# US 10,482,993 B2 · Analyzing copy number variation in the detection of cancer

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- Number: US 10,482,993 B2
- Document ID: US10482993B2
- Status: Granted
- Kind: B2
- Application number: 15/017,475
- Filed: 2016-02-05
- Published: 2016-08-11
- Granted: 2019-11-19
- Earliest claimed: 2010-01-19
- Assignees: Verinata Health, Inc.
- Applicants: Verinata Health, Inc.
- Inventors: Richard P. Rava; Brian K. Rhees
- Web page: https://patentgrep.com/patent/US10482993B2

## CPC

- G16H 50/30: for calculating health indices; for individual health risk assessment
- A61P 35/00: Antineoplastic agents
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C12Q 1/6869: Methods for sequencing
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B 30/10: Sequence alignment; Homology search
- G16H 50/20: for computer-aided diagnosis, e.g. based on medical expert systems
- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
- G16H: HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

The invention provides a method for determining copy number variations (CNV) of a sequence of interest in a test sample that comprises a mixture of nucleic acids that are known or are suspected to differ in the amount of one or more sequence of interest. The method comprises a statistical approach that accounts for accrued variability stemming from process-related, interchromosomal and inter-sequencing variability. The method is applicable to determining CNV of any fetal aneuploidy, and CNVs known or suspected to be associated with a variety of medical conditions. CNV that can be determined according to the method include trisomies and monosomies of any one or more of chromosomes 1-22, X and Y, other chromosomal polysomies, and deletions and/or duplications of segments of any one or more of the chromosomes, which can be detected by sequencing only once the nucleic acids of a test sample.

## Claims

### Claim 1

A method, implemented using a computer system comprising one or more processors and system memory, for identifying the presence of a cancer and/or an increased risk of a cancer in a mammal, said method comprising:
(a) providing, to the computer system, at least 10,000 sequence reads of nucleic acids in a test sample from said mammal, wherein said test sample comprises both nucleic acids from cancerous or precancerous cells and nucleic acids from constitutive cells, wherein the sequence reads are provided in an electronic format;
(b) aligning, by the one or more processors, the at least 10,000 sequence reads to one or more reference sequences using a computing apparatus and thereby providing sequence tags corresponding to the sequence reads;
(c) identifying, by the one or more processors, a number of sequence tags from the nucleic acids for one or more sequences of interest amplifications of which or deletions of which are known to be associated with cancers, wherein said sequences of interest are selected from chromosomes 1-22, X, and Y and segments thereof and identifying, by the one or more processors, a number of sequence tags for at least one normalizing sequence for each of the one or more sequences of interest;
(d) calculating, by the one or more processors and using said number of sequence tags identified for each of said one or more sequences of interest and said number of sequence tags identified for each said normalizing sequence, a single sequence dose for each of said one or more sequences of interest,
wherein the at least one normalizing sequence is a sequence that yields sequence doses for the sequence of interest having: (a) the smallest variability among unaffected samples, (b) the greatest differentiability between affected and unaffected samples, (c) the smallest variability and the greatest differentiability, or (d) an optimal combination of small variability and large differentiability;
(e) evaluating said single sequence dose for each of said one or more sequences of interest, and thereby detecting aneuploidies or copy number variations in said sample; and
(f) determining the presence of a cancer and/or an increased risk of a cancer based on the detected aneuploidies or copy number variations.

### Claim 2 (depends on 1)

The method of claim 1, wherein (f) comprises determining the presence of the cancer and/or the increased risk of the cancer based on the presence, an increased number, and/or an increased severity of the aneuploidies or copy number variations.

### Claim 3 (depends on 1)

The method of claim 1, wherein evaluating said single sequence dose comprises comparing the single sequence dose to a threshold value for a sequence of interest.

### Claim 4 (depends on 1)

The method of claim 1, wherein the at least one normalizing sequence is a sequence that was selected for a sequence of interest by (i) identifying a plurality of unaffected samples; and (ii) selecting a normalizing sequence giving sequence doses of unaffected samples having: (a) the smallest variability, (b) the greatest differentiability, (c) the smallest variability and the greatest differentiability, or (d) an optimal combination of small variability and large differentiability.

### Claim 5 (depends on 1)

The method of claim 1, wherein said aneuploidies comprise a whole chromosome gain or a whole chromosome loss as shown in Table 1.

### Claim 6 (depends on 1)

The method of claim 1, wherein said sequence of interest are substantially arm-level segments comprising a p arm or a q arm of any one or more of chromosomes 1-22, X and Y.

### Claim 7 (depends on 1)

The method of claim 1, wherein said sequences of interest are segment that comprise a region and/or a gene shown in Table 3 and/or Table 5 and/or Table 4 and/or Table 6.

### Claim 8 (depends on 7)

The method of claim 7, wherein said aneuploidies comprise an amplification of a region and/or a gene shown in Table 3 and/or Table 5.

### Claim 9 (depends on 7)

The method of claim 7, wherein said aneuploidies comprise a deletion of a region and/or a gene shown in Table 4 and/or Table 6.

### Claim 10 (depends on 1)

The method of claim 1, wherein said sequences of interest contain one or more oncogenes and/or one or more tumor suppressor genes.

### Claim 11 (depends on 10)

The method of claim 10, wherein said aneuploidies comprise an amplification of one or more regions selected from the group consisting of 20Q13, 19q12, 1q21-1q23, 8p11-p12, and the ErbB2.

### Claim 12 (depends on 10)

The method of claim 10, wherein said aneuploidies comprise an amplification of one or more regions comprising a gene selected from the group consisting of MYC, ERBB2 (EFGR), CCND1 (Cyclin D1), FGFR1, FGFR2, HRAS, KRAS, MYB, MDM2, CCNE, KRAS, MET, ERBB1, CDK4, MYCB, ERBB2, AKT2, MDM2 and CDK4.

### Claim 13 (depends on 10)

The method of claim 10, wherein said cancer is a cancer selected from the group consisting of leukemia, ALL, brain cancer, breast cancer, colorectal cancer, dedifferentiated liposarcoma, esophageal adenocarcinoma, esophageal squamous cell cancer, GIST, glioma, HCC, hepatocellular cancer, lung cancer, lung NSC, lung SC, medulloblastoma, melanoma, MPD, myeloproliferative disorder, cervical cancer, ovarian cancer, prostate cancer, and renal cancer.

### Claim 14 (depends on 1)

The method of claim 1, wherein said test sample comprises a sample selected from the group consisting of whole blood, a blood fraction, saliva/oral fluid, urine, a tissue biopsy, pleural fluid, pericardial fluid, cerebral spinal fluid, and peritoneal fluid.

### Claim 15 (depends on 1)

The method of claim 1, wherein a sequence dose for a selected sequence of interest is calculated as a ratio of the number of sequence tags identified for the selected sequence of interest and the number of sequence tags identified for the at least one normalizing sequence for the sequence of interest.

### Claim 16 (depends on 1)

The method of claim 1, wherein at least one normalizing sequence is a sequence selected for an associated sequence of interest by (i) identifying a plurality of qualified samples for the sequence of interest; (ii) repeatedly calculating chromosome doses for the associated sequence of interest using multiple potential normalizing sequences; and (iii) selecting the normalizing sequence alone or in a combination giving the smallest variability and/or greatest differentiability in calculated chromosome doses.

### Claim 17 (depends on 1)

The method of claim 1, further comprising calculating a normalized sequence value (NSV), wherein said NSV relates said sequence dose to the mean of a corresponding sequence dose in a set of qualified samples as:
NSV
ij
=
x
ij
-
μ
^
j
σ
^
j
where {circumflex over (μ)}j and {circumflex over (σ)}j are the estimated mean and standard deviation, respectively, for the j-th sequence dose in a set of qualified samples, and xij is the sequence dose calculated in (c) for the sequence of interest.

### Claim 18 (depends on 1)

The method of claim 1, wherein the nucleic acids in the test sample comprise cell free DNA.

### Claim 19 (depends on 1)

The method of claim 1, wherein the detected aneuploidies or copy number variations comprises a component in a differential diagnosis for cancer.

### Claim 20 (depends on 1)

The method of claim 1, wherein the detection of aneuploidies or copy number variations indicates a positive result and said method further comprises prescribing, initiating, and/or altering treatment of the mammal.

### Claim 21 (depends on 20)

The method of claim 20, wherein said prescribing, initiating, and/or altering treatment of the mammal comprises prescribing and/or performing further diagnostics to determine the presence and/or severity of a cancer.

### Claim 22 (depends on 21)

The method of claim 21, wherein said further diagnostics comprise screening a sample from said mammal for a biomarker of a cancer, and/or imaging said mammal for a cancer.

### Claim 23 (depends on 20)

The method of claim 20, wherein, when said method indicates the presence of neoplastic cells in said mammal, the treatment comprises treating said mammal, or causing said mammal to be treated, to remove and/or to inhibit a growth or proliferation of said neoplastic cells.

### Claim 24 (depends on 23)

The method of claim 23, wherein said treating said mammal comprises surgically removing said cells.

### Claim 25 (depends on 24)

The method of claim 24, wherein said treating said mammal comprises performing radiotherapy or causing radiotherapy to be performed on said mammal to kill said neoplastic cells.

### Claim 26 (depends on 25)

The method of claim 25, wherein said treating said mammal comprises administering or causing to be administered to said mammal an anti-cancer drug.

### Claim 27 (depends on 26)

The method of claim 26, wherein said anti-cancer drug is a drug selected from the group consisting of matuzumab, erbitux, vectibix, nimotuzumab, matuzumab, panitumumab, flourouracil, capecitabine, 5-trifluoromethyl-2′-deoxyuridine, methotrexate, raltitrexed, pemetrexed, cytosine arabinoside, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine, cladribine, floxuridine, cyclophosphamide, neosar, ifosfamide, thiotepa, 1,3-bis(2-chloroethyl)-1-nitrosourea, 1,-(2-chloroethyl)-3-cyclohexyl-nitrosourea, hexamethylmelamine, busulfan, procarbazine, dacarbazine, chlorambucil, melphalan, cisplatin, carboplatin, oxaliplatin, bendamustine, carmustine, chloromethine, dacarbazine, fotemustine, lomustine, mannosulfan, nedaplatin, nimustine, prednimustine, ranimustine, satraplatin, semustine, streptozocin, temozolomide, treosulfan, triaziquone, triethylene melamine, thiotepa, triplatin tetranitrate, trofosfamide, uramustine, doxorubicin, daunorubicin, mitoxantrone, etoposide, topotecan, teniposide, irinotecan, camptosar, camptothecin, belotecan, rubitecan, vincristine, vinblastine, vinorelbine, vindesine, paclitaxel, docetaxel, abraxane, ixabepilone, larotaxel, ortataxel, tesetaxel, vinflunine, imatinib mesylate, sunitinib malate, sorafenib tosylate, nilotinib hydrochloride monohydrate/, tasigna, semaxanib, vandetanib, vatalanib, retinoic acid, and a retinoic acid derivative.

### Claim 28 (depends on 1)

A method of monitoring a treatment of a subject for a cancer said method comprising:
performing the method of claim 1 on a sample from said subject or receiving results of such a method performed on said sample before or during said treatment, thereby obtaining a first measurement of the aneuploidies or the copy number variations;
performing the method of claim 1 on a second sample from said subject or receiving results of such a method performed on said second sample at a later time during or after said treatment, thereby obtaining a second measurement of the aneuploidies or the copy number variations, wherein a reduced number or severity of aneuploidy or copy number variation in said second measurement is an indicator of a positive course of treatment and the same or increased number or severity of aneuploidy or copy number variation in said second measurement is an indicator of a negative course of treatment; and
adjusting, when said indicator is negative, said treatment to a more aggressive treatment regimen or a palliative treatment regimen.

### Claim 29

A non-transitory computer readable medium for use in determining the presence of a cancer and/or an increased risk of a cancer in a mammal, said program product comprising:
(a) code for providing at least 10,000 sequence reads of nucleic acids in a test sample from said mammal, wherein said test sample comprises both nucleic acids from cancerous or precancerous cells and nucleic acids from constitutive cells, wherein the sequence reads are provided in an electronic format;
(b) code for aligning the at least 10,000 sequence reads to one or more reference sequences using a computing apparatus and thereby providing sequence tags corresponding to the sequence reads;
(c) code for identifying a number of sequence tags from the nucleic acids for one or more sequences of interest amplifications of which or deletions of which are known to be associated with cancers, wherein said sequences of interest are selected from chromosomes 1-22, X, and Y and segments thereof and identifying a number of sequence tags for at least one normalizing sequence for each of the one or more sequences of interest;
(d) code for calculating, using said number of sequence tags identified for each of said one or more sequences of interest and said number of sequence tags identified for each said normalizing sequence, a single sequence dose for each of said one or more sequences of interest,
wherein the at least one normalizing sequence is a sequence that yields sequence doses for the sequence of interest having: (a) the smallest variability among unaffected samples, (b) the greatest differentiability between affected and unaffected samples, (c) the smallest variability and the greatest differentiability, or (d) an optimal combination of small variability and large differentiability; and
(e) code for evaluating said single sequence dose for each of said one or more sequences of interest, and thereby detecting aneuploidies or copy number variations in said sample.