JP5688497B2 - Methods and compositions for predicting postoperative prognosis in patients with lung adenocarcinoma - Google Patents

Description

The present invention relates to methods and compositions for predicting postoperative prognosis of lung adenocarcinoma patients.
The present invention also relates to a method for classifying lung adenocarcinoma into its subtypes.

  Lung cancer is the leading cause of cancer-related death in developed countries (Non-patent Documents 1 and 2). Currently, tumor classification is large and based on microscopic histological features, while significant changes in clinical judgment are sometimes evident, and adenocarcinoma is the highest, even within certain tissue types It is known to show a degree of morphological and clinical diversity (Non-Patent Document 3). Therefore, a more detailed and accurate means to classify non-small cell lung cancer (NSCLC), especially adenocarcinoma, has improved not only a better understanding of the etiology but also today's inappropriate diagnostic capabilities. It is highly expected to enable more effective treatments.

  Recent development of microarray technology has made it possible to correlate various clinical parameters with gene expression profiles of individual cases (Non-Patent Documents 4 to 9). To date, various other groups, including our group, have reported that expression profiling can reproduce the morphological classification of NSCLC, and several studies have shown that adenocarcinoma is further sub- It is shown that classification is possible (Non-Patent Documents 10 to 13).

  However, the results of these sub-classifications reported so far are quite different from each other, it is difficult to simply adapt to those findings, and it is also difficult to get some clear conclusions. It should also be pointed out that conventional expression profiling studies provide very little information about the relationship between expression profiles and basic genetic changes (Non-Patent Document 14). Such information is inherently known to be important in clarifying the pathogenesis of lung cancer.

Incidentally, the discovery of an activating mutation of epidermal growth factor receptor (EGFR) is one of the most noteworthy findings in the field of lung cancer research today (Non-patent Documents 15 and 16). EGFR mutations are present in a subset of lung adenocarcinoma (Non-Patent Documents 15-20) and tumors with this mutation are highly sensitive to gefitinib (selective EGFR tyrosine kinase inhibitor; eg Iressa ^™ ) Is shown (Non-patent Documents 15 to 17, 19). A good clinical response to gefitinib has been observed most frequently in non-smoking adenocarcinoma patients of Japanese and other Asian women (Non-Patent Documents 21 and 22). The inventors have also found that EGFR mutations are frequently detected in lung adenocarcinoma, a so-called terminal respiratory unit (TRU) adenocarcinoma, which is morphologically considered to be derived from peripheral airway epithelial cells. (Non-Patent Documents 23 and 24).

Minna JD et al. (Edit), Harrison's Principals of Internal Medicine (16th edition), McGraw-Hill, 2001, pp 506-516. Statistics and Information Department, Minister's Secretariat: Vital Statistics of Japan 2001 Vol. 3, Ministry of Health, Labor and Welfare, Japan, 2003, pp 384-411 Travis WD, et al., Pathology and genetics of tumors of the lang, pleura, thymus and heart, World Health Organization of AR4, L Perou CM et al., Nature 406: 747-52, 2000 Alizadeh AA et al., Nature 403: 503-11, 2000. Shipp MA et al., Nat Med 8: 68-74, 2002. Singh D et al., Cancer Cell 1: 203-9, 2002 van't Veer LJ et al., Nature 415: 530-6, 2002 Hedenfalk I et al., Proc Natl Acad Sci USA 100: 2532-7, 2003 Garber ME et al., Proc Natl Acad Sci USA 98: 13784-9, 2001 Bhattacharjee A et al., Proc Natl Acad Sci USA 98: 13790-5, 2001 Beer DG et al., Nat Med 8: 816-24, 2002 Tomida S et al., Oncogene 23: 5360-70, 2004 Meyerson M et al., J Clin Oncol 23: 3219-26, 2005 Paez JG et al., Science 304: 1497-500, 2004. Lynch TJ et al., N Engl J Med 350: 2129-39, 2004. Pao W et al., Proc Natl Acad Sci USA 101: 13306-11, 2004. Kosaka T et al., Cancer Res 64: 8919-23, 2004 Mitsumi T et al., J Clin Oncol 23: 2513-20, 2005 Shigematsu H et al., J Natl Cancer Inst 97: 339-46, 2005 Fukuoka M et al., J Clin Oncol 21: 2237-46, 2003. Kris MG et al., Jama 290: 2149-58, 2003. Yatabe Y et al., Am J Surg Pathol 29: 633-639, 2005. Yatabe Y et al., Am J Surg Pathol 26: 767-73, 2002.

  Conventionally, in an actual clinical setting, human lung cancer has been diagnosed based on clinical and pathological classification. That is, since the conventional diagnosis relies on morphological diagnosis by microscopic observation, it is not objective and information on the prognosis of the patient is extremely limited. In addition, in the attempt of diagnosis based on comprehensive and systematic expression profiles, information that should have genetic abnormalities closely related to the development and progression of lung cancer is integrated with the expression profile and incorporated into the diagnosis in a useful form. I couldn't do it.

  In this situation, we analyzed simultaneously a broad expression profile and EGFR, p53 and K-ras mutation status in 149 NSCLC cases, including 90 adenocarcinomas, The purpose of this study was to establish a taxonomy defined by expression profiles related to clinicopathology.

  In addition, we have used this taxonomy to significantly increase prevalence and clear EGFR mutations in one of the two major subtypes identified in lung adenocarcinoma (TRU type). The purpose was to clarify the effects on prognosis.

  In the present application, the present inventors have found four gene sets identified by comprehensive gene analysis of human lung cancer, and by using these sets, lung adenocarcinoma is objectively clinically based on its expression profile. It was found that the group can be broadly divided into two main groups that are meaningful in terms of molecular biology, and one of them can be divided into two subgroups.

  As a result of further research based on these findings, the use of these gene sets enables prediction of postoperative prognosis in patients with lung adenocarcinoma and is important for the development and progression of lung cancer. We have succeeded in detecting the relationship between mutations in the EGFR gene, which plays a role, and prognosis. Such new findings have made it possible to provide important information for determining NSCLC, particularly lung adenocarcinoma treatment.

  Therefore, in summary, the present invention has the following features.

In the first aspect, the present invention relates to a method for predicting the postoperative prognosis of a patient having lung adenocarcinoma in vitro, wherein the method is a subtype of TRU type or non-TRU type. And then identify TRU type lung adenocarcinoma as either TRU-a type or TRU-b type, and if TRU-b type, postoperative Including determining that the prognosis is good, or that the TRU-a type or non-TRU type has a poor postoperative prognosis, wherein the TRU type, non-TRU type, TRU-a type, and TRU type -b type is a TRU-type adenocarcinoma or a TRU-a type adenocarcinoma for one or more genes of the following corresponding gene set in the biological sample of the patient Identified by measuring the relative expression level difference between TRU-b type adenocarcinoma That, as well as,
The gene set belonging to the TRU type has the following Unigene registration number:
Hs.512690, Hs.153322, Hs.218366, Hs.220629, Hs.436996, Hs.435759, Hs.104555, Hs.247824, Hs.127821, Hs.480281, Hs.529117, Hs.545862, Hs. 391561, Hs.479372, Hs.533055, Hs.550526, Hs.322854, Hs.465720, Hs.356664, Hs.26630, Hs.534496, Hs.85962, Hs.211267, Hs.128041, Hs.534458, Hs.495774, Hs.437806, Hs.133062, Hs.501758, Hs.444535, Hs.495480, Hs.326561, Hs.483906, Hs.169943, Hs.271285, Hs.158339, Hs.62604, Hs. 469359, Hs.436657, Hs.8417, Hs.155538, Hs.533526, Hs.512756, Hs.87191, Hs.463079, Hs.513779, Hs.476209, Hs.279580, Hs.351544, Hs.269408, Hs.134807, Hs.482417, Hs.176626, Hs.465643, Hs.183390, Hs.411299, Hs.234027, Hs.109358, Hs.103983, Hs.26216, Hs.534352, Hs.240457, Hs. 516036, Hs.144875, Hs.411312, Hs.103989, Hs.537722, Hs.333130, Hs.517962, Hs.90250, Hs.478930, Hs.121629, Hs.194061, Hs.520627, Hs.348012, Hs.522836, Hs.1376, Hs.520049, Hs.512856, Hs.355236, Hs.349470, Hs.476231, Hs.137556, Hs.390567, Hs .368353, Hs.412792, Hs.449207, Hs.527095, Hs.118722, Hs.377090, Hs.232696, Hs.447544, Hs.372773, Hs.2222055, Hs.511839, Hs.153299, Hs.434374 , Hs.287729, Hs.553740, Hs.127189, Hs.497723, Hs.181973, Hs.173656, Hs.451956, Hs.184507, Hs.532492, Hs.370904, Hs.460468, Hs.520612, Hs .436667, Hs.125116, Hs.459391, Hs.450320, Hs.149769, Hs.325890, Hs.356820, Hs.289319, Hs.73893, Hs.129493, Hs.515069, Hs.34560, Hs.477278 , Hs.351571, Hs.112087, Hs.154224, Hs.125950, Hs.438016, Hs.367956, Hs.553778, Hs.329266, Hs.479658, Hs.458713, Hs.249196, Hs.467529, Hs .145061, Hs.49653, Hs.129227, Hs.313343, Hs.194554, Hs.123114, Hs.126561, Hs.42091, Hs.369385, Hs.98661, Hs.458306, Hs.148584, Hs.501684 , Hs.422466, Hs.523732, Hs.525557, Hs.1372, Hs.379097, Hs.208124, Hs.389311, Hs.2561, Hs.117545, Hs.446388, Hs.2813, Hs.473894, Hs .502092, Hs.524479, Hs.314261, Hs.382306, Hs.458252, Hs.380222, Hs.379636, Hs.302034, Hs.253495, Hs.3 45877, Hs.259563, Hs.528569, Hs.152337, Hs.436317, Hs.546408, Hs.46700, Hs.1027, Hs.151219, Hs.279611, Hs.310456, Hs.520319, Hs.406976, Hs.181245, Hs.449621, Hs.515465, Hs.310540, Hs.554891, Hs.449601, Hs.355394, Hs.380710, Hs.171995, Hs.449585, Hs.522484, Hs.298023, Hs. 520339, a gene having Hs.121443, or a variant, homologue or derivative thereof,
The gene set belonging to the non-TRU type is the following UniGene registration number:
Hs.62661, Hs.200804, Hs.533185, Hs.461329, Hs.479270, Hs.446201, Hs.35086, Hs.500761, Hs.44298, Hs.469030, Hs.309767, Hs.441047, Hs. 98309, Hs.31409, Hs.518299, Hs.532870, Hs.196534, Hs.108106, Hs.289319, Hs.69771, Hs.374378, Hs.369422, Hs.368641, Hs.302963, Hs.530461, Hs.1955, Hs.513726, Hs.148767, Hs.523220, Hs.525796, Hs.271264, Hs.69321, Hs.231367, Hs.500761, Hs.528304, Hs.148685, Hs.87417, Hs. 164060, Hs.514843, Hs.418416, Hs.126521, Hs.519839, Hs.103834, Hs.279840, Hs.497741, Hs.531457, Hs.226390, Hs.480143, Hs.473721, Hs.369762, Hs.514527, Hs.204238, Hs.3104, Hs.519873, Hs.519909, Hs.179718, Hs.103183, Hs.520210, Hs.444683, Hs.234545, Hs.80976, Hs.311187, Hs. 89497, Hs.444118, Hs.541635, Hs.477898, Hs.511776, Hs.434886, Hs.117299, Hs.252451, Hs.468058, Hs.21554, Hs.165904, Hs.445244, Hs.413924, Hs.99120, Hs.521171, Hs.462379, Hs.481860, Hs.489207, Hs.414407, Hs.505575, Hs.516826, Hs.62180, Hs.368934 Hs. 530509, Hs. 278906, Hs. 511987, Hs. 444082, Hs. 471873, Hs. 24583, or a variant, homologue or derivative thereof,
The gene set belonging to the TRU-a type has the following UniGene registration number:
Hs.220629, Hs.153322, Hs.127821, Hs.218366, Hs.436996, Hs.247824, Hs.550526, Hs.62604, Hs.104555, Hs.465720, Hs.480281, Hs.495480, Hs. 545862, Hs.1051, Hs.133062, Hs.115263, Hs.349470, Hs.495774, Hs.155538, Hs.435759, Hs.128041, Hs.465643, Hs.169943, Hs.534496, Hs.379010, Hs.184507, Hs.469359, Hs.2012, Hs.109358, Hs.438016, Hs.326561, Hs.367956, Hs.271285, Hs.176626, Hs.137556, Hs.437806, Hs.240457, Hs. 533055, Hs.532492, Hs.85962, Hs.268698, Hs.520627, Hs.524479, Hs.449585, Hs.158339, Hs.522836, Hs.279580, Hs.444535, Hs.446388, Hs.149769, Hs.513779, Hs.103983, Hs.512756, Hs.348012, Hs.467529, Hs.434374, Hs.389311, Hs.49653, Hs.424542, Hs.436667, Hs.516036, Hs.533526, Hs. 463079, Hs.310456, Hs.125950, Hs.351571, Hs.478930, Hs.483906, Hs.259563, Hs.249196, Hs.412792, Hs.183390, Hs.87191, Hs.171995, Hs.117545, Hs.554891, Hs.351544, Hs.368353, Hs.482417, Hs.126561, Hs.154224, Hs.232696, Hs.520319, Hs.153299, Hs.449621, Hs.502092, Hs.537722, Hs.127189, Hs.525589, Hs.476231, Hs.527095, Hs.511839, Hs.372773, Hs.112087, Hs.458252, Hs.181973, Hs. 289319, Hs.25333, Hs.513075, Hs.356820, Hs.549577, Hs.380222, Hs.46700, Hs.129227, Hs.100431, Hs.129493, Hs.173656, Hs.9613, Hs.301478, Hs.553740, Hs.151219, Hs.369385, Hs.525383, Hs.473721, Hs.375624, Hs.355236, Hs.116724, Hs.9613, Hs.418055, or variants or homologues thereof Or a derivative, and
The gene set belonging to the TRU-b type has the following UniGene registration number:
Hs.334873, Hs.180878, Hs.126521, Hs.519033, Hs.187636, Hs.518448, Hs.322761, Hs.31409, Hs.524513, Hs.253495, Hs.436437, Hs.148989, Hs. 279575, Hs.75668, Hs.470791, Hs.104476, Hs.494496, Hs.517549, Hs.278906, Hs.498586, Hs.183617, Hs.499758, Hs.350065, Hs.511138, Hs.150793, Hs.129174, Hs.212606, Hs.275775, Hs.279611, Hs.496414, Hs.436142, Hs.282984, Hs.32417, Hs.69321, Hs.414629, Hs.502618, Hs.405755, Hs. 480143, Hs.90250, Hs.436317, Hs.60371, Hs.283683, Hs.208093, Hs.336768, Hs.1116459, Hs.131673, Hs.42091, Hs.32417, Hs.75812, Hs.355394 A gene having, or a variant, homologue or derivative thereof,
The method is provided.

  In this specification, TRU (Terminal Respiratory Unit) is a unit composed of airway epithelium located in the periphery particularly in the lung, and has a character generated from or similar to those constituent units. Adenocarcinoma is referred to as TRU-type adenocarcinoma. Non-TRU type adenocarcinoma refers to a subtype other than TRU type.

  In the present specification, postoperative prognosis means that the prognosis of a patient after surgery for lung adenocarcinoma is typically determined by the survival rate at 5 years after the operation. Prognosis is closely related to cancer metastasis, and poor prognosis means that the cancer is highly invasive, metastatic, or highly advanced, while good prognosis or good prognosis is such invasiveness. , Means less metastatic or less advanced.

  In the present specification, the mutant of the gene includes, for example, a mutant caused by a biological event such as mutation, gene polymorphism, alternative splicing, or the degeneracy of the genetic code. A mutant has one or more mutations such as substitution, deletion, addition, and insertion on the original nucleotide sequence of the gene.

  In the present specification, homologues are partially different in sequence from the gene for reasons such as different species, but have the same biological function or action as the gene, and therefore belong to the same family. Refers to the nucleic acid to which it belongs.

  Mutants and homologues are 70% or more, preferably 80% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more at the nucleotide level with each of the corresponding genes. Have the same identity. Here, the identity (%) can be determined using a known BLAST program into which a gap is introduced. In general, identity (%) can be calculated as a percentage of the number of matched bases relative to the total number of bases.

  In the present specification, a derivative refers to the gene containing a chemical modification of a base, and such chemical modification includes, for example, methylation, acetylation, thiolation, carboxymethylation, methoxylation and the like. .

  In this specification, a patient refers to mammals including humans, dogs, and cats, and preferred mammals are humans.

In one embodiment of the invention, the UniGene registration number:
Hs.512690, Hs.153322, Hs.218366, Hs.220629, Hs.436996, Hs.435759, Hs.104555, Hs.247824, Hs.127821, Hs.480281, Hs.529117, Hs.545862, Hs. 391561, Hs.479372, Hs.533055, Hs.550526, Hs.322854, Hs.465720, Hs.356664, Hs.26630, Hs.534496, Hs.85962, Hs.211267, Hs.128041, Hs.534458, Hs.495774, Hs.437806, Hs.133062, Hs.501758, Hs.444535, Hs.495480, Hs.326561, Hs.483906, Hs.169943, Hs.271285, Hs.158339, Hs.62604, Hs. 469359, Hs.436657, Hs.8417, Hs.155538, Hs.533526, Hs.512756, Hs.87191, Hs.463079, Hs.513779, Hs.476209, Hs.279580, Hs.351544, Hs.269408, Hs.134807, Hs.482417, Hs.176626, Hs.465643, Hs.183390, Hs.411299, Hs.234027, Hs.109358, Hs.103983, Hs.26216, Hs.534352, Hs.240457, Hs. 516036, Hs.144875, Hs.411312, Hs.103989, Hs.537722, Hs.333130, Hs.517962, Hs.90250, Hs.478930, Hs.121629, Hs.194061, Hs.520627, Hs.348012, Hs.522836, Hs.1376, Hs.520049, Hs.512856, Hs.355236, Hs.349470, Hs.476231, Hs.137556, Hs.390567, Hs .368353, Hs.412792, Hs.449207, Hs.527095, Hs.118722, Hs.377090, Hs.232696, Hs.447544, Hs.372773, Hs.2222055, Hs.511839, Hs.153299, Hs.434374 , Hs.287729, Hs.553740, Hs.127189, Hs.497723, Hs.181973, Hs.173656, Hs.451956, Hs.184507, Hs.532492, Hs.370904, Hs.460468, Hs.520612, Hs .436667, Hs.125116, Hs.459391, Hs.450320, Hs.149769, Hs.325890, Hs.356820, Hs.289319, Hs.73893, Hs.129493, Hs.515069, Hs.34560, Hs.477278 , Hs.351571, Hs.112087, Hs.154224, Hs.125950, Hs.438016, Hs.367956, Hs.553778, Hs.329266, Hs.479658, Hs.458713, Hs.249196, Hs.467529, Hs .145061, Hs.49653, Hs.129227, Hs.313343, Hs.194554, Hs.123114, Hs.126561, Hs.42091, Hs.369385, Hs.98661, Hs.458306, Hs.148584, Hs.501684 , Hs.422466, Hs.523732, Hs.525557, Hs.1372, Hs.379097, Hs.208124, Hs.389311, Hs.2561, Hs.117545, Hs.446388, Hs.2813, Hs.473894, Hs .502092, Hs.524479, Hs.314261, Hs.382306, Hs.458252, Hs.380222, Hs.379636, Hs.302034, Hs.253495, Hs.3 45877, Hs.259563, Hs.528569, Hs.152337, Hs.436317, Hs.546408, Hs.46700, Hs.1027, Hs.151219, Hs.279611, Hs.310456, Hs.520319, Hs.406976, Hs.181245, Hs.449621, Hs.515465, Hs.310540, Hs.554891, Hs.449601, Hs.355394, Hs.380710, Hs.171995, Hs.449585, Hs.522484, Hs.298023, Hs. 520339, Hs.121443,
Hs.62661, Hs.200804, Hs.533185, Hs.461329, Hs.479270, Hs.446201, Hs.35086, Hs.500761, Hs.44298, Hs.469030, Hs.309767, Hs.441047, Hs. 98309, Hs.31409, Hs.518299, Hs.532870, Hs.196534, Hs.108106, Hs.289319, Hs.69771, Hs.374378, Hs.369422, Hs.368641, Hs.302963, Hs.530461, Hs.1955, Hs.513726, Hs.148767, Hs.523220, Hs.525796, Hs.271264, Hs.69321, Hs.231367, Hs.500761, Hs.528304, Hs.148685, Hs.87417, Hs. 164060, Hs.514843, Hs.418416, Hs.126521, Hs.519839, Hs.103834, Hs.279840, Hs.497741, Hs.531457, Hs.226390, Hs.480143, Hs.473721, Hs.369762, Hs.514527, Hs.204238, Hs.3104, Hs.519873, Hs.519909, Hs.179718, Hs.103183, Hs.520210, Hs.444683, Hs.234545, Hs.80976, Hs.311187, Hs. 89497, Hs.444118, Hs.541635, Hs.477898, Hs.511776, Hs.434886, Hs.117299, Hs.252451, Hs.468058, Hs.21554, Hs.165904, Hs.445244, Hs.413924, Hs.99120, Hs.521171, Hs.462379, Hs.481860, Hs.489207, Hs.414407, Hs.505575, Hs.516826, Hs.62180, Hs.368934 , Hs.530509, Hs.278906, Hs.511987, Hs.444082, Hs.471873, Hs.24583,
Hs.1051, Hs.115263, Hs.379010, Hs.2012, Hs.268698, Hs.49653, Hs.424542, Hs.525589, Hs.25333, Hs.513075, Hs.549577, Hs.100431, Hs. 9613, Hs.301478, Hs.553740, Hs.525383, Hs.473721, Hs.375624, Hs.116724, Hs.9613, Hs.418055,
Hs.334873, Hs.180878, Hs.126521, Hs.519033, Hs.187636, Hs.518448, Hs.322761, Hs.31409, Hs.524513, Hs.436437, Hs.148989, Hs.279575, Hs. 75668, Hs.470791, Hs.104476, Hs.494496, Hs.517549, Hs.278906, Hs.498586, Hs.183617, Hs.499758, Hs.350065, Hs.511138, Hs.150793, Hs.129174, Hs.212606, Hs.275775, Hs.496414, Hs.436142, Hs.282984, Hs.32417, Hs.69321, Hs.414629, Hs.502618, Hs.405755, Hs.480143, Hs.60371, Hs. 283683, Hs.208093, Hs.336768, Hs.1116459, Hs.131673, Hs.32417, Hs.75812
Each of the genes having the sequence is characterized by including the sequence shown in SEQ ID NOs: 1 to 351 or a complementary sequence thereof.

In another embodiment, when the expression level of the gene belonging to the TRU type is relatively high between the TRU type adenocarcinoma and the non-TRU type adenocarcinoma, the adenocarcinoma is determined as the TRU type adenocarcinoma, When the expression level of the gene belonging to the non-TRU type is relatively high, the adenocarcinoma is determined as a non-TRU type adenocarcinoma .

Alternatively , when the expression level of the gene belonging to the TRU-a type is relatively high between the TRU-a type adenocarcinoma and the TRU-b type adenocarcinoma, the adenocarcinoma is determined as the TRU-a type adenocarcinoma. On the other hand, when the expression level of the gene belonging to the TRU-b type is relatively high, the adenocarcinoma is determined as a TRU-b type adenocarcinoma .

  Since such a difference in expression level is a relative difference between TRU-type adenocarcinoma and non-TRU-type adenocarcinoma, or between TRU-a type adenocarcinoma and TRU-type-b adenocarcinoma, It depends on whether it is larger or smaller than the other. Preferably, such a difference is 1.5 times or more, more preferably 2.0 times or more.

  In another embodiment, the expression level of the gene is determined by measuring the presence or amount of nucleic acid or protein corresponding to the gene.

  In the present specification, the nucleic acid is either DNA or RNA, and includes, for example, a transcription product (mRNA), cDNA, cRNA and the like of each gene.

  The protein means a protein encoded by each gene, a variant thereof or a fragment thereof. Variants include variants based on splicing and polymorphisms. A fragment is a protein fragment that is naturally generated by the action of a protease or peptidase in vivo, and includes fragments of any size as long as it can be identified as a fragment of the target protein. The preferred size of the fragment is 20 or more amino acids, more preferably 30 or more amino acids, and even more preferably 50 or more amino acids.

  In still another embodiment, the gene expression level can be measured by a hybridization method or a gene amplification method.

  The hybridization method includes, for example, a microarray method, a blot method, such as a Northern blot or a Southern blot method, a Northern or Southern hybridization method, an in situ hybridization, and the like. Preferred hybridization methods are microarray methods such as DNA microarrays, oligonucleotide microarrays and protein microarrays. The gene amplification method includes a polymerase chain reaction (PCR) method, an ICAN method, a LAMP method, a PALSAR method, etc., obtained by converting cDNA converted from RNA with a reverse transcriptase. A preferred gene amplification method is the PCR method.

  In another embodiment, the expression level of the gene can alternatively be measured by immunological methods.

  The immunological method includes detecting an immunological complex of a specific antibody against a protein encoded by the gene or a fragment thereof and a target protein. Here, the protein or a fragment thereof can be preferably obtained by a conventional cDNA cloning method based on a sequence registered in a gene bank and a protein synthesis method using an expression vector / host system. Immunological methods include enzyme immunoassays (EIA, ELISA, etc.), fluorescent antibody methods, radioimmunoassays, agglutination methods, turbidimetric methods, and the like. In addition, the antigen-antibody reaction can be performed under conditions of uniform or heterogeneous, or solid phase or non-solid phase, but a reaction by a solid phase method is desirable. Furthermore, a sandwich method using a labeled secondary antibody is also preferably used.

  In yet another embodiment, the prognostic method of the present invention includes the above-mentioned TRU-type lung adenocarcinoma, wherein, when the epidermal growth factor receptor (EGFR) gene contains a mutation, It can further comprise predicting that the postoperative prognosis of the patient is poor compared to.

  Whether or not a mutation is included in the EGFR gene is determined by, for example, Kosaka T et al., Cancer Res 64: 8919-23, 2004 (Non-Patent Document 18), Mitsudomi T et al., J clin Oncol 23: 2513-20, 2005 (Non-Patent Documents). It can be detected by the method described in literature 19). Briefly, in such a method, the gene region can be amplified using the PCR method, and the mutation can be detected using gel electrophoresis or determination of the base sequence.

In the second aspect, the present invention also classifies lung adenocarcinoma as either TRU-type or non-TRU-type subtype, and then classifies TRU-type adenocarcinoma as TRU-a type or TRU-b type. A method, wherein the method is for TRU-type adenocarcinoma or TRU-a type adenocarcinoma for one or more genes of the following corresponding gene set in a biological sample of a patient: A gene that measures the difference in relative expression level between adenocarcinoma and TRU-b type adenocarcinoma,
(A) The following UniGene registration number:
Hs.512690, Hs.153322, Hs.218366, Hs.220629, Hs.436996, Hs.435759, Hs.104555, Hs.247824, Hs.127821, Hs.480281, Hs.529117, Hs.545862, Hs. 391561, Hs.479372, Hs.533055, Hs.550526, Hs.322854, Hs.465720, Hs.356664, Hs.26630, Hs.534496, Hs.85962, Hs.211267, Hs.128041, Hs.534458, Hs.495774, Hs.437806, Hs.133062, Hs.501758, Hs.444535, Hs.495480, Hs.326561, Hs.483906, Hs.169943, Hs.271285, Hs.158339, Hs.62604, Hs. 469359, Hs.436657, Hs.8417, Hs.155538, Hs.533526, Hs.512756, Hs.87191, Hs.463079, Hs.513779, Hs.476209, Hs.279580, Hs.351544, Hs.269408, Hs.134807, Hs.482417, Hs.176626, Hs.465643, Hs.183390, Hs.411299, Hs.234027, Hs.109358, Hs.103983, Hs.26216, Hs.534352, Hs.240457, Hs. 516036, Hs.144875, Hs.411312, Hs.103989, Hs.537722, Hs.333130, Hs.517962, Hs.90250, Hs.478930, Hs.121629, Hs.194061, Hs.520627, Hs.348012, Hs.522836, Hs.1376, Hs.520049, Hs.512856, Hs.355236, Hs.349470, Hs.476231, Hs.137556, Hs.390567, Hs .368353, Hs.412792, Hs.449207, Hs.527095, Hs.118722, Hs.377090, Hs.232696, Hs.447544, Hs.372773, Hs.2222055, Hs.511839, Hs.153299, Hs.434374 , Hs.287729, Hs.553740, Hs.127189, Hs.497723, Hs.181973, Hs.173656, Hs.451956, Hs.184507, Hs.532492, Hs.370904, Hs.460468, Hs.520612, Hs .436667, Hs.125116, Hs.459391, Hs.450320, Hs.149769, Hs.325890, Hs.356820, Hs.289319, Hs.73893, Hs.129493, Hs.515069, Hs.34560, Hs.477278 , Hs.351571, Hs.112087, Hs.154224, Hs.125950, Hs.438016, Hs.367956, Hs.553778, Hs.329266, Hs.479658, Hs.458713, Hs.249196, Hs.467529, Hs .145061, Hs.49653, Hs.129227, Hs.313343, Hs.194554, Hs.123114, Hs.126561, Hs.42091, Hs.369385, Hs.98661, Hs.458306, Hs.148584, Hs.501684 , Hs.422466, Hs.523732, Hs.525557, Hs.1372, Hs.379097, Hs.208124, Hs.389311, Hs.2561, Hs.117545, Hs.446388, Hs.2813, Hs.473894, Hs .502092, Hs.524479, Hs.314261, Hs.382306, Hs.458252, Hs.380222, Hs.379636, Hs.302034, Hs.253495, Hs.3 45877, Hs.259563, Hs.528569, Hs.152337, Hs.436317, Hs.546408, Hs.46700, Hs.1027, Hs.151219, Hs.279611, Hs.310456, Hs.520319, Hs.406976, Hs.181245, Hs.449621, Hs.515465, Hs.310540, Hs.554891, Hs.449601, Hs.355394, Hs.380710, Hs.171995, Hs.449585, Hs.522484, Hs.298023, Hs. 520339, if it is a gene in a gene set consisting of a gene having Hs.121443, or a variant, homologue or derivative thereof, the lung adenocarcinoma is determined to be TRU type, or
(B) The following UniGene registration number:
Hs.62661, Hs.200804, Hs.533185, Hs.461329, Hs.479270, Hs.446201, Hs.35086, Hs.500761, Hs.44298, Hs.469030, Hs.309767, Hs.441047, Hs. 98309, Hs.31409, Hs.518299, Hs.532870, Hs.196534, Hs.108106, Hs.289319, Hs.69771, Hs.374378, Hs.369422, Hs.368641, Hs.302963, Hs.530461, Hs.1955, Hs.513726, Hs.148767, Hs.523220, Hs.525796, Hs.271264, Hs.69321, Hs.231367, Hs.500761, Hs.528304, Hs.148685, Hs.87417, Hs. 164060, Hs.514843, Hs.418416, Hs.126521, Hs.519839, Hs.103834, Hs.279840, Hs.497741, Hs.531457, Hs.226390, Hs.480143, Hs.473721, Hs.369762, Hs.514527, Hs.204238, Hs.3104, Hs.519873, Hs.519909, Hs.179718, Hs.103183, Hs.520210, Hs.444683, Hs.234545, Hs.80976, Hs.311187, Hs. 89497, Hs.444118, Hs.541635, Hs.477898, Hs.511776, Hs.434886, Hs.117299, Hs.252451, Hs.468058, Hs.21554, Hs.165904, Hs.445244, Hs.413924, Hs.99120, Hs.521171, Hs.462379, Hs.481860, Hs.489207, Hs.414407, Hs.505575, Hs.516826, Hs.62180, Hs.368934 , Hs. 530509, Hs. 278906, Hs. 511987, Hs. 444082, Hs. 471873, Hs. 24583, or a gene in a gene set comprising a variant, homologue or derivative thereof, the lung Determine that the adenocarcinoma is non-TRU, or
(C) The following UniGene registration numbers:
Hs.220629, Hs.153322, Hs.127821, Hs.218366, Hs.436996, Hs.247824, Hs.550526, Hs.62604, Hs.104555, Hs.465720, Hs.480281, Hs.495480, Hs. 545862, Hs.1051, Hs.133062, Hs.115263, Hs.349470, Hs.495774, Hs.155538, Hs.435759, Hs.128041, Hs.465643, Hs.169943, Hs.534496, Hs.379010, Hs.184507, Hs.469359, Hs.2012, Hs.109358, Hs.438016, Hs.326561, Hs.367956, Hs.271285, Hs.176626, Hs.137556, Hs.437806, Hs.240457, Hs. 533055, Hs.532492, Hs.85962, Hs.268698, Hs.520627, Hs.524479, Hs.449585, Hs.158339, Hs.522836, Hs.279580, Hs.444535, Hs.446388, Hs.149769, Hs.513779, Hs.103983, Hs.512756, Hs.348012, Hs.467529, Hs.434374, Hs.389311, Hs.49653, Hs.424542, Hs.436667, Hs.516036, Hs.533526, Hs. 463079, Hs.310456, Hs.125950, Hs.351571, Hs.478930, Hs.483906, Hs.259563, Hs.249196, Hs.412792, Hs.183390, Hs.87191, Hs.171995, Hs.117545, Hs.554891, Hs.351544, Hs.368353, Hs.482417, Hs.126561, Hs.154224, Hs.232696, Hs.520319, Hs.153299, Hs.449621, Hs.502092, Hs.537722, Hs.127189, Hs.525589, Hs.476231, Hs.527095, Hs.511839, Hs.372773, Hs.112087, Hs.458252, Hs.181973, Hs. 289319, Hs.25333, Hs.513075, Hs.356820, Hs.549577, Hs.380222, Hs.46700, Hs.129227, Hs.100431, Hs.129493, Hs.173656, Hs.9613, Hs.301478, Hs.553740, Hs.151219, Hs.369385, Hs.525383, Hs.473721, Hs.375624, Hs.355236, Hs.116724, Hs.9613, Hs.418055, or variants or homologues thereof Or if it is a gene in a gene set consisting of derivatives, the lung adenocarcinoma is determined to be TRU-a, or
(D) The following UniGene registration numbers:
Hs.334873, Hs.180878, Hs.126521, Hs.519033, Hs.187636, Hs.518448, Hs.322761, Hs.31409, Hs.524513, Hs.253495, Hs.436437, Hs.148989, Hs. 279575, Hs.75668, Hs.470791, Hs.104476, Hs.494496, Hs.517549, Hs.278906, Hs.498586, Hs.183617, Hs.499758, Hs.350065, Hs.511138, Hs.150793, Hs.129174, Hs.212606, Hs.275775, Hs.279611, Hs.496414, Hs.436142, Hs.282984, Hs.32417, Hs.69321, Hs.414629, Hs.502618, Hs.405755, Hs. 480143, Hs.90250, Hs.436317, Hs.60371, Hs.283683, Hs.208093, Hs.336768, Hs.1116459, Hs.131673, Hs.42091, Hs.32417, Hs.75812, Hs.355394 The lung adenocarcinoma is determined to be TRU-b type in the case of a gene in a gene set consisting of a gene having or a variant, homologue or derivative thereof,
Providing the method.

Here, TRU, non-TRU, mutant, homologue and derivative are as defined above.
Further, here, when the expression level of the gene belonging to the TRU type is relatively large between the TRU type adenocarcinoma and the non-TRU type adenocarcinoma, the adenocarcinoma is determined as the TRU type adenocarcinoma, When the expression level of a gene belonging to the non-TRU type is relatively high, the adenocarcinoma is determined as a non-TRU type adenocarcinoma .

Furthermore, when the expression level of the gene belonging to the TRU-a type is relatively high between the TRU-a type adenocarcinoma and the TRU-b type adenocarcinoma, the adenocarcinoma is determined as the TRU-a type adenocarcinoma. On the other hand, when the expression level of the gene belonging to the TRU-b type is relatively high, the adenocarcinoma is determined as a TRU-b type adenocarcinoma .

In one embodiment thereof, the UniGene registration number:
Hs.512690, Hs.153322, Hs.218366, Hs.220629, Hs.436996, Hs.435759, Hs.104555, Hs.247824, Hs.127821, Hs.480281, Hs.529117, Hs.545862, Hs. 391561, Hs.479372, Hs.533055, Hs.550526, Hs.322854, Hs.465720, Hs.356664, Hs.26630, Hs.534496, Hs.85962, Hs.211267, Hs.128041, Hs.534458, Hs.495774, Hs.437806, Hs.133062, Hs.501758, Hs.444535, Hs.495480, Hs.326561, Hs.483906, Hs.169943, Hs.271285, Hs.158339, Hs.62604, Hs. 469359, Hs.436657, Hs.8417, Hs.155538, Hs.533526, Hs.512756, Hs.87191, Hs.463079, Hs.513779, Hs.476209, Hs.279580, Hs.351544, Hs.269408, Hs.134807, Hs.482417, Hs.176626, Hs.465643, Hs.183390, Hs.411299, Hs.234027, Hs.109358, Hs.103983, Hs.26216, Hs.534352, Hs.240457, Hs. 516036, Hs.144875, Hs.411312, Hs.103989, Hs.537722, Hs.333130, Hs.517962, Hs.90250, Hs.478930, Hs.121629, Hs.194061, Hs.520627, Hs.348012, Hs.522836, Hs.1376, Hs.520049, Hs.512856, Hs.355236, Hs.349470, Hs.476231, Hs.137556, Hs.390567, Hs .368353, Hs.412792, Hs.449207, Hs.527095, Hs.118722, Hs.377090, Hs.232696, Hs.447544, Hs.372773, Hs.2222055, Hs.511839, Hs.153299, Hs.434374 , Hs.287729, Hs.553740, Hs.127189, Hs.497723, Hs.181973, Hs.173656, Hs.451956, Hs.184507, Hs.532492, Hs.370904, Hs.460468, Hs.520612, Hs .436667, Hs.125116, Hs.459391, Hs.450320, Hs.149769, Hs.325890, Hs.356820, Hs.289319, Hs.73893, Hs.129493, Hs.515069, Hs.34560, Hs.477278 , Hs.351571, Hs.112087, Hs.154224, Hs.125950, Hs.438016, Hs.367956, Hs.553778, Hs.329266, Hs.479658, Hs.458713, Hs.249196, Hs.467529, Hs .145061, Hs.49653, Hs.129227, Hs.313343, Hs.194554, Hs.123114, Hs.126561, Hs.42091, Hs.369385, Hs.98661, Hs.458306, Hs.148584, Hs.501684 , Hs.422466, Hs.523732, Hs.525557, Hs.1372, Hs.379097, Hs.208124, Hs.389311, Hs.2561, Hs.117545, Hs.446388, Hs.2813, Hs.473894, Hs .502092, Hs.524479, Hs.314261, Hs.382306, Hs.458252, Hs.380222, Hs.379636, Hs.302034, Hs.253495, Hs.3 45877, Hs.259563, Hs.528569, Hs.152337, Hs.436317, Hs.546408, Hs.46700, Hs.1027, Hs.151219, Hs.279611, Hs.310456, Hs.520319, Hs.406976, Hs.181245, Hs.449621, Hs.515465, Hs.310540, Hs.554891, Hs.449601, Hs.355394, Hs.380710, Hs.171995, Hs.449585, Hs.522484, Hs.298023, Hs. 520339, Hs.121443,
Hs.62661, Hs.200804, Hs.533185, Hs.461329, Hs.479270, Hs.446201, Hs.35086, Hs.500761, Hs.44298, Hs.469030, Hs.309767, Hs.441047, Hs. 98309, Hs.31409, Hs.518299, Hs.532870, Hs.196534, Hs.108106, Hs.289319, Hs.69771, Hs.374378, Hs.369422, Hs.368641, Hs.302963, Hs.530461, Hs.1955, Hs.513726, Hs.148767, Hs.523220, Hs.525796, Hs.271264, Hs.69321, Hs.231367, Hs.500761, Hs.528304, Hs.148685, Hs.87417, Hs. 164060, Hs.514843, Hs.418416, Hs.126521, Hs.519839, Hs.103834, Hs.279840, Hs.497741, Hs.531457, Hs.226390, Hs.480143, Hs.473721, Hs.369762, Hs.514527, Hs.204238, Hs.3104, Hs.519873, Hs.519909, Hs.179718, Hs.103183, Hs.520210, Hs.444683, Hs.234545, Hs.80976, Hs.311187, Hs. 89497, Hs.444118, Hs.541635, Hs.477898, Hs.511776, Hs.434886, Hs.117299, Hs.252451, Hs.468058, Hs.21554, Hs.165904, Hs.445244, Hs.413924, Hs.99120, Hs.521171, Hs.462379, Hs.481860, Hs.489207, Hs.414407, Hs.505575, Hs.516826, Hs.62180, Hs.368934 , Hs.530509, Hs.278906, Hs.511987, Hs.444082, Hs.471873, Hs.24583,
Hs.1051, Hs.115263, Hs.379010, Hs.2012, Hs.268698, Hs.49653, Hs.424542, Hs.525589, Hs.25333, Hs.513075, Hs.549577, Hs.100431, Hs. 9613, Hs.301478, Hs.553740, Hs.525383, Hs.473721, Hs.375624, Hs.116724, Hs.9613, Hs.418055,
Hs.334873, Hs.180878, Hs.126521, Hs.519033, Hs.187636, Hs.518448, Hs.322761, Hs.31409, Hs.524513, Hs.436437, Hs.148989, Hs.279575, Hs. 75668, Hs.470791, Hs.104476, Hs.494496, Hs.517549, Hs.278906, Hs.498586, Hs.183617, Hs.499758, Hs.350065, Hs.511138, Hs.150793, Hs.129174, Hs.212606, Hs.275775, Hs.496414, Hs.436142, Hs.282984, Hs.32417, Hs.69321, Hs.414629, Hs.502618, Hs.405755, Hs.480143, Hs.60371, Hs. 283683, Hs.208093, Hs.336768, Hs.1116459, Hs.131673, Hs.32417, Hs.75812
Each of the genes having the sequence includes a sequence shown in SEQ ID NOs: 1 to 351 or a complementary sequence thereof.

  In another embodiment, the expression level of the gene can be measured by a hybridization method.

  Examples of the hybridization method include the same methods as described above, for example, a microarray method, or a blot method such as Northern blot or Southern blot. For this purpose, a microarray can be preferably used.

In the third aspect, the present invention provides the following UniGene registration number:
Hs.512690, Hs.153322, Hs.218366, Hs.220629, Hs.436996, Hs.435759, Hs.104555, Hs.247824, Hs.127821, Hs.480281, Hs.529117, Hs.545862, Hs. 391561, Hs.479372, Hs.533055, Hs.550526, Hs.322854, Hs.465720, Hs.356664, Hs.26630, Hs.534496, Hs.85962, Hs.211267, Hs.128041, Hs.534458, Hs.495774, Hs.437806, Hs.133062, Hs.501758, Hs.444535, Hs.495480, Hs.326561, Hs.483906, Hs.169943, Hs.271285, Hs.158339, Hs.62604, Hs. 469359, Hs.436657, Hs.8417, Hs.155538, Hs.533526, Hs.512756, Hs.87191, Hs.463079, Hs.513779, Hs.476209, Hs.279580, Hs.351544, Hs.269408, Hs.134807, Hs.482417, Hs.176626, Hs.465643, Hs.183390, Hs.411299, Hs.234027, Hs.109358, Hs.103983, Hs.26216, Hs.534352, Hs.240457, Hs. 516036, Hs.144875, Hs.411312, Hs.103989, Hs.537722, Hs.333130, Hs.517962, Hs.90250, Hs.478930, Hs.121629, Hs.194061, Hs.520627, Hs.348012, Hs.522836, Hs.1376, Hs.520049, Hs.512856, Hs.355236, Hs.349470, Hs.476231, Hs.137556, Hs.390567, Hs .368353, Hs.412792, Hs.449207, Hs.527095, Hs.118722, Hs.377090, Hs.232696, Hs.447544, Hs.372773, Hs.2222055, Hs.511839, Hs.153299, Hs.434374 , Hs.287729, Hs.553740, Hs.127189, Hs.497723, Hs.181973, Hs.173656, Hs.451956, Hs.184507, Hs.532492, Hs.370904, Hs.460468, Hs.520612, Hs .436667, Hs.125116, Hs.459391, Hs.450320, Hs.149769, Hs.325890, Hs.356820, Hs.289319, Hs.73893, Hs.129493, Hs.515069, Hs.34560, Hs.477278 , Hs.351571, Hs.112087, Hs.154224, Hs.125950, Hs.438016, Hs.367956, Hs.553778, Hs.329266, Hs.479658, Hs.458713, Hs.249196, Hs.467529, Hs .145061, Hs.49653, Hs.129227, Hs.313343, Hs.194554, Hs.123114, Hs.126561, Hs.42091, Hs.369385, Hs.98661, Hs.458306, Hs.148584, Hs.501684 , Hs.422466, Hs.523732, Hs.525557, Hs.1372, Hs.379097, Hs.208124, Hs.389311, Hs.2561, Hs.117545, Hs.446388, Hs.2813, Hs.473894, Hs .502092, Hs.524479, Hs.314261, Hs.382306, Hs.458252, Hs.380222, Hs.379636, Hs.302034, Hs.253495, Hs.3 45877, Hs.259563, Hs.528569, Hs.152337, Hs.436317, Hs.546408, Hs.46700, Hs.1027, Hs.151219, Hs.279611, Hs.310456, Hs.520319, Hs.406976, Hs.181245, Hs.449621, Hs.515465, Hs.310540, Hs.554891, Hs.449601, Hs.355394, Hs.380710, Hs.171995, Hs.449585, Hs.522484, Hs.298023, Hs. 520339, Hs.121443,
Hs.62661, Hs.200804, Hs.533185, Hs.461329, Hs.479270, Hs.446201, Hs.35086, Hs.500761, Hs.44298, Hs.469030, Hs.309767, Hs.441047, Hs. 98309, Hs.31409, Hs.518299, Hs.532870, Hs.196534, Hs.108106, Hs.289319, Hs.69771, Hs.374378, Hs.369422, Hs.368641, Hs.302963, Hs.530461, Hs.1955, Hs.513726, Hs.148767, Hs.523220, Hs.525796, Hs.271264, Hs.69321, Hs.231367, Hs.500761, Hs.528304, Hs.148685, Hs.87417, Hs. 164060, Hs.514843, Hs.418416, Hs.126521, Hs.519839, Hs.103834, Hs.279840, Hs.497741, Hs.531457, Hs.226390, Hs.480143, Hs.473721, Hs.369762, Hs.514527, Hs.204238, Hs.3104, Hs.519873, Hs.519909, Hs.179718, Hs.103183, Hs.520210, Hs.444683, Hs.234545, Hs.80976, Hs.311187, Hs. 89497, Hs.444118, Hs.541635, Hs.477898, Hs.511776, Hs.434886, Hs.117299, Hs.252451, Hs.468058, Hs.21554, Hs.165904, Hs.445244, Hs.413924, Hs.99120, Hs.521171, Hs.462379, Hs.481860, Hs.489207, Hs.414407, Hs.505575, Hs.516826, Hs.62180, Hs.368934 , Hs.530509, Hs.278906, Hs.511987, Hs.444082, Hs.471873, Hs.24583,
Hs.1051, Hs.115263, Hs.379010, Hs.2012, Hs.268698, Hs.49653, Hs.424542, Hs.525589, Hs.25333, Hs.513075, Hs.549577, Hs.100431, Hs. 9613, Hs.301478, Hs.553740, Hs.525383, Hs.473721, Hs.375624, Hs.116724, Hs.9613, Hs.418055,
Hs.334873, Hs.180878, Hs.126521, Hs.519033, Hs.187636, Hs.518448, Hs.322761, Hs.31409, Hs.524513, Hs.436437, Hs.148989, Hs.279575, Hs. 75668, Hs.470791, Hs.104476, Hs.494496, Hs.517549, Hs.278906, Hs.498586, Hs.183617, Hs.499758, Hs.350065, Hs.511138, Hs.150793, Hs.129174, Hs.212606, Hs.275775, Hs.496414, Hs.436142, Hs.282984, Hs.32417, Hs.69321, Hs.414629, Hs.502618, Hs.405755, Hs.480143, Hs.60371, Hs. 283683, Hs.208093, Hs.336768, Hs.1116459, Hs.131673, Hs.32417, Hs.75812
A nucleic acid capable of detecting the expression of a gene having or a variant, homologue or derivative thereof,
(1) a nucleotide sequence represented by SEQ ID NO: 1 to 351,
(2) a nucleotide sequence comprising the nucleotide sequence shown in SEQ ID NOs: 1 to 351,
(3) a nucleotide sequence complementary to the nucleotide sequence of (1) or (2),
(4) a nucleotide sequence that hybridizes under stringent conditions with any of the sequences (1), (2), or (3), and
(5) a partial sequence of less than the total number of bases from 15 bases of the nucleotide sequence of (1), (3) or (4),
Post-surgical treatment for patients with lung adenocarcinoma comprising a nucleic acid having a sequence selected from the group consisting of: Compositions for predicting prognosis in vitro or for classifying lung adenocarcinoma into any subtype of TRU, non-TRU, TRU-a or TRU-b are provided.

  The composition of the present invention can be used in vitro to predict postoperative prognosis in patients with lung adenocarcinoma as described above, as well as to classify lung adenocarcinoma into four types as described above.

  In the present specification, stringent conditions mean hybridization and washing conditions such that nucleotide sequences having at least 80%, preferably at least 95% identity, hybridize to each other. For example, hybridization in microarray analysis And washing conditions were hybridization in 1 M sodium chloride / 0.5% (W / V) sarkosyl / 30% formamide at 60 ° C. for 17 hours, followed by 6 × SSC / 0.005% (W / V) Triton. Once in the X-102 solution at room temperature for 10 minutes, and once in 5 minutes while maintaining at 0 to 4 ° C. in the 0.1 × SSC / 0.005% (W / V) Triton X-102 solution. Is the condition for cleaning. Here, 1 × SSC is 150 mM sodium chloride and 15 mM sodium citrate aqueous solution (pH 7.2). For hybridization, see Ausbel FM et al., Short Protocols in Molecular Biology (3rd edition) A Compendium of Methods in Current Protocols in Molecular Biology, 1995, Joh. (United States).

In another embodiment, the present invention also comprises a nucleic acid or an antibody or fragment thereof constituting the composition (also referred to as a DNA chip) kit or DNA microarray or protein array also that provide.

  According to the present invention, lung adenocarcinoma is classified into its subtypes (TRU, non-TRU type, TRU-a and TRU-b), the relationship between each subtype and postoperative prognosis, and TRU type lung adenocarcinoma The relationship between EGFR gene mutation and postoperative prognosis became clear. By using the method of the present invention, it is possible to make a treatment plan for improving the survival rate by selecting patients who are likely to recur after surgery, or clinically used as a molecular target drug for lung cancer. A patient with a particularly poor prognosis among patients having an EGFR mutation known to be involved in the reactivity of gefitinib (trade name Iressa), and can be selected as a subject for postoperative treatment with an EGFR inhibitor It becomes possible.

  Of the cases diagnosed as non-small cell lung cancer (NSCLC) at Aichi Cancer Center (Nagoya), 90 cases of adenocarcinoma, 35 cases of squamous cell carcinoma, 18 cases of large cell carcinoma, 4 cases of squamous cancer For tumor specimens from 149 cases, including epithelial adenocarcinoma and 2 large cell neuroendocrine carcinomas, a double-stranded cDNA followed by cRNA was prepared and used using a microarray linking 18,175 unique genes • A systematic expression profile was obtained. Furthermore, the clustering of genes and cases was performed using the CLUSTER program, the results were displayed by TREEVIEW (Eisen MB et al., Proc Natl Acad Sci USA 95: 14863-8, 1998), and the microarray significance analysis method (SAM ) Was used to score genes specific to each of the patient subtypes (Tusher VG et al., Proc Natl Acad Sci USA 98: 5116-21, 2001).

  Analysis using gene ontology (GO) (Ashburner M et al., Nat Genet 25: 25-9, 2000) reveals functionally distinct biological characteristics of each specific gene set of patient subtypes. Revealed.

  In addition, multivariate logistic regression analysis was performed to investigate the relationship between various clinical parameters and the expression of the subtypes defined by the expression profile, and the postoperative survival rate was determined using the Kaplan-Meier method. evaluated.

  As a result, we found that lung adenocarcinoma cases could be classified into two main sub-clusters, which were called TRU type and non-TRU type. Two main dendrograms (TRU and non-TRU types) and right-hand dendrogram, with taxonomic clustering performed with 4,138 transcripts most variably expressed in adenocarcinoma The presence of two further subclusters (TRU-a type and TRU-b type) was revealed (FIG. 2A).

  To obtain further knowledge about the dendrogram and biological properties of lung adenocarcinoma subtypes defined by these two major expression profiles, namely non-TRU and TRU types, a SAM analysis was performed, and 0. 1,657 differentially expressed genes were extracted in pre-filtering with a significance level of false positive rate less than 1%. Of these genes, 286 genes showed a difference in expression level between TRU-type and non-TRU-type by a factor of 2 or more. The 286 genes found this time consisted of 194 genes with higher expression in the TRU type and 92 genes with higher expression in the non-TRU type. As a result of investigating basic functional differences between TRU type and non-TRU type, TRU type showed a clear relationship with normal lung function, whereas non-TRU type had a relationship with cell cycle and proliferation. It turned out to show a relationship.

  Furthermore, as a result of investigating the prognosis after surgery, TRU-a type showed a poor prognosis similar to that of non-TRU type, whereas TRU-b type prognosis was more significant than non-TRU type prognosis. (Fig. 4). SAM analysis was performed according to the same standard (false positive rate of less than 0.1%), and 169 genes showing differences in expression level between TRU-a type and TRU-b type at a magnification of 2 times or more were extracted. The 169 genes consisted of 119 genes with higher expression in TRU-a type and 50 genes with higher expression in TRU-b type.

  Tables 1 to 4 show gene sets belonging to adenocarcinoma subtypes of TRU type, non-TRU type, TRU-a and TRU-b, respectively. In the table, Uni-Gene symbol and UniID are respectively the symbol and sequence registration number of the gene registered in the Uni-Gene data bank, and GBID represents the registration number of the gene sequence registered in GenBank. At the same time, the gene name (or protein name or feature) is also displayed.

  Therefore, according to one aspect of the present invention, a method for discriminating and classifying lung adenocarcinoma as TRU type or non-TRU type, and then identifying and classifying TRU type adenocarcinoma as TRU-a type or TRU-b type I will provide a.

  This method may be used for one or more genes in each of the above gene sets in a biological sample of a patient, between TRU-type adenocarcinoma and non-TRU-type adenocarcinoma, or TRU-a type adenocarcinoma and TRU-b. When the difference in relative expression level between type adenocarcinomas is measured and the gene showing the difference in expression level is one of the genes listed in Table 1, the lung adenocarcinoma is TRU type If any of the gene groups listed in Table 2 is determined, the lung adenocarcinoma is determined to be non-TRU type. Furthermore, if the adenocarcinoma determined as TRU type is one of the gene groups listed in Table 3, it is determined that the lung adenocarcinoma is TRU-a type, and the gene groups listed in Table 4 Determining that the lung adenocarcinoma is TRU-b type.

More specifically, when the expression level of the gene belonging to the TRU type is relatively high between the TRU type adenocarcinoma and the non-TRU type adenocarcinoma, the adenocarcinoma is determined as the TRU type adenocarcinoma, When the expression level of the gene belonging to the non-TRU type is relatively high, the adenocarcinoma is determined as a non-TRU type adenocarcinoma .

Furthermore, when the expression level of the gene belonging to the TRU-a type is relatively high between the TRU-a type adenocarcinoma and the TRU-b type adenocarcinoma, the adenocarcinoma is determined as the TRU-a type adenocarcinoma. On the other hand, when the expression level of the gene belonging to the TRU-b type is relatively high, the adenocarcinoma is determined as a TRU-b type adenocarcinoma .

  For example, for genes A, B and C belonging to TRU type adenocarcinoma and genes X, Y and Z belonging to non-TRU type adenocarcinoma, the expression levels of gene A in adenocarcinoma sample 1 and sample 2 are 130 and 80 ( The expression level of gene B in adenocarcinoma sample 1 and sample 2 is 25 and 20 (arbitrary unit), respectively, and the expression level of gene C in adenocarcinoma sample 1 and sample 2 is respectively 1050 and 950 (arbitrary units), the expression levels of gene X in adenocarcinoma sample 1 and sample 2 are 600 and 650 (arbitrary units), respectively, and gene Y in adenocarcinoma sample 1 and sample 2 When the expression levels are 350 and 450 (arbitrary units), respectively, and the expression levels of gene Z in the same sample 1 and sample 2 are 25 and 50 (arbitrary units), respectively, sample 1 is a TRU adenocarcinoma, Identify sample 2 as non-TRU adenocarcinoma.

    The invention further provides, according to another aspect, the TRU type, non-TRU type, TRU-a type or TRU-b type of lung adenocarcinoma listed in Tables 1-4 above in a patient biological sample. In the same manner as in the above classification method, one or more genes of the gene set of 1 or two or more, homologues or derivatives thereof, between TRU-type adenocarcinoma and non-TRU-type adenocarcinoma, Provided is a method for predicting a patient's postoperative prognosis comprising identifying by measuring the difference in relative expression levels between TRU-b adenocarcinoma.

  According to this method, lung adenocarcinoma is first identified as TRU-type or non-TRU-type, and if it is determined to be TRU-type adenocarcinoma, TRU-type is further identified as TRU-a type or TRU-b type. If the subtype of lung adenocarcinoma is TRU-b type, the postoperative prognosis is good, or if it is TRU-a type or non-TRU type, the postoperative prognosis is judged to be poor. including.

  Furthermore, we found that the presence of EGFR mutations was significantly more observed in TRU-type adenocarcinoma than in non-TRU-type adenocarcinoma (45.3% vs. 21.6%), and EGFR mutations Interesting findings were obtained that the postoperative prognosis of patients with TRU-type adenocarcinoma including was poorer than the prognosis of patients with TRU adenocarcinoma containing wild-type non-mutated EGFR. It is known that lung cancer having EGFR gene mutation, particularly NSCLC, is highly sensitive to gefitinib (EGFR tyrosine kinase inhibitor). Can be used.

  That is, according to the present invention, in the prognosis prediction method described above, the postoperative prognosis of a patient is poor compared to lung adenocarcinoma containing a wild type EGFR gene, particularly when the EGFR gene contains a mutation in TRU-type lung adenocarcinoma. Further comprising predicting. Such prediction in TRU-type lung adenocarcinoma is considered to be capable of diagnosis or determination with higher accuracy and high clinical application value by combining with the prediction based on the relationship between the above-mentioned subtypes and prognosis.

  Also, in all adenocarcinomas, 5 genes with EGFR mutations and more than 2-fold up-regulation within adenocarcinoma, and 1.5-2 fold up-regulation associated with the presence of EGFR mutations 11 genes are identified and further searched for genes that are differentially and differentially expressed in TRU-type adenocarcinoma with EGFR mutations that occur frequently, and doubled in the presence of EGFR mutations The GGTLA4 gene having the above up-regulation and the RAMP1, APOH, PEX3, EST and DHRS7 genes showing 1.5 to 2-fold up-regulation were identified.

  Furthermore, non-TRU-type adenocarcinoma cases contain the highest percentage of tumors with p53 and / or K-ras mutations (41% for p53; 16% for K-ras), followed by TRU-a type ( 29% and 12%, respectively, followed by TRU-b type (21% and 0%, respectively). This indicates that non-TRU-type adenocarcinoma contains relatively high p53 and / or K-ras mutations compared to other subtypes, particularly TRU-a and TRU-b type adenocarcinoma. Show.

  These findings are also considered to be useful for prognosis determination, when used in combination with the postoperative prognosis determination of lung adenocarcinoma patients as well.

  In the method of the present invention, first, a nucleic acid for measuring the expression of a gene included in each gene set listed in Tables 1 to 4 or a mutant, homologue or derivative thereof is prepared.

Such nucleic acids have the following UniGene accession numbers:
Hs.512690, Hs.153322, Hs.218366, Hs.220629, Hs.436996, Hs.435759, Hs.104555, Hs.247824, Hs.127821, Hs.480281, Hs.529117, Hs.545862, Hs. 391561, Hs.479372, Hs.533055, Hs.550526, Hs.322854, Hs.465720, Hs.356664, Hs.26630, Hs.534496, Hs.85962, Hs.211267, Hs.128041, Hs.534458, Hs.495774, Hs.437806, Hs.133062, Hs.501758, Hs.444535, Hs.495480, Hs.326561, Hs.483906, Hs.169943, Hs.271285, Hs.158339, Hs.62604, Hs. 469359, Hs.436657, Hs.8417, Hs.155538, Hs.533526, Hs.512756, Hs.87191, Hs.463079, Hs.513779, Hs.476209, Hs.279580, Hs.351544, Hs.269408, Hs.134807, Hs.482417, Hs.176626, Hs.465643, Hs.183390, Hs.411299, Hs.234027, Hs.109358, Hs.103983, Hs.26216, Hs.534352, Hs.240457, Hs. 516036, Hs.144875, Hs.411312, Hs.103989, Hs.537722, Hs.333130, Hs.517962, Hs.90250, Hs.478930, Hs.121629, Hs.194061, Hs.520627, Hs.348012, Hs.522836, Hs.1376, Hs.520049, Hs.512856, Hs.355236, Hs.349470, Hs.476231, Hs.137556, Hs.390567, Hs .368353, Hs.412792, Hs.449207, Hs.527095, Hs.118722, Hs.377090, Hs.232696, Hs.447544, Hs.372773, Hs.2222055, Hs.511839, Hs.153299, Hs.434374 , Hs.287729, Hs.553740, Hs.127189, Hs.497723, Hs.181973, Hs.173656, Hs.451956, Hs.184507, Hs.532492, Hs.370904, Hs.460468, Hs.520612, Hs .436667, Hs.125116, Hs.459391, Hs.450320, Hs.149769, Hs.325890, Hs.356820, Hs.289319, Hs.73893, Hs.129493, Hs.515069, Hs.34560, Hs.477278 , Hs.351571, Hs.112087, Hs.154224, Hs.125950, Hs.438016, Hs.367956, Hs.553778, Hs.329266, Hs.479658, Hs.458713, Hs.249196, Hs.467529, Hs .145061, Hs.49653, Hs.129227, Hs.313343, Hs.194554, Hs.123114, Hs.126561, Hs.42091, Hs.369385, Hs.98661, Hs.458306, Hs.148584, Hs.501684 , Hs.422466, Hs.523732, Hs.525557, Hs.1372, Hs.379097, Hs.208124, Hs.389311, Hs.2561, Hs.117545, Hs.446388, Hs.2813, Hs.473894, Hs .502092, Hs.524479, Hs.314261, Hs.382306, Hs.458252, Hs.380222, Hs.379636, Hs.302034, Hs.253495, Hs.3 45877, Hs.259563, Hs.528569, Hs.152337, Hs.436317, Hs.546408, Hs.46700, Hs.1027, Hs.151219, Hs.279611, Hs.310456, Hs.520319, Hs.406976, Hs.181245, Hs.449621, Hs.515465, Hs.310540, Hs.554891, Hs.449601, Hs.355394, Hs.380710, Hs.171995, Hs.449585, Hs.522484, Hs.298023, Hs. 520339, Hs.121443,
Hs.62661, Hs.200804, Hs.533185, Hs.461329, Hs.479270, Hs.446201, Hs.35086, Hs.500761, Hs.44298, Hs.469030, Hs.309767, Hs.441047, Hs. 98309, Hs.31409, Hs.518299, Hs.532870, Hs.196534, Hs.108106, Hs.289319, Hs.69771, Hs.374378, Hs.369422, Hs.368641, Hs.302963, Hs.530461, Hs.1955, Hs.513726, Hs.148767, Hs.523220, Hs.525796, Hs.271264, Hs.69321, Hs.231367, Hs.500761, Hs.528304, Hs.148685, Hs.87417, Hs. 164060, Hs.514843, Hs.418416, Hs.126521, Hs.519839, Hs.103834, Hs.279840, Hs.497741, Hs.531457, Hs.226390, Hs.480143, Hs.473721, Hs.369762, Hs.514527, Hs.204238, Hs.3104, Hs.519873, Hs.519909, Hs.179718, Hs.103183, Hs.520210, Hs.444683, Hs.234545, Hs.80976, Hs.311187, Hs. 89497, Hs.444118, Hs.541635, Hs.477898, Hs.511776, Hs.434886, Hs.117299, Hs.252451, Hs.468058, Hs.21554, Hs.165904, Hs.445244, Hs.413924, Hs.99120, Hs.521171, Hs.462379, Hs.481860, Hs.489207, Hs.414407, Hs.505575, Hs.516826, Hs.62180, Hs.368934 , Hs.530509, Hs.278906, Hs.511987, Hs.444082, Hs.471873, Hs.24583,
Hs.1051, Hs.115263, Hs.379010, Hs.2012, Hs.268698, Hs.49653, Hs.424542, Hs.525589, Hs.25333, Hs.513075, Hs.549577, Hs.100431, Hs. 9613, Hs.301478, Hs.553740, Hs.525383, Hs.473721, Hs.375624, Hs.116724, Hs.9613, Hs.418055,
Hs.334873, Hs.180878, Hs.126521, Hs.519033, Hs.187636, Hs.518448, Hs.322761, Hs.31409, Hs.524513, Hs.436437, Hs.148989, Hs.279575, Hs. 75668, Hs.470791, Hs.104476, Hs.494496, Hs.517549, Hs.278906, Hs.498586, Hs.183617, Hs.499758, Hs.350065, Hs.511138, Hs.150793, Hs.129174, Hs.212606, Hs.275775, Hs.496414, Hs.436142, Hs.282984, Hs.32417, Hs.69321, Hs.414629, Hs.502618, Hs.405755, Hs.480143, Hs.60371, Hs. 283683, Hs.208093, Hs.336768, Hs.1116459, Hs.131673, Hs.32417, Hs.75812
A nucleic acid capable of detecting the expression of a gene having or a variant, homologue or derivative thereof,
(1) a nucleotide sequence represented by SEQ ID NO: 1 to 351,
(2) a nucleotide sequence comprising the nucleotide sequence shown in SEQ ID NOs: 1 to 351,
(3) a nucleotide sequence complementary to the nucleotide sequence of (1) or (2),
(4) a nucleotide sequence that hybridizes under stringent conditions with any of the sequences (1), (2), or (3), and
(5) a partial sequence of less than the total number of bases from 15 bases of the nucleotide sequence of (1), (3) or (4),
One or more nucleic acids having a sequence selected from the group consisting of:

  For example, if the nucleic acid is a DNA molecule of about 100 bases or less, it can be synthesized using an automatic DNA synthesizer (for example, Applied Biosystems, USA) using the phosphoramidite method. Alternatively, the nucleic acid can be produced by cDNA cloning. After obtaining total RNA from tumor lung tissue and obtaining poly A (+) RNA by oligo dT cellulose column treatment, a cDNA library was prepared by reverse transcriptase-polymerase chain reaction (RT-PCR) method. A cDNA clone can be obtained from a library by hybridization with a probe (15 or more, preferably 30 or more, more preferably 50 to 100 or more base length) prepared in advance based on a sequence registered in a gene bank. it can. The obtained clone is inserted into an appropriate host cell such as Escherichia coli or Bacillus subtilis after incorporation into a commercially available expression vector, and the host cell is propagated or based on a sequence registered in the gene bank. Amplification can be performed by polymerase chain reaction (PCR) using a primer prepared in advance (usually 15 to 30 bases, preferably 17 to 25 bases long) and using the cDNA clone as a template. For specific procedures and reagents of cDNA cloning and PCR methods, commercially available kits, devices, and reagents can be used. For example, Sambrook J et al., Molecular Cloning A Laboratory Manual, 1989, Cold Spring Harbor Laboratory. Press (USA); Ausbel FM, et al., Short Protocols in Molecular Biology (3rd edition) A Compendium of Methods from Current Protocols in Molecular Biology, 1995 & Jon. (United States).

  In the nucleic acid of (4) above, the nucleic acid containing a nucleotide sequence that hybridizes under stringent conditions is any nucleic acid that hybridizes with any nucleic acid containing the sequence shown in SEQ ID NOs: 1 to 351 or a complementary sequence thereof. It is a nucleic acid. Such nucleic acids are DNA or RNA.

  Stringent conditions include the conditions defined and exemplified above. That is, an example of such conditions is hybridization in 1 M sodium chloride / 0.5% (W / V) sarkosyl / 30% formamide at 60 ° C. for 17 hours, followed by 6 × SSC / 0.005% ( (W / V) Triton X-102 solution at room temperature, once for 10 minutes, and further maintained at 0-4 ° C. in 0.1 × SSC / 0.005% (W / V) Triton X-102 solution. The condition for washing is one time for 5 minutes. Here, 1 × SSC is 150 mM sodium chloride and 15 mM sodium citrate aqueous solution (pH 7.2).

  Hybridization includes microarray methods, blotting methods such as Northern or Southern blots, Northern or Southern hybridization methods, in situ hybridization, quantitative RT-PCR methods and the like.

  In the nucleic acid (5), the nucleic acid fragment having the nucleotide sequence (1), (3) or (4) has a size of 15 bases to less than the total number of bases. Fragments can have any number of bases within this range, for example, 20 bases or more, 30 bases or more, 50 bases or more, 70 bases or more, 100 bases or more, 150 bases or more, 200 bases or more, 300 bases or more, 400 bases or more, 500 bases The number of bases as described above.

  According to the present invention, for one or more genes of each gene set in the above table in a biological sample of a patient, between TRU-type adenocarcinoma and non-TRU-type adenocarcinoma, or TRU-a type adenocarcinoma The difference in relative expression levels between TRU and TRU-b adenocarcinoma is measured.

  Here, the difference in relative expression level is a specific gene having the above gene set between TRU-type adenocarcinoma and non-TRU-type adenocarcinoma or between TRU-a type adenocarcinoma and TRU-b type adenocarcinoma. Means the difference in expression level that the gene exhibits between the above-mentioned subtypes. In the present invention, a preferable difference in expression level for a specific gene is 1.5 times or more, more preferably 2.0 times or more.

  By identifying a gene showing a difference in expression level, the relationship between the gene and a subtype of lung adenocarcinoma (ie, TRU, TRU-a, TRU-b, or non-TRU type) can be clarified. Thus, for example, the postoperative prognosis of a patient can be predicted by identifying a subtype of lung adenocarcinoma, or based on the above relationship between a gene set containing the identified gene and the subtype Lung adenocarcinoma can be classified into four types.

  According to the present invention, the difference in gene expression level can be made by measuring the presence or amount of nucleic acid or protein corresponding to the gene.

  Biological samples include lung cancer tissues or cells of lung cancer patients, such as cancer tissues excised by surgery, tissues or cells obtained by biopsy, and the like.

  Hereinafter, these two different methods will be described in detail.

(Method using nucleic acid)
In order to detect 351 genetic markers involved in the present invention, the above nucleic acids that hybridize with each of these markers are used. The number of genes to be detected is 1 or 2 or more for each gene set, preferably 2 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 60 or more. The greater the number of genes, the better the accuracy of identifying lung adenocarcinoma subtypes.

  Hybridization can be performed by a microarray method, a blotting method such as Northern or Southern blotting, Northern or Southern hybridization method, in situ hybridization method, quantitative RT-PCR method or the like. Preferred hybridization methods are microarray, quantitative RT-PCR or blotting. Examples of preferred microarrays are DNA microarrays and protein microarrays.

  In the DNA microarray method, a DNA chip in which nucleic acid probes that hybridize with the genes (1 to all) listed in Tables 1 to 4 are bound to a substrate is prepared and used.

  As the DNA chip, any type of substrate can be used as long as the nucleic acid probe can be immobilized. The solid phase includes, for example, glass, a polymer, and a spacer or a crosslinker including a reactive group for covalently binding a nucleic acid can be introduced. Since such chips are commercially available, it is desirable to use them.

  The solid phase immobilization of the nucleic acid probe is not particularly limited, but a general method such as a method of spotting DNA using a high-density dispenser called a spotter or an arrayer, an ink jet method of ejecting droplets from a nozzle, or the like This method can be used.

  A nucleic acid obtained by labeling a nucleic acid such as DNA or RNA in a biological sample, cDNA or cRNA derived therefrom with a fluorescent substance such as Cy dye (Cr3 or Cy5) is hybridized with a probe on a DNA chip. The fluorescence intensity is read using a reading device by laser scanning, and the data is analyzed by a computer.

In the blotting method, the nucleic acid probe of the present invention is labeled with a radioisotope (for example, ³² P and ³⁵ S) or a fluorescent substance (rhodamine derivative, Cy dye, etc.) and then transferred to a polymer membrane such as nylon. Hybridization is performed with DNA or RNA in the sample, and nucleic acids such as cDNA and cRNA derived therefrom. The signal is detected using a radiation detector or a fluorescence detector and its intensity is measured.

  In the quantitative RT-PCR method, PCR is performed by annealing the primer with cDNA so that each target gene region can be amplified using cDNA prepared from RNA in a biological sample as a template. Is detected. Detect and quantify target genes by pre-labeling primers with radioisotopes or fluorescent materials, or by electrophoresis of PCR products on agarose gel and staining double-stranded DNA with ethidium bromide can do.

PCR conditions are, for example, denaturation: 92 to 94 ° C. for 30 to 60 seconds; annealing: 50 to 55 ° C. for 30 to 60 seconds; extension: 68 to 72 ° C. for 30 to 60 seconds, and 30 to 40 cycles of reaction. including. As the reverse transcriptase, commercially available enzymes such as SuperScript ^™ III (Invitrogen, USA), AMV Reverse Transcriptase (Promega, USA), M-MLV (RNaseH ⁻ ) (Takara Shuzo, Kyoto) and the like can be used.

(Protein method)
An alternative method for measuring the expression level of the gene is an immunological method.
In this method, a protein or fragment thereof corresponding to each gene is synthesized using protein synthesis or gene recombination techniques, and the resulting protein or fragment thereof is used as an antigen for rabbit, mouse, rat, horse, cow, Animals such as goats and sheep are immunized, and antibodies against these antigens are produced and purified.
The antibody includes a polyclonal antibody, a monoclonal antibody, an anti-peptide antibody and the like.

  The polyclonal antibody is obtained by immunizing the animal subcutaneously with about 10 to 300 μg of the antigen, further immunizing about 2 weeks later, collecting blood about 3 weeks to 1 month after the first immunization, and obtaining the desired polyclonal antibody from the antiserum. The IgG component can be produced by a method including separation using ammonium sulfate fractionation and ion exchange chromatography. In order to increase specificity, the obtained IgG is bound to a column made by binding the target protein to a carrier such as cellulose or agarose, and then eluted with a high salt buffer for dialysis or ultrafiltration. A specific polyclonal antibody can be obtained by desalting by a method such as The antibody titer can be measured by a conventional immunoassay, for example, an enzyme immunoassay (EIA, ELISA), a radioimmunoassay (RIA), a fluorescent antibody method, or the like.

A monoclonal antibody can be prepared, for example, by the following general method.
The target protein or a fragment thereof is administered subcutaneously to mice or rats (for example, Balb / c mice) in the same manner as polyclonal antibody production, and booster immunization is performed about 2 to 4 times at 1 to 4 week intervals. When the antibody titer reaches a peak, the antigen is injected intravenously or intraperitoneally to obtain the final immunization. After 2 to 5 days, antibody-producing cells (eg spleen cells or lymph node cells) are collected. The antibody-producing cells are then fused to a myeloma cell line (preferably a hypoxanthine / guanine / phosphoribosyl transferase (HGPRT) deficient cell line) to generate hybridoma cells, and HAT (hypoxanthine, aminopterin, thymidine ) selection is performed. Do. For cell fusion, antibody-producing cells and myeloma cell lines are mixed in a ratio of about 1: 1 to 20: 1 in animal cell culture media such as serum-free DMEM, RPMI-1640 medium, and the like. It is carried out in the presence of a cell fusion promoter such as Confirmation of the target antibody can be performed by the immunoassay described above. Further, in order to proliferate the hybridoma, about 10 million hybridomas are administered into the abdominal cavity of the mouse, and after the hybridoma is proliferated, ascites is collected after 1 to 2 weeks. Antibody purification can be performed by appropriately combining methods such as ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and gel chromatography.

  Anti-peptide antibodies are antibodies against linear peptides on the surface of proteins and can increase immunological specificity. Such peptides include, for example, the estimation of hydrophilic-hydrophobic regions by Kyte-Doolittle, et al., The probability of being located on the surface of a specific peptide site on a protein molecule by Emini et al., The degree of bending of the polypeptide chain, eg Chou-Fasman Can be estimated using an α helix, β sheet, secondary structure prediction of a protein indicating a turn, etc. alone or in combination. The estimated peptide can then be synthesized using a peptide synthesizer.

  Here, the synthesis of the target protein (encoded by the genes shown in Tables 1 to 4 above) is performed by incorporating a cDNA clone into an expression vector and culturing a prokaryotic or eukaryotic host cell transformed or transfected with the vector. Can be obtained from the cells or culture supernatant. A commercially available expression vector can be used. Host cells include prokaryotic cells such as bacteria (eg, E. coli, Bacillus subtilis, Pseudomonas bacteria), yeast (eg, Saccharomyces, Pichia, etc.), insect cells (eg, Sf cells), mammalian cells (eg, CHO, COS, BHK, HEK293, etc.). The vector is composed of a plasmid, cosmid, phage or the like, and may contain DNA encoding the target protein, promoter, enhancer if necessary, polyadenylation signal, ribosome binding site, replication origin, terminator, selection marker, and the like. To facilitate polypeptide purification, a DNA sequence encoding a labeled peptide, such as a 6-10 residue histidine tag, FLAG, GFP polypeptide, etc., can also be included. The gene recombination techniques are described in Sambrook et al. (Above), Ausbel et al. (Above), and the techniques described therein can be used for the present invention.

  The target protein obtained as described above is appropriately combined with gel filtration, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, isoelectric focusing, electrophoresis, ultrafiltration, salting out, dialysis, etc. And can be purified.

  The sequence of the target protein or fragment thereof can be obtained by accessing the NCBI HomePage based on the GenBank accession numbers listed in Tables 1-4 above.

  The antibodies described above can be used for the detection of target proteins or fragments thereof in biological samples. Detect or quantify a large number of target proteins at a time by creating a protein microarray in which a large number of antibodies are bound on a microarray substrate, or by spotting a large number of antibodies in a dot pattern on a filter such as a PVDF membrane. It becomes possible. Or, conventional immunological measurement methods such as enzyme immunoassay (ELISA, EIA), fluorescent antibody method, radioimmunoassay (RIA), luminescence immunoassay, immunoturbidimetric method, latex agglutination, latex turbidimetric The target protein or a fragment thereof in a biological sample can be detected or quantified by a method, a hemagglutination reaction, a particle agglutination reaction, a Western blot method or the like.

  When the reaction is performed on a solid phase, solid phase carriers include films (filters) of polymers such as polystyrene, polycarbonate, and polyethylene, plates, tubes, strips, and particles such as latex and magnetic materials. The solid phase can be physically or chemically performed. For chemical coupling, the solid phase can be treated with a reagent such as a maleating reagent or cyanogen bromide to introduce a functional group that reacts with the amino group of the protein into the solid phase.

Examples of the label include enzymes such as horseradish peroxidase and alkaline phosphatase, fluorescent materials such as fluorescein, rhodamine and derivatives thereof, luminescent materials such as luciferase and luminol, and radioisotopes such as ³² P and ¹²⁵ I. . Labeling includes, for example, glutaraldehyde method, maleimide method, pyridyl disulfide method, chloramine T method, Bolton Hunter method and the like.

  The present invention also provides the nucleic acid (1) to (5) for measuring the expression of a gene included in each gene set listed in Tables 1 to 4 above, or a mutant, homologue or derivative thereof, For predicting the postoperative prognosis of a patient with lung adenocarcinoma in vitro or comprising a TRU for lung adenocarcinoma, comprising an antibody against the protein or fragment encoded by the gene, or a variant, homologue or derivative thereof Compositions are provided for classification into any subtype of type, non-TRU type, TRU-a type or TRU-b type.

According to an embodiment of the present invention , a kit or a microarray comprising the nucleic acid or antibody or fragment thereof constituting the composition is provided .
In the kit of the present invention, the nucleic acid shown in the above (1) to (5), which is 1 from each TRU type, non-TRU type, TRU-a type and TRU-b gene set (see Tables 1 to 4, respectively) Alternatively, nucleic acids capable of detecting a total number of genes from two or more are packaged for each gene set.

  Another kit of the present invention comprises a gene contained in each gene set listed in Tables 1 to 4 above, or an antibody or fragment thereof against a protein encoded by a variant, homologue or derivative thereof, or a fragment thereof. Package for each protein set corresponding to the gene set.

The antibody contained in the composition of the present invention is a polyclonal antibody, a monoclonal antibody, an anti-peptide antibody or the like produced by the above method, but is not limited thereto. The type of antibody may be any type, class, subclass, and includes, for example, IgG, IgM, IgE, IgD, IgA and the like. Antibody fragments include Fab, (Fab ′) ₂ , Fv, and the like.

  The kit may further contain reagents for performing hybridization, such as a buffer, a reverse transcriptase, a labeled secondary antibody, and the like.

  The microarray of the present invention is a DNA microarray (also referred to as a DNA chip) or a protein microarray.

  Each of these microarray chips is bound with the above-described nucleic acids (1) to (5) or the above-described antibodies or fragments thereof. That is, on the surface of the chip, a nucleic acid capable of hybridizing with a gene included in the gene set listed in Tables 1 to 4 above or a variant, homologue or derivative thereof, or a protein encoded by these genes Or an antibody or fragment thereof that reacts immunologically specifically with a variant or derivative thereof.

  Variants have 70% or more, preferably 80% or more, more preferably 90% or more, 95% or more or 98% or more identity with the fully mature sequence of the gene or protein at the nucleotide or amino acid level. is there. Here, the identity (%) can be determined using a known BLAST or FASTA program into which a gap is introduced. In general, identity (%) can be calculated as a percentage of the number of matched bases relative to the total number of bases.

  Protein derivatives include, for example, chemically modified derivatives such as glycosylation, phosphorylation, sulfation, alkylation, acylation and the like.

  As the substrate of the chip, glass or resin (polymer) is usually used, and for example, poly L-lysine, silane or densified amino groups are introduced on the surface thereof.

  The nucleic acid or antibody is bound to the substrate by the spot method or the ink jet method as described above.

  The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples.

A. Materials and methods
Aichi Prefecture has developed a series of 149 NSCLC cases, including 90 patients with adenocarcinoma, 35 squamous cell carcinomas, 18 large cell carcinomas, 4 squamous cell carcinomas, and 2 large cell neuroendocrine cancers. Obtained from a file of successful patients who underwent curative resection between December 1995 and December 1999 at the Department of Thoracic Surgery (Nagoya). For post-surgical survival analysis, 82 cases with adenocarcinoma and follow-up range from 6 to 108 months (median 77.0 months; average 65.1 months) used. The remaining 8 cases were excluded because they received gefitinib treatment. All tumor specimens were embedded in OCT compounds and stored at −80 ° C. after obtaining the required approval from the formal review department and written informed consent from the patient.

Obtaining an expression profile The frozen tissue of the tumor specimen was subjected to rough microdissection under the guidance of a pathologist using all 10th sections stained with Giemsa. Total RNA was extracted using RNeasy kit (Qiagen, USA) and then treated with DNase I. Large batches of reference RNA were prepared by using 20 lung cell lines, all representing major tissue type lung cancer. Double-stranded cDNA was synthesized from 250 ng of total RNA using a poly dT primer incorporating a T7 promoter and MMLV-RT. cRNA was generated and labeled with Cy3 or Cy5 (Cy dye; Amersham Pharmacia Biotech, USA) using a low RNA Fluorescent Linear Amplification kit (Agilent Technologies, USA). Cy5-sample cRNA and Cy3-regular reference cRNA were hybridized with a custom-made Agilent oligonucleotide microarray containing a total of 21,619 spots corresponding to 18,175 unique genes, followed by confocal laser scanning ( (Agilent Technologies, USA). The fluorescence intensity on the scanned image was quantified and the value was corrected and normalized to the background value.

In order to filter out potential noise during bioinformatics analysis analysis, genes that did not provide sufficient expression signal in more than 10 samples were excluded from further analysis. In addition, genes whose expression levels changed less than 3-fold throughout the sample set of interest were excluded because such genes could not be considered to provide useful information. Using the CLUSTER program, mean association order clustering of both genes and cases was performed using median centering and normalization, followed by TREEVIEW (Eisen MB et al., Proc Natl Acad Sci USA 95: 14863-8, 1998). The result was displayed. SAM, a characteristic signal extraction technique, was used to score genes specific to each of the patient's lung adenocarcinoma subtypes (Tusher VG et al., Proc Natl Acad Sci USA 98: 5116-21, 2001). .

Mutation analysis of EGFR, p53 and K-ras genes p53 (exons 4-10), EGFR (exons 15-24) and K-ras (exons 1-2) genes were identical RNAs used for microarray analysis Amplified from The resulting PCR product was directly sequenced as previously described (Kosaka T et al., Cancer Res 64: 8919-23, 2004).

Identification based on GO items and other statistical analysis Gene ontology (GO) (Ashburner M et al., Nat Genet 25: 25-9, 2000) analysis, specific to each of the patient's adenocarcinoma subtypes Biological characteristics of different functions of the gene set were elucidated. The database file used for this GO analysis was downloaded from UniGene's ftp site (ftp://ftp.ncbi.nih.gov/repository/UniGene/). The Unigene ID corresponding to the spot on the microarray is obtained with the help of a program written in Perl newly developed by the present inventors. seq. Connected to the corresponding GO item via LocusLink ID by analyzing the database file containing all, His data and LL_templ. Eventually, 12,745 known genes out of 18,175 unique genes on the microarray were associated with approximately 67,000 GO items. Among these items, items that frequently appear in the target gene set were identified by Fisher's exact test.

For the frequency analysis, χ ² test or Fisher exact test was used. Multivariate logistic regression analysis was performed to examine the relationship between various clinical parameters and the expression of the subtypes defined by the expression profile. Survival was evaluated as a function of time using the Kaplan-Meier method and the difference in survival rate was analyzed with the log-rank test. An independent factor affecting survival was analyzed using the Cox proportional hazards model. All analyzes were performed using Stata software (version 7; Stat Corp, USA), with a two-sided significance level set at P <0.05.

B. result
All 149 samples were classified using the non-small cell lung cancer expression profile classification and the unsupervised phylogenetic clustering method. At this time, a molecular classification method based on the similarity of genome-wide expression patterns in individual tumors was established using 4,834 most variably expressed transcripts. The resulting cluster accurately reproduced the well-established and widely used NSCLC histological classification (FIG. 1). SAM analysis reveals the existence of a different set of genes with up-regulation or down-regulation for each cluster, and this gene set was also identified by the inventors (Tomida S et al., Oncogene 23: 5360-70, 2004) and A gene set similar to that previously reported by other investigators (Garber ME et al., Proc Natl Acad Sci USA 98: 13784-9, 2001; Bhattercharje A et al., Proc Natl Acad Sci USA 98: 13790-5, 2001) Turned out to be.

  In this study, we conducted an extensive search for mutations in the EGFR, p53, and K-ras genes that are strongly thought to be responsible for the development of lung cancer. Or it was investigated whether it was related to non-existence. A clear and accurate reproduction of the conventional NSCLC histological classification and the highly significant relationship between the EGFR mutation and the corresponding adenocarcinoma tree, as predicted from previous reports on these three genetic changes, (36% vs. 0%; P <0.001) and between the p53 mutation and a dendrogram consisting mostly of squamous cell carcinoma and large cell carcinoma (67% vs. 33%; P <0 .001).

Two adenocarcinoma tumor types identified by expression profile During this study, we have two major subclusters, while adenocarcinoma cases are clustered together as a large single dendrogram I noticed that there is. This finding was performed in a separate analysis of adenocarcinoma because of the strong features of other NSCLC tissue types that could obscure differences within the adenocarcinoma. To this end, a taxonomic clustering performed with the 4,138 transcripts most variably expressed in adenocarcinoma resulted in two major dendrograms and a right dendrogram. The presence of two additional subclusters was clearly shown (FIG. 2A). Morphological analysis shows that bronchioloalveolar carcinoma is located in the right-handed subcluster, but other adenocarcinoma subtypes or variants that follow the WHO classification are clearly clustered in any particular dendrogram It was shown not to be. However, we found that the case features in the right dendrogram were TRU-type adenocarcinoma, ie different adenocarcinoma subsets (Yatabe Y et al., Am J Surg Pathol 29: 633-639, 2005; Yata Y Y et al., Am J Surg Pathl 26: 767-73, 2002; Yata Y, Elsevier Science / Academic Press, wN I noticed. (Note: For convenience, the tumors in the right and left tree diagrams are referred to as TRU adenocarcinoma and non-TRU adenocarcinoma, respectively).

  To gain further insight into the dendrogram and biological properties of adenocarcinoma subtypes defined by these two major expression profiles, namely non-TRU and TRU types, SAM analysis was first performed and differential The gene expressed in was selected. In the SAM analysis, 1,657 genes were extracted by pre-filtering at a significant level of false positive rate of less than 0.1%, and 286 of these genes were compared to TRU-type and non-TRU at a magnification of 2 or more. Differences were shown between their expression levels with the TRU type. These 286 genes consisted of 194 genes with higher expression in the TRU form and 92 genes with higher expression in the non-TRU form (data not shown). In order to better understand the basic functional differences between TRU and non-TRU, the SAM identified gene set was analyzed using GO items. This method was developed in our laboratory for this study. Using this identification method, GO items related to 9 biological processes, 6 molecular functions, and 2 cellular components were extracted as being significantly more frequent in TRU adenocarcinoma, A clear relationship with normal lung function was shown (Table 5).

  In contrast, GO items identified as specific for non-TRU types include those related to cell cycle and proliferation, which indicates the inherent aggressive nature of non-TRU type adenocarcinoma. Suggests.

  Identification of the subtypes identified by these very robust expression profiles of adenocarcinoma was performed by similar unsupervised clustering analysis using independent data sets. A Stanford data set consisting of 34 cases of lung adenocarcinoma (Garber ME et al., Proc Natl Acad Sci USA 98: 13784-9, 2001) was analyzed for differential expression in non-TRU, TRU-a and TRU-b types. Analysis was performed by unsupervised classification systematic clustering based on the expression profiles of the 30 top ranked genes. This analysis resulted in a clear visualization of the two main dendrograms with subclusters. This dendrogram appeared to correspond well to the adenocarcinoma subtype identified by the three expression profiles identified in this study (FIG. 3).

Significant relationship between adenocarcinoma subtype identified by expression profile and clinicopathological features Next, various clinicopathological properties and adenocarcinoma subtype identified by two expression profiles, ie TRU adenocarcinoma And the relationship between non-TRU adenocarcinoma (Table 6).

  TRU-type adenocarcinoma was significantly more frequently observed in females (P = 0.005) and non-smokers (P <0.001) than non-TRU-type. On the other hand, by detailed microscopic examination, cell cycle-related GO items in which the presence of invasive growth and necrosis, which are indicators of higher malignancy / appearance and rapid proliferation, are distinguished from non-TRU type tumors And according to the dominant growth, it was characteristically widespread in non-TRU types (P <0.001 for both invasive growth and necrosis). Multivariate logistic regression analysis with age, gender, smoking status and pathological stage as variables identified a non-smoking status as a significantly related variable (P = 0.001). Regarding the prognosis after surgery, we have found that cases belonging to the two obvious clusters under the dendrogram of TRU-type adenocarcinoma seen in FIG. 2, namely TRU-a and TRU-b, I noticed that it was differentiated in terms of later prognosis. TRU-a type has a prognosis similar to that of non-TRU type, whereas TRU-b type adenocarcinoma has a significantly better prognosis than non-TRU type (P = 0.021; FIG. 4). This is consistent with the fact that microscopic examination showed that explicit invasive growth occurred in the TRU-b type adenocarcinoma much less frequently than in other adenocarcinoma types. It seems (P <0.001).

Significant relationship between adenocarcinoma subtypes identified by expression profile and EGFR mutation status Any of the three major genetic changes in NSCLC are significantly associated with adenocarcinoma subtypes identified by expression profile It was investigated whether it was possible (FIG. 2B). The presence of EGFR mutations was found to be significantly more frequent in TRU-type adenocarcinoma than in non-TRU-type adenocarcinoma (45.3% vs 21.6%; P = 0.026). We also found that the two obvious clusters under the TRU-type adenocarcinoma dendrogram, TRU-a and TRU-b, are slightly different in terms of the frequency of EGFR mutations, It was noticed that it showed a higher EGFR mutation frequency for TRU-b type adenocarcinoma (52.6%) than for TRU-a type adenocarcinoma (41.2%). In contrast, K-ras and p53 did not correlate with adenocarcinoma subtypes (P = 0.33 for p53; P = 0.17 for K-ras), but an interesting reversal of mutation frequency A correlation was observed between these genetic changes and EGFR mutations. Non-TRU-type adenocarcinoma contains the highest percentage of tumors with p53 and / or K-ras mutations (41% for p53; 16% for K-ras), followed by TRU-a type (29% and 12%) and TRU-b type (21% and 0%, respectively).

Prognostic significance of EGFR mutations in TRU-type adenocarcinoma We and other researchers have reported that the presence of EGFR mutations does not affect the postoperative prognosis of NSCLC patients (Kosaka) T et al., Cancer Res 64: 8919-23, 2004; Shigematsu H et al., J Natl Cancer Inst 97: 339-46, 2005), which was confirmed in this independent data set (FIG. 5A; P = 0). .42). Based on the reason that this finding may indicate a more important role of EGFR mutations in the development of TRU-type adenocarcinoma than in the development of other lung cancer types, the present inventors Et al. Performed a separate analysis of TRU-type adenocarcinoma cases to investigate the potential relationship between EGFR mutations and postoperative prognosis. A significant relationship was detected between postoperative bad prognosis and the presence of EGFR mutations in TRU-type adenocarcinoma (FIG. 5B; P = 0.024). This relationship was further confirmed by the results of multivariable Cox regression analysis (Table 7).

  The presence of EGFR mutations in TRU-type adenocarcinoma is indicated by the stage of the disease (HR = 7.61; P = 0.001) and the histological subtype (HR = 8.25; P = 0.003), showed independent prognostic factors (HR = 7.71; P = 0.003), while gender, age and smoking status were any significant No relationship was shown.

Search for genes with significant differential expression in the presence or absence of EGFR mutations In all adenocarcinoma cases, a significant 5% false positive rate was chosen to select genes associated with the presence of EGFR mutations Levels were used because they were not selected at all when using a 1% false positive rate (Table 8).

  Five genes with EGFR mutations and more than 2-fold upregulation in the tumor were identified, while an additional 11 genes had 1.5-2 fold upregulation associated with the presence of EGFR mutations Indicated. We further searched for genes that are differentially and differentially expressed in TRU-type adenocarcinoma with EGFR mutations that appear significantly more frequently. Although it was necessary to use a fairly high false positive rate (10%) to select genes by SAM, a single gene with more than 2-fold up-regulation in the presence of the EGFR mutation was identified, namely GGTLA4 . In addition, 8 genes showed 1.5-2 fold up-regulation at the same significant difference level (Table 5).

Discussion The expression profile in a given tumor can be considered as a result of a complex effect. Such effects are the result of accumulated genetic changes important in pathogenesis, as well as the differentiation and commitment of progenitor cells. In this study seeking that understanding, we succeeded in establishing a very robust adenocarcinoma taxonomy identified by the expression profile. Its relevance to the method is clearly supported by the inclusion of various distinct molecular genetic and clinicopathological features. The two main subtypes, TRU and non-TRU, are characterized by differential expression of a large number of genes and thus show their significant differences in gene usage (Yatabe Y et al., Am J Surg Pathol 29: 633-639, 2005). The results obtained using our GO item identification method further support their different properties. TRU-type tumors are characterized by biological processes that are important for the management of peripheral lung function (Veldhuizen EJ et al., Biochim Biophys Acta 1467: 255-70, 2000). Similarly, molecular functions associated with TRU types appear to reflect the retention of their progenitor cell characteristics. In contrast, many non-TRU-type related GO items are associated with cell cycle and cell proliferation and appear to be consistent with high-grade microscopic findings.

  The marked differences in clinical features between TRU and non-TRU subtypes also support the robustness of the taxonomy identified in the expression profile of the present invention. A notable clinical feature of the TRU type is a significantly higher proportion of women and non-smokers, while multivariate analysis results indicate non-smoking status as an independently related factor. It is not showing gender. That is, this tumor type appears to arise from peripheral lung airway cells in a much lesser effect of smoking, and seems to retain its ancestral features as shown by GO item-based analysis It is to be. TRU-b has the most favorable prognosis, has a low frequency of invasive growth, and expresses higher levels of various differentiation markers even when compared to TRU-a. This suggests that TRU-b type retains the characteristics of normal peripheral lung airway cells better than other types but may progress to TRU-a type.

  This study clearly shows that the presence of EGFR mutations is significantly associated with TRU-type adenocarcinoma. In fact, 45.3% of TRU-type adenocarcinoma cases have an EGFR mutation, while the non-TRU-type has a 21.6% incidence. On the other hand, p53 and K-ras did not show a significant difference in their mutation frequency, but there was an interesting reverse trend of the highest incidence in non-TRU types. The presence of EGFR mutations was significantly associated with short postoperative survival regardless of disease stage, especially for TRU-type adenocarcinoma. Indeed, the 5-year survival rate for patients with TRU-type stage II / III disease was found to be 25% in the presence of the EGFR mutation and 78% in the absence. These findings indicate the possible clinical utility of simultaneous analysis of subtypes identified in expression profiles and EGFR mutation status to select candidate drugs for intensive adjuvant therapy with gefitinib if possible Suggests.

  In conclusion, this study shed light on the establishment of a molecular taxonomy identified by genetic and clinicopathologically relevant expression profiles for the presence of heterogeneity in lung cancer, especially adenocarcinoma. .

C. Identification Example lung adenocarcinoma TRU adenocarcinoma and non TRU adenocarcinoma to classify the TRU and non TRU type, the signal - noise function (signal-to-noise metrics, Golub et al., Science, Vol.286, pp531 to 537, 1999) was used. When the lung adenocarcinoma case group is defined as class1 for TRU type and class2 for non-TRU type, the weight S which is a signal-noise statistical value is calculated by the following equation (FIG. 6).
S = (μ _class1 −μ _class2 / σ _class1 + σ _class2 )

Here, for each gene, μ _class1 represents the average value of all expression intensity data of _class1 , μ _class2 represents the average value of all expression intensity data of class2, and σ _class1 represents the standard deviation of all expression intensity data of class1. Σ _class2 indicates the standard deviation of all expression intensity data of class2.

Next, a weighted vote for gene x is calculated using the following weighted-voting formula (FIG. 7).
_{V x = S (G x -b} x)

Here, V _X represents a weighted vote for gene x, S represents a weight calculated by the above formula, G _X represents the expression intensity (or expression level) of gene x, and b _X is
b _X = (μ1 + μ2) / 2
(Where μ1 and μ2 indicate the average of the average values of class1 and class2, respectively), and indicate the centers (ie, centroids) of the two groups.

The technique will by adding the weights in accordance with a deviation from the center of gravity, when the sum is greater than 0 V _X of each group, adenocarcinomas are classified as class1, when the sum of V _X is smaller than 0, adenocarcinomas It can be classified as class2.

For example, for samples 1, 2, 3, 4 and 5 belonging to class 1 and samples 6, 7, 8, 9 and 10 belonging to class 2, genes (Gene) A, B, C (hereinafter TRU characteristic genes), genes (Gene ) Measure the expression intensity (or expression level) of X, Y, Z (and above, non-TRU genes), and calculate the mean value (μ) and standard deviation (σ) of the expression intensity of each gene in the five samples of each group Further, the weight (S) and the center of gravity (b _X ) are calculated from the above formula. The results are shown in Table 9.

For sampleA and sampleB to be typed, measure the expression intensity (or expression level) of each gene, calculate the weight (S) and G _x -b _x from the above formula, obtain each V _X , and then add 6 genes The sum of V _X is calculated (Fig. 8).

As a result, sample A is identified as the TRU type because the sum of V _X is greater than zero. Also, sampleB is identified as a non-TRU type because the sum of V _X is smaller than 0.

  The present invention makes it possible to predict the relationship between adenocarcinoma subtypes and postoperative prognosis in pulmonary adenocarcinoma patients, and the relationship between EGFR gene mutation of a subtype (TRU type) and postoperative prognosis. A treatment plan suitable for the later patient can be made. Thus, the present invention can contribute to the medical industry for the treatment of lung adenocarcinoma.

Shows unsupervised classification systematic clustering and analysis results of four major gene changes in 149 NSCLC cases. Boxes in the histology row (“Hist”) are squamous cell carcinoma (blue; SQ), large cell carcinoma (red, LA), adenocarcinoma (orange, AD), squamous adenocarcinoma (grey), and large Represents cell endocrine cancer (yellow). Black boxes indicate the presence of EGFR, p53 and K-ras mutations in their individual rows. The expression index of the transcript sequence (row) in the sample (column) is indicated by the color code (see the expression index in the bottom bar in the figure). 4 shows unsupervised classification systematic clustering (TRU, non-TRU, TRU-a, TRU-b) and analysis results of four major gene changes in 90 lung adenocarcinoma cases. FIG. 2A shows the results of classification systematic clustering and search for mutations. The expression index is the same as in FIG. FIG. 2B is a graph showing the frequency of EGFR, p53 and K-ras mutations (%). Black boxes indicate the presence of EGFR, p53 and K-ras mutations in their individual rows. Prominent invasive growth is indicated by the black box, localized invasive growth is indicated by the gray box, and negative or negligible invasive growth is indicated by the white box. Lung adenocarcinoma identified by profiling identified using non-TRU, TRU-a or TRU-b, and Stanford data sets (Garber ME et al., Proc Natl Acad Sci USA 98: 13784-9, 2001) FIG. 6 shows the results of unsupervised classification systematic clustering based on the expression profiles of 30 genes ranked higher in terms of differential expression in a subset. The expression index is the same as in FIG. This figure shows that adenocarcinoma cases are systematically classified into TRU type and non-TRU type, and cases identified as TRU type are further classified into TRU-a type or TRU-b type. . Figure 3 shows Kaplan-Meier survival curves for lung adenocarcinoma subtypes identified by expression profile. The horizontal axis shows the number of months after surgery (Months after surgery), and the vertical axis shows the survival rate (Survaival). In addition, the number of risk patients classified into non-TRU type, TRU-a type, and TRU-b type in each postoperative observation month is also shown. From the figure, TRU-b adenocarcinoma had a significantly better prognosis than non-TRU type, but the prognosis of TRU-a type was similar to that of non-TRU type. Figure 3 shows Kaplan-Meier survival curves for the presence or absence of EGFR mutations in all cases of lung adenocarcinoma and TRU-type lung adenocarcinoma. FIG. 5A shows postoperative survival curves for adenocarcinoma cases with or without EGFR gene mutation. FIG. 5B shows a postoperative survival curve for the presence or absence of EGFR gene mutations in TRU-type adenocarcinoma. The horizontal axis shows the number of months after surgery (Months after surgery), and the vertical axis shows the survival rate (Survaival). In addition, the number of risk patients with wild-type or mutant EGFR (wt-EGFR and mut-EGFR, respectively) in each postoperative observation month is also shown. The formula for calculating the weight (S) is shown below. Shows the weighted-voting formula and its meaning. The discrimination model by Weighted-Voting is shown.

Claims (14)

A method for predicting the postoperative prognosis of a patient with lung adenocarcinoma in vitro, wherein the method identifies lung adenocarcinoma as one of its subtypes, TRU type or non-TRU type, and then TRU type The TRU-type lung adenocarcinoma is further identified as either TRU-a type or TRU-b type, and if TRU-b type, the postoperative prognosis is good, or TRU- evaluation of postoperative prognosis is poor if type a or non-TRU, wherein the TRU type, non-TRU type, TRU-a type and TRU-b type are related to the organism of the patient The expression level of one or more genes selected from each of the following gene sets in a biological sample is measured, and belongs to the TRU type of the gene set in TRU type lung adenocarcinoma compared to non-TRU type lung adenocarcinoma using an index called the expression level of the gene is relatively large Adenocarcinoma was identified as TRU lung adenocarcinoma, whereas, the expression levels of genes belonging in non TRU lung adenocarcinoma in comparison with TRU lung adenocarcinoma in non TRU form of the gene set is relatively large The lung adenocarcinoma is identified as a non-TRU lung adenocarcinoma using the index , or the TRU-a type in the TRU-a type lung adenocarcinoma compared to the TRU-b type lung adenocarcinoma. using an index expression levels belonging genes say relatively large identify lung adenocarcinoma that the TRU-a lung adenocarcinoma, whereas, TRU-b lung compared to TRU-a lung adenocarcinoma in adenocarcinoma using an index called the expression levels of genes belonging to TRU-b form of the gene set is relatively large for identifying lung adenocarcinoma that the TRU-b lung adenocarcinoma, and,
The gene set belonging to the TRU type is a gene consisting of the nucleotide sequence of SEQ ID NO: 1 to 195, or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence,
The gene set belonging to the non-TRU type is a gene consisting of the nucleotide sequence of SEQ ID NOS: 196 to 255, 257 to 280, 282 to 286, or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence,
The gene set belonging to the TRU-a type is SEQ ID NO: 4, 2, 9, 3, 5, 8, 16, 37, 7, 18, 10, 31, 12, 287, 28, 288, 81, 26, 41. 6, 24, 54, 34, 21, 289, 105, 38, 290, 58, 127, 32, 128, 35, 53, 83, 27, 62, 15, 106, 22, 291, 74, 161, 187 36, 76, 48, 30, 157, 114, 46, 59, 43, 75, 134, 97, 154, 292, 293, 110, 63, 42, 45, 179, 126, 123, 71, 33, 170 133, 86, 55, 44, 190, 156, 186, 49, 85, 52, 141, 125, 91, 180, 96, 183, 160, 67, 100, 294, 82, 88, 95, 93, 124 164 , 102, 117, 295, 296, 116, 297, 165, 175, 137, 298, 119, 103, 299, 300, 301, 177, 143, 302, 303, 304, 80, 305, 306, 307 A gene comprising a sequence or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence, and
The gene set belonging to the TRU-b type is a nucleotide sequence of SEQ ID NOs: 308 to 316, 168, 317 to 334, 178, 335 to 343, 70, 173, 344 to 349, 142, 350, 351, 188, or A gene comprising a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence,
Said method.   The method of claim 1, wherein the expression level of the gene is determined by measuring the presence or amount of a nucleic acid or protein corresponding to the gene.   The method according to claim 1 or 2, wherein the expression level of the gene is measured by a hybridization method.   The method according to claim 3, wherein the hybridization method is a microarray method or a blot method.   The method according to claim 1 or 2, wherein the expression level of the gene is measured by an immunological method.   6. The method according to claim 5, wherein the immunological method comprises detecting an immunological complex of a specific antibody against a protein encoded by the gene or a fragment thereof and a target protein.   In the TRU-type lung adenocarcinoma, when the epidermal growth factor receptor (EGFR) gene contains a mutation, it is predicted that the postoperative prognosis of the patient is poor compared to the lung adenocarcinoma containing the wild-type EGFR gene The method according to any one of claims 1 to 6, further comprising: A method of classifying lung adenocarcinoma into either TRU-type or non-TRR-type subtype, and then classifying TRU-type into TRU-a or TRU-b, the method comprising: The expression level of one or more genes selected from each of the following gene sets in an experimental sample , and a gene belonging to the TRU type of the gene set in TRU-type lung adenocarcinoma compared to non-TRU-type lung adenocarcinoma lung adenocarcinoma classified into TRU lung adenocarcinoma using an index of expression levels rather relatively large, while non-TRU of said gene set in comparison with TRU lung adenocarcinoma in non TRU lung adenocarcinoma classifying lung adenocarcinoma in non TRU lung adenocarcinoma using an indicator gene expression levels that belong to the type say a relatively large or, TRU-a Katahaisen compared with TRU-b lung adenocarcinoma Genes belonging to TRU-a type of the gene set in cancer Lung adenocarcinoma classified as TRU-a lung adenocarcinoma with an index of expression levels rather relatively large, whereas, the in TRU-b lung adenocarcinoma in comparison with TRU-a lung adenocarcinoma comprises classifying lung adenocarcinoma TRU-b lung adenocarcinoma using an indicator gene expression levels belonging to TRU-b-type gene sets say relatively large, however,
(A) The gene set belonging to the TRU type is a gene consisting of the nucleotide sequence of SEQ ID NO: 1 to 195, or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence,
(B) The gene set belonging to the non-TRU type is a gene comprising a nucleotide sequence of SEQ ID NOS: 196 to 255, 257 to 280, 282 to 286, or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence ,
(C) The gene set belonging to the TRU-a type is SEQ ID NO: 4, 2, 9, 3, 5, 8, 16, 37, 7, 18, 10, 31, 12, 287, 28, 288, 81, 26, 41, 6, 24, 54, 34, 21, 289, 105, 38, 290, 58, 127, 32, 128, 35, 53, 83, 27, 62, 15, 106, 22, 291, 74, 161, 187, 36, 76, 48, 30, 157, 114, 46, 59, 43, 75, 134, 97, 154, 292, 293, 110, 63, 42, 45, 179, 126, 123, 71, 33, 170, 133, 86, 55, 44, 190, 156, 186, 49, 85, 52, 141, 125, 91, 180, 96, 183, 160, 67, 100, 294, 82, 88, 95, 93,124 164, 102, 117, 295, 296, 116, 297, 165, 175, 137, 298, 119, 103, 299, 300, 301, 177, 143, 302, 303, 304, 80, 305, 306, 307 A gene comprising a nucleotide sequence or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence;
(D) The nucleotide set of the gene set belonging to the TRU-b type is SEQ ID NO: 308 to 316, 168, 317 to 334, 178, 335 to 343, 70, 173, 344 to 349, 142, 350, 351, 188 Or a gene comprising a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence,
Said method comprising:   The method according to claim 8, wherein the expression level of the gene is measured by a hybridization method.   The method according to claim 9, wherein the hybridization method is a microarray method or a blot method. A plurality of nucleic acids capable of detecting the expression of a gene comprising a nucleotide sequence of SEQ ID NOs: 1 to 255, 257 to 280, 282 to 351 or a nucleotide sequence having 90% or more sequence identity with the sequence,
(1) a nucleotide sequence represented by SEQ ID NOs: 1 to 255, 257 to 280, 282 to 351,
(2) a nucleotide sequence comprising the nucleotide sequence shown in SEQ ID NOs: 1 to 255, 257 to 280, 282 to 351,
(3) a nucleotide sequence complementary to the nucleotide sequence of (1) or (2),
(4) a nucleotide sequence having 90% or more sequence identity with any one of the nucleotide sequences of (1), (2) or (3), and
(5) a partial sequence of less than the total number of bases from 15 bases of the nucleotide sequence of (1), (3) or (4),
The method according to any one of claims 1 to 10, comprising the plurality of nucleic acids having a sequence selected from the group consisting of: or a plurality of antibodies to proteins encoded by the genes or protein-binding fragments thereof. To predict the postoperative prognosis of patients with lung adenocarcinoma in vitro or to classify lung adenocarcinoma into any subtype of TRU, non-TRU, TRU-a or TRU-b Composition. A plurality of nucleic acids capable of detecting the expression of a gene comprising a nucleotide sequence of SEQ ID NOs: 1 to 255, 257 to 280, 282 to 351 or a nucleotide sequence having 90% or more sequence identity with the sequence,
(1) a nucleotide sequence represented by SEQ ID NOs: 1 to 255, 257 to 280, 282 to 351,
(2) a nucleotide sequence comprising the nucleotide sequence shown in SEQ ID NOs: 1 to 255, 257 to 280, 282 to 351,
(3) a nucleotide sequence complementary to the nucleotide sequence of (1) or (2),
(4) a nucleotide sequence having 90% or more sequence identity with any one of the nucleotide sequences of (1), (2) or (3), and
(5) a partial sequence of less than the total number of bases from 15 bases of the nucleotide sequence of (1), (3) or (4),
The method according to any one of claims 1 to 10, comprising the plurality of nucleic acids having a sequence selected from the group consisting of: or a plurality of antibodies to proteins encoded by the genes or protein-binding fragments thereof. To predict the postoperative prognosis of patients with lung adenocarcinoma in vitro or to classify lung adenocarcinoma into any subtype of TRU, non-TRU, TRU-a or TRU-b Kit. A plurality of nucleic acids capable of detecting the expression of a gene comprising a nucleotide sequence of SEQ ID NOs: 1 to 255, 257 to 280, 282 to 351 or a nucleotide sequence having 90% or more sequence identity with the sequence,
(1) a nucleotide sequence represented by SEQ ID NOs: 1 to 255, 257 to 280, 282 to 351,
(2) a nucleotide sequence comprising the nucleotide sequence shown in SEQ ID NOs: 1 to 255, 257 to 280, 282 to 351,
(3) a nucleotide sequence complementary to the nucleotide sequence of (1) or (2),
(4) a nucleotide sequence having 90% or more sequence identity with any one of the nucleotide sequences of (1), (2) or (3), and
(5) a partial sequence of less than the total number of bases from 15 bases of the nucleotide sequence of (1), (3) or (4),
Comprising the plurality of nucleic acid having a sequence selected from the group consisting of, by a method according to any one of claims 1 to 10, for predicting the postoperative prognosis of patients with lung adenocarcinoma in vitro Alternatively, a DNA chip for classifying lung adenocarcinoma into any subtype of TRU, non-TRU, TRU-a, or TRU-b. A plurality of antibodies or protein binding fragments thereof against the protein encoded by the nucleotide sequence or Ranaru gene having a nucleotide sequence or sequences at least 90% sequence identity to the SEQ ID NO: 1～255,257～280,282～351 The method according to any one of claims 1 to 10, wherein the postoperative prognosis of a patient with lung adenocarcinoma is predicted in vitro or the lung adenocarcinoma is TRU-type, non-TRU-type, TRU- A protein array for classifying into either a type or TRU-b type.

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