[EUS-FNA: how to improve biopsy results? An evidence based review]. / EUS-geführte Feinnadelbiopsie (EUS-FNP): Wie gelingen gute Biopsien? Eine evidenzbasierte Analyse.

Endosonography with fine-needle aspiration biopsy (EUS-FNA) has become a widespreadly available clinical tool to diagnose numerous different lesions in humans. EUS-FNA is frequently used for tissue-based diagnoses such as lymphatic diseases (ranging from tuberculosis / sarcoidosis to malignant lymphoma) or solid tumors (such as pancreatic carcinoma, neuroendocrine tumors, sub-epithelial gastrointestinal tumors and others). Outcomes of EUS-FNA results, however, vary which is caused by several different factors ranging from experience of the endoscopist over technical factors such as use of stylet or suction for puncture through the skills of the cyto-pathologist who takes care of the specimen obtained by EUS-FNA. Though introduced since more than 20 years ago EUS-FNA has still not yet been perfectionized and several issues remain controversial among endoscopist. These issues include needle size and type (FNA versus TNB needles), use of a stylet and suction for FNA sampling, pure cytologic assessment versus cyto-histologic techniques, grading of the investigator´s and pathologist´s experience and improvement of EUS training for novices. In this report we briefly review the actual literature and summarize the available evidence on some controversely discussed issues. The results support the view that use of a stylet rarely aids to increase the amount of tissue obtained during EUS-FNA, whereas use of suction can be helpful in certain situations. Novel cutting needles may potentially improve number and size of core biopsies that can be rendered for special histologic tissue processing techniques. An in-room-cytopathologist not necessarily improves outcome of EUS-FNA results but may have a role during build-up of EUS units to become more successful. EUS-FNA education requires skilled endoscopists on both sides and can presumably be improved by objective testing of practical expertise by peer review and introducing objective sampling parameters. Novel techniques and equipment are about to evolve in the near future.

MALDI MS imaging as a powerful tool for investigating synovial tissue.

OBJECTIVE: To identify and image protein biomarker candidates in the synovial tissue of patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA). METHODS: A novel matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) technique was applied to the analysis of synovial tissue. Patients were classified according to the American College of Rheumatology (ACR) criteria for RA. Frozen sections were stained to obtain morphological data. Serial sections were desiccated, and spotted with matrix for MALDI analysis. Ions generated by laser irradiation of the tissue were separated in time, based on their m/z ratio, and were subsequently detected. IMS was used in a 'profiling' mode to detect discrete spots for rapid evaluation of proteomic patterns in various tissue compartments. Photomicrographs of the stained tissue images were reviewed by a pathologist. Areas of interest (10 discrete areas/compartment) were marked digitally and the histology-annotated images were merged to form a photomicrograph of the section taken before the MALDI measurement. Pixel coordinates of these areas were transferred to a robotic spotter, the matrix was spotted, and the coordinates of the spots were transferred to a mass spectrometer for spectral acquisition. The data generated were then subjected to biocomputation analysis to reveal the biomarker candidates. RESULTS: Several peaks (m/z) consistent in mass with calgranulins, defensins, and thymosins were detected and their distribution in various synovial compartments (synovial lining and sublining layer) was demonstrated. CONCLUSION: MALDI IMS is a powerful tool for the rapid detection of numerous proteins (in situ proteomics) and was applied here for the analysis of the distribution of proteins in synovial tissue sections.

Laparoscopic deroofing of hepatic cysts: The most effective treatment option.

OBJECTIVES: With advances in laparoscopic surgery, laparoscopic deroofing has gained wide acceptance in the surgical community to treat symptomatic non-parasitic hepatic cysts. Published non-surgical data still favour aspiration and sclerotherapy as treatment in these cases, though morbidity is higher and recurrence rates are not acceptable. We reviewed all patients that had been treated by laparoscopic deroofing in our department over a period of 6 years in order to find out if the surgical approach should be considered the standard treatment. MATERIALS AND METHODS: Over a 6 year period, 34 laparoscopic deroofings were performed in 21 patients with symptomatic cysts. Patients underwent laparoscopy followed by radical deroofing using an Ultracision device. RESULTS: All cases were completed laparoscopically, no intraoperative adverse events were recorded. Mean operation time was 101 ± 22.7 min. The mean size of treated cysts was 9.7 ± 2.18 cm. Follow up showed only one symptomatic recurrence (3.3%) two months after surgery. CONCLUSION: Laparoscopic deroofing of hepatic cysts is a safe and effective treatment option. Recurrence rates are unprecedentedly low. Our data suggest that the risk of operation is justified and that the method is superior to sclerotherapy.

Protein profiling of non-malignant and malignant ascites by SELDI-TOF MS: proof of principle.

Ascites is a common clinical symptom in liver cirrhosis, inflammatory disorders of the abdomen and a major late manifestation of metastatic malignancies. Standard cytopathological techniques and immunocytochemistry have specificities and sensitivities of approximately 95 and 60%, respectively for the presence of tumor cells. Development of faster and more accurate screening methods would be of great clinical utility. In this work we examined differential analysis of the unbound proteins in the supernatant of ascites fluid by Protein-Chip SELDI mass spectrometry. There were 21 tumor cell-positive and 34 tumor cell-negative samples. We used principal component analysis coupled with linear regression applied to the mass spectra of the samples to distinguish between the sample groups. Two sample sets for statistical analysis were created after randomization, a training set with 37 samples and a validation set with 18 samples resulting in a specificity of 93% and a sensitivity of 83% on the training set. The validation set yielded a specificity and sensitivity of 75%. This study suggests that SELDI-TOF mass spectrometry appears to have great potential as a surrogate diagnostic tool to evaluate effusion specimens.

[Solitary peritoneal carcinomatosis in prostate cancer]. / Solitäre Peritonealkarzinose eines Prostatakarzinoms.

INTRODUCTION: Peritoneal carcinomatosis is a rare finding in metastatic prostate cancer. In the literature peritoneal carcinomatosis is usually reported in its final stages with multiple metastases. A single peritoneal carcinomatosis with no further metastases is a very rare finding. CASE REPORT: We report the case of a 75-year-old patient with initial ischuria. A prostate cancer could be confirmed and the further diagnostics showed no metastasis. In a transperitoneal approach for laparoscopic pelvic lymphadenectomy a peritoneal carcinomatosis from prostate cancer was proven. A complete antiandrogen therapy was started and PSA decreased for more than 14 months to a stable level of < 1 microg/L. CONCLUSION: An isolated peritoneal carcinomatosis from prostate cancer is a very rare finding. The complete antiandrogen therapy is effective.

[Terminally ill tumor patients in emergency medical care]. / Der finale Tumorpatient in der notfallmedizinischen Versorgung.

We report about an emergency case of a female patient with terminal carcinoma of the ovary. On the basis of this case it becomes evident that palliative care questions are also important in emergency medicine. In this situation cooperation of the medical disciplines involved appears urgently necessary. This may allow the possibility for terminally ill patients to stay at home in the last days of life.

Pouchitis in ulcerative colitis: correlation between predictors from colectomy specimens and clinico-histological features.

Pouchitis after restorative proctocolectomy for ulcerative colitis is usually of ill-defined etiology and is encountered with sclerosing cholangitis, bacterial overgrowth, and ischemia. Recently, appendiceal involvement, ileitis, and fissures in the colectomy specimen have been associated with short- and long-term development of pouchitis. To corroborate these recent findings, the histology of 40 colectomies (70% males; mean age 46.3 years, age range 20-70 years; mean follow-up period 3.7 years, range 1-13 years) with yearly follow-up biopsies was correlated with pouchitis and clinical symptoms. Appendicitis, fissures, and ileitis were present in 47, 45 and 5% of the patients, respectively. Pouchitis in patients with appendicitis or with fissures was noted in 44 and 50% at first biopsy and in 70 and 58% during follow-up (p = NS). Of the patients without appendicitis or without fissures, 33 and 33% demonstrated pouchitis at the first biopsy and 30 and 55% during follow-up (p = NS). Clinico-histological correlation revealed normal/near-normal biopsies with the lowest clinical severity score in 77% and with the highest clinical score in 43% (p < 0.025). The histological findings of appendiceal involvement, fissuring ulcers, and ileitis in colectomies for ulcerative colitis do not correlate with the finding of pouchitis in early or late pouch biopsies. A high clinical suspicion score is frequently not correlated with significant inflammation of the pouch.

GSTP1 hypermethylation as a molecular marker in the diagnosis of prostatic cancer: is there a correlation with clinical stage, Gleason grade, PSA value or age?

OBJECTIVES: Epigenetic events such as promoter hypermethylation have been implicated in prostate carcinogenesis. We present a real-time, methylation specific protocol to detect hypermethylation in the promoter region of the GSTP1 gene in benign hyperplasia and adenocarcinoma of the prostate. METHODS: In our preliminary study, 31 prostate cancer and 5 benign prostatic hyperplasia (BPH) tissue samples were analyzed. Genomic DNA was isolated from formalin-fixed and paraffin-embedded specimens and subjected to sodium bisulfite modification, followed by real-time, methylation specific PCR. Patients with prostatic cancer were also subdivided according to their Gleason score, PSA, age and TNM Staging. Prostate cancer cell lines (LNCaP, DU145, PC3) and a BPH cell line (BPH-1) were also tested as controls. RESULTS: GSTP1 promotor hypermethylation was detected in 28 of the 31 prostate cancer cases (90.3%) and none of the five (0%) BPH cases. Statistical analysis did not reveal a significant correlation between GSTP1 hypermethylation and Gleason score, PSA, age or TNM staging. All prostate cancer cell lines were testes positive for GSTP1 promotor hypermethylation, whereas the BPH cell line (BPH-1) was tested negative. CONCLUSION: GSTP1 promotor hypermethylation occurs during carcinogenesis and is considered to be a major event of prostate carcinogenesis. Our data support this thesis and shows that GSTP1 hypermethylation reliably distinguishes between prostate cancer and BPH . Although it is not yet clear at what time during carcinogenesis hypermethylation of the GSTP1 promotor occurs it seems to provide valuable information for prostate cancer screening and diagnosis. Larger studies are underway to determine the potential role for GSTP1 hypermethylation in clinical settings.

CSE1L/CAS: its role in proliferation and apoptosis.

CAS/CSE1L is the human homologue of the yeast gene CSE1. It was first cloned while searching for genes that rendered breast cancer cells resistant towards toxin induced apoptosis. Since depletion of CSE1 leads to cell-cycle arrest, CAS is thought to be involved in proliferation. CAS functions in the mitotic spindle checkpoint. CAS is located on chromosome 20q13, a locus often amplified in cancers of various origin, e.g. colonic or breast cancer. Since genetic instability is a hallmark of cancer, amplification or over expression of the CAS gene might interfere with or override its role in the mitotic spindle checkpoint. CAS is also implicated in the nuclear to cytoplasmic reshuffling of importin alpha, which itself is necessary for the nuclear transport of several proliferation activating proteins, transcription factors, oncogene and tumor suppressor gene products such as p53 and BRCA1. Inhibition of MEK1 mediated phosphorylation has been shown to enhance paclitaxel (Taxol) induced apoptosis in breast, ovarian, and lung tumor cell lines in-vitro. Since CAS is also phosphorylated (activated) by MEK1, and since the anti-cancer drug Taxol alters the microtubule assembly and activates pro-apoptotic signaling pathways, altering the activity/phosphorylation status of CAS via MEK1 inhibition may present a potential strategy in experimental cancer therapy.

Presence of genetic alterations in microdissected stroma of human colon and breast cancers.

BACKGROUND: Human carcinomas not only consist of neoplastic epithelial cells but also of tumor stroma, which may play an important role in tumor-progression. Whereas the tumor surrounding stroma is generally believed to represent a reactive component induced by tumor cell derived factors, a contribution of neoplastic cells to stroma formation via epithelium-mesenchyme transition during tumor invasion has become a novel concept in recent years. MATERIALS, METHODS AND RESULTS: We here show, by laser-assisted microdissection, that frequent genetic alterations in non-hereditary invasive human colon and breast cancers (loss of heterozygosity and TP53 mutations) occur not only in the neoplastic epithelial cells, but also in the adjacent fibroblastic stroma and that both components can share clonal features. CONCLUSION: Tumor cell-mesenchyme transitions are among the possible explanations for these findings and could actually occur during tumor invasion in vivo.

Implication of the proliferation and apoptosis associated CSE1L/CAS gene for breast cancer development.

The CSEIL/CAS protein (CAS) is a Ran-binding protein with a function as a nuclear transport (export) factor. CSEIL/CAS, similar to Ran and other ran-binding proteins, plays at the same time an important role in the mitotic spindle checkpoint, which assures genomic stability during cell division. This checkpoint is frequently disturbed in neoplasms of various origin, including breast, hepatic and colonic tumors. CAS is located on chromosome 20ql3 and amplified in several cell lines, including breast, colon and bladder cancer. MEKl phosphorylation is known to be a reason for different CAS localization and activity. We evaluated the expression of CAS in 50 benign and malignant tumors of the breast by immunohistochemistry. Benign lesions of the breast (n=13) revealed a weak, predominantly cytoplasmatic CAS positivity. In ductal and lobular in situ carcinomas (n=17), 70-90% of the tumor cells were positive for anti-CAS staining which was predominantly cytoplasmatic. In invasive ductal and lobular carcinomas (n =20), 70-90% of the tumor cells stained positive with anti-CAS in a predominantly nuclear pattern. Different localization of CAS might affect its role not only for chromosome segregation, proliferation and apoptosis, but also its function in nuclear transport of proteins like retinoblastoma-gene-product, p53 and BRCAl. A different regulation in this checkpoint might contribute to the invasive potential in malignant carcinomas of the breast. Alteration of CAS-activity, possibly via MEKl-inhibition, might therefore be a possible option for breast cancer therapy.

Downregulation of clusterin expression in testicular germ cell tumours.

OBJECTIVES: Clusterin is implicated in many biological processes including cell adhesion, apoptosis and transformation. Clusterin expression was demonstrated during sperm maturation and is overexpressed in different malignancies including breast and prostate carcinomas and anaplastic large-cell lymphomas. METHODS: The aim of this study was to determine the expression of clusterin in a series of different germ cell tumours by immunohistochemistry. Twenty-two seminomas, 27 embryonal carcinomas, 22 mature and immature teratomas, 8 yolk sac tumours and 1 chorion carcinoma as well as 30 normal testes were analysed using a monoclonal antibody. RESULTS: In normal testes strong signals were seen in the maturing germ cells and in Leydig cells. In most tumours examined no clusterin expression was detected (84%). Only a focal weak expression was seen in some teratomas (32%), embryonal carcinomas (15%) and yolk sac tumours (25%). CONCLUSIONS: Our results suggest that clusterin is associated with normal germ cell development and that the loss of clusterin expression might play a role in the malignant transformation of germ cells.

Invasive properties of serous human epithelial ovarian tumors are related to Ets-1, MMP-1 and MMP-9 expression.

The invasive potential of serous epithelial ovarian tumors is the main factor determining their biological behaviour. In contrast to invasive serous ovarian carcinomas serous borderline tumors generally present without stromal invasion and without or non-invading peritoneal implants. Little is known about the reasons underlying these differences. In the present study we found that two matrix-degrading metalloproteinases, collagenases 1 and 4 (MMPs 1 and 9), as well as the Ets-1 transcription factor are expressed at very low levels in serous benign cystadenomas, upregulated in the fibroblastic stroma, but not in the epithelium of borderline tumors and most strongly expressed in both stromal and epithelial tumor cells of serous invasive carcinomas. Since expression of Ets-1 and of MMPs 1 and 9 are topographically related, a transcriptional regulation of both proteases by Ets-1 is suggested. Upregulation of MMPs 1 and 9 within fibroblastic stromal cells of borderline tumors might be related to matrix remodelling and additional expression of both enzymes by the neoplastic cells of invasive carcinomas could then allow invasive propagation. The different expression patterns might supports the view, that no transition of serous borderline tumors into invasive carcinomas occurs.

Glut-1 expression in dysplastic and regenerative lesions of the colon.

Monosaccaride transporter proteins are responsible for transmembrane transport of monosaccarides into cells. Glucose transporter protein 1 (Glut-1) is most prevalent in the cell membranes of erythrocytes and facilitates transport of glucose in tissues with barrier functions, i.e. blood brain barrier. Expression of Glut-1 in malignant tumors is increased due to increased metabolic need of the proliferating cell populations. In colorectal adenomas and carcinomas, membranous expression of Glut-1 has been associated with higher grade of tumors and decreased survival time. We studied the expression of Glut-1 in dysplastic proliferations of the colon which included sporadic adenomas and dysplasia associated lesions (DALM) in patients with ulcerative colitis and reactive/regenerative proliferations of the colon, including non-dysplastic chronic colitis, acute colitis and ischemia. Two patterns of Glut-1 expression were detected. Most adenomas and DALMs showed at least focal membranous expression of Glut-1. In addition a second staining pattern was recognized which consisted of prominent supranuclear dots. This pattern of staining was not only seen in adenomas and DALM but also in non-dysplastic areas immediately surrounding sporadic adenomas, in regenerative chronic colitis and in areas surrounding acute inflammation. Areas away from dysplasia did not show any positive staining for Glut-1. We conclude that two distinct patterns of Glut-1 expression may be found in colonic epithelial proliferation: membranous staining, associated with dysplasia, and, heretofore not described, supranuclear staining which may be related to Glut-1 expression secondary to expression of specific growth factors and not necessarily related to dysplasia.

The Ets-1 transcription factor is up-regulated together with MMP 1 and MMP 9 in the stroma of pre-invasive breast cancer.

The first steps of stroma generation are of pivotal importance for carcinogenesis because at this stage are initiated both angiogenesis, the prerequisite for continuous tumour growth, and the proliferation of stromal fibroblasts. These developments contribute to the onset of tumour invasion by secreting several matrix-degrading proteases. Both angiogenesis and the production of proteases are tightly controlled at several levels; of significant importance is transcription. The Ets-1 transcription factor transactivates several genes encoding matrix-degrading proteases and is thought to be involved in both tumour vascularization and invasion. This study therefore investigated, by in situ hybridization and immunohistochemistry, the expression of Ets-1 and of two of its target genes, encoding matrix metalloproteinase (MMP) 1 and MMP 9, in order to demonstrate a topographical in vivo correlation between the expression of these three genes during breast cancer formation. All three genes were first expressed within both endothelial cells and stromal fibroblasts during the onset of stroma generation around intraductal and intralobular in situ carcinomas and they were significantly up-regulated in the stroma of invasive ductal and lobular cancers. The results of this study further support the suggested in vivo role of Ets-1 for both angiogenesis and tumour invasion, via matrix-degrading proteases which are already expressed during the early stages of breast carcinogenesis.

High expression of the proliferation and apoptosis associated CSE1L/CAS gene in hepatitis and liver neoplasms: correlation with tumor progression.

The CSE1L/CAS protein (CAS) is a Ran-binding protein with a function as nuclear transport (export) factor. Like recently observed for ran and other ran-binding proteins, CSE1L/CAS simultaneously plays a role in the mitotic spindle checkpoint, which assures genomic stability during cell division. This checkpoint is frequently disturbed in neoplasias of various origin, including hepatic tumors. We have evaluated by immunohistology the expression of CAS in adult and embryonic liver, hepatitis, and in liver hyperplasias. Normal hepatocytes revealed no CAS expression while embryonic liver showed strong expression in all parenchymal cells. Bile ducts stained positive with anti-CAS antibodies, and strong CAS expression was also detected at the interface between bile ducts and hepatocytes under conditions associated with regenerative proliferation. The localization of these CAS expressing cells correlated with the distribution of putative liver stem-cells. In active viral (but not in inactive) hepatitis, strong hepatocytal CAS expression correlates in site and intensity with degree of inflammation. Neoplastic liver demonstrated different degrees of CAS expression: no remarkable expression in adenomas, moderate expression in a narrow rim of hepatocytes and in periseptal cholangiolar proliferations in focal nodular hyperplasia, and strong CAS expression in hepatocellular carcinoma. Less differentiated tumors stain stronger than well differentiated. Cholangio-cellular carcinomas show even stronger CAS expression than hepatocellular carcinomas. Our observation of strong expression of CAS in liver cells that are committed for proliferation among them possibly liver stem cells, and in liver neoplasms, is consistant with the fact that CAS functions not solely as a nuclear transport factor but that it is also essential for cell proliferation, particularly for the mitotic spindle checkpoint. Interestingly, genomic instability is frequently observed in hepatic tumors which we have shown here to express large amounts of CAS. Since the degree of CAS-expression correlates with the grade of tumor dedifferentiation, we suggest that CAS should also be further investigated as prognostic marker for hepatic neoplasms.