Looking at tardigrades in a new light: using epifluorescence to interpret structure.

The use of epifluorescence microscopy coupled with ultraviolet (UV) autofluorescence is suggested as a means to view and interpret tardigrade structures. Endogenous fluorochromes are a known component of tardigrade cuticle, claws and bucco-pharyngeal apparatus. By imaging the autofluorescence from tardigrades, it is possible to document these structures in detail, including the subdivisions and boundaries of echiniscid (heterotardigrade) plates and the nature and spatial relationships of the texture (pores, granules, papillae and tubercles) on the various plates. This allows the determination of taxonomic features not easily seen with other microscopic techniques.

Trends in the distribution of breast cancer over time in the southeast of Scotland and review of the literature.

INTRODUCTION: Breast cancer is the most common form of malignancy in Scottish women, and its incidence appears to be increasing with time. It is therefore important to identify factors associated with risk and outcome. Whilst breast cancer occurs equally in the right and left breasts, tumours most commonly affect the upper outer quadrant (UOQ) of the breast. However, there is only limited information as to whether the incidence has changed over time. MATERIALS AND PATIENTS: We investigated two cohorts of women diagnosed with breast cancer in the south-east of Scotland between either 1957-1959 or 1997-1999 (i.e., 40 years apart). The earlier cohorts represent 1158 of 1207 women referred to radiation oncologists in the region and the latter group comprised 1477 of about 1600 women referred to the Edinburgh Breast Unit. RESULTS: Whilst the mean age, menopausal status, and laterality of the patients were similar in both groups, the tumour size and tumour location within the breast were significantly different in the two groups. Thus, there was significant reduction in T stage with year of diagnosis (p < 0.0001), the incidence of T1, T2, and T3/4 being 15.6%, 51.9%, and 25.6% in the earlier cohort compared with 49.3%, 36.8%, and 13.7% in the later cohort. The overall distribution within the breast was significantly different by chi-squared analysis ( p < 0.0001). In terms of individual quadrants 469 of 1158 (40.5%) tumours were located in the UOQ, whereas in the more recent cohort it was 788 of 1477 (53.4%), this increase in proportion being statistically significant ( p < 0.0001). Occurrence in the lower outer quadrant also significantly increased (p < 0.028) but was significantly reduced in the upper inner quadrant and centrally (both p < 0.0001). CONCLUSION: Analysing data on location for each T stage separately showed that the increased incidence in the UOQ with time was apparent for each subgroup. The increased incidence in UOQ tumours over time is therefore not a simple reflection of decreased size between the two time groups. The underlying reason(s) for this change in distribution with time requires further study.

Sequential changes in gene expression profiles in breast cancers during treatment with the aromatase inhibitor, letrozole.

The study aim was to identify early (within 14 days) and late changes (by 3 months) in breast cancer gene expression profiles associated with neoadjuvant therapy with letrozole. RNA from sequential tumour biopsies in 54 patients was analyzed on microarrays; changes were determined by frequency, magnitude and significance analyses. Substantially more genes were changed at 3 months (1503) than at 14 days (237). Early changed genes were associated with cell cycle (downregulation), blood vessel development and extracellular matrix (upregulation); late changes included 'cellular metabolic process', 'generation of precursor metabolites and energy' (decreased) and 'cell adhesion' 'biological adhesion' (increased). A striking difference between the early and late changes was the general location of downregulated genes-nuclear structures at 14 days and mitochondria after 3 months. These changes in gene expression profiles provide a new and important database by which to understand molecular mechanisms of letrozole in breast cancers.

Molecular endocrinology and breast cancer--a reed before the wind lives on (a tribute to Professor Mike Reed).

Oestrogens in breast cancers are derived from both uptake from the circulation and in situ synthesis. Third generation aromatase inhibitors (AIs) effectively block aromatase activity within the breast. The effects of AIs on the molecular biology of breast cancers may be monitored in patients given neoadjuvant therapy. Changes in tumour gene expression associated with AIs is influenced by time of drug exposure and gene expression profiles may provide important information on tumour response/ resistance to AIs.

Clinical, pathological, proliferative and molecular responses associated with neoadjuvant aromatase inhibitor treatment in breast cancer.

Neoadjuvant treatment provides an exceptional setting in which to monitor clinical, pathological, proliferative and molecular responses to aromatase inhibitors. Sequential measurements of the primary tumour provide an accurate assessment of clinical changes and the relatively easy access to the tumour within the breast means that biopsies are available for histological and molecular measurements before and during treatment. Large randomised trials (P024 and IMPACT) together with informative non-randomised studies have demonstrated clinical responses to third generation aromatase inhibitors in 40-70% of ER-positive tumours, rates generally significantly higher than observed with tamoxifen. Pathological responses in terms of reduced cellularity/increased fibrosis are also seen in 65-75% of cases. Whilst these are more often seen in clinically responding tumours, the correlation between clinical and pathological response is not absolute. A marked feature of treatment with third generation inhibitors is a reduction in cellular proliferation. Using Ki67 as a marker, this may be observed as early as 10-14 days into treatment. Reduction in proliferation with treatment may be seen in both clinically responding and non-responding tumours, although incidence and degree of effect are higher in responding cases. Aromatase inhibitor treatment frequently fails to reduce proliferation in tumours over-expressing HER-2. In terms of molecular events, aromatase inhibitor treatment is associated with changes in expression of genes classically associated with oestrogen regulation (KIAA0101, ZWINT, IRS1 and TFF1) and cell cycle progression, most notably mitotic phase proteins (CDC2, CCNB1 and CKS2). Changes occur both in clinically responding and non-responding tumours. Although expression of no individual gene correlates absolutely with response status, expression signatures can be produced which distinguish between responding and non-responding tumours. In terms of gene ontology, terms relating to macro-molecular biosynthesis, translation and structural components of ribosomes are significantly enriched. Finally, molecular signatures can be used to illustrate the relative homogeneity of responding tumours and the disparate nature of non-responding tumours suggesting multiple and diverse pathways associated with resistance.

Nuclear receptor co-activators and HER-2/neu are upregulated in breast cancer patients during neo-adjuvant treatment with aromatase inhibitors.

BACKGROUND: Acquired resistance to endocrine therapy in breast cancer is poorly understood. Characterisation of the molecular response to aromatase inhibitors in breast cancer tissue may provide important information regarding development of oestrogen hypersensitivity. METHODS: We examined the expression levels of nuclear receptor co-regulators, the orphan nuclear receptor liver receptor homologue-1 and HER-2/neu growth factor receptor using real-time RT-PCR before and after 13-16 weeks of primary medical treatment with the aromatase inhibitors anastrozole or letrozole. RESULTS: mRNA expression of the steroid receptor co-activator 1 (SRC-1) and peroxisome-proliferator-activated receptor gamma co-activator-1alpha (PGC-1alpha) was correlated (P=0.002), and both co-activators increased during treatment in the patient group as a whole (P=0.008 and P=0.032, respectively), as well as in the subgroup of patients achieving an objective treatment response (P=0.002 and P=0.006). Although we recorded no significant change in SRC-3/amplified in breast cancer 1 level, the expression correlated positively to the change of SRC-1 (P=0.002). Notably, we recorded an increase in HER-2/neu levels during therapy in the total patient group (18 out of 26; P=0.016), but in particular among responders (15 out of 21; P=0.008). CONCLUSION: Our results show an upregulation of co-activator mRNA and HER-2/neu during treatment with aromatase inhibitors. These mechanisms may represent an early adaption of the breast cancer cells to oestrogen deprivation in vivo.

Aromatase and the breast: regulation and clinical aspects.

The aromatase enzyme is unique to the pathway of oestrogen biosynthesis and converts androgen precursors into oestrogens, major stimulatory factors for breast cancer proliferation. Although there is only a single gene for aromatase and a single protein for the enzyme, transcriptional control is complex using different promoters which are in part tissue-specific. These generate different mRNA transcripts that vary in the presence/absence of individual untranslated exon 1s. In breast cancers, species vary between individual tumours, types I.3 and I.4 being the major species in some tumours but type II predominates in the majority. Since the type II promoter is regulated by prostaglandins/cyclic AMP, agents signalling through these systems seem largely responsible for local regulation of intratumoural oestrogen biosynthesis. Autocrine production of these factors would account for the high activity in breast cancers and paracrine secretion for the raised activity in breast fat associated with the local presence of cancer. Given the central role of oestrogen in normal development and pathological processes, there has been great interest in controlling aromatase activity by the use of specific inhibitors. Clinically, this is particularly evident in the management of postmenopausal women with breast cancer.

Oestrogen receptor beta and neoadjuvant therapy with tamoxifen: prediction of response and effects of treatment.

In order to elucidate the relative importance of oestrogen receptor (ER)alpha, ERbeta and an ERbeta variant (ERbeta2/betacx) in the response of breast cancers to tamoxifen, tumour levels of each receptor were assessed in 36 patients before and after 3 months of neoadjuvant treatment with tamoxifen (20 mg daily). All patients were postmenopausal women presenting with large ERalpha-positive breast cancers. Clinical response to treatment was assessed by tumour volume changes as determined from sequential ultrasounds and pathological response by comparison of the tumour morphology before and after treatment. Of 33 cases, 23 (70%) were classified as having a clinical response and 16 (48%) as having a response pathologically. All tumours stained positively for ERalpha and ERbeta and 15 out of 33 (45%) for ERbeta2/betacx. There were no significant differences in quantitative expression of any receptor between tumours that subsequently responded and that did not, whether response was assessed clinically or pathologically. Tamoxifen treatment was associated with a decrease in ERalpha, but an increase was the most frequent change (17 out of 33) in ERbeta, and no consistent change was evident in staining of the ERbeta2/betacx variant. In summary, ERbeta1 and ERbeta2/betacx variant protein are detected in ERalpha-positive breast tumours but their expression is not associated with a response to tamoxifen. Differential changes in ERalpha and ERbeta were seen with treatment.

Proliferation, steroid receptors and clinical/pathological response in breast cancer treated with letrozole.

Sixty-three postmenopausal women with large primary breast cancers were treated with neoadjuvant letrozole (2.5 mg daily) for 3 months. Tumour samples were taken at diagnosis and after 10-14 days and 3 months treatment. Immunohistochemical staining for Ki67, oestrogen receptor (ER) and progesterone receptor (PgR) was performed and related to clinical (ClinR) and pathological responses (PathR) after 3 months treatment. ClinR was observed in 48 of 63 cases (76.2%) and PathR in 47 of 62 (75.8%). Pretreatment Ki67 scores were similar in responders (R) and non-responders (NR). Highly significant Ki67 decreases occurred in all tumour subgroups at 10-14 days (P<0.005). A significant difference in Ki67 scores at 10-14 days (P<0.007) was found between PathR and PathNR but not between ClinR and ClinNR. At 3 months, decreases from pretreatment Ki67 scores were highly significant in all tumour subgroups irrespective of response status. However, whereas Ki67 scores were significantly different between pathological R and NR (P = 0.009), the corresponding comparison of ClinR status was not. Significant decreases between 10-14 days and 3 months were found only in ClinR and PathR (P = 0.02 and 0.045, respectively). Treatment significantly reduced PgR expression at 14 days and 3 months (both P<0.0001), but the level of changes was not different between response status groups. In summary, letrozole produces rapid and profound decreases in expression of Ki67 and PgR but changes do not always correlate with clinical and pathological responses.

Aromatase inhibitors and breast cancer.

Oestrogens are involved in risk to, and progression of, breast cancer. Drugs that inhibit the production of oestrogens (aromatase inhibitors, AIs), are therefore extremely attractive strategies both to prevent the disease and to treat established tumours. AIs now have a central role in the treatment of established breast cancer and are being considered for prevention. Third generation agents have been derived from rational drug design. They are able to block oestrogen production and reduce oestrogen levels to a degree that has not been observed previously and without affecting levels of other hormones. Such selective reduction of endogenous oestrogen provides targeted therapy for hormone-dependent breast cancer. This has led to improved clinical benefits in patients with these tumours. Anastrozole, letrozole and exemestane all have impressive antitumour effects in postmenopausal women with breast cancer and they are at least as beneficial as or better than other established endocrine agents when used to treat hormone-sensitive cancer in the advanced setting or as an adjuvant to surgery in earlier stages of the disease; ongoing trials are exploring the use of AIs in the preventative setting. Third generation inhibitors are well tolerated, having no greater side effects than might be expected from oestrogen suppression. Important differences in endocrinological and molecular effects exist between AIs and SERMs. These have implications for the preferred drug sequence and setting in which AIs are used. Since the major obstacle to more widespread use is primary/acquired resistance, discovery of the mechanisms by which resistance occurs offers hope for the future. More detailed study of AIs will yield important information about the involvement of oestrogen on the development and progression of breast cancer. Consequently AIs offer major clinical benefits to patients with breast cancer and the promise of relatively nontoxic intervention in women at high risk of the disease.

Consensus statement. Workshop on therapeutic resistance in breast cancer: impact of growth factor signalling pathways and implications for future treatment.

Anti-hormones (notably tamoxifen), chemotherapy and modern radiotherapeutic approaches are invaluable in the management of breast cancer, and collectively have contributed substantially to the improved survival in this disease. Moreover, there is promise that these successes will continue with the emergence of other endocrine agents (for example, aromatase inhibitors and pure anti-oestrogens). However, de novo and acquired resistance comprises a significant problem with all treatment approaches examined to date. This Workshop aimed to evaluate the contribution made by growth factor signalling pathways in the various resistant states, primarily focusing on resistance to anti-hormonal strategies and spanning experimental models and, where possible, clinical breast cancer data. The successes and limitations of therapeutic targeting of these pathways with various signal transduction inhibitors (STIs) were evaluated in model systems and from emerging clinical trials (including epidermal growth factor receptor inhibitors such as gefitinib). It was concluded that growth factor signalling is an important contributor in the development of endocrine resistance in breast cancer and that use of STIs provides a promising therapeutic strategy for this disease. However, the cancer cell is clearly able to harness alternative growth factor signalling pathways for growth and cell survival in the presence of STI monotherapy and, as a consequence, the efficacy of STIs is likely to be limited by the acquisition of resistance. A number of strategies were proposed from studies in model systems that appeared to enhance anti-tumour actions of existing STI monotherapy, notably including combination therapies targeting multiple pathways. With the increased availability of diverse STIs and improved drug delivery, there is much hope that the more complex therapeutic strategies proposed may ultimately be achievable in clinical practice.

Growth factor signalling in clinical breast cancer and its impact on response to conventional therapies: the Edinburgh experience.

Neoadjuvant endocrine treatment in which therapy is given while the primary tumour is still in the breast provides a highly useful model system by which to identify mechanisms associated with de novo resistance and signs of early acquired resistance. Most importantly, the model is clinically relevant. It has been confirmed that the absence of tumour oestrogen receptors confers resistance to endocrine therapy. Early changes in tumour cell proliferation following neoadjuvant treatment with the third-generation aromatase inhibitor, letrozole, do not predict accurately for subsequent clinical response. Additionally, changes in proliferation seen at later times can be the consequence of response and may be associated with early resistance. High expression of c-erbB2 does not reduce tumour responses to neoadjuvant treatment with aromatase inhibitors, but is associated with high tumour proliferation before and during treatment. It remains to be determined whether these characteristics confer subsequent resistance to treatment and early relapse in the adjuvant setting.

Role of aromatase inhibitors in breast cancer.

Primarily, the role of the aromatase inhibitors has been investigated in postmenopausal women with breast cancer, although it is also now being assessed in premenopausal patients following ovarian ablation/suppression. Aromatase inhibitors markedly suppress endogenous oestrogens without directly interacting with oestrogen receptors, and thus have a different mechanism of action to the antioestrogen, tamoxifen. The inhibitors may be divided into subgroups according to their structure (steroidal and nonsteroidal), and there appears to be a lack of cross-resistance between the classes of aromatase inhibitors enabling them to be used sequentially and potentially to prolong endocrine hormone therapy. In addition, with increased efficacy and favourable safety and tolerability profiles, the aromatase inhibitors are starting to challenge tamoxifen as first choice endocrine treatment in a number of settings. Potential differences in side-effect profiles may appear between the steroidal and nonsteroidal aromatase inhibitors when used in long-term settings. Thus, it has been suggested that the steroidal agents have favourable end organ effects; for example, the steroidal inhibitor, exemestane, has minimal negative effects on bone and lipid metabolism in animal and clinical studies. This paper provides an overview of the current and future roles of aromatase inhibitors for breast cancer treatment.

The validation of new aromatase monoclonal antibodies for immunohistochemistry--a correlation with biochemical activities in 46 cases of breast cancer.

Intratumoral aromatase is a therapeutic target for the treatment of post-menopausal estrogen-dependent breast cancers. Therefore, reliable methods should be developed for routine application for the detection of intratumoral aromatase. Immunohistochemistry (IHC) is considered one of the most suitable methods in this regard. A multi-centre collaborative group has been established to generate and validate new aromatase monoclonal antibodies. We have selected two monoclonal antibodies, #677 against native aromatase protein and F2 against formalin-fixed protein for this purpose. With these two monoclonal antibodies 43 cases of invasive ductal carcinoma, which had been previously assayed for aromatase activity by product isolation methodology, were immunostained in three laboratories in UK, USA and Japan and independently evaluated by three pathologists (H.S., T.A. and S.G.S.). Staining of malignant epithelium, adipose tissue, normal/benign and stromal compartments of the tumors were assessed by estimating the proportion of positive staining cells and the relative intensity of staining in this fashion. Immunoreactivity could be detected in each component of the tissue specimens but a significant positive correlation with biochemical activity was detected only in malignant epithelium stained with 677 not in other components with #677 and not in any of the components. Staining using F2 as a primary antibody did not produce a positive correlation in any components with aromatase activity. These results suggest that we now have a monoclonal antibody against aromatase (#677) which may be used to stain archival materials. A methodology and scoring system is recommended whereby staining significantly correlates with aromatase activity of the resected tissue specimens of breast cancer.

Aromatase expression in ovarian epithelial cancers.

Our study focused on aromatase cytochrome P450 (CYP19) expression in ovarian epithelial normal and cancer cells and tissues. Aromatase mRNA expression was analyzed by real-time PCR in ovarian epithelial cancer cell lines, in human ovarian surface epithelial (HOSE) cell primary cultures, and in ovarian tissue specimens (n=94), including normal ovaries, ovarian cysts and cancers. Aromatase mRNA was found to be expressed in HOSE cells, in BG1, PEO4 and PEO14, but not in SKOV3 and NIH:OVCAR-3 ovarian cancer cell lines. Correlation analysis of aromatase expression was performed according to clinical, histological and biological parameters. Aromatase expression in ovarian tissue specimens was higher in normal ovaries and cysts than in cancers (P<0.0001). Using laser capture microdissection in normal postmenopausal ovaries, aromatase was found to be predominantly expressed in epithelial cells as compared to stromal component. Using immunohistochemistry (IHC), aromatase was also detected in the epithelium component. There was an inverse correlation between aromatase and ERalpha expression in ovarian tissues (P<0.001, r=-0.34). In the cancer group, no significant differences in aromatase expression were observed according to tumor histotype, grade, stage and survival. Aromatase activity was evaluated in ovarian epithelial cancer (OEC) cell lines by the tritiated water assay and the effects of third-generation aromatase inhibitors (AIs) on aromatase activity and growth were studied. Letrozole and exemestane were able to completely inhibit aromatase activity in BG1 and PEO14 cell lines. Interestingly, both AI showed an antiproliferative effect on the estrogen responsive BG1 cell line co-expressing aromatase and ERalpha. Aromatase expression was found in ovarian epithelial normal tissues and in some ovarian epithelial cancer cells and tissues. This finding raises the possibility that some tumors may respond to estrogen and provides a basis for ascertaining an antimitogenic effect of AI in a subgroup of ovarian epithelial cancers.

Anastrozole demonstrates clinical and biological effectiveness in oestrogen receptor-positive breast cancers, irrespective of the erbB2 status.

Overexpression of erbB2 in breast tumours can predict resistance to tamoxifen therapy. We conducted a small trial to determine if erbB2 status correlates with tumour response and biochemical changes in postmenopausal women receiving neoadjuvant therapy with the aromatase inhibitor, anastrozole. Twenty-four postmenopausal women with oestrogen receptor (ER)-rich, large, operable breast tumours received three months of neoadjuvant anastrozole, 1 or 10 mg daily, then surgery, followed by another five years of anastrozole 1 mg daily. Response to the treatment was based on changes in clinical and ultrasound measurements of tumour volume and changes in tumour proliferation and progesterone receptor (PgR) status. After follow-up for a median duration of four years therapy, there was no apparent difference between erbB2 0/1+ and erbB2 3+ tumours in clinical response or changes in proliferation and PgR expression. In conclusion, anastrozole appears to be an effective endocrine option in this patient population, irrespective of the erbB2 status.

Concentrations of parabens in human breast tumours.

Parabens are used as preservatives in many thousands of cosmetic, food and pharmaceutical products to which the human population is exposed. Although recent reports of the oestrogenic properties of parabens have challenged current concepts of their toxicity in these consumer products, the question remains as to whether any of the parabens can accumulate intact in the body from the long-term, low-dose levels to which humans are exposed. Initial studies reported here show that parabens can be extracted from human breast tissue and detected by thin-layer chromatography. More detailed studies enabled identification and measurement of mean concentrations of individual parabens in samples of 20 human breast tumours by high-pressure liquid chromatography followed by tandem mass spectrometry. The mean concentration of parabens in these 20 human breast tumours was found to be 20.6 +/- 4.2 ng x g(-1) tissue. Comparison of individual parabens showed that methylparaben was present at the highest level (with a mean value of 12.8 +/- 2.2 ng x g(-1) tissue) and represents 62% of the total paraben recovered in the extractions. These studies demonstrate that parabens can be found intact in the human breast and this should open the way technically for more detailed information to be obtained on body burdens of parabens and in particular whether body burdens are different in cancer from those in normal tissues.

Biological rationale for endocrine therapy in breast cancer.

Oestrogens are heavily implicated in the risk to, and progression of, breast cancer. Therapeutic strategies targeted at the oestrogenic stimulus to the breast and hormone-sensitive breast cancers are extremely attractive measures both to prevent the disease and to treat established tumours. The present review outlines the biological rationale for such endocrine therapy and traces the evolution whereby irreversible surgical procedures have been replaced by potent and specific drugs. In particular, the development of the latest generation of agents which inhibit oestrogen biosynthesis (aromatase inhibitors) is considered by defining the central role of the aromatase enzyme, its regulation and contribution to circulating and tumour endogenous oestrogens. The nature of response and resistance which may be elicited following the use of endocrine therapy is also described as this may determine the optimal use of aromatase inhibitors and, more generally, anti-hormone therapy in the management of women at high risk to, or with, breast cancer.

The pharmacology of letrozole.

Recent clinical trials indicate that the third-generation aromatase inhibitors may be more effective than tamoxifen as first line endocrine therapy in ER+ metastatic breast cancer in postmenopausal women. This review will focus exclusively on the pharmacology of the non-steroidal inhibitor letrozole. Aromatase derived from a variety of sources is inhibited at low nM concentrations of the drug. In non-cellular systems, letrozole is 2-5 times more potent than anastrozole and exemestane in its inhibition of aromatase, whilst in cellular systems it is 10-20 times more potent. Anti-tumour effects of letrozole have been demonstrated in several animal models. In postmenopausal women, letrozole commonly suppresses circulating concentrations of estrone and estradiol to below the sensitivity limit of the assays used to measure them. In a recent randomized cross-over study, letrozole (2.5mg daily) achieved a significantly greater suppression of the plasma concentrations of both estrone and estrone sulphate than anastrozole (1mg daily) and a greater inhibition of in vivo aromatization. Letrozole appears to have a small effect on adrenal steroidogenesis such that a small number of patients exhibit an abnormal response to synthetic ACTH during letrozole therapy. This is unlikely to have any clinical significance. In short-term studies letrozole has been shown to increase markers of bone resorption indicating the need to monitor bone integrity when the drug is used for extended periods of time. A consistent effect of letrozole on serum lipids has not been demonstrated.