Need for objective and reproducible criteria in histopathological assessment of total mesorectal excision specimens: lessons from a national improvement project.

AIM: Data on quality control of the pathologic evaluation of total mesorectal excision (TME) specimens are scarce. We aimed to assess differences between evaluation by local pathologists participating in PROject on CAncer of the REctum (PROCARE; a Belgian improvement project on rectal cancer) and by a review panel of experts. METHOD: Based on photographic material and histopathology slides, a Review Committee of gastrointestinal expert pathologists re-evaluated the mesorectal plane, the tumour differentiation grade, the (y)pT stage and the tumour regression grade in 444 patients previously routinely assessed by local pathologists. RESULTS: The surgical plane was reported in 89% of patients and the circumferential resection margin in 88% of patients by the local pathologist. The median number of lymph nodes harvested in patients undergoing neoadjuvant radiochemotherapy was 11 and 14 in the other patients. The Review Committee downgraded the surgical plane from (intra)mesorectal to intramuscular in 17% of patients, and upgraded it from intramuscular to (intra)mesorectal in 27%. Tumour differentiation grade, T stage and tumour regression grade differed between local pathologists and the Review Committee in 15%, 10% and 38%, respectively, of patients. T stage was upgraded, mainly from T2 to T3, in 8% of patients. Tumour regression was judged by the Review Committee to be less advanced in 15% of patients. CONCLUSION: Acknowledging some shortcomings, this study gives a realistic view of clinical practice. There are differences in interpretation with regard to both macroscopic and microscopic analysis of TME specimens. These findings indicate a need for more objective and reproducible criteria in histopathology. Being aware of this is a first step for improvement.

Cleanrooms and tissue banking how happy I could be with either GMP or GTP?

The regulatory framework of tissue banking introduces a number of requirements for monitoring cleanrooms for processing tissue or cell grafts. Although a number of requirements were clearly defined, some requirements are open for interpretation. This study aims to contribute to the interpretation of GMP or GTP guidelines for tissue banking. Based on the experience of the participating centers, the results of the monitoring program were evaluated to determine the feasibility of a cleanroom in tissue banking and the monitoring program. Also the microbial efficacy of a laminar airflow cabinet and an incubator in a cleanroom environment was evaluated. This study indicated that a monitoring program of a cleanroom at rest in combination with (final) product testing is a feasible approach. Although no statistical significance (0.90 < p < 0.95) was found there is a strong indication that a Grade D environment is not the ideal background environment for a Grade A obtained through a laminar airflow cabinet. The microbial contamination of an incubator in a cleanroom is limited but requires closed containers for tissue and cell products.

International and national initiatives in biobanking.

Translational research and biobanking are "in", also in Flanders and in Belgium. In Flanders the Advice report 120 from the Flemish Council for Science and innovation, entitled "Extension of translational research in Flanders" paved the way for the Center for Medical Innovation. The Center for Medical Innovation aims at promoting collaboration between Flemish Universities, university hospitals, pharma and biotech industry and the Flemish Government specifically in the domain of translational research. The Initiative # 27 of the Cancer plan from the Federal Government aims at financing a virtual interuniversity tumor bank in order to promote "cancer" translational research in a collaborative network between academic structures, general hospitals en different industrial partners (pharmacy, biotechnology, diagnostics, ...) active in research in Belgium. However, the scientific interest in the human tissues is not new, at all. This text aims at giving an overview of the development and evolutions of "biobanking" initiatives.

Epidemiology of mucormycosis: review of 18 cases in a tertiary care hospital.

Mucormycosis is an angio-invasive mycosis with high morbidity and mortality rates which mainly affects immunocompromised patients. It is no longer an uncommon disease due to the increased incidence of diabetes and use of immunosuppressive agents in the current era. Our objective was to review all cases of proven and probable mucormycosis--according to EORTC criteria--diagnosed from 2000 until 2007 at the University Hospitals Leuven, a 1900-bed tertiary care hospital, to assess the changing epidemiology of the disease. In 45 patients there was microbiological or histopathological evidence for the presence of a member of Mucorales during the hospital stay of which 12 cases fulfilled the criteria for proven mucormycosis and 6 for probable mucormycosis. The overall incidence was 0.042 cases per 10,000 patient days. A slight although not statistically significant increase in incidence was noticeable during the study period. The major site of infection was the lungs (78% of the cases), with haematological malignancy the most common underlying disorder and Rhizopus species the most often suspected etiologic agent. Overall mortality was 55% and co-infections with Aspergillus species, proven or probable, noted in 44% of cases. The highest survival rate was achieved with surgery combined with antifungal therapy.

Influence of postmortem time on the outcome of blood cultures among cadaveric tissue donors.

Tissue banks provide tissues of human cadaver donors for transplantation. The maximal time limit for tissue retrieval has been set at 24 h postmortem. This study aimed at evaluating the evidence for this limit from a microbiological point of view. The delay of growth in postmortem blood cultures, the identification of the species isolated and clinical/environmental factors were investigated among 100 potential tissue donors. No significant difference was found in the rate of donors with grown blood cultures within (25/65=38%) compared with after (24/65=37%) 24 h of death. Coagulase-negative staphylococci and gastro-intestinal microorganisms were isolated within and after 24 h of death. Two factors--antimicrobial therapy and "delay before body cooling"--were significantly inversely related with donors' blood culture results. From a microbiological point of view, there is no evidence for avoiding tissue retrieval among donors after 24 h of death.

Effectiveness of antibiotics and antiseptics on coagulase-negative staphylococci for the decontamination of bone allografts.

Bone allografts retrieved from multi-organ donors can be decontaminated with minimally aggressive methods. Therefore, we evaluated the efficacy of antibiotics and antiseptics in the decontamination of bone fragments actively contaminated with coagulase-negative staphylococci. Gentamicin (512/1,024 microg/mL), rifampicin (400/1,000 microg/mL), chlorhexidine in alcohol and chlorhexidine soap were tested with different contact times and temperatures and a delay in starting decontamination. Gentamicin-susceptible strains dried on bone could be removed by gentamicin 512 microg/mL after 19 h of contact, while strains not dried on bone could be eliminated by soaking bone for 60 min in gentamicin 512 microg/mL. Rifampicin-susceptible strains could be eliminated by soaking bone for 60 min in rifampicin 1,000 microg/mL. In none of the experimental conditions could gentamicin/rifampicin-resistant staphylococci be eliminated. Antiseptics could not eliminate staphylococci from bone. Different antibiotics need different protocols in order to decontaminate bone allografts.

Bacteriostasis testing on allograft tissue inoculated in Wilkins-Chalgren broth.

Tissue banks culture tissue specimens to confirm the absence of viable micro-organisms after decontamination with antibiotics. It is possible that antibiotic residues attached to decontaminated tissue are introduced into enrichment culture media. These could have an inhibitory effect on the culture results and generate false-negative results. Our aim was to detect bacteriostasis in Wilkins-Chalgren broth inoculated with bone and tendon remnants. These remnants had been soaked in a solution containing gentamicin as part of the tissue-processing procedure. We used the United States Pharmacopeia method for bacteriostasis testing with gentamicin-susceptible Pseudomonas aeruginosa American Type Culture Collection (ATCC) 15442, Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633 as test strains, and gentamicin-resistant Candida albicans ATCC 90029 as control. The residual gentamicin concentration in the broths was determined and gentamicin-soaked tissue was placed on Mueller-Hinton agar inoculated with a staphylococcal suspension. Bacteriostasis was present in 53-75% of the reference test strains. Tendon remnants had a significantly higher rate of bacteriostasis (85%) than bone remnants (28%). Broths inoculated with tendon remnants had the highest residual gentamicin concentrations.

Determinants of complications and adequacy of surgical resection in laparoscopic versus open total gastrectomy for adenocarcinoma.

BACKGROUND: The role of laparoscopic total gastrectomy (LTG) in the treatment of gastric cancer is controversial. The present study analyzed the morbidity and adequacy of resection in LTG versus open total gastrectomy (OTG) for gastric adenocarcinoma. METHODS: Between 2003 and 2006, clinical data of 38 consecutive patients who underwent LTG for gastric adenocarcinoma were collected prospectively. The same data-entry form was used for retrospective data collection from 22 consecutive patients who underwent OTG within the same time period. Logistic regression models were used in univariate and multivariate analyses to identify the optimally combined factors related to the occurrence of postoperative complications and to the number of harvested lymph nodes. RESULTS: Postoperative complications occurred in 24 patients with subsequent mortality in two. Median (range) length of hospital stay was 11 (6-73) days and comparable after LTG versus OTG (p = 0.847). The occurrence of postoperative complications was related (p = 0.004) to the first year of surgery and patients' medical condition before surgery [American Society of Anaesthesiologists (ASA) physical status III]. Microscopic tumor-free margins were obtained in all but two patients. The number of harvested lymph nodes was 17 (0-90), and determined by tumor wall penetration (p = 0.001). CONCLUSIONS: The occurrence of complications after total gastrectomy is determined by the patients' medical condition before surgery and the surgical expertise, but not by the approach. LTG and OTG can result in adequate tumor-free resection margins and lymph node yield, which is related to the tumor wall penetration. The role of LTG in gastric cancer needs further evaluation in randomized controlled trials with large patient series.

Review article: the nature of oesophageal injury in gastro-oesophageal reflux disease.

The purpose of this review was to explore issues relating to the nature of oesophageal injury in gastro-oesophageal reflux disease. Several structural and functional elements of the oesophageal epithelium provide for an inherent resistance against refluxed material. It is only when this defence is overcome that reflux-induced damage ensues. The light microscopic changes in reflux oesophagitis are manifold. Early changes are confined to the epithelium and consist of reactive changes and inflammatory cell infiltration. When the epithelial integrity can no longer be maintained, erosions and ulcers develop. Recently, dilatation of the intercellular spaces was described as a sensitive early marker for gastro-oesophageal reflux disease. This parameter was first identified by electron microscopy but can also be assessed by light microscopy in routinely stained tissue sections. Some of the changes occurring in early gastro-oesophageal reflux disease can be reproduced by incubating oesophageal mucosal biopsies in gastrointestinal fluids or their components. Activated pepsin, trypsin and conjugated bile acids at an acidic pH have been identified as probable causes of reflux-induced damage.

Utility of positron emission tomography for the staging of patients with potentially operable esophageal carcinoma.

PURPOSE: A prospective study of preoperative tumor-node-metastasis staging of patients with esophageal cancer (EC) was designed to compare the accuracy of 18-F-fluoro-deoxy-D-glucose (FDG) positron emission tomography (PET) with conventional noninvasive modalities. PATIENTS AND METHODS: Seventy-four patients with carcinomas of the esophagus (n = 43) or gastroesophageal junction (n = 31) were studied. All patients underwent attenuation-corrected FDG-PET imaging, a spiral computed tomography (CT) scan, and an endoscopic ultrasound (EUS). RESULTS: FDG-PET demonstrated increased activity in the primary tumor in 70 of 74 patients (sensitivity: 95%). False-negative PET images were found in four patients with T1 lesions. Thirty-four patients (46%) had stage IV disease. FDG-PET had a higher accuracy for diagnosing stage IV disease compared with the combination of CT and EUS (82% v 64%, respectively; P: =.004). FDG-PET had additional diagnostic value in 16 (22%) of 74 patients by upstaging 11 (15%) and downstaging five (7%) patients. Thirty-nine (53%) of the 74 patients underwent a 2- or 3-field lymphadenectomy in conjunction with primary curative esophagectomy. In these patients, tumoral involvement was found in 21 local and 35 regional or distant lymph nodes (LN). For local LN, the sensitivity of FDG-PET was lower than EUS (33% v 81%, respectively; P: =.027), but the specificity may have been higher (89% v 67%, respectively; P: = not significant [NS]). For the assessment of regional and distant LN involvement, compared with the combined use of CT and EUS, FDG-PET had a higher specificity (90% v 98%, respectively; P: =. 025) and a similar sensitivity (46% v 43%, respectively; P: = NS). CONCLUSION: PET significantly improves the detection of stage IV disease in EC compared with the conventional staging modalities. PET improves diagnostic specificity for LN staging.

Chemo-radiotherapy versus radiotherapy alone in the pre-operative treatment of resectable rectal cancer.

AIM: To determine the differences in downstaging, local control (LC), disease free survival (DFS) and overall survival (OS) between combined pre-operative chemoradiation and pre-operative radiotherapy alone in the treatment of resectable rectal cancer. METHODS: One hundred and ten patients who underwent pre-operative radiotherapy or chemo-radiotherapy were reviewed. Fifty-seven patients were treated with radiotherapy (30 Gy/3 Gy) alone and 53 patients with chemo-radiotherapy (bolus 5FU+45 Gy/1.8 Gy). The median interval between the end of neo-adjuvant treatment and surgery was 28 and 46 days for the patients treated with radiotherapy alone and chemo-radiotherapy. RESULTS: The groups were homogeneously distributed for all characteristics except for cN-stage with more clinically node positive patients in the combined modality treatment group (47 vs 73%). A significant downstaging for tumour and/or lymph node status was observed in both groups. More ypT0-x-is were observed after chemoradiation than after radiotherapy alone (26 vs 7%; p=0.02). The local control rate at 3 years was 94% for both groups. DFS after radiation and chemoradiation was comparable with a 3-year DFS of 83 and 88%, respectively. CONCLUSION: Both pre-operative schemes have similar outcomes concerning DFS, OS and LC. Tumour downstaging is associated with improved survival.

Primary diffuse large B cell lymphoma of the stomach: analysis of somatic mutations in the rearranged immunoglobulin heavy chain variable genes indicates antigen selection.

Gastric low grade MALT lymphomas show a pattern of somatic mutations in their rearranged immunoglobulin genes, indicative of antigen selection. This provides evidence for antigen stimulation in the lymphomagenesis. Gastric diffuse large B cell lymphomas develop secondary to low grade MALT lymphoma or de novo. To study whether antigen-selection is also a feature of primary diffuse large B cell lymphomas, we analysed somatic mutations in the rearranged immunoglobulin heavy chain (IgH) variable genes (VH). The rearranged VH genes of six cases of gastric primary diffuse large B cell lymphoma were amplified from genomic or complementary DNA by a VH gene family-specific polymerase chain reaction method. The PCR products were directly sequenced and were compared to published germline sequences to analyse somatic mutations. Similarly to low grade MALT lymphomas 5/6 primary diffuse large B cell lymphomas show a pattern of somatic mutation in their rearranged VH genes, indicative of antigen selection and suggesting a role for antigens in lymphomagenesis. One case showed bi-allelic VH gene rearrangements, which were non-functional due to extensive deletions. Antigen selection could not be demonstrated or excluded. Antigen selection is a common feature in most analysed primary diffuse large B cell lymphomas, although some heterogeneity in the mechanisms involved in the lymphomagenesis of gastric primary diffuse large B cell lymphomas has not been excluded entirely (case 4).

Are carcinomas of the cardia oesophageal or gastric adenocarcinomas?

There is a clear relationship between Barrett's oesophagus and oesophageal adenocarcinoma, and between Helicobacter pylori and gastric cancer, but the histogenesis of cardiac adenocarcinomas is unknown. Some clues as to possible disease associations may be provided by the pattern of gastritis. In our study, we analysed gastritis associated with oesophageal, cardiac and gastric adenocarcinomas according to the Sydney classification. Chronic gastritis was more common in gastric (88%) than in cardiac (56%) and oesophageal adenocarcinomas (38%). H. pylori was significantly more prevalent in gastric (73%) than in cardiac (34%) or oesophageal (21%) adenocarcinomas. Our results show that factors other than H. pylori must be involved in the histogenesis of cardiac adenocarcinomas. As the pattern of gastritis and the clinical features of cardiac adenocarcinomas are more comparable to oesophageal carcinomas than gastric carcinomas, we speculate that most of these tumours share similar aetiological factors with oesophageal carcinomas.

Is calcifying fibrous pseudotumor a late sclerosing stage of inflammatory myofibroblastic tumor?

Calcifying fibrous pseudotumor is a recently described distinctive lesion, characterized by the presence of abundant hyalinized collagen with psammomatous or dystrophic calcifications and a lymphoplasmacytic infiltrate. The cause and pathogenesis are unclear, but a possible relationship with other pseudotumors, like nodular fasciitis or inflammatory myofibroblastic tumor, has been proposed by some authors. However, cases with overlapping histologic features have not been reported. A 17-year-old girl with multiple peritoneal calcifying fibrous pseudotumors and inflammatory myofibroblastic tumors (inflammatory pseudotumors) is described. Some multinodular lesions showed calcifying fibrous pseudotumors next to inflammatory myofibroblastic tumors. Transitional stages between calcifying fibrous pseudotumor and inflammatory myofibroblastic tumor were also present. This case clearly illustrates a histogenetic relationship between calcifying fibrous pseudotumor and inflammatory myofibroblastic tumor, and it suggests that calcifying fibrous pseudotumor is a late sclerosing stage of inflammatory myofibroblastic tumor, at least in some cases.

Abnormalities in mucin gene expression in Crohn's disease.

Alterations in the structure and/or quantity of mucins could alter the barrier function of mucus and play a role in initiating and maintaining mucosal inflammation in Crohn's disease. To investigate the hypothesis of a mucin gene defect in Crohn's disease, we analyzed the expression of the different mucin genes in the ileal mucosa of patients with Crohn's disease and controls. mRNA expression levels were assessed by a quantitative dot blot analysis and compared (i) between healthy and involved ileal mucosa of patients with Crohn's disease and (ii) between healthy mucosa of patients with Crohn's disease and controls. Expression of the different mucin genes was heterogeneous among controls and patients with Crohn's disease, except for MUC6 in controls. Nevertheless, MUC1 mRNA expression was significantly decreased in the involved ileal mucosa of patients with Crohn's disease when compared to the healthy mucosa (p = 0.02). Moreover, the expression levels of MUC3, MUC4, and MUC5B were significantly lower in both healthy and involved ileal mucosa of patients with Crohn's disease compared to controls (p < or = 0.05). The decrease of expression levels of some mucin genes (more particularly MUC3, MUC4, and MUC5B) in both healthy and involved ileal mucosa suggests a primary or very early mucosal defect of these genes in CD.

Mucin histochemistry of heterotopic gastric mucosa of the upper esophagus in adults: possible pathogenic implications.

The mucin profile of 24 endoscopic biopsies of heterotopic gastric mucosa (HGM) of the upper esophagus in adults and a control group of ten cases of Meckel's diverticula containing heterotopic gastric mucosa were studied with two combined histochemical methods: alcian blue pH 2.5/PAS and high iron diamine/alcian blue pH 2.5. The clinical and light microscopic features of the 24 HGM cases were also reviewed. In addition to overall secretion of neutral mucins by the 24 HGM cases, mucin histochemistry showed prominent secretion of acidic mucins in 19 of 24 HGM cases (79%), with sulphomucins in 11 of 24 HGM cases (45.8%). This mucin profile of HGM was unlike that of either normal gastric mucosa or heterotopic gastric mucosa in Meckel's diverticula. Moreover, a comparison between the mucin profile and clinical features of HGM and Barrett's esophagus showed certain similarities. The data suggest a physiopathologic link between HGM and Barrett's esophagus.

Extracapsular lymph node involvement is a negative prognostic factor in T3 adenocarcinoma of the distal esophagus and gastroesophageal junction.

OBJECTIVE: To assess prognosis according to whether lymph node involvement is intracapsular or with extracapsular breakthrough in adenocarcinoma of the distal esophagus and gastroesophageal junction. Materials and methods One hundred ninety-five consecutive patients with T3 adenocarcinoma of the distal esophagus and gastroesophageal junction between 1990 and 1999 were studied. All patients underwent primary R0 esophagectomy. The mean number of resected nodes per patient was 36.9. Survival was analyzed according to intracapsular and extracapsular involvement. RESULTS: In N0 patients 5-year survival was 57% and 9-year survival was 38.7%. In patients with positive nodes these figures were 26.2% and 18.1%, respectively (P =.0069). Intracapsular and extracapsular node involvement showed 5- and 10-year survival of 40.9% and 21.7% versus 18% and 15.7%, respectively. There was no significant difference in 5- and 10-year survival between N0 and intracapsular node involvement (P =.43). However, there was a significant difference in survival between N0 and extracapsular node involvement (P =.002) and between intracapsular and extracapsular node involvement (P =.0001). CONCLUSIONS: This study shows a significant difference in survival according to whether lymph node involvement was intracapsular or extracapsular. Patients with intracapsular lymph node involvement have similar survival rates as N0 patients. Extracapsular lymph node involvement is a bad prognostic factor, independent of the number of involved lymph nodes. The number of involved lymph nodes has an additive negative effect. These data may have an impact on treatment strategies.

A pancreatic mucinous cystadenoma in a man with mesenchymal stroma, expressing oestrogen and progesterone receptors.

A 43-year-old man presented with abdominal discomfort caused by relapsing pancreatitis. Radiological examination revealed a multilocular cystic mass in the tail of the pancreas, which was resected. Gross examination showed a multilocular cystic lesion measuring 2.5 cm in diameter and containing clear fluid. Microscopically, a mucinous cystadenoma with mesenchymal stroma was diagnosed. The lesion showed two different components: a cyst lined by a columnar, mucin-secreting epithelium and a moderate cellular stroma composed of spindle cells. The stromal element appeared similar to primitive mesenchyme. Immunohistochemical staining confirmed this origin through vimentin expression and showed moderate to strong nuclear staining with oestrogen and progesterone receptor antibodies. Cystadenomas are rare tumours of the pancreas, but mesenchymal stroma is uncommon in such tumours; it is more frequently described in the liver and the bile ducts, and primarily in women.

Multifocal epithelioid angiosarcoma of the small intestine.

A 67-year-old man presented with weight loss, intermittent severe abdominal pain and melaena. Initial radiology (including abdominal ultrasonography), gastroscopy and colonoscopy did not demonstrate any lesions that could explain the complaints. Three weeks later, upper gastrointestinal and small-bowel barium studies revealed two areas in the small intestine with an abnormal mucosal pattern. Explorative laparotomy revealed three tumoral lesions. Three partial enterectomies were performed. Gross examination showed centrally depressed dark reddish tumoral lesions extending from the mucosa throughout the full thickness of the bowel wall (diameter varying between 1.6 cm and 2.2 cm). The tumours, composed of large, plump, polygonal cells showing little architectural differentiation, were mainly situated in submucosa and muscularis propria. The growth pattern appeared rather solid. The epithelioid cells showed pronounced nuclear pleomorphism and atypia with central large nucleoli. There were several small blood vessels with occasional anaplastic endothelial cells. Immunohistochemical staining demonstrated an intense expression of CD 31, CD 34, factor VIII related antigen and keratin. This supported the diagnosis of an epithelioid angiosarcoma. The patient died 3 months after diagnosis. Tumours of the small intestine are very rare, and angiosarcomas of the small intestine are even more rare. Epithelioid variants have only been described in two patients and only one of these had a multifocal presentation. The prognosis is very poor. Because of the epithelioid growth pattern and the cytokeratin expression, these tumours may erroneously be diagnosed as a carcinoma.

Surgery for gastric remnant carcinoma following Billroth II gastrectomy.

Between January 1983 and December 1995, 31 patients with gastric remnant carcinoma were operated on 6-45 years after a Billroth II resection for peptic ulcer disease. Total gastrectomy with Roux-en-Y reconstruction was performed in all cases. In 16 patients (52%) extended resection with removal of one or more adjacent organs was necessary for oncological reasons. In this elderly population with a high incidence of pre-operative risk factors (55%), most tumours were classified as stage III or IV (45%). Although total gastrectomy should be the surgical option of first choice for gastric remnant carcinoma, it resulted in high levels of post-operative mortality and morbidity (13% and 35%, respectively), especially in patients with stage III and IV tumours who underwent resection of an adjacent organ. Despite extended surgery, if necessary, the recurrence rate after 'curative' surgery is high (8/20 patients) and the cumulative disease-free 5-year survival rate was 48%. Detection of the tumour at an earlier stage not only limits the extent of resection and lowers the complication rate, but also improves survival.