Association of PD-L1 expression by immunohistochemistry and gene microarray with molecular subtypes of ovarian tumors.

Identifying patients who respond to immune checkpoint blockade (ICB) is a significant challenge in oncology. While PD-L1 expression by immunohistochemistry (IHC) is the current diagnostic gold standard for patient selection, it nevertheless does not capture all patients who may respond to ICB. Recent gene expression studies in high-grade serous ovarian carcinoma have defined an immunoreactive molecular subtype that has a measurable difference in patient survival compared with non-immunoreactive subtypes, but no studies have yet demonstrated its impact on predicting response to ICB. As a step toward establishing the predictive value of gene expression classifiers in ICB, we assessed the relationship between PD-L1 IHC and molecular subtypes of ovarian epithelial cancer. This was done by analyzing a total of 93 tissue specimens from patients with stage III and IV disease, and comparing PD-L1 IHC with gene expression by Agilent microarrays using TCGA-defined subtypes. We showed that ovarian tumors with elevated IHC PD-L1 expression are most strongly associated with immunoreactive subtype as compared with other molecular subtypes, reaching statistical significance against differentiated (Dunn's test, 33.39, p = 0.0003) and mesenchymal (39.63, p < 0.0001) subtypes. Comparing PD-L1 scoring with CPS vs. TPS showed similar trends, but with stronger correlation strength when using CPS (Kruskal-Wallis, H = 27.52, p < 0.0001), as opposed to TPS (H = 25.04, p < 0.0001). Interestingly, while PD-L1 gene expression by microarray was significantly increased in the immunoreactive subtype (H = 20.25, p = 0.0002), it showed a positive but relatively poor correlation to IHC. Overall, the results demonstrate potential value in use of the molecular classifier to select patients for ICB, pending further studies that assess its ability to predict treatment outcomes. In the future, integration of cellular, protein, and genomic biomarkers in the tumor and tumor microenvironment may improve current methods of predicting treatment response.

Intratumoral immune cell infiltrates, FoxP3, and indoleamine 2,3-dioxygenase in patients with melanoma undergoing CTLA4 blockade.

PURPOSE: CTL-associated antigen 4 (CTLA4)-blocking monoclonal antibodies induce long-term regression of metastatic melanoma in some patients, but the exact mechanism is unknown. In this study, biopsies of selected accessible tumor lesions from patients treated with tremelimumab were examined to further elucidate the mechanism of its antitumor activity. EXPERIMENTAL DESIGN: Fifteen tumor biopsies from 7 patients who had been treated with tremelimumab (CP-675,206) were collected. Samples were analyzed for melanoma markers, immune cell subset markers, the presence of the T regulatory-specific transcription factor FoxP3 and the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO). RESULTS: Clinically responding lesions had diffuse intratumoral infiltrates of CD8(+) T cells that were markedly increased in cases where comparison with a baseline biopsy was available. Nonregressing lesions had sparse, patchy CD8(+) intratumoral infiltrates. Patients with regressing lesions had an increased frequency of CD8(+) cells with or without a concomitant increase in CD4(+) cells. Two of 3 responding patients with paired samples showed a slight increase in the number of FoxP3(+) cells in the postdosing biopsies. In patients with regressing lesions who had paired samples, the intensity of IDO staining in macrophages and/or melanoma cells showed no clear pattern of change postdosing. CONCLUSIONS: Administration of tremelimumab was associated with massive intratumoral infiltrates of CD8(+) CTLs in patients with regressing tumors but had varying effects on intratumoral infiltrates of CD4(+) and FoxP3(+) cells or intratumoral expression of IDO.

The effect of delay in fixation, different fixatives, and duration of fixation in estrogen and progesterone receptor results in breast carcinoma.

Accurate determination of estrogen receptor (ER) and progesterone receptor (PR) status in breast carcinoma is essential. Preanalytic variation may contribute to discordant results. Recently, American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) made recommendations to normalize fixation for breast biomarkers. To evaluate this, a 4-cm invasive lobular carcinoma was processed according to ASCO/CAP guidelines. The remainder was stored fresh at 4°C for 4 days and cut into biopsy-sized pieces. Each was fixed in 10% formalin, Pen-Fix (Richard-Allan Scientific, Kalamazoo, MI), Bouin solution, Sakura Molecular Fixative (Sakura Tissue-Tek Xpress, Torrance, CA), zinc formalin, or 15% formaldehyde for times ranging between 1 and 168 hours. Immunohistochemical studies for ER and PR were performed and interpreted. After 4 days at 4°C, all samples showed no degradation or ER/PR staining differences, except 2 Bouin-fixed samples, in comparison with the patient's sample processed according to ASCO/CAP guidelines. In our study, the preanalytic variables of fixative type, fixation time, and 4 days of ischemic time did not affect immunohistochemical accuracy for ER/PR.