Angel Lanas

Lars Aabakken

Jorge Fonseca

Zeynel A Mungan

George V Papatheodoridis

Hubert Piessevaux

Livio Cipolletta

Javier Nuevo

Monica Tafalla

Clinical Predictors of Poor Outcomes Among Patients with Non-Variceal Upper Gastrointestinal Bleeding in Europe

Keywords: Non-variceal bleeding < Topics, X keyword = no topic, Y

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Collecting data on presence or absence of co-morbidity means that severity is not taken into account. It would be probable that a NYHA IV heart failure would have a greater impact than NYHA I heart failure although this information would be difficult to gain accurately retrospectively. The referee is right. We have preferred to include this information as 'yes or no' and the number of comorbidities because of accuracy in retrospective collection of data. We have included this aspect as one of the weaknesses of the study in the discussion. Page: 15 We are given no information on what therapy was delivered (injection, clip, thermal, combination) or to which lesions with which stigmata making it difficult to draw any conclusions from the analysis of patients receiving endoscopic therapy. This information is now provided in the Results text. Page:9

Was haemodynamic instability not associated with adverse outcome? This is at odds with a body of literature so should be discussed if this is the case. It was indeed. Haemodynamic instability can be shown or expressed in different ways (hypotension, tachycardia, shock , etc.). The inclusion of variables involved in that concept have shown shock as the actual variable associated with poor outcomes.

Table 3 presents data for a different sub group to that presented in figures 2 and 3. A consistent approach should be made to data presentation. Either figures 2 and 3 should be combined in a table or the data from table 3 should be presented in two figures. Our aim was to provide the relevant primary analysis as Figures 2 and3 for a better and visual understanding of the primary results. Those of Table 3 were secondary analysis of a subset of patients undergoing therapeutic endoscopy, and we have therefore presented these in a Table . We believe that it is not unusual to present data as a mix of Figures and Tables. We would prefer to avoid inclusion of a third Figure of similar appearance to Figures 2 and3, as this may not allow easy discrimination of the subset analysis from the primary analysis. We can report all as tables or figures if the referee or editors considered this important.

It is completely unnecessary to state that AstraZeneca is "the manufacturer of esomeprazole" in the acknowledgements. This is blatant advertising and must be removed. Our intention was to be fully transparent to the reader with respect to the association between the corporate funding for the study, and the marketed product used to manage gastric disorder. This was added in order to comply with the recommended acknowledgement text advised in the GPP2 guidelines. We are sorry this was misunderstood as an attempt to advertise, as this was not the intention. We have removed the sentence.

Reviewer: 2

Comments for Transmission to the Authors In the past few years, AP&T has published several relevant papers about upper GI bleeding, which could augment the Discussion. E.g. Button LA, Roberts SE, Evans PA, Goldacre MJ, Akbari A, Dsilva R, Macey S, Williams JG. Aliment Pharmacol Ther. 2011 Jan;33(1):64-76.

Fletcher EH, Johnston DE, Fisher CR, Koerner RJ, Newton JL, Gray CS. Aliment Pharmacol Ther. 2010 Oct;32 (7):831-9.

We have included the first of the references in the discussion, since we believe it has relationship with our paper. Thanks. Page:15 The paper retrospectively investigates in a subset of European countries clinical predictors of poor outcomes in pts with NVUGIB The idea to evaluate in "real life" this critical and cost-driven condition is valid and the derived informations are useful.

Reviewer: 3 Comments for Transmission to the Authors

However, the methodology of the study is questionable. In fact, there is a geographical unbalance between European included countries i.e. mediterraneans are prevalent, 5 out of 7. These aspects can be reflected on the logistic and routine practice for the patients care. In fact, as stated in mat and meth ( page 7 lines 3-4) the management of patients was performed according to local routine practice , and these can be greatly different . A criteria to limit this problem should be to included only teaching hospitals or not-teaching hospital or hospitals with GI services or without . There is a predominance of Mediterranean countries included in the study. We did not intend to reflect a picture of the whole of Europe. Since Health Systems in Europe are in general homogeneous in the sense that they are public and universal or highly subsidised by the states, we believe that the homogeneity across countries is much higher than in other regions of the world. However, we wanted to know whether differences in local management of NVUGIB events could impact outcomes. All these potential variables had been included in the multivariate analysis and the results were precisely that within this regional setting, local differences in management did not affect hard outcomes. This was reflected in the Results section ( Page: 10) .In any case, we have conducted additional analysis as requested by the reviewer and the conclusions were the same, (please see below the predictors of rebleeding/bleeding continuation and mortality for large teaching hospitals ) which seems logical since large hospitals and other factors that may differentiate teaching hospitals (six GI services,) were included in the model. In any case we have included a reference to these analyses in the Result section We agree that some countries participated with less centres than others although the number of patients was the same and that this could have induced an imbalance. As commented above, this potential bias has been controlled in the logistic regression model since country, and size of hospital variables, among other potential confounders have been introduced in the models. We have conducted analysis according to size of centres and found no differences. This potential bias has been included in the discussion (Page 15), but we do not present these data in the Ms core text since subgroup analyses reduce dramatically the estimated sample size, and post-hoc analysis could be mislead the main goal of the study and be misinterpreted.

Finally the lack of generalisation of the study conclusion ( page 15, conlusion last para) weakens the study We had a very conservative approach in our conclusion due to the retrospective nature of the study. We do believe however that the extension of the study allows us to expand the scope of the conclusions and have modified this accordingly. America, Europe and Asia most commonly place the incidence of continued and/or re-bleeding and mortality following acute NVUGIB between 2-17%. [START_REF] Muller | Non-variceal upper GI bleeding in patients already hospitalized for another condition[END_REF][START_REF] Cipolletta | Outpatient management for low-risk nonvariceal upper GI bleeding: a randomized controlled trial[END_REF][START_REF] Marmo | Predictive factors of mortality from nonvariceal upper gastrointestinal hemorrhage: a multicenter study[END_REF][START_REF] Sandel | Nonvariceal upper gastrointestinal bleeding: differences in outcome for patients admitted to internal medicine and gastroenterological services[END_REF][START_REF] Viviane | Estimates of costs of hospital stay for variceal and nonvariceal upper gastrointestinal bleeding in the United States[END_REF][START_REF] Barkun | The Canadian Registry on Nonvariceal Upper Gastrointestinal Bleeding and Endoscopy (RUGBE): Endoscopic hemostasis and proton pump inhibition are associated with improved outcomes in a real-life setting[END_REF][START_REF] Sung | Causes of mortality in patients with peptic ulcer bleeding: a prospective cohort study of 10,428 cases[END_REF] Recent data from randomised, controlled trials and meta-analyses have shown that appropriate endoscopic and pharmacological treatments can reduce the incidence of re-bleeding 6, 11, 13, 14 and surgery, [START_REF] Leontiadis | Systematic review and meta-analysis of proton pump inhibitor therapy in peptic ulcer bleeding[END_REF] duration of hospitalisation [START_REF] Sung | Intravenous esomeprazole for prevention of recurrent peptic ulcer bleeding: a randomized trial[END_REF][START_REF] Leontiadis | Systematic review and meta-analysis: proton-pump inhibitor treatment for ulcer bleeding reduces transfusion requirements and hospital stay--results from the Cochrane Collaboration[END_REF] and number of blood units transfused. [START_REF] Sung | Intravenous esomeprazole for prevention of recurrent peptic ulcer bleeding: a randomized trial[END_REF][START_REF] Leontiadis | Systematic review and meta-analysis: proton-pump inhibitor treatment for ulcer bleeding reduces transfusion requirements and hospital stay--results from the Cochrane Collaboration[END_REF] However, mortality associated with the bleeding event seems to be much more difficult to improve. [START_REF] Sung | Intravenous esomeprazole for prevention of recurrent peptic ulcer bleeding: a randomized trial[END_REF][START_REF] Leontiadis | Systematic review and meta-analysis of proton pump inhibitor therapy in peptic ulcer bleeding[END_REF] For example, in a population-based study of patients hospitalised due to GI complications in Spain, the age-and sex-adjusted mortality rate associated with upper GI bleeding events decreased between 1996 and 2005, but the case fatality rate has remained unchanged over this time period. [START_REF] Lanas | Time trends and impact of upper and lower gastrointestinal bleeding and perforation in clinical practice[END_REF] The reasons for this are unclear, but progressive population aging and increased frequency of comorbidities may be relevant. Furthermore, the outcomes of NVUGIB, and factors associated with these outcomes in clinical practice, may be very different from those reported in randomised clinical trials. Also, factors unrelated to endoscopic and pharmacological treatments, such as organisational factors, early endoscopy, specialty-based management, and the use of predetermined care protocols may have a major impact on clinical outcomes. In order to further improve the management of NVUGIB, precise information regarding these issues is needed in clinical practice.

F

Outcome definition

The main outcome variables were continued bleeding following therapeutic endoscopy, re-bleeding, need for surgical procedures to control bleeding (other than endoscopic treatment) and in-hospital and 30-day all-cause mortality. Bleeding continuation was defined as one of the following: spurting from an artery at the initial endoscopic examination, which did not respond to endoscopic therapy or persisted following initial endoscopy; red bloody nasogastric aspirate; shock with pulse >100 beats/min, systolic blood pressure <100 mmHg, or both; or need for substantial replacement of blood and fluid volume (transfusion of >3 units of blood within 4 hours) following endoscopy. Re-bleeding was defined as recurrent vomiting of fresh blood, melaena, or both with either shock or a decrease in haemoglobin level of ≥20 g/L following initial successful treatment.

This report focuses on clinical factors predictive of a poor outcome of an initial NVUGIB episode, defined as bleeding continuation/re-bleeding or all-cause mortality within 30 days.

Statistical analysis

Multivariable analyses were performed using random-intercept mixed-model logistic regression to account for clustering by study centre. Model creation and selection was based on a priori clinical and biological considerations. PPI use before endoscopy was used as an adjustment factor in every model. Raw (univariate) and multivariable adjusted odds ratios and 95% confidence intervals (CIs) were calculated from the logistic regression coefficients and standard errors. The significance level was 5% for keeping independent predictors in the models.

Sample size calculation

A target sample size of 400 patients per country was established in order to obtain country-specific estimates of the incidence of 30-day bleeding continuation/rebleeding with 95% CIs of width ±3.5%, based on an expected combined endpoint incidence of 14.1%. 11

Results

Patients and initial management of NVUGIB

A total of 123 centres participated in the study, with the following distribution per country: Belgium (26 centres), Greece (10), Italy (11)

Outcomes of NVUGIB

Overall, 12.3% of patients experienced bleeding continuation/re-bleeding within 30 days of the initial NVUGIB episode. Continued bleeding was observed in 7.3% of patients, while 10.5% of patients experienced re-bleeding within 30 days. A secondlook endoscopy was performed in 28.6% of patients, but in most cases no therapeutic endoscopy was performed. The proportion of patients undergoing surgery (other than endoscopy) to control bleeding was low at 3.3%. The mortality rate within 30 days of the bleeding event was 5.2%. There were significant unadjusted differences across countries in theses outcomes (p= 0.017 for bleeding continuation/re-bleeding and p=0.005 for any death) (Figure 1).

Clinical predictors of poor outcomes

Predictors for bleeding continuation and re-bleeding were very similar (data not shown) and therefore, since these two outcomes imply a failure to control bleeding, here we report the combined outcome. The determinants for bleeding continuation/rebleeding are summarised in Figure 2. The multivariate analysis did not detect differences across countries in this outcome. No differences were also observed concerning the type of team managing the bleeding event or size of hospital (defined as those with > 500 beds). However, the number of comorbidities, bleeding during hospitalisation for other non-GI bleeding reasons, age >65 years and a history of NVUGIB were associated with an increased risk of bleeding continuation/re-bleeding.

Bleeding from a gastric or duodenal ulcer was associated with a worst outcome; however, bleeding from acute mucosal lesions, or associated with anticoagulants or use of non-steroidal anti-inflammatory drugs (NSAIDs) had a lower risk. The multivariate analysis for predictors of death showed no differences by country and only Spain was close to significance for a lower risk with Belgium as a reference group. Also, the size of the hospital, the type of team managing the bleeding event, the type of lesion causing the bleeding event or therapy were not linked to this outcome. Only the number of comorbidities, age >65 years, alcohol abuse, presence of haematemesis at admission, duodenal ulcer as the cause of bleeding, and bleeding during hospitalisation for other non-GI bleeding reasons were associated with increased risk of death. On the contrary, use of low-dose acetylsalicylic acid (ASA; aspirin) was associated with decreased risk of death (Figure 3). When data where analysed to identify the determinants of poor outcomes in large teaching hospitals (which represented 63.2% of all hospitals of the study) similar results were found (data not shown).

When the analysis was restricted to patients undergoing therapeutic endoscopy (patients with high-risk stigmata for rebleeding) the rate of bleeding continuation/rebleeding was 18.8% (n = 178) and surgery to control bleeding was carried out in 5.6%

(n = 49) of cases; 6.0% of patients (n = 57) died. Table 3 summarises the predictors of bleeding continuation/re-bleeding in this subset of patients. Country differences, teams who managed the episode of bleeding or PPI treatment were not identified as independent predictors. Unlike the whole patient cohort, hospital size was associated with an increased risk of re-bleeding.

Discussion

In this 'real-life' study, the overall rate of the combined endpoint of bleeding continuation/re-bleeding, need of surgery to control bleeding and mortality rates were found to be within the lower range of previously reported findings. [START_REF] Muller | Non-variceal upper GI bleeding in patients already hospitalized for another condition[END_REF][START_REF] Marmo | Predictive factors of mortality from nonvariceal upper gastrointestinal hemorrhage: a multicenter study[END_REF][START_REF] Sandel | Nonvariceal upper gastrointestinal bleeding: differences in outcome for patients admitted to internal medicine and gastroenterological services[END_REF][START_REF] Barkun | The Canadian Registry on Nonvariceal Upper Gastrointestinal Bleeding and Endoscopy (RUGBE): Endoscopic hemostasis and proton pump inhibition are associated with improved outcomes in a real-life setting[END_REF][START_REF] Sung | Causes of mortality in patients with peptic ulcer bleeding: a prospective cohort study of 10,428 cases[END_REF][START_REF] Manguso | In-hospital mortality in non-variceal upper gastrointestinal bleeding Forrest 1 patients[END_REF][START_REF] Marmo | Predicting mortality in non-variceal upper gastrointestinal bleeders: validation of the Italian PNED score and prospective comparison with the Rockall score[END_REF] The ENERGIB study therefore confirms that mortality associated with NVUGIB has not decreased substantially in recent years, despite advances in endoscopic and pharmacological therapies. We have reported bleeding continuation and rebleeding as a single outcome due to several reasons. First, both outcomes represent a similar concept which is a failure to control bleeding with medical and endoscopy therapy;

second, the retrospective collection of data may not always distinguish clearly between these two events; third, by doing so, we avoid double counting, and finally the analysis of the determinants for each event report very similar results.

To our knowledge, this is the first study to characterise the outcomes of NVUGIB, and factors predictive of poor outcomes, in a 'real-world', within a European setting.

By recruiting a significant number of centres from each participating country, and including all consecutive patients with NVUGIB within a limited period of time, we have made a picture of patient characteristics, type of management and outcomes in the participating countries that probably reflects outcomes of bleeding events in clinical practice. By doing so, we have detected important differences in the main outcomes, including death, of NVUGIB events across countries. The hypothesis was that these differences could be attributed to differences in patient management and teams dealing with bleeding patients between countries, or even within the same country depending of the size of the hospital, for example. However, further analysis of the data, in a multivariate context, has shown that these differences are basically dependent on patients' characteristics rather than differences in the clinical management of the bleeding event. One explanation for this may be the fact that the two major factors affecting outcome-related bleeding -use of therapeutic endoscopy for peptic ulcer bleeding and use of parenteral PPI after endoscopy -were high in the whole cohort and differences between countries were minor. The analysis carried out in the subgroup of patients at the highest risk for re-bleeding or death (i.e. those with high-risk stigmata undergoing therapeutic endoscopy) have shown that country or management factors were not associated with these outcomes. There were some countries (e.g Belgium) which showed clearly a higher mortality rate when compared to others, but this difference disappeared when adjusted for other important variables such as comorbidities or in-hospital vs extra-hospital bleeding. The size of the hospital (> 500 beds) was linked to a higher risk of re-bleeding when the analysis was restricted to patients with high-risk stigmata, which may be confounded by the fact that these hospitals may receive complex referral patients.

Risk factors that have been previously identified to be predictive of bleeding continuation and re-bleeding include: presence of comorbidities; 6 endoscopy-observed high-risk stigmata of bleeding; [START_REF] Kapsoritakis | Acute upper gastrointestinal bleeding in central Greece: the role of clinical and endoscopic variables in bleeding outcome[END_REF][START_REF] Muller | Non-variceal upper GI bleeding in patients already hospitalized for another condition[END_REF][START_REF] Barkun | The Canadian Registry on Nonvariceal Upper Gastrointestinal Bleeding and Endoscopy (RUGBE): Endoscopic hemostasis and proton pump inhibition are associated with improved outcomes in a real-life setting[END_REF] worse health status at admission; reasons) were associated with a significantly lower risk of poor outcomes. The latter may be explained by a potentially worse health status and the presence of comorbidities among patients who were already hospitalised.

Previously characterised predictors of mortality include: older age; [START_REF] Barkun | The Canadian Registry on Nonvariceal Upper Gastrointestinal Bleeding and Endoscopy (RUGBE): Endoscopic hemostasis and proton pump inhibition are associated with improved outcomes in a real-life setting[END_REF] presence of, and increasing number of, comorbidities; [START_REF] Muller | Non-variceal upper GI bleeding in patients already hospitalized for another condition[END_REF][START_REF] Barkun | The Canadian Registry on Nonvariceal Upper Gastrointestinal Bleeding and Endoscopy (RUGBE): Endoscopic hemostasis and proton pump inhibition are associated with improved outcomes in a real-life setting[END_REF] continued bleeding and/or re-bleeding; [START_REF] Kapsoritakis | Acute upper gastrointestinal bleeding in central Greece: the role of clinical and endoscopic variables in bleeding outcome[END_REF][START_REF] Muller | Non-variceal upper GI bleeding in patients already hospitalized for another condition[END_REF] and a finding of bright blood in the nasogastric tube aspirate. [START_REF] Muller | Non-variceal upper GI bleeding in patients already hospitalized for another condition[END_REF] The predictive validity of older age and the presence of comorbidities were confirmed in ENERGIB; in fact, the presence of comorbidities was by far the strongest predictor of mortality in this patient population. Other factors identified to be significantly predictive of mortality in this study were presentation with clinical symptoms of acute upper GI bleeding (haematemesis) and alcohol abuse. Patients' use of either low-(< 100 mg/day) or higher dose of ASA (33% of ASA use was > 100 mg/day and one third of that for ischaemic heart disease) was found to be protective against mortality, which could be related to the findings of Sung et al. that almost 80% of deaths following acute peptic ulcer bleeding were non-bleeding related. [START_REF] Sung | Causes of mortality in patients with peptic ulcer bleeding: a prospective cohort study of 10,428 cases[END_REF] Unfortunately, we do not have data on the prescription of low-dose ASA after the bleeding event in terms of whether this could have contributed to the finding of decreased mortality.

The strength of this study is its observational nature and the finding that differences in outcomes of NVUGIB reported across countries does not seem to be due to differences in clinical management or health organisations attending these GI complications, at least within a European setting. While data obtained through highquality, randomised controlled trials are very valuable; there is a difference between outcomes in such studies and outcomes that can be observed in everyday clinical practice in the general population. As all patients included in the ENERGIB study However, the methodological limitations inherent in the design of all observational, retrospective studies must also be considered. In this way, the variable described as alcohol abuse is not well defined and may represent a wide spectrum of alcohol use.

However, it is reasonable to assume that patients with no or sporadic use have not been ascribed here. The variable was associated with increased risk of mortality and not with rebleeding, which we believe makes sense, since mortality is linked to morbidity rather than to the bleeding event in most cases [START_REF] Sung | Causes of mortality in patients with peptic ulcer bleeding: a prospective cohort study of 10,428 cases[END_REF] . Also, the severity of the comorbidities has not been taken into account, which could have determined a more specific profile for poor outcomes, but the retrospective collection of data does not allow this. Other factors that have been linked to fatality rates in upper GI bleeding such as social deprivation, day of the week of hospitalisation etc [START_REF] Button | Hospitalized incidence and case fatality for upper gastrointestinal bleeding from 1999 to 2007: a record linkage study[END_REF] have not been considered. Another limitation is that some countries are represented with less number of centres, but with the total patient number overlapping each other, which may impact the results by over-representing different clinical expertise. The inclusion of these variables in the logistic regression model limit this impact but may not overcome all potential bias. Finally, we need to point out that we may have missed bleeding events with no endoscopy study. Although we believe these to be very few, they may represent either very severe or very mild events. The potential for large differences in medical record keeping across the sites and European studies included in ENERGIB may also have impacted the results. As this study design is not

  mortality and agree with the data reported by Sung et al. in Annals of Internal Medicine 2010). We have included a comment in the discussion. Page: 14

5

 5 The aim of the European Survey of Non-variceal Upper Gastrointestinal Bleeding (ENERGIB) was to report on outcomes across different countries and evaluate a number of variables possibly associated with outcomes of acute NVUGIB including clinical predictors, management strategies and healthcare resource utilisation in a 'real-world', European, clinical practice setting. This report focuses on the predictors of poor outcomes of NVUGIB in terms of bleeding continuation/re-bleeding and mortality. .gov Identifier: NCT00797641; study code: NIS-GEU-DUM-2008/2) was an observational, retrospective cohort study carried out across multiple centres in seven countries (Belgium, Greece, Italy, Norway, Portugal, Spain and Turkey). Eligible patients were adult (aged 18 years or older) men and women who underwent endoscopy due to overt NVUGIB at participating hospitals. Both patients admitted to the hospital because of NVUGIB and those admitted for another reason who developed NVUGIB during hospitalisation were eligible. Overt NVUGIB was defined as haematemesis/coffee ground vomiting, melaena, haematochezia and other clinical or laboratory evidence of acute blood loss from the upper GI tract confirmed by upper GI endoscopy. Patients with incomplete medical records were excluded from the study.The initial enrolment period was between October 1 and November 30, 2008. In order to avoid bias in patient selection, all consecutive patients admitted to hospital with NVUGIB were identified by hospital computer records based on ICD-9 codes[START_REF] Lanas | A nationwide study of mortality associated with hospital admission due to severe gastrointestinal events and those associated with nonsteroidal antiinflammatory drug use[END_REF] Based on a previous internal survey and in order to have a representative sample from each country, we estimated that 20 consecutive patients per centre matching the required diagnosis would require around 20 centres per country and around 2 months for large general hospitals. Data were extracted by specialists from participating centres. If the number of patients identified during the initial recruitment period was lower than the recruitment target for each centre, further selection windows were used to ensure adequate enrolment of patients. If the number of patients was greater than the recruitment target, study participants were selected at random among eligible study was designed in line with the ethical principles described in the Declaration of Helsinki, and the final study protocol was approved by local ethics committees in each participating country. As this was a non-interventional study, the management of patients was according to routine practice at each centre. Data were collected retrospectively from patient medical records in relation to the initial NVUGIB episode and up to 30 days afterwards.

  to the standard clinical care protocol at each participating study site, the results of this study are able to represent a 'real-world' scenario of the outcomes of NVUGIB in the community. The observational, pan-European nature of ENERGIB means important information based on a large and diverse group of patients is accessible.

Figure 1 .

 1 Figure 1. Inter-country differences in rates of bleeding continuation/re-bleeding and

Figure 2 .

 2 Figure 2. Factors significantly predictive of the combined endpoint of bleeding

Figure 3 .

 3 Figure 3. Factors significantly predictive of mortality within 30 days of the initial

  Analysis of predictors of bleeding continuation/re-bleeding and/or mortality in Hospitals ≥ 500 beds (n = 1680)Another imbalance methodological factor is that three countries are represented with at least 24 centres and the others with half of the centres but with the total patients number overlapping each other except Grece.( i.e Belgium 26 centres total pts =426 vs Portugal 12 centres , pts n= 404 ). These variabilty probably means that different Hospitals type are included reflecting expertise, clinical skillness ect I suggest to the authors to revised your data in order to make a more appropiate comparison at least between similar Hospital

	Comparison of significant predictors of bleeding continuation/re-
	bleeding and/or mortality, between all patients vs. patients from
	Hospitals ≥ 500 beds (OR and 95%CI)
			Bleeding continuation / rebleeding (n = 219) Bleeding continuation / rebleeding		Mortality (n = 84) Mortality
			OR All patients 95% CI	p-value Big Hospitals	OR	95% CI All patients	p-value Big Hospitals
	Country		N=327		N=219		N=138	N=84
	Greece (vs. Belgium) Country		0.66	(0.33 -1.35)	0.27	0.51	(0.16 -1.55)	0.24
	Italy (vs. Belgium) Greece (vs. Belgium)		0.54	(0.28 -1.06)	0.08	0.86	(0.35 -2.15)	0.76
	F o r Norway (vs. Belgium) Portugal (vs. Belgium) Spain (vs. Belgium) Turkey (vs. Belgium) Sex (male vs. female) F o r Italy (vs. Belgium) Norway (vs. Belgium) Portugal (vs. Belgium) Spain (vs. Belgium) Turkey (vs. Belgium) F o r	0.52 0.75 0.60 0.54 1.14	(0.22 -1.25) (0.34 -1.65) (0.32 -1.12) (0.27 -1.08) (0.83 -1.57)	0.15 0.48 0.12 0.09 0.43	1.32 0.65 0.46 0.67 0.79	(0.44 -3.91) (0.22 -1.99) (0.18 -1.16) (0.25 -1.85) (0.51 -1.23)	0.62 0.46 0.11 0.45 0.30
	(Page 10-11) Age (>65 vs. ≤65 years) History of alcohol abuse Comorbidity (per comorbidity) History of NVUGIB Sex (male vs. female) Age (>65 vs. ≤65 years) History of alcohol abuse Admission (acute vs. no) Hematemesis Comorbidity (per comorbidity) History of NVUGIB Admission (acute vs. no)	P e e r 1.27 (0.91 -1.78) 1.16 (0.75 -1.79) 1.23 (1.07 -1.41) 1.36 (0.94 -1.95) P e 1.56 (1.18 -2.07) P e 1.66 (1.11 -2.48) 1.45 (1.08 -1.95) e r 1.22 (1.09 -1.36) 1.23 (1.07 -1.41) 0.16 0.50 0.003 0.10 0.01 0.02 1.47 (1.09 -1.98) 1.59 (1.16 -2.18) 1.66 (1.11 -2.48) e r	1.53 2.61 1.74 1.40 1.75 (1.18 -2.61) (0.93 -2.52) (1.50 -4.55) (1.45 -2.08) (0.84 -2.33) 2.69 (1.77 -4.09) 2.16 (1.32 -3.53) 1.41 (0.92 -2.14) 1.75 (1.52 -2.00) 1.96 (1.34 -2.86)	0.09 0.001 <0.001 2.61 (1.50 -4.55) 0.21 0.002 0.12 1.74 (1.45 -2.08) 2.16 (1.32 -3.53)
	High-dose ASA (>100 mg/day) Low-dose ASA (≤100 mg/day) NSAIDS Warfarin Oesophagitis diagnosis Gastric ulcer diagnosis Gastritis \ Erosions diagnosis Duodenal ulcer diagnosis Erosive duodenitis diagnosis Intra-hospital management by GI Intra-hospital management by GI bleeding team / Gastroenterology Hematemesis High-dose ASA (>100 mg/day) Low-dose ASA (≤100 mg/day) NSAIDS Warfarin Oesophagitis diagnosis Gastric ulcer diagnosis Gastritis \ Erosions diagnosis Duodenal ulcer diagnosis Erosive duodenitis diagnosis	R e v i e w (0.51 -1.44) 0.56 0.16 (0.05 -0.58) (0.69 -1.44) 0.97 0.26 (0.13 -0.52) 0.63 (0.41 -0.98) 0.86 0.99 0.04 0.45 (0.22 -0.93) 1.02 (0.63 -1.65) 0.93 0.87 (0.45 -1.68) 0.36 (0.19 -0.67) 0.002 0.68 (0.33 -1.41) 0.99 (0.70 -1.40) 0.95 1.38 (0.86 -2.22) 0.71 (0.44 -1.15) 0.16 0.95 (0.49 -1.86) 1.93 (1.40 -2.67) <0.001 1.45 (0.91 -2.32) 0.48 (0.20 -1.19) 0.12 0.63 (0.21 -1.86) 0.79 (0.55 -1.13) 0.20 0.69 (0.40 -1.18) R e v i e w 1.40 (1.10 -1.78) 1.45 (1.08 -1.95) 1.60 (1.16 -2.22) 0.20 (0.09 -0.45) R 0.39 (0.25 -0.62) 0.66 (0.47 -0.93) 0.63 (0.41 -0.98) e 0.65 (0.43 -0.98) 0.43 (0.27 -0.70) 0.36 (0.19 -0.67) v i e 0.58 (0.38 -0.87) 1.77 (1.36 -2.31) 1.93 (1.40 -2.67) 1.53 (1.06 -2.19) w	0.005 <0.001 0.03 0.67 0.30 0.19 0.89 0.12 0.40 0.17 0.16 (0.05 -0.58) 0.26 (0.13 -0.52) 0.45 (0.22 -0.93)
	bleeding team / Gastroenterology
	PPI use before endoscopy was used as adjustment factor.
	PPI use before endoscopy was used as adjustment factor.
	Less significant factors in patients from Hospitals ≥ 500 beds, probably due to the reduced sample size.

Clinical Predictors of Poor Outcomes Among Patients with Non-Variceal Upper Gastrointestinal Bleeding in Europe Running head: Clinical predictors of NVUGIB outcomes in Europe Angel Lanas, 1 Lars Aabakken, 2 Jorge Fonseca, 3 Zeynel A Mungan, 4 George V Papatheodoridis, 5 Hubert Piessevaux, 6 Livio Cipolletta, 7 Javier Nuevo, 8 Monica Tafalla 8

  To report outcomes and predictive factors for bleeding continuation/re-bleeding and mortality of NVUGIB in clinical practice in different European countries. Differences in the outcomes of NVUGIB in clinical practice across some European countries are explained mainly by patient-related factors, and not

	Introduction
	Acute upper gastrointestinal (GI) bleeding is a common cause of emergency
	management factors. hospitalisation worldwide. 1-3 While considerable advances in the prevention of peptic
	[Word limit 250: Current word count 239 excluding section headings] ulcer complications have meant that the incidence of acute non-variceal upper GI
	bleeding (NVUGIB) has decreased substantially in the past decade, 4, 5 NVUGIB is
	still associated with considerable morbidity and mortality. Studies from North
	Keywords: non-variceal upper gastrointestinal bleeding, predictors, rebleeding,
	mortality	o r F o r F o r F o r
		P P P P
		e e e e
		e r e r e r e r
		R R R R
		e e e e
		v i e v i e v i e v i e
	w w Significant differences across countries in bleeding continuation/re-bleeding (range: w w
	9-15.8%) or death (2.5-8%) at 30 days were explained by clinical factors (number of
	comorbidities, age >65 years, history of bleeding ulcers, in-hospital bleeding, type of
	lesion or type of concomitant medication). Other factors (country, size of hospital,
	profile of team managing the event, or endoscopic/pharmacological therapy received)
	did not affect these outcomes. Similar predictors were observed in patients with high-
	risk stigmata.

1 Title:

[START_REF] Kapsoritakis | Acute upper gastrointestinal bleeding in central Greece: the role of clinical and endoscopic variables in bleeding outcome[END_REF]

Gastroenterology, University of Zaragoza, I+CS. CIBERehd, Zaragoza, Spain;

2 Department of Medicine, Oslo University Hospital, Oslo, Norway;

[START_REF] Blatchford | Acute upper gastrointestinal haemorrhage in west of Scotland: case ascertainment study[END_REF]

Service of Gastroenterology, Hospital Garcia de Orta, CiiEM -Instituto Superior do Ciências da Saúde Egas Moniz, Almada, Portugal; 4 Department of Gastroenterohepatology, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey; 5 2 nd Department of Internal Medicine, Athens University Medical School, Hippokration Hospital of Athens, Athens, Greece; 6 Service de Gastroentérologie, Cliniques Universitaires St-Luc, Brussels, Belgium; 7 Division of Gastroenterology, Maresca Hospital, Torre del Greco, Italy; 8 Epidemiology Unit, AstraZeneca, Madrid, Spain Address for correspondence: Angel Lanas, Servicio de Aparato Digestivo. Hospital Clinico Universitario Lozano Blesa, C/ Domingo Miral s/n. 50009 Zaragoza, Spain. Telephone: 34976765786 Fax: 34 976 765787 Email: angel.lanas@gmail.com

2 SUMMARY Background: Non-variceal upper gastrointestinal bleeding (NVUGIB) is a common medical emergency associated with substantial morbidity and mortality. Despite advances in endoscopic and pharmacological treatment during the past two decades, the incidence of mortality associated with NVUGIB has remained relatively constant. Aim: Methods: This observational, retrospective cohort study (NCT00797641; ENERGIB) was conducted in Belgium, Greece, Italy, Norway, Portugal, Spain and Turkey. Eligible patients were hospitalised (new admissions or inpatients), presenting with overt NVUGIB with endoscopy from 1 October to 30 November, 2008. Patients were managed according to routine care, and data regarding bleeding continuation/rebleeding, pharmacological treatment, surgery and mortality during 30-days after the initial bleed were collected. A multivariate analysis of clinical factors predictive of poor outcomes was conducted.

Results: Overall, 2660 patients (64.7% men; mean age 67.7 years) were evaluable. 3 Conclusions:

Table 2

 2

	, Norway (16), Portugal (12),

. A gastroenterology team managed the event in 46.7% of cases with great variability across countries (19.4% in Portugal vs 64.8% in Turkey), as was the case for PPI before endoscopy (66.0% overall, but 32.6% in Belgium vs. 87.7% in

Table 1 . Summary of patient baseline characteristics, by country a

 1 new predictive factors for bleeding continuation/re-bleeding and mortality need to be reassessed in an independent, prospective setting. In any case we believe that the sample size of the data collected and the results obtained represent those of each country and probably they can be expanded to, at least, part of Europe, since although there was a predominance of Southern European Mediterranean countries, there were 2 others such as Belgium and Norway that can be considered Demographic characteristics were missing for one patient. NVUGIB, non-variceal upper gastrointestinal bleeding; SD, standard deviation.

	16
	experimental, any North European countries.
	F o r
	P
	e
	e r
	R
	e
	v i e
	w

a

Table 2 . Summary of bleeding episode characteristics and management strategies Overall (n = 2655) Bleeding episode characteristics

 2

	Bleeding episode occurring outside of hospital, %	85.3
	Haemodynamically stable (systolic blood pressure > 100 mm Hg and pulse < 100), %	62.4
	Haematemesis, %	42.6
	Helicobacter pylori test, % performed	32.4
	Esophagitis, %	11.0
	Stomach gastritis/erosion(s), %	15.7
	Gastric ulcer, %	28.7
	Erosive duodenitis, %	6.4
	Duodenal ulcer, %	34.6
	Management strategy	(n = 2644)
	General medical ward, %	37.0
	Emergency observation, %	14.3
	Gastroenterology team, %	46.7
	Mean duration of hospitalisation in days (SD)*	6.9 (5.9)
	Therapeutic procedure during endoscopy, %	35.8
	Pre-endoscopy PPI treatment, %	66.0
	Post-endoscopy PPI treatment, %	92.6
	*Overall (n = 2444).
	SD, standard deviation; PPI, proton pump inhibitor.

Table 3 . Significant predictors of bleeding continuation/re-bleeding and/or mortality among patients undergoing therapeutic endoscopy (n = 951)

 3 Other non-significant factors included in the models: Country, Sex (male vs. female), History of alcohol abuse, History of NVUGIB, Hematemesis, NSAIDS, Warfarin, Oesophagitis diagnosis, Gastric ulcer diagnosis, Gastritis \ Erosions diagnosis, Erosive duodenitis diagnosis, Intra-hospital management by GI bleeding team / Gastroenterology. PPI use before endoscopy was used as adjustment factor in these models.

		Bleeding continuation/ re-bleeding (n = 178)	Mortality (n = 57)
		OR (95% CI)	P value	OR (95% CI)	P value
	Age (>65 vs ≤65 years)	1.81 (1.21-2.70)	0.004	2.66 (1.46-4.83)	0.002
	Hospital size (large vs other)	1.59 (1.02-2.47)	0.04	0.74 (0.33-1.67)	0.48
	Admission (inpatient vs outpatient)	1.55 (0.97-2.47)	0.07	2.92 (1.69-5.06)	<0.001
	Duodenal ulcer diagnosis	1.48 (1.01-2.18)	0.05	1.17 (0.68-2.00)	0.58
	History of alcohol abuse	1.36 (0.83-2.25)	0.23	4.32 (2.38-7.85)	<0.001
	Comorbidities (per comorbidity)	1.13 (0.96-1.33)	0.14	1.72 (1.40-2.12)	<0.001
	Low-dose ASA (≤100 mg/day)	0.89 (0.57-1.38)	0.61	0.39 (0.21-0.73)	0.004
	High-dose ASA (>100 mg/day)	0.83 (0.47-1.46)	0.52	0.13 (0.04-0.41)	0.001
	ASA, acetylsalicylic acid (aspirin); CI, confidence interval; OR, odds ratio.

Alimentary Pharmacology & Therapeutic

Acknowledgements

This study was supported by AstraZeneca. We thank Jackie Campbell, from inScience Communications, a Wolters Kluwer business, who provided editorial support funded by AstraZeneca. The authors were responsible for data interpretation and writing of the first and subsequent drafts.

Disclosures of Interest

Angel Lanas: consultancy for Pfizer, AstraZeneca, Nicox and Bayer, Lars Aabakken: none to declare, Jorge Fonseca: none to declare, Zeynel A Mungan: consultancy and speaker fees for AstraZeneca, Turkey, George V Papatheodoridis: consultancy and travel grants for AstraZeneca, Greece, Hubert Piessevaux: none to declare, Livio Cipolletta: none to declare, Javier Nuevo: employee of AstraZeneca, Monica Tafalla: employee of AstraZeneca


Oskar Koperek

Reza Asari

Bruno Niederle

MD Klaus Kaserer
email: klaus.kaserer@meduniwien.ac.at

Desmoplastic stroma reaction in papillary thyroid microcarcinoma

Keywords: papillary thyroid microcarcinoma, desmoplasia, lymph node metastasis papillary thyroid microcarcinoma, desmoplasia, lymph node metastasis

Aims: To evaluate the value of desmoplastic stroma reaction (desmoplasia) as an indicator of metastasis in papillary thyroid microcarcinoma (PTC).

Methods and result: Hundred and nine cases of papillary thyroid microcarcinoma from the pathological archive of the Medical University of Vienna, Austria, were analyzed for the presence of desmoplasia in relation to other morphologic and clinicopathologic parameters 81/109 papillary microcarcinomas showed a desmoplastic stroma reaction. The desmoplasia was significantly associated with lymph node metastases (p = 0.001) and tumour diameter (p < 0.001). In addition "invasive parameters", like peritumoral and vascular invasion were associated with the presence of a desmoplastic stroma reaction (p < 0.001 and p = 0.006, respectively) and all pT3b tumours according to the UICC classification of 2002 (=with perithyroidal infiltration) showed a desmoplasia. Of all morphologic parameters the best indicator of lymph node metastasis was tumour calcification (p < 0.001).

Conclusion:

Our data indicate that desmoplastic stroma reaction seems to be an indicator of an invasive behaviour of papillary thyroid microcarcinomas significantly associated with lymph node metastases. Papillary microtumours without signs of invasion, e.g. desmoplasia should not be regarded as invasive carcinoma as they are more likely thyroidal intraepithelial neoplasias.

Introduction

Papillary thyroid microcarcinoma is defined as a papillary thyroid carcinoma (PTC) with a tumour diameter of 10 mm or less. [START_REF] Hedinger | Histologic Typing of Thyroid Tumors[END_REF][START_REF] Delellis | Pathology and Genetics of Tumors of the Endocrine Organs[END_REF] In the past microcarcinomas were often found incidentally after pathologic examination of thyroidectomy specimens operated for other reasons, e.g. multinodular goiter, follicular adenoma or Graves disease, but with the increasing use of ultrasonography and fine needle aspiration biopsy in nodules < 10 mm, clinically unapparent microcarcinoma were also detected more often preoperatively. Not surprisingly the incidence of surgically removed thyroid microcarcinomas increased in the last years. [START_REF] Davies | Increasing incidence of thyroid cancer in the United States, 1973-2002[END_REF][START_REF] Colonna | time trend analysis of papillary and follicular cancers as a function of tumour size: a study of data from six cancer registries in France (1983-2000)[END_REF] The frequency of these small tumours in autopsy studies and in surgical thyroidectomies varies between 5 and 36%. [START_REF] Lang | Occult carcinomas of the thyroid. Evaluation of 1,020 sequential autopsies[END_REF][START_REF] De Matos | Prevalence of papillary microcarcinoma of the in Brazilian autopsy and surgical series[END_REF][START_REF] Harach | Occult papillary carcinoma of the thyroid. A "normal" finding in Finland. A systematic autopsy study[END_REF][START_REF] Yamamoto | Occult papillary carcinoma of the thyroid. A study of 408 autopsy cases[END_REF][START_REF] Baloch | Microcarcinoma of the thyroid[END_REF] In many studies papillary thyroid microcarcinomas exhibit an excellent clinical course with a low disease related mortality rate, however some of the papillary microcarcinomas were associated with local recurrence or distant metastases, and lymph node metastasis are detected in a significant portion (up to 50%). [START_REF] Roti | Thyroid papillary microcarcinoma: a descriptive and meta-analysis study[END_REF][START_REF] Pazaitou-Panayiotou | Clinical features and therapeutic implication of papillary thyroid microcarcinoma[END_REF][START_REF] Hay | Papillary thyroid microcarcinoma: a study of 535 cases observed in a 50-year period[END_REF][START_REF] Baudin | Microcarcinoma of the thyroid gland: the Gustave-Roussy Institute experience[END_REF][START_REF] Chow | Papillary microcarcinoma of the significance of lymph node metastasis and multifocality[END_REF][START_REF] Sugino | Papillary microcarcinoma of the thyroid[END_REF] Regarding morphologic parameters in papillary microcarcinoma some studies found multifocality, tumour size or extension through the thyroid capsule significantly associated with lymph node metastasis or recurrent disease but others did not. [START_REF] Pazaitou-Panayiotou | Clinical features and therapeutic implication of papillary thyroid microcarcinoma[END_REF][START_REF] Chow | Papillary microcarcinoma of the significance of lymph node metastasis and multifocality[END_REF][START_REF] Roti | and histological characteristics of papillary thyroid microcarcinoma: results of a retrospective study in 243 patients[END_REF][START_REF] Kim | parameters for recurrence of papillary thyroid microcarcinoma[END_REF] Investigating the metastatic behaviour in other thyroid tumours, namely medullary carcinoma we found that tumours without a desmoplastic stroma reaction (desmoplasia) did not show lymph node metastasis. [START_REF] Kaserer | Sporadic versus familial medullary thyroid microcarcinoma: a histopathologic study of 50 consecutive patients[END_REF][START_REF] Koperek | Desmoplasia in medullary thyroid carcinoma: a reliable indicator of metastatic potential[END_REF] Characterizing the desmoplastic stroma of medullary carcinoma we found a similar stroma reaction in a few control cases of other thyroid carcinomas including PTC. [START_REF] Koperek | Molecular characterization of the desmoplastic tumor stroma in medullary thyroid carcinoma[END_REF] A subsequent in silico expression analysis of stromal components, namely fibroblast activation protein and tenascin C revealed an 4.

Material and Methods

We analyzed 109 cases of histological proven papillary microcarcinoma (tumour diameter: Representative slices from the upper, middle and bottom region of each lobe and additionally from every suspect lesion were embedded in paraffin.

In all cases resected lymph nodes at least from the central cervical compartment were available for histological analysis. In case of histologically proven lymph node metastasis of a papillary thyroid carcinoma and macroscopically inconspicuous thyroid gland, the whole organ was embedded in 2-3 mm thick slices and processed for histological evaluation. 3 µm sections of each block were cut and stained with haematoxylin and eosin.

All papillary thyroid carcinomas were classified according to the World Health Organization criteria outlined in 2004 2 and staged according to the International Union Against Cancer (UICC) 2002 guidelines and its supplement 2003.

21, 22

Histology Desmoplastic stroma reaction (desmoplasia) was defined, as the presence of a newly formed fibrotic (collagenous) stroma surrounding the invasive epithelial tumour cells not found in the non-neoplastic thyroid parenchyma.

Results

Patient Data and morphologic parameters:

Patient data, morphologic characteristics of the tumours and correlations with metastatic behaviour are listed in table 1.

Of the 109 cases of papillary microcarcinomas lymph node metastases were found in 25.7%

(n = 28), 92.6% (n = 101) of the tumours were classified as pT1a whereas 7.4% (n= 8) of the microtumours infiltrated the perithyroidal tissue and were hence classified as pT3b according to the UICC guidelines. Multifocal PTC was present in 37.6% (n = 41) of the patients.

A desmoplastic stroma reaction (see figure 1a) was found either in the entire tumour or focally in a majority of the cases (74.3 %; n = 81) with the remaining cases (25.7%; n = 28)

showing no desmoplasia (see figure 1b). Interobserver variability (author OK vs. author KK) in assessing desmoplasia was low. A concordant result was found in 93.6% (n = 102; Cohen kappa coefficient = 0.841).

Associated thyroid pathology revealed nodular goiter in 77.1% (n = 84), lymphocytic thyroiditis in 37.6% (n = 41) and other malignant thyroid neoplasms in 20.2% (n = 22; seventeen medullary thyroid carcinoma, four follicular thyroid carcinoma and one MALTlymphoma). Follicular thyroid adenoma was detected in 7.3% (n = 8).

Correlation of desmoplastic stroma reaction with clinicopathologic and morphologic parameters

The desmoplastic stroma reaction was significantly associated with vascular invasion, tumour

Discussion

The desmoplastic stroma reaction in cancer is composed of activated fibroblasts expressing α-smooth muscle actin, fibroblast activation factor (FAP) and extracellular matrix proteins, e.g. tenascin C, analogous to the wound healing stroma. [START_REF] Koperek | Molecular characterization of the desmoplastic tumor stroma in medullary thyroid carcinoma[END_REF] It has been suggested to represent an important player in the development of the invasion process. 23, 24 Using in silico expression analyses we observed upregulation of FAP and tenascin C expression in metastasizing vs. not metastasizing PTC. In the present study we investigated if desmoplastic stroma reaction is seen early in tumorigenesis of PTC and if the absence of desmoplastic stroma reaction indicates a "preinvasive" form of a PTC. In our cases of papillary thyroid microcarcinoma the presence of desmoplasia was significantly associated with morphologic parameters of invasion, namely peritumoral invasion and vascular invasion as well as lymph node metastasis. The absence of a desmoplastic stroma on the other hand was strongly indicative of a non invasive, non metastasizing tumour, and can probably be used as a diagnostic/prognostic parameter in assessing the metastatic potential of papillary microcarcinoma.

A link of stroma reaction and invasive growth in PTC was also indicated by other authors.

Harach et al. reported that significant fibrosis was found in 79% of occult PTCs with invasive growth pattern as opposed to their circumscribed tumours which showed only slight fibrosis. [START_REF] Harach | Occult papillary carcinoma of the thyroid. A "normal" finding in Finland. A systematic autopsy study[END_REF] A study by Mai et al. compared so called "infiltrating" PTC defined by a sclerotic stroma with non infiltrating tumours and found that only one encapsulated non infiltrating tumour showed lymph node metastases. [START_REF] Mai | Infiltrating papillary thyroid carcinoma: review of 134 cases of papillary carcinoma[END_REF] Unfortunately they do not describe the size of the tumour or if the tumour was completely embedded for histological examination to find focal infiltrative growth pattern. In our series only one case of papillary thyroid microcarcinoma without desmoplasia showed a lymph node metastasis. Revaluation of the lymph node metastasis displayed a follicular arrangement of thyroid cells (see figure 2b) which might also be interpreted as ectopic thyroid tissue. Although the existence of ectopic thyroid tissue in a lymph node is discussed controversially [START_REF] Rosai | Atlas of Tumor Pathology: Tumors of the Thyroid Gland[END_REF] , we think we that possibility in this case. A more likely explanation however could be that we failed to detect a second PTC lesion with desmoplasia in the area of postoperative necrosis, bleeding and inflammation (see figure 2c).

Similar to other studies the presence of lymph node metastases as well as desmoplasia correlated with the tumour diameter. [START_REF] Roti | and histological characteristics of papillary thyroid microcarcinoma: results of a retrospective study in 243 patients[END_REF][START_REF] Wada | Lymph node metastasis from 259 papillary thyroid microcarcinomas: frequency, pattern of occurrence and recurrence, and optimal strategy for neck dissection[END_REF] This indicates a possible progression of a papillary thyroid neoplasia without desmoplasia i. e. without invasive behaviour into an invasive tumour with desmoplasia and the possibility of lymph node metastasis. Yamamoto et al. found a similar result classifying occult PTC in three types, namely encapsulated, non encapsulated sclerosing and nonsclerosing types. The mean size of non sclerosing PTC was significantly smaller than the tumour size of the sclerosing type. In this study only 2/42 tumours showed lymph node metastasis, both of the sclerosing type with calcification. [START_REF] Yamamoto | Occult papillary carcinoma of the thyroid. A study of 408 autopsy cases[END_REF] In our study tumour calcification showed the highest level of significance of all morphologic parameter associated with lymph node metastasis (p < 0,001). Similar observations were made in a recent study investigating calcification in papillary thyroid carcinoma. [START_REF] Bai | Survival impact of psammoma body, stromal calcification, and bone formation in papillary thyroid carcinoma[END_REF] They show that psammoma bodies and stromal calcification in the primary tumour are strongly associated to the presence of gross lymph node metastasis. However since calcification or psamomma bodies were not seen in the absence of desmoplasia we consider calcification not as an independent parameter but rather a consequence of the stromal desmoplasia.

The presence of lymphocytic thyroiditis was inversely correlated with lymph node metastasis.

These findings corroborate previously observations in Hashimoto`s thyroiditis that an autoimmune reaction might be protective of the development of lymph node metastases. Titles and legends to figures

  10mm or less) retrieved from the archives of the Department of Pathology of the Medical University of Vienna diagnosed between 2000 and 2006. Only cases with clinicopathologic data including sex, age, tumour size and lymph node status were selected. Total thyroidectomy was performed in all patients. The thyroid samples were routinely processed at the Department of Pathology, Medical University of Vienna. The entire organ was sectioned horizontally in slices of approximately 3-5 mm and formalin fixed.

Figure 1 .

 1 Figure 1. Papillary thyroid microcarcinoma and desmoplasia

Figure 2 .

 2 Figure 2. Papillary thyroid microcarcinoma without desmoplasia but with lymph node

  of the British Division of the International Academy of Pathology Histopathology

  All but one of the cases with lymph node metastasis showed a desmoplastic stroma reaction in the primary tumour. This one patient was operated primarily because of hyperplastic parathyroid glands. In the postoperative work up a macroscopically inconspicuous lymph node from the central neck showed a group of thyroid follicles within its parenchyma interpreted as an incidental micrometastasis of a PTC. The subsequently removed thyroid gland displayed extensive postoperative bleeding, necrosis, and a small (0.4 millimetre in diameter) papillary microcarcinoma without desmoplastic stroma reaction (see figure2).

	Koperek et al -9-.
	0.001). All pT3 tumours (=with perithyroidal infiltration) showed a desmoplastic stroma
	reaction.
	F o F o
	r r
	P Correlation of lymph node metastasis with clinicopathologic and morphologic P
	e e r In addition to desmoplastic stroma reaction the presence of lymph node metastasis was parameters (see table 1) e e r
	R e v i e w significantly associated with tumour calcification, tumour diameter, vascular invasion, peritumoral invasion, perithyroidal invasion (pT1 vs. pT3b tumours), multifocality, absence of lymphocytic thyroiditis, and younger age (p calcification < 0.001; p tumour diameter = 0.036, p vascular invasion = 0.003; p peritumoral invasion = 0.003, p perithyroidal invasion = 0.029; p multifocality = 0.023; p lymphocytic thyroiditis = 0.013; p age = 0.03). R e v i e w

calcification, peritumoral infiltration, tumour diameter and lymph node metastasis (p vascular invasion = 0.006, p calcification = 0.007, p peritumoral infiltration < 0.001, p tumour diameter < 0.001; p lymph node =

Page 10 of 18 Published on behalf of the British Division of the International Academy of Pathology

	Koperek et al -11-. Koperek et al -15-.
	In conclusion our data indicate that desmoplastic stroma reaction seems to be a reliable
	indicator of an invasive behaviour of papillary thyroid microcarcinomas. In particular in
	combination with calcification desmoplasia is significantly associated with lymph node
	metastases. Our study suggests that papillary microtumours without signs of invasion, e.g.
	desmoplasia, should probably not be regarded as invasive carcinoma but rather as thyroidal
	intraepithelial neoplasms.
	F o F o F o F o F o
	r r r r r
	P P P P P
	e e r e e r e e r e e r e e r
	R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w

Table 1 .

 1 Clinicopathologic/Morphologic Parameters and Lymph Node Metastasis Koperek et al -16-.

	mPTC	mPTC with	mPTC without	Significant
	total	LN-Meta	LN-Meta	p-values

Published on behalf of the British Division of the International Academy of Pathology Histopathology

Published on behalf the British Division of the International Academy of Pathology Histopathology

Published on behalf of the British Division of the International of Pathology Histopathology

The presence of a desmoplastic stroma reaction within the tumour was determined retrospectively on HE-stained sections by author OK. For interobserver analyses the presence of desmoplastic stroma reaction was classified by author KK in a blinded manner.

Both authors are pathologists with special expertise in thyroid pathology.

Additionally we assessed growth patterns, the peritumoral infiltration pattern, vascular invasion, presence of intratumoural lymphocytes and calcification.

Statistical Evaluation

For statistical analysis, the SPSS program (SPSS, Chicago, IL) was used. For statistical analysis of the interobserver reliability the Cohen-Kappa-coefficient was calculated. The value 1 means total agreement whereas the value 0 means that the agreement does not exceed the random measure. For comparative analysis chi-square calculations and the Mann