Faculty Bibliography

Substantial progress has been made in understanding how tumors escape immune surveillance. However, few measures to counteract tumor immune evasion have been developed. Suppression of tumor antigen expression is a common adaptive mechanism that cancers use to evade detection and destruction by the immune system. Epigenetic modifications play a critical role in various aspects of immune invasion, including the regulation of tumor antigen expression. To identify epigenetic regulators of tumor antigen expression, we established a transplantable syngeneic tumor model of immune escape with silenced antigen expression and used this system as a platform for a CRISPR-Cas9 suppressor screen for genes encoding epigenetic modifiers. We found that disruption of the genes encoding either of the chromatin modifiers activating transcription factor 7-interacting protein (Atf7ip) or its interacting partner SET domain bifurcated histone lysine methyltransferase 1 (Setdb1) in tumor cells restored tumor antigen expression. This resulted in augmented tumor immunogenicity concomitant with elevated endogenous retroviral (ERV) antigens and mRNA intron retention. ERV disinhibition was associated with a robust type I interferon response and increased T-cell infiltration, leading to rejection of cells lacking intact Atf7ip or Setdb1. ATF7IP or SETDB1 expression inversely correlated with antigen processing and presentation pathways, interferon signaling, and T-cell infiltration and cytotoxicity in human cancers. Our results provide a rationale for targeting Atf7ip or Setdb1 in cancer immunotherapy.

Inhibition of mTORC1 signaling has been shown to diminish growth of meningiomas and schwannomas in preclinical studies, and clinical data suggest that everolimus, an orally administered mTORC1 inhibitor, may slow tumor progression in a subset of NF2 patients with vestibular schwannoma (VS). To assess the pharmacokinetics, pharmacodynamics and potential mechanisms of treatment resistance, we performed a pre-surgical (phase 0) clinical trial of everolimus in patients undergoing elective surgery for VS or meningiomas. Eligible patients with meningioma or VS requiring tumor resection enrolled on study received everolimus 10 mg daily for 10 days immediately prior to surgery. Everolimus blood levels were determined immediately prior to and after surgery. Tumor samples were collected intraoperatively. Ten patients completed protocol therapy. Median pre- and post-operative blood levels of everolimus were found to be in a high therapeutic range (17.4 ng/ml and 9.4 ng/ml, respectively). Median tumor tissue drug concentration determined by mass spectrometry was 24.3 pg/mg (range 9.2-169.2). We observed only partial inhibition of phospho-S6 in the treated tumors, indicating incomplete target inhibition compared to control tissues from untreated patients (p=0.025). Everolimus led to incomplete inhibition of mTORC1 and downstream signaling. These data may explain the limited anti-tumor effect of everolimus observed in clinical studies for NF2 patients and will inform the design of future pre-clinical and clinical studies targeting mTORC1 in meningiomas and schwannomas.

Bodies have continuous reticular networks, comprising collagens, elastin, glycosaminoglycans, and other extracellular matrix components, through all tissues and organs. Fibrous coverings of nerves and blood vessels create structural continuity beyond organ boundaries. We recently validated fluid flow through human fibrous tissues, though whether these interstitial spaces are continuous through the body or discontinuous, confined within individual organs, remains unclear. Here we show evidence for continuity of interstitial spaces using two approaches. Non-biological particles (tattoo pigment, colloidal silver) were tracked within colon and skin interstitial spaces and into adjacent fascia. Hyaluronic acid, a macromolecular component of interstitial spaces, was also visualized. Both techniques demonstrate interstitial continuity within and between organs including within perineurium and vascular adventitia traversing organs and the spaces between them. We suggest that there is a body-wide network of fluid-filled interstitial spaces that has significant implications for molecular signaling, cell trafficking, and the spread of malignant and infectious disease.

Epilepsy is a heterogenous group of disorders defined by recurrent seizure activity due to abnormal synchronized activity of neurons. A growing number of epilepsy cases are believed to be caused by genetic factors and copy number variants (CNV) contribute to up to 5% of epilepsy cases. However, CNVs in epilepsy are usually large deletions or duplications involving multiple neurodevelopmental genes. In patients who underwent seizure focus resection for treatment-resistant epilepsy, whole genome DNA methylation profiling identified 3 main clusters of which one showed strong association with receptor tyrosine kinase (RTK) genes. We identified focal copy number gains involving epidermal growth factor receptor (EGFR) and PDGFRA loci. The dysplastic neurons of cases with amplifications showed marked overexpression of EGFR and PDGFRA, while glial and endothelial cells were negative. Targeted sequencing of regulatory regions and DNA methylation analysis revealed that only enhancer regions of EGFR and gene promoter of PDGFRA were amplified, while coding regions did not show copy number abnormalities or somatic mutations. Somatic focal copy number gains of noncoding regulatory represent a previously unrecognized genetic driver in epilepsy and a mechanism of abnormal activation of RTK genes. Upregulated RTKs provide a potential avenue for therapy in seizure disorders.

Immunohistochemical (IHC) stain for PD-L1 as a biomarker for immunotherapy is recommended in non-small cell lung cancer (NSCLC). Under the FDA, the selection of patients for pembrolizumab requires companion diagnostic testing using the Dako Agilent PD-L1 IHC 22C3 pharmDx kit performed on the Dako Autostainer Link 48 platform. However, because it is not widely available, there is need for cross-platform validation. Existing studies provide incomplete protocol detail. In our study, 73 lung tumors were stained using the FDA-approved test ('gold standard'). The same blocks were stained using two different models of the Ventana DISCOVERY platform (ULTRA, n = 73 and XT, n = 70) using different parameters, and interpreted by three pathologists. The ULTRA group met College of American Pathologists (CAP) validation criteria (concordance 91.8%) while the XT group did not (concordance 67.1%). Using tumor proportion score (TPS) ≥1% and TPS ≥50% as cut-offs, the ULTRA protocol had higher sensitivity (97.8% and 91.7%) than XT (73.3% and 60.9%) and similar specificity (ULTRA 88.9% and 100%, XT 88% and 100%). Discordance between ULTRA and XT was 27%, and in all these cases ULTRA was concordant with gold standard. Interobserver reliability was substantial for ULTRA and almost perfect for XT, providing evidence that staining rather than observer variability accounts for XT's inferior performance. Cross-validation of the clinically used 22C3 anti PD-L1 antibody test with substantial interobserver agreement is possible on the commonly used the Ventana DISCOVERY ULTRA automated instrument, while the validation failed on the XT. Cautious attention to detail must be paid when choosing cross-validation parameters.

Multiplexed immunohistochemical techniques give insight into contextual cellular relationships by offering the ability to collect cell-specific data with spatial information from formalin-fixed, paraffin-embedded tissue sections. We established an automated sequential elution-stripping multiplex immunohistochemical assay to address two controversial scientific questions in the field of hepatopathology: 1) whether epithelial-to-mesenchymal transition or mesenchymal-to-epithelial transition occurs during liver injury and repair of a chronic liver disease and 2) if there is a stromal:epithelial relationship along the canals of Hering that would support the concept of this biliary structure being a stem/progenitor cell niche. Our 4-plex assay includes both epithelial and mesenchymal clinical immunohistochemical markers and was performed on clinical human liver specimens in patients with primary biliary cholangitis. The assay demonstrated that in each specimen, co-expression of epithelial and mesenchymal markers was observed in extraportal cholangiocytes. In regard to possible mesenchymal components in a stem cell niche, 82.3% ± 5.5% of extraportal cholangiocytes were intimately associated with a vimentin-positive cell. Co-expression of epithelial and mesenchymal markers by extraportal cholangiocytes is evidence for epithelial to mesenchymal transition in primary biliary cholangitis. Vimentin-positive stromal cells are frequently juxtaposed to extraportal cholangiocytes, supporting an epithelial:mesenchymal relationship within the hepatobiliary stem cell niche. Our automated sequential elution-stripping multiplex immunohistochemical assay is a cost-effective multiplexing technique that can be readily applied to a small series of clinical pathology samples in order to answer scientific questions involving cell:cell relationships and cellular antibody expression.