WO2011028980A2 - Markers for detecting cancer cells - Google Patents

Abstract

Detecting presence of cancer cells in a subject using one or more of fibronectin 1, S100 calcium binding protein A7, and carboxy-terminal domain (RNA polymerase II, polypeptide A) small phosphatase-like protein as a marker.

Description

MARKERS FOR DETECTING CANCER CELLS

CROSS-REFRENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 61/239,534, filed on September 3, 2009, the content of which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Carcinoma, developed from epithelial cells, accounts for around 85% of human cancers. It has been reported that carcinoma cells exist in the bone marrow and peripheral blood of cancer patients.

Methods such as immunocytology and flow cytometry have been developed and employed in commercial instruments (e.g., CellSearch, Veridex, NJ) to detect circulating tumor cells (CTC) in peripheral blood. See US Patent No. 6,670,197; Budd, et al, Clin. Cancer Res., 2006, 12:

6403-6409; and Riethdorf, et al, Int. J. Caner, 2008, 123: 1991-2006).

These methods all use antibodies specifically targeting epithelial cell surface markers (e.g., EpCAM) to detect carcinoma cells. Their sensitivity is limited by the affinity of the antibody to the CTC cell surface marker it binds, and they require a large amount of blood and yield a high rate of false negative results. See Ring, et al, Br. J. Cancer, 2005, 92: 906-912.

Compared with immunocytology and flow cytometry, polymerase chain reaction (PCR) is more facile and less expensive to perform. Peck, et al. developed a scoring method, using quantitative PCR (qPCR), by calculating cancer cell load (Lc) in peripheral blood sample (Peck, et al., U.S. Patent No. 7,507,534) and the Lc value correlated well with the cell count of CTC.

To detect CTCs at high sensitivity and high accuracy, new marker genes as well as qPCR control genes are needed. SUMMARY OF THE INVENTION

The present application is based on, at least in part, on the discovery of novel marker genes and a control gene for detecting the presence of circulating tumor cells (CTCs) in the blood of cancer patients.

Accordingly, provided herein are methods for detecting cancer cells in a subject (e.g., a human) or a sample. This methods include (i) providing a sample (e.g., a peripheral blood sample) of the subject that is suspected of containing cancer cells, (ii) determining in the sample the expression level of one or more of marker genes, (iii) determining in the sample the expression level of a control gene, (iv) normalizing the expression level(s) of the one or more marker genes against that of the control gene to obtain an expression profile of the one or more marker genes, and (v) determining whether the sample contains cancer cells based on the expression profile. An expression profile representing an expression level of the one or more marker genes higher than that in a cancer- free subject indicates that the sample contains cancer cells.

The marker genes include fibronectin 1 (FN1), SI 00 calcium binding protein A7 (S100A7), and carboxy-terminal domain (R A polymerase II, polypeptide A) small phosphatase-like protein (CTDSPL). In some cases, the expression levels of all three marker genes in the sample are determined. In one example, the cancer cells to be detected are circulating lung cancer cells (e.g., non-small cell lung cancer cells). The expression levels of the marker genes and the control gene (e.g., DEAD box polypeptide 5; DDX5) can be determined by real-time quantitative PCR (qPCR) analysis.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of an example, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

1 is a diagram depicting an examplary screening process for identifying marker genes useful in detecting circulating lung cancer cells ("CLCC") in the peripheral blood of lung cancer patients.

FIGs. 2A-C are graphs showing distribution of cancer cell load (Lc) values of marker genes for lung cancer patients and normal PBMC controls. (A) FNl; (B) S100A7; and (C) CTDSPL.

FIGs. 3A and B are two graphs demonstrating positive CLCC detection rates using FNl, S100A7, and/or CTDSPL as markers. (A) positive CLCC detection rates using FNl, S100A7, or CTDSPL as a marker. (B) positive CLCC detection rates using a combination of FNl, S100A7, and CTDSPL as a marker.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are, inter alia, methods for detecting cancer cells, particularly circulating tumor cells, based on the expression level of one or more of marker genes FNl, S100A7, and CTDSPL, normalized against that of a control gene (e.g., DDX5).

Circulating Tumor Cells

Circulating tumor cells (CTCs) are believed to be cells that have detached from a primary tumor and then released into circulation. Studies suggest that analysis of CTCs can help monitoring and predicting cancer progression and evaluating response to treatments. See, e.g., Peck et al, Cancer Research, 1998, 58: 2761-2765; and Budd et al., Clin Cancer Res, 2006,12(21): 6403-6409.

Marker and Control Genes

Novel marker and control genes useful for detecting CTCs using quantitative PCR are described herein. Shown below are the gene sequences of FNl, S100A7, CTDSPL, and DDX5: FN1 (GenBank Accession No.: NM_212482; SEQ ID NO: l):

1 gcccgcgccg gctgtgctgc acagggggag gagagggaac cccaggcgcg agcgggaaga 61 ggggacctgc agccacaact tctctggtcc tctgcatccc ttctgtccct ccacccgtcc 121 ccttccccac cctctggccc ccaccttctt ggaggcgaca acccccggga ggcattagaa 181 gggatttttc ccgcaggttg cgaagggaag caaacttggt ggcaacttgc ctcccggtgc 241 gggcgtctct cccccaccgt ctcaacatgc ttaggggtcc ggggcccggg ctgctgctgc 301 tggccgtcca gtgcctgggg acagcggtgc cctccacggg agcctcgaag agcaagaggc 361 aggctcagca aatggttcag ccccagtccc cggtggctgt cagtcaaagc aagcccggtt 421 gttatgacaa tggaaaacac tatcagataa atcaacagtg ggagcggacc tacctaggca 481 atgcgttggt ttgtacttgt tatggaggaa gccgaggttt taactgcgag agtaaacctg 541 aagctgaaga gacttgcttt gacaagtaca ctgggaacac ttaccgagtg ggtgacactt 601 atgagcgtcc taaagactcc atgatctggg actgtacctg catcggggct gggcgaggga 661 gaataagctg taccatcgca aaccgctgcc atgaaggggg tcagtcctac aagattggtg 721 acacctggag gagaccacat gagactggtg gttacatgtt agagtgtgtg tgtcttggta 781 atggaaaagg agaatggacc tgcaagccca tagctgagaa gtgttttgat catgctgctg 841 ggacttccta tgtggtcgga gaaacgtggg agaagcccta ccaaggctgg atgatggtag 901 attgtacttg cctgggagaa ggcagcggac gcatcacttg cacttctaga aatagatgca 961 acgatcagga cacaaggaca tcctatagaa ttggagacac ctggagcaag aaggataatc 1021 gaggaaacct gctccagtgc atctgcacag gcaacggccg aggagagtgg aagtgtgaga 1081 ggcacacctc tgtgcagacc acatcgagcg gatctggccc cttcaccgat gttcgtgcag 1141 ctgtttacca accgcagcct cacccccagc ctcctcccta tggccactgt gtcacagaca 1201 gtggtgtggt ctactctgtg gggatgcagt ggctgaagac acaaggaaat aagcaaatgc 1261 tttgcacgtg cctgggcaac ggagtcagct gccaagagac agctgtaacc cagacttacg 1321 gtggcaactc aaatggagag ccatgtgtct taccattcac ctacaatggc aggacgttct 1381 actcctgcac cacagaaggg cgacaggacg gacatctttg gtgcagcaca acttcgaatt 1441 atgagcagga ccagaaatac tctttctgca cagaccacac tgttttggtt cagactcgag 1501 gaggaaattc caatggtgcc ttgtgccact tccccttcct atacaacaac cacaattaca 1561 ctgattgcac ttctgagggc agaagagaca acatgaagtg gtgtgggacc acacagaact 1621 atgatgccga ccagaagttt gggttctgcc ccatggctgc ccacgaggaa atctgcacaa 1681 ccaatgaagg ggtcatgtac cgcattggag atcagtggga taagcagcat gacatgggtc 1741 acatgatgag gtgcacgtgt gttgggaatg gtcgtgggga atggacatgc attgcctact 1801 cgcagcttcg agatcagtgc attgttgatg acatcactta caatgtgaac gacacattcc 1861 acaagcgtca tgaagagggg cacatgctga actgtacatg cttcggtcag ggtcggggca 1921 ggtggaagtg tgatcccgtc gaccaatgcc aggattcaga gactgggacg ttttatcaaa 1981 ttggagattc atgggagaag tatgtgcatg gtgtcagata ccagtgctac tgctatggcc

2041 gtggcattgg ggagtggcat tgccaacctt tacagaccta tccaagctca agtggtcctg 2101 tcgaagtatt tatcactgag actccgagtc agcccaactc ccaccccatc cagtggaatg 2161 caccacagcc atctcacatt tccaagtaca ttctcaggtg gagacctaaa aattctgtag 2221 gccgttggaa ggaagctacc ataccaggcc acttaaactc ctacaccatc aaaggcctga 2281 agcctggtgt ggtatacgag ggccagctca tcagcatcca gcagtacggc caccaagaag 2341 tgactcgctt tgacttcacc accaccagca ccagcacacc tgtgaccagc aacaccgtga 2401 caggagagac gactcccttt tctcctcttg tggccacttc tgaatctgtg accgaaatca 2461 cagccagtag ctttgtggtc tcctgggtct cagcttccga caccgtgtcg ggattccggg 2521 tggaatatga gctgagtgag gagggagatg agccacagta cctggatctt ccaagcacag 2581 ccacttctgt gaacatccct gacctgcttc ctggccgaaa atacattgta aatgtctatc

2641 agatatctga ggatggggag cagagtttga tcctgtctac ttcacaaaca acagcgcctg 2701 atgcccctcc tgacccgact gtggaccaag ttgatgacac ctcaattgtt gttcgctgga 2761 gcagacccca ggctcccatc acagggtaca gaatagtcta ttcgccatca gtagaaggta 2821 gcagcacaga actcaacctt cctgaaactg caaactccgt caccctcagt gacttgcaac 2881 ctggtgttca gtataacatc actatctatg ctgtggaaga aaatcaagaa agtacacctg 2941 ttgtcattca acaagaaacc actggcaccc cacgctcaga tacagtgccc tctcccaggg 3001 acctgcagtt tgtggaagtg acagacgtga aggtcaccat catgtggaca ccgcctgaga

3061 gtgcagtgac cggctaccgt gtggatgtga tccccgtcaa cctgcctggc gagcacgggc 3121 agaggctgcc catcagcagg aacacctttg cagaagtcac cgggctgtcc cctggggtca

3181 cctattactt caaagtcttt gcagtgagcc atgggaggga gagcaagcct ctgactgctc 3241 aacagacaac caaactggat gctcccacta acctccagtt tgtcaatgaa actgattcta 3301 ctgtcctggt gagatggact ccacctcggg cccagataac aggataccga ctgaccgtgg 3361 gccttacccg aagaggacag cccaggcagt acaatgtggg tccctctgtc tccaagtacc 3421 cactgaggaa tctgcagcct gcatctgagt acaccgtatc cctcgtggcc ataaagggca 3481 accaagagag ccccaaagcc actggagtct ttaccacact gcagcctggg agctctattc 3541 caccttacaa caccgaggtg actgagacca ccattgtgat cacatggacg cctgctccaa 3601 gaattggttt taagctgggt gtacgaccaa gccagggagg agaggcacca cgagaagtga 3661 cttcagactc aggaagcatc gttgtgtccg gcttgactcc aggagtagaa tacgtctaca 3721 ccatccaagt cctgagagat ggacaggaaa gagatgcgcc aattgtaaac aaagtggtga 3781 caccattgtc tccaccaaca aacttgcatc tggaggcaaa ccctgacact ggagtgctca 3841 cagtctcctg ggagaggagc accaccccag acattactgg ttatagaatt accacaaccc 3901 ctacaaacgg ccagcaggga aattctttgg aagaagtggt ccatgctgat cagagctcct 3961 gcacttttga taacctgagt cccggcctgg agtacaatgt cagtgtttac actgtcaagg 4021 atgacaagga aagtgtccct atctctgata ccatcatccc agaggtgccc caactcactg 4081 acctaagctt tgttgatata accgattcaa gcatcggcct gaggtggacc ccgctaaact 4141 cttccaccat tattgggtac cgcatcacag tagttgcggc aggagaaggt atccctattt 4201 ttgaagattt tgtggactcc tcagtaggat actacacagt cacagggctg gagccgggca 4261 ttgactatga tatcagcgtt atcactctca ttaatggcgg cgagagtgcc cctactacac 4321 tgacacaaca aacggctgtt cctcctccca ctgacctgcg attcaccaac attggtccag 4381 acaccatgcg tgtcacctgg gctccacccc catccattga tttaaccaac ttcctggtgc 4441 gttactcacc tgtgaaaaat gaggaagatg ttgcagagtt gtcaatttct ccttcagaca 4501 atgcagtggt cttaacaaat ctcctgcctg gtacagaata tgtagtgagt gtctccagtg 4561 tctacgaaca acatgagagc acacctctta gaggaagaca gaaaacaggt cttgattccc 4621 caactggcat tgacttttct gatattactg ccaactcttt tactgtgcac tggattgctc 4681 ctcgagccac catcactggc tacaggatcc gccatcatcc cgagcacttc agtgggagac 4741 ctcgagaaga tcgggtgccc cactctcgga attccatcac cctcaccaac ctcactccag 4801 gcacagagta tgtggtcagc atcgttgctc ttaatggcag agaggaaagt cccttattga 4861 ttggccaaca atcaacagtt tctgatgttc cgagggacct ggaagttgtt gctgcgaccc 4921 ccaccagcct actgatcagc tgggatgctc ctgctgtcac agtgagatat tacaggatca 4981 cttacggaga gacaggagga aatagccctg tccaggagtt cactgtgcct gggagcaagt 5041 ctacagctac catcagcggc cttaaacctg gagttgatta taccatcact gtgtatgctg 5101 tcactggccg tggagacagc cccgcaagca gcaagccaat ttccattaat taccgaacag 5161 aaattgacaa accatcccag atgcaagtga ccgatgttca ggacaacagc attagtgtca 5221 agtggctgcc ttcaagttcc cctgttactg gttacagagt aaccaccact cccaaaaatg 5281 gaccaggacc aacaaaaact aaaactgcag gtccagatca aacagaaatg actattgaag 5341 gcttgcagcc cacagtggag tatgtggtta gtgtctatgc tcagaatcca agcggagaga 5401 gtcagcctct ggttcagact gcagtaacca acattgatcg ccctaaagga ctggcattca 5461 ctgatgtgga tgtcgattcc atcaaaattg cttgggaaag cccacagggg caagtttcca 5521 ggtacagggt gacctactcg agccctgagg atggaatcca tgagctattc cctgcacctg 5581 atggtgaaga agacactgca gagctgcaag gcctcagacc gggttctgag tacacagtca 5641 gtgtggttgc cttgcacgat gatatggaga gccagcccct gattggaacc cagtccacag 5701 ctattcctgc accaactgac ctgaagttca ctcaggtcac acccacaagc ctgagcgccc 5761 agtggacacc acccaatgtt cagctcactg gatatcgagt gcgggtgacc cccaaggaga 5821 agaccggacc aatgaaagaa atcaaccttg ctcctgacag ctcatccgtg gttgtatcag 5881 gacttatggt ggccaccaaa tatgaagtga gtgtctatgc tcttaaggac actttgacaa 5941 gcagaccagc tcagggagtt gtcaccactc tggagaatgt cagcccacca agaagggctc 6001 gtgtgacaga tgctactgag accaccatca ccattagctg gagaaccaag actgagacga 6061 tcactggctt ccaagttgat gccgttccag ccaatggcca gactccaatc cagagaacca 6121 tcaagccaga tgtcagaagc tacaccatca caggtttaca accaggcact gactacaaga 6181 tctacctgta caccttgaat gacaatgctc ggagctcccc tgtggtcatc gacgcctcca 6241 ctgccattga tgcaccatcc aacctgcgtt tcctggccac cacacccaat tccttgctgg 6301 tatcatggca gccgccacgt gccaggatta ccggctacat catcaagtat gagaagcctg 6361 ggtctcctcc cagagaagtg gtccctcggc cccgccctgg tgtcacagag gctactatta 6421 ctggcctgga accgggaacc gaatatacaa tttatgtcat tgccctgaag aataatcaga 6481 agagcgagcc cctgattgga aggaaaaaga cagacgagct tccccaactg gtaacccttc 6541 cacaccccaa tcttcatgga ccagagatct tggatgttcc ttccacagtt caaaagaccc 6601 ctttcgtcac ccaccctggg tatgacactg gaaatggtat tcagcttcct ggcacttctg 6661 gtcagcaacc cagtgttggg caacaaatga tctttgagga acatggtttt aggcggacca 6721 caccgcccac aacggccacc cccataaggc ataggccaag accatacccg ccgaatgtag 6781 gtgaggaaat ccaaattggt cacatcccca gggaagatgt agactatcac ctgtacccac 6841 acggtccggg actcaatcca aatgcctcta caggacaaga agctctctct cagacaacca 6901 tctcatgggc cccattccag gacacttctg agtacatcat ttcatgtcat cctgttggca 6961 ctgatgaaga acccttacag ttcagggttc ctggaacttc taccagtgcc actctgacag 7021 gcctcaccag aggtgccacc tacaacatca tagtggaggc actgaaagac cagcagaggc 7081 ataaggttcg ggaagaggtt gttaccgtgg gcaactctgt caacgaaggc ttgaaccaac 7141 ctacggatga ctcgtgcttt gacccctaca cagtttccca ttatgccgtt ggagatgagt 7201 gggaacgaat gtctgaatca ggctttaaac tgttgtgcca gtgcttaggc tttggaagtg 7261 gtcatttcag atgtgattca tctagatggt gccatgacaa tggtgtgaac tacaagattg 7321 gagagaagtg ggaccgtcag ggagaaaatg gccagatgat gagctgcaca tgtcttggga 7381 acggaaaagg agaattcaag tgtgaccctc atgaggcaac gtgttatgat gatgggaaga 7441 cataccacgt aggagaacag tggcagaagg aatatctcgg tgccatttgc tcctgcacat 7501 gctttggagg ccagcggggc tggcgctgtg acaactgccg cagacctggg ggtgaaccca 7561 gtcccgaagg cactactggc cagtcctaca accagtattc tcagagatac catcagagaa 7621 caaacactaa tgttaattgc ccaattgagt gcttcatgcc tttagatgta caggctgaca 7681 gagaagattc ccgagagtaa atcatctttc caatccagag gaacaagcat gtctctctgc 7741 caagatccat ctaaactgga gtgatgttag cagacccagc ttagagttct tctttctttc 7801 ttaagccctt tgctctggag gaagttctcc agcttcagct caactcacag cttctccaag 7861 catcaccctg ggagtttcct gagggttttc tcataaatga gggctgcaca ttgcctgttc 7921 tgcttcgaag tattcaatac cgctcagtat tttaaatgaa gtgattctaa gatttggttt 7981 gggatcaata ggaaagcata tgcagccaac caagatgcaa atgttttgaa atgatatgac 8041 caaaatttta agtaggaaag tcacccaaac acttctgctt tcacttaagt gtctggcccg 8101 caatactgta ggaacaagca tgatcttgtt actgtgatat tttaaatatc cacagtactc 8161 actttttcca aatgatccta gtaattgcct agaaatatct ttctcttacc tgttatttat 8221 caatttttcc cagtattttt atacggaaaa aattgtattg aaaacactta gtatgcagtt 8281 gataagagga atttggtata attatggtgg gtgattattt tttatactgt atgtgccaaa 3341 gctttactac tgtggaaaga caactgtttt aataaaagat ttacattcca caacttgaag 3401 ttcatctatt tgatataaga caccttcggg ggaaataatt cctgtgaata ttctttttca 3461 attcagcaaa catttgaaaa tctatgatgt gcaagtctaa ttgttgattt cagtacaaga 3521 ttttctaaat cagttgctac aaaaactgat tggtttttgt cacttcatct cttcactaat 3581 ggagatagct ttacactttc tgctttaata gatttaagtg gaccccaata tttattaaaa 3641 ttgctagttt accgttcaga agtataatag aaataatctt tagttgctct tttctaacca 3701 ttgtaattct tcccttcttc cctccacctt tccttcattg aataaacctc tgttcaaaga 3761 gattgcctgc aagggaaata aaaatgacta agatattaaa aaaaaaaaaa aaaaa S100A7 (GenBank Accession No.: NM_002963; SEQ ID NO:2)

1 gtccaaacac acacatctca ctcatccttc tactcgtgac gcttcccagc tctggctttt

61 tgaaagcaaa gatgagcaac actcaagctg agaggtccat aataggcatg atcgacatgt

121 ttcacaaata caccagacgt gatgacaaga ttgagaagcc aagcctgctg acgatgatga

181 aggagaactt ccccaacttc cttagtgcct gtgacaaaaa gggcacaaat tacctcgccg 241 atgtctttga gaaaaaggac aagaatgagg ataagaagat tgatttttct gagtttctgt

301 ccttgctggg agacatagcc acagactacc acaagcagag ccatggagca gcgccctgtt

361 ccgggggcag ccagtgaccc agccccacca atgggcctcc agagacccca ggaacaataa

421 aatgtcttct cccaccagaa aaaaaaaaaa

CTDSPL (GenBank Accession No.: NM 001008392; SEQ ID NO:3)

1 gcggccgccg cgccgcgcac ccatggacgg cccggccatc atcacccagg tgaccaaccc

61 caaggaggac gagggccggt tgccgggcgc gggcgagaaa gcctcccagt gcaacgtcag

121 cttaaagaag cagaggagcc gcagcatcct tagctccttc ttctgctgct tccgtgatta

181 caatgtggag gcccctccac ccagcagccc cagtgtgctt ccgccactgg tggaggagaa 241 tggtgggctt cagaagggtg accagaggca ggtcattccc ataccaagtc caccagctaa

301 gtaccttctt ccagaggtga cggtgcttga ctatggaaag aaatgtgtgg tcattgattt

361 agatgaaaca ttggtgcaca gttcgtttaa gcctattagt aatgctgatt ttattgttcc

421 ggttgaaatc gatggaacta tacatcaggt gtatgtgctg aagcggccac atgtggacga

481 gttcctccag aggatggggc agctttttga atgtgtgctc tttactgcca gcttggccaa 541 gtatgcagac cctgtggctg acctcctaga ccgctggggt gtgttccggg cccggctctt

601 cagagaatca tgtgtttttc atcgtgggaa ctacgtgaag gacctgagtc gccttgggcg 661 ggagctgagc aaagtgatca ttgttgacaa ttcccctgcc tcatacatct tccatcctga 721 gaatgcagtg cctgtgcagt cctggttcga tgacatgacg gacacggagc tgctggacct 781 catccccttc tttgagggcc tgagccggga ggacgacgtg tacagcatgc tgcacagact 841 ctgcaatagg tagccctggc ctctgcctgc ctcccgcctg tgcactctgg aacctctggc 901 ctcaggggac ctgcctgtcc tcagctccct gggagctgaa agtgaggata ctccgtgctc 961 caggccacag ggtgaatgtg gccatgccta cctgttttgt ttttttaaga acagaaacaa 1021 ctattttaaa agaactcttt taagaaattt cataaaggga catgcatttt actgggtttg 1081 cttttcttaa aacataccaa aaaagaaaaa aatagaaaaa aaaaaaaaaa aagcttgatc 1141 tctatcagac ttcctttcaa ctgtcctccc ctccaagcag accacctgtc cccttctatc 1201 ccagctcaga gcagctgacc caactcagaa tctctttcct acaggatgaa gtgccttttg 1261 aatgttattt taagccgaga gttaattttt ctacacaaca tatttccaga catcttttag 1321 tcttttattg tcttagatac tataagaaga tgaacatgac aattttctag aacctggtag 1381 cgtgtgtgtg tgtggcgggg ggtgctgagg gaggggagtg agtcacagga gcctgtcccc 1441 caacaggtgt gactgctctg acaacctgtg gcatgctgca gggtcaggct cctgatagga

1501 ggatttcatg actatgtcat tgtctccact catttttgac ccagtttgga atgtatctgc 1561 aattgtgtgg ctcaacactt taggaaacaa tagattattt tatattatta tttctgatgg 1621 tgacaagttt gtcttgaggt cacattttct ccttgaaaag tgacatcctg tcacttctgc 1681 tctcacacta ctgccataca tttgtgtttt ttgttgttat tgtttgggta gagcagttac 1741 aagaaaccct aaaacccttg gatataaaag aaatctgttt attgattttt aaatctttcc 1801 tttccaaaag ctggatacac atggagctgt ttgggaattt tccttgctgc taccgcgctg 1861 ccaccaaatg gaattgacca gcggctgtta cactgttctt tgccactgtg cctatgctca 1921 gaatatgctc actgctaagc tacaaactcg gacagggtca gaaacagagg tgtcccatcc 1981 cattgcagcc tccaccacct gtaacccctt cctggcattg gccactgaag ggtacaaagg 2041 caaaaggacc acagcaccac ttaggtgtag catggatttt aaactgcagt cagtatcaga 2101 tcctgtttga taaataagct gactgttctc tcttgagaac ctgtggcctc aaccagccac 2161 caagctgatg tggcccaagt ccatctcttg gtcttctcct ttgaagcaca gcctatttct 2221 gagccaaggg ttggggaagc ctgtctagat gtgggactca ttgccccaaa ccagggagag 2281 gaagagctcc cacagggaga gcccaggctc tctttgcagc ctttcccagt ttggtgttta 2341 agcagtgcca tgttccttgt ttgacaacaa gacagtctgt aaagtattgc tcttaaaaac 2401 aattaaaaag aaccctttca tattggcacc attgccttag tcctctgtgg gttggtcttc 2461 agccagcatt ctggtgggag tgactggcat taacaagact ggaaatcggg ggtcaaagta 2521 aaatatcttt gttttgcttt cattcacaaa gtaatgaagc cagctgccaa ttacatcctc 2581 ccaacagcac tttggtctgt ggactgctgt gtgaatattc agaagggaag taagtattca 2641 gggggtaaac aggtctccca gcattctgag tgttccaaac cagtaatcca catgccaatt 2701 caaatagaac agccccttgc tagatattac cacagataat gacagttaca tggtagaact 2761 gcccatgcca caaatattta tttggaaaag tagtcattaa atgaacccac tgccttaaat 2821 gtcttgaatg ttgcagtcaa gtgtctgtca tgtgttgata tccacacaga attaggccct 2881 aatgagagcc ttagaccctc aaccatgccc ccttcgttgg catcacaggg ccttatttgg 2941 aagagcgggc aaagaggatg gaaatcataa aatatttcat gggaatcgaa cctagggata 3001 gtgctccact tctgacgatg gagtgaagac acttggcaga cttgagccag acacttcacc 3061 tagtagttcc tgaaactgtg agcaccactg cactaagcca gtgcggagct gttagggacg 3121 ggcccagctc ctgcaccacg gacacagaat gtctggagag ggccagcagg ccctctgagg 3181 gttctggaat ctgtgcacct tatttgacca cactccaaaa ttctgttttt attttaaccc 3241 ttgaatctgc tttatgtaca taatcaaaat atctatatct atatctatat ctatatctat 3301 atatttttaa tcatctacat gtaaatgaag caatagaatt ctaacataag gccaagaaat 3361 gagacgaatg tttggggttt atgtttttta aggtaaatac gggtattgtt tttaattatt 3421 accatgtatt aaattgtggg ctttgaaacc taatgaaacc tgttagccac ttctctgtgc 3481 catatacttc ccatgttacc aaaatacccc caactcttta gccaaaagag aaccctgacc 3541 tcctgagttt ccatgctcct ttctgtacca ggtttaaatg tagtcttctg gagaagtatt 3601 tttgacattg agctctggga caggacacct tgggtttgtg gactgcagcc cactatgatg 3661 ttattacttc tctggccagg cctccagtgg aagtgcacag gcactcccaa tgttgttaat 3721 gctctgtctt ccatttgttc tggaatccta cgtgttggtc tgtggttcca tgcattagct 3781 gtttgtaaat aatgcatttg catactgaaa aaggaatgcc acctgccaca gttgatggtg 3841 aggaagctcc tttgacgtgg tgcaattttg atgagatgtc tctggggaca cgaggatgcc 3901 ctaatgatgc tgacttgtca tggttgcagc atttgaactt ttggtgttaa aaaaaaaaac 3961 ctgtaagtct gtaacctggc aacattttac aaccctgtat ttttaaagat ggctttctaa 4021 taaaaaatcc agaaccacac agccctatgg tcaaacaatc ctacgtttgt gcctctgctt 4081 ttaaaggtgc tgtgctggac agttggcatg ccagggttcg agaagagtga atggcttgac 4141 gtacttgcag ttaactgtgc aaaattggct ggctgcctct gttcctactg tactgtaact 4201 ttgatcatgt ctgttcctgt tccattctcc caggagcttc tctgcagact gacacaccct 4261 cccccacccc gggtagtgga gatgctggtg tctgggtagt catggatttc tgctggacat 4321 ttgaatgtga taaacaatcc agcattactt aggaaatgct acatgcggaa tgtgcacgtt 4381 tccaggggcg agtattgtca atcaaaaggt ttgcaatgat ttccttcctg ccaaaaataa 4441 acatgtgaaa ctgca

DDX5 (GenBank Accession No.: NM 004396; SEQ ID NO:4)

1 atagagcggc tcccagcgtt ccctgcggcg taggaggcgg tccagactat aaaagcggct

61 gccggaaagc ggccggcacc tcattcattt ctaccggtct ctagtagtgc agcttcggct

121 ggtgtcatcg gtgtccttcc tccgctgccg cccccgcaag gcttcgccgt catcgaggcc

181 atttccagcg acttgtcgca cgcttttcta tatacttcgt tccccgccaa ccgcaaccat

241 tgacgccatg tcgggttatt cgagtgaccg agaccgcggc cgggaccgag ggtttggtgc 301 acctcgattt ggaggaagta gggcagggcc cttatctgga aagaagtttg gaaaccctgg

361 ggagaaatta gttaaaaaga agtggaatct tgatgagctg cctaaatttg agaagaattt

421 ttatcaagag caccctgatt tggctaggcg cacagcacaa gaggtggaaa catacagaag

481 aagcaaggaa attacagtta gaggtcacaa ctgcccgaag ccagttctaa atttttatga

541 agccaatttc cctgcaaatg tcatggatgt tattgcaaga cagaatttca ctgaacccac 601 tgctattcaa gctcagggat ggccagttgc tctaagtgga ttggatatgg ttggagtggc

661 acagactgga tctgggaaaa cattgtctta tttgcttcct gccattgtcc acatcaatca 721 tcagccattc ctagagagag gcgatgggcc tatttgtttg gtgctggcac caactcggga 781 actggcccaa caggtgcagc aagtagctgc tgaatattgt agagcatgtc gcttgaagtc 841 tacttgtatc tacggtggtg ctcctaaggg accacaaata cgtgatttgg agagaggtgt 901 ggaaatctgt attgcaacac ctggaagact gattgacttt ttagagtgtg gaaaaaccaa 961 tctgagaaga acaacctacc ttgtccttga tgaagcagat agaatgcttg atatgggctt 1021 tgaaccccaa ataaggaaga ttgtggatca aataagacct gataggcaaa ctctaatgtg 1081 gagtgcgact tggccaaaag aagtaagaca gcttgctgaa gatttcctga aagactatat 1141 tcatataaac attggtgcac ttgaactgag tgcaaaccac aacattcttc agattgtgga 1201 tgtgtgtcat gacgtagaaa aggatgaaaa acttattcgt ctaatggaag agatcatgag 1261 tgagaaggag aataaaacca ttgtttttgt ggaaaccaaa agaagatgtg atgagcttac 1321 cagaaaaatg aggagagatg ggtggcctgc catgggtatc catggtgaca agagtcaaca 1381 agagcgtgac tgggttctaa atgaattcaa acatggaaaa gctcctattc tgattgctac 1441 agatgtggcc tccagagggc tagatgtgga agatgtgaaa tttgtcatca attatgacta 1501 ccctaactcc tcagaggatt atattcatcg aattggaaga actgctcgca gtaccaaaac 1561 aggcacagca tacactttct ttacacctaa taacataaag caagtgagcg accttatctc 1621 tgtgcttcgt gaagctaatc aagcaattaa tcccaagttg cttcagttgg tcgaagacag 1681 aggttcaggt cgttccaggg gtagaggagg catgaaggat gaccgtcggg acagatactc 1741 tgcgggcaaa aggggtggat ttaatacctt tagagacagg gaaaattatg acagaggtta 1801 ctctagcctg cttaaaagag attttggggc aaaaactcag aatggtgttt acagtgctgc 1861 aaattacacc aatgggagct ttggaagtaa ttttgtgtct gctggtatac agaccagttt 1921 taggactggt aatccaacag ggacttacca gaatggttat gatagcactc agcaatacgg 1981 aagtaatgtt ccaaatatgc acaatggtat gaaccaacag gcatatgcat atcctgctac 2041 tgcagctgca cctatgattg gttatccaat gccaacagga tattcccaat aagactttag 2101 aagtatatgt aaatgtctgt ttttcataat tgctctttat attgtgtgtt atctgacaag 2161 atagttattt aagaaacatg ggaattgcag aaatgactgc agtgcagcag taattatggt 2221 gcactttttc gctatttaag ttggatattt ctctacattc ctgaaacaat ttttaggttt 2281 tttttgtact agaaaatgca ggcagtgttt tcacaaaagt aaatgtacag tgatttgaaa 2341 tacaataaat gaaggcaatg catggccttc caataaaaaa tatttgaaga ctgaattaag 2401 tggaaattgt actttatttt atataatgtc atgtaaaact ttgcttaaga tggtctggtt 2461 ttttttttgt ttttgtttgg tttttttttt ccatgaaaac aaatgactgt tcctttttat 2521 ttaatttggg aggcaggggg aatcagaagg cccttcttta taatgagcta ttcatattgc 2581 aggagtcaga atgaattgat acaggtgaat ttttagttac aggctaaatt gcataaaagc 2641 tttgtcagct tccagcatca ggggagtcat ttaatagcct ttttccttat ttgctagtat 2701 ggttaaatga gaaaatagta aaatagatac aaagtcatct atatagtgtg agaacgtggg 2761 tgactttttc aaagtttata atttaaaaag ctccaaataa ctggcttttt caagagactt 2821 atactcatgc tcttggctat actgtgaatt actgaaatgt tgaacaaacc tgtgaaagac 2881 atacattagc cctttaagat ggccaggagc taagcttgag tctcctttac tgaatttcgt 2941 tcttagtgca ggttacttgt agattctagt cttcacaggc tccctggggc tcttaactag 3001 tcacactggg agtcatgaat gtctttccaa taattcaggg aattctagag atcctcaaac 3061 tgtaaggtct attcatactc aacacaagga aaaaacctca ttaaaattaa tgactaatca 3121 ggaggcaacg taaccaaaag cacagtgaat gaaagttttc atggtaggtt caacatgggt 3181 ttattgctag aaagatccag gggatagctt taggtttaac ttcggctcac caacgtaact 3241 ttctaatcat ttatttcagt aatagctaga agtgggtctg aatgttttcc cagagtctga 3301 tacgtgtttt tttttgccag aagagaggtc ttcaggagac ttcatttaaa ttctgattat 3361 taaactgagg ctttaattga tgttaatgcc ttatgtcaaa tgtaaagtta gaatttgcta 3421 gggctgggat agggagtgat atttctagga cttagacatt gaaaactaat tcagcctgta 3481 gtaacctgga tggttttcaa tggcatggtt agtcaaattc atggttttaa acttagaagc 3541 agctttcggg ggagagggta ggttggagca tttattacat attttactgt ttaatgtctt 3601 aaccgtgggc cttttaattt gtaaacactg aaatgattgt tgggctgtgg aaaacattta 3661 cctatttacc ttggaagttt taaaagacag tccacttttt agcatgtgtg ttgtgtccag 3721 cctgtggtcg tcttaactaa taaatgtgat ttttctccca aaaaaaaaa

Diagnostic Methods

The present methods can be used to detect the presence of CTCs in a subject (e.g., a human subject) suspected or diagnosed as having cancer.

A test sample suspected of or known as containing tumor cells (e.g., a blood sample, a bone marrow sample, or a lymph node sample) is obtained from a human subject and the expression level of one or more marker genes FN1, S100A7, and CTDSPL, as well as that of a control gene can be determined by conventional methods. See, e.g., McDoniels-Silvers et al, Clin. Cancer Res, 2002, 8: 1127-1138; Fleischhacker et al, Ann. N. Y. Acad. Sci., 2001, 945: 179-188. It may be advantageous to determine the expression levels of all three marker genes in the same sample, as the data described herein suggest that accurate detection of CTCs is increased when the expression of all three marker genes is taken into account.

In one example, the expression level of a marker gene is determined by real-time quantitative PCR using a kit containing a set of primers specific to the one or more of the marker genes and the control gene. Alternatively, those of ordinary skill in the art would be able to design primers specific to the marker and control genes useful for real-time quantitative PCR.

The data indicating the expression level of a marker gene is first normalized against that of a control gene and the normalized data is then processed by, e.g., computational analysis, to generate an expression profile that characterizes the expression level of the marker gene. This profile is compared with an expression profile representing the expression level of the same marker gene, normalized against that of the same control gene, in a cancer-free human subject. If the expression profile obtained from the test sample indicates a higher expression level of the marker gene than that of the cancer-free patient, it indicates that the sample contains cancer cells.

Conventionally statistical analysis (e.g., the t test) can be employed to determine whether a sample from a particular subject (e.g., one who is suspected of having cancer) has a meaningfully higher expression level of a marker gene as compared to that of a sample from a cancer-free subject.

The relative expression level of a marker gene (normalized against that of a control gene) in peripheral blood can be calculated following the formula below:

(control gene) ^ (marker gene)

L-T(marker)— -

_CT(control gene) ^ ^(marker gene) _refer ^ ^ _{qpCR mreshold cyde numbers 0}f _me control gene and the marker gene, respectively. If the fluorescence signal of a marker gene were not detected after 55 cycles, 55 was assigned as the CT value of that marker gene.

The differential expression level (a test sample versus a healthy control sample) of a marker gene (Q) can be calculated as follows:

Q = ACx(test) - (mean Of AC T(healthy control)),

in which AC_T(t_est) and (mean of AC_T( eaithy control)) refer to qPCR differential threshold cycle numbers obtained for a candidate cancer patient (a test sample) and the mean differential threshold cycle numbers obtained from healthy controls, respectively.

The normalized differential expression of marker gene j in test sample i (Ej_j) can be calculated by the equation Ey = ——— , in which (¾ refers to Q of marker gene j in test sample i; Q , refers to the mean of Q of marker gene j in healthy controls; and σ _j refers to the standard deviation of Q of marker gene j in the healthy controls.

The load of cancer cells in the circulation, Lc, which has been shown to closely correlate with CTC numbers (see Peck et al., U.S. Patent No.

7,507,534), can also be calculated. A higher Lc score obtained from the sample of a suspected cancer patient (as compared to the Lc score obtained from the sample of a cancer- free subject) is indicative of the presence of cancer cells in the patient. Lc is defined as the sum of the E values of all markers:

L_c , where n is the number of marker genes.

The method described herein can be used to detect the presence of tumor cells (e.g., lung cancer cells, breast cancer cells, colorectal cancer cells, and prostate cancer cells), particularly CTCs, in a patient. The present methods are useful for diagnosing cancer in patients suspected of having cancer. For those patients already diagnosed with cancer, the present methods can be used to stage the cancer, monitor cancer progression, detect relapse and evaluate response to treatments.

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific example is, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference.

EXAMPLE

This example describes the identification and verification of novel marker and control genes for detecting CTCs.

Materials and Methods

Peripheral blood samples were obtained from 45 patients with histologically documented non-small cell lung cancer (NSCLC). These patients were treated with chemotherapy. 21 healthy volunteers free of cancer were chosen as normal controls. Two blood samples were collected from each subject. The first 1-2 ml of peripheral blood was collected in the Vacutainer® (Becton Dickinson, Rutherford, NJ) tube containing EDTA, which was later discarded, and a subsequent 5 ml of blood in another

Vacutainer® blood collection tube. Total RNAs were extracted from the 5 mo blood using the QIAamp RNeasy Blood Mini kit (Qiagen, Hiden,

Germany) within 2 hours after the blood samples were collected. The RNA amount was quantified by UV/VIS spectrophotometer and the RNAs were stored in a -80°C freezer. The first-strand cDNA was generated as follows. 1 to 2 μg of the total RNAs was incubated with 2.5 μΜ oligo (dT)i₈ primer at 70°C for 5 min and then chilled immediately on ice for 5 min. The reverse transcription reaction was conducted by adding 4 μΐ of 5x First-Strand Buffer, 5 mM DTT, 0.5 mM dNTP, 200 units of Superscript® III reverse transcriptase, and 40 units of RNaseOUT™ Recombinant RNase Inhibitor (Invitrogen, Carlsbad, CA) to the aforementioned RNA and primer mix solution to a final volume of 20 μΐ and allowed the reverse transcription reaction to proceed for 1.5 h at 50°C. The enzyme activity was inactivated by heating at 70°C for 20 min.

Real-time quantitative PCR (qPCR) was used to determine the amount of a CTC marker gene transcript. The qPCR primers and probes were selected from the Universal ProbeLibrary (UPL; Roche Diagnostics, Penzberg, Germany). qPCR reactions were performed using the LightCycler 480® (Roche Diagnostics, Penzberg, Germany). Reaction mixtures contained 5 μΐ of 2 Probes Master, 100 nM of UPL and 200 nM of primer (each) in a total volume of 10 μΐ. The qPCR conditions were: 95°C for 10 min, followed by 60 cycles at 95°C for 10 s, 60°C for 20 s, and 72°C for 2 s. Data thus obtained were analyzed by LC480 software.

Results

Identification ofDDX5 as a control for detecting CLCC by qPCR analysis The GAPDH gene is often used in qPCR assay as a control to measure the differential threshold cycle number, ACT, between the control and the target genes to account for the variations in the sample processing procedures. Other than GAPDH, housekeeping gene such as ACTB (β-actin), 18S ribosomal RNA, and several others have been used as control genes in qPCR assay. Others, such as DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 (DDX5), has been reported to serve as a control in analyzing gene expression profiles of cancer cell lines, cancerous tissues, and adjacent normal tissues. See Su, et al, BMC Genomics, 2007, 8: 140. It is unknown what control gene can be used in qPCR analysis to identify CTCs from a large pool of mononuclear cells in peripheral blood.

To identify control genes for CTCs, three genes, GAPDH, β-actin (ACTB), and DDX5, were tested for their use as controls in detecting CLCC. Their expression levels in 28 lung cancer patients and 16 healthy controls were determined by qPCR. Table 1 below shows the coefficient of variation (CV) of the expression levels of these genes in the cancer patients relative to those in the healthy controls.

Table 1. Expression Level Variations of GAPDH, ACTB, and DDX5 in Lung Cancer Patient Versus Healthy Controls qPCR control gene GAPDH ACTB DDX5

Coefficient of variation 1.69% 1.68% 1.2%

P value of unpaired t test 0.92 0.13 0.03

Among the three control genes, the CV of DDX5 (1.21%) is lower than GAPDH (1.69%) and ACTB (1.68%), indicating that the DDX5 expression level is more consistent in both lung cancer patients receiving chemotherapy and healthy volunteers.

To further test which control gene among the three yields better results in identifying CTCs from the overwhelmingly large number of PBMCs in blood plasma, we examined the expression level of CTDSPL and normalized it with that of GAPDH, ACTB, or DDX5 in both healthy control and lung cancer patient samples. The P values obtained from unpaired t test shown in Table 1 indicate that DDX5 is the best internal control gene to be used in a qPCR assay for detecting CLCCs. The DDX5-specific primers used in this study are listed in Table 2 below.

Identification and verification of marker genes useful in detecting CLCCs

In the circulation system, the number of peripheral blood mononuclear cells (PBMC) is 5 to 6 times greater than that of circulating tumor cells in the peripheral blood. It is challenging to identify marker genes that are expressed in tumor cells but are not expressed or are expressed at a much lower level in blood cells. Keratin 19 has been employed as a marker for CTC detection. The marker facilitates a detection limit of 1 CTC in 1 ml of blood and a positive detection rate around 50% (Peck, et al, Cancer Res., 1998, 58: 2761-2765).

To identify novel marker genes, candidate marker genes were first selected based on the Cancer Genome Anatomy Project (CGAP) database (an expressed sequence tag database) provided by the National Cancer Institute and the Gene Expression Omnibus (GEO) microarray database (an gene expression database) provided by the National Center for Biotechnology Information. FIG. 1 shows the validation and screening process used in this example for identifying candidate marker genes. Genes having differential expression ratios between cancerous lung tissues/cancer cell lines and normal leukocytes were selected from the databases as candidate marker genes. These selected genes were further compared with literature reports to exclude those that have already been reported for use as markers in detecting CLCCs (see Sheu, et al, Int. J. Cancer, 2006, 119: 1419-1426; Mitas, J. Mol. Diagn., 2003, 5: 237-242). The remaining candidate marker genes were analyzed by qPCR to determining their expression levels in lung cancer cell lines and in lung cancer tissues. Those having high expression levels in cancer cells/tissues were further analyzed by qPCR to determining their expression levels in peripheral blood mononuclear cells (PBMCs). 66 candidate marker genes were identified as exhibiting high expression levels in cancer cells and low expression levels in PBMCs. Among them, three marker genes, fibronection 1 (FNl), SI 00 calcium binding protein A7 (S100A7), and CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase-like (CTDSPL) were found to be useful in detecting CLCCs from lung cancer patients. The primers and probes for determining the mRNA levels of these three genes and the control gene are listed in Table 2 below:

Table 2. The primer and probe sequences specific to FNl, SlOOAl, CTDSPL, and DDX5

*Locked nucleic acid (LNA) sequence

Semi-quantification of CTCs and statistical analysis

The relative expression level of a marker gene (normalized against that of a control gene) in peripheral blood was calculated following the formula below:

AC_T(marker) = CT^(DDX5) - C_T ^{(marker gene)}

C_T ^(DDX5) and c_T ^(markergene) refer to the qPCR threshold cycle numbers of DDX5 and the marker gene, respectively. If the fluorescence signal of a marker gene were not detected after 55 cycles, 55 was assigned as the C_T value of that marker gene.

The differential expression level (a test sample versus a healthy control sample) of a marker gene (Q) is calculated as follows:

Q = ACx(test) - (mean of ACx(healthy control)),

in which ACx(_test) and (mean of

control)) refer to qPCR differential threshold cycle numbers obtained for a candidate lung cancer patient (a test sample) and the mean differential threshold cycle numbers obtained from healthy controls, respectively.

The normalized differential expression of marker gene j in test sample i (Ej_j) is calculated by the equation Ey = ——— , in which (¾ refers to Q of marker gene j in test sample i; Q_j refers to the mean of Q of marker gene j in healthy controls; and σ _j refers to the standard deviation of Q of marker gene j in the healthy controls.

The load of cancer cells in the circulation, Lc, which has been proven to closely correlated with CTC numbers (see Peck et al, US Patent

7,507,534), is defined as the sum of the E values of all markers:

L_c , where n is the number of marker genes.

Verification of the markers and enhancement of positive detection rate with multiple marker genes

As shown in Figs 2A-C, FN1 , CTDSPL, and S 100A7 exhibited higher

Lc scores in lung cancer patients than in healthy controls and the results were statistically significant (P< 0.05). Determined by the t test, the P values of

FN1 , CTDSPL, S 100A7 are 2.6E-02, 2.8E-02, and 6.5E-03, respectively.

The positive CLCC detection rates using these three marker genes individually are 47% (FN1), 64% (S100A7), and 64% (CTDSPL) in NSCLC patients.

See Fig. 3A. As shown in Fig. 2B, the positive CLCC detection rate based on CTDSPL alone is 64%, based on the combination of CTDSPL and S 100A7 is 87%o, and based on all three marker genes is 96%. Figs. 3A and 3B also show that positive detection increases with the number of the marker genes, i.e., from 64% (CTDSPL alone) to 87% (CTDSPL and S 100A7) and 96%. OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be

5 replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing l o from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

Claims

What is claimed is:

1. A method for detecting cancer cells in a subject, comprising providing a sample of the subject that is suspected of containing cancer cells,

determining in the sample an expression level of one or more marker genes selected from the group consisting of fibronectin 1 (FNl), SI 00 calcium binding protein A7 (S100A7), and carboxy-terminal domain (RNA

polymerase II, polypeptide A) small phosphatase-like protein (CTDSPL), determining in the sample an expression level of a control gene, normalizing the expression level of the one or more marker genes against that of the control gene to obtain an expression profile of the one or more marker genes, and

determining whether the sample contains cancer cells based on the expression profile;

wherein an expression profile representing an expression level of the one or more marker genes higher than that in a cancer-free subject indicates that the subject has cancer cells.

2. The method of claim 1, wherein the sample is a peripheral blood sample.

3. The method of claim 1, wherein the sample is suspected of containing lung cancer cells.

4. The method of claim 3, wherein the lung cancer cells are non- small cell lung cancer cells.

5. The method of claim 1, wherein the expression levels of FNl, S100A7, and CTDSPL are determined.

6. The method of claim 5, wherein the expression levels of FN1, S100A7, and CTDSPL are determined by real-time quantitative PCR (qPCR).

7. The method of claim 1 , wherein the control gene is DEAD box polypeptide 5 (DDX5).

8. The method of claim 7, wherein the expression level of DDX5 is determined by real-time qPCR.

9. The method of claim 5, wherein the control gene is DDX5.

10. The method of claim 9, wherein the expression level of DDX5 is determined by real-time qPCR.

1 1. The method of claim 2, wherein the sample is suspected of containing lung cancer cells.

12. The method of claim 11 , wherein the expression levels of FN 1 , S100A7, and CTDSPL are determined.

13. The method of claim 12 , wherein the expression levels of FN 1 , S100A7, and CTDSPL are determined by real-time quantitative PCR (qPCR).

14. The method of claim 11 , wherein the control gene is DEAD box polypeptide 5 (DDX5).

15. The method of claim 14, wherein the expression level of DDX5 is determined by real-time qPCR.

16. The method of claim 12, wherein the control gene is DDX5.

17. The method of claim 13, wherein the control gene is DDX5 and its expression level is determined by real-time qPCR.

18. The method of claim 6, further comprising calculating a cancer cell load (Lc) in the sample.

19. The method of claim 13, further comprising calculating a cancer cell load (Lc) in the sample.