Long-term follow-up after complete resection of well-differentiated cancer confined to the thyroid gland

BACKGROUND: Papillary or follicular thyroid carcinomas exhibit a relatively benign course. Hence, long-term follow-up studies with well-defined disease stages and treatment details are needed to evaluate treatment strategies. METHODS: Patients who underwent complete resection of well-differentiated thyroid carcinoma (WDTC) confined to the thyroid gland between 1972 and 1990 identified from a prospective database were assessed. Follow-up was performed by interview, review of patient charts, and analysis of the Death Registry. Primary endpoints were overall survival (OS) and disease-specific survival (DSS). Review of histology was performed and extent of thyroid resection, postoperative therapy, and recognized prognostic factors but not lymphadenectomy were evaluated. RESULTS: Of 2,867 patients, 213 had complete resection of WDTC confined to the thyroid gland. Follow-up was completed in 166 patients with median age 54.2 (range, 20-85) years, and median follow-up of 27.2 (range, 15.6-34.5) years. The 10- and 20-year OS was 71 and 55%, respectively. DSS at 10 and 20 years was 81 and 69%, respectively, and correlated with age, histology, tumor size, radio-iodide ablation (RIA), and external beam irradiation (EBR) treatment. No patient died of WDTC more than 18 years after resection. Total or near-total thyroidectomy without lymphadenectomy was not superior to partial thyroidectomy. In multivariate analysis for DSS, age was the dominant factor, which correlated with histology. CONCLUSION: After a median follow-up of 27 years, about one-third of patients died of WDTC. Age, histology and postoperative therapy but not extent of thyroid resection determined DSS.

Diffusion-weighted imaging of the parotid gland: Influence of the choice of b-values on the apparent diffusion coefficient value

PURPOSE: To determine how the ADC value of parotid glands is influenced by the choice of b-values. MATERIALS AND METHODS: In eight healthy volunteers, diffusion-weighted echo-planar imaging (DW-EPI) was performed on a 1.5 T system, with b-values (in seconds/mm2) of 0, 50, 100, 150, 200, 250, 300, 500, 750, and 1000. ADC values were calculated by two alternative methods (exponential vs. logarithmic fit) from five different sets of b-values: (A) all b-values; (B) b=0, 50, and 100; (C) b=0 and 750; (D) b=0, 500, and 1000; and (E) b=500, 750, and 1000. RESULTS: The mean ADC values for the different settings were (in 10(-3) mm2/second, exponential fit): (A) 0.732+/-0.019, (B) 2.074+/-0.084, (C) 0.947+/-0.020, (D) 0.890+/-0.023, and (E) 0.581+/-0.021. ADC values were significantly (P <0.001) different for all pairwise comparisons of settings (A-E) of b-values, except for A vs. D (P=0.172) and C vs. D (P=0.380). The ADC(B) was significantly higher than ADC(C) or ADC(D), which was significantly higher than ADC(E). ADC values from exponential vs. logarithmic fit (P=0.542), as well as left vs. right parotid gland (P=0.962), were indistinguishable. CONCLUSION: The ADC values calculated from low b-value settings were significantly higher than those calculated from high b-value settings. These results suggest that not only true diffusion but also perfusion and saliva flow may contribute to the ADC.

Apoptotic cell death in the lactating mammary gland is enhanced by a folding variant of alpha-lactalbumin

Apoptosis is essential to eliminate secretory epithelial cells during the involution of the mammary gland. The environmental regulation of this process is however, poorly understood. This study tested the effect of HAMLET (human alpha-lactalbumin made lethal to tumor cells) on mammary cells. Plastic pellets containing HAMLET were implanted into the fourth inguinal mammary gland of lactating mice for 3 days. Exposure of mammary tissue to HAMLET resulted in morphological changes typical for apoptosis and in a stimulation of caspase-3 activity in alveolar epithelial cells near the HAMLET pellets but not more distant to the pellet or in contralateral glands. The effect was specific for HAMLET and no effects were observed when mammary glands were exposed to native a-lactalbumin or fatty acid alone. HAMLET also induced cell death in vitro in a mouse mammary epithelial cell line. The results suggest that HAMLET can mediate apoptotic cell death in mammary gland tissue.

Differential expression and localization of lipid transporters in the bovine mammary gland during the pregnancy-lactation cycle

The transport of lipids across mammary gland epithelial cells (MEC) determines milk lipid content and composition. We investigated the expression of lipid transporters and their regulators in comparison to blood metabolites during lactation and dry period (DP) in dairy cows. Repeated mammary gland biopsies and blood samples were taken from 10 animals at 7 stages of the pregnancy-lactation cycle. Expression levels of the specific mRNAs were determined by quantitative reverse transcription-PCR, whereas ABCA1 was localized by immunohistochemistry. Blood serum metabolites were determined by common enzymatic chemistries. Elevated mRNA profiles of ABCA1 and ABCA7 were found during DP as compared with lactation and were inversely associated with blood cholesterol levels. Elevated levels of ABCG2, NPC1, SREBP1, SREBP2, LXR alpha, and PPAR gamma were found postpartum, whereas ABCG1 did not differ between the functional stages of the mammary gland. The ABCA1 protein was localized in MEC and showed differential activity between DP and lactation suggesting a role of ABCA1 in the removal of excess cellular cholesterol from MEC during the DP. The expression profiles of ABCA7 and NPC1 may reflect a role of these transporters in the clearance of apoptotic cells and the intracellular redistribution of cholesterol, respectively. Regulation of lipid transporters in the mammary gland is partially associated with transcription factors that control lipid homeostasis.

Deregulated ephrin-B2 expression in the mammary gland interferes with the development of both the glandular epithelium and vasculature and promotes metastasis formation

Eph receptor tyrosine kinases and their membrane-bound ephrin ligands play key roles during morphogenesis and adult tissue homeostasis. Receptor-ligand interactions result in forward and reverse signalling from the receptor and ligand respectively. To delineate the role(s) of forward and reverse signalling in mammary gland biology we have established transgenic mice exhibiting mammary epithelial-specific overexpression of either the native ephrin-B2 or a dominant negative ephrin-B2 mutant incapable of reverse signalling. During pregnancy and lactation overexpression of the native ephrin-B2 resulted in precocious differentiation, whereas overexpression of mutated ephrin-B2 caused delayed epithelial differentiation and in disturbed tissue architecture. Both transgenes affected also mammary vascularisation. Whereas ephrin-B2 induced superfluous but organised capillaries, mutant ephrin-B2 overexpression resulted in an irregular vasculature with blind-ending capillaries. Mammary tumours were not observed in either transgenic line, however, the crossing with NeuT transgenic animals revealed that mutated ephrin-B2 expression significantly accelerated tumour growth and imposed a metastatic phenotype.

Role of amylase, mucin, IgA and albumin on salivary protein buffering capacity: a pilot study

It has been suggested that proteins serve as major salivary buffers below pH5. It remains unclear, however, which salivary proteins are responsible for these buffering properties. The aim of this pilot study was to evaluate the correlation between salivary concentration of total protein, amylase, mucin, immunoglobulin A (IgA), albumin and total salivary protein buffering capacity at a pH range of 4-5. In addition, the buffering capacity and the number of carboxylic acid moieties of single proteins were assessed. Stimulated saliva samples were collected at 9:00, 13:00 and 17:00 from 4 healthy volunteers on 3 successive days. The buffering capacities were measured for total salivary protein or for specific proteins. Also, the concentration of total protein, amylase, mucin, IgA and albumin were analysed. Within the limits of the current study, it was found that salivary protein buffering capacity was highly positively correlated with total protein, amylase and IgA concentrations. A weak correlation was observed for both albumin and mucin individually. Furthermore, the results suggest that amylase contributed to 35 percent of the salivary protein buffering capacity in the pH range of 4-5.

A new optical detection method to assess the erosion inhibition by in vitro salivary pellicle layer

OBJECTIVES Application of the recently developed optical method based on the monitoring of the specular reflection intensity to study the protective potential of the salivary pellicle layer against early enamel erosion. METHODS The erosion progression was compared between two treatment groups: enamel samples coated by the 15 h-in vitro-formed salivary pellicle layer (group P, n=90) and the non-coated enamel surfaces (control group C, n=90). Different severity of the erosive impact was modelled by the enamel incubation in 1% citric acid (pH=3.6) for 2, 4, 8, 10 or 15 min. Erosion quantification was performed by the optical method as well as by the microhardness and calcium release analyses. RESULTS Optical assessment of the erosion progression showed erosion inhibition by the in vitro salivary pellicle in short term acidic treatments (≤ 4 min) which was also confirmed by microhardness measurements proving significantly less (p<0.05) enamel softening in the group P at 2 and 4 min of erosion compared to the group C. SEM images demonstrated less etched enamel interfaces in the group P at short erosion durations as well. CONCLUSIONS Monitoring of the specular reflection intensity can be successfully applied to quantify early erosion progression in comparative studies. In vitro salivary pellicle (2h) provides erosion inhibition but only in short term acidic exposures. CLINICAL SIGNIFICANCE The proposed optical technique is a promising tool for the fast and non-invasive erosion quantification in clinical studies.

Characteristics and functional relevance of apolipoprotein-A1 and cholesterol binding in mammary gland tissues and epithelial cells

Cholesterol in milk is derived from the circulating blood through a complex transport process involving the mammary alveolar epithelium. Details of the mechanisms involved in this transfer are unclear. Apolipoprotein-AI (apoA-I) is an acceptor of cellular cholesterol effluxed by the ATP-binding cassette (ABC) transporter A1 (ABCA1). We aimed to 1) determine the binding characteristics of (125)I-apoA-I and (3)H-cholesterol to enriched plasma membrane vesicles (EPM) isolated from lactating and non-lactating bovine mammary glands (MG), 2) optimize the components of an in vitro model describing cellular (3)H-cholesterol efflux in primary bovine mammary epithelial cells (MeBo), and 3) assess the vectorial cholesterol transport in MeBo using Transwell(®) plates. The amounts of isolated EPM and the maximal binding capacity of (125)I-apoA-I to EPM differed depending on the MG's physiological state, while the kinetics of (3)H-cholesterol and (125)I-apoA-I binding were similar. (3)H-cholesterol incorporated maximally to EPM after 25±9 min. The time to achieve the half-maximum binding of (125)I-apoA-I at equilibrium was 3.3±0.6 min. The dissociation constant (KD) of (125)I-apoA-I ranged between 40-74 nmol/L. Cholesterol loading to EPM increased both cholesterol content and (125)I-apoA-I binding. The ABCA1 inhibitor Probucol displaced (125)I-apoA-I binding to EPM and reduced (3)H-cholesterol efflux in MeBo. Time-dependent (3)H-cholesterol uptake and efflux showed inverse patterns. The defined binding characteristics of cholesterol and apoA-I served to establish an efficient and significantly shorter cholesterol efflux protocol that had been used in MeBo. The application of this protocol in Transwell(®) plates with the upper chamber mimicking the apical (milk-facing) and the bottom chamber corresponding to the basolateral (blood-facing) side of cells showed that the degree of (3)H-cholesterol efflux in MeBo differed significantly between the apical and basolateral aspects. Our findings support the importance of the apoA-I/ABCA1 pathway in MG cholesterol transport and suggest its role in influencing milk composition and directing cholesterol back into the bloodstream.

Cholesterol Transport and Regulation in the Mammary Gland.

The milk-producing alveolar epithelial cells secrete milk that remains after birth the principal source of nutrients for neonates. Milk secretion and composition are highly regulated processes via integrated actions of hormones and local factors which involve specific receptors and downstream signal transduction pathways. Overall milk composition is similar among mammalian species, although the content of individual constituents such as lipids may significantly differ from one species to another. The milk lipid fraction is essentially composed of triglycerides, which represent more than 95 % of the total lipids in human and commercialized bovine milk. Though sterols, including cholesterol, which is the major milk sterol, represent less than 0.5 % of the total milk lipid fraction, they are of key importance for several biological processes. Cholesterol is required for the formation of biological membranes especially in rapidly growing organisms, and for the synthesis of sterol-based compounds. Cholesterol found in milk originates predominantly from blood uptake and, to a certain extent, from local synthesis in the mammary tissue. The present review summarizes current knowledge on cellular mechanisms and regulatory processes determining intra- and transcellular cholesterol transport in the mammary gland. Cholesterol exchanges between the blood, the mammary alveolar cells and the milk, and the likely role of active cholesterol transporters in these processes are discussed. In this context, the hormonal regulation and signal transduction pathways promoting active cholesterol transport as well as potential regulatory crosstalks are highlighted.

Nutrient Transport in the Mammary Gland: Calcium, Trace Minerals and Water Soluble Vitamins.

Milk nutrients are secreted by epithelial cells in the alveoli of the mammary gland by several complex and highly coordinated systems. Many of these nutrients are transported from the blood to the milk via transcellular pathways that involve the concerted activity of transport proteins on the apical and basolateral membranes of mammary epithelial cells. In this review, we focus on transport mechanisms that contribute to the secretion of calcium, trace minerals and water soluble vitamins into milk with particular focus on the role of transporters of the SLC series as well as calcium transport proteins (ion channels and pumps). Numerous members of the SLC family are involved in the regulation of essential nutrients in the milk, such as the divalent metal transporter-1 (SLC11A2), ferroportin-1 (SLC40A1) and the copper transporter CTR1 (SLC31A1). A deeper understanding of the physiology and pathophysiology of these transporters will be of great value for drug discovery and treatment of breast diseases.

IMPROVING QUANTITATIVE TREATMENT RESPONSE MONITORING WITH DEFORMABLE IMAGE REGISTRATION

Quantitative imaging with 18F-FDG PET/CT has the potential to provide an in vivo assessment of response to radiotherapy (RT). However, comparing tissue tracer uptake in longitudinal studies is often confounded by variations in patient setup and potential treatment induced gross anatomic changes. These variations make true response monitoring for the same anatomic volume a challenge, not only for tumors, but also for normal organs-at-risk (OAR). The central hypothesis of this study is that more accurate image registration will lead to improved quantitation of tissue response to RT with 18F-FDG PET/CT. Employing an in-house developed “demons” based deformable image registration algorithm, pre-RT tumor and parotid gland volumes can be more accurately mapped to serial functional images. To test the hypothesis, specific aim 1 was designed to analyze whether deformably mapping tumor volumes rather than aligning to bony structures leads to superior tumor response assessment. We found that deformable mapping of the most metabolically avid regions improved response prediction (P<0.05). The positive predictive power for residual disease was 63% compared to 50% for contrast enhanced post-RT CT. Specific aim 2 was designed to use parotid gland standardized uptake value (SUV) as an objective imaging biomarker for salivary toxicity. We found that relative change in parotid gland SUV correlated strongly with salivary toxicity as defined by the RTOG/EORTC late effects analytic scale (Spearman’s ρ = -0.96, P<0.01). Finally, the goal of specific aim 3 was to create a phenomenological dose-SUV response model for the human parotid glands. Utilizing only baseline metabolic function and the planned dose distribution, predicting parotid SUV change or salivary toxicity, based upon specific aim 2, became possible. We found that the predicted and observed parotid SUV relative changes were significantly correlated (Spearman’s ρ = 0.94, P<0.01). The application of deformable image registration to quantitative treatment response monitoring with 18F-FDG PET/CT could have a profound impact on patient management. Accurate and early identification of residual disease may allow for more timely intervention, while the ability to quantify and predict toxicity of normal OAR might permit individualized refinement of radiation treatment plan designs.

Estrogen action during early mouse mammary gland development

Estrogens have been implicated in the normal and neoplastic development of the mammary gland. Although estradiol is essential for early mammary differentiation, its role in postnatal ductal morphogenesis is poorly defined. We have found that neonatal estradiol exposure promotes precocious ductal outgrowth and terminal end bud formation in 21 day-old female mice. In contrast to this precocious phenotype, day 21 estradiol-treated epithelium, transplanted into control host fatpads, grows more slowly than control epithelium. Western and immunohistochemical (IHC) analyses indicate that neonatally-estrogenized glands have significantly less total ER than controls at days 7 and 21, and significantly more stromal ER at day 35. Estrogen receptor α (ER) is present in the gland when treatment is initiated at day 1. We propose that the premature activation of ER by neonatal estradiol exposure, during this critical perinatal period, is a key factor in the alteration of mammary growth and ER expression. ^ To address the role of ER function in mammary morphogenesis, we have developed an in vitro system to study the effect of estradiol exposure in vivo. Keratin and ER-positive mammary epithelial cell lines from 7, 21 and 35 day-old oil or estradiol treated mice have been established. Cell lines derived from estradiol-treated mice grow significantly slower than cells from control glands. Although the level of ER expressed by each cell line is correlated to its rate of growth, epithelial growth in vitro is estradiol-independent and antiestrogen-insensitive. Estradiol-induced transcription from an ERE-reporter in transiently-transfected cell lines confirms the functionality of the ER detected by western and IHC. However, there are no differences in estradiol-stimulated transcription between cell lines. ^ In conclusion, neonatal estradiol treatment alters the pattern of ER expression in mammary epithelial and stromal cells in vivo, and the growth of mammary epithelial cells in vivo and in vitro. When grown outside of the estrogenized host, exposed epithelium grows more slowly than the control. Therefore, an extra-epithelial factor is necessary for enhanced epithelial growth. Our model, which couples an in vivo-in vitro approach, can be used in the future to identify factors involved in the period of early mammary outgrowth and carcinogen susceptibility. ^