Mucosal Tuft Cell Density Is Increased in Diarrhea-Predominant Irritable Bowel Syndrome Colonic Biopsies.

Tuft cells are rare chemosensory sentinels found in the gut epithelium. When triggered by helminth infection, tuft cells secrete interleukin-25 (IL-25) basolaterally and subsequently evoke an immune response. Irritable bowel syndrome (IBS) is a common and heterogeneous disorder characterized by bowel dysfunction and visceral pain sensitivity. Dysfunctional gut-brain communication and immune activation contribute to the pathophysiology of this disorder. The study aims were to investigate changes in tuft cell density in non-post-infectious IBS patients. Immunofluorescent labeling of DCLK1-positive tuft cells was carried out in mucosal biopsies from the distal colons of diarrhea and constipation-predominant IBS patients and healthy controls. Tuft cell numbers were also assessed in animal models. Concentrations of interleukin-25 (IL-25) secreted from colonic biopsies and in plasma samples were analyzed using an immunoassay. The density of tuft cells was increased in diarrhea-but not constipation-predominant IBS patient colonic biopsies. Biopsy secretions and plasma concentrations of IL-25 were elevated in diarrhea-but not constipation-predominant IBS participants. Tuft cell hyperplasia was detected in a rat model of IBS but not in mice exposed to chronic stress. Tuft cell hyperplasia is an innate immune response to helminth exposure. However, the patients with diarrhea-predominant IBS have not reported any incidents of enteric infection. Moreover, rats exhibiting IBS-like symptoms displayed increased tuft cell density but were not exposed to helminths. Our findings suggest that factors other than helminth exposure or chronic stress lead to tuft cell hyperplasia in IBS colonic biopsies.

Glucagon-Like Peptide-1 Secreting L-Cells Coupled to Sensory Nerves Translate Microbial Signals to the Host Rat Nervous System.

An intact gut epithelium preserves the immunological exclusion of "non-self" entities in the external environment of the gut lumen. Nonetheless, information flows continuously across this interface, with the host immune, endocrine, and neural systems all involved in monitoring the luminal environment of the gut. Both pathogenic and commensal gastrointestinal (GI) bacteria can modulate centrally-regulated behaviors and brain neurochemistry and, although the vagus nerve has been implicated in the microbiota-gut-brain signaling axis, the cellular and molecular machinery that facilitates this communication is unclear. Studies were carried out in healthy Sprague-Dawley rats to understand cross-barrier communication in the absence of disease. A novel colonic-nerve electrophysiological technique was used to examine gut-to-brain vagal signaling by bacterial products. Calcium imaging and immunofluorescent labeling were used to explore the activation of colonic submucosal neurons by bacterial products. The findings demonstrate that the neuromodulatory molecule, glucagon-like peptide-1 (GLP-1), secreted by colonic enteroendocrine L-cells in response to the bacterial metabolite, indole, stimulated colonic vagal afferent activity. At a local level indole modified the sensitivity of submucosal neurons to GLP-1. These findings elucidate a cellular mechanism by which sensory L-cells act as cross-barrier signal transducers between microbial products in the gut lumen and the host peripheral nervous system.

GHSR-1 agonist sensitizes rat colonic intrinsic and extrinsic neurons to exendin-4: A role in the manifestation of postprandial gastrointestinal symptoms in irritable bowel syndrome?

BACKGROUND: Patients with irritable bowel syndrome (IBS) may experience postprandial symptom exacerbation. Nutrients stimulate intestinal release of glucagon-like peptide 1 (GLP-1), an incretin hormone with known gastrointestinal effects. However, prior to the postprandial rise in GLP-1, levels of the hunger hormone, ghrelin, peak. The aims of this study were to determine if ghrelin sensitizes colonic intrinsic and extrinsic neurons to the stimulatory actions of a GLP-1 receptor agonist, and if this differs in a rat model of IBS. METHODS: Calcium imaging of enteric neurons was compared between Sprague Dawley and Wistar Kyoto rats. Colonic contractile activity and vagal nerve recordings were also compared between strains. KEY RESULTS: Circulating GLP-1 concentrations differ between IBS subtypes. Mechanistically, we have provided evidence that calcium responses evoked by exendin-4, a GLP-1 receptor agonist, are potentiated by a ghrelin receptor (GHSR-1) agonist, in both submucosal and myenteric neurons. Although basal patterns of colonic contractility varied between Sprague Dawley and Wister Kyoto rats, the capacity of exendin-4 to alter smooth muscle function was modified by a GHSR-1 agonist in both strains. Gut-brain signaling via GLP-1-mediated activation of vagal afferents was also potentiated by the GHSR-1 agonist. CONCLUSIONS & INFERENCES: These findings support a temporal interaction between ghrelin and GLP-1, where the preprandial peak in ghrelin may temporarily sensitize colonic intrinsic and extrinsic neurons to the neurostimulatory actions of GLP-1. While the sensitizing effects of the GHSR-1 agonist were identified in both rat strains, in the rat model of IBS, underlying contractile activity was aberrant.

Reassessing risk models for atypical hyperplasia: age may not matter.

PURPOSE: The aim of this study was to investigate the influence of age at diagnosis of atypical hyperplasia ("atypia", ductal [ADH], lobular [ALH], or severe ADH) on the risk of developing subsequent invasive breast cancer or ductal carcinoma in situ (DCIS). METHODS: Using standard survival analysis methods, we retrospectively analyzed 1353 women not treated with chemoprevention among a cohort of 2370 women diagnosed with atypical hyperplasia to determine the risk relationship between age at diagnosis and subsequent breast cancer. RESULTS: For all atypia diagnoses combined, our cohort showed a 5-, 10-, and 15-year risk of invasive breast cancer or DCIS of 0.56, 1.25, and 1.30, respectively, with no significant difference in the (65,75] year age group. For women aged (35,75] years, we observed no significant difference in the 15-year risk of invasive breast cancer or DCIS after atypical hyperplasia, although the baseline risk for a 40-year-old woman is approximately 1/8 the risk of a 70-year-old woman. The risks associated with invasive breast cancer or DCIS for women in our cohort diagnosed with ADH, severe ADH, or ALH, regardless of age, were 7.6% (95% CI 5.9-9.3%) at 5 years, 25.1% (20.7-29.2%) at 10 years, and 40.1% (32.8-46.6%) at 15 years. CONCLUSION: In contrast to current risk prediction models (e.g., Gail, Tyrer-Cuzick) which assume that the risk of developing breast cancer increases in relation to age at diagnosis of atypia, we found the 15-year cancer risk in our cohort was not significantly different for women between the ages of 35 (excluded) and 75. This implies that the "hits" received by the breast tissue along the "high-risk pathway" to cancer might possibly supersede other factors such as age.

Using machine learning to parse breast pathology reports.

PURPOSE: Extracting information from electronic medical record is a time-consuming and expensive process when done manually. Rule-based and machine learning techniques are two approaches to solving this problem. In this study, we trained a machine learning model on pathology reports to extract pertinent tumor characteristics, which enabled us to create a large database of attribute searchable pathology reports. This database can be used to identify cohorts of patients with characteristics of interest. METHODS: We collected a total of 91,505 breast pathology reports from three Partners hospitals: Massachusetts General Hospital, Brigham and Women's Hospital, and Newton-Wellesley Hospital, covering the period from 1978 to 2016. We trained our system with annotations from two datasets, consisting of 6295 and 10,841 manually annotated reports. The system extracts 20 separate categories of information, including atypia types and various tumor characteristics such as receptors. We also report a learning curve analysis to show how much annotation our model needs to perform reasonably. RESULTS: The model accuracy was tested on 500 reports that did not overlap with the training set. The model achieved accuracy of 90% for correctly parsing all carcinoma and atypia categories for a given patient. The average accuracy for individual categories was 97%. Using this classifier, we created a database of 91,505 parsed pathology reports. CONCLUSIONS: Our learning curve analysis shows that the model can achieve reasonable results even when trained on a few annotations. We developed a user-friendly interface to the database that allows physicians to easily identify patients with target characteristics and export the matching cohort. This model has the potential to reduce the effort required for analyzing large amounts of data from medical records, and to minimize the cost and time required to glean scientific insight from these data.

Factors Associated with Recurrence Rates and Long-Term Survival in Women Diagnosed with Breast Cancer Ages 40 and Younger.

BACKGROUND: Young age at breast cancer diagnosis has been associated with increased risk of recurrence and mortality. We reevaluated this assumption in a large, modern cohort of women diagnosed with breast cancer at age ≤40 years. METHODS: We identified women with breast cancer at age ≤40 years at a single institution from 1996-2008. We assessed locoregional recurrence (LRR), distant recurrence, disease-free survival (DFS), and overall survival (OS), and correlated patient and tumor characteristics with outcomes. RESULTS: We identified 584 women aged ≤40 years with breast cancer. Median age was 37 years, and median follow-up was 124 months; 61.5 % were stages 0-I and 38.5 % were stages II-III. Overall, 57.4 % had lumpectomies and 42.5 % mastectomies. DFS was 93 % at 5 years and 84.5 % at 10 years. OS was 93 % at 5 years and 86.5 % at 10 years. On multivariate analysis, worse DFS was associated with positive nodes (p = 0.002); worse OS was associated with larger tumor size (p = 0.042). When stratified by lumpectomy versus mastectomy, there were no significant differences in survival or recurrence. For lumpectomy patients, DFS was 96 % at 5 years and 88 % at 10 years; OS was 96 % at 5 years and 89 % at 10 years. For mastectomy patients, DFS was 89.5 % at 5 years and 79 % at 10 years; OS was 90 % at 5 years and 83 % at 10 years. Lumpectomy LRR rates were 1 % at 5 years and 4 % at 10 years. Mastectomy LRR rates were 3.5 % at 5 years and 8.7 % at 10 years. CONCLUSIONS: Outcomes for women with breast cancer at age ≤40 years have improved. Lumpectomy recurrence rates are low, suggesting that lumpectomy is oncologically safe for young breast cancer patients.

Intraoperative micro-computed tomography (micro-CT): a novel method for determination of primary tumour dimensions in breast cancer specimens.

OBJECTIVES: Micro-CT is a promising modality to determine breast tumour size in three dimensions in intact lumpectomy specimens. We compared the accuracy of tumour size measurements using specimen micro-CT with measurements using multimodality pre-operative imaging. METHODS: A tabletop micro-CT was used to image breast lumpectomy specimens. The largest tumour dimension on three-dimensional reconstructed micro-CT images of the specimen was compared with the measurements determined by pre-operative mammography, ultrasound and MRI. The largest dimension of pathologic invasive cancer size was used as the gold standard reference to assess the accuracy of imaging assessments. RESULTS: 50 invasive breast cancer specimens in 50 patients had micro-CT imaging. 42 were invasive ductal carcinoma, 6 were invasive lobular carcinoma and 2 were other invasive cancer. Median patient age was 63 years (range 33-82 years). When compared with the largest pathologic tumour dimension, micro-CT measurements had the best correlation coefficient (r = 0.82, p < 0.001) followed by MRI (r = 0.78, p < 0.001), ultrasound (r = 0.61, p < 0.001) and mammography (r = 0.40, p < 0.01). When compared with pre-operative modalities, micro-CT had the best correlation coefficient (r = 0.86, p < 0.001) with MRI, followed by ultrasound (r = 0.60, p < 0.001) and mammography (r = 0.54, p < 0.001). Overall, mammography and ultrasound tended to underestimate the largest tumour dimension, while MRI and micro-CT overestimated the largest tumour dimension more frequently. CONCLUSION: Micro-CT is a potentially useful tool for accurate assessment of tumour dimensions within a lumpectomy specimen. Future studies need to be carried out to see if this technology could have a role in margin assessment. ADVANCES IN KNOWLEDGE: Micro-CT is a promising new technique which could potentially be used for rapid assessment of breast cancer dimensions in an intact lumpectomy specimen in order to guide surgical excision.

A pilot study evaluating shaved cavity margins with micro-computed tomography: a novel method for predicting lumpectomy margin status intraoperatively.

Microscopically clear lumpectomy margins are essential in breast conservation, as involved margins increase local recurrence. Currently, 18-50% of lumpectomies have close or positive margins that require re-excision. We assessed the ability of micro-computed tomography (micro-CT) to evaluate lumpectomy shaved cavity margins (SCM) intraoperatively to determine if this technology could rapidly identify margin involvement by tumor and reduce re-excision rates. Twenty-five SCM from six lumpectomies were evaluated with a Skyscan 1173 table top micro-CT scanner (Skyscan, Belgium). Micro-CT results were compared to histopathological results. We scanned three SCM at once with a 7-minute scanning protocol, and studied a total of 25 SCM from six lumpectomies. Images of the SCM were evaluated for radiographic signs of breast cancer including clustered microcalcifications and spiculated masses. SCM were negative by micro-CT in 19/25 (76%) and negative (≥2 mm) by histopathology in 19/25 (76%). Margin status by micro-CT was concordant with histopathology in 23/25 (92%). Micro-CT overestimated margin involvement in 1/25 and underestimated margin involvement in 1/25. Micro-CT had an 83.3% positive predictive value, a 94.7% negative predictive value, 83.3% sensitivity, and 94.7% specificity for evaluation of SCM. Evaluation of SCM by micro-CT is an accurate and promising method of intraoperative margin assessment in breast cancer patients. The scanning time required is short enough to permit real-time feedback to the operating surgeon, allowing immediate directed re-excision.

Micro-computed tomography (Micro-CT): a novel approach for intraoperative breast cancer specimen imaging.

Intraoperative radiographic examination of breast specimens is commonly performed to confirm excision of image-detected breast lesions, but it is not reliable for assessing margin status. A more accurate method of intraoperative breast specimen imaging is needed. Micro-CT provides quantitative imaging parameters, image rotation, and virtual "slicing" of intact breast specimens. We explored the use of micro-CT for assessment of a variety of clinical breast specimens. Specimens were evaluated with a table top micro-CT scanner, Skyscan 1173 (Skyscan, Belgium), with a 40-130 kV, 8 W X-ray source. Skyscan software for 3D image analysis (Dataviewer and CTVox) was employed to review 3D graphics of specimens. Scanning for 7 min and another 7 min for image reconstruction provided the desired resolution for breast specimens. Breast lumpectomy specimens, shaved cavity margins, mastectomy specimens, and axillary lymph nodes were imaged by micro-CT. The micro-CT images could be rotated in all directions and cross sections of internal portions of specimens could be visualized from any angle. This provided information about spatial orientation of masses and calcifications relative to margins in intact lumpectomy specimens. Micro-CT is a potentially useful tool for assessment of breast cancer specimens, allowing real-time analysis of tumor location in breast lumpectomy specimens or shaved cavity margins. Micro-CT may also be useful for assessing sentinel lymph nodes and mastectomy specimens.

The role of chemoprevention in modifying the risk of breast cancer in women with atypical breast lesions.

Women with atypical ductal hyperplasia (ADH), atypical lobular hyperplasia (ALH), lobular carcinoma in situ (LCIS), and severe ADH are at increased risk of breast cancer, but a systematic quantification of this risk and the efficacy of chemoprevention in the clinical setting is still lacking. The objective of this study is to evaluate a woman's risk of breast cancer based on atypia type and to determine the effect of chemoprevention in decreasing this risk. Review of 76,333 breast pathology reports from three institutions within Partners Healthcare System, Boston, from 1987 to 2010 using natural language processing was carried out. This approach identified 2,938 women diagnosed with atypical breast lesions. The main outcome of this study is breast cancer occurrence. Of the 2,938 patients with atypical breast lesions, 1,658 were documented to have received no chemoprevention, and 184/1,658 (11.1 %) developed breast cancer at a mean follow-up of 68 months. Estimated 10-year cancer risks were 17.3 % with ADH, 20.7 % with ALH, 23.7 % with LCIS, and 26.0 % with severe ADH. In a subset of patients treated from 1999 on (the chemoprevention era), those who received no chemoprevention had an estimated 10-year breast cancer risk of 21.3 %, whereas those treated with chemoprevention had a 10-year risk of 7.5 % (p < 0.001). Chemoprevention use significantly reduced breast cancer risk for all atypia types (p < 0.05). The risk of breast cancer with atypical breast lesions is substantial. Physicians should counsel patients with ADH, ALH, LCIS, and severe ADH about the benefit of chemoprevention in decreasing their breast cancer risk.

The feasibility of using natural language processing to extract clinical information from breast pathology reports.

OBJECTIVE: The opportunity to integrate clinical decision support systems into clinical practice is limited due to the lack of structured, machine readable data in the current format of the electronic health record. Natural language processing has been designed to convert free text into machine readable data. The aim of the current study was to ascertain the feasibility of using natural language processing to extract clinical information from >76,000 breast pathology reports. APPROACH AND PROCEDURE: Breast pathology reports from three institutions were analyzed using natural language processing software (Clearforest, Waltham, MA) to extract information on a variety of pathologic diagnoses of interest. Data tables were created from the extracted information according to date of surgery, side of surgery, and medical record number. The variety of ways in which each diagnosis could be represented was recorded, as a means of demonstrating the complexity of machine interpretation of free text. RESULTS: There was widespread variation in how pathologists reported common pathologic diagnoses. We report, for example, 124 ways of saying invasive ductal carcinoma and 95 ways of saying invasive lobular carcinoma. There were >4000 ways of saying invasive ductal carcinoma was not present. Natural language processor sensitivity and specificity were 99.1% and 96.5% when compared to expert human coders. CONCLUSION: We have demonstrated how a large body of free text medical information such as seen in breast pathology reports, can be converted to a machine readable format using natural language processing, and described the inherent complexities of the task.

Increased susceptibility of ST2-deficient mice to polymicrobial sepsis is associated with an impaired bactericidal function.

ST2, a member of the Toll/IL-1R superfamily, negatively regulates both TLR2 and TLR4 signaling. In this study, we report that ST2-deficient mice were more susceptible to polymicrobial sepsis than their wild-type littermates, with increased production of proinflammatory cytokines. Bacterial clearance from the circulation and visceral organs following polymicrobial infection was markedly impaired in ST2-deficient mice. This was associated with substantially reduced uptake, phagocytosis, and intracellular killing of both Gram-positive and Gram-negative bacteria by ST2-deficient phagocytes. Consistent with a reduced antimicrobial response, phagocytes lacking ST2 displayed a defect in bactericidal activity in response to bacterial challenges with severely impaired phagosome maturation and NOX2 function. Thus, ST2-deficient mice exhibit an increased susceptibility to polymicrobial infection with impaired bacterial clearance, which is associated with defects in phagosome maturation and NOX2-derived production of reactive oxygen species characterized in ST2-deficient phagocytes.

Lumpectomy cavity shaved margins do not impact re-excision rates in breast cancer patients.

BACKGROUND: The benefits of taking shaved cavity margins (SCM) at the time of lumpectomy are unclear. We sought to determine if taking SCM decreases re-excision rates by increasing the total breast tissue volume excised. METHODS: We undertook a retrospective review of breast cancer patients who underwent lumpectomy from 2004 to 2006. Patients were divided into three groups. Group 1 had lumpectomy alone, group 2 had lumpectomy plus select (1-3) SCM, and group 3 had lumpectomy plus complete (≥4) SCM. Pathologic findings and surgical outcomes were compared between groups. RESULTS: 773 cancers treated by lumpectomy were included in this study; 197 were in group 1, 130 were in group 2, and 446 were in group 3. The mean total volume of breast tissue excised in group 1 (106.6 cm(3)) was significantly larger than the volume excised in groups 2 (79.3 cm(3)) and 3 (76.3 cm(3)). Rates of re-excision and successful breast-conservation therapy (BCT) were not significantly different between groups. Despite a lower total volume of breast tissue excised in groups 2 and 3, there was no significant increase in locoregional recurrence rates (LRR) at median follow-up of 54 months. CONCLUSIONS: Taking additional SCM during lumpectomy resulted in a significantly lower overall volume of breast tissue excised, with no increase in LRR. Contrary to prior studies, we found that SCM did not decrease re-excision rates or impact the success of BCT. Therefore, the main advantage of taking SCM appears to be that less breast tissue is excised, which could potentially improve cosmetic outcomes.

ST2 negatively regulates TLR2 signaling, but is not required for bacterial lipoprotein-induced tolerance.

Activation of TLR signaling is critical for host innate immunity against bacterial infection. Previous studies reported that the ST2 receptor, a member of the Toll/IL-1 receptor superfamily, functions as a negative regulator of TLR4 signaling and maintains LPS tolerance. However, it is undetermined whether ST2 negatively regulates TLR2 signaling and furthermore, whether a TLR2 agonist, bacterial lipoprotein (BLP)-induced tolerance is dependent on ST2. In this study, we show that BLP stimulation-induced production of proinflammatory cytokines and immunocomplex formation of TLR2-MyD88 and MyD88-IL-1R-associated kinase (IRAK) were significantly enhanced in ST2-deficient macrophages compared with those in wild-type controls. Furthermore, overexpression of ST2 dose-dependently attenuated BLP-induced NF-kappaB activation, suggesting a negative regulatory role of ST2 in TLR2 signaling. A moderate but significantly attenuated production of TNF-alpha and IL-6 on a second BLP stimulation was observed in BLP-pretreated, ST2-deficient macrophages, which is associated with substantially reduced IRAK-1 protein expression and downregulated TLR2-MyD88 and MyD88-IRAK immunocomplex formation. ST2-deficient mice, when pretreated with a nonlethal dose of BLP, benefitted from an improved survival against a subsequent lethal BLP challenge, indicating BLP tolerance develops in the absence of the ST2 receptor. Taken together, our results demonstrate that ST2 acts as a negative regulator of TLR2 signaling, but is not required for BLP-induced tolerance.

Cellular reprogramming by gram-positive bacterial components: a review.

LPS tolerance has been the focus of extensive scientific and clinical research over the last several decades in an attempt to elucidate the sequence of changes that occur at a molecular level in tolerized cells. Tolerance to components of gram-positive bacterial cell walls such as bacterial lipoprotein and lipoteichoic acid is a much lesser studied, although equally important, phenomenon. This review will focus on cellular reprogramming by gram-positive bacterial components and examines the alterations in cell surface receptor expression, changes in intracellular signaling, gene expression and cytokine production, and the phenomenon of cross-tolerance.