10 mM Na(2)HPO(4) at pH 9.6, a temperature of 25°C and an applied voltage of 25 kV. Good linearities for all eight analytes were obtained with correlation coefficients higher than 0.99. The LODs were between 0.03 and 5.05 µg/mL and the RSD values of the migration times were found to be less than 1%. The optimal separation conditions were then used for the identification and the quantitation of polyphenolic compounds in Cypriot wine samples using six different sample preparation procedures. In particular, two direct injection methods (without any extraction step), three different liquid-liquid extraction procedures and an SPE procedure were examined. These sample pre-treatment methods were also compared in order to determine the one that is the most effective, in regard to analyte recovery, time, difficulty, and reproducibility. Liquid-liquid extraction using diethyl ether as the organic solvent proved to be the most effective.

Pericardial involvement as an atypical manifestation of giant cell arteritis: report of a clinical case and literature review.

PURPOSE: Pericardial effusion has been known to be a rare manifestation of giant cell arteritis. During the last six decades, only 24 cases have been cited in the literature. In this report, we describe the case of a patient presenting with nonspecific symptoms and development of pericardial effusion. PROCEDURES AND FINDINGS: A 71-year-old woman was admitted to the hospital with low-grade fever, exertion breathlessness, atypical diffuse muscular pain, and weight loss over a period of about 5 weeks. Pericardial effusion and giant cell arteritis were diagnosed by echocardiography and left temporal artery biopsy, respectively. Treatment with corticosteroids resulted in remarkable improvement of symptoms and complete remission of pericardial effusion. One year after admission, the patient remained in a stable good condition, under low steroid maintenance dosage. CONCLUSIONS: The diversity of clinical manifestations (such as pericardial effusion) in such a potentially severe disease should alert the physician to prompt diagnosis and treatment in view of impending irreparable vascular damages, even in cases in which the initial presentation is quite uncommon.

Report of an atypical case of leishmaniasis presented as acute tonsillitis in an immunocompetent patient.

Visceral leishmaniasis typically presents with symptoms such as fever, enlargement of the spleen and the liver, hypergammaglobulinaemia and infection of the bone marrow resulting in anaemia and leukopenia. The disease is sporadic in the countries of the Mediterranean basin. We report an unusual case of acute tonsillitis due to tonsillar leishmaniasis, in an immunocompetent 34-y-old male patient. Diagnosis was confirmed by serological tests and histopathological examination following biopsy of the right tonsil. The patient was successfully treated with liposomal amphotericin-B.

The relationship between five kinds of laparoscopic knots and five types of suture materials and histological findings in tissues: an experimental study on rabbits.

The aim of the current study was to examine the slipping and the tightening of laparoscopic knots with various kinds of sutures, as well as the histologic alterations in tissues. Fifty rabbits and five kinds of sutures were used-silk, polyglactine-910 (Vicryl), Polydioxanone (PDS), Polyglycol (Dexon), and cat-gut chromic-and five laparoscopic knots were used-Tayside, Roeder, Melzer, Cross, and Blood. The knots were performed extracorporeally and were used to ligate a part of the omentum. Sliding and tightening of the knots were evaluated. The omentum, the suture, and the knots were checked 10 days and 1 month after operation. Histologic examination was performed 1 month after surgery. Polyglactine-910 (Vicryl) and silk were the most qualitative sutures used in Tayside, Roeder, and Blood knots and the least harmful for the tissues. Catgut chromic and Polydioxanone (PDS) were the most defective sutures. The most efficient laparoscopic slipknots are Tayside, Roeder, and Blood, especially when constructed with silk and polyglactine-910 (Vicryl).