<p>Supplemental Figure 2. The concentration of plasma cfDNA in previous described patients with cancer according to stage. (A) The concentration of cfDNA in patients of all cancer types (9). The concentration of cfDNA in patients with Stage III cancers was significantly higher than that of patients with Stage I cancers (p < 0.01). (B) The concentration of cfDNA in individual cancer types. Stage was classified according to AJCC 7th edition. The concentration of cfDNA in patients with Stage III breast cancer was significantly higher than that of patients with Stage I breast cancer (p < 0.05). For both panels, data were derived from the previously published CancerSEEK study (9).</p>
The analysis of cell-free DNA (cfDNA) from plasma offers great promise for the earlier detection of cancer. At present, changes in DNA sequence, methylation, or copy number are the most sensitive ways to detect the presence of cancer. To further increase the sensitivity of such assays with limited amounts of sample, it would be useful to be able to evaluate the same template molecules for all these changes. Here, we report an approach, called MethylSaferSeqS, that achieves this goal, and can be applied to any standard library preparation method suitable for massively parallel sequencing. The innovative step was to copy both strands of each DNA-barcoded molecule with a primer that allows the subsequent separation of the original strands (retaining their 5-methylcytosine residues) from the copied strands (in which the 5-methylcytosine residues are replaced with unmodified cytosine residues). The epigenetic and genetic alterations present in the DNA molecules can then be obtained from the original and copied strands, respectively. We applied this approach to plasma from 265 individuals, including 198 with cancers of the pancreas, ovary, lung, and colon, and found the expected patterns of mutations, copy number alterations, and methylation. Furthermore, we could determine which original template DNA molecules were methylated and/or mutated. MethylSaferSeqS should be useful for addressing a variety of questions relating genetics and epigenetics.
<p>Supplemental Figure 4. Methylation profiles using quadratic programming vs. non-negative least- squares regression using the reference matrix described in Sun et al. (3). Pearson’s correlation coefficient and p values are presented at the bottom of this figure, showing the derived contributions from each of the 12 tissue types that could be assessed.</p>
<p>Supplemental Figure 9. Plasma AST and ALT levels before and ~24 hours after surgery for pancreatic cancer. AST and ALT levels substantially increased in all five patients.</p>
<p>Supplemental Figure 6. Correlation between the cfDNA derived from colon epithelial cells, lung cells, and pancreatic cells, and the fraction of cfDNA derived from neoplastic colon epithelial cells, lung cells, or pancreatic cells.</p>
Current non-invasive approaches for detection of urothelial cancers are suboptimal. We developed a test to detect urothelial neoplasms using DNA recovered from cells shed into urine. UroSEEK incorporates massive parallel sequencing assays for mutations in 11 genes and copy number changes on 39 chromosome arms. In 570 patients at risk for bladder cancer (BC), UroSEEK was positive in 83% of those who developed BC. Combined with cytology, UroSEEK detected 95% of patients who developed BC. Of 56 patients with upper tract urothelial cancer, 75% tested positive by UroSEEK, including 79% of those with non-invasive tumors. UroSEEK detected genetic abnormalities in 68% of urines obtained from BC patients under surveillance who demonstrated clinical evidence of recurrence. The advantages of UroSEEK over cytology were evident in low-grade BCs; UroSEEK detected 67% of cases whereas cytology detected none. These results establish the foundation for a new non-invasive approach for detection of urothelial cancer.
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