Evaluation of long-term outcome following therapeutic mammaplasty: the effect of wound complication on initiation of adjuvant therapy and subsequent oncological outcome.

INTRODUCTION: Therapeutic mammaplasty (TM) facilitates large tumour resection while maintaining optimal aesthetic outcome. It carries higher wound complication risks, which may delay adjuvant therapy initiation. Whether this delay affects oncological outcome requires evaluation. METHODS: Data were collected for consecutive patients receiving TM at the Leeds breast unit (2009-2017). A prospectively maintained database was used to determine tumour characteristics, wound complication rates, receipt of adjuvant therapy and breast cancer recurrence or death. RESULTS: In total 112 patients (median age of 54 years) underwent 114 TM procedures. The most common histological subtypes were invasive ductal carcinoma (61.4%), invasive lobular carcinoma (13.2%) and ductal carcinoma in situ (13.2%). Of the patients, 88.2% had oestrogen receptor-positive cancer and 14% had human epidermal growth factor receptor-positive cancer; 26.3% had multifocal cancer. The median tumour size was 30mm. The median Nottingham Prognostic Index was 4.2. The local recurrence rate was 3.5% (median follow-up of 8.6 years). The 5- and 10-year disease-free survival (DFS) was 88.5% and 83.5%, and the equivalent overall survival (OS) rates were 94% and 83.5%. Wound complication rate was 23.6% (n=27), the commonest being wound infection (11.4%; n=13) and T-junction wound breakdown (10.5%; n=12). The median time to adjuvant therapy was 72 days (interquartile range [IQR] 56-90) for patients with wound complications, and 51 days (IQR 42-58) for those without. However, this delay did not affect DFS or OS (log-rank test; p=0.58 and p=0.94, respectively). This was confirmed on Cox regression analysis. CONCLUSION: Our study finding demonstrates that although wound complications after TM leads to a modest delay to adjuvant therapy, the long-term oncological outcomes were comparable with those in patients without wound complications.

Multi-layer graphene as a selective detector for future lung cancer biosensing platforms.

Highly selective, fast detection of specific lung-cancer biomarkers (CMs) in exhaled human breath is vital to the development of enhanced sensing devices. Today, e-nose is a promising approach for the diagnosis of lung cancer. Nevertheless, considerable challenges to early-stage disease diagnostics still remain: e.g. decrease in sensor sensitivities in the presence of water vapor, sensor drift leading to the inability to calibrate exactly, relatively short sensor lifetimes, and difficulty discriminating between multiple diseases. However, there is a wide scope for breath diagnostics techniques, and all advanced electrodes applicable to e-nose devices will benefit them. Here, we present the promising sensing capabilities of bare multi-layer graphene (MLG) as a proof of concept for advanced e-nose devices and demonstrate its utility for biomolecule discrimination of the most common lung CMs (ethanol, isopropanol, and acetone). We report on a comparative study involving exposure of the three CM solutions on flat MLG (f-MLG) and patterned MLG (p-MLG) electrodes, where the electrical conductivity of p-MLG is significantly increased while applying acetone. Based on sensitivity tests, we demonstrate the ability to monitor the electrical response of graphene electrodes employing graphene of various wettabilities. Specifically, the f-MLG electrode displays almost 2 times higher sheet resistance (30 Ω sq-1) compared to the hydrophilic p-MLG (12 Ω sq-1). We show significant sensitivity to selected specific molecules of pristine f-MLG and p-MLG while applying CM solutions with a 1.4 × 105 ppm concentration. Finally, we show the selectivity of f-MLG and p-MLG-based sensors when exposed to 2.0 × 105 ppm solutions containing different CM combinations. Both sensors were selective in particular to acetone, since the presence of acetone leads to a sheet resistance increase. We demonstrate that an advanced e-nose approach integrated with MLG electrodes has significant potential as a design concept for utilization of molecular detection at variable concentrations such as in early-stage disease diagnosis. This early-stage approach will provide convenient and reusable complex monitoring of CMs compared to typical contact sensors which require target analysis and are limited by disposable measuring. Moreover, further integration of the Internet of Things will introduce advanced e-nose devices as a biotechnological innovation for disease resilience with the potential for commercialization.

Epithelial stem/progenitor cells in lung postnatal growth, maintenance, and repair.

The adult lung consists of a trachea leading into a system of branched airways ending in millions of alveolar sacs. It contains many different epithelial cell types arranged in precise patterns along the proximodistal axis. Each region of the lung has the capacity to repair through the proliferation of different epithelial cell types. However, the precise identity of the cells mediating repair is not fully resolved. To address this problem, we are using genetic lineage-labeling techniques in the mouse. The tools we have made will also be useful for understanding how progenitor cell behavior is regulated under normal and pathological conditions.

Summary: present and future challenges for stem cell research.

Stem cell research is being driven forward at an intense pace by creative interactions among scientists working in different fields. These include developmental and reproductive biology, regeneration, genomics, live cell imaging, RNA biology, and cancer biology, to name a few. Numerous model systems and techniques are being exploited, and lab scientists are teaming up with bioengineers and clinicians. The ferment of ideas that makes the field so exciting was in full evidence throughout the Symposium. However, many challenges still need to be overcome to translate basic discoveries into therapeutic outcomes that will save lives and fulfill the promises that have been made. This chapter summarizes some of the highlights of the Symposium and indicates future directions that are being taken by leaders in the field.

A microscale in vitro physiological model of the liver: predictive screens for drug metabolism and enzyme induction.

In vitro models of the liver using isolated primary hepatocytes have been used as screens for measuring the metabolism, toxicity and efficacy of xenobiotics, for studying hepatocyte proliferation, and as bioartificial liver support systems. Yet, primary isolated hepatocytes rapidly lose liver specific functions when maintained under standard in vitro cell culture conditions. Many modifications to conventional culture methods have been developed to foster retention of hepatocyte function. Still, not all of the important functions -- especially the biotransformation functions of the liver -- can as yet be replicated at desired levels, prompting continued development of new culture systems. In the first part of this article, we review primary hepatocyte in vitro systems used in metabolism and enzyme induction studies. We then describe a scalable microreactor system that fosters development of 3D-perfused micro-tissue units and show that primary rat cells cultured in this system are substantially closer to native liver compared to cells cultured by other in vitro methods, as assessed by a broad spectrum of gene expression, protein expression and biochemical activity metrics. These results provide a foundation for extension of this culture model to other applications in drug discovery -- as a model to study drug-drug interactions, as a model for the assessment of acute and chronic liver toxicity arising from exposure to drugs or environmental agents; and as a disease model for the study of viral hepatitis infection and cancer metastasis.

Auditing a new lung cancer service.

The Adelaide and Meath Hospital (AMNCH) is a new hospital, with the respiratory service established in 1999. We report on our experience with lung cancer in this short time. We conducted a retrospective chart audit, identifying patients from the Oncology Nurse patient list, of lung cancer cases diagnosed in 2001-2002. In 2001, 47 cases were diagnosed, with 71 cases diagnosed in 2002. Time to tissue diagnosis was 14.8, 2-46 (mean, range, one subject skewing the data) days. Diagnosis by tissue type was as follows: non-small cell carcinoma (NSCLC) n=92 (77.9%), small cell lung carcinoma (SCLC) n=17 (14.4%), mesothelioma n=2 (17%), carcinoid n=1 (0.8%) and no tissue diagnosis n=6 (5.1%). Staging of NSCLC: stage 1a n=3 (3.2%), stage 1b n=6 (6.5%), stage 2a n=5 (5.4%), stage 2b n=2 (2.2%), stage 3a n=10 (10.9%), stage 3b n=22 (23.9%) and stage 4 n=44 (47.8%). Staging of SCLC: limited stage n=5 (29.4%), extensive stage n=12 (70.6%). Positive emission tomography (PET) was employed in the staging of 22 patients. In these patients PET up-staged the disease in 8 patients, and management was unchanged in 8 patients. We conclude that 1. the relatively new respiratory service in AMNCH is handling increasing numbers of lung cancer cases, 2. cases are diagnosed within an acceptable time-frame, 3. rate of tissue diagnosis is comparable to national figures, 4. most patients present with advanced disease and 5. PET is a useful imaging modality for detection of cancer spread in selected patients.

Hepatic metastases from an unknown primary neoplasm (UPN): survival, prognostic indicators and value of extensive investigations.

AIM: The objectives of this study were to identify prognostic features for patients with hepatic metastases and unknown primary neoplasms (UPN), determine the common primary tumours, assess the value of diagnostic tests in finding these tumours, and evaluate the impact of therapy and knowledge of the primary tumour on patient survival. MATERIALS AND METHODS: Eighty-eight patients with UPN and liver biopsy proven hepatic metastases over a 10-year period were reviewed (M:F, 58:30; age range 27-91 years, median 64.5 years). Histopathology, diagnostic investigations and success at identifying the primary neoplasm were recorded. In addition, in 70 patients with adenocarcinoma histology (M:F, 48:22; age range 27-91 years, median 65 years), treatment and survival data from the date of biopsy were recorded. RESULTS: The histological spectrum included adenocarcinoma in 70, neuroendocrine in four, squamous cell carcinoma in four, small cell carcinoma in four, carcinoid in two, hepatoma in one and three others. Extensive investigation identified a primary neoplasm in 16/88 patients (18%) including colorectal in six, gastric in two, lung in four, oesophageal in two, prostate in one and carcinoid in one. In the adenocarcinoma group survival data were available for 62/70 patients. Sixteen of 62 patients received active treatment with either surgery, chemotherapy, radiotherapy or a combination protocol. Forty-six of 62 patients received palliative care alone. Median survival for the adenocarcinoma group overall was 49 days. The median survival for treated patients (49 days) versus untreated patients (52 days) was not significantly different (P=0.128). Patients <65 years were more likely to receive active treatment than those >65 years (P=0.006). Age with a hazard ratio (HR) of 1.01 (P=0.178), active treatment (HR=0.65;P=0.194), knowledge of the primary neoplasm (HR=0.60;P=0.213) and male gender (HR=0.88;P=0.642) had no significant effect on survival. CONCLUSION: Although hepatic metastases are associated with poor prognosis, it is essential that a liver biopsy be performed to obtain a histological diagnosis. Adenocarcinoma metastases carry a dismal prognosis, and no prognostic factors, including knowledge of the primary tumour, are significant for patient survival. Extensive investigation is not warranted in patients with adenocarcinoma liver metastases.

Immune function, telomerase, and angiogenesis in patients with primary, operable nonsmall cell lung carcinoma: tumor size and lymph node status remain the most important prognostic features.

BACKGROUND: Lung carcinoma usually is advanced at the time of presentation and frequently shows metastatic spread. In recent times, prognostic factors such as c-erbB-2 in patients with breast carcinoma have provided useful information and beneficial therapeutic targets. The objective of this study was to evaluate angiogenesis, immune function, and telomerase expression in patients with nonsmall cell lung carcinoma (NSCLC) to determine their prognostic significance. METHODS: Immunohistochemistry was used to evaluate the expression of human telomerase reverse transcriptase (hTERT; n = 115 patients), interleukin-2r (IL-2r; n = 40 patients), microvessel density (MVD; n = 81 patients), and vascular endothelial growth factor (VEGF; n = 61 patients). Three-year survival follow-up information was available for most patients, and a comprehensive review of clinicopathologic features was carried out. RESULTS: Fifty percent of tumors showed nuclear staining for hTERT, 55% of tumors showed some degree of lymphocyte IL-2r expression, 33% of tumors were recorded with an MVD that was higher than average, and VEGF staining was detected in 85% of tumors. None of the parameters measured had an impact on survival. hTERT expression was correlated with lymph node status. Lymph node status and tumor size were identified as independent prognostic factors. CONCLUSIONS: This study failed to identify a marker of prognosis for patients with NSCLC other than tumor size and lymph node status in this population. Telomerase expression was associated with metastases, raising the possibility that this enzyme is involved in the metastatic process. Tumor cell VEGF expression was identified frequently: This growth factor may have potential as a target for antiangiogenic therapy. Lung carcinoma typically is the result of large numbers of mutations. Further understanding of the biologic implications of these mutations will lead to the development of effective prognostic markers and treatments for patients with NSCLC.

Cellular and molecular responses of the uterus to embryo implantation can be elicited by locally applied growth factors.

The implantation of a blastocyst into a receptive uterus is associated with a series of events, namely the attachment reaction followed by decidualization of the stroma. Previous studies established that the gene encoding heparin-binding EGF-like growth factor (HB-EGF) is expressed in the luminal epithelium solely at the site of blastocyst apposition preceding the attachment reaction. We report here the expression during implantation of 21 genes encoding other signaling proteins, including those belonging to the Bone morphogenetic protein (BMP), fibroblast growth factor (FGF), WNT, and Hedgehog (HH) pathways. We find that the attachment reaction is associated with a localized stromal induction of genes encoding BMP-2, FGF-2, and WNT-4. Despite efforts by many investigators, a simple in vitro model of implantation is not yet available to study either the hierarchy of the events triggered in the uterus by the embryo or the function of individual signaling proteins. We have therefore approached these questions by introducing beads loaded with purified factors into the receptive uterus. We show that beads soaked in HB-EGF or insulin-like growth factor-1 (IGF-1), but not other proteins, induce many of the same discrete local responses elicited by the blastocyst, including increased localized vascular permeability, decidualization, and expression of Bmp2 at the sites of the beads. By contrast, the expression domains of Indian hedgehog (Ihh), patched, and noggin become restricted as decidualization proceeds. Significantly, beads containing BMP-2 do not themselves elicit an implantation response but affect the spacing of implantation sites induced by blastocysts cotransferred with the beads.

The ultrasound investigation of scrotal swelling.

The objectives of this study were to: (1) establish the causes of scrotal swelling in the hospital catchment population; (2) define the role of high frequency ultrasound examination in the management of scrotal swelling. A retrospective study of 582 patients who had high frequency ultrasound examination was carried out. Those requiring more information on perfusion had colour doppler examination. Forty-four per cent of examinations were performed for scrotal swelling. The cause of the scrotal swelling was mainly extratesticular (75% of all scrotal swellings), hydrocele being the commonest. Of the intratesticular causes, infection (50.8%) and tumour (20.6%) were the commonest. In conclusion ultrasound examination distinguishes extratesticular (almost always benign) from intratesticular (potentially malignant) causes of scrotal swelling. Infection, trauma and torsion mimic the ultrasound appearance of tumour as do rare benign entities.

Bone morphogenetic protein 4 regulates the budding site and elongation of the mouse ureter.

In the normal mouse embryo, Bmp4 is expressed in mesenchymal cells surrounding the Wolffian duct (WD) and ureter stalk, whereas bone morphogenetic protein (BMP) type I receptor genes are transcribed either ubiquitously (Alk3) or exclusively in the WD and ureter epithelium (Alk6). Bmp4 heterozygous null mutant mice display, with high penetrance, abnormalities that mimic human congenital anomalies of the kidney and urinary tract (CAKUT), including hypo/dysplastic kidneys, hydroureter, ectopic ureterovesical (UV) junction, and double collecting system. Analysis of mutant embryos suggests that the kidney hypo/dysplasia results from reduced branching of the ureter, whereas the ectopic UV junction and double collecting system are due to ectopic ureteral budding from the WD and accessory budding from the main ureter, respectively. In the cultured metanephros deprived of sulfated glycosaminoglycans (S-GAGs), BMP4-loaded beads partially rescue growth and elongation of the ureter. By contrast, when S-GAGs synthesis is not inhibited, BMP4 beads inhibit ureter branching and expression of Wnt 11, a target of glial cell-derived neurotrophic factor signaling. Thus, Bmp4 has 2 functions in the early morphogenesis of the kidney and urinary tract. One is to inhibit ectopic budding from the WD or the ureter stalk by antagonizing inductive signals from the metanephric mesenchyme to the illegitimate sites on the WD. The other is to promote the elongation of the branching ureter within the metanephros, thereby promoting kidney morphogenesis.

Bone morphogenetic protein-6 is a marker of serous acinar cell differentiation in normal and neoplastic human salivary gland.

Bone morphogenetic protein (BMP-6, also known as vegetal-pale-gene-related and decaplentaplegic-vegetal-related) is a member of the transforming growth factor-beta superfamily of multifunctional signaling molecules. BMP-6 appears to play various biological roles in developing tissues, including regulation of epithelial differentiation. To study the possible involvement of BMP-6 in normal and neoplastic human salivary glands, we compared its mRNA and protein expression in 4 fetal and 15 adult salivary glands and in 22 benign and 32 malignant salivary gland tumors. In situ hybridization and Northern blot analysis indicated that BMP-6 transcripts are expressed at low levels in acinar cells of adult submandibular glands but not in ductal or stromal cells. BMP-6 was immunolocated specifically in serous acini of parotid and submandibular glands. None was found in primitive fetal acini or any other types of cell in adult salivary glands, including mucous acini and epithelial cells of intercalated, striated, and excretory ducts. All 16 cases of acinic cell carcinoma consistently exhibited cytoplasmic BMP-6 staining in the acinar tumor cells. Other cell types in these tumors, including intercalated duct-like cells, clear, vacuolated cells, and nonspecific glandular cells, exhibited no cytoplasmic BMP-6 staining. Other benign and malignant salivary gland tumors lacked BMP-6 immunoreactivity, except in areas of squamous differentiation. The results indicate that in salivary glands, BMP-6 expression is uniquely associated with acinar cell differentiation and suggest that BMP-6 may play a role in salivary gland function. More importantly, our experience of differential diagnostic problems related to salivary gland tumors suggests that the demonstration of consistent and specific BMP-6 immunoreactivity in acinic cell carcinoma is likely to be of clinical value.

Attenuated host resistance against Mycobacterium bovis BCG infection in mice lacking osteopontin.

Expression of the cytokine osteopontin (OPN) is elevated in granulomas caused by Mycobacterium tuberculosis. We tested the hypothesis that OPN contributes to host protection in a mouse model of mycobacterial infection. When infected with Mycobacterium bovis BCG, mice lacking a functional OPN gene had more severe infections characterized by heavier bacterial loads and a delayed clearance of the bacteria. The OPN-null mice had greater granuloma burdens consistent with the elevated bacterial load. The ability of osteopontin to facilitate the clearance of mycobacteria was most pronounced early after infection and appeared to be independent of known mediators of resistance to infection by mycobacteria: antigen-specific T-cell immunity, gamma interferon production, and nitric oxide production. BCG grew more rapidly in macrophages derived from OPN-null mice than in those from wild-type mice, demonstrating that the null phenotype was due to an intrinsic macrophage defect. These results indicate that osteopontin augments the host response against a mycobacterial infection and that it acts independently from other antimycobacterial resistance mechanisms.

BMP4 is essential for lens induction in the mouse embryo.

Vertebrate lens development is a classical model system for studying embryonic tissue interactions. Little is known, however, about the molecules mediating such inductive events. Here, we show that Bmp4, which is expressed strongly in the optic vesicle and weakly in the surrounding mesenchyme and surface ectoderm, has crucial roles during lens induction. In Bmp4(tm1) homozygous null mutant embryos, lens induction is absent, but the process can be rescued by exogenous BMP4 protein applied into the optic vesicle in explant cultures. This is associated with rescue of ectodermal expression of Sox2, an early lens placode marker. Substituting the optic vesicle in explant cultures with BMP4-carrying beads, however, does not lead to lens induction, indicating that other factors produced by the optic vesicle are involved. BMP4 appears to regulate expression of a putative downstream gene, Msx2, in the optic vesicle. No change in Pax6 expression is seen in Bmp4(tm1) mutant eyes, and Bmp4 expression appears unaffected in the eyes of homozygous Pax6(Sey-1Neu), suggesting that PAX6 and BMP4 function independently. Based on these results we propose that BMP4 is required for the optic vesicle to manifest its lens-inducing activity, by regulating downstream genes and/or serving as one component of multiple inductive signals.

Formation of Rathke's pouch requires dual induction from the diencephalon.

Targeted disruption of the homeobox gene T/ebp (Nkx2.1, Ttf1, Titf1) in mice results in ablation of the pituitary. Paradoxically, while T/ebp is expressed in the ventral diencephalon during forebrain formation, it is not expressed in Rathke's pouch or in the pituitary gland at any time of embryogenesis. Examination of pituitary development in the T/ebp homozygous null mutant embryos revealed that a pouch rudiment is initially formed but is eliminated by programmed cell death before formation of a definitive pouch. In the diencephalon of the mutant, Bmp4 expression is maintained, whereas Fgf8 expression is not detectable. These data and additional genetic and molecular observations suggest that Rathke's pouch develops in a two-step process that requires at least two sequential inductive signals from the diencephalon. First, BMP4 is required for induction and formation of the pouch rudiment, a role confirmed by analysis of Bmp4 homozygous null mutant embryos. Second, FGF8 is necessary for activation of the key regulatory gene Lhx3 and subsequent development of the pouch rudiment into a definitive pouch. This study provides firm molecular genetic evidence that morphogenesis of the pituitary primordium is induced in vivo by signals from the adjacent diencephalon.

Mice deficient for the secreted glycoprotein SPARC/osteonectin/BM40 develop normally but show severe age-onset cataract formation and disruption of the lens.

SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin/BM40) is a secreted Ca2+-binding glycoprotein that interacts with a range of extracellular matrix molecules, including collagen IV. It is widely expressed during embryogenesis, and in vitro studies have suggested roles in the regulation of cell adhesion and proliferation, and in the modulation of cytokine activity. In order to analyse the function of this protein in vivo, the endogenous Sparc locus was disrupted by homologous recombination in murine embryonic stem cells. SPARC-deficient mice (Sparctm1Cam) appear normal and fertile until around 6 months of age, when they develop severe eye pathology characterized by cataract formation and rupture of the lens capsule. The first sign of lens pathology occurs in the equatorial bow region where vacuoles gradually form within differentiating epithelial cells and fibre cells. The lens capsule, however, shows no qualitative changes in the major basal lamina proteins laminin, collagen IV, perlecan or entactin. These mice are an excellent resource for further studies on how SPARC affects cell behaviour in vivo.