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Nucleolar organizer region-associated proteins (AgNOR) and proliferating cell nuclear antigen (PCNA) have been studied by means of a silver staining technique and immunohistochemistry, in paraffin-embedded, gastrectomy specimens of 12 low-grade and 13 high-grade gastric MALT lymphomas respectively. A significant difference was found between the AgNOR count and PCNA index of low-grade lymphomas (mean AgNOR count 2.5 and mean PCNA index 8.33%) and high-grade lymphomas (mean AgNOR count 8.67 and mean PCNA index 49.7%). It is suggested that both methods are useful adjuncts to histopathology for the distinction between low and high grade gastric MALT lymphomas. We also found heterogeneity in AgNOR counts and PCNA index among individual cases of either low or high grade MALT gastric lymphomas. This suggests that the AgNOR count and PCNA index is helpful in the individual approach of the proliferation rate of each tumour, a parameter of potential importance for predicting the biological behaviour of the tumour and the prognosis of the disease. Pathol Res Pract

Epidermal growth factor (EGF) and its receptor (EGFr) constitute an important and well-characterized mitogenic system in various ectodermal tissues. We evaluated the expression of EGFr and examined possible EGFr gene alterations in 18 formalin-fixed, paraffin-embedded squamous cell lung carcinomas (SCLC) by an immunohistochemical assay, Southern blotting and differential polymerase chain reaction (DPCR). The immunohistochemical study employing the F4 and EGF-R1 monoclonal antibodies, directed against the intra- and extra-cellular portion of the receptor respectively, showed EGFr over-expression in 89% of the SCLC cases examined. All cases showed positive immunostaining for both antibodies, thus excluding the possibility of truncated receptors. In addition, analysis of the EGFr gene was carried out by Southern blotting and DPCR on paraffin extracted DNA from the same carcinoma cases. We found amplification of the EGFr gene in 5/18 (27%) SCLCs. All 5 positive cases showed EGFr over-expression, suggesting a possible correlation between the presence of EGFr gene amplification and over-expression of receptor protein. No correlation was observed among EGFr staining, EGFr gene amplification and differentiation of carcinomas. In addition, Southern blot analysis with HER-A2, a probe which hybridizes a sequence of the receptor's intracellular domain, revealed three novel EcoRI restriction fragment patterns. We suggest that these patterns correspond to EcoRI polymorphic sites of the receptor's tyrosine kinase domain.

A total of 366 consecutive modified radical mastectomy specimens were studied for determination of multicentricity. The authors found that 187 samples (49.1%) were multicentric. Ten specimens contained in situ carcinoma without an infiltrating component; eight of them were multicentric. Multicentricity was correlated with various laboratory and clinical features, including patient age, tumor size, histologic type of breast cancer, tumor grade, presence and values of estrogen and progesterone receptors, the amount of solid tissue in the breast, and the family history. The data were organized in eight independent dimensions, four ordinal and four cardinal. Correlation analysis was applied to a cross tabulation supplemented with other statistical tests. The authors found that the factors related to multicentricity were the age of the patient, the size and the histologic type of the tumor, levels of the progesterone receptors more than 50 fmol/mg of protein, and the amount of solid tissue in the breasts. Tumor grade, estrogen receptors levels, and family history were not related to multicentricity. It was concluded that multicentricity is a frequent property of breast cancer. It is more common in young and perimenopausal women. Multicentricity occurs in small tumors but is, more common in larger ones.

Summary Immunohistochemical study for epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) was performed on paraffin-embedded tissue specimens from 39 colorectal and 24 gastric carcinomas. The carcinomas were placed in one of the following 3 groups: group 1, neither EGF nor EGFR was stained (11 gastric and 21 colorectal carcinomas); group 2, either EGF or EGFR was stained (4 gastric and 4 colorectal carcinomas); and group 3, both EGF and EGFR were stained (9 gastric and 14 colorectal carcinomas). Compared with the carcinomas in groups 1 and 2, those in group 3 had significantly higher rates of lymph node spread and serosal invasion of the gastrointestinal wall. In contrast, no significant differences were found between the EGF and/or EGFR expression and histological differentiation of carcinomas. These results suggest that gastrointestinal carcinomas expressing both EGF and EGFR display pathological features of more aggressive disease. Furthermores the synchronous expression of EGF and EGFR indicates that these carcinomas may regulate their growth by an autocrine and/or paracrine mechanism.

BACKGROUND AND AIM: Given that anorectal human papillomavirus (HPV) infection has been related to anal intraepithelial neoplasia (AIN) and rectal cancer, we conducted this study to evaluate the role of cytology of anal smears in the diagnosis of intraanal disease and related AIN and to correlate it to HPV genotypes. METHOD: A total of 72 patients (58 males and 14 females) with perianal warts underwent anoscopy with biopsies and anal cytologic examination. Cytology was carried out for the identification of any dysplasia according to the Bethesda system. All specimens were examined with polymerase chain reaction (PCR) for HPV DNA identification. Exclusion criteria included immunosuppression and high-grade squamous intraepitheliel lesion (HGSIL) or SCC in anal specimens. RESULTS: Seven patients were excluded from the study. Intraanal warts were detected with anoscopy in 57 out of 65 patients, whereas histology showed HPV infection in 56 out of 65 patients and cytology was positive in 52 out of 65 low-grade squamous intraepitheliel lesion (LGSIL) patients. In 43 out of 52 positive patients, simple HPV infection was detected whereas in 9 out of 52 positive patients AIN I. HPV DNA was detected in 51 out of 65 patients, whereas 3 specimens were characterized as invalids. In the majority, HPV 6 could be identified (39/48, 81%), whereas HPV 16 was detected in 4 patients (4/48, 8.3%). One fourth of the positive patients had been infected with more than 1 HPV types (13/48, 27%). Cytology presented a sensitivity 87.5% and specificity 67% in comparison with the histology. CONCLUSIONS: Cytology is highly sensitive in the diagnosis of intraanal warts comparable with histopathology. The combination of the 3 examinations (anoscopy, cytology, and PCR HPV typing) improves diagnostic accuracy and offers a global picture of the anorectal HPV disease.

p53 (gene location: 17p13.1) overexpression is a common event in pancreatic ductal adenocarcinoma (PDAC), a highly aggressive malignant neoplasm. Although specific mechanisms of p53 gene deregulation have been identified, correlation between p53 expression and chromosome 17 gross numerical imbalances (aneuploidy) are under investigation.Using tissue microarray technology, 60 paraffin-embedded tissue samples of histologically confirmed primary PDACs were cored and re-embedded to the final recipient block. Immunohistochemistry (IHC) for p53 expression and chromogenic in situ hybridization (CISH) for chromosome 17 numerical alterations were performed. Digital image analysis was applied for p53 expression levels evaluation (Nuclear Labelling Index-NLIs).p53 overexpression was detected in 38/60 (63.3%), whereas chromosome 17 aneuploidy was observed in 21/60 (35%) cases, respectively. Polysomy was identified in 19 cases, whereas monosomy in 2 of them. p53 overall expression was strongly correlated to the stage of the examined tumors (p=0.02). Chromosome aneuploidy was not associated to tumors' stage and grade (p=0.42, p=0.71, respectively). Although overall chromosome 17 centromeric imbalances were not correlated with p53 overexpression (p=0.32), both cases with monosomy demonstrated high expression levels.p53 overexpression combined with chromosome 17 numerical imbalances characterizes a significant proportion of PDACs. Because commercially available antip53 antibodies detect mutant and also wild-type protein expression levels, chromosome 17 monosomy maybe is a gross genetic criterion for discriminating them due to point mutation that frequently affects the remaining allele.

Adult intussusception is a rare clinical entity accounting for 5% of all intussusceptions. Symptoms and signs are often vague and non-specific making a preoperative diagnosis difficult. The purpose of this study is to present a rare case of a jejuno-jejunal intussusception due to primary intestinal non-Hodgkin's lymphoma in a patient with an unusual clinical course.A 78-year-old man presented with a 1-month history of abdominal pain, nausea, epigastric fullness, and weight loss. Computed tomography scan and ultrasonography findings were suggestive of small bowel intussusception.Laparotomy revealed a jejuno-jejunal intussusception caused by a primary B cell non-Hodgkin's lymphoma 20 cm distal to the ligament of Treitz. Resection without prior reduction was performed. The patient refused postoperative adjuvant chemotherapy. Seven months later, he presented with upper gastrointestinal bleeding, and the diagnostic evaluation revealed gastric infiltration of large B cell non-Hodgkin's lymphoma. Despite chemotherapy, he died of disseminated progressive disease 7 months later.Adult jejuno-jejunal intussusception due to primary non-Hodgkin's lymphoma is a rare clinical entity. A high index of suspicion is needed as symptoms and signs are not pathognomonic. Appropriate investigations can lead to a prompt preoperative diagnosis. Resection without prior reduction is the treatment of choice. Our patient's refusal of postoperative adjuvant chemotherapy likely resulted in relapse of the disease in another part of the gastrointestinal tract.

Estrogen receptor alpha–encoded by ESR1 gene–overexpression correlates with prognosis and response to specific chemotherapy in breast adenocarcinoma cases. Mechanisms of ESR-1 deregulation in carcinomas remain under investigation. To analyze ESR1 in carcinomas of different histogenesis. Using tissue microarray technology, 172 primary carcinomas including breast ductal adenocarcinomas (n = 60), hepatocellular carcinomas (n = 52), and colon adenocarcinomas (n = 60) were cored and re-embedded in three paraffin blocks. Initial diagnosis was based on liquid based cytology (LiquiPrep/ThinPrep). Immunohistochemistry and fluorescence in situ hybridization were performed. Quantitative evaluation of ER-a protein levels was assessed by applying digital image analysis. ER-a overexpression was observed in 41/60 (68.3%), 23/52 (44.2%) and 4/60 (6.6%) cases, respectively. ESR1 gene multiple copies were confirmed in 13/60 (21.6%) breast adenocarcinomas, but high amplification only in 8/13 (62.8%). Allelic absence was identified in 3/52 (5.7%) hepatocellular carcinomas, whereas colon adenocarcinomas demonstrated gene gains in 5/60 (8.3%) cases referred to chr 6 aneuploidy and not to amplification. ER-a overall expression was associated strongly to ESR1 gene copies only in breast carcinoma (P = 0.036). ESR-1 gene overexpression happens frequently in breast cancer, but only a subset of them are high amplified cases correlated to increased response rates in hormonal therapy (tamoxifen). Absence of this mechanism in hepatocellular and colon carcinomas maybe is a negative factor for applying this therapy. This is a pattern of histo-genetic depended targeted therapeutic strategy.

Overexpression of Epidermal Growth Factor Receptor (EGFR) and also of cell cycle control proteins, such as cyclin D1 is a frequent event in squamous cell carcinoma of the larynx (LSSC). Our aim was to correlate their protein levels with telomerase catalytic subunit (h-TERT) expression. Using tissue microarray technology, fifty-five paraffin embedded histologically confirmed primary LSSCs and also ten dysplastic lesions were cored at a diameter of 1.5 mm. Immunohistochemistry (IHC) was performed by the use of anti-EGFR, anti-cyclin D1, and anti-h TERT monoclonal antibodies. Chromogenic in situ hybridization (CISH) analysis was also applied using EGFR gene and chromosome 7 probes, respectively. EGFR, cyclin D1 and h-TERT protein overexpression was observed in 48/55 (87.2%), 19/55 (34.5%) and 21/55 (38.1%) carcinoma cases, respectively. EGFR protein expression was statistically associated with grade (P = 0.01), and also with stage (P = 0.001) of the examined tumors. Borderline statistical significance was assessed correlating overall cyclin D1 expression to h TERT expression (P = 0.06). Simultaneous up regulation of the three proteins was established in 7/55 (12.7%) cases, correlated to the stage of the tumors (P = 0.05). EGFR gene amplification was observed in 7/65 (10.7%) carcinomas and dysplasias, whereas chromosome 7 aneuploidy was detected in 4/65 (6.1%) of those cases.Simultaneous up regulation of EGFR, cyclin D1 and h TERT proteins correlates with advanced stage in LSCC. EGFR gene amplification and not only protein over expression maybe is the eligible criterion for targeted therapeutic strategies in those patients.

Summary Paraffin-embedded tissue specimens from 46 cases ofHodgkin's disease (HD) were studied by in situ hybridization with biotinylated probes for the presence of EBV genomes. EBV specific DNA sequences were detected in the nuclei of Reed-Sternberg (RS) and Hodgkin cells (H), in 14 of these 46 cases. There was no correlation between positive hybridization and morphological subtype or site of tumor. By demonstrating an exclusive localization of the viral DNA in the tumor cells of HD, our study adds to the growing body of evidence to suggest an involvement of EBV in the pathogenesis of at least some cases of HD.

Retroperitoneal liposarcoma is a rare tumor that may grow to a considerable size before causing clinical symptoms. Mixed-type retroperitoneal liposarcoma is a very rare clinical entity.We herein describe a rare case of a 54-year-old female who was diagnosed with a giant retroperitoneal liposarcoma arising from the right perinephric space.Radical nephrectomy and right salpingo-oophorectomy were necessary to achieve complete tumor excision. Histological examination revealed a mixed-type liposarcoma consisting of well-differentiated and pleomorphic elements and deep invasion into the renal parenchyma, which is quite infrequent. The patient was well and disease-free 12 months after surgery.Mixed-type retroperitoneal liposarcoma is a rare tumor. Aggressive surgical resection of the tumor together with adjacent structures, if necessary, is the mainstay of treatment.

KEYWORDS: Matrix metalloproteinases (MMPs) are endopeptidases considered to participate in the transient invasive property of trophoblastic cells during embryo implantation and placentation. The same molecules play an important role in the invasive and metastatic potential of cancer cells. The aim of this study was to compare the immunohistochemical expression of MMP2, 7and 9between clearly invasive carcinomas and "in situ" trophoblast invasion in an effort to illuminate their distinct roles in uncontrolled and controlled invasion. We performed an immunohistochemical analysis of 45 clearly invasive carcinomas of various organs (colorectal, gastric, breast, pulmonary, renal) and 40 first trimester gestation specimens (before the 9th week of gestation). The markers expression was evaluated semiquantitavely, seperately in cancer parenchymal and gestational trophoblastic cells as well as cancer stromal and decidual cells, according to apercentage scale (0 %, 50% of cells) and according to staining intensity (0, +, ++, +++). MMP9 was expressed more often in the malignant parenchymal as well as in the malignant stromal component of carcinomas than in the trophoblastic (p=0, 0118) and decidual (p=0,017) component of gestations respectively. Although all carcinomas and almost all gestation specimens stained for MMP2 and MMP7, the immunostaining for both molecules was statistically more extensive and intense in trophoblasts and decidual cells by comparison to cancerous elements. In conclusion, although there seems to be adirect link between cancer invasion and MMP9 immunohistochemical expression, the role of MMP2 and MMP7 appears to be more complicated underlining the complexity of the mechanisms involved in cancer spreading.

Since its discovery in 1997, survivin has garnered significant interest due to its putative role as an inhibitor of apoptosis. Few studies have investigated the immunohistochemical status of survivin in urothelial bladder carcinoma. The subcellular localization of survivin (nuclear, cytoplasmic) and its differential predictive value is a parameter that previous studies have almost ignored. The aim of this study was to investigate the expression pattern of survivin in order to determine its potential prognostic significance.Archival tumor tissue from 80 patients with urothelial carcinoma were analysed by immunohistochemistry.Nuclear and cytoplasmic positive scores of 61.25% (49/80) and 22.5% (18/80), respectively were found. Nuclear positive staining correlated strongly with increased grade (p=0.001), stage (p=0.039) and the probability of tumor recurrence (p=0.029). No relationship was found between the cytoplasmic survivin level and the clinicopathological parameters. Nuclear expression was identified as a significant independent predictor of relapse-free survival (p=0.016).Nuclear expression of survivin reflects an adverse disease outcome.

This study was conducted to evaluate the quantitative assessment of HER2/neu immunohistochemical expression in urothelial bladder cancer in order to determine its prognostic significance.Archival tumor tissue from 80 patients with primary urothelial carcinoma were analysed for HER2/neu immunohistochemical expression. A highly reproducible standardized procedure on a Bond-X automated slide stainer was used.HER2 protein was overexpressed in 41 of 80 patients (51.25%), demonstrating an increase in the expression rate corresponding to progressively advanced tumor stage (p=0.032) and tumor grade (p=0.0001). Kaplan-Meier analyses showed that positive membranous expression of HER2/neu was not associated with an increased probability of tumor recurrence (p=0.362). In contrast, HER2 scores correlated strongly with specific survival probability (p=0.002) and overall survival (p=0.025). Multivariate analysis revealed that only stage was an independent predictor of specific survival (p=0.016). HER2 expression was an independent predictor of specific survival with borderline statistical significance (p=0.08).HER2 overexpression represents a prognostic factor for adverse disease outcome.

Although Epidermal Growth Factor Receptor (EGFR) over expression is a frequent event in colon adenocarcinoma (CA), identification of EGFR gene deregulation mechanisms--combined to k-ras mutations--remains the basic criterion for rational application of anti-EGFR targeted therapeutic strategies.To detect EGFR gene numerical alterations in CA based on a combination of intra-operative imprints and the corresponding tissue microarrays.60 paraffin embedded primary CAs were cored at 1.5 mm diameter and transferred to the final microarray block. Chromogenic in situ hybridization (CISH) was performed using EGFR gene and chromosome 7 centromeric probes in the tissue microarray and also in the corresponding intra-operative imprints.CISH analysis detected 4/60 (6.6%) EGFR gene amplified cases, whereas chromosome 7 aneuploidy was identified in 11/60 (18.3%) cases. Significant association was established by correlating stage to chromosome 7 (p=0.024). A high value of concordance (kappa=1) was observed comparing overall gene status based on the tissue cores and the corresponding imprints, whereas EGFR/CEN 7 copies were more numerous in imprints than in tissue microarrays (p=0.03).Intra-operative imprint cytology provides accurate and fast results in detecting EGFR gene/chromosome 7 centromeric signals by CISH due to the nuclear integrity and monolayer formation of the examined cells. Based on this molecular analysis, gastroenterologists and oncologists can handle those patients in a rational way regarding targeted therapies. Furthermore, chromosome 7 aneuploidy is associated with a more advanced stage in CA.

Heterotopic mesenteric ossification is a rare disorder. Only a few cases have been reported in the literature, associated with previous abdominal surgery or trauma. We report a case of heterotopic mesenteric ossification leading to abdominal sepsis, after abdominal operation for recurrent gastric bleeding, due to gastric antral vascular ectasia (GAVE), otherwise called "watermelon stomach", another rare disorder.

BackgroundEpidermal growth factor receptor (EGFR) overexpression is observed in significant proportions of non-small cell lung carcinomas (NSCLC). Furthermore, overactivation of vascular endothelial growth factor (VEGF) leads to increased angiogenesis implicated as an important factor in vascularization of those tumors.Patients and MethodsUsing tissue microarray technology, forty-paraffin ( n = 40) embedded, histologically confirmed primary NSCLCs were cored and re-embedded into a recipient block. Immunohistochemistry was performed for the determination of EGFR and VEGF protein levels which were evaluated by the performance of computerized image analysis. EGFR gene amplification was studied by chromogenic in situ hybridization based on the use of EGFR gene and chromosome 7 centromeric probes.ResultsEGFR overexpression was observed in 23/40 (57.5%) cases and was correlated to the stage of the tumors ( p = 0.001), whereas VEGF was overexpressed in 35/40 (87.5%) cases and was correlated to the stage of the tumors ( p = 0.005) and to the smoking history of the patients ( p = 0.016). Statistical significance was assessed comparing the protein levels of EGFR and VEGF ( p = 0.043, k = 0.846). EGFR gene amplification was identified in 2/40 (5%) cases demonstrating no association to its overall protein levels ( p = 0.241), whereas chromosome 7 aneuploidy was detected in 7/40 (17.5%) cases correlating to smoking history of the patients ( p = 0.013).ConclusionsA significant subset of NSCLC is characterized by EGFR and VEGF simultaneous overexpression and maybe this is the eligible target group for the application of combined anti-EGFR/VEGF targeted therapies at the basis of genetic deregulation (especially gene amplification for EGFR).

In the present study, we carried out a comparative immunohistochemical analysis of aurora-A and aurora-B expression in 40 patients with primary glioblastomas, and attempted to identify any associations with Ki-67 index and the patients' clinical features. The impact of various treatment modalities and proliferative activity on patient outcome was also assessed. Immunohistochemistry was carried out using formalin-fixed and paraffin-embedded tissue sections. Aurora-A expression was higher in tumors with high Ki-67 expression (p=0.01) and was positively, though marginally, related to aurora-B expression (p=0.085). Aurora-B expression was not linked to Ki-67 expression (p=0.182). Lower aurora-A immunohistochemical expression, chemotherapy administration, and tumor localization in one lobe of the brain implied a greater probability of patient survival in univariate analysis (p=0.044, p=0.008, p=0.041, respectively). Ki-67 and aurora-B immunoreactivities were not associated with patient survival (p=0.918 and p=0.539, respectively). To our knowledge, for the first time, the association between aurora-A and aurora-B expression, the correlation of aurora-A with Ki-67 index, and the prognostic impact of aurora-A expression were assessed in glioblastomas. Although we addressed a prognostic connotation of aurora-A, we presume that aurora-A and aurora-B play a complicated role within glioblastomas. Further examinations of larger series are required, so that definite conclusions can be drawn.

Background Recent evidence has underscored the role of hypoxia and angiogenesis in the pathogenesis of idiopathic fibrotic lung disease. Inhibitor of growth family member 4 (ING4) has recently attracted much attention as a tumor suppressor gene, due to its ability to inhibit cancer cell proliferation, migration and angiogenesis. The aim of our study was to investigate the role of ING4 in the pathogenesis of pulmonary fibrosis both in the bleomycin (BLM)-model and in two different types of human pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF) and cryptogenic organizing pneumonia (COP). Methods Experimental model of pulmonary fibrosis was induced by a single tail vein injection of bleomycin in 6- to 8-wk-old C57BL/6mice. Tissue microarrays coupled with qRT-PCR and immunohistochemistry were applied in whole lung samples and paraffin-embedded tissue sections of 30 patients with IPF, 20 with COP and 20 control subjects. Results A gradual decline of ING4 expression in both mRNA and protein levels was reported in the BLM-model. ING4 was also found down-regulated in IPF patients compared to COP and control subjects. Immunolocalization analyses revealed increased expression in areas of normal epithelium and in alveolar epithelium surrounding Masson bodies, in COP lung, whereas showed no expression within areas of active fibrosis within IPF and COP lung. In addition, ING4 expression levels were negatively correlated with pulmonary function parameters in IPF patients. Conclusion Our data suggest a potential role for ING4 in lung fibrogenesis. ING4 down-regulation may facilitate aberrant vascular remodelling and fibroblast proliferation and migration leading to progressive disease.

Topoisomerase II alpha (Topo IIa gene location 17q21) is a nucleic enzyme involved in the DNA replication, transcription and chromosome topological formation. Topo IIa inhibition strategies include specific chemotherapeutic agents such as anthracyclines. Our aim was to investigate potential protein alterations of the enzyme comparing them to ki 67 proliferation marker expression in papillary thyroid carcinoma (PTC).Using tissue microarray (TMA) technology, 50 specimens consisting of histologically confirmed PTCs (n=20), multi-nodular goiters (n=20) and also normal thyroid epithelia (n=10) were cored and re-embedded in the final paraffin block. Immunohistochemical analysis was performed using monoclonal anti-Topo IIa and anti-ki 67 (MIB-1) antibodies. Digital image analysis assay was also applied for the evaluation of the protein expression results (Nuclear Labeling Index-NLI).Topo IIa and ki 67 proteins were overexpressed in 4/20 (20%) and 14/20 (70%) cases, respectively. Concerning multi-nodular goiters, overexpression was observed in 2/20 and 4/20 specimens, respectively. Statistical association was assessed correlating ki 67 expression to pathology type, capsular invasion and also to vascular infiltration (p=0.001, p=0.008, and p=0.012, respectively). Topo IIa protein expression was strongly correlated only to capsular invasion (p=0.004). Overall expression of the examined markers demonstrated a medium concordance (kappa=0.27), but a strong association (p=0.001).Topo IIa and also ki 67 overexpression are correlated to an aggressive phenotype in PTC. Topo IIa overexpression maybe is a reliable marker for a rational application of targeted chemotherapeutic strategies in some subgroups of patients.

In the present study, we carried out a comparative immunohistochemical analysis of aurora-A and aurora-B expression in 40 patients with primary glioblastomas, and attempted to identify any associations with Ki-67 index and the patients' clinical features. The impact of various treatment modalities and proliferative activity on patient outcome was also assessed. Immunohistochemistry was carried out using formalin-fixed and paraffin-embedded tissue sections. Aurora-A expression was higher in tumors with high Ki-67 expression (p=0.01) and was positively, though marginally, related to aurora-B expression (p=0.085). Aurora-B expression was not linked to Ki-67 expression (p=0.182). Lower aurora-A immunohistochemical expression, chemotherapy administration, and tumor localization in one lobe of the brain implied a greater probability of patient survival in univariate analysis (p=0.044, p=0.008, p=0.041, respectively). Ki-67 and aurora-B immunoreactivities were not associated with patient survival (p=0.918 and p=0.539, respectively). To our knowledge, for the first time, the association between aurora-A and aurora-B expression, the correlation of aurora-A with Ki-67 index, and the prognostic impact of aurora-A expression were assessed in glioblastomas. Although we addressed a prognostic connotation of aurora-A, we presume that aurora-A and aurora-B play a complicated role within glioblastomas. Further examinations of larger series are required, so that definite conclusions can be drawn. © 2009 Elsevier GmbH. All rights reserved.

Background: Despite intense research efforts, the aetiology and pathogenesis of idiopathic pulmonary fibrosis remain poorly understood. Gelsolin, an actin-binding protein that modulates cytoskeletal dynamics, was recently highlighted as a likely disease modifier through comparative expression profiling and target prioritisation. Methods: To decipher the possible role of gelsolin in pulmonary inflammation and fibrosis, immunocytochemistry on tissue microarrays of human patient samples was performed followed by computerised image analysis. The results were validated in the bleomycin-induced animal model of pulmonary inflammation and fibrosis using genetically-modified mice lacking gelsolin expression. Moreover, to gain mechanistic insights into the mode of gelsolin activity, a series of biochemical analyses was performed ex vivo in mouse embryonic fibroblasts. Results: Increased gelsolin expression was detected in lung samples of patients with idiopathic interstitial pneumonia as well as in modelled pulmonary inflammation and fibrosis. Genetic ablation of gelsolin protected mice from the development of modelled pulmonary inflammation and fibrosis attributed to attenuated epithelial apoptosis. Conclusions: Gelsolin expression is necessary for the development of modelled pulmonary inflammation and fibrosis, while the caspase-3-mediated gelsolin fragmentation was shown to be an apoptotic effector mechanism in disease pathogenesis and a marker of lung injury.

Heterotopic mesenteric ossification is a rare disorder. Only a few cases have been reported in the literature, associated with previous abdominal surgery or trauma. We report a case of heterotopic mesenteric ossification leading to abdominal sepsis, after abdominal operation for recurrent gastric bleeding, due to gastric antral vascular ectasia (GAVE), otherwise called "watermelon stomach", another rare disorder. J Gastrointestin Liver Dis

Although Epidermal Growth Factor Receptor (EGFR) overexpression is observed frequently in hepatocellular carcinomas (HCC), specific gene deregulation mechanisms remain unknown. Our aim was to investigate the prognostic significance of the combined protein and gene/chromosome 7 numerical alterations. Using tissue microarray technology, thirty-five (n=35) paraffin embedded histologically confirmed HCCs were cored and re-embedded in a paraffin block. Immunohistochemistry was performed for the determination of EGFR protein levels and evaluated by the performance of digital image analysis. Chromogenic in situ hybridization was also performed based on the use of EGFR gene and chromosome 7 centromeric probes, respectively. EGFR overexpression was observed in 26/35 (74.2%) cases and was correlated to the grade of the tumors and also to the history of the patients (p=0.013, p=0.036, respectively). Numerical alterations regarding gene and chromosome 7 were identified in 4/35 (11.4%) and 12/35 (43.2%) cases associated to the grade of the tumors (p=0.019, p=0.001, respectively) and to the survival rate of the patients (p=0.037, p=0.001, respectively). EGFR overall expression was also correlated to the gene copies (p=0.020). EGFR gene numerical alterations -although rare- and also chromosome 7 aneuploidy maybe affect prognosis in HCC patients. To our knowledge this is the first chromogenic in situ hybridization analysis based on tissue microarrays in hepatocellular carcinoma. © 2009 Arányi Lajos Foundation.

A silver colloid technique to identify nucleolar organizer region associated protein (AGNORs) has been applied to paraffin sections in a total of 43 endometrial hyperplasias (24 adenomatous and 19 adenocystic) 26 endometrial carcinomas and 22 normal endometria (11 of proliferative and 11 of secretory phase). A morphometric analysis of highly magnified photographic images of AGNORs in light microscopic preparations was performed. Malignant tumor cells showed significantly higher AGNOR numbers, maximum diameter and mean area compared with normal and hyperplastic endometrium, with the exception of adenocystic hyperplasia whose Dmax and mean area were significantly larger. Regarding the distribution pattern of AGNOR dots in the cases studied, it was found that normal and hyperplastic endometrium had a mainly clustered distribution while endometrial adenocarcinomas revealed a scattered one. The significant differences observed in the number of AGNORs, their size and mean area between benign and malignant endometrial epithelia suggest that the AG-NOR staining technique is of diagnostic importance in distinguishing between these two groups.

Journal URL: http://www3.interscience.wiley.com/journal/1130/home

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Journal URL: http://www.sciencedirect.com/science/journal/03440338

p53 protein promotes apoptosis, whereas Bcl-2 family proteins have an antiapoptotic function. This study determines the predictive value of selected clinical and histopathological factors in correlation with the expression of p53, Bcl-2, and Bcl-X(L) proteins in esophageal squamous cell carcinomas (SCCs). Paraffin-embedded sections from 19 surgically resected primary esophageal SCCs were examined by immunohistochemistry. p53 expression was related to degree of tumor differentiation (P = 0.044). Bcl-2 expression was associated with regional lymph node metastasis (P = 0.053), whereas Bcl-X(L) expression was correlated with distant metastasis (P = 0.060) and with the expression of Bcl-2 protein (P = 0.068). p53 and Bcl-2 family proteins may help to estimate the properties of esophageal SCCs and provide useful information to the oncologist for the selection of patients for intensive combined therapy modalities with curative intention or for palliative therapy.

Although Epidermal Growth Factor Receptor (EGFR) overexpression is observed frequently in hepatocellular carcinomas (HCC), specific gene deregulation mechanisms remain unknown. Our aim was to investigate the prognostic significance of the combined protein and gene/chromosome 7 numerical alterations. Using tissue microarray technology, thirty-five (n = 35) paraffin embedded histologically confirmed HCCs were cored and re-embedded in a paraffin block. Immunohistochemistry was performed for the determination of EGFR protein levels and evaluated by the performance of digital image analysis. Chromogenic in situ hybridization was also performed based on the use of EGFR gene and chromosome 7 centromeric probes, respectively. EGFR overexpression was observed in 26/35 (74.2%) cases and was correlated to the grade of the tumors and also to the history of the patients (p = 0.013, p = 0.036, respectively). Numerical alterations regarding gene and chromosome 7 were identified in 4/35 (11.4%) and 12/35 (43.2%) cases associated to the grade of the tumors (p = 0.019, p = 0.001, respectively) and to the survival rate of the patients (p = 0.037, p = 0.001, respectively). EGFR overall expression was also correlated to the gene copies (p = 0.020). EGFR gene numerical alterations -although rare- and also chromosome 7 aneuploidy maybe affect prognosis in HCC patients. To our knowledge this is the first chromogenic in situ hybridization analysis based on tissue microarrays in hepatocellular carcinoma.

Our knowledge regarding the biology of the gastroesophageal junction adenocarcinomas is still incomplete. Paraffin-embedded sections from 31 surgically resected primary cardia adenocarcinomas were examined by immunohistochemistry. Statistical analysis showed that Bcl-2 expression was significantly correlated with the age of the patients (P = 0.043), whereas Bcl-X(L) expression was inversely correlated with Bcl-2 expression (P = 0.021). An inverse correlation of high statistical significance was also found between p53 and Bcl-2 expression (P = 0.000). Fas expression was highly correlated with tumor stage (P = 0.006), degree of differentiation (P = 0.044), and the stage of the disease (P = 0.029). A significant correlation was also observed between the expression levels of WAF1 and Fas (P = 0.037), Fas and Bcl-X(L) (P = 0.018), and WAF1 and p53 (P = 0.018). These proteins may contribute to the estimation of the properties of adenocarcinomas of the gastroesophageal junction, facilitating prognosis of cancer patients treated by multimode therapy.

Gastrointestinal stromal tumors (GISTs) are rare mesenchymal neoplasms of the digestive tract. Synchronous occurrence of a gastrointestinal stromal tumor with a tumor of different histogenesis is very rare and has been documented in the literature mainly in case reports. We present the case of a 78-year old female patient who underwent surgery for an advanced gastric carcinoma during which a gastric stromal tumor was incidentally discovered. A review of the literature is also conducted on the extremely rare synchronous occurrence of malignant tumors of different histogenesis in the stomach.

p53 protein promotes apoptosis, whereas Bcl-2 family proteins have an antiapoptotic function. This study determines the predictive value of selected clinical and histopathological factors in correlation with the expression of p53, Bcl-2, and Bcl-X(L) proteins in esophageal squamous cell carcinomas (SCCs). Paraffin-embedded sections from 19 surgically resected primary esophageal SCCs were examined by immunohistochemistry. p53 expression was related to degree of tumor differentiation (P = 0.044). Bcl-2 expression was associated with regional lymph node metastasis (P = 0.053), whereas Bcl-X(L) expression was correlated with distant metastasis (P = 0.060) and with the expression of Bcl-2 protein (P = 0.068). p53 and Bcl-2 family proteins may help to estimate the properties of esophageal SCCs and provide useful information to the oncologist for the selection of patients for intensive combined therapy modalities with curative intention or for palliative therapy. Int Surg

Our knowledge regarding the biology of the gastroesophageal junction adenocarcinomas is still incomplete. Paraffin-embedded sections from 31 surgically resected primary cardia adenocarcinomas were examined by immunohistochemistry. Statistical analysis showed that Bcl-2 expression was significantly correlated with the age of the patients (P = 0.043), whereas Bcl-X(L) expression was inversely correlated with Bcl-2 expression (P = 0.021). An inverse correlation of high statistical significance was also found between p53 and Bcl-2 expression (P = 0.000). Fas expression was highly correlated with tumor stage (P = 0.006), degree of differentiation (P = 0.044), and the stage of the disease (P = 0.029). A significant correlation was also observed between the expression levels of WAF1 and Fas (P = 0.037), Fas and Bcl-X(L) (P = 0.018), and WAF1 and p53 (P = 0.018). These proteins may contribute to the estimation of the properties of adenocarcinomas of the gastroesophageal junction, facilitating prognosis of cancer patients treated by multimode therapy. Int Surg

Background: The prognostic value of vascular endothelial growth factor (VEGF) expression in colorectal cancer is still unclear, as shown by the discordant results still reported in the literature. The aim of the study was to examine the expression of VEGF in colorectal adenocarcinomas and investigate its prognostic relevance. Materials and methods: VEGF expression was investigated by immunohistochemistry performed on tissue microarrays, in a series of 117 colorectal cancer specimens. The VEGF staining intensity in the cytoplasm of tumor cells was quantified using a semi-automated computerized image analysis and correlated with various clinicopathological characteristics and survival. Results: All tumors evaluated showed expression of VEGF, which were further divided into 49 high expression and 68 low expression tumors by their staining intensity. In tumors with lymph node metastasis, the intensity of staining of VEGF was more intense than in those without (P < 0.0001). Furthermore, the intensity of VEGF staining was more intense in Stage III tumors than those in Stage I/II (P < 0.0001). The mean number of involved lymph nodes in tumors with high VEGF staining intensity was significantly greater than in those with low staining intensity (P = 0.031). Survival analysis showed a significant correlation between high levels of VEGF staining intensity and poor disease-specific survival (P < 0.0001), with independent prognostic significance in multivariate analysis (RR = 3.5, P < 0.0001). In addition, patients with Stage II disease and high staining intensity of VEGF had a significantly worse disease-specific survival than those with low staining intensity (P = 0.001). Conclusions: VEGF expression in colorectal cancer seems to be an independent prognostic marker of tumor behavior and may be useful to identify patients with unfavorable clinical outcome. © 2008 Elsevier Inc. All rights reserved.

Aim: Topoisomerases represent a class of nucleic enzymes involved in the DNA replication, transcription and also chromosome topological formation. Topoisomerase IIa (Topo Iia - gene location 17q21) inhibition promotes cell death and for this reason, it is a target for specific chemotherapy (anthracyclines, podophyllotoxines). Our aim was to investigate the potential prognostic significance of its protein expression in squamous cell carcinomas of the tongue (TSCCs) based on a quantitative, digital image analysis method. Materials and methods: We analysed immunohistochemically eighty-seven (n = 87) archival, primary TSCCs using a monoclonal anti-Topo IIa antibody. A computerised image analysis assay was applied for the evaluation of the results [nuclear labellingindex(NLI)]. Survival analysiswasalso performed. Results: Topo IIa protein was overexpressed in 48/87 (55.1%) cases. High NLI values were detected in 17/48 (35.4%), whereas moderate levels of expression were detected in 31/48 (64.6%) cases. Statistical significance was not assessed correlating overall protein expression to grade or stage of the examined tumours (P = 0.88, P = 0.73, respectively), but only to the anatomical region (P = 0.04). Cox regression analysis showed that Topo IIa and also the size of the tumours were strongly associated to the survival of the patients (P = 0.01, P = 0.02, respectively). Conclusions: Topo IIa protein overexpression is a frequent event in TSCCs. Topo IIa may be used as a prognostic factor and also as a basis for targeted chemotherapy strategies in TSCCs. Furthermore, digital image analysis improves the quantification of immunohistochemical stains in an accurate and fast way. © 2008 The Authors. Journal compilation © 2008 Blackwell Munksgaard.

Deregulation of apoptotic pathways in cutaneous malignant melanoma appears to be correlated with chemoresistance and poor prognosis. Furthermore, telomerase (especially h-TERT) expression induces proliferation and also represents a potential target for vaccination regarding some types of malignancies.Using tissue microarrays (TMA) technology, 25 paraffin-embedded tissue samples of histologically confirmed malignant melanomas were cored at a diameter of 2 mm and re-embedded into one recipient block (final TMA density 24/25-96%). Immunohistochemistry (IHC) was performed by the use of anti-bcl-2, anti-caspase 3, anti-caspase 8 and anti- h-TERT antibodies. Protein expression levels were evaluated using a computerized image analysis system (CIA). SPSS (chi square test and inter-rater kappa) was used for statistical analysis.Strong protein expression was observed in 1/24 (4.1%), 1/24 (4.1%), 2/24 (8.2%), and 4/24 (16.4%) cases regarding h-TERT, caspase 3, caspase 8 and bcl-2, respectively. Moderate was observed in 7/24 (29.1%), 8/24 (32.2%), 5/24 (20.2%), and 8/24(32.2%) cases, whereas reduced or absent expression demonstrated 16/24 (65%), 15/24 (60.2%), 17/24 (68.5%), and 12/24 (50 %) cases. Statistical significance was assessed correlating age to caspase 3 (p=0.05), Breslow's thickness to telomerase (p=0.013) and to bcl-2 (p=0.053), Clark's level to telomerase (p=0.008) and to bcl-2 (p=0.022), and finally ulceration to telomerase expression (p=0.007).bcl-2 and telomerase expression are correlated to critical parameters of malignant melanoma, affecting its biological behavior. Furthermore, downregulation of proteins such as caspases 3/8, which normally induce apoptosis, is perhaps associated with resistance of the applied chemotherapeutic strategies in this type of malignancy.

BACKGROUND: Peritoneal mesothelioma is a rare disease associated with poor prognosis. Acute abdomen as the first presentation is an extremely rare occurrence. We report an exceptional case of a patient who was found to have a jejunal perforation due to infiltration of peritoneal mesothelioma. METHODS: A 62-year-old man was admitted with clinical signs of peritonitis. Computerized tomographic scans showed a mass distal to the ligament of Treitz, thickening of the mesentery and a small amount of ascites. RESULTS: Emergency laparotomy revealed a perforated tumor 15 cm distal to the ligament of Treitz and diffuse peritoneal disease. Segmental small bowel resection and suboptimal cytoreduction were performed. Histopathology and immunohistochemistry showed infiltration of malignant mesothelioma. During the postoperative period pleural mesothelioma was also diagnosed. Despite adjuvant chemotherapy, the patient died of disseminated progressive disease 7 months after surgery. CONCLUSIONS: Peritoneal mesothelioma is a rare malignancy with grim prognosis. Small bowel involvement is a poor prognostic indicator. Our case of a small bowel perforation due to direct infiltration by peritoneal mesothelioma appears to be the first reported in the English literature.

Despite intense research efforts, the etiology and pathogenesis of idiopathic pulmonary fibrosis remain poorly understood.To discover novel genes and/or cellular pathways involved in the pathogenesis of the disease.We performed expression profiling of disease progression in a well-characterized animal model of the disease. Differentially expressed genes that were identified were compared with all publicly available expression profiles both from human patients and animal models. The role of hypoxia-inducible factor (HIF)-1alpha in disease pathogenesis was examined with a series of immunostainings, both in the animal model as well as in tissue microarrays containing tissue samples of human patients, followed by computerized image analysis.Comparative expression profiling produced a prioritized gene list of high statistical significance, which consisted of the most likely disease modifiers identified so far in pulmonary fibrosis. Extending beyond target identification, a series of meta-analyses produced a number of biological hypotheses on disease pathogenesis. Among them, the role of HIF-1 signaling was further explored to reveal HIF-1alpha overexpression in the hyperplastic epithelium of fibrotic lungs, colocalized with its target genes p53 and Vegf.Comparative expression profiling was shown to be a highly efficient method in identifying deregulated genes and pathways. Moreover, tissue microarrays and computerized image analysis allowed for the high-throughput and unbiased assessment of histopathologic sections, adding substantial confidence in pathologic evaluations. More importantly, our results suggest an early primary role of HIF-1 in alveolar epithelial cell homeostasis and disease pathogenesis, provide insights on the pathophysiologic differences of different interstitial pneumonias, and indicate the importance of assessing the efficacy of pharmacologic inhibitors of HIF-1 activity in the treatment of pulmonary fibrosis.

Ulcerative colitis (UC) constitutes a chronic inflammatory process of the colon of unknown etiology. Current data support a pivotal role of apoptosis in the evolution of pathogenesis of UC. We performed a prospective study in order to determine the role of Bcl-2, Bax and Bcl-x in the apoptotic pathway in UC.We included 23 patients with UC and 11 controls. Histological severity of the disease was assessed according to the Sidney classification system. Patients in the UC group were divided in 2 groups according to histological severity of the disease. The TUNEL method was used for the in situ evaluation of apoptosis. Immunohistochemical staining was used for the detection of Bax, Bcl-2, Bcl-x. For the assessment of cellular proliferation we used the monoclonal antibody Ki67. Appropriate statistical methods were applied.Overall 77 specimens were assessed; 57 from UC patients and 20 from controls. Bcl-2, Bax and Bcl-x were upregulated in the group of patients with UC compared to controls. Nevertheless, Bax in epithelial cells and Bcl-x in lymphocytes were downregulated in patients with moderate/severe disease (p = 0.029 and 0.04 respectively). A weak correlation between epithelial apoptosis and Bcl-x expression in lymphocytes (r = 0.31, p = 0.02) was found. An even weaker correlation was also noticed between the epithelial component apoptosis and Bax in lymphocytes (r = 0.02, p = 0.07).Bcl-2/Bax system does not appear to be involved in the induction of apoptosis in UC. Activation of intraepithelial lymphocytes may be associated with epithelial apoptosis or simply represent epiphenomena related to the inflammatory process.

Deregulation of cell cycle control molecules, such as cyclins and their inhibitors, is a crucial event in the carcinogenetic process. Our aim was to identify potential correlations between p16 and cyclin D1 expression in pancreatic ductal adenocarcinoma (PDAC) that affect the biological behavior of this neoplasm.Using tissue microarray (TMA) technology, 50 paraffin-embedded tissue samples of histologically confirmed primary PDACs were cored twice and re-embedded to the final recipient block. Immunohistochemistry (IHC) was performed using monoclonal anti-p16 and anti-cyclin D1 antibodies. Protein expression levels were determined by performing computerized image analysis (CIA; estimation of Nuclear Labeling Index-NLI). SPSS (chi square test and interrater Cohen's kappa) was used for statistical analysis.Cyclin D1 overexpression was observed in 24/50 (48%) of the examined carcinomas, whereas p16 loss or reduced expression was detected in 40/50 (80%) cases. Statistical significance was noted when correlating grade to cyclin D1 (p=0.038), stage to p16 (p=0.012) and also to cyclin D1 (p=0.011). Interestingly, combined protein alterations (p16 loss and cyclin D1 overexpression) were observed in 23/50 (46%) cases associated with advanced stage (p=0.019). Overall combined expression of the two molecules demonstrated a significantly low value (kappa=0.012; 95% confidence interval-CI: 0.010-0.014).A significant proportion of PDACs is characterized by simultaneous protein alterations regarding p16 and cyclin D1 genes. This mechanism of genetic deregulation in cell cycle potentially explains in part the aggressive phenotype of this neoplasm.

Here is reported an extremely rare case of a large intraparotid facial nerve schwannoma in a 32-year-old female who presented with a parotid mass. There had been a long clinical course and sudden onset of facial weakness. Diagnostic evaluation and surgical management are discussed along with a brief review of the literature.

Purpose: Tumor cell metastasis constitutes a major problem in the treatment of cancer. Because the cure rate of metastatic tumors is very low, new therapeutic approaches are needed. Heat shock protein 90 (HSP90) is a molecular chaperone that is recognized as a new target for the treatment of cancer. Here, we examine the value of a monoclonal antibody (mAb) against HSP90, mAb 4C5, as a potential marker in malignant melanomas. Moreover, we investigate the possibility to use mAb 4C5 as an inhibitor of melanoma cell invasion and metastasis. Experimental Design: Paraffin blocks of formalin-fixed human melanoma tumor tissues were used to prepare tissue microarrays. The B16 F10 melanoma cell line was used in all the in vitro experiments. To assess melanoma cell invasion, the wound-healing assay and the Matrigel invasion assay were applied. To evaluate the effect of mAb 4C5 on tumor metastasis, we used an experimental model of metastatic melanoma. Results: Immunohistochemical studies done on a panel of malignant melanomas showed positive immunostaining with mAb 4C5 in all cases. mAb 4C5 inhibits B16 F10 cell invasion by binding to surface HSP90 because it is not internalized. mAb 4C5 significantly inhibits melanoma metastasis in C57BL/6 mice inoculated with B16 F10 cells. Conclusions: mAb 4C5 could be potentially used as a novel specific marker for malignant melanomas. mAb 4C5 inhibits melanoma cell invasion in vitro by binding to cell surface HSP90 expressed on B16 F10 melanoma cells. Finally, this antibody significantly inhibits melanoma metastasis, thus rendering it a potential therapeutic agent for the treatment of cancer metastasis.

Telomerase activation plays a crucial role in tumorigenesis by sustaining cellular immortality. It consists of two main components which include a RNA subunit (h-TERC) and a catalytic protein subunit (h-TERT). Similarly, amplification or deletion correlating with overexpression of c-myc is a common event in various neoplasias, including non-small cell lung carcinoma (NSCLC). Because c-myc activates telomerase by inducing expression of its catalytic subunit, our aim was to correlate the expression of these two proteins with the biological behavior in NSCLC.Using tissue microarrays technology (TMA) we evaluated by computerized image analysis (CIA) the results of h-TERT and c-myc immuno-histochemistry (IHC) in 40 NSCLCs, which were cored and re-embedded into one TMA block.Co-overexpression (moderate or high levels of NLI: Nuclear Labeling Index) of h-TERT and c-myc was observed in the majority of cases and found to be statistically significant (p=0.001). The results showed also strong association between c-myc and h-TERT overexpression correlating with stage (p=0.001 for both of them), but not with grade (p=0.206 and p=0.313, respectively).Our combined study showed that there is a strong correlation between the activation and expression of these two genes and maybe this co-deregulation could be used as a prognostic factor for the evaluation of biological behavior in NSCLCs.

The Fas/FasL system plays an important role in the regulation of tumor apoptosis. This study examined the correlation between the expression levels of Fas and WAF1 with the clinicopathological characteristics of esophageal squamous cell carcinoma (SCC) tumors. Paraffin-embedded sections from 19 surgically resected primary esophageal squamous cell carcinomas were examined by immunohistochemistry. At low expression levels (5% to 24%), a 4-fold higher expression of Fas relative to WAF1 expression was observed. High expression levels (50% to 74%) of Fas were not recorded, whereas such levels of expression for WAF1 were retained in a proportion of cells20%. Proteins WAF1 and Fas were both expressed in all lesions of SCCs. Lesions showing high expression levels of WAF1 have a high degree of differentiation, but a sample that expresses WAF1 and belongs to the morphology I category probably has a medium degree of differentiation.