Cancer mutation signature can be reproducibly detected in blood in patients with stage II or III breast cancer.

a: Blood samples 1–8 and 10 in case-1 and 1–3 in case-2 were frozen, thawed, and centrifuged. DNA was extracted from the cell pellet and from the supernatant, which contained plasma and lysed red cells. Blood cells for time points 9, 11, and 12 were centrifuged when fresh and the plasma was removed.

b: The patient (case-1 with c.720_721insAGT in p53 gene) remained disease free at the end of follow-up. The other patient (case-2 with c.165_166delTG in p53 gene) died of progressive disease.

c: The number of positive signals appearing in the total number of MAP reactions per time point.

d: The expected average number of mutants per reaction is estimated using a formula (the frequency of zero mutants per reaction = e^−x, where x is the average number of mutants per reaction), assuming that: the mutant distributes in the reaction according to a Poisson distribution; if one or more mutants are in the reaction, the amplification is positive; and if zero mutants are in the reaction, it is negative.

e: Follow-up assays were performed with 1 ml plasma instead of 3 ml, as in the earlier assays. Fewer reactions were run, perhaps decreasing the analytical sensitivity of the measurement.