[CMV-associated enterocolitis due to primary CMV infection in the immunocompetent]. / CMV-assoziierte Enterokolitis im Rahmen einer primären CMV-Infektion im Immunkompetenten.

Cytomegalovirus (CMV) plays an important role in non-immunocompetent patients due to its high seroprevalence and life-long persistence. However, cases of severe CMV infections are also described in the immunocompetent. Here in particular, the gastrointestinal involvement is of major importance. We describe the case of a 29-year-old immunocompetent young man, who presented with a primary CMV infection mainly of the colon with clinical signs of bloody diarrhoea, fever, hepatitis and haemolysis. The diagnosis was established on the basis of a suspicious endoscopic finding with immunohistochemical detection of CMV in the colonic mucosa, a positive CMV viral load in the peripheral blood and an immune system response typical for primary infection. Based on this case and previous publications, we suggest that a colonoscopy and diagnostic procedures for CMV should be considered if the patient presents with gastrointestinal symptoms like (bloody) diarrhoea, fever, and hepatitis. In a severe case, we recommend antiviral therapy due to a high mortality that has been reported for CMV colitis in immunocompetent individuals.

Liver transplantation in the rat: single-center experience with technique, long-term survival, and functional and histologic findings.

OBJECTIVE: Orthotopic liver transplantation (OLT) in rats is frequently used as an experimental model. Numerous surgical techniques have been developed that enable the investigator to conduct clinically relevant studies. The objective of this study was to develop a rat model of acute and chronic rejection, to explicitly study technical modifications of vascular anastomoses with precision, and to examine histopathologic and functional changes in the graft. MATERIALS AND METHODS: With DA-(RT1av1) rats as donors and Lewis-(RT1) rats as recipients, arterialized OLT was performed using a combined suture, cuff, and splint method. Recipients were divided into 5 groups: syngeneic control rats (group 1), allogeneic control rats (group 2), allogeneic OLT rats with low-dose tacrolimus (FK506) immunosuppression (group 3), allogeneic OLT rats with high-dose tacrolimus immunosuppression (group 4), and allogeneic OLT rats with high-dose tacrolimus immunosuppression and retrograde reperfusion via the infrahepatic caval vein (group 5). After OLT, serum parameters were determined and hepatic biopsy specimens were sampled. We examined the effects of acute rejection with or without immunosuppression therapy at histopathologic evaluation. RESULTS: Liver grafts in syngeneic and allogeneic rats (groups 1, 2, 4, and 5) demonstrated normal serum parameters and histopathologic findings at 10 days after OLT, and 93% survival at 3 months. The simplified technique using 1 suture and 2 cuff anastomoses provided the best short- and long-term survival after OLT in all groups. Retrograde perfusion via the infrahepatic caval vein resulted in lower postoperative liver enzyme values. CONCLUSION: The present model is feasible, enabling comprehensive preclinical experimental research on liver transplantation. Furthermore, we provide helpful instructions for learning this surgical technique.

Microsurgical technique of simultaneous pancreas/kidney transplantation in the rat: clinical experience and review of the literature.

BACKGROUND: For experimental basic research, standardized transplantation models reflecting technical and immunologic aspects are necessary. This article describes an experimental model of combined pancreas/kidney transplantation (PKTx) in detail. MATERIALS AND METHODS: Donor rats underwent en bloc pancreatectomy and nephrectomy. Revascularization was performed using the aorta with the superior mesenteric artery and the inferior vena cava with the portal vein. Exocrine drainage of the pancreas took place over a segment of the duodenum which was transplanted side-to-side to the jejunum. The kidney vessels were transplanted end-to-side. The ureter was anastomosed by patch technique. Postoperatively, serum parameters were monitored daily. Biopsies for histopathology were taken on days 5, 8 and 12. RESULTS: All 12 recipients survived the combined PKTx without serious surgical complications. One thrombosis of the portal vein led to organ failure. Blood glucose levels were normal by the 3rd postoperative day. The transplanted duodenal segment showed slight villous atrophy, and the kidneys were well perfused without vascular complications. The anastomosis between ureter and bladder was leakproof. CONCLUSIONS: Excellent graft function and survival rates can be achieved due to simplified operation technique and short operation time. It may thus have high clinical relevance to immunologic issues within the scope of basic research.

[Mammography screening. Concept, quality assurance and interdisciplinary cooperation]. / Mammographie-Screening. Konzept, Qualitätssicherung und interdisziplinäre Zusammenarbeit.

In 2005/2006 the German National Mammography Screening Program was initiated and has now become established. The objective is to reduce breast cancer mortality and the early diagnosis and therapy of small cancers. The program follows the European guidelines and is controlled by over 30 parameters of quality. All trained members of the team document each step of the screening chain electronically. Histological assessment (HA) is recommended in up to 2% of examinations, 90% of HAs are performed by core needle biopsy (CNB) or by stereotactic vacuum-assisted biopsy (VABB). Open diagnostic biopsies are performed in <10% of all HAs and therapy is successful in some of the B3 lesions. Mammograms are interpreted by two independent readers. Recommendations of the regular interdisciplinary conferences, preoperative and postoperative, follow the European guidelines. About 45% of all breast cancers detected by screening are in-situ or less than 10 mm in size. The 17% alterations diagnosed by needle biopsy are B3 or B4 lesions and impose high demands on the pathologists and the interdisciplinary team. Due to the many early and discrete lesions counterchecking of representative biopsies is crucial. Problems may be caused by sampling error or partial volume effects. Interdisciplinary conferences and knowledge of the limitations of each discipline and method are needed to optimize diagnostic and therapeutic decisions.

[Practical problems in breast screening. Columnar cell lesions including flat epithelial atypia and lobular neoplasia]. / Praktische Probleme im Mammographie-Screening. Kolumnarzellläsionen einschliesslich flache Epithelatypie und lobuläre Neoplasien.

Columnar cell lesions (CCL) and lobular neoplasia (LN) are encountered with increasing frequency in breast screening biopsies. CCLs are frequently associated with microcalcifications, whereas LN is an incidental finding in most cases. Flat epithelia atypia (FEA) the atypical variant of CLL, LN and atypical ductal hyperplasia (ADH) are frequently associated lesions. Molecular genetic studies of CCL, ductal carcinoma in situ (DCIS) and low grade invasive carcinomas revealed similar chromosomal alterations supporting the assumption that CCLs are neoplastic proliferations. The frequent association of FEA together with well differentiated invasive carcinomas provides further evidence of this concept. There is no internationally accepted classification of CCLs at present. CDH1-gene mutations are the cardinal feature of LN and invasive lobular carcinoma. In immunohistochemically CDH1-positive cases, alternative genetic alterations of the CDH1 pathway can lead to functional loss of CDH1. In our opinion morphologically and immunohistochemically hybrid lesions may represent this group of lobular lesions. Recent follow-up data suggest a higher rate of ipsilateral carcinomas in patients with previously diagnosed LN. It is currently an open question whether FEA and LN are members of a common family of intralobular proliferations, which are non-obligatory precursors of a low nuclear grade breast neoplasia family.

Differential expression of c-Met, its ligand HGF/SF and HER2/neu in DCIS and adjacent normal breast tissue.

AIMS: Tyrosine kinase receptors Her2/neu and c-Met play an important role in breast cancer development and progression. Our aim was to determine the expression of c-Met, its ligand hepatocyte growth factor/scatter factor (HGF/SF) and Her2/neu in ductal carcinoma in situ (DCIS) lesions of the breast (n = 39) by two different immunocytochemical techniques, classical immunohistochemistry and immunofluorescence, and to correlate their expression levels with histopathological and clinical characteristics. METHODS AND RESULTS: Both methods revealed similar c-Met staining patterns in both the in situ component and the adjacent normal tissue (P < 0.001). However, an imbalance in c-Met expression between tumour and surrounding normal tissue was correlated with high-grade DCIS (Van Nuys Grade 3). No correlation existed between Her2/neu and c-Met expression. High HGF/SF immunoreactivity was observed in 43.6% of the cases, yet the adjacent cellular stroma revealed only low levels of HGF/SF. No correlation existed between c-Met, Her2/neu or HGF/SF expression and clinicopathological factors. CONCLUSION: An imbalance in c-Met expression between tumour and surrounding normal tissue is associated with an aggressive DCIS phenotype. Moreover, c-Met and HGF/SF may contribute to tumour development by different means than those controlled by Her2/neu.

Quantitative protein analysis from formalin-fixed tissues: implications for translational clinical research and nanoscale molecular diagnosis.

Owing to its cross-linking effects, it is currently believed that formalin fixation of routinely processed tissues in the clinic prevents protein extraction and profiling. The aim of our study was to develop a robust, fast, standardized, and easy to use technique for the solubilization of non-degraded, full length, and immunoreactive proteins from formalin-fixed tissues for western blot and protein microarray analysis. Sections of routinely processed formalin-fixed and paraffin-embedded tissues of various origin were analysed. After deparaffination, tissues were manually dissected from the slides and transferred into an optimized protein extraction buffer system. Proteins were solubilized and subsequently analysed by western blot and reverse phase protein microarrays. We succeeded in isolating non-degraded, soluble, and immunoreactive proteins from routinely processed formalin-fixed tissues. We were able to detect membrane, cytoplasmic and nuclear proteins at the expected molecular weight. No differences were found in the protein yield and protein abundances between fresh frozen and formalin-fixed tissues. Using western blots and reverse phase protein microarrays, the receptor tyrosine kinase HER2, an important protein target for antibody based cancer treatment, was reliably measured in formalin-fixed breast cancer biopsy samples when compared with measurement by immunohistochemistry and fluorescence in situ hybridization; remarkably, immunohistochemically equivocal cases (score 2+) can be categorized according to HER2 protein abundance. Our new clinically orientated multiplexed protein measurement system may be generally applicable to determine the relative abundances of known disease-related proteins in small amounts of routinely processed formalin-fixed tissue samples for research and diagnosis. This technique may also be used to identify, characterize, and validate known and new protein markers in a variety of human diseases.

[Experiences of the Bavarian mammography screening program]. / Erfahrungen aus dem Bayerischen Mammographie-Screening-Programm.

The Bavarian Mammography Screening Program started in April 2003. A detailed analysis of the consistency of diagnosis in the evaluation of vacuum-assisted stereotactic or core needle breast biopsies is presented. A total of 32 pathologists participated in a blinded evaluation of the biopsies. Each case was evaluated independently by two participating pathologists. A total of 1,357 cases were reviewed. The histopathological reports of the biopsies made by the two consulting pathologists were compared. The concordance rate of the first and second consulting pathologist was 93% for the B-classification. In general, the level of diagnostic agreement was very high for well defined, benign and malignant lesions. Some of the discrepancies resulted from the incorrect application of the B-classification. Discrepancies in the reports were also due to divergent interpretation of benign and "borderline" lesions. The protocol for the blinded evaluation of breast biopsies in two rounds assured a high level of quality. In conclusion, prerequisites for the success of a mammography screening program are interdisciplinary consensus conferences and audit rounds involving pathologists.

Prognostic significance of expression patterns of c-erbB-2, p53, p16INK4A, p27KIP1, cyclin D1 and epidermal growth factor receptor in oesophageal adenocarcinoma: a tissue microarray study.

AIMS: To correlate immunohistochemical expression patterns and prognosis in oesophageal adenocarcinoma. METHODS: The expression of c-erbB-2, p53, p16INK4A, p27KIP1, cyclin D1 and epidermal growth factor receptor (EGFR) was studied in a series of 137 primarily resected oesophageal adenocarcinoma samples. The expression analysis on protein level was performed on routine paraffin wax-embedded material, with immunohistochemical staining of the samples, assembled on a tissue microarray. The results were correlated with clinicopathological features (pT, pN and G) and survival. RESULTS: 22 (16%) tumours showed an overexpression of the c-erbB-2 oncoprotein. Expression of EGFR was observed in 72 (55%) cases, accumulation of p53 in 68 (52%) cases and of cyclin D1 in 102 (77%) cases. Loss of p16INK4A expression was observed in 101 (76%) cases and low expression of p27KIP1 in 91 (71%) cases. Expression of these proteins did not correlate with tumour stage, grade, Lauren's or World Health Organization classification or lymph node status. On univariate survival analysis, more advanced tumour stage (p = 0.002), lymph node involvement (p = 0.003), high tumour grade (p = 0.017) and lack of EGFR expression (p = 0.034) were found to be associated with poorer survival. On multiple regression analysis, only tumour stage (p = 0.03) and lymph node involvement (p = 0.004) were shown to have an association with the survival of the patient. CONCLUSION: The immunohistochemical expression of c-erbB-2 oncoprotein, cylin D1, p16INK4A, p27KIP1, p53 and EGFR in most oesophageal adenocarcinomas suggests their implication in the pathogenesis of this entity. None of the molecular markers assessed, however, was of prognostic value. Identification of any marker superior to or even approaching the prognostic value of conventional histopathological markers (pT and pN) was therefore not possible.

Heterogeneous chromosomal aberrations in intraductal breast lesions adjacent to invasive carcinoma.

There is evidence that breast cancer is a heterogeneous disease phenotypically as well as molecular biologically. So far, heterogeneity on the molecular biological level has not been investigated in potential precursor lesions, such as ductal hyperplasia (DH) and ductal carcinoma in situ (DCIS). In this study we applied comparative genomic hybridization (CGH) to formalin-fixed, paraffin-embedded breast tissue with DH and DCIS, adjacent to invasive ductal carcinoma (IDC), to screen these potential precursor lesions for whole genomic chromosomal imbalances. Laser-microdissection was used to select pure cell populations from the sections. Isolated DNA was amplified by degenerate oligonucleotide primed PCR (DOP-PCR) and further processed for CGH analysis. Investigating multiple samples (n = 25) from four patients we found an average of 5.6 +/- 0.9 (mean +/- SEM) chromosomal imbalances already present in DH. In the twelve DCIS lesions an average of 10.8 (+/- 0.9) aberrations was identified with 14.8 (+/- 0.8) aberrations in the four adjacent IDC lesions. The increasing number of chromosomal changes in parallel with the histopathological sequence corroborate the hypothesis, that the carcinomas may have developed through a sequential progression from normal to proliferative epithelium and eventually into carcinoma. However, heterogeneous results were identified in the multiple samples per entity from the same patient, demonstrated mainly in the DCIS samples in the chromosomal regions 6p, 9p, 11q, 16p and 17q, in the DH samples by 3p, 16p and 17q. This heterogeneous findings were most pronounced within the DH and was less in the DCIS and IDC samples. The only aberration consistently found in all samples-even in all DH sample-was amplification of the 20q13 region. Our results demonstrate, that the applied combination of laser-microdissection, DOP-PCR and CGH, may serve to analyse breast carcinogenesis pathways in suitable histological material. However, so far, it is unclear how to handle heterogeneous results and these make identification of relevant changes more difficult. Setting a threshold and evaluating only those chromosomal changes which are present in a majority of samples may be one possibility. This involves however, the risk that infrequent but possibly significant aberrations may be missed. Figures on http://www.esacp.org/acp/2000/20-1/aubele. htm.

Comparison of different histopathological methods for the examination of sentinel lymph nodes in breast cancer.

BACKGROUND: Sentinel lymphonodectomy is a new method for the classification of axillary lymph nodes in breast cancer. The optimum technique for the pathological examination of sentinel lymph nodes (SLNs) is still under debate. MATERIALS AND METHODS: Different histopathological techniques were evaluated in order to study their diagnostic accuracy regarding the detection of metastases in 49 SLNs of 40 breast cancer patients. RESULTS: In single hematoxylin and eosin (HE) stained paraffin sections 18 out of 40 patients showed positive SLNs and 8 out of 40 showed positive axillary lymph nodes (ALNs). Serial sections with a spacing of 150 microns between following sections revealed an additional 4 and 5 cases with positive SLNs and ALNs, respectively. Single HE frozen sections showed 11 tumor positive SLNs among 25 patients. The ultrarapid-immunohistochemistry on single frozen sections confirmed these data and detected one additional case with isolated tumor cells in the lymph node sinus. CONCLUSION: Histopathological nodal staging of SLNs should include serial sections with a spacing of 150 microns between sections as well as immunohistochemistry. The ultrarapid-immunohistochemistry is a sensitive method for the detection of minimal metastatic disease in SLNs and can be applied intraoperatively.

Detection of micrometastases in sentinel lymph nodes of the breast applying monoclonal antibodies AE1/AE3 to pancytokeratins.

Sentinel lymph node excision in breast cancer is a minimally invasive diagnostic procedure for accurate staging of the axilla and for avoiding unnecessary axillary dissection. In patients with palpable breast cancer we injected microcolloidal particles of human serum albumin labelled with technetium-99m the day before surgery. The sentinel node was detected intraoperatively with a handheld gammaprobe and then removed. Complete axillary dissection was performed and the nodes inspected by routine histological examination. The axillary lymph node status was correctly predicted by the sentinel node technique in 32 of 33 breast cancer patients. Two cases of micrometastases escaped routine histopathological detection but were identified by immunohistochemical analysis applying the antibody AE1/AE3 to pancytokeratins. Immunohistochemical examination of the sentinel node improves the diagnostic security of patients with breast carcinoma by detection of micrometastases.

Positron emission tomography using [(18)F]Fluorodeoxyglucose for monitoring primary chemotherapy in breast cancer.

PURPOSE: To address the role of positron emission tomography (PET) using [(18)F]fluorodeoxyglucose (FDG) to monitor primary (neoadjuvant) chemotherapy in patients with locally advanced breast cancer. PATIENTS AND METHODS: Quantification of regional FDG uptake of the breast acquired after the first and second courses of chemotherapy was compared with the baseline scan in 22 patients with a total of 24 breast carcinomas. To evaluate the predictive value of PET imaging, histopathologic response after completion of chemotherapy classified as gross residual disease (GRD) or minimal residual disease (MRD) served as the gold standard. RESULTS: Significant differences in tracer uptake between nonresponding tumors (GRD) and responding lesions (MRD) were observed (P <.05) as early as after the first course of chemotherapy. Tracer uptake showed little change in tumors with GRD found later in pathologic analysis but decreased sharply to the background level in most tumors with MRD. After the first course, all responders were correctly identified (sensitivity 100%, specificity 85%) by a standardized uptake value decrease below 55% of the baseline scan. At this threshold, histopathologic response could be predicted with an accuracy of 88% and 91% after the first and second courses of therapy, respectively. CONCLUSION: This study demonstrates that in patients with advanced breast cancer undergoing primary chemotherapy, FDG-PET differentiates responders from nonresponders early in the course of therapy. This may help improve patient management by avoiding ineffective chemotherapy and supporting the decision to continue dose-intensive preoperative chemotherapy in responding patients.

Protocol for ultrarapid immunostaining of frozen sections.

Rapid immunostaining of frozen sections within a tolerable time span would be very helpful for intraoperative diagnosis. A protocol was therefore established using the enhanced polymer one-step staining (EPOS) system (Dako) with antibodies against leucocyte common antigen (LCA), cytokeratin (CK), and anti-melanoma (MEL). Best results with reliable and specific immunostaining and a labelling intensity comparable to standard immunostaining protocols were achieved with fixation of samples in 100% acetone for 20 seconds (CK, LCA) or two minutes (MEL), followed by incubation of the primary antibody and development of the chromogen reaction with 3,3'diaminobenzidine (DAB) for three and five minutes at 37 degrees C, respectively. The total procedure takes only 12 minutes, thus enabling rapid immunostaining on intraoperative frozen sections. Apart from its use in tumour classification, this method is especially useful in detecting tumour cells in sentinel lymph nodes.