Aligning tumor mutational burden (TMB) quantification across diagnostic platforms: phase II of the Friends of Cancer Research TMB Harmonization Project.

BACKGROUND: Tumor mutational burden (TMB) measurements aid in identifying patients who are likely to benefit from immunotherapy; however, there is empirical variability across panel assays and factors contributing to this variability have not been comprehensively investigated. Identifying sources of variability can help facilitate comparability across different panel assays, which may aid in broader adoption of panel assays and development of clinical applications. MATERIALS AND METHODS: Twenty-nine tumor samples and 10 human-derived cell lines were processed and distributed to 16 laboratories; each used their own bioinformatics pipelines to calculate TMB and compare to whole exome results. Additionally, theoretical positive percent agreement (PPA) and negative percent agreement (NPA) of TMB were estimated. The impact of filtering pathogenic and germline variants on TMB estimates was assessed. Calibration curves specific to each panel assay were developed to facilitate translation of panel TMB values to whole exome sequencing (WES) TMB values. RESULTS: Panel sizes >667 Kb are necessary to maintain adequate PPA and NPA for calling TMB high versus TMB low across the range of cut-offs used in practice. Failure to filter out pathogenic variants when estimating panel TMB resulted in overestimating TMB relative to WES for all assays. Filtering out potential germline variants at >0% population minor allele frequency resulted in the strongest correlation to WES TMB. Application of a calibration approach derived from The Cancer Genome Atlas data, tailored to each panel assay, reduced the spread of panel TMB values around the WES TMB as reflected in lower root mean squared error (RMSE) for 26/29 (90%) of the clinical samples. CONCLUSIONS: Estimation of TMB varies across different panels, with panel size, gene content, and bioinformatics pipelines contributing to empirical variability. Statistical calibration can achieve more consistent results across panels and allows for comparison of TMB values across various panel assays. To promote reproducibility and comparability across assays, a software tool was developed and made publicly available.

Validation of a Multiplexed Gene Signature Assay for Diagnosis of Canine Cancers from Formalin-Fixed Paraffin-Embedded Tissues.

BACKGROUND: Use of molecular-based diagnostics for companion animals is impeded by availability of technology platforms, tissue acquisition requirements, and species-specific reagents. HYPOTHESIS/OBJECTIVES: To validate a quantitative nuclease protection assay (qNPA) to simultaneously measure RNA expression of multiple genes in archived formalin-fixed paraffin-embedded (FFPE) tumors from dogs. ANIMALS: All tumor biopsy samples were collected retrospectively from surgical biopsies and in the care of veterinarians. METHODS: Retrospective case series. A qNPA 96-well ArrayPlate was built using 30 canine-specific genes, 5 housekeeping genes, positive and negative controls with qualified gene-specific oligonucleotides. Pearson's correlation, coefficient of variation (CV), and multivariate analysis were used to determine analytical performance using 40 FFPE dog tumors. Once validated, 70 FFPE dog tumors were analyzed for differences in gene expression using hierarchical clustering and analysis of variance of log transformed data. Immunohistochemistry (IHC) was performed to correlate gene expression and protein expression in a subset of tumors. RESULTS: The assay was linear with decreasing sample input (R2 = 0.978), reproducible within and between 96-well plates (r = 0.988 and 0.95, respectively) and between different laboratories (CV = 0.96). Hierarchical cluster analysis showed grouping of tumors by histogenesis and oncogenes. Significant differences were found between BCl2, E2F transcription factor 1, MDM2, COX-2, MET proto-oncogene receptor kinase, and other biologically relevant gene expression in tumor subtypes. Immunohistochemistry confirmed protein expression. CONCLUSIONS AND CLINICAL IMPLICATIONS: Because this technology works reliably on FFPE specimens, it can help expedite the broad introduction of multiplexed genomic information for improved diagnostics and discovery of new targets for therapies in veterinary oncology.

Cyclooxygenase-1 and 2 in normal and malignant human ovarian epithelium.

OBJECTIVES: Cyclooxygenase-1 and 2 (COX-1 and COX-2) play important roles in normal physiology and are often dysregulated in neoplastic tissues. The present study determines whether COX-1 and COX-2 are expressed in ovarian cancers and whether the pattern of expression of these enzymes reveals clues to their roles in this cancer. METHODS: The expression of COX-1 and COX-2 proteins in 9 normal human ovaries, in 137 cases of ovarian cancers of epithelial origin (83 primary and 54 metastatic), and in 7 ovarian cancer cell lines was examined by immunohistochemistry and western analysis. RESULTS: COX-1 protein was present in 95/137 (69.3%) of the total cancers studied, with 55/83 (66.3%) of the primary cancers and 40/54 (74.1%) of the metastatic cancers positive for protein. COX-2 was present in 97/137 (70.8%) of all cancers studied, with 53/83 (63.9%) of the primary cancers and 44/54 (81.5%) of the metastatic cancers positive for protein. Notably, the quickscores for COX-2-positive staining were significantly higher in metastatic cancers. Moreover, COX-2 immunostaining was frequently found at the advancing margin of tumor invasion or in new metastatic loci. COX-1 protein expression was observed in the ovarian surface epithelial cells, especially that of the inclusion cysts. COX-1 was also detected by western blot in seven of nine ovarian cancer cell lines. However, no COX-2 was detected in either normal epithelium or cancer cell lines. CONCLUSION: COX-1 and COX-2 were expressed in every type of ovarian epithelial cancer, suggesting that each may contribute to the cancer development or progression.

Effects of TCDD upon IkappaB and IKK subunits localized in microsomes by proteomics.

Biochemical studies have shown that microsomes represent an important subcellular fraction for determining 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) effects. Proteomic analysis by two-dimensional gel-mass spectrometry of liver microsomes was undertaken to gain new insight into the actions of TCDD in male and female rats. Proteomic analysis showed TCDD induced several xenobiotic metabolism enzymes as well as a protein at 90kDa identified by mass spectrometry as IkappaB kinase beta/IKK2. This observation led to the discovery of other NF-kappaB binding proteins and kinases in microsomes and effects by TCDD. Western blotting for IKK and IkappaB family members in microsomes showed a distinct pattern from cytosol. IKK1 and IKK2 were both present in microsomes and were catalytically active although, unlike cytosol, IKKgamma/NEMO was not detectable. TCDD exposure produced an elevation in cytosolic and microsomal IKK activity of both genders. The NF-kappaB binding proteins IkappaBbeta and IkappaBgamma were prevalent in microsomes, while IkappaBalpha and IkappaB epsilon proteins were absent. TCDD treatment produced hyperphosphorylation of microsomal IkappaBbeta in both sexes with females being most sensitive. In cytosol, IkappaBalpha, IkappaBbeta, and IkappaB epsilon, but not IkappaBgamma, were clearly observed but were not changed by TCDD. Overall, proteomic analysis indicated the presence of NF-kappaB pathway members in microsomes, selectively altered by dioxin, which may influence immune and inflammatory responses within the liver.