Identification of microbial agents in tissue specimens of ocular and periocular sarcoidosis using a metagenomics approach

Background: Metagenomic sequencing has the potential to identify a wide range of pathogens in human tissue samples. Sarcoidosis is a complex disorder whose etiology remains unknown and for which a variety of infectious causes have been hypothesized. We sought to conduct metagenomic sequencing on cases of ocular and periocular sarcoidosis, none of them with previously identified infectious causes. Methods: Archival tissue specimens of 16 subjects with biopsies of ocular and periocular tissues that were positive for non-caseating granulomas were used as cases. Four archival tissue specimens that did not demonstrate non-caseating granulomas were also included as controls. Genomic DNA was extracted from tissue sections. DNA libraries were generated from the extracted genomic DNA and the libraries underwent next-generation sequencing. Results: We generated between 4.8 and 20.7 million reads for each of the 16 cases plus four control samples. For eight of the cases, we identified microbial pathogens that were present well above the background, with one potential pathogen identified for seven of the cases and two possible pathogens for one of the cases. Five of the eight cases were associated with bacteria (Campylobacter concisus, Neisseria elongata, Streptococcus salivarius, Pseudopropionibacterium propionicum, and Paracoccus yeei), two cases with fungi (Exophiala oligosperma, Lomentospora prolificans and Aspergillus versicolor) and one case with a virus (Mupapillomavirus 1). Interestingly, four of the five bacterial species are also part of the human oral microbiome. Conclusions: Using a metagenomic sequencing we identified possible infectious causes in half of the ocular and periocular sarcoidosis cases analyzed. Our findings support the proposition that sarcoidosis could be an etiologically heterogenous disease. Because these are previously banked samples, direct follow-up in the respective patients is impossible, but these results suggest that sequencing may be a valuable tool in better understanding the etiopathogenesis of sarcoidosis and in diagnosing and treating this disease.

Safety and efficacy of rogaratinib in combination with atezolizumab in cisplatin-ineligible patients (pts) with locally advanced or metastatic urothelial cancer (UC) and FGFR mRNA overexpression in the phase Ib/II FORT-2 study.

4521 Background: Rogaratinib (R) is a novel pan-FGFR inhibitor that showed promising efficacy and safety in a Phase I trial in pts with advanced solid tumors, including UC, with FGFR1-3 mRNA overexpression. The Phase Ib/II FORT-2 study (NCT03473756) of R plus atezolizumab (A) in pts with first-line cisplatin-ineligible, FGFR-positive, advanced/metastatic UC previously identified a maximum tolerated dose of R 600 mg twice daily (BID) plus A (1200 mg every 3 weeks) . We report updated safety, efficacy, and the recommended Phase II dose (RP2D) for combination therapy from the Phase Ib study. Methods: Pts with cisplatin-ineligible, locally advanced/metastatic UC with FGFR1/3 mRNA overexpression detected by RNA in situ hybridization of archival tissue (RNAscope) received oral R 600 mg BID plus A 1200 mg on day 1 of a 21-day cycle. Archival tissue was examined for programmed cell-death ligand 1 (PD-L1) protein expression levels, FGFR3-activating mutations via a targeted Illumina NGS panel, and FGFR fusions via an Archer fusion plex assay. Primary objectives were safety, tolerability, and determination of the RP2D. Results: 26 pts (enrolled May 25, 2018 to Nov 25, 2020) were treated; 89% were male, median age was 76 years (range 47-85), 58% had an ECOG performance status of 1, and 77% displayed low or absent (negative or non-detectable) PD-L1 expression (combined positive score < 10%). Common treatment-emergent adverse events (TEAEs) included diarrhea (n = 17, 65%; 1 grade [G] 3), hyperphosphatemia (n = 15, 58%; all G1 or 2), and nausea (n = 11, 42%; 1 G3). The most common G3/4 TEAEs were elevated lipase without pancreatitis (n = 5, 19%), elevated amylase (n = 3, 12%), and rash and syncope (n = 2, 8% each). TEAEs led to interruption/reduction/discontinuation of R in 69%/46%/19% of pts. R-related unique TEAEs were hyperphosphatemia in 15 pts (58%) and retinal pigment epithelium detachment in 1 pt (4%). G5 events occurred in 3 pts (12%), unrelated to treatment. 13 of 24 evaluable pts (54%) had an objective response (OR) per RECIST v1.1. The disease control rate was 83%, including 3 pts (13%) with a complete response (CR), 10 (42%) with a partial response (PR), and 7 (29%) with stable disease. Median duration of response was not reached. OR rate was 56% (2 CRs and 7 PRs) in the 16 pts with tumors having low/negative PD-L1 protein and FGFR3 mRNA overexpression without mutation. The RP2D for R+A was 600 mg BID. Conclusions: First-line treatment with the RP2D of R+A achieved favorable clinical efficacy and tolerability in pts with cisplatin-ineligible, metastatic UC characterized by high FGFR1/3 mRNA expression and generally low/negative PD-L1 expression. Encouraging efficacy was observed regardless of PD-L1 expression or FGFR3 mutation status, warranting future investigation. Clinical trial information: NCT03473756.

Micro-region transcriptomics of fixed human tissue using Pick-Seq

ABSTRACTSpatial transcriptomics and multiplexed imaging are complementary methods for studying tissue biology and disease. Recently developed spatial transcriptomic methods use fresh-frozen specimens but most diagnostic specimens, clinical trials, and tissue archives rely on formaldehyde-fixed tissue. Here we describe the Pick-Seq method for deep spatial transcriptional profiling of fixed tissue. Pick-Seq is a form of micro-region sequencing in which small regions of tissue, containing 5-20 cells, are mechanically isolated on a microscope and then sequenced. We demonstrate the use of Pick-Seq with several different fixed and frozen human specimens. Application of Pick-Seq to a human melanoma with complex histology reveals significant differences in transcriptional programs associated with tumor invasion, proliferation, and immuno-editing. Parallel imaging confirms changes in immuno-phenotypes and cancer cell states. This work demonstrates the ability of Pick-Seq to generate deep spatial transcriptomic data from fixed and archival tissue with multiplexed imaging in parallel.

In-silico performance, validation, and modeling of the Nanostring Banff Human Organ transplant gene panel using archival data from human kidney transplants

Background RNA gene expression of renal transplantation biopsies is commonly used to identify the immunological patterns of graft rejection. Mostly done with microarrays, seminal findings defined the patterns of gene sets associated with rejection and non-rejection kidney allograft diagnoses. To make gene expression more accessible, the Molecular Diagnostics Working Group of the Banff Foundation for Allograft Pathology and NanoString Technologies partnered to create the Banff Human Organ Transplant Panel (BHOT), a gene panel set of 770 genes as a surrogate for microarrays (~ 50,000 genes). The advantage of this platform is that gene expressions are quantifiable on formalin fixed and paraffin embedded archival tissue samples, making gene expression analyses more accessible. The purpose of this report is to test in silico the utility of the BHOT panel as a surrogate for microarrays on archival microarray data and test the performance of the modelled BHOT data. Methods BHOT genes as a subset of genes from downloaded archival public microarray data on human renal allograft gene expression were analyzed and modelled by a variety of statistical methods. Results Three methods of parsing genes verify that the BHOT panel readily identifies renal rejection and non-rejection diagnoses using in silico statistical analyses of seminal archival databases. Multiple modelling algorithms show a highly variable pattern of misclassifications per sample, either between differently constructed principal components or between modelling algorithms. The misclassifications are related to the gene expression heterogeneity within a given diagnosis because clustering the data into 9 groups modelled with fewer misclassifications. Conclusion This report supports using the Banff Human Organ Transplant Panel for gene expression of human renal allografts as a surrogate for microarrays on archival tissue. The data modelled satisfactorily with aggregate diagnoses although with limited per sample accuracy and, thereby, reflects and confirms the modelling complexity and the challenges of modelling gene expression as previously reported.

Tissue and plasma tumor mutation burden (TMB) as predictive biomarkers in the CO.26 trial of durvalumab + tremelimumab (D+T) versus best supportive care (BSC) in metastatic colorectal cancer (mCRC).

61 Background: Pembrolizumab was recently granted tissue agnostic FDA accelerated approval for metastatic cancers with TMB≥10 mut/Mb. However, limited data supports immunotherapy in microsatellite stable (MSS) mCRC with TMB≥10 mut/Mb. We assessed tissue TMB and contrasted it to plasma derived TMB in the CO.26 trial. Methods: CO.26 was a phase 2 trial (2-sided ⍺ = 0.1 and 80% power) that randomized 180 patients (pts) 2:1 to D+T or BSC in refractory mCRC. Pre-treatment plasma was sequenced with the GuardantOMNI assay and archival tissue underwent exome sequencing with TMB assessed per the TMB harmonization project. MSI-H cases were excluded. For plasma TMB, we used a previously published cut point (≥28). Results: Overall survival (OS) but not progression free survival (PFS) was improved with D+T in the entire population. Of 180 pts, 163 were evaluable for plasma and 110 for tissue TMB. Median time between archival tissue and plasma collection was 3.1 yrs (IQR 1.9-5.1). Median tissue TMB was 6.6 muts/Mb (IQR 4.1-12.0), while median plasma TMB was 16.3 muts/Mb (IQR 9.4-25.9). Tissue and plasma TMB (r = -0.039, P = 0.69) were not correlated. Tissue TMB≥10 was not prognostic in the BSC arm (HR 1.01, 90%CI 0.52-1.92, P = 0.99) and OS was not improved in pts with tissue TMB≥10 (32/110 pts) following D+T vs BSC. A test of interaction suggested this threshold was not predictive (P = 0.85). Using a minimum P-value approach, no threshold supported high tissue TMB as predictive in MSS mCRC. In fact, the optimal cut point suggested low tissue TMB ( < 4.1 muts/Mb) had the greatest benefit from D+T (P-interaction = 0.048) and pts with TMB ≥4.1 mut/Mb (HR 0.50, 90%CI 0.26-0.96, P = 0.083) trended to better OS in the BSC arm. In contrast, 35/163 pts (21%) were identified in a high plasma TMB group associated with worse OS (HR 2.56, 90%CI 1.45-4.54, P = 0.007) in the BSC arm but improved OS following D+T compared to BSC with P-interaction = 0.082. Only 1 response was noted following D+T in a pt with tissue TMB = 16 mut/Mb and plasma TMB = 13 mut/Mb. Conclusions: Archival tissue TMB≥10 mut/Mb does not appear predictive of D+T benefit in MSS mCRC. Plasma derived TMB may better reflect evolutionary changes following intervening therapy than archival tissue. Clinical trial information: NCT02870920. [Table: see text]