Genomic Analysis of Human Brain Metastases Treated with Stereotactic Radiosurgery Under the Phase-II Clinical Trial (NCT03398694) Reveals DNA Damage Repair at the Peripheral Tumor Edge

Abstract

Stereotactic Radiosurgery (SRS) is one of the leading treatment modalities for oligo brain metastasis (BM), however no comprehensive genomic data assessing the effect of radiation on BM in humans exist. Leveraging a unique opportunity, as part of the clinical trial (NCT03398694), we collected post-SRS, delivered via Gamma-knife or LINAC, tumor samples from core and peripheral-edges of the resected tumor to characterize the genomic effects of overall SRS as well as the SRS delivery modality. Using these rare patient samples, we show that SRS results in significant genomic changes at DNA and RNA levels throughout the tumor. Mutations and expression profiles of peripheral tumor samples indicated interaction with surrounding brain tissue as well as elevated DNA damage repair. Central samples show GSEA enrichment for cellular apoptosis while peripheral samples carried an increase in tumor suppressor mutations. There are significant differences in the transcriptomic profile at the periphery between Gamma-knife vs LINAC.

Figure 1:. Clinical trial details and patient characteristics:

A) Characteristics of the 34 patients enrolled in the clinical trial who contributed samples to the analyses contained within the manuscript. B) Schematic depicting diagnosis, radiosurgery timeline, and surgery and sample collection for clinical trial participants. C) Infographic depicting the primary tumor type of clinical trial participants and components of center or peripheral DNA and/or RNA contributed to the analyses.

Figure 2:. Pre-operative stereotactic radiosurgery induces DNA damage leading to cellular changes in brain metastasis:

A) Schematic depicting analysis of samples with and without radiosurgery compared between common primary tumor locations (lung) or across primary tumor locations. B) Quantification of total damage marks and type of damage detected by variant calling between radiated and non-radiated samples. C) Comparison of high impact deletions or insertions among radiated and non-radiated lung cancer samples. D) 3D plot of all samples organized by chromosome (x), number of variants detected (y), and patient (z). E) Chromosome visualization plot of mutated genes across non-radiated and radiated samples. F) Single base substitutions present in radiated samples. G) Single base substitutions present grouped by primary tumor location across radiated samples. H) ALLEZ GSEA waterfall plot with previous term exclusion applied to genes differentially expressed among tumors metastasized from lung. I) Heatmap visualization of differentially expressed genes among non-radiated samples (left/blue) and radiated samples from tumors metastasized from lung (right/brown). J) Heatmap visualizing expression of genes associated with GO term “DNA Repair” among non-radiated and radiated tumors metastasized from lung. K) Heatmap visualizing expression of genes associated with GO term “Double Strand Break” among non-radiated and radiated tumors metastasized from lung. Comparison between samples done using students t- test (B), differential expression analysis was done within DESeq2 or ALLEZ using adjusted p-value < 0.05 (H, I, J, K). **** p< 0.0001

Figure 3:. Radiation dosing fall-off along peripheral edges of brain metastasis induces DNA damage repair and enrichment of cellular growth genes.

A) Schematic depicting isolation of central and peripheral tumor samples during surgery post SRS with comparisons being made across and between tumor types. B) Quantification of total somatic variants detected among central and peripheral biopsy locations. C) Quantification of shared or unique somatic variants detected among central and peripheral biopsy locations. D) Chromosome level visualization of variants from central or peripheral biopsy locations summed (top) or individualized (below). E) Heatmap visualization of variants per gene per sample among all central and peripheral biopsy locations across primary tumor location and SRS delivery modality. F) Heatmap of differential isoform enrichment analyses conducted by DIFFUSE across central and peripheral biopsy locations. G) ALLEZ GSEA waterfall plot with previous term exclusion conducted on differentially expressed isoforms between central and peripheral biopsy locations. H) GSEA of differentially expressed genes using ClusterProfiler between central and peripheral biopsy locations restricted to terms included in DNA damage or repair. I) Quantification of mutations on genes annotated to be tumor suppressors across central and peripheral biopsy locations. J) FARDEEP deconvolution analysis using brain cell type references conducted on central and peripheral biopsy locations. K) FARDEEP deconvolution analysis using immune cell type references conducted on central and peripheral biopsy locations. L) In-vitro visualization of CD45+ cell invasion using IHC on central and peripheral tumor biopsy locations. Statistical comparisons between groups were conducted with ANOVA with Bonferroni correction (L). Differential expression analysis was done within DESeq2, ALLEZ, ClusterProfiler, and DIFFUSE using adjusted p-value < 0.05 (E, F, G, H, I). * p< 0.05

Figure 4:. Radiation delivery modality and primary tumor location influence genetic signatures and relevant DNA damage measured in brain metastases.

A) Schematic depicting comparison between GK and LINAC treated samples with representative images for dose contouring on individual patients. B) Chromosome visualization of mutations between GK and LINAC treated samples across central and peripheral biopsy locations. C) Quantification of total variants detected among peripheral samples between GK and LINAC SRS delivery. D) Volcano plot of DESeq2 determined differentially expressed genes between GK and LINAC treated samples. E) Visualization of insertion or deletion variants detected among all primary tumor locations. F) Heatmap displaying differentially expressed genes between BM’s of breast of lung primary tumor origin. G) Venn diagram of differentially expressed genes across radiosurgery v no-radiosurgery comparisons and central and peripheral comparisons. H) Venn diagram of high impact mutations across the 4 patients within the clinical trial with documented local failure. I) Schematic depicting the in vitro validation of bioinformatic gene hits using QPCR on both isolated peripheral and central biopsy samples as well as radiated A549 primary tumor cells. J) QPCR quantification of relative expression of CENPB, PSG1, and POM121, in radiated and non-radiated A549 tumor cells. K) QPCR quantification of relative expression of POM121, PSG1, and SPINK13, in isolated central or peripheral tumor biopsies. Statistical comparison between two groups conducted with students T-test (C, J), between more than two groups using ANOVA with Bonferroni correction (K). Differential expression analysis was done within DESeq2, ALLEZ, ClusterProfiler, and DIFFUSE using adjusted p-value < 0.05 (D.F). * p<0.05, *** p<0.001, **** p<0.0001