METHODS AND SYSTEMS FOR DETERMINING BLOOD TUMOR MUTATIONAL BURDEN IN A LIQUID BIOPSY ASSAY

1 . A method of determining a liquid biopsy tumor mutational burden (lTMB) for a test subject comprising:

at a computer system having one or more processors, and memory storing one or more programs for execution by the one or more processors:

A) obtaining, from a panel-enriched sequencing reaction, a plurality of nucleic acid sequences comprising a corresponding sequence for each cell-free DNA fragment in a first plurality of cell-free DNA fragments obtained from a liquid biopsy sample from the test subject, wherein each respective cell-free DNA fragment in the first plurality of cell-free DNA fragments corresponds to a respective probe sequence in a plurality of probe sequences used to enrich cell-free DNA fragments in the liquid biopsy sample in the panel-enriched sequencing reaction; and

B) determining, using the panel-enriched sequencing reaction, that a circulating tumor fraction (ctFE) is above a threshold ctFE value;

C) responsive to determining that the ctFE is above the threshold, calculating the lTMB for the test subject from the panel-enriched sequencing reaction; and

D) reporting the lTMB for the test subject.

2 . The method according to claim 1 , wherein the panel-enriched sequencing reaction is performed at a read depth of at least 500×.

3 . (canceled)

4 . The method of claim 1 , wherein the plurality of probe sequences used to enrich cell-free DNA fragments in the liquid biopsy sample in the panel-enriched sequencing reaction collectively map to from 25 different genes to 150 different genes in a human reference genome.

5 . The method of claim 1 , wherein the panel-enriched sequencing reaction uses a sequencing panel that enriches for at least 10 genes listed in Table 1, List 1, or List 2.

6 - 7 . (canceled)

8 . The method of claim 1 , wherein the calculating C) comprises:

determining a count of a plurality of genetic variants present in the plurality of nucleic acid sequences, wherein the count of the plurality of genetic variants present in the plurality of nucleic acid sequences is a count of unique genetic variants present in the plurality of nucleic acid sequences that satisfy one or more qualifying criterion in a set of qualifying criteria; and

normalizing the count of the plurality of genetic variants present in the plurality of nucleic acid sequences by a coverage of the plurality of probe sequences.

9 - 12 . (canceled)

13 . The method of claim 8 , wherein a qualifying criterion in the set of qualifying criteria is a requirement that the respective genetic variant is a missense variant, a combination of a missense variant and a splice region variant, a frameshift variant, a stop loss variant, a splice acceptor variant, an in frame insertion variant, an in frame deletion variant, a combination of a frameshift variant and a splice region variant, a disruptive in frame insertion variant, or a disruptive in frame deletion variant.

14 . The method of claim 8 , wherein a qualifying criterion in the set of qualifying criteria is a requirement that the respective genetic variant has a variant allele frequency in the liquid biopsy sample that is greater than 0.005 (0.5%).

15 . (canceled)

16 . The method of claim 12 , wherein a qualifying criterion in the set of qualifying criteria is a requirement that the respective genetic variant has a variant allele frequency (VAF) in the liquid biopsy sample that is any one of:

(i) greater than 0.01 (1%) and less than 0.4 (40%),

(ii) greater than 0.6 (60%) and less than 0.9 (90%),

(iii) greater than 0.4 (40%) and less than 0.60 (60%) with the proviso that

❘

"\[LeftBracketingBar]"

VAF

-

ctFE

❘

"\[RightBracketingBar]"

ctFE

<

1

,

or

(iv) greater than 0.9 (90%) with the proviso that

❘

"\[LeftBracketingBar]"

VAF

-

ctFE

❘

"\[RightBracketingBar]"

ctFE

<

1.

17 . (canceled)

18 . The method of claim 1 , wherein the liquid biopsy sample is a blood sample.

19 . The method of claim 1 , wherein the method further comprises:

using the lTMB to identify a concordant lTMB based on a predetermined correlation between (i) detection of somatic mutations in cell-free DNA from a liquid biopsy sample from each training subject in a cohort of training subjects and (ii) detection of somatic mutations in genomic DNA from solid tumor biopsy samples from the cohort of training subjects; and

the reporting D) further comprises reporting the concordant lTMB.

20 . The method of claim 1 , wherein the reporting D) further comprises

determining that the lTMB satisfies a therapeutic threshold and reporting a matched therapeutic recommendation for the test subject.

21 . The method of claim 1 , the method further comprising:

determining that the lTMB for the test subject satisfies a therapeutic threshold, and

administering an immunotherapeutic agent to the test subject.

22 . The method of claim 1 , wherein the reporting D) further comprises:

determining that the lTMB satisfies a clinical trial threshold; and

reporting a matched clinical trial recommendation for the test subject.

23 . The method of claim 1 , further comprising

determining that the lTMB for the test subject satisfies a clinical trial threshold; and

enrolling the test subject in a clinical trial associated with the clinical trial threshold.

24 . The method of claim 1 , wherein the reporting D) comprises comparing the lTMB for the subject to a severity threshold and reporting a qualitative status of either lTMB high (lTMB-H) or lTMB low (lTMB-L) based on the comparing.

25 . (canceled)

26 . The method of claim 1 , further comprising updating an electronic health record for the test subject to include the lTMB for the test subject.

27 . The method of claim 12 , wherein a qualifying criterion in the set of qualifying criteria is selection of a genetic variant present in the plurality of nucleic acid sequences by a medical professional.

28 . The method of claim 1 , wherein the plurality of probe sequences map to no more than 150 genes in the human genome

29 . A computer system comprising:

one or more processors; and

a non-transitory computer-readable medium including computer-executable instructions that, when executed by the one or more processors, cause the processors to perform a method of determining a liquid biopsy tumor mutational burden (lTMB) for a test subject comprising:

A) obtaining, from a panel-enriched sequencing reaction, a plurality of nucleic acid sequences comprising a corresponding sequence for each cell-free DNA fragment in a first plurality of cell-free DNA fragments obtained from a liquid biopsy sample from the test subject, wherein each respective cell-free DNA fragment in the first plurality of cell-free DNA fragments corresponds to a respective probe sequence in a plurality of probe sequences used to enrich cell-free DNA fragments in the liquid biopsy sample in the panel-enriched sequencing reaction; and

B) determining, using the panel-enriched sequencing reaction, that a circulating tumor fraction (ctFE) is above a threshold ctFE value;

C) responsive to determining that the ctFE is above the threshold, calculating the lTMB for the test subject from the panel-enriched sequencing reaction; and

D) reporting the lTMB for the test subject.

30 . A non-transitory computer-readable storage medium having stored thereon program code instructions that, when executed by a processor, cause the processor to perform a method of determining a liquid biopsy tumor mutational burden (lTMB) for a test subject comprising:

A) obtaining, from a panel-enriched sequencing reaction, a plurality of nucleic acid sequences comprising a corresponding sequence for each cell-free DNA fragment in a first plurality of cell-free DNA fragments obtained from a liquid biopsy sample from the test subject, wherein each respective cell-free DNA fragment in the first plurality of cell-free DNA fragments corresponds to a respective probe sequence in a plurality of probe sequences used to enrich cell-free DNA fragments in the liquid biopsy sample in the panel-enriched sequencing reaction; and

B) determining, using the panel-enriched sequencing reaction, that a circulating tumor fraction (ctFE) is above a threshold ctFE value;

C) responsive to determining that the ctFE is above the threshold, calculating the lTMB for the test subject from the panel-enriched sequencing reaction; and

D) reporting the lTMB for the test subject.