Christoph Kakies

SOX2 amplification is a common event in squamous cell carcinomas of different organ sites

Acquired chromosomal aberrations, including gene copy number alterations, are involved in the development and progression of human malignancies. SOX2, a transcription factor-coding gene located at 3q26.33, is known to be recurrently and specifically amplified in squamous cell carcinomas of the lung, the esophagus, and the oral cavity. In these organs, the SOX2 protein plays an important role in tumorigenesis and tumor survival. The aim of this study was to determine whether SOX2 amplification is also found in squamous cell carcinomas in other organs commonly affected by this tumor entity. In addition, we examined a large spectrum of lung cancer entities with neuroendocrine differentiation (ie, small cell cancers, large cell cancers, typical and atypical carcinoids) for SOX2 and TTF1 copy number gains to reveal potential molecular ties to squamous cell carcinomas or adenocarcinomas of the lung. Applying fluorescence in situ hybridization, we assessed squamous cell carcinomas of the cervix uteri (n = 47), the skin (n = 57), and the penis (n = 53) for SOX2 copy number alterations and detected amplifications in 28%, 28%, and 32% of tumors, respectively. Furthermore, we performed immunohistochemical SOX2 staining and found that SOX2 amplification is significantly associated with overexpression of the corresponding protein in squamous cell carcinomas (P < .001). Of the lung cancer entities with neuroendocrine differentiation, only small cell cancers and large cell cancers exhibited SOX2 or TTF1 amplifications at significant frequencies, indicating that at least a subset of these might be dedifferentiated forms of squamous cell carcinomas or adenocarcinomas of the lung. We conclude that SOX2 amplification and consequent SOX2 protein overexpression may represent important mechanisms of tumor initiation and progression in a considerable subset of squamous cell carcinomas.

Alterations in the tumor suppressor gene p16(INK4A) are associated with aggressive behavior of penile carcinomas.

Alterations in the p16/cyclinD1/Rb and ARF/Mdm2/p53 pathways are frequent events in the pathogenesis of squamous cell carcinomas. Different mechanisms of p16 regulation have been described for penile carcinomas so far. Therefore, expression of p16 and p53 was immunohistochemically detected with monoclonal antibodies in 52 primary invasive penile squamous cell carcinomas. The carcinomas were analyzed for allelic loss (LOH) in p16INK4A and p53, as well as for mutations in the p16INK4A and the p53 gene. In addition, we examined the promoter status of p16INK4A by methylation-specific PCR. The presence of human papilloma virus (HPV) 6/11, HPV 16 and HPV 18 DNA was analyzed by PCR. Data were compared to clinical data. Concerning p16, 26 (50%) tumors showed positive immunohistochemistry, 32 (62%) tumors showed allelic loss and 22 tumors (42%) showed promoter hypermethylation. All tumors with negative p16 immunohistochemistry showed LOH near the p16INK4A locus and/or hypermethylation of the p16INK4A promoter. HPV 16 DNA was detected in 17 tumors, ten of them with positive p16 immunostaining. The remaining seven tumors with negative p16 staining showed allelic loss and/or promoter hypermethylation. Evidence of lymph node metastasis was significantly associated with negative p16 immunohistochemistry as well as with combined LOH and promoter hypermethylation (p = 0.003 and p = 0.018, respectively). Allelic loss around p53 was found in 22 tumors (42%), and seven mutations of the p53 gene could be demonstrated in our tumors. No correlations could be found between any p53 alteration and clinical parameters.

Expression of p53, p21 and cyclin D1 in penile cancer: p53 predicts poor prognosis

Aims To evaluate the role of p53, p21 and cyclin D1 expression in patients with penile cancer (PC). Methods Paraffin-embedded tissues from PC specimens from six pathology departments were subjected to a central histopathological review performed by one pathologist. The tissue microarray technique was used for immunostaining which was evaluated by two independent pathologists and correlated with cancer-specific survival (CSS). κ-statistics were used to assess interobserver variability. Uni- and multivariable Cox proportional hazards analysis was applied to assess the independent effects of several prognostic factors on CSS over a median of 32 months (IQR 6–66 months). Results Specimens and clinical data from 110 men treated surgically for primary PC were collected. p53 staining was positive in 30 and negative in 62 specimens. κ-statistics showed substantial interobserver reproducibility of p53 staining evaluation (κ=0.73; p<0.001). The 5-year CSS rate for the entire study cohort was 74%. Five-year CSS was 84% in p53-negative and 51% in p53-positive PC patients (p=0.003). Multivariable analysis showed p53 (HR=3.20; p=0.041) and pT-stage (HR=4.29; p<0.001) as independent significant prognostic factors for CSS. Cyclin D1 and p21 expression were not correlated with survival. However, incorporating p21 into a multivariable Cox model did contribute to improved model quality for predicting CSS. Conclusions In patients with PC, the expression of p53 in the primary tumour specimen can be reproducibly assessed and is negatively associated with cancer specific survival.

Scoring the percentage of Ki67 positive nuclei is superior to mitotic count and the mitosis marker phosphohistone H3 (PHH3) in terms of differentiating flat lesions of the bladder mucosa

Aims To systematically compare different approaches for evaluating mucosal proliferative activity regarding their diagnostic role for delineating flat lesions of the bladder mucosa. Methods 32 carcinoma in situ (CIS) and 31 flat non-CIS conditions (low-grade dysplasia and reactive atypia) of the bladder mucosa were assessed by two independent pathologists in two rounds in terms of their proliferative activity assessed by the mitotic counts on H&E-stained sections (mitoses per mm2) and immunohistochemically using the MIB-1 antibody and the mitosis marker phosphohistone H3 (PHH3). Two different approaches for immunoscoring (percentage of stained nuclei vs dichotomised height of mucosal staining considering lower half vs full-thickness marker expression) were applied. κ statistics were used to evaluate interobserver and intraobserver reproducibility. Results Scoring the percentage of Ki67 expressing cell nuclei seems to be superior to dichotomisation of the height of mucosal staining as well as to PHH3 immunostaining and conventional mitotic counts in terms of delineating CIS from flat non-CIS conditions. This approach shows substantial (κ=0.62–0.65; p<0.001) interobserver and substantial to almost perfect (κ=0.67–0.83; p<0.001) intraobserver reproducibility. Conclusions The MIB-1 antibody is a useful adjunct in the differential diagnosis of conventionally challenging flat lesions of the bladder mucosa. In particular, 16% or more Ki67 positive cell nuclei favours CIS over flat non-CIS conditions, whereas 15% or less Ki67 positive cell nuclei is supportive of non-CIS conditions. However, due to some important limitations of MIB-1 staining, the MIB-1 antibody should be used as a component of a panel.

Different HER2 Protein Expression Profiles Aid in the Histologic Differential Diagnosis Between Urothelial Carcinoma In Situ (CIS) and Non-CIS Conditions (Dysplasia and Reactive Atypia) of the Urinary Bladder Mucosa

We evaluated HER2 expression profiles in 32 carcinoma in situ (CIS) and 31 non-CIS conditions (5 dysplasia and 26 reactive atypia) of the urinary bladder mucosa by applying breast cancer scoring rules. In situ hybridization was performed on tissue microarrays to assess HER2 gene amplification status. Our immunoprofiling data disclosed moderate to strong HER2 expression in CIS, including the basal layer of the urothelium, and absent to weak HER2 expression in non-CIS conditions. From the histologic differential diagnostic standpoint, immunostaining for HER2 protein represents a useful adjunct to aid in the delineation between CIS and non-CIS conditions of the bladder mucosa. Pathogenically, aberrant HER2 protein expression in CIS seems to be more commonly associated with polysomy than with gene amplification. From a therapeutic viewpoint, prospective clinical studies should investigate the potential benefit of HER2-targeted therapies in CIS, particularly in cases unresponsive to conventional therapeutic regimens.

Lewis y antigen (blood group 8, BG8) is a useful marker in the histopathological differential diagnosis of flat urothelial lesions of the urinary bladder

Background and aims The cell-surface carbohydrate Lewisy antigen (blood group 8, BG8) has recently been investigated in bladder cancer, but its role in the differential diagnosis of flat urothelial lesions of the bladder has not yet been systematically evaluated. Methods 30 carcinoma in situ (CIS) and 30 non-CIS conditions of the bladder mucosa (four dysplasia and 26 reactive atypia according to consensus diagnoses) were comparatively assessed in terms of their Lewisy antigen staining profiles by two independent clinical pathologists. Results Lewisy antigen expression differed significantly (p<0.001) between CIS (full thickness expression throughout the entire urothelium including the basal cell layer) and non-CIS conditions (patchy discontinuous expression restricted to individual cells scattered singly throughout the urothelial mucosa). The four dysplastic study cases showed Lewisy antigen expression more closely related to the staining profiles observed in most of the reactive urothelial atypia. κ statistics showed excellent inter-observer agreement between both raters in terms of Lewisy antigen staining evaluation (κ=0.9, p<0.001). Conclusions The data hint at the cell-surface carbohydrate Lewisy antigen as a so far neglected diagnostically useful marker to aid in the histological classification of conventionally equivocal flat urothelial lesions of the bladder in contemporary surgical pathology practice.