Immunological differences between primary and metastatic breast cancer.

Background: Little is known about how the immune microenvironment of breast cancer evolves during disease progression. Patients and methods: We compared tumor infiltrating lymphocyte (TIL) count, programmed death-ligand 1 (PD-L1) protein expression by immunohistochemistry and mRNA levels of 730 immune-related genes using Nanostring technology in primary and metastatic cancer samples. Results: TIL counts and PD-L1 positivity were significantly lower in metastases. Immune cell metagenes corresponding to CD8, T-helper, T-reg, Cytotoxic T, Dendritic and Mastoid cells, and expression of 13 of 29 immuno-oncology therapeutic targets in clinical development including PD1, PD-L1, and CTLA4 were significantly lower in metastases. There was also coordinated down regulation of chemoattractant ligand/receptor pairs (CCL19/CCR7, CXCL9/CXCR3, IL15/IL15R), interferon regulated genes (STAT1, IRF-1,-4,-7, IFI-27,-35), granzyme/granulysin, MHC class I and immune proteasome (PSMB-8,-9,-10) expression in metastases. Immunotherapy response predictive signatures were also lower. The expression of macrophage markers (CD163, CCL2/CCR2, CSF1/CSFR1, CXCR4/CXCL12), protumorigenic toll-like receptor pathway genes (CD14/TLR-1,-2,-4,-5,-6/MyD88), HLA-E, ecto-nuclease CD73/NT5E and inhibitory complement receptors (CD-59,-55,-46) remained high in metastases and represent potential therapeutic targets. Conclusions: Metastatic breast cancers are immunologically more inert than the corresponding primary tumors but some immune-oncology targets and macrophage and angiogenesis signatures show preserved expression and suggest therapeutic combinations for clinical testing.

Pathway level alterations rather than mutations in single genes predict response to HER2-targeted therapies in the neo-ALTTO trial.

Background: We performed whole-exome sequencing of pretreatment biopsies and examined whether genome-wide metrics of overall mutational load, clonal heterogeneity or alterations at variant, gene, and pathway levels are associated with treatment response and survival. Patients and Methods: Two hundred and three biopsies from the NeoALTTO trial were analyzed. Mutations were called with MuTect, and Strelka, using pooled normal DNA. Associations between DNA alterations and outcome were evaluated by logistic and Cox-proportional hazards regression. Results: There were no recurrent single gene mutations significantly associated with pathologic complete response (pCR), except PIK3CA [odds ratio (OR) = 0.42, P = 0.0185]. Mutations in 33 of 714 pathways were significantly associated with response, but different genes were affected in different individuals. PIK3CA was present in 23 of these pathways defining a 'trastuzumab resistance-network' of 459 genes. Cases with mutations in this network had low pCR rates to trastuzumab (2/50, 4%) compared with cases with no mutations (9/16, 56%), OR = 0.035; P < 0.001. Mutations in the 'Regulation of RhoA activity' pathway were associated with higher pCR rate to lapatinib (OR = 14.8, adjusted P = 0.001), lapatinib + trastuzumab (OR = 3.0, adjusted P = 0.09), and all arms combined (OR = 3.77, adjusted P = 0.02). Patients (n = 124) with mutations in the trastuzumab resistance network but intact RhoA pathway had 2% (1/41) pCR rate with trastuzumab alone (OR = 0.026, P = 0.001) but adding lapatinib increased pCR rate to 45% (17/38, OR = 1.68, P = 0.3). Patients (n = 46) who had no mutations in either gene set had 6% pCR rate (1/15) with lapatinib, but had the highest pCR rate, 52% (8/15) with trastuzumab alone. Conclusions: Mutations in the RhoA pathway are associated with pCR to lapatinib and mutations in a PIK3CA-related network are associated with resistance to trastuzumab. The combined mutation status of these two pathways could define patients with very low response rate to trastuzumab alone that can be augmented by adding lapatinib or substituting trastuzumab with lapatinib.

Efficacy and tolerability of combination cisplatin and ifosfamide chemotherapy with vaginal cuff brachytherapy in the first line treatment of uterine carcinosarcoma.

PURPOSE OF INVESTIGATION: A retrospective study to evaluate six cycles of cisplatin 40 mg/m2 on day 1 and ifosfamide 1,200 mg/m2 daily on days 1 to 4 with Mesna every four weeks as first line treatment for 29 patients with a diagnosis of uterine carcinosarcoma. MATERIALS AND METHODS: A total of 23 of 29 patients received high dose rate intracavitary vaginal cuff brachytherapy (VCBT) with two fractions of seven Gy each. Median age was 65 years (range 40-82); 13 (44.8%) had Stage I disease, three (10.3%) had Stage II, eight (27.6%) had Stage III, and five (17.2%) patients had Stage IV disease. RESULTS: Most common toxicities were anemia grade 1 (35%)/grade 2 (45%), and neutropenia grade 3 (17%)/grade 4 (6.9%). Eleven dose modifications, four treatment discontinuations, and one patient withdrawal occurred. At a median follow up of 45 months (range 9 to 144), Progression free survival (PFS) was 20% and overall survival (OS) was 40% for Stage IV, PFS 75% and OS 62.5% for Stage III, compared to a PFS 75% and OS 72.2% for Stages I-II. Median OS for the entire group was 12.43 years (95% CI 3.69 to inf); for Stage I-III 12.4 years (6.1 to inf), and for Stage IV 15.6 months (95% CI 9.4 to inf). CONCLUSIONS: Cisplatin and ifosfamide chemotherapy with VCBT was well tolerated and has promising activity in uterine carcinosarcoma.

Metabolic isoenzyme shifts in cancer as potential novel therapeutic targets.

The functional redundancy of metabolic enzyme expression may present a new strategy for developing targeted therapies in cancer. To satisfy the increased metabolic demand required during neoplastic transformations and proliferation, cancer cells may rely on additional isoforms of a metabolic enzyme to satisfy the increased demand for metabolic precursors, which could subsequently render cancer cells more vulnerable to isoform-specific inhibitors. In this review, we provide a survey of common isoenzyme shifts that have been reported to be important in cancer metabolism and link those to metabolic pathways that currently have drugs in various stages of development. This phenomenon suggests a potentially new therapeutic strategy for the treatment of cancer by identifying shifts in the expression of metabolic isoenzymes between cancer and normal cells. We also delineate other putative metabolic isoenzymes that could be targets for novel targeted therapies for cancer. Changes in isoenzyme expression that occur during neoplastic transformations or in response to environmental pressure in cancer cells may result in isoenzyme diversity that may subsequently render cancer cells more vulnerable to isoform-specific inhibitors due to reliance on a single isoform to perform a vital enzymatic function.

Prevalence of obesity and physical inactivity among farmers from Crete (Greece), four decades after the Seven Countries Study.

BACKGROUND AND AIM: As first shown 40 years ago farmers from Crete had one of the healthiest lifestyles compared to other participants of the Seven Countries Study. Taking the above into account we investigated the prevalence of obesity and its indexes among farmers in Crete in 2005. METHODS AND RESULTS: 502 farmers (18-79 years old) from the Valley of Messara in Crete were randomly selected and examined. Body Mass Index (BMI), waist circumference (WC), waist-to-hip ratio (W/Hip Ratio), waist-to-height ratio (W/Height Ratio), conicity index, percentage of body fat and hours of daily light physical activity (LPhA) and moderate-to-vigorous physical activity (MVPhA) were calculated for each subject. 86,1% of the study population was overweight and/or obese. Specifically 42.9% had a BMI of 25.1-30 kg/m(2) and were overweight and 43.2% were obese with a BMI>30 kg/m(2). The percentage of body fat was estimated at 27.3% of total body weight among males and 39.3% among females, while all obesity indexes were found to differ between genders. CONCLUSIONS: In comparison to middle aged male farmers from Crete in the 1960s, mean weight has increased by 20 kg (83 kg vs. 63 kg), which has lead to a 7 kg/m(2) in mean BMI (22.9 kg/m(2) vs. 29.8 kg/m(2)), findings that support the fact that the prevalence of obesity in Greece has risen dramatically over the years, even among farmers from Crete, a population historically known for being the gold standard of health status globally.

Adipose tissue fatty acids in breast cancer patients versus healthy control women from Crete.

BACKGROUND: Few studies have implemented biomarkers of fatty acid intake in relation to breast cancer. AIMS: To examine possible differences in adipose tissue fatty acid composition between breast cancer patients and healthy control women. The relationship between tumor promotion and adipose tissue fatty acid synthesis was also investigated. METHODS: The study was conducted at the University of Crete. Subjects included 94 women with clinically diagnosed cancer of the breast and 131 healthy control women. Histological tumor grading and breast cancer staging were assessed. Fatty acids were determined by gas chromatography in gluteal adipose tissue. RESULTS: Conditional logistic regression analysis controlling for potential confounders indicated that elevated adipose monounsaturated fatty acids and oleic acid are associated with reduced odds of breast cancer [OR (T2 vs. T1) 0.15; 95% CI 0.03-0.64, and OR (T2 vs. T1) 0.18; 95% CI 0.04-0.71, respectively]. Adipose myristic acid was associated with an increase in breast cancer risk [OR (T3 vs. T1) 5.66; 95% CI 1.3-23.9]. CONCLUSIONS: Adipose oleic acid is inversely related, whereas adipose myristic acid is positively related to breast cancer risk. These relations could be mediated by Her-2/neu and FAS oncogenes.

Hormone receptor status and pathologic response of HER2-positive breast cancer treated with neoadjuvant chemotherapy and trastuzumab.

BACKGROUND: The aim of this study was to compare the extent of pathologic response in patients with HER2-positive (HER2+) breast cancer treated with standard neoadjuvant chemotherapy, with or without trastuzumab (H), according to hormone receptor (HR) status. PATIENTS AND METHODS: We included 199 patients with HER2+ breast cancer from three successive cohorts of neo-adjuvant chemotherapy on the basis of paclitaxel (Taxol) (P) administered weekly (w) or three weekly (3-w), followed by 5-fluorouracil (F), doxorubicin (A) or epirubicin (E), and cyclophosphamide (C). Residual cancer burden (RCB) was determined from pathologic review of the primary tumor and lymph nodes and was classified as pathologic complete response (pCR) or minimal (RCB-I), moderate (RCB-II), or extensive (RCB-III) residual disease. RESULTS: In HR-positive (HR+) cancers, a higher rate of pathologic response (pCR/RCB-I) was observed with concurrent H + 3-wP/FEC (73%) than with 3-wP/FEC (34%, P = 0.002) or wP/FAC (47%; P = 0.02) chemotherapy alone. In HR-negative (HR-) cancers, there were no significant differences in the rate of pathologic response (pCR/RCB-I) from 3-wP/FAC (50%), wP/FAC (68%), or concurrent H + 3-wP/FEC (72%). CONCLUSIONS: Patients with HR+/HER2+ breast cancer obtained significant benefit from addition of trastuzumab to P/FEC chemotherapy; pathologic response rate was similar to that seen in HR-/HER2+ breast cancers.

Prevalence and independent predictors of insulin resistance in children from Crete, Greece: the Children Study.

BACKGROUND: It has been proposed that insulin resistance (IR) is associated with the development of Type 2 diabetes mellitus and cardiovascular disease. The aim of this study is to determine the prevalence of IR in Greek schoolchildren and to investigate factors associated with IR. METHODS: Between October 2005 and March 2006, 522 children were recruited from Crete. Physical activity and dietary habits, anthropometric and biochemical characteristics, as well as medical history of pupils' parents were recorded. IR was estimated using the homeostasis model assessment (HOMA-IR), fasting glucose-to-insulin ratio (FGIR) and quantitative insulin sensitivity check index (QUICKI). Multiple linear regression was used to determine independent predictors for IR. RESULTS: Fasting insulin levels and HOMA-IR scores were higher in obese children and girls compared with their normal-weight peers (P < 0.001). Moreover, the former had lower values in FGIR and QUICKI indices compared with the latter, indicating that obese children and girls are more insulin resistant compared with their counterparts (P < 0.001). The prevalence of IR was 9.2% (2.9% in normal-weight, 10.5% in overweight and 31.0% in obese children), using as a threshold HOMA-IR > 2.10 97.5th percentile of normal-weight participants). Multiple linear regression revealed that central adiposity, female gender and intake of simple carbohydrates is associated positively with HOMA-IR values, even after controlling for many other factors. CONCLUSION: These findings demonstrate that girls and obese children, particularly those with central adiposity, are at high risk of developing IR. Therefore, these groups should be targets of Type 2 diabetes mellitus and cardiovascular disease preventive interventions.

Depression and long chain n-3 fatty acids in adipose tissue in adults from Crete.

BACKGROUND: Only one study has investigated the relationship of essential fatty acids in the adipose tissue with depression in adults and suggested an inverse relationship between docosahexaenoic acid (22:6 n-3) (DHA) and depression. OBJECTIVE: To examine the relation between adipose tissue polyunsaturated fatty acids especially n-3 and n-6 fatty acids, an index of long-term or habitual fatty acid intake, and depression in adults. DESIGN: Cross-sectional study of healthy adults from the island of Crete. SETTING: The Preventive Medicine and Nutrition Clinic, University of Crete, Greece. SUBJECTS: A total of 130 healthy adults (59 males, 71 females) aged 22-58 years. The sample was a sub-sample of the Greek ApoEurope study group. METHODS: Fatty acids were determined by gas chromatography in adipose tissue. Information about depression was obtained through the Zung Self-rating Depression Scale. RESULTS: Adipose tissue DHA was inversely related with depression. Multiple linear regression analysis taking into account the possible confounding effect of age, gender, body mass index, smoking and educational level confirmed this association. CONCLUSIONS: The inverse relationship between adipose DHA and depression in adults, replicates findings of a previous study. This relationship indicates that a low long-term dietary intake of DHA is associated with an increased risk for depression in adults.

Differences in postprandial lipaemic response between Northern and Southern Europeans.

Postprandial lipaemic responses to two test meals were investigated in 30 Northern (15 British and 15 Irish), and 30 Southern (Greeks from Crete) healthy male Europeans. The meals were a saturated fatty acid (SFA) meal, which resembled the fatty acid composition of an average UK diet, and a monounsaturated fatty acid (MUFA) meal in which the fat consisted of olive oil. Habitual diets of the two groups differed, with higher total fat, (P < 0.03) and MUFA (P < 0.0001) and lower polyunsaturated fatty acid (PUFA) (P < 0.0001) intakes in Southern than Northern Europeans. Levels of total MUFA (P < 0.02) and oleic acid (P < 0.004) were also higher in adipose tissue of Southern in comparison to Northern Europeans. In both European groups there were no significant differences in postprandial triglyceride response between the two meal types, SFA or MUFA. However, Northern and Southern Europeans showed significant differences in their patterns of postprandial response in plasma triglycerides (P < 0.0001), apolipoprotein B-48 (P < 0.0001), NEFA (P < 0.0001), insulin (P < 0.0007), and factor VII activity (P-0.03). In the case of NEFA, areas under the response curve were higher following the SFA than the MUFA meal for both groups, (P < 0.003) and were greater in Southern than Northern Europeans (P < 0.002) and apo B-48 responses were lower (P < 0.005). Some of these differences may reflect differences in fasting levels since fasting apolipoprotein B-48 levels were lower (P < 0.01) and fasting NEFA (P < 0.02) and insulin (P < 0.005) were higher in the Southern than in the Northern Europeans. In addition, 9 h postprandial post-heparin lipoprotein lipase activity was lower in the Southern than in the Northern Europeans (P < 0.0006). This is the first report of differences in postprandial lipid, factor VII and insulin responses in Southern and Northern Europeans which may be of importance in explaining the different susceptibilities of these two populations to risk of coronary artery disease.

Biochemical engineering analysis of critical process factors in the biomass-to-ethanol technology.

Ethanol from cellulosic biomass is a promising renewable liquid transportation fuel. Applied research in the area of biomass conversion to ethanol in the last 20 years has answered most of the major challenges on the road to commercialization but, as with any new technology, there is still room for performance improvement. A verified mathematical model was used to examine the most critical biochemical engineering aspects of ethanol production in this study. Extensive simulations of the simultaneous saccharification and fermentation (SSF) of cellulose were conducted to identify the effects of operating conditions, pretreatment effectiveness, microorganism parameters, and enzyme characteristics on ethanol production. The results clearly show that the biomass-enzyme interaction plays a dominant role in determining the performance of SSF in batch and continuous operating modes. In particular, the digestibility of the substrate (as a result of pretreatment) and the cellulase enzyme dosage, specific activity, and composition had a profound effect on ethanol yield. This investigation verified the conclusion that R&D emphasis should be placed on developing more effective pretreatment methods and producing cellulase preparations of high specific activity (low cost per enzyme unit) to realize gains from any development of advanced hexose/pentose-fermenting organisms.

Multistaged corpuscular models of microbial growth: Monte Carlo simulations.

A new framework is developed by extending the existing population balance framework for modeling the growth of microbial populations. The new class of multistaged corpuscular models allows further structuring of the microbial life cycle into separate phases or stages and thus facilitates the incorporation of cell cycle phenomena to population models. These multistaged models consist of systems of population balance equations coupled by appropriate boundary conditions. The specific form of the equations depend on the assumed forms for the transition rate functions, the growth rate functions, and the partitioning function, which determines how the biological material is distributed at division. A growth model for ciliated protozoa is formulated to demonstrate the proposed framework. To obtain a solution to the system of the partial integro differential equations that results from such formulation, we adopted a Monte Carlo simulation technique which is very stable, versatile, and insensitive to the complexity of the model. The theory and implementation of the Monte Carlo simulation algorithm is analyzed and results from the simulation of the ciliate growth model are presented. The proposed approach seems to be promising for integrating single-cell mechanisms into population models.

A discrete, stochastic model for microbial filter feeding: a model for feeding of ciliated protists on spatially uniform, nondepletable suspensions.

Suspension-feeding ciliates, either bacteriovorous or planktonic, are adapted to feed on particulate food matter of size much smaller than their own size. These microorganisms collect their prey by generating water currents that draw prey toward their capture surfaces. Under such conditions food particles are treated in bulk, and captures of individual food particles from a suspension by individual single-celled organisms are discrete events that occur at random intervals of time. Each such event is followed by a sequence of additional events that also occur at random intervals of time. This sequence culminates in the incorporation of the digestible portion of the food particle into the cell's cytoplasm and the expulsion of the indigestible portion from the cell. In theory, the rate of the overall ingestion-digestion process can be limited by the passage of particles through any stage of this sequence of events. In this paper, we assume that only the initial events in the sequence, those that occur in the oral region of the cell, limit the rate of the ingestion-digestion process, and we develop a discrete, stochastic model of filter feeding based on that assumption. We use the model to show how advanced instrumentation, such as flow cytometry, can be used to measure parameters of the model and also to answer a number of important questions about the mechanism of filter feeding. We show also how the model can be applied to nonhomogeneous cell populations for which parameters of the model are distributed.

A statistical analysis of flow cytometric determinations of phagocytosis rates.

Uptake of particles by phagocytosing cells is a process that exhibits variability of its rate. This variability is inherent in the mechanism of particle uptake and in the mechanisms that determine the distribution of physiological states within a population of phagocytosing cells. When numbers of particles ingested by cells are determined flow cytometrically an additional measurement variability is superimposed on and interacts with the aforementioned biological variability. In one method of determining population phagocytosis parameters, which involves fitting theoretical equations to experimental time course data on the fractions of cells which have ingested 0, 1, 2, ... particles, the effects of measurement variability are circumvented, although this usually has the cost of not using all the sample data obtained. However, in a second, simpler, method which is based on determining the time course of the number of particles ingested by an average cell, measurement variability is not circumvented and its effects must be considered. An analysis of the combined effects of biological and measurement variability on the results obtained with the simpler method is presented in this paper. Experimental results for phagocytosis of latex microspheres of uniform size and fluorochrome content by populations of the ciliate Tetrahymena pyriformis show that, for this system, measurement variability is entirely negligible in comparison with biological variability. This conclusion might not apply to other systems, however, and situations which might make measurement variability of some significance are mentioned in the paper. The equations given can be used for the analysis of such situations.

Feeding heterogeneity in ciliate populations: Effects of culture age and nutritional state.

We have used a novel approach in conjunction with flow cytometry to quantify the biological heterogeneity of populations of the ciliate Tetrahymena pyriformis. It was found that the rate of particle uptake of exponentially growing cells is not uniform among cells and partially correlated with cell size. The physiological state and growth history of the culture was found to affect to a large degree the population's feeding heterogeneity. Stationary phase populations exhibited more uniform feeding behavior, as cell aging affects all cells and effectively reduces their ability to feed. Cells that were removed from the growth medium and resuspended in nonnutritive medium exhibited a more heterogeneous feeding behavior. The starved cells were stimulated to feed at considerably higher rates, and the stimulatory effect was more pronounced for larger cells. It is therefore demonstrated that population heterogeneity has to be evaluated in conjunction with the populations growth state as it is determined by the history of the population's growth and nutritional state. (c) 1994 John Wiley & Sons, Inc.