Identification of a Sacral, Visceral Sensory Transcriptome in Embryonic and Adult Mice.

Visceral sensory neurons encode distinct sensations from healthy organs and initiate pain states that are resistant to common analgesics. Transcriptome analysis is transforming our understanding of sensory neuron subtypes but has generally focused on somatic sensory neurons or the total population of neurons in which visceral neurons form the minority. Our aim was to define transcripts specifically expressed by sacral visceral sensory neurons, as a step towards understanding the unique biology of these neurons and potentially leading to identification of new analgesic targets for pelvic visceral pain. Our strategy was to identify genes differentially expressed between sacral dorsal root ganglia (DRG) that include somatic neurons and sacral visceral neurons, and adjacent lumbar DRG that comprise exclusively of somatic sensory neurons. This was performed in adult and E18.5 male and female mice. By developing a method to restrict analyses to nociceptive Trpv1 neurons, a larger group of genes were detected as differentially expressed between spinal levels. We identified many novel genes that had not previously been associated with pelvic visceral sensation or nociception. Limited sex differences were detected across the transcriptome of sensory ganglia, but more were revealed in sacral levels and especially in Trpv1 nociceptive neurons. These data will facilitate development of new tools to modify mature and developing sensory neurons and nociceptive pathways.

Fungal-derived cues promote ocular autoimmunity through a Dectin-2/Card9-mediated mechanism.

Uveitis (intraocular inflammation) is a leading cause of loss of vision. Although its aetiology is largely speculative, it is thought to arise from complex genetic-environmental interactions that break immune tolerance to generate eye-specific autoreactive T cells. Experimental autoimmune uveitis (EAU), induced by immunization with the ocular antigen, interphotoreceptor retinoid binding protein (IRBP), in combination with mycobacteria-containing complete Freund's adjuvant (CFA), has many clinical and histopathological features of human posterior uveitis. Studies in EAU have focused on defining pathogenic CD4+ T cell effector responses, such as those of T helper type 17 (Th17) cells, but the innate receptor pathways precipitating development of autoreactive, eye-specific T cells remain poorly defined. In this study, we found that fungal-derived antigens possess autoimmune uveitis-promoting function akin to CFA in conventional EAU. The capacity of commensal fungi such as Candida albicans or Saccharomyces cerevisae to promote IRBP-triggered EAU was mediated by Card9. Because Card9 is an essential signalling molecule of a subgroup of C-type lectin receptors (CLRs) important in host defence, we evaluated further the proximal Card9-activating CLRs. Using single receptor-deficient mice we identified Dectin-2, but not Mincle or Dectin-1, as a predominant mediator of fungal-promoted uveitis. Conversely, Dectin-2 activation by α-mannan reproduced the uveitic phenotype of EAU sufficiently, in a process mediated by the Card9-coupled signalling axis and interleukin (IL)-17 production. Taken together, this report relates the potential of the Dectin-2/Card9-coupled pathway in ocular autoimmunity. Not only does it contribute to understanding of how innate immune receptors orchestrate T cell-mediated autoimmunity, it also reveals a previously unappreciated ability of fungal-derived signals to promote autoimmunity.

Prognostic impact of macrometastasis linear size in sentinel node biopsy for breast carcinoma.

AIM: The aim of the present study was to evaluate the risk of axillary non-sentinel lymph-node metastases (ALN) in breast cancer patients presenting macrometastasis (Mac-m) in the sentinel lymph node (SN). MATERIALS AND METHODS: A retrospective series of 1464 breast cancers from patients who underwent ALN dissection following the diagnosis of Mac-m in the sentinel node (SN) was studied. In all the cases the MAC-m linear size was evaluated and correlated with presence or absence of non-SN ALN metastases. RESULTS: Non-SN metastases were detected in 644∖1464 cases (43.98%). The risk of further axillary metastases ranged from 20.2% (37/183) in cases with Mac-m between 2 and 2.9 mm, to 65.3% (262/401) in cases with Mac-m measuring > 10 mm. The risk of non-SN ALN metastases showed a 3% increase, parallel to each mm increment in SN metastasis size. The data evaluated with the receiver operating characteristic (ROC) curve showed that the Mac-m could be subdivided according to a new cut-off of 7 mm. pT1 tumours, with Mac-m < 7 mm had a risk of non-SN ALN metastases of <30%. Furthermore 109/127 of these (85.8%) had 3 or less non-SN ALN -metastases. CONCLUSIONS: The present data give a detailed description on the risk of non-SN ALN involvement, that may be useful in the evaluation of breast cancer patients. It is suggested that a Mac-m size of <7 mm is related to a low residual axillary disease burden in breast cancer patients with small (pT1) tumours.

Mesenchymal Stromal Cells are Readily Recoverable from Lung Tissue, but not the Alveolar Space, in Healthy Humans.

Stromal support is critical for lung homeostasis and the maintenance of an effective epithelial barrier. Despite this, previous studies have found a positive association between the number of mesenchymal stromal cells (MSCs) isolated from the alveolar compartment and human lung diseases associated with epithelial dysfunction. We hypothesised that bronchoalveolar lavage derived MSCs (BAL-MSCs) are dysfunctional and distinct from resident lung tissue MSCs (LT-MSCs). In this study, we comprehensively interrogated the phenotype and transcriptome of human BAL-MSCs and LT-MSCs. We found that MSCs were rarely recoverable from the alveolar space in healthy humans, but could be readily isolated from lung transplant recipients by bronchoalveolar lavage. BAL-MSCs exhibited a CD90Hi , CD73Hi , CD45Neg , CD105Lo immunophenotype and were bipotent, lacking adipogenic potential. In contrast, MSCs were readily recoverable from healthy human lung tissue and were CD90Hi or Lo , CD73Hi , CD45Neg , CD105Int and had full tri-lineage potential. Transcriptional profiling of the two populations confirmed their status as bona fide MSCs and revealed a high degree of similarity between each other and the archetypal bone-marrow MSC. 105 genes were differentially expressed; 76 of which were increased in BAL-MSCs including genes involved in fibroblast activation, extracellular matrix deposition and tissue remodelling. Finally, we found the fibroblast markers collagen 1A1 and α-smooth muscle actin were increased in BAL-MSCs. Our data suggests that in healthy humans, lung MSCs reside within the tissue, but in disease can differentiate to acquire a profibrotic phenotype and migrate from their in-tissue niche into the alveolar space. Stem Cells 2016;34:2548-2558.

Consistency in recognizing microinvasion in breast carcinomas is improved by immunohistochemistry for myoepithelial markers.

Microinvasion is the smallest morphologically identifiable stage of invasion. Its presence and distinction from in situ carcinoma may have therapeutic implications, and clinical staging also requires the recognition of this phenomenon. Microinvasion is established on the basis of several morphological criteria, which may be difficult and not perfectly reproducible among pathologists. The aim of this study was to assess the consistency of diagnosing microinvasion in the breast on traditional haematoxylin and eosin (HE) stained slides and to evaluate whether immunohistochemistry (IHC) for myoepithelial markers could improve this. Digital images were generated from representative areas of 50 cases stained with HE and IHC for myoepithelial markers. Cases were specifically selected from the spectrum of in situ to microinvasive cancers. Twenty-eight dedicated breast pathologists assessed these cases at different magnifications through a web-based platform in two rounds: first HE only and after a washout period by both HE and IHC. Consistency in the recognition of microinvasion significantly improved with the use of IHC. Concordance rates increased from 0.85 to 0.96, kappa from 0.5 to 0.85, the number of cases with 100% agreement rose from 9/50 to 25/50 with IHC and the certainty of diagnosis also increased. The use of IHC markedly improves the consistency of identifying microinvasion. This corroborates previous recommendations to use IHC for myoepithelial markers to clarify cases where uncertainty exists about the presence of microinvasion. Microinvasive carcinoma is a rare entity, and seeking a second opinion may avoid overdiagnosis.

The requirements of a specialist Breast Centre.

INTRODUCTION: In recognition of the advances and evidence based changes in clinical practice that have occurred in recent years and taking into account the knowledge and experience accumulated through the voluntary breast unit certification programme, Eusoma has produced this up-dated and revised guidelines on the requirements of a Specialist Breast Centre (BC). METHODS: The content of these guidelines is based on evidence from the recent relevant peer reviewed literature and the consensus of a multidisciplinary team of European experts. The guidelines define the requirements for each breast service and for the specialists who work in specialist Breast Centres. RESULTS: The guidelines identify the minimum requirements needed to set up a BC, these being an integrated Breast Centre, dealing with a sufficient number of cases to allow effective working and continuing expertise, dedicated specialists working with a multidisciplinary approach, providing all services throughout the patients pathway and data collection and audit. It is essential that the BC also guarantees the continuity of care for patients with advanced (metastatic) disease offering treatments according to multidisciplinary competencies and a high quality palliative care service. The BC must ensure that comprehensive support and expertise may be needed, not only through the core BC team, but also ensure that all other medical and paramedical expertise that may be necessary depending on the individual case are freely available, referring the patient to the specific care provider depending on the problem. CONCLUSIONS: Applying minimum requirements and quality indicators is essential to improve organisation, performance and outcome in breast care. Efficacy and compliance have to be constantly monitored to evaluate the quality of patient care and to allow appropriate corrective actions leading to improvements in patient care.

Predictive markers in breast cancer--the present.

Breast cancer is a heterogeneous disease and there is a continual drive to identify markers that will aid in predicting prognosis and response to therapy. To date, relatively few markers have established prognostic power. Oestrogen receptor (ER) is probably the most powerful predictive marker in breast cancer management, both in determining prognosis and in predicting response to hormone therapies. Progesterone receptor (PR) is also a widely used marker, although its value is less well established. HER-2 status has also become a routine prognostic and predictive factor in breast cancer. Given the importance of these biological markers in patient management, it is essential that assays are robust and quality controlled, and that interpretation is standardized. Furthermore, it is important to be aware of the limitations in their predictive power, and how this may be refined through addition of further biological markers. The aim of this review is to provide an overview of the established role of ER, PR and HER-2 in patient management, the current standards for assessing these markers, as well as highlighting the controversies that still surround their use and methods of assessment.

The role of radiotherapy in treating small early invasive breast cancer.

AIM: The aim of the study was to identify if radiotherapy can be safely avoided in a selected subgroup of largely screening detected small invasive breast cancer. METHODS: One hundred and eighty-eight patients with node negative invasive early breast cancer < or =1cm (< or =T1b) treated in our centre between 1990 and 2004 were retrospectively followed for local, regional and distant recurrences. Treatment involved adequate local excision by breast conserving surgery (BCS). Axillary staging was performed by a four node axillary sampling until 2000, following which sentinel lymph node sampling was employed. All sections were assessed histologically by haematoxylin and eosin stained sections. The inked margins were reported as being involved, close and clear. Radiotherapy (RT) was employed only if the resected margins were inadequate, and in those with involved axillary nodes who refused further completion axillary clearance. RESULTS: Ninety-four patients (Group A) had BCS alone and 79 patients (Group B) had both BCS and RT. There was no ipsilateral breast tumour recurrence (IBTR) in 88 patients in Group A, corresponding to an actuarial freedom from IBTR of 96%, 91% and 88.1% at 5 years, 8 years and 9 years. In Group B, there was no IBTR in 75 patients corresponding to an actuarial freedom from IBTR of 97%, 94.9% and 90.6% at 5 years, 8 years and 10 years. CONCLUSION: Our experience over 14 years has shown that it is possible to safely avoid radiotherapy in a selected subgroup of small invasive breast cancer.

Non-operative breast pathology: apocrine lesions.

Apocrine metaplasia is a very common finding in the female breast after the age of 25. It is so common that many people regard it as a normal component of the breast. This, however, is only really the case in apocrine sweat glands of the axilla and in the peri-areolar apocrine glands. The apocrine cell does, however, contribute to a number of different breast lesions, some of which are very taxing diagnostically; apocrine variants of both in-situ and invasive cancer are encountered. This review considers the common apocrine metaplastic lesions seen in fibrocystic change as well as apocrine adenoma, apocrine change within sclerosing adenosis, atypical apocrine lesions and apocrine malignancies.

Methylene blue dye versus combined dye-radioactive tracer technique for sentinel lymph node localisation in early breast cancer.

AIM: The study compared the accuracy and success rate of two techniques, methylene blue alone versus combined methylene blue and radioactive colloid in sentinel lymph node localisation in the management early breast cancer. METHODS: Three hundred and twenty-nine patients with tumours less than 2 cm on ultrasound assessment were prospectively evaluated. One hundred and seventy-three patients (Group A) underwent sentinel lymph node localisation using 1 ml of 1% methylene blue. A combined technique of both methylene blue and radioactive colloid was used in 156 patients (Group B). Application of both was subdermal and subareolar. Sentinel lymph nodes were examined by standard microscopy. Patients underwent breast conservation surgery or mastectomy and sentinel node guided four node axillary sampling+/-clearance. RESULTS: In Group A, the sentinel lymph node identification rate was 96.5%. The negative predictive value was 96.3%, with false negative of 3.7% and accuracy of 87.4%. In group B the identification rate for sentinel lymph node was 98.7%, with false negative of 4.1%, negative predictive value of 96%, and accuracy of 83.8%. CONCLUSION: Sentinel lymph node localisation using methylene blue or combined dye and radioactive tracer technique predicts the axillary lymph node status in early breast cancer with comparable success rates, accuracy and false negative rates. The combined technique facilitates quicker identification of sentinel lymph node; however the dye technique alone can be used successfully in centres without nuclear medicine facilities.

Best Practice No 179. Guidelines for breast needle core biopsy handling and reporting in breast screening assessment.

Non-operative diagnosis has become the norm in breast disease assessment and, until relatively recently, fine needle aspiration cytology has been the sampling method of choice. The introduction of automated core biopsy guns in the mid 1990s led to the additional introduction of core biopsy in assessment units. This paper presents a summary of the guidance on handling and routine reporting of breast needle core biopsy specimens in the context of breast disease multidisciplinary assessment. This guidance has been produced by the UK National Coordinating Committee for Breast Screening Pathology and is endorsed by the European Commission working group on breast screening pathology.

Consistency of staining and reporting of oestrogen receptor immunocytochemistry within the European Union--an inter-laboratory study.

To assess the variability of oestrogen receptor (ER) testing using immunocytochemistry, centrally stained and unstained slides from breast cancers were circulated to the members of the European Working Group for Breast Screening Pathology, who were asked to report on both slides. The results showed that there was almost complete concordance among readers (kappa=0.95) in ER-negative tumours on the stained slide and excellent concordance among readers (kappa=0.82) on the slides stained in each individual laboratory. Tumours showing strong positivity were reasonably well assessed (kappa=0.57 and 0.4, respectively), but there was less concordance in tumours with moderate and low levels of ER, especially when these were heterogeneous in their staining. Because of the variation, the Working Group recommends that laboratories performing these stains should take part in a external quality assurance scheme for immunocytochemistry, should include a tumour with low ER levels as a weak positive control and should audit the percentage positive tumours in their laboratory against the accepted norms annually. The Quick score method of receptor assessment may also have too many categories for good concordance, and grouping of these into fewer categories may remove some of the variation among laboratories.

Discrepancies in current practice of pathological evaluation of sentinel lymph nodes in breast cancer. Results of a questionnaire based survey by the European Working Group for Breast Screening Pathology.

AIMS: To evaluate aspects of the current practice of sentinel lymph node (SLN) pathology in breast cancer via a questionnaire based survey, to recognise major issues that the European guidelines for mammography screening should address in the next revision. METHODS: A questionnaire was circulated by mail or electronically by the authors in their respective countries. Replies from pathology units dealing with SLN specimens were evaluated further. RESULTS: Of the 382 respondents, 240 European pathology units were dealing with SLN specimens. Sixty per cent of these units carried out intraoperative assessment, most commonly consisting of frozen sections. Most units slice larger SLNs into pieces and only 12% assess these slices on a single haematoxylin and eosin (HE) stained slide. Seventy one per cent of the units routinely use immunohistochemistry in all cases negative by HE. The terms micrometastasis, submicrometastasis, and isolated tumour cells (ITCs) are used in 93%, 22%, and 71% of units, respectively, but have a rather heterogeneous interpretation. Molecular SLN staging was reported by only 10 units (4%). Most institutions have their own guidelines for SLN processing, but some countries also have well recognised national guidelines. CONCLUSIONS: Pathological examination of SLNs throughout Europe varies considerably and is not standardised. The European guidelines should focus on standardising examination. They should recommend techniques that identify metastases > 2 mm as a minimum standard. Uniform reporting of additional findings may also be important, because micrometastases and ITCs may in the future be shown to have clinical relevance.

Integrin expression and survival in human breast cancer.

BACKGROUND: Integrin cell adhesion molecules are fundamental to numerous cellular functions including anchorage, differentiation and proliferation. Reduced expression of certain alpha and beta integrin subunits in primary breast cancer cells has been correlated with increased invasion and metastasis. Conversely, over-expression of the alpha6 subunit has been linked to poorer survival. The objective of this study was to measure the survival of a cohort with breast carcinoma in relation to integrin expression and to evaluate their potential as prognostic indicators. METHOD: Integrin expression on samples from 99 consecutive patients with breast cancer was assayed using monoclonal antibodies to the subunits alpha(1,2,3,6,V) and beta(1,3,4,5). This cohort has now been followed prospectively for almost five years allowing for early assessment of survival in relation to integrin expression. RESULTS: Whilst analysis of the data confirmed the relation of survival to proven predictors of tumour grade, tumour size and vascular invasion, statistical significance was not demonstrated with regard to both lymph node status and all integrin subunits studied. CONCLUSION: Previous research correlating certain integrin subunits with survival has not been confirmed in this study. Despite proven molecular importance in tumour cell adhesion, invasion and metastasis, integrin expression would appear not to translate clinically as independent indicators of prognosis, at least in the short-term.

Inadequate rates are lower when FNAC samples are taken by cytopathologists.

Inadequate rates (IR) in FNAC from different sources were compared. The rates were lowest when FNAC was performed by a cytopathologist (12%) and highest when done by a non-cytopathologist (32%). These differences were mirrored in high IRs in breast cancer cases. IR was not significantly improved when non-cytopathologist FNAC was attended by a cytotechnician.

Pathological work-up of sentinel lymph nodes in breast cancer. Review of current data to be considered for the formulation of guidelines.

Controversies and inconsistencies regarding the pathological work-up of sentinel lymph nodes (SNs) led the European Working Group for Breast Screening Pathology (EWGBSP) to review published data and current evidence that can promote the formulation of European guidelines for the pathological work-up of SNs. After an evaluation of the accuracy of SN biopsy as a staging procedure, the yields of different sectioning methods and the immunohistochemical detection of metastatic cells are reviewed. Currently published data do not allow the significance of micrometastases or isolated tumour cells to be established, but it is suggested that approximately 18% of the cases may be associated with further nodal (non-SN) metastases, i.e. approximately 2% of all patients initially staged by SN biopsy. The methods for the intraoperative and molecular assessment of SNs are also surveyed.

How significant is detection of ductal carcinoma in situ in a breast screening programme?

PURPOSE: To compare the histological grades of screen detected and non-screen detected ductal carcinoma in situ (DCIS) and to identify any differences that might support the contention that DCIS found by breast screening represents an over-diagnosis. The aim was also to establish whether any particular mammographic features of DCIS can be used to predict tumour grade reliably. MATERIALS AND METHODS: Biopsy proven cases of DCIS (n=153) were reviewed with respect to grade and subdivided into high, intermediate and low grades using the Van Nuys classification. A more aggressive subset of DCIS (microinvasive and interval cancers) were similarly analysed. Mammograms were reviewed with regard to abnormal features and distribution, and the appearances correlated with grade. RESULTS: Fifty-four percent (53/98) of screen detected and 62% (34/52) of non-screen detected DCIS were high grade. The rest were equally intermediate and low grade, with no statistical difference between the two groups. Eighty-four percent of the aggressive subset of tumours were high grade. Micro-calcification was present in 90% and in 10% there were soft tissue changes alone. Seventy-six percent of linear branching calcification was associated with high grade DCIS. Only 13% of high grade DCIS demonstrated punctate micro-calcification; however, 38% of cases of punctate micro-calfication were associated with high grade tumours and there was a great deal of overlap between the groups. CONCLUSION: Most cases of DCIS in both screen and non-screen detected groups were high grade. Only one in five was low grade. Analysis of the aggressive subgroup underlines the significance of high grade DCIS. Mammographic patterns are not always reliable in the prediction of tumour grade. The detection of DCIS in screening programmes is important and should not be regarded as over-diagnosis.

Axillary node status in breast cancer patients prior to surgery by imaging with Tc-99m humanised anti-PEM monoclonal antibody, hHMFG1.

In early breast cancer axillary nodes are usually impalpable and over 50% of such patients may have an axillary clearance when no nodes are involved. This work identifies axillary node status by imaging with a Tc-99m radiolabelled anti-Polymorphic Epithelial Mucin, humanised monoclonal antibody (human milk fat globule 1), prior to surgery in 30 patients. Change detection analysis of image data with probability mapping is undertaken. A specificity of 93% and positive predictive value of 92% (both 100% if a second cancer in the axilla with negative nodes is considered) were found. A strategy for combining negative imaging with the sentinel node procedure is presented.

Androgen receptor expression in ductal carcinoma in situ of the breast: relation to oestrogen and progesterone receptors.

AIMS: Ductal carcinoma in situ (DCIS) of the breast has been diagnosed increasingly since the advent of mammographic screening. In contrast to the situation in invasive breast carcinoma, there are no reports on androgen receptor (AR) status in DCIS and few reports on oestrogen (ER) and progesterone (PR) receptors. METHODS: AR expression was examined in 57 cases of DCIS of the breast and correlated to the degree of differentiation and ER/PR status using immunohistochemical methods. RESULTS: AR positivity was noted in 19 of the cases, whereas the other 38 cases were negative. There was no significant association between AR expression and the degree of differentiation of DCIS; three of the 13 well differentiated DCIS cases, 10 of the 19 intermediately differentiated cases, and six of the 25 poorly differentiated cases were positive (p = 0.093). However, a strong association was shown between the expression of ER (p < 0.0001) and PR (p = 0.002) and the degree of differentiation of DCIS. In addition, no significant association was found between the expression of AR and the expression of ER (p = 0.26) or PR (p = 0.57) in DCIS of the breast. CONCLUSIONS: A large number of cases of DCIS of the breast express AR and this may be associated with apocrine differentiation, which may impact on accurate typing of DCIS. Moreover, the expression of AR (but not ER or PR) in DCIS does not appear to be associated with the degree of differentiation.