Patent Grant
9758827
MicroRNAs (miRNA) are a recently discovered class of small non-coding RNAs (17-14 nucleotides). Due to their function as regulators of gene expression they play a critical role both in physiological and in pathological processes, such as cancer (Calin and Croce 2006; Esquela-Kerscher and Slack 2006; Zhang, Pan et al. 2007; Sassen, Miska et al. 2008).
There is increasing evidence that miRNAs are not only found in tissues but also in human blood both as free circulating nucleic acids (also called circulating miRNAs) and in mononuclear cells. A recent proof-of-principle study demonstrated miRNA expression pattern in pooled blood sera and pooled blood cells, both in healthy individuals and in cancer patients including patients with lung cancer (Chen, Ba et al. 2008). In addition, a remarkable stability of miRNAs in human sera was recently demonstrated (Chen, Ba et al. 2008; Gilad, Meiri et al. 2008). These findings make miRNA a potential tool for diagnostics for various types of diseases based on blood analysis.
Lung cancer is the leading cause of cancer death worldwide (Jemal, Siegel et al. 2008). Its five-year survival rate is among the lowest of all cancer types and is markedly correlated to the stage at the time of diagnosis (Scott, Howington et al. 2007). Using currently existing techniques, more than two-thirds of lung cancers are diagnosed at late stages, when the relative survival rate is low (Henschke and Yankelevitz 2008). This reality calls for the search of new biomarkers that are able to catch lung cancer while it is still small and locally defined.
Various markers have been proposed to indicate specific types of disorders and in particular cancer. However, there is still a need for more efficient and effective methods and compositions for the diagnosis of diseases and in particular cancer.
The present invention provides novel methods for diagnosing diseases based on the determination of specific miRNAs that have altered expression levels in disease states compared to healthy or other relevant controls. The present invention particularly provides novel methods for the diagnosis and/or prognosis and/or monitoring of lung cancer or related diseases in human individuals based on miRNA analysis from samples derived from blood.
Subject-matter of the invention is a method for diagnosing lung cancer, comprising the steps
A “biological sample” in terms of the invention means a sample of biological tissue or fluid. Examples of biological samples are sections of tissues, blood, blood fractions, plasma, serum, etc. A biological sample may be provided by removing a sample of cells from a subject, but can also be provided by using a previously isolated sample. For example, a tissue sample can be removed from a subject suspected of having a disease by conventional biopsy techniques. In a preferred embodiment, a blood sample is taken from the subject. In one embodiment, the blood or tissue sample is obtained from the subject prior to initiation of radiotherapy, chemotherapy or other therapeutic treatment. According to the invention, the biological sample preferably is a blood, plasma, PBMC (peripheral blood mononuclear cell) or a serum sample. Further, it is also preferred to use blood cells, e.g. erythrocytes, leukocytes or thrombocytes.
A biological sample from a patient means a sample from a subject suspected to be affected by a disease. As used herein, the term “subject” refers to any mammal, including both human and other mammals. Preferably, the methods of the present invention are applied to human subjects.
In step (a) of the method of the invention, an expression profile of a predetermined set of miRNAs is determined. The determination may be carried out by any convenient means for determining nucleic acids. For expression profiling, qualitative, semi-quantitative and preferably quantitative detection methods can be used. A variety of techniques are well known to those of skill in the art. In particular, the determination may comprise nucleic acid hybridization and/or nucleic acid amplification steps.
Nucleic acid hybridization may for example be performed using a solid phase nucleic acid biochip array, in particular a microarray, beads, or in situ hybridization. The miRNA microarray technology affords the analysis of a complex biological sample for all expressed miRNAs. Nucleotides with complementarity to the corresponding miRNAs are spotted or synthesized on coated carriers. E.g., miRNAs isolated from the sample of interest may be labelled, e.g. fluorescently labelled, so that upon hybridization of the miRNAs to the complementary sequences on the carrier the resulting signal indicates the occurrence of a distinct miRNA. Preferably, microarray methods are employed that do not require a labeling of the miRNAs prior to hybridization (
Further, real-time or quantitative real-time polymerase chain reaction (RT-RCR or qRT-PCR) can be used to detect also low abandoned miRNAs. Furthermore, bead-based assays, e.g. the Luminex platform, are suitable.
Alternative methods for obtaining expression profiles may also contain sequencing, next generation sequencing or mass spectroscopy.
The predetermined set of miRNAs in step (a) of the method of the invention depends on the disease to be diagnosed. The inventors found out that single miRNA biomarkers lack sufficient accuracy, specificity and sensitivity, and therefore it is preferred to analyze more complex miRNA expression patterns, so-called miRNA signatures. The predetermined set of miRNAs comprises one or more, preferably a larger number of miRNAs (miRNA signatures) that are differentially regulated in samples of a patient affected by a particular disease compared to healthy or other relevant controls.
The expression profile determined in step (a) is subsequently compared to a reference expression profile in step (b). The reference expression profile is the expression profile of the same set of miRNAs in a biological sample originating from the same source as the biological sample from a patient but obtained from a healthy subject. Preferably, both the reference expression profile and the expression profile of step (a) are determined in a blood or serum sample including whole blood, plasma, serum or fractions thereof, or in a sample of peripheral blood mononuclear cells, of erythrocytes, leukocytes and/or thrombocytes. It is understood that the reference expression profile is not necessarily obtained from a single healthy subject but may be an average expression profile of a plurality of healthy subjects. It is preferred to use a reference expression profile obtained from a person of the same gender, and a similar age as the patient. It is also understood that the reference expression profile is not necessarily determined for each test. Appropriate reference profiles stored in databases may also be used. These stored references profiles may, e.g., be derived from previous tests. The reference expression profile may also be a mathematical function or algorithm developed on the basis of a plurality of reference expression profiles.
The method of the invention is suitable for diagnosing lung cancer. The diagnosis may comprise determining type, rate and/or stage of lung cancer. The course of the disease and the success of therapy such as chemotherapy may be monitored. The method of the invention provides a prognosis on the survivor rate and enables to determine a patient's response to drugs.
The inventors succeeded in developing a generally applicable approach to arrive at miRNA signatures that are correlated with a particular disease. The general work flow is depicted in
These techniques include but are not restricted to:
The inventors surprisingly found out that the described approach yields in miRNA signatures that provide high diagnostic accuracy, specificity and sensitivity in the determination of lung cancer.
According to the invention, the disease to be determined is lung cancer, e.g. lung carcinoid, lung pleural mesothelioma or lung squamous cell carcinoma, in particular non-small cell lung carcinoma.
The inventors succeeded in determining miRNAs that are differentially regulated in samples from lung cancer patients as compared to healthy controls. A complete overview of all miRNAs that are found to be differentially regulated in blood samples of lung cancer patients is provided in the tables shown in
According to the invention mutual information is considered as the reduction in uncertainty about the class labels “0” for controls and “1” for tumor samples due to the knowledge of the miRNA expression. The higher the value of the MI of a miRNA, the higher is the diagnostic content of the respective miRNA. The computation of the MI of each miRNA is explained in the experimental section below.
For example, the predetermined set of miRNAs representative for lung cancer comprises at least 1, 7, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100 of the miRNAs selected from the group consisting of hsa-miR-126, hsa-miR-423-5p, hsa-let-7i, hsa-let-7d, hsa-miR-22, hsa-miR-15a, hsa-miR-98, hsa-miR-19a, hsa-miR-574-5p, hsa-miR-324-3p, hsa-miR-20b, hsa-miR-25, hsa-miR-195, hsa-let-7e, hsa-let-7c, hsa-let-7f, hsa-let-7a, hsa-let-7g, hsa-miR-140-3p, hsa-miR-339-5p, hsa-miR-361-5p, hsa-miR-1283, hsa-miR-18a*, hsa-miR-26b, hsa-miR-604, hsa-miR-423-3p, hsa-miR-93*, hsa-miR-29a, hsa-miR-1248, hsa-miR-210, hsa-miR-19b, hsa-miR-453, hsa-miR-126*, hsa-miR-188-3p, hsa-miR-624*, hsa-miR-505*, hsa-miR-425, hsa-miR-339-3p, hsa-miR-668, hsa-miR-363*, hsa-miR-15b*, hsa-miR-29c*, hsa-miR-550*, hsa-miR-34c-3p, hsa-miR-20a, hsa-miR-374a, hsa-miR-145*, hsa-miR-302b, hsa-miR-106a, hsa-miR-30e, hsa-miR-223, hsa-miR-1269, hsa-let-7b, hsa-miR-542-3p, hsa-miR-516b*, hsa-miR-451, hsa-miR-519c-3p, hsa-miR-1244, hsa-miR-602, hsa-miR-361-3p, hsa-miR-19a*, hsa-miR-433, hsa-miR-1200, hsa-miR-522, hsa-miR-520f, hsa-miR-519c-5p, hsa-miR-192, hsa-miR-1245, hsa-miR-151-5p, hsa-miR-1288, hsa-miR-503, hsa-miR-563, hsa-miR-663b, hsa-let-7d*, hsa-miR-199a-5p, hsa-miR-720, hsa-miR-1246, hsa-miR-338-5p, hsa-miR-297, hsa-miR-1261, hsa-miR-922, hsa-miR-185, hsa-miR-611, hsa-miR-1272, hsa-miR-1299, hsa-miR-335*, hsa-miR-497, hsa-miR-1207-3p, hsa-miR-16, hsa-miR-1, hsa-miR-1291, hsa-miR-138-2*, hsa-miR-136, hsa-miR-548d-3p, hsa-miR-561, hsa-miR-548h, hsa-miR-331-3p, hsa-miR-186*, hsa-miR-145, hsa-miR-17, hsa-miR-30b, hsa-let-7f-1*, hsa-miR-1305, hsa-miR-129-5p, hsa-miR-1204, hsa-miR-106b*, hsa-miR-619, hsa-miR-34a*, hsa-miR-652, hsa-miR-1256, hsa-miR-20b*, hsa-miR-424*, hsa-miR-517a, hsa-miR-1284, hsa-miR-199b-3p, hsa-miR-599, hsa-miR-411, hsa-miR-23b, hsa-miR-1302, hsa-miR-449a, hsa-miR-548f, hsa-miR-597, hsa-miR-603, hsa-miR-1247, hsa-miR-1539, hsa-miR-1911, hsa-miR-325, hsa-miR-409-5p, hsa-miR-182, hsa-miR-658, hsa-miR-215, hsa-miR-147b, hsa-miR-30d, hsa-miR-378*, hsa-miR-221*, hsa-miR-34b, hsa-miR-593*, hsa-miR-552, hsa-miR-378, hsa-miR-143*, hsa-miR-1266, hsa-miR-554, hsa-miR-631, hsa-miR-609, hsa-miR-30c, hsa-miR-28-5p, hsa-miR-23a, hsa-miR-645, hsa-miR-647, hsa-miR-302b*, hsa-miR-607, hsa-miR-1289, hsa-miR-1324, hsa-miR-513a-3p, hsa-miR-939, hsa-miR-29b, hsa-miR-665, hsa-miR-18a, hsa-miR-1224-5p, hsa-miR-10a*, hsa-miR-181a*, hsa-miR-218-2*, hsa-miR-371-3p, hsa-miR-377, hsa-miR-140-5p, hsa-miR-301a, hsa-miR-1277, hsa-miR-130a*, hsa-miR-1912, hsa-miR-193b, hsa-miR-214*, hsa-miR-216b, hsa-miR-302f, hsa-miR-522*, hsa-miR-548j, hsa-miR-568, hsa-miR-648, hsa-miR-662, hsa-miR-222, hsa-miR-1287, hsa-miR-891b, hsa-miR-342-3p, hsa-miR-512-3p, hsa-miR-623, hsa-miR-208b, hsa-miR-16-1*, hsa-miR-551b, hsa-miR-146b-3p, hsa-miR-520b, hsa-miR-449b, hsa-miR-520g, hsa-miR-24-2*, hsa-miR-518f, hsa-miR-649, hsa-miR-32, hsa-miR-151-3p, hsa-miR-454, hsa-miR-101, hsa-miR-19b-1*, hsa-miR-509-5p, hsa-miR-144, hsa-miR-508-5p, hsa-miR-569, hsa-miR-636, hsa-miR-937, hsa-miR-346, hsa-miR-506, hsa-miR-379*, hsa-miR-1184, hsa-miR-579, hsa-miR-23b*, hsa-miR-1262, hsa-miR-153, hsa-miR-520e, hsa-miR-632, hsa-miR-106a*, hsa-miR-31*, hsa-miR-33b*, hsa-miR-654-3p, hsa-miR-99b*, hsa-miR-1278, hsa-miR-135b, hsa-let-7c*, hsa-miR-1468, hsa-miR-374b*, hsa-miR-514, hsa-miR-590-3p, hsa-miR-606, hsa-miR-369-3p, hsa-miR-488, hsa-miR-128, hsa-miR-362-5p, hsa-miR-671-5p, hsa-miR-874, hsa-miR-1911*, hsa-miR-1292, hsa-miR-194, hsa-miR-15b, hsa-miR-342-5p, hsa-miR-125b-2*, hsa-miR-1297, hsa-miR-933, hsa-miR-493*, hsa-miR-105, hsa-miR-141, hsa-miR-181c*, hsa-miR-193a-3p, hsa-miR-302c, hsa-miR-485-5p, hsa-miR-499-3p, hsa-miR-545, hsa-miR-548b-5p, hsa-miR-549, hsa-miR-576-5p, hsa-miR-577, hsa-miR-583, hsa-miR-587, hsa-miR-624, hsa-miR-646, hsa-miR-655, hsa-miR-885-5p, hsa-miR-194*, hsa-miR-299-5p, hsa-miR-337-3p, hsa-miR-493, hsa-miR-497*, hsa-miR-519a, hsa-miR-99a*, hsa-miR-1280, hsa-miR-523*, hsa-miR-198, hsa-miR-934, hsa-miR-30d*, hsa-miR-452*, hsa-miR-548b-3p, hsa-miR-586, hsa-miR-92b, hsa-miR-517b, hsa-miR-548a-3p, hsa-miR-875-5p, hsa-miR-431*, hsa-miR-384, hsa-miR-644, hsa-miR-1185, hsa-miR-29b-2*, hsa-miR-489, hsa-miR-566, hsa-miR-1538, hsa-miR-28-3p, hsa-let-7f-2*, hsa-miR-1322, hsa-miR-1827, hsa-miR-192*, hsa-miR-302e, hsa-miR-411*, hsa-miR-424, hsa-miR-582-3p, hsa-miR-629*, hsa-miR-491-3p, hsa-miR-519b-3p, hsa-miR-1197, hsa-miR-127-5p, hsa-miR-1286, hsa-miR-132*, hsa-miR-33b, hsa-miR-553, hsa-miR-620, hsa-miR-708, hsa-miR-892b, hsa-miR-520h, hsa-miR-500*, hsa-miR-551b*, hsa-miR-186, hsa-miR-558, hsa-miR-26a, hsa-miR-1263, hsa-miR-211, hsa-miR-1304, hsa-miR-220b, hsa-miR-891a, hsa-miR-1253, hsa-miR-1205, hsa-miR-137, hsa-miR-154*, hsa-miR-555, hsa-miR-887, hsa-miR-363, hsa-miR-1537, hsa-miR-219-1-3p, hsa-miR-220a, hsa-miR-222*, hsa-miR-323-3p, hsa-miR-376b, hsa-miR-490-5p, hsa-miR-523, hsa-miR-302a*, hsa-miR-27b*, hsa-miR-591, hsa-miR-888, hsa-miR-376a*, hsa-miR-618, hsa-miR-1182, hsa-miR-532-3p, hsa-miR-181b, hsa-miR-521, hsa-miR-545*, hsa-miR-9*, hsa-miR-920, hsa-miR-571, hsa-miR-635, hsa-miR-200b, hsa-miR-455-5p, hsa-miR-876-3p, hsa-miR-373*, hsa-miR-146a*, hsa-miR-122*, hsa-miR-450b-3p, hsa-miR-24, hsa-miR-484, hsa-miR-103-as, hsa-miR-380, hsa-miR-513a-5p, hsa-miR-509-3-5p, hsa-miR-873, hsa-miR-556-5p, hsa-miR-369-5p, hsa-miR-653, hsa-miR-767-3p, hsa-miR-516a-3p, hsa-miR-520c-3p, hsa-miR-708*, hsa-miR-924, hsa-miR-520d-5p, hsa-miR-512-5p, hsa-miR-374a*, hsa-miR-921, hsa-miR-1206, hsa-miR-1259, hsa-miR-525-5p, hsa-miR-200a*, hsa-miR-1293, hsa-miR-372, hsa-miR-548a-5p, hsa-miR-548k, hsa-miR-1300, hsa-miR-1264, hsa-miR-551a, hsa-miR-196b, hsa-miR-32*, hsa-miR-33a, hsa-miR-548d-5p, hsa-miR-616, hsa-miR-876-5p, hsa-miR-508-3p, hsa-miR-26a-2*, hsa-miR-187, hsa-miR-199a-3p, hsa-miR-96*, hsa-miR-18b, hsa-miR-432*, hsa-miR-509-3p, hsa-miR-1183, hsa-miR-626, hsa-miR-513b, hsa-miR-617, hsa-miR-9, hsa-miR-519e, hsa-miR-204, hsa-miR-29c, hsa-miR-1268, hsa-miR-122, hsa-miR-7-2*, hsa-miR-15a*, hsa-miR-181d, hsa-miR-219-5p, hsa-miR-302d, hsa-miR-34a, hsa-miR-410, hsa-miR-33a*, hsa-miR-502-3p, hsa-miR-379, hsa-miR-498, hsa-miR-518d-5p, hsa-miR-556-3p, hsa-miR-502-5p, hsa-miR-31, hsa-miR-100, hsa-miR-296-3p, hsa-miR-615-5p, hsa-miR-21*, hsa-miR-657, hsa-miR-651, hsa-miR-765, hsa-miR-548m, hsa-miR-219-2-3p, hsa-miR-501-3p, hsa-miR-302a, hsa-miR-202*, hsa-miR-206, hsa-miR-520d-3p, hsa-miR-548i, hsa-miR-511, hsa-miR-30a, hsa-miR-1224-3p, hsa-miR-525-3p, hsa-miR-1225-5p, hsa-miR-223*, hsa-miR-615-3p, hsa-miR-570, hsa-miR-320a, hsa-miR-770-5p, hsa-miR-582-5p, hsa-miR-590-5p, hsa-miR-659, hsa-miR-1251, hsa-miR-664, hsa-miR-488*, hsa-miR-548g, hsa-miR-802, hsa-miR-542-5p, hsa-miR-190, hsa-miR-218-1*, hsa-miR-367*, hsa-miR-450a, hsa-miR-367, hsa-miR-124, hsa-miR-767-5p, hsa-miR-200c, hsa-miR-572, hsa-miR-526a, hsa-miR-936, hsa-miR-548n, hsa-miR-21, hsa-miR-182*, hsa-miR-34c-5p, hsa-miR-429, hsa-miR-628-5p, hsa-miR-29a*, hsa-miR-370, hsa-let-7a*, hsa-miR-101*, hsa-miR-559, hsa-miR-217, hsa-miR-519b-5p, hsa-miR-30e*, hsa-miR-147, hsa-miR-487b, hsa-miR-888*, hsa-miR-205, hsa-miR-1257, hsa-miR-7, hsa-miR-296-5p, hsa-miR-1255a, hsa-miR-380*, hsa-miR-1275, hsa-miR-330-5p, hsa-miR-1243, hsa-miR-136*, hsa-miR-141*, hsa-miR-517c, hsa-miR-621, hsa-miR-1915*, hsa-miR-541, hsa-miR-543, hsa-miR-942, hsa-miR-26a-1*, hsa-miR-567, hsa-miR-184, hsa-miR-376a, hsa-miR-124*, hsa-miR-1254, hsa-miR-1207-5p, hsa-miR-580, hsa-let-7b*, hsa-miR-539, hsa-miR-520a-3p, hsa-miR-585, hsa-miR-675b, hsa-miR-943, hsa-miR-573, hsa-miR-93, hsa-miR-27a*, hsa-miR-613, hsa-miR-220c, hsa-miR-524-3p, hsa-miR-500, hsa-miR-1201, hsa-miR-20a*, hsa-miR-1914*, hsa-miR-425*, hsa-miR-515-3p, hsa-miR-377*, hsa-miR-504, hsa-miR-548c-3p, hsa-miR-1276, hsa-miR-138, hsa-miR-431, hsa-miR-494, hsa-miR-448, hsa-miR-633, hsa-miR-487a, hsa-miR-149, hsa-miR-300, hsa-miR-1826, hsa-miR-127-3p, hsa-miR-486-5p, hsa-miR-148a, hsa-miR-1294, hsa-miR-548l, hsa-miR-142-5p, hsa-miR-889, hsa-miR-365, hsa-miR-99b, hsa-miR-200b*, hsa-miR-200a, hsa-miR-518e, hsa-miR-612, hsa-miR-183*, hsa-miR-148b, hsa-miR-103, hsa-miR-548o, hsa-miR-1203, hsa-miR-135a*, hsa-miR-383, hsa-miR-1913, hsa-miR-373, hsa-miR-371-5p, hsa-miR-298, hsa-miR-758, hsa-miR-412, hsa-miR-518c, hsa-miR-589*, hsa-miR-643, hsa-miR-592, hsa-miR-892a, hsa-miR-944, hsa-miR-576-3p, hsa-miR-581, hsa-miR-625*, hsa-miR-1260, hsa-miR-1281, hsa-miR-337-5p, hsa-miR-133b, hsa-miR-92a-2*, hsa-miR-100*, hsa-miR-589, hsa-miR-218, hsa-miR-224, hsa-miR-16-2*, hsa-miR-301b, hsa-miR-190b, hsa-miR-375, hsa-miR-548p, hsa-miR-185*, hsa-miR-519d, hsa-miR-605, hsa-miR-877, hsa-miR-125a-3p, hsa-miR-744*, hsa-miR-520c-5p, hsa-miR-148a*, hsa-miR-212, hsa-miR-505, hsa-miR-496, hsa-miR-1323, hsa-miR-548e, hsa-miR-628-3p, hsa-miR-1914, hsa-miR-584, hsa-miR-135b*, hsa-miR-1295, hsa-miR-95, hsa-miR-133a, hsa-miR-485-3p, hsa-miR-541*, hsa-miR-374b, hsa-miR-329, hsa-miR-483-5p, hsa-miR-885-3p, hsa-let-7i*, hsa-miR-935, hsa-miR-130b, hsa-miR-1274a, hsa-miR-1226, hsa-miR-518e*, hsa-miR-1225-3p, hsa-miR-923, hsa-miR-196a*, hsa-miR-1270, hsa-miR-1271, hsa-miR-610, hsa-miR-574-3p, hsa-miR-1282, hsa-miR-10b*, hsa-miR-216a, hsa-miR-144*, hsa-miR-23a*, hsa-miR-499-5p, hsa-miR-183, hsa-miR-490-3p, hsa-miR-330-3p, hsa-let-7g*, hsa-miR-483-3p, hsa-miR-214, hsa-miR-34b*, hsa-miR-302d*, hsa-miR-382, hsa-miR-454*, hsa-miR-1202, hsa-miR-202, hsa-miR-544, hsa-miR-593, hsa-miR-760, hsa-miR-940, hsa-let-7e*, hsa-miR-1237, hsa-miR-18b*, hsa-miR-630, hsa-miR-519e*, hsa-miR-452, hsa-miR-26b*, hsa-miR-516b, hsa-miR-299-3p, hsa-miR-381, hsa-miR-340, hsa-miR-132, hsa-miR-142-3p, hsa-miR-125b-1*, hsa-miR-30c-2*, hsa-miR-627, hsa-miR-1908, hsa-miR-1267, hsa-miR-507, hsa-miR-188-5p, hsa-miR-486-3p, hsa-miR-596, hsa-miR-193a-5p, hsa-miR-671-3p, hsa-miR-24-1*, hsa-miR-19b-2*, hsa-miR-1308, hsa-miR-208a, hsa-miR-135a, hsa-miR-331-5p, hsa-miR-181c, hsa-miR-640, hsa-miR-1909, hsa-miR-629, hsa-miR-10a, hsa-miR-491-5p, hsa-miR-492, hsa-miR-516a-5p, hsa-miR-510, hsa-miR-1915, hsa-miR-518c*, hsa-miR-1273, hsa-miR-25*, hsa-miR-744, hsa-miR-550, hsa-miR-890, hsa-miR-1303, hsa-miR-650, hsa-miR-1227, hsa-miR-595, hsa-miR-1255b, hsa-miR-1252, hsa-miR-455-3p, hsa-miR-345, hsa-miR-96, hsa-miR-1321, hsa-miR-513c, hsa-miR-548c-5p, hsa-miR-663, hsa-miR-320c, hsa-miR-320b, hsa-miR-654-5p, hsa-miR-326, hsa-miR-1825, hsa-miR-328, hsa-miR-146b-5p, hsa-miR-886-3p, hsa-miR-1909*, hsa-miR-1469, hsa-miR-338-3p, hsa-miR-886-5p, hsa-miR-601, hsa-miR-1298, hsa-miR-1910, hsa-miR-1226*, hsa-miR-421, hsa-miR-1471, hsa-miR-150*, hsa-miR-1229, hsa-miR-17*, hsa-miR-320d, hsa-miR-10b, hsa-miR-766, hsa-miR-600, hsa-miR-641, hsa-miR-340*, hsa-miR-616*, hsa-miR-520a-5p, hsa-miR-1179, hsa-miR-1178, hsa-miR-30b*, hsa-miR-155*, hsa-miR-138-1*, hsa-miR-501-5p, hsa-miR-191, hsa-miR-107, hsa-miR-639, hsa-miR-518d-3p, hsa-miR-106b, hsa-miR-129-3p, hsa-miR-1306, hsa-miR-187*, hsa-miR-125b, hsa-miR-642, hsa-miR-30a*, hsa-miR-139-5p, hsa-miR-1307, hsa-miR-769-3p, hsa-miR-532-5p, hsa-miR-7-1*, hsa-miR-196a, hsa-miR-1296, hsa-miR-191*, hsa-miR-221, hsa-miR-92a-1*, hsa-miR-1285, hsa-miR-518f*, hsa-miR-1233, hsa-miR-1290, hsa-miR-598, hsa-miR-769-5p, hsa-miR-614, hsa-miR-578, hsa-miR-1301, hsa-miR-515-5p, hsa-miR-564, hsa-miR-634, hsa-miR-518b, hsa-miR-941, hsa-miR-376c, hsa-miR-195*, hsa-miR-518a-5p, hsa-miR-557, hsa-miR-1228*, hsa-miR-22*, hsa-miR-1234, hsa-miR-149*, hsa-miR-30c-1*, hsa-miR-200c*, hsa-miR-1181, hsa-miR-323-5p, hsa-miR-1231, hsa-miR-203, hsa-miR-302c*, hsa-miR-99a, hsa-miR-146a, hsa-miR-656, hsa-miR-526b*, hsa-miR-148b*, hsa-miR-181a, hsa-miR-622, hsa-miR-125a-5p, hsa-miR-152, hsa-miR-197, hsa-miR-27b, hsa-miR-1236, hsa-miR-495, hsa-miR-143, hsa-miR-362-3p, hsa-miR-675, hsa-miR-1274b, hsa-miR-139-3p, hsa-miR-130b*, hsa-miR-1228, hsa-miR-1180, hsa-miR-575, hsa-miR-134, hsa-miR-875-3p, hsa-miR-92b*, hsa-miR-660, hsa-miR-526b, hsa-miR-422a, hsa-miR-1250, hsa-miR-938, hsa-miR-608, hsa-miR-1279, hsa-miR-1249, hsa-miR-661, hsa-miR-1208, hsa-miR-130a, hsa-miR-450b-5p, hsa-miR-432, hsa-miR-409-3p, hsa-miR-527, hsa-miR-877*, hsa-miR-1238, hsa-miR-517*, hsa-miR-193b*, hsa-miR-524-5p, hsa-miR-1258, hsa-miR-154, hsa-miR-637, hsa-miR-588, hsa-miR-155, hsa-miR-664*, hsa-miR-1470, hsa-miR-105*, hsa-miR-324-5p, hsa-miR-129*, hsa-miR-625, hsa-miR-519a*, hsa-miR-181a-2*, hsa-miR-199b-5p, hsa-miR-27a, hsa-miR-518a-3p, hsa-miR-1265, hsa-miR-92a, hsa-miR-29b-1*, hsa-miR-150, hsa-miR-335, hsa-miR-638.
The miRNAs that provide the highest mutual information in samples from lung cancer patients compared to healthy controls are hsa-miR-361-5p, hsa-miR-23b, hsa-miR-126, hsa-miR-527, hsa-miR-29a, hsa-let-7i, hsa-miR-19a, hsa-miR-28-5p, hsa-miR-185*, hsa-miR-23a, hsa-miR-1914*, hsa-miR-29c, hsa-miR-505*, hsa-let-7d, hsa-miR-378, hsa-miR-29b, hsa-miR-604, hsa-miR-29b, hsa-let-7b, hsa-miR-299-3p, hsa-miR-423-3p, hsa-miR-18a*, hsa-miR-1909, hsa-let-7c, hsa-miR-15a, hsa-miR-425, hsa-miR-93*, hsa-miR-665, hsa-miR-30e, hsa-miR-339-3p, hsa-miR-1307, hsa-miR-625*, hsa-miR-193a-5p, hsa-miR-130b, hsa-miR-17*, hsa-miR-574-5p, hsa-miR-324-3p (group (a)).
Further, the measured miRNA profiles of
The miRNAs given above that have been grouped in the order of their performance in the t-tests or in the order of their MI-values provide the highest diagnostic power. Thus, preferably the predetermined set of miRNAs for the diagnosis of lung cancer comprises one or more nucleic acids selected from the above groups (a) and (b) of miRNAs. The predetermined set of miRNAs should preferably comprise at least 7, preferably at least 10, 15, 20 or 24 of the indicated nucleic acids. Most preferably, all of the above indicated miRNAs are included in the predetermined set of miRNAs. It is particularly preferred to include the 24, 20, 15, 10 or at least 7 of the first mentioned miRNAs in the order of their performance in the t-tests or of their MI-values. A comparison of the results obtained by determining 4, 8, 10, 16, 20, 24, 28 or 40 miRNAs provided in
In a particularly preferred embodiment of the method of the invention, the predetermined set of miRNAs includes the miRNAs hsa-miR-126, hsa-miR-423-5p, hsa-let-7i, hsa-let-7d, hsa-miR-22, hsa-miR-15a, hsa-miR-98, hsa-miR-19a, hsa-miR-574-5p, hsa-miR-324-3p, hsa-miR-20b, hsa-miR-25, hsa-miR-195, hsa-let-7e, hsa-let-7c, hsa-let-7f, hsa-let-7a, hsa-let-7g, hsa-miR-140-3p, hsa-miR-339-5p, hsa-miR-361-5p, hsa-miR-1283, hsa-miR-18a* and hsa-miR-26b.
In a further particularly preferred embodiment of the method of the invention, the miRNAs are selected from the miRNAs shown in
In another embodiment, the predetermined set of miRNAs for the diagnosis of lung cancer comprises at least one preferred signature L1-251 as shown in
For the diagnosis of different types of diseases, such as for a different type of cancer, a different predetermined set of miRNAs should be determined in step (a) of the method of the invention. The relevant miRNA signatures can be obtained according to the workflow depicted in
Another embodiment of the present invention is a kit for diagnosing a disease, comprising means for determining an expression profile of a predetermined set of miRNAs in a biological sample, in particular in a blood, plasma, and/or serum sample including whole blood, plasma, serum or fractions thereof, or in a sample comprising peripheral blood mononuclear cells, erythrocytes, leukocytes and/or thrombocytes. Preferably, one or more reference expression profiles are also provided which show the expression profile of the same set of miRNAs in the same type of biological sample, in particular in a blood and/or serum sample, obtained from one or more healthy subjects. A comparison to said reference expression profile(s) allows for the diagnosis of the disease.
The kit is preferably a test kit for detecting a predetermined set of miRNAs in sample by nucleic acid hybridisation and optionally amplification such as PCR or RT-PCR. The kit preferably comprises probes and/or primers for detecting a predetermined set of miRNAs. Further, the kit may comprise enzymes and reagents including reagents for cDNA synthesis from miRNAs prior to realtime PCR.
A kit for diagnosing lung cancer preferably comprises means for determining the expression profile of one or more miRNAs selected from the group (a) consisting of hsa-miR-361-5p, hsa-miR-23b, hsa-miR-126, hsa-miR-527, hsa-miR-29a, hsa-let-7i, hsa-miR-19a, hsa-miR-28-5p, hsa-miR-185*, hsa-miR-23a, hsa-miR-1914*, hsa-miR-29c, hsa-miR-505*, hsa-let-7d, hsa-miR-378, hsa-miR-29b, hsa-miR-604, hsa-miR-29b, hsa-let-7b, hsa-miR-299-3p, hsa-miR-423-3p, hsa-miR-18a*, hsa-miR-1909, hsa-let-7c, hsa-miR-15a, hsa-miR-425, hsa-miR-93*, hsa-miR-665, hsa-miR-30e, hsa-miR-339-3p, hsa-miR-1307, hsa-miR-625*, hsa-miR-193a-5p, hsa-miR-130b, hsa-miR-17*, hsa-miR-574-5p and hsa-miR-324-3p.
According to another embodiment of the invention, the kit for diagnosing lung cancer preferably comprises means for determining the expression profile of one or more miRNAs selected from the group (b) consisting of hsa-miR-126, hsa-miR-423-5p, hsa-let-7i, hsa-let-7d, hsa-miR-22, hsa-miR-15a, hsa-miR-98, hsa-miR-19a, hsa-miR-574-5p, hsa-miR-324-3p, hsa-miR-20b, hsa-miR-25, hsa-miR-195, hsa-let-7e, hsa-let-7c, hsa-let-7f, hsa-let-7a, hsa-let-7g, hsa-miR-140-3p, hsa-miR-339-5p, hsa-miR-361-5p, hsa-miR-1283, hsa-miR-18a*, hsa-miR-26b, hsa-miR-604, hsa-miR-423-3p and hsa-miR-93*.
In a preferred embodiment, the kit comprises means for determining at least 7, preferably at least 10, 15, 20 or 24 of the indicated groups of miRNAs. It is particularly preferred to include means for determining the 24, 20, 15, 10 or at least 7 of the first mentioned miRNAs in the order of their MI-values or their performance in the t-tests as shown in the tables in
In a particularly preferred embodiment, the kit comprises means for determining the miRNAs hsa-miR-126, hsa-miR-423-5p, hsa-let-7i, hsa-let-7d, hsa-miR-22, hsa-miR-15a, hsa-miR-98, hsa-miR-19a, hsa-miR-574-5p, hsa-miR-324-3p, hsa-miR-20b, hsa-miR-25, hsa-miR-195, hsa-let-7e, hsa-let-7c, hsa-let-7f, hsa-let-7a, hsa-let-7g, hsa-miR-140-3p, hsa-miR-339-5p, hsa-miR-361-5p, hsa-miR-1283, hsa-miR-18a* and hsa-miR-26b.
The means for determining a predetermined set of miRNAs may for example comprise a microarray comprising miRNA-specific oligonucleotide probes. In a preferred embodiment, the microarray comprises miRNA-specific oligonucleotide probes for one or more miRNAs selected from the group consisting of (a) hsa-miR-361-5p, hsa-miR-23b, hsa-miR-126, hsa-miR-527, hsa-miR-29a, hsa-let-7i, hsa-miR-19a, hsa-miR-28-5p, hsa-miR-185*, hsa-miR-23a, hsa-miR-1914*, hsa-miR-29c, hsa-miR-505*, hsa-let-7d, hsa-miR-378, hsa-miR-29b, hsa-miR-604, hsa-miR-29b, hsa-let-7b, hsa-miR-299-3p, hsa-miR-423-3p, hsa-miR-18a*, hsa-miR-1909, hsa-let-7c, hsa-miR-15a, hsa-miR-425, hsa-miR-93*, hsa-miR-665, hsa-miR-30e, hsa-miR-339-3p, hsa-miR-1307, hsa-miR-625*, hsa-miR-193a-5p, hsa-miR-130b, hsa-miR-17*, hsa-miR-574-5p and hsa-miR-324-3p or (b) hsa-miR-126, hsa-miR-423-5p, hsa-let-7i, hsa-let-7d, hsa-miR-22, hsa-miR-15a, hsa-miR-98, hsa-miR-19a, hsa-miR-574-5p, hsa-miR-324-3p, hsa-miR-20b, hsa-miR-25, hsa-miR-195, hsa-let-7e, hsa-let-7c, hsa-let-7f, hsa-let-7a, hsa-let-7g, hsa-miR-140-3p, hsa-miR-339-5p, hsa-miR-361-5p, hsa-miR-1283, hsa-miR-18a*, hsa-miR-26b, hsa-miR-604, hsa-miR-423-3p and hsa-miR-93*. In a preferred embodiment, the microarray comprises oligonucleotide probes for determining at least 7, preferably at least 10, 15, 20 or 24 of the indicated groups (a) and (b) of miRNAs. It is particularly preferred to include oligonucleotide probes for determining the 24, 20, 15, 10 or at least 7 of the first mentioned miRNAs in the order of their MI-values or their performance in the t-tests as shown in the tables in
In a particularly preferred embodiment, the microarray comprises oligonucleotide probes for determining the miRNAs hsa-miR-126, hsa-miR-423-5p, hsa-let-7i, hsa-let-7d, hsa-miR-22, hsa-miR-15a, hsa-miR-98, hsa-miR-19a, hsa-miR-574-5p, hsa-miR-324-3p, hsa-miR-20b, hsa-miR-25, hsa-miR-195, hsa-let-7e, hsa-let-7c, hsa-let-7f, hsa-let-7a, hsa-let-7g, hsa-miR-140-3p, hsa-miR-339-5p, hsa-miR-361-5p, hsa-miR-1283, hsa-miR-18a* and hsa-miR-26b.
The microarray can comprise oligonucleotide probes obtained from known or predicted miRNA sequences. The array may contain different oligonucleotide probes for each miRNA, for example one containing the active mature sequence and another being specific for the precursor of the miRNA. The array may also contain controls such as one or more sequences differing from the human orthologs by only a few bases, which can serve as controls for hybridization stringency conditions. It is also possible to include viral miRNAs or putative miRNAs as predicted from bioinformatic tools. Further, it is possible to include appropriate controls for non-specific hybridization on the microarray.
The invention also relates to sets of oligo- or polynucleotides for diagnosing lung cancer comprising the sequences of at least 5, preferably at least 7, 10, 15, 20 or all of the indicated miRNAs, and/or the complement of such sequences. It is particularly preferred to include oligo- or polynucleotides for detecting of the most significant miRNAs, which are represented by their order in the table depicted in
Another embodiment of the present invention relates to a method for the assessment of a clinical condition related to lung cancer of a patient.
Recent developments have shown that there is a tendency towards smaller sets of biomarkers for the detection of diseases. However, for single biomarkers and small biomarker sets, there is only a basic understanding whether these biomarkers are specific for only the single diseases or whether they occur in any other disease.
Therefore, the present inventors developed a novel class of diagnostic tests improving the current test scenarios. The inventors found out that a variety of diseases are correlated with a specific expression profile of miRNAs. In case a patient is affected by a particular disease, several miRNAs are present in larger amounts compared to a healthy normal control, whereas the amount of other miRNAs is decreased. Interestingly, the amount of some miRNAs is deregulated, i.e. increased or decreased, in more than one disease. The miRNA profile for a particular disease therefore shows conformity with the miRNA profile of other diseases in regard of individual miRNAs while other miRNAs show significant differences. If the expression profile of a large variety of miRNAs in a biological sample of a patient is measured, the comparison of the expression profile with a variety of reference expression profiles which are each characteristic for different diseases makes it possible to obtain information about the clinical condition of a certain patient and to determine, which disease(s) is/are present in said patient.
A further subject matter of the invention is a method for the assessment of a clinical condition related to lung cancer of a patient comprising the steps
The inventors found out that the above method for the assessment of a clinical condition makes it possible to carry out an integrative diagnosis of a wide variety of diseases, particularly including lung cancer. Comparing a miRNA profile obtained from a biological sample of a patient whose clinical condition is not known with a plurality of reference profiles characteristic for different diseases enables the diagnosis of a wide variety of diseases with high specificity and sensitivity.
A “biological sample” in terms of the invention means a sample of biological tissue or fluid as described hereinabove. Examples of biological samples are sections of tissues, blood, blood fractions, plasma, serum, urine or samples from other peripheral sources. Preferred biological samples are blood, plasma and/or serum samples including blood fractions such as PBMC.
The set of miRNAs determined in step (d) preferably includes a large number of different miRNAs. It is particularly preferred to use at least 10, 20, 30, 50, preferably at least 100, 200, 500 or 1,000 miRNAs. Most preferably, all known miRNAs are included in the set of miRNAs determined in step (b) Such a complex set of miRNA-biomarkers enables a diagnosis with higher specificity and sensitivity compared to single biomarkers or sets of only a few dozens of such markers.
The determination of the set of miRNAs can be done as described herein above. Preferably, the determination is done on an experimental platform which shows a high degree of automation to minimize experimental variations, measure results time- and cost-efficiently, measures results highly reproduceably and be able for measuring more than one sample at once in order to ensure a high throughput.
Step (c) preferably includes a comparison of the miRNA profile measured for a patient with a large number of different reference profiles to provide information about the presence of as many different diseases as possible. The reference expression profiles may be laid down in a database, e.g. an Internet database, a centralized or a decentralized database. The reference profiles do not necessarily have to include information about all miRNAs included in step (b), which are determined in the sample of the patient. It is, according to the invention, sufficient if the reference profile provides information on those miRNAs which are altered to a large extent compared to the condition of a healthy individual in case of the presence of a disease. Alternatively, the said relevant reference may be a mathematical function or algorithm.
Preferably, an miRNA reference profile or the relevant reference according to the invention provides information on miRNA expression characteristic for a particular disease in the same type of biological sample as used in step (b) for determining a predetermined set of miRNAs in a sample from a patient. This means that, if a patient with an unknown disease is to be classified with the analysis of a blood sample, the comparison is preferably made with miRNA reference expression profiles, which do also relate to the miRNA expression pattern in a blood sample.
The reference profiles or the relevant reference characteristic for particular diseases provide information on one or more miRNAs, which are, in case of the disease, highly deregulated, for example strongly increased or decreased, as compared to a healthy condition. It is not necessary for the reference profiles to provide information about all miRNAs included in the set of biomarkers determined in step (b). However, the more miRNAs are included in the reference profile or relevant reference, the more precise the diagnosis will be. If, for example, a reference profile for lung cancer is included, it is preferred to include the characteristic miRNAs for lung cancer.
Another embodiment of this aspect of the invention is a kit for the assessment of a clinical condition related to lung cancer of a patient comprising
(a) means for determining a predetermined set of miRNAs in a biological sample from a patient, and
(b) a plurality of miRNA reference expression profiles characteristic for different diseases or a mathematical function that allows for the diagnosis on the basis of the data derived from the miRNA expression profiles of a patient.
The set of miRNAs to be determined in a biological sample from a patient preferably includes a large number of different miRNAs. It is particularly preferred to include all known miRNAs in the set of miRNAs to be determined. In each case, said predetermined set of miRNAs should include those miRNAs for which information is provided in the reference profiles characteristic for particular diseases. It is understood that only in case the set of miRNAs determined in a biological sample from a patient comprises those miRNAs included in the reference profile/reference for a disease, a diagnosis regarding this particular disease can be provided or otherwise the diagnosis may be less informative.
The assessment of a clinical condition of a patient according to the invention is suitable for diagnosing any diseases which are correlated with a characteristic miRNA profile. Accordingly, the kit for the assessment of a clinical condition preferably includes reference profiles/references for a plurality of diseases that are correlated with a characteristic miRNA profile. It is understood that all miRNAs that are significantly deregulated in the disease states for which reference profiles are provided should be included in the set of miRNAs to be determined in a biological sample from a patient. If the kit for the assessment of a clinical condition of a patient should provide information regarding, e.g. lung cancer or multiple sclerosis, a reference profile should be available providing information about the significantly deregulated miRNAs compared to a normal or any other relevant control individual or any other relevant control individual(s). A kit for the assessment of a clinical condition shall provide information on the presence of lung cancer, a reference profile characteristic for lung cancer should be included. Said reference profile preferably includes information on those miRNAs that are most significantly deregulated in the case of lung cancer. The relevant miRNAs are as disclosed hereinabove.
The invention will now be illustrated by the following figures and the non-limiting experimental examples.
Scheme of a miRNA hybridization assay for use in the invention.
miRNA capture probes consist of 1 miRNA probe sequence stretch that is linked to support via 3′-end or alternatively by 5′-end (not depicted here)
the miRNA probe sequence stretches are complementary to miRNA target sequences
each miRNA capture probe can bind 1 miRNA target sequences
the miRNA target sequences are labeled prior to hybridisation (e.g. by biotin labeling)
Scheme of an miRNA tandem hybridization assay for use in the invention
miRNA capture probes consist of 2 DNA-based miRNA probe sequence stretches that are linked to each other by a spacer element
the miRNA probe sequence stretches are complementary to miRNA target sequences
each miRNA capture probe can bind 2 miRNA target sequences
the spacer sequence consists of 0-8 nucleotides
the miRNA target sequences are labeled prior to hybridisation (e.g. by biotin labeling)
miRNA RAKE-Assay for use in the invention (PT Nelson et al., Nature Methods, 2004, 1(2), 1)
the miRNA capture probes consist of one miRNA probe sequence stretch (white) and one elongation element “(gray)”
probes are oriented 5′→3′, presenting a free terminal 3′-OH
the miRNA probe sequence stretch (white) is complementary to miRNA target sequences
the elongation sequences (gray) can be freely chosen and is typically between 1-12 nucleotides long, preferably a homomeric sequence
each miRNA capture probe can bind 1 miRNA target sequences
the miRNA target sequences are NOT labeled prior to hybridisation
Labeling occurs after hybridisation during elongation by polymerase extention reaction
Biochip is not reusable due to exonuclease treatment
miRNA MPEA-Assay for use in the invention (Vorwerk S. et al., Microfluidic-based enzymatic on-chip labeling of miRNAs, N. Biotechnol. 2008; 25(2-3):142-9. Epub 2008 Aug. 20)
the miRNA capture probes consist of one miRNA probe sequence stretch (white) and one elongation element (gray)
probes are oriented 3′→5′, presenting a free terminal 5′-OH
the miRNA probe sequence stretch (white) is complementary to miRNA target sequences
the elongation sequences (gray) can be freely chosen and is typically between 1-12 nucleotides long, preferably a homomeric sequence
each miRNA capture probe can bind 1 miRNA target sequences
the miRNA target sequences are NOT labeled prior to hybridisation
Labeling occurs after hybridisation during elongation by polymerase extention reaction
Biochip is reusable after removal of target/elongated target
miRNA capture probe design
Depicted is the design of a capture probe (SEQ ID NO:866) for the exemplary miRNA human mature miRNA let-7a (SEQ ID NO:17) for use in the various types of hybridization assays shown in
Spacer Element.
Capture probes (SEQ ID NO:866) for use in e.g. a tandem hybridization assay as shown in
Elongation element
A capture probe (SEQ ID NO:866), e.g. for use in a RAKE or MPEA assay as shown in
Diagram describing the general approach for determining miRNA signatures for use as biomarkers in disease diagnosis.
Overview of all miRNAs that are found to be differentially regulated in blood samples of lung cancer patients, grouped according to their mutual information (MI).
Overview of all miRNAs that are found to be differentially regulated in blood samples of lung cancer patients, grouped according to their results in t-tests.
Overview of preferred miRNAs that are found to be significantly (p<0.1) differentially regulated in blood samples of lung cancer patients.
Overview of preferred signatures of miRNAs for the diagnosis of lung cancer.
Expression of some relevant miRNAs. The bar-chart shows for 15 deregulated miRNAs the median value of cancer samples and normal samples. Here, left bars correspond to cancer samples while right bars controls.
Bar diagrams showing a classification of the accuracy, specificity and sensitivity of the diagnosis of lung cancer based on blood samples using different sizes of subsets of miRNAs. Blue bars represent accuracy, specificity and sensitivity of the diagnosis using the indicated biomarkers and red bars represent the results of the same experiments of random classifications. The relevant value is the population median (horizontal black lines inside the bars).
hsa-miR-126, hsa-miR-423-5p, hsa-let-7i and hsa-let-7d;
hsa-miR-126, hsa-miR-423-5p, hsa-let-7i; hsa-let-7d, hsa-miR-22, hsa-miR-15a, hsa-miR-98, and hsa-miR-19a;
hsa-miR-126, hsa-miR-423-5p, hsa-let-7i; hsa-let-7d, hsa-miR-22, hsa-miR-15a, hsa-miR-98, hsa-miR-19a, hsa-miR-574-5p, and hsa-miR-324-3p;
hsa-miR-126, hsa-miR-423-5p, hsa-let-7i; hsa-let-7d, hsa-miR-22, hsa-miR-15a, hsa-miR-98, hsa-miR-19a; hsa-miR-574-5p; hsa-miR-324-3p, hsa-miR-20b, hsa-miR-25, hsa-miR-195, hsa-let-7e, hsa-let-7c, and has-let-7f;
hsa-miR-126, hsa-miR-423-5p, hsa-let-7i; hsa-let-7d, hsa-miR-22, hsa-miR-15a, hsa-miR-98, hsa-miR-19a; hsa-miR-574-5p; hsa-miR-324-3p, hsa-miR-20b, hsa-miR-25, hsa-miR-195, hsa-let-7e, hsa-let-7c, hsa-let-7f; hsa-let-7a, hsa-let-7g, hsa-miR-140-3p and hsa-miR-339-5p;
hsa-miR-126, hsa-miR-423-5p, hsa-let-7i; hsa-let-7d, hsa-miR-22, hsa-miR-15a, hsa-miR-98, hsa-miR-19a; hsa-miR-574-5p; hsa-miR-324-3p, hsa-miR-20b, hsa-miR-25, hsa-miR-195, hsa-let-7e, hsa-let-7c, hsa-let-7f; hsa-let-7a, hsa-let-7g, hsa-miR-140-3p, hsa-miR-339-5p, hsa-miR-36′-5p, hsa-miR-1283, hsa-miR-18a*, hsa-miR-26b, hsa-miR-604, hsa-miR-423-3p, hsa-miR-93*, and hsa-miR-29a;
hsa-miR-126, hsa-miR-423-5p, hsa-let-71; hsa-let-7d, hsa-miR-22, hsa-miR-15a, hsa-miR-98, hsa-miR-19a; hsa-miR-574-5p; hsa-miR-324-3p, hsa-miR-20b, hsa-miR-25, hsa-miR-195, hsa-let-7e, hsa-let-7c, hsa-let-7f; hsa-let-7a, hsa-let-7g, hsa-miR-140-3p, hsa-miR-339-5p, hsa-miR-36′-5p, hsa-miR-1283, hsa-miR-18a*, hsa-miR-26b, hsa-miR-604, hsa-miR-423-3p, hsa-miR-93*, hsa-miR-29a, hsa-miR-1248, hsa-miR-210, hsa-miR-19b, hsa-miR-453, hsa-miR-126*, hsa-miR-188-3p, hsa-miR-624*, hsa-miR-505*, hsa-miR-425, hsa-miR-339-3p, hsa-miR-668, and hsa-miR-363*.
Classification of cancer samples versus controls for two individual miRNAs (miR-126 and miR-196). Blue bars correspond to cancer samples, while red bars correspond to controls.
Scatterplot of fold quotients of rt-qPCR (x-axis) and microarray experiments (y-axis).
The mutual information of all miRNAs that have higher information content than the best permutation test (upper red line). The middle red line denotes the 95% quantile of the 1000 permutation tests and the bottom red line the mean of the permutation experiments, corresponding to the background MI.
Box plots of the classification accuracy, specificity and sensitivity of the set of 24 best miRNAs (obtained with radial basis function support vector machine). These miRNAs allow for the discrimination between blood cells of lung cancer patients and blood cells of controls with an accuracy of 95.4% [94.9%-95.9%], a specificity of 98.1%[97.3%-98.8%], and a sensitivity of 92.5%[91.8%-92.5%]. The permutation tests showed significantly decreased accuracy, specificity and sensitivity with 94.2% [47.2%-51.3%], 56.9%[54.5%-59.3%] and 40.6%[37.9%-43.4%], respectively, providing evidence that the obtained results are not due to an overfit of the statistical model on the miRNA fingerprints.
Lung Cancer
1. Material and Methods
1.1 Samples
Blood samples were obtained with patients' informed consent. The patient samples stem from 17 patients with non-small cell lung carcinoma and normal controls. Normal samples were obtained from 19 different volunteers. More detailed information of patients and controls is given in Table 1.
1.2 miRNA Microarray Screening
Blood of lung cancer patients and volunteers without known disease was extracted in blood collection tubes which prevent RNA degradation (PAXgene Blood RNA tubes BD, Franklin Lakes, N.J. USA). For each blood donor, 5 ml of peripheral blood were obtained. Total RNA was extracted from blood cells using the miRNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and the RNA has been stored at−70.degree. C. Samples were analyzed with the Geniom Realtime Analyzer (GRTA, febit gmbh, Heidelberg, Germany) using the Geniom Biochip miRNA homo sapiens. Each array contains 7 replicates of 866 miRNAs and miRNA star sequences as annotated in the Sanger mirBase 12.0 (Griffiths-Jones, Moxon at al. 2005; Griffiths-Jones, Saini et al. 2008). Sample labelling with Biotin has been carried out either by using the miRVANA™ miRNA Labelling Kit (Applied Biosystems Inc, Foster City, Calif. USA) or by multifluidic-based enzymatic on-chip labelling of miRNAs (MPEA (Vorwerk, Canter et al. 2008), incorporated herein by reference).
Following hybridization for 16 hours at 42° C. the biochip was washed automatically and a program for signal enhancement was processed with the GRTA. The resulting detection pictures were evaluated using the Geniom Wizard Software. For each array, the median signal intensity was extracted from the raw data file such that for each miRNA seven intensity values have been calculated corresponding to each replicate copy of mirBase on the array. Following background correction, the seven replicate intensity values of each miRNA were summarized by their median value. To normalize the data across different arrays, quantile normalization (Bolstad, Irizarry et al, 2003) was applied and all further analyses were carried out using the normalized and background subtracted intensity values.
1.3 Statistical Analysis
After having verified the normal distribution of the measured data, parametric t-tests (unpaired, two-tailed) were carried out for each miRNA separately, to detect miRNAs that show a different behavior in different groups of blood donors. The resulting p-values were adjusted for multiple testing by Benjamini-Hochberg (Hochberg 1988; Benjamini and Hochberg 1995) adjustment. Moreover, the Mutual Information (MI) (Shannon 1984) was computed as a measure to access the diagnostic value of single miRNA biomarkers. To this end, all biomarkers were transformed to z-scores and binned in three bins before the MI values of each biomarker, and the information whether the marker has been measured from a normal or lung cancer sample, was computed. In addition to the single biomarker analysis classification of samples using miRNA patterns was carried out using Support Vector Machines (SVM, (Vapnik 2000)) as implemented in the R (Team 2008) e1071 package. In detail, different kernel (linear, polynomial, sigmoid, radial basis function) Support Vector Machines were evaluated, where the cost parameter was sampled from 0.01 to 10 in decimal powers. The measured miRNA profiles were classified using 100 repetitions of standard 10-fold cross-validation. As a subset selection technique a filter approach based on t-test was applied. In detail, the s miRNAs with lowest p-values were computed on the training set in each fold of the cross validation, where s was sampled from 1 to 866. The respective subset was used to train the SVM and to carry out the prediction of the test samples. As result, the mean accuracy, specificity, and sensitivity were calculated together with the 95% Confidence Intervals (95% CI) for each subset size. To check for overtraining permutation tests were applied. Here the class labels were sampled randomly and classifications were carried out using the permuted class labels. All statistical analyzes were performed using R (Team 2008).
2. Results
2.1 miRNA Experiments
The expression of 866 miRNAs and miRNA star sequences was analyzed in blood cells of 17 patients with NSCLC. As a control blood cells of 19 volunteers without known disease were used (see also Materials and Methods).
Following RNA isolation and labeling by miRVANA™ miRNA Labeling Kit, the miRNA expression profiles were measured by the Geniom Bioship miRNA homo sapiens in the GRTA (febit gmbh, Heidelberg). Following intensity value computation and quantile normalization of the miRNA profiles (Bolstad, Irizarry et al. 2003), a mean correlation value of 0.97 for technical replicates was determined by using purchased total RNA from Ambion (four heart and four liver replicates). For the biological replicates the different tumor samples were compared between each other and the different normal samples between each other. The biological replicates showed a mean correlation of 0.87 and a variance of 0.009.
2.2 Ruling Out the Influence of Age and Gender
To cross-check that age and gender do not have an influence on our analysis, t-tests were computed for the normal samples. In the case of males versus females there was no statistically significant deregulated miRNA. The most significant miRNA, hsa-miR-423, showed an adjusted significance level of 0.78.
To test for the influence of donor age the profiles obtained from samples obtained from the oldest versus youngest patients were compared by splitting the group in half based on age. Here, the most significant miRNA, miR-890, obtained an adjusted p-value of 0.87. As for gender, there were no deregulated miRNAs, thus providing evidence that age and gender do not have a substantial influence on the miRNA profiles.
2.3 Single Deregulated miRNAs
Hypothesis testing was applied to identify miRNAs deregulated in the blood cells of lung cancer patients as compared to the blood cells of the controls.
Following verification of an approximately normal distribution, two-tailed unpaired t-tests were performed for each miRNA. The respective p-values were adjusted for multiple testing by the Benjamini-Hochberg approach (Hochberg 1988; Benjamini and Hochberg 1995). In total 27 miRNAs significantly deregulated in blood cells of lung cancer patients as compared to the controls were detected. A complete list of deregulated miRNAs is given in the tables in
To validate the findings, the miRNA profiling was repeated using an enzymatic on-chip labeling technique termed MPEA (Microfluidic-based enzymatic on-chip labeling of miRNAs). For this control experiment, 4 out of the 17 lung cancer patients and 10 of the controls were used. Hereby, 100 differentially regulated miRNAs were detected. The miRNAs that were most significantly deregulated include hsa-miR-1253 with a p-value of 0.001, hsa-miR-126 with a p-value of 0.006, hsa-let-7d with a p-value of 0.006, and hsa-let-7f with a p-value of 0.006. Of the previously identified 27 miRNAs 12 were detected to be significant in the second experiment, while the remaining miRNAs showed increased p-values. The correlation of fold changes was 0.62. Also other members of the let-7 family were confirmed as deregulated in blood cells of lung cancer patients. Furthermore, it was confirmed that the majority of the deregulated miRNAs were down-regulated in patients' blood samples. Here, 62% of the deregulated miRNAs showed decreased intensity values in lung cancer samples.
As a further control experiment an expression analysis by qRT-PCR was performed. As a test sample the fold changes of has-miR-106b, miR-98, miR-140-3p, let-7d, mir-126, and miR-22 were analyzed in blood cells of eight tumor patients and five controls. The fold quotients detected by the Geniom Biochip experiments agreed very well with the qRT-PCR experiments, as demonstrated by an excellent R2 value of 0.994. The fold quotients are presented as a scatterplot together with the R2 value and the regression line in
2.4 Diagnostic Value of miRNA Biomarkers
Mutual Information (MI) (Shannon 1984) is an adequate measure to estimate the overall diagnostic information content of single biomarkers (Keller, Ludwig et al. 2006). In the present study, Mutual Information is considered as the reduction in uncertainty about the class labels ‘0’ for controls and ‘1’ for tumor samples due to the knowledge of the miRNA expression. The higher the value of the MI of a miRNA, the higher is the diagnostic content of the respective miRNA.
The MI of each miRNA with the class labels was computed. First, a permutation test was carried out to determine the background noise of the miRNAs, e.g. the random information content of each miRNA. 1000 miRNAs (with replacements) were randomly selected and the class labels were sampled for each miRNA. These permutation tests yielded a mean MI value of 0.029, a 95% quantile of 0.096 and a value of 0.217 for the highest random MI. Second, the MI values were calculated for the comparison between the miRNAs in blood cells of tumor patients and controls. The overall comparison of the 866 miRNAs yielded significantly increased MI values with a two-tailed p-value of ≦10−10 as shown by an unpaired Wilcoxon Mann-Whitney test (Wilcoxon 1945; Mann and Wilcoxon 1947). The miRNA hsa-miR-361-5p showed the highest MI with a value of 0.446. The miRNAs with the best significance values as computed by the t-test, namely hsa-miR-126 and hsa-miR-98, were also among the miRNAs showing the highest MI values. In total 37 miRNAs with MI values higher than the highest of 1000 permuted miRNAs and 200 miRNAs with MI values higher than the 95% quantile were detected (
2.5 Evaluating Complex Fingerprints
Even single miRNAs with highest MI values are not sufficient to differentiate between blood cells of tumor patients as compared to controls with high specificity. For example, the has-miR-126 separates blood cells of tumor patients from blood cells of healthy individuals with a specificity of 68%, only. In order to improve the classification accuracy the predictive power of multiple miRNAs was combined by using statistical learning techniques. In detail, Support Vector Machines with different kernels (linear, polynomial, sigmoid, radial basis function) were applied to the data and a hypothesis test was carried out based subset selection as described in Material and Methods. To gain statistical significance 100 repetitions of 10-fold cross validation were carried out. Likewise, 100 repetitions for the permutation tests were computed.
The best results were obtained with radial basis function Support Vector Machines and a subset of 24 miRNAs. These miRNAs allowed for the discrimination between blood cells of lung tumor patients and blood cells of controls with an accuracy of 95.4% [94.9%-95.9%], a specificity of 98.1% [97.3%-98.8%], and a sensitivity of 92.5% [91.8%-92.5%]. The permutation tests showed significantly decreased accuracy, specificity, and sensitivity with 49.2% [47.2%-51.3%], 56.9% [54.5%-59.3%] and 40.6% [37.9%-43.4%], respectively (
3. Discussion
While complex miRNA expression patterns have been reported for a huge variety of human tumors, information there was only one study analyzing miRNA expression in blood cells derived from tumor patients. In the following the present miRNA expression profiling is related to both the miRNA expression in blood cells and in cancer cells of non-small cell lung cancer patients. A significant down-regulation of has-miR-126 was found that was recently detected in blood cells of healthy individuals, but not in blood cells of lung cancer patients (Chen, Ba et al. 2008). Down-regulation of has-miR-126 was also found in lung cancer tissue in this study. Functional studies on has-miR-126 revealed this miRNA as a regulator of the endothelial expression of vascular cell adhesion molecule 1 (VCAM-1), which is an intercellular adhesion molecule expressed by endothelial cells focuses on the identification of miRNAs in serum of patients with cancer and other diseases or healthy controls. Since most miRNAs are expressed in both, serum and blood cells of healthy controls, most serum miRNAs are likely derived from circulating blood cells. Since there was only a weak correlation between the miRNA expression in serum and blood cell, miRNA expression appears to be deregulated in either serum or blood cells of cancer patients. The present experimental example focused on the analysis of miRNA expression in blood cells of non-small cell lung cancer patients and healthy controls. Significant downregulation of has-miR-126 was found that was recently detected in blood cells of healthy individuals, but not in blood cells of lung cancer patients (Harris, YamakuchiChen, Ba et al. 2008). Downregulation of has-miR-126 was also found in lung cancer tissue (Yanaihara, Caplen et al. 2006). Functional studies on has-miR-126 revealed this miRNA as regulator of the endothelial expression of vascular cell adhesion molecule 1 (VCAM-1), which is an intercellular adhesion molecule expressed by endothelial cells (Harris, Yamakuchi et al. 2008). hsa-miR-126 is also reported to be an inhibitor of cell invasion in non-small cell lung cancer cell lines, and down-regulation of this miRNA 126 might be a mechanism of lung cancer cells to evade these inhibitory effects (Crawford, Brawner et al. 2008). Members of the has-let-7 family that were found down-regulated in the present invention were the first miRNAs reported as de-regulated in lung cancer (Johnson, Grosshans et al. 2005). This down-regulation of the let-7 family in lung cancer was confirmed by several independent studies (Takamizawa, Konishi et al. 2004; Stahlhut Espinosa and Slack 2006; Tong 2006; Zhang, Wang et al. 2007; Williams 2008). The present data are also in agreement with a recent study showing the down-regulation of has-let-7a, has-let-7d, has-let-7f, has-let-7g, and has-let-7i in blood cells of lung cancer patients (Chen, Ba et al. 2008). Notably, down-regulation of let-7 in lung cancer was strongly associated with poor clinical outcome (Takamizawa, Konishi et al. 2004). The let-7 family members negatively regulate oncogene RAS (Johnson, Grosshans et al. 2005). The miRNA has-miR-22 that showed a high MI value and up-regulation in the present study, was recently also reported to be up-regulated in blood cells of lung cancer patients (Chen, Ba et al. 2008). The miRNA has-miR-19a that also showed a high MI value and up-regulation in the present study was reported to be up-regulated in lung cancer tissue (Hayashita, Osada et al. 2005; Cahn and Croce 2006). In contrast, has-miR-20a, which is significantly down-regulated in the present experiments, was reported as up-regulated in lung cancer tissue (Hayashita, Osada et al. 2005; Calin and Croce 2006). The up-regulation of has-miR-20a was found in small-cell lung cancer cell lines, the present study investigated only NSCLC. In summary, there is a high degree of consistency between miRNA expression found in the peripheral blood cells of lung cancer patients and miRNA expression in lung cancer tissue (Takamizawa, Konishi et al. 2004; Hayashita, Osada et al. 2005; Lu, Getz et al. 2005; Calin and Croce 2006; Stahlhut Espinosa and Slack 2006; Tong 2006; Volinia, Calin et al. 2006; Yanaihara, Caplen et al. 2006; Zhang, Wang et al. 2007; Williams 2008).
Some of the deregulated miRNAs identified in the present invention are also reported as de-regulated in other cancer entities, e.g. has-miR-346 in gastritic cancer, has-miR-145 in bladder cancer, and has-miR-19a in hepatocellular carcinoma and B-cell leukemia (Alvarez-Garcia and Miska 2005; He, Thomson et al. 2005; Feitelson and Lee 2007; Guo, Huang et al. 2008; Ichimi, Enokida et al. 2009). In addition, miRNAs with high diagnostic potential e.g. high MI value, were found that were not yet related to cancer as for example has-miR-527 or has-mir-361-5p that were both up-regulated in blood cells of lung cancer patients.
Besides the deregulation of single miRNAs, the overall expression pattern of miRNAs in peripheral blood cells of lung cancer patients were analyzed in comparison to the pattern in blood cells of healthy controls. Recently, Chen et al. (Chen, Ba et al. 2008) reported a high correlation of 0.9205 between miRNA profiles in serum and miRNA profiles in blood cells, both in healthy individuals. The correlation of the miRNA profiles between serum and blood cells in lung cancer patients were significantly lower (0.4492). These results are indicative of deregulated miRNAs in blood and/or serum of patients and are in agreement with the present data that show the deregulation of miRNAs in the blood cells of lung carcinoma patients. These deregulated miRNAs can be used to differentiate patients with lung cancer from normal controls with high specificity and sensitivity. This is the first evidence for the diagnostic potential of miRNA expression profiles in peripheral blood cells of cancer patients and healthy individuals.
| Number | Date | Country | Kind |
|---|---|---|---|
| 09015668 | Dec 2009 | EP | regional |
This application is a 35 U.S.C. 371 National Phase Entry Application from PCT/EP2010/057942, filed Jun. 7, 2010, which claims the benefit of U.S. Provisional Applications Nos. 61/184,452 filed Jun. 5, 2009, 61/213,971 filed Aug. 3, 2009, 61/287,521 filed Dec. 17, 2009 and European Patent Application No. 09015668.8 filed on Dec. 17, 2009, the disclosures of which are incorporated herein in their entirety by reference.
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| PCT/EP2010/057942 | 6/7/2010 | WO | 00 | 1/19/2012 |
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| WO2010/139810 | 12/9/2010 | WO | A |
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| 20070050146 | Bentwich et al. | Mar 2007 | A1 |
| 20100184034 | Bankaitis-Davis | Jul 2010 | A1 |
| 20110251098 | Showe | Oct 2011 | A1 |
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| 2009036332 | Mar 2009 | WO |
| 2009055979 | May 2009 | WO |
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