The HE4 (WFDC2) gene encodes a WAP-type four disulphide core domain-containing protein with a presumptive role in natural immunity. Multiple studies have consistently identified upregulation of HE4 gene expression in carcinomas of the ovary; however, the expression in normal and malignant adult tissues has not been examined in detail. Here, we examined the expression of the HE4 gene and protein in a large series of normal and malignant adult tissues by oligonucleotide microarray and tissue microarray, respectively. HE4 gene expression was highest in normal human trachea and salivary gland, and to a lesser extent, lung, prostate, pituitary gland, thyroid, and kidney. In a series of 175 human adult tumors, gene expression was highest in ovarian serous carcinomas. However, adenocarcinomas of the lung, and occasional breast, transitional cell and pancreatic carcinomas had moderate or high levels of HE4 expression. Using tissue microarrays and full tissue sections of normal and 448 neoplastic tissues, HE4 immunoreactivity was found in normal glandular epithelium of the female genital tract and breast, the epididymis and vas deferens, respiratory epithelium, distal renal tubules, colonic mucosa, and salivary glands, consistent with HE4 gene expression. In addition to consistent positivity in ovarian carcinoma, some pulmonary, endometrial, and breast adenocarcinomas, mesotheliomas, and less often, gastrointestinal, renal and transitional cell carcinomas were also positive. Knowledge of the expression patterns of HE4 in our survey is useful for application in histopathologic diagnosis, and should be taken into consideration in future studies that examine the role of HE4 as a serological tumor biomarker or as a target for gene-based therapy.

Histopathologic diagnosis of cervical biopsies determines clinical management of patients with an abnormal cervical cancer-screening test yet is prone to poor inter-observer reproducibility. Immunohistochemical staining for biomarkers related to the different stages of cervical carcinogenesis may provide objective standards to reduce diagnostic variability of cervical biopsy evaluations but systematic, rigorous evaluations of their potential clinical utility are lacking. To address diagnostic utility of HPV L1, p16 INK4a , and Ki-67 immunohistochemical staining for improving diagnostic accuracy, we conducted a community-and population-based evaluation using 1455 consecutive cervical biopsies submitted to the Department of Pathology at the University of Virginia during a period of 14 months. Thin-sections of each biopsy from 1451 of 1455 (99.7%) biopsies underwent evaluation of immunohistochemical stains for three biomarkers, masked to the original diagnosis, and the results were compared to an adjudicated, consensus diagnosis by 3 pathologists. p16 INK4a immunostaining, using the strongest staining as the cutpoint, was 86.7% sensitive and 82.8% specific for cervical intraepithelial neoplasia grade 2 or more severe (CIN2+) diagnoses. The p16 INK4a performance was more sensitive (p < 0.001), less specific (p < 0.001), and of similar overall accuracy for CIN2+ compared to the combined performance of all pathologist reviews in routine clinical diagnostic service (sensitivity = 68.9%, specificity = 97.2%). Ki-67 immunostaining was also strongly associated with a CIN2+ diagnosis but its performance at all staining intensities was inferior to p16 INK4a immunostaining, and did not increase the accuracy of CIN2+ diagnosis when combined with p16 INK4a immunostaining compared to p16 INK4a immunostaining alone. We found no utility for L1 immunostaining in distinguishing between CIN and non-CIN. In conclusion, with a rigorous evaluation, we found immunohistochemical staining for p16 INK4a to be a useful and reliable diagnostic adjunct for distinguishing biopsies with and without CIN2+.

Protein kinase C-related kinases (PRKs) are regulated by PI-3 kinase and Rho family GTPases. The isoform PRK1 has been characterized in detail in prostate cancer, but not in other carcinomas. We analyzed our prior microarray data for PRK1 gene expression in 175 carcinomas, and evaluated tissue microarrays for protein expression in 251 carcinomas and a comprehensive group of normal tissues. We also used immunoblotting to determine the levels and phospho-activation status of PRK1, PRK2, and PDK1 in 12 ovarian serous carcinomas, SKOV3 cells, and three samples of normal ovarian surface epithelium. The highest average level of PRK1 mRNAwas observed in ovarian serous carcinomas compared to all other carcinomas, including those of the prostate, bladder/ureter, breast, colon, stomach/esophagus, kidney, liver, pancreas, and lung (p=0.05). By immunohistochemistry, PRK1 was observed in selected normal cells including epithelium from the gynecological tract and hematolymphoid elements. All serous ovarian and endometrial endometrioid adenocarcinomas, and mesotheliomas were immunoreactive for PRK1. Nonserous ovarian and a majority of carcinomas from the prostate, breast, and pancreas were also positive but less consistently so. In comparison to ovarian surface epithelium, the serous carcinomas typically had greater pPRK1/total PRK1 (p=0.02) as well as greater pPDK/total PDK (p=0.01). The relative phosphorylation status of these two kinases correlated within each sample. In summary, PRK1 is present in various malignancies, but especially in serous carcinomas, where the increased activation status of PRK1 and its upstream regulator, PDK, as compared to normal ovarian surface epithelium suggests a role in ovarian cancer development or progression.

A sclerotic or hyalinized stroma with a trabecular growth pattern may be seen in a number of different thyroid lesions and, when seen, is usually a focal feature of a lesion other than HTA. Immunohistochemistry may be of assistance as cases of FVPC with prominent hyalinization and trabeculation will show immunoreactivity for HBME1 and CK19, whereas HTAs and other thyroid lesions with hyalinization and trabeculation will not.

Podoplanin, D2-40, is often used for highlighting lymphatics. However, it has been described in a variety of normal and neoplastic tissues. We evaluated the expression of D2-40 in breast, salivary gland, skin, and mucosa, all organs rich in myoepithelial cells and basal cells. This study assessed the utility of using D2-40 to highlight basal/myoepithelial cells and to identify potential pitfalls in misinterpretation of lymphatic invasion. Our results showed that myoepithelial cells in breast and salivary gland and basal cells in prostate consistently demonstrate D2-40 immunoreactivity, but typically less intensely than lymphatics. Cutaneous and mucosal-based basal cells also have D2-40 expression, but often in a patchy, focal manner. In addition, many salivary gland tumors and squamous cell carcinomas had strong D2-40 expression, sometimes making distinction of lymphatics versus tumor edge staining difficult. D2-40 is excellent for assessing lymphatic invasion in breast, prostate, and cervix as long as the pathologist is aware of the expression in myoepithelial cells/basal cells to avoid misinterpretation of ductal carcinoma in situ or normal prostate glands or tumor stroma retraction as tumor lymphatic invasion. Given the patchy positivity in basal cells of skin and mucosa and the reactivity in squamous cell carcinoma, D2-40 was not helpful in assessing for microinvasion of squamous cell carcinoma.