Relapse Autopsy Worksheet Pdf ((BETTER))
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a) This identification process is shown in the illustration above. Patient samples (pointed by arrows) were sequenced in triplicate. For each patient, hotspots were identified in all three replicates that matched the same distinct subclonal population. Combining these hotspots with the matching subclonal tumor phylogeny reveals common subclonal "trunk" which is differentiated into branch subclonal mutations. Highlighted subclonal clusters (red) correspond to specific signature mutations.
Application of the Informative Automated Variant Analysis tool #9 to a hotspot identified in a relapse biopsy revealed that only a subset of this subclonal branch also exists in the metastatic lesion ( #21 ). In this example, only L861Q is identified in the metastatic lesion (highlighted in yellow) and so the clonal and subclonal proportions are matched between lesion and biopsy. The subclonal branch identified in D456 (blue) does not exist in the metastatic lesion, suggesting that the subclonal branch was already de-evolved prior to metastasis and was not responsible for relapse or disease progression. Alternatively, if the subclonal branch remains intact in the metastasis, the observed VAF in the metastatic lesion would have decreased, which is not consistent with the data.
b) Additional metastatic samples at other time points may be used to determine if a distinct subclonal population is responsible for cancer progression. The #9 analysis tool was also used to detect and differentiate subclonal mutations in other metastatic samples sequenced at time points D29 and D45. This analysis revealed two new subclonal recurrent branches, identified as R1219 | C1490 and A2177V | E972V, that were not identified in the two earliest metastatic samples sequenced. Each of these subclones has a unique phylogenetic tree that matches each of the metastatic lesion with a unique M-seq derived tumor phylogeny.
i) Reconstructed tumors. Tumors were derived from relapse (day 433) and primary (day 0) biopsies for each patient by extraction of single nuclei and singlecell whole genome amplification. Tumor tissue was accurately matched to tumor derived material for both. Includes matched primary, relapse ctDNA and relapse tumors followed by the matched primary tumor and ctDNA. j) Tumor derived ctDNA was successfully amplified by Whole Genome Amplification (WGA) from 6/6 matched pairs of primary and relapse tumors. k) DNA sequencing was completed for six libraries representing primary, relapse, primary sequencing data, ctDNA derived from primary and relapse biopsies. Depth of coverage by sequencing was between 147X and 188X sequencing depth. Detection of somatic mutations in pre-operative ctDNA (located on chromosome 19, 13, and 5) and tumor DNA (chromosome 2 and 16) are shown graphically by a-i). d2c66b5586