[Acute "difficult" gastrointestinal bleeding]

Gastrointestinal bleeding with its point of origin outside the reach of conventional gastro- and colonoscopy represents an extraordinary diagnostic and therapeutic challenge. Bleeding may originate from the small bowel distal to the duodenojejunal junction (middle gastrointestinal bleeding) or from the biliary tree (haemobilia) or from the pancreatic ductal system (haemosuccus pancreaticus). This particular type of gastrointestinal bleeding is often intermittend and caused by a variety of different pathologies. Angiography is the diagnostic method of choice for further investigation. It allows precise localization of the bleeding site and simultaneous interventional therapy (embolization/coiling). The importance of further diagnostic modalities such as scintigraphy, capsule endoscopy, push-enteroscopy and double-balloon-enteroscopy is discussed.

Cellular differentiation determines the expression of the hypoxia-inducible protein NDRG1 in pancreatic cancer

N-myc downstream-regulated gene-1 (NDRG1) is a recently described hypoxia-inducible protein that is upregulated in various human cancers. Pancreatic ductal adenocarcinoma, called pancreatic cancer, is a highly aggressive cancer that is characterised by its avascular structure, which results in a severe hypoxic environment. In this study, we investigated whether NDRG1 is upregulated in these tumours, thus providing a novel marker for malignant cells in the pancreas. By immunohistochemistry, we observed that NDRG1 was highly expressed in well-differentiated cells of pancreatic cancer, whereas the poorly differentiated tumour cells were negative. In addition, hyperplastic islets and ducts of nonquiescent pancreatic tissue were positive. To further explore its selective expression in tumours, two well-established pancreatic cancer cell lines of unequal differentiation status were exposed to 2% oxygen. NDRG1 mRNA and protein were upregulated by hypoxia in the moderately differentiated Capan-1 cells; however, its levels remained unchanged in the poorly differentiated Panc-1 cell line. Taken together, our data suggest that NDRG1 will not serve as a reliable marker of tumour cells in the pancreas, but may serve as a marker of differentiation. Furthermore, we present the novel finding that cellular differentiation may be an important factor that determines the hypoxia-induced regulation of NDRG1.

MR cholangiopancreatography: 1.5T versus 3T

Soon after its introduction in 1991, MR cholangiopancreatography has become an established diagnostic tool for the evaluation of the pancreaticobiliary ductal system at a field strength of 1.5T. It remains unclear whether MR cholangiopancreatography performed at 3T will benefit from the higher magnetic field strength or whether a field strength of 1.5T should continue to be considered the gold standard for MR cholangiopancreatography. This article reviews the current literature on the benefits and drawbacks of MR cholangiopancreatography at 3T compared with a standard field strength of 1.5T. Field strength-related artifacts that affect MR cholangiopancreatography at 3T also are discussed.

Gene expression variation between distinct areas of breast cancer measured from paraffin-embedded tissue cores

BACKGROUND: Diagnosis and prognosis in breast cancer are mainly based on histology and immunohistochemistry of formalin-fixed, paraffin-embedded (FFPE) material. Recently, gene expression analysis was shown to elucidate the biological variance between tumors and molecular markers were identified that led to new classification systems that provided better prognostic and predictive parameters. Archived FFPE samples represent an ideal source of tissue for translational research, as millions of tissue blocks exist from routine diagnostics and from clinical studies. These should be exploited to provide clinicians with more accurate prognostic and predictive information. Unfortunately, RNA derived from FFPE material is partially degraded and chemically modified and reliable gene expression measurement has only become successful after implementing novel and optimized procedures for RNA isolation, demodification and detection. METHODS: In this study we used tissue cylinders as known from the construction of tissue microarrays. RNA was isolated with a robust protocol recently developed for RNA derived from FFPE material. Gene expression was measured by quantitative reverse transcription PCR. RESULTS: Sixteen tissue blocks from 7 patients diagnosed with multiple histological subtypes of breast cancer were available for this study. After verification of appropriate localization, sufficient RNA yield and quality, 30 tissue cores were available for gene expression measurement on TaqMan(R) Low Density Arrays (16 invasive ductal carcinoma (IDC), 8 ductal carcinoma in situ (DCIS) and 6 normal tissue), and 14 tissue cores were lost. Gene expression values were used to calculate scores representing the proliferation status (PRO), the estrogen receptor status and the HER2 status. The PRO scores measured from entire sections were similar to PRO scores determined from IDC tissue cores. Scores determined from normal tissue cores consistently revealed lower PRO scores than cores derived from IDC or DCIS of the same block or from different blocks of the same patient. CONCLUSION: We have developed optimized protocols for RNA isolation from histologically distinct areas. RNA prepared from FFPE tissue cores is suitable for gene expression measurement by quantitative PCR. Distinct molecular scores could be determined from different cores of the same tumor specimen.

Infiltrating Lobular Carcinoma of the Breast: Systemic Treatment

Invasive lobular carcinoma (ILC) is the second most common type of breast cancer after invasive ductal carcinoma (IDC). It is characterized by unique clinical, biological and molecular properties. ILC is almost always positive for the estrogen receptor and is typically of a lower grade compared with IDC. We have reviewed selected literature on preoperative (neoadjuvant) and adjuvant systemic therapy of breast cancer focusing on the differential therapy of ILC. Despite the importance of this type of breast cancer, information about its specific treatment is sparse, in particular with regard to adjuvant systemic chemotherapy. ILC has significantly lower rates of response to neoadjuvant chemotherapy compared with IDC; however, the low chemosensitivity seems not to result in a survival disadvantage. Adjuvant hormonal therapy studies do not distinguish between ILC and IDC. Thus, recommendations about endocrine therapies are made using the same criteria as for IDC.

Mitotic figure counts are significantly overestimated in resection specimens of invasive breast carcinomas

Several authors have demonstrated an increased number of mitotic figures in breast cancer resection specimen when compared with biopsy material. This has been ascribed to a sampling artifact where biopsies are (i) either too small to allow formal mitotic figure counting or (ii) not necessarily taken form the proliferating tumor periphery. Herein, we propose a different explanation for this phenomenon. Biopsy and resection material of 52 invasive ductal carcinomas was studied. We counted mitotic figures in 10 representative high power fields and quantified MIB-1 immunohistochemistry by visual estimation, counting and image analysis. We found that mitotic figures were elevated by more than three-fold on average in resection specimen over biopsy material from the same tumors (20±6 vs 6±2 mitoses per 10 high power fields, P=0.008), and that this resulted in a relative diminution of post-metaphase figures (anaphase/telophase), which made up 7% of all mitotic figures in biopsies but only 3% in resection specimen (P<0.005). At the same time, the percentages of MIB-1 immunostained tumor cells among total tumor cells were comparable in biopsy and resection material, irrespective of the mode of MIB-1 quantification. Finally, we found no association between the size of the biopsy material and the relative increase of mitotic figures in resection specimen. We propose that the increase in mitotic figures in resection specimen and the significant shift towards metaphase figures is not due to a sampling artifact, but reflects ongoing cell cycle activity in the resected tumor tissue due to fixation delay. The dwindling energy supply will eventually arrest tumor cells in metaphase, where they are readily identified by the diagnostic pathologist. Taken together, we suggest that the rapidly fixed biopsy material better represents true tumor biology and should be privileged as predictive marker of putative response to cytotoxic chemotherapy.

Variation in precursor lesions of pancreatic cancer among high-risk groups

PURPOSE Pancreatic ductal adenocarcinoma (PDAC) surveillance programs are currently offered to high-risk individuals aiming to detect precursor lesions or PDAC at an early stage. We assessed differences in frequency and behavior of precursor lesions and PDAC between two high-risk groups. EXPERIMENTAL DESIGN Individuals with a p16-Leiden germline mutation (N = 116; median age 54 years) and individuals from familial pancreatic cancer (FPC) families (N = 125; median age 47 years) were offered annual surveillance by MRI and magnetic resonance cholangiopancreatography (MRCP) with or without endoscopic ultrasound (EUS) for a median surveillance period of 34 months (0-127 months) or 36 months (0-110 months), respectively. Detailed information was collected on pancreatic cystic lesions detected on MRCP and precursor lesions in surgical specimens of patients who underwent pancreatic surgery. RESULTS Cystic lesions were more common in the FPC cohort (42% vs. 16% in p16-Leiden cohort), whereas PDAC was more common in the p16-Leiden cohort (7% vs. 0.8% in FPC cohort). Intraductal papillary mucinous neoplasm (IPMN) was a common finding in surgical specimens of FPC-individuals, and was only found in two patients of the p16-Leiden cohort. In the p16-Leiden cohort, a substantial proportion of cystic lesions showed growth or malignant transformation during follow-up, whereas in FPC individuals most cystic lesions remain stable. CONCLUSION In p16-Leiden mutation carriers, cystic lesions have a higher malignant potential than in FPC-individuals. On the basis of these findings, a more intensive surveillance program may be considered in this high-risk group.

Glucagon-like-peptide-1 receptor expression in normal and diseased human thyroid and pancreas.

Glucagon-like-peptide-1 (GLP1) analogs may induce thyroid or pancreatic diseases in animals, raising questions about their use in diabetic patients. There is, however, controversy regarding expression of GLP1 receptors (GLP1R) in human normal and diseased thyroid and pancreas. Here, 221 human thyroid and pancreas samples were analyzed for GLP1R immunohistochemistry and compared with quantitative in vitro GLP1R autoradiography. Neither normal nor hyperplastic human thyroids containing parafollicular C cells express GLP1R with either method. Papillary thyroid cancer do not, and medullary thyroid carcinomas rarely express GLP1R. Insulin- and somatostatin-producing cells in the normal pancreas express a high density of GLP1R, whereas acinar cells express them in low amounts. Ductal epithelial cells do not express GLP1R. All benign insulinomas express high densities of GLP1R, whereas malignant insulinomas rarely express them. All ductal pancreatic carcinomas are GLP1R negative, whereas 6/20 PanIN 1/2 and 0/12 PanIN 3 express GLP1R. Therefore, normal thyroid, including normal and hyperplastic C cells, or papillary thyroid cancer are not targets for GLP1 analogs in humans. Conversely, all pancreatic insulin- and somatostatin-producing cells are physiological GLP1 targets, as well as most acini. As normal ductal epithelial cells or PanIN 3 or ductal pancreatic carcinomas do not express GLP1R, it seems unlikely that GLP1R is related to neoplastic transformation in pancreas. GLP1R-positive medullary thyroid carcinomas and all benign insulinomas are candidates for in vivo GLP1R targeting.Modern Pathology advance online publication, 12 September 2014; doi:10.1038/modpathol.2014.113.

Overexpression of EphB4 in the mammary epithelium shifts the differentiation pathway of progenitor cells and promotes branching activity and vascularization

Postnatally, the mammary gland undergoes continuous morphogenesis and thereby is especially prone to malignant transformation. Thus, the maintenance of the epithelium depends on a tight control of stem cell recruitment. We have previously shown that epithelial overexpression of the EphB4 receptor results in defective mammary epithelial development and conferred a metastasizing tumor phenotype on experimental mouse mammary tumors accompanied by a preponderance of progenitor cells. To analyze the effect of EphB4 overexpression on mammary epithelial cell fate, we have used Fluorescence Activated Cell Sorting (FACS) analyses to quantify epithelial sub-populations and repopulation assays of cleared fat pads to investigate their regenerative potential. These experiments revealed that deregulated EphB4 expression leads to an augmentation of bi-potent progenitor cells and to a shift of the differentiation pathway towards the luminal lineage. The analyses of the ductal outgrowths indicated that EphB4 overexpression leads to enforced branching activity, impedes ductal differentiation and stimulates angiogenesis. To elucidate the mechanisms forwarding EphB4 signals, we have compared the expression profile of defined cell populations between EphB4 transgene and wild type mammary glands concentrating on the wnt signaling pathway and on genes implicated in cell migration. With respect to wnt signaling, the progenitor cell population was the most affected, whereas the stem cell-enriched population showed the most pronounced deregulation of migration-associated genes. Thus, the luminal epithelial EphB4 signaling contributes, most likely via wnt signaling, to the regulation of migration and cell fate of early progenitors and is involved in the determination of branching points along the ductal tree.

Chondroitin sulfate proteoglycan CSPG4 as a novel hypoxia-sensitive marker in pancreatic tumors.

CSPG4 marks pericytes, undifferentiated precursors and tumor cells. We assessed whether the shed ectodomain of CSPG4 (sCSPG4) might circulate and reflect potential changes in CSPG4 tissue expression (pCSPG4) due to desmoplastic and malignant aberrations occurring in pancreatic tumors. Serum sCSPG4 was measured using ELISA in test (n = 83) and validation (n = 221) cohorts comprising donors (n = 11+26) and patients with chronic pancreatitis (n = 11+20) or neoplasms: benign (serous cystadenoma SCA, n = 13+20), premalignant (intraductal dysplastic IPMNs, n = 9+55), and malignant (IPMN-associated invasive carcinomas, n = 4+14; ductal adenocarcinomas, n = 35+86). Pancreatic pCSPG4 expression was evaluated using qRT-PCR (n = 139), western blot analysis and immunohistochemistry. sCSPG4 was found in circulation, but its level was significantly lower in pancreatic patients than in donors. Selective maintenance was observed in advanced IPMNs and PDACs and showed a nodal association while lacking prognostic relevance. Pancreatic pCSPG4 expression was preserved or elevated, whereby neoplastic cells lacked pCSPG4 or tended to overexpress without shedding. Extreme pancreatic overexpression, membranous exposure and tissue(high)/sera(low)-discordance highlighted stroma-poor benign cystic neoplasm. SCA is known to display hypoxic markers and coincide with von-Hippel-Lindau and Peutz-Jeghers syndromes, in which pVHL and LBK1 mutations affect hypoxic signaling pathways. In vitro testing confined pCSPG4 overexpression to normal mesenchymal but not epithelial cells, and a third of tested carcinoma cell lines; however, only the latter showed pCSPG4-responsiveness to chronic hypoxia. siRNA-based knockdowns failed to reduce the malignant potential of either normoxic or hypoxic cells. Thus, overexpression of the newly established conditional hypoxic indicator, CSPG4, is apparently non-pathogenic in pancreatic malignancies but might mark distinct epithelial lineage and contribute to cell polarity disorders. Surficial retention on tumor cells renders CSPG4 an attractive therapeutic target. Systemic 'drop and restoration' alterations accompanying IPMN and PDAC progression indicate that the interference of pancreatic diseases with local and remote shedding/release of sCSPG4 into circulation deserves broad diagnostic exploration.

The mammary gland in small ruminants: major morphological and functional events underlying milk production – a review

The importance of small ruminants to the dairy industry has increased in recent years, especially in developing countries, where it has a high economic and social impact. Interestingly and despite the fact that the mammary gland is the specialised milk production organ, very few authors studied the modifications occurring in the mammary gland through the lactation period in production animals, particularly in the small ruminants, sheep (Ovis aries) and goat (Capra hircus). Nevertheless, understanding the different mammary gland patterns throughout lactation is essential to improve dairy production. In addition, associating these patterns with different milking frequencies, lactation number or different diets is also of high importance, directly affecting the dairy industry. The mammary gland is commonly composed of parenchyma and stroma, which includes the ductal system, with individual proportions of each changing during the different periods and yields in a lactation cycle. Indeed, during late gestation, as well as during early to mid-lactation, mammary gland expansion occurs, with an increase in the number of epithelial cells and lumen area, which leads to increment of the parenchyma tissue, as well as a reduction of stroma, corresponding macroscopically to the increase in mammary gland volume. Throughout late lactation, the mammary gland volume decreases owing to the regression of the secretory structure. In general, common mammary gland patterns have been shown for both goats and sheep throughout the several lactation stages, although the number of studies is limited. The main objective of this manuscript is to review the colostrogenesis and lactogenesis processes as well as to highlight the mammary gland morphological patterns underlying milk production during the lactation cycle for small ruminants, and to describe potential differences between goats and sheep, hence contributing to a better description of mammary gland development during lactation for these two poorly studied species.

Implications of Imaging Criteria for the Management and Treatment of Intraductal Papillary Mucinous Neoplasms - Benign versus Malignant Findings

OBJECTIVES Evaluation of computed tomography (CT) and magnetic resonance imaging (MRI) for differentiation of pancreatic intraductal papillary mucinous neoplasm (IPMN) subtypes based on objective imaging criteria. METHODS Fifty-eight patients with 60 histologically confirmed IPMNs were included in this retrospective study. Eighty-three imaging studies (CT,n = 42; MRI,n = 41) were analysed by three independent blinded observers (O1-O3), using established imaging criteria to assess likelihood of malignancy (-5, very likely benign; 5, very likely malignant) and histological subtype (i.e., low-grade (LGD), moderate-grade (MGD), high-grade dysplasia (HGD), early invasive carcinoma (IPMC), solid carcinoma (CA) arising from IPMN). RESULTS Forty-one benign (LGD IPMN,n = 20; MGD IPMN,n = 21) and 19 malignant (HGD IPMN,n = 3; IPMC,n = 6; solid CA,n = 10) IPMNs located in the main duct (n = 6), branch duct (n = 37), or both (n = 17) were evaluated. Overall accuracy of differentiation between benign and malignant IPMNs was 86/92 % (CT/MRI). Exclusion of overtly malignant cases (solid CA) resulted in overall accuracy of 83/90 % (CT/MRI). The presence of mural nodules and ductal lesion size ≥30 mm were significant indicators of malignancy (p = 0.02 and p < 0.001, respectively). CONCLUSIONS Invasive IPMN can be identified with high confidence and sensitivity using CT and MRI. The diagnostic problem that remains is the accurate radiological differentiation of premalignant and non-invasive subtypes. KEY POINTS • CT and MRI can differentiate benign from malignant forms of IPMN. • Identifying (pre)malignant histological IPMN subtypes by CT and MRI is difficult. • Overall, diagnostic performance with MRI was slightly (not significantly) superior to CT.

Expression of E-cadherin repressors SNAIL, ZEB1 and ZEB2 by tumour and stromal cells influences tumour-budding phenotype and suggests heterogeneity of stromal cells in pancreatic cancer.

BACKGROUND There is evidence that tumour-stroma interactions have a major role in the neoplastic progression of pancreatic ductal adenocarcinoma (PDAC). Tumour budding is thought to reflect the process of epithelial-mesenchymal transition (EMT); however, the relationship between tumour buds and EMT remains unclear. Here we characterize the tumour-budding- and stromal cells in PDAC at protein and mRNA levels concerning factors involved in EMT. METHODS mRNA in situ hybridisation and immunostaining for E-cadherin, β-catenin, SNAIL1, ZEB1, ZEB2, N-cadherin and TWIST1 were assessed in the main tumour, tumour buds and tumour stroma on multipunch tissue microarrays from 120 well-characterised PDACs and associated with the clinicopathological features, including peritumoural (PTB) and intratumoural (ITB) budding. RESULTS Tumour-budding cells showed increased levels of ZEB1 (P<0.0001) and ZEB2 (P=0.0119) and reduced E-cadherin and β-catenin (P<0.0001, each) compared with the main tumour. Loss of membranous β-catenin in the main tumour (P=0.0009) and tumour buds (P=0.0053), without nuclear translocation, as well as increased SNAIL1 in tumour and stromal cells (P=0.0002, each) correlated with high PTB. ZEB1 overexpression in the main tumour-budding and stromal cells was associated with high ITB (P=0.0084; 0.0250 and 0.0029, respectively) and high PTB (P=0.0005; 0.0392 and 0.0007, respectively). ZEB2 overexpression in stromal cells correlated with higher pT stage (P=0.03), lymphatic invasion (P=0.0172) and lymph node metastasis (P=0.0152). CONCLUSIONS In the tumour microenvironment of phenotypically aggressive PDAC, tumour-budding cells express EMT hallmarks at protein and mRNA levels underlining their EMT-type character and are surrounded by stromal cells expressing high levels of the E-cadherin repressors ZEB1, ZEB2 and SNAIL1, this being strongly associated with the tumour-budding phenotype. Moreover, our findings suggest the existence of subtypes of stromal cells in PDAC with phenotypical and functional heterogeneity.

5-year results of accelerated partial breast irradiation using sole interstitial multicatheter brachytherapy versus whole-breast irradiation with boost after breast-conserving surgery for low-risk invasive and in-situ carcinoma of the female breast: a randomised, phase 3, non-inferiority trial.

BACKGROUND In a phase 3, randomised, non-inferiority trial, accelerated partial breast irradiation (APBI) for patients with stage 0, I, and IIA breast cancer who underwent breast-conserving treatment was compared with whole-breast irradiation. Here, we present 5-year follow-up results. METHODS We did a phase 3, randomised, non-inferiority trial at 16 hospitals and medical centres in seven European countries. 1184 patients with low-risk invasive and ductal carcinoma in situ treated with breast-conserving surgery were centrally randomised to either whole-breast irradiation or APBI using multicatheter brachytherapy. The primary endpoint was local recurrence. Analysis was done according to treatment received. This trial is registered with ClinicalTrials.gov, number NCT00402519. FINDINGS Between April 20, 2004, and July 30, 2009, 551 patients had whole-breast irradiation with tumour-bed boost and 633 patients received APBI using interstitial multicatheter brachytherapy. At 5-year follow-up, nine patients treated with APBI and five patients receiving whole-breast irradiation had a local recurrence; the cumulative incidence of local recurrence was 1·44% (95% CI 0·51-2·38) with APBI and 0·92% (0·12-1·73) with whole-breast irradiation (difference 0·52%, 95% CI -0·72 to 1·75; p=0·42). No grade 4 late side-effects were reported. The 5-year risk of grade 2-3 late side-effects to the skin was 3·2% with APBI versus 5·7% with whole-breast irradiation (p=0·08), and 5-year risk of grade 2-3 subcutaneous tissue late side-effects was 7·6% versus 6·3% (p=0·53). The risk of severe (grade 3) fibrosis at 5 years was 0·2% with whole-breast irradiation and 0% with APBI (p=0·46). INTERPRETATION The difference between treatments was below the relevance margin of 3 percentage points. Therefore, adjuvant APBI using multicatheter brachytherapy after breast-conserving surgery in patients with early breast cancer is not inferior to adjuvant whole-breast irradiation with respect to 5-year local control, disease-free survival, and overall survival. FUNDING German Cancer Aid.