International survey on invasive lobular breast cancer identifies priority research questions.

There is growing awareness of the unique etiology, biology, and clinical presentation of invasive lobular breast cancer (ILC), but additional research is needed to ensure translation of findings into management and treatment guidelines. We conducted a survey with input from breast cancer physicians, laboratory-based researchers, and patients to analyze the current understanding of ILC, and identify consensus research questions. 1774 participants from 66 countries respondents self-identified as clinicians (N = 413), researchers (N = 376), and breast cancer patients and advocates (N = 1120), with some belonging to more than one category. The majority of physicians reported being very/extremely (41%) to moderately (42%) confident in describing the differences between ILC and invasive breast cancer of no special type (NST). Knowledge of histology was seen as important (73%) and as affecting treatment decisions (51%), and most agreed that refining treatment guidelines would be valuable (76%). 85% of clinicians have never powered a clinical trial to allow subset analysis for histological subtypes, but the majority would consider it, and would participate in an ILC clinical trials consortium. The majority of laboratory researchers, reported being and very/extremely (48%) to moderately (29%) confident in describing differences between ILC and NST. They reported that ILCs are inadequately presented in large genomic data sets, and that ILC models are insufficient. The majority have adequate access to tissue or blood from patients with ILC. The majority of patients and advocates (52%) thought that their health care providers did not sufficiently explain the unique features of ILC. They identified improvement of ILC screening/early detection, and identification of better imaging tools as top research priorities. In contrast, both researchers and clinicians identified understanding of endocrine resistance and identifying novel drugs that can be tested in clinical trials as top research priority. In summary, we have gathered information from an international community of physicians, researchers, and patients/advocates that we expect will lay the foundation for a community-informed collaborative research agenda, with the goal of improving management and personalizing treatment for patients with ILC.

Reporting on invasive lobular breast cancer in clinical trials: a systematic review.

Invasive lobular breast cancer (ILC) differs from invasive breast cancer of no special type in many ways. Evidence on treatment efficacy for ILC is, however, lacking. We studied the degree of documentation and representation of ILC in phase III/IV clinical trials for novel breast cancer treatments. Trials were identified on Pubmed and clinicaltrials.gov. Inclusion/exclusion criteria were reviewed for requirements on histological subtype and tumor measurability. Documentation of ILC was assessed and ILC inclusion rate, central pathology and subgroup analyses were evaluated. Inclusion restrictions concerning tumor measurability were found in 39/93 manuscripts. Inclusion rates for ILC were documented in 13/93 manuscripts and varied between 2.0 and 26.0%. No central pathology for ILC was reported and 3/13 manuscripts had ILC sub-analyses. ILC is largely disregarded in most trials with poor representation and documentation. The current inclusion criteria using RECIST v1.1, fall short in recognizing the unique non-measurable metastatic infiltration of ILC.

CD9 co-operation with syndecan-1 is required for a major staphylococcal adhesion pathway.

Epithelial colonization is a critical first step in bacterial pathogenesis. Staphylococcus aureus can utilize several host factors to associate with cells, including α5ß1 integrin and heparan sulfate proteoglycans, such as the syndecans. Here, we demonstrate that a partner protein of both integrins and syndecans, the host membrane adapter protein tetraspanin CD9, is essential for syndecan-mediated staphylococcal adhesion. Fibronectin is also essential in this process, while integrins are only critical for post-adhesion entry into human epithelial cells. Treatment of epithelial cells with CD9-derived peptide or heparin caused significant reductions in staphylococcal adherence, dependent on both CD9 and syndecan-1. Exogenous fibronectin caused a CD9-dependent increase in staphylococcal adhesion, whereas blockade of ß1 integrins did not affect adhesion but did reduce the subsequent internalization of adhered bacteria. CD9 disruption or deletion increased ß1 integrin-mediated internalization, suggesting that CD9 coordinates sequential staphylococcal adhesion and internalization. CD9 controls staphylococcal adhesion through syndecan-1, using a mechanism that likely requires CD9-mediated syndecan organization to correctly display fibronectin at the host cell surface. We propose that CD9-derived peptides or heparin analogs could be developed as anti-adhesion treatments to inhibit the initial stages of staphylococcal pathogenesis. IMPORTANCE Staphylococcus aureus infection is a significant cause of disease and morbidity. Staphylococci utilize multiple adhesion pathways to associate with epithelial cells, including interactions with proteoglycans or ß1 integrins through a fibronectin bridge. Interference with another host protein, tetraspanin CD9, halves staphylococcal adherence to epithelial cells, although CD9 does not interact directly with bacteria. Here, we define the role of CD9 in staphylococcal adherence and uptake, observing that CD9 coordinates syndecan-1, fibronectin, and ß1 integrins to allow efficient staphylococcal infection. Two treatments that disrupt this action are effective and may provide an alternative to antibiotics. We provide insights into the mechanisms that underlie staphylococcal infection of host cells, linking two known adhesion pathways together through CD9 for the first time.

Multi-Attribute Monitoring Method for Process Development of Engineered Antibody for Site-Specific Conjugation.

Antibody drug conjugates, a class of biotherapeutic proteins, have been extensively developed in recent years, resulting in new approvals and improved standard of care for cancer patients. Among the numerous strategies of conjugating cytotoxic payloads to monoclonal antibodies, insertion of a cysteine residue achieves a tightly controlled, site-specific drug to antibody ratio. Tailored analytical tools are required to direct the development of processes capable of manufacturing novel antibody scaffolds with the desired product quality. Here, we describe the development of a 12 min, mass-spectrometry-based method capable of monitoring four distinct quality attributes simultaneously: variations in the thiol state of the inserted cysteines, N-linked glycosylation, reduction of interchain disulfide bonds, and polypeptide fragmentation. This method provides new insight into the properties of the antibody intermediate and associated manufacturing processes. Oxidized thiol states are formed within the bioreactor, of which a variant containing an additional disulfide bond was produced and remained relatively constant throughout the fed-batch process; reduced thiol variants were introduced upon harvest. Nearly 20 percent of N-linked glycans contained sialic acid, substantially higher than anticipated for wildtype IgG1. Lastly, previously unreported polypeptide fragmentation sites were identified in the C239i constant domain, and the relationship between fragmentation and glycoform were explored. This work illustrates the utility of applying a high-throughput liquid chromatography-mass spectrometry multi-attribute monitoring method to support the development of engineered antibody scaffolds.

Urinary Volatile Organic Compound Testing in Fast-Track Patients with Suspected Colorectal Cancer.

Colorectal symptoms are common but only infrequently represent serious pathology, including colorectal cancer (CRC). A large number of invasive tests are presently performed for reassurance. We investigated the feasibility of urinary volatile organic compound (VOC) testing as a potential triage tool in patients fast-tracked for assessment for possible CRC. A prospective, multi-center, observational feasibility study was performed across three sites. Patients referred to NHS fast-track pathways for potential CRC provided a urine sample that underwent Gas Chromatography-Mass Spectrometry (GC-MS), Field Asymmetric Ion Mobility Spectrometry (FAIMS), and Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) analysis. Patients underwent colonoscopy and/or CT colonography and were grouped as either CRC, adenomatous polyp(s), or controls to explore the diagnostic accuracy of VOC output data supported by an artificial neural network (ANN) model. 558 patients participated with 23 (4%) CRC diagnosed. 59% of colonoscopies and 86% of CT colonographies showed no abnormalities. Urinary VOC testing was feasible, acceptable to patients, and applicable within the clinical fast track pathway. GC-MS showed the highest clinical utility for CRC and polyp detection vs. controls (sensitivity = 0.878, specificity = 0.882, AUROC = 0.896) but it is labour intensive. Urinary VOC testing and analysis are feasible within NHS fast-track CRC pathways. Clinically meaningful differences between patients with cancer, polyps, or no pathology were identified suggesting VOC analysis may have future utility as a triage tool.

How Researchers, Clinicians and Patient Advocates Can Accelerate Lobular Breast Cancer Research.

Breast cancer research and therapies have significantly advanced in recent years. However, Invasive Lobular Carcinoma (ILC), the second most common histological type of breast cancer and the sixth most frequently diagnosed cancer of women, has not always benefited from critical analysis, missing opportunities to better understand this important subtype. Recent progress understanding the biological and behavioral differences of ILC demonstrates that it is a unique subtype of breast cancer which can respond differently to common therapies. These new insights have increased interest in researching lobular breast disease. Concurrently, the formation of motivated patient-led advocacy organizations working in partnership with basic, translational and clinical researchers creates new opportunities, including connecting a dispersed patient population to research, encouraging new research funding and connecting patient advocates to researchers to advance common goals. This commentary will explore the unprecedented opportunity to drive multidisciplinary, multicenter and international collaborative research into lobular breast cancer that builds on recent research progress. Collaborative research partnerships that include advocates can result in a better understanding of ILC, identify targeted therapies and refine standard of care therapies that are currently equally applied to all breast cancers, resulting in improvements in the diagnosis, treatment and follow-up care for patients with ILC.

Extracellular bacterial lymphatic metastasis drives Streptococcus pyogenes systemic infection.

Unassisted metastasis through the lymphatic system is a mechanism of dissemination thus far ascribed only to cancer cells. Here, we report that Streptococcus pyogenes also hijack lymphatic vessels to escape a local infection site, transiting through sequential lymph nodes and efferent lymphatic vessels to enter the bloodstream. Contrasting with previously reported mechanisms of intracellular pathogen carriage by phagocytes, we show S. pyogenes remain extracellular during transit, first in afferent and then efferent lymphatics that carry the bacteria through successive draining lymph nodes. We identify streptococcal virulence mechanisms important for bacterial lymphatic dissemination and show that metastatic streptococci within infected lymph nodes resist and subvert clearance by phagocytes, enabling replication that can seed intense bloodstream infection. The findings establish the lymphatic system as both a survival niche and conduit to the bloodstream for S. pyogenes, explaining the phenomenon of occult bacteraemia. This work provides new perspectives in streptococcal pathogenesis with implications for immunity.

Mutations in NLRP5 are associated with reproductive wastage and multilocus imprinting disorders in humans.

Human-imprinting disorders are congenital disorders of growth, development and metabolism, associated with disturbance of parent of origin-specific DNA methylation at imprinted loci across the genome. Some imprinting disorders have higher than expected prevalence of monozygotic twinning, of assisted reproductive technology among parents, and of disturbance of multiple imprinted loci, for which few causative trans-acting mutations have been found. Here we report mutations in NLRP5 in five mothers of individuals affected by multilocus imprinting disturbance. Maternal-effect mutations of other human NLRP genes, NLRP7 and NLRP2, cause familial biparental hydatidiform mole and multilocus imprinting disturbance, respectively. Offspring of mothers with NLRP5 mutations have heterogenous clinical and epigenetic features, but cases include a discordant monozygotic twin pair, individuals with idiopathic developmental delay and autism, and families affected by infertility and reproductive wastage. NLRP5 mutations suggest connections between maternal reproductive fitness, early zygotic development and genomic imprinting.

Use of the Analysis of the Volatile Faecal Metabolome in Screening for Colorectal Cancer.

Diagnosis of colorectal cancer is an invasive and expensive colonoscopy, which is usually carried out after a positive screening test. Unfortunately, existing screening tests lack specificity and sensitivity, hence many unnecessary colonoscopies are performed. Here we report on a potential new screening test for colorectal cancer based on the analysis of volatile organic compounds (VOCs) in the headspace of faecal samples. Faecal samples were obtained from subjects who had a positive faecal occult blood sample (FOBT). Subjects subsequently had colonoscopies performed to classify them into low risk (non-cancer) and high risk (colorectal cancer) groups. Volatile organic compounds were analysed by selected ion flow tube mass spectrometry (SIFT-MS) and then data were analysed using both univariate and multivariate statistical methods. Ions most likely from hydrogen sulphide, dimethyl sulphide and dimethyl disulphide are statistically significantly higher in samples from high risk rather than low risk subjects. Results using multivariate methods show that the test gives a correct classification of 75% with 78% specificity and 72% sensitivity on FOBT positive samples, offering a potentially effective alternative to FOBT.

Analysis of volatile organic compounds of bacterial origin in chronic gastrointestinal diseases.

BACKGROUND: The aim of this study was to determine whether volatile organic compounds (VOCs) present in the headspace of feces could be used to diagnose or distinguish between chronic diseases of the gastrointestinal tract and apparently healthy volunteers. METHODS: A total of 87 people were recruited, divided between 4 categories: healthy volunteers (n = 19), Crohn's disease (n = 22), ulcerative colitis (n = 20), and irritable bowel syndrome (n = 26). They each supplied fecal samples before, and except for the healthy volunteers, after treatment. Fecal samples were incubated in a sample bag with added purified air at 40°C and headspace samples were taken and concentrated on thermal sorption tubes. Gas chromatography-mass spectrometry then desorbed and analyzed these. The concentrations of a selection of high-abundance compounds were determined and assessed for differences in concentration between the groups. RESULTS: Crohn's disease samples showed significant elevations in the concentrations of ester and alcohol derivates of short-chain fatty acids and indole compared with the other groups; indole and phenol were elevated in ulcerative colitis and irritable bowel syndrome but not at a statistically significant level. After treatment, the levels of many of the VOCs were significantly reduced and were more similar to those concentrations in healthy controls. CONCLUSIONS: The abundance of a number of VOCs in feces differs markedly between Crohn's disease and other gastrointestinal conditions. Following treatment, the VOC profile is altered to more closely resemble that of healthy volunteers.

Dominant missense mutations in ABCC9 cause Cantú syndrome.

Cantú syndrome is characterized by congenital hypertrichosis, distinctive facial features, osteochondrodysplasia and cardiac defects. By using family-based exome sequencing, we identified a de novo mutation in ABCC9. Subsequently, we discovered novel dominant missense mutations in ABCC9 in 14 of the 16 individuals with Cantú syndrome examined. The ABCC9 protein is part of an ATP-dependent potassium (K(ATP)) channel that couples the metabolic state of a cell with its electrical activity. All mutations altered amino acids in or close to the transmembrane domains of ABCC9. Using electrophysiological measurements, we show that mutations in ABCC9 reduce the ATP-mediated potassium channel inhibition, resulting in channel opening. Moreover, similarities between the phenotype of individuals with Cantú syndrome and side effects from the K(ATP) channel agonist minoxidil indicate that the mutations in ABCC9 result in channel opening. Given the availability of ABCC9 antagonists, our findings may have direct implications for the treatment of individuals with Cantú syndrome.

Cantú syndrome: report of nine new cases and expansion of the clinical phenotype.

Cantú syndrome, a rare disorder of congenital hypertrichosis, characteristic facial anomalies, cardiomegaly, and osteochondrodysplasia was first described in 1982 by Cantú. Twenty-three cases of Cantú syndrome have been reported to date. The pathogenesis of this rare autosomal dominant condition is unknown. We describe 10 patients with Cantú syndrome (9 new cases and the long-term follow-up of a 10th case reported by Robertson in 1999) comparing the phenotype with that of the previously reported cases. We describe how the distinctive facial appearance evolves with time and report several new findings including recurrent infections with low immunoglobulin levels and gastric bleeding in some of our patients. The cardiac manifestations include patent ductus arteriosus, septal hypertrophy, pulmonary hypertension, and pericardial effusions. They may follow a benign course, but of the 10 cases we report, 4 patients required surgical closure of the patent ductus arteriosus and 1 patient a pericardectomy. Long-term follow-up of these patients has shown reassuring neuro-developmental outcome and the emergence of a behavior phenotype including obsessive traits and anxiety.

Evaluation of a gas sensor array and pattern recognition for the identification of bladder cancer from urine headspace.

Previous studies have indicated that volatile compounds specific to bladder cancer may exist in urine headspace, raising the possibility that headspace analysis could be used for diagnosis of this particular cancer. In this paper, we evaluate the use of a commercially available gas sensor array coupled with a specifically designed pattern recognition algorithm for this purpose. The best diagnostic performance that we were able to obtain with independent test data provided by healthy volunteers and bladder cancer patients was 70% overall accuracy (70% sensitivity and 70% specificity). When the data of patients suffering from other non-cancerous urological diseases were added to those of the healthy controls, the classification accuracy fell to 65% with 60% sensitivity and 67% specificity. While this is not sufficient for a diagnostic test, it is significantly better than random chance, leading us to conclude that there is useful information in the urine headspace but that a more informative analytical technique, such as mass spectrometry, is required if this is to be exploited fully.

Understanding the fate and transport of petroleum hydrocarbons from coal tar within gasholders.

Coal tars have been identified as posing a threat to human health due to their toxic, mutagenic and carcinogenic characteristics. Workers involved in former gasholders decommissioning are potentially exposed to relevant concentrations of volatile and semi-volatile hydrocarbons upon opening up derelict tanks and during tar excavation/removal. While information on contaminated sites air-quality and its implications on medium-long term exposure is available, acute exposure issues associated with the execution of critical tasks are less understood. Calculations indicated that the concentration of a given contaminant in the gasholder vapour phase only depends on the coal tar composition, being only barely affected by the presence of water in the gasholder and the tar volume/void space ratio. Fugacity modelling suggested that risk-critical compounds such as benzene, naphthalene and other monocyclic and polycyclic aromatic hydrocarbons may gather in the gasholder air phase at significant concentrations. Gasholder emissions were measured on-site and compared with the workplace exposure limits (WELs) currently in use in UK. While levels for most of the toxic compounds were far lower than WELs, benzene air-concentrations where found to be above the accepted threshold. In addition due to the long exposure periods involved in gasholder decommissioning and the significant contribution given by naphthalene to the total coal tar vapour concentration, the adoption of a WEL for naphthalene may need to be considered to support operators in preventing human health risk at the workplace. The Level I fugacity approach used in this study demonstrated its suitability for applications to sealed environments such as gasholders and its further refining could provide a useful tool for land remediation risk assessors.

Mutations in phospholipase C epsilon 1 are not sufficient to cause diffuse mesangial sclerosis.

Diffuse mesangial sclerosis occurs as an isolated abnormality or as a part of a syndrome. Recently, mutations in phospholipase C epsilon 1 (PLCE1) were found to cause a nonsyndromic, autosomal recessive form of this disease. Here we describe three children from one consanguineous kindred of Pakistani origin with diffuse mesangial sclerosis who presented with congenital or infantile nephrotic syndrome. Homozygous mutations in PLCE1 (also known as KIAA1516, PLCE, or NPHS3) were identified following genome-wide mapping of single-nucleotide polymorphisms. All affected children were homozygous for a four-basepair deletion in exon 3, which created a premature translational stop codon. Analysis of the asymptomatic father of two of the children revealed that he was also homozygous for the same mutation. We conclude this nonpenetrance may be due to compensatory mutations at a second locus and that mutation within PLCE1 is not always sufficient to cause diffuse mesangial sclerosis.

A longitudinal study of ammonia, acetone and propanol in the exhaled breath of 30 subjects using selected ion flow tube mass spectrometry, SIFT-MS.

Selected ion flow tube mass spectrometry, SIFT-MS, has been used to monitor the volatile compounds in the exhaled breath of 30 volunteers (19 males, 11 females) over a 6 month period. Volunteers provided breath samples each week between 8:45 am and 1 pm (before lunch), and the concentrations of several trace compounds were obtained. In this paper the focus is on ammonia, acetone and propanol. It was found that the concentration distributions of these compounds in breath were close to log-normal. The median ammonia level estimated as a geometric mean for all samples was 833 parts per billion (ppb) with a multiplicative standard deviation of 1.62, the values ranging from 248 to 2935 ppb. Breath ammonia clearly increased with increasing age in this volunteer cohort. The geometric mean acetone level for all samples was 477 parts per billion (ppb) with a multiplicative standard deviation of 1.58, the values ranging from 148 to 2744 ppb. The median propanol level for all samples was 18 ppb, the values ranging from 0 to 135 ppb. A weak but significant correlation between breath propanol and acetone levels is apparent in the data. The findings indicate the potential value of SIFT-MS as a non-invasive breath analysis technique for investigating volatile compounds in human health and in the diseased state.

A longitudinal study of ethanol and acetaldehyde in the exhaled breath of healthy volunteers using selected-ion flow-tube mass spectrometry.

Selected-ion flow-tube mass spectrometry (SIFT-MS) has been used to monitor the volatile compounds in the exhaled breath of 30 volunteers (19 male, 11 female) over a 6-month period. Volunteers provided breath samples each week between 8:45 and 13:00 (before lunch), and the concentrations of several trace compounds were obtained. In this paper the focus is on ethanol and acetaldehyde, which were simultaneously quantified by SIFT-MS using H3O+ precursor ions. The mean ethanol level for all samples was 196 parts-per-billion (ppb) with a standard deviation of 244 ppb, and the range of values for breath samples analysed is 0 to 1663 ppb. The mean acetaldehyde level for all samples was 24 ppb with a standard deviation of 17 ppb, and the range of values for breath samples analysed is 0 to 104 ppb. Background (ambient air) levels of ethanol were around 50 ppb, whereas any background acetaldehyde was usually undetectable. Increased ethanol levels were observed if sweet drink/food had been consumed within the 2 h prior to providing the breath samples, but no increase was apparent when alcohol had been consumed the previous evening. The measured endogenous breath ethanol and acetaldehyde levels were not correlated. These data relating to healthy individuals are a prelude to using breath analysis for clinical diagnosis, for example, the recognition of bacterial overload in the gut (ethanol) or the possibly of detecting tumours in the body (acetaldehyde).