Methods and systems for adjusting tumor mutational burden by tumor fraction and coverage

A method of treating a subject having cancer, comprising administering an immunotherapy agent to the subject, wherein the subject has been identified for immunotherapy from a tumor mutational burden (TMB) status of the subject determined by:

• a. determining an observed mutational count from sequence information obtained from one or more nucleic acids in a cell-free DNA (cfDNA) sample from the subject;
• b. determining a tumor fraction and a coverage of the nucleic acids to generate sequencing parameters, wherein the tumor fraction is below about 1% of all nucleic acids in the cfDNA sample;
• c. determining an expected mutational fraction and/or an expected distribution of the expected mutational fraction given the sequencing parameters to generate an expected result,
• wherein determining the expected mutation fraction and/or the expected distribution of the expected mutational fraction comprises calculating a probability of identifying a mutation in a given mutant allele fraction (MAF) across a distribution of expected MAFs,
• wherein the distribution of expected MAFs is determined from the expected distribution of relative MAFs obtained from a control sample dataset comprising at least about 5000 control samples;
• d. adjusting the observed mutational count given the expected result to generate an adjusted mutational count;
• e. determining a TMB score using the adjusted mutational count;
• and
• f. classifying the TMB status of the sample as TMB-High when TMB score exceeds a threshold (thresholdTMB score), wherein the thresholdTMB score is set to indicate a subject who is likely to positively respond to immunotherapy.