# US 2017/0283883 A1 · Methods for lung cancer detection

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- Number: US 2017/0283883 A1
- Document ID: US20170283883A1
- Status: Published application
- Kind: A1
- Application number: 15/509,690
- Filed: 2015-09-09
- Published: 2017-10-05
- Earliest claimed: 2014-09-09
- Assignees: Fondazione Istituto Firc Di Oncologia Molecolare; Universita' Degli Studi Di Milano; Ieo - Istituto Europeo Di Oncologia S.R.L.
- Applicants: Fondazione Istituto Firc Di Oncologia Molecolare; Universita Degli Studi Di Milano; IEO - Istituto Europeo di Oncologia S.r.l.
- Inventors: Pier Paolo Di FIORE; Fabrizio BIANCHI; Francesco NICASSIO; Matteo Jacopo Luca Nicolo MARZI; Francesca MONTANI
- Web page: https://patentgrep.com/patent/US20170283883A1

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- G16B 20/00: ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B 20/20: Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
- C12Q 2600/158: Expression markers
- C12Q 2600/178: miRNA, siRNA or ncRNA
- G16B 15/00: ICT specially adapted for analysing two-dimensional [2D] or three-dimensional [3D] molecular structures, e.g. structural or functional relations or structure alignment
- 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 disclosure describes a method for diagnosing lung cancer in a subject by detecting in a biological sample obtained from that patient a miRNA signature, the presence of which provides an earlier indication of cancer than alternative art-recognized methods, including, but not limited to, low-dose computed tomography (LDCT).

## Claims

### Claim 1

A method of diagnosing lung cancer in a subject in need thereof comprising
(a) detecting in a biological sample from the subject a decrease in the abundance of each of hsa-mir-92a-3p, hsa-mir-30b-5p, hsa-mir-191-5p, hsa-mir-484, hsa-mir-328-3p, hsa-mir-30c-5p, hsa-mir-374a-5p, hsa-mir-7d-5p, and hsa-mir-331-3p compared to a control abundance value corresponding to each of hsa-mir-92a-3p, hsa-mir-30b-5p, hsa-mir-191-5p, hsa-mir-484, has-mir-328-3p, hsa-mir-30c-5p, hsa-mir-374a-5p, hsa-mir-7d-5p, and hsa-mir-331-3p from a control sample;
(b) detecting in the biological sample an increase in the abundance of each of hsa-mir-29a-3p, hsa-mir-148a-3p, hsa-mir-223-3p, and hsa-mir-140-5p compared to a control abundance value corresponding to each of hsa-mir-29a-3p, hsa-mir-148a-3p, hsa-mir-223-3p, and hsa-mir-140-5p from a control sample; and
(c) diagnosing the subject with lung cancer, when a decreased abundance in each of the miRNA in (a) is detected and an increased abundance of each of the miRNA in (b) is detected.

### Claim 2 (depends on 1)

The method of claim 1, wherein the biological sample comprises a biological fluid.

### Claim 3 (depends on 2)

The method of claim 2, wherein the biological fluid is saliva, urine, blood, or lymph fluid.

### Claim 4 (depends on 1)

The method of claim 1, wherein biological sample comprises blood, whole blood, blood plasma and/or blood serum.

### Claim 5 (depends on 1)

The method of claim 1, wherein the biological sample comprises blood serum.

### Claim 6 (depends on 1)

The method of claim 1, wherein the decrease is a statistically significant difference from the control value, or the increase is a statistically significant difference from the control value, or both.

### Claim 7 (depends on 6)

The method of claim 6, wherein a statistically significant difference is characterized by a p-value of less than 0.05.

### Claim 8 (depends on 7)

The method of claim 7, wherein a statistically significant difference is characterized by a p-value of less than 0.001.

### Claim 9 (depends on 8)

The method of claim 8, wherein a statistically significant difference is characterized by a p-value of less than 0.0001.

### Claim 10

(canceled)

### Claim 11 (depends on 1)

The method of claim 1, further comprising the step of calculating a risk score.

### Claim 12 (depends on 11)

The method of claim 11, wherein the risk score is a product of
(a) a fold decrease and a weight coefficient, or
(b) a fold increase and a weight coefficient,
wherein the weight coefficient is determined by a diagonal linear discriminant analysis (DLDA).

### Claim 13 (depends on 1)

The method of claim 1, wherein the control value for each miRNA is determined by a method comprising detecting in a control biological sample from a normal subject an abundance of each of hsa-mir-92a-3p, hsa-mir-30b-5p, hsa-mir-191-5p, hsa-mir-484, hsa-mir-328-3p, has-mir-30c-5p, hsa-mir-374a-5p, hsa-mir-7d-5p, hsa-mir-331-3p, hsa-mir-29a-3p, hsa-mir-148a-3p, hsa-mir-223-3p, and hsa-mir-140-5p.

### Claim 14 (depends on 13)

The method of claim 13, wherein the normal subject does not have cancer.

### Claim 15 (depends on 13)

The method of claim 13, wherein the normal subject does not have lung cancer.

### Claim 16 (depends on 13)

The method of claim 13, wherein the control biological sample comprises a biological fluid.

### Claim 17 (depends on 16)

The method of claim 16, wherein the control biological fluid is saliva, urine, blood, or lymph fluid.

### Claim 18 (depends on 13)

The method of claim 13, wherein the control biological sample comprises blood, whole blood, blood plasma and/or blood serum.

### Claim 19 (depends on 13)

The method of claim 13, wherein the control biological sample comprises blood serum.

### Claim 20 (depends on 13)

The method of claim 13, wherein the control biological sample comprises blood, whole blood, blood plasma and/or blood serum obtained from at least two normal subjects.

### Claim 21 (depends on 19)

The method of claim 19, wherein the control biological sample comprises blood serum obtained from at least two normal subjects.

### Claim at position 22

22-23. (canceled)

### Claim 24 (depends on 1)

The method of claim 1, wherein the subject has stage I lung cancer.

### Claim 25 (depends on 1)

The method of claim 1, wherein the subject has stage II lung cancer.

### Claim at position 25

26-29. (canceled)

### Claim 30 (depends on 1)

The method of claim 1, further comprising the step of performing low-dose computed tomography (LDCT) or referring the subject for LDCT if the subject is diagnosed with lung cancer.

### Claim 31 (depends on 1)

The method of claim 1, further comprising the step of providing a treatment or referring the subject for treatment if the subject is diagnosed with lung cancer.

### Claim 32 (depends on 1)

The method of claim 1, wherein the detecting step of (a) and/or (b) comprises
transcribing each of the miRNA in (a) and/or (b) using at least one human miRNA stem-loop primer specific for at least one miRNA of (a) or (b) to generate a complementary DNA (cDNA) corresponding to each of the miRNA,
amplifying each of the cDNA, and determining a relative abundance of each of the cDNA compared to at least one housekeeping miRNA.

### Claim 33 (depends on 32)

The method of claim 32, wherein the at least one human miRNA stem-loop primer specific for at least one miRNA and the at least one miRNA hybridize to form at least one duplex.

### Claim 34 (depends on 32)

The method of claim 32, wherein the amplifying step comprises performing quantitative real-time polymerase chain reaction (qRT-PCR).

### Claim 35 (depends on 32)

The method of claim 32, wherein the at least one housekeeping miRNA comprise miR-197, miR-19b, miR-24, miR-146, miR-15b, or miR-19a.

### Claim 36 (depends on 33)

The method of claim 33, wherein the housekeeping miRNA are miR-197, miR-19b, miR-24, miR-146, miR-15b, and miR-19a.

### Claim 37 (depends on 33)

The method of claim 33, wherein the relative abundance of each of the cDNA is determined by adding a scaling factor to the raw cycle threshold (CT) of each of the cDNA to generate a normalized (CT) value.

### Claim 38 (depends on 37)

The method of claim 37, wherein the scaling factor is determined by determining a mean cycle threshold of the at least two housekeeping miRNA selected from the group consisting of miR-197, miR-19b, miR-24, miR-146, miR-15b, and miR-19a and subtracting the mean CT from a constant value (K).

### Claim 39 (depends on 37)

The method of claim 37, wherein the constant value (K) equals 21.646.

### Claim 40 (depends on 37)

The method of claim 37, wherein the normalized CT value is used to determine a weight coefficient.

### Claim 41 (depends on 1)

The method of claim 1, further comprising the step of determining a risk score for the subject.

### Claim 42 (depends on 41)

The method of claim 41, wherein the risk score is calculated according to: RS=−(Σiwixi−THRES),
wherein i corresponds to each of the miRNAs of (a) and (b), respectively, and
wherein THRES=−261.779, wi is the weight coefficient of the ith miRNA, and xi is the expression value of the ith miRNA.

### Claim 43 (depends on 42)

The method of claim 42, wherein the expression value of the ith miRNA is the raw CT of the ith miRNA.

### Claim 44 (depends on 41)

The method of claim 41, wherein the risk score is greater than or equal to 5, indicating that the subject has a high risk of developing cancer.

### Claim 45 (depends on 41)

The method of claim 41, wherein the risk score is less than 5 and greater than or equal to −5, indicating that the subject has an intermediate risk of developing cancer.

### Claim 46 (depends on 41)

The method of claim 41, wherein the risk score is less than −5, indicating that the subject has a low high risk of developing cancer.