WO2006053442A1 - Calml3 a specific and sensitive target for lung cancer diagnosis, prognosis and/or theranosis - Google Patents

Abstract

The present invention relates in general to lung cancer and to the CALML3 gene. The present invention more specifically relates to methods of diagnosing lung cancer in a biological sample by detecting CALML3 nucleic acid or protein in the sample. In one particular embodiment, the invention relates to a method for diagnosing lung cancer in a human biological sample, the method comprising: analyzing the human biological sample for the presence of CALML3 polynucleotide, the detection of an elevated level of CALML3 indicating the presence of lung cancer, as compared to the level thereof in a normal human biological sample. In one embodiment, the biological sample is obtained by a non-invasive method. In yet another embodiment of the present invention the human biological sample is selected from a sputum, a bronchial aspirate, and a coughing sample. The present invention also relates to kits for diagnosing lung cancer.

Description

TITLE OF THE INVENTION

[0001] CALML3 A SPECIFIC AND SENSITIVE TARGET FOR LUNG

CANCER DIAGNOSIS, PROGNOSIS AND/OR THERANOSIS

FIELD OF THE INVENTION

[0002] The present invention relates generally to CALML3, a specific and sensitive target for lung cancer diagnosis, prognosis and/or theranosis. More specifically, the present invention relates to CALML3 polynucleotides and polypeptides and uses thereof in methods and assay kits for the detection of lung cancer.

BACKGROUND OF THE INVENTION

[0003] Cancer is a significant health problem throughout the world.

Approximately 650 000 lung cancer cases are diagnosed annually worldwide. This type of cancer is currently the primary cause of cancer death in industrial countries with a 5-year survival rate among all lung cancer patients, regardless of the state of the disease, of only 15%. The survival rate is 49% for cases detected when the disease is still localized, but only 16% of lung cancers are discovered that early.

[0004] The World Health Organization classifies lung cancer into four major histological types (1 ) squamous cell carcinoma (SCC), (2) adenocarcinoma, (3) large cell carcinoma and (4) small cell lung carcinoma (SCLC). However, there is a great deal of tumor heterogeneity even within the various subtypes, and it is not uncommon for lung cancer to have features of more than one subtype. The term non-small cell lung carcinoma (NSCLC) includes squamous, adenocarcinoma and large cell carcinoma and represents approximately 80% of all lung cancers.

[0005] Histological differentiation between SCLC and NSCLC as well of staging of NSCLC are of great importance for the choice of therapy. For patients diagnosed with NSCLC, surgical resection frequently offers the only chance of meaningful survival. On the other hand, SCLC is the most malignant and fastest growing form of lung cancer and accounts for approximately 20% of new cases of lung cancer. The primary tumor is generally responsive to chemotherapy but is followed by wide spread metastasis.

[0006] Tumor markers are frequently found in a biological sample of cancer patients at elevated concentrations as compared to that of healthy patients. These markers are often proteins or nucleic acids encoding such protein, but can also be non-coding nucleic acid molecules. They are useful for detection, staging, monitoring and follow-up of tumor patients. A correlation is often found between the tumor marker and tumor mass in a given type of cancer.

[0007] The change of the tumor marker level as well as its absolute level as compared to the average level thereof in healthy people are often used for monitoring cancer therapy. A persistent rise in this level or a value above a defined cut-off value is indicative of recurrent cancer. In some cases, tumor markers are used for screening persons suspected of having cancer, such tumor markers being often elevated before the appearance of any clinical evidence of the disease.

[0008] The identification of tumor markers or antigens associated with lung cancer has stimulated considerable interest because of their use in screening, diagnosis, clinical management and potential treatment of lung cancer. Tumor markers frequently used for surveillance, follow up and monitoring of lung cancer include carcinoembryonic antigen (CEA) and neuron specific enolase (NSE).

Among these markers, NSE is very specific for SCLC whereas the other does not display high sensitivity at an acceptable specificity level for NSCLC. These markers are often used in combination with other markers called proliferation markers, which show high sensitivity but relatively low specificity for NSCLC. Moreover, for screening persons suspected of having lung cancer and monitoring lung cancer patients, it is at present necessary to determine the level of multiple lung cancer markers in order to have reliable data on the presence and progression of lung cancer.

[0009] Early detection of lung cancer, while currently essential, is difficult since clinical symptoms are often not seen until the disease has reached an advanced stage. Thus, in spite of considerable research into therapies and diagnostic methods, lung cancer remains difficult to diagnose and to treat effectively. This has raised the global interest for characterizing lung cancer cells in order to identify molecular markers and potential therapeutic targets that could be used for disease detection and rational therapy of lung cancer.

[0010] The desire to identify such markers and targets has led several companies and institutions into a race to identify lung cancer specific polynucleotides and polypeptide sequences. By comparing molecular expression profiles between lung tumor cells and normal cells, several thousands genes have been reported to be specifically modulated in lung cancer.

[0011] One such gene is Calmodulin-like 3 (CALML3). This gene is part of the Calmodulin family of proteins, which participate, for example, in cyclic nucleotide metabolism, protein phosphorylation and dephosphorylation cascades, Ca²⁺ and other ion transport and cell growth and proliferation as well as differentiation. CALML3 is a 148 amino acid protein containing 4 helix-loop-helix Ca²⁺ binding motifs, and shares 85% identity with vertebrate calmodulin. The CALML3 gene is localized on chromosome 10p13-ter and its expression appears to be highly restricted to particular cell-types (e.g., epithelial) in vivo. CALML3 gene expression has been reported to be increased during terminal differentiation and is strongly decreased upon malignant transformation. CALML3 is also known as CLP (Rogers and Strehler, 2001, Exp. Cell Res. 267:216-224). CALML3 and Calmodulin, bind Ca²⁺ in a similar fashion while the affinity of the former has been described as being lower than that of the latter (Rogers and Strehler, 2001 , Exp. Cell Res. 267:216-224 and references 11 and 12 cited therein). It should be noted that CALML3 has been shown to be expressed in skin (keratinocytes) as well as in a number of epitheliums such as those of thyroid, breast, prostate and kidney, (based on Online Mendelian Inheritance in Man (OMIM) and Berchtold et al., 1993).

[0012] WO03042661 describes thousands of genes whose expression is up-regulated or down-regulated in specific cancers or other diseases, or are otherwise regulated in disease. Methods and compositions that can be used for diagnosis, prognosis, and treatment of those medical conditions are disclosed in WO03042661. Also described therein are methods that can be used to identify modulators of these selected conditions. Of note, CALML3 does not figure among the genes specifically overexpressed in lung cancer.

[0013] WO0194629 describes the identification of thousands of genes modulated (either overexpressed or downregulated) in several types of cancer. More particularly, processes for assaying potential antitumor agents based on the modulation of the expression of specified genes, or sets, of suspected cancer cell genes are disclosed. In addition, methods for diagnosing cancerous, or potentially cancerous, conditions as a result of the expression, or patterns of expression, of such genes, or sets of genes, are taught. Also disclosed are methods for determining functionally related genes, or gene sets, as well as methods for treating cancer, based on targeting expression products of such genes, or gene sets, and determining genes involved in the cancerous process.

[0014] Although these expression profile studies are useful for understanding disease biology and for identifying potential tumor markers, they do not assess the specificity or the usefulness of such markers as diagnostic tools. In addition, it should be recognized that the identification of differential expression of genes, by bioinformatics methods does not imply that those genes can actually form the basis of a clinical test for cancer.

[0015] In view of the fact that lung cancer remains a life threatening disease reaching a very significant proportion of the human population, there remains a need for the development of new treatment and diagnostic modalities for such cancer.

[0016] More particularly, there remains a need to identify molecular markers for the diagnosis of lung cancer and for the development of useful, specific and sensitive diagnostic tests for the early detection of lung cancer. The present invention is aimed at fulfilling these and other needs and aims at providing other related advantages.

[0017] The present description refers to a number of documents, the content of which is herein incorporated by reference in its entirety.

SUMMARY OF THE INVENTION

[0018] The present invention relates to the discovery that CALML3 polynucleotide sequences are selectively and specifically expressed at higher levels in biological samples derived from lung cancer patients. More specifically, the present invention relates to the identification of CALML3 gene sequences, as useful, specific and sensitive targets for new diagnostic, prognostic and theranostic developments in the field of lung cancer.

[0019] In one embodiment, the present invention relates to non-small cell lung carcinoma (NSCLC) diagnosis. In another embodiment, the present invention relates to CALML3 sequences as validated markers for the diagnosis of lung cancer, more particularly of the squamous cell carcinoma subtype.

[0020] In another embodiment, the present invention relates to a method of detecting CALML3 polynucleotides in a biological sample. In another aspect, the present invention relates to a method of detecting polynucleotides encoding CALML3 in a biological sample.

[0021] In a related aspect, the present invention concerns nucleic acids for the specific detection of the presence of CALML3 polynucleotides or polynucleotides encoding CALML3 proteins or polypeptides in a biological sample.

[0022] The invention thus further provides a method of diagnosing the presence of, or predisposition to develop, lung cancer in a patient. The present invention provides methods of diagnosis, for in vitro or ex vivo (e.g., using a sample from a patient and analyzing same in a laboratory or the like), and in vivo use (e.g., detecting a tracer [e.g., a label] administered to a patient and directed at CALML3, using an appropriate detection method).

[0023] In addition, by enabling diagnosis of lung cancer, the present invention enables a clinician to which the present invention pertains, to rationally adapt a therapeutic treatment or regimen according to the patient parameters and/or to that of the disease. Non-limiting examples of such parameters include the age, sex and fitness of the patient, the grade of the cancer, family history, and the like.

[0024] In a related aspect, the present invention also relates to theranostic methods, i.e., use of the molecular test of the present invention to diagnose the disease, choose or adapt the correct or most appropriate treatment regimen and monitor the patient's response to therapy. [0025] The present invention also concerns a lung cancer diagnostic, theranostic and prognostic kit for detecting the presence of CALML3 nucleic acid in a sample. Such a kit can comprise a first container means having disposed therein at least one oligonucleotide probe or primer that hybridizes to a CALML3 nucleic acid (e.g., CALML3 mRNA). In another embodiment, a second container means contains a probe, which specifically hybridizes to the CALML3 amplification product. Of course, numerous kits can be designed and adapted by a person skilled in the art.

[0026] In yet another embodiment, the kit further includes other containers comprising additional components such as an additional oligonucleotide(s) or primer(s) (e.g. additional lung cancer markers, internal control designed for normalizing the number of lung cells and/or the amplification and detection methods such as primers/probes for PBGD, 18S RNA and/or SFTPC, etc.), and/or one or more of the following: buffers, reagents to be used in the assay (e.g., wash reagents, polymerases, internal control nucleic acid or cells or else) and reagents capable of detecting the presence of bound nucleic acid probe(s)/primer(s). Of course the separation or assembly of reagents in same or different container means is dictated by the types of extraction, amplification or hybridization methods, and detection methods used as well as other parameters including stability, need for preservation etc. It will be understood that different permutations of containers and reagents of the above and foregoing are also covered by the present invention. The kit may also include instructions regarding each particular possible diagnosis, prognosis or use, correlating a corresponding range of CALML3 expression level as well as information on the experimental protocol to be used.

[0027] In another aspect, the present invention relates to a method of detecting a CALML3 polypeptide in a biological sample. In one aspect, the biological sample is lung aerosol and CALML3 protein (or nucleic acid) is detected in that lung aerosol sample. In one particular embodiment, the lung aerosol is a cough sample from a patient. In one particular embodiment, the cough sample is collected in a device or by means adapted to receive such samples.

[0028] In one embodiment of the present invention, CALML3 RNA is detected using an RNA amplification method. In one such embodiment, the RNA amplification method is coupled to real-time detection of the amplified products using fluorescence specific probes. In yet a further embodiment, the amplification method is PCR. In an additional embodiment the PCR is real-time PCR or a related method enabling a detection in real-time of the amplified products.

[0029] In one embodiment, the cells collected from the sample are harvested and a total nucleic acid extraction is carried out. In one particular embodiment, total nucleic acid extraction is performed using a solid phase band method on silica beads, as described by BOOM et a/ (J. Clin. Microbiol. 1990, 28:495-503). Of course, it should be understood that numerous nucleic acid extraction methods exists and thus, that other methods could be used in accordance with the present invention. One non-limiting example is a phenol/chloroform extraction method. Other such methods are described in herein referenced textbooks. Of course, it also should be understood that the amplification method, or other method of the invention, can be performed on the cells without a nucleic acid extraction method, or with only a partial nucleic acid extraction method. Such amplification methods are also well known in the art.

[0030] In one additional embodiment, RNA encoded by the CALML3 gene is detected by an in vitro RNA amplification method named Nucleic Acid based Amplification (NASBA). Of course other RNA amplification methods are known and the instant methods and kits are therefore not limited to NASBA. Non- limiting examples of such RNA amplification methods include polymerase chain reaction (PCR), transcriptase mediated amplification (TMA) and ligase chain reaction (LCR).

[0031] In one embodiment, the amplified products are detected in a homogenous phase using a fluorescent probe by the Beacon approach. In another embodiment, the product is detected on solid phase using fluorescent or colorimetric method. It should be understood that numerous fluorescent, colorimetric, or enzymatic methods could be used in accordance with the present invention to detect and/or quantify the targeted RNAs. Such fluorescent, colorimetric or enzymatic methods are well known in the art.

[0032] The invention further relates to a method of detecting antibodies that bind selectively to a CALML3 protein, polypeptide or epitope.

[0033] The invention further concerns a lung cancer diagnostic kit comprising a first container means containing an antibody that specifically binds to CALML3 polypeptide. In another embodiment the kit comprises a second container means containing a conjugate comprising a binding partner of the antibody (e.g., monoclonal antibody) and a label. Of course the separation or assembly of reagents in same or different container means is dictated by the types of CALML3 polypeptide (or fragment thereof) detection methods used, as well as other parameters including stability, need for preservation, types of control reactions included etc. The person skilled in the art can easily adapt and design kits to suit particular needs.

[0034] The invention further relates to diagnostic, prognostic and theranostic methods for human lung cancer. Preferably, a method of diagnosing the presence or predisposition to develop lung cancer in a patient is disclosed herein. Also disclosed is a method for monitoring the progression of lung cancer in a patient. Of course, the diagnostic, prognostic and theranostic methods of the present invention may optionally be employed in conjunction with other markers for lung cancer such as CEA, NSE, p53, etc.

[0035] Unless defined otherwise, the scientific and technical terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains. Commonly understood definitions of molecular biology terms can be found for example in Dictionary of Microbiology and Molecular Biology, 2nd ed. (Singleton et al., 1994, John Wiley & Sons, New York, NY), the Harper Collins Dictionary of Biology, Hale & Marham, 1991, Harper Perennial, New York, NY); Rieger et al., Glossary of genetics: Classical and molecular, 5^th edition, Springer-Verlag, New- York, 1991 ; Alberts et al., Molecular Biology of the Cell, 4^th edition, Garland science, New-York, 2002; and, Lewin, Genes VIl, Oxford University Press, New-York, 2000. Generally, the procedures of molecular biology methods and the like are common methods used in the art. Such standard techniques can be found in reference manuals such as for example Sambrook et al. (cf., Molecular Cloning: A Laboratory Manual, Third Edition, edited by Cold Spring Harbor Laboratory, 2000); and Ausubel et al. (1994, Current Protocols in Molecular Biology, Wiley, New York).

[0036] It should be understood by a person of ordinary skill, that numerous statistical methods can be used in the context of the present invention to determine if the test is positive or negative.

[0037] In the present description, a number of terms are extensively utilized. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

DEFINITIONS

[0038] Nucleotide sequences are presented herein by single strand, in the 5' to 3' direction, from left to right, using the one-letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission.

[0039] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one" but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0040] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. Routinely a 10% to 15% deviation preferably 10% is within the scope of the term "about".

[0041] As used herein, "nucleic acid molecule" or "polynucleotides", refers to a polymer of nucleotides. Non-limiting examples thereof include DNA (e.g., genomic DNA, cDNA), RNA molecules (e.g., mRNA) and chimeras thereof. The nucleic acid molecule can be obtained by cloning techniques or synthesized. DNA can be double-stranded or single-stranded (coding strand or non-coding strand [antisense]). Conventional ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) are included in the term "nucleic acid" and polynucleotides as are analogs thereof. A nucleic acid backbone may comprise a variety of linkages known in the art, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (referred to as "peptide nucleic acids" (PNA); Hydig-Hielsen et al., PCT Int'l Pub. No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages or combinations thereof. Sugar moieties of the nucleic acid may be ribose or deoxyribose, or similar compounds having known substitutions, e.g., 2' methoxy substitutions (containing a 2'-O-methylribofuranosyl moiety; see PCT No. WO 98/02582) and/or 2' halide substitutions. Nitrogenous bases may be conventional bases (A, G, C, T, U), known analogs thereof (e.g., inosine or others; see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11^th ed., 1992), or known derivatives of purine or pyrimidine bases (see, Cook, PCT Int'l Pub. No. WO 93/13121) or "abasic" residues in which the backbone includes no nitrogenous base for one or more residues (Arnold et al., U.S. Pat. No. 5,585,481). A nucleic acid may comprise only conventional sugars, bases and linkages, as found in RNA and DNA, or may include both conventional components and substitutions (e.g., conventional bases linked via a methoxy backbone, or a nucleic acid including conventional bases and one or more base analogs).

[0042] The terminology "CALML3 nucleic acid" or "CALML3 polynucleotide" refers to a native CALML3 nucleic acid sequence. In one embodiment, the CALML3 nucleic acid sequence has the sequence set forth in

SEQ ID NO: 9. In one particular embodiment, the CALML3 nucleic acid encodes

CALML3 protein (SEQ ID NO: 10). In yet a further embodiment, the CALML3 sequence which is targeted by the CALML3 sequences encompassed by the present invention, is a natural CALML3 sequence found in a patient sample, which has significant conservation as compared to the sequences of SEQ ID NOs: 9 or

10.

[0043] Isolated nucleic acid molecule. An "isolated nucleic acid molecule", as is generally understood and used herein, refers to a polymer of nucleotides, and includes but should not be limited to DNA and RNA. The "isolated" nucleic acid molecule is purified from its natural in vivo state.

[0044] A nucleic acid segment. A DNA or RNA segment (or chimera), as is generally understood and used herein, refers to a molecule comprising a linear stretch of nucleotides wherein the nucleotides are present in a sequence that can encode, through the genetic code when applicable (not all segments being coding sequences), a molecule comprising a linear sequence of amino acid residues that is referred to as a protein, a protein fragment or a polypeptide. [0045] ^' As used herein, "protein" or "polypeptide" means any peptide- linked chain of amino acids, regardless of postranslational modifications (e.g., phosphorylation, glycosylation, sulfatation, sumoylation, prenylation, ubiquitination etc). A "CALML3 protein" or a "CALML3 polypeptide" is an expression product of CALML3 nucleic acid (e.g., CALML3 gene) such as native CALML3 protein (SEQ ID NO: 10) or a CALML3 protein homolog that shares at least 60% (but preferably, at least 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%) amino acid sequence identity with CALML3 and displays functional activity of native CALML3 protein. For the sake of brevity, the units (e.g., 66, 67...81 , 82%...) have not been specifically recited but are nevertheless considered within the scope of the present invention. An "isolated protein" or "isolated polypeptide" is purified from its natural in vivo state.

[0046] A functional activity of a polypeptide or protein is any activity associated with a structural, biochemical or physiological activity of the protein (either structural or functional). For instance, one non-limiting example of a functional activity of CALML3 protein includes calcium binding. In addition, CALML3 can interact with a number of proteins which interact with Calmodulin. CALML3 protein also binds with proteins which do not bind to Calmodulin. For example, in the presence of calcium, CALML3 interacts with high affinity to Myosin X (Rogers and Strehler, 2001, J Biol. Chem. 276:12182-12189), a protein which does not bind to Calmodulin. The high affinity interaction between Myosin X and CALML3 has been shown by a double-hybrid system and is thus predicted to occur in vivo in cell lines. Thus, one non-limiting example of a functional assay based on CALML3 could be a two-hybrid system or another system (e.g., in vitro binding assay) that monitors the interaction of CALML3 (or a fragment thereof) with Myosin X (or fragment thereof).

[0047] Gene. A DNA sequence related to a polypeptide chain or protein, and as used herein includes the 5' and 3' untranslated ends. The polypeptide can be encoded by a full-length sequence or any portion of the coding sequence, so long as the functional activity of the protein is retained.

[0048] When referring to nucleic acid molecules, proteins or polypeptides, the term native refers to a naturally occurring nucleic acid or polypeptide. A homolog is a gene sequence encoding a polypeptide isolated from an organism other than a human being. Similarly, a homolog of a native polypeptide is an expression product of a gene homolog. Of course, the non- coding portion of a gene can also find a homolog portion in another organism.

[0049] Complementary DNA (cDNA). Recombinant nucleic acid molecules synthesized by reverse transcription of messenger RNA ("imRNA").

[0050] Agarose Gel Electrophoresis. The most commonly used technique (though not the only one) for fractionating double strand DNA is agarose gel electrophoresis. The principle of this method is that DNA molecules migrate through the gel as though it were a sieve that retards the movement of the largest molecules to the greatest extent and the movement of the smallest molecules to the least extent. Note that the smaller the DNA fragment, the greater the mobility under electrophoresis in the agarose gel.

[0051] The DNA fragments fractionated by agarose gel electrophoresis can be visualized directly by a staining procedure (e.g., EtBr) if the number of fragments included in the pattern is small. In order to visualize a small subset of these fragments, a methodology referred to as the Southern hybridization procedure can be applied.

[0052] Southern Transfer Procedure. The purpose of the Southern transfer procedure (also referred to as blotting) is to physically transfer DNA fractionated by agarose gel electrophoresis onto a nitrocellulose filter paper or another appropriate surface or method, while retaining the relative positions of DNA fragments resulting from the fractionation procedure. The methodology used to accomplish the transfer from agarose gel to nitrocellulose involves drawing the DNA from the gel onto the nitrocellulose paper by capillary action, or other action.

[0053] Nucleic Acid Hybridization. Nucleic acid hybridization depends on the principle that two single-stranded nucleic acid molecules that have complementary base sequences will reform the thermodynamically favored double-stranded structure if they are mixed under the proper conditions. The double-stranded structure will be formed between two complementary single- stranded nucleic acids even if one is immobilized on a nitrocellulose filter. In the Southern or Northern hybridization procedures, the latter situation occurs. The DNA/RNA of the individual to be tested may be digested with a restriction endonuclease if applicable, prior to its fractionation by agarose gel electrophoresis, conversion to the single-stranded form, and transfer to nitrocellulose paper, making it available for reannealing to the hybridization probe. Non-limiting examples of hybridization conditions can be found in Ausubel, F. M. et al., Current protocols in Molecular Biology, John Wiley & Sons, Inc., New York, NY (1994). For purposes of illustration, an example of moderately stringent conditions for testing the hybridization of a polynucleotide of the present invention with other polynucleotides, include prewashing, in a solution of 5X SSC, 0.5% SDS, 1mM EDTA (pH 8.0); hybridizing at 50 °C-60 ⁰C, 5X SSC and 100 μg/ml denatured salmon sperm DNA overnight (12-16 hours); followed by washing twice at 6O⁰C for 15 minutes with each of 2X SSC, 0.5X SSC and 0.2X SSC containing 0.1 % SDS. For example for highly stringent hybridization conditions, the hybridization temperature is changed to 65, 66, 67 or 68 ⁰C. One skilled in the art will understand that the stringency of hybridization can be readily manipulated, such as by altering the salt and SDS concentration of the hybridizing and washing solutions and/or temperature at which the hybridization is performed. The temperature and salt concentration selected is determined based on the melting temperature (Tm) of the DNA hybrid. Other protocols or commercially available hybridization kits using different annealing and washing solutions can also be used as well known in the art. The use of formamide in different mixtures to lower the melting temperature is well known in the art.

[0054] A "probe" is meant to include a nucleic acid oligomer that hybridizes specifically to a target sequence in a nucleic acid or its complement, under conditions that promote hybridization, thereby allowing detection of the target sequence or its amplified nucleic acid. Detection may either be direct (i.e., resulting from a probe hybridizing directly to the target or amplified sequence) or indirect (i.e., resulting from a probe hybridizing to an intermediate molecular structure that links the probe to the target or amplified sequence). A probe's "target" generally refers to a sequence within an amplified nucleic acid sequence (i.e., a subset of the amplified sequence) that hybridizes specifically to at least a portion of the probe sequence by standard hydrogen bonding or "base pairing." Sequences that are "sufficiently complementary" allow stable hybridization of a probe sequence to a target sequence, even if the two sequences are not completely complementary. A probe may be labeled or unlabeled. A probe is generally produced by molecular cloning of a specific DNA sequence. Of course, a probe can also be synthesized. Numerous primers and probes which can be designed and used in the context of the present invention can be readily determined by a person of ordinary skill in the art to which the present invention pertains. Non-limiting examples of primers and probes are shown in Table 1. A person skilled in the art can design numerous other probes and primers based on the teachings herein and the general knowledge.

[0055] By "sufficiently complementary" is meant a contiguous nucleic acid base sequence that is capable of hybridizing to another sequence by hydrogen bonding between a series of complementary bases. Complementary base sequences may be complementary at each position in sequence by using standard base pairing (e.g., G:C, A:T or A:U pairing) non standard base pairing (e.g., I:C) or may contain one or more residues (including a basic residues) that are not complementary by using standard base pairing, but which allow the entire sequence to specifically hybridize with another base sequence in appropriate hybridization conditions. Contiguous bases of an oligomer are preferably at least about 80% (81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100%), more preferably at least about 90% complementary to the sequence to which the oligomer specifically hybridizes. Appropriate hybridization conditions are well known to those skilled in the art, can be predicted readily based on sequence composition and conditions, or can be determined empirically by using routine testing (see Sambrook et al., (cf., Molecular Cloning: A Laboratory Manual, Third Edition,^' edited by Cold Spring Harbor Laboratory, 2000) at §§ 1.90-1.91 , 7.37- 7.57, 9.47-9.51 and 11.47-11.57, particularly at §§ 9.50-9.51 , 11.12-11.13, 11.45- 11.47 and 11.55-11.57).

[0056] Nucleic acid sequences may be detected by using hybridization with a complementary sequence (e.g., oligonucleotide probes) (see U.S. Patent Nos. 5,503,980 (Cantor), 5,202,231 (Drmanac et al.), 5,149,625 (Church et al.), 5,112,736 (Caldwell et al.), 5,068,176 (Vijg et al.), and 5,002,867 (Macevicz)). Hybridization detection methods may use an array of probes (e.g., on a DNA chip) to provide sequence information about the target nucleic acid which selectively hybridizes to an exactly complementary probe sequence in a set of four related probe sequences that differ by one nucleotide (see U.S. Patent Nos. 5,837,832 and 5,861 ,242 (Chee et al.).

[0057] A detection step may use any of a variety of known methods to detect the presence of nucleic acid by hybridization to a probe oligonucleotide. One specific example of a detection step uses a homogeneous detection method such as described in detail previously in Arnold et al. Clinical Chemistry 35:1588- 1594 (1989), and U.S. Patent Nos. 5,658,737 (Nelson et al.), and 5,118,801 and 5,312,728 (Lizardi et al.).

[0058] The types of detection methods in which probes can be used include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection). Labeled proteins could also be used to detect a particular nucleic acid sequence to which it binds (e.g., protein detection by far western technology: Guichet et al., 1997, Nature 385(6616): 548-552; and Schwartz et al., 2001 , EMBO 20(3): 510-519). Other detection methods include kits containing reagents of the present invention on a dipstick setup and the like. Of course, it might be preferable to use a detection method which is amenable to automation. A non-limiting example thereof includes a chip or other support comprising one or more (e.g., an array) different probes.

[0059] A "label" refers to a molecular moiety or compound that can be detected or can lead to a detectable signal. A label is joined, directly or indirectly, to a nucleic acid probe or the nucleic acid to be detected (e.g., an amplified sequence). Direct labeling can occur through bonds or interactions that link the label to the nucleic acid (e.g., covalent bonds or non-covalent interactions), whereas indirect labeling can occur through the use of a "linker" or bridging moiety, such as additional oligonucleotide(s), which is either directly or indirectly labeled. Bridging moieties may amplify a detectable signal. Labels can include any detectable moiety (e.g., a radionucleotide, ligand such as biotin or avidin, enzyme or enzyme substrate, reactive group, chromophore such as a dye or colored particle, luminescent compound including a bioluminescent, phosphorescent or chemiluminescent compound, and fluorescent compound). In one particular embodiment, the label on a labeled probe is detectable in a homogeneous assay system, i.e., in a mixture, the bound label exhibits a detectable change compared to an unbound label.

[0060] Other methods of labeling nucleic acids are known whereby a label is attached to a nucleic acid strand as it is fragmented, which is useful for labeling nucleic acids to be detected by hybridization to an array of immobilized DNA probes (e.g., see PCT No. PCT/IB99/02073).

[0061] A "homogeneous detectable label" refers to a label whose presence can be detected in a homogeneous fashion based upon whether the labeled probe is hybridized to a target sequence. A homogeneous detectable label can be detected without physically removing hybridized from unhybridized forms of the labeled probe. Homogeneous detectable labels and methods of detecting them have been described in detail elsewhere (e.g., see U.S. Pat. Nos. 5,283,174, 5,656,207 and 5,658,737).

[0062] As used herein, "oligonucleotides" or "oligos" define a molecule having two or more nucleotides (ribo or deoxyribonucleotides). The size of the oligo will be dictated by the particular situation and ultimately on the particular use thereof and adapted accordingly by the person of ordinary skill. An oligonucleotide can be synthesized chemically or derived by cloning according to well-known methods. While they are usually in a single-stranded form, they can be in a double- stranded form and even contain a "regulatory region". They can contain natural, rare or synthetic nucleotides. They can be designed to enhance a chosen criterion like stability, for example. Chimeras of deoxyribonucleotides and ribonucleotides may also be within the scope of the present invention.

[0063] Sequence Amplification. A method for generating large amounts of a target sequence. In general, one or more amplification primers are annealed to a nucleic acid sequence. Using appropriate enzymes, sequences found adjacent to, or in between the primers are amplified.

[0064] "Amplification" refers to any known in vitro procedure for obtaining multiple copies ("amplicons") of a target nucleic acid sequence or its complement or fragments thereof. In vitro amplification refers to the production of an amplified nucleic acid that may contain less than the complete target region sequence or its complement. Known in vitro amplification methods include, e.g., transcription-mediated amplification, replicase-mediated amplification, polymerase chain reaction (PCR) amplification, ligase chain reaction (LCR) amplification, nucleic acid sequence-based amplification (NASBA), and strand-displacement amplification (SDA). Replicase-mediated amplification uses self-replicating RNA molecules, and a replicase such as Qβ-replicase (e.g., Kramer et al., U.S. Pat. No. 4,786,600). PCR amplification is well known and uses DNA polymerase, primers and thermal cycling to synthesize multiple copies of the two complementary strands of DNA or cDNA (e.g., MuIHs et al., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). LCR amplification uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation (e.g., EP Pat. App. Pub. No. 0 320 308). SDA is a method in which a primer contains a recognition site for a restriction endonuclease that permits the endonuclease to nick one strand of a hemimodified DNA duplex that includes the target sequence, followed by amplification in a series of primer extension and strand displacement steps (e.g., Walker et al., U.S. Pat. No. 5,422,252). Another known strand-displacement amplification method does not require endonuclease nicking (Dattagupta et al., U.S. Patent No. 6,087,133). Transcription-mediated amplification (TMA) can also be used in the present invention. In one embodiment, TMA and NASBA isothermic methods of nucleic acid amplification are used. Those skilled in the art will understand that the oligonucleotide primer sequences of the present invention may be readily used in any in vitro amplification method based on primer extension by a polymerase (see generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14-25 and (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi et al., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods MoI. Biol., 28:253-260; and Sambrook et al., (cf., Molecular Cloning: A Laboratory Manual, Third Edition, edited by Cold Spring Harbor Laboratory, 2000). As commonly known in the art, the oligos are designed to bind to a complementary sequence under selected conditions. [0065] As used herein, a "primer" defines an oligonucleotide which is capable of annealing to a target sequence, thereby creating a double stranded region which can serve as an initiation point for nucleic acid synthesis under suitable conditions. Primers can be, for example, designed to be specific for certain alleles so as to be used in an allele-specific amplification system. The primer's 5¹ region may be non-complementary to the target nucleic acid sequence and include additional bases, such as a promoter sequence (which is referred to as a "promoter primer"). Those skilled in the art will appreciate that any oligomer that can function as a primer can be modified to include a 5' promoter sequence, and thus function as a promoter primer. Similarly, any promoter primer can serve as a primer, independent of its functional promoter sequence. Of course the design of a primer from a known nucleic acid sequence is well known in the art. As for the oligonucleotides, it can comprise a number of types of different nucleotides. Skilled artisans can easily assess the specificity of selected primers or probes (e.g., CALML3, PBGD, 18S RNA, SFTPC specific primers and probes) by performing computer alignments/searches using well-known databases (e.g., GenBank™).

[0066] Polymerase chain reaction (PCR). PCR is carried out in accordance with known techniques. See, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188 (the disclosures of all three U.S. Patents are incorporated herein by reference). In general, PCR involves a treatment of a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) under hybridizing conditions, with one oligonucleotide primer for each strand of the specific sequence to be detected. An extension product of each primer which is synthesized is complementary to each of the two nucleic acid strands, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith. The extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers. Following a sufficient number of rounds of synthesis of extension products, the sample is analyzed to assess whether the sequence or sequences to be detected are present. Detection of the amplified sequence may be carried out by visualization following like, for example, EtBr staining of the DNA following gel electrophoresis, or using a detectable label in accordance with known techniques, and the like. For a review on PCR techniques (see for example "PCR Protocols, A Guide to Methods and Amplifications", Michael et al. Eds, Acad. Press, 1990).

[0067] Ligase chain reaction (LCR). Another example of amplification technique is LCR. It is carried out in accordance with known techniques (Weiss, 1991, Science 254:1292). Adaptation of the protocol to meet the desired needs can be carried out by a person of ordinary skill. Strand displacement amplification (SDA) is also carried out in accordance with known techniques or adaptations thereof to meet the particular needs (Walker et al., 1992, Proc. Natl. Acad. Sci. USA 89:392-396; and ibid., 1992, Nucleic Acids Res. 20:1691-1696).

[0068] Transcription-associated amplification. Amplifying a target nucleic acid sequence by using at least two primers can be accomplished using a variety of known nucleic acid amplification methods, but preferably uses a transcription-associated amplification reaction that is substantially isothermal. By using such an in vitro amplification method, many strands of nucleic acid are produced from a single copy of target nucleic acid, thus permitting detection of the target in the sample by specifically binding the amplified sequences to one or more detection probes. Transcription-associated amplification methods have been described in detail elsewhere (e.g., U.S. Pat. Nos. 5,399,491 and 5,554,516). Briefly, transcription-associated amplification uses two types of primers (one being a promoter primer because it contains a promoter sequence for an RNA polymerase), two enzyme activities (a reverse transcriptase (RT) and an RNA polymerase), substrates (deoxyribonucleoside triphosphates, ribonucleoside triphosphates) and appropriate salts and buffers in solution to produce multiple RNA transcripts from a nucleic acid template. Initially, a promoter primer hybridizes specifically to a target sequence (e.g., RNA) and reverse transcriptase creates a first complementary DNA strand (cDNA) by extension from the 3¹ end of the promoter primer. The cDNA is made available for hybridization with the second primer by any of a variety of methods, such as, by denaturing the target- cDNA duplex or using RNase H activity supplied by the RT that degrades RNA in a DNA:RNA duplex. A second primer binds to the cDNA and a new strand of DNA is synthesized from the end of the second primer using the RT activity to create a double-stranded DNA (dsDNA) having a functional promoter sequence at one end. An RNA polymerase binds to the dsDNA promoter sequence and transcription produces multiple transcripts ("amplicons"). Amplicons are used in subsequent steps or cycles of the transcription-associated amplification process by serving as a new template for replication, thus generating many copies of amplified nucleic acid (i.e., about 100 to 3,000 copies of RNA are synthesized from each template).

[0069] Nucleic Acid Sequence-Based Amplification (NASBA). The

NASBA reaction is based on the simultaneous activity of avian myeloblastosis virus (AMV) reverse transcriptase (RT), Rnase H and T7 RNA polymerase with two oligonucleotide primers to produce amplification of the desired fragment more than 10¹² fold in 90 to 120 minutes (Kievits et al., Journal of Virological methods 1991 :273-286 and Compton, Nature 1991: 91-92). In a NASBA reaction, nucleic acids are a template for the amplification reaction only if they are single stranded and located in the primer-binding region. Because the NASBA reaction is isothermal (41 ⁰C), specific amplification of ssRNA is possible if denaturation of dsDNA is prevented in the sample preparation procedure. It is thus possible to pick up RNA in a dsDNA background without getting false positive results caused by genomic dsDNA. By using molecular beacons with the NASBA amplification, a real time detection and quantification system can be generated. Molecular beacons are stem-and-loop-structured oligonucleotides with a fluorescent label at the 5' end and a universal quencher at the 3' end. They are highly specific for their target and hybridize with their target RNA when present in a NASBA amplification reaction to form stable hybrids at relatively low temperature (41 °C) (Polstra et al., BMC infectious disease, 2002 : 18-27).

[0070] Target capture. In one embodiment, target capture is included in the method to increase the concentration or purity of the target nucleic acid before in vitro amplification. Preferably, target capture involves a relatively simple method of hybridizing and isolating the target nucleic acid, as described in detail elsewhere (e.g., see US Pat. Nos. 6, 110,678, 6,280,952, and 6,534,273). Generally speaking, target capture can be divided in two families, sequence specific and non-sequence specific. In the non-specific method, a reagent (e.g., silica beads) is used to capture non specifically nucleic acids. In the sequence specific method an oligonucleotide attached to a solid support is contacted with a mixture containing the target nucleic acid under appropriate hybridization conditions to allow the target nucleic acid to be attached to the solid support to allow purification of the target from other sample components. Target capture may result from direct hybridization between the target nucleic acid and an oligonucleotide attached to the solid support, but preferably results from indirect hybridization with an oligonucleotide that forms a hybridization complex that links the target nucleic acid to the oligonucleotide on the solid support. The solid support is preferably a particle that can be separated from the solution, more preferably a paramagnetic particle that can be retrieved by applying a magnetic field to the vessel. After separation, the target nucleic acid linked to the solid support is washed and amplified when the target sequence is contacted with appropriate primers, substrates and enzymes in an in vitro amplification reaction.

[0071] Generally, capture oligomer sequences include a sequence that specifically binds to the target sequence, when the capture method is indeed specific, and a "tail" sequence that links the complex to an immobilized sequence by hybridization. That is, the capture oligomer includes a sequence that binds specifically to CALML3 or to another Lung specific marker (non-limiting examples include NSE, CYFRA21 or CEA) target sequence and a covalently attached 3' tail sequence (e.g., a homopolymer complementary to an immobilized homopolymer sequence). It will be understood from the fact that it is possible to use CEA as another lung specific marker, that the absolute specificity to lung cancer is not essential. Indeed, CEA can also be found in colon cancer. The tail sequence which is, for example, 5 to 50 nucleotides long, hybridizes to the immobilized sequence to link the target-containing complex to the solid support and thus purify the hybridized target nucleic acid from other sample components. A capture oligomer may use any backbone linkage, but some embodiments include one or more 2'- methoxy linkages. Of course, other capture methods are well known in the art. The capture method on the cap structure (Edery et al., 1988, gene 74(2): 517-525, US 5,219,989) and the silica-based method are two non-limiting examples of capture methods.

[0072] An "immobilized probe" or "immobilized nucleic acid" refers to a nucleic acid that joins, directly or indirectly, a capture oligomer to a solid support. An immobilized probe is an oligomer joined to a solid support that facilitates separation of bound target sequence from unbound material in a sample. Any known solid support may be used, such as matrices and particles free in solution, made of any known material (e.g., nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane polypropylene and metal particles, preferably paramagnetic particles). Preferred supports are monodisperse paramagnetic spheres (i.e., uniform in size ± about 5%), thereby providing consistent results, to which an immobilized probe is stably joined directly (e.g., via a direct covalent linkage, chelation, or ionic interaction), or indirectly (e.g., via one or more linkers), permitting hybridization to another nucleic acid in solution.

[0073] Vector. A plasmid or phage DNA or other DNA sequence into which DNA can be inserted to be cloned. The vector can replicate autonomously in a host cell, and can be further characterized by one or a small number of endonuclease recognition sites at which such DNA sequences can be cut in a determinable fashion and into which DNA can be inserted. The vector can further contain a marker suitable for use in the identification of cells transformed with the vector. Markers, for example, are antibiotic resistance markers such as tetracycline resistance or ampicillin resistance. The words "cloning vehicle" are sometimes used for "vector."

[0074] Purified. As used herein, the term "purified" refers to a molecule or molecules having been separated from a component of the composition in which it was originally contained. Thus, for example, a "purified protein" or a "purified nucleic" acid has been purified to a level not found in nature. A "substantially pure" molecule is a molecule that is lacking in most other components (e.g., 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% free of contaminants). By opposition, the term "crude" means molecules that have not been separated from the components of the original composition in which it was present. For the sake of brevity, the units (e.g., 66, 67...81, 82,...91, 92%....) have not been specifically recited but are considered nevertheless within the scope of the present invention.

[0075] Expression. By the term "expression" is meant the process by which a gene or otherwise nucleic acid sequence produces a polypeptide. It involves transcription of the gene into mRNA, and the translation of such mRNA into polypeptide(s). When referring to a RNA nucleic acid, the term expression relates to its translation into a polypeptide(s).

[0076] Expression Vector. A vector or vehicle similar to a cloning vector but which is capable of expressing a gene which has been cloned into it, after transformation into a host. The cloned gene (or nucleic acid sequence) is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences. [0077] Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene (or nucleic acid sequence) in a prokaryotic and/or eukaryotic host and can additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites. Vectors which can be used both in prokaryotic and eukaryotic cells are often called shuttle vectors.

[0078] Polyacrylamide Gel Electrophoresis (PAGE). The most commonly used technique (though not the only one) for achieving a fractionation of polypeptides on the basis of size is polyacrylamide gel electrophoresis. The principle of this method is that polypeptide molecules migrate through the gel as though it were a sieve that retards the movement of the largest molecules to the greatest extent and the movement of the smallest molecules to the least extent. The smaller the polypeptide fragment, the greater the mobility under electrophoresis in the polyacrylamide gel. Both before and during electrophoresis, the polypeptides typically are continuously exposed to the detergent sodium dodecyl sulfate (SDS), under which conditions the polypeptides are denatured. Native gels are run in the absence of SDS. The polypeptides fractionated by polyacrylamide gel electrophoresis can be visualized directly by a staining procedure if the number of polypeptide components is small.

[0079] Western blotting Procedure. The purpose of the Western transfer procedure (also referred to as blotting) is to physically transfer polypeptides fractionated by polyacrylamide gel electrophoresis onto a nitrocellulose filter paper or another appropriate surface or method, while retaining the relative positions of polypeptides resulting from the fractionation procedure. The blot is then probed with an antibody that specifically binds to the polypeptide of interest. [0080] CALML3 marker. A CALML3 marker is defined as any molecule whose presence in a biological sample indicates that CALML3 is expressed from the CALML3 gene. CALML3 markers include CALML3 nucleic acids (e.g., mRNA) and CALML3 proteins. A biological sample expresses CALML3 from the CALML3 gene when it contains a detectable level of CALML3 nucleic acids (e.g., CALML3 mRNA) or CALML3 protein.

[0081] As used herein the terminology "Lung specific marker" relates to any molecule whose presence in the sample indicates that such sample contains lung cells (or a marker therefrom). Therefore a "lung specific sequence" refers to a nucleic acid or protein sequence specifically found in lung cells and usually not in other tissues which could "contaminate" a particular sample used to determine the presence of the CALML3 marker. One non-limiting example of a lung specific marker is surfactant protein C (SFTPC - SEQ ID NO:18, GenBank™ accession number NM_003018.2). For certainty, when a sputum sample is used, for example, the second lung specific marker according to the present invention does not have to be solely expressed in the lung. In fact markers which are solely expressed in one organ or tissue are very rare. However, should the second lung specific marker be expressed in non-lung tissue, this non-lung tissue expression will not jeopardized the specificity of this second marker, provided that it occurs in cells of tissues or organs which are not normally present in the selected sample, sputum or other sample in which CALML3 is to be used for detection. It should be understood that while the monitoring of a second lung-specific marker provides an advantage, the invention can also be carried out by way of a detection of CALML3 and a more ubiquitous marker, enabling a better validation that a negative result for CALML3 is not a false negative, for example. In one particular embodiment, the ubiquitous marker used as a second marker is porphobilinogen deaminase (PBGD: SEQ ID NO:17, corresponding to Genbank™ accession number X04808), or 18S RNA. Of course other ubiquitous markers (housekeeping genes), well known in the art can also be used. It will also be understood that in addition to CALML3, other markers (lung specific and/or ubiquitous) could be monitored.

[0082] Thus, the present invention also relates to a detection of

CALML3 mRNA together with another mRNA sequence to obtain an internal control of mRNA amount in the sample. In one particular embodiment a normalized ratio of CALML3 mRNA over the second marker detected (lung specific or more ubiquitous,; SFTPC or PGBD, for example) provides the result as to lung cancer presence or predisposition thereto. In one particular embodiment the intensity of the second marker (or amount) enables a normalization of the intensity (or amount) of CALML3. In this particular embodiment, it is not the value or intensity of CALML3 which is compared to same between cancerous and non-cancerous samples, but the normalized ratio of CALML3/second marker. Different normalized values are used in order to select the threshold (cut-off value) normalized ratio which is the most appropriate for differentiating between cancerous and non¬ cancerous states, between good or bad prognosis, as well as for good or bad response to a particular treatment. One aim is to select the threshold value such that higher and lower levels of the ratio value obtained from the sample, as compared to the normalized ratio, enable a distinction between the most possible numbers of cancerous versus non-cancerous samples at an acceptable specificity rate. Thus, one skilled in the art would choose a particular threshold value which translates into the desired specificity and sensitivity for the test.

[0083] CALML3 antibody. As used herein, the term "CALML3 antibody" or "CALML3 specific antibody" refers to an antibody that specifically binds to (interacts with) a CALML3 protein and displays no substantial binding to other naturally occurring proteins other than the ones sharing the same antigenic determinants as the CALML3 protein. CALML3 antibodies include polyclonal, monoclonal, humanized as well as chimeric antibodies.

[0084] Control sample. By the term "control sample" or "normal sample" is meant here a sample that does not contain a specifically chosen cancer. In a particular embodiment, the control sample does not contain lung cancer or is indicative of the absence of lung cancer. Control samples can be obtained from patients/individuals not afflicted with lung cancer. Alternatively, a control sample can be taken from a non-afflicted tissue of a suspected cancer patient. Other types of control samples may also be used. For example, a lung specific marker can be used as to make sure that the sample contains lung specific cells (this marker is generally described herein as the second lung-specific marker). In a related aspect, a control reaction may be designed to control the method itself (e.g., the cell extraction, the capture, the amplification reaction or detection method, number of cells present in the sample, a combination thereof or any step which could be monitored to positively validate that the absence of a signal (e.g., the absence of CALML3 signal) is not the result of a defect in one ore more of the steps). Once a cut-off value is determined, a control sample giving a signal characteristic of said predetermined cut-off value can also be designed and used in the methods of the present invention.

[0085] Diagnosis/prognosis tests are commonly characterized by the following 4 performance indicators: sensitivity (Se), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV). The following table presents the data used in calculating the 4 performance indicators.

Table 1 :

[0086] Sensitivity corresponds to the proportion of subjects having a positive diagnostic test who truly have the disease or condition (Se=a/a+c). Specificity relates to the proportion of subjects having a negative diagnostic test and who do not have the disease or condition (Sp=d/b+d). The positive predictive value concerns the probability of actually having the disease or condition (e.g., lung cancer) when the diagnostic test is positive (PPV=a/a+b). Finally, the negative predictive value is indicative of the probability of truly not having the disease/condition when the diagnostic test is negative (NPV=c/c+d). The values are generally expressed in %. Se and Sp generally relate to the precision of the test while PPV and NPV concern its clinical utility.

[0087] Cut-off value. The cut-off value for the predisposition or presence of lung cancer is the average mean signal plus n standard deviations obtained when the level of CALML3 polypeptide, polynucleotide or fragments thereof is assessed in biological samples of patients without lung cancer. Alternatively, the cut-off value may be determined by comparing the signal obtained in a normal and a suspected cancerous sample from the same patient. For example lung biopsies from an afflicted area and from a non-afflicted area of the lung in the same patient can be compared. As mentioned earlier, cut-off values indicative of the presence or predisposition to develop lung cancer may be the same or alternatively, they may be different from each other. Since tumor markers are in many instances not solely produced by tumor cells, deriving clinical utility from a given marker often entails finding a balance between sensitivity and specificity. Such a compromise is often reached at a specific threshold « cut-off » value, which is empirically based on collected data. It should thus be understood that a person skilled in the art, to which the present invention pertains, will be able, with routine experimentation, to select a particular cut-off value based on the desired specificity. and sensitivity, the type of sample used, the preparation thereof, the stage of the cancer, the fact that a ratio is used rather than an absolute level of expression of CALML3, and other such factors described herein. More specifically, in the CALML3 case, the person of skill in the art can choose the cut¬ off value to be higher or lower than the exemplified ratio value of 4, described herein. Without specifically listing all useful lower and higher values which can be selected for CALML3/PGBD, and which are within the scope of the present invention, it should be understood that a normalized ratio of 2, 3, 5, 6, etc. could be selected by the skilled artisan in order to choose a useful level of specificity and sensitivity. It should be understood, that the level of the second marker used to obtain the ratio value is important. For example, choosing a second marker which is expressed to lower or higher levels than the non-limiting exemplified PGBD marker, would affect the normalization of the ratio and, hence the particular cut-off value of the ratio. To draw a parallel, the commonly used cut-off value of 4 ng/ml for circulating levels of PSA (prostate-specific antigen), while allowing a quite sensitive detection of prostate cancer, shows a poorer level of specificity due to elevated expression in other non-malignant conditions such as benign prostatic hyperplasia and prostatitis. Thus, a cut-off value of 10 or 20 ng/ml for circulating levels of PSA, for example could be selected in certain conditions to increase the level of specificity, while lowering that of sensitivity. For certainty, it should be clear that the skilled artisan to which the present invention pertains can select the cut-off value (whether absolute level or ratio is used) to obtain a chosen useful sensitivity and specificity.

[0088] Binding agent. A binding agent is a molecule or compound that specifically binds to or interacts with a CALML3 polypeptide. Non-limiting examples of binding agents include antibodies, interacting partners (e.g., Myosin-X), ligands (e.g., Ca²⁺), substrates and the like. It will be understood that such binding agents can be natural, recombinant or synthetic.

[0089] The revised international system for the staging of Lung cancer, based on information from a clinical database of more than 5,000 patients, was adopted in 1997 by the American Joint Committee on Cancer (AJCC) and the Union Internationale Contre Ie Cancer (UICC). Each stage is characterized by the TNM classification (T: Primary tumor; N: Regional lymph nodes; and M: distant metastasis). Each category of the TNM classification is divided into subcategories representative of its particular state. For example, primary tumors may be classified into:

Tx (cannot be assessed);

TO (no evidence of primary tumor);

Tis (carcinoma in situ);

T1 (A tumor that is < 3 cm in greatest dimension, is surrounded by lung or visceral pleura, and is without bronchoscopic evidence of invasion more proximal than the lobar bronchus);

T2 (a tumor with any of the following feature: > 3 cm; involves the main bronchus and is > 2 cm from the carina; invades the visceral pleura and associated with atelectasis or obstructive pneumonitis that extends to the hilar region but does not involve the entire lung);

T3 (Tumor of any size that invades any of the following: chest wall, diaphragm, mediastinal pleura, parietal pericardium; or tumor in the main bronchus <2 cm distal to the carina but without involvement of the carina; or, associated atelectasis or obstructive pneumonitis of the entire lung); or T4 (tumor of any size that invades any of the following: mediastinum, heart, great vessels, trachea, esophagus, vertebral body, carina; or, separate tumor nodules in the same lobe; or, tumor with a malignant pleural effusion).

[0090] Regional lymph nodes (N) and distal metastasis (M) are also divided into several categories based on their status (N: Nx, NO, N1, N2, N3 and M: Mx, MO, M1). To each cancer stage (0, IA, IB, HA, MB, HA, MIB and IV) is associated a particular combination of TNM classification. For example, Stage 0 is characterized by the following combination: Tis, NO, MO. Thus, all cancer stages are characterized by a particular combination of T, N and M. All cancer stages and TNM categories are well known in the art and are described in Lung in: American Joint Committee on Cancer: AJCC Cancer Staging Manual. 6th edition New-York, NY: Springer, 2002 pp: 167-181.

[0091] Variant. The term "variant" refers herein to a protein or nucleic acid molecule which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention, to maintain at least one of its biological activity. Thus, provided that two molecules possess a common activity and can substitute for each other, they are considered variants as that term is used herein even if the composition, or secondary, tertiary or quaternary structure of one molecule is not identical to that found in the other, or if the amino acid sequence or nucleotide sequence is not identical. More particularly, variants of the present invention differ from the polynucleotide and polypeptide sequences disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying the polypeptides and evaluating their immunogenic and/or functional activity using a number of techniques well known in the art. For more certainty a variant of CALML3, lung cancer marker or the like, refers to a protein or nucleic acid variant thereof which is substantially similar to a CALML3 sequence marker defined herein (see the definition of CALML3 marker above). When referring to a variant of the mRNA of CALML3, and especially when the portion of CALML3 mRNA which is to be detected is a non-coding region of CALML3 mRNA, the tolerance for variation can usually be higher than when a coding region of CALML3 mRNA is targeted, as well known from the evolution-based conservation of sequences.

[0092] The terminology "subsequent point in time" can be adapted by the skilled person to which the present invention pertains (e.g., the clinician), to seek its particular needs. Non-limiting factors which can influence the determination of that subsequent point in time include the gravity of the disease, familial history, environment factors (e.g., smoking habits), or other factors. Thus, in a broad sense, the terminology is meant to cover any subsequent time that might furnish clinical or other information concerning the progression of the disease, the initiation thereof, its remission, or its status quo. Non-limiting examples of subsequent time points include: following a curative or prophylactic treatment, after one, two, three or four weeks. Following two, three or four months. After half a year, one year, two years or five years. Thus, it will be clear that the subsequent time point can be adapted as seen fit, in order to monitor the level of CALML3 in time. Of course, other time points not specifically listed above are also encompassed by the broad definition of "subsequent point in time".

[0093] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of illustrative embodiments thereof, given by way of example only with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Having thus generally described the invention, reference will be made to the accompanying drawings, showing by way of illustration only an illustrative embodiment thereof and in which:

[0095] Figure 1 shows representative results of the analysis of CALML3 gene expression in lung cancer. Total RNA was extracted from 4 frozen sections of surgically removed squamous cell carcinomas of the lung. Tumor samples were paired with non-tumor (« normal ») lung tissues from the same patient. The expression of the CALML3 gene was assessed by RT-PCR analysis.

A control reaction with 18S rRNA was used to normalize for the amount of total RNA. Each sample was analyzed at two RNA concentrations by RT-PCR, and once by PCR with the highest concentration (left track of each trio). The absence of bands in PCR reactions indicated that signals did not originate from genomic DNA.

[0096] Figure 2 shows the clinical performances of CALML3 on tissues from a lung cancer tissue bank. The expression of the CALML3 gene was evaluated on about 60 sample pairs. Specific bands for CALML3 and for the control gene (18S rRNA) were quantified by densitometric scanning of gel photographs. The expression level of CALML3 was calculated as the CALML3 / 18S ratio; the positivity threshold for that ratio was arbitrarily set at zero.

[0097] Figure 3 shows diagnostic performances of CALML3 with bronchial aspirates and washings. Bronchial aspirates or washings were routinely collected for cytological examination, and duplicates of those were also obtained for the current study. Total RNA was extracted, and the expression of the CALML3 gene and the control gene PBGD was assessed by RT-PCR analysis. Specific bands were quantitated by densitometric scanning of gel photographs. Individual CALML3 signals were normalized over PBGD signals. Setting the threshold of the

CALML3/PBGD ratio at a value of 4 yielded the current performances. Results with

CALML3 were compared to those of cytology, and the combination of CALML3 with cytology was also examined. It should be clear that the present invention is not limited to a normalized ratio of 4, and that persons of skill in the art can adapt the ratio to numerous factors (e.g., to modulate the threshold of specificity and/or sensitivity, depending on the second marker used, depending on the clinical state of the patient, etc.).

[0098] Figure 4 shows the gene expression analysis of CALML3 mRNA in correlation with lung cancer tumor stage. Samples from 59 normal subjects, 11 stage 1 lung cancer patients, 31 stage 2 lung cancer patients, 3 stage 3 lung cancer patients and 5 stage 4 lung cancer patients were analyzed. The level of CALML3 expression in the samples was normalized with the expression level of the 18S ribosomal RNA.

[0099] Figure 5 shows the gene expression analysis of CALML3 mRNA in correlation with regional lymph node (RLN) status. Samples from 37 subjects with a NO RLN status, 9 subjects with a N1 RLN status and 2 subjects with N2 RLN status were analyzed. NO= no regional lymph node metastasis; N1= metastasis to ipsilateral peribronchial and/or ipsilateral hilar lymph nodes and intrapulmonary nodes, including involvement by direct extension of the primary tumor), and N2= metastasis to ipsilateral mediastinal and/or subcarinal lymph nodes. The level of

CALML3 expression in the samples was normalized with the expression level of the 18S ribosomal RNA.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

/. Synthesis of Nucleic Acid

[0100] Isolated nucleic acid molecules of the present invention are meant to include those that result from any known method, such as chemicalsynthesis. Similarly, an oligomer which corresponds to the nucleic acid molecule or to each of the divided fragments, can be synthesized. Such synthetic oligonucleotides can be prepared, for example, by the triester method of Matteucci et al., J. Am. Chem. Soc. "/03:3185-3191 (1981) or by using an automated DNA synthesizer.

[0101] An oligonucleotide can be derived synthetically or by cloning. If necessary, the 5'-ends of the oligomers can be phosphorylated using T4 polynucleotide kinase. Kinasing of single strands prior to annealing or for labeling can be achieved using an excess of the enzyme. If kinasing is for the labeling of probe, the ATP can contain high specific activity radioisotopes. Then, the DNA oligomer can be subjected to annealing and ligation with T4 ligase or the like. //. A Nucleic Acid for the Specific Detection of CALML3 Nucleic Acid

[0102] The present invention relates to a nucleic acid for the specific detection, in a sample, of the presence of CALML3 nucleic acid sequences which are associated with lung cancer, comprising the herein-described nucleic acid molecules or at least a fragment thereof which binds under stringent conditions to CALML3 nucleic acid.

[0103] In one preferred embodiment, the present invention relates to oligomers which specifically target and enable amplification (i.e., at least one primer) of CALML3 RNA sequences associated with lung cancer.

[0104] In one embodiment, the amplified product can be detected following hybridizing with a probe which hybridizes preferentially to an amplified product which originated from CALML3 RNA associated with lung cancer, but preferentially not the CALML3 gene. In embodiments, the nucleic acid probe is, or is complementary to, a nucleotide sequence consisting of at least 10 consecutive nucleotides (preferably 12, 15, 18, 20, 25, or 30) from the nucleic acid molecule comprising a polynucleotide sequence at least 90% identical to a sequence selected from the group consisting of: i) a polynucleotide according to SEQ ID NO 9 ; ii) a polynucleotide encoding CALML3 protein according to SEQ ID NO 10; iii) a polynucleotide sequence that hybridizes under high stringency conditions to the nucleotide sequence in i) or ii); and iv) a polynucleotide sequence fully complementary to i), ii) or iii).

[0105] Complementary sequences are also known as antisense nucleic acids when they comprise sequences which are complementary to the coding (+) strand. Herein, SEQ ID NO: 9 is arbitrarily defined as being the coding (+) strand.

[0106] While the present invention can be carried out without the use of a probe which targets CALML3 sequences, in accordance with the present invention, such probes can add a further specificity to the methods and kits of the present invention.

[0107] The test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids. The sample used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample which is compatible with the method utilized. In embodiments, the sample is a coughing sample, a bronchial aspirate, a bronchial washing or sputum.

///. Methods for Detecting the Presence of the CALML3 Marker in a Sample

[0108] The invention encompasses methods for detecting the presence of a CALML3 nucleic acid or CALML3 protein in a biological sample as well as methods for measuring the level of a CALML3 nucleic acid or CALML3 protein in said sample. Such methods are useful for the diagnostic and monitoring of lung cancers associated with CALML3 overexpression.

[0109] In general, the predisposition or presence of lung cancer may be detected in a patient based on the presence of an elevated amount of CALML3 proteins or polynucleotides in a biological sample obtained from a patient. In other words, CALML3 polynucleotides or polypeptides may be used as markers to indicate the presence or predisposition to develop lung cancer, preferably of Non- Small Cell Lung cancers, more preferably of squamous cell cancer subtype. Polynucleotide primers and probes may be used to detect the level of CALML3 mRNA (encoding or not CALML3 protein), which is indicative of the predisposition, presence or absence of lung cancer. Alternatively, binding agents or ligands (e.g., an antibody directed against CALML3 protein variant or alleles thereof) may be used to determine the level of CALML3 protein that binds to the agent in a biological sample. Non-limiting examples of variants include post-translationally modified proteins or differentially expressed CALML3 nucleic acids or proteins. In general, the elevated expression of a CALML3 marker in a biological sample as compared to a normal control sample indicates that the sample is from a patient who has lung cancer or is susceptible to develop lung cancer.

[0110] In one embodiment, the CALML3 marker of the present invention is a nucleic acid such as CALML3 mRNA, cDNA or native CALML3 nucleic acid, or fragment thereof associated with lung cancer. The native CALML3 nucleic acid can have the nucleotide sequence disclosed in SEQ ID NO: 9. The

CALML3 marker can also be a CALML3 protein or polypeptide such as native

CALML3 protein having the amino acid sequence of SEQ ID NO: 10. Of course, it will be understood that portions, variants or fragments of CALML3 (e.g., CALML3 polypeptides or nucleic acids) are also considered as CALML3 markers.

[0111] One non-limiting example of a method to detect CALML3 nucleic acid (e.g., CALML3 mRNA) in a biological sample is by (1) contacting a biological sample with at least one oligonucleotide probe or primer that hybridizes to a CALML3 polynucleotide; and (2) detecting in the biological sample a level of oligonucleotide (i.e., probe(s) or primer(s)) that hybridizes to the CALML3 polynucleotide. The amount of CALML3 polynucleotide detected can be compared with a predetermined cut-off value, and therefrom the predisposition, presence or absence of a lung cancer in the patient is determined. In one non-limiting embodiment in which only one primer specifically hybridizes to CALML3 is used, a second primer which binds to polyA is used. In another embodiment, the present invention relates to a method of detecting the presence of lung cancer specific CALML3 nucleic acid in a sample comprising: (1) contacting the sample with at least one of the above-described nucleic acid primers, under specific amplification conditions, and (2) detecting the presence of the amplified product. One skilled in the art would select the nucleic acid primers according to techniques known in the art as described above. In another embodiment a probe is used to identify the amplification product. Samples to be tested include but should not be limited to RNA samples from human tissue.

[0112] Within certain embodiments, the amount of mRNA may be detected via a RT-PCR based assay. In RT-PCR, the polymerase chain reaction (PCR) is applied in conjunction with reverse transcription. In such an assay, at least two oligonucleotide primers may be used to amplify a portion of CALML3 cDNA derived from a biological sample, wherein at least one oligonucleotide is specific for (i.e., hybridizes to) a polynucleotide encoding CALML3 protein. Of course, it is also possible to target a non-coding portion (or portions) of CALML3. PCA3 is a non-limiting example of a marker which can serve as a validated cancer marker, even in the portion thereof where no predicted ORF is identified (WO 98/45420). The amplified cDNA may then be separated and detected using techniques that are well known in the art such as gel electrophoresis and ethidium bromide staining. Alternatively, real-time PCR may be used to enable the direct detection of amplified prodμcts. Amplification may be performed on biological samples taken from a test patient and an individual who is not afflicted with a lung cancer (control sample), or using other types of control samples (see above). The amplification reaction may be performed on several dilutions of cDNA (or directly on several dilutions of the biological sample) spanning, for example, two orders of magnitude. A value above a predetermined cut-off value is indicative of the presence or predisposition to develop lung cancer. In general, the elevated expression of CALML3 nucleic acid in a biological sample as compared to control samples indicates the presence or, alternatively, the predisposition to develop lung cancer. [0113] In further embodiments, CALML3 mRNA (encoding CALML3 protein or not) is detected in a nucleic acid extract from a biological sample by an in vitro RNA amplification method named Nucleic Acid Sequence-Based Amplification (NASBA). Numerous other amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill. Non-limiting examples of amplification techniques include strand displacement amplification (SDA), transcription-based amplification, the Qβ replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi et al., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods MoI. Biol., 28:253-260; and Sambrook et al., 2000, supra). Other non- limiting examples of amplification methods include rolling circle amplification (RCA); signal mediated amplification of RNA technology (SMART); split complex amplification reaction (SCAR); and split promoter amplification of RNA (SPAR).

[0114] Non-limiting examples of suitable methods to detect the presence of the amplified products include the followings: agarose or polyacrylamide gel, addition of DNA labeling dye in the amplification reaction (such as ethidium bromide, picogreen, SYBER™ green, etc.), use of labeled primers/probes (e.g., by the addition of fluorescent moieties) and detection with a suitable apparatus (fluorometer in most cases). Other suitable methods include sequencing reaction (either manual or automated); restriction analysis (provided restriction sites were built into the amplified sequences), or any method involving hybridization with a sequence specific probe (Southern or Northern blot, TaqMan™ probes, molecular beacons, and the like). Of course, other amplification methods are encompassed by the present invention.

[0115] Alternatively, oligonucleotide probes that specifically hybridize under stringent conditions to a CALML3 nucleic acid may be used in a nucleic acid hybridization assay (e.g., Southern and Northern blots, dot blot, slot blot, in situ hybridization and the like) to determine the presence and/or amount of CALML3 polynucleotide in a biological sample.

[0116] In a further embodiment, oligonucleotides and primers could be designed to directly sequence and assess the presence of lung cancer specific CALML3 sequences in the patient sample following an amplification step. Such sequencing-based diagnostic methods are automatable and are encompassed by the present invention.

[0117] In one embodiment, the present invention has taken advantage of technological advances in methods for detecting and identifying nucleic acids. Therefore, the present invention is suitable for detection by any one of a number of detection methods including molecular beacons.

[0118] Molecular beacons are single-stranded oligonucleotide hybridization probes/primers that form a stem loop structure. The loop contains a probe sequence that is complementary to a target sequence, and the stem is formed by the annealing of complementary arm sequences that are located on either side of the probe/primer sequence. A fluorophore is covalently linked to the end of one arm and a quencher is covalently linked to the end of the other arm. Molecular beacons do not fluoresce when they are free in solution. However, when they hybridize to a nucleic acid strand containing a target sequence they undergo conformational change that enables them to fluoresce brightly (see US Patent 5,925,517, and 6,037,130). Molecular beacons can be used as amplicon detector probes/primers in diagnostic assays. Because nonhybridized molecular beacons are dark, it is not necessary to isolate the probe-target hybrids to determine for example, the number of amplicons synthesized during an assay. Therefore, molecular beacons simplify the manipulations that are often required when traditional detection and identifications means are used.

[0119] By using different colored fluorophores, molecular beacons can also be used in multiplex amplification assays such as assays that target the simultaneous amplification and detection of CALML3 nucleic acid and of a control sequence (e.g., SFTPC) and optionally of one or more lung cancer marker(s). The design of molecular beacons probes/primers is well known in the art and softwares dedicated to help their design are commercially available (e.g., Beacon designer from Premier Biosoft International). Molecular beacon probes/primers can be used in a variety of hybridization and amplification assays (e.g., NASBA and PCR).

[0120] In accordance with one embodiment of the present invention, the amplified product can either be directly detected using molecular beacons (or other labeled probes/primers) as primers for the amplification assay (e.g., real-time multiplex NASBA or PCR assays) or indirectly using, internal to the primer pair binding sites, a molecular beacon probe of 18 to 25 nucleotides long (e.g., 18, 19, 20, 21, 22, 23, 24, 25) which specifically hybridizes to the amplification product. Labeled probes or primers having a length comprised between 18 and 25 nucleotides are preferred when used according to the present invention (Tyagi et al., 1996, Nature Biotechnol. 14: 303-308). Shorter fragments could result in a less fluorescent signal, whereas longer fragments often do not increase significantly the signal. Of course shorter or longer probes and primers could nevertheless be used.

[0121] In a related embodiment, probes and primers of the present invention may be detectably labeled by other methods than molecular beacons. A label or reporter group, as generally understood and used herein, means a molecule, which provides directly or indirectly a detectable signal. Various labels may be employed such as radiolabels (³²P, ³H, ¹⁴C, ³⁵S etc.), biotinylated derivatives, enzymes (e.g., alcaline phosphatase, horseradish peroxidase) or fluorescent moieties, (e.g., molecular beacons). The CALML3 mRNA or cDNA may also be detected using colorimetric methods. It should be understood that numerous fluorescent, colorimetric or enzymatic methods may be used in A2005/001760

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accordance with the present invention to detect and/or quantify CALML3 mRNAs.

[0122] As commonly known, radioactive nucleotides can be incorporated into probes of the invention by several methods. Non-limiting examples thereof include kinasing the 5^! ends of the probes using gamma ³²P ATP and polynucleotide kinase, using the Klenow fragment of Pol I of E. coli in the presence of radioactive dNTP (e.g., uniformly labeled DNA probe using random oligonucleotide primers), using the SP6/T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and the like.

[0123] In one embodiment, the label used in the present invention in a homogenous detection assay is a chemiluminescent compound (e.g., U.S. Pat. Nos. 5,656,207, 5,658,737 and 5,639,604), in another an acridinium ester ("AE") compound, such as standard AE or derivatives thereof. Methods of attaching labels to nucleic acids and detecting labels are well known (e.g., see Sambrook et al., (cf., Molecular Cloning: A Laboratory Manual, Third Edition, edited by Cold Spring Harbor Laboratory, 2000), Chapt. 10; U.S. Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174 and 4,581,333; and European Pat. App. No. 0 747 706). Preferred methods of labeling a probe with an AE compound attached via a linker have been previously described in detail (e.g., see U.S. Pat. No 5,639,604, Example 8).

[0124] To enable hybridization to occur under the assay conditions of the present invention, oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least 70% (at least 71%, 72%, 73%, 74%), preferably at least 75% (75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%) and more preferably at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identity to a portion of a CALML3 polynucleotide. Probes and primers of the present invention are those that hybridize to CALML3 nucleic acid (e.g., cDNA or mRNA) sequence (SEQ ID NO: 9) under stringent hybridization conditions and those that hybridize to CALML3 gene homologs under at least moderately stringent conditions. In certain embodiments probes and primers of the present invention have complete sequence identity to CALML3 gene sequence (e.g., cDNA or mRNA). However, probes and primers differing from the native CALML3 gene sequence that keep the ability to hybridize to native CALML3 gene sequence under stringent conditions may also be used in the present invention. It should be understood that other probes and primers could be easily designed and used in the present invention based on the CALML3 nucleic acid sequence disclosed herein by using methods of computer alignment and sequence analysis known in the art (cf., Molecular Cloning: A Laboratory Manual, Third Edition, edited by Cold Spring Harbor Laboratory, 2000).

[0125] In a preferred embodiment, the oligonucleotide probes and primers of the present invention comprise at least 10 contiguous nucleotides (preferably, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32) of a CALML3 nucleic acid molecule or its complementary sequence. Longer probes and primers are also within the scope of the present invention as well known in the art. Primers having more than 30, more than 40, more than 50 nucleotides and probes having more than 100, more than 200, more than 300, more than 500 more than 800 and more than 1000 nucleotides in length are also covered by the present invention. As is well known in the art, probes ranging from 20 to more than 2000 nucleotides in length can be used in the methods of the present invention. Thus, for example, probes consisting of 10 to 1000 nucleotides, 10 to 400, 10 to 100, 10 to 50, 10 to 35, 20 to 1000, 20 to 400, 15 to 100, 15 to 200, 15 to 350, 15 to 500, 15 to 600, 20 to 100, 20 to 50, or 20 to 35, which hybridize to a CALML3 nucleic acid are within the scope of the present invention. As described when referring to the % of identity described above, non- specifically described sizes and ranges of sizes of probes and primers (e.g., 16, 17, 31, 24, 39, 350, 450, 550, 900, 1240 nucleotides) are also within the scope of the present invention.

[0126] Probes and primers of the present invention are designed in order to specifically hybridize to CALML3 polynucleotides. Preferably, probes and primers of the present invention are designed in order to specifically hybridize to CALML3 polynucleotides over other calmodulin related sequences. Thus these probes and primers can be referred to generally as CALML3 specific probes or primers.

[0127] For examples, probes and/or primers that may be used in accordance with the present invention are presented in Table 2.

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Table 2: Examples of CALML3 nucleic acid sequences from which primers and probes of variable length can be derived.

SEQ Number # Size Position according to SEQ ID NO: 9

(NM__005185)

SEQ NO: 1 22 350-371

SEQ NO: 2 22 531-552

SEQ NO: 5 20 1172-1191

SEQ NO: 6 23 1296-1302ⁿ

SEQ NO: 11 24 162-185

SEQ NO: 12 24 764-787

SEQ NO: 13 24 697-720

SEQ NO:14 24 1228-1251

SEQ NO 15 39 751-787

SEQ NO 16 32 271-302 the 7 last nucleotides plus 16 first A's of polyA tail

[0128] Since the CALML3 gene is intronless, primers and probes of the present invention, specific for CALML3, may be designed in order to distinguish between genomic DNA and mRNA (or its corresponding cDNA). For example, a primer with specificity for the 3' extremity of the CALML3 mRNA, including the polyA tail, which is absent from the genomic sequence may be used in accordance with the present invention. Alternatively, a prior DNAse treatment or purification of mRNAs from other types of nucleic acids contained in a sample may be used. Of course, when amplifying control sequences which contain at least one intron, primers and probes spanning intron/exon junctions may be used in order to distinguish between genomic DNA and mRNA amplification. Alternatively or additionally an amplification reaction without the prior reverse transcription of mRNA may be used in order to control for the amplification of genomic DNA.

[0129] In a related aspect, the present invention provides methods for monitoring the progression of a lung cancer in a patient comprising in one embodiment the steps of: (1) contacting a biological sample from a lung cancer patient with at least one oligonucleotide (probe(s) or primer(s)) that hybridize to a CALML3 polynucleotide; (2) detecting in the biological sample a level of oligonucleotide (probe(s) or primer(s)) that hybridize to a CALML3 polynucleotide; (3) repeating steps (1) to (2) using a biological sample from the patient at a subsequent point in time; and (4) comparing the relative amount (i.e., relatively to the amount of cells or cell components (e.g., protein or nucleic acids present therein)) of polynucleotide detected in step (3) with the relative amount detected in step (2) wherein an increase in the amount of CALML3 is indicative of a progression of the lung cancer in the patient. In another embodiment, the present invention relates to a method of monitoring the progression of a lung cancer in a patient comprising: (1) contacting a biological sample from a lung cancer patient with at least one of the above-described nucleic acid primers, under specific amplification conditions; (2) detecting the presence of the amplified product; (3) repeating step (1) to (2) using a biological sample from the patient at a subsequent point in time; and (4) comparing the relative amount (i.e., relatively to the amount of cells or cell components (e.g., protein or nucleic acids present therein)) of polynucleotide detected in step (3) with the relative amount detected in step (2), wherein an increase in the amount of CALML3 over time is indicative of a progression of the lung cancer in the patient. As mentioned above, the amount of CALML3 nucleic acid may be detected via hybridization procedures or any nucleic acid sequence amplification assay well known in the art. Non-limiting examples include Southern, Northern, slot blots, dot blots, in situ hybridization, RT-PCR based assay (standard or real time), NASBA (standard or real time), ligase chain reaction, transcriptase-mediated amplification, strand displacement amplification 2005/001760

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(SDA) and, the Qβ replicase system and the like. It will be understood that the monitoring of a level of CALML3 over time enables a monitoring of the effect of a chosen therapeutic regimen that a patient is undergoing.

[0130] In a further embodiment, cells contained in a biological test sample are harvested and lysed in a lysis buffer. Nucleic acids are extracted (e.g., from the lysate by solid phase extraction on silica beads, for example). Detection of the presence of RNA encoded by the CALML3 gene in the nucleic acid extract is done by an in vitro specific RNA amplification coupled to real-time detection of amplified products by fluorescent specific probes. In this method, simultaneously to the amplification of the CALML3 lung cancer specific RNA undergoes the amplification of the second lung-specific marker (e.g., SFTPC) as a control for the presence of lung cells in the sample.

[0131] The screening, diagnostic, prognostic and theranostic methods of the invention do not require that the entire CALML3 RNA sequence be detected. Rather, it is only necessary to detect a fragment or length of nucleic acid that is sufficient to detect the presence of the CALML3 nucleic acid from a normal or affected individual, the absence of such nucleic acid, or an altered structure of such nucleic acid (such as an aberrant splicing pattern). For this purpose, any of the probes or primers as described above may be used, and many more can be designed as conventionally known in the art based on the sequences described herein and others known in the art.

[0132] In one embodiment an internal control is included in the methods of the present invention. It is possible to verify the efficiency of nucleic acid amplification and/or detection only, by performing external control reaction(s) using highly purified control target nucleic acids added to the amplification and/or detection reaction mixture. Alternatively, the efficiency of nucleic acid recovery from cells and/or organelles, the level of nucleic acid amplification and/or detection inhibition (if present) can be verified and estimated by adding to each test sample control cells or organelles (e.g., a define number of cells from a lung cancer cell line expressing CALML3 and/or second lung specific marker) by comparison with external control reaction(s). To verify the efficiency of both, sample preparation and amplification and/or detection, such external control reaction(s) may be performed using a reference test sample or a blank sample spiked with cells, organelles and/or viral particles carrying the control nucleic acid sequence(s). For example, a signal from the internal control (IC) sequences present into the cells, viruses and/or organelles added to each test sample that is lower than the signal observed with the external control reaction(s) may be explained by incomplete lysis and/or inhibition of the amplification and/or detection processes for a given test sample. On the other hand, a signal from the IC sequences that is similar to the signal observed with the external control reaction(s), would confirm that the sample preparation including cell lysis is efficient and that there is no significant inhibition of the amplification and/or detection processes for a given test sample. Alternatively, verification of the efficiency of sample preparation only may be performed using external control(s) analyzed by methods other than nucleic acid testing (e.g., analysis using microscopy, mass spectrometry or immunological assays).

[0133] Therefore, in one particular embodiment, the methods of the present invention use purified nucleic acids, lung cells or viral particles containing nucleic acid sequences serving as targets for an internal control (IC) in nucleic acid test assays to verify the efficiency of cell lysis and of sample preparation as well as the performance of nucleic acid amplification and/or detection. More broadly, the IC serves to verify any chosen step of the process of the present invention.

[0134] IC in PCR or related amplification techniques can be highly purified plasmid DNA either supercoiled, or linearized by digestion with a restriction endonuclease and repurified. Supercoiled IC templates are amplified much less A2005/001760

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efficiently (about 100 fold) and in a less reproducible manner than linearized and repurified IC nucleic acid templates. Consequently, IC controls for amplification and detection of the present invention are preferably performed with linearized and repurified IC nucleic acid templates when such types of IC are used.

[0135] The nucleic acids, cells, and/or organelles are incorporated into each test sample at the appropriate concentration to obtain an efficient and reproducible amplification/detection of the IC, based on testing during the assay optimization. The optimal number of control cells (or nucleic acids or organelles) added, which is dependent on the assay, is preferentially the minimal number of cells (or nucleic acids or organelles) which allows a highly reproducible IC detection signal without having any significant detrimental effect on the amplification and/or detection of the other genetic target(s) of the nucleic acid- based assay. A sample to which is added the purified linearized nucleic acids, cells, viral particles or organelles is generally referred to as a "spiked sample".

[0136] According to the invention, presymptomatic screening of an individual in need of such screening is now possible using DNA encoding the CALML3 protein or the CALML3 gene of the invention or fragments thereof. The screening method of the invention allows a presymptomatic diagnosis, including prenatal diagnosis, of the presence of an aberrantly expressed CALML3 gene in individuals, and thus an opinion concerning the likelihood that such individual would develop or has developed a CALML3-associated disease. Early diagnosis is also desired to maximize appropriate timely intervention.

[0137] Lung cancer (or a predisposition thereto) may also be detected based on the level of CALML3 proteins or polypeptides present in a sample. Therefore, the present invention also relates to diagnosing lung cancer in a patient or determining whether a patient has a predisposition to lung cancer by detecting a CALML3 polypeptide (directly or indirectly) in a sample from that patient. Accordingly, the present invention provides methods, both direct and indirect, of detecting CALML3 proteins or polypeptides in a sample. One non-limiting method to detect CALML3 polypeptide in a biological sample comprises: (1) contacting a biological sample with a CALML3 specific binding agent (e.g., an antibody or other polypeptide or ligand that specifically recognizes/interacts with CALML3 protein), (2) indirectly or directly detecting and quantifying the presence of CALML3 polypeptide; and (3) comparing the level of CALML3 polypeptide with that of a predetermined cut-off value (control value). Since tumor markers are in many instances not solely produced by tumor cells, deriving clinical utility from a given marker often entails finding a balance between sensitivity and specificity. Such a compromise is often reached at a specific threshold « cut-off » value, which is empirically based on collected data. For example, the commonly used cut-off value of 4 ng/ml for circulating levels of PSA (prostate-specific antigen), while allowing a quite sensitive detection of prostate cancer, shows poor sensitivity due to elevated expression in other non-malignant conditions such as benign prostatic hyperplasia and prostatitis. It should thus be understood that a person skilled in the art, to which the present invention pertains, will be able, without undue routine experimentation, to select a particular cut-off value based on the desired specificity and sensitivity, the type of sample used, the preparation thereof, the stage of the cancer, and other such factors described herein. In detail, in a certain embodiment, the method can comprise incubating a biological test sample with one or more of the antibodies that specifically recognizes CALML3 polypeptides and assaying whether the antibody binds to the test sample. A value above the predetermined cut-off value is indicative of the presence or predisposition to develop lung cancer. In general, the elevated expression of CALML3 in a biological sample as compared to control samples indicates the presence or alternatively the predisposition to develop lung cancer.

[0138] In a further embodiment, the present invention relates to a method of detecting a CALML3 antibody in a biological sample, comprising: (1) contacting the biological sample with a CALML3 protein (or suspected of containing same), under conditions such that immunocomplexes form; and (2) detecting the presence of the protein bound to the antibody or antibody bound to the protein. In detail, such an embodiment of the method comprises incubating a biological test sample with CALML3 polypeptides and assaying whether the antibody binds to the test sample.

[0139] In general, techniques for preparing antibodies (including monoclonal antibodies and hybridomas) and for detecting antigens using antibodies are well known in the art (Howard et Bethell, Basic methods in Antibody production and characterization, Interpharm/CRC press, Boca Raton FL, (2000)), Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1999). In the methods of the present invention, polyclonal, monoclonal antibodies, or humanized versions thereof, chimeric antibodies and the like may be used. The invention further includes single chain antibodies. Antibody fragments which contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')₂ fragment; the Fab' fragments, Fab fragments, and Fv fragments.

[0140] Humanized antibodies can be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non- immunogenic portion (i.e., chimeric antibodies) (Robinson, R. R. et al., International Patent Publication PCT/US86/02269; Akira, K. et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171 ,496; Morrison, S. L. et al., European Patent Application 173,494; Neuberger, M.S. et al., PCT Application WO 86/01533; Cabilly, S. et al., European Patent Application 125,023; Better, M. et al., Science 240:1041-1043 (1988); Liu, A.Y. et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Liu, A.Y. et al., J. Immunol. 739:3521-3526 (1987); Sun, L.K. et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Nishimura, Y. et al., Cane. Res. 47:999-1005 (1987); Wood, CR. et al., Nature 374:446-449 (1985)); T/CA2005/001760

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Shaw et al., J. Natl.Cancer Inst. 80:1553-1559 (1988). General reviews of "humanized" chimeric antibodies are provided by Morrison, S. L. (Science, 229:1202-1207 (1985)) and by Oi, VT. etal., BioTechniques 4:214 (1986)). Suitable "humanized" antibodies can be alternatively produced by CDR or CEA substitution (Jones, PT. et al., Nature 321:552-525 (1986); Verhoeyan et al., Science 239:1534 (1988); Beidler, CB. et al., J. Immunol. 141:4053-4060 (1988)).

[0141] Any animal (mouse, rabbit, and the like) that is known to produce antibodies can be immunized with the selected polypeptide. Methods for immunization are well known in the art. Such methods include subcutaneous or interperitoneal injection of the polypeptide. One skilled in the art will recognize that the amount of polypeptide used for immunization will vary based on the animal which is immunized, the antigenicity of the polypeptide and the site of injection.

[0142] The polypeptide can be modified or administered in an adjuvant in order to increase the peptide antigenicity. Methods of increasing the antigenicity of a polypeptide are well known in the art. Such procedures include coupling the antigen with a heterologous protein (such as globulin or /?-galactosidase) or through the inclusion of an adjuvant during immunization.

[0143] For monoclonal antibodies, spleen cells from the immunized animals are removed, fused with myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells. Any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al., Exp. Cell Res. -/75:109-124 (1988)).

[0144] Hybridomas secreting the desired antibodies are cloned and the class and subclass is determined using procedures known in the art (Campbell, Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, supra (1984)).

[0145] For polyclonal antibodies, antisera containing antibody is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures.

[0146] Furthermore, one skilled in the art can readily adapt currently available procedures, as well as the techniques, methods and kits disclosed above with regard to antibodies, to generate peptides capable of binding to a specific peptide sequence in order to generate rationally designed antipeptide peptides, for example see Hurby et al., "Application of Synthetic Peptides: Antisense Peptides", In Synthetic Peptides, A User's Guide, W.H. Freeman, NY, pp. 289-307 (1992), and Kaspczak et al., Biochemistry 28:9230-8 (1989).

[0147] The anti-peptide peptides can be generated for example by replacing the basic amino acid residues found in the CALML3 peptide sequence with acidic residues, while maintaining hydrophobic and uncharged polar groups. For example, lysine, arginine, and/or histidine residues are replaced with aspartic acid or glutamic acid and glutamic acid residues are replaced by lysine, arginine or histidine.

[0148] Conditions for incubating an antibody with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the antibody used in the assay. One skilled in the art will recognize that any one of the commonly available immunological assay formats (such as radioimmunoassays, enzyme-linked immunosorbent assays, diffusion based Ouchterlony, immunoprecipitation, western blot or rocket immunofluorescent assays) can readily be adapted to detect the CALML3 protein of the present invention. Examples of such assays can be found in Chard, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock et al., Techniques in Immunocytochemistry, Academic Press, Orlando, FL (1997); Tijssen, Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985); Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New-York (1997).

[0149] In one embodiment, the assay involves the use of a binding agent immobilized on a solid support to bind and remove the polypeptide from the remainder of the biological sample. The bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex. Such detection reagents includes, for example, a binding agent that specifically binds to the CALML3 polypeptide or an antibody or other agents that specifically binds to the binding agent, such as an antiimmunoglobulin, protein A, protein G or lectin. Alternatively, the binding agent (e.g., an antibody or other) may be directly labeled.

[0150] In another embodiment, a competitive assay may be used. In such assay, the CALML3 polypeptide is labeled and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide (e.g., CALML3 polypeptides) to the binding agent (e.g., an antibody recognizing specifically CALML3 polypeptides) is indicative of the reactivity of the sample with the immobilized binding agent. Suitable polypeptides for use in such assays include full length CALML3 protein or fragment thereof to which the binding agent binds.

[0151] The labels of the present invention can be any detectable label such as radioisotopes, affinity labels (such as biotin, avidin, and the like), enzymatic labels (such as horse radish peroxidase, alkaline phosphatase, and the like) fluorescent labels (such as FITC or rhodamine, and the like), paramagnetic atoms, and the like. Procedures for accomplishing such labeling are well known in the art, for example, see Sternberger et al., J. Histochem. Cytochem. 18:315 (1970); Bayer et al., Meth. Enzym. 62:308 (1979); Engval et al., Immunol. 109:129 (1972); Goding, J. Immunol. Meth. -/3:215 (1976). Such labels may be attached to proteins as well as to nucleotides, as well known in the art.

[0152] The solid support used in the above-described assay may be any material known to those of ordinary skill in the art to which CALML3 protein or binding agent (e.g., an antibody that specifically binds to CALML3 protein or fragments thereof) may be immobilized. Examples of such solid supports include glass, fiberglass, plastics such as polycarbonate, polystyrene or polyvinylchloride, complex carbohydrates such as agarose and sepharose, acrylic resins such as polyacrylamide and latex beads. Other suitable solid supports include microtiter plates, magnetic particles or a nitrocellulose or other membranes. Techniques for coupling antibodies to such solid supports are well known in the art (Weir et al., "Handbook of Experimental Immunology" 5th Ed., Blackwell Scientific Publications, Oxford, England, (1996); Jacoby et al., Meth. Enzym. 34 Academic Press, N.Y. (1974)).

[0153] In the context of the above-described assay, the term immobilization refers to both non-covalent association, such as- adsorption and, covalent association (e.g., a direct linkage between the CALML3 protein or binding agent and functional groups on the solid support or indirect linkage via a cross linking agent).

[0154] To assess the presence, predisposition, absence or progression of lung cancer, the signal detected from the reporter group or label is generally compared to the signal that corresponds to a predetermined cut-off value. In one embodiment, the cut-off value for the detection of lung cancer is the average mean signal plus n standard deviations obtained when the binding agent (CALML3 antibody) is incubated with samples of patients without cancer (or in the case of a biopsy, an unaffected area of the lung from the patient). In general, a sample generating a signal that is higher than the cut-off value is considered as containing lung cancer. Alternatively, a normal (i.e., control) and an afflicted biological sample obtained from the same patient may also be compared to determine a predisposition, presence, or absence of lung cancer.

[0155] As will be recognized, numerous types of immunoassays are available for use in the present invention. For instance, direct or indirect binding assays, competitive assays, sandwich assays can readily be used in the context of the present invention. Other techniques for detection of a CALML3 protein include immunofluorescence, Western blotting, immunoprecipitation and ELISAs (enzyme- linked immunosorbant assays). These immunoassays are well known in the art and described in numerous publications.

[0156] In a related aspect, CALML3 polypeptides may be used as markers for the progression of cancer. In this embodiment, the assays described above are performed over time and the change in the level of CALML3 polypeptides present in a biological sample is evaluated. In general, a cancer is considered progressing if the relative level (i.e., relative to the amount of cells or cell components (e.g., protein or nucleic acids present therein)) of polypeptide detected increases with time. In contrast, the cancer is not progressing (e.g., regressing or being stable) when the relative level of CALML3 polypeptide either decreases or remains constant over time. In another embodiment, antibodies that specifically bind to CALML3 protein, or fragments thereof, which are present in a biological sample of a suspected lung cancer patient may also be used as markers for the progression of lung cancer. To improve sensitivity, the above described assays for CALML3 polynucleotides, polypeptides or antibodies may be combined with assays for other known lung tumor markers such as CEA, NSE, CYFRA 21. Therefore, multiple lung tumor markers (polynucleotides or polypeptides or a combination thereof) may be assayed within a given biological test sample. In such assays, multiple binding agents, probes and primers specific for different polypeptides or polynucleotides may be combined in a single assay. The selection of tumor markers to be assayed may be based on routine experiments to determine the combination that results in optimal sensitivity. Furthermore, the detection of a lung cell marker may be used in the methods of the present invention to control for the presence of lung cells in a sample. Internal controls IC may also be included in the method of the present invention to verify any chosen step of the process (e.g., number of cells or portions thereof (e.g., organelles) present in the sample, cell extraction methods, amplification or hybridization methods, detection methods etc). Of course, the detection of other lung tumor markers or lung cell marker can be measured separately from that of CALML3.

[0157] In the above described methods, biological and kits the terminology "sample", "biological sample", clinical sample" and the like is meant to include any tissue or material derived from a living or dead human which may contain the CALML3 target nucleic acid or protein. Non limiting examples of samples include any tissue or material that may contain cells expressing the CALML3 target or contain CALML3 nucleic acid or protein such as blood or fraction thereof, lung biopsies, bronchial aspiration, bronchoalveolar lavage, brushing, sputum, saliva or coughing samples from test patients (suspected cancer patients and control patients) or other body fluids or tissue that might be tested for CALML3 expression. In one embodiment the sample is a bronchial aspirate. In another embodiment, the biological sample of the present invention is a crude sample (i.e., unpurified). In another embodiment, the biological sample is semi- purified or substantially purified (e.g., a nucleic acid extract). The biological sample may be treated to physically disrupt tissue or cell structure, thus releasing intracellular components into a solution which may further contain enzymes, buffers, salts, detergents, and the like which are used to prepare the sample for analysis. Biological samples to be tested include but should not be limited to samples from mammalian (e.g., human) or any other sources. Of course, human samples are preferred biological samples in accordance with the present invention.

[0158] In one particularly preferred embodiment, the clinical sample from the patient is not obtained through an invasive method. Non-limiting examples of such clinical samples include bronchial aspiration, bronchoalveolar lavage, brushing, sputum, saliva or coughing samples from a patient. The terminology "sputum" is meant to cover an expectoration from a patient. It usually is a mostly liquid, non-homogenous and viscous sample comprising saliva and discharges from the respiratory passages, such as mucus. The terminology "aerosol", "cough sample", "coughing sample" and the like, are used herein to refer to a clinical sample which is relatively dry (i.e., aerosol) and comprises cellular elements and cells which are present in the pulmonary air. Multiple techniques can be performed during lung bronchoscopy in order to collect samples that will be used for diagnostic, prognostic or theranostic purposes. Bronchial aspiration refers to a technique, which is a standard part of the bronchoscopy procedure aimed at improving visibility, whereby the fluid present in the lungs is simply aspirated and collected. Bronchial lavage or washing refers to the aspiration of the fluid present in the lungs following addition of a small quantity (e.g., about 20 ml) of a suitable fluid (e.g., saline). Bronchoalveolar lavage or washing is a more specific technique that involves the addition of a saline fluid (or other suitable fluids) and a wedging of the bronchoscope into more distal segments of the lung before aspirating and collecting the fluid. Brushing of an abnormal area visualized through the bronchoscope is another standard technique that may produce a higher cell yield than bronchial aspiration, which might be advantageous under certain conditions. In one embodiment the clinical sample is obtained via a bronchoalveolar lavage (BAL), which is also referred to as a "liquid biopsy" of the distal airways and alveoli. In BAL, the quantity of fluid used is generally larger than for a bronchial lavage (e.g., about 400 ml). The supernatant fluid or the cell pellet of the BAL can be used in the diagnosis assay (see for example The Merck Manual 6^th Edition, page 625, for a general textbook on the collection of clinical samples that can be used in accordance with the present invention). Numerous clinical textbook and articles exist and are well known in the art concerning means of obtaining clinical samples and treatment thereof prior to use in the molecular diagnosis or cytology methods of the present invention. One non-limiting example of such article is Funahashi et al., 1979 (Chest 76: 514-517).

IV. A Diagnostic Kit for Detecting the Presence of CALML3 Nucleic Acid in a Sample

[0159] In another embodiment, the present invention relates to a kit for detecting the presence of CALML3 nucleic acid in a sample. Such kit generally comprises a first container means having disposed therein at least one oligonucleotide probe or primer that hybridizes to a nucleic acid encoding CALML3 protein. The kit may further include oligonucleotide probes or primers specific for the amplification and/or detection of a lung specific marker such as SFTPC (e.g., SEQ ID NOs:19 and 20) or other sequences enabling the normalization of CALML3 expression levels (e.g., 18S RNA, GAPDH, actin, internal control or other housekeeping genes well known in the art).

[0160] Oligonucleotides probes or primers of the present kit may be used, for example, within a NASBA, PCR or hybridization assay. In a preferred embodiment, the kit further includes other containers comprising additional components such as a second oligonucleotide or primer and/or one or more of the following: buffers, reagents to be used in the assay (e.g., wash reagents, polymerases or else) and reagents capable of detecting the presence of bound nucleic acid probe. Examples of detection reagents include, but are not limited to radiolabeled probes, enzymatic labeled probes (horse radish peroxidase, alkaline phosphatase), fluorescent probes rhodamine (ROX), fluoresceine (FAM), or the like, and affinity labeled probes (biotin, avidin, or steptavidin). In one embodiment, the detection reagents are molecular beacon probes which specifically hybridize to the amplification products. In another embodiment, the detection reagents are chemiluminescent compounds such as Acridinium Ester (AE).

[0161] For example, a compartmentalized kit of the present invention includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include for example, a container which will accept the test sample, a container which contains the probe or primers used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, and the like), and containers which contain the reagents used to detect the hybridized probe, bound antibody, amplified product, or the like. As mentioned above, the separation or combination of reagents can be adapted by the person of ordinary skill to which this invention pertain, according to the type of kit which is preferred (e.g., a diagnostic kit based on amplification or hybridization methods or both), the types of reagents used and their stability or trther intrinsic properties. In one embodiment, one container contains the amplification reagents and a separate container contains the detection reagent. In another embodiment, amplification and detection reagents are contained in the same container. Of course it will be understood that numerous permutations of primers and reagents in different containers can be designed by a person skilled in the art.

[0162] One skilled in the art will readily recognize that the nucleic acid probes and primers described in the present invention can readily be incorporated into one of the established kit formats which are well known in the art. [0163] Kits may also contain oligonucleotides that serve as capture oligomers for purifying the target nucleic acids from a sample. Examples of capture oligomers have sequences of at least 15 nucleotides complementary to a portion of the CALML3 target nucleic acid. Embodiments of capture oligomers may have additional bases attached to a 3¹ or 5' end the sequence that is complementary to the CALML3 target sequence which may act functionally in a hybridization step for capturing the target nucleic acid. Such additional sequences are preferably a homopolymeric tail sequence, such as a poly-A or poly-T sequence, although other embodiments of tail sequences are included in capture oligomers of the present invention. In one embodiment, CAP binding protein (e.g., elF4G or elF4E) or part thereof may be used to capture cap-structure containing mRNAs (Edery et al., 1987, Gene 74(2): 517-525). In another embodiment, a non¬ specific capture reagent is used (e.g., silica beads). Capture oligomers or other purifying methods which target a lung specific marker (e.g., SFTPC) may also be included in kits of the present invention.

[0164] Kits useful for practicing the methods of the present invention may include those that comprise any of the amplification oligonucleotides and/or detection probes disclosed herein which are packaged in combination with each other. Kits may also include capture oligomers for purifying the CALML3 target nucleic acid from a sample, which capture oligomers may be packaged in combination with the amplification oligonucleotides and/or detection probes. Finally, the kits may further include instructions for practicing the diagnostic, theranostic and/or prognostic methods of the present invention. Such instructions can concern details relating to the experimental protocol as well as to the cut-off values that may be used.

V. A Diagnostic Kit Comprising CALML3 Polypeptide or Antibody.

[0165] In another embodiment of the present invention, a kit is provided which contains all the necessary reagents to carry out the previously described methods of detection.

[0166] The kit can comprise: (1) a first container means containing a

CALML3 specific antibody or fragment thereof, and (2) a second container means containing a conjugate comprising a binding partner of the antibody or fragment thereof and a label. As with diagnostic kits for detecting the presence of CALML3 mRNA in a sample, the kit can further include antibodies for detecting the presence of a lung specific marker (e.g., SFTPC) or antibodies specific for housekeeping genes (e.g., GAPDH) which enable the normalization of CALML3 expression levels.

[0167] The kit can also comprise: (1) a first container means containing CALML3 protein or fragments thereof, and in one embodiment, (2) a second container means containing a conjugate comprising a binding partner of the CALML3 protein (or fragments thereof) and a label. More specifically, a diagnostic kit comprises CALML3 protein as described above, to detect antibodies in the serum of potentially infected animals or humans.

[0168] In another embodiment, the kit further comprises one or more other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound antibodies. Examples of detection reagents include, but are not limited to, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the chromophoric, enzymatic, or antibody binding reagents, which are capable of reacting with the labeled antibody. The compartmentalized kit can be as described above for nucleic acid probe kits.

[0169] Further descriptive embodiments of the present invention will be more specifically illustrated in the examples herein below. These embodiments are described as examples only and should not be considered as limitative. EXAMPLE 1

ATTEMPTS AT VALIDATING MARKERS HAVING BEEN IDENTIFIED AS CANCER SPECIFIC. BY MOLECULAR PROFILING

[0170] The collagen-11 alpha-1 gene (Wang et al., 2002, Oncogene 21: 7598-7604; and WO194629), a marker having been associated with cancer, by the use of multiple arrays, was tested herein. The collagen-11 alpha-1 gene had been reported to be overexpressed in most non-small cell lung carcinomas.

However, collagen-11 alpha-1 gene sequences could not be detected in RNA purified from bronchial aspirate of 6 patients, five of which having been diagnosed with non-small cell lung carcinoma (data not shown). Conversely, 3 out of 10 samples from patients with non-cancerous pathologies were positive for that marker (data not shown).

[0171] SIM2 has been described as differentially expressed in cancer.

Recent publications from Narayanan's group (US Patent Application 2002/00816113, De Young et al., 2002, and De Young et al., 2003) describe the SIM2 (Single Minded 2) gene, previously identified by Chrast et al., (1997) as being a good tumor marker specifically in the case of colon, pancreas and prostate cancers. Further WO04012847, associates SIM2 to lung cancer. The applicant of the instant invention confirmed quantitatively the differential expression of SIM2 in lung cancer, using frozen tissue sections (data not shown). However, while the applicant confirmed with RNA from frozen tissue sections that the long transcript of the SIM2 gene (Genbank™ U80456) was overexpressed in lung cancer (data not shown), it failed to demonstrate overexpression of that transcript in bronchial aspirates or washings from cancerous versus non-cancerous patients (data not shown).

[0172] It follows that data from molecular profiling that potentially identifies a marker as being associated with cancer often does not predict the association of this marker in clinical specimens or in a clinical situation (as exposed above).

EXAMPLE 2

SPECIFIC RNA EXPRESSION OF CALML3 IN LUNG CANCER [0173] Approximately sixty pairs of frozen specimens from lung cancer surgery were selected from a tissue bank at a nearby hospital. Each pair consisted of a sample of non-small cell lung carcinoma and a sample of normal-appearing (non-tumor) lung specimen from the same patient. For each sample, ten 20- micrometer sections were cut, immediately homogenized in Trizol™ (from Invitrogen), and kept at 4⁰C until purification of the RNA. A single supplementary section was put on a slide and stained with hematoxylin and eosin according to standard methods. Prior acceptance from the patients was obtained before actually accessing to the sample. In all cases diagnosis of both tumor and non- tumor samples was confirmed independently by an experienced pathologist.

[0174] Total RNA was purified from the tissue homogenates according to the manufacturer of Trizol™, and those samples were thereafter treated with DNase. No direct quantification of total RNA was attempted, but the amount of purified material was estimated on the basis of the band intensity of RT-PCR products run on ethidium bromide-stained agarose gels (below).

[0175] The presence of the Calmodulin-like 3 (CALML3) mRNA was assessed by RT-PCR analysis, using the OneStep™ RT-PCR kit from Qiagen. The sequence of forward and reverse primers is given below (SEQ ID NO: 1 and SEQ ID NO: 2 respectively). The amplification program was as follows: After a 30 min RT (room temperature) reaction at 5O⁰C, the reactions were heated at 95⁰C for 15 min, then submitted to 27-35 cycles of amplification (1 min at 94°C / 1 min at 58⁰C / 1 min at 72⁰C). The actual number of cycles was set according to results with 5 001760

68

previous 18S control reactions (details below). After the thermal cycling steps, tubes were heated 10 min at 72⁰C for final extension. The expected size for the RT-PCR product, based on Genbank™ sequence NM_005185, was 203 base pairs. As mentioned, control reactions were made using 18S rRNA-specific primers (SEQ ID NO: 3 and 4), under similar conditions to those for CALML3, except for the number of cycles (20-25) and the annealing temperature (64⁰C).

[0176] RT-PCR products from all samples of the complete panel were obtained as above and analyzed on ethidium bromide-stained agarose gels. For each sample, the two concentrations were chosen so that no band saturation was observed and that a decrease in signal correlated with decreasing concentration of RNA sample. The assumption was made that under such conditions the intensity of bands was indicative of the initial amount of target RNA, a requirement for the appropriate estimation of CALML3 gene expression. All bands were quantified using a Gel Doc™ analyzer (from BioRad).

[0177] The ratios of CALML3 band intensities over 18S band intensities were calculated and correlations with regards to the confirmed diagnosis were examined (Figures 1 and 2). CALML3 gene expression was also analyzed in correlation with lung cancer tumor stage (Figure 4) and regional lymph node status (Figure 5). These figures show that CALML3 expression tends to be the highest in tumors from stage III disease and that no correlation can be made with regard to nodule metastasis. However, the correlation between CALML3 expression level and tumor stage was found to be not statistically significative. Results obtained for the quantification of RT-PCR products indicated that CALML3 gene expression could be detected in most non-small cell lung carcinomas of the squamous cell carcinoma subtype. EXAMPLE 3

DETECTION OF CALML3 TRANSCRIPTS IN BRONCHIAL ASPIRATES

[0178] Clinical samples were obtained from the bronchology department of a collaborating hospital, in accordance with the recommendations of the relevant ethics committee. All patients signed an informed consent agreement form for their voluntary inclusion in the study. The hospital provided us with the final diagnosis of patients based on various clinical observations, as well as results from the cytological examination of their bronchial aspirations/washings. Bronchial aspiration samples were obtained by a bronchologist in parallel with the samples routinely collected for cytological examinations. For each patient, a first bronchial aspiration sample was obtained for the usual cytological examination (above), and immediately after a second sample from the same anatomical site was collected for the purpose of the herein presented study. That latter sample was quickly stabilized with the addition of 30 ml of a phosphate-buffered saline solution provided by us (Na₂HPO₄ 0,06 M, NaH₂PO₄ 0,04 M, NaCI 0,3 M, final pH 7.0). Of course other stabilization buffers could be used, as well known in the art. Prior to aspiration, blood samples were drawn according to standard procedures (e.g., arm puncture), one sample being collected with anticoagulant, one without. Aspirates and blood samples were sent to our laboratories on the same day with one-hour delivery service at ambient temperature, and either processed immediately for RNA extraction (below), or put at 4⁰C and processed the next morning. Since the sample can be kept at 4⁰C overnight it will be understood by a person of ordinary skill in the art that the methods of the present invention can readily be adapted to suit particular needs. Patients were also given three plastic sampling bottles and asked to collect a first sputum (expectoration) on rising for the next three mornings following bronchoscopy. Instruction was given to quickly (immediately, if possible) add the content of a provided 30 ml bottle of phosphate-buffered saline to each expectorate (once again it should be recognized that the stabilization of the sample is the aim of the addition of the buffered solution and that numerous buffers and 01760

70

stabilization could be used as known in the art). Samples were kept in the refrigerator (4⁰C) until the third sample was collected, after which the three samples were sent to us as above.

[0179] Total RNA was purified from samples according to the following methods. Cells from bronchial aspirates stabilized in phosphate-buffered saline (PBS) were collected by centrifugation. RNA was then extracted from the pellet using 3 volumes of Trizol™ LS reagent (Invitrogen), following the instructions of the manufacturer. Total RNA was purified either from whole blood (tube with anticoagulant) using Trizol™ LS Reagent, or from nucleated cells following treatment with the Red Blood Cell Lysis Buffer (Roche). Total RNA from the plasma was isolated (following a brief centrifugation) using Trizol™ LS Reagent. Adding glycogen (Roche) in the process improved recovery at the nucleic acids precipitation step. Finally, cells from sputa were isolated by centrifugation, followed by homogenization of the cells/debris pellet with Trizol™ LS Reagent and further purification of the RNA according to the manufacturer. In all instances, the resulting RNA pellet was dissolved in nuclease-free water and frozen until used in RT-PCR assays. Given the possible scarcity of RNA in some samples above, no DNAse treatment was carried on. Of course, should samples provide more RNA or be under conditions of significant RNA stabilization, a DNAse step could be added. It should be recognized from the above that the methods and kits of the present inventions can be adapted according to the type of sample, the quality of the sample, and the like. For instance, depending on the type of sample and the amount of macromolecule it contains (nucleic acid or protein), the skilled artisan can routinely adapt the method and kit to use the crude sample, semi purified sample, or substantially purified sample (e.g., cells for example), crude macromolecule preparation, semi purified macromolecule preparation, or substantially purified macromolecule preparation (e.g., protein preparation, RNA preparation). [0180] RNA samples were diluted and analyzed by RT-PCR. Given the lack of introns in the genomic sequence of CALML3, a new pair of primers was designed with specificity for the 3' extremity of the CALML3 mRNA, including the polyA tail, which is absent from the genomic sequence. As before, control reactions without the RT step were carried out in order to rule out the contribution of genomic DNA to the observed signals. The absence of DNase treatment precluded the use of 18S rRNA as a reference, since both the rRNA and the intronless 18S gene could be amplified and give identical signals on a gel. Consequently, the mRNA levels for porphobilinogen deaminase (PBGD) were assessed as a reference (housekeeping) gene with the help of specific primers (SEQ ID NOs: 7-8) and the same technique. Of course other primers specific for PBGD or other control genes (e.g., SFTPC) could be used in accordance with the present invention. As well, differentially expressed CALML3 mRNA could be targeted instead of the CALML3 sequence or part thereof defined in SEQ ID NO: 9. Amplification products were analyzed and quantified as above, with both products always being deposited and quantified on the same gel in order to derive meaningful ratios for the expression of CALML3 over the second lung-specific marker ( e.g., PBGD).

[0181] In addition, similar experiments using SFTPC (a lung specific marker) as a control for the normalization of CALML3 expression were conducted. RNA was purified from 40 cancer samples (including 30 NSCLC) and 19 non¬ cancerous samples and analyzed by RT-PCR as described above. CALML3 mRNA was amplified using the oligonucleotides set forth in SEQ ID NOs:5 and 6 and SFTPC mRNA was amplified using the oligonucleotides set forth in SEQ ID NOs: 19 and 20. The expected size for the SFTPC amplified product was 172 base pairs, based on GenBank™ sequence NM_003018.2. The specific PCR protocol was as described above except that the number of cycles used was 40. The expression level of CALML3 was normalized with that of SFTPC in all samples. When cytology was also considered, the sensitivity of the diagnosis test increased (Se: approximately 90%).The following parameters were used when assessing clinical performances of a diagnostic test based on the combination of a cytology analysis and on the detection of CALML3 normalized with SFTPC: CALML3 + and cytology + -> presence of cancer; CALML3 + and cytology - → presence of cancer; CALML3 - and cytology + →presence of cancer and CALML3- and cytology - → absence of cancer.

[0182] Results with the clinical samples of 31 patients confirmed the validation of CALML3 as a diagnostic marker for lung cancer (Figure 3). With an appropriately selected threshold (in this particular case, a normalized ratio of 4 was chosen), cancers could be distinguished from non-cancerous samples with a sensitivity of 79% and a specificity of 65%. This level of sensitivity compared favorably to cytology, which showed a sensitivity of 43% (for a 100% selectivity). The combination of CALML3 expression with cytology moderately increased the sensitivity of CALML3 alone, while greatly improving the detection of cancer with cytology alone.

[0183] Thus, CALML3 represents a useful validated and sensitive tool for the diagnosis of lung cancer in clinical samples. Of course, having identified a molecular marker which is on its own specific and selective, in one embodiment the present invention also provides a diagnosis based on a combination of the detection of CALML3 and cytology, which enables a powerful diagnostic assay for lung cancer.

[0184] Although the present invention has been described hereinabove by way of illustrative embodiments thereof, it will be appreciated by one skilled in the art from reading of this disclosure that various changes in form and detail can be made without departing from the spirit and nature of the invention as defined in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for diagnosing lung cancer in a human biological sample, the method comprising: analyzing the human biological sample for the presence of a CALML3 polynucleotide, a detection of an elevated level of CALML3 as compared to the level thereof in a normal human biological sample is indicative of the presence of, or predisposition to develop lung cancer.

2. The method of claim 1, wherein the human biological sample is selected from a sputum, a bronchial aspirate, bronchial lavage, bronchoalveolar lavage and a coughing sample.

3. The method of claim 2, wherein said aspirate is carried out following a bronchial aspirate.

4. The method of claim 1, wherein the human biological sample is selected from a lung biopsy and blood.

5. The method of any one of claims 1 to 4, wherein the cancer is a NSCLC.

6. The method of any one of claims 1 to 5, wherein CALML3 RNA is detected.

7. A method for determining a predisposition to or presence of lung cancer in a patient comprising: a) contacting a biological sample of said patient with at least one oligonucleotide that hybridizes to a CALML3 polynucleotide selected from the group consisting of: i) a polynucleotide according to SEQ ID NO:9; ii) a polynucleotide encoding CALML3 protein according to SEQ ID NO:10; iii) a polynucleotide sequence that hybridizes under high stringency conditions to the polynucleotide sequence in i) or ii); and iv) a polynucleotide sequence fully complementary to i), ii) or iii); b) detecting in said biological sample an amount of CALML3 polynucleotide; and c) comparing the amount of said CALML3 polynucleotide that hybridizes to the oligonucleotide to a predetermined cut-off value, and therefrom determining the presence or absence of lung cancer in said patient or its predisposition to develop same.

8. The method of claim 7, wherein the biological sample is selected from the group consisting of: a) blood or fraction thereof; b) sputum; c) coughing sample; d) bronchial aspiration; e) urine; f) saliva; g) brushing; h) lung biopsy; i) bronchial lavage; and j) bronchoalveolar lavage.

9. The method according to claim 7 or 8, wherein said sample is selected from a crude clinical sample, a semi purified clinical sample and a substantially pure clinical sample.

10. The method according to claim 9, wherein an amplification reaction is used.

11. A method according to claim 10, wherein the amount of an amplified CALML3 polynucleotide is determined using a probe which specifically hybridizes thereto.

12. A method according to claim 7, 8, 9, 10, or 11 , wherein the amount of a CALML3 polynucleotide that hybridizes with the oligonucleotide is determined using a nucleic acid sequence-based amplification assay (NASBA) or a polymerase chain reaction.

13. A method of monitoring a progression of lung cancer in a patient comprising: a) contacting a biological sample of said patient with at least one oligonucleotide that hybridizes with a CALML3 polynucleotide selected from the group consisting of : i) a polynucleotide according to SEQ ID NO:9 ; ii) a polynucleotide encoding CALML3 protein according to SEQ ID

NO:10; iii) a polynucleotide sequence that hybridizes under high stringency conditions to the nucleotide sequence in i) or ii); and iv) a polynucleotide sequence fully complementary to i), ii) or iii); b) detecting in said biological sample an amount of CALML3 polynucleotide; c) repeating steps (a) and (b) using a biological sample from said patient at a subsequent point in time; and d) comparing the relative amount of CALML3 polynucleotide detected in step (c) to the relative amount of CALML3 polynucleotide detected in step (b) and therefrom monitoring the progression of the lung cancer in said patient.

14. A method to determine the predisposition, presence or progression of lung cancer in a biological sample of a patient comprising: detecting in said biological sample the presence of the CALML3 mRNA by an in vitro amplification assay, the detection of an elevated amount of CALML3 in said biological sample as compared to that in control sample indicating the predisposition, presence or progression of lung cancer.

15. A method according to claim 13, wherein the amount of CALML3 polynucleotide that hybridizes with the oligonucleotide is determined using an assay selected from the group consisting of: a) polymerase chain reaction; b) a hybridization assay; c) nucleic acid sequence-based amplification assay (NASBA); d) transcription mediated amplification assay (TMA); and e) ligase chain reaction assay (LCR).

16. The method of claim 13 or 15, wherein said amount of CALML3 detected in steps b) or c) is compared to an amount of CALML3 detected in a control sample.

17. A method for determining the predisposition, or presence of lung cancer in a patient comprising: a) contacting a biological sample of a patient with a binding agent that binds to a CALML3 protein, wherein said CALML3 protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of : i) a polynucleotide according to SEQ ID NO:9; ii) a polynucleotide encoding CALML3 protein according to SEQ ID NO:10; and iii) a polynucleotide that hybridizes under high stringency conditions to the complement of a polynucleotide in i) and ii); b) detecting in said biological sample an amount of CALML3 protein that binds to the binding agent; and c) comparing the amount of CALML3 protein that binds to the binding agent to a predetermined cut-off value, and therefrom determining the presence or absence of lung cancer in said patient.

18. The method of claim 17, wherein the binding agent is an antibody.

19. The method of claim 18, wherein said antibody is selected from the group consisting of: a) a polyclonal CALML3 antibody; b) a monoclonal CALML3 antibody; c) a recombinant CALML3 antibody; and d) a humanized CALML3 antibody.

20. A method of monitoring a progression of lung cancer in a patient comprising: a) contacting a biological sample of said patient with a binding agent that binds to a CALML3 protein, wherein CALML3 protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of : i) a polynucleotide according to SEQ ID NO:9; ii) a polynucleotide encoding CALML3 protein according to SEQ

ID NO:10; and iii) a polynucleotide variant or allele of polynucleotide i), or iii) wherein said variant or allele is associated with lung cancer. b) detecting in said biological sample an amount of CALML3 protein that binds to the binding agent; c) repeating steps (a) and (b) using a biological sample from said patient at a subsequent point in time; and d) comparing the relative amount of CALML3 protein detected in step (c) to the relative amount of CALML3 protein detected in step (b) and therefrom monitoring the progression of the lung cancer in said sample.

21. The method of claim 19, wherein the binding agent is an antibody.

22. The method of claim 19, wherein said antibody is selected from the group consisting of: a) a polyclonal CALML3 antibody; b) a monoclonal CALML3 antibody; c) a recombinant CALML3 antibody; and d) a humanized CALML3 antibody.

23. A diagnostic kit for the detection of lung cancer comprising at least a container means having disposed therein at least one oligonucleotide probe or primer that hybridizes to one of: a) a CALML3 nucleic acid sequence according to SEQ ID NO:9; b) a polynucleotide encoding CALML3 protein according to SEQ ID NO:10; c) a sequence which is fully complementary to a) or b); and d) a sequence which hybridizes under high stringency conditions to a), b) or c); and e) instructions for the diagnostic of lung cancer based on the detection of a particular amount of CALML3 mRNA.

24. A diagnostic kit for the detection of lung cancer comprising: a) one or more specific CALML3 antibody; and b) a detection reagent comprising a reporter group and/or a label; and c) instructions for the diagnostic of lung cancer based on the detection of a particular amount of CALML3 mRNA.

25. A diagnostic kit according to claim 24, wherein the label is selected from the group consisting of: a) radioisotopes; b) enzymes; c) fluorescent groups; d) biotin; e) chemiluminescent groups; and f) dye particles, and wherein said reporter group is selected from the group consisting of: g) antiimmunoglobulin; h) protein A; and i) protein G.

26. The kit according to claim 23 further comprising a reagent for detecting a second lung specific marker.

27. The method of any one of claims 7 to 22, wherein the cancer is a NSCLC.

28. The method of claim 17, wherein the human biological sample is selected from the group consisting of: a) a sputum; b) a coughing sample; c) a lung biopsy; d) a blood sample or fraction thereof; e) urine; f) a bronchial aspiration; g) a brushing h) saliva; i) bronchial lavage, and j) a bronchoalveolar lavage.

29. The method according to claim 28, wherein said sample is selected from a crude clinical sample, a semi purified clinical sample and a substantially pure clinical sample.

30. A method according to any one of claims 1 to 22, wherein an amount of a further lung tumor marker is detected in addition to CALML3.

31. A method according to any one of claims 1 to 22, wherein an amount of a lung-specific marker is also detected.

32. The method of claim 31, wherein said lung-specific marker is SFTPC.

33. The method of claim 32, wherein a ratio of CALM L3/S FTPC is determined.

34. A method according to any one of claims 1 to 22, wherein an amount of a gene whose expression does not vary in lung cancer is also detected.

35. The method of claim 34, wherein said gene is PBGD.

36. The method of claim 35, wherein a ratio of CALML3/PBGD is determined.

37. A kit adapted to perform the method of claim 33.

38. The method of claim 13, 15 or 16, wherein said patient undergoes a lung cancer treatment between said steps a) and c) thereby monitoring an effect of said treatment on the progression of lung cancer.