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Supplemental Digital Content is available in the text., SP142 programmed cell death ligand 1 (PD-L1) status predicts response to atezolizumab in triple-negative breast carcinoma (TNBC). Prevalence of VENTANA PD-L1 (SP142) Assay positivity, concordance with the VENTANA PD-L1 (SP263) Assay and Dako PD-L1 IHC 22C3 pharmDx assay, and association with clinicopathologic features were assessed in 447 TNBCs. SP142 PD-L1 intraobserver and interobserver agreement was investigated in a subset of 60 TNBCs, with scores enriched around the 1% cutoff. The effect of a 1-hour training video on pretraining and posttraining scores was ascertained. At a 1% cutoff, 34.2% of tumors were SP142 PD-L1 positive. SP142 PD-L1 positivity was significantly associated with tumor-infiltrating lymphocytes (P

Incorporation of genome and exome sequencing into fetal and neonatal autopsy investigations has been shown to improve diagnostic yield. This requires deoxyribonucleic acid (DNA) to be extracted from either the placenta or autopsy tissue for molecular testing. However, the sources and quality of DNA obtained are highly variable and there are no adequate published data on what tissue is most ideal to sample for DNA extraction in this setting. Here we compare the quality of DNA extracted from sampling the placenta and various solid organs at fetal and neonatal autopsy, thereby determining the optimal tissue from which to source DNA for ancillary testing as part of the modern perinatal autopsy. A total of 898 tissue samples were obtained at autopsy from 176 fetuses (gestational ages 17-40 weeks) and 44 neonates (age range 0-28 days) at our tertiary institution. Fetal tissue was processed using the QIAsymphony DSP DNA Mini kit and placental tissue was extracted using the New iGENatal Kit. DNA concentration was quantified using the Qubit dsDNA BR Assay Kit. DNA integrity, as stratified by gel electrophoresis was classified as high (≥5 kb) or low quality (

Breast cancer is the dominant cancer among women as it accounts for about one-quarter of all cancer cases in females. The digitized images of Hematoxylin and Eosin (H&E) stained slides of breast cancer specimens carry valuable diagnostic information. However, inspecting these slides manually is a non-trivial task prone to subjective interpretation. Digital pathology (DP) and artificial intelligence (AI) open an opportunity for objective interpretation of the image data. It is challenging to automate the segmentation process in the whole slide images due to the visual complexity of tissue appearance without the need for tedious and time-consuming fine annotations. Many algorithms classify the tissue regions into different types instead of segmenting them, as the classification algorithms require coarse annotations that are easier to acquire. In this paper, we propose a new segmentation framework that combines the simple non-iterative clustering algorithm with a standard convolutional neural network (CNN) classifier to segment whole slide images into different tissue types. In addition, a graph-based post-processing step is applied to improve the framework segmentation performance further. The results show promising improvement to the CNN classifier based coarse segmentation, which would give better feasibility to quantify and study tissues’ mutual relationships.

High stage and recurrent ovarian clear cell carcinoma (OCC) are associated with poor prognosis and resistance to chemotherapy. A distinguishing histological feature of OCC is abundant cytoplasmic stores of glucose, in the form of glycogen, that can be mobilized for cellular metabolism. Here, we report the effect on preclinical models of OCC of disrupting glycogen utilization using the glucose analogue 2-deoxy-D-glucose (2DG). At concentrations significantly lower than previously reported for other cancers, 2DG markedly improves the efficacy in vitro of carboplatin chemotherapy against chemo-sensitive TOV21G and chemo-resistant OVTOKO OCC cell lines, and this is accompanied by the depletion of glycogen. Of note, 2DG doses&mdash, of more than 10-fold lower than previously reported for other cancers&mdash, significantly improve the efficacy of carboplatin against cell line and patient-derived xenograft models in mice that mimic the chemo-responsiveness of OCC. These findings are encouraging, in that 2DG doses, which are substantially lower than previously reported to cause adverse events in cancer patients, can safely and significantly improve the efficacy of carboplatin against OCC. Our results thus justify clinical trials to evaluate whether low dose 2DG improves the efficacy of carboplatin in OCC patients.

Background Recent pre-clinical studies indicate that activated progesterone receptor (PR) (particularly the PR-B isoform) binds to oestrogen receptor-α (ER) and reprogrammes transcription toward better breast cancer outcomes. We investigated whether ER and PR-B interactions were present in breast tumours and associated with clinical parameters including response to aromatase inhibitors. Methods We developed a proximity ligation assay to detect ER and PR-B (ER:PR-B) interactions in formalin-fixed paraffin-embedded tissues. The assay was validated in a cell line and patient-derived breast cancer explants and applied to a cohort of 229 patients with ER-positive and HER2-negative breast cancer with axillary nodal disease. Results Higher frequency of ER:PR-B interaction correlated with increasing patient age, lower tumour grade and mitotic index. A low frequency of ER:PR-B interaction was associated with higher risk of relapse. In multivariate analysis, ER:PR-B interaction frequency was an independent predictive factor for relapse, whereas PR expression was not. In subset analysis, low frequency of ER:PR-B interaction was predictive of relapse on adjuvant aromatase inhibitor (HR 4.831, p = 0.001), but not on tamoxifen (HR 1.043, p = 0.939). Conclusions This study demonstrates that ER:PR-B interactions have utility in predicting patient response to adjuvant AI therapy.

Summary: LINE-1 (L1) retrotransposons are a source of insertional mutagenesis in tumor cells. However, the clinical significance of L1 mobilization during tumorigenesis remains unclear. Here, we applied retrotransposon capture sequencing (RC-seq) to multiple single-cell clones isolated from five ovarian cancer cell lines and HeLa cells and detected endogenous L1 retrotransposition in vitro. We then applied RC-seq to ovarian tumor and matched blood samples from 19 patients and identified 88 tumor-specific L1 insertions. In one tumor, an intronic de novo L1 insertion supplied a novel cis-enhancer to the putative chemoresistance gene STC1. Notably, the tumor subclone carrying the STC1 L1 mutation increased in prevalence after chemotherapy, further increasing STC1 expression. We also identified hypomethylated donor L1s responsible for new L1 insertions in tumors and cultivated cancer cells. These congruent in vitro and in vivo results highlight L1 insertional mutagenesis as a common component of ovarian tumorigenesis and cancer genome heterogeneity. : Acquired chemoresistance drives ovarian cancer mortality. Nguyen et al. show that L1 retrotransposons, a type of mobile genetic element, can escape silencing in ovarian tumor cells. They find a tumor-specific L1 insertion that enhances STC1 oncogene expression and is selected for during chemotherapy. L1-driven chemoresistance may therefore affect clinical outcomes. Keywords: ovarian cancer, retrotransposon, LINE-1, L1, chemoresistance

Objective To measure association between endometrial carcinoma ER and PR status and endometrial cancer (EC) survival, accounting for inter-observer variation. Methods The intensity and proportion of tumor cell expression of ER and PR in ECs were assessed independently and semi-quantitatively by two pathologists using digital images of duplicate tumor tissue microarrays (TMAs). Cases with inconsistent initial assessment were reviewed and final scoring agreed. The association between overall and EC-specific survival and hormone receptor expression (intensity, proportion and combined) was assessed using Cox regression analysis. The C-index was used to evaluate model discrimination with addition of ER and PR status. Results Tumor ER and PR analysis was possible in 659 TMAs from 255 patients, and in 459 TMAs from 243 patients, respectively. Initial ER and PR scoring was consistent in 82% and 80% of cases, respectively. In multivariate analyses decreased ER and PR expression was associated with increased tumor-related mortality. Associations reached statistical significance for ER proportion score (P=0.05), ER intensity score (P=0.003), and PR combined score (P=0.04). Decreased expression of combined ER/PR expression was associated with poorer EC-specific survival than decreased expression of either hormone receptor alone (P=0.005). However, hormone receptor status did not significantly improve mortality prediction in individual cases. Conclusion ER and PR expression combined, using cut-points that capture variation in scoring and across cores, is significantly associated with EC-specific survival in analyses adjusting for known prognostic factors. However, at the individual level, ER and PR expression does not improve mortality prediction.

The authors regret that Donal Brennan was missing from the author list. The details shown above are now correct with Dr Brennan included. Dr. Brennan contributed to conceptualisation and data curation and final review of manuscript. Dr. Brennan does not have any conflicts to disclose. The authors would like to apologise for any inconvenience caused.

Introduction: Ectopic thyroid tissue can be found anywhere along the embryologic path of thyroid descent. Intralaryngo-tracheal thyroid tissue is the least common site of ectopia and can present with upper airways obstruction. Its presentation in the neonate is exceptional. Case Report: We describe a term female neonate with subglottic thyroid tissue causing near-total occlusion of the larynx, which led to upper airways obstruction and neonatal death. Conclusion: This emphasizes the importance of considering intralaryngo-tracheal tumors as a cause of acute and otherwise unexplainable respiratory distress immediately after birth. The cause of this neonatal death would not have been elucidated without careful autopsy examination.

Papillary lesions of the breast are most commonly diagnosed via mammographic screening. The standard practice has been to excise these lesions, since a subset of papillary lesions are neoplastic. However, this approach leads to a high proportion of negative excisions. In order to identify papillary lesions which could be managed by surveillance alone, we assessed the outcome of 103 papillary lesions diagnosed on core needle biopsy in a public screening program. Subsequent excision biopsy led to an upgrade to malignancy in 30% of cases. Segregation via presence or absence of atypia stratified the outcome into 72% upgrade, compared with 7% upgrade, respectively. Further, in the latter group (i.e., no atypia on core needle biopsy with 7% upgrade to malignancy), the neoplasia found in the targeted excision area was low to intermediate grade ductal carcinoma in situ only, with no invasive neoplasia (4 cases). Of the lesions identified due to microcalcification, the microcalcification was present within an adjacent benign lesion in 35% of cases and hence the papillary lesion was detected incidentally. Overall therefore, we have identified a cohort of papillary lesions in which conservative management, rather than excision, could be considered, i.e., those without atypia, including those without atypia in which the papillary lesion was found incidental to microcalcification in an adjacent benign lesion.

There are no data on the epidemiology of amyloidosis in Australia. Most international epidemiological studies are based on death certificate data [1–4]. This study aims to describe the incidence an...

Ventricular noncompaction cardiomyopathy is a rare form of congenital cardiomyopathy with increasing evidence of genetic etiology, especially when presenting in childhood. Fetal presentation is rare. We describe a case of fetal hydrops, presenting at 24 weeks gestation and leading to intrapartum death at 26 weeks gestation. Autopsy examination revealed characteristic features of left ventricular noncompaction. A genetic analysis identified a constellation of variants of unknown significance in MYH6, TNNC1, and MYBPC3, genes known to be important in sarcomeric function. Additionally, the variant in MYBPC3 was homozygous. While this case did not demonstrate a conventional single-gene mutation as the cause of the ventricular noncompaction, a broader genomic investigation revealed several variants in sarcomeric genes which may act synergistically to impact cardiac function.

CardioCel is a bovine pericardium that is subjected to a novel anticalcification tissue-engineering process. We present the histopathologic findings of human explants of CardioCel that were used in operations for congenital heart disease in children. Six explants were identified from 140 patients undergoing CardioCel implants from October 2012 to March 2015. CardioCel explants were evaluated histologically using hematoxylin and eosin, Masson trichrome, and immunohistochemical staining. A variable inflammatory response was seen in the surrounding native tissue, but not within the CardioCel graft in any of the explants. A neointimal layer of varying thickness developed on the visceral surface of 5 CardioCel explants with endothelialization of the longest duration explant. A granulation tissue layer developed on the parietal surface of the graft (consistently thicker than the neointima). Maintained collagen fiber architecture (laminated) and variable fibroblastic invasion (which increased with the age of the implant) were identified in all 6 cases. Scattered capillary vessels were noted in the majority of the explants with new collagen fibers in one, suggesting early remodeling. Calcium was seen in 1 explant at the interface of the graft and inflammatory response on its parietal surface. Evidence of graft remodeling was noted in the majority of the explants without inflammatory cells or calcification within the explanted graft material. A noticeable feature was the differential thickness of the host reaction to the parietal compared with the visceral surface of the graft. We will continue to evaluate CardioCel as a cardiovascular substitute for extracardiac and intracardiac reconstructions.

Childhood Interstitial lung disease (chILD) is an umbrella term used to define a broad range of rare, diffuse pulmonary disorders with altered interstitial structure that leads to abnormal gas exchange. Presentation of chILD in infancy can be difficult to differentiate from other common causes of diffuse lung disease. This article aimed at paediatricians provides an overview of interstitial lung disease presenting in infancy and includes key clinical features, a suggested approach to investigation and a summary of management. An overview of three clinical cases has been included to demonstrate the diagnostic approach, characteristic investigation findings and varied clinical outcomes.

Background: Development of targeted therapies for high-grade serous ovarian cancer (HGSC) remains challenging, as contributing molecular pathways are poorly defined or expressed heterogeneously. CUB-domain containing protein 1 (CDCP1) is a cell-surface protein elevated in lung, colorectal, pancreas, renal and clear cell ovarian cancer. Methods: CUB-domain containing protein 1 was examined by immunohistochemistry in HGSC and fallopian tube. The impact of targeting CDCP1 on cell growth and migration in vitro, and intraperitoneal xenograft growth in mice was examined. Three patient-derived xenograft (PDX) mouse models were developed and characterised for CDCP1 expression. The effect of a monoclonal anti-CDCP1 antibody on PDX growth was examined. Src activation was assessed by western blot analysis. Results: Elevated CDCP1 was observed in 77% of HGSC cases. Silencing of CDCP1 reduced migration and non-adherent cell growth in vitro and tumour burden in vivo. Expression of CDCP1 in patient samples was maintained in PDX models. Antibody blockade of CDCP1 significantly reduced growth of an HGSC PDX. The CDCP1-mediated activation of Src was observed in cultured cells and mouse xenografts. Conclusions: CUB-domain containing protein 1 is over-expressed by the majority of HGSCs. In vitro and mouse model data indicate that CDCP1 has a role in HGSC and that it can be targeted to inhibit progression of this cancer.

Published studies on the epidemiology of amyloidosis have relied on death certificate data for case ascertainment. We estimated the incidence and mortality burden of amyloidosis among residents of the Australian state, Queensland, aged ≥20 years for the years 1999-2013 based on case ascertainment from histopathology reports. Information systems for participating laboratories were scrutinised to identify histopathology reports that documented a diagnosis of amyloidosis. Case mortality status was determined via linkage to the National Death Index. A total of 447 cases of amyloidosis were identified, with a median age at diagnosis of 66 years. A plasma cell dyscrasia was identified in 72% of patients who had paraprotein studies performed. The estimated incidence for Queenslanders aged ≥20 years was 12·1 cases per million person years. The median survival was 2·45 years. Age at diagnosis, presence of a paraprotein, earlier year of diagnosis, and inner regional location of residence (compared with residence in a major city) were independently associated with reduced survival. Our data confirms previously reported incidence data for amyloidosis of approximately 10 cases per million patient years and indicates that survival for Queensland patients with amyloidosis is improving, though it remains poor for the elderly and patients with AL amyloidosis.

Death in the fetal, perinatal, and early infant age-group has a multitude of causes, a proportion of which is presumed to be genetic. Defining a specific genetic aberration leading to the death is problematic at this young age, due to limited phenotype–genotype correlation inherent in the underdeveloped phenotype, the inability to assess certain phenotypic traits after death, and the problems of dealing with rare disorders. In this study, our aim was to increase the yield of identification of a defined genetic cause of an early death. Therefore, we employed whole genome sequencing and bioinformatic filtering techniques as a comprehensive, unbiased genetic investigation into 16 fetal, perinatal, and early infant deaths, which had undergone a full autopsy. A likely genetic cause was identified in two cases (in genes; COL2A1 and RYR1) and a speculative genetic cause in a further six cases (in genes: ARHGAP35, BBS7, CASZ1, CRIM1, DHCR7, HADHB, HAPLN3, HSPG2, MYO18B, and SRGAP2). This investigation indicates that whole genome sequencing is a significantly enabling technology when determining genetic causes of early death.

Ventricular noncompaction cardiomyopathy is a rare form of congenital cardiomyopathy with increasing evidence of genetic etiology, especially when presenting in childhood. Fetal presentation is rare. We describe a case of fetal hydrops, presenting at 24 weeks gestation and leading to intrapartum death at 26 weeks gestation. Autopsy examination revealed characteristic features of left ventricular noncompaction. A genetic analysis identified a constellation of variants of unknown significance in MYH6, TNNC1, and MYBPC3, genes known to be important in sarcomeric function. Additionally, the variant in MYBPC3 was homozygous. While this case did not demonstrate a conventional single-gene mutation as the cause of the ventricular noncompaction, a broader genomic investigation revealed several variants in sarcomeric genes which may act synergistically to impact cardiac function.

Many cancers are dependent on inappropriate activation of epidermal growth factor receptor (EGFR), and drugs targeting this receptor can improve patient survival, although benefits are generally short-lived. We reveal a novel mechanism linking EGFR and the membrane-spanning, cancer-promoting protein CDCP1 (CUB domain-containing protein 1). Under basal conditions, cell surface CDCP1 constitutively internalizes and undergoes palmitoylation-dependent degradation by a mechanism in which it is palmitoylated in at least one of its four cytoplasmic cysteines. This mechanism is functional in vivo as CDCP1 is elevated and palmitoylated in high-grade serous ovarian tumors. Interestingly, activation of the EGFR system with EGF inhibits proteasome-mediated, palmitoylation-dependent degradation of CDCP1, promoting recycling of CDCP1 to the cell surface where it is available to mediate its procancer effects. We also show that mechanisms inducing relocalization of CDCP1 to the cell surface, including disruption of its palmitoylation and EGF treatment, promote cell migration. Our data provide the first evidence that the EGFR system can function to increase the lifespan of a protein and also promote its recycling to the cell surface. This information may be useful for understanding mechanisms of resistance to EGFR therapies and assist in the design of treatments for EGFR-dependent cancers.

AimsProgesterone receptor (PR) expression is prognostic in early stage breast cancer. There are several reports of discordant expression between primary tumour and axillary lymph node (ALN) metastasis expression of oestrogen receptor (ER) and PR. We sought to determine whether expression of these biomarkers in the synchronous ALN metastases of ER positive (+), HER2 negative (−) breast cancer could provide more accurate prognostic information.MethodsThe retrospective cohort included 229 patients from a single institution with ER+, HER2− breast cancer who had synchronous ALN metastatic disease (2005–2014). PR expression was correlated with relapse-free survival, and subset analysis was performed for patients who received adjuvant tamoxifen or an aromatase inhibitor.ResultsOne patient had an ER+ primary tumour, which was ER− in the ALN metastasis. 27 (11.3%) were PR− in the primary tumour and 56 (23.6%) in the ALN metastasis. The predominant change was from PR+ in the primary tumour to PR− in the lymph node. Absence of PR expression in the ALN was significantly associated with relapse; however, this was not the case in the primary tumour. In a subset analysis of patients taking adjuvant endocrine therapy, poorer prognosis was limited to those with PR− metastases on tamoxifen (HR=5.203, 95% CI 1.649 to 16.416, p=0.005). No significant prognostic effect of PR− metastases in patients taking aromatase inhibitors was seen (HR=1.519, 95% CI 0.675 to 3.418, p=0.312).ConclusionsEvaluation of PR expression in ALN metastasis may enable prediction of patients who are less likely to benefit from adjuvant tamoxifen. This study should be replicated in other cohorts.

We describe an infant with congenital mydriasis, patent ductus arteriosus (PDA), pulmonary hypertension, and cystic lung disease. She had all the major components of multisystemic smooth muscle dysfunction syndrome. Due to progressive respiratory deterioration, she required surgical PDA interruption, extracorporeal life support, and subsequent prolonged respiratory support. Genetic testing revealed ACTA2 R179H mutation and cystic lung disease on biopsy.

Microcystic stromal tumor (MST) is a rare tumor of presumed sex-cord stromal differentiation. We present a case of MST arising within a patient with constitutional 5q deletion syndrome, whose deletion encompassed the APC gene. Genomic analysis of the MST revealed a point mutation in the remaining APC allele, predicted to result in abnormal splicing of Exon 7. Subsequent clinical investigation revealed multiple gastrointestinal polyps qualifying for a diagnosis of familial adenomatous polyposis. This case emphasizes the importance of an aberrant Wnt/β-catenin pathway in the development of MST and adds credence to the inclusion of MST as a rare phenotype of familial adenomatous polyposis. In a search for additional genetic aberrations which may contribute to the development of this rare tumor, genomic analysis revealed a frameshift mutation in FANCD2, a protein which plays a key role in DNA repair. This protein is expressed in human ovarian stromal cells and FANCD2-knockout mice are known to develop sex cord-stromal tumors, factors which further support a possible role of aberrant FANCD2 in the development of MST.

Background Many women who develop endometrial cancer (EC) or endometrial hyperplasia with atypia are obese and therefore at high risk of surgical complications. Recently clinical trials have been initiated offering non-surgical treatment to these women, but not all may agree to participate in such trials. This paper aims to describe the patient characteristics, and surgical outcomes of women with suspected early stage endometrial cancer and body mass index (BMI) of 30 or greater, who declined enrolment in the feMMe trial, which offers non-surgical hormonal treatment, hormonal plus metformin or hormonal plus weight loss as primary treatment. Methods Consecutive case series from a tertiary gynaecological oncology unit. Over the course of the first 2 years of the feMMe trial, 27 patients met the initial eligibility screening, but declined enrolment in the feMMe trial and opted for upfront surgery. The main surgical outcome measures were type of surgical approach, need for conversion from laparoscopic to open approach, length of stay in hospital and adverse events. Results Patients’ median age was 63 years (range 40 to 86); median BMI was 37.3 kg/m2 (range 30.7 to 54.7); median medical co-morbidities were six (range 3–10). Of the 26/27 surgeries planned to be undertaken laparoscopically, 2/26 patients had to be converted (7 %). Overall, the average hospital stay was 4.5 days, and 11/27 (41 %) of the patients developed one or more adverse events grade 2+ rated according to the Common Toxicity Criteria Version 3. Conclusions Adverse surgical outcomes are common in multi-morbid, obese or morbidly obese patients diagnosed with early stage EC or endometrial hyperplasia with atypia and who have a hysterectomy.

To determine the factors associated with symptom-detected breast cancers in a population offered screening.We interviewed 1,459 Australian women aged 40-69, 946 with symptom-detected and 513 with mammogram-detected invasive breast cancers ≥ 1.1 cm in diameter about their personal, mammogram, and breast histories before diagnosis and reviewed medical records for tumor characteristics and mammogram dates, calculating ORs and 95% confidence intervals (CIs) for symptom- versus mammogram-detected cancers in logistic regression models.Lack of regular mammograms (2 mammograms in the 4.5 years before diagnosis) was the strongest correlate of symptom-detected breast cancer (OR = 3.04 for irregular or no mammograms). In women who had regular mammograms (≥ 2 mammograms in the 4.5 years before diagnosis), the independent correlates of symptom-detected cancers were low BMI (OR25 kg/m(2) vs. ≥ 30 kg/m(2) = 2.18, 95% CI 1.23-3.84; p = 0.008), increased breast density (available in 498 women) (OR highest quarter vs. lowest = 3.50, 95% CI 1.76-6.97; p (trend) = 0.004), high-grade cancer, and a larger cancer (each p0.01). In women who did not have regular mammograms, the independent correlates were age50 years, a first cancer, and a ≥ 2-cm cancer. Smoking appeared to modify the association of symptom-detected cancer with low BMI (higher ORs for low BMI in current smokers) and estrogen receptor (ER) status (higher ORs for low BMI in ER cancers).Women with low BMI may benefit from a tailored approach to breast cancer detection, particularly if they smoke.

The IL-6 cytokine family, which signals via the shared gp130 coreceptor, is linked with the pathogenesis of emphysema. However, the definitive mechanisms by which these cytokines cause emphysema remain ill-defined. We took an in vivo genetic complementation approach to identify the specific IL-6 cytokine family members and gp130-regulated cellular processes that cause emphysema. We used gp130(F/F) mice homozygous for a subtle knock-in mutation in gp130 that deregulates intracellular signaling by the IL-6 cytokine family. The gp130(F/F) mice spontaneously develop emphysema by age 6 months. Within the IL-6 cytokine family, only IL-6 was significantly up-regulated in the lungs of gp130(F/F) mice, and the genetic targeting of IL-6 in gp130(F/F) mice (gp130(F/F):IL-6(-/-)) prevented emphysema. By contrast, the genetic ablation of receptor signaling via IL-11, which like IL-6 signals via a gp130 homodimer and uses the same signaling machinery, failed to ameliorate emphysema in gp130(F/F) mice. Among the disease-associated processes examined, emphysema strongly correlated with elevated alveolar cell apoptosis. Acute (4-day) exposure to cigarette smoke (CS) further augmented the expression of IL-6 in lungs of gp130(F/F) mice, and subchronic (6-week) exposure to CS exacerbated emphysematous and apoptotic changes in the lungs of gp130(F/F) but not gp130(F/F): IL-6(-/-) mice. IL-6 is the main causative agent of IL-6 cytokine family-induced emphysema, and operates to induce apoptosis in the lung. We propose that the discrete targeting of IL-6 signaling may provide an effective therapeutic strategy against human lung disease.

Alternatively activated M2 macrophages are implicated as both regulators and agents of lung disease, but their control is poorly understood. SHIP-1 is a 5′ inositol phosphatase that negatively regulates the PI3K signaling pathway implicated in inflammation. SHIP-1–deficient mice have defects in hematopoiesis and B cell development, and die prematurely due to consolidation of lungs with M2-skewed macrophages. SHIP-1 is thought to restrain M2 macrophage polarization, with deregulated M2 skewing coinciding with severe lung disease in SHIP-1–deficient mice. To determine the influence of genetic background on the lung phenotype in SHIP-1−/− mice, we backcrossed the SHIP-1 null mutation onto C57BL/6 (Th2-resistant) and BALB/c (Th2-prone) backgrounds. Remarkably, we found that inflammatory lung disease was severe in C57.SHIP-1−/− mice, but absent in BALB.SHIP-1−/− mice. C57.SHIP-1−/−, but not BALB.SHIP-1−/− mice had greatly increased myeloid progenitors, myeloid hyperplasia, markedly enhanced numbers of activated alveolar macrophages, and elevated amounts of Th2 and proinflammatory cytokines in bronchoalveolar lavage fluid and serum, suggesting that deregulated cytokine production induced disease. C57.SHIP-1−/− mice also developed severe B cell-dependent autoimmune disease, which was markedly attenuated on the BALB/c background. These data demonstrate that, contrary to current concepts, loss of SHIP-1 alone is not sufficient to cause lung inflammation, with disease only manifest on a permissive genetic background. This finding questions the nature of the lung disease in SHIP-1−/− mice, suggesting that its M2 classification is not strictly correct. Future identification of disease-promoting loci might reveal determinants of comorbid lung disease and autoimmunity and uncover potentially useful therapeutic targets.

In August 2006, the Australian government announced a decision to subsidize trastuzumab therapy for early breast cancer, to commence 6 weeks later. It was mandated that HER2 gene amplification, determined by in situ hybridization (ISH), be shown, and that the sponsor company, Roche Products Pty Ltd, should fund this testing. This announcement potentially required provision of ISH testing for HER2 for every newly diagnosed breast cancer, where previously HER2 testing had been performed by immunohistochemistry with support from a single fluorescence ISH (FISH) reference laboratory for indeterminate cases. The Australian HER2 Testing Advisory Board, an independent expert group, responded to the challenge of rapidly providing accurate nationwide ISH testing. Bright-field ISH was selected as the testing platform and a decentralized testing model, with support from a central FISH laboratory, was adopted. An implementation plan was developed addressing standards for training, accreditation, and quality assurance. Within 6 weeks, 8 pathology laboratories were accredited for ISH testing and by September 2008, 2 years after the announcement, 22 ISH testing laboratories were taking part in the national program and almost 20,000 ISH tests had been performed. This article describes the design and rapid implementation of a nationwide program of bright-field ISH as the first-line testing platform for HER2 status in early breast cancer. We believe that this model for the coordinated and large-scale implementation of a new biomarker test has wide application, given that accurate assessment of a range of novel biomarkers is being used increasingly to determine eligibility for new targeted treatment modalities.

There is a high frequency of serous gynaecological and basal breast cancers in patients with germline BRCA1 mutations. These patients often undergo prophylactic salpingo-oophorectomies, where small tumours may be identified in which morphological distinction between primary serous and metastatic basal breast cancer may be problematic. These two cancer types share similar molecular genetics and have immunophenotypic overlap. We aimed to develop an immunohistochemical panel which could differentiate between these two tumour types.Immunohistochemistry was performed on a training set of 20 serous gynaecological and 20 basal breast cancers, from which a small differential panel was developed. This differential panel was tested in an additional validation set of serous and basal cancers.WT1, ER and CK5/6 expression successfully differentiated serous gynaecological and basal breast cancers. WT1 and ER expression was significantly more common in serous cancers and CK5/6 expression more common in basal cancers.There is considerable genetic, morphological and immunophenotypic overlap between serous gynaecological and basal breast cancers. A limited panel of WT1, ER and CK5/6 was found to optimally differentiate these tumour types. Differences in the expression of these proteins appear to reflect the expression pattern of their cell of origin, rather than identify aberrant oncogenic pathways.

Ovarian cancer is often asymptomatic and is diagnosed at an advanced stage with poor survival rates, thus there is an urgent need to develop biomarkers for earlier detection of ovarian cancer. In the present study, we demonstrate for the first time that the previously reported metastasis-inducing protein AGR2 (anterior gradient protein 2) can be detected in the blood of ovarian cancer patients. Using a newly developed ELISA, we show significantly increased concentrations of AGR2 protein in plasma from cancer patients relative to normal controls. Plasma AGR2 concentrations were highest in stages II and III ovarian cancer patients and were similarly elevated in patients with both serous and non-serous tumours. The identification of elevated plasma concentrations of AGR2 may provide a useful biomarker to aid in the discrimination of normal and ovarian cancer patients particularly when used in combination with CA125.

Rates of large breast cancers should decrease in a population that is offered mammography screening, but women continue to present with them. We sought an explanation in a population-based epidemiological study of 1,459 women diagnosed with invasive breast cancer in 2002-2003 in Australia; breast cancers wereor =2 cm in 766 women (53%) and 11-1.9 cm in a comparison group (693, 47%). We interviewed the women about their personal, mammogram and breast histories in the years before diagnosis and collected biological characteristics of tumors and mammogram dates from medical records. The strongest correlate of breast cancer size at diagnosis was the method of detection: the odds of aor =2 cm breast cancer was substantially lower for detection by a screening mammogram (OR = 0.27, 95% CI 0.21-0.34; p0.001) than for detection after a breast symptom. Higher BMI (ORs approximately 1.6 foror =25 kg/m(2)), higher cancer grade (ORs of 1.6 for moderate, 2.89 for high grade) and lobular type (OR 2.09, 95% CI 1.45-3.0) were also independent correlates (p0.001) of aor =2 cm breast cancer. HRT use strongly reduced the odds but only in cancers detected after a breast symptom (OR = 0.49, 95% CI 0.33-0.74; p = 0.002), not in those detected by a screening mammogram. As assessed from their proportional contribution toor =2 cm breast cancers in our study population, lack of mammogram detection, BMIor =25 kg/m(2) and moderate or high grade of the cancer were the most important factors with population attributable fractions of 42%, 11% and 29% respectively; the first 2 are amenable to intervention.

To examine the contribution of life event and social support factors to diagnosis with aor= 2 cm breast cancer.We studied 1,459 Australian women aged 40-69 diagnosed in 2002-2003 with a first primary invasive breast cancer 1.1 cm or larger. We measured stressful life events, perceived stress levels, and social support in the year before diagnosis and collected information on other potential risk factors and confounders.The odds of aor= 2 cm breast cancer relative to a 1.1-1.9 cm breast cancer were reduced in women who reported tension or change in an intimate relationship in the year before diagnosis (OR=0.71 95% CI 0.54-0.92; p=0.009); the reduction was greatest in women living with a partner (OR=0.64 95% CI 0.47-0.88; p=0.006) and was largely unaffected by adjustment for other variables independently associated with aor= 2 cm breast cancer in our study. There was no evidence that the total number or severity of all studied life events influenced cancer size. Low partner support increased the odds of aor= 2 cm cancer but only in women not living with a partner.Intimate relationship stress may reduce risk of aor= 2 cm breast cancer. Suppression by stress of estrogen synthesis and metabolism is a possible mechanism.

Pagetoid squamous cell carcinoma in situ (PSSCIS) is a variant of squamous cell intraepithelial neoplasia. Although PSSCIS is well-documented in the cutaneous skin and esophagus, cases of vulvar PSSCIS are rare. In the vulva, the main differential diagnosis is extramammary Paget disease (EMPD). We report 2 cases of vulvar PSSCIS along with the immunohistochemical and human papillomavirus (HPV) status of this disease compared with primary cutaneous EMPD of the vulva. Although PSSCIS and EMPD share CK7 and CK19 expression, PSSCIS is consistently mucin and carcinoembryonic antigen negative. In contrast to EMPD, both cases of PSSCIS strongly expressed p16 (INK4A) protein, consistent with RB1 protein dysregulation. However, integration of high-risk HPV was found in only 1 of the 2 PSSCIS cases. Given the morphological and immunohistochemical findings, we suggest that PSSCIS arises from a bidirectional stem cell capable of both squamous and glandular differentiation. Additionally, as with nonpagetoid squamous cell neoplasias of the vulvar, integration of high-risk HPV may occur in some, but not all, cases of PSSCIS.

Childhood Interstitial lung disease (chILD) is an umbrella term used to define a broad range of rare, diffuse pulmonary disorders with altered interstitial structure that leads to abnormal gas exchange. Presentation of chILD in infancy can be difficult to differentiate from other common causes of diffuse lung disease. This article aimed at paediatricians provides an overview of interstitial lung disease presenting in infancy and includes key clinical features, a suggested approach to investigation and a summary of management. An overview of three clinical cases has been included to demonstrate the diagnostic approach, characteristic investigation findings and varied clinical outcomes.

An unusual anatomic configuration of segmental tracheal agenesis/atresia with esophageal duplication on autopsy in a fetus that demised in utero at 29 weeks is reported. The mother was scanned initially for a cardiac anomaly at 20 weeks and on follow-up scan at 27 weeks had polyhydramnios and underwent amnioreduction. The final autopsy diagnosis was vertebral, ano-rectal, cardiac, tracheoesophageal, renal, and limb malformations (VACTERL). We discuss the autopsy findings along with the embryological mechanisms and compare the configuration with Floyd's classification for tracheal agenesis. The difficulties in prenatal diagnosis are discussed.

A comprehensive differential gene expression screen on a panel of 54 breast tumors and >200 normal tissue samples using DNA microarrays revealed 15 genes specifically overexpressed in breast cancer. One of the most prevalent genes found was trichorhinophalangeal syndrome type 1 (TRPS-1), a gene previously shown to be associated with three rare autosomal dominant genetic disorders known as the trichorhinophalangeal syndromes. A number of corroborating methodologies, includingin situhybridization, e-Northern analysis using ORF EST (ORESTES) and Unigene EST abundance analysis, immunoblot and immunofluorescence analysis of breast tumor cell lines, and immunohistochemistry, confirmed the microarray findings. Immunohistochemistry analysis found TRPS-1 protein expressed in >90% of early- and late-stage breast cancer, including ductal carcinomain situand invasive ductal, lobular, and papillary carcinomas. The TRPS-1 gene is also immunogenic with processed and presented peptides activating T cells found after vaccination of HLA-A2.1 transgenic mouse. Human T cell lines from HLA-A*0201+female donors exhibiting TRPS-1-specific cytotoxic T lymphocyte activity could also be generated.

Recently, the tissue origin of MDA-MB-435 cell line has been the subject of considerable debate. In this study, we set out to determine whether MDA-MB-435-DTP cells shown to express melanoma-specific genes were identical to various other MDA-MB-435 cell stocks worldwide. CGH-microarray, genetic polymorphism genotyping, microsatellite fingerprint analysis and/or chromosomal number confirmed that the MDA-MB-435 cells maintained at the Lombardi Comprehensive Cancer Center (MDA-MB-435-LCC) are almost identical to the MDA-MB-435-DTP cells, and showed a very similar profile to those obtained from the same original source (MD Anderson Cancer Center) but maintained independently (MDA-MB-435-PMCC). Gene expression profile analysis confirmed common expression of genes among different MDA-MB-435-LCC cell stocks, and identified some unique gene products in MDA-MB-435-PMCC cells. RT-PCR analysis confirmed the expression of the melanoma marker tyrosinase across multiple MDA-MB-435 cell stocks. Collectively, our results show that the MDA-MB-435 cells used widely have identical origins to those that exhibit a melanoma-like gene expression signature, but exhibit a small degree of genotypic and phenotypic drift.

Loss of genetic material from chromosome arm 8p occurs commonly in breast carcinomas, suggesting that this region is the site of one or more tumor-suppressor genes (TSGs). Comparative genomic hybridization analysis showed that 8p loss is more common in breast cancers from pre-menopausal compared with post-menopausal patients, as well as in high-grade breast cancers, regardless of the menopausal status. Subsequent high-resolution gene expression profiling of genes mapped to chromosome arm 8p, on an extended cohort of clinical tumor samples, indicated a similar dichotomy of breast cancer clinicopathologic types. Some of these genes showed differential downregulation in early-onset and later-onset, high-grade cancers compared with lower-grade, later-onset cancers. Three such genes were analysed further by in situ technologies, performed on tissue microarrays representing breast tumor and normal tissue samples. PCM1, which encodes a centrosomal protein, and DUSP4/MKP-2, which encodes a MAP kinase phosphatase, both showed frequent gene and protein loss in carcinomas. In contrast, there was an excess of cases showing loss of expression in the absence of reduced gene copy number of SFRP1, which encodes a dominant-negative receptor for Wnt-family ligands. These candidate TSGs may constitute some of the molecular drivers of chromosome arm 8p loss in breast carcinogenesis.

The incidence of ductal carcinoma in situ (DCIS) has increased substantially since the advent of widespread screening mammography. Identification of histological markers that predict recurrent disease is essential for optimal treatment management. To assist the clinico-pathological stratification of DCIS, we sought to determine histological markers of recurrence in DCIS in an Australian population-based series.In a study of all DCIS reported in Victoria between 1988 and 1992, managed by breast conserving therapy (wide local excision or subtotal mastectomy) with or without adjuvant radiotherapy and/or hormonal therapy, the histological features of DCIS lesions with subsequent ipsilateral recurrence as in situ or invasive breast cancer were compared with a similarly managed control group of DCIS without recurrence.Large lesion size, presence of nuclear pleomorphism, absence of cellular polarisation and extensive necrosis were all significant predictors of recurrence (P0.05). Primary and recurrent DCIS lesions had similar morphological features, and invasive recurrence was characterised by ductal type with high nuclear grade.This study identifies histological markers in DCIS associated with recurrence in an Australian population, and demonstrates similar histological appearances between primary and secondary lesions. These histological characteristics may be used to stratify DCIS subtypes and facilitate the future optimisation of disease management.

To define the location of potential oncogenes and tumor suppressor genes in ocular melanoma we carried out comparative genomic hybridization (CGH) analysis on a population-based series of 25 formalin-fixed, paraffin-embedded primary tumors comprising 17 choroidal, 2 ciliary body, 4 iris, and 2 conjunctival melanomas. Twelve (48%) of the 25 melanomas showed no chromosomal changes and 13 (52%) had at least one chromosomal gain or loss. The mean number of CGH changes in all tumors was 3.3, with similar mean numbers of chromosomal gains (1.5) and losses (1.8). The highest number of chromosomal changes (i.e., nine) occurred in a conjunctival melanoma and included four changes not observed in tumors at any other ocular site (gains in 22q and 11p and losses in 6p and 17p). The most frequent gains in all primary ocular melanomas were on chromosome arm 8q (69%), 6p (31%) and 8p (23%) and the most frequent losses were on 6q (38%), 10q (23%), and 16q (23%). The most common pairing was gain in 8p and gain in 8q, implying a whole chromosome copy number increase; gains in 8p occurred only in conjunction with gains in 8q. The smallest regions of copy number alteration were mapped to gain of 8q21 and loss of 6q21, 10q21, and 16q22. Sublocalization of these chromosomal changes to single-band resolution should accelerate the identification of genes involved in the genesis of ocular melanoma.

Genetic testing for cancer predisposing mutations in BRCA1 and BRCA2 has been of benefit to many individuals from breast and ovarian cancer-prone kindreds. However, a function has not been assigned to many of the domains that make up these complex proteins and hence, the significance of many sequence variants, including missense mutations, splice-site mutations, and in-frame deletions/insertions, remains unclear. We identified a putative splice site mutation (IVS6-2delA) in BRCA1 in a family attending a Familial Cancer Centre that had a significant history of both breast and ovarian cancer. This sequence variant was not novel but the exact effect on mRNA splicing and hence the biological impact of this sequence variation was unclear and therefore the finding was unable to be used in genetic counseling of the family. Via the construction of novel GFP-based expression fusion constructs, we demonstrated that this sequence variation prevented normal splicing of the BRCA1 transcript. By combining these data with an assessment of the histopathological features of the breast carcinomas in this family and mutation penetrance estimate we were able to conclude that this BRCA1 variant conveyed an increased risk of breast cancer.

The tests that are currently available for the measurement of overexpression of the human epidermal growth factor-2 (HER2) in breast cancer have shown considerable problems in accuracy and interlaboratory reproducibility. Although these problems are partly alleviated by the use of validated, standardised 'kits', there may be considerable cost involved in their use. Prior to testing it may therefore be an advantage to be able to predict from basic pathology data whether a cancer is likely to overexpress HER2. In this study, we have correlated pathology features of cancers with the frequency of HER2 overexpression assessed by immunohistochemistry (IHC) using HercepTest (Dako). In addition, fluorescence in situ hybridisation (FISH) has been used to re-test the equivocal cancers and interobserver variation in assessing HER2 overexpression has been examined by a slide circulation scheme. Of the 1536 cancers, 1144 (74.5%) did not overexpress HER2. Unequivocal overexpression (3+ by IHC) was seen in 186 cancers (12%) and an equivocal result (2+ by IHC ) was seen in 206 cancers (13%). Of the 156 IHC 3+ cancers for which complete data was available, 149 (95.5%) were ductal NST and 152 (97%) were histological grade 2 or 3. Only 1 of 124 infiltrating lobular carcinomas (0.8%) showed HER2 overexpression. None of the 49 'special types' of carcinoma showed HER2 overexpression. Re-testing by FISH of a proportion of the IHC 2+ cancers showed that only 25 (23%) of those assessable exhibited HER2 gene amplification, but 46 of the 47 IHC 3+ cancers (98%) were confirmed as showing gene amplification. Circulating slides for the assessment of HER2 score showed a moderate level of agreement between pathologists (kappa 0.4). As a result of this study we would advocate consideration of a triage approach to HER2 testing. Infiltrating lobular and special types of carcinoma may not need to be routinely tested at presentation nor may grade 1 NST carcinomas in which only 1.4% have been shown to overexpress HER2. Testing of these carcinomas may be performed when HER2 status is required to assist in therapeutic or other clinical/prognostic decision-making. The highest yield of HER2 overexpressing carcinomas is seen in the grade 3 NST subgroup in which 24% are positive by IHC.

The optimal management of ductal carcinoma in situ (DCIS) is controversial, due in part to our poor understanding of its natural history. We undertook to identify subgroups of DCIS based on the expression of biomarkers, which were related to the likelihood of clinical recurrence. Biomarker expression of a total of 95 DCIS lesions in a nested case–control study within a population-based cohort with up to 135 months follow-up data (median 101 months) was analysed using immunohistochemistry. ERBB2-positivity and bcl-2-, oestrogen receptor (ER)- and progesterone receptor (PR)-negativity were individually associated with the risk of clinical recurrence. The predictive value of these biomarkers was independent of cytonuclear grade. ERBB2, bcl-2, ER and PR expression were conserved in the recurrent lesions, including subsequent invasive cancers. p21-positive DCIS was also associated with clinical recurrence, independently of the associations with ERBB2/bcl-2/ER/PR expression. These data identify clinically and biologically relevant subcategories of DCIS lesions, an essential basis for improving management.

Genetic ablation of the Lyn tyrosine kinase has revealed unique inhibitory roles in B lymphocyte signaling. We now report the consequences of sustained activation of Lyn in vivo using a targeted gain-of-function mutation (Lynup/up mice). Lynup/up mice have reduced numbers of conventional B lymphocytes, down-regulated surface immunoglobulin M and costimulatory molecules, and elevated numbers of B1a B cells. Lynup/up B cells are characterized by the constitutive phosphorylation of negative regulators of B cell antigen receptor (BCR) signaling including CD22, SHP-1, and SHIP-1, and display attributes of lymphocytes rendered tolerant by constitutive engagement of the antigen receptor. However, exaggerated positive signaling is also apparent as evidenced by the constitutive phosphorylation of Syk and phospholipase Cγ2 in resting Lynup/up B cells. Similarly, Lynup/up B cells show a heightened calcium flux in response to BCR stimulation. Surprisingly, Lynup/up mice develop circulating autoreactive antibodies and lethal autoimmune glomerulonephritis, suggesting that enhanced positive signaling eventually overrides constitutive negative signaling. These studies highlight the difficulty in maintaining tolerance in the face of chronic stimulation and emphasize the pivotal role of Lyn in B cell signaling.

Squamous cell carcinoma of the vulva is a disease of significant clinical importance, which arises in the presence or absence of human papillomavirus. We used comparative genomic hybridisation to document non-random chromosomal gains and losses within human papillomavirus positive and negative vulvar cancers. Gain of 3q was significantly more common in human papillomavirus-positive cancers compared to human papillomavirus-negative cancers. The smallest area of gain was 3q22–25, a chromosome region which is frequently gained in other human papillomavirus-related cancers. Chromosome 8q was more commonly gained in human papillomavirus-negative compared to human papillomavirus-positive cancers. 8q21 was the smallest region of gain, which has been identified in other, non-human papillomavirus-related cancers. Chromosome arms 3p and 11q were lost in both categories of vulvar cancer. This study has demonstrated chromosome locations important in the development of vulvar squamous cell carcinoma. Additionally, taken together with previous studies of human papillomavirus-positive cancers of other anogenital sites, the data indicate that one or more oncogenes important in the development and progression of human papillomavirus-induced carcinomas are located on 3q. The different genetic changes seen in human papillomavirus-positive and negative vulvar squamous cell carcinomas support the clinicopathological data indicating that these are different cancer types. British Journal of Cancer (2002) 86, 924–928. DOI: 10.1038/sj/bjc/6600112 www.bjcancer.com © 2002 Cancer Research UK