Marcus J. Trunk

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), κ 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.

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.

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.

Enhancing tissue repair in annulus fibrosus defects of the intervertebral disc: analysis of a bio‐integrative annulus implant in an in‐vivo ovine model

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.

Molekulare Pathogenese des Zervixkarzinoms und seiner Vorstufen

ZusammenfassungBestimmte 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 Bevölkerung sehr weit verbreitet, jedoch entwickelt sich nach einer HPV-Infektion eher selten eine klinisch relevante Läsion. Für 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 Beeinträchtigung der Zellzyklusregulation, des mitotischen Spindelapparats und der Zentrosomenfunktionen zur Störung der Chromosomenverteilung und -struktur während der Mitose kommen. Dies führt zur chromosomalen Instabilität und bereitet den Boden für die anschließende Karzinogenese. Die Expression des HPV-E7-Onkoproteins bewirkt eine massive Überexpression des zellulären p16INK4a-Proteins, das sich daher zunehmend als geeigneter Surrogatmarker für die Diagnostik in Zytologie und Histologie erweist. Diese Übersicht fasst die Mechanismen der HPV-assoziierten Transformation zusammen und legt die sich daraus ergebende Möglichkeiten für die Diagnostik des Zerivxkarzinoms dar.AbstractSpecific types of the human papillomaviruses (high risk human papillomaviruses) play an essential role in the pathogenesis of cervical cancer. Although infections by these viruses are very common in the general population, only few result in clinically relevant lesions. Continuous and deregulated expression of two viral oncoproteins E6 and E7 in basal or parabasal cells are required to induce and maintain neoplastic growth. In the course of an acute HPV-infection these genes are exclusively expressed in cell cycle arrested, terminally differentiated cells in the intermediate or superficial layers of the epithelium. Accidental activation of these genes in proliferating cells in the basal or parabasal cell layers results in interference with the cell cycle regulation, disturbances of the mitotic spindle apparatus and centrosome functions. This results in numerical and structural chromosome aberrations, chromosomal instability, increasing aneuploidy and initiates cervical carcinogenesis. The deregulated expression of the viral oncogene E7 is indicated by strong over-expression of the cellular p16INK4a gene product. This finding may have significant influence on novel strategies in cervical cancer diagnosis and screening. This review summarizes the basic molecular mechanisms of how papillomaviruses contribute to cellular transformation and how this can influence future diagnostic applications.Specific types of the human papillomaviruses (high risk human papillomaviruses) play an essential role in the pathogenesis of cervical cancer. Although infections by these viruses are very common in the general population, only few result in clinically relevant lesions. Continuous and deregulated expression of two viral oncoproteins E6 and E7 in basal or parabasal cells are required to induce and maintain neoplastic growth. In the course of an acute HPV-infection these genes are exclusively expressed in cell cycle arrested, terminally differentiated cells in the intermediate or superficial layers of the epithelium. Accidental activation of these genes in proliferating cells in the basal or parabasal cell layers results in interference with the cell cycle regulation, disturbances of the mitotic spindle apparatus and centrosome functions. This results in numerical and structural chromosome aberrations, chromosomal instability, increasing aneuploidy and initiates cervical carcinogenesis. The deregulated expression of the viral oncogene E7 is indicated by strong over-expression of the cellular p16(INK4a) gene product. This finding may have significant influence on novel strategies in cervical cancer diagnosis and screening. This review summarizes the basic molecular mechanisms of how papillomaviruses contribute to cellular transformation and how this can influence future diagnostic applications.

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

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).

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

BackgroundWhile 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.MethodsWe 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.ResultsWe 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.ConclusionsLocal 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.

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.

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.

Association between tumor response and postoperative morbidity after neoadjuvant chemotherapy for gastroesophageal adenocarcinoma

1Department of Surgery, University Medical Centre Mannheim, Medical Faculty Mannheim of the University of Heidelberg, 68167 Mannheim, Germany. 2Institute of Pathology, University Medical Centre Mannheim, Medical Faculty Mannheim of the University of Heidelberg, 68167 Mannheim, Germany. 3Department of Trauma Surgery, Hannover Medical School, University of Hannover, 30060 Hannover, Germany. 4Day Treatment Center (TTZ), Interdisciplinary Tumor Center Mannheim (ITM) & 3rd Department of Medicine, University Medical Centre Mannheim, Medical Faculty Mannheim of the University of Heidelberg, 68167 Mannheim, Germany.