Analytic and Diagnostic Performances of Human Papillomavirus E6/E7 mRNA Test on up-to 11-Year-Old Liquid-Based Cervical Samples. A Biobank-Based Longitudinal Study.

Human Papillomavirus (HPV) E6/E7 mRNA test demonstrated high specificity in detecting HPV infections, but studies assessing its efficacy in terms of cancer risk stratification are lacking. Follow-up studies are arduous and expensive. Biobank would be the answer to the problem, although data investigating the effects of long-term storage on RNA preservation are still needed. We addressed these issues by retrieving 202 residual liquid-based cervical specimens, collected from 149 women attending cervical cancer screening during the years 2001-2012. Samples were stored in Adriatic Biobank at room temperature and without any handing. After calculation of RNA yield and purity, E6/E7 mRNA test was retrospectively performed on each samples, to assess analytic and diagnostic performances. Using automated extraction procedures, RNA of good quantity and quality was obtained. The mean value of RNA concentration was 27.5 ng/µL. The mean A260/A280 ratio was 2.1. An invalid mRNA test result was found in 11.9% of the specimens. Neither RNA integrity, nor analytic performances of mRNA test were influenced by the year of sample collection. In total, 62.4% of the specimens tested as mRNA positive; among these, 89.2% were CIN2+. E6/E7 mRNA was detected in all Squamous Cervical Cancer (SCC) cases. Percentage of positive samples increased with the severity of histological diagnosis. mRNA testing, showing specificity and predictive values of 75.6% and 84.4%, respectively, significantly improved the corresponding values for DNA testing. Thus, the reflex mRNA test was demonstrated to be suitable to triage women with persistent cervical lesions. A "one sample for all" approach is possible, with practical benefits for Biobank-based long-term longitudinal studies, diseases prevention, prediction, diagnosis and treatment.

Role of E6/E7 mRNA test in the diagnostic algorithm of HPV-positive patients showing ASCUS and LSIL: clinical and economic implications in a publicly financed healthcare system.

OBJECTIVE: Colposcopy is widely used to triage women with mild cervical abnormalities. However, this approach is associated with low specificity and predictive value. The efficacy of E6/E7 mRNA test for this purpose has been demonstrated, but studies estimating its cost-effectiveness are still lacking. Given the limited healthcare financial resources, such an evaluation is a priority. METHODS: We analyzed the clinical history of 432 women referred to colposcopy and colposcopy-directed biopsy for persisting ASCUS and LSIL, and compared three alternative triage protocols: immediate colposcopy; reflex HPV DNA testing and HPV DNA plus mRNA tests in sequence. RESULTS: Molecular tests in sequence significantly reduce colposcopy referral, cost for assessed women, and cost for CIN2 detected. On the other hand, incremental cost-effectiveness ratio of this protocol was the highest. CONCLUSION: Our preliminary data, providing an estimation of the economic burden deriving from the introduction of E6/E7 mRNA test in the triage algorithm of patients with mild cervical abnormalities, may be useful for future healthcare policy.

Expression of PTEN in endometrial liquid-based cytology.

OBJECTIVES: Endometrial cytology offers a reliable alternative to biopsy in endometrial cancer detection and it may be useful in obtaining material to study prognostic and predictive markers. Over the years, new sampling devices have been developed. Molecular alterations in endometrial cancers were previously described using formalin-fixed paraffin-embedded tissues with particular attention, in endometrioid carcinomas, to the PTEN-PI3K pathway. PTEN evaluation could be useful in endometrial carcinomas for selecting patients for target therapies. STUDY DESIGN: We studied 51 endometrial samples collected using the Endogyn device and 71 obtained with the Endoflower dispositive device, and processed using liquid-based cytology. Most of the cases were matched with a corresponding histological biopsy. The overall accuracy of Endoflower was 100%. Immunohistochemistry (IHC) and immunocytochemistry (ICC) for PTEN were performed using monoclonal antibody 6H2.1 from DAKO. RESULTS: The IHC showed PTEN-null glands in 4 cases. The same cancers were negative in ICC. Among the 10 carcinomas on cytology, PTEN-null glands were found in 1 case. All the normal endometrium control cases were positive in cytology and histology. CONCLUSIONS: Our results suggest that endometrial devices provide useful material for the diagnosis and evaluation of PTEN expression.

A very rare case of HPV-53-related cervical cancer, in a 79-year-old woman with a previous history of negative Pap cytology.

The introduction of organized cervical cancer (CC) screening programs has drastically reduced the prevalence of CC. However the incidence is still too high, especially among elderly women. All guidelines strongly recommend a regular Papanicolaou (Pap) testing for young and middle-aged patients. On the other hand, many international professional societies no longer advise screening in women who have undergone hysterectomy, and in women aged 65 years and above, who have a previous history of regular Pap smears. Here we report the case of poorly differentiated CC, involving the pelvic lymph nodes and urinary bladder, occurring in a 79-year-old woman who regularly underwent Pap tests, with no reported cytological abnormalities. In this very rare case, the CC cells, as well as cells from metastatic lymph nodes and cells from urinary specimens, molecularly showed human papilloma virus (HPV)-53. With the limitations of a single case, this report brings important information to prevent CC in elderly patients: the utility of molecular tests to increase sensitivity of Pap smears in postmenopausal women; the importance of HPV-53 as one of the four "emergent" genotypes having a possible role in oncogenesis; and the presence of HPV-53 in lymph node metastases from cervical carcinoma, which would support the role of this virus in the maintenance of malignant status.

Chromogenic in situ hybridization and p16/Ki67 dual staining on formalin-fixed paraffin-embedded cervical specimens: correlation with HPV-DNA test, E6/E7 mRNA test, and potential clinical applications.

Although HPV-DNA test and E6/E7 mRNA analyses remain the current standard for the confirmation of human papillomavirus (HPV) infections in cytological specimens, no universally adopted techniques exist for the detection of HPV in formalin-fixed paraffin-embedded samples. Particularly, in routine laboratories, molecular assays are still time-consuming and would require a high level of expertise. In this study, we investigated the possible use of a novel HPV tyramide-based chromogenic in situ hybridization (CISH) technology to locate HPV on tissue specimens. Then, we evaluate the potential usefulness of p16(INK4a)/Ki-67 double stain on histological samples, to identify cervical cells expressing HPV E6/E7 oncogenes. In our series, CISH showed a clear signal in 95.2% of the specimens and reached a sensitivity of 86.5%. CISH positivity always matched with HPV-DNA positivity, while 100% of cases with punctated signal joined with cervical intraepithelial neoplasia grade 2 or worse (CIN2+). p16/Ki67 immunohistochemistry gave an interpretable result in 100% of the cases. The use of dual stain significantly increased the agreement between pathologists, which reached 100%. Concordance between dual stain and E6/E7 mRNA test was 89%. In our series, both CISH and p16(INK4a)/Ki67 dual stain demonstrated high grade of performances. In particular, CISH would help to distinguish episomal from integrated HPV, in order to allow conclusions regarding the prognosis of the lesion, while p16(INK4a)/Ki67 dual stain approach would confer a high level of standardization to the diagnostic procedure.

Prognostic value of HPV E6/E7 mRNA assay in women with negative colposcopy or CIN1 histology result: a follow-up study.

Pap test, and especially HPV DNA test, identify a large group of women who do not have any clinically relevant lesions, i.e., CIN2+ (Cervical Intraepithelial Neoplasia grade 2 or worse), but who are at greater risk of getting lesions in the future. The follow up of these women needs new biomarkers with prognostic value. The objective of this study is to evaluate the prognostic value of E6/E7 mRNA over-expression assay (PreTect HPV-Proofer, Norchip) for 5 HR-HPV types (16, 18, 31, 33, and 45) for progression to CIN2+ after a negative colposcopy. This prospective study, conducted at four Italian centres, enrolled 673 women with either a negative colposcopy or a negative or CIN1 histology. The clinical end-point was histological confirmation of CIN2+. Women were classified at baseline according to mRNA results and managed according to local colposcopy protocols. At least one conclusive follow-up test was obtained for 347 women (25 months average lapse since recruitment, range 5-74). Only seven CIN2+ were detected during follow up, three among the 82 women positive for mRNA at baseline, two among the 250 negative (Fisher exact test, p = 0.02), and two among the 12 with an invalid test. Absolute CIN2+ risk was 6.7/1,000 person/years in the whole cohort. The absolute CIN2+ risk was 18.4/1,000 person/years and 3.6/1,000 person/years in mRNA-positive and mRNA-negative women, respectively. In conclusion, E6/E7 mRNA over-expression appears to be a good candidate as a prognostic biomarker to manage HR-HPV DNA-positive women with negative colposcopy or histology, particularly in order to decrease follow-up intensity in those who are negative.

Comparison of oncogenic HPV type-specific viral DNA load and E6/E7 mRNA detection in cervical samples: results from a multicenter study.

High-risk human papillomavirus (HR-HPV) genotype viral load and E6/E7 mRNA detection are proposed as surrogate markers of malignant cervical lesion progression. Currently, the use of commercially available DNA-based or mRNA-based tests is under investigation. In this study, the viral DNA load and E6/E7 mRNA detection of the five most common HR-HPV types detected in cervical cancer worldwide were compared in 308 cervical samples by using in-house type-specific quantitative real-time PCR assays and PreTect HPV-Proofer test, respectively. Sensitivity and negative predictive values were higher for the HPV-DNA assays combined (95.0% and 96.0%, respectively) than the RNA assays (77.0% and 88.0%, respectively); conversely, the mRNA test showed a higher specificity and higher positive predictive value (81.7% and 66.9%, respectively) than the DNA test (58.6% and 52.5%, respectively) for detecting histology-confirmed high-grade cervical intraepithelial neoplasia. A significantly higher association between viral DNA load and severity of disease was observed for HPV 16 and 31 (γ = 0.62 and γ = 0.40, respectively) than for the other HPV types screened. A good degree of association between the two assays was found for detection of HPV 16 (k = 0.83), HPV 18 (k = 0.72), HPV 33 (k = 0.66), and HPV 45 (k = 0.60) but not for HPV 31 (k = 0.24). Sequence analysis in L1 and E6-LCR regions of HPV 31 genotypes showed a high level of intra-type variation. HR-HPV viral DNA load was significantly higher in E6/E7 mRNA positive than negative samples (P < 0.001), except for HPV 31. These findings suggest that transcriptional and replicative activities can coexist within the same sample.

Molecular alterations in endometrial archived liquid-based cytology.

Endometrial cancer is one of the most common gynecological malignancy worldwide and its prevalence is increasing. The introduction of liquid-based cytology (LBC) and endoflower dispositive in routine practice gives the possibility to examine endometrial cells by cytological diagnosis and may also release the opportunity to study molecular alterations, in endometrioid type cancer in which carcinogenesis is well known. We gathered 72 cases of endometrial LBC samples and corresponding formalin-fixed paraffin-embedded (FFPE) blocks, collected from 2004 to 2010. DNA was isolated from both samples using standard protocols. DNA quality and quantity were assessed using Nanodrop and BIOMED2 multiplex PCR. Mutations in exon 5 of PTEN and exon 20 of PI3K were studied using Sanger sequencing. DNA with good quality and amount was isolated from 67/72 FFPE cases. In these samples, two cases were found to harbor mutations in exon 5 of PTEN. No PI3K mutations were identified. LBC samples were then assessed to verify the concordance with the FFPE DNA results. The results obtained were concordant, that is the wild type cases in FFPE were also wild type in LBC and vice versa for the mutated case. Unfortunately, the second case of mutation in PTEN could not be confirmed in LBC due to low amount of DNA obtained. Detection of molecular alterations in LBC will open a new era for the detection in asymptomatic women of precursor lesions that could evolve into cancer and for endometrial cancer diagnosis and screening in selected high-risk women.

Implementing specificity of HPV-DNA primary screening in a successful organised cervical cancer prevention programme.

OBJECTIVE: This two-arm longitudinal study was performed within a regional organized cervical-cancer-prevention program in which HPV-DNA test is used in primary screening. The aim was to analyze the diagnostic performances of p16INK4a/Ki-67 dual-test and E6/E7-mRNA test in identifying CIN2+ lesion among HPV-DNA positive (HPV-DNAve) women triaged for LSIL-or-worse liquid based cytology (LBC). METHODS: Thirty-six thousand thirty-one women participated to HPV-DNA screening program pilot study. Three thousand six hundred forty-one resulted HPV-DNAve; among these, 43% were LSIL-or-worse (LSIL+). HPV-DNAve/LSIL+ patients were submitted to colposcopy and histological assessment of any visible lesions. Dual-test was performed on 794 residual LBC specimens. In 405 cases, dual-test result was related to histology, considering CIN2+ as endpoint. mRNA test has been carried out retrospectively, on a subset of 173 residual LBC specimens. RESULTS: Agreement between dual-test and histological diagnosis was 59%. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of cytology-plus-dual-test approach were 62.3%, 76.8%, 63.1% and 84.2%, respectively. Dual-test improved specificity, PPV and NPV of cytological triage Agreement between mRNA testing and histology was 65%. Cytology-plus mRNA testing showing sensitivity, specificity, PPV and NPV reaching 32.1%, 94.9%, 75% and 50%, respectively; implemented specificity and PPV of cytology alone in triaging DNA-ve/LSIL+ patients (p<0.01). CONCLUSIONS: We provided promising data indicating the important role that p16(INK4)/Ki-67 dual-test, and mostly E6/E7 mRNA test, might have in triaging HPV-DNAve. These approaches would exclude the occurrence of cervical cancer and would avoid overtreatment, at the same time. Further longitudinal analysis has to be considered.

Detection of residual/recurrent cervical disease after successful LEEP conization: the possible role of mRNA-HPV test.

BACKGROUND: Loop Electrosurgical Excision Procedure (LEEP) represents the mainstay technique for CIN2+ removal. The major concern in conservative treatment is to verify whether CIN eradication was complete, since incomplete excision is associated with an increased risk of cervical cancer. The histopathologic evaluation of resection margins status is far from perfect, since cervical lesions may recur in 5-15% of patients who had conisation specimens with clean margins. Current follow-up protocol of patients treated by conisation for high grade CIN is manly based on the combination of cytology-plus- HPV-DNA testing. This approach showed high sensitivity but low specificity level in detecting recurrence. The consequence were overdiagnosis and overtreatment, especially in youngest women, in which spontaneous regression rate of CIN is substantial. In this longitudinal study we investigated whether patient's age, cone depth and pre-conisation HPV-load level, may be used as predictive markers for residual/recurrent CIN after conisation. Then we aimed to examined the role of E6/E7 mRNA testing during post-conization follow-up. METHODS: The study, focused on the outcome of 116 patients treated for CIN by LEEP, included three consecutive steps. Firstly, the authors analysed the prevalence of residual/recurrence disease after conization; then, they investigated which factors may influence treatment failure even when resection margins were clean; finally, they evaluated the diagnostic accuracy of E6/E7 mRNA test as predictive marker of recurrence. RESULTS: HPV infection was detected in 31% of patients at 6-month follow-up and in 11.2% of patients, at 24-month follow-up. Younger women showed higher rate of recurrence than older ones. The risk of residual/recurrent infection did not correlate with cone-depth. Recurrence is higher in patients with low viral load level than in those having high load levels. mRNA test showed higher specificity and positive predictive value than the combination cytology-plus-HPV-DNA test. CONCLUSION: The inclusion of mRNA test within the current protocol of follow-up would efficiently and earlier predict the risk of residual/ recurrent cervical abnormalities after conisation. This molecular strategy would also reduce overtreatment, particularly in patients above 30 years of age.

Effective assessment of egfr mutation status in bronchoalveolar lavage and pleural fluids by next-generation sequencing.

PURPOSE: The therapeutic choice for patients with lung adenocarcinoma depends on the presence of EGF receptor (EGFR) mutations. In many cases, only cytologic samples are available for molecular diagnosis. Bronchoalveolar lavage (BAL) and pleural fluid, which represent a considerable proportion of cytologic specimens, cannot always be used for molecular testing because of low rate of tumor cells. EXPERIMENTAL DESIGN: We tested the feasibility of EGFR mutation analysis on BAL and pleural fluid samples by next-generation sequencing (NGS), an innovative and extremely sensitive platform. The study was devised to extend the EGFR test to those patients who could not get it due to the paucity of biologic material. A series of 830 lung cytology specimens was used to select 48 samples (BAL and pleural fluid) from patients with EGFR mutations in resected tumors. These samples included 36 cases with 0.3% to 9% of neoplastic cells (series A) and 12 cases without evidence of tumor (series B). All samples were analyzed by Sanger sequencing and NGS on 454 Roche platform. A mean of 21,130 ± 2,370 sequences per sample were obtained by NGS. RESULTS: In series A, EGFR mutations were detected in 16% of cases by Sanger sequencing and in 81% of cases by NGS. Seventy-seven percent of cases found to be negative by Sanger sequencing showed mutations by NGS. In series B, all samples were negative for EGFR mutation by Sanger sequencing whereas 42% of them were positive by NGS. CONCLUSIONS: The very sensitive EGFR-NGS assay may open up to the possibility of specific treatments for patients otherwise doomed to re-biopsies or nontargeted therapies.

Detection of nuclear and membrane antigens by liquid-based cytology following long-term storage of d1 cells, karpas cells, and peripheral blood mononuclear cells.

Immunofluorescence is the most frequently utilized technique to analyze protein expression. Fixed immunofluorescent cell suspensions, however, can only be stored for a week. We investigated whether liquid-based cytology could be used to detect antigens in cultured cells after a long storage period. Murine and human cells were fixed in PreservCyt solution, stored for various periods, and then used to perform an automated immunocytochemical analysis. Phosphorylation of the nuclear transcription factor Stat-5 induced by IL-7 was detected up to 4 months after IL-7 stimulation. Simultaneous nuclear positivity for the proliferation index MIB-1 and membrane positivity for the CD30 antigen were evident three months after fixation. Liquid-based cytology thus ensures long-lasting nuclear and membrane antigen immunoreactivity and permits the storage of cells from laborious experiments at room temperature for future analyses.

Sensitivity, specificity, and clinical value of human papillomavirus (HPV) E6/E7 mRNA assay as a triage test for cervical cytology and HPV DNA test.

There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2+). The proportion of positive results and the sensitivity and specificity for CIN2+ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] = 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI = 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI = 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI = 74 to 83%) and 69% (95% CI = 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI = 32 to 44%) and by 15% (95% CI = 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI = 60 to 66%), having 68% sensitivity (95% CI = 61 to 75%), whereas cytology at the ASC-US+ threshold reduced colposcopies by 23% (95% CI = 20 to 26%), showing 92% sensitivity (95% CI = 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of HPV DNA positive-mRNA negative cases.

Thin-layer cytopathology of a gastrointestinal stromal tumor (GIST) in effusion: diagnostic dilemmas.

Although gastrointestinal stromal tumors (GISTs) are uncommon, they represent the most frequent mesenchymal neoplasms of the gastrointestinal tract. During recent years, considerable information has been published about the pathogenesis, molecular biology, histological criteria, surgery, and adjuvant pharmacological treatment of GISTs, but there have been few reports about the cytologic diagnosis of GISTs, particularly in effusions; in such specimens these neoplasms cause a wide range of potential pitfalls. In this case report, we show that by combining morphological and immunocytochemical studies on thin layer slide preparations, the cytologic diagnosis of GISTs can be both accurate and efficient.

IL-12 can target human lung adenocarcinoma cells and normal bronchial epithelial cells surrounding tumor lesions.

BACKGROUND: Non small cell lung cancer (NSCLC) is a leading cause of cancer death. We have shown previously that IL-12rb2 KO mice develop spontaneously lung adenocarcinomas or bronchioalveolar carcinomas. Aim of the study was to investigate i) IL-12Rbeta2 expression in human primary lung adenocarcinomas and in their counterparts, i.e. normal bronchial epithelial cells (NBEC), ii) the direct anti-tumor activity of IL-12 on lung adenocarcinoma cells in vitro and vivo, and the mechanisms involved, and iii) IL-12 activity on NBEC. METHODOLOGY/PRINCIPAL FINDINGS: Stage I lung adenocarcinomas showed significantly (P = 0.012) higher frequency of IL-12Rbeta2 expressing samples than stage II/III tumors. IL-12 treatment of IL-12R(+) neoplastic cells isolated from primary adenocarcinoma (n = 6) inhibited angiogenesis in vitro through down-regulation of different pro-angiogenic genes (e.g. IL-6, VEGF-C, VEGF-D, and laminin-5), as assessed by chorioallantoic membrane (CAM) assay and PCR array. In order to perform in vivo studies, the Calu6 NSCLC cell line was transfected with the IL-12RB2 containing plasmid (Calu6/beta2). Similar to that observed in primary tumors, IL-12 treatment of Calu6/beta2(+) cells inhibited angiogenesis in vitro. Tumors formed by Calu6/beta2 cells in SCID/NOD mice, inoculated subcutaneously or orthotopically, were significantly smaller following IL-12 vs PBS treatment due to inhibition of angiogenesis, and of IL-6 and VEGF-C production. Explanted tumors were studied by histology, immuno-histochemistry and PCR array. NBEC cells were isolated and cultured from lung specimens of non neoplastic origin. NBEC expressed IL-12R and released constitutively tumor promoting cytokines (e.g. IL-6 and CCL2). Treatment of NBEC with IL-12 down-regulated production of these cytokines. CONCLUSIONS: This study demonstrates that IL-12 inhibits directly the growth of human lung adenocarcinoma and targets the adjacent NBEC. These novel anti-tumor activities of IL-12 add to the well known immune-modulatory properties of the cytokine and may provide a rational basis for the development of a clinical trial.

The lack of epithelial interleukin-7 and BAFF/BLyS gene expression in prostate cancer as a possible mechanism of tumor escape from immunosurveillance.

PURPOSE: The human prostate is endowed with intraepithelial and stromal lymphocytes, which may develop lymphoid follicles (LF) and allow a local immune response. We sought to investigate whether interleukin (IL)-7 and BAFF/BLyS, two fundamental survival factors for T and B cells, are expressed in the normal and neoplastic prostate and affect intraprostatic lymphocyte homeostasis. EXPERIMENTAL DESIGN: We have used real-time reverse transcription-PCR of microdissected prostatic glands and confocal microscopy to detect cytokine production, combined with immunohistochemistry to characterize intraprostatic lymphocytes. RESULTS: Prostatic epithelia constitutively produce IL-7 and, to a lesser extent, BAFF/BLyS. Indeed, we show that IL-7 receptor alpha is expressed by intraepithelial T lymphocytes and parafollicular T cells, whereas BAFF-R is found on periglandular B lymphocytes and mantle zone B cells of LFs. Prostate-homing B and T lymphocytes are scarcely proliferating, whereas most of them express the antiapoptotic protein bcl-2 and reveal a low apoptotic index in the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The transition from normal to neoplastic glands in prostate cancer (PCa) is marked by a dramatic decline of IL-7 and BAFF/BLyS production. Accordingly, PCa is characterized by a significant reduction of intraepithelial lymphocytes and loss of LFs. B-cell and T-cell expression of bcl-2 decrease, whereas the apoptotic events increase. The remaining PCa-infiltrating lymphocytes are mostly CD8(+) T cells that lack terminal differentiation and barely penetrate neoplastic glands. CONCLUSIONS: These results suggest that epithelial IL-7 and BAFF/BLyS production support intraprostatic lymphocyte survival. Its loss in PCa is associated with a severe depletion of prostate-associated lymphocytes and points to a novel tumor escape mechanism.

Cervical cancer screening: from molecular basis to diagnostic practice, going through new technologies.

In the era of Human Papillomavirus (HPV) vaccination, a lot of misunderstanding still exists among healthcare professionals and patients regarding HPV infection. The purpose of this review is to synthesize the clinical molecular mechanisms that contribute to HPV-mediated cervical carcinogenesis, as well as to appraise the current status of new biomarkers and technologies in terms of available data on clinical applications and future promises.

Mutational analysis of the HER2 gene in lung tumors from Caucasian patients: mutations are mainly present in adenocarcinomas with bronchioloalveolar features.

Activating mutations in the tyrosine kinase domain of the HER2 gene have recently been reported in lung adenocarcinomas, mainly in East Asian patients. Our study was devised to evaluate the prevalence and nature of HER2 mutations in lung adenocarcinomas from Caucasian patients. The mutational status of the HER2 gene was evaluated in 403 lung adenocarcinomas by PCR-single strand conformation polymorphism analysis and direct sequencing of Exons 19 and 20. We found HER2 mutations in 9 (2.2%) cases. Seven (78%) of the mutations were in frame duplications/insertions at codons 776-779 (YVMA), the other 2 were base substitutions resulting in aminoacid changes. The hotspot mutation at bases 776-779 was previously found to be the most frequent HER2 mutation in Asiatic patients. The distribution of mutations was significantly different between conventional lung adenocarcinomas (CLAs) and lung adenocarcinomas with bronchioloalveolar features (ABAFs). Seven (6.2%) of 113 ABAFs and 2 (0.7%) of 290 CLA were mutated (p = 0.0025). In addition, the frequency of HER2 mutations was slightly higher in females (4.1%) than in males (1.8%) and in never smokers (3.1%) than in smokers (1.9%), but differences were not statistically significant. This series of tumors was also investigated for EGFR and K-ras mutations. EGFR mutations were observed in 43 (10.7%) cases, and K-ras mutations in 110 (27.3%) cases. EGFR, HER2 and K-ras mutations were found to be mutually exclusive events. The presence of HER2 mutations in a subset of patients with lung adenocarcinoma raise hope to treat these patients with HER2 specific kinase inhibitors.

Int6 expression can predict survival in early-stage non-small cell lung cancer patients.

PURPOSE: The Int6 gene was originally identified as a common insertion site for the mouse mammary tumor virus in virally induced mouse mammary tumors. Recent studies indicate that Int6 is a multifaceted protein involved in the regulation of protein translation and degradation through binding with three complexes: the eukaryotic translation initiation factor 3, the proteasome regulatory lid, and the constitutive photomorphogenesis 9 signalosome. This study aimed to investigate the prognostic role of Int6 in a large series of stage I non-small cell lung cancers (NSCLC) patients with long-term follow-up. EXPERIMENTAL DESIGN: We determined the methylation status of Int6 DNA by methylation-specific PCR and the steady-state levels of Int6 RNA by quantitative real-time reverse transcription-PCR in 101 NSCLCs and matched normal lung tissues. RESULTS: In 27% of the tumors, Int6 RNA levels were reduced relative to normal tissue. In 85% of the tumors with reduced Int6 expression, the transcription promoter and first exon were hypermethylated, whereas only 4% of the tumors with elevated Int6 RNA levels were hypermethylated (P <0.000001). Low levels of Int6 RNA were found a significant predictor of overall and disease-free survival (P=0.0004 and P=0.0020, respectively). A multivariate analysis confirmed that low Int6 expression was the only independent factor to predict poor prognosis, for both overall (P=0.0006) and disease-free (P=0.024) survival. CONCLUSIONS: Our results suggest that Int6 expression, evaluated by quantitative real-time PCR, may represent a new prognostic factor in patients with stage I NSCLC.