Determination of lung cancer using MIRNA ratios

Abstract

The present disclosure relates to the determination of lung cancer using miRNA ratios. The present invention provides methods of determining the presence of a lung tumor in a subject. Also provided are methods of determining the presence of an invasive lung tumor in a subject. In addition, methods of determining the risk of developing a lung tumor in a subject are provided. Also provided are methods of determining the risk of developing an invasive lung tumor in a subject. Also provided are methods of predicting the risk of developing or having a lung tumor in a subject. Also provided are methods of establishing lung cancer treatment options.

Description

This application is a divisional application of the invention patent filed on December 31, 2014, with application number 201480076735.0 and invention title "Using MIRNA Ratio to Determine Lung Cancer".

[0001] Cross-reference of related applications This application claims priority and benefit to U.S. Provisional Application No. 61 / 923,758, filed January 5, 2014, and U.S. Provisional Application No. 61 / 926,323, filed January 12, 2014, both of which are incorporated herein by reference in their entirety. Technical Field

[0002] This application generally relates to the diagnosis of lung cancer and the determination of the risk of lung cancer. More specifically, this application relates to using miRNA expression rates to determine the risk of developing or invasive lung tumors, and to determine the presence of or invasive lung tumors. Background Technology Lung cancer is a leading cause of cancer death worldwide (Jemal et al., CA Cancer J Clin, 61:69-9, 2011). Currently, most lung cancers are detected at an advanced stage, at which point treatment has limited efficacy and survival rates are low. Early detection of lung cancer can significantly reduce mortality.

[0003] European randomized lung cancer screening trials, using observational control groups but of limited size, have so far failed to demonstrate a reduction in mortality (Infante et al., Am J Respir Crit Care Med 180:445-453, 2009; Saghir et al., Thorax 67:296-301, 2012; Pastorino et al., Eur J Cancer Prev 21:308-315, 2012); however, results from the large NCI-initiated National Lung Screening Trial (NSLT) indicate that screening high-risk individuals with a history of ≥30 pack-years and ≤15 years since quitting smoking using low-dose computed tomography (LDCT) resulted in a 20% reduction in mortality compared to annual chest radiographs (Aberle et al., N Engl J Med 365:395-409, 2011). High false positive rates, the cost of screening a large number of high-risk individuals (estimated at $3.5 million in the United States), and potential harms associated with LDCT screening remain considerations in clinical settings (Aberle et al., 2011; Goulart et al., J Natl Compr Canc Netw 10:267-275, 2012).

[0004] Therefore, there is a need for alternative methods to improve the prediction, prognosis, and diagnosis of lung cancer. This invention fulfills that need. Summary of the Invention This invention is based in part on the discovery that assay methods can utilize circulating miRNA biomarkers to provide a three-level classifier for the determination of overall “risk of developing or having lung cancer”.

[0005] Therefore, in some implementations, a method is provided for determining the presence of a lung tumor in a subject. The method includes: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cut-off value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then a positive score is assigned to the ratio; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then a non-positive score is assigned to the ratio. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 9 miRNA pairs are assigned a positive score for their expression ratio, or (2) fewer than 9 miRNA pairs are assigned a positive score after comparing the expression ratios of 27 miRNA pairs. The miRNA pairs mentioned include 106a / 140-5p, 106a / 142-3p, 126 / 140-5p, 126 / 142-3p, 133a / 142-3p, 140-5p / 17, 142-3p / 148a, 142-3p / 15b, 142-3p / 17, 142-3p / 21, 142-3p / 221, 142-3p / 30b, or 320 / 660, or their inverse ratios; and (e) The presence of lung tumors is classified as follows: (i) positive if at least 9 miRNAs are assigned a positive score for the expression ratio; or (ii) negative if fewer than 9 miRNAs are assigned a positive score for the expression ratio.

[0006] According to another implementation plan, the method for determining the presence of aggressive lung tumors in subjects includes: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cutoff value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then a positive score is assigned to the ratio; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then a non-positive score is assigned to the ratio. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 14 miRNA pairs are assigned a positive score for their expression ratio, or (2) fewer than 14 miRNA pairs are assigned a positive score after comparing the expression ratios of 28 miRNA pairs. The miRNA pairs mentioned include 106a / 16, 106 / 660, 16 / 17, 16 / 320, 17 / 660, 197 / 30b, 197 / 30c, 320 / 451, 320 / 486-5p, or 320 / 660, or their inverses; and (e) The presence of invasive lung tumors is classified as follows: (i) positive if at least 14 miRNAs are assigned a positive score for the expression ratio; or (ii) negative if fewer than 14 miRNAs are assigned a positive score for the expression ratio.

[0007] According to yet another implementation plan, the methods for determining the risk of developing lung cancer in subjects include: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cutoff value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then a positive score is assigned to the ratio; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then a non-positive score is assigned to the ratio. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 10 miRNA pairs are assigned a positive score for their expression ratio, or (2) after comparing the expression ratios of 27 miRNA pairs, fewer than 10 miRNA pairs are assigned a positive score for their expression ratio. The miRNA pairs mentioned include 133a / 92a, 15b / 21, 15b / 30b, 15b / 30c, 16 / 197, or 28-3p / 451, or their inverses; and (e) The risk of developing lung cancer is classified as follows: (i) if at least 10 miRNAs are assigned a positive score for their expression ratio, it is positive; or (ii) if fewer than 10 miRNAs are assigned a positive score for their expression ratio, it is negative.

[0008] Other methods for determining the risk of developing aggressive lung tumors, based on other implementation schemes, include: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cutoff value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then a positive score is assigned to the ratio; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then a non-positive score is assigned to the ratio. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 14 miRNA pairs are assigned a positive score for their expression ratio, or (2) fewer than 14 miRNA pairs are assigned a positive score after comparing the expression ratios of 28 miRNA pairs. The miRNA pairs mentioned include 106a / 142-3p, 126 / 142-3p, 126 / 21, 126 / 92a, 142-3p / 17, 142-3p / 197, 142-3p / 28-3p, 197 / 19b, 197 / 660 or 28-3p / 660, or their inverses; and (e) Risk classification for the occurrence of invasive lung tumors as follows: (i) positive if at least 14 miRNAs are assigned a positive score for the expression ratio; or (ii) negative if fewer than 14 miRNAs are assigned a positive score for the expression ratio.

[0009] In other implementation methods, methods for predicting the risk of developing or having lung cancer in subjects include: (a) Determine the presence of the lung tumor label as described above; (b) Determine the presence of the aforementioned invasive lung tumor label; (c) Determine the risk labels for lung cancer mentioned above; (d) Determine the risk labels for the occurrence of aggressive lung tumors as described above; (e) If none of the labels in steps (a)-(d) are classified as positive, the subject is classified as low risk of developing or having lung cancer. (f) If at least the lung tumor presence label in step (a) is positive, or the risk label for the occurrence of lung tumor in step (c) is positive; and both the presence label for invasive lung tumor in step (b) and the risk label for the occurrence of invasive lung tumor in step (d) are negative, then the subject is classified as having an intermediate risk of developing or having lung tumor; and (g) If the presence of invasive lung tumor in at least step (b) is labeled positive or the risk of developing invasive lung tumor in step (d) is labeled positive, the subject is classified as being at high risk of developing or having lung tumor.

[0010] Additionally, a method for establishing treatment options for a subject is provided. This method includes testing the subject using any of the methods described above and determining treatment options based on the results of the methods. The method may further include administering treatment to the subject who requires it.

[0011] Although this disclosure has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative and not limiting of the scope of this disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0012] The patents and scientific literature cited herein provide knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated by reference. Genbank and NCBI submissions indicated by search numbers cited herein are incorporated by reference. All other published references, documents, manuscripts, and scientific literature cited herein are incorporated by reference. Attached Figure Description Figure 1 This is a simplified diagram describing the characteristics of the sample group in Example 1, Example 1: Multicentric Italian Lung Detection (MILD) Study 2005-2012.

[0013] Figure 2 These are graphs and tables showing the three-year survival rate of all subjects from the date of blood sample collection, based on miRNA tag classifiers (MSCs). Detailed Implementation According to one embodiment, the present invention utilizes multiple ratios of miRNA expression levels to determine (a) the risk of developing lung tumor, (b) the risk of developing invasive lung tumor, (c) the presence of lung tumor, (d) the presence of invasive tumor, and, when combining the risks detected in steps (a)-(d), to determine the overall risk of having or developing lung tumor.

[0014] This invention may include removing samples with detectable levels of hemolysis, for example by including the use of miRNA hemolysis classifiers; using a three-level (low, intermediate, or high) "disease risk" classifier instead of a two-level (low, high) classifier; or using a control plasma sample from a single subject instead of a pool. Three miRNAs may be included: miR-101, miR-145, and miR-133a, which were excluded in previous assay validation steps due to high variability in the control pool.

[0015] According to the exemplary method of the present invention, the “microRNA tag classifier” (MSC) provides a screening sensitivity of 87% for MSCs alone and 98% when combined with low-dose computed tomography (LDCT) screening. Therefore, MSCs can be used alone or in synergistic combination with other methods (e.g., LDCT) to improve the effectiveness of LDCT for lung cancer screening by avoiding more rounds of LDCT and unnecessary invasive diagnostic follow-ups in a large proportion of subjects. The prognostic and diagnostic performance of MSCs alone, detailed in this application, demonstrates that, regardless of tumor stage, miRNAs measured within MSCs are not merely an output of tumor load, but rather an indicator of pathogenesis related to tumor invasiveness.

[0016] The development of MSCs was based on an unbiased computer method that screened 4,950 ratios of 100 different plasma miRNAs to select the optimal set of miRNA ratios associated with lung cancer detection and poor prognosis (Boeri et al., ProcNatl Acad Sci USA 108:3713-3718, 2011). These miRNA ratios reflect the competitive mechanisms of miRNA regulation within different cellular components of the tumor and the regulation between the surrounding microenvironment. Unbound by any particular theory, stromal cells are activated by the inflamed lung microenvironment, releasing specific miRNAs into circulation that can functionally participate in the regulation of target genes related to tumor transformation.

[0017] MSCs utilize robust assays of plasma-derived miRNA tags. MSCs possess diagnostic capabilities for the presence of malignant disease, the risk of future malignancy, and the ability to differentiate lung cancer from most benign LDCT-detected lung nodules. This article details the specific tags available to MSCs.

[0018] "Presence of Lung Tumors" tag In some implementations, a method is provided to determine the presence of a lung tumor in a subject. The method includes: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cutoff value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then a positive score is assigned to the ratio; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then a non-positive score is assigned to the ratio. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 9 miRNA pairs are assigned a positive score for their expression ratio, or (2) fewer than 9 miRNA pairs are assigned a positive score after comparing the expression ratios of 27 miRNA pairs. The miRNA pairs mentioned include 106a / 140-5p, 106a / 142-3p, 126 / 140-5p, 126 / 142-3p, 133a / 142-3p, 140-5p / 17, 142-3p / 148a, 142-3p / 15b, 142-3p / 17, 142-3p / 21, 142-3p / 221, 142-3p / 30b, or 320 / 660, or their inverse ratios; and (e) The presence of lung tumors is classified as follows: (i) positive if at least 9 miRNAs are assigned a positive score for the expression ratio; or (ii) negative if fewer than 9 miRNAs are assigned a positive score for the expression ratio.

[0019] In some embodiments, the miRNA pairs of the assay include ratios of 106a / 140-5p, 106a / 142-3p, 126 / 140-5p, 126 / 142-3p, 133a / 142-3p, 140-5p / 17, 142-3p / 148a, 142-3p / 15b, 142-3p / 17, 142-3p / 21, 142-3p / 221, 142-3p / 30b, and 320 / 660 or their inverse ratios, exceeding 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or each.

[0020] In other embodiments, the miRNA pairs may further include ratios of 106a / 660, 106a / 92a, 126 / 660, 140-5p / 197, 140-5p / 28-3p, 142-3p / 145, 142-3p / 197, 142-3p / 28-3p, 17 / 660, 17 / 92a, 197 / 660, 197 / 92a, 19b / 660, or 28-3p / 660, or the inverse of those ratios.

[0021] In other embodiments, the miRNA pairs include ratios of 106a / 140-5p, 106a / 142-3p, 126 / 140-5p, 126 / 142-3p, 133a / 142-3p, 140-5p / 17, 142-3p / 148a, 142-3p / 15b, 142-3p / 17, 142-3p / 21, 142-3p / 221, 142-3p / 30b, 320 / 660, 106a / 660, 106a / 92a, 126 / 660, 140-5p / 1 At least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, or each of these 27 ratios. Those skilled in the art will understand that utilizing some, but not all, of these 27 ratios will be expected to provide a method for accurately identifying the presence of a tumor.

[0022] In some embodiments, the present invention provides the use of a plurality of primers or probes for detecting any one of 27 miRNA pairs in the preparation of a diagnostic reagent for determining the presence of lung tumors in a subject, wherein said detection includes: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cutoff value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then a positive score is assigned to the ratio; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then a non-positive score is assigned to the ratio. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 9 miRNA pairs are assigned a positive score for their expression ratio, or (2) fewer than 9 miRNA pairs are assigned a positive score after comparing the expression ratios of 27 miRNA pairs. The miRNA pairs mentioned include 106a / 140-5p, 106a / 142-3p, 126 / 140-5p, 126 / 142-3p, 133a / 142-3p, 140-5p / 17, 142-3p / 148a, 142-3p / 15b, 142-3p / 17, 142-3p / 21, 142-3p / 221, 142-3p / 30b, or 320 / 660, or their inverse ratios; and (e) The presence of lung tumors is classified as follows: (i) positive if at least 9 miRNAs are assigned a positive score for the expression ratio; or (ii) negative if fewer than 9 miRNAs are assigned a positive score for the expression ratio.

[0023] In some embodiments, the plurality of primers or probes are used to detect miRNAs expressing ratios of 106a / 140-5p, 106a / 142-3p, 126 / 140-5p, 126 / 142-3p, 133a / 142-3p, 140-5p / 17, 142-3p / 148a, 142-3p / 15b, 142-3p / 17, 142-3p / 21, 142-3p / 221, 142-3p / 30b, and 320 / 660 or their inverse ratios, exceeding 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or each of these ratios.

[0024] In other embodiments, the plurality of primers or probes are further used to detect miRNA expression ratios of 106a / 660, 106a / 92a, 126 / 660, 140-5p / 197, 140-5p / 28-3p, 142-3p / 145, 142-3p / 197, 142-3p / 28-3p, 17 / 660, 17 / 92a, 197 / 660, 197 / 92a, 19b / 660, or 28-3p / 660, or their inverse ratios.

[0025] In other embodiments, the plurality of primers or probes are used to detect miRNA expression ratios of 106a / 140-5p, 106a / 142-3p, 126 / 140-5p, 126 / 142-3p, 133a / 142-3p, 140-5p / 17, 142-3p / 148a, 142-3p / 15b, 142-3p / 17, 142-3p / 21, 142-3p / 221, 142-3p / 30b, 320 / 660, 106a / 660, 106a / 92a, 126 / 660, 14 At least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, or each of the following 27 ratios: 0-5p / 197, 140-5p / 28-3p, 142-3p / 145, 142-3p / 197, 142-3p / 28-3p, 17 / 660, 17 / 92a, 197 / 660, 197 / 92a, 19b / 660, and 28-3p / 660 or their inverse ratios. Those skilled in the art will understand that utilizing some, but not all, of these 27 ratios will be expected to provide a method for accurately identifying the presence of a tumor.

[0026] In some embodiments, at least one of the plurality of primers and probes is capable of selectively binding to at least one miRNA of a miRNA pair in the sample. In some embodiments, the plurality of primers or probes includes those capable of selectively binding to miRNA pairs with expression ratios of 106a / 140-5p, 106a / 142-3p, 126 / 140-5p, 126 / 142-3p, 133a / 142-3p, 140-5p / 17, 142-3p / 148a, 142-3p / 15b, 142-3p / 17, 142-3p / 21, 142-3p / 221, 142-3p / 30b, 320 / 660, 106a / 660, 106a / 92a, 126 / 660, and 140-5p. At least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26 miRNAs of each of the following types: / 197, 140-5p / 28-3p, 142-3p / 145, 142-3p / 197, 142-3p / 28-3p, 17 / 660, 17 / 92a, 197 / 660, 197 / 92a, 19b / 660, and 28-3p / 660.

[0027] In some embodiments, the present invention provides a kit for determining the presence of lung tumors in a subject. The kit includes a plurality of primers or probes for detecting any one of 27 miRNA pairs in the preparation of diagnostic reagents, and instructions for using said plurality of primers or probes to determine the presence of lung tumors in a subject. The instructions include: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cutoff value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then a positive score is assigned to the ratio; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then a non-positive score is assigned to the ratio. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 9 miRNA pairs are assigned a positive score for their expression ratio, or (2) fewer than 9 miRNA pairs are assigned a positive score after comparing the expression ratios of 27 miRNA pairs. The miRNA pairs mentioned include 106a / 140-5p, 106a / 142-3p, 126 / 140-5p, 126 / 142-3p, 133a / 142-3p, 140-5p / 17, 142-3p / 148a, 142-3p / 15b, 142-3p / 17, 142-3p / 21, 142-3p / 221, 142-3p / 30b, or 320 / 660, or their inverse ratios; and (e) The presence of lung tumors is classified as follows: (i) positive if at least 9 miRNAs are assigned a positive score for the expression ratio; or (ii) negative if fewer than 9 miRNAs are assigned a positive score for the expression ratio.

[0028] In some implementations, multiple primers or probes provided in the kit can be used to detect miRNAs expressing ratios of 106a / 140-5p, 106a / 142-3p, 126 / 140-5p, 126 / 142-3p, 133a / 142-3p, 140-5p / 17, 142-3p / 148a, 142-3p / 15b, 142-3p / 17, 142-3p / 21, 142-3p / 221, 142-3p / 30b, and 320 / 660 or their inverse ratios, exceeding 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or each of these ratios.

[0029] In other embodiments, the multiple primers or probes provided in the kit can be further used to detect miRNA expression ratios of 106a / 660, 106a / 92a, 126 / 660, 140-5p / 197, 140-5p / 28-3p, 142-3p / 145, 142-3p / 197, 142-3p / 28-3p, 17 / 660, 17 / 92a, 197 / 660, 197 / 92a, 19b / 660, or 28-3p / 660, or their inverse ratios.

[0030] In other implementations, the multiple primers or probes provided in the kit can be used to detect miRNA expression ratios of 106a / 140-5p, 106a / 142-3p, 126 / 140-5p, 126 / 142-3p, 133a / 142-3p, 140-5p / 17, 142-3p / 148a, 142-3p / 15b, 142-3p / 17, 142-3p / 21, 142-3p / 221, 142-3p / 30b, 320 / 660, 106a / 660, 106a / 92a, and 126 / 660. 140-5p / 197, 140-5p / 28-3p, 142-3p / 145, 142-3p / 197, 142-3p / 28-3p, 17 / 660, 17 / 92a, 197 / 660, 197 / 92a, 19b / 660 and 28-3p / 660 or their inverse ratios of at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26 or each.

[0031] In some embodiments, at least one primer or probe of the plurality of primers and probes provided in the kit is capable of selectively binding to at least one miRNA of a miRNA pair in the sample. In some embodiments, the plurality of primers or probes provided in the kit include those capable of selectively binding to miRNA pairs with expression ratios of 106a / 140-5p, 106a / 142-3p, 126 / 140-5p, 126 / 142-3p, 133a / 142-3p, 140-5p / 17, 142-3p / 148a, 142-3p / 15b, 142-3p / 17, 142-3p / 21, 142-3p / 221, 142-3p / 30b, 320 / 660, 106a / 660, 106a / 92a, 126 / 660, 140- At least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26 miRNAs of 5p / 197, 140-5p / 28-3p, 142-3p / 145, 142-3p / 197, 142-3p / 28-3p, 17 / 660, 17 / 92a, 197 / 660, 197 / 92a, 19b / 660 and 28-3p / 660, or at least 26 each of these, with at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 26.

[0032] The tag "Presence of aggressive lung tumors" In other embodiments, a method is provided for determining the presence of aggressive lung tumors in a subject. The method includes: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cutoff value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then a positive score is assigned to the ratio; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then a non-positive score is assigned to the ratio. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 14 miRNA pairs are assigned a positive score for their expression ratio, or (2) fewer than 14 miRNA pairs are assigned a positive score after comparing the expression ratios of 28 miRNA pairs. The miRNA pairs mentioned include 106a / 16, 106 / 660, 16 / 17, 16 / 320, 17 / 660, 197 / 30b, 197 / 30c, 320 / 451, 320 / 486-5p, or 320 / 660, or their inverses; and (e) The presence of invasive lung tumors is classified as follows: (i) positive if at least 14 miRNAs are assigned a positive score for the expression ratio; or (ii) negative if fewer than 14 miRNAs are assigned a positive score for the expression ratio.

[0033] In some embodiments, the miRNA pairs of the assay include ratios of 106a / 16, 106 / 660, 16 / 17, 16 / 320, 17 / 660, 197 / 30b, 197 / 30c, 320 / 451, 320 / 486-5p, and 320 / 660 or their inverse ratios, exceeding 2, 3, 4, 5, 6, 7, 8, 9, or each.

[0034] In other embodiments, the miRNA pair may further include ratios of 106a / 451, 106a / 486-5p, 126 / 451, 126 / 486-5p, 126 / 660, 140-5p / 197, 16 / 197, 17 / 451, 17 / 486-5p, 197 / 451, 197 / 486-5p, 197 / 660, 197 / 92a, 19b / 451, 19b / 486-5p, 19b / 660, 28-3p / 451 or 28-3p / 486-5p, or the inverse of those ratios.

[0035] In other embodiments, the miRNA pairs include ratios of 106a / 16, 106 / 660, 16 / 17, 16 / 320, 17 / 660, 197 / 30b, 197 / 30c, 320 / 451, 320 / 486-5p, 320 / 660, 106a / 451, 106a / 486-5p, 126 / 451, 126 / 486-5p, 126 / 660, 140-5p / 197, 16 / 197, 17 / 451, At least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, or each of the following 28 ratios: 17 / 486-5p, 197 / 451, 197 / 486-5p, 197 / 660, 28-3p / 451 or 28-3p / 486-5p or their inverse ratios. Those skilled in the art will understand that utilizing some, but not all, of these 28 ratios will be expected to provide a method for accurately identifying the presence of invasive tumors.

[0036] In other embodiments, the present invention provides the use of a plurality of primers or probes for detecting any one of 28 miRNA pairs in the preparation of a diagnostic reagent for determining the presence of invasive lung tumors in a subject, wherein said detection includes: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cutoff value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then a positive score is assigned to the ratio; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then a non-positive score is assigned to the ratio. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 14 miRNA pairs are assigned a positive score for their expression ratio, or (2) fewer than 14 miRNA pairs are assigned a positive score after comparing the expression ratios of 28 miRNA pairs. The miRNA pairs mentioned include 106a / 16, 106 / 660, 16 / 17, 16 / 320, 17 / 660, 197 / 30b, 197 / 30c, 320 / 451, 320 / 486-5p, or 320 / 660, or their inverses; and (e) The presence of invasive lung tumors is classified as follows: (i) positive if at least 14 miRNAs are assigned a positive score for the expression ratio; or (ii) negative if fewer than 14 miRNAs are assigned a positive score for the expression ratio.

[0037] In some implementations, multiple primers or probes can be used to detect miRNAs with expression ratios of 106a / 16, 106 / 660, 16 / 17, 16 / 320, 17 / 660, 197 / 30b, 197 / 30c, 320 / 451, 320 / 486-5p, and 320 / 660 or their inverse ratios, exceeding 2, 3, 4, 5, 6, 7, 8, 9, or each.

[0038] In other embodiments, multiple primers or probes may be further used to detect miRNA expression ratios of 106a / 451, 106a / 486-5p, 126 / 451, 126 / 486-5p, 126 / 660, 140-5p / 197, 16 / 197, 17 / 451, 17 / 486-5p, 197 / 451, 197 / 486-5p, 197 / 660, 197 / 92a, 19b / 451, 19b / 486-5p, 19b / 660, 28-3p / 451, or 28-3p / 486-5p, or their inverse ratios.

[0039] In other implementations, multiple primers or probes can be used to detect miRNA expression ratios of 106a / 16, 106 / 660, 16 / 17, 16 / 320, 17 / 660, 197 / 30b, 197 / 30c, 320 / 451, 320 / 486-5p, 320 / 660, 106a / 451, 106a / 486-5p, 126 / 451, 126 / 486-5p, 126 / 660, 140-5p / 197, 16 / 197, 1 7 / 451, 17 / 486-5p, 197 / 451, 197 / 486-5p, 197 / 660, 197 / 92a, 19b / 451, 19b / 486-5p, 19b / 660, 28-3p / 451 or 28-3p / 486-5p or their inverse ratios of at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27 or each of these 28 ratios. Those skilled in the art will understand that utilizing some, but not all, of these 28 ratios will be expected to provide a method for accurately identifying the presence of invasive tumors.

[0040] In some embodiments, at least one primer or probe among a plurality of primers and probes is capable of selectively binding to at least one miRNA of a miRNA pair in the sample. In some embodiments, the plurality of primers or probes includes those capable of selectively binding to miRNA pair expression ratios of 106a / 16, 106 / 660, 16 / 17, 16 / 320, 17 / 660, 197 / 30b, 197 / 30c, 320 / 451, 320 / 486-5p, 320 / 660, 106a / 451, 106a / 486-5p, 126 / 451, 126 / 486-5p, 126 / 660, 140-5p / 197, 16 / 197, 17 / 451. At least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27 miRNAs of 17 / 486-5p, 197 / 451, 197 / 486-5p, 197 / 660, 197 / 92a, 19b / 451, 19b / 486-5p, 19b / 660, 28-3p / 451 or 28-3p / 486-5p, or at least 18 miRNAs of 28-3p / 486-5p, or at least 27 miRNAs of 28-3p / 486-5p, or at least 17 / 486-5p, 197 / 451, 19b / 486-5p, 19b / 660, 28-3p / 451 or 28-3p / 486-5p, or at least 18-29, 20-20, 21-22, 23-24, 25-26 ...8-29

[0041] In other embodiments, the present invention provides a kit for determining the presence of invasive lung tumors in a subject. The kit includes a plurality of primers or probes for detecting any one of 28 miRNA pairs in the preparation of diagnostic reagents, and instructions for using said plurality of primers or probes to determine the presence of invasive lung tumors in a subject. The instructions include: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cutoff value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then a positive score is assigned to the ratio; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then a non-positive score is assigned to the ratio. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 14 miRNA pairs are assigned a positive score for their expression ratio, or (2) fewer than 14 miRNA pairs are assigned a positive score after comparing the expression ratios of 28 miRNA pairs. The miRNA pairs mentioned include 106a / 16, 106 / 660, 16 / 17, 16 / 320, 17 / 660, 197 / 30b, 197 / 30c, 320 / 451, 320 / 486-5p, or 320 / 660, or their inverses; and (e) The presence of invasive lung tumors is classified as follows: (i) positive if at least 14 miRNAs are assigned a positive score for the expression ratio; or (ii) negative if fewer than 14 miRNAs are assigned a positive score for the expression ratio.

[0042] In some implementations, multiple primers or probes can be used to detect miRNAs with expression ratios of 106a / 16, 106 / 660, 16 / 17, 16 / 320, 17 / 660, 197 / 30b, 197 / 30c, 320 / 451, 320 / 486-5p, and 320 / 660 or their inverse ratios, exceeding 2, 3, 4, 5, 6, 7, 8, 9, or each.

[0043] In other embodiments, multiple primers or probes may be further used to detect miRNA expression ratios of 106a / 451, 106a / 486-5p, 126 / 451, 126 / 486-5p, 126 / 660, 140-5p / 197, 16 / 197, 17 / 451, 17 / 486-5p, 197 / 451, 197 / 486-5p, 197 / 660, 197 / 92a, 19b / 451, 19b / 486-5p, 19b / 660, 28-3p / 451, or 28-3p / 486-5p, or their inverse ratios.

[0044] In other implementations, multiple primers or probes can be used to detect miRNA expression ratios of 106a / 16, 106 / 660, 16 / 17, 16 / 320, 17 / 660, 197 / 30b, 197 / 30c, 320 / 451, 320 / 486-5p, 320 / 660, 106a / 451, 106a / 486-5p, 126 / 451, 126 / 486-5p, 126 / 660, 140-5p / 197, 16 / 197, 1 7 / 451, 17 / 486-5p, 197 / 451, 197 / 486-5p, 197 / 660, 197 / 92a, 19b / 451, 19b / 486-5p, 19b / 660, 28-3p / 451 or 28-3p / 486-5p or their inverse, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27 or each of them.

[0045] In some embodiments, at least one primer or probe among a plurality of primers and probes provided in the kit is capable of selectively binding to at least one miRNA of a miRNA pair in the sample. In some embodiments, the plurality of primers or probes provided in the kit include those capable of selectively binding to miRNA pairs with expression ratios of 106a / 16, 106 / 660, 16 / 17, 16 / 320, 17 / 660, 197 / 30b, 197 / 30c, 320 / 451, 320 / 486-5p, 320 / 660, 106a / 451, 106a / 486-5p, 126 / 451, 126 / 486-5p, 126 / 660, 140-5p / 197, 16 / 197, 17 / At least one primer or probe of at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27 miRNAs of 451, 17 / 486-5p, 197 / 451, 197 / 486-5p, 197 / 660, 197 / 92a, 19b / 451, 19b / 486-5p, 19b / 660, 28-3p / 451 or 28-3p / 486-5p.

[0046] Together with the two diagnostic labels mentioned above ("Presence of Lung Cancer" and "Presence of Invasive Lung Cancer"), two prognostic labels are provided – a "Risk of Developing Lung Cancer" label and a "Risk of Developing Invasive Lung Cancer" label. These prognostic labels can be used to determine the risk at any time after sample acquisition, such as 3 months, 6 months, 12 months, 18 months, 24 months, 36 months, or any time outside or in between these time periods.

[0047] "Risk of developing lung cancer" label Therefore, in a further embodiment, a method for determining the risk of developing lung tumors in a subject is provided. The method includes: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cutoff value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then the ratio is assigned a positive score; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then the ratio is assigned a non-positive score. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 10 miRNA pairs are assigned a positive score for their expression ratio, or (2) fewer than 10 miRNA pairs are assigned a positive score after comparing the expression ratios of 27 miRNA pairs. The miRNA pairs mentioned include 133a / 92a, 15b / 21, 15b / 30b, 15b / 30c, 16 / 197, or 28-3p / 451, or their inverses; and (e) The risk of developing lung cancer is classified as follows: (i) if at least 10 miRNAs are assigned a positive score for their expression ratio, it is positive; or (ii) if fewer than 10 miRNAs are assigned a positive score for their expression ratio, it is negative.

[0048] In some embodiments, the miRNA pairs of the assay include more than 2, more than 3, more than 4, more than 5, or each of the ratios 133a / 92a, 15b / 21, 15b / 30b, 15b / 30c, 16 / 197, and 28-3p / 451 or their inverse ratios.

[0049] In other embodiments, the miRNA pairs may further include ratios of 101 / 140-3p, 106a / 451, 106a / 660, 106a / 92a, 126 / 660, 133a / 451, 133a / 660, 140-3p / 660, 142-3p / 15b, 15b / 451, 15b / 660, 17 / 451, 17 / 660, 17 / 92a, 197 / 19b, 197 / 451, 197 / 660, 197 / 92a, 19b / 660, 28-3p / 660, or 320 / 660, or the inverse of these ratios.

[0050] In other embodiments, the miRNA pairs include ratios of 133a / 92a, 15b / 21, 15b / 30b, 15b / 30c, 16 / 197, 28-3p / 451, 101 / 140-3p, 106a / 451, 106a / 660, 106a / 92a, 126 / 660, 133a / 451, 133a / 660, 140-3p / 660, 142-3p / 15b, 15b / 451, 15b / 660, At least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, or each of the following 27 ratios: 17 / 451, 17 / 660, 17 / 92a, 197 / 19b, 197 / 451, 197 / 660, 197 / 92a, 19b / 660, 28-3p / 660, or 320 / 660 or their inverse ratios. Those skilled in the art will understand that utilizing some, but not all, of these 27 ratios will be expected to provide an accurate assay for identifying the risk of developing lung tumors.

[0051] In some embodiments, the present invention provides the use of a plurality of primers or probes for detecting any one of 27 miRNA pairs in the preparation of a diagnostic or prognostic reagent for determining the risk of developing lung cancer in a subject, wherein said detection includes: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cutoff value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then a positive score is assigned to the ratio; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then a non-positive score is assigned to the ratio. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 10 miRNA pairs are assigned a positive score for their expression ratio, or (2) after comparing the expression ratios of 27 miRNA pairs, fewer than 10 miRNA pairs are assigned a positive score for their expression ratio. The miRNA pairs mentioned include 133a / 92a, 15b / 21, 15b / 30b, 15b / 30c, 16 / 197, or 28-3p / 451, or their inverses; and (e) The risk of developing lung cancer is classified as follows: (i) if at least 10 miRNAs are assigned a positive score for their expression ratio, it is positive; or (ii) if fewer than 10 miRNAs are assigned a positive score for their expression ratio, it is negative.

[0052] In some implementations, multiple primers or probes can be used to detect more than 2, more than 3, more than 4, more than 5, or each of the miRNAs expressing ratios of 133a / 92a, 15b / 21, 15b / 30b, 15b / 30c, 16 / 197, and 28-3p / 451 or their inverse ratios.

[0053] In other embodiments, multiple primers or probes may be further used to detect miRNA expression ratios of 101 / 140-3p, 106a / 451, 106a / 660, 106a / 92a, 126 / 660, 133a / 451, 133a / 660, 140-3p / 660, 142-3p / 15b, 15b / 451, 15b / 660, 17 / 451, 17 / 660, 17 / 92a, 197 / 19b, 197 / 451, 197 / 660, 197 / 92a, 19b / 660, 28-3p / 660, or 320 / 660, or their inverse ratios.

[0054] In other implementations, multiple primers or probes can be used to detect miRNAs at expression ratios of 133a / 92a, 15b / 21, 15b / 30b, 15b / 30c, 16 / 197, 28-3p / 451, 101 / 140-3p, 106a / 451, 106a / 660, 106a / 92a, 126 / 660, 133a / 451, 133a / 660, 140-3p / 660, 142-3p / 15b, 15b / 451, 15 b / 660, 17 / 451, 17 / 660, 17 / 92a, 197 / 19b, 197 / 451, 197 / 660, 197 / 92a, 19b / 660, 28-3p / 660 or 320 / 660 or their inverse ratios of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26 or each of these 27 ratios. Those skilled in the art will understand that utilizing some, but not all, of these 27 ratios will be expected to provide an accurate assay for identifying the risk of developing lung tumors.

[0055] In some embodiments, at least one primer or probe among a plurality of primers and probes is capable of selectively binding to at least one miRNA of a miRNA pair in the sample. In some embodiments, the plurality of primers or probes includes those capable of selectively binding to miRNA pair expression ratios of 133a / 92a, 15b / 21, 15b / 30b, 15b / 30c, 16 / 197, 28-3p / 451, 101 / 140-3p, 106a / 451, 106a / 660, 106a / 92a, 126 / 660, 133a / 451, 133a / 660, 140-3p / 660, 142-3p / 15b, 15b / 451, 15b / 660. At least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26 miRNAs of each of the following types: 17 / 451, 17 / 660, 17 / 92a, 197 / 19b, 197 / 451, 197 / 660, 197 / 92a, 19b / 660, 28-3p / 660, or 320 / 660.

[0056] In other embodiments, the present invention provides a kit for determining the risk of developing lung cancer in a subject. The kit includes a plurality of primers or probes for detecting any one of 27 miRNA pairs in the preparation of diagnostic or prognostic reagents, and instructions for using said plurality of primers or probes to determine the risk of developing lung cancer in a subject. The instructions include: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cutoff value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then a positive score is assigned to the ratio; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then a non-positive score is assigned to the ratio. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 10 miRNA pairs are assigned a positive score for their expression ratio, or (2) after comparing the expression ratios of 27 miRNA pairs, fewer than 10 miRNA pairs are assigned a positive score for their expression ratio. The miRNA pairs mentioned include 133a / 92a, 15b / 21, 15b / 30b, 15b / 30c, 16 / 197, or 28-3p / 451, or their inverses; and (e) The risk of developing lung cancer is classified as follows: (i) if at least 10 miRNAs are assigned a positive score for their expression ratio, it is positive; or (ii) if fewer than 10 miRNAs are assigned a positive score for their expression ratio, it is negative.

[0057] In some implementations, multiple primers or probes can be used to detect more than 2, more than 3, more than 4, more than 5, or each of the miRNAs expressing ratios of 133a / 92a, 15b / 21, 15b / 30b, 15b / 30c, 16 / 197, and 28-3p / 451 or their inverse ratios.

[0058] In other embodiments, multiple primers or probes may be further used to detect miRNA expression ratios of 101 / 140-3p, 106a / 451, 106a / 660, 106a / 92a, 126 / 660, 133a / 451, 133a / 660, 140-3p / 660, 142-3p / 15b, 15b / 451, 15b / 660, 17 / 451, 17 / 660, 17 / 92a, 197 / 19b, 197 / 451, 197 / 660, 197 / 92a, 19b / 660, 28-3p / 660, or 320 / 660, or their inverse ratios.

[0059] In other implementations, multiple primers or probes can be used to detect miRNAs at expression ratios of 133a / 92a, 15b / 21, 15b / 30b, 15b / 30c, 16 / 197, 28-3p / 451, 101 / 140-3p, 106a / 451, 106a / 660, 106a / 92a, 126 / 660, 133a / 451, 133a / 660, 140-3p / 660, 142-3p / 15b, 15b / 451, 15 b / 660, 17 / 451, 17 / 660, 17 / 92a, 197 / 19b, 197 / 451, 197 / 660, 197 / 92a, 19b / 660, 28-3p / 660 or 320 / 660 or their inverse ratios of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26 or each.

[0060] In some embodiments, at least one primer or probe among a plurality of primers and probes provided in the kit is capable of selectively binding to at least one miRNA of a miRNA pair in the sample. In some embodiments, the plurality of primers or probes provided in the kit include those capable of selectively binding to miRNA pairs with expression ratios of 133a / 92a, 15b / 21, 15b / 30b, 15b / 30c, 16 / 197, 28-3p / 451, 101 / 140-3p, 106a / 451, 106a / 660, 106a / 92a, 126 / 660, 133a / 451, 133a / 660, 140-3p / 660, 142-3p / 15b, 15b / 451, 15b / At least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26 miRNAs of 660, 17 / 451, 17 / 660, 17 / 92a, 197 / 19b, 197 / 451, 197 / 660, 197 / 92a, 19b / 660, 28-3p / 660 or 320 / 660, or at least 19 / 20 / 26, or each of them, with at least one primer or probe.

[0061] "Risk of developing aggressive lung cancer" tag This article also provides a method for determining the risk of developing aggressive lung tumors in subjects. The method includes: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cutoff value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then a positive score is assigned to the ratio; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then a non-positive score is assigned to the ratio. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 14 miRNA pairs are assigned a positive score for their expression ratio, or (2) fewer than 14 miRNA pairs are assigned a positive score after comparing the expression ratios of 28 miRNA pairs. The miRNA pairs mentioned include 106a / 142-3p, 126 / 142-3p, 126 / 21, 126 / 92a, 142-3p / 17, 142-3p / 197, 142-3p / 28-3p, 197 / 19b, 197 / 660 or 28-3p / 660, or their inverses; and (e) Risk classification for the occurrence of invasive lung tumors as follows: (i) positive if at least 14 miRNAs are assigned a positive score for the expression ratio; or (ii) negative if fewer than 14 miRNAs are assigned a positive score for the expression ratio.

[0062] In some embodiments, the miRNA pairs of the assay include ratios of 106a / 142-3p, 126 / 142-3p, 126 / 21, 126 / 92a, 142-3p / 17, 142-3p / 197, 142-3p / 28-3p, 197 / 19b, 197 / 660 and 28-3p / 660 or their inverse ratios, exceeding 2, exceeding 3, exceeding 4, exceeding 5, exceeding 6, exceeding 7, exceeding 8, exceeding 9 or each.

[0063] In other embodiments, the miRNA pairs may further include ratios of 106a / 451, 126 / 451, 145 / 197, 17 / 451, 197 / 21, 197 / 30b, 197 / 30c, 197 / 451, 197 / 92a, 19b / 451, 21 / 221, 21 / 28-3p, 28-3p / 30b, 28-3p / 30c, 28-3p / 451, 28-3p / 92a, 320 / 451 or 320 / 92a, or the inverse of those ratios.

[0064] In other embodiments, the miRNA pairs include ratios of 106a / 142-3p, 126 / 142-3p, 126 / 21, 126 / 92a, 142-3p / 17, 142-3p / 197, 142-3p / 28-3p, 197 / 19b, 197 / 660, 28-3p / 660, 106a / 451, 126 / 451, 145 / 197, 17 / 451, 197 / 21, 197 / 30b, 197 / 30c, 197 / 451, 197 / 92a, 19b / 451, 21 / 221, 21 / 28-3p, 28-3p / 30b, 28-3p / 30c, 28-3p / 451, 28-3p / 92a, 320 / 451 and 320 / 92a, or their inverse ratios of at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, or each of these 28 ratios. Those skilled in the art will understand that utilizing some, but not all, of these 28 ratios will be expected to provide an accurate assay for identifying the risk of developing invasive tumors.

[0065] In some embodiments, the present invention provides the use of a plurality of primers or probes for detecting any one of 28 miRNA pairs in the preparation of a diagnostic or prognostic reagent for determining the risk of developing aggressive lung tumors in a subject, wherein said detection includes: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cutoff value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then a positive score is assigned to the ratio; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then a non-positive score is assigned to the ratio. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 14 miRNA pairs are assigned a positive score for their expression ratio, or (2) fewer than 14 miRNA pairs are assigned a positive score after comparing the expression ratios of 28 miRNA pairs. The miRNA pairs mentioned include 106a / 142-3p, 126 / 142-3p, 126 / 21, 126 / 92a, 142-3p / 17, 142-3p / 197, 142-3p / 28-3p, 197 / 19b, 197 / 660 or 28-3p / 660, or their inverses; and (e) Risk classification for the occurrence of invasive lung tumors as follows: (i) positive if at least 14 miRNAs are assigned a positive score for the expression ratio; or (ii) negative if fewer than 14 miRNAs are assigned a positive score for the expression ratio.

[0066] In some implementations, multiple primers or probes can be used to detect miRNAs expressing more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, or each of the following ratios: 106a / 142-3p, 126 / 142-3p, 126 / 21, 126 / 92a, 142-3p / 17, 142-3p / 197, 142-3p / 28-3p, 197 / 19b, 197 / 660, and 28-3p / 660, or their inverse ratios.

[0067] In other embodiments, multiple primers or probes may be further used to detect miRNA expression ratios of 106a / 451, 126 / 451, 145 / 197, 17 / 451, 197 / 21, 197 / 30b, 197 / 30c, 197 / 451, 197 / 92a, 19b / 451, 21 / 221, 21 / 28-3p, 28-3p / 30b, 28-3p / 30c, 28-3p / 451, 28-3p / 92a, 320 / 451, or 320 / 92a, or their inverse ratios.

[0068] In other embodiments, multiple primers or probes can be used to detect miRNA expression ratios of 106a / 142-3p, 126 / 142-3p, 126 / 21, 126 / 92a, 142-3p / 17, 142-3p / 197, 142-3p / 28-3p, 197 / 19b, 197 / 660, 28-3p / 660, 106a / 451, 126 / 451, 145 / 197, 17 / 451, 197 / 21, 197 / 30b, 19 At least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, or each of the following 28 ratios: 7 / 30c, 197 / 451, 197 / 92a, 19b / 451, 21 / 28-3p, 28-3p / 30b, 28-3p / 30c, 28-3p / 451, 28-3p / 92a, 320 / 451 and 320 / 92a, or their inverse ratios. Those skilled in the art will understand that utilizing some, but not all, of these 28 ratios will be expected to provide an accurate assay for identifying the risk of developing aggressive lung tumors.

[0069] In other embodiments, the present invention provides a kit for determining the risk of developing aggressive lung cancer in a subject. The kit includes a plurality of primers or probes for detecting any one of 28 miRNA pairs in the preparation of diagnostic or prognostic reagents, and instructions for using said plurality of primers or probes to determine the risk of developing aggressive lung cancer in a subject. The instructions include: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cutoff value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then a positive score is assigned to the ratio; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then a non-positive score is assigned to the ratio. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 14 miRNA pairs are assigned a positive score for their expression ratio, or (2) fewer than 14 miRNA pairs are assigned a positive score after comparing the expression ratios of 28 miRNA pairs. The miRNA pairs mentioned include 106a / 142-3p, 126 / 142-3p, 126 / 21, 126 / 92a, 142-3p / 17, 142-3p / 197, 142-3p / 28-3p, 197 / 19b, 197 / 660 or 28-3p / 660, or their inverses; and (e) Risk classification for the occurrence of invasive lung tumors as follows: (i) positive if at least 14 miRNAs are assigned a positive score for the expression ratio; or (ii) negative if fewer than 14 miRNAs are assigned a positive score for the expression ratio.

[0070] In some implementations, multiple primers or probes can be used to detect miRNAs expressing more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, or each of the following ratios: 106a / 142-3p, 126 / 142-3p, 126 / 21, 126 / 92a, 142-3p / 17, 142-3p / 197, 142-3p / 28-3p, 197 / 19b, 197 / 660, and 28-3p / 660, or their inverse ratios.

[0071] In other embodiments, multiple primers or probes may be further used to detect miRNA expression ratios of 106a / 451, 126 / 451, 145 / 197, 17 / 451, 197 / 21, 197 / 30b, 197 / 30c, 197 / 451, 197 / 92a, 19b / 451, 21 / 221, 21 / 28-3p, 28-3p / 30b, 28-3p / 30c, 28-3p / 451, 28-3p / 92a, 320 / 451, or 320 / 92a, or their inverse ratios.

[0072] In other embodiments, multiple primers or probes can be used to detect miRNA expression ratios of 106a / 142-3p, 126 / 142-3p, 126 / 21, 126 / 92a, 142-3p / 17, 142-3p / 197, 142-3p / 28-3p, 197 / 19b, 197 / 660, 28-3p / 660, 106a / 451, 126 / 451, 145 / 197, 17 / 451, 197 / 21, 197 / 30b, 19 7 / 30c, 197 / 451, 197 / 92a, 19b / 451, 21 / 221, 21 / 28-3p, 28-3p / 30b, 28-3p / 30c, 28-3p / 451, 28-3p / 92a, 320 / 451 and 320 / 92a or their inverses of at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27 or each.

[0073] In some embodiments, at least one primer or probe among a plurality of primers and probes provided in the kit is capable of selectively binding to at least one miRNA of a miRNA pair in the sample. In some embodiments, the plurality of primers or probes provided in the kit include those capable of selectively binding to miRNA pairs with expression ratios of 106a / 142-3p, 126 / 142-3p, 126 / 21, 126 / 92a, 142-3p / 17, 142-3p / 197, 142-3p / 28-3p, 197 / 19b, 197 / 660, 28-3p / 660, 106a / 451, 126 / 451, 145 / 197, 17 / 451, 197 / 21, 197 / 30b, 197 / At least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27 miRNAs of 30c, 197 / 451, 197 / 92a, 19b / 451, 21 / 221, 21 / 28-3p, 28-3p / 30b, 28-3p / 30c, 28-3p / 451, 28-3p / 92a, 320 / 451 and 320 / 92a, or at least 27 of each.

[0074] "Risk of developing or having lung cancer" category The four methods described above can be combined with methods for predicting a subject's risk of developing or having lung cancer. This method includes: (a) Determine the presence of the aforementioned lung tumor label; (b) Determine the presence of the aforementioned invasive lung tumor label; (c) Determine the risk labels for developing lung cancer as described above; (d) Identify the risk labels for the development of aggressive lung tumors mentioned above; (e) If none of the labels in steps (a)-(d) are classified as positive, the subject is classified as low risk of developing or having lung cancer. (f) If at least the lung tumor presence label in step (a) is positive or the risk label for lung tumor development in step (c) is positive; and both the presence label for invasive lung tumor in step (b) and the risk label for invasive lung tumor development in step (d) are negative, then the subject is classified as having an intermediate risk of developing or having lung tumor; and (g) If the presence of invasive lung tumor in at least step (b) is labeled positive or the risk of developing invasive lung tumor in step (d) is labeled positive, the subject is classified as being at high risk of developing or having lung tumor.

[0075] The four uses described above can be combined in conjunction with the use of predicting the risk of a subject developing or having lung cancer. This use includes: (a) Use multiple primers or probes for detecting any one of the 27 miRNA pairs in the preparation of diagnostic reagents for determining the presence of the aforementioned lung tumors. (b) Use multiple primers or probes for detecting any one of the 28 miRNA pairs in the preparation of diagnostic reagents for determining the presence of the above-mentioned invasive lung tumor label. (c) Use multiple primers or probes for detecting any one of the 27 miRNA pairs in the preparation of diagnostic or prognostic reagents for determining the risk labels for the occurrence of lung tumors described above. (d) Use multiple primers or probes for detecting any one of the 28 miRNA pairs in the preparation of diagnostic or prognostic reagents for determining the risk labels for the occurrence of the above-mentioned invasive lung tumors. (e) If none of the labels in steps (a)-(d) are classified as positive, the subject is classified as low risk of developing or having lung cancer. (f) If at least the lung tumor presence label in step (a) is positive or the risk label for lung tumor development in step (c) is positive; and both the presence label for invasive lung tumor in step (b) and the risk label for invasive lung tumor development in step (d) are negative, then the subject is classified as having an intermediate risk of developing or having lung tumor; and (g) If the presence of invasive lung tumor in at least step (b) is labeled positive or the risk of developing invasive lung tumor in step (d) is labeled positive, the subject is classified as being at high risk of developing or having lung tumor.

[0076] A. definition The term "lung tumor" as used in this article can refer to benign or malignant lung tumors. Lung tumors can be associated with one or more lung conditions and can take the form of, for example, lung nodules or lung masses.

[0077] As used herein, the term "pulmonary condition" refers to a disease, event, or change in the health status of the lungs, including, for example, lung cancer and various non-cancerous conditions. Examples of non-cancerous pulmonary conditions include chronic obstructive pulmonary disease (COPD), benign lung tumors or cellular masses (e.g., hamartoma, fibroma, neurofibroma), granulomas, sarcoidosis, and infections caused by bacterial (e.g., tuberculosis) or fungal (e.g., histoplasmosis) pathogens. In some embodiments, a pulmonary condition may be associated with the presence of radiographic lung nodules.

[0078] The methods described above are not narrowly limited to diagnosing and / or predicting any specific lung cancer. As used herein, "lung cancer" preferably refers to cancer of the lungs, but may include any disease or other condition of the respiratory system in humans or other mammals. Respiratory system tumors include, for example, small cell carcinoma or small cell lung cancer (SCLC), non-small cell carcinoma or non-small cell lung cancer (NSCLC), squamous cell carcinoma (SCC), adenocarcinoma, bronchoalveolar carcinoma (BAC), mixed lung cancer, malignant pleural mesothelioma, undifferentiated large cell carcinoma, giant cell carcinoma, synchronous tumor, large cell neuroendocrine carcinoma, adenosquamous carcinoma, undifferentiated carcinoma; and small cell carcinoma, including oat cell carcinoma, mixed small cell / large cell carcinoma (LC), large cell carcinoma, and combined small cell carcinoma; as well as adenoid cystic carcinoma, hamartoma, myxoepidermoid tumor, typical carcinoid lung tumor, atypical carcinoid lung tumor, peripheral carcinoid lung tumor, central carcinoid lung tumor, pleural mesothelioma, undifferentiated lung cancer, and secondary cancers originating outside the lung, such as those metastasizing to the lung from other parts of the body, or any other lung cancer now known or later discovered. Lung cancer can be at any stage or grade.

[0079] As used herein, the term "biological sample from a subject" refers to a sample from any subject, including smokers, non-smokers, former smokers, cancer patients (e.g., for diagnosis and / or monitoring of cancer recurrence after treatment), and former cancer patients. In a specific implementation, the biological sample is derived from a smoker who, at the time of sample collection, does not have a lung tumor if imaging diagnostic methods are used; particularly, smokers who, if spiral CT scans are performed, do not have nodules larger than 5 mm.

[0080] As used herein, the term "biological sample" can refer to any tissue that provides an accurate measurement of a subject's miRNA profile. In some embodiments, the biological sample is a biological fluid. These embodiments are not narrowly limited to any particular body fluid, as miRNAs are present in virtually all body fluids (Weber et al., The microRNA spectrum in 12 body fluids, Clin Chem 56:1733-1741, 2010; De Guire et al., Clin Biochem 46:846-860, 2013; see also Rodriguez-Dorantes et al., Meth Mol Biol 1165:81-87, 2014). Examples of useful body fluids include peripheral blood, serum, plasma, ascites, urine, sputum, saliva, bronchoalveolar lavage fluid, cystic fluid, pleural fluid, peritoneal fluid, lymph, pus, lavage fluid from the sinoatrial ventricles, bronchopulmonary aspirate, and bone marrow aspirate. A technician can determine whether any particular body fluid is suitable for any particular application without excessive experimentation. In some implementations, the body fluid is plasma or serum. In some implementations, the biological sample is a tissue sample.

[0081] The singular forms “a,” “one,” and “the” used in this document are intended to include the plural forms as well, unless the context clearly indicates otherwise. Additionally, the use of “or” is intended to include “and / or”, unless the context clearly indicates otherwise.

[0082] As used herein, “individual,” “subject,” “patient,” or “subject in need” refers to an individual at risk of developing a tumor or invasive tumor, or an individual who may have, is likely to have, or has been diagnosed with a tumor or invasive tumor. These terms are used interchangeably. Preferably, the individual is a mammal. A mammal can be any mammal, such as a human, primate, bird, mouse, rat, poultry, dog, cat, cow, horse, goat, camel, sheep, or pig. Preferably, the mammal is a human.

[0083] The term "primer" or "probe" in the multiple primers or probes used herein refers to nucleic acid molecules. Primers or probes can be DNA, RNA, or cDNA. Primers or probes can be naturally occurring or synthetic. Primers or probes can be at least 5, 10, 15, 20, 25, 50, 75, or 100 nucleic acid lengths. Preferably, at least one primer or probe among the multiple primers and probes is capable of selectively binding at least one miRNA of a miRNA pair in the sample. Preferably, the multiple primers or probes comprise at least one primer or probe capable of selectively binding at least one miRNA of at least one miRNA of all miRNA pair expression ratios in the sample. The term "selective binding" means that the primer or probe has a high affinity for binding at least one miRNA of a miRNA pair in the sample, or a higher affinity for binding at least one miRNA of a miRNA pair compared to any other miRNA in the sample. The term "primer" means a nucleic acid molecule that can be used as a starting point for DNA synthesis. That is, DNA polymerase can replicate from the 3' end of the primer.

[0084] Using any of the methods described above, the term "miRNA pair expression ratio" as used herein refers to the ratio of the expression levels of two specific miRNAs. It should be understood that the ratio of any miRNA pair specified in any of the methods described herein can be calculated as the inverse of the specified ratio. As a non-limiting example, when the miRNA pairs of a specific method are “106a / 140-5p, 106a / 142-3p, 126 / 140-5p, 126 / 142-3p, 133a / 142-3p, 140-5p / 17, 142-3p / 148a, 142-3p / 15b, 142-3p / 17, 142-3p / 21, 142-3p / 221, 142-3p / 30b and 320 / 660, or their inverse ratios”, the expression ratios of any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of these miRNA pairs can be calculated as inverse ratios, with the cutoff values ​​adjusted accordingly.

[0085] The term "cutoff value" as used in this article can be negative or positive. For some ratios, "exceeding the cutoff value" expresses a ratio that is quantitatively less than the cutoff value. For other ratios, "exceeding the cutoff value" expresses a ratio that is quantitatively greater than the cutoff value. See, for example, Table 9, where ">" indicates that numbers quantitatively greater than the cutoff value represent ratios exceeding the cutoff value, and "<" indicates that numbers quantitatively less than the cutoff value represent ratios exceeding the cutoff value.

[0086] Under the definition of “exceeding the cutoff value”, (i) for ratio 133a / 92a, the value of -4.50 exceeds the cutoff value of -4.18 listed in Table 9, (ii) for ratio 17 / 451, the value of 3.50 exceeds the cutoff value of 3.33 listed in Table 9, (iii) for ratio 142-3p / 17, the value of -1.80 exceeds the cutoff value of -1.95 listed in Table 9, and (iv) for ratio 16 / 197, the value of 4.50 exceeds the cutoff value of 5.00 listed in Table 9.

[0087] In any of the methods described herein, the cutoff value for the expression ratio of any particular miRNA pair is determined from the average ratio of multiple corresponding miRNA pairs from multiple control samples. The term "multiple control samples" as used herein can be from any defined population, such as healthy subjects with no history of cancer, healthy subjects with no history of lung cancer, subjects with lung cancer, subjects who have never smoked, subjects who have smoked but no longer smoke, etc. In a preferred embodiment, the multiple control samples are from healthy subjects, and the cutoff value is the average ratio of multiple corresponding miRNA pairs from the multiple control samples. In some of these embodiments, the term "mean" as used herein can be the mean or median. In other embodiments, the cutoff value is the mean plus / minus one standard deviation or any smaller or larger percentage of the standard deviation, such as plus / minus one standard deviation. 1 / 2 standard deviations, + / - 3 / 4 standard deviations, + / -1.5 standard deviations, + / -2 standard deviations, or any larger, smaller, or intermediate percentage of the standard deviation.

[0088] For any number of control samples from any control population, the cutoff value can be determined using known methods without excessive experimentation, such as by using the training set described in the examples. The cutoff value is established to obtain the desired sensitivity (SE), specificity (SP), positive predictive value (PPV), and negative predictive value (NPV).

[0089] To evaluate these diagnostic methods (presence of tumor; presence of invasive tumor) and prognostic methods (risk of developing tumor; risk of developing invasive tumor), categorical analysis of test results in a clinical context was performed. The diagnostic or prognostic value of a method can be defined by its SE, SP, PPV, and NPV. Any test method will produce true positives (TP), false negatives (FN), false positives (FP), and true negatives (TN). The test's "sensitivity" is the percentage of all patients with a positive test who have disease or a response, or (TP / TP+FN) × 100%. The test's "specificity" is the percentage of all patients with a negative test who have no disease or no response, or (TN / FP+TN) × 100%. The test's "predictive value" or "PV" is a measure (%) of the number of times a value (positive or negative) is the true value, i.e., the percentage of all positive tests that are true positives is the positive predictive value (PV+) or (TP / TP+FP) × 100%. The negative predictive value (PV) is the percentage of patients with a negative test who do not respond, or (TN / FN+TN)×100%. Another measure, the test's accuracy or validity, is the percentage of times the test gives a correct answer compared to the total number of tests, or (TP+TN / TP+TN+FP+FN)×100%. The error rate is calculated from those patients predicted to respond but not to, and those predicted not to respond but to, or (FP+FN / TP+TN+FP+FN)×100%. Overall test specificity is a measure of the accuracy of sensitivity, and the test's specificity does not change with the overall probability of disease in the population, while the predictive value can change. PV varies with the physician's clinical assessment of the presence or absence of disease or the presence or absence of a clinical response in a given patient.

[0090] For any given test, TP, FN, FP, and TN can be determined and adjusted by a technician without excessive experimentation, for example, by adjusting the cutoff value, adjusting the statistical significance (e.g., P-value) used to make prognostic or diagnostic decisions, and adjusting the accuracy of the test procedure.

[0091] B. method The expression level of miRNAs can be determined by any method known in the art. In some embodiments, the expression level is determined by preparing cDNA copies of each miRNA in each miRNA pair using reverse transcriptase-polymerase chain reaction (RT-PCR). In various embodiments, the expression ratio is further determined using real-time PCR. In specific embodiments of these methods, the expression ratio is further determined using a TaqMan probe. However, the methods are not limited to these embodiments, and any suitable method now known or hereafter discovered for determining miRNA expression levels can be used.

[0092] In some embodiments of the method of the present invention, miRNA is amplified before measurement. In other embodiments, the levels of these nucleic acids are measured during the amplification process. In still other methods, nucleic acids are not amplified before measurement.

[0093] Numerous methods exist for amplifying miRNA nucleic acid sequences, such as mature miRNAs, precursor miRNAs, and primary miRNAs. Suitable nucleic acid polymerization and amplification techniques include reverse transcription (RT), polymerase chain reaction (PCR), real-time PCR (quantitative PCR (q-PCR)), sequence-based amplification (NASBA), ligase chain reaction, multiplexable probe amplification, third wave technology, rolling circle amplification, in vitro transcription (IVT), strand displacement amplification, transcription-mediated amplification (TMA), RNA (Eberwine) amplification, and other methods known to those skilled in the art. In some embodiments, more than one amplification method is used, such as reverse transcription, followed by real-time quantitative PCR (qRT-PCR). In PCR and q-PCR methods, for example, a set of primers is used for each target sequence. In some embodiments, the primer length depends on many factors, including but not limited to the primers, the required hybridization temperature between the target nucleic acid sequences, and the complexity of the different target nucleic acid sequences to be amplified. In some embodiments, the primers are about 15 to about 35 nucleotides long. In other embodiments, the primers are equal to or less than 15, 20, 25, 30, or 35 nucleotides long. In another implementation, the primer is at least 35 nucleotides long.

[0094] In a further aspect, the forward primer may contain at least one sequence annealed to the miRNA, and alternatively may contain an additional 5′ non-complementary region. On the other hand, the reverse primer may be designed to anneal to the complement of the reverse-transcribed miRNA. The reverse primer may be independent of the miRNA sequence, and multiple miRNA biomarkers can be amplified using the same reverse primer. Alternatively, the reverse primer may be specific to the miRNA biomarker.

[0095] qRT-PCR reactions can be further combined with reverse transcription reactions by including both reverse transcriptase and a thermostable DNA polymerase based on DNA. When using two polymerases, a “hot-start” approach can be used to maximize assay performance (US Patent Nos. 5,411,876 and 5,985,619). For example, components of the reverse transcriptase reaction and PCR reaction can be isolated using one or more thermal activation methods or chemical alterations to improve polymerization efficiency (US Patent Nos. 5,550,044, 5,413,924, and 6,403,341).

[0096] In some implementations, tags, dyes, or labeled probes and / or primers are used to detect amplified or unamplified miRNAs. Those skilled in the art will recognize which detection method is 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, depending on the sensitivity of the detection method and the abundance of the target. Those skilled in the art will recognize that miRNA amplification is a preferred detection method.

[0097] Probes or primers may include Watson-Crick bases or modified bases. Modified bases include, but are not limited to, AEGIS bases (from Eragen Biosciences), which have been described, for example, in U.S. Patent Nos. 5,432,272, 5,965,364, and 6,001,983. In some respects, the bases are linked by native phosphodiester bonds or different chemical bonds. Different chemical bonds include, but are not limited to, peptide bonds or locked nucleic acid (LNA) bonds, which have been described, for example, in U.S. Patent No. 7,060,809.

[0098] In a further aspect, oligonucleotide probes or primers present in the amplification reaction are suitable for monitoring the amount of amplification products generated over time. In some respects, probes with different single-stranded versus double-stranded characteristics are used for nucleic acid detection. Probes include, but are not limited to, 5′-exonuclease assays (e.g., TaqMan). TM ) probes (see U.S. Patent No. 5,538,848), stem-loop molecular beacons (see, for example, U.S. Patent Nos. 6,103,476 and 5,925,517), stemless or linear beacons (see, for example, WO9921881, U.S. Patent Nos. 6,485,901 and 6,649,349), peptide nucleic acid (PNA) molecular beacons (see, for example, U.S. Patent Nos. 6,355,421 and 6,593,091), linear PNA beacons (see, for example, U.S. Patent No. 6,329,144), non-FRET probes (see, for example, U.S. Patent No. 6,150,097), Sunrise TM / AmplifluorB TM Probes (see, for example, U.S. Patent No. 6,548,250), stem-loop, and double-stranded Scorpion TM Probes (see, for example, U.S. Patent No. 6,589,743), convex ring probes (see, for example, U.S. Patent No. 6,590,091), pseudo-junction probes (see, for example, U.S. Patent No. 6,548,250), cyclicons (see, for example, U.S. Patent No. 6,383,752), MGB Eclipse TMProbes (Epoch Biosciences), hairpin probes (see, for example, U.S. Patent No. 6,596,490), PNA illuminating probes, anti-primer quenching probes (Li et al., Clin. Chem. 53:624-633 (2006)), self-assembled nanoparticle probes, and ferrocene-modified probes described, for example, U.S. Patent No. 6,485,901.

[0099] In some embodiments, one or more primers in the amplification reaction include tags. In other embodiments, different probes or primers include detectable tags that distinguish them from each other. In some embodiments, nucleic acids, such as probes or primers, can be tagged with two or more distinguishable tags.

[0100] In some implementations, the concentration of miRNA is measured using a microarray or another vector.

[0101] A “microarray” is a linear, two-dimensional, or three-dimensional (and solid-phase) array of discrete regions, each having a defined area formed on the surface of a solid support, such as, but not limited to, glass, plastic, or synthetic membranes. The density of the discrete regions on the microarray is determined by the total number of detectable polynucleotides immobilized on the surface of a single solid-phase support, for example, at least about 50 / cm³. 2 At least approximately 100 / cm 2 Or at least about 500 / cm 2 Up to approximately 1,000 / cm 2 Or higher. The array may contain fewer than approximately 500, approximately 1000, approximately 1500, approximately 2000, approximately 2500, approximately 3000 or more fixed polynucleotides. The DNA microarrays used in this paper are arrays of oligonucleotide or polynucleotide probes placed on a chip or other surface for hybridization to polynucleotides amplified or cloned from the sample. Because the positions of specific probe sets on the array are known, the identity of sample polynucleotides can be determined based on their binding to specific positions on the microarray.

[0102] As an alternative to using microarrays, arrays of any size on a vector can be used to implement the present disclosure, including arrangements of one or more sites in two-dimensional or three-dimensional arrays, to detect miRNA expression.

[0103] In some respects, the tag is attached to one or more probes, and the tag has one or more of the following properties: (i) providing a detectable signal; (ii) interacting with a second tag to alter the detectable signal provided by the second tag, such as FRET (fluorescence resonance energy transfer); (iii) stabilizing hybridization, such as double-strand formation; and (iv) providing a member of the binding complex or affinity group, such as affinity, antibody-antigen, ionic complex, hapten-ligand (e.g., biotin-avidin). In other respects, the use of the tag can be achieved using any of a variety of known techniques, employing known tags, linkages, linking groups, reagents, reaction conditions, and analytical and purification methods.

[0104] miRNAs can be detected by direct or indirect methods. In direct detection methods, one or more miRNAs are detected via a detectable tag linked to a nucleic acid molecule. In such methods, the miRNA can be labeled before binding to a probe. Therefore, binding is detected by screening for labeled miRNAs that bind to the probe. The probe may optionally be linked to a bead in the reaction volume.

[0105] In some embodiments, nucleic acids are detected by direct binding to labeled probes, and the probes are subsequently detected. In one embodiment of the invention, nucleic acids, such as amplified miRNAs, are detected using FIexMAP Microspheres (Luminex) conjugated with probes to capture the desired nucleic acid. Some methods may include, for example, detection with fluorescently labeled polynucleotide probes or branched DNA (bDNA) detection.

[0106] In other embodiments, nucleic acids are detected by indirect detection methods. For example, a biotinylated probe can be combined with a streptavidin-conjugated dye to detect the bound nucleic acid. The streptavidin molecule binds to a biotin tag on an amplified miRNA, and the bound miRNA is detected by detecting a dye molecule linked to the streptavidin molecule. In one embodiment, the streptavidin-conjugated dye molecule includes... Streptomycin R-phycoerythrin (PROzyme). Other conjugated dye molecules are known to those skilled in the art.

[0107] Labels include, but are not limited to, light-emitting, light-scattering, and light-absorbing compounds that generate or quench fluorescence, chemiluminescence, or bioluminescence signals (see, for example, Kricka, L., Nonisotopic DNA Probe Techniques, Academic Press, San Diego (1992) and Garman A., Non-Radioactive Labeling, Academic Press (1997)). Fluorescent reporter dyes used as tags include, but are not limited to, fluorescein (see, for example, U.S. Patent Nos. 5,188,934, 6,008,379, and 6,020,481), rhodamine (see, for example, U.S. Patent Nos. 5,366,860, 5,847,162, 5,936,087, 6,051,719, and 6,191,278), and benzophenoxazine (see, for example, U.S. Patent No. 6,140,500); energy-transfer fluorescent dyes, including donor and acceptor pairs (see, for example, U.S. Patent Nos. 5,863,727; 5,800,996; and 5,945,526), ​​and anthocyanins (see, for example, WO 9745539), lissamine, phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham), Alexa 350, and Alexa. 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA, tetramethylrhodamine and / or Texas Red, and any other fluorescent fractions capable of producing a detectable signal. Examples of fluorescein dyes include, but are not limited to, 6-carboxyfluorescein; 2′,4′,1,4-tetrachlorofluorescein; and 2′,4′,5′,7′,1,4-hexachlorofluorescein. In some respects, the fluorescent tag is selected from SYBR-Green, 6-carboxyfluorescein (“FAM”), TET, ROX, VICTM, and JOE. For example, in some embodiments, the tag is a different fluorophore (e.g., 4-different-color fluorophores) capable of emitting light at different spectrally resolvable wavelengths; certain probes with such tags are known in the art and described above and in U.S. Patent No. 6,140,054.In some implementations, dual-labeled fluorescent probes comprising both a reporter fluorophore and a quencher fluorophore are used. It should be understood that fluorophore pairs with different emission spectra are selected so that they can be easily distinguished.

[0108] On the other hand, tags are hybridization stabilizing parts that are used to enhance, stabilize, or influence hybridization of duplexes, such as intercalating agents and intercalating dyes (including, but not limited to, ethidium bromide and SYBR-Green), minor groove binders, and cross-linking functional groups (see, for example, Blackburn et al., ed., “DNA and RNA Structure,” in Nucleic Acids in Chemistry and Biology (1996)).

[0109] In other respects, hybridization- and / or ligation-dependent methods for quantifying miRNAs can be used, including oligonucleotide ligation (OLA) methods and methods that allow distinguishable probes hybridized to the target nucleic acid sequence to be separated from unbound probes. As an example, the HARP-like probe disclosed in U.S. Publication No. 2006 / 0078894 can be used to measure the number of miRNAs.

[0110] In another embodiment of the method, probe ligation reactions can be used to quantify miRNAs. In multiplexed ligation-dependent probe amplification (MLPA) technology (Schouten et al., Nucleic Acids Research 30:e57 (2002)), probe pairs that hybridize directly adjacent to each other on the target nucleic acid ligate each other only in the presence of the target nucleic acid. In some respects, MLPA probes have side-linked PCR primer binding sites. MLPA probes can be amplified only if they are already ligated, thus allowing the detection and quantification of miRNA biomarkers.

[0111] C. Hemolysis detection Hemolysis of erythrocytes and platelets releases miRNAs, which can interfere with the methods described above (Kirschner et al., PLoS One 6:e24145, 2011; Pritchard et al., Cancer Prev Res (Phila) 5:492-497, 2012). Therefore, in some embodiments of the methods described above, hemolysis in the biological sample is measured, and if hemolysis is detected, the sample is not subjected to the methods described.

[0112] Hemolysis can be determined in subject samples by any method known in the art. In some embodiments, hemolysis is determined by spectrophotometry, for example by measuring absorbance at different wavelengths (414 nm, 541 nm, 576 nm) to identify the presence and amount of free hemoglobin in the sample (Kirschner et al., 2011).

[0113] In other embodiments, hemolysis is determined by analyzing the incrementally regulated expression levels of hemolysis-associated miRNAs in samples where hemolysis has occurred. In some of these embodiments, any one of the miRNAs miR-451, miR-486-5p, miR-16, miR-92a, or miR-140-3p is used, exhibiting a significantly incremental regulation in samples where hemolysis has occurred compared to samples where hemolysis has not occurred. In various aspects of these embodiments, the hemolysis-associated miRNAs are miR-451, miR-486-5p, miR-16, and miR-92a.

[0114] In a further embodiment, the expression levels of several non-incrementally regulated corrective miRNAs are determined in the hemolysis-affected sample, and the expression levels of hemolysis-associated miRNAs are normalized. These corrective miRNAs can be used as a ratio to the hemolysis-associated miRNAs to normalize the determined expression levels of the latter miRNAs. In some of these embodiments, the corrective miRNAs include miR-126, miR-15b, miR-221, and miR-30b.

[0115] "Hemolysis" label In some implementation schemes, hemolysis is further assessed by the following assays: (a) Determine the expression ratio of each of the 16 miRNA pairs in the sample, consisting of each of miR-451, miR-486-5p, miR-16, and miR-92a paired with each of miR-126, miR-15b, miR-221, and miR-30b (i.e., miR-451 / miR-126, miR-451 / miR-15b, miR-451 / miR-221, miR-451 / miR-30b, miR-486-5p / miR-12). 6.miR-486-5p / miR-15b,miR-486-5p / miR-221,miR-486-5p / miR-30b,miR-16 / miR-126,miR-16 / miR-15b,miR-1 6 / miR-221,miR-16 / miR-30b,miR-92a / miR-126,miR-92a / miR-15b,miR-92a / miR-221 andmiR-92a / miR-30b or their inverse ratio); (b) Compare each of the 16 expression ratios from step (a) to the cutoff value of each expression ratio determination for the average ratio of multiple corresponding miRNA pairs from multiple control samples. (c) For each of the 16 miRNA pairs, if the expression rate in step (a) exceeds the cutoff value in step (b), a positive score is assigned to the rate; or if the expression rate in step (a) does not exceed the cutoff value in step (b), a non-positive score is assigned to the rate; and (d) The samples are classified as follows: (i) if 8 or more of the 16 ratios exceed the cutoff value, then hemolysis is present; or (ii) if fewer than 8 of the 16 ratios exceed the cutoff value, then hemolysis is not present.

[0116] Regarding the diagnostic and prognostic assays described above, the cutoff value for this hemolysis assay depends on the control population. For example, the cutoff value for multiple serum samples without hemolysis used as controls will differ from the cutoff value for multiple serum samples with hemolysis used as controls. See the examples. Additionally, the cutoff value can be adjusted, for example, to achieve desired SE, SP, PPV, and NPV values. In some embodiments where serum samples without hemolysis are used as controls, the cutoff value will be the average (mean or median) ratio of multiple control samples. Optionally, the cutoff value has a standard deviation of + / - proportion to account for differences in variability among the samples used to determine the cutoff value.

[0117] In various implementations, any of the above-described hemolysis assays using miRNA expression levels are also combined with another hemolysis assay, such as the spectrophotometric assay described in Kirschner et al., 2011.

[0118] D. Diagnostic combination As described in the examples, combining the MSC tag described herein with low-dose computed tomography (LDCT) allows for the detection of other cancers that are undetectable by LDCT, improving screening sensitivity from 87% for MSC alone and 84% for LDCT alone to 98% when LDCT is combined with MSC. When combined with cancer screening assays other than LDCT, the diagnostic and prognostic methods described above are expected to show similar improvements in diagnostic sensitivity.

[0119] The healthcare costs of LDCT screening and related follow-up procedures are significant. Recent budget impact models considering the widespread adoption of LDCT in the United States suggest that LDCT screening would prevent up to 8,100 premature lung cancer deaths, a screening rate of 75%, requiring an additional $240,000 in screening costs to prevent each lung cancer death (Goulart et al., 2012). By reducing downstream costs and supplementing LDCT screening with non-invasive biomarker tests, the number of individuals enrolled in LDCT screening can be increased (Peres, J Natl Cancer Inst 105:1-2, 2013).

[0120] Therefore, any of the above-described diagnostic or prognostic methods can be combined with screening subjects for lung cancer using a system that does not include miRNA expression analysis. This system can be used before, during, or after miRNA expression analysis. Any system now known or later discovered will be expected to benefit from additional analysis using MSCs. Non-limiting examples of such systems include blood tests, X-rays, computed tomography (CT), positron emission tomography (PET), thoracentesis, bronchoscopy, fine-needle aspiration biopsy, thoracotomy, thoracotomy, or mediastinoscopy. In some embodiments, the system is low-dose computed tomography (LDCT).

[0121] E. monitor Any of the methods described above can be used to monitor the presence of lung cancer and / or the risk of developing lung cancer in a subject. Monitoring of the subject is performed when any of the above methods are applied to at least two different biological samples obtained from the subject at different times. In some embodiments, at least one of the at least two different biological samples is obtained from the subject after treatment for lung cancer. Such monitoring assesses the recurrence or risk of relapse of lung cancer.

[0122] F. treat The methods described above can be used to select treatment options. The term "treatment" as used herein refers to the administration of a medication to eliminate signs or symptoms of lung cancer or to reduce its severity. Alternatively or additionally, a condition that can occur in multiple sites is treated if the therapy is applied to the condition in at least one of the multiple sites.

[0123] According to one implementation, the above method can be used to select treatment options, and if a negative condition or risk is determined, or if a low risk is determined in a combination of methods used to predict the risk of developing or having a tumor, the subject does not receive treatment for lung cancer. In another implementation, if a positive condition or risk is determined, or if an intermediate or high risk is determined in a combination of methods, the subject receives treatment for lung cancer.

[0124] Therefore, a method for establishing treatment options for lung cancer in subjects is also provided. This method includes testing the subject using the diagnostic and / or prognostic methods described above and determining treatment options based on the results of said methods. The method may further include administering treatment to subjects in need.

[0125] G. miRNA A total of 24 miRNAs can be used to construct lung tumor presence (PD) tags, aggressive lung tumor presence (PAD) tags, lung tumor risk (RD) tags, and aggressive lung tumor risk (RAD) tags. Table 1 lists these 24 miRNAs that can be present in each tag.

[0126] Table 1 miRNA hsa-miR-16 hsa-miR-17 hsa-miR-21 hsa-miR-101 hsa-miR-126 hsa-miR-145 hsa-miR-197 hsa-miR-221 hsa-miR-320 hsa-miR-451 hsa-miR-660 hsa-miR-106a hsa-miR-133a hsa-miR-140-3p hsa-miR-140-5p hsa-miR-142-3p hsa-miR-148a hsa-miR-15b hsa-miR-19b hsa-miR-28-3p hsa-miR-30b hsa-miR-30c hsa-miR-486-5p hsa-miR-92a Table 2 provides an overview of miRNAs used in all aspects of this invention.

[0127] Table 2 The following examples are provided to better illustrate the claimed invention and should not be considered as limiting the scope of the invention. References to specific materials are for illustrative purposes only and are not intended to limit the invention. Equivalent tools or reagents can be developed by those skilled in the art without inventive effort and without departing from the scope of the invention. Example

[0128] The following examples relate to the clinical utility of plasma-based microRNA tag classifiers in computed tomography (CT) lung cancer screening, also known as the relevant MILD trial study. Results from a validation study are provided, which retrospectively evaluated the diagnostic performance of a pre-specified miRNA tag classifier (MSC) algorithm in 939 subjects in a randomized, multicenter Italian Lung Detection (MILD) clinical trial comparing LDCT with observational studies (Pastorino et al., 2012). We demonstrated that MSCs exhibited significant diagnostic and prognostic performance.

[0129] Example 1: Method A. Study Population. The multicenter Italian Lung Detection (MILD) trial was a randomized prospective clinical trial initiated in 2005 and recruited 4,099 current or former smokers, at least 50 years of age, and without a history of cancer in the previous five years at the Istituto Nazionaledei Tumori: 2,376 (58%) were randomized to the LDCT group (1,190 annually, 1,186 biennially) and 1,723 (42%) to the observation group (Pastorino et al., 2012). Whole blood was collected at all volunteer recruitment (baseline) and at recalls, annually or biennially, as described by the Istituto Nazionaledei Tumori's Internal Review and the Ethics Boards (Boeri et al., 2011).

[0130] For this study, 1,000 consecutive plasma samples collected from lung cancer-free individuals recruited for the trial between June 2009 and July 2010 were used to determine the specificity of MSCs. Hemolysis of the plasma samples was first determined (see below) to remove samples from patients who might be contaminated with blood cell miRNAs (Kirschner et al., 2011; Pritchard et al., 2012). Of these 1,000 samples, 130 were unevaluable due to hemolysis. Of the remaining 870 subjects, 594 (68%) were in the LDCT group and 276 (32%) were in the observation group. To obtain a panel for determining the sensitivity performance of MSCs, plasma samples were obtained from almost all patients diagnosed with lung cancer by September 2012 (N=85). We preferred to measure the negative predictive value (NPV) in a large unselected subject series instead of matched cases and controls (which greatly reduced the number of controls and study capacity). For 69 of these 85 patients, at least one evaluable sample was collected. For all patients, we considered samples from LDCT scans that were closest to yielding a cancer diagnosis. Specifically, samples were available at diagnosis for 50 patients and samples were available before the disease was diagnosed for 19 patients. Figure 1 Pre-disease samples were collected 8–35 months prior to lung cancer detection, with a median lag time of 18 months.

[0131] B. MicroRNA Profile Analysis. Total RNA was extracted from 200 μl plasma samples using the mirVana PARIS Kit (Life Technologies, Ambion) and eluted in 50 μl buffer. For miRNA expression, in 3 μl of eluted RNA, the expression was measured on a custom microfluidic card (Life Technologies, Applied Biosystems) containing two copies of each miRNA sample, using the Multiplex Pools Protocol. For each sample, the Ct of individual miRNAs was determined using ViiA7 software (Life Technologies, Applied Biosystems) with a threshold of 0.15 and automated baseline. For input to MSCs, the mean of the two copies of the predetermined miRNA ratio was calculated as previously described (Boeri et al., 2011).

[0132] C. Statistical Analysis. Clinical outcome-blinded MSC risk scores for individual subjects were submitted to an independent research center, the Mario Negri Institute in Milan (Istituto Mario Negri), and data analysis was performed according to a pre-specified statistical analysis plan. Sensitivity (SE), specificity (SP), positive predictive value (PPV), and negative predictive value (NPV) of MSCs were computer-processed to evaluate the diagnostic performance of MSCs in classifying patients diagnosed with lung cancer against disease-free subjects, both in the study and in the observation group (LDCT). For diagnostic performance, individuals classified in the MSC-low-risk group were compared with those in the MSC-intermediate or MSC-high-risk groups. Considering single-positive and double-positive tests, we also computer-processed SE and SP to combine binary MSCs and LDCTs.

[0133] To explain the time dependence of MSCs as a predictor of disease progression, SE, SP, PPV, and NPV were evaluated at different time intervals (6, 12, 18, and 24 months) from autologous blood sample collection to lung cancer diagnosis using the methods described in Heagerty et al., Biometrics 56:337-344 (2000) and Zheng and Heagerty, Biometrics 63:332-341 (2007).

[0134] To determine the prognostic performance of MSCs, we examined all three risk groups and, across all 939 subjects, Kaplan-Meier estimates of MSC survival curves based on the date of autologous blood sample collection. We evaluated MSC survival inconsistencies using a Cox proportional incident model, also considering models further adjusted for age and sex, and performed a χ²1 test computationally between high / intermediate and low MSC levels.

[0135] D. Evaluation of the impact of hemolysis on plasma samples. Plasma samples exhibiting hemolysis were removed from subsequent analyses because the release of contaminated miRNAs from hemolyzed blood cells such as red blood cells (RBCs) or platelets (Kirschner et al., 2011; Pritchard et al., 2012). Two quality control (QC) measurements were used for this evaluation. First, in a pre-analysis step prior to RNA extraction, spectrophotometric analysis was performed by measuring absorbance at different wavelengths (414 nm, 541 nm, 576 nm) to identify the presence and amount of free hemoglobin in the sample (Kirschner et al., 2011).

[0136] For all samples, a second QC step was performed to obtain even greater hemolysis sensitivity by analyzing the expression levels of hemolysis-associated miRNAs contained within the miRNA tag classifier (MSC; mir-451,486-5p,16,92a). Plasma samples with hemolysis-associated miRNA expression levels exceeding 2 standard deviations from the overall mean of all samples were excluded from subsequent analyses. Samples with measurable hemolysis as measured spectrophotometrically were also excluded in this second QC step. No differences in the frequency or amount of hemolysis as measured spectrophotometrically or by hemolysis-associated miRNA analysis were observed in the cancer control samples.

[0137] Aside from the possibility of contaminating miRNAs released during hemolysis, differentially expressed miRNAs in plasma can simply reflect different blood cell counts (Pritchard, 2012). To analyze this hypothesis, the ratio of miRNAs composed of those expressed by neutrophils and those expressed by RBCs (Id.) was compared with the ratios of neutrophil and RBC levels obtained from complete blood counts (CBCs) of 23 lung cancer patients from this study. As reported in Table 3, none of the miRNA ratios present in the tags were found to have a significant correlation with their respective CBC ratios.

[0138] Table 3. Correlation between plasma miRNA ratio and blood cell count in 23 lung cancer patients. The Pearson correlation coefficients between neutrophil-to-RBC miRNA ratio and neutrophil-to-RBC count in the 23 samples are shown in the table. The results showed a statistically significant correlation (two-tailed p-value < 0.05). + The tags mentioned in this article exist. The two-tailed p-value of the Pearson correlation was <0.05.

[0139] The second QC step, which includes the analysis of hemolysis-related miRNAs, may additionally or alternatively utilize hemolytic miRNA tags, including miRNAs contained in MSCs as described herein.

[0140] i. Detection of hemolysis using miRNA tagging: plasma collection Collect whole blood samples, add EDTA, and store at room temperature for no more than 1–2 hours before processing. Storage at lower temperatures should be avoided as this can lead to nonspecific release of miRNAs. Centrifuge the samples at approximately 1250 x g for 10 minutes at 4°C to separate the plasma, carefully transferring it while avoiding material closest to the lymphocyte loop. Centrifuge the plasma again under the same conditions and then aliquot, without precipitating the material, and store at -80°C for up to 1 year or longer.

[0141] ii. miRNA expression levels in samples that underwent hemolysis compared to samples that did not. To develop miRNA tags for detecting hemolysis, 24 plasma samples were hemolyzed. Hemolysis was evaluated by visual inspection. A custom microfluidic card was used to analyze the profile of hemolyzed samples for the expression of a set of miRNAs. The expression of miRNAs in the hemolyzed samples was compared with the expression in non-hemolyzed (visually assessed) samples from 98 disease-free individuals.

[0142] Four miRNAs (miR-16, miR-451, miR-486-5p, and miR-92a) were included in the development of the miRNA tag. The original Ct values ​​(measures of expression levels) are shown in Table 4. Compared to samples without hemolysis, in samples with hemolysis, all four miRNAs, except miR-140-3p, were also significantly incrementally regulated (p < 0.001).

[0143] Table 4. Original Ct values ​​of miRNAs in samples that underwent hemolysis compared to samples that did not.

[0144] iii. The ratio of miRNAs produced in hemolytic tags To differentiate between hemolyzed and non-hemolyzed samples using miRNA ratios, miRNA expression levels in normal plasma samples containing serially diluted in vitro hemolyzed plasma samples were analyzed. The ratio of each miRNA to each of the four incrementally regulated miRNAs (miR-16, miR-451, miR-486-5p, and miR-92a) shown in Table 4 was calculated. MiR-126, miR-15b, miR-221, and miR-30b were identified as the miRNAs with the best hemolytic correlation to the ratios of the four incrementally regulated miRNAs (Table 5).

[0145] Table 5. Ratio of plasma miRNA expression levels representing in vitro hemolysis.

[0146] Each value is represented as log2 (ratio). The mean baseline miRNA ratio was calculated using the following formula: the mean ratio of non-hemolytic plasma samples from 98 disease-free individuals + 1.5 SD (standard deviation). These baseline ratios were set as cutoff values ​​for each of the 16 miRNA ratios in the development of hemolytic miRNA tags.

[0147] iv. Hemolysis was detected using hemolytic miRNA tags. Hemolytic miRNA tags can be used to detect hemolysis in plasma samples. Table 6 shows a list of 16 ratios consisting of eight miRNAs (miR-126, 15b, 30b, 221, 451, 16, 486-5p, and 92a). Specifically, four miRNAs, miR-451, miR-486-5p, miR-16, and miR-92a, were all overexpressed in samples where hemolysis occurred. This overexpression can be used as an indicator of hemolysis in plasma samples. Furthermore, if eight or more of the 16 ratios shown in Table 6 exceed the cutoff value, the plasma sample is considered hemolytic positive.

[0148] Table 6. Hemolytic miRNA Tags v. Comparison of hemolytic miRNA tags with hemoglobin Absorbance method for detecting hemolysis Hemoglobin is known in the art to have an absorbance wavelength of 414 nm. A standard method used in both scientific and clinical practice to analyze hemolysis is the spectral measurement of free hemoglobin in plasma samples at a wavelength of 414 nm (using an absorbance threshold of 0.2). In this experiment, the absorbance at 414 nm is normalized against the absorbance at 375 nm to overcome the high background signal in some samples (e.g., in lipemic samples). A cutoff value of 1.4 is set for the ratio of absorbance at 414 nm to absorbance at 375 nm.

[0149] As shown in the last row of Table 5, samples with an A414nm / A375nm value greater than 1.40 corresponded to miRNA tags for at least 8 of the 16 miRNA ratios exceeding their respective cutoff values. These settings were used to evaluate the ability of miRNA tags to distinguish 24 plasma samples that underwent hemolysis from 98 non-hemolytic (visually inspected) plasma samples. The miRNA tagging method showed a sensitivity of 0.88 and a specificity of 0.93. Regarding sensitivity, it is evident that the 24 samples that underwent hemolysis underwent visual assessment did indeed show hemolysis.

[0150] Hemolytic miRNA tagging and spectrophotometric methods were compared using an independent series consisting of 60 visually hemolytic samples (red, orange, or dark yellow) and 43 samples that did not show hemolysis based on visual inspection. Regarding sensitivity (Table 7), all five most hemolytic (red) samples were identified as hemolytic positive using both methods. However, the miRNA tagging method was more sensitive (>90% compared to 76% for the spectrophotometric method) when evaluating orange and dark yellow samples.

[0151] Table 7. Comparison of spectrophotometric measurements and miRNA results to evaluate hemolysis in 60 visually hemolytic plasma samples.

[0152] () The total percentage of hemolysis was determined by various methods. On the other hand, among the 43 plasma samples that did not show hemolysis based on visual examination (Table 8), 3 (7%) samples were hemolysis-positive when using the two methods: 1 (2%) samples were positive using the spectrophotometric method alone; and 2 (5%) samples were positive using the miRNA tagging method alone. There was hemolysis classification consistency in 40 (93%) samples between the two methods. Except for the 3 cases that were positive using both methods, the spectrophotometric method had 97.5% specificity and the miRNA tagging method had 95% specificity.

[0153] Table 8. Comparison of spectrophotometric measurements and miRNA results to evaluate hemolysis in 43 plasma samples that did not undergo hemolysis (based on visual inspection).

[0154] () Total percentage; κ statistic = 0.63 E. miRNA Tag Classifier (MSC) Algorithm. This paper describes the MSC algorithm in detail. To develop this algorithm, starting with 4,950 ratios of 100 different miRNAs circulating (stable) in plasma, and generating different gene expression ratios of 24 different miRNAs, the algorithm was developed from samples prospectively collected prior to the previously described INT / IEO lung cancer screening trial (Boeri et al., 2011) or at the time of diagnosis from the INT / IEO lung cancer screening trial. The pre-specified MSC algorithm used in this study was refined from previous studies in two ways.

[0155] First, samples showing detectable hemolysis were removed from the original training set (Boeri et al., 2011). Following that publication, researchers reported that hemolysis affects the accurate measurement of miRNA expression in plasma and serum samples. Therefore, to generate the optimal training set for MSCs, five samples from the original training set were excluded due to increased levels of detectable hemoglobin- and hemolysis-related miRNAs as described herein (by spectrophotometric analysis). The miRNA ratios were then used in the optimal training set to develop miRNA tags for disease risk (RD), disease presence (PD), invasive disease risk (RAD), and invasive disease presence (PAD; Table 9).

[0156] Table 9. Refined miRNA-ratio tags and corresponding cutoff values ​​(log2).

[0157] RD: Disease risk; RAD: Risk of invasive disease; PD: Presence of disease; PAD: Presence of invasive disease.

[0158] Establish ratio cutoff values ​​for plasma samples stored at -80°C for at least 1 year and up to 5 years.

[0159] A second approach to developing the MSC algorithm, different from previous studies, involves using plasma samples from a training set of 84 disease-free individuals not included in the validation group of 939 subjects from the MILD trial to generate a predetermined ratio cutoff value to achieve ≥80% specificity.

[0160] Using plasma samples from single subjects as a substitute library (as used in Boeri et al., 2011) allowed us to consider all 24 miRNAs initially identified in the training set and generate accurate cutoff values. Therefore, this study could include mir-101, 145, and 133a, which were excluded from the previous validation set due to high variability among subjects within the control library used in this study.

[0161] To establish a three-level risk classification for the disease (MSC low, intermediate, and high), the training set was also used to establish the minimum proportions exceeding the respective cutoff values ​​required to be considered positive: 10 / 27 for RD, 9 / 27 for PD, 14 / 28 for RAD, and 14 / 28 for PAD. The three-level MSCs were then defined as follows: low risk (L) if RDneg ∩ PDneg ∩ RADneg ∩ PADneg; intermediate risk (I) if RDpos ≠ PDpos ∩ RADneg ∩ PADneg; or high risk (H) if RADpos ≠ PADpos. These pre-specified risk groups were then used to test diagnostic and prognostic performance in an independent set of 939 subjects from the MILD screening trial.

[0162] F. Subject Characteristics. The characteristics of the 939 subjects with evaluable plasma samples recruited in the MILD trial (2005–2012; LDCT: N = 652; observations: N = 287) included 69 patients with lung cancer and 870 subjects without lung cancer, indicated by age, sex, smoking status, duration, and cigarettes / day (Table 10). Patients with lung cancer were older than those without (p < 0.0001) and had a higher proportion of males (81.2% vs. 63.3%; p = 0.0029). Smoking status did not differ significantly between the cancer-prescribing and cancer-free groups, but subjects who developed cancer had smoked for a longer period (p < 0.0001).

[0163] Table 10: Distribution of 69 lung cancer participants and 870 non-lung cancer participants by age, sex, and smoking. Multicenter Italian Lung Detection (MILD) study, 2005–2012. () Percentage of subjects with or without lung cancer in each category *via χ 2 Test estimation

[0164] For cancer patients, the median time from randomization to diagnosis was 29 months (range 1–82) and the median time from plasma collection to diagnosis was 2 months (range 0–35). In subjects without lung cancer, the median time from randomization to plasma collection was 44 months (range 0–58) and the median time from plasma collection to last follow-up was 27 months (range 3–41).

[0165] Example 2: Diagnostic and prognostic performance of MSCs For evaluable plasma samples obtained from 939 subjects across the LDCT and observation groups before or at diagnosis, analysis was performed using a pre-specified MSC algorithm for low, intermediate, and high risk in the cancer group, based on a real-time RT-PCR-based assay. MSC risk groups were examined for all 939 subjects according to lung cancer occurrence, lung cancer death, and tumor stage (Table 11). Intermediate and high MSC risk correctly classified 60 of the 69 lung cancer patients, with 87% SE, 81% SP, 27% PPV, and 99% NPV. Of the 19 lung cancer patients who died during follow-up, 18 were MSC-positive, with 95% SE, 81% SP, 10% PPV, and 100% NPV. No deaths due to causes other than lung cancer were observed during follow-up. The diagnostic performance of MSC for lung cancer detection was similar in both groups, with 88% SE, 80% SP, 31% PPV, and 99% NPV in the LDCT and observation groups, and 82% SE, 83% SP, 16% PPV, and 99% NPV in the observation group.

[0166] Table 11. Distribution of 69 lung cancer participants and 870 non-lung cancer participants based on lung cancer prevalence, lung cancer mortality, lung cancer stage, and miRNA tag classifier (MSC), along with corresponding sensitivity (SE), specificity (SP), positive predictive value (PPV), and negative predictive value (NPV). Multicenter Italian Lung Detection (MILD) study, 2005–2012. Percentage of subjects in the MSC risk groups (L, I, &H) within the total number of subjects in each category. * The calculation of SE, SP, PPV, and NPV combines pre-specified high and medium risk MSCs with low risk. + P = 0.0366, according to the Cochran-Armitage test, for the trend of mortality rates across different MSC risk groups in lung cancer subjects. Plasma samples obtained 30 months prior to disease detection Tumor stage information is not available in a single patient; for the association between MSCs and tumor stage, p = 0.49.

[0167] A clear trend in the proportion of deaths due to disease was observed across all three MSC risk groups, with an increased proportion of lung cancer deaths associated with low, intermediate, and high risk, respectively (p = 0.0336). MSC risk groups were not significantly associated with any tumor stage (I, II-III, or IV; Table 11) (p = 0.40).

[0168] Between MSC risk groups and histological subtypes (χ²) 2 =1.60, p=0.4485) and between adenocarcinoma and squamous cell carcinoma (χ² = 1.60, p=0.4485) 2 No significant difference was observed (p=0.55, p=0.759). Time-dependent analysis of the diagnostic performance of MSCs showed similar values ​​for SE, SP, PPV, and NPV at 6, 12, 18, and 24-month intervals between blood collection and lung cancer diagnosis (Table 12).

[0169] Table 12. Time-dependent analysis of miRNA tag classifiers (MSCs), and the calculated sensitivity (SE), specificity (SP), positive predictive value (PPV), and negative predictive value (NPV) at 6, 12, 18, and 24 months from blood collection to lung cancer detection.

[0170] Example 3: Complementary Diagnostic Performance of LDCT and MSC The analysis was limited to a total of 652 subjects in the LDCT group. LDCT identified 46 of the 58 subjects with lung cancer, missing 3 patients among the 251 subjects with no detectable pulmonary nodules and 9 patients with interphase cancer due to 79% SE (Table 13). The three cancers without pulmonary nodules included one non-solid lesion, one mediastinal adenosis, and one pleural effusion. The pre-specified MSC binary risk group (considering high and intermediate versus low) identified 40 of the 46 LDCT-detected cancers, 8 of the 9 interphase cancers, and all 3 subjects without pulmonary nodules.

[0171] LDCT had an 81% SP rate and an associated false positive rate of 19.4% (115 / 594) in the clinically ineffective subgroup for non-calcified nodules >5 mm (Table 13). When considering subjects with double positivity (LDCT and MSC), the false positive rate decreased to 3.7% (22 / 594), with a reduced SE rate (40 / 58, 69%). Conversely, when considering subjects with at least one positive test (LDCT or MSC) as positive, the combined use of LDCT and MSC identified 57 out of 58 cases, with an SE rate of 98% and an SP rate of 65%. On the other hand, MSC detected 9 out of 11 (82%) lung cancers present in the observation group (Table 13).

[0172] Example 4: Association between MSC risk groups and survival rate Plasma samples collected from all subjects (N=939) with three-year follow-up were analyzed to determine prognostic performance in three predefined MSC risk groups to predict overall survival. The two-year survival rate was 100% for subjects with low MSCs, 98% for those with intermediate MSCs, and 87% for those with high MSCs, while the three-year survival rates were 100%, 97%, and 77% for low, intermediate, and high MSCs, respectively. Figure 2 The difference in survival rates between high / intermediate and low MSCs was statistically significant (χ²). 2 =49.53, P<0.0001). The inconsistency remained significant after adjusting for age and sex (χ²). 2 =12.57, P=0.0004).

[0173] Example 5: Analysis The high sensitivity of diagnostic features associated with 99% of NPV indicates that MSCs are a clinically validated screening test. Furthermore, the diagnostic performance of MSCs as a predictor of lung cancer development was confirmed by time-dependent analysis.

[0174] MSCs identified subjects with a high probability of disease-related death. As a binary diagnosis, MSCs had a 95% SE and a 100% NPV for disease-related death across 939 subjects in two groups. Furthermore, the MSC risk group was associated with significantly different 3-year survival rates across the entire group of 939 subjects (100%, 97%, and 77% for low, intermediate, and high MSCs, respectively). These findings confirm that plasma miRNAs identify both the malignancy and invasiveness of tumors.

[0175] Three small European randomized trials, including MILD, have reported non-significant reductions in mortality to date (Infante et al., 2009; Saghir et al., 2012; Pastorino et al., 2012). The National Lung Screening Trial (NSLT), a randomized clinical screening trial that recruited 53,454 individuals for annual screening using three rounds of contrast chest radiographs with LDCT, demonstrated a 20% reduction in lung cancer mortality (Aberle et al., 2011). After three rounds of screening, 24.2% of participants were classified as positive, of which 96.4% were false positives, requiring screening of 320 participants to prevent one death from lung cancer.

[0176] In a systematic review of all randomized clinical trials examining the benefits and harms of LDCT screening, the mean nodal detection rate was 20%, with 90% of nodules being benign (Bach et al., JAMA 307:2418-2429, 2012). In this study, MSCs categorized 74% (485) of the 652 individuals in the LDCT group as low risk: 478 were true negatives and 7 were false negatives. MSCs were able to identify 8 of the 9 interphase cancers that were not detected by LDCT. Therefore, the integration of MSCs and LDCT (with at least one positive test) would improve screening sensitivity from 87% for MSCs alone and 84% for LDCT alone to 98% (57 / 58), with a false positive rate of 35%. Conversely, considering double-positive subjects (positive for both MSCs and LDCT), the integration of MSCs with LDCT would reduce the false positive rate of LDCT screening by more than 5-fold. In particular, the frequency of double false positive MSC and LDCT was only 3.7% (22 / 594) compared to 19.7% (115 / 594) for LDCT alone, but the SE was reduced to 69%.

[0177] Therefore, MSCs can complement LDCT screening by reducing false positives and enabling greater standardization of diagnostic algorithms, thereby reducing healthcare costs. Furthermore, given the lack of 3-year mortality for subjects with low MSCs, it could be proposed to repeat MSCs instead of LDCT for individuals with low MSCs. This pre-specified, plasma-based, blinded study represents the largest study testing biomarkers in LDCT screening trials and confirms the excellent diagnostic performance of LDCT when combined with MSCs.

Claims

1. A method for determining the presence of lung tumors in a subject, the method comprising: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cutoff value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then a positive score is assigned to the ratio; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then a non-positive score is assigned to the ratio. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 9 miRNA pairs are assigned a positive score for their expression ratio, or (2) after comparing the expression ratios of 27 miRNA pairs, fewer than 9 miRNA pairs are assigned a positive score for their expression ratio. The miRNA pairs mentioned include 106a / 140-5p, 106a / 142-3p, 126 / 140-5p, 126 / 142-3p, 133a / 142-3p, 140-5p / 17, 142-3p / 148a, 142-3p / 15b, 142-3p / 17, 142-3p / 21, 142-3p / 221, 142-3p / 30b, or 320 / 660, or their inverse ratios; and (e) The presence of lung tumors is classified as follows: (i) positive if at least 9 miRNAs are assigned a positive score for the expression ratio; or (ii) negative if fewer than 9 miRNAs are assigned a positive score for the expression ratio.

2. The method of claim 1, wherein the miRNA pair comprises 106a / 140-5p, 106a / 142-3p, 126 / 140-5p, 126 / 142-3p, 133a / 142-3p, 140-5p / 17, 142-3p / 148a, 142-3p / 15b, 142-3p / 17, 142-3p / 21, 142-3p / 221, 142-3p / 30b, and 320 / 660, or inverse ratios thereof.

3. The method of claim 1, wherein the miRNA pair further comprises 106a / 660, 106a / 92a, 126 / 660, 140-5p / 197, 140-5p / 28-3p, 142-3p / 145, 142-3p / 197, 142-3p / 28-3p, 17 / 660, 17 / 92a, 197 / 660, 197 / 92a, 19b / 660 or 28-3p / 660, or the inverse of the above.

4. The method of claim 2, wherein the miRNA pair further comprises 106a / 660, 106a / 92a, 126 / 660, 140-5p / 197, 140-5p / 28-3p, 142-3p / 145, 142-3p / 197, 142-3p / 28-3p, 17 / 660, 17 / 92a, 197 / 660, 197 / 92a, 19b / 660, and 28-3p / 660, or inverse ratios thereof.

5. Methods for determining the presence of aggressive lung tumors in subjects, including: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cutoff value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then a positive score is assigned to the ratio; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then a non-positive score is assigned to the ratio. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 14 miRNA pairs are assigned a positive score for their expression ratio, or (2) after comparing the expression ratios of 28 miRNA pairs, fewer than 14 miRNA pairs are assigned a positive score for their expression ratio. The miRNA pairs mentioned therein include 106a / 16, 106 / 660, 16 / 17, 16 / 320, 17 / 660, 197 / 30b, 197 / 30c, 320 / 451, 320 / 486-5p or 320 / 660, or their inverse ratios; and (e) The presence of invasive lung tumors is classified as follows: (i) positive if at least 14 miRNAs are assigned a positive score for the expression ratio; or (ii) negative if fewer than 14 miRNAs are assigned a positive score for the expression ratio.

6. The method of claim 5, wherein the miRNA pair comprises 106a / 16, 106 / 660, 16 / 17, 16 / 320, 17 / 660, 197 / 30b, 197 / 30c, 320 / 451, 320 / 486-5p, and 320 / 660, or inverse ratios thereof.

7. The method of claim 5, wherein the miRNA pair further comprises 106a / 451, 106a / 486-5p, 126 / 451, 126 / 486-5p, 126 / 660, 140-5p / 197, 16 / 197, 17 / 451, 17 / 486-5p, 197 / 451, 197 / 486-5p, 197 / 660, 197 / 92a, 19b / 451, 19b / 486-5p, 19b / 660, 28-3p / 451 or 28-3p / 486-5p, or the inverse of the above.

8. The method of claim 6, wherein the miRNA pair further comprises 106a / 451, 106a / 486-5p, 126 / 451, 126 / 486-5p, 126 / 660, 140-5p / 197, 16 / 197, 17 / 451, 17 / 486-5p, 197 / 451, 197 / 486-5p, 197 / 660, 197 / 92a, 19b / 451, 19b / 486-5p, 19b / 660, 28-3p / 451 or 28-3p / 486-5p, or the inverse of the above.

9. A method for determining the risk of developing lung cancer in a subject, the method comprising: (a) Determine the expression ratio of miRNA pairs in biological samples from subjects; (b) Compare the expression ratio from step (a) with the cutoff value determined by the average ratio of multiple corresponding miRNA pairs from multiple control samples; (c) If the expression ratio of the miRNA pair in step (a) exceeds the cutoff value in step (b), then a positive score is assigned to the ratio; or if the expression ratio of the miRNA pair in step (a) does not exceed the cutoff value in step (b), then a non-positive score is assigned to the ratio. (d) Repeat steps (a) to (c) for the other miRNA pairs until (1) at least 10 miRNA pairs are assigned a positive score for their expression ratio, or (2) after comparing the expression ratios of 27 miRNA pairs, fewer than 10 miRNA pairs are assigned a positive score for their expression ratio. The miRNA pairs mentioned therein include 133a / 92a, 15b / 21, 15b / 30b, 15b / 30c, 16 / 197 or 28-3p / 451, or their inverses; and (e) The risk of developing lung cancer is classified as follows: (i) if at least 10 miRNAs are assigned a positive score for their expression ratio, it is positive; or (ii) if fewer than 10 miRNAs are assigned a positive score for their expression ratio, it is negative.

10. The method of claim 9, wherein the miRNA pair comprises 133a / 92a, 15b / 21, 15b / 30b, 15b / 30c, 16 / 197 and 28-3p / 451, or inverse ratios thereof.

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