Guido Coggi

p53 protein accumulation and p53 gene mutation in esophageal carcinoma. A molecular and immunohistochemical study with clinicopathologic correlations.

p53 gene mutation and p53 protein accumulation are common in human cancer. However, their clinical significance is controversial and p53 accumulation may not correlate with gene mutation. The current study investigates the occurrence of p53 alterations in esophageal carcinoma, the correlation between the analyses at the gene and protein level, and their prognostic significance.

bcl-2 oncoprotein in colorectal hyperplastic polyps, adenomas, and adenocarcinomas

The bcl-2 gene is an oncogene that inhibits programmed cell death (apoptosis). We investigated by immunocytochemistry bcl-2 expression in normal colonic mucosa, hyperplastic polyps, adenomas, and adenocarcinomas of the large bowel. The purpose of the investigation was twofold; to assess the possible role of bcl-2 in colorectal tumorigenesis and to evaluate its clinical significance. The cases studied included 24 hyperplastic polyps, 49 adenomas, and 205 colorectal carcinomas. In both normal mucosa and hyperplastic polyps bcl-2 immunoreactivity was detected only in the proliferative cells of the colonic crypts. Conversely, bcl-2 immunoreactivity was noted in all adenomas irrespective of the degree of dysplastic change; it was diffuse in 84% of adenomas and focal in the remaining cases. In colorectal carcinomas bcl-2 expression was undetectable in 50% and focal (less than 50% immunostained neoplastic cells) in 38% of tumors. The remaining 12% of the carcinomas displayed diffuse (more than 50% immunostained neoplastic cells) bcl-2 immunoreactivity. In colorectal carcinomas bcl-2 expression was not correlated with relevant clinicopathologic parameters, including disease stage, tumor location and growth fraction, DNA ploidy, and p53 protein accumulation, and had no prognostic significance by univariate or multivariate analysis. These results suggest that bcl-2 oncoprotein may play a role in colorectal tumorigenesis, probably in the early phases of the adenoma-carcinoma sequence. bcl-2 expression in established tumors has no prognostic significance.

Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors

Predicting drug response in cancer patients remains a major challenge in the clinic. We have perfected an ex vivo, reproducible, rapid and personalized culture method to investigate antitumoral pharmacological properties that preserves the original cancer microenvironment. Response to signal transduction inhibitors in cancer is determined not only by properties of the drug target but also by mutations in other signaling molecules and the tumor microenvironment. As a proof of concept, we, therefore, focused on the PI3K/Akt signaling pathway, because it plays a prominent role in cancer and its activity is affected by epithelial–stromal interactions. Our results show that this culture model preserves tissue 3D architecture, cell viability, pathway activity, and global gene-expression profiles up to 5 days ex vivo. In addition, we show pathway modulation in tumor cells resulting from pharmacologic intervention in ex vivo culture. This technology may have a significant impact on patient selection for clinical trials and in predicting response to small-molecule inhibitor therapy.

Inhibitors of apoptosis proteins (IAPs) expression and their prognostic significance in hepatocellular carcinoma

BackgroundSimilarly to other tumor types, an imbalance between unrestrained cell proliferation and impaired apoptosis appears to be a major unfavorable feature of hepatocellular carcinoma (HCC). The members of IAP family are key regulators of apoptosis, cytokinesis and signal transduction. IAP survival action is antagonized by specific binding of Smac/DIABLO and XAF1. This study aimed to investigate the gene and protein expression pattern of IAP family members and their antagonists in a series of human HCCs and to assess their clinical significance.MethodsRelative quantification of IAPs and their antagonist genes was assessed by quantitative Real Time RT-PCR (qPCR) in 80 patients who underwent surgical resection for HCC. The expression ratios of XIAP/XAF1 and of XIAP/Smac were also evaluated. Survivin, XIAP and XAF1 protein expression were investigated by immunohistochemistry. Correlations between mRNA levels, protein expression and clinicopathological features were assessed. Follow-up data were available for 69 HCC patients. The overall survival analysis was estimated according to the Kaplan-Meier method.ResultsSurvivin and Livin/ML-IAP mRNAs were significantly over-expressed in cancer tissues compared to non-neoplastic counterparts. Although Survivin immunoreactivity did not correlate with qPCR data, a significant relation was found between higher Survivin mRNA level and tumor stage, tumor grade and vascular invasion.The mRNA ratio XIAP/XAF1 was significantly higher in HCCs than in cirrhotic tissues. Moreover, high XIAP/XAF1 ratio was an indicator of poor prognosis when overall survival was estimated and elevated XIAP immunoreactivity was significantly associated with shorter survival.ConclusionOur study demonstrates that alterations in the expression of IAP family members, including Survivin and Livin/ML-IAP, are frequent in HCCs. Of interest, we could determine that an imbalance in XIAP/XAF1 mRNA expression levels correlated to overall patient survival, and that high XIAP immunoreactivity was a poor prognostic factor.

Cytokeratin-immunoreactive cells of human lymph nodes and spleen in normal and pathological conditions - An immunocytochemical study

The occurrence and the distribution of cytokeratin (CK)-immunoreactive reticulum cells in a series of normal and pathological human lymph nodes and spleens are documented. The immunoreactive cells exhibit morphological and immunophenotypic features of so-called fibroblastic reticulum cells, with or without myoid differentiation. They invariably co-express vimentin and, to a lesser extent, desmin and muscle-specific actin isoforms. These CK-immunoreactive cells are apparently a normal subpopulation of reticulum cells, being detectable from the early stages of spleen and lymph node development. They are distributed mainly in the paracortical and medullary regions of the lymph nodes and at the periphery of the white pulp in the spleen. Their number and distribution are highly variable in different neoplastic and non-neoplastic pathological conditions but the changes are not disease specific. CK-immunoreactive reticulum cells are easily identifiable in both frozen and fixed lymphoid tissue and in cytological smears of fine-needle aspirates, provided that monoclonal antibodies whose spectrum of reactivity includes cytokeratins 8 and 18 are used. The awareness of the occurrence of CK-immunoreactive cells in normal lymphoid tissues is of particular relevance in the search for micrometastatic foci using anti-CK antibodies.

Banking together. A unified model of informed consent for biobanking

D uring the past 10 years, human biological material—body fluids, cells, tissues, intracellular substances or DNA—and the related data have become an important resource for academic medical research, and for the industrial development of diagnostics and therapeutics (Godard et al, 2003). The increasing creation and use of biobanks that store both the material and the related data bears witness to their scientific value, but there is still no consensus— either internationally, or at the European or national levels—about the regulations that should govern biobanks in ethical or legal terms (Cambon-Thomsen et al, 2007; Kaye, 2005). In particular, consent models designed to appropriately regulate biobankbased research are characterized by a maze of laws, policies and ethical recommendations that range from strict (specific informed consent) to basically unrestricted use (broad consent; Boggio et al, 2007).

Glial fibrillary acidic protein immunoreactivity in normal and diseased human breast

Immunostaining for glial fibrillary acidic protein (GFAP) identifies a minor subpopulation of immunoreactive myoepithelial cells in the normal resting human breast. The GFAP-immunoreactive cells also express a panel of myoepithelial cell markers, including cytokeratin 14 (CK 14), vimentin, smooth-muscle-specific actin isoforms, nerve growth factor receptor (NGFR) and common acute lymphoblastic leukaemia antigen (CALLA). The percentage of GFAP-immunoreactive myoepithelial cells is greatly increased in various neoplastic and non-neoplastic diseases of the breast, being highest in adenomyoepitheliomas. Furthermore, in all the instances of fibroadenoma, phyllodes tumour, epitheliosis and gynaecomastia, a variable number of epithelial cells also acquires immunoreactivity for GFAP, vimentin, CK 14, NGFR and, to a lesser extent, for CALLA. Conversely, GFAP immunoreactivity has never been encountered in the malignant cells of the different types of breast carcinoma. These findings suggest that the expression of GFAP might be a (possibly transient) feature of proliferating epithelial and myoepithelial cells in breast diseases other than carcinomas.

Detection of p53 mutations by single-strand conformation polymorphisms (SSCP) gel electrophoresis. A comparative study of radioactive and nonradioactive silver-stained SSCP analysis.

p53 mutations are the most common genetic abnormality in human tumors, but their clinical significance remains to he precisely elucidated. Conventional single-strand conformation polymorphism (SSCP) analysis, a well-established technique for detecting p53 mutations, uses radioactively labeled polymerase chain reaction (PCR) products, which migrate abnormally in the presence of mutations. We performed radioactive PCR-SSCP analysis in a series of 30 formalin-fixed, paraffin-embedded ovarian carcinomas and two cell lines (SW480 and Caov4) harboring known homozygous p53 mutations and compared the results with nonradioactive silver-stained SSCP. The purpose was to assess whether nonradioactive SSCP is suitable for detecting p53 mutations in a rapid, sensitive, cost-effective fashion, without the need of radioactive isotopes. We accomplished PCR amplification of p53 exons 5 through 8 in 26 carcinomas, and radioactive SSCP detected p53 mutations in 13 tumors: three mutations were localized in exon 5, six in exon 6, two in exon 7, and two in exon 8. All mutations were correctly identified with nonradioactive SSCP, except for one exon 8 mutation. To establish the sensitivity of nonradioactive SSCP, DNA samples of SW480 and Caov4 were mixed with increasing amounts (0–90%) of normal DNA and subjected to PCR-SSCP analysis. Mutations were detected until the concentration of SW480 and Caov4 was 15% and 10% respectively, of the total sample. The results of our investigation demonstrate that nonradioactive silver-stained SSCP is a sensitive, rapid, and simple technique to detect p53 mutations, even in formalin-fixed tissues, and could be easily used to investigate large series of patients to assess the clinical significance of p53 mutations in human tumors.

The anterior spinal artery: the main arterial supply of the human spinal cord--a preliminary anatomic study.

Paraplegia is the most feared complication of surgery of the thoracic aorta. Controversy continues regarding the continuity of the anterior spinal artery (ASA). We studied the arterial vascularization of human spinal cord to indagate ASA continuity and possible anatomic variations of the arteria radicularis magna (ARM). Methods. From July 1998 to January 1999, 31 spinal cords from adult cadavers of both sexes were studied (mean age 72 ± 12 years). The cause of death was well established in each case and no one had spinal, cerebral, or significant aortic disease. The abdomen, thoracic viscera, and vessels were removed after 24 to 36 hours from death; only the upper trunks of the aorta were left in situ. The anterior vertebral spinal column was exposed and the attached muscles were divided. The vertebral bodies were removed with an electrical oscillating saw. The spinal cord in situ was exposed after a longitudinal paramedian incision of the dura mater. In all cases the course of the ASA was visualized and the distribuFrom the Department of Cardiovascular Surgery, Centro Cardiologico “I Monzino” Foundation IRCCS,a and II Department of Pathology,b University of Milan, Milan, Italy. Received for publication April 6, 1999; accepted for publication Sept 20, 1999. Address for reprints: Maurizio Roberto, MD, Department of Cardiovascular Surgery, “I Monzino” Foundation IRCCS, via Parea 4, 20138 Milan, Italy. J Thorac Cardiovasc Surg 2000;119:376-9 Copyright

Quantitative PCR and HER2 Testing in Breast Cancer : A Technical and Cost-Effectiveness Analysis

We performed a technical and cost-effectiveness analysis of quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) for the assessment of HER2 in breast cancer. We evaluated 44 frozen and 55 formalin-fixed paraffin-embedded (FFPE) breast carcinoma specimens by Q-RT-PCR, immunohistochemical analysis, and fluorescent in situ hybridization (FISH). Immunohistochemical and FISH analyses were performed on individual slides and on tissue microarray. Costs of techniques were calculated to study 1 case and 10 or 40 cases. Q-RT-PCR provided reliable data in frozen and FFPE specimens, which were significantly correlated. HER2 messenger RNA levels were significantly stratified in agreement with immunohistochemical data (P < .05). There was complete concordance between Q-RT-PCR and immunohistochemical results for negative and strongly positive (3+) cases. The intermediate immunohistochemical group (2+), including FISH+ and FISH- cancers, could also be stratified by Q-RT-PCR. Cost analysis documented the advantage of Q-RT-PCR in all US Food and Drug Administration-approved assays. Our data support the use of Q-RT-PCR for testing breast cancer specimens to select patients for HER2 inhibitory therapy.