# US 11,441,188 B2 · Methods for detecting DNA mutations using Mitra tip extraction

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- Number: US 11,441,188 B2
- Document ID: US11441188B2
- Status: Granted
- Kind: B2
- Application number: 15/811,515
- Filed: 2017-11-13
- Published: 2018-05-17
- Granted: 2022-09-13
- Earliest claimed: 2016-11-15
- Assignees: Quest Diagnostics Investments Incorporated
- Applicants: Quest Diagnostics Investments LLC
- Inventors: Heather Sanders; Nigel J. Clarke
- Web page: https://patentgrep.com/patent/US11441188B2

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12N 15/1058: {Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms}
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- G01N 33/4875: {Details of handling test elements, e.g. dispensing or storage, not specific to a particular test method (test-elements per se B01L, automatic analysers G01N35/00, in-vivo analysis on the human body for medical diagnosis A61B)}
- G01N 33/49: Blood {(chemical methods for determining blood cell populations G01N33/5094; chemical analysis of blood groups or blood types G01N33/80)}
- G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites}
- G01N 33/533: with fluorescent label
- C12Q 2527/125: Specific component of sample, medium or buffer
- C12Q 2600/112: Disease subtyping, staging or classification
- C12Q 2600/118: Prognosis of disease development
- C12Q 2600/156: Polymorphic or mutational markers
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present disclosure provides rapid and non-invasive methods for determining whether a patient exhibiting cancer symptoms, or at risk for hereditary cancers such as breast cancer, ovarian cancer, colon cancer, or skin cancer, will benefit from treatment with one or more therapeutic agents. These methods are based on detecting hereditary cancer-related mutations in small-volume dried biological fluid samples that are collected using a volumetric absorptive microsampling device (e.g., MITRA Tip). Kits for use in practicing the methods are also provided.

## Claims

### Claim 1

A method for detecting the presence or absence of mutations in a plurality of hereditary cancer-related genes in a pooled sample comprising:
(a) eluting at least two dried biological fluid samples, each containing no more than 400 ng of genomic DNA, from at least two absorbent tips of microsampling devices, each of the at least absorbent tips comprising a separate dried biological fluid sample, by contacting the least two absorbent tips of the microsampling devices with a Proteinase K and a lysis buffer comprising guanidine hydrochloride, Tris.Cl, EDTA, Tween 20, and Triton X-100;
(b) extracting genomic DNA from the at least two dried biological fluid samples; to obtain at least two nucleic acid samples;
(c) generating at least two libraries of amplicons from each of the at least two nucleic acid samples, wherein each of the at least two libraries of amplicons comprises sequences corresponding to at least six and up to all of APC, ATM, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2, EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1, POLE, PTEN, RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, and VHL, wherein an index sequence is ligated to at least one end of the amplicons;
(d) pooling the at least two libraries of amplicons to form a pooled library of amplicons; and
(e) detecting the presence or absence of mutations in the pooled library of amplicons using high throughput massive parallel sequencing.

### Claim 2 (depends on 1)

The method of claim 1, wherein the dried biological fluid sample is obtained from a patient having or suspected of having a hereditary cancer.

### Claim 3 (depends on 1)

The method of claim 1, wherein the dried biological fluid sample is dried plasma, dried serum, or dried whole blood.

### Claim 4 (depends on 1)

The method of claim 1, wherein the dried biological fluid sample is collected from a patient via fingerstick.

### Claim 5 (depends on 1)

The method of claim 1, wherein elution of the dried biological fluid sample is performed by contacting the absorbent tip of the microsampling device with the lysis buffer for up to 15 minutes at 90° C.

### Claim 6 (depends on 1)

The method of claim 1, wherein elution of the dried biological fluid sample is performed by contacting the absorbent tip of the microsampling device with Proteinase K for up to 1 hour at 56° C.

### Claim 7 (depends on 1)

The method of claim 1, wherein elution of the dried biological fluid sample is performed by contacting the absorbent tip of the microsampling device with Proteinase K for up to 18 hours at 56° C.

### Claim 8 (depends on 1)

The method of claim 1, wherein the sample volume of the microsampling device is no more than 20 μL.

### Claim 9 (depends on 2)

The method of claim 2, wherein the hereditary cancer is breast cancer, ovarian cancer, colon cancer, or skin cancer.

### Claim 10 (depends on 1)

The method of claim 1, wherein about 100 ng to about 400 ng of genomic DNA is eluted from the absorbent tip of the microsampling device.

### Claim 11 (depends on 1)

The method of claim 1, wherein the high throughput massive parallel sequencing is performed using pyrosequencing, reversible dye-terminator sequencing, SOLiD sequencing, Ion semiconductor sequencing, Helioscope single molecule sequencing, sequencing by synthesis, sequencing by ligation, or SMRT™ sequencing.

### Claim 12 (depends on 1)

The method of claim 1, wherein the adapter sequence is a P5 adapter, P7 adapter, P1 adapter, A adapter, or Ion Xpress™ barcode adapter.

### Claim 13 (depends on 1)

The method of claim 1, wherein the plurality of amplicons are enriched using a bait set comprising nucleic acid sequences that are complementary to at least one of the amplicons in the plurality of amplicons.

### Claim 14 (depends on 13)

The method of claim 13, wherein the nucleic acid sequences of the bait set are RNA baits, DNA baits, or a combination thereof.

### Claim 15

A method for detecting the presence or absence of mutations in a plurality of hereditary cancer-related genes in a pooled nucleic acid sample comprising generating a library from a pooled nucleic acid sample comprising amplicons corresponding to at least six and up to all of APC, ATM, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2, EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1, POLE, PTEN, RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, and VHL, and detecting the presence of absence of at least one mutation in the plurality of hereditary cancer-related genes using high throughput massive parallel sequencing; wherein the pooled nucleic acid sample was extracted from dried biological fluid samples obtained from at least two subjects and eluted from an absorbent tip of a microsampling device by contacting the absorbent tip of the microsampling device with (i) a lysis buffer comprising guanidine hydrochloride, Tris.Cl, EDTA, Tween 20, and Triton X-100 and (ii) Proteinase K, wherein no more than 400 ng of genomic DNA is eluted from the absorbent tip of the microsampling device.

### Claim 16

A method for selecting a patient exhibiting cancer symptoms, or a patient at risk for hereditary cancer, for treatment with an anti-cancer therapeutic agent comprising
(a) eluting a dried blood sample from a absorbent tip of a volumetric microsampling device by contacting the absorbent tip with a Proteinase K and a lysis buffer comprising guanidine hydrochloride, Tris.Cl, EDTA, Tween 20, and Triton X-100 under conditions that result in the release of genomic DNA from blood cells, wherein the dried blood sample was collected from the patient with the volumetric absorptive microsampling device, wherein no more than 400 ng of genomic DNA is eluted from the absorbent tip of the microsampling device;
(b) isolating genomic DNA from the eluted dried blood sample;
(c) generating a library of amplicons corresponding to at least six and up to all of APC, ATM, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2, EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1, POLE, PTEN, RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, and VHL, wherein an index sequence is ligated to at least one end of the amplicons;
(d) pooling the library of amplicons generated from the patient with at least one other library of amplicons to form a pooled library of amplicons;
(e) detecting the presence or absence of mutations in the pooled library of amplicons using high throughput massive parallel sequencing; and
(f) selecting the patient for treatment with an anti-cancer therapeutic agent, if a mutation in at least one of the library of amplicons is detected.

### Claim 17 (depends on 16)

The method of claim 16, wherein the patient has or is at risk of developing a hereditary cancer selected from the group consisting of breast cancer, ovarian cancer, skin cancer, or colon cancer.

### Claim 18 (depends on 16)

The method of claim 16, wherein the anti-cancer therapeutic agent is one or more agents selected from the group consisting of cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserlin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, tykerb, anthracyclines, bevacizumab, aldesleukin, cobimetinib, dabrafenib, dacarbazine, talimogene laherparepvec, imiquimod, recombinant Interferon Alfa-2b, ipilimumab, pembrolizumab, trametinib, nivolumab, peginterferon Alfa-2b, sonidegib, vismodegib, vemurafenib, cetuximab, irinotecan hydrochloride, leucovorin calcium, trifluridine and tipiracil hydrochloride, oxaliplatin, panitumumab, ramucirumab, regorafenib, and ziv-aflibercept.