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Background and Objectives: General practitioners (GPs) play a central role in healthcare, serving as the first point of contact, making appropriate referrals and coordinating care for chronic conditions such as heart failure (HF). We sought to determine healthcare use by people with HF in primary care., Method: In this Study of Heart failure in the Australian Primary carE setting (SHAPE), we analysed records of 1.93 million adult patients who attended a total of 43 practices between 1 July 2013 and 30 June 2018. We identified and examined the data of 20,219 patients with HF to describe the frequency of visits and use of Medicare Benefits Schedule items., Results: Patients with HF saw GPs 14.4 times per annum on average; 59.5% had a General Practice Management Plan (GPMP), 2.9% of GPMPs were reviewed annually or more frequently, and 46.8% of patients had been referred to a cardiologist. A total of 3761 had coexisting anxiety or depression, and of these 37.1% had a mental health plan., Discussion: Patients with HF visit their GP frequently, with many not reaching guideline therapy nor referred to cardiologists. Low use of care planning and reviews presents an opportunity for GPs to improve care.

Aims: Heart failure (HF) causes significant morbidity and mortality, but the rates and characteristics of people with HF in Australia are not well studied. SHAPE set out to describe the characteristics of HF patients seen in the real-world setting., Methods: We analysed anonymized patient data extracted from the clinical software of 43 participating GP clinics for the 5 year period from 1 July 2013 to 30 June 2018. Patients were stratified into 'definite' and 'probable' HF based on a hierarchy of selection criteria and analysed for their clinical characteristics. Symptoms and signs of HF and ejection fraction data were searched for within the free text of the medical notes., Results: Of the 1.12 million adults seen regularly, 20 219 were classified as having definite or probable HF. The mean age of the population was 69.8 years, 50.6% were female, and mean body mass index was 31.2 kg/m 2 . Fewer than 1 in 6 had the HF diagnosis optimally recorded. Only 3.2% (650 patients) had their left ventricular ejection fraction (EF) quantified: 40.9% had an EF ≥50% and 59.1% had an EF <50%. The most common comorbidities in people with HF were hypertension (41.1%), chronic obstructive pulmonary disease/asthma (25.1%) and depression/anxiety (18.4%). Hypotension (2.3%), bradycardia (6.3%), severe renal impairment (6.4%) and hyperkalaemia (2.0%) were uncommon. Just over one-third (37.8%) had iron deficiency. Loop diuretic use was common (56.7%) but only 33.7% were on a guideline recommended beta-blockers. Use of ivabradine (1.4%) and sacubitril/valsartan (1.2%) was very low, while 39.9% had been prescribed an angiotensin-converting enzyme inhibitor, 31.6% an angiotensin receptor blocker and 16.0% spironolactone. Many patients were prescribed medications that may worsen HF or are relatively contraindicated, such as macrolide antibiotics (29.9%), corticosteroids (25.8%), nonsteroidal anti-inflammatory drugs (23.9%), and tricyclic antidepressants (9.4%)., Conclusions: Heart failure is poorly documented in general practice records and may be contributing to untoward downstream effects, such as low documentation of echocardiography, poor use of guideline recommended therapies and frequent use of medications that may worsen HF., (© 2021 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Aims: At present, there is no robust information on the prevalence and incidence of heart failure (HF) in the general Australian community. The present study of primary care data sought to estimate the prevalence and incidence of HF in the community and to describe the demographic and clinical profile of Australians with HF., Methods and Results: We undertook a retrospective cohort study based on analysis of anonymized medical records of adult patients cared for at 43 Australian general practices between 1 July 2013 and 30 June 2018. Data were extracted from coded and uncoded fields in electronic medical records. The prevalence and annual incidence of HF were calculated, along with 95% confidence intervals, using the 'active' population of people who were regular attenders at the practices. Age-standardized estimates were also derived using the 2017 Australian population as reference. The mean age of the population with HF was 69.8 years, 50.6% were female, and mean body mass index was 31.2 kg/m 2 . The age-standardized prevalence was 2.199% [95% confidence interval (CI): 2.168-2.23%], and the age-standardized annual incidence was 0.348% (95% CI: 0.342-0.354%). These estimates accord with almost 420 000 people living with HF in Australia in 2017, and >66 000 new cases of HF occurring that year. Only 18.9% of patients with definite HF had this formally captured as a 'diagnosis' in their medical record. HF was more frequent among those of lower socio-economic status., Conclusions: HF is common in Australia. The majority of HF patients do not have this diagnosis optimally noted in their primary care medical records., (© 2020 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Background: There is a paucity of information on the epidemiology of heart failure (HF) in Australia. The Study of Heart failure in the Australian Primary carE setting (SHAPE) study aims to estimate the prevalence and annual incidence of HF in the general Australian community and to describe the demographic and key clinical profile of Australians with HF., Methods: We undertook a retrospective cohort study based on analysis of non-identifiable medical records of adult patients cared for at 43 general practices between 1 July 2013 and 30 June 2018. Data were extracted from coded (diagnosis, pathology and prescription fields) and uncoded fields (clinical notes) in the medical records. The latter searches of free text looked for common synonyms relevant to HF. The population was stratified into three groups based on a hierarchy of selection criteria: (1) definite HF, (2) probable HF and (3) possible HF. The prevalence and annual incidence of HF were calculated, along with 95% confidence intervals., Results: The practices provided care to 2.3 million individual patients over the five-year study period, of whom 1.93 million were adults and 1.12 million were regular patients. Of these patients 15,468 were classified as having 'definite HF', 4751 as having 'probable HF' and 33,556 as having 'possible HF'. A further 39,247 were identified as having an aetiological condition associated with HF. A formal HF diagnosis, HF terms recorded as text in the notes and HF-specific medication were the most common methods to identify 'definite' HF patients. Typical signs and symptoms in combination with a diuretic prescription was the most common method to identify 'probable HF' patients. The majority of 'possible' HF patients were identified by the presence of 2 or more of the typical signs or symptoms. Dyspnoea was the commonest recorded symptom and an elevated jugular venous pressure the commonest recorded sign., Conclusions: This novel approach to undertaking retrospective research of primary care data successfully analysed a combination of coded and uncoded data from the electronic medical records of patients routinely managed in the GP setting. SHAPE is the first real-world study of the epidemiology of HF in the general Australian community setting.

Evidence suggests that patient-centred medical home (PCMH) is more effective than standard general practitioner care in improving patient outcomes in primary care. This paper reports on the design, early implementation experiences, and early findings of the 12-month PCMH model called 'WellNet' delivered across six primary care practices in Sydney, Australia. The WellNet study sample comprises 589 consented participants in the intervention group receiving enhanced primary care in the form of patient-tailored chronic disease management plan, improved self-management support, and regular monitoring by general practitioners (GPs) and trained clinical coordinators. The comparison group consisted of 7750 patients who were matched based on age, gender, type and number of chronic diseases who received standard GP care. Data collected include sociodemographic characteristics, clinical measures, and self-reported health assessments at baseline and 12 months. Early study findings show the mean age of the study participants was 70 years with nearly even gender distribution of males (49.7%) and females (50.3%). The most prevalent chronic diseases in descending order were circulatory system disorders (69.8%), diabetes (47.4%), musculoskeletal disorders (43.5%), respiratory diseases (28.7%), mental illness (18.8%), and cancer (13.6%). To our knowledge, the WellNet study is the first study in Australia to generate evidence on the feasibility of design, recruitment, and implementation of a comprehensive PCMH model. Lessons learned from WellNet study may inform other medical home models in Australian primary care settings.

PDGF-CC is a member of the platelet-derived growth factor (PDGF) family that stimulates PDGFRα phosphorylation and thereby activates intracellular signalling events essential for development but also in cancer, fibrosis and neuropathologies involving blood-brain barrier (BBB) disruption. In order to elucidate the biological and pathological role(s) of PDGF-CC signalling, we have generated high affinity neutralizing monoclonal antibodies (mAbs) recognizing human PDGF-CC. We determined the complementarity determining regions (CDRs) of the selected clones, and mapped the binding epitope for clone 6B3. Using the monoclonal 6B3, we determined the expression pattern for PDGF-CC in different human primary tumours and control tissues, and explored its ability to neutralize PDGF-CC-induced phosphorylation of PDGFRα. In addition, we showed that PDGF-CC induced disruption of the blood-retinal barrier (BRB) was significantly reduced upon intraperitoneal administration of a chimeric anti-PDGF-CC antibody. In summary, we report on high affinity monoclonal antibodies against PDGF-CC that have therapeutic efficacy in vivo., Competing Interests: H.L., A.M.S., L.A. and U.E. have submitted a patent application (Methods and compositions for PDGF-CC inhibition, PCT/US2017/040170) on these antibodies. A.M.S. and U.E. are shareholders of a company (Paracrine Therapeutics AB), developing these antibodies. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: At present, it is difficult to predict which patients with ductal carcinoma-in-situ (DCIS) will subsequently develop frank invasive breast cancer (IDC). A recent survey by our group has shown that NY-ESO-1 and MAGEA are both expressed in DCIS. This study was aimed at determining whether expression of these antigens was related to the later development of IDC., Results: 14 of 42 (33%) of patients developed invasive breast cancer during the follow up period. Only one of those DCIS cases that relapsed was positive for NYESO-1 at diagnosis. In contrast, DCIS samples of 15 of the 28 (54%) of those patients who remained disease-free expressed NY-ESO-1. (Permutation chi square p=0.0033)., Methods: We identified 42 patients with DCIS, and followed them up for more than 10 years. NY-ESO-1 and MAGEA were demonstrated by immunostaining as were CD8+ infiltrates on all sections together with the conventional markers, ER, PR, and HER2., Conclusions: Expression of NY-ESO-1 may predict those patients who will not subsequently develop invasive breast cancer and could therefore potentially be helpful in defining prognosis in patients with DCIS., Competing Interests: CONFLICTS OF INTEREST Raoul C. Coombes declares that he has no conflict of interest. Otavia L. Caballero declares that she has no conflict of interest, Sami. Shousha declares that he has no conflict of interest. Sadaf Ghaem-Maghami declares that she has no conflict of interest, Laura Woodley-Barker declares that she has no conflict of interest. Charlotte S. Wilhelm-Benartzi declares that she has no conflict of interest, and A Munro Neville declares that he has no conflict of interest.

Studies using patient-level data to determine the attributable cost of invasive fungal diseases (IFDs) are few. Using a case-control study with activity-based costing of patients admitted to a quaternary hospital from 2002 to 2007, we determined attributable hospitalization cost (and 12 weeks thereafter), length of stay (LOS), and costly antifungal treatment (C-AT; liposomal amphotericin B, voriconazole, posaconazole, caspofungin), expressed as defined daily doses (DDDs) per IFD episode, in patients with hematological malignancies and hematopoietic stem cell recipients. Matching criteria and median regression modeling controlled for confounding variables, including LOS prior to IFD onset. Multiple mycoses were identified in 43 matched case-control pairs (n=86). A separate sensitivity analysis included 22 unmatched patients. IFD status was associated with a median excess cost of AU$30,957 (95% confidence interval [CI]=AU$2,368 to AU$59,546; P=0.034), approximating at purchasing power parity US$21,203 (95% CI=US$1,622 to US$40,784) and €15,788 (95% CI=€1,208 to €30,368), increasing to AU$80,291 (95% CI=AU$33,636 to AU$126,946; P=0.001), i.e., US$54,993 (95% CI=US$23,038 to US$86,948) and €40,948 (95% CI=€17,154 to €64,742), with intensive care unit (ICU) requirement. Cost determinants were pharmacy costs (64%; P<0.001) inclusive of antifungal treatment (27%; P<0.001) and ward costs (27%; P=0.091), with proportions persisting through 12 weeks for 25 surviving matched pairs (pharmacy, 60% [P=0.12]; ward, 31% [P=0.21]). Median LOS was not significantly increased unless unmatched patients were included (8 days, 95% CI=1.8 to 14 days; P=0.012). Excess C-ATs were 17 DDDs (95% CI=15 to 19 DDDs; P<0.001) per case patient and 19 DDDs (95% CI=16 to 22 DDDs; P<0.001) per ICU patient. The sensitivity analysis was confirmatory (for median cost, AU$29,441, 95% CI=AU$5,571 to AU$53,310, P=0.016; for C-AT, 17 DDDs, 95% CI=16 to 18 DDDs, P<0.001). IFD results in increased hospital and ICU costs, with pharmacy costs, including antifungal treatment, being major determinants. Consumption of costly antifungal drugs may be a novel resource metric with wider generalizability than cost alone.

Background: Cancer/testis (CT) antigens are protein antigens normally expressed only in germ cells of testis, and yet are expressed in a proportion of a wide variety of human cancers. CT antigens can elicit spontaneous immune responses in cancer patients with CT-positive cancers, and CT antigen-based therapeutic cancer vaccine trials are ongoing for "CT-rich" tumors. Although some previous studies found breast cancer to be "CT-poor", our recent analysis identified increased CT mRNA transcripts in the ER-negative subset of breast cancer., Methodology/principal Findings: In this study, we performed a comprehensive immunohistochemical study to investigate the protein expression of eight CT genes in 454 invasive ductal carcinomas, including 225 ER/PR/HER2-negative (triple-negative) carcinomas. We found significantly more frequent expression of all eight CT antigens in ER-negative cancers, and five of them--MAGEA, CT7, NY-ESO-1, CT10 and CT45, were expressed in 12-24% of ER-negative cancers, versus 2-6% of ER-positive cancers (p<0.001 to 0.003). In comparison, GAGE, SAGE1 and NXF2 were only expressed in 3-5% of ER-negative and 0-2% of ER-positive cancers. ER-negative cancers were also more likely to simultaneously co-express multiple CT antigens, with 27% (34/125) of ER-negative, CT-positive tumors expressing three or more CT antigens. HER2 status had no consistent effect on CT expression, and triple-negative carcinomas showed similar frequencies of MAGEA and NY-ESO-1 expression as ER-negative/HER2-positive carcinomas. More frequent CT expression was also found in tumors with higher nuclear grade (p<0.001 to p = 0.01) and larger in size (>2 cm)., Conclusions/significance: CT antigens are preferentially expressed in hormone receptor-negative and high-grade breast cancer. Considering the limited treatment options for ER/PR/HER2 triple-negative breast cancer, the potential of CT-based immunotherapy should be explored.

Background: cancer-testis (CT) antigens, frequently expressed in human germline cells but not in somatic tissues, may become aberrantly reexpressed in different cancer types. The aim of this study was to investigate the expression of CT antigens in breast cancer., Patients and Methods: a total of 100 selected invasive breast cancers, including 50 estrogen receptor (ER) positive/HER2 negative and 50 triple negative (TN), were examined for NY-ESO-1 and MAGE-A expression by immunohistochemistry., Results: a significantly higher expression of MAGE-A and NY-ESO-1 was detected in TN breast cancers compared with ER-positive tumors (P = 0.04). MAGE-A expression was detected in 13 (26%) TN cancers compared with 5 (10%) ER-positive tumors (P = 0.07). NY-ESO-1 expression was confirmed in nine (18%) TN tumor samples compared with two (4%) ER-positive tumors., Conclusions: MAGE-A and NY-ESO-1 CT antigens are expressed in a substantial proportion of TN breast cancers. Because of the limited therapeutic options for this group of patients, CT antigen-based vaccines might prove to be useful for patients with this phenotype of breast cancer.

Introduction: Breast cancer currently accounts for more than one-quarter of all female cancers and, despite the great progress in treatment observed in the past few years, the need for identification of new gene targets that can be used for diagnosis, prognosis and therapy is evident. A previous study identified the transcription factor NR4A1 as a gene upregulated in primary breast cancer compared with normal tissue by microarray analysis and sequencing technologies. The purpose of the study was to identify the role of NR4A1 in normal mammary epithelial and breast cancer cell biology., Methods: NR4A1 expression in breast tumours was assessed by semiquantitative and real-time PCR using RNA from normal and tumour samples or breast cancer cell lines. Immunohistochemistry on tissue microarrays was performed to check NR4A1 protein expression in breast tumours. MCF-10A and 226L normal mammary epithelial cells as well as the tumour lines PMC42, ZR-75-1 and MDA-MB-231 were transduced with full-length NR4A1, and the ability of NR4A1-overexpressing cells to migrate was tested using scratch wound or transwell migration assays. Proliferation was measured using the MTT and BrdU assays, while apoptosis was determined by the Annexin V assay. The ability of the cells to adhere to extracellular matrix was tested by adhesion assays and integrin cell surface expression was measured by flow cytometry. Activation of the FAK as well as ERK1/2 and PI3K pathways was checked by western blotting., Results: Breast tissue microarray analysis showed NR4A1 expression in primary tumours, which was reduced in higher grade and metastatic tumours. Ectopic expression of NR4A1 in MCF-10A, 226L, PMC42 and ZR-75-1 cells led to reduced ability of the cells to migrate, while no differences were observed in their proliferation and apoptotic index. NR4A1 expression altered the ability of the MCF-10A cells to adhere to the extracellular matrix and affected cell surface expression of integrins., Conclusions: NR4A1 acts as an antimigratory factor in two normal mammary epithelial and two breast cancer cell lines tested. It is therefore possible that NR4A1 acts as an antimigratory factor in breast tumours, and further studies should be conducted to understand the mechanisms involved.

Cancer/testis (CT) genes are predominantly expressed in human germ line cells, but not somatic tissues, and frequently become activated in different cancer types. Several CT antigens have already proved to be useful biomarkers and are promising targets for therapeutic cancer vaccines. The aim of the present study was to investigate the expression of CT antigens in breast cancer. Using previously generated massively parallel signature sequencing (MPSS) data, together with 9 publicly available gene expression datasets, the expression pattern of CT antigens located on the X chromosome (CT-X) was interrogated. Whereas a minority of unselected breast cancers was found to contain CT-X transcripts, a significantly higher expression frequency was detected in estrogen and progesterone receptor (ER) negative breast cancer cell lines and primary breast carcinomas. A coordinated pattern of CT-X antigen expression was observed, with MAGEA and NY-ESO-1/CTAG1B being the most prevalent antigens. Immunohistochemical staining confirmed the correlation of CT-X antigen expression and ER negativity in breast tumors and demonstrated a trend for their coexpression with basal cell markers. Because of the limited therapeutic options for ER-negative breast cancers, vaccines based on CT-X antigens might prove to be useful.

Background: More than 20% of human transcripts have naturally occurring antisense products (or natural antisense transcripts--NATs), some of which may play a key role in a range of human diseases. To date, several databases of in silico defined human sense-antisense (SAS) pairs have appeared, however no study has focused on differential expression of SAS pairs in breast tissue. We therefore investigated the expression levels of sense and antisense transcripts in normal and malignant human breast epithelia using the Affymetrix HG-U133 Plus 2.0 and Almac Diagnostics Breast Cancer DSA microarray technologies as well as massively parallel signature sequencing (MPSS) data., Results: The expression of more than 2500 antisense transcripts were detected in normal breast duct luminal cells and in primary breast tumors substantially enriched for their epithelial cell content by DSA microarray. Expression of 431 NATs were confirmed by either of the other two technologies. A corresponding sense transcript could be identified on DSA for 257 antisense transcripts. Of these SAS pairs, 163 have not been previously reported. A positive correlation of differential expression between normal and malignant breast samples was observed for most SAS pairs. Orientation specific RT-QPCR of selected SAS pairs validated their expression in several breast cancer cell lines and solid breast tumours., Conclusion: Disease-focused and antisense enriched microarray platforms (such as Breast Cancer DSA) confirm the assumption that antisense transcription in the human breast is more prevalent than previously anticipated. Expression of a proportion of these NATs has already been confirmed by other technologies while the true existence of the remaining ones has to be validated. Nevertheless, future studies will reveal whether the relative abundances of antisense and sense transcripts have regulatory influences on the translation of these mRNAs.

Introduction: Diverse microarray and sequencing technologies have been widely used to characterise the molecular changes in malignant epithelial cells in breast cancers. Such gene expression studies to identify markers and targets in tumour cells are, however, compromised by the cellular heterogeneity of solid breast tumours and by the lack of appropriate counterparts representing normal breast epithelial cells., Methods: Malignant neoplastic epithelial cells from primary breast cancers and luminal and myoepithelial cells isolated from normal human breast tissue were isolated by immunomagnetic separation methods. Pools of RNA from highly enriched preparations of these cell types were subjected to expression profiling using massively parallel signature sequencing (MPSS) and four different genome wide microarray platforms. Functional related transcripts of the differential tumour epithelial transcriptome were used for gene set enrichment analysis to identify enrichment of luminal and myoepithelial type genes. Clinical pathological validation of a small number of genes was performed on tissue microarrays., Results: MPSS identified 6,553 differentially expressed genes between the pool of normal luminal cells and that of primary tumours substantially enriched for epithelial cells, of which 98% were represented and 60% were confirmed by microarray profiling. Significant expression level changes between these two samples detected only by microarray technology were shown by 4,149 transcripts, resulting in a combined differential tumour epithelial transcriptome of 8,051 genes. Microarray gene signatures identified a comprehensive list of 907 and 955 transcripts whose expression differed between luminal epithelial cells and myoepithelial cells, respectively. Functional annotation and gene set enrichment analysis highlighted a group of genes related to skeletal development that were associated with the myoepithelial/basal cells and upregulated in the tumour sample. One of the most highly overexpressed genes in this category, that encoding periostin, was analysed immunohistochemically on breast cancer tissue microarrays and its expression in neoplastic cells correlated with poor outcome in a cohort of poor prognosis estrogen receptor-positive tumours., Conclusion: Using highly enriched cell populations in combination with multiplatform gene expression profiling studies, a comprehensive analysis of molecular changes between the normal and malignant breast tissue was established. This study provides a basis for the identification of novel and potentially important targets for diagnosis, prognosis and therapy in breast cancer.

The normal duct-lobular system of the breast is lined by two epithelial cell types, inner luminal secretory cells and outer contractile myoepithelial cells. We have generated comprehensive expression profiles of the two normal cell types, using immunomagnetic cell separation and gene expression microarray analysis. The cell-type specificity was confirmed at the protein level by immunohistochemistry in normal breast tissue. New prognostic markers for survival were identified when the luminal- and myoepithelial-specific molecules were evaluated on breast tumor tissue microarrays. Nuclear expression of luminal epithelial marker galectin 3 correlated with a shorter overall survival in these patients, and the expression of SPARC (osteonectin), a myoepithelial marker, was an independent marker of poor prognosis in breast cancers as a whole. These data provide a framework for the interpretation of breast cancer molecular profiling experiments, the identification of potential new diagnostic markers, and development of novel indicators of prognosis.

Whereas genome sequencing defines the genetic potential of an organism, transcript sequencing defines the utilization of this potential and links the genome with most areas of biology. To exploit the information within the human genome in the fight against cancer, we have deposited some two million expressed sequence tags (ESTs) from human tumors and their corresponding normal tissues in the public databases. The data currently define approximately 23,500 genes, of which only approximately 1,250 are still represented only by ESTs. Examination of the EST coverage of known cancer-related (CR) genes reveals that <1% do not have corresponding ESTs, indicating that the representation of genes associated with commonly studied tumors is high. The careful recording of the origin of all ESTs we have produced has enabled detailed definition of where the genes they represent are expressed in the human body. More than 100,000 ESTs are available for seven tissues, indicating a surprising variability of gene usage that has led to the discovery of a significant number of genes with restricted expression, and that may thus be therapeutically useful. The ESTs also reveal novel nonsynonymous germline variants (although the one-pass nature of the data necessitates careful validation) and many alternatively spliced transcripts. Although widely exploited by the scientific community, vindicating our totally open source policy, the EST data generated still provide extensive information that remains to be systematically explored, and that may further facilitate progress toward both the understanding and treatment of human cancers.

To investigate changes in gene expression associated with ERBB2, expression profiling of immortalized human mammary luminal epithelial cells and variants expressing a moderate and high level of ERBB2 has been carried out using cDNA microarrays corresponding to approximately 6000 unique genes/ESTs. A total of 61 significantly up- or downregulated (2.0-fold) genes were identified and further validated by RT-PCR analysis as well as microarray comparisons with a spontaneously ERBB2- overexpressing breast cancer cell line and ERBB2-positive primary breast tumors. The expression and clinical relevance of proteins predicted to be associated with ERBB2 overexpression in breast cancers were analysed together with their clinical relevance by antibody screening using a tissue array. Differentially regulated genes include those involved in cell-matrix interactions including proline 4-hydroxylase (P4HA2), galectin 1 (LGALS1) and galectin 3 (LGALS3), fibronectin 1 (FN1) and p-cadherin (CDH3), and cell proliferation (CRIP1, IGFBP3) and transformation (S100P, S100A4). A number of genes associated with MYC signalling were also differentially expressed, including NDRG1, USF2 and the epithelial membrane proteins 1 and 3 (EMP1, EMP3). These data represent profiles of the transcriptional changes associated with ERBB2-related pathways in the breast, and identify novel and potentially useful targets for prognosis and therapy.

Whereas information is rapidly accumulating about the structure and position of genes encoded in the human genome, less is known about the complexity and relative abundance of their expression in individual human cells and tissues. Here, we describe the characteristics of the transcriptomes of two cultured cell lines, HB4a (normal breast epithelium) and HCT-116 (colon adenocarcinoma), using massively parallel signature sequencing (MPSS). We generated in excess of 10(7) short signature sequences per cell line, thus providing a comprehensive snapshot of gene expression, within the technical limitations of the method. The number of genes expressed at one copy per cell or more in either of the lines was estimated to be between 10,000 and 15,000. The vast majority of the transcripts found in these cells can be mapped to known genes and their polyadenylation variants. Among the genes that could be identified from their signature sequences, approximately 8,500 were expressed by both cell lines, whereas 6,000 showed cellular specificity. Taking into account sequence tags that map uniquely to the genome but not to known transcripts, overall the data are consistent with an upper limit of 17,000 for the total number of genes expressed at more than one copy per cell in one or both of the two cell lines examined.

Reports differ as to whether reconstitution of telomerase activity alone is sufficient for immortalization of different types of human somatic cells or whether additional activities encoded by other "immortalizing" genes are also required. Here we show that ectopic expression of either the catalytic subunit of human telomerase (hTERT) or a temperature-sensitive mutant (U19tsA58) of simian virus 40 large-tumor antigen alone was not sufficient for immortalization of freshly isolated normal adult human mammary fibroblasts and endothelial cells. However, a combination of both genes resulted in the efficient generation of immortal cell lines irrespective of the order in which they were introduced or whether they were introduced early or late in the normal proliferative lifespan of the cultures. The order and timing of transduction, however, did influence genomic stability. Karyotype analysis indicated that introduction of both transgenes at early passage, with hTERT first, yielded diploid cell lines. Temperature-shift experiments revealed that maintenance of the immortalized state depended on continued expression of functional U19tsA58 large-tumor antigen, with hTERT alone unable to maintain growth at nonpermissive temperatures for U19tsA58 large-tumor antigen. Such conditional diploid lines may provide a useful resource for both cell engineering and for studies on immortalization and in vitro transformation.

The presence of a high number of infiltrating CD1a+ cells in malignant neoplasms has been reported to be associated with an improved prognosis, reduced tumour recurrence and fewer metastases. This study identified a population of CD1a+ cells within the lymphoid cell infiltrate in human ductal breast carcinoma (n = 52), which was significantly different from normal breast tissue, in which only two out of nine cases expressed CD1a+ cells (P = 0.0192). In the majority of cases, the infiltrate was low compared with the number of macrophages and T cells present (results not shown). There was no correlation between the number of CD1a+ cells and tumour grade, with all tumour grades expressing similar numbers of infiltrating CD1a+ cells. There was clear evidence, however, that the CD1a+ cells were closely associated with tumour cells. It is likely that CD1a+ cells have a role in antigen capture and presentation in human tumours, and this study documents the density of CD1a+ cells in a large sample of all histological grades of human breast carcinomas.

In order to determine the mechanisms of relapse following response to endocrine therapy, we have measured the oestrogen receptor (RE) content of biopsies of breast cancer in patients receiving various types of endocrine treatment. RE content fell in responding (means of 260.2 to 12 fmol/mg protein) and in nonresponding (means of 155.1 to 31.8 fmol/mg protein) patients who had measurable receptor at the start of treatment. Some of these patients, and a further group of responders to endocrine therapy, were monitored until relapse. Tumour biopsies at the time of relapse showed that 10/14 tumour samples contained significant RE (mean of 86.7 fmol/mg protein; range less than 10-271 fmol/mg protein) after successful endocrine therapy. No relationship could be found between RE content and plasma gonadotrophin or steroid-hormone concentration, but the fall in RE content correlated with reduced numbers of tumour cells in the biopsy. These results indicate that relapse following successful endocrine therapy in breast cancer does not appear to be due to the emergence of RE-negative tumour cells. The fall in RE content during response to endocrine therapy may be due to reduced tumour-cell content of the biopsy.

A xenograft line, HX 53, has been established in immune-suppressed mice from a specimen of a lymph node metastasis in a patient with a histological diagnosis of seminoma but with markedly raised circulating levels of alpha-fetoprotein. Histological, immunocytochemical, and ultrastructural studies of this xenograft line have suggested that a solid variant of yolk sac carcinoma may exist, which morphologically resembles seminoma, or that a continuum of differentiation exists between seminoma and yolk sac carcinoma.

The preparation of highly purified monolayer cultures of human cytotrophoblast cells essentially free of stromal and syncytial cells is described. Such cells subsequently form multinucleate syncytial cells in vitro. This is accompanied by the synthesis of heat-stable alkaline phosphatase and beta-HCG. A significant proportion of the multinucleate cells that form in monolayer cultures of early placentae arise as a result of an amitosis. It is suggested that this in vitro behavior may reflect an in vivo process.

An immunocytochemical method for fixed and paraffin-embedded human breast biopsies is reported for the detection of myoepithelial and epithelial cells using antibodies to myosin and keratin, respectively, and of basement membranes using antibodies to laminin and type IV collagen. Using these markers, myoepithelial cells can be clearly distinguished in the normal breast and in the benign breast diseases sclerosing adenosis, epitheliosis, and fibroadenoma. In sclerosing adenosis, myoepithelial cells form a major cellular component. A stromally derived spindle cell is identified which stains with myosin but not with keratin antibodies (myofibroblast). These cells are seen in one-fifth of the fibroadenomas. Although cells staining with myosin antibodies are seen in the infiltrating component of all 18 carcinomas examined, elongated cells staining with both myosin and keratin antibodies (myoepithelial-like) are seen in only one infiltrating carcinoma where they are interposed at the stromal-epithelial junction of the infiltrating tumor cells. In contrast to the situation in benign breast diseases, mature myoepithelial cells form a very minor component of the majority of infiltrating ductal carcinomas. Basement membrane proteins, laminin, and type IV collagen are present in normal breast, benign breast disease, and grade I infiltrating ductal carcinomas but are absent in carcinomas of grades II and III.

Gonadotrophins, oestradiol, androstenedione, testosterone and dehydroepiandrosterone sulphate (DHAS) were measured sequentially in 72 patients with advanced breast cancer receiving endocrine therapy of various types. Tamoxifen significantly reduced gonadotrophins but did not effect other hormones. Danazol also reduced gonadotrophins. Aminoglutethimide (AGT) reduced oestradiol and DHAS but had not effect on gonadotrophins. The effects of administering tamoxifen, AGT and danazol together (TAD) together were therefore examined. This combination reduced gonadotrophins, oestradiol and DHAS, but no further than tamoxifen and AGT alone. The degree and duration of hormone suppression were similar in both responders and non-responders to tamoxifen, AGT or TAD, though patients responding to AGT showed more complete suppression at the end of the course of treatment, perhaps because they were treated longer. On relapse, adequate gonadotrophin and steroid suppression was demonstrated in patients receiving tamoxifen and AGT respectively. We conclude that (a) response to endocrine therapy is unlikely to be related to the degree of endocrine suppression produced by the therapy; (b) combination endocrine therapy does not further reduce serum-hormone concentrations and (c) relapse is unlikely to be due to escape from the hormone-inhibitory effects of endocrine agents.

We have devised a method utilizing a monoclonal antibody-toxin conjugate (LICR-LON-Fib75/abrin A-chain) for ridding bone marrow of infiltrating breast cancer cells to rescue patients with autologous bone marrow following high dose therapy. Initially we examined the activity of this conjugate in vitro. Five of seven human breast cancer cell lines were killed following exposure at 10(-8) M for 2 h; this concentration only reduced bone marrow colony formation to 83% (range, 50-100%) of control bone marrow. We then examined the pattern of bone marrow recovery after high dose melphalan (200 mg/m2) in patients with advanced breast cancer who were in remission following combination chemotherapy. To do this we compared the time of recovery of the blood count in three patients who received treated marrow and seven who received untreated marrow. Mean time to recovery of the peripheral white count (greater than 1.5 X 10(9)/liter) was 16.7 days (treated) and 18.3 days (untreated), respectively. Mean time to recovery of peripheral platelet count (greater than 50 X 10(9)/liter) was 23.7 days (treated) and 18.9 days (untreated), respectively. Patients continued in remission for 1-greater than 14 mo after high dose melphalan, and remission duration was similar in patients who received treated (6.2 mo) and untreated (7.3 mo) bone marrow. These findings indicate that treatment of bone marrow with LICR-LON-Fib75/abrin A-chain conjugate does not significantly impair bone marrow recovery, and it is, therefore, possible to rescue breast cancer patients with bone marrow that has been cleansed of infiltrating cancer cells. This may have an application in patients with poor-risk primary breast cancer who have micrometastases and who may benefit from intensive therapy, but it has minimal application in patients with more advanced disease.

A reliable immunoperoxidase schedule for the cellular demonstration of tumour and normal cell products which can be completed in well under two hours is described. The preparations are as clear and readable as those stained by routine histological techniques and should form a valuable adjunct in research and diagnostic studies.

Immune-suppressed mice carrying xenografts of several different types of human germ-cell tumours were injected with a radiolabelled monoclonal antibody (LICR LON/HT13) raised against membrane components of a human germ-cell tumour (HX39). Subsequent assessment of radioactivity in excised organs and tumours showed a selective accretion of antibody in the tumour. Quantitative autoradiography supported the results of radiolocalization observed in vivo in different tumours, and also showed that the antibody localized to viable tumour cells and in close association with their cell membrane. The vascular architecture of tumours was found to be an important factor governing antibody distribution. No localization occurred with radiolabelled normal mouse IgG.

A new human antigen, designated epithelial membrane antigen (EMA), has recently been described on surface membranes of a wide variety of normal epithelium but not on connective tissue cells. The antigen is only weakly expressed on normal or reactive mesothelium. Increased expression of the antigen has been observed in most neoplasms of epithelial origin and in malignant mesothelioma. We have investigated the possibility of using this difference in the expression of the antigen to distinguish between mesothelial cells and malignant cells in cytological smears of serous effusions. This distinction cannot always be made on morphological grounds alone and problems of differential diagnosis are encountered in about 15% of all specimens of serous effusions sent for cytological examination. Using antisera to EMA we have applied an indirect immunoalkaline phosphatase technique to alcohol-fixed smears prepared from serous effusions and have found that intense staining of the antigen is confined to effusions from patients in whom there is either clinical or cytological evidence of malignancy. The technique proved to be especially useful in cytologically equivocal cases, where there were problems of differential diagnosis.