Elena Gabusi

Increased expression of p16(INK4a) and p27(Kip1) cyclin-dependent kinase inhibitor genes in aging human kidney and chronic allograft nephropathy

BACKGROUND The goal of the current study was to examine the potential value of p16(INK4a) and p27(Kip1) cyclin-dependent kinase inhibitor (CDKI) genes in the process of human kidney aging in vivo, and in the development of chronic allograft nephropathy (CAN). METHODS Expression of p16(INK4a) and p27(Kip1) CDKI genes was evaluated and compared in 20 normal human kidney tissues of different ages (range, 21 to 80 years) and in 9 chronically rejected kidney grafts. Age dependency of marker expression was analyzed by the Pearson correlation and linear regression. RESULTS Expression of p16 in cortical tubular (CTS) and interstitial (CIS) cells of normal kidney was age dependent (correlation coefficients: 0.608 and 0.726, 95% confidence interval [CI]: 0.227 to 0.828 and 0.417 to 0.884, respectively). Cortical tubular expression of p27 was also correlated with increasing age (0.672, 95% CI: 0.327 to 0.859). Linear regression analyses confirmed the linearity of marker relationship with age (coefficient of determination R(2):0.370, 0.452, and 0.527 for CIS p16, CTS p27, and CTS p16, respectively). The mean chronological and predicted graft ages (53 +/- 21 and 76 +/- 8.9 years, respectively) were significantly different (P = 0.0126). The glomeruli, tubules, and interstitial cells of rejected grafts expressed significantly higher levels of p16 and p27 than normal kidneys. Expression of p16 in glomerular and cortical interstitial cells was higher in grade 3 of CAN than in grade 2 (P = 0.013 and 0.004, respectively). CONCLUSION The results of the current study show that expression of p16(INK4a) and p27(Kip1) CDKI genes is increased in cortical cells of the aging human kidney and in chronic allograft rejection, supporting the senescence theory of CAN.

Diagnostic role of circulating free plasma DNA detection in patients with localized prostate cancer

To analyze the potential diagnostic relevance of free plasma DNA (FPDNA), we enrolled 64 patients with localized prostate cancer (CaP). FPDNA was quantified by real-time polymerase chain reaction assessment of the HTERT gene in blood samples from 64 patients with CaP and 45 healthy males. Methylation of the GSTP1 gene was used to confirm the neoplastic origin of FPDNA in selected cases. The mean +/- SD levels of FPDNA were higher in patients with CaP (15.4 +/- 10.9 ng/mL) than in control subjects (5.5 +/- 3.5 ng/mL; P <.001). By using the best cutoff value, the sensitivity of the test was 80%, the specificity was 82%, the area under the receiver operating characteristic curve, 0.881. High FPDNA values were significantly associated with pathologic T3 stage (P = . 035). Methylation of the GSTP1 gene was found in 4 (25%) of 16 FPDNA samples and 15 (94%) of 16 tissue samples. Quantification of FPDNA discriminates between patients with CaP and healthy subjects and correlates with pathologic tumor stage. FPDNA is a candidate biomarker for early diagnosis and monitoring of CaP.

Investigation of ErbB1 and ErbB2 expression for therapeutic targeting in primary liver tumours

BACKGROUND Molecular targets are needed for primary liver tumours. AIMS ErbB1 and ErbB2 expression was analysed in neoplastic and surrounding tissue in surgical specimens from 52 hepatocellular carcinomas and 48 intrahepatic cholangiocarcinomas, randomly chosen from cases surgically treated in this institution. METHODS ErbB1 and ErbB2 expression were evaluated immunohistochemically, the latter by Herceptest. Gene amplification of ErbB2 was tested by chromogenic in situ hybridisation. RESULTS In normal/cirrhotic non-neoplastic tissue, the ErbB1 (but not ErbB2) antibody commonly stained normal hepatocytes and mature intrahepatic ducts. In neoplastic tissue, moderate/strong ErbB1 immunostaining occurred in 43/52 (85%) hepatocellular carcinomas and 39/48 (81%) intra-hepatic cholangiocarcinomas. With ErbB2 Herceptest, 0/52 (0%) hepatocellular carcinomas and 2/48 (4%) intra-hepatic cholangiocarcinomas had treatable scores of 2+/3+ (chromogenic in situ hybridisation confirmed gene amplification in the latter two cases only). Neither ErbB1 nor ErbB2 expression correlated with any of the main clinical-pathologic features or survival. CONCLUSIONS Although not related to prognosis, ErbB1 could be a molecular target in a large percentage of patients with hepatocellular carcinoma or intrahepatic cholangiocarcinoma. Inclusion of anti-ErbB1 drugs such as ZD 1839 and c225 (and possibly also anti-ErbB2 drugs like Trastuzumab for a small subset of patients) in clinical trials is suggested.

Predictive Value of Biological Markers for Hepatocellular Carcinoma Patients Treated with Orthotopic Liver Transplantation

Purpose: To help stratify candidates with hepatocellular carcinoma (HCC) for orthotopic liver transplantation (OLT), biomarkers are needed that are capable of predicting recurrence of disease (ROD). We investigated the prognostic role in this setting of immunohistochemical markers reported previously to predict poor prognosis in HCC patients treated with resection. Experimental Design: Eighty-three patients with HCC who underwent OLT between 1987 and 2001 with a minimum clinical follow up of 12 months were included in this retrospective study. We analyzed immunohistochemical expression of the adhesion molecules E-cadherin and β-catenin (membrane/nuclear localization), MIB-1 proliferative index and the cyclin-dependent kinase inhibitor p27, alongside the main clinical-pathological variables. Results: At univariate analysis, vascular thrombosis, high MIB-1 index, lower membrane expression of E-cadherin and β-catenin, and nuclear β-catenin localization were associated with ROD. At multivariate analysis, only MIB-1 index, low equal E-cadherin (with respect to non-neoplastic surrounding tissue), and nuclear β-catenin appeared as independent predictors of ROD. The logistic regression analysis model indicated that detection of any one parameter was associated with at least 88% estimated risk of ROD (up to 99% for all three). Conclusions: We propose these three molecular parameters as an additional tool for rational selection of OLT candidates among HCC patients (stratification according to the risk of ROD might help provide a similar life expectancy for cirrhotic candidates with and without HCC).

Detection of the M30 neoepitope as a new tool to quantify liver apoptosis: timing and patterns of positivity on frozen and paraffin-embedded sections.

One of the first stages of apoptosis is cytokeratin cleavage mediated by caspases, which is associated with the expression of a neoepitope, the cleavage site of cytokeratin 18, identifiable by the M30 monoclonal antibody. The aim of this study was to evaluate the timing of neoantigen expression and its modifications in the various morphologic stages of apoptosis on frozen and paraffin-embedded sections from liver biopsies of patients with chronic hepatitis or transplanted liver. The appearance of this neoepitope coincides with the gradual disappearance of cytokeratins, with the appearance of nuclear DNA fragmentation, and with the presence of Councilman bodies. The staining patterns on paraffin-embedded sections of liver specimens were similar to those found in frozen sections, with a reduced sensitivity. The M30 antibody is correlated with apoptosis, and its specificity for epithelial cells makes this method the first choice for routine evaluation of apoptosis in liver epithelial cells.

Real time RT-PCR approach for the evaluation of ERBB2 overexpression in breast cancer archival samples: a comparative study with FISH, SISH, and immunohistochemistry.

We tested the reliability of real time reverse transcription polymerase chain reactions as an alternative method for the assessment of ERBB2 status in paraffin-embedded tissues of 83 patients with breast cancer and 20 non-neoplastic controls. PCR was also compared with the immunohistochemical (IHC) HercepTest score and with fluorescence (FISH) and silver (SISH) in-situ hybridization, in 42 selected cases. ERBB2 mRNA was overexpressed in 26/83 (31%) breast cancer samples, using a cutoff calculated as the mean value of the controls plus 3 SD or with the receiver operating curve. The PCR test showed a 96% sensitivity and a 100% specificity when compared with FISH, with an area under the receiver operating curve of 98.4%. Overexpression of ERBB2 at PCR was also significantly correlated with amplification in FISH (P<0.001, Mann-Whitney test) and in SISH (P<0.001, Mann-Whitney test), and with the IHC HercepTest scores 2 or 3 (P<0.001, Spearman rank correlation). FISH, SISH, and IHC were also compared with each other. ERBB2 amplification in FISH significantly correlated with that in SISH (P=0.002, χ2 test with a concordance of the 87%), but not with IHC HercepTest scores (P=0.214, χ2 test). Real time PCR is a reliable and cost-effective method for the assessment of ERBB2 status in archival breast cancer samples, compared with FISH. Its introduction in routine diagnostic pathology practice is feasible even if it requires amendments to the current clinical oncology protocols.

Detection of the M30 Neoepitope as a New Tool to Quantify Liver Apoptosis

One of the first stages of apoptosis is cytokeratin cleavage mediated by caspases, which is associated with the expression of a neoepitope, the cleavage site of cytokeratin 18, identifiable by the M30 monoclonal antibody. The aim of this study was to evaluate the timing of neoantigen expression and its modifications in the various morphologic stages of apoptosis on frozen and paraffin-embedded sections from liver biopsies of patients with chronic hepatitis or transplanted liver. The appearance of this neoepitope coincides with the gradual disappearance of cytokeratins, with the appearance of nuclear DNA fragmentation, and with the presence of Councilman bodies. The staining patterns on paraffin-embedded sections of liver specimens were similar to those found in frozen sections, with a reduced sensitivity. The M30 antibody is correlated with apoptosis, and its specificity for epithelial cells makes this method the first choice for routine evaluation of apoptosis in liver epithelial cells.

Solitary Fibrous Tumour of the Urinary Bladder with Expression of bcl–2, CD34, and Insulin–Like Growth Factor Type II

We describe a solitary fibrous tumour of the urinary bladder wall removed from a 50–year–old man with a history of pelvic pain, dysuria, and urinary bleeding. Anamnesis revealed a weight increase during the preceding 3 months, but no apparent episodes of biochemical hypoglycaemia or hormonal abnormalities. The patient is alive and well 18 months after surgery. Pathological examination revealed a 6.5–cm well–circumscribed nodular mass composed of uniform spindle cells arranged in bundles and fascicles with varying amounts of collagen and a typical haemangiopericytoma–like vascular pattern. The tumour cells were positive for bcl–2, CD34, and vimentin and ultrastructurally showed mesenchymal–myofibroblastic traits. These cells produced insulin–like growth factor type II mRNA as demonstrated by non–isotopic in situ hybridization. This rare case with a solitary fibrous tumor suggests that insulin–like growth factor type II could join CD34 and bcl–2 as markers for postoperative differential diagnosis.

Quantification of free plasma DNA before and after chemotherapy in patients with advanced epithelial ovarian cancer

Objectives A nonrandomized trial was planned to investigate the role of free plasma DNA (FPDNA) in patients with epithelial ovarian cancer before and after chemotherapy. Twenty-two patients with advanced stage ovarian cancer not suitable for debulking were treated with a neoadjuvant platinum/taxanes chemotherapy. Patients with clinical complete or partial response underwent radical hystero-oophorectomy, omentectomy, and lymphadenectomy and were followed up every 3 to 6 months. Methods Blood samples were obtained from each patient before chemotherapy, before each cycle, before and after surgery. FPDNA was quantified by real-time quantitative polymerase chain reaction using the Quantifiler Human Quantification Kit and expressed in ng/mL. Fifty female healthy blood donor volunteers were used as controls. Results Median FPDNA quantities discriminated between patients before chemotherapy (29.6±22.7 ng/mL) and controls (6.4±4.0 ng/mL) using a 14.5 ng/mL cutoff with 77% sensitivity and 96% specificity (P<0.001). Mean FPDNA concentrations significantly decreased after chemotherapy (17.9±14.5 ng/mL, P=0.001). A peak of FPDNA levels (66.2±45.2 ng/mL) was observed in association with surgery (P<0.001). Median follow-up and median progression-free survival time were 13.4±5.1 and 11.7±5.6 months, respectively. Eight patients with FPDNA values above the cutoff after chemotherapy showed disease progression or died, whereas 7 patients with FPDNA below the cutoff were free from disease. Patients with FPDNA levels above and below the cutoff showed significantly separated progression-free survival curves (P=0.007, log-rank test). Conclusions FPDNA quantification significantly discriminates between cancer patients and controls and correlates with response to chemotherapy. Although performed in a limited series, we demonstrated a correlation between FPDNA values and clinical behavior of ovarian cancer patients.

Application of a fluorescent PCR method for molecular diagnosis of posttransplant lymphoproliferative disorders on routine tissue sections

Molecular detection of monoclonality can play an important role in the diagnosis of posttransplantation lymphoproliferative disorders (PTLD). To permit accurate molecular diagnosis of PTLD even on very small amounts of DNA extracted from routinely embedded histologic material, we adapted a commercially available PCR protocol (for FR-1, -2 and -3 regions), originally designed for use on fresh/frozen samples. We applied this approach on routine biopsy/surgical material of 10 PTLD (from nine patients). All three FR regions were always amplified, indicating that the extracted DNA was of medium quality. All five PTLD morphologically classified as lymphomas were monoclonal in at least one FR region. Thus, using the WHO histologic, immunohistochemical, and clinical criteria as the reference standard, the approach provided 100% sensitivity for detection of monoclonal malignancies, supporting the validity of the method. Of five specimens classified morphologically as polymorphic PTLD, three displayed a solitary IgH gene rearrangement peak, consistent with the presence of a monoclonal B-cell population (ie, monoclonal polymorphic PTLD). This rapid and straightforward procedure, which allows identification of a wide range of IgH rearrangements, could facilitate molecular analysis of PTLD in routine practice, while limiting consumption of valuable diagnostic material.