Aoife Maguire

Spatial variation of the colonic microbiota in patients with ulcerative colitis and control volunteers

Objectives The relevance of spatial composition in the microbial changes associated with UC is unclear. We coupled luminal brush samples, mucosal biopsies and laser capture microdissection with deep sequencing of the gut microbiota to develop an integrated spatial assessment of the microbial community in controls and UC. Design A total of 98 samples were sequenced to a mean depth of 31 642 reads from nine individuals, four control volunteers undergoing routine colonoscopy and five patients undergoing surgical colectomy for medically-refractory UC. Samples were retrieved at four colorectal locations, incorporating the luminal microbiota, mucus gel layer and whole mucosal biopsies. Results Interpersonal variability accounted for approximately half of the total variance. Surprisingly, within individuals, asymmetric Eigenvector map analysis demonstrated differentiation between the luminal and mucus gel microbiota, in both controls and UC, with no differentiation between colorectal regions. At a taxonomic level, differentiation was evident between both cohorts, as well as between the luminal and mucosal compartments, with a small group of taxa uniquely discriminating the luminal and mucosal microbiota in colitis. There was no correlation between regional inflammation and a breakdown in this spatial differentiation or bacterial diversity. Conclusions Our study demonstrates a conserved spatial structure to the colonic microbiota, differentiating the luminal and mucosal communities, within the context of marked interpersonal variability. While elements of this structure overlap between UC and control volunteers, there are differences between the two groups, both in terms of the overall taxonomic composition and how spatial structure is ascribable to distinct taxa.

Low MAD2 expression levels associate with reduced progression-free survival in patients with high-grade serous epithelial ovarian cancer

Epithelial ovarian cancer (EOC) has an innate susceptibility to become chemoresistant. Up to 30% of patients do not respond to conventional chemotherapy [paclitaxel (Taxol®) in combination with carboplatin] and, of those who have an initial response, many patients relapse. Therefore, an understanding of the molecular mechanisms that regulate cellular chemotherapeutic responses in EOC cells has the potential to impact significantly on patient outcome. The mitotic arrest deficiency protein 2 (MAD2), is a centrally important mediator of the cellular response to paclitaxel. MAD2 immunohistochemical analysis was performed on 82 high‐grade serous EOC samples. A multivariate Cox regression analysis of nuclear MAD2 IHC intensity adjusting for stage, tumour grade and optimum surgical debulking revealed that low MAD2 IHC staining intensity was significantly associated with reduced progression‐free survival (PFS) (p = 0.0003), with a hazard ratio of 4.689. The in vitro analyses of five ovarian cancer cell lines demonstrated that cells with low MAD2 expression were less sensitive to paclitaxel. Furthermore, paclitaxel‐induced activation of the spindle assembly checkpoint (SAC) and apoptotic cell death was abrogated in cells transfected with MAD2 siRNA. In silico analysis identified a miR‐433 binding domain in the MAD2 3′ UTR, which was verified in a series of experiments. Firstly, MAD2 protein expression levels were down‐regulated in pre‐miR‐433 transfected A2780 cells. Secondly, pre‐miR‐433 suppressed the activity of a reporter construct containing the 3′‐UTR of MAD2. Thirdly, blocking miR‐433 binding to the MAD2 3′ UTR protected MAD2 from miR‐433 induced protein down‐regulation. Importantly, reduced MAD2 protein expression in pre‐miR‐433‐transfected A2780 cells rendered these cells less sensitive to paclitaxel. In conclusion, loss of MAD2 protein expression results in increased resistance to paclitaxel in EOC cells. Measuring MAD2 IHC staining intensity may predict paclitaxel responses in women presenting with high‐grade serous EOC. Copyright

Collagenous sprue: a clinicopathologic study of 12 cases.

Collagenous sprue is a rare form of small bowel enteropathy characterized by chronic diarrhea and progressive malabsorption with little data available on its natural history. The pathologic lesion consists of subepithelial collagen deposition associated with variable alterations in villous architecture. The small bowel biopsies of 12 cases were reviewed. Clinical details, celiac serology, and T-cell receptor gene rearrangement study results, when available, were collated. There were 8 females and 4 males (age ranged from 41 to 84 y) who presented with chronic diarrhea and weight loss. Small intestinal biopsies showed subepithelial collagen deposition with varying degrees of villous atrophy and varying numbers of intraepithelial lymphocytes. Four patients had previous biopsies showing enteropathic changes without collagen deposition. Seven cases were associated with collagenous colitis and 1 also had features of lymphocytic colitis. Three patients also had collagen deposition in gastric biopsies. One case was associated with lymphocytic gastritis. Celiac disease (CD, gluten-sensitive enteropathy) was documented in 4 patients. Five patients made a clinical improvement with combinations of a gluten-free diet and immunosuppressive therapy. Two patients died of complications of malnutrition and 1 of another illness. Clonal T-cell populations were identified in 5 of 6 cases tested. Four of these patients improved clinically after treatment but 1 has died. Collagenous sprue evolved on a background of CD in 4 cases. There was no history of CD in others and these cases may be the result of a biologic insult other than gluten sensitivity. None has developed clinical evidence of lymphoma to date.

Controversies in the pathological assessment of colorectal cancer.

Pathologic assessment of colorectal cancer specimens plays an essential role in patient management, informing prognosis and contributing to therapeutic decision making. The tumor-node-metastasis (TNM) staging system is a key component of the colorectal cancer pathology report and provides important prognostic information. However there is significant variation in outcome of patients within the same tumor stage. Many other histological features such as tumor budding, vascular invasion, perineural invasion, tumor grade and rectal tumor regression grade that may be of prognostic value are not part of TNM staging. Assessment of extramural tumor deposits and peritoneal involvement contributes to TNM staging but there are some difficulties with the definition of both of these features. Controversies in colorectal cancer pathology reporting include the subjective nature of some of the elements assessed, poor reporting rates and reproducibility and the need for standardized examination protocols and reporting. Molecular pathology is becoming increasingly important in prognostication and prediction of response to targeted therapies but accurate morphology still has a key role to play in colorectal cancer pathology reporting.

Reproducibility of the Rapid Bud Count Method for Assessment of Tumor Budding in Stage II Colorectal Cancer

To the Editor: Tumor budding, which is manifested by the migration of small discrete groups of tumor cells into desmoplastic stroma at the invasive tumor front, has been shown to be a negative prognostic factor in patients with stage II colorectal carcinoma (CRC). It is postulated to be a manifestation of the epithelialmesenchymal transition, with epithelial cells migrating as fibroblast-like cells. A recent study in our department has compared a new rapid bud count method (RBCM) for counting tumour buds with the conventional bud count method (CBCM). This has shown the RBCM to be equivalent to the CBCM, and has shown pT3N0 tumors to be divisible into ‘‘high budding’’ and ‘‘low budding’’ groups with significant differences in cancer-specific survival. We aimed in this study to assess the reproducibility of the RBCM between a group of practicing surgical pathologists of varied levels of experience in a busy academic surgical pathology department. The 40 stage II CRC cases used in the reproducibility component of an earlier reported study formed the basis for this study. Each case comprised between 3 and 9 H&E-stained slides, representing slides from all of the tumor blocks. A tumor bud was defined as a group of less than 5 detached tumor cells, usually at the invasive front (Fig. 1). Each case had been examined by LW and KS and classified, by consensus, as either high or low budding by the ‘‘gold standard’’ CBCM. By this method, each slide was initially scanned at 40 magnification to identify 5 areas with the highest concentration of tumor buds. These areas were then reexamined at 200 magnification. Tumor buds were formally counted in each of the five 200 fields, yielding 5 bud scores per slide. The process was repeated for each slide in the case, resulting in a total of between 15 and 45 bud scores per case. All of the bud scores from a case were collected and a median value calculated. Cases with a median bud score of Z1 were classified as high budding, whereas those with a median bud score of <1 were classified as low budding. The original study found that this definition achieved the best cut-off for stratifying survival data between the 2 groups. In essence, it meant that once a case showed tumor buds in more than half of the examined 200 microscopic fields, it was classified as high budding. In addition to the consensus CBCM, both LW and KS had individually applied the RBCM to the 40 cases. This method was identical to the CBCM except that when the 5 selected areas per slide were reexamined at 200 , the absolute number of tumor buds was not counted. Instead, the presence of any tumor bud in a given 200 microscopic field was recorded as positive (denoted by bud score ‘‘1’’) and similarly when no tumor bud was observed, it was recorded as negative (denoted by bud score ‘‘0’’). A median bud score of <1 defined a case as low budding by this method, with highbudding cases having a median bud value of 1. Each of the 4 study participants (BH, AM, NC, DG) underwent brief training in assessing tumor budding by the RBCM comprising review of a PowerPoint presentation, review of the original paper and review of a set of sample cases with one of the authors (KS). Results were recorded on a standardized form, and agreement was measured using percentages and free-marginal k statistics for multiple raters of dichotomous data. There was substantial agreement between the 4 pathologists (k=0.617) on the presence of low or high budding (Table 1). There was complete agreement (4:0) in 62.5% of cases (25/40), and complete disagreement (2:2) in one case (2.5%). In the remaining 14 cases (35%) there was 3-way agreement. In 10 of these cases the disagreeing pathologist underestimated budding relative to the other participants, and in 4 cases there was overestimation. The mean deviation in the number of bud-positive 200 microscopic fields scored by the disagreeing pathologist was low and was similar in cases of underestimation (4.1 or 28%) and overestimation (4.125 or 33%). In 3 of the cases of underestimation, the degree of deviation in the number of positive fields was marginal (less than 1, or 4%), FIGURE 1. Tumor buds in a desmoplastic stroma. LETTERS TO THE EDITOR

Inhibition of Dendritic Cell Maturation by the Tumor Microenvironment Correlates with the Survival of Colorectal Cancer Patients following Bevacizumab Treatment

Development of bevacizumab has improved survival in colorectal cancer, however, currently there are no biomarkers that predict response to bevacizumab and it is unknown how it influences the immune system in colorectal cancer patients. Dendritic cells are important for the induction of an antitumor immune response; however tumors are capable of disabling dendritic cells and escaping immune surveillance. The aim of this study was to assess the numbers of CD11c+ cells infiltrating tumor tissue and to examine the effects of tumor conditioned media (TCM) and bevacizumab conditioned media (BCM) on dendritic cell maturation and correlate our findings with patient survival. colorectal cancer explant tissues were cultured with or without bevacizumab, to generate BCM and TCM, which were used to treat dendritic cells. CD80, CD86, CD83, CD54, HLA-DR, and CD1d expression was measured by flow cytometry. Interleukin (IL)-10 and IL-12p70 were measured by ELISA. The Cox proportional hazards model was used to associate survival with dendritic cell inhibition. TCM and BCM inhibited lipopolysaccharide (LPS)-induced dendritic cell maturation and IL-12p70 secretion (P < 0.0001), while increasing IL-10 secretion (P = 0.0033 and 0.0220, respectively). Inhibition of LPS-induced CD1d (P = 0.021, HR = 1.096) and CD83 (P = 0.017, HR = 1.083) by TCM and inhibition of CD1d (P = 0.017, HR = 1.067), CD83 (P = 0.032, HR = 1.035), and IL-12p70 (P = 0.037, HR = 1.036) by BCM was associated with poor survival in colorectal cancer patients. CD11c expression was elevated in tumor tissue compared with normal tissue (P < 0.001), but this did not correlate with survival. In conclusion, TCM and BCM inhibit dendritic cells, and this inhibition correlates with survival of colorectal cancer patients receiving bevacizumab. Mol Cancer Ther; 11(8); 1829–37. ©2012 AACR.

Pathology of oesophagitis

Maguire A & Sheahan K 
(2012) Histopathology 60, 864–879
Pathology of oesophagitis

Spindle assembly checkpoint protein expression correlates with cellular proliferation and shorter time to recurrence in ovarian cancer

Ovarian carcinoma (OC) is the most lethal of the gynecological malignancies, often presenting at an advanced stage. Treatment is hampered by high levels of drug resistance. The taxanes are microtubule stabilizing agents, used as first-line agents in the treatment of OC that exert their apoptotic effects through the spindle assembly checkpoint. BUB1-related protein kinase (BUBR1) and mitotic arrest deficient 2 (MAD2), essential spindle assembly checkpoint components, play a key role in response to taxanes. BUBR1, MAD2, and Ki-67 were assessed on an OC tissue microarray platform representing 72 OC tumors of varying histologic subtypes. Sixty-one of these patients received paclitaxel and platinum agents combined; 11 received platinum alone. Overall survival was available for all 72 patients, whereas recurrence-free survival (RFS) was available for 66 patients. Increased BUBR1 expression was seen in serous carcinomas, compared with other histologies (P = .03). Increased BUBR1 was significantly associated with tumors of advanced stage (P = .05). Increased MAD2 and BUBR1 expression also correlated with increased cellular proliferation (P < .0002 and P = .02, respectively). Reduced MAD2 nuclear intensity was associated with a shorter RFS (P = .03), in ovarian tumors of differing histologic subtype (n = 66). In this subgroup, for those women who received paclitaxel and platinum agents combined (n = 57), reduced MAD2 intensity also identified women with a shorter RFS (P < .007). For the entire cohort of patients, irrespective of histologic subtype or treatment, MAD2 nuclear intensity retained independent significance in a multivariate model, with tumors showing reduced nuclear MAD2 intensity identifying patients with a poorer RFS (P = .05).

Deoxycholic acid promotes development of gastroesophageal reflux disease and Barrett's oesophagus by modulating integrin‐αv trafficking

The fundamental mechanisms underlying erosive oesophagitis and subsequent development of Barretts oesophagus (BO) are poorly understood. Here, we investigated the contribution of specific components of the gastric refluxate on adhesion molecules involved in epithelial barrier maintenance. Cell line models of squamous epithelium (HET‐1A) and BO (QH) were used to examine the effects of bile acids on cell adhesion to extracellular matrix proteins (Collagen, laminin, vitronectin, fibronectin) and expression of integrin ligands (α3, α4, α5, α6 and αν). Experimental findings were validated in human explant oesophageal biopsies, a rat model of gastroesophageal reflux disease (GORD) and in patient tissue microarrays. The bile acid deoxycholic acid (DCA) specifically reduced adhesion of HET‐1A cells to vitronectin and reduced cell‐surface expression of integrin‐αν via effects on endocytic recycling processes. Increased expression of integrin‐αv was observed in ulcerated tissue in a rat model of GORD and in oesophagitis and Barretts intestinal metaplasia patient tissue compared to normal squamous epithelium. Increased expression of integrin‐αν was observed in QH BO cells compared to HET‐1A cells. QH cells were resistant to DCA‐mediated loss of adhesion and reduction in cell‐surface expression of integrin‐αν. We demonstrated that a specific component of the gastric refluxate, DCA, affects the epithelial barrier through modulation of integrin αν expression, providing a novel mechanism for bile acid‐mediated erosion of oesophageal squamous epithelium and promotion of BO. Strategies aimed at preventing bile acid‐mediated erosion should be considered in the clinical management of patients with GORD.

Primary small bowel adenomas and adenocarcinomas—recent advances

The small intestine represents 75% of the length and 90% of the absorptive surface area of the gastrointestinal tract (GIT), yet only 2% of digestive system cancers occur at this site. Adenocarcinoma accounts for half of small bowel malignancies. There have been a number of important recent advances in our understanding, classification and treatment of small bowel tumours. Over recent years, ampullary tumours have become recognised as a form of small bowel carcinoma, distinct from head of pancreas and lower biliary tract tumours. This is reflected in separate TNM systems and increasing interest in separating intestinal from pancreatobiliary subtypes. The recognition of the importance of microsatellite (MSI) status and the advent of molecular pathology has also changed our approach to these neoplasms.