Probes for INS

Targeted inhibition of programmed cell death-1 (PD-1) and its ligand (PD-L1) has emerged as first-line therapy for advanced non-small cell lung cancer. While patients with high PD-L1 expression have improved outcomes with anti-PD-1/PD-L1 directed therapies, use as a predictive biomarker is complicated by robust responses in some patients with low-level expression. Furthermore, reported PD-L1 levels in lung cancers vary widely and discrepancies exist with different antibodies. PD-L1 expression was thus compared by immunohistochemistry (IHC) versus RNA in situ hybridization (ISH) in 112 lung cancers by tissue microarray: 51 adenocarcinoma, 42 squamous cell carcinoma, 9 adenosquamous carcinoma, 5 carcinoid, 3 undifferentiated large-cell carcinoma, 1 large-cell neuroendocrine carcinoma, and 1 small cell carcinoma. At least 1% tumor cell staining was considered positive in each modality. A positive concordance of only 60% (67/112) was found between IHC and ISH. 50% (56/112) were positive by IHC and 50% (56/112) by ISH, however 20% (22/112) were ISH positive but IHC negative. Conversely, 21% (23/112) were IHC positive but ISH negative. There was no significant stratification of PD-L1 positivity by histologic subtype. A trend of more PD-L1 positive stage I cancers identified by ISH versus IHC was observed, however was not statistically significant [50% (27/54) by IHC and 64% (35/55) by ISH, P=.18]. No significant difference in survival was identified, with an average of 5.3months in IHC versus 5.2months in ISH positive cases. The results demonstrate discordance between PD-L1 RNA levels and protein expression in non-small cell lung cancers, warranting comparison as predictive biomarkers.

Recently three different cyclin-dependent kinase 4 and 6 (CDK4/6) dual inhibitors were approved for the treatment of breast cancer (palbociclib, ribociclib and abemaciclib), all of which offer comparable therapeutic benefits. Their safety profiles however are different. For example, neutropenia is observed at varying incidences in patients treated with these drugs; however it is the most common adverse event for palbociclib and ribociclib, whereas diarrhea is the most common adverse event observed in patients treated with abemaciclib. In order to understand the mechanism of diarrhea observed with these drugs and in an effort to guide the development of safer drugs, we compared the effects of oral administration of palbociclib, ribociclib and abemaciclib on the gastrointestinal tract of rats using doses intended to produce comparable CDK4/6 inhibition. Rats administered abemaciclib, but not palbociclib or ribociclib, had fecal alterations, unique histopathological findings and distinctive changes in intestinal gene expression. Morphologic changes in the intestine were characterized by proliferation of crypt cells, loss of goblet cells, poorly differentiated and degenerating enterocytes with loss of microvilli and mucosal inflammation. In the jejunum of abemaciclib-treated rats, down-regulation of enterocyte membrane transporters and up-regulation of genes associated with cell proliferation were observed, consistent with activation of the Wnt pathway and downstream transcriptional regulation. Among these CDK4/6 inhibitors, intestinal toxicity was unique to rats treated with abemaciclib, suggesting a mechanism of toxicity not due to primary pharmacology (CDK4/6 inhibition), but to activity at secondary pharmacological targets.

Abstract

BACKGROUND:

Programmed death receptor and programmed death ligand (PD-L1) are immunoregulatory proteins. Nonsmall cell lung cancer bypasses the immune system through the induction of protumorigenic immunosuppressive changes. The better understanding of immunology and antitumor immune responses has brought the promising development of novel immunotherapy agents like programmed death receptor checkpoint inhibitors. The aim of this study was to investigate the expression of PD-L1 in lung adenocarcinoma (ADC), comparing 2 different technologies: immunohistochemistry (IHC) by 2 methods versus RNA in situ hybridization (RISH).

METHODOLOGY:

In total, 20 cases of ADC of the lung and 4 samples of metastatic colon ADC were selected. Evaluation of PD-L1 expression was performed by IHC and RISH. RISH was performed using RNAscope. Both methods were scored in tumor cells and quantified using combined intensity and proportion scores.

RESULTS:

Eight of 20 (40%) lung ADC and 2 of 4 (50%) colon ADC were positive for PD-L1 with Cell Signaling IHC, and 65% lung ADC were positive by Dako IHC (13/20). All 4 cases of colon ADC were negative. When evaluated by RISH, 12 lung ADC (60%) and 1 colon ADC (25%) were PD-L1 positive.

CONCLUSIONS:

RNAscope probes provide sensitive and specific detection of PD-L1 in lung ADC. Both IHC methods (Cell Signaling and Dako) show PD-L1 expression, with the Dako method more sensitive (40% vs. 65%). This study illustrates the utility of RISH and Cell Signaling IHC as complementary diagnostic tests, and Food and Drug Administration approved Dako IHC as a companion diagnostic test.

Aim: The presence of cells within meningioma (MG) that express embryonic stem cell (ESC) markers has been previously reported. However, the precise location of these cells has yet to be determined.

Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on 11 WHO grade I MG tissue samples for the expression of the ESC markers OCT4, NANOG, SOX2, KLF4 and c-MYC. Immunofluorescence (IF) IHC staining was performed to investigate the localization of each of these ESC markers. NanoString and colorimetric in situ hybridization (CISH) mRNA expression analyses were performed on six snap-frozen MG tissue samples to confirm transcriptional activation of these proteins, respectively.

Results: DAB IHC staining demonstrated expression of OCT4, NANOG, SOX2, KLF4, and c-MYC within all 11 MG tissue samples. IF IHC staining demonstrated the expression of the ESC markers OCT4, NANOG, SOX2, KLF4, and c-MYC on both the endothelial and pericyte layers of the microvessels. NanoString and CISH mRNA analyses confirmed transcription activation of these ESC markers.

Conclusion: This novel finding of the expression of all aforementioned ESC markers in WHO grade I MG infers the presence of a putative stem cells population which may give rise to MG.