# US 2023/0054587 A1 · Multiplexed Assay Using Differential Fragment Size to Identify Cancer Specific Cell-Free DNA

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- Number: US 2023/0054587 A1
- Document ID: US20230054587A1
- Status: Published application
- Kind: A1
- Application number: 17/786,969
- Filed: 2020-12-18
- Published: 2023-02-23
- Earliest claimed: 2019-12-19
- Assignees: Cadex Genomics, Corp.
- Applicants: Cadex Genomics, Corp.
- Inventors: Sudhir SINHA; Gary SPITZER; Hiromi BROWN; Patrick HALL
- Web page: https://patentgrep.com/patent/US20230054587A1

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 1/6851: Quantitative amplification
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 2600/118: Prognosis of disease development
- C12Q 2600/16: Primer sets for multiplex assays
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A retrotransposable element-based multiplexed quantitative polymerase chain reaction (qPCR) assay system to quantitate and distinguish cell free DNA integrity and concentration in blood, plasma, and serum as a measure of minimum residual disease, therapeutic effectiveness, neoadjuvant effectiveness in a patient having stage I, stage II, stage III, or stage IV cancer, and disease progression, thereby improving patient outcomes.

## Claims

### Claim 1

A method to quantitate the integrity of circulating cell free human DNA and implement a treatment of a patient, said method comprising:
(a) providing a first and second sample of serum, plasma, urine, or other biological fluid from a subject wherein the first and second samples are obtained at least one week apart, at least 2 weeks apart, at least 3 weeks apart or at least 4 weeks apart, the first and second samples comprising cell free human DNA (cfDNA), the cfDNA comprising (i) a first and second short retrotransposable interspersed element (RE) target sequence having a length of between about 60 base pairs to about 135 base pairs and (ii) a long RE target sequence having a length of between 200 bp and 300 bp, wherein the first and second short targets differ in length;
(b) quantitating each of the short and long RE targets in the first and second samples using a quantitative polymerase chain reaction (qPCR) method;
(c) obtaining for each of the quantitated RE targets in the first and second samples a threshold cycle number;
(d) comparing the threshold cycle number of each quantitated RE target with a standard curve to determine an amount of each of the quantitated RE targets that were present in the samples, wherein the amount of short RE targets in the second samples is indicative of the integrity of the circulating cell free DNA;
(e) determining an increase in the amount of short RE targets in the second sample as compared to the first sample above a threshold level, and
(f) implement a treatment of a patient having an increase in the amount of short RE targets in the second sample as compared to the first sample above a threshold level.

### Claim 2 (depends on 1)

The method of claim 1 wherein the sample is a plasma sample.

### Claim 3 (depends on 1)

The method of claim 1 wherein the first sample is obtained from the subject prior to administration of a neoadjuvant and the second sample is obtained from the subject after the neoadjuvant is administered and before another therapy is administered.

### Claim 4 (depends on 3)

The method of claim 3, wherein the subject has a stage I, stage II or stage III cancer.

### Claim 5 (depends on 1)

The method of claim 1 wherein the first sample is obtained from the subject prior to administration of a cycle of therapy and the second sample is obtained from the subject after the cycle of therapy is administered and before a next cycle of therapy is administered.

### Claim 6 (depends on 1)

The method of claim 1, wherein steps (a) through (f) are repeated through multiple cycles of therapy.

### Claim 7 (depends on 6)

The method of claim 6, wherein an increase in the amount of short RE targets in the second sample as compared to the first sample above a threshold level identifies the therapy as ineffective.

### Claim 8 (depends on 1)

The method of claim 1, wherein an increase in the amount of short RE targets in the second sample as compared to the first sample above a threshold level identifies the patient as having progressive disease.

### Claim 9 (depends on 1)

The method of claim 1, wherein an increase in the amount of short RE targets in the second sample as compared to the first sample above a threshold level identifies the patient as having minimum residual disease (MRD).

### Claim 10 (depends on 1)

The method of claim 1, wherein the retrotransposable interspersed element is an ALU, SVA, or LINE element.

### Claim 11 (depends on 1)

The method of claim 1, wherein the retrotransposable interspersed element has a copy number in excess of 1000 copies per genome.

### Claim 12 (depends on 1)

The method of claim 1 wherein the short RE targets has a length from 70 bp to about 130 bp, or from 60 bp to 120 bp.

### Claim 13 (depends on 1)

The method of claim 1 wherein the first short RE targets has a length between 70 and 80 bp, and the second short RE target has a length between 105 and 120 bp.

### Claim 14 (depends on 1)

The method of claim 1 wherein the qPCR method uses primer pairs set forth in Table 2A, 2B or 2C.

### Claim 15 (depends on 1)

The method of claim 1, wherein the forward primer and reverse primer pair for amplifying the short target sequence are selected from the following forward and reverse primer pairs,
Fragment
Primer
SEQ ID
name
Size
Type
Primer Sequence
NO
Yb8-80bp
80bp
Forward
GGAAGCGGAGCTTGCAGTGA
1
Reverse
AGACGGAGTCTCGCTCTGTCGC
2
Yb8-71bp
71bp
Forward
CTTGCAGTGAGCCGAGATT
4
Reverse
GAGACGGAGTCTCGCTCTGTC
5
Yb8-97bp
97bp
Forward
GTGGCTCACGCCTGTAAT
7
Reverse
GTGGCTCACGCCTGTAAT
7
Yb8-105bp
105bp
Forward
AGGCAGGAGAATGGCGTGAACC
10
Reverse
AGACGGAGTCTCGCTCTGTCGC
11
Yb8-120bp
120bp
Forward
TGGATCATGAGGTCAGGAGAT
15
Reverse
CCGAGTAGCTGGGACTACA
16
SVA-100bp
100bp
Forward
AATGGCGGCTTTGTGGAATA
20
Reverse
GTCTCCCATGTCTACTTCTTTCTAC
21
SVA-101bp
101bp
Forward
AACCCTGTGCTCTCTGAAAC
23
Reverse
ACGCTGCCTTCAAGCAT
24
SVA-103bp
103bp
Forward
GCCCAACAGCTCATTGAGAA
25
Reverse
ACGGCAACCATCCGATTT
26
SVA-104bp
104bp
Forward
TGTCCACTCAGGGTTAAATGG
27
Reverse
GATTAGGGATTGGTGATAACTCTTA
28
SVA-106bp
106bp
Forward
TGTGTCCACTCAGGGTTAAAT
30
Reverse
GATTAGGGATTGGTGATGACTCT
31
SVA-106bp-v2
106bp
Forward
TGTGCCCAACAGCTCATT
33
Reverse
ACGGCAACCATCCGATTT
34
SVA-116bp
116bp
Forward
CTGTGTCCACTCAGGGTTAAATG
35
Reverse
ATTACTTGAGATTAGGGATTGGTGATG
36
SVA-116bp-v2
116bp
Forward
CCCAACAGCTCATTGAGAACG
38
Reverse
CTTTCTACACAGACACGGCAA
39
SVA-118bp
118bp
Forward
CTCTCTGAAACATGTGCTGTGT
40
Reverse
GGGATTGGTGATGACTCTTAACG
41
SVA-118bp-v2
118bp
Forward
CTGTGTCCACTCAGGGTTAAAT
43
Reverse
TGATTACTTGAGATTAGGGATTGGT
44
SVA-126bp
126bp
Forward
CTGTGTCCACTCAGGGTTAAAT
46
Reverse
TGTGTCCCTGATTACTTGAGATTAG
47
SVA-126bp-
126bp
Forward
CCTGTTGATCTGTGACCTTACC
48
V2
Reverse
ACGCTGCCTTCAAGCAT
49
SVA-128bp
128bp
Forward
GTTGCCGTGTCTGTGTAGAA
51
Reverse
TTTCAGAGAGCACAGGGTTG
52
SVA-132bp
132bp
Forward
AACCCTGTGCTCTCTGAAAC
54
Reverse
GATTAGGGATTGGTGATAACTCTTA
55
and the forward primer and reverse primer pairs for amplifying the long target sequence are selected from the following forward and reverse primer pairs,
Primer
SEQ
Name
Size
Type
Primer Sequence
ID NO
Yb8-105bp
105bp
Forward
AGGCAGGAGAATGGCGTGAACC
10
Reverse
AGACGGAGTCTCGCTCTGTCGC
11
Yb8-120bp
120bp
Forward
TGGATCATGAGGTCAGGAGAT
15
Reverse
CCGAGTAGCTGGGACTACA
16
SVA-257bp
257bp
Forward
CCTGTGCTCTCTGAAACATGTGCT
60
Reverse
GATTTGGCAGGGTCATGGGACAAT
61
SVA-265bp
265bp
Forward
ATGTGCTGTGTCCACTCAGGGTTA
63
Reverse
ATTCTTGGGTGTTTCTCACAGAGG
64
Line1-252bp
252bp
Forward
CACAATAGCAAAGACTTGGAACC
77
Reverse
CCCTTCCTGTGTCCATGTG
78
Probe
CCTTTGTAGGGACATGGATGAAAGTGGA
79
Line1-257bp
257bp
Forward
GACTTGGAACCAACCCAAATG
80
Reverse
CCCAGAGTGTGACGTTCC
81
Probe
AGTGAGAACACATGGACACAGGAAGG
82
Line1-262bp
262bp
Forward
GTGGCACATATACACCATGGAA
83
Reverse
CGTTAGGTATATCTCCCAATGCTATC
84
Probe
TGAGAACACATGGACACAGGAAGGG
85
Line1-266bp
266bp
Forward
ACTTGGAACCAACCCAAATG
86
Reverse
CACAACAGTCCCCAGAGTG
87
Probe
TGAGAACACATGGACACAGGAAGGG
88
Line1-267bp
267bp
Forward
CATGGAATACTATGCAGCCATAAA
89
Reverse
CCCACTAACTCGTCATCTAGC
90
Probe
TGAGAACACATGGACACAGGAAGGG
91

### Claim 16 (depends on 1)

The method of claim 1, further comprising a step of adding a synthetic DNA sequence as an internal positive control prior to step (b), quantitating the internal positive control in step (b), and utilizing the quantitative internal positive control result in the comparing step to improve the accuracy and reliability of the comparing step.

### Claim 17 (depends on 16)

The method of claim 16, the use of the internal positive control enabling a determination of a concentration of cell free DNA in the sample.

### Claim 18 (depends on 1)

The method of claim 1, the providing and using steps being carried out in a single tube or well.

### Claim 19 (depends on 1)

The method of claim 1, the providing step further comprising providing a hybridization probe that hybridizes to the RE target.

### Claim 20 (depends on 10)

The method of claim 10, the probe including an observable label.

### Claim 21 (depends on 11)

The method of claim 11, the observable label being a fluorescent label.

### Claim 22 (depends on 10)

The method of claim 10, wherein the observable label is detected with a microfluidic device.

### Claim 23 (depends on 1)

The method of claim 1, wherein the amount of the short and long RE targets in the first and second samples amplified in the quantitative polymerase chain reaction (qPCR) method are measured using an electrical biosensor.

### Claim 24 (depends on 1)

The method of claim 1, wherein the patient is suffering from cancer, is in remission from cancer, is at high risk for developing cancer, is categorized as having a complete response (“CR”), is categorized as having stable disease (“SD”), is categorized as having partial response (“PR”), is categorized as having progressive disease (“PD”), is characterized as having a stage I, stage II, stage III or stage IV cancer, has had surgery to remove a tumor, has undergone a targeted therapy to treat a cancer has undergone chemotherapy to treat a cancer, has undergone immunotherapy to treat a cancer, or has undergone radiotherapy to treat a cancer or the patient has a minimum residual disease diagnosis.

### Claim 25 (depends on 1)

The method of claim 1, wherein the quantitated short RE target amount represents one cancer cell in 500,000 total cells or greater, 1,000,000 total cells or greater or 1,500,000 cells or greater in the patient.

### Claim 26 (depends on 1)

The method of claim 1, wherein the treatment of the patient is a neoadjuvant, a targeted therapy, a cancer chemotherapy, immunotherapy or radiotherapy.

### Claim 27 (depends on 26)

The method of claim 26, wherein the treatment is selected from the group including antineoplastic agents, alkylating agents, topoisomerase inhibitors, mitotic inhibitors, methotrexate, vinca alkaloids, antimetabolites, antifolates, pyrimidine antagonists, purine analogs, purine antagonists, proteasome inhibitors, tyrosine kinase inhibitors, nitrogen mustards, immunotherapy, or another cancer therapy.

### Claim 28

A multiplexed system for identifying an ineffective neoadjuvant or cancer treatment, or for characterizing cancer or MRD in humans, the system comprising:
a. a sample of serum, plasma, urine, or other biological fluid from a human, the sample comprising cell free DNA, the cell free DNA comprising two short RE targets and a long RE target, the short RE targets having a length between 60 bp and 135 bp with the proviso that the two short RE targets differ in size sufficiently to distinguish their amplicons, the long RE target having a length between 200 and 300 bp, between 207 bp and 270 bp, or between 260 and 265 bp, the short RE targets and the long RE target being independent of each other, the sample further comprising an internal positive control comprising synthetic DNA;
b. a TaqMan® probe corresponding to each of the short RE targets, the long RE target and the IPC, each probe comprising a detectable label that is distinct from the labels incorporated into the other probes;
c. a forward primer and a reverse primer for amplifying each of the short RE target, the long RE target and the IPC;
d. a DNA standard for generating standard curves for RE targets;
e. a qPCR system for simultaneously amplifying the short RE targets, the long RE target and the IPC and for producing a threshold cycle number for each target; and
f. a qPCR data analysis system for producing DNA quantitation values for each RE target by interpolation using threshold cycle numbers and standard curves and for using the DNA quantitation values to produce an indication of the integrity of the cell free DNA.

### Claim 29 (depends on 28)

The method of claim 28 wherein the two short RE targets differ in length by at least 10 bp, 15 bp, 20 bp, or 25 bp.

### Claim 30 (depends on 28)

The multiplexed method of claim 28, wherein the primers for amplifying the targets are set forth in Table 2A, 2B and 2C.