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PDF file 100K, S2: Generation of CRC cell lines with high expression of CXCR4 and/or CXCR7

Purpose: In colorectal cancer, increased expression of the CXC chemokine receptor 4 (CXCR4) has been shown to provoke metastatic disease due to the interaction with its ligand stromal cell-derived factor-1 (SDF-1). Recently, a second SDF-1 receptor, CXCR7, was found to enhance tumor growth in solid tumors. Albeit signaling cascades via SDF-1/CXCR4 have been intensively studied, the significance of the SDF-1/CXCR7–induced intracellular communication triggering malignancy is still only marginally understood.Experimental Design: In tumor tissue of 52 patients with colorectal cancer, we observed that expression of CXCR7 and CXCR4 increased with tumor stage and tumor size. Asking whether activation of CXCR4 or CXCR7 might result in a similar expression pattern, we performed microarray expression analyses using lentivirally CXCR4- and/or CXCR7-overexpressing SW480 colon cancer cell lines with and without stimulation by SDF-1α.Results: Gene regulation via SDF-1α/CXCR4 and SDF-1α/CXCR7 was completely different and partly antidromic. Differentially regulated genes were assigned by gene ontology to migration, proliferation, and lipid metabolic processes. Expressions of AKR1C3, AXL, C5, IGFBP7, IL24, RRAS, and TNNC1 were confirmed by quantitative real-time PCR. Using the in silico gene set enrichment analysis, we showed that expressions of miR-217 and miR-218 were increased in CXCR4 and reduced in CXCR7 cells after stimulation with SDF-1α. Functionally, exposure to SDF-1α increased invasiveness of CXCR4 and CXCR7 cells, AXL knockdown hampered invasion. Compared with controls, CXCR4 cells showed increased sensitivity against 5-FU, whereas CXCR7 cells were more chemoresistant.Conclusions: These opposing results for CXCR4- or CXCR7-overexpressing colon carcinoma cells demand an unexpected attention in the clinical application of chemokine receptor antagonists such as plerixafor. Clin Cancer Res; 20(3); 604–16. ©2013 AACR.

Pancreatic cysts or dilated pancreatic ducts are often found by cross-sectional imaging, but only mucinous lesions can become malignant. Therefore, distinction between mucinous and non-mucinous lesions is crucial for adequate patient management. We performed a prospective study including targeted next generation sequencing (NGS) of cell-free DNA in the diagnostic endoscopic ultrasound (EUS)-guided workup. Pancreatic cyst(s) or main duct fluid obtained by EUS-guided FNA was analysed by carcinoembryonic antigen (CEA), cytology and deep targeted NGS of 14 known gastrointestinal cancer genes (AKT1, BRAF, CTNNB1, EGFR, ERBB2, FBXW7, GNAS, KRAS, MAP2K1, NRAS, PIK3CA, SMAD4, TP53, APC) with a limit of detection down to variant allele frequency of 0.01%. Results were correlated to histopathology and clinical follow-up. One hundred and thirteen patients with pancreatic cyst(s) and/or a dilated pancreatic main duct (≥5 mm) were screened. Sixty-six patients had to be excluded, mainly due to inoperability or small cyst size (≤10 mm). Forty-seven patients were enrolled for further analysis. A final diagnosis was available in 27 cases including 8 negative controls. In 43/47 (91.5%) of patients a KRAS- and/or GNAS-mutation was diagnosed by NGS. 27.0% of the KRAS-mutated and 10.0% of the GNAS-mutated lesions harbored multiple mutations. KRAS/GNAS-testing by NGS, cytology, and CEA had a sensitivity and specificity of 94.7/100%, 38.1/100%, and 42.1/75.0%, respectively. KRAS/GNAS-testing was significantly superior to CEA (P = .0209) and cytology (P = .0016). In conclusion, KRAS/GNAS-testing by deep targeted NGS is a suitable method to distinguish mucinous from non-mucinous pancreatic lesions, suggesting its usage as a single diagnostic test. Results must be confirmed in a larger cohort.

Introduction: Survival in gastric cancer is often limited by peritoneal carcinomatosis, which supposedly develops from serosal tumor infiltration or tumor cell spread during gastrectomy with lymphadenectomy. To eliminate peritoneal tumor cells, extensive intraperitoneal lavage (EIPL) has been suggested. Impressive results have been achieved in Japanese trials. In this trial, we assessed EIPL in Western patients. Methods: This prospective trial included patients with non-metastatic gastric adenocarcinoma undergoing gastrectomy with D2 lymphadenectomy. Peritoneal fluid samples at laparotomy, after lymphadenectomy, and after EIPL were analyzed for tumor cells using cytology and EpCAM antibodies. The primary endpoint was peritoneal conversion rate (PCR; proportion of patients in whom EIPL eliminated tumor cells after lymphadenectomy). Secondary endpoints were peritoneal release rate (PRR; proportion of patients with peritoneal tumor cells after gastrectomy/lymphadenectomy among all patients without cells before gastrectomy/lymphadenectomy) and prevalence of peritoneal tumor cells before resection. EIPL was considered ineffective if PCR ⩽ 0.2 and warranted further exploration if PCR ⩾ 0.5. Clinicaltrials.gov identifier is NCT01476553. Results: The trial was stopped early because tumor cells after gastrectomy/lymphadenectomy were detected in only 3/27 (11.1%) patients. In none of these did EIPL eliminate tumor cells (PCR 0, 95% confidence interval [CI] 0%–12.5%). In 8/27 (29.6%) patients, tumor cells were detected after EIPL. PRR was 11.1% (95% CI 2.4%–29.2%). There were no perioperative complications higher than Clavien-Dindo grade 3a. Conclusions: In Western patients, free peritoneal tumor cells after gastrectomy with D2 lymphadenectomy for gastric cancer were detected only sporadically. Although based on few cases, the findings suggest that EIPL spreads tumor cells into the peritoneal cavity, thus being potentially harmful. Therefore, EIPL cannot be generally recommended.

We analyzed the performance of p16INK4a immunocytochemistry on a series of 810 retrospectively collected atypical squamous cells of undetermined significance (ASC-US) and low-grade squamous intraepithelial lesion (LSIL) cases with available biopsy follow-up data, including 94 cases of cervical intraepithelial neoplasia (CIN) 2 and 128 cases of CIN 3. Human papillomavirus (HPV) testing was performed from the same residual liquid-based cytologic specimen, and results for both tests were correlated with histologic follow-up data. Sensitivity values for high-grade CIN (HGCIN) confirmed on biopsy within 6 months were 92.6% (ASC-US) and 92.2% (LSIL) for cytotechnologists’ reviews of p16 cytology and 90.1% (ASC-US) and 95.7% (LSIL) for HPV testing. Sensitivity rates of initial pathologists’ reviews were slightly lower, 76.4% to 80.1%, with levels comparable to cytotechnologists’ results after adjudication. The specificity of p16 cytology for HGCIN detection was significantly higher than for HPV testing for cytotechnologists and pathologists: 63.2% to 71.1% (p16 cytology) vs 37.8% for HPV in ASC-US (P < .001) and 37.3% to 53.3% (p16 cytology) vs 18.5% for HPV in LSIL (P < .001). This evaluation of the diagnostic performance of p16 cytology confirms the potential of this stain for the efficient triage of ASC-US and LSIL cytologic results.

Background: While intensity modulated radiotherapy (IMRT) in anal cancer is feasible and improves high-dose conformality, the current RTOG/AGITG contouring atlas and planning guidelines lack specific instructions on how to proceed with external genitalia. Meanwhile, the RTOG-Protocol 0529 explicitly recommends genital sparing on the basis of specific genital dose constraints. Recent pattern-of-relapse studies based on conventional techniques suggest that marginal miss might be a potential consequence of genital sparing. Our goal is to outline the potential scope and increase the awareness for this clinical issue. Methods: We present and discuss four patients with perigenital spread in anal cancer in both early and advanced stages (three at time of first diagnosis and one in form of relapse). Genital/perigenital spread was observed once as direct genital infiltration and thrice in form of perigenital lymphatic spread. Results: We review the available data regarding the potential consequences of genital sparing in anal cancer. Pattern-of-relapse studies in anal cancer after conventional radiotherapy and the current use of IMRT in anal cancer are equivocal but suggest that genital sparing may occasionally result in marginal miss. An obvious hypothesis suggested by our report is that perigenital lymphovascular invasion might be associated with manifest inguinal N+ disease. Conclusions: Local failure has low salvage rates in recent anal cancer treatment series. Perigenital spread may pose a risk of marginal misses in IMRT in anal cancer. To prevent marginal misses, meticulous pattern-of-relapse analyses of controlled IMRT-series are warranted. Until their publication, genital sparing should be applied with caution, PET/CT should be used when possible and meeting genital dose constraints should not be prioritized over CTV coverage, especially (but not only) in stage T3/4 and N+ disease.

The purpose of the study is to test the hypothesis that expression of cell cycle regulatory proteins p16(INK4a) and pRb is significantly associated with prognosis in ovarian carcinomas. We performed immunohistochemical analysis of p16(INK4a) and pRb expression and correlated with survival in a series of 300 patients with FIGO stage IIb-IV ovarian carcinoma which were enrolled in a randomized prospective trial evaluating two different platinum and paxlitaxel chemotherapy combinations after radical surgery. p16(INK4a) negative tumours (17/300; 6%) had a significantly worse prognosis (univariate analysis, P0.001; multivariate analysis: odds ratio 2.41, P=0.009). Among p16(INK4a)-positive tumours (283 out of 300; 94%), survival was better for patients with intermediate expression as compared to low or high expression levels (P=0.001). High expression levels of pRb were associated with an incremental deterioration of prognosis (univariate analysis, P=0.004; multivariate analysis: odds ratio 2.98, P=0.002). This observation held also true in the subgroup of optimally debulked patients (n=82), in whom the most important established prognostic factor, postoperative residual tumour cannot be applied. In conclusion p16(INK4a) and pRb are independent prognostic factors in advanced-stage ovarian carcinomas after radical surgery and postoperative chemotherapy. High pRb expression is a significant prognosticator in optimally debulked patients and may hold potential for subgroup stratification in postoperative treatment.

Bestimmte Typen der humanen Papillomviren (so genannte humane Papillomviren vom Hochrisikotyp: HR-HPV). spielen eine zentrale Rolle bei der Entstehung des Zervixkarzinoms. Zwar sind diese Viren in der Bevolkerung sehr weit verbreitet, jedoch entwickelt sich nach einer HPV-Infektion eher selten eine klinisch relevante Lasion. Fur die Entstehung einer Dysplasie ist die deregulierte Expression der viralen Onkoproteine E6 und E7 in epithelialen Stammzellen erforderlich. Im Verlauf einer akuten Infektion werden diese Gene aber normalerweise nur in differenzierten und zellzyklusarretierten Zellen der oberen Schichten des Epithels exprimiert. Kommt es dagegen zur Aktivierung dieser Gene in den proliferierenden Basal- und Parabasalzellen, kann es durch Beeintrachtigung der Zellzyklusregulation, des mitotischen Spindelapparats und der Zentrosomenfunktionen zur Storung der Chromosomenverteilung und -struktur wahrend der Mitose kommen. Dies fuhrt zur chromosomalen Instabilitat und bereitet den Boden fur die anschliesende Karzinogenese. Die Expression des HPV-E7-Onkoproteins bewirkt eine massive Uberexpression des zellularen p16INK4a-Proteins, das sich daher zunehmend als geeigneter Surrogatmarker fur die Diagnostik in Zytologie und Histologie erweist. Diese Ubersicht fasst die Mechanismen der HPV-assoziierten Transformation zusammen und legt die sich daraus ergebende Moglichkeiten fur die Diagnostik des Zerivxkarzinoms dar.

Objective Transitional cell carcinoma (TCC) of the ovary is a less well recognized histological type of ovarian carcinoma resembling TCC of the urinary bladder. A better prognosis due to a better chemosensitivity of ovarian TCC has been suggested. It was the aim of the present retrospective study to compare incidence and outcome of patients with TCCs and other subtypes of ovarian carcinoma from a large homogeneous collective of patients with primary advanced-stage ovarian carcinoma. Methods H&E-stained sections from a total of 302 cases from a prospective randomized, multi-center, phase III study of patients with ovarian cancer, FIGO-stages IIB–IV, comparing cisplatin plus paclitaxel (PT) with paclitaxel plus carboplatin (TC) were available for histological retyping of ovarian carcinomas applying current WHO criteria. Kaplan–Meier survival analysis was performed. Results 16 of 302 tumors (5.3%) were diagnosed as TCC. Only 1 of the 16 TCCs had been previously diagnosed as such by referring pathologists. TCCs were associated with smaller preoperative extraovarian tumor and with smaller postoperative residual tumor. 5-year survival of patients with TCC was 57% as compared to 31% for patients with ovarian carcinomas of other types ( P = 0.03). Conclusion TCC of the ovary seems to be a less well recognized entity. In the current series, TCCs had a significantly better prognosis as compared to all other types of ovarian carcinomas after standardized chemotherapy. A propensity for micronodular rather than macronodular extraovarian spread and better surgical resectability of TCC might contribute to the survival benefit.

OBJECTIVE: Overexpression of p16 INK4a has been proposed as a biomarker helpful for the identification of dysplastic cervical epithelial cells on histologic slides as well as in cervical smears. Since a few nontransformed cells in the genital tract in some instances may also express p16 INK4a , we evaluated whether applying established morphologic criteria for cervical dysplasia allows a distinction of dysplastic from nondysplastic p16 INK4a -stained cells in cytologic samples. STUDY DESIGN: Liquid-based cytology samples were obtained from a screening population (n = 50), and from patients attending a dysplasia clinic (n = 40). Slides prepared from these samples were stained with the conventional Papanicolaou stain procedure. From each specimen, a second slide was prepared in parallel and immunostained for p16 INK4a . Cytologic diagnoses for most patients attending the dysplasia clinic could be compared to the reported histologic diagnoses on punch biopsy samples taken from the patients at the time of colposcopy. This allowed a comparison of the cytology and p16 INK4a immunostaining results with subsequent hematoxylin and eosin- based histologic diagnoses. RESULTS: Overall, in 10% of slides obtained from patients with nonsuspicious smears, few p16 INK4a -positive cells were found. Using established morphologic criteria and applying these criteria on cells showing any p16 INK4a immunoreactivity, p16 INK4a -positive normal or metaplastic cells could be discriminated from p16 INK4a -expressing dysplastic cells. In 21 of 22 cases (95%) of high grade lesions (cervical intraepithelial neoplasia 2 or higher in follow-up histology), easily recognizable p16 INK4a -positive dysplastic cells could be detected, with the remaining case lacking dysplastic cells in the thin-layer slide used for p16 INK4a immunostaining. CONCLUSION: Established morphologic criteria for cervical dysplasia can be readily applied to p16 INK4a -immunostatined cytologic specimens. Thus, p16 INK4a immunostaining may help to avoid ambiguities in the interpretation of cervical cytology samples and facilitate more rapid diagnosis and possibly even automated screening of cytologic slides.

Objective Natural orifice transluminal endoscopic surgery (NOTES) is a surgical approach that uses natural orifices to gain access to areas of the body. In the present article, we describe the first transgastric pure NOTES salpingo-oophorectomy, which we call peroral endoscopic salpingo-oophorectomy (POESY). Methods A woman with BRCA1 mutation presented for prophylactic bilateral salpingo-oophorectomy. We offered her the transgastric approach, having performed more than 25 transgastric appendectomies. After gastroscopic incision in the corpus wall, we advanced the gastroscope into the abdominal cavity. Salpingo-oophorectomy was performed with the help of an intrauterine manipulator and a transvaginally introduced 5-mm trocar. The posterior colpotomy was dilated and the specimens were extracted. The gastrotomy was closed with an over-the-scope clip, and the colpotomy with a running suture. Results The gastroscope provided excellent optical control and good tissue preparation. Prophylactic bilateral salpingo-oophorectomy was performed successfully via POESY. The patient recovered quickly and was discharged on the third day, with an uneventful follow-up. Conclusion The present case demonstrates the feasibility of transgastric access. The gastroscope provided excellent optical control and good tissue preparation. Therefore, we expect an increasing role of transgastric procedures for diseases in the pelvic region, particularly if new endoscopic platforms with better means of instrumentation and tissue management become available.

Annulus fibrosus repair techniques for the intervertebral disc (IVD) address the unsolved problem of reherniation after IVD herniation and might facilitate the development of nucleus pulposus replacement techniques for IVD diseases. This study investigates the suitability of a bio-integrative annulus implant.Standardized box defects were applied to the annulus L3/4 and L4/5 of 16 sheep, followed by randomized insertion of the textile polyglycolic acid/polyvinylidene fluoride annulus implant in one of the defects. Explantation was conducted after 2, 6 and 12 weeks, followed by provocative pressure testing and histological analysis. At 2 weeks' follow-up, all specimens of the control defect group demonstrated uncontained herniated nucleus pulposus tissue in the annulus defects. For the treated specimens, the annulus implant consistently provided an effective barrier for herniating nucleus pulposus tissue, with no implant dislocation at all time-points. After 2 weeks, a homogeneous cell infiltration of the annulus implant was observed, leading to a progressive directional matrix build-up. Repair tissue thickness was significantly stronger with the annulus implant at all follow-ups (p < 0.01). No pronounced foreign body reaction and no difference in the amount of supra-annular scar tissue over the defect sites were observed. The implantation procedure inflicted annulus damage adjacent to the defect. At later time-points, however, no difference in comparison with the control defect group was evident. The investigated biointegrative annulus implant showed promising results with regard to biointegration, enhancement of repair tissue and function as a mechanical barrier in an ovine model.

The histopathologic interpretation of cervical intraepithelial neoplasia (CIN) is subject to a high level of interobserver variability and a substantial number of false-positive and false-negative results. We assessed the impact of the conjunctive interpretation of p16INK4a-immunostained slides on the accuracy of community-based pathologists in diagnosing high-grade cervical intraepithelial neoplasia (CIN; CIN 2 and CIN 3) in biopsy specimens. Twelve pathologists rendered independent diagnoses on a set of 500 H&E-stained cervical punch and conization specimens. Results were compared with a dichotomized “gold standard” established by consensus of 3 gynecopathology experts. When p16INK4a-immunostained slides were added and conjunctively interpreted with the H&E-stained slides, a significant increase in diagnostic accuracy for the detection of high-grade CIN was observed (P = .0004). Sensitivity for high-grade CIN was increased by 13%, cutting the rate of false-negative results in half. Agreement of community-based pathologists in diagnosing high-grade CIN was significantly improved (mean κ values advanced from 0.566 to 0.749; P < .0001). Reproducibility of p16INK4a stain interpretation was excellent (κ = 0.899). Our results show that conjunctive interpretation of p16INK4a-stained slides could significantly improve the routine interpretation of cervical histopathology.

Human papillomavirus-induced cervical carcinomas often show impaired expression of MHC class I molecules resulting in the inability of tumor cells to directly present viral peptides to cytotoxic T lymphocytes. Loss of MHC class I expression combined with the expression of activating NK cell receptor ligands renders tumor cells potentially susceptible to NK cell attack. Thus, in this study, we analyzed the expression of activating NK cell receptor ligands, NK cell accumulation and activation status in situ in normal ectocervical tissue (NCT), cervical intraepithelial neoplasia (CIN) and squamous cervical carcinoma (CxCa). We observed that expression of the DNAM-1 ligand CD155 was frequently upregulated in CxCa, but not in CIN. The NKG2D ligand MICA was upregulated in fewer CxCa biopsies. In contrast, another NKG2D ligand ULBP2 was preferentially expressed in differentiated epithelial cells of NCT. Increased numbers of NK cells were detected in CIN as compared to NCT and CxCa. Expression of activating NK cell receptor ligands combined with loss of MHC class I was not correlated with enhanced NK cell accumulation or activation status. Furthermore, we demonstrate that cervical cancer cell lines are killed by the NK cell line, NKL, in a NKG2D- and DNAM-1-dependent manner in vitro. Since a significant number of CxCa biopsies showed low MHC class I expression combined with high expression of one or more of the tested activating NK cell receptor ligands, we conclude that CxCa might be a promising target for NK cell-based adoptive immunotherapy.

The quality of cervical histopathology is critical to cervical cancer prevention, cancer treatment, and research programs. On the basis of the histology results further patient management is determined. However, the diagnostic interpretation of histologic hematoxylin-eosin (H&E)-stained slides is affected by substantial rates of discordance among pathologists. Overexpression of the cyclin-dependent kinase inhibitor p16(INK4a), a cell cycle regulating protein, has been shown to be strongly correlated with dysplastic lesions of the cervix uteri. In this study.. we assessed whether p16(INK4a) immunohistochemistry may increase the performance of pathologists in diagnosing squamous lesions in cervical punch and cone biopsies. When using a consecutive p 16(INK4a)-stained slide in conjunction to the H&E-stained slide, interobserver agreement between 6 pathologists improved significantly for both cervical punch and cone biopsies (P < 0.001). For punch biopsies (n = 247), K value increased from 0.49 (moderate agreement) to 0.64 indicating substantial agreement, and interobserver agreement for cone biopsies (n = 249) improved from 0.63 (conventional H&E slide reading) to 0.70 when H&E-stained slides were read conjunctively with p16(INK4a)-stained slides. In comparison to a common consensus diagnosis established by 3 independent experts, 4 pathologists reached an improvement with the conjunctive p16(INK4a) test, 2 of them showing significantly better agreement (P < 0.001 and P = 0.002, respectively). P-16INK4a immunohistochemistry as an adjunct to conventional H&E-stained specimens thus contributes to a more reproducible diagnosis of cervical intraepithelial neoplasia, and may be a valuable aid for the interpretation of cervical histology. (Less)

BACKGROUND. Current cervical cancer screening approaches are based on cytology supplemented by human papillomavirus (HPV) testing in some settings. Whereas cytology is laborious and depends on the cytologists' experience, HPV testing has limited specificity when it is used to detect high-grade lesions. A dichotomous test to identify high-grade lesions with greater specificity may be a useful tool for cervical cancer screening. p16INK4a is a cell-cycle regulator that has demonstrated strong overexpression in cervical precancer cells and cervical cancer induced by the deregulated expression of HPV oncogenes. METHODS. The authors used a sandwich enzyme-linked immunosorbent assay (ELISA) to quantify the amount of solubilized p16INK4a protein in lysates that were prepared from cervical samples to detect high-grade cervical lesions. In total, 187 specimens that were obtained after sampling for conventional cytology in women who attended a cervical colposcopy clinic were analyzed. Seventy-six women underwent a biopsy, and 45 of those women showed histologically confirmed, high-grade cervical intraepithelial neoplasia. RESULTS. For 76 women with biopsy-proven diagnoses, receiver operating characteristic (ROC) analysis of different cutoff values showed an area under the ROC curve of 0.89 for the detection of high-grade cervical dysplasia. At a cutoff value of 8 U/mL, the sensitivity of the p16INK4a ELISA for detecting high-grade dysplastic cervical lesions was 96%. CONCLUSIONS. The data obtained in this study suggested that ELISA-based quantification of solubilized p16INK4a protein may have high sensitivity for detecting cervical precancer. Further population-based studies will be necessary to analyze the specificity and predictive values of p16INK4a protein quantification in cervical samples. Cancer 2006. © 2006 American Cancer Society.

Overexpression of p16INK4a has been proposed as a biomarker helpful for the identification of dysplastic cervical epithelial cells on histologic slides as well as in cervical smears. Since a few nontransformed cells in the genital tract in some instances may also express p16INK4a, we evaluated whether applying established morphologic criteria for cervical dysplasia allows a distinction of dysplastic from nondysplastic p16INK4a-stained cells in cytologic samples.Liquid-based cytology samples were obtained from a screening population (n=50), and from patients attending a dysplasia clinic (n=40). Slides prepared from these samples were stained with the conventional Papanicolaou stain procedure. From each specimen, a second slide was prepared in parallel and immunostained for p16INK4a. Cytologic diagnoses for most patients attending the dysplasia clinic could be compared to the reported histologic diagnoses on punch biopsy samples taken from the patients at the time of colposcopy. This allowed a comparison of the cytology and p16INK4a immunostaining results with subsequent hematoxylin and eosin-based histologic diagnoses.Overall, in 10% of slides obtained from patients with nonsuspicious smears, few p16INK4a-positive cells were found. Using established morphologic criteria and applying these criteria on cells showing any p16INK4a immunoreactivity, p16INK4a-positive normal or metaplastic cells could be discriminated from p16INK4a-expressing dysplastic cells. In 21 of 22 cases (95%) of high grade lesions (cervical intraepithelial neoplasia 2 or higher in follow-up histology), easily recognizable p16INK4a-positive dysplastic cells could be detected, with the remaining case lacking dysplastic cells in the thin-layer slide used for p16INK4a immunostaining.Established morphologic criteria for cervical dysplasia can be readily applied to p16INK4a-immunostained cytologic specimens. Thus, p16INK4a immunostaining may help to avoid ambiguities in the interpretation of cervical cytology samples and facilitate more rapid diagnosis and possibly even automated screening of cytologic slides.

Disadvantages of the traditional Papanicolaou-method for the cytological detection of cervical carcinomas and their precursors can be overcome by the use of specific molecular markers for nuclear attypicality. High grade HR-HPV induced cervical dysplasia is initiated by deregulated expression of viral oncogenes in replicating epithelial stem cells. Here, the E6-E7 gene products gain control of cell cycle and mitotic activity first and induce multistep mutagenesis with severe genomic instability in successin. The detailed molecular analysis of these activities has allowed the development of biomarkers for dysplastic cervical cells. The marked over-expression of the cyclin dependent kinase inhibitor p16INK4a is regularly observed in HR-HPV induced malignant lesions and indicates an active expression of the viral oncogene E7 in dysplastic cells. Morphologically, these molecular deregulations are reflected mainly in an altered nuclear-cytoplasmic ratio, anisonucleosis, and hyperchromasia. With p16INK4a immunostaining--as reported in the literature--dysplastic and atypical cells can be easily detected even under low magnification and differentiated by higher magnification from occasional positive atrophic or metaplastic cells by their atypical nuclear structure. In questionable cases the additional use of proliferation markers could eliminate false interpretation. The results with these new molecular techniques can by further optimized by applying the ThinPrep-method for the preparation of the cytological slides to ensure overlying blood, mucus or inflammatory cells do not mask atypical cells. With these new methods we can expect to lower the rate of false-positive and false-negative cytology tests as experienced with the traditional Papanicolaou-method, of not eliminate them completely, gaining thereby great advantages for patients and for cost-efficiency.

e15515 Background: Conflicting results regarding the (over-)expression of receptors of the HER-family in carcinoma of the urinary bladder (UCB) have been reported. In view off limited treatment opt...

4065 Background: Evaluation of the HPV infection- and transformation-status as a predictor of the response to definitive radio-chemotherapy for anal cancer. Methods: 80 patients (54 fm, 26 m) with histologically confirmed anal cancer and known HPV-Infection- (determined by PCR) and p16-expression-status (determined by immunohistochemistry) were analyzed. All pts. were treated with definitive radio-chemotherapy (RCT) with 5-FU/MMC, median age 60ys (35–86), median follow up 54mo (4–180). 41 pts. were HPV+ and p16+ (group 1), 10 pts. were HPV-/p16+ (group 2), 9 pts. were HPV+/p16- (group 3) and 17 pts. were HPV+/p16- (group 4). Endpoints were local control (LC) at 5ys and overall survival (OS) at 5ys. In addition to HPV/p16 status, the influence of T-stage and tumor localization (canal vs. margin) was analyzed. Results: More women than men were HPV+ (fm 77% vs. m 33%) while gender was evenly distributed among HPV-pts. (fm 48% vs. m 53%). Upon univariate analysis, gender, HPV+ and p16+ were significant predictors of both LC and OS (p