Analysis of gene amplification and prognostic markers in ovarian cancer using comparative genomic hybridization for microarrays and immunohistochemical analysis for tissue microarrays.

To identify oncogene amplification involved in ovarian carcinogenesis, we studied 21 ovarian carcinomas and 5 serous borderline tumors using conventional comparative genomic hybridization (CGH) and CGH to a genomic DNA microarray. Immunohistochemical analysis of the proteins encoded by the genes that were amplified frequently (FGF3/4, FGFR1, CCNE1, PAK1, JUNB, and MDM2) was performed on a tissue microarray comprising 254 cases of ovarian neoplasms. Regarding histologic type, characteristic patterns of copy number changes were revealed. They correlated with histologic tumor type and with intratumoral heterogeneity. Gain of FGF3/4 and CCNE1 was found in all serous carcinomas. Endometrioid carcinomas most frequently showed gain of JUNB (83%), KRAS2 (67%), MYCN (50%), ESR (50%), and CCND2 (50%). Of the serous borderline tumors, 80% harbored amplification of FGFR1 and MDM2 and a 75% gain of PIK3CA. Only CCNE1 immunoreactivity was significantly correlated with CGH results (P < .05) and postoperative survival (P < .05). Microarray-based genomic analysis in combination with immunohistochemical analysis was found to be a powerful technique for identification of clinically relevant gene amplification in human ovarian cancer.

Recurrence of lymphangioleiomyomatosis after single lung transplantation: new insights into pathogenesis.

Lymphangioleiomyomatosis (LAM) is a rare disease found primarily in white women of childbearing age. The present study describes a case of recurrent LAM after single lung transplantation. Double-staining nonisotopic in situ hybridization, immunohistochemistry, and short tandem repeat loci analysis demonstrated that the recurrent LAM lesions originated from the recipient. The data strongly support that metastatic spread of LAM cells or migration of progenitor cells plays an important role in the pathogenesis of LAM.

Microsatellite instability, loss of heterozygosity, and loss of hMLH1 and hMSH2 protein expression in endometrial carcinoma.

Microsatellite instability (MSI) due to replication errors occurs frequently in hereditary tumors. Association with functional inactivation of the mismatch repair (MMR) genes and lack of protein expression has been described. In endometrial carcinoma (EC), the prevalence and clinical significance of these phenomena are not well known. Therefore, DNA samples from 89 EC and 5 metachronous tumors were analyzed with polymerase chain reaction, using 5 microsatellite markers and a DNA sequencer for amplicon detection. The results were correlated with immunohistochemistry of hMLH1 and hMSH2. MSI at >or=2 loci (MSI-H) was detected in 10/89 EC (11%); 1 of 10 showed loss of both hMLH1 and hMSH2, and 5 of 10 showed loss of hMLH1 (P < 0.0001). MSI-H was observed frequently in tumors with mucinous differentiation (P = 0.048), >10% of solid-cribriform pattern (P = 0.037), International Federation of Obstetrics and Gynecology (FIGO) stage III to IV (4 of 13; P = 0.016), and necrosis >5% (P = 0.07). Loss of heterozygosity (LOH) in >or=1 loci was found in 17 of 156 (11%). Survival (Kaplan-Meier) was longer for patients with endometrioid tumors with predominant glandular pattern, <5% necrosis, low FIGO stage and grade, superficial myometrial infiltration, no lymph-vascular invasion (LVI), and loss of hMLH1 expression (all P

CAS (cellular apoptosis susceptibility) gene expression in ovarian carcinoma: Correlation with 20q13.2 copy number and cyclin D1, p53, and Rb protein expression.

We immunohistochemically analyzed cellular apoptosis susceptibility (CAS) protein expression and compared it with 20q13.2 copy number and the expression of cell cycle-associated proteins retinoblastoma (Rb), cyclin D1, and p53 and prognosis on paraffin-embedded tissue from 69 ovarian carcinomas (OCs). CAS protein reactivity was present in 100%, Rb in 54%, cyclin D1 in 47%, and p53 in 49%. Significant reciprocal correlation was observed between high levels of CAS and histologic type, FIGO (International Federation of Obstetrics and Gynecology) stage III and grade 3, residual tumor (>2 cm), 20q13.2 (ZNF217 gene) amplification (>4 copies in >20% cells), and high expression of cyclin D1 (all P < .05). No association was found between cyclin D1, p53, or Rb levels with clinicopathologic factors. In univariate analysis, residual tumor, FIGO stage and grade, ZNF217 amplification, and CAS levels predicted outcome (all P < .05). In multivariate analysis, stage, grade, amount of residual tumor, and ZNF217 amplification showed independent prognostic value (all P < .05). In OC, alteration of CAS and ZNF217 genes, both located at 20q13, is frequent and relevant prognostically. Cyclin D1, Rb, and p53 seem to have a secondary role.

Are gastrointestinal mucosal mast cells increased in patients with systemic mastocytosis?

In patients with mastocytosis, gastrointestinal symptoms are a frequent phenomenon. However, there are only limited data about the quantity and distribution pattern of mast cells in the gastrointestinal mucosa. We stained gastroduodenal biopsy specimens from 27 patients with mastocytosis and 48 control subjects for mast cell tryptase, CD117, and CD25. The numbers of mucosal mast cells per high-power field showed wide variation in all groups and were decreased markedly in biopsy specimens of corpus and duodenum and statistically significantly decreased in antrum biopsy specimens from patients with systemic mastocytosis compared with patients with pure urticaria pigmentosa and with control subjects. Staining for tryptase showed highly significant correlation with staining for CD117. All mast cells were negative for CD25, which is expressed characteristically by neoplastic mast cells. Causes of the decrease of mucosal mast cells remain enigmatic, but our results show that gastrointestinal symptoms of patients with mastocytosis are most likely mediator-related and not due to an increase of local mast cells.

Regeneration in chronic sialadenitis: an analysis of proliferation and apoptosis based on double immunohistochemical labelling.

The understanding of regeneration in salivary glands as a finely tuned balance of cellular proliferation, differentiation and apoptosis has been limited by the difficulty of identifying proliferating cells. This has been overcome in the present investigation by double immunohistochemical labelling for the proliferation-associated antigen Ki67 and for different cell-type-specific antigens applied to 8 specimens of normal parotids and 16 specimens of chronic parotid sialadenitis with particular reference to acini and intercalated ducts. In comparison with low baseline rates of proliferation in normal parotids, proliferative indices were significantly increased in chronic sialadenitis in mature acinar cells, intercalated ductal cells and myoepithelial cells without evidence of proliferation by an additional population of cells. In accordance with findings in glands of experimental animals, the present data do not support the previously postulated concept of regeneration of acini and intercalated ducts by a hypothetical population of uncommitted ductal stem cells. The demonstration of a profound capacity for intrinsic glandular regeneration from differentiated cells represents a biological basis for the good results obtained from conservative therapy of chronic sialadenitis and offers hope for novel therapies designed to reconstitute impaired salivary flow.

Fas/FasL and perforin/granzyme pathway in acute rejection and diffuse alveolar damage after allogeneic lung transplantation-a human biopsy study.

Acute rejection and diffuse alveolar damage are major problems during the early time after transplantation. Against this background, lung biopsies after allogeneic lung transplantation were studied using immunohistochemistry. Biopsies with acute rejection, diffuse alveolar damage and morphological inconspicuous biopsies were chosen. The objectives of this study were to ascertain: (a) if and how CD4 and CD8 T cells contribute to allograft rejection and diffuse alveolar damage, (b) whether there is a correlation of the chemoattractant regulated on activation normal T cells (RANTES) with the mononuclear infiltrate and (c) whether perforin/granzyme and Fas/FasL pathways contribute to lung injury after lung transplantation. Our results show that CD4(+) and CD8(+) T cells were increased in biopsies with acute rejection and, to a minor extent, also in biopsies with diffuse alveolar damage due to reperfusion injury. RANTES expression of T cells was increased in biopsies with acute rejection. Perforin seemed to have a dual role in the alloimmune response. In one regard, it had a cytolytic function in the acute rejection process, and, in contrast, it may be responsible for downregulating both CD4- and CD8-mediated alloimmune responses. The FasL/Fas pathway is not only important for induction of apoptosis during rejection but is also a mechanism of lung injury in the development of diffuse alveolar damage.

A morphogenetic concept of salivary duct regeneration and metaplasia.

The exact mechanisms of physiological regeneration and of metaplastic processes of the salivary duct have not been definitely established, although regeneration from a putative uncommitted stem cell population has long been favored. In the present study, double immunohistochemical labeling for Ki 67 and alpha-actin or different cytokeratin subtypes, respectively, made possible an exact localization and quantification of cellular proliferation in the regular salivary duct and in different types of metaplasia. Our data demonstrate a baseline proliferative capacity in all five cell types of the salivary duct. Luminal secretory cells of the acinus and intercalated duct regenerate independently from myoepithelial or basal cells. In contrast, the renewal of oxyphilic cells in the striated and excretory duct is maintained by proliferation and differentiation of basal cells. The great majority of metaplasias develops from uncommitted, Bcl-2 positive basal cells of striated/excretory ducts which possess an enormous capacity for pluridirectional morphogenetic differentiation. Despite this important role of basal cells, our findings demonstrate that all cell types principally have to be considered as potential progenitor cells for salivary gland tumors. The improved insight into regenerative and metaplastic processes of the salivary duct may contribute to a better understanding of the complex formal carcinogenesis.

The role of graft-resident Kupffer cells and lymphocytes of donor type during the time course after liver transplantation--a clinico-pathological study.

Although graft-resident passenger leukocytes are known to mediate acute rejection by triggering direct allorecognition, they may also act in an immunomodulatory fashion and play an important role in tolerance induction. The present study evaluated by non-isotopic in situ hybridization and immunohistochemistry if and during which time period after transplantation Kupffer cells (KC) and lymphocytes of the liver were replaced by recipient cells and if there is any correlation with the occurrence of rejection episodes and the clinical course. A successive re-population of the liver by recipient lymphocytes and KC was observed after transplantation but a smaller portion of lymphocytes and KC with donor genotype was detectable during the whole time course studied. There was no correlation between the portion of recipient-derived KC and donor-derived lymphocytes and histopathological alterations of the liver tissue. The biopsy content of KC with recipient origin has had no prognostic significance for the probability of survival, but patients with a low portion of donor lymphocytes in the liver biopsy obtained during the first week after transplantation have had a better prognosis for survival. The present results indicate that graft-resident KC and lymphocytes are potentially not the main cell types involved in tolerance induction after liver transplantation.

Cellular protein and mRNA expression patterns of matrix metalloproteinases-2, -3 and -9 in human breast cancer: correlation with tumour growth.

The expression of matrix metalloproteinases (MMPs) with type IV collagenase activity has been associated with tumour invasion and metastatic potential in experimental models. We studied whether the cellular localization of MMP expression provides useful information on tumour behaviour in human breast cancer. Immunohistochemistry and non-radioisotopic-detected in situ hybridization were used to study protein and mRNA expression profiles for MMPs-2, -3 and -9 in paraffin sections of 70 invasive breast carcinomas. Protein and mRNA expression of the MMPs was observed in tumour as well as in peritumoural stromal cells. MMP protein expression was increased at the invasive border (p < 0.05). Grade 3 carcinomas expressed MMP-2 mRNA in significantly more tumour cells than grade 2 carcinomas (p = 0.006). Ductal carcinomas with an extensive intraductal component were characterized by the lowest percentages of MMPs-2 and -3 mRNA expressing peritumoural stromal cells (p < 0.05). No correlation was observed between MMP protein/mRNA expression and pTNM classification. In conclusion our results indicate that the expression of MMPs is associated with tumour behaviour. The correlation of MMPs-2 and -3 expression in peritumoural stromal cells with tumour type, shown for the first time, suggests that transcriptional regulation of these MMPs in stromal cells is important for the growth pattern of breast cancer.

Differential diagnosis of salivary acinic cell carcinoma and adenocarcinoma (NOS). A comparison of (immuno-)histochemical markers.

A correct histologic differential diagnosis between salivary acinic cell carcinoma (ACC) and adenocarcinoma not otherwise specified (AC-NOS) is highly relevant because of the strikingly different biologic behavior and related therapeutical strategies. The distinction between both tumor types can be difficult because of an enormous variation in histologic appearance, with either type showing partially overlapping morphologic features. Owing to a lack of approved markers, the expression of PAS-staining, alpha-Amylase, alpha-1 Anti-trypsin, cytokeratin (CK)-subtypes 7/18 and Ki-67 was evaluated in 16 cases of ACC and 16 cases of AC-NOS. CK 7 is identified as the most reliable marker with strong positivity in AC-NOS, and complete or preponderant negativity in ACC. The characteristic membranous staining pattern of CK 18 in ACC, in contrast to a diffuse cytoplasmic pattern in AC-NOS, proved to be an additional valuable criterion. PAS and alpha-Amylase are only of little value when ACC is diagnosed, as many cases are only faintly positive or completely negative. The proliferation index (Ki-67) proved to be significantly higher in AC-NOS; however, the diagnostic usefulness is limited by a relevant overlap. In conclusion, we recommend CK 7 and 18 as the most valuable markers in cases with difficult differential diagnosis between ACC and AC-NOS.

Multifocal manifestation does not affect vascular invasion of hepatocellular carcinoma: implications for patient selection in liver transplantation.

BACKGROUND AND AIMS: Liver transplantation (OLT) for hepatocellular carcinoma (HCC) improves patient survival when tumor size and number are limited according to the Milan criteria. However, the impact of tumor size vs. the number of lesions for tumor recurrence after OLT is unclear. Microvascular invasion appears to be a significant risk factor for tumor recurrence. Therefore, it was the aim of this study to investigate tumor differentiation and microvascular invasion in relation to tumor number and size and their impact on survival after transplantation. PATIENTS AND METHODS: In 97 adult HCC patients who underwent OLT between June 1985 and December 2005 the incidence of microvascular invasion, tumor differentiation, and the number and size of tumor lesions were analyzed retrospectively. Their impact on survival was studied by multivariate analysis. RESULTS: Microvascular invasion was the only independent negative predictor of survival after OLT for HCC (p = 0.025). Tumor size > 5 cm was predictive for microvascular invasion (p = 0.007). In contrast, tumor number did not affect the incidence of microvascular invasion or cumulative survival. CONCLUSION: The size of the largest HCC lesion, but not the number of tumors, determined microvascular invasion, a predictor of the outcome following OLT for HCC. Thus, the number of HCC lesions should not be applied to patient selection prior to OLT. These data support the extension of the Milan criteria for the selection of HCC patients for OLT with regard to tumor number, but not tumor size.

KRAS and BRAF mutations in ovarian tumors: a comprehensive study of invasive carcinomas, borderline tumors and extraovarian implants.

OBJECTIVE: Mutations of BRAF, a downstream mediator of K-RAS, have been described in serous borderline tumors of the ovary. Data concerning other types of ovarian tumors are scarce. Therefore, we assessed KRAS and BRAF mutation in a series of more than 100 different ovarian tumors. METHODS: Paraffin-embedded material, including invasive carcinomas, borderline tumors, benign lesions and implants, was used. BRAF codon 600 in exon 15 and K-RAS codon 12 in exon 2 were analysed. RESULTS: 92 cases (92%), including all serous carcinomas (100%), did not show a mutation of BRAF. Eight cases (8.0%), including five serous borderline tumors (31.25%), contained a mutation. In all serous borderline tumors, codon 600 was affected. The remaining three cases were invasive carcinomas of endometrioid (mutation on codon 600), mucinous (mutation on codon 600) and clear cell (mutation on codon 615) subtype. There was no BRAF mutation in mucinous borderline tumors. Regarding K-RAS, 89 cases (87.25%) did not show an aberration. The 11 positive borderline tumors (10.7%) were of serous (22.2%) and of mucinous type (46.6%). There was a KRAS mutation in a serous and a mucinous invasive carcinoma each. BRAF and K-RAS mutations were mutually exclusive and not seen in implants. CONCLUSION: Mutation of either K-RAS or BRAF is frequent in borderline tumors but is not found in invasive serous carcinomas and is very rare in other invasive subtypes. This supports the notion of different pathological pathways. For the development of extraovarian implants, further studies are observed.

Mutations of the BRAF gene in ulcerative colitis-related colorectal carcinoma.

The Raf/MEK/ERK (MAPK) signal transduction cascade is an important mediator of a number of cellular fates including growth, proliferation and survival. The BRAF gene is activated by oncogenic Ras, leading to cooperative effects in cells responding to growth factor signals. Our study was performed to elucidate a possible function of BRAF in ulcerative colitis (UC)-related colorectal carcinogenesis. Mutations of BRAF and KRAS were determined in 33 UC-related colorectal cancers by direct DNA sequencing analyses after microdissection. Mismatch-repair deficiency was assessed by immunohistochemistry for major mismatch-repair proteins hMLH1, hMSH2 and hMSH6 and microsatellite analyses of the BAT25 and BAT26 loci. Hypermethylation of the hMLH1 promoter was also tested. The results obtained were correlated with histopathologic variables. Activating BRAF missense mutations were identified in 3/33 UC-related cancers (9%), 2 of which exhibited a loss of hMLH1-protein expression and hypermethylation of the hMLH1 promoter. Corresponding nondysplastic UC-mucosa of these patients did not show BRAF mutations. KRAS mutations were found in 6/33 (18%) UC cancers. All 6 UC cancers with KRAS mutations had an intact BRAF gene as the 3 cancers with BRAF mutations had an intact KRAS gene. There was no significant correlation between BRAF or KRAS status and clinicopathologic variables. Our data indicate that BRAF mutations are not an initiating event in UC-related carcinogenesis and are associated with mismatch-repair deficiency through hMLH1-promoter hypermethylation. Disruption of the Raf/MEK/ERK (MAPK) kinase pathway-either through RAS or BRAF mutation-was detected in 27% of all UC-related cancers and thus plays an important role in UC-related carcinogenesis.

K-RAS mutations in ovarian and extraovarian lesions of serous tumors of borderline malignancy.

K-RAS mutations are the most frequent molecular genetic alteration in serous ovarian tumors of borderline malignancy (SBOT). The pathogenesis of associated contralateral tumors and extraovarian implants and Müllerian inclusion cysts is obscure. We hypothesized that the comparison of K-RAS mutations in these lesions might help to distinguish multifocal from metastatic foci. Eight cases of SBOT with known K-RAS mutation (RAS+) and two cases without mutation (RAS-) were analyzed for comparison. DNA was extracted from multiple samples of 58 paraffin-embedded and laser-microdissected ovarian and extraovarian lesions (10 ovarian borderline tumors, 8 contralateral tumors, 25 implants, 15 inclusion cysts; total: 97 samples). K-RAS exon 1 was amplified by PCR and analyzed by denaturing gradient gel electrophoresis and cycle sequencing. In 12 of 14 SBOT and in 2 of 2 extraovarian implants, the K-RAS mutation could be found in different areas of the same lesion, indicating monoclonality. All RAS+ ovarian borderline tumors with contralateral tumors (six of six) harbored an identical mutation in both ovaries (in one case, a separate surface borderline tumor component contained a different mutation in addition). In 4 of 5 RAS+ ovarian tumors with extraovarian lesions, RAS mutations were also found in implants (15/21 implants [71%]) and more rarely in inclusion cysts (3 of 12 lesions [25%]). These extraovarian mutations were always identical to the one in the ovary (18 of 18 [100%]). Regarding the contralateral and extraovarian lesions of the two RAS- SBOT, only one extraovarian implant contained a RAS mutation. The demonstration of K-RAS mutations in Müllerian inclusion cysts and implants of SBOT suggests that K-RAS mutations represent a pivotal event during neoplastic transformation of ovarian and extraovarian serous epithelium. Considering our observations, the two putative pathogenetic mechanisms for the development of implants and endosalpingiosis-multifocal tumorigenesis and spread from the ovarian primary tumor-seem to coexist.

Endothelial cells but not epithelial cells or cardiomyocytes are partially replaced by donor cells after allogeneic bone marrow and stem cell transplantation.

Recent studies have convincingly demonstrated that adult bone marrow contains cells capable of differentiating into a variety of cell types. To investigate whether such bone marrow-derived cells participate on self-renewal and proliferation of nonhematopoietic tissues, we studied tissue obtained by autopsy from female recipients after sex-mismatched allogeneic bone marrow and stem cell transplantation for the presence of donor-derived cells. Epithelial, endothelial, and smooth muscle cells and hematopoietic cells were characterized by double-staining immunohistochemistry with a panel of antibodies and nonisotopic in situ hybridization with a Y-chromosome-specific probe. The present study showed that the capillary endothelium was the only nonhematopoietic cell type that was replaced in a significant amount by donor cells after allogeneic stem cell and bone marrow transplantation. We could not demonstrate any participation of graft-derived cells on repopulation of cardiomyocytes or epithelial cells of the skin and gastrointestinal mucosa and of hepatocytes.

EGFR, HER-2/neu, cyclin D1, p21 and p53 in correlation to cell proliferation and steroid hormone receptor status in ductal carcinoma in situ of the breast.

Abnormalities in G1/S transition in cell cultures have been attributed to alterations in ErbB (erythroblastic leukaemia viral [v-erb-b] oncogene homologue, avian) signalling, cyclin D1 overexpression or disturbance of the p21(WAF1) (p21)-mediated cell cycle arrest induced by p53. To investigate the significance of these mechanisms on an early stage of human breast tumour growth, we studied the expression of EGFR (ErbB1), HER-2/neu (ErbB2), cyclin D1, p21 and p53 as well as oestrogen (ER) and progesterone receptor (PgR) in paraffin sections of 45 ductal carcinoma in situ (DCIS) by immunohistochemistry. Cell proliferation was assessed by immunohistochemical quantification of Ki-67. Five cases with cyclin D1 overexpression were analysed by FISH for CCND1 amplification. Increased proliferative activity was observed in 46% of DCIS. It was correlated with the expression of EGFR and HER-2/neu (p < 0.05), but neither with cyclin D1 and p21 overexpression nor with p53 accumulation. ErbB positive status was associated with p21 overexpression (p < 0.05). In addition we found a correlation between the overexpression of p21 and cyclin D1 restricted to ErbB-positive cases (p = 0.013). ErbB-negative tumours with increased proliferative activity were ER and cyclin D1 positive. No CCND1 amplification was detected in the analysed cases. In conclusion, our data support that EGFR and HER-2/neu play an important role in cell cycle control in DCIS. p21 appears to be a potential mediator of ErbB signalling. We propose that cyclin D1 could be indirectly induced by ErbB signalling through p21. Besides, ER-mediated upregulation of cyclin D1 seems to be a possible mechanism of maintaining cell proliferation in DCIS in case of EGFR- and HER-2/neu-negativity.

Generalized crystal-storing histiocytosis associated with monoclonal gammopathy: molecular analysis of a disorder with rapid clinical course and review of the literature.

Crystal-storing histiocytosis (CSH) is a rare event in disorders associated with monoclonal gammopathy. The intracellular crystal formation is almost always accompanied by the expression of kappa light chains. However, the exact mechanism for the storage has not been clarified until now. We report a case of generalized CSH in a 73-year-old man who presented with IgA kappa paraproteinemia and paraproteinuria. The initially observed CSH in the bone marrow biopsy was associated with the clinical and pathomorphologic features of a monoclonal gammopathy of undetermined significance. The progression of disease could not be affected by steroid therapy and the patient died of septic shock 7 months after detection of CSH. At the time of autopsy there was evidence for multiple myeloma and generalized CSH. Two-dimensional gel electrophoresis of liver tissue combined with immunoblotting revealed the massive storage of heavy chains of alpha type and light chains of kappa type, each in a monoclonal pattern. Analysis of the stored kappa light chain by nanoelectrospray-ionization mass spectrometry indicated that it belongs to the variable (kappa)I variability subgroup. We identified some unusual amino acid substitutions including Leu59, usually important for hydrophobic interactions within a protein, at a position where it has never been previously described in plasma cell disorders. In conclusion, we present the first case of CSH with molecular identification of the stored kappa subgroup and detection of unusual amino acid substitutions. Our results suggest that conformational alterations induced by amino acid exchanges represent a crucial pathogenic factor in CSH.