[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.

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.

Involvement of CDKN2 (p16INK4A/MTS1) and p15INK4B/MTS2 in human leukemias and lymphomas.

CDKN2 (p16INK4A/MTS1) and p15INK4B/MTS2 have been shown recently to be potent inhibitors of the cyclin D/cyclin-dependent kinase 4 complex. Both genes are candidates for the putative tumor suppressor gene located at chromosome 9p21. We examined a series of 14 hematopoietic cell lines and 117 primary lymphoid tumors for deletion and mutation of these genes. The primary tumors included 65 T-cell malignancies and 52 B-cell malignancies. The cell line study revealed 4 of 4 T-ALL lines to have homozygous deletions of CDKN2. Two of the 4 lines also showed homozygous deletions of MTS2, while the remaining 2 lines retained both MTS2 alleles. In the primary tumors, homozygous deletions of both CDKN2 and MTS2 were found in 35% of the T-ALL/lymphoblastic lymphoma (8 of 23). Homozygous deletions of both genes also occurred in 1 of 3 precursor B-ALLs. PCR-single strand conformational polymorphism analysis of CDKN2 exons 2 and 3 and MTS2 exon 2 failed to demonstrate mutations in either CDKN2 or MTS2 in any of the T- or B-cell malignancies, with two possible exceptions. These results are consistent with a role for CDKN2 and/or MTS2 in the pathogenesis of some lymphoid leukemia/lymphomas, particularly in T-ALL/lymphoblastic lymphoma.

Absence of p18 mutations or deletions in lymphoid malignancies.

p18 is a recently described cyclin-dependent kinase inhibitor (CDK-I) wih homology to p16 and p15. The latter two CDK-Is have been implicated as possible tumor suppressor genes in a wide variety of human tumors, including hematological malignancies. Because of p18's structural and functional homology to p16 and p15, we hypothesized that it may also function as a tumor suppressor gene in some lymphoid malignancies. To explore this possibility we examined 81 primary lymphoid tumors for deletion and mutation p18. The primary tumors included 40 T cell malignancies and 41 B cell malignancies. None of the lymphoid tumors studied possessed deletions of p18, including a group of lymphoblastic lymphomas which we previously reported to have deletions of p16 and p15. PCR-SSCP analysis of the p18 gene identified a single polymorphism of codon 114, but failed to demonstrate mutations in any of the lymphoid tumors. These results do not support a role for p18 in the pathogenesis of the lymphoid neoplasms studied.

Allelic loss of 11q13 as detected by MEN1-FISH is not associated with mutation of the MEN1 gene in lymphoid neoplasms.

Deletions and rearrangements involving the long arm of chromosome 11 are not infrequent occurrences in the non-Hodgkin's lymphomas. Recently, a tumor suppressor gene, the multiple endocrine neoplasia type 1 gene (MEN1) was cloned and mapped to chromosome 11q13. To assess the potential involvement of this gene in lymphomagenesis, we examined 94 primary cases of lymphoma and 12 cell lines by a combination of fluorescent in situ hybridization and PCR-SSCP analysis. In our initial analysis of 41 primary B or T lymphomas, MEN1 FISH analysis revealed allelic deletions in 15 cases (three of four B cell chronic lymphocytic leukemias, six of 15 follicular lymphomas, three of nine diffuse large B cell lymphomas, two of five mantle cell lymphomas, one of four Burkitt's lymphoma). To discern whether the MEN1 gene was in fact the target of the deletions, we assessed 20 of these 41 cases and an additional 74 primary lymphomas and 12 cell lines for MEN1 gene mutations using PCR-SSCP analysis. Abnormal SSCP patterns were found in exon 2 in two of the primary lymphoma cases and in one of the cell lines, but not in any of the original cases that showed MEN1 deletions by FISH. Furthermore, sequencing analysis revealed that the abnormal SSCP patterns in exon 2 were the result of a previously described genetic polymorphism (S145S: AGC --> ACT), and in one sample, the result of this S145S polymorphism associated with a second nucleotide substitution at position 498 which left the encoded amino acid unchanged. Our study indicates that the 11q13 locus is a frequent target of deletion in lymphoid neoplasms, but that there are no associated mutations of the MEN1 gene. This suggests that the 11q deletions either target another gene in lymphomas, or that the MEN1 gene is inactivated through means other than mutation.

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

We 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 tumor stroma and that both components can share clonal features. Tumor cell-mesenchyme transitions are among the possible explanations for these findings.

Monoclonal antibodies against inhibin represent key markers of adult granulosa cell tumors of the ovary even in their metastases. A report of three cases with late metastasis, being previously misinterpreted as hemangiopericytoma.

Antibodies against human inhibin, a peptide hormone produced by ovarian granulosa cells to inhibit FSH, are widely applied to determine serum inhibin levels. Recently, they were, however, proved also to stain follicle cells in ovarian tissue by immunoreactions in histological sections. The commercially available inhibin antibody produced by Serotec, applied to sections of paraffin blocks, stained follicle epithelia in 6/6 samples of ovarian tissue from females under the age of 40 recruited from the archives. Adult granulosa cell tumor tissue samples from primary tumors of the ovary showed positive reaction in 6/6 cases. No positive reaction was found in staining tissues from hemangiopericytomas from males (0/3), leiomyomas, leiomyosarcomas, and a malignant melanoma (0/5), serving as negative controls. No positive reactions could be observed in tumor cells of 10 ovarian carcinomas, whereas in two of these cases single cells of the specialized ovarian stroma stained positively with inhibin. Positive immunostainings were revealed in three late metastases (two within the liver) from granulosa cell tumors in females, primarily misinterpreted as hemangiopericytomas or leiomyosarcomas, because the previously resected primaries of the ovary were not known at the time of liver surgery. The recognition of granulosa cell tumors, especially the distinction of the sarcomatoid growth type from soft tissue tumors, may be difficult, even if immunostaining for intermediate filaments are applied. Immunostaining by antibodies against inhibin, which can be applied reliably in histopathology, may therefore provide a useful tool to distinguish between granulosa cell tumors and genuine soft tissue tumors. This is also of clinical importance, because treatment of the former by cisplatin-based polychemotherapy and antisex hormone therapy proved to be helpful. Furthermore, the inhibin antibody can be used as an early serum marker for detecting tumor recurrence months before clinical evidence.

bc1-1 rearrangement and cyclin D1 protein expression in multiple lymphomatous polyposis.

Multiple lymphomatous polyposis (MLP), characterized by multiple polyps involving long segments of the gastrointestinal (GI) tract, is believed to represent GI involvement by mantle cell lymphoma (MCL), primarily based on its histologic and immunophenotypic similarities with MCL. However, rearrangement of the bcl-1 locus, the molecular lesion characteristic of MCL, has not been investigated in this group of patients. The authors evaluated the morphologic, immunophenotypic, and molecular features of 18 cases of MLP and 8 B-cell lymphomas involving the GI tract (including 6 MALT lymphomas). All MLP cases presented with GI disease, and were histologically similar to MCL. DNA extracted from formalin-fixed, paraffin-embedded tissue was analyzed for evidence of bcl-1 rearrangement by PCR, using chromosome 11 specific and consensus JH primers. Amplifiable DNA was obtained in 24 of 26 cases (16 of 18 MLP cases and 8 of 8 controls). bcl-1 rearrangement was detected in 6 of 16 cases (38%), subsequently confirmed by sequencing of the breakpoint region, and in 0 of 8 controls. Immunostaining for cyclin D1 was positive in 14 of 18 MLPs, including the 6 bcl-1 rearranged cases and negative in 6 of 6 evaluable controls. The detection of bcl-1 rearrangement and cyclin D1 expression in cases of MLP supports the view that MLP represents primary MCL, of the GI tract. These techniques may also be helpful in differentiating MLP from other GI lymphomas, particularly low grade lymphomas of MALT, when only small routinely fixed endoscopic biopsies are available.

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.

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.

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.

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.

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.