Clinical role of p16INK4a expression in liquid-based cervical cytology: correlation with HPV testing and histologic diagnosis.

p16INK4a is overexpressed in high-risk human papillomavirus (HR-HPV)-infected preneoplastic and neoplastic lesions of the uterine cervix. Our aim was to verify whether p16 is a diagnostic marker also in cervical liquid-based cytology. We performed p16 immunocytochemical analysis and the Hybrid Capture 2 (HC2) test (Digene, Gaithersburg, MD) for HR-HPV infection in 471 ThinPrep-processed (Cytyc, Boxborough, MA) cervicovaginal samples and correlated the results with histologic findings. A total of 32.3% of the specimens showed p16 immunoreactivity, whereas the HC2 test was positive in 41.2% of the cases (65.2% concordance rate). Correlating the cytologic, p16, and HPV results with histologic findings revealed HC2 as the most sensitive test for a diagnosis of cervical intraepithelial neoplasia 2 or worse, whereas cytologic examination was the most specific. The positive predictive value was significantly higher for cytologic examination than for p16 and HR-HPV testing. These data suggest that p16 evaluation in ThinPrep samples does not have better clinical effectiveness for identifying high-grade lesions than conventional morphologic examination and HPV testing.

HLA-A, -B, -C expression in colon carcinoma mimics that of the normal colonic mucosa and is prognostically relevant.

Whether human leukocyte antigen (HLA)-A, -B, -C expression has any predictive value on the prognosis of human malignancies remains controversial. Herein, monoclonal antibodies with preferential reactivity for HLA-A, HLA-B, and HLA-C (HCA2, HC10, and L31) were used to stain an archival collection of 291 formalin-fixed/paraffin-embedded tissues, comprising neoplastic lesions from stages II and III colon carcinoma patients (n=165), and the uninvolved, morphologically normal mucosae from a subset (n=126) of these patients. Marked staining variability was detected not only in the tumors as in previous studies, but also in the normal paired mucosae. HLA-A, -B, -C expression was similar in approximately two thirds of the available 126 normal/neoplastic pairs, confirming in vivo our previous observation that most tumor cells mimic the HLA phenotypes of their normal counterparts. Both up and down-regulation occurred in the remaining third of the pairs, but did not coincide with high and low expression, respectively, conventionally evaluated on the tumor lesion only. Remarkably, a "paired" evaluation, but not high or low expression in the tumor, was predictive of the clinical outcome. Deviations from the expression in the normal paired mucosa (both increases and decreases) of HCA2-reactive class I molecules (possibly HLA-A), and down-regulation of L31-reactive class I molecules (possibly HLA-C), particularly in tumors from stage II patients, correlated with poor 5-year overall and disease-free survival, hazard risk ranging from 2 to 6, approximately. Thus, a paired immunohistochemical comparison reveals a novel immune evasion strategy that may impact on the prognosis of colon carcinoma.

Non-Hodgkin lymphomas concurrent with HHV8-associated Kaposi's sarcoma in the same lymph node in AIDS and non-AIDS patients.

BACKGROUND: The association between lymphomas and Kaposi's sarcoma has been described since 1920. The simultaneous presence of the 2 pathologic entities within the same lymph node is a rare and interesting occurrence. In the few cases described, the presence of human herpesvirus 8 (HHV8) and Epstein-Barr virus (EBV) in the different neoplastic areas was investigated only by immunohistochemistry and in situ hybridization studies. METHODS: Two cases of concurrent non-Hodgkin lymphoma and Kaposi's sarcoma in the same lymph node are described: a diffuse large B cell lymphoma in an AIDS patient and a T cell-rich large B cell lymphoma in a HIV-negative patient, complete with the clinical, immunohistological and molecular features, the latter ones defined after isolation of the different neoplastic areas by laser capture microdissection. RESULTS: Polymerase chain reaction assays revealed HHV8 DNA sequences only in the microdissected Kaposi's sarcoma areas and EBV DNA sequences only in the lymphomatous areas in both cases, confirming the HHV8 infection only in the neoplastic sarcomatous cells and evidencing the EBV infection only in the lymphomatous cells. CONCLUSION: This study represents a further confirmation of the supposed different etiopathogenic mechanisms of the 2 neoplasias, suggesting a coincidental occurrence even when localized in the same lymph node, independently from HIV infection.

Simultaneous determination of 16 anti-HIV drugs in human plasma by high-performance liquid chromatography.

Therapeutic drug monitoring (TDM) is pivotal to improve the management of HIV infection. Here, a HPLC-UV method has been developed to quantify simultaneously seven HIV protease inhibitors (amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir; PIs), seven nucleoside reverse transcriptase inhibitors (abacavir, didanosine, emtricitabine, lamivudine, stavudine, zalcitabine, and zidovudine; NRTIs), and two non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine; NNRTIs) in human plasma. The volume of the plasma sample was 600 microL. This method involved automated solid-phase extraction with Oasis HLB Cartridge 1 cc (divinylbenzene and N-vinylpyrrolidone) and evaporation in a water bath under nitrogen stream. The extracted samples were reconstituted with 100 microL methanol. Twenty microliters of these samples were injected into a HPLC-UV system, the analytes were eluted on an analytical C(18) Symmetry column (250 mm x 4.6mm I.D.) with a particle size of 5 microm. The mobile phase (0.01 M KH(2)PO(4) and acetonitrile) was delivered at 1.0 mL/min with linear gradient elution. The total run time for a single analysis was 35 min, the anti-HIV drugs were detected by UV at 240 and 260 nm. The calibration curves were linear up to 10 microg/mL. The absolute recovery ranged between 88 and 120%. The in vitro stability of anti-HIV drugs (0.005-10 microg/mL) in plasma has been studied at 24.0 degrees C. On these bases, a two to four analyte method has been tailored to the individual needs of the HIV-infected patient. The HPLC-UV method here reported has been validated and is currently applied to monitor PIs, NRTIs, and NNRTIs in plasma of HIV-infected patients. It allows to monitor the largest number of anti-HIV drugs simultaneously, appearing useful in a routine laboratory, and represents an essential step to elucidate the utility of a formal therapeutic drug monitoring for the optimal follow-up of HIV-infected patients.

DNA demethylation is directly related to tumour progression: evidence in normal, pre-malignant and malignant cells from uterine cervix samples.

A computer-assisted assay based on the quantitative analysis of DNA methylation in individual interphase nuclei by indirect immunolabelling with anti-5-methylcytosine antibodies was recently developed in our laboratory. In situ analyses were performed on individual nuclei from normal and experimentally hypo- or hypermethylated cultured cells as well as on human peripheral blood B-lymphocytes from normal and chronic lymphoid leukemia (CLL) samples. We present the results obtained on cells from patients affected by different degrees of preneoplastic or neoplastic changes of the uterine cervix as compared to normal controls. The analysis of DNA methylation in individual cells from cytofuge samples was performed as follows: within each nucleus the eu- and heterochromatin methylation levels were quantified in the grey scale range by dedicated software in terms of numbers, areas and optical densities (ODs) of the immunolabeled dense heterochromatic regions ("spots"), and of the optical density of nuclear background, i.e., of nuclear euchromatin. Analogously, in randomly chosen microscope fields of tissue sections from paraffin-embedded samples, progressive tissue demethylation was observed in dysplastic and cancer cells as compared to normal ones. Both methods showed significant and progressive DNA hypomethylation in dysplastic and cancer cells as compared to control specimens.

Role and prognostic significance of CD44s expression in colorectal cancer.

UNLABELLED: The purpose of this study was to clarify the role and the predictive strength of the adhesion molecule CD44s (standard isoform) in colorectal carcinogenesis. MATERIALS AND METHODS: CD44s immunohistochemical expression was evaluated in 100 patients with colon adenoma and 100 patients with colon adenocarcinoma and adjacent non-neoplastic mucosa (ANNM). The patients were followed-up for five years. RESULTS: CD44s immunoreactivity was expressed in low-moderate-high-grade dysplasia adenomas and associated with adenocarcinoma (p = 0.01), ANNM (p = 0.05) and pTNM stage (p = 0.00001). Univariate analysis revealed that CD44s expression was associated with overall survival (OS) in carcinomas (p = 0.01) and ANNM (p = 0.05). Bivariate analysis revealed that CD44s was associated with OS in stages I and II patients (p = 0.03). Multivariate analysis revealed that stage (p = 0.0001) and CD44s expression (p = 0.05) were independent predictors of OS. CONCLUSION: CD44s is involved in colon carcinogenesis and is associated with aggressive carcinomas. The immunohistochemical expression of CD44s may reveal cells that have lost their adhesion ability and therefore detect carcinomas with high metastatic power.

Fatty acid synthase (FAS) is a marker of increased risk of recurrence in lung carcinoma.

BACKGROUND: We explored the expression of Fatty Acid Synthase (FAS) in lung carcinomas and its association with clinico-pathological features and prognosis. FAS is a recently discovered molecule involved in the energy supply of normal cells. FAS is also overexpressed in neoplastic tissues because of their increased necessity for energy. PATIENTS AND METHODS: One hundred and six patients with non-small cell lung carcinoma were followed-up for an average period of 5 years. FAS expression was detected immunohistochemically. RESULTS: FAS staining was observed in 61 out of 106 cases (57.54%). Statistical analysis revealed that FAS had an overall low prognostic value (p = 0.14), while FAS-negative expression in stage I patients showed a trend for better survival (p = 0.10). PTNM stage (p < 0.0001) was the only significant prognostic marker for overall survival. CONCLUSION: FAS is a reliable marker of low-stage clinically aggressive lung carcinomas. The determination of FAS expression in lung carcinomas may stratify patients and determine therapeutic approaches for their care.

Endothelial NOS, estrogen receptor beta, and HIFs cooperate in the activation of a prognostic transcriptional pattern in aggressive human prostate cancer.

The identification of biomarkers that distinguish between aggressive and indolent forms of prostate cancer (PCa) is crucial for diagnosis and treatment. In this study, we used cultured cells derived from prostate tissue from patients with PCa to define a molecular mechanism underlying the most aggressive form of PCa that involves the functional activation of eNOS and HIFs in association with estrogen receptor beta (ERbeta). Cells from patients with poor prognosis exhibited a constitutively hypoxic phenotype and increased NO production. Upon estrogen treatment, formation of ERbeta/eNOS, ERbeta/HIF-1alpha, or ERbeta/HIF-2alpha combinatorial complexes led to chromatin remodeling and transcriptional induction of prognostic genes. Tissue microarray analysis, using an independent cohort of patients, established a hierarchical predictive power for these proteins, with expression of eNOS plus ERbeta and nuclear eNOS plus HIF-2alpha being the most relevant indicators of adverse clinical outcome. Genetic or pharmacologic modulation of eNOS expression and activity resulted in reciprocal conversion of the transcriptional signature in cells from patients with bad or good outcome, respectively, highlighting the relevance of eNOS in PCa progression. Our work has considerable clinical relevance, since it may enable the earlier diagnosis of aggressive PCa through routine biopsy assessment of eNOS, ERbeta, and HIF-2alpha expression. Furthermore, proposing eNOS as a therapeutic target fosters innovative therapies for PCa with NO inhibitors, which are employed in preclinical trials in non-oncological diseases.

HPV prevalence among healthy Italian male sexual partners of women with cervical HPV infection.

Genital human papillomavirus (HPV) is the causative agent of cervical cancer and is the most common sexually transmitted infection. Only limited and controversial data are available regarding HPV transmission in male sexual partners of women with cervical intraepithelial neoplasia (CIN). The aim of this study was to investigate the prevalence and the genotype distribution of HPV in penile scrapings of a series of Italian men, who had no visible penile lesions and were partners of women who were affected, or had been affected previously by cervical intraepithelial neoplasia or who were infected with HPV. The concordance of the viral group in the infected partners was determined. A total of 77 penile scrapings were screened for HPV infection by the polymerase chain reaction, while 59 cervicovaginal brushings of their female partners were tested. 35% of evaluable male samples and 64% of female sexual partners were found to be HPV positive. In the 55 simultaneously evaluable couples, a concordance of 45% was found, 11 couples (20%) with both partners being HPV negative and 14 couples (25%) with both partners HPV positive (P=0.001). Six out of the 14 couples (43%), where both partners were HPV positive, harbored the same HPV genotype group. These data, although preliminary, could support further the hypothesis that male HPV infection is more frequent in sexual partners of HPV positive or women with cervical intraepithelial neoplasia indicating that men could represent an important source of HPV transmission between sex partners.

Chromogenic in situ hybridization to detect HER-2/neu gene amplification in histological and ThinPrep-processed breast cancer fine-needle aspirates: a sensitive and practical method in the trastuzumab era.

The increasing evidence of trastuzumab efficacy in breast cancer (BC) patients means that an accurate and reproducible evaluation of HER-2 statusis of paramount importance in histological and in cytological samples. Currently, the two main methods used to analyze HER-2 amplification or overexpression are fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Although the two methods are strongly correlated for histological tissue, the evaluation of tumor morphology through FISH may be difficult and fluorescence fades quickly. These limitations can be overcome by chromogenic in situ hybridization (CISH), which can visualize the amplification product along with morphological features. In view of this, in the present study, we analyzed the usefulness of CISH on formalin-fixed, paraffin-embedded (FFPE) BC specimens and investigated whether CISH can be a valid technique in the determination of HER-2 status for fine-needle aspirates (FNAs) processed by liquid-based cytology. The results we obtained in a retrospective series of 111 FFPE BC specimens demonstrated good concordance between CISH and IHC and between CISH and FISH. The former concordance was comparable with that observed between FISH and IHC. When CISH was applied to a prospective series of 53 FNAs, from surgically removed BC, our data showed evidence of a higher concordance of results between liquid-based cytology and the companion FFPE tissues using CISH rather than HercepTesttrade mark. Therefore, CISH analysis, which is avaluable and reproducible alternative to FISH for selecting breast cancer patients for trastuzumab therapy, can lower false-positive immunocytochemistry findings in ThinPrep-processed FNAs.

Fenretinide: a p53-independent way to kill cancer cells.

The synthetic retinoid fenretinide [N-(4 hydroxyphenyl)retinamide] induces apoptosis of cancer cells and acts synergistically with chemotherapeutic drugs, thus providing opportunities for novel approaches to cancer therapy. The upstream signaling events induced by fenretinide include an increase in intracellular levels of ceramide, which is subsequently metabolized to GD3. This ganglioside triggers the activation of 12-Lox (12-lipoxygenase) leading to oxidative stress and apoptosis via the induction of the transcription factor Gadd153 and the Bcl-2-family member protein Bak. Increased evidence suggests that the apoptotic pathway activated by fenretinide is p53-independent and this may represent a novel way to treat tumors resistant to DNA-damaging chemotherapeutic agents. Therefore, fenretinide offers increased clinical benefit as a novel agent for cancer therapy, able to complement the action of existing chemotherapeutic treatment regimes. Furthermore, synergy between fenretinide and chemotherapeutic drugs may facilitate the use of chemotherapeutic drugs at lower concentrations, with possible reduction in treatment-associated morbidity.

Phenotypic changes of p53, HER2, and FAS system in multiple normal tissues surrounding breast cancer.

To determine whether phenotypic field changes occur in tissues adjacent to carcinoma, we assayed, by immunohistochemistry, the expression of HER-2, p53, Fas, and FasL in 72 breast cancers (BC) and multiple autologous peritumoral tissues (PTTs) sampled up to 5 cm distance and in 44 benign breast tumors (BBTs). About 5% and 3% of the PTTs and 4.5% and 6.8% of BBTs showed alterations in HER2 and p53 expression, respectively. Of interest, gene amplification was observed in 50% of HER2 positive PTTs, but not in any HER2 positive BBTs. Fas, highly expressed in BBTs and downregulated in BC, maintained its expression in PTTs, whereas FasL, usually negative in BBTs, was upregulated in BC as well as in the PTTs closest (1 cm) to the invasive lesion. Our data suggest that FasL could be a potential novel biomarker of transformation, which may identify, along with HER2 and p53, precursor lesions in a genetically altered breast tissue.

Gangliosides link the acidic sphingomyelinase-mediated induction of ceramide to 12-lipoxygenase-dependent apoptosis of neuroblastoma in response to fenretinide.

BACKGROUND: The lipid second messenger ceramide, which is generated by acidic and neutral sphingomyelinases or ceramide synthases, is a common intermediate of many apoptotic pathways. Metabolism of ceramide involves several enzymes, including glucosylceramide synthase and GD3 synthase, and results in the formation of gangliosides (GM3, GD3, and GT3), which in turn promote the generation of reactive oxygen species (ROS) and apoptosis. Fenretinide, a retinoic acid derivative, is thought to induce apoptosis via increases in ceramide levels, but the link between ceramide and subsequent apoptosis in neuroblastoma cells is unclear. METHODS: SH-SY5Y and HTLA230 neuroblastoma cells were treated with fenretinide in the presence or absence of inhibitors of enzymes important in ceramide metabolism (fumonisin B1, inhibitor of ceramide synthase; desipramine, inhibitor of acidic and neutral sphingomyelinases; and PDMP, inhibitor of glucosylceramide). Small interfering RNAs were used to specifically block acidic sphingomyelinase or GD3 synthase activities. Apoptosis, ROS, and GD3 expression were measured by flow cytometry. RESULTS: In neuroblastoma cells, ROS generation and apoptosis were associated with fenretinide-induced increased levels of ceramide, glucosylceramide synthase activity, GD3 synthase activity, and GD3. Fenretinide also induced increased levels of GD2, a ganglioside derived from GD3. Inhibition of acidic sphingomyelinase but not of neutral sphingomyelinase or ceramide synthase, blocked fenretinide-induced increases in ceramide, ROS, and apoptosis. Exogenous GD3 induced ROS and apoptosis in SH-SY5Y cells but not in SH-SY5Y cells treated with baicalein, a specific 12-lipoxygenase inhibitor. Exogenous GD2 did not induce apoptosis. CONCLUSIONS: A novel pathway of fenretinide-induced apoptosis is mediated by acidic sphingomyelinase, glucosylceramide synthase, and GD3 synthase, which may represent targets for future drug development. GD3 may be a key signaling intermediate leading to apoptosis via the activation of 12-lipoxygenase.