Expression of E-cadherin repressors SNAIL, ZEB1 and ZEB2 by tumour and stromal cells influences tumour-budding phenotype and suggests heterogeneity of stromal cells in pancreatic cancer.

BACKGROUND: There is evidence that tumour-stroma interactions have a major role in the neoplastic progression of pancreatic ductal adenocarcinoma (PDAC). Tumour budding is thought to reflect the process of epithelial-mesenchymal transition (EMT); however, the relationship between tumour buds and EMT remains unclear. Here we characterize the tumour-budding- and stromal cells in PDAC at protein and mRNA levels concerning factors involved in EMT. METHODS: mRNA in situ hybridisation and immunostaining for E-cadherin, ß-catenin, SNAIL1, ZEB1, ZEB2, N-cadherin and TWIST1 were assessed in the main tumour, tumour buds and tumour stroma on multipunch tissue microarrays from 120 well-characterised PDACs and associated with the clinicopathological features, including peritumoural (PTB) and intratumoural (ITB) budding. RESULTS: Tumour-budding cells showed increased levels of ZEB1 (P<0.0001) and ZEB2 (P=0.0119) and reduced E-cadherin and ß-catenin (P<0.0001, each) compared with the main tumour. Loss of membranous ß-catenin in the main tumour (P=0.0009) and tumour buds (P=0.0053), without nuclear translocation, as well as increased SNAIL1 in tumour and stromal cells (P=0.0002, each) correlated with high PTB. ZEB1 overexpression in the main tumour-budding and stromal cells was associated with high ITB (P=0.0084; 0.0250 and 0.0029, respectively) and high PTB (P=0.0005; 0.0392 and 0.0007, respectively). ZEB2 overexpression in stromal cells correlated with higher pT stage (P=0.03), lymphatic invasion (P=0.0172) and lymph node metastasis (P=0.0152). CONCLUSIONS: In the tumour microenvironment of phenotypically aggressive PDAC, tumour-budding cells express EMT hallmarks at protein and mRNA levels underlining their EMT-type character and are surrounded by stromal cells expressing high levels of the E-cadherin repressors ZEB1, ZEB2 and SNAIL1, this being strongly associated with the tumour-budding phenotype. Moreover, our findings suggest the existence of subtypes of stromal cells in PDAC with phenotypical and functional heterogeneity.

Increased number of non-degranulated mast cells in pancreatic ductal adenocarcinoma but not in acute pancreatitis.

Increasing evidence indicates that tumor microenvironment (TME) is crucial in tumor survival and metastases. Inflammatory cells accumulate around tumors and strangely appear to be permissive to their growth. One key stroma cell is the mast cell (MC), which can secrete numerous pro- and antitumor molecules. We investigated the presence and degranulation state of MC in pancreatic ductal adenocarcinoma (PDAC) as compared to acute ancreatitis (AP). Three different detection methods: (a) toluidine blue staining, as well as immunohistochemistry for (b) tryptase and (c) c-kit, were utilized to assess the number and extent of degranulation of MC in PDAC tissue (n=7), uninvolved pancreatic tissue derived from tumor-free margins (n=7) and tissue form AP (n=4). The number of MC detected with all three methods was significantly increased in PDAC, as compared to normal pancreatic tissue derived from tumor-free margins (p<0.05). The highest number of MC was identified by c-kit, 22.2∓7.5 per high power field (HPF) in PDAC vs 9.7∓5.1 per HPF in normal tissue. Contrary to MC in AP, where most of the detected MC were found degranulated, MC in PDAC appeared intact. In conclusion, MC are increased in number, but not degranulated in PDAC, suggesting that they may contribute to cancer growth by permitting selective release of pro-tumorogenic molecules.

Geographic analysis of RKIP expression and its clinical relevance in colorectal cancer.

BACKGROUND: This study evaluates the geographic expression pattern of Raf-1 Kinase Inhibitor Protein (RKIP) in colorectal cancer (CRC) in correlation with clinicopathological and molecular features, markers of epithelial-mesenchymal transition (EMT) and survival outcome. METHODS: Whole-tissue sections of 220 well-characterised CRCs were immunostained for RKIP. NF-κB and E-Cadherin expression was assessed using a matched multi-punch tissue microarray. Analysis of mismatch repair (MMR) protein expression, B-Raf and KRAS mutations was performed. RKIP expression in normal mucosa, tumour centre, invasion front and tumour buds was each assessed for clinical relevance. RESULTS: RKIP was diffusely expressed in normal mucosa and progressively lost towards tumour centre and front (P<0.0001). Only 0.9% of tumour buds were RKIP-positive. In the tumour centre, RKIP deficiency predicted metastatic disease (P=0.0307), vascular invasion (P=0.0506), tumour budding (P=0.0112) and an invasive border configuration (P=0.0084). Loss of RKIP correlated with NF-κB activation (P=0.0002) and loss of E-Cadherin (P<0.0001). Absence of RKIP was more common in MMR-deficient cancers (P=0.0191), while no impact of KRAS and B-Raf mutation was observed. RKIP in the tumour centre was identified as a strong prognostic indicator (HR (95% CI): 2.13 (1.27-3.56); P=0.0042) independently of TNM classification and therapy (P=0.0474). CONCLUSION: The clinical relevance of RKIP expression as an independent prognostic factor is restricted to the tumour centre. Loss of RKIP predicts features of EMT and correlates with frequent distant metastasis.

Tumour budding: a promising parameter in colorectal cancer.

In 2011, the Tumour Node Metastasis (TNM) staging system still remains the gold standard for stratifying colorectal cancer (CRC) patients into prognostic subgroups, and is considered a solid basis for treatment management. Nevertheless, there is still a challenge with regard to therapeutic strategy; stage II patients are not typically selected for postoperative adjuvant chemotherapy, although some stage II patients have a comparable outcome to stage III patients who, themselves do receive such treatment. Consequently, there has been an inundation of 'prognostic biomarker' studies aiming to improve the prognostic stratification power of the TNM staging system. Most proposed biomarkers are not implemented because of lack of reproducibility, validation and standardisation. This problem can be partially resolved by following the REMARK guidelines. In search of novel prognostic factors for patients with CRC, one might glance at a table in the book entitled 'Prognostic Factors in Cancer' published by the International Union against Cancer (UICC) in 2006, in which TNM stage, L and V classifications are considered 'essential' prognostic factors, whereas tumour grade, perineural invasion, tumour budding and tumour-border configuration among others are proposed as 'additional' prognostic factors. Histopathology reports normally include the 'essential' features and are accompanied by tumour grade, histological subtype and information on perineural invasion, but interestingly, the tumour-border configuration (i.e., growth pattern) and especially tumour budding are rarely reported. Although scoring systems such as the 'BRE' in breast and 'Gleason' in prostate cancer are solidly based on histomorphological features and used in daily practice, no such additional scoring system to complement TNM staging is available for CRC. Regardless of differences in study design and methods for tumour-budding assessment, the prognostic power of tumour budding has been confirmed by dozens of study groups worldwide, suggesting that tumour budding may be a valuable candidate for inclusion into a future prognostic scoring system for CRC. This mini-review therefore attempts to present a short and concise overview on tumour budding, including morphological, molecular and prognostic aspects underlining its inter-disciplinary relevance.

CD8+ lymphocytes/ tumour-budding index: an independent prognostic factor representing a 'pro-/anti-tumour' approach to tumour host interaction in colorectal cancer.

BACKGROUND: The tumour-host interaction at the invasive front of colorectal cancer, including the epithelial-mesenchymal transition and its hallmark 'tumour budding', is an important area of investigation in terms of prognosis. The aim of this study was to determine the prognostic impact of a 'pro-/anti-tumour' approach defined by an established 'pro-tumour' (tumour budding) and host-related 'anti-tumour' factor of the adaptive immunological microenvironment (CD8+ lymphocytes). METHODS: Double immunostaining for CK22/CD8 on whole tissue sections (n=279; Cohort 1) and immunohistochemistry for CD8+ using tissue microarrays (n=191; Cohort 2) was carried out. Tumour buds, CD8+ and CD8+ T-lymphocytes : tumour buds indices were evaluated per high-power field. RESULTS: In Cohort 1, a low-CD8+/ buds index was associated with lymph node metastasis (P<0.001), vascular invasion (P=0.009), worse survival in univariate (P<0.001) and multivariable (P<0.001) analysis, and furthermore in lymph node-negative patients (P=0.002). In Cohort 2, the CD8+/ buds index was associated with T stage (P<0.001), N stage (P=0.041), vascular invasion (P=0.005) and survival in patients with TNM stage II (P=0.019), stage III (P=0.004), and adjuvantly untreated (P=0.009) and treated patients (P<0.001). CONCLUSION: The CD8+ lymphocyte : tumour-budding index is an independent prognostic factor in colorectal cancer and a promising approach for a future prognostic score for patients with this disease.

Prognostic significance of MIB-1, p53, and bcl-2 immunoreactivity in meningiomas.

Sixty biopsy specimens of meningiomas, including 37 benign, 10 atypical, and 13 malignant meningiomas, were examined immunohistochemically using the monoclonal antibodies MIB-1 (a cell proliferation marker), p53, and bcl-2 (two apoptosis-associated markers). Benign meningiomas were subdivided into two groups: group 1, 29 tumors without recurrence; and group 2, eight tumors with recurrence after complete surgical resection. The mean MIB-1 labeling index (LI) values+/-SD were 1.3+/-3.2% for the benign, 9.3+/-6.9% for the atypical, and 15.0+/-16.9% for the anaplastic meningiomas. The mean MIB-1 LI+/-SD in group 1 tumors (n = 29) was 1.06+/-1.15%, and in group 2 tumors (n = 8), 2.3+/-4.76% (P = .028). p53 protein expression was found in 10.8% of the benign (10.34% of group 1 and 12.5% of group 2), 50% of the atypical, and 77% of the anaplastic meningiomas. bcl-2 protein expression was observed in 21.6% of the benign, 20% of the atypical, and 46.1% of the anaplastic meningiomas. Among the benign meningiomas, group 2 tumors expressed significantly more often bcl-2 protein (62.5%) than group 1 neoplasms (10.3%). Our results indicate that (1) in meningiomas, a good correlation exists between histological grading, MIB-1 and p53 protein expression, and (2) in benign meningiomas, the presence of bcl-2 protein expression together with high MIB-1 LI are associated with unfavorable prognosis of the disease.

Ganglioglioma of the brain stem: neurological dysfunction of 16-year duration.

An autopsy case of a brain stem ganglioglioma in a 38-year-old male patient with neurological dysfunction of 16-year duration is reported. Immunohistochemical investigation of the tumor was performed using a panel of antibodies against neurofilament protein (NFP), synaptophysin (SY 38), beta-tubulin (TUJ1), neuron specific enolase (NSE), and glial fibrillary acidic protein (GFAP). The value of these markers in the establishment of the diagnosis, as well as the general features, the prognosis and the therapeutic approach of the gangliogliomas are discussed.

Overview of quantitative methods in toxicologic pathology.

Today's stringent requirements for new drugs make it necessary to prove their safety by the use of ever more refined techniques, in particular methods to quantify morphologic findings to determine accurately, for example, the highest non-toxic dose in animal studies or to correlate morphological effects with other parameters. Increased cell proliferation due to subtle cytotoxicity can lead to epigenetic tumorigenicity, which can be recognized early by cytokinetic investigations using bromodeoxyuridine (BrdU) incorporation and/or proliferating cell nuclear antigen (PCNA) expression, or by malignancy-associated nuclear texture changes. Morphometric criteria are also used to increase diagnostic accuracy in experimental animal pathology. The various applications of morphometry in toxicopathology are reviewed, and examples illustrating these applications are presented, most of them unpublished.

Proliferating cell nuclear antigen expression in Wistar rat livers. A retrospective immunohistochemical study of normal and neoplastic livers.

Formalin-fixed, paraffin-embedded liver specimens from 27 2-year-old Wistar rats, including 10 normal livers, 11 hepatocellular adenomas, 2 hepatocellular carcinomas, and 4 cystic cholangiomas, were immunostained using the streptavidin/peroxidase method and the PC10 monoclonal antibody (Mab), which recognizes an epitope on the proliferating cell nuclear antigen (PCNA). The following PCNA labeling index (LI) mean values were found for the above four groups of liver specimens: normal livers, 0.43 +/- 0.31%; hepatocellular adenomas, 1.51 +/- 0.59%; hepatocellular carcinomas, 24.80% +/- 10.28%; and cystic cholangiomas, 0.61 +/- 0.21%. Our findings indicate that PCNA LI clearly separates liver malignancies from other benign liver tumors, as well as from normal, non-neoplastic, liver tissues. Although the mean PCNA LI values seemed to reflect histological grading (i. e. normal, neoplastic benign, neoplastic malignant), overlapping between normal livers and hepatocellular adenomas was observed in five cases (i. e. in 2 normal livers and 3 hepatocellular adenomas, where the PCNA LI values varied between 0.74% and 0.96%). It thus appears that PCNA immunohistochemistry represents a promising tool for investigating liver cell proliferation in laboratory rats, and permits distinguishing between benign and malignant liver parenchymal tumors.

Pretreatment with sevoflurane attenuates direct lung injury.

BACKGROUND: Sevoflurane exerts effects on pulmonary cells that could protect against lung injury. We evaluated the potential of pretreatment with sevoflurane to attenuate lipopolysaccharide (LPS)-induced lung injury. METHODS: LPS was administered intratracheally in Wistar rats to induce lung injury. Sevoflurane was administered for 30 min at 0.25, 0.5 or 1.0 MAC 15 min before LPS or for 30 min at 0.5 MAC 24 hours before LPS. After initial analysis of bronchoalveolar lavage fluid (BALF) cells and total protein, the group of 0.5 MAC 15 min before LPS was further analyzed for surfactant aggregates subfractions, plasma malondialdehyde levels and lung histology. RESULTS: LPS instillation resulted in neutrophils sequestration in the lungs, loss of alveolar macrophages, increased BALF total protein and decreased large surfactant aggregates. Only inhalation of sevoflurane for 30 min at 0.5 MAC 15 min before LPS installation effectively reduced neutrophil accumulation, preserved alveolar epithelial cells and reduced total protein content in BALF. This regimen also reduced plasma malondialdehyde levels and increased large surfactant aggregates, despite the application of mechanical ventilation. This effect was preserved after LPS instillation and the favorable composition of surfactant was maintained. CONCLUSION: Pretreatment with sevoflurane effectively attenuates direct severe lung injury, possibly by inhibition of neutrophil accumulation and alteration of the surfactant composition.

Tumour budding is a strong and independent prognostic factor in pancreatic cancer.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer that escapes detection and resists treatment. Tumour budding, defined as the presence of de-differentiated single tumour cells or small cell clusters at the invasive front of gastrointestinal carcinomas like colorectal, oesophageal, gastric and ampullary, is linked to adverse prognosis. Tumour budding has not yet been reported in PDAC. AIM: To assess the frequency and prognostic impact of tumour budding in PDAC. METHODS: Whole-tissue sections of 117 PDACs with full clinico-pathological and follow-up information, including postoperative therapy, were stained using a pancytokeratin marker. Tumour budding was assessed in 10 high-power fields (HPFs) by two pathologists. High-grade budding was defined as an average of >10buds across 10HPFs. Measurements were correlated to patient and tumour characteristics. The study was performed according to the REMARK guidelines. RESULTS: Inter-observer agreement was considered strong (ICC=0.72). Low-grade budding was observed in 29.7% and high-grade budding in 70.3% cases. High-grade budding was linked to advanced pT classification (p=0.0463), lymphatic invasion (p=0.0192) and decreased disease-free (p=0.0005) and overall survival (p<0.0001). There was no association with pN, pM, R-status or blood vessel invasion. In multivariate analysis, the prognostic effect of tumour budding was independent of lymph node metastasis, lymphatic invasion and R-status (p<0.0001; HR (95% CI): 3.65 (2.1-6.4)). CONCLUSIONS: Our results show that high-grade tumour budding occurs frequently in PDAC and is a strong, independent and reproducible, highly unfavourable prognostic factor that could be used to guide future individualised therapeutic approaches.

Loss of E-cadherin independently predicts the lymph node status in colorectal cancer.

BACKGROUND: The identification of biomarkers that improve risk stratification in patients with colorectal cancer (CRC) is still a challenge. The objective of our study was to identify independent protein markers as predictors of lymph node (N) stage in CRC. METHODS: Tumour specimens from 221 CRC patients were mounted onto a multiple-punch tissue microarray and evaluated for 21 tumour related factors and one host related factor involved in CRC carcinogenesis, namely ß-catenin, E-cadherin, EGFR, pERK, RHAMM, pAKT, pSMAD2, p21, p16, Bcl-2, Ki-67, APAF-1, MST1, RKIP, VEGF, EphB2, MMP7, Laminin5γ2, MUC1, CDX2, caspase-3 as well as intra-tumoural and stromal CD8+ tumour infiltrating lymphocytes (iTILs and sTILs). RESULTS: Node positive cancers showed significant losses for p21 (p = 0.026), Bcl-2 (p = 0.027), APAF-1 (p = 0.033), EphB2 (p = 0.006), E-cadherin (p < 0.001), RKIP (p = 0.019), CD8+ iTILs and sTILs (p < 0.001 and p = 0.008, respectively) and cytoplasmic MST1 (p = 0.014). Based on the area under the receiver operating characteristic curve (AUC) EphB2, E-cadherin, iTILs and sTILs were identified as potential predictors of N stage (AUC values >0.6), but only loss of E-cadherin was an independent predictor in multivariate analysis. CONCLUSIONS: E-cadherin appears to be a strong predictor of N stage in CRC and should be considered in pre-operative and post-operative management of colon and rectal cancer patients.

p53 protein expression in central nervous system tumors: an immunohistochemical study with CM1 polyvalent and DO-7 monoclonal antibodies.

Formalin-fixed, paraffin-embedded surgical specimens from 137 primary central nervous system tumors, including 26 astrocytomas (21 fibrillary, 1 protoplasmic, 1 gemistocytic and 3 pilocytic), 26 anaplastic astrocytomas, 9 glioblastomas, 1 gliosarcoma, 8 oligodendrogliomas, 4 ependymomas, 1 anaplastic ependymoma, 2 subependymomas, 3 paragangliomas, and 57 meningiomas, were immunostained with the CM1 polyclonal (pAb) and the DO-7 monoclonal (mAb) antibodies against the p53 protein, using the streptavidin/peroxidase method. In addition, two series of 17 and 9 medulloblastomas were also immunostained with the above pAb and mAb, respectively. p53 protein expression was observed in 7 fibrillary astrocytomas, 17 anaplastic astrocytomas, 5 glioblastomas, 1 gliosarcoma, 1 oligodendroglioma, 1 anaplastic ependymoma, and 4 meningiomas with the CM1 pAb. An additional 10 cases (i.e., 3 anaplastic astrocytomas and 7 meningiomas) were found to be p53 protein positive with the DO-7 mAb. Of the medulloblastomas, 8 (of the 17) and 4 (of the 9) were found to express p53 protein with CM1 pAb and DO-7 mAb, respectively. Our results indicate that p53 protein is expressed in a number of central nervous system neoplasms, and suggest that in astrocytic tumors a possible association may exist between p53 protein expression and tumor progression through increasing histological grades of malignancy.

p53 protein immunoreactivity in extrahepatic bile duct and gallbladder cancer: correlation with tumor grade and survival.

Forty-five extrahepatic bile duct carcinomas (i.e., tumors of the region proximal to the duct junction, including Klatskin tumors, tumors of the lower mid-region, and tumors of the ampulla of Vater) and 11 gallbladder carcinomas were immunohistochemically examined for p53 protein expression, using the DO-7 monoclonal (mAb) and the CM-1 polyvalent (pAb) antibodies and an antigen retrieval method. Because the DO-7 mAb was found to be significantly more reliable than the CM-1 pAb in detecting p53 immunoreactivity, the immunohistochemical results obtained with the former antibody were used for comparing p53 protein immunoreactivity with tumor site, tumor grade, and survival of patients. Approximately one third (3 of 10) of the proximal tumors were found to express weak p53 immunopositivity, whereas moderate immunopositivity and higher rate (18 of 29) was observed in tumors of the lower mid-region. Finally, moderate and marked p53 immunopositivity was observed in tumors of the ampulla (5 of 6) and gallbladder (8 of 11). In tumors of the lower mid-region of the ampulla and the gallbladder, a significantly higher p53 positivity was noted in high-grade compared with low-grade neoplasms. For the cases in which complete follow-up was available (11 tumors of the low mid-region), median survival of patients with p53-negative tumors was 25.7 months, whereas survival of those with p53-positive tumors was 5.2 months.(ABSTRACT TRUNCATED AT 250 WORDS)

Ki-67 immunoreactivity in human central nervous system tumors: a study with MIB 1 monoclonal antibody on archival material.

Paraffin-embedded surgical specimens from 136 primary human central nervous system (CNS) tumors, including 50 meningiomas, 24 astrocytomas, 26 anaplastic astrocytomas, 9 glioblastomas, 8 oligodendrogliomas, 4 ependymomas, 1 anaplastic ependymoma, 2 subependymomas, 9 medulloblastomas, and 3 paragangliomas, were immunostained, following microwave processing, using a streptavidin/peroxidase method and the MIB 1 monoclonal antibody (mAb) against the Ki-67 antigen. The following mean Ki-67 labeling index (LI) values +/- SD were found: meningiomas, 2.47 +/- 1.83; astrocytomas, 2.03 +/- 2.03; anaplastic astrocytomas, 12.80 +/- 6.29; glioblastomas, 14.57 +/- 6.77; oligodendrogliomas, 5.06 +/- 4.78; ependymomas, 2.63 +/- 2.58; anaplastic ependymoma, 6.89; subependymomas, 1.79 +/- 1.54; medulloblastomas, 18.77 +/- 9.65; and paragangliomas, 2.19 +/- 2.51. Our findings indicate that while malignant CNS tumors always exhibited high Ki-67 LI values, and benign CNS tumors generally displayed lower values, increased immunoreactivity for Ki-67 epitopes (Ki-67 LI higher than 4) was noted in a number of meningiomas, astrocytomas, ependymomas, oligodendrogliomas and paragangliomas, contrasting with their benign histological features. Further investigations of the Ki-67 immunoreactivity in CNS tumors and systematic correlation with the postoperative follow-up of patients are necessary to determine the value of Ki-67 LI in predicting the biological behavior of CNS neoplasms.

Proliferating cell nuclear antigen immunoreactivity in human central nervous system neoplasms.

Formalin-fixed, paraffin-embedded surgical specimens from 140 primary human central nervous system tumors, including 51 meningiomas, 26 astrocytomas, 26 anaplastic astrocytomas, 9 glioblastomas, 1 gliosarcoma, 8 oligodendrogliomas, 5 ependymomas, 2 subependymomas, 9 medulloblastomas, and 3 paragangliomas, were immunostained using a streptavidin/peroxidase method and the PC10 monoclonal antibody, which recognizes an epitope on the proliferating cell nuclear antigen (PCNA). The following PCNA labeling index (LI) mean values were found for the above neoplasms: meningiomas, 3.80 +/- 7.35%; astrocytomas, 0.65 +/- 1.03%; anaplastic astrocytomas, 8.46 +/- 7.95%; glioblastomas, 10.26 +/- 11.21; gliosarcoma, 46.34%; oligodendrogliomas, 2.31 +/- 3.59%; ependymomas, 1.12 +/- 2.10%; medulloblastomas, 23.91 +/- 11.95%; and paragangliomas, 2.07 +/- 1.86%. Collectively, our findings indicate that while benign central nervous system tumors generally have low PCNA LI values, consistent over-expression of PCNA epitopes was noted in some examples, especially in a number of meningiomas. Among the malignant neuroectodermal tumors, medulloblastomas were found to have the highest PCNA LI values, corresponding to their histological grade of malignancy, and malignant glial tumors generally displayed significantly higher PCNA LI values, than their benign counterparts. Although in our study mean PCNA LI values seemed to reflect histological grading, large discrepancies were noted in all tumor groups. Our data, therefore, suggest than PCNA immunoreactivity can not be considered reliable for predicting the prognosis of the disease in individual cases.

Cat scratch disease simulating a malignant process of the chest wall with coexistent osteomyelitis.

A 6-y-old girl developed fever, soft-tissue mass in the right chest wall, osteomyelitis of the 10th rib and hepatic granuloma. Cat scratch disease was diagnosed by histological examination of the mass and serological tests. The patient was treated successfully with antibiotics and recovered completely, as shown by a 10 month follow-up.

p53 protein expression in colorectal adenomas: an immunohistochemical study using an antigen retrieval system.

The immunohistochemical expression of the p53 gene product was examined in 91 colorectal adenomas from patients without (group 1, 50 cases) or with (group 2, 41 cases) concurrent sporadic colorectal carcinoma, and in 15 additional cases of randomly selected carcinomas from group 2 patients. Immunohistochemical reactions were performed with the DO-7 monoclonal and the CM1 polyclonal antibodies, following microwave irradiation of the tissues in an antigen retrieval solution, and the proportion of the immunoreactive cells was semiquantitatively assessed. p53 protein immunoreactivity was present in 46.1% (42, of 91, i.e., 20 out of 50 of group 1 and 22 out of 41 of group 2) and 33% (30 of 91, i.e. 14 out of 50 of group 1 and 16 out of 41 of group 2) of the adenomas using DO-7 and CM1 antibody, respectively. High p53 expression (i.e. immunolabelling of more than 30% of the tumour cell nuclei) was found in 13.2% of the adenomas (12 of 91, i.e. three out of 50 of group 1 and nine out of 41 of group 2; P = 0.025 using the chi 2 test) using the DO-7 antibody, and in 6.6% of the cases (six of 91, i.e. two out of 50 of group 1 and four out of 41 of group 2) using the CM1 antibody. In carcinomas, 80% of the cases (i.e. 12 of 15) were found to express p53 protein with both antibodies. p53 immunoreactivity in colorectal adenomas increased with the degree of dysplasia: only five (17.8%) of the 28 adenomas with mild dysplasia were found to be DO-7 positive, while all of them remained CM1 negative. From the 50 adenomas exhibiting moderate dysplasia, 28 (56%) were DO-7 positive, and 22 (44%) were CM1 positive. Finally, from the 13 adenomas with severe dysplasia, nine (69.2%) and eight (61.5%) were found to be positive with the DO-7 and the CM1 antibody, respectively. Our results indicate that an increased number of group 2 adenomas express p53 protein, when compared with group 1 adenomas, and suggest that a strong correlation exists between p53 protein expression and the degree of dysplasia in colorectal neoplasms.

Activation of the receptor protein tyrosine kinase EphB4 in endometrial hyperplasia and endometrial carcinoma.

BACKGROUND: Members of the Eph family of tyrosine kinases have been implicated in embryonic pattern formation and vascular development; however, little is known about their role in the adult organism. We have observed estrogen-dependent EphB4 expression in the normal breast suggesting its implication in the hormone-controlled homeostasis of this organ. Since the endometrium is a similarly hormone dependent organ and endometrial carcinoma is thought to result from estrogenic stimulation, we have investigated EphB4 expression in normal human endometrium and during its carcinogenesis. PATIENTS AND METHODS: EphB4 expression was analyzed immunohistochemically in 26 normal endometrium specimens, 15 hyperplasias and 102 endometrioid adenocarcinomas and correlated with clinical and prognostic tumor characteristics. RESULTS: In normal endometrial tissue no EphB4 protein was detected. Strikingly, we observed a drastic increase (P <0.0001) in the number of EphB4 protein-expressing glandular epithelial cells in the majority of hyperplasias and carcinomas. Moreover, we found a statistically highly significant positive correlation between EphB4 expression and post-menopausal stage of the patient (P = 0.007). CONCLUSIONS: These findings indicate that in the endometrium, EphB4 is an early indicator of malignant development and, thus, EphB4 may represent a potent tool for diagnosis and therapeutic intervention.