Ovarian Clear Cell Carcinoma: From Morphology to Molecular Biology Ovarian clear cell carcinoma (oCCC) is a distinctive subtype of ovarian carcinoma, with peculiar genetic and environmental risk factors, precursor lesions, molecular events during oncogenesis, patterns of spread, and response to treatment. Because of low response to chemotherapy and poor prognosis in advanced stages, there is growing interest in investigating the molecular pathways involved in oCCC development, in order to individualize novel/molecular targeted therapies. Until now, the main molecular genetic changes associated with oCCC remain to be identified, and, although several molecular changes have been reported in clear cell tumors, most studies have analyzed a limited number of cases; therefore, the true prevalence of those changes is not known. The present review will present the clinicopathologic features of oCCC, from morphology to molecular biology, discussing the diagnostic and treatment challenges of this intriguing ovarian carcinoma.

Unexpected PAX8 Immunoreactivity in Metastatic High-grade Breast Cancer Immunohistochemistry (IHC) is often critical for distinction between metastatic carcinomas of Mullerian organ and breast origin. Paired box family protein 8 (PAX8) has been described as a transcription factor highly specific to neoplasms derived from Mullerian organs, thyroid, and kidney. PAX8 IHC with polyclonal and monoclonal antibody reagents was performed on 27 primary and 22 metastatic breast carcinomas. Eight of 27 primary breast carcinomas (30%) were positive for PAX8 with the monoclonal antibody reagent only; 0 of 22 were polyclonal anti-PAX8 immunoreactive. Substantial numbers of metastases had positive immunoreactivity for polyclonal anti-PAX8 (23%). Each of these metastases and additional cases (45% total) also had positive immunoreactivity for monoclonal anti-PAX8, including 5 of 7 brain metastases. IHC with monoclonal anti-PAX8 was positive on 6 of 7 primary breast carcinomas corresponding to PAX8-positive metastases. Together, these results indicate a significant fraction of breast carcinoma metastases and corresponding primary neoplasms have immunoreactivity for PAX8, and positivity rates depend on the antibody used. Diagnoses of metastatic breast carcinoma were achieved with the aid of clinical history and additional IHC in cases of PAX8 immunoreactivity. Contextual interpretation is imperative for PAX8 IHC, particularly when the differential diagnosis includes metastatic breast carcinoma with limited diagnostic material available.

nCounter NanoString Assay Shows Variable Concordance With Immunohistochemistry-based Algorithms in Classifying Cases of Diffuse Large B-Cell Lymphoma According to the Cell-of-Origin Classifying diffuse large B-cell lymphoma (DLBCL) according to the cell-of-origin (COO) was first proposed using gene expression profiling; accordingly, DLBCL is classified into germinal-center B-cell type and activated B-cell type. Immunohistochemistry (IHC)-based classification using different algorithms is used widely due to the ability to use formalin-fixed paraffin-embedded tissue. Recently, newer techniques using RNA expression from formalin-fixed paraffin-embedded were introduced including the nCounter NanoString platform assay. In this brief report, we study the degree of concordance between the NanoString assay and 6 commonly utilized IHC-based algorithms to classify DLBCL cases by COO. Stains for CD10, BCL2, BCL6, FOXP-1, MUM-1, and LOM2 were used to classify a cohort of DLBCL by COO according to the respective IHC-algorithms. Then, RNA was extracted from the same cases for NanoString assay classification. The degree of concordance was calculated between the NanoString classification and each IHC-algorithm as well as among the different IHC-algorithm themselves. The concordance in COO classification of DLBCL between NanonoString assay and IHC-based algorithms is variable depending on the used IHC-algorithm; the highest concordance is seen with the Visco algorithm (κ=0.69; P=0.001). Therefore, discrepancies between the recently introduced NanoString assay and the commonly utilized IHC-algorithms are expected to some extent and should be taken into consideration when interpreting conflicting results.

Immunohistochemical Expression of Microfibrillar-associated Protein 5 (MFAP5) in Invasive Breast Carcinoma of No Special Type Breast cancer (BC) remains the most prevalent female cancer in Egypt and worldwide. Microfibrillar-associated protein 5 (MFAP5) is a multifunctional glycoprotein. Although MFAP5 gene was among the genes that found globally expressed in human cancers, it had been only recently reported in few cancer research studies. This is a retrospective study that has been conducted on 66 Egyptian patients who had invasive carcinoma of no special type. Immunohistochemical staining for MFAP5 was applied on the archival formalin-fixed paraffin-embedded blocks. Staining was assessed semiquantitatively and correlated with the available clinicopathologic parameters and immunohistochemical subtypes of BC. MFAP5 epithelial cytoplasmic expression was observed in 89.4% (59/66) of cases. In contrast, nuclear expression was seen in non-neoplastic breast lobules and premalignant lesions adjacent to tumors that also exhibited constant staining in myoepithelial layer. Statistical analysis of epithelial cytoplasmic expression revealed association of MFAP5 expression with tumor size (P=0.046), high histologic grade (P=0.007), presence of lymph node metastasis (P=0.014), poor Nottingham Prognostic Index (NPI) (P=0.001), late stage (P=0.008), immunohistochemical subtypes of BC (P=0.018), and increased microvessel density using CD34 immunostianing (P=0.04). MFAP5 cytoplasmic expression was also observed in an adjacent duct carcinoma in situ component in 37/45 cases (82.2%). This study showed that MFAP5 is a novel myoepithelial cell marker that appears to be upregulated in duct epithelium in duct carcinoma in situ and invasive carcinoma of no special type during tumorogenesis and that its cytoplasmic expression in invasive tumors seems to have a poor prognostic role manifested by its association with poor prognostic parameters such as high grade, late stage, lymph node invasion, and increased microvessel density.

Correlation of p16 Expression on Cancer and Stromal Cells With Clinicopathologic and Immunohistochemical Features of Lobular Breast Carcinoma Background: Cancer-associated fibroblasts, play a central role in the tumor-stroma interaction and promote tumorigenesis. However, it is still unclear how these processes are regulated. The aim of this study is to investigate p16 expression in cancer and stromal cells of invasive lobular carcinoma (ILC). Design: Clinicopathologic parameters and immunohistochemical stains for estrogen receptor (ER), progesterone receptor, E-cadherin, and human epidermal growth factor receptor 2 of 70 ILC cases were retrieved. In addition, immunohistochemical were performed for p53, p16, and cyclin D1. The p16 expression in cancer and stromal cells were correlated with different clinicopathologic parameters. Results: Of the 70 cases, 8 cases were p16− cancer and stromal cells, 14 cases p16− cancer and p16+ stromal cells, 14 cases p16+ cancer and p16− stromal cells, and 34 cases p16+ cancer and stromal cells. Thirty-one of the 59 cases showed axillary lymph node metastases. Nodal involvement, recurrence, and metastasis of ILC with p16+ cancer cells and p16− stromal cells were more frequent compared with other groups. ILC with p16+ cancer and p16− stromal cells were frequently negative for ER, progesterone receptor, and cyclin D1, p53 positive and triple negative compared with other groups. There was no recurrence and metastasis in ILC with p16− cancer and p16+ stromal cells. ILC with p16+ cancer and stromal cells were significantly node negative and were positive for ER and cyclin D1 compared with other groups. Conclusions: ILC with p16+ cancer and p16− stromal cells were characterized by frequent nodal involvement, recurrence, and metastatic propensity. These results suggest that p16, has novel anticancer properties capable of suppressing cancer cell migration and invasion and pharmacologic restoration of p16 level in stromal fibroblasts may be exploited as therapeutic strategy to prevent nodal or distant metastasis.

Comparison of 22C3 PharmDx and SP263 Assays to Test PD-L1 Expression in NSCLC Immunohistochemical assays for programmed cell death ligand 1 (PD-L1) expression in non–small cell lung cancer (NSCLC) are either required or recommended to guide therapy with immune checkpoint inhibitors. Four commercially available immunohistochemical assays are currently available as either complimentary or companion diagnostic assay for their counterpart therapy. Harmonization or exchangeability of one assay for the other is a highly sought for goal. The aim of this study was to compare one assay, 22C3, with another, SP263, and examine whether they can be exchanged one for the other. Seventy samples from 70 patients with NSCLC were tested for PD-L1 using the SP263 and then the 22C3 antibody clones according to the manufacturer’s instructions in case of the SP263 assay and according to a previously described and reported method for the 22C3 assay on the Ventana’s ultra immunstainer. Results were evaluable in 51 cases, which were interpreted independently by 2 different pathologists on 2 different occasions for each case. The cases were given a percentage score based on the tumor proportion score. The Pearson correlation coefficient was calculated. A high concordance rate was found between the 2 assays. The Pearson correlation coefficient was 0.95, which indicates an almost perfect correlation (95% confidence limits, 0.92-0.97 and P<0.0001). The findings indicate that SP263 assay can be used in place of the 22C3 assay for PD-L1 assay in NSCLC, and it can be used on the Ventana platform.

Lepidic, Papillary Components and EGFR Mutations are Frequent in Patients With Lung Adenocarcinoma Who are Over 75 Years Old Treatment for lung adenocarcinoma frequently diverges from standard treatment in older patients. Clinical, pathologic, and molecular characteristics of lung cancer in patients over 75 years old have not been fully described. The aim of our work was to describe the rate of EGFR, KRAS, BRAFV600, and HER2 mutations, and ALK rearrangement and pathologic characteristics in patients with lung adenocarcinoma over 75, compared with patients below 75 years old. This is a retrospective study from 2 cohorts: a histopathologic cohort of all consecutively resected lung adenocarcinoma in our institution for patients over 75 (n=54, from 2006 to 2017) compared with patients below 75 years old (n=148, from 2014 to 2017) and a molecular cohort of all stage IIIb or IV lung adenocarcinoma from 2009 to 2017 (n=1611). Papillary and lepidic components were more frequently found in patients over 75 years old (P=0.046 and 0.0078, respectively). The rate of current smokers was lower in older patients (P<0.0001). EGFR mutations were more frequent in patients over 75 than in younger patients: 17% versus 8.1% (P<0.0001). The mutually exclusive KRAS mutation was more frequent in patients below 75 years old than in older patients: 25.8% versus 12.8% (P<0.0001). There was no difference for the proportion of the 2 most frequent EGFR mutations (exon 19 deletion and L858R mutation) (P=0.85) or KRAS-mutated codon (P=0.22) between tumors in younger or older patients. There was no statistically significant difference for the presence of BRAFV600, HER2 mutations, and ALK rearrangement (P=0.44, 0.71, and 1, respectively). Our work highlights the fact that EGFR mutations are more frequent in patients over 75 years old in our population. We can hypothesize that this difference might be mainly caused by the less frequent occurrence of tobacco-smoking–related lung cancers in the elderly and the presence of a lepidic or papillary component in this age group.

Clinical and Economic Value of p16INK4a for the Differential Diagnosis of Morphologic Cervical Intraepithelial Neoplasia 2 The detection of high-grade intraepithelial lesions requires highly sensitive and specific methods that allow more accurate diagnoses. This contributes to a proper management of preneoplastic lesions, thus avoiding overtreatment. The purpose of this study was to analyze the value of immunostaining for p16INK4a in the morphologic assessment of cervical intraepithelial neoplasia 2 lesions, to help differentiate between low-grade (p16-negative) and high-grade (p16-positive) squamous intraepithelial lesions. The direct medical cost of the treatment of cervical intraepithelial neoplasia 2 morphologic lesions was estimated. A retrospective observational cross-sectional study was carried out. This study analyzed 46 patients treated with excisional procedures because of cervical intraepithelial neoplasia 2 lesions, using loop electrosurgical excision procedures. Immunostaining for the biomarker was performed. For the estimation of overtreatment, percentages (%) and their 95% confidence interval were calculated. Of the 41 patients analyzed, 32 (78%) showed overexpression of p16 and 9 (22%) were negative (95% confidence interval, 11%-38%). Mean follow-up was 2.9 years, using cervical cytology testing (Pap) and colposcopy. High-risk human papillomavirus DNA tests were performed in 83% of patients. These retrospective results reveal the need for larger biopsy samples, which would allow a more accurate prediction of lesion risk. Considering the cost of p16 staining, and assuming the proper management of the low-grade lesion, an average of US$919 could be saved for each patient with a p16-negative result, which represents a global direct cost reduction of 10%.

Mismatch Repair Deficiency in Endometrial Cancer: Immunohistochemistry Staining and Clinical Implications Introduction: DNA mismatch repair (MMR) deficiency is associated with increased risk of developing several types of cancer and is the most common cause of hereditary endometrial cancer. Identification of the microsatellite instability (MSI) phenotype in endometrial carcinoma is important given that such tumors are frequent. Objective: The objective of this study was to assess the utility of immunohistochemistry (IHC), a simple and fast technique, in detecting MSI status in endometrial carcinoma and evaluate the correlation between the MSI phenotype and the various anatomo-clinical parameters. Methods: IHC expression of 4 markers (MLH1, MSH2, PMS2, and MSH6) was studied. For all IHC markers, a combined score based on the intensity of nuclear labeling and the percentage of labeled cells was defined to establish a score. Correlation between MSI phenotype and different clinicopathologic parameters was evaluated using statistical analysis (software STATA and the Fisher exact test). Results: The mean age of the patients was 58.6 years. Positive staining was highly extended (score 3) with 79% to 100% of marked cells. Less than 10% of positive tumor cells were seen in 3% of cases for MSH6 and PMS2. Abnormal MMR IHC was detected in 10 cases (22.22%). Seven tumors showed loss of MLH1/PMS2. The loss of MSH2/MSH6 was observed in 1 case. The loss of MLH1 or PMS2 was seen only in 2 cases. The number of MSI positive status was 10 cases (22.7%). Correlation between clinicopathologic parameters showed MMR deficiency was significantly associated with low-grade tumor and localized stage. There was no positive correlation between age, histologic subtype, or myometrium invasion. Conclusions: In summary, detection of DNA MMR deficiencies by IHC can effectively diagnose the MSI phenotype in endometrial carcinoma. Correlation between clinicopathologic parameters showed MMR deficiency was significantly associated with low-grade tumor and localized stage.

Carcinoma-associated Fibroblasts are a Common Finding in the Microenvironment of HPV-positive Oropharyngeal Squamous Cell Carcinoma The important role of the human papillomavirus (HPV) is widely established in oropharyngeal squamous cell carcinoma (OPSCC). The behavior of a OPSCCs especially induced by HPV might be influenced by the tissue microenvironment and its changes according to the tumor nature. Recognition of the role of the tumor microenvironment on the behavior of neoplastic cells has led to utilization of the microenvironment to use as therapeutic target. Carcinoma-associated fibroblasts (CAFs), the most abundant cells in the tumor microenvironment, show wide-spread expression of alpha-smooth muscle actin (α-SMA). We focused on CAFs, its presence in OPSCC and the relationship with HPV for the first time. Expression of α-SMA protein in CAFs of the tumor microenvironment of the 44 formalin-fixed paraffin-embedded tissue blocks from the primary tumor of OPSCC evaluated by immunohistochemistry between HPV-positive and HPV-negative tumors separated by nested polymerase chain reaction. In 44 samples 23 HPV-positive cases were detected. Statistically there were significant differences between histopathologic grade, percent and final score of α-SMA and HPV expression. Significant difference between HPV expression and inflammation, intensity, and clinical parameters was not identified in the present study. Our results indicate that CAFs are a common finding in the microenvironment of HPV-positive OPSCC and associated with higher histopathologic grade. Therapeutic strategies to use CAF-mediated drugs need to be considered and evaluated more for treatment of HPV-positive OPSCC.