Extensive intraperitoneal lavage to eliminate intraperitoneal tumor cells in gastrectomy with D2 lymphadenectomy for gastric cancer.

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.

Genital invasion or perigenital spread may pose a risk of marginal misses for Intensity Modulated Radiotherapy (IMRT) in anal cancer.

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.

Prognostic Relevance of HPV Infection and p16 Overexpression in Squamous Cell Anal Cancer.

PURPOSE: Human papillomavirus (HPV) DNA and p16 status have both been reported as prognostic factors in anal cancer, but the prognostic relevance of combined detection and particularly HPV-/p16+ and HPV+/p16- signatures is unknown. We evaluated combined HPV DNA and p16 status as a prognostic factor of treatment response in anal cancer. METHODS: 106 patients treated with radiochemotherapy (RCT+5-FU/MMC) with available paraffin-embedded tumor tissue specimens were evaluated regarding local control (LC) and overall survival (OS) at 5 years. In addition to HPV DNA/p16 status, the influence of age, gender, previous surgery, initial recurrence, T stage, N status, and tumor localization was analyzed. RESULTS: 63 patients were HPV+/p16+, 9 were HPV+/p16-, 11 were HPV-/p16+, and 23 were HPV-/p16-. In univariate analysis, LC was significantly better in patients with T1/2 stage, female gender, and HPV/p16 status. HPV+/p16+ was associated with significantly better LC (88.1%; 95% confidence interval [CI]: 78.89-97.31) compared with HPV-/p16+ (63.6%; 95% CI: 35.18-92.02; P=.021) and especially HPV-/p16- (55.8%; 95% CI: 33.46-78.14; P=.002) but not with HPV+/p16- (77.8%; 95% CI: 50.56-105.04; P=.270). OS was influenced by T stage and LC. HPV+/p16+ patients showed a trend toward better OS compared with HPV-/p16- patients (HPV+/p16+: 81.1%; 95% CI: 70.12-92.08 vs HPV-/p16-: 68.8%; 95%CI: 47.44-90.16; P=.138). On multivariate analysis, T3/4 stage and HPV/p16 status (HPV-/p16+, HPV-/p16- vs HPV+/p16+) predicted poorer LC (T3/4: 50.3% vs T1/2: 86.6%, hazard ratio [HR] 0.22; 95% CI: 0.09-0.53; P<.001; HPV+/p16+ vs HPV-/p16+: HR 4.73; 95% CI: 1.33-16.82; P=.016, and HPV+/p16+ vs HPV-/p16-: HR 6.40; 95% CI: 2.23-18.35; P<.001), whereas local relapse dramatically influenced OS. CONCLUSION: Our data suggest that HPV/p16 signature determines prognosis. HPV+/p16+ patients had the best prognosis, and HPV-/p16+ and HPV-/p16- patients showed the worst outcome and therefore require therapy optimization, particularly given that LC is the most important factor for OS.

Microvascular invasion of testicular nonseminomatous germ cell tumors: implications of separate evaluation of lymphatic and blood vessels.

PURPOSE: We separately evaluated the lymphatic and blood vascular systems to assess the diagnostic accuracy of microvascular invasion and identify predictive markers for occult metastasis of testicular nonseminomatous germ cell tumors. MATERIALS AND METHODS: Tissue samples of 86 patients treated for testicular nonseminomatous germ cell tumors (stage 1 in 48 and stage greater than 1 in 38) were stained using the lymphatic endothelial cell specific marker LYVE-1 and the blood vessel endothelial cell marker von Willebrand factor. We assessed lymph vessel density in LYVE-1 stained sections and blood vessel density in von Willebrand factor stained sections. Lymphovascular invasion in LYVE-1 stained sections and blood vascular invasion in von Willebrand factor stained sections were documented. Parameters were correlated with standard clinicopathological data. RESULTS: Blood vessel density in von Willebrand factor sections was significantly greater than lymphatic vessel density in LYVE-1 sections (p<0.001). Peritumor and nontumor lymphatic vessel density in LYVE-1 sections was associated with metastasis at diagnosis (OR 1.277/U, p=0.020 and OR 1.113/U, p=0.095). Lymphovascular invasion in LYVE-1 sections was significantly associated with metastasis (OR=4.517, p=0.002) but blood vascular invasion in von Willebrand factor sections was only slightly significant (OR 2.261, p=0.071). Only lymphovascular invasion in LYVE-1 stained sections was significantly associated with metastasis in a multiple logistic regression model. Microvascular invasion in hematoxylin and eosin stained sections was not associated with metastasis but microvascular invasion evaluated in LYVE-1 and von Willebrand factor stained sections was associated with metastasis (OR 3.506, p=0.016). CONCLUSIONS: Lymphovascular invasion in LYVE-1 stained sections was the most important predictive parameter for metastasis at diagnosis, suggesting greater relevance of the lymphatic system in metastatic dissemination of testicular nonseminomatous germ cell tumors. Vascular endothelial cell specific markers provide higher diagnostic accuracy for microvascular invasion. Our results may impact the current concept of microvascular invasion used for risk stratification of clinical stage 1 testicular nonseminomatous germ cell tumors.

Pure natural orifice transluminal endoscopic surgery (NOTES) involving peroral endoscopic salpingo-oophorectomy (POESY).

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.

The disparate twins: a comparative study of CXCR4 and CXCR7 in SDF-1α-induced gene expression, invasion and chemosensitivity of colon cancer.

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.

The sensitivity and specificity of p16(INK4a) cytology vs HPV testing for detecting high-grade cervical disease in the triage of ASC-US and LSIL pap cytology results.

We analyzed the performance of p16(INK4a) 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.

Conjunctive p16INK4a testing significantly increases accuracy in diagnosing high-grade cervical intraepithelial neoplasia.

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 p16(INK4a)-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 p16(INK4a)-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 kappa values advanced from 0.566 to 0.749; P < .0001). Reproducibility of p16(INK4a) stain interpretation was excellent (kappa = 0.899). Our results show that conjunctive interpretation of p16(INK4a)-stained slides could significantly improve the routine interpretation of cervical histopathology.

Activating NK cell receptor ligands are differentially expressed during progression to cervical cancer.

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.

Immunostaining for p16INK4a used as a conjunctive tool improves interobserver agreement of the histologic diagnosis of cervical intraepithelial neoplasia.

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 p16INK4a, 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 p16INK4a immunohistochemistry may increase the performance of pathologists in diagnosing squamous lesions in cervical punch and cone biopsies. When using a consecutive p16INK4a-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), kappa 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 p16INK4a-stained slides. In comparison to a common consensus diagnosis established by 3 independent experts, 4 pathologists reached an improvement with the conjunctive p16INK4a test, 2 of them showing significantly better agreement (P < 0.001 and P = 0.002, respectively), p16INK4a 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.

Identification of high-grade cervical dysplasia by the detection of p16INK4a in cell lysates obtained from cervical samples.

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. p16(INK4a) 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 p16(INK4a) 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 p16(INK4a) ELISA for detecting high-grade dysplastic cervical lesions was 96%. CONCLUSIONS: The data obtained in this study suggested that ELISA-based quantification of solubilized p16(INK4a) protein may have high sensitivity for detecting cervical precancer. Further population-based studies will be necessary to analyze the specificity and predictive values of p16(INK4a) protein quantification in cervical samples.

Survival benefit for patients with advanced-stage transitional cell carcinomas vs. other subtypes of ovarian carcinoma after chemotherapy with platinum and paclitaxel.

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 and 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.

Morphologic characteristics of p16INK4a-positive cells in cervical cytology samples.

OBJECTIVE: 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. 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 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. RESULTS: 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. CONCLUSION: 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.

Traditional and new molecular methods for early detection of cervical cancer.

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.