Faculty Bibliography

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.

Background/Purpose: Tubulointerstitial damage in lupus nephritis (LN) is a strong predictor of progression to chronic kidney disease (CKD) and end stage renal disease (ESRD). While prior studies showed complement activation mediates glomerular injury, the role of complement in renal tubular damage has not been evaluated. The objective of this study is to investigate the association between complement activation as measured by tubular complement 9 (C9) deposition with interstitial fibrosis and tubular atrophy (IFTA) in LN.
Method(s): LN biopsies from July 2014 to July 2016 were evaluated. Chromogenic immunohistochemistry was performed on formalin-fixed, paraffin-embedded, 4-mum human renal biopsy sections using unconjugated, murine anti-human Complement C9 (Hycult Biotech, clone X197) as a marker of the membrane attack complex (MAC) activation. C3 glomerulopathy was used as a positive control and normal kidney served as a negative control. Tubular basement membrane C9 staining intensity was analyzed as absent (0) versus present (1 to 3) scored on a semi-quantitative scale by a renal pathologist. IFTA was categorized as 0-10%, 11-20%, and >20%. Clinical parameters were assessed at the time of biopsy and 6 months post biopsy.
Result(s): Renal biopsies from 30 LN patients were studied, of which 23 (77%) were proliferative LN. There were 24 (80%) women, mean age 33 (standard deviation) (12) years. Positive tubular C9 staining (C9+) was observed in 7 (23%) biopsies. At the time of renal biopsy, C9+ patients had significantly higher proteinuria, compared to C9- patients: median (interquartile range) 6.2g (3.3-13.1) vs. 2.4g (1.3-4.6), p< 0.01. The differences persisted at 6 months after induction therapy: 1.08g (1.0-8.3) in C9+ vs. 0.68g (0.2-2.1) in C9- patients, p=0.06. Tubular C9 deposition was associated with the finding of interstitial fibrosis on biopsy: 3 out of 7 (42.9%) had >20% interstitial fibrosis as compared to none in the C9- group, p=< 0.01. Higher proportion of C9+ patients had moderate NIH Chronicity index: 3 out of 7 (42.9%) vs 2 out of 23 (8.7%) in the C9- group, p=0.07. There was no significant difference in eGFR at renal biopsy between the two groups.
Conclusion(s): Tubular C9 deposition is associated with proteinuria, interstitial fibrosis and increased chronicity which are predictors of progression to ESRD. Complement activation may play an important role in tubulointerstitial damage in LN

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.