Personalized ctDNA disease monitoring via representative DNA sequencing
Disclosed herein is a method of deriving a plurality of genetic variants from a homogenized input sample. Also disclosed herein are methods of identifying a plurality of genetic variants in a sample comprising: homogenizing one or more input samples to provide a homogenized sample; preparing genomic material isolated from the homogenized input sample for sequencing; and identifying the plurality of genetic variants within sequencing data derived after sequencing the prepared genomic material.
1 . A method comprising:
(a) homogenizing a fixed tumor sample from a patient diagnosed with cancer to provide a homogenized sample, wherein the fixed tumor sample is residual tumor tissue, wherein any aliquot removed from the homogenized sample comprises a plurality of genetic mutations at a proportion at which they existed within the residual tumor tissue;
(b) sequencing genomic material derived from the homogenized sample to obtain a sequencing data set for the homogenized sample;
(c) identifying the plurality of genetic mutations within the obtained sequencing data set,
(d) identifying one or more common genetic mutations of the plurality of genetic mutations that are present in all cells in the homogenized sample;
(e) identifying one or more specific genetic mutations of the plurality of genetic mutations that are present in one or more subpopulations of cells within the homogenized sample but not in all cells in the homogenized sample;
(f) preparing a ctDNA monitoring panel specific for the patient diagnosed with cancer and based on (i) the identified one or more common genetic mutations present in all cells in the homogenized sample, and (ii) the identified one or more specific genetic mutations present in the one or more subpopulations of cells within the homogenized sample but not in all cells in the homogenized sample; and
(g) using the prepared ctDNA monitoring panel, repeatedly determining in one or more ctDNA samples obtained from the patient diagnosed with cancer which of the identified one or more specific genetic mutations present in the one or more subpopulations of cells has recurred or is resistant to therapy.
2 . The method of claim 1 , further comprising determining a response to therapy.
3 . The method of claim 1 , further comprising predicting a response to a future therapeutic strategy.
4 . The method of claim 1 , further comprising ascertaining a presence or absence of cancer in a patient during or following therapy.
5 . The method of claim 1 , further comprising detecting a minimal residual disease following surgical removal of a primary tumor.
6 . The method of claim 1 , further comprising detecting a minimal residual disease following surgical removal of a metastatic tumor.
7 . The method of claim 1 , wherein the homogenized sample includes genetic mutations from multiple residual tumor tissues from a single patient.
8 . The method of claim 1 , further comprising computing a clonal structure based on the identified plurality of genetic mutations, wherein the computing of the clonal structure comprises (i) calculating cancer cell fraction estimates for each of the plurality of identified genetic mutations; and (ii) grouping the calculated cancer cell fraction estimates into mutational clusters.
9 . The method of claim 1 , further comprising sorting cellular particles within the homogenized sample prior to the sequencing of the genomic material.
10 . The method of claim 9 , wherein the sorting of the cellular particles is based on size.
11 . The method of claim 9 , wherein the sorting of the cellular particles is based on a presence of one or more biomarkers.
12 . The method of claim 1 , further comprising determining a therapeutic strategy based on the identification of the one or more specific genetic mutations of the plurality of genetic mutations that are present in the one or more subpopulations of cells within the homogenized sample but not in all the cells in the homogenized sample.
13 . The method of claim 1 , wherein the sequencing with the sequencing comprises whole genome sequencing (WGS), whole exome sequencing (WES), single nucleotide polymorphism (SNP) analysis, deep sequencing, targeted gene sequencing, polymerase chain reaction (PCR), or any combination thereof.
14 . The method of claim 1 , wherein the residual tumor tissue is homogenized with a mechanical shearing apparatus.
15 . The method of claim 14 , wherein the mechanical shearing apparatus is an ultra sonicator.
16 . The method of claim 14 , wherein the mechanical shearing apparatus is a blender.
17 . The method of claim 16 , wherein the residual tumor tissue is mechanically sheared in the blender for about 2 minutes.
18 . The method of claim 16 , wherein the residual tumor tissue is mechanically sheared in the blender at about 15,000 rpm.
19 . The method of claim 14 , wherein the residual tumor tissue is mechanically sheared for a time period and at an energy based on the collagen content of the residual tumor tissue.
20 . The method of claim 14 , further comprising dissociating the homogenized sample to provide dissociated cells nuclei, and/or small tissue aggregates.
21 . The method of claim 20 , wherein the dissociation comprises mechanical dissociation, chemical dissociation, or enzymatic dissociation.
22 . The method of claim 1 , further comprising filtering the homogenized sample.