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  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

• lung cancer has the highest incidence and the highest mortality rate of all cancers (http://seer.cancer.gov/statistics/). Lung cancer is divided into two major classes: small cell lung cancer (SCLC) and non- small cell lung cancer (NSCLC), the former affecting 20% of patients and the latter 80%.
• SCLC small cell lung cancer
• NSCLC non- small cell lung cancer
• NSCLC consists of three major subtypes: adenocarcinoma, squamous cell carcinoma (SCC), and large cell carcinoma, with adenocarcinoma and squamous cell carcinoma accounting for the vast majority (Sekido et al., Biochim Biophys Acta, 1378:F21-59; Forgacs et al., Pathol Oncol Res, 7:6-13 (2001)). An estimated 215,000 new cases of lung cancer and approximately 162,000 deaths from lung cancer occurred in the United States during 2008 (Jemal et al., J Natl Cancer Inst, 100:1672-94 (2008)).
• the high mortality rate of lung cancer has been attributed in part to the fact that more than 75% of lung cancer patients are diagnosed with regional or distant metastasis at first presentation, and in part to the lack of highly effective therapies for lung cancer.
• the current treatment options for lung cancer are limited, it has been shown that patients who are diagnosed at the earliest stage have a better chance of 5-yr survival (50%) than those in later stages ( ⁇ 10%) (see, e.g., http://seer.cancer.gov/statistics;
• Risk factors for lung cancer include age and tobacco consumption by smoking (first hand and second hand). Approximately 90% of all lung cancer cases occur in smokers. Additionally, significant differences exist in both incidence and mortality of lung cancer between males and females, suggesting the possibility of inherent biological processes between the two sexes. These differences are among the most consistently reported significant risk factors in lung cancer (Visbal et al, Ann Thorac Surg, 78:209-15 (2004)).
• a chest X-ray may reveal a mass in the lung that may be biopsied for verification and
• Circulating biomarkers offer an alternative to imaging with the following advantages: 1) they are found in a minimally-invasive, easy to collect specimen type (blood or blood-derived fluids), 2) they can be monitored frequently over time in a subject to establish an accurate baseline, making it easy to detect changes over time, 3) they can be provided at a reasonably low cost, 4) they may limit the number of patients undergoing repeated expensive and potentially harmful CT scans, and/or 5) unlike CT scans, biomarkers may potentially distinguish indolent from more aggressive lung lesions (see, e.g., Greenberg and Lee, Opin PuIm Med, 13:249-55 (2007)).
• Circulating nucleic acids such as DNA and mRNA have also been evaluated as possible diagnostic markers for lung cancer. These studies are based on the observations that circulating nucleic acids show differentia! expression that is suggestive of cancer.
• circulating nucleic acids show differentia! expression that is suggestive of cancer.
• the invention relates to the detection or monitoring of lung diseases such as small cell lung cancer or non-small cell lung cancer by detecting miRNAs from serum or plasma.
• the methods of the invention include detection of biomarkers that can be used to diagnose disease and/or evaluate the prognosis or aggressiveness of a lung disease. Further, the methods may be used to characterize the progression of a lung disease. In certain embodiments, the methods of the invention may be used to determine whether a lung tumor or lesion in a patient is cancerous or benign. The patients tested using the methods of the invention may also be tested using other known methods in the art.
• the diagnosis or prognosis may be achieved by measuring the amount of a miRNA that is present in elevated or reduced levels in the serum or plasma of a subject with lung disease.
• one serum or plasma miRNA may be detected (e.g., amplified and measured) to characterize lung disease, while in other embodiments, two or more miRNAs are detected from serum or plasma.
• Some embodiments include detecting a pair of miRNAs. In some instances, one miRNA in the pair is elevated in serum or plasma of patients with lung disease or lung cancer, and the other miRNA in the pair is reduced. In other circumstances, both miRNAs in the pair can be elevated or both reduced.
• biomarkers such as protein markers may also be measured.
• Some embodiments of the invention relate to diagnosis or prognosis of lung cancer, or determining the type of lung cancer in a patient. In some embodiments, the patient has previously been screened for lung disease.
• FIG. 1 is a schematic outline of the study design for the mouse model for lung cancer.
• One hundred fifteen A/J mice were administered with an oral gavage of benzo(a)pyrene at the indicated dose at time (0) and at 10 weeks post the first gavage.
• Ten control mice received an oral gavage of 200 ⁇ l cottonseed oil with no carcinogen at week/day 0.
• the number of mice sacrificed at each time point is shown below the arrows. The remaining mice were bled every two weeks until 18 weeks and every four weeks thereafter. At the end of 34 weeks, all remaining mice were sacrificed.
• Figure 2A and Figure 2B show temporal changes of miR-183 and miR-182 (Figure 2A) and miR-365 and miR-141 ( Figure 2B) plasma miRNA expression in mice that developed lung tumors and in control mice.
• Figure 4 shows performance estimates of training and test data from the Linear Discriminate Analysis of Example 5.
• SENS sensitivity
• SPEC specificity
• NPV negative predictive value
• PPV positive predictive value.
• Six differential miRNA pairs were used: miR-142-5p and miR181d; miR-142-3p and miR181d; miR-142-3p and miR-422a; miR-142-5p and miR-422a; miR-92 and miR-27b; and miR-24 and miR-27a.
• Figure 5 shows human precursor miRNA (pre-miRNA) sequences (SEQ ID NOS 1-323, respectively, in order of appearance), as provided by
• the methods of the invention provide assays for amplifying and measuring the amount of a miRNA in a serum or plasma sample, thereby characterizing a lung disease.
• microRNA includes human miRNAs, mature single stranded miRNAs, precursor miRNAs (pre-miR), and variants thereof, which may be naturally occurring.
• miRNA also includes primary miRNA transcripts and duplex miRNAs.
• miRNA-122a refers to a mature miRNA sequence derived from pre-miR-122.
• the sequences for particular miRNAs, including human mature and precursor sequences, are reported in the miRBase::Sequences Database (http://microrna.sanger.ac.uk (version 15 released April 2010); Griffiths-Jones et al., Nucleic Acids Research, 2008, 36, Database Issue, D154-D158; Griffiths-Jones et al., Nucleic Acids Research, 2006, 34, Database Issue, D140-D144; Griffiths-Jones, Nucleic Acids Research, 2004, 32, Database Issue, D109-D111).
• miRNAs For certain miRNAs, a single precursor contains more than one mature miRNA sequence. In other instances, multiple precursor miRNAs contain the same mature sequence. In some instances, mature miRNAs have been re-named based on new scientific consensus. For example, miR-213, as used herein, refers to a mature miRNA from pre-miR-181a- 1 , and is also called miR-181a * . Other miRNAs that have been re-named include miR-189 (also called miR-24*), which comes from pre-miR-24-1 ; miR-368 (also called miR-376c); and miR-422b (also called miR-378 * ). The skilled artisan will appreciate that scientific consensus regarding the precise nucleic acid sequence for a given miRNA, in particular for mature forms of the miRNAs, may change with time. MiRNAs detected by assays of this application include naturally occurring sequences for the miRNAs.
• the term "characterizing" is used herein to encompass detection and prognosis, and it includes detection of a miRNA for making diagnostic or prognostic determinations or predictions of disease. In some instances, the characterization will identify whether a subject has a lung disease such as cancer, or will determine the disease state. Additionally, detection of a miRNA according to the methods herein includes measuring the amount of a miRNA that can used to distinguish patients with lung cancer from patients having other lung diseases, or determine whether a patient with a lung tumor has cancer. In other
• characterizing includes detection of a miRNA for determining the stage or aggressiveness of a disease state such as lung cancer, or determining an appropriate treatment method for lung disease.
• Serum is typically the fluid, non-cellular portion of coagulated blood. Plasma is also a non-cellular blood sample, but unlike serum, plasma contains clotting factors.
• serum or plasma samples are obtained from a human patient previously screened for lung disease using another diagnostic method.
• serum or plasma samples are obtained from patients that have tested positive for a tumor or lung lesion.
• the patient has undergone imaging detection, e.g., by chest X-ray or CT scan.
• the methods involve detection of miRNA in patients with a positive imaging result for lung disease.
• samples are obtained from patients that have a lung tumor or lesion. The tumor or lesion may have been detected by chest X-ray or CT scan, or by other imaging or detection methods known in the art. Additional embodiments include
• the sample is from a patient suspected of having lung cancer or at risk of developing lung cancer.
• the volume of plasma or serum obtained and used for the assay may be varied depending upon clinical intent.
• clotting activators such as silica particles are added to the blood collection tube.
• the blood is not treated to facilitate clotting.
• Blood collection tubes are commercially available from many sources and in a variety of formats (e.g., Becton Dickenson Vacutainer ® tubes-SSTTM, glass serum tubes, or plastic serum tubes).
• the blood is collected by venipuncture and processed within three hours after drawing to minimize hemolysis and minimize the release of miRNAs from intact cells in the blood.
• blood is kept on ice until use.
• the blood may be fractionated by centrifugation to remove cellular components.
• centrifugation to prepare serum can be at a speed of at least 500, 1000, 2000, 3000, 4000, or 5000 X G.
• the blood can be incubated for at least 10, 20, 30, 40, 50, 60, 90, 120, or 150 minutes to allow clotting. In other embodiments, the blood is incubated for at most 3 hours.
• plasma the blood is not permitted to coagulate prior to separation of the cellular and acellular components. Serum or plasma can be frozen after separation from the cellular portion of blood until further assayed.
• RNA may be extracted from serum or plasma and purified using methods known in the art. Many methods are known for isolating total RNA, or for specifically extracting small RNAs, including miRNAs.
• the RNA may be extracted using commercially-available kits (e.g., Perfect RNA Total RNA Isolation Kit, Five Prime-Three Prime, Inc.; mirVanaTM kits, Ambion, Inc.).
• RNA or viral RNA may be adapted, either as published or with modification, for extraction of RNA from plasma and serum.
• RNA may be extracted from plasma or serum using silica particles, glass beads, or diatoms, as in the method or adaptations described in U.S. Publication No. 2008/0057502. II. miRNA MARKERS FOR LUNG DISEASE
• Certain embodiments of the invention provide serum or plasma miRNAs as markers for lung disease.
• miRNAs that are present at elevated levels in the serum and/or plasma of patients with lung disease are used as markers.
• miRNAs that have reduced levels are used as markers.
• more than one miRNA from serum or plasma will be used as markers. When more than one miRNA biomarker is used, the miRNAs may all have elevated levels, all have reduced levels, or a mixture of miRNAs with elevated and reduced levels may be used.
• reduced levels refer to the amount of a miRNA in a serum or plasma sample from a patient compared to the amount of the miRNA in serum or plasma from a cohort or cohorts that do not have the lung disease that the patient is being tested for.
• a miRNA that has reduced levels in the sera of lung cancer patients is present at lower amounts in lung cancer patient sera than in serum from a donor who does not have lung cancer (e.g., patients with benign tumors or normal patients).
• elevated levels in a patient serum or plasma sample indicates presence or prognosis for a lung disease.
• Other miRNAs are present in reduced levels in patients with lung disease.
• Lung disease includes cancer and benign conditions.
• Lung cancer refers to malignant tumors of the lung, and can be classified as small ceil or non- small cell lung cancer.
• non-small cell lung cancer can be further characterized as adenocarcinoma, squamous cell carcinoma (SCC), and large cell carcinoma.
• SCC squamous cell carcinoma
• cancers can be classified based on X-ray or CT scanning results, aggressiveness, pathology, and measurements of non-miRNA biomarkers, as well as other methods known in the art.
• the lung cancer is classified by TNM principles (T-primary tumor, N-regional lymph nodes, M-distant metastasis) and/or stage 0, IA, IB, HA, HB, HIA, IHB or IV.
• TNM principles T-primary tumor, N-regional lymph nodes, M-distant metastasis
• stage 0, IA, IB, HA, HB, HIA, IHB or IV See, e.g., Lababede et al., Chest, 115:233-235 (1999).
• the methods described herein can be used to characterize a lung disease in a patient with at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sensitivity.
• the degree of sensitivity indicates the percentage of patients with a disease who are positively characterized as having the disease.
• the methods have at least 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% specificity (e.g., the percentage of non-diseased patients who are correctly characterized).
• the assay parameters can be adjusted to optimize for both sensitivity and specificity.
• the level of the miRNA marker will be compared to a control to determine whether the level is reduced or elevated.
• the control may be an external control, such as a miRNA in a serum or plasma sample from a subject known to be free of lung disease.
• the external control may be a sample from a normal (non-diseased) subject or from a patient with benign lung disease.
• the external control may be a miRNA from a non-serum sample like a tissue sample or a known amount of a synthetic RNA.
• the external control may be a pooled, average, or individual sample; it may be the same or different miRNA as one being measured.
• An internal control is a marker from the same serum or plasma sample being tested, such as a miRNA control. See, e.g., US Publication No. US 2009/0075258, which is incorporated by reference in its entirety.
• Table 1 lists miRNAs that have elevated or reduced levels in serum from patients with lung disease. These miRNAs may be used in accordance with the invention. Some of the miRNAs are useful for characterizing lung cancer, including distinguishing the type of cancer and/or distinguishing cancer from benign lung disease. In addition, some miRNAs may be used to predict the aggressiveness or outcome of lung cancer.
• Table 1 miRNAs with elevated or reduced levels in serum from patients with lung cancer. Levels are of miRNA in lung cancer patients compared to patients with benign tumors or lesions.
• one serum miRNA is used to detect, diagnose, characterize, or monitor lung disease and/or lung cancer. In other embodiments, more than one miRNA is used as a marker. In additional embodiments, two or more miRNAs are used to characterize lung disease. In certain embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNAs are detected in the methods of the invention. In certain methods, miRNAs that have reduced levels in serum or plasma from patients with lung disease are used as biomarkers. In other embodiments, a miRNA with elevated levels in serum or plasma can be used as a biomarker. In certain embodiments, the patient has a lung tumor or lesion. In additional embodiments, the patient has previously been screened for lung disease.
• a miRNA for diagnosing lung cancer is chosen from miR-375, miR-499, miR-22, miR-122a, miR-206, miR-103, miR-24, miR-26a, miR-498, miR-205, miR-222, and let-7c.
• a miRNA is chosen from let-7a, let-7b, let- 7c, let-7d, let-7e, let-7f, let-7g, miR-106a, miR-106b, miR-125a, miR-126, miR- 130b, miR-132, miR-133a, miR-133b, miR-140, miR-142-3p, miR-143, miR-146a, miR-150, miR-151 , miR-155, miR-15a, miR-15b, miR-16, miR-181a, miR-181 b, miR-181d, miR-186, miR-18a, miR-190, miR-191 , miR-195, miR-197, miR-19b, miR-202, miR-206, miR-20a, miR-210, miR-214, miR-22, miR-221 , miR-222, miR- 223, miR-23a, miR-24, mi
• a miRNA is chosen from let-7f, let-7g, let-7i, miR-106b, miR-126, miR-126 * , miR-140, miR-142-3p, miR-142-5p, miR- 143, miR-145, miR-150, miR-15a, miR-15b, miR-181a, miR-181b, miR-181d, miR- 202, miR-214, miR-27a, miR-27b, miR-30e-5p, miR-320, miR-324-3p, miR-340, miR-342, miR-345, miR-374, miR-378, miR-422a, miR-486, miR-518b, and miR- 92.
• At least one miRNA is chosen from let-7b, let- 7c, let-7d, let-7e, miR-10a, miR-10b, miR-130b, miR-132, miR-133b, miR-139, miR-143, miR-152, miR-155, miR-15b, miR-17-5p, miR-193, miR-194, miR-195, miR-196b, miR-199a * , miR-19b, miR-202, miR-204, miR-205, miR-206, miR-20b, miR-21 , miR-210, miR-214, miR-221 , miR-27a, miR-27b, miR-296, miR-29a, miR- 301 , miR-324-3p, miR-324-5p, miR-339, miR-346, miR-365, miR-378, miR-422a, miR-432, miR-485-3p, miR-4
• a miRNA is chosen from miR-106a, miR- 106b, miR-126 * , miR-142-3p, miR-15b, miR-181c, miR-182, miR-26b, miR-30b, miR-30e-5p, miR-422b, let-7i, and let-7g.
• a miRNA is chosen from miR-24, miR-92, miR-142-3p, miR-142-5p, miR-181d, miR-27a, miR-27b, miR-422a, miR-29b, miR-15a, miR-106b, miR-126, miR-140, and miR-202.
• 2, 3, 4, 5, 6, 7, or 8 of these miRs can be used to distinguish patients with lung cancer from patients with benign lung tumors or lesions.
• 2, 3, 4, 5, 6, 7, or 8 miRNAs are chosen from miR-24, miR-92, miR- 142-3p, miR-142-5p, miR-181d, miR-27a, miR-27b, and miR-422a.
• a miRNA for characterizing lung cancer vs. non-cancer samples is chosen from miR-15b, miR-182, miR-15a, miR-30b, miR- 26b, miR-106b, let-7g, miR-142-3p, miR-301 , miR-181c, miR-126, miR-346, miR- 422b, and miR-92.
• Non-cancer samples include samples from subjects with benign lung tumors or lesions, or from normal subjects.
• Certain embodiments include a method for characterizing lung disease and/or lung cancer in a patient comprising the steps of measuring the level of a miRNA in a serum sample, wherein the miRNA is chosen from let-7b, let-7c, let-7d, let-7e, miR-10a, miR-10b, miR-130b, miR-132, miR-133b, miR-139, miR-143, miR-152, miR-155, miR-15b, miR-17-5p, miR-193, miR-194, miR-195, miR-196b, miR-199a * , miR-19b, miR-202, miR-204, miR-205, miR-206, miR-20b, miR-21 , miR-210, miR-214, miR-221 , miR-27a, miR-27b, miR-296, miR-29a, miR- 301 , miR-324-3p, miR-324-5p, miR-339, miR
• Table 2 lists miRNAs that have elevated or reduced levels in plasma from patients with lung disease. These miRNAs may be used in accordance with the invention. Table 2. miRNAs with elevated or reduced levels in plasma from patients with lung disease. Levels are of miRNA in lung cancer patients compared to patients without lung cancer.
• a single plasma miRNA may be used to characterize lung cancer.
• one of the miRNAs from Table 2 may be used to characterize lung cancer, either alone or in combination with one or more additional miRNA markers.
• the methods distinguish lung cancer from benign lung disease.
• at least one measured miRNA is elevated in the serum or plasma of lung cancer patients compared to patients with benign conditions or no disease.
• at least one measured miRNA is reduced.
• at least one measured miRNA is elevated and at least one miRNA is reduced.
• at least two elevated miRNAs or at least two reduced miRNAs are measured.
• one of the following miRNAs is used in combination with at least one other miRNA biomarker to determine whether a patient has lung cancer: let-7a, let-7b, let-7d, Iet-7f, let-7g, let-7i, miR-101 , miR- 106a, miR-106b, miR-125a, miR-126, miR-126*, miR-130b, miR-132, miR-133b, miR-140, miR-142-3p, miR-142-5p, miR-145, miR-146a, miR-146b, miR-148b, miR-150, miR-151 , miR-15a, m ⁇ R-15b, miR-181a, miR-181b, miR-181d, miR-185, miR-186, miR-190, miR-191 , miR-193a, miR-199a*, miR-202, miR-210, miR-214, miR-222
• one of the following miRNAs is used in combination with at least one other miRNA biomarker to determine whether a patient has lung cancer or to distinguish iung cancer from benign lung disease: miR-422a; miR-29b; miR-92; miR-142-5p; miR-142-3p; miR-181d; miR-27b; miR- 378; miR-27a; miR-30e-5p; miR-181a; miR-126; miR-342; miR-140; miR-15a; miR-324-3p; miR-374; miR-486; miR-518b; miR-106b; miR-145; miR-150; miR- 191 ; miR-345; miR-126*; miR-148b; miR-214; miR-320; let-7g; let-7i; miR-146a; miR-15b; miR-185; miR-186; miR-23a; miR-24; miR-30a
• one of the following miRNAs is used in combination with at least one other miRNA biomarker to determine whether a patient has lung cancer: let-7g, miR-106b, miR-126, miR-126 * , miR-132, miR- 140, miR-142-3p, miR-146a, miR-150, miR-15a, miR-15b, miR-181a, miR-181b, miR-181d, miR-214, miR-24, miR-30a-5p, miR-320, miR-342, miR-345, miR-374, miR-422a, miR-422b, miR-486, or miR-92.
• Some embodiments of the invention relate to amplifying and measuring at least a pair of miRNAs from serum.
• Table 3 includes pairs that may be used to characterize lung disease. These pairs may be used in combination with other lung disease biomarkers. Table 3. miRNA pairs measured in serum samples.
• the pair of miRNAs is chosen from miR-202 and miR-29b; miR-142-5p and miR-422a; miR-24 and miR-27a; miR-27b and miR-422a; miR-140 and miR-422a; miR-185 and miR-93; miR-126 and miR-181d; miR-142-3p and miR-422a; miR-30e-5p and miR-345; miR-29b and miR-422a; miR-324-5p and miR-422a; miR-374 and miR-422a; miR-140 and miR-345; miR- 23a and miR-27a; miR-29b and miR-378; miR-30e-5p and miR-422a; miR-30e-5p and miR-92; miR-660 and miR-92; miR-106b and miR-422a; let-7g and miR-422a; miR-101 and miR-
• the pair of miRNAs is chosen from let-7a and miR-181a; let-7a and miR-181d; let-7b and miR-150; let-7b and miR-181d; let-7b and miR-92; let-7c and miR-150; let-7c and miR-181d; let-7c and miR-92; let-7e and miR-378; let-7f and miR-181a; let-7f and miR-181d; let-7f and miR-342; let-7f and miR-92; let-7g and miR-150; let-7g and miR-181a; let-7g and miR-181d; let-7g and miR-342; let-7g and miR-92; let-7i and miR-486; let-7i and miR-92; miR-106b and miR-150; miR-106b and miR-181d; miR-106b and miR-92; miR- 125a and miR-142-3p; mi
• the pair of miRNAs is chosen from miR-142- 5p and miR181d; miR-142-3p and miR181d; miR-142-3p and miR-422a; miR-142- 5p and miR-422a; miR-92 and miR-27b; and miR-24 and miR-27a.
• the pair of miRNAs is chosen from miR-106a and miR-422b; miR- 126* and miR-26b; miR-106a and miR-26b; miR-15b and miR-30a-5p; let-7a and miR-26b; let-7g and miR-106a; miR-126 * and miR-15b; miR-126 * and miR-30b; and miR-106a and miR-106b.
• the pair is measured in a serum sample.
• one or more additional miRNAs are measured.
• certain miRNA pairs may be used to characterize lung disease in female or male patients (Table 4).
• the methods detect sex-specific miRNA biomarkers.
• Table 4 miRNA pairs for characterizing lung disease in female or male patients
• Some embodiments of the invention relate to amplifying and measuring two or more miRNAs from plasma.
• One of the following miRNA plasma biomarkers may be used in combination with at least one other miRNA: miR-10b, miR-192, miR-206, miR-101 , miR-205, miR-16, miR-151 , miR-137, miR- 215, miR-181a, miR-218, miR-126 * , miR-125b, miR-326, miR-100, miR-31 , miR- 197, miR-222, miR-191 , miR-200c, miR-186, miR-145, miR-155, miR-29c, let-7c, miR-181c, miR-125a, miR-134, miR-181d, let-7b, miR-127, miR-146a, miR-139, miR-152, miR-190, miR-30e-5p, miR-106b, miR-10a, miR
• RNA levels or amounts of miRNAs are contemplated. Any reliable, sensitive, and specific method can be used.
• a miRNA is amplified prior to measurement.
• the level of miRNA is measured during the amplification process.
• the miRNA is not amplified prior to measurement.
• nucleic acid sequences such as mature miRNAs, precursor miRNAs, and primary miRNAs.
• Suitable nucleic acid polymerization and amplification techniques include reverse
• RT transcription
• PCR polymerase chain reaction
• q-PCR quantitative PCR
• NASBA nucleic acid sequence-base amplification
• ligase chain reaction multiplex Iigatable probe amplification
• IVT in vitro transcription
• IVT in vitro transcription
• TMA transcription-mediated amplification
• RNA Edberwine amplification
• more than one amplification method is used, such as reverse transcription followed by real time quantitative PCR (qRT-PCR) (Chen et al., Nucleic Acids Research, 33(20):e179 (2005)).
• a typical PCR reaction includes multiple amplification steps, or cycles that selectively amplify target nucleic acid species: a denaturing step in which a target nucleic acid is denatured; an annealing step in which a set of PCR primers (forward and reverse primers) anneal to complementary DNA strands; and an elongation step in which a thermostable DNA polymerase elongates the primers. By repeating these steps multiple times, a DNA fragment is amplified to produce an amplicon, corresponding to the target DNA sequence.
• Typical PCR reactions include 20 or more cycles of denaturation, annealing, and elongation. In many cases, the annealing and elongation steps can be performed concurrently, in which case the cycle contains only two steps. Since mature miRNAs are single-stranded, a reverse transcription reaction (which produces a
• Reverse transcription reactions include the use of, e.g., a RNA-based DNA polymerase (reverse transcriptase) and a primer.
• a set of primers is used for each target sequence.
• the lengths of the primers depends on many factors, including, but not limited to, the desired hybridization temperature between the primers, the target nucleic acid sequence, and the complexity of the different target nucleic acid sequences to be amplified.
• a primer is about 15 to about 35 nucleotides in length. In other embodiments, a primer is equal to or fewer than 15, 20, 25, 30, or 35 nucleotides in length. In additional embodiments, a primer is at least 35 nucleotides in length.
• a forward primer can comprise at least one sequence that anneals to a miRNA biomarker and alternatively can comprise an additional 5' non-complementary region.
• a reverse primer can be designed to anneal to the complement of a reverse transcribed miRNA. The reverse primer may be independent of the miRNA biomarker sequence, and multiple miRNA biomarkers may be amplified using the same reverse primer. Alternatively, a reverse primer may be specific for a miRNA biomarker.
• two or more miRNAs are amplified in a single reaction volume.
• One aspect includes multiplex q-PCR, such as qRT-PCR, which enables simultaneous amplification and quantification of at least two miRNAs of interest in one reaction volume by using more than one pair of primers and/or more than one probe.
• the primer pairs comprise at least one amplification primer that uniquely binds each miRNA, and the probes are labeled such that they are distinguishable from one another, thus allowing simultaneous quantification of multiple miRNAs.
• Multiplex qRT-PCR has research and diagnostic uses, including but not limited to detection of miRNAs for diagnostic, prognostic, and therapeutic applications.
• the qRT-PCR reaction may further be combined with the reverse transcription reaction by including both a reverse transcriptase and a DNA-based thermostable DNA polymerase.
• a "hot start” approach may be used to maximize assay performance (U.S. Patent Nos.
• transcriptase reaction and a PCR reaction may be sequestered using one or more thermoactivation methods or chemical alteration to improve polymerization efficiency (U.S. Patent Nos. 5,550,044, 5,413,924, and 6,403,341).
• labels, dyes, or labeled probes and/or primers are used to detect amplified or unamplified miRNAs.
• detection methods are appropriate based on the sensitivity of the detection method and the abundance of the target.
• amplification may or may not be required prior to detection.
• miRNA amplification is preferred.
• a probe or primer may include Watson-Crick bases or modified bases.
• Modified bases include, but are not limited to, the AEGIS bases (from Eragen Biosciences), which have been described, e.g., in U.S. Patent Nos.
• bases are joined by a natural phosphodiester bond or a different chemical linkage.
• Different chemical linkages include, but are not limited to, a peptide bond or a Locked Nucleic Acid (LNA) linkage, which is described, e.g., in U.S. Patent No. 7,060,809.
• LNA Locked Nucleic Acid
• oligonucleotide probes or primers present in an amplification reaction are suitable for monitoring the amount of amplification product produced as a function of time.
• probes having different single stranded versus double stranded character are used to detect the nucleic acid.
• Probes include, but are not limited to, the 5'-exonuclease assay (e.g., TaqManTM) probes (see U.S. Patent No. 5,538,848), stem-loop molecular beacons (see, e.g., U.S. Patent Nos.
• stemless or linear beacons see, e.g., WO 9921881 , U.S. Patent Nos. 6,485,901 and 6,649,349), peptide nucleic acid (PNA) Molecular Beacons (see, e.g., U.S. Patent Nos.
• amplification reaction can include a label.
• different probes or primers comprise detectable labels that are distinguishable from one another.
• a nucleic acid, such as the probe or primer may be labeled with two or more distinguishable labels.
• a label is attached to one or more probes and has one or more of the following properties: (i) provides a detectable signal; (ii) interacts with a second label to modify the detectable signal provided by the second label, e.g., FRET (Fluorescent Resonance Energy Transfer); (iii) stabilizes hybridization, e.g., duplex formation; and (iv) provides a member of a binding complex or affinity set, e.g., affinity, antibody-antigen, ionic complexes, hapten- ligand (e.g., biotin-avidin).
• FRET Fluorescent Resonance Energy Transfer
• MiRNAs can be detected by direct or indirect methods.
• a direct detection method one or more miRNAs are detected by a detectable label that is linked to a nucleic acid molecule.
• the miRNAs may be labeled prior to binding to the probe. Therefore, binding is detected by screening for the labeled miRNA that is bound to the probe.
• the probe is optionally linked to a bead in the reaction volume.
• nucleic acids are detected by direct binding with a labeled probe, and the probe is subsequently detected.
• the nucleic acids such as amplified miRNAs
• FlexMAP Microspheres Luminex
• Some methods may involve detection with polynucleotide probes modified with fluorescent labels or branched DNA (bDNA) detection, for example.
• nucleic acids are detected by indirect detection methods.
• a biotinylated probe may be combined with a streptavidin-conjugated dye to detect the bound nucleic acid.
• the streptavidin molecule binds a biotin label on amplified miRNA, and the bound miRNA is detected by detecting the dye molecule attached to the streptavidin molecule.
• the streptavidin-conjugated dye molecule comprises Phycolink® Streptavidin R-Phycoerythrin (PROzyme). Other conjugated dye molecules are known to persons skilled in the art.
• Labels include, but are not limited to: light-emitting, light-scattering, and light-absorbing compounds which generate or quench a detectable
• Fluorescent reporter dyes useful as labels include, but are not limited to, fluoresceins (see, e.g., U.S. Patent Nos. 5,188,934, 6,008,379, and 6,020,481), rhodamines (see, e.g., U.S. Patent Nos. 5,366,860, 5,847,162, 5,936,087, 6,051 ,719, and
• Tetramethylrhodamine, and/or Texas Red as well as any other fluorescent moiety capable of generating a detectable signal.
• fluorescein dyes include, but are not limited to, 6-carboxyfluorescein; 2',4',1 ,4,-tetrachlorofluorescein; and 2 l ,4 l ,5',7',1 ,4-hexachlorofluorescein.
• the fluorescent label is selected from SYBR-Green, 6-carboxyfluorescein ("FAM”), TET, ROX, VICTM, and JOE.
• labels are different fluorophores capable of emitting light at different, spectrally-resolvable wavelengths (e.g., 4- differently colored fluorophores); certain such labeled probes are known in the art and described above, and in U.S. Patent No. 6,140,054.
• a dual labeled fluorescent probe that includes a reporter fluorophore and a quencher fluorophore is used in some embodiments. It will be appreciated that pairs of fluorophores are chosen that have distinct emission spectra so that they can be easily
• labels are hybridization-stabilizing moieties which serve to enhance, stabilize, or influence hybridization of duplexes, e.g., intercalators and intercalating dyes (including, but not limited to, ethidium bromide and SYBR-Green), minor-groove binders, and cross-linking functional groups (see, e.g., Blackburn et al., eds. "DNA and RNA Structure” in Nucleic Acids in Chemistry and Biology (1996)).
• intercalators and intercalating dyes including, but not limited to, ethidium bromide and SYBR-Green
• minor-groove binders include, but not limited to, ethidium bromide and SYBR-Green
• cross-linking functional groups see, e.g., Blackburn et al., eds. "DNA and RNA Structure” in Nucleic Acids in Chemistry and Biology (1996)).
• methods relying on hybridization and/or ligation to quantify miRNAs may be used, including oligonucleotide ligation (OLA) methods and methods that allow a distinguishable probe that hybridizes to the target nucleic acid sequence to be separated from an unbound probe.
• OLA oligonucleotide ligation
• HARP-Iike probes as disclosed in U.S. Publication No. 2006/0078894 may be used to measure the quantity of miRNAs.
• the probe is modified to measure the quantity of miRNAs.
• a probe inactivation region comprises a subset of nucleotides within the target hybridization region of the probe.
• a post-hybridization probe inactivation step is carried out using an agent which is able to distinguish between a HARP probe that is hybridized to its targeted nucleic acid sequence and the corresponding unhybridized HARP probe. The agent is able to inactivate or modify the unhybridized HARP probe such that it cannot be amplified.
• a probe ligation reaction may be used to quantify miRNAs.
• MLPA Multiplex Ligation-dependent Probe Amplification
• pairs of probes which hybridize immediately adjacent to each other on the target nucleic acid are ligated to each other only in the presence of the target nucleic acid.
• MLPA probes have flanking PCR primer binding sites. MLPA probes can only be amplified if they have been ligated, thus allowing for detection and quantification of miRNA biomarkers.
• the examples described herein include the use of qRT-PCR, which includes real-time monitoring of PCR products during the exponential phase instead of by an end-point measurement.
• the threshold cycle (C t ) measurements in the examples refer to the number of cycles it takes to reach a pre-defined point in the fluorescent signal.
• ROC Receiver Operator Characteristic
• ROC analysis captures the continuum of sensitivity and specificity, but it can be summarized as a single quantity, i.e., the area under the curve (AUC) of the ROC.
• AUC area under the curve
• Advantages of the ROC technique include (1) it does not assume a parametric form of the class probability as required in the logistic regression method, (2) it is adaptable to outcome-dependent samplings, e.g. the case-control design, which are widely used in medical studies, and (3) it is relatively straightforward to assign different 'costs' to false positives and false negatives (Dodd 2003; Pepe 2006).
• A/J mouse model was used to chemically induce lung tumors and monitor the miRNA expression profile in plasma.
• Benzo[a]pyrene (Sigma- Aldrich, St. Louis, MO, USA; cat. no. 48564) served as a carcinogen to
• mice (67 male and 68 female) were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA; Stock Number 000646) and sent to Perry Scientific Inc. (San Diego, CA, USA), at the age of six weeks.
• Perry Scientific performed all the animal related experiments including carcinogen administration, animal monitoring, blood withdrawal and plasma processing.
• mice were bled via orbital sinus and 10 mice were sacrificed by isofluorane inhalation and bled out by cardiac puncture.
• mice in the experimental group were dosed with carcinogen via oral gavage at a dose equivalent to 20 ⁇ M in 200 ⁇ l (250 mg/kg body weight) in cottonseed oil (Sigma- Aldrich, cat. no. CIlQl).
• Ten control mice (5 males and 5 females) received an oral gavage of 200 ⁇ l cottonseed oil with no carcinogen.
• a second carcinogen dose (250 mg/kg body weight) was administered to the test group at 10 weeks post the first gavage, with the control animals receiving a second gavage of cottonseed oil as described above.
• mice were sacrificed at specific time points (Figure 1), and the remaining mice were bled every two weeks ( Figure 1). At termination, animals were weighed, bled via cardiac puncture, and lung lobes collected. Control mice were bled throughout the study and sacrificed at week 34 post-carcinogen administration.
• Plasma samples were processed individually into plasma.
• blood samples were collected into BD Vacutainer® K2EDTA tubes (Becton, Dickinson and Company; Franklin Lakes, NJ, USA; cat. no. 367841). Blood was centrifuged at 2,000 x g for 10 minutes, after which the plasma layer was aspirated and put into a fresh tube and centrifuged at 2,500 x g for 10 minutes. The resulting plasma was immediately stabilized by the addition of 2X denaturing buffer from the m/ ⁇ /anaTM miRNA Isolation Kit (Ambion Inc., Austin, TX, USA; cat no. AM1560 ) to achieve a final concentration of 1X. Plasma and buffer were mixed by vortexing and immediately frozen at -80 0 C until shipment to Asuragen on dry ice. Cell pellets were also frozen at -80 0 C and shipped to Asuragen with plasma samples.
• Plasma RNA was purified using the organic extraction of the mirVana PARISTM Kit (Ambion, Inc.; Part No. AM1556), with the following modifications. Following the addition of acid phenokchloroform and vortexing, samples were incubated on ice for 5 min then centrifuged at 13,000 x g for 10 min at 4 0 C. The aqueous layer was removed, extracted with chloroform, and centrifuged again. The aqueous layer was removed from the second extraction, and 3M NaOAc (1/10 volume), glycogen (5 mg/ml), and 100% ethanol (1.5 volume) were added to the samples.
• Lysate/ethanol mixtures were passed through a mirVana PARISTM filter cartridge, and filters were washed once with 650 ⁇ l of Wash 1 buffer and twice with 650 ⁇ l of Wash 2/3 buffer. RNA was eluted with two aliquots of nuclease free water (50 ⁇ l) and stored at -80 0 C.
• FFPE hematoxylin and eosin
• H&E-stained slides were sent to Asuragen and analyzed by a board certified pathologist and classified as normal, hyperplasia, adenoma, or
• Table 6 shows the number of visible tumors observed in each sacrificed animals, and the pathological diagnosis in each of the lung lobes.
• Table 6 Lung tumors and pathological diagnosis from experimental and control mice. SCC, squamous cell carcinoma.
• miRNA expression was evaluated in plasma samples from mice with pathology-confirmed adenocarcinoma of the lung and from control mice that did not receive carcinogen. Control mice were age matched to the mice with adenocarcinoma. Expression levels of 329 miRNAs, were determined by qRT- PCR using TaqMan® MicroRNA Assays (Applied Biosystems) specific for each miRNA. A subset of the miRNAs (170) have identical mature sequences in both mice and humans and the remainder are mouse homologues of corresponding human miRNAs. Reverse transcription (RT) reaction components (Table 7) were assembled on ice prior to the addition of RNA template, and included ⁇ 1 ng plasma RNA per each 10 ⁇ l reaction.
• RT Reverse transcription
• RT reactions were incubated in a 384-well GeneAmp® PCR System 9700 (Applied Biosystems) at 16 0 C for 30 minutes, then at 42 0 C for 30 minutes, then at 85 0 C for 5 minutes. RT reactions were then frozen at -20 0 C. Table 7. Reverse transcription reaction components.
• PCR components (Table 8) were assembled on ice prior to the addition of cDNA (2 ⁇ l) from the RT reaction. Reactions were incubated in an ABI PRISMTM 7900HT Fast Real-Time PCR system (Applied Biosystems) at 95 0 C for 1 minute, then for 50 cycles at 95 0 C for 5 seconds and 60 0 C for 30 seconds. Results were analyzed with the 7900HT Fast Real-Time PCR system SDS V2.3 software (Applied Biosystems).
• Table 9 shows the average Ct data and ddCt values for cancer vs. control mice from the qRT-PCR experiment. miRNAs with p-values of 0.1 or less are shown.
• FIG. 2A and Figure 2B show the changes in expression of selected miRNAs over time, prior to the detection of tumors. This figure shows that differential expression of miRNAs can be detected as early as 10 weeks after carcinogen administration-at least 6 weeks prior to the detection of tumors.
• samples were incubated on ice for 5 min then centrifuged at 13,000 x g for 10 min at 4 0 C. The aqueous layer was removed, extracted with chloroform, and centrifuged again. The aqueous layer was removed from the second extraction, and 3M NaOAc (1/10 volume), glycogen (5mg/ml), and 100% ethanol (1.5 volume) were added to the samples.
• Lysate/ethanol mixtures were passed through a mirVana PARISTM filter cartridge, and filters were washed once with 650 ⁇ l of Wash 1 buffer and twice with 650 ⁇ l of Wash 2/3 buffer. RNA was eluted with two aliquots of nuclease free water (50 ⁇ l) and stored at -80 0 C.
• miRNA expression levels were determined by qRT-PCR using TaqMan® MicroRNA Assays (Applied Biosystems; Foster City, CA, USA) specific for each miRNA.
• Reverse transcription (RT) reactions were performed using the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA; cat. no. 4366597).
• Reaction components listed in Table 10 were assembled on ice prior to the addition of RNA template. All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) as a kit with multiple components. Serum RNA (1 ng total RNA per reaction) was added and mixed.
• RT reactions were incubated in a 384-well GeneAmp® PCR System 9700 (Applied Biosystems) at 16 0 C for 30 minutes, then at 42 0 C for 30 minutes, then at 85 0 C for 5 minutes. RT reactions were then frozen at -20 0 C.
• PCR components (Table 11) were assembled on ice prior to the addition of cDNA (4 ⁇ l) from the RT reaction. Reactions were incubated in an ABI PRISMTM 7900HT Fast Real-Time PCR system (Applied Biosystems) at 95 °C for 1 minute, then for 50 cycles at 95 0 C for 5 seconds and 60 0 C for 30 seconds. Results were analyzed with the 7900HT Fast Real-Time PCR system SDS V2.3 software (Applied Biosystems).
• Table 12 provides average dCt values for cancer and normal samples for each miRNA. In addition, ddCt values represent the difference in expression between cancer and normal samples.
• Table 12 Expression of miRNAs in serum from patients with and without lung cancer.
• adenocarcinoma SCC for squamous cell carcinoma
• R for right
• RUL for right upper lobe
• RML for right mid lobe
• RLL for right lower lobe
• LUL for left upper lobe
• LLL for left lower lobe
• Table 13 Histopathological and patient information for lung cancer and benign specimens.
• Table 14 provides differential expression data for miRNAs in the form of average dCt values for cancer and benign samples.
• ddCt values represent the difference in expression between cancer and benign samples.
• Table 14 Expression of miRNAs in serum from patients with lung cancer or benign lung conditions.
• Table 15 includes miRNA pairs that distinguish patients with lung cancer from those with benign lung conditions. The miRNA pairs in Table 15 are listed in order of decreasing ROC AUC. Serum miRNA biomarker pairs for classifying patients with cancer vs. patients with benign lung conditions are presented. Mean values represent the average difference in Gt values between the two miRNAs in the pair.
• “Benign mean” represents the mean of data from 12 subjects with benign lung conditions; “Cancer mean” is the mean for data from 16 lung cancer patients; “Benign SD” is the standard deviation for data from patients with benign lung conditions; “Cancer SD” is the standard deviation for data from lung cancer patients; “Benign vs Cancer Assoc.” refers to the p- value associated with the diagnosis of benign/lung cancer; ROC stands for receiver operating characteristic; and AUC is area under curve.
• Tables 15 and 16 show miRNAs and miRNA biomarker pairs that can distinguish patients with benign conditions of the lung from patients with lung cancer in serum samples, irrespective of patient sex or cancer type. These miRNAs are useful for diagnosis of lung cancer.
• the serum samples from cancer patients were from patients with early stage I through stage III lung cancer. These results show that miRNAs are suited to detect lung cancer at an early stage and are effective for screening patients and distinguishing patients with benign lung conditions from those with lung cancer.
• Table 18 microRNA pairs that are differentially expressed in serum of male patients with benign lung conditions as compared to male lung cancer patients.
• Table 19 Histopathological and patient information for lung cancer and benign specimens.
• Table 21 shows differentially expressed miRNA biomarker pairs that distinguish samples from lung cancer patients from normal patients and patients with benign tumors. Such miRNA and biomarker pairs can be used for screening and diagnosis of lung cancer.
• Table 22 shows miRNAs that can be used in combination with other miRNA biomarkers to characterize lung disease, as well as the prevalence of those biomarkers in pairs from Table 21.
• Table 22 Prevalence of serum biomarkers in paired analysis of cancer vs.
• An initial training set of 14 miRNAs were selected from pairs in Table 15 to further verify their diagnostic potential. These miRNAs were shown above to have diagnostic potential for distinguishing patients with benign lung conditions from patients with lung cancer, as evidenced by the high AUC ROC scores for miRNA biomarker pairs that include these miRNAs. From a group of 20 benign lung samples and 34 lung cancer samples (Table 23), a set of training samples (12 benign, 16 lung cancer) was chosen from patients that were age and sex matched. qRT-PCR was performed as described above in Example 2.
• Table 23 Histopathological and patient information for lung cancer and benign specimens.
• a separate test sample set was selected from Table 23 that was composed of 8 benign and 18 LuCa samples.
• miRNA biomarker selection and classifier evaluation was generated by performing 25 repetitions of 5-fold cross validation on the training samples, and measuring the AUC ROC values as a function of the number of miRNA biomarkers (features). A range of biomarkers was used in order to determine the optimal number of features for classification.
• Five-fold cross- validation is a process where the training set is subdivided into an 80% sample set and a 20% sample set, and feature selection and classifier training is performed on the 80% training samples. Performance is measured by classifying the remaining 20% samples not used for feature selection and classifier training.
• Figure 3 shows the distribution of AUC values for the LDA classifier as a function of the number of biomarkers. This procedure determines the optimal number of biomarkers to use for the classifier, and performance expectations moving forward.
• a classifier of 6 differentially expressed miRNA pairs (miR-142-5p and miR181d; miR-142-3p and miR181d; miR-142-3p and miR-422a; miR-142-5p and miR-422a; miR-92 and miR-27b; miR-24 and miR-27a) was identified as the optimal classifier for diagnosis of lung cancer in this experiment. This classifier is marked by the vertical dotted line in Figure 3.
• a cutoff for the LDA classifier was set so that the continuous valued classification probabilities were converted into calls diagnosing patients as having lung cancer (classifier-positive) or benign (classifier-negative) tumors. For simplicity, a cutpoint of 0.5 was chosen as the threshold. This value produced a near maximum of sensitivity and specificity. Performance estimates of training and test data using a cutoff of 0.5 of the LDA classifier trained on six biomarker pairs is provided in Figure 4. The sensitivity (SENS), specificity (SPEC), negative predictive value (NPV) and positive predictive value (PPV) are as indicated.
• any of the six pairs identified as part of the classifier in this experiment may be used to diagnose lung cancer. Predictive accuracy may increase with the use of more than one pair from the optimal classifier set.
• Plasma RNA was purified using the organic extraction of the mirVana PARISTM Kit (Part No. AM1556; Applied Biosystems/Ambion; Austin, TX, USA), with the following modifications. After thawing plasma on ice, an equal volume of 2X denaturing solution from the mirVana PARISTM Kit was added and the mixture was incubated on ice for five minutes. An equal volume of acid phenol:chloroform was added, then the mixture was vortexed for one minute, and incubated on ice for five minutes. Tubes were centrifuged at 13,000 x g for 15 minutes at 4 0 C, and the aqueous phase was removed to a fresh tube.
• the mirVana PARISTM Kit Part No. AM1556; Applied Biosystems/Ambion; Austin, TX, USA
• RNA template (Table 25).
• 0.25 ⁇ l of plasma RNA template per reaction was added and mixed.
• RT reactions were incubated in a 384-well GeneAmp® PCR System 9700 (Applied Biosystems) at 16 0 C for 30 minutes, then at 42 0 C for 30 minutes, then at 85 0 C for 5 minutes, and then were frozen in a -20 0 C freezer.
• PCR components were assembled on ice prior to the addition of the cDNA from the RT reactions (Table 26). PCRs were incubated in an ABI
• PRISMTM 7900HT Fast Real-Time PCR system (Applied Biosystems) at 95 0 C for 1 minute, then for 50 cycles at 95 0 C for 5 seconds, and then at 60 0 C for 30 seconds.
• Initial data analysis was done using the 7500 Fast System SDS V2.3 software (Applied Biosystems). Table 26. Real-time PCR components.
• the qRT-PCR data were initially assessed for outliers. All miRNAs in a given sample with raw Ct values of 50 were eliminated from further analysis. All data from samples with fewer than 150 miRNAs that had raw Ct values less than 50 were eliminated. The average raw Ct for 50 miRNAs that were detected in each plasma sample was calculated for each individual sample. The average Ct for a given sample was subtracted from the raw Ct values for each miRNA in the corresponding sample to produce a dCt for each miRNA that was detected. These normalized measures were used to identify miRNAs that were present at significantly different levels in the plasma samples from normal donors and lung cancer patients.
• a second set of plasma samples, isolated from a distinct set of lung cancer and normal patients was used to perform an additional comparison of miRNAs expressed in lung cancer and normal patients.
• TNM tumor, node, metastasis stage is described in Sobin and Wittekind, New Jersey: John Wiley & Sons, 2002.
• the following abbreviations are used in Table 28: adenocarcinoma (ADCA); bronchoalveolar (BA), squamous cell carcinoma (SCC), and not available (NA).
• Table 28 Human Lung Cancer and Normal Patient Information and Tumor Pathology. .
• Plasma RNA was isolated as described above in Example 6. qRT- PCR reactions were performed using TaqMan® MicroRNA Assays (Applied Biosystems; Foster City, CA, USA) specific for each individual miRNA. Reverse transcription reactions were assembled on ice prior to the addition of RNA template (Table 29). Next, 0.25 ⁇ l of plasma RNA template per reaction was added and mixed. RT reactions were incubated in a 384-well GeneAmp® PCR System 9700 (Applied Biosystems) at 16 0 C for 30 minutes, then at 42 0 C for 30 minutes, then at 85 0 C for 5 minutes, and then were frozen in a -20 0 C freezer.
• PCR components were assembled on ice prior to the addition of the cDNA from the RT reactions (Table 30). PCRs were incubated in an ABI
• PRISMTM 7900HT Fast Real-Time PCR system (Applied Biosystems) at 95 0 C for 1 minute, then for 50 cycles at 95 0 C for 5 seconds and then at 60 0 C for 30 seconds.
• Initial data analysis was done using the 7500 Fast System SDS V2.3 software (Applied Biosystems).
• the qRT-PCR data were initially assessed for outliers. All miRNAs in a given sample with raw Ct values of 50 were eliminated from further analysis. All data from samples with fewer than 150 miRNAs that had raw Ct values less than 50 were eliminated. The average raw Ct for 50 miRNAs that were detected in each plasma sample was calculated for each individual sample. The average Ct for a given sample was subtracted from the raw Ct values for each miRNA in the corresponding sample to produce a dCt for each miRNA that was detected. These normalized measures were used to identify miRNAs that were present at significantly different levels in the plasma samples from normal donors and lung cancer patients.
• the average dCt values for each miRNA in the normal donor and lung cancer patient samples were calculated.
• the average dCt values for the lung cancer patient samples were subtracted from the average dCt values for the normal donors to determine the variance in the levels of the various miRNAs between the two patient sets.
• the student t-test was then used to evaluate the capacity of the various miRNAs to distinguish the plasmas of lung cancer patients and normal donors.
• Table 31 provides dCt values for normal and cancer patients, as well as ddCt values for cancer vs normal samples for each miRNA.
• Table 31 miRNA Biomarkers Differentially Expressed Between Plasma Samples of Lung Cancer Patients and Plasma Samples of Normal Patients.
• Example 8 Combinations of miRNAs that Distinguish Plasma from Lung Cancer Patients and Plasma from Normal Patients
• ROC Characteristic
• Table 33 Biomarkers that can be used in combination to identify plasma from lung cancer patients, and the prevalence of each miRNA.
• ADCA adenocarcinoma
• SCCA squamous cell carcinoma
• Table 35 Expression of miRNAs in serum from patients having lung cancer and from normal subjects.
• Table 36 Lung cancer patient, tumor pathology, and normal subject information.
• TNM Stage Sobin and Wittekind, 2002
• AJCC Stage Greene, 2002
• ADCA adenocarcinoma
• ADCA/BA adenocarcinoma of the bronchoalveolar
• SCCA squamous cell carcinoma
• NA not applicable.
• the 27 miRNAs were evaluated for their ability to distinguish lung cancer patient samples from normal subject samples shown in Table 36.
• Table 37 shows the mean Ct values and standard deviation for each miRNA for the cancer and the normal group.
• the dCt values represent the difference of the Cts between the cancer and the normal group.
• the miRNAs are arranged in increasing t-test p-value. Twenty-four of the 27 miRNAs were differentially expressed at a high statistical significance level, having t-test p-values of less than 0.001. These twenty-four miRNAs can be used for diagnosing lung cancer.
• Table 37 Expression of miRNAs in serum from patients having lung cancer or from normal subjects.
• Receiver Operator Characteristic (ROC) analysis was used to identify the miRNA pairs having the capacity to distinguish serum samples of lung cancer patients from those of normal patients. Select differentially-expressed pairs that distinguished normal patients from cancer patients are shown in Table 38 and are arranged in decreasing AUC ranging from 0.99 to 0.74.
• Table 38 miRNA biomarker pairs that can be used to identify serum from lung cancer patients.
• Table 39 Performance of classification models and feature selection in training set and test set of normal subjects and lung cancer patients.
• the model's performance was estimated on training data by performing 25 repetitions of 5-fold cross validation on the training samples and measuring the ROC AUC values as previously described in Example 5.
• the frequency of appearance of miRNA pairs in each of the classification models is shown in Table 40.
• Table 40 Prevalence of paired biomarkers in classifier generation. The classification models are as indicated.
• Table 41 miRNAs that can be used to distinguish patients with lung cancer from cancer free subjects

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