WO2011149943A1 - Method for differentiation of non-small cellung carcinoma - Google Patents

Abstract

Disclosed herein are methods for diagnosing lung carcinoma type in a subject by differentiating subtypes of non-small cell lung carcinoma (NSCLC), for example, diagnosing a squamous cell type lung carcinoma or a non-squamous type lung carcinoma in the subject. In some embodiments, the methods include determining a first amount of serpin B13 protein in the sample, determining a second amount of cytokeratin 5 and/or cytokeratin 6 protein in the sample, determining a third amount of thyroid transcription factor- 1 protein in the sample, and determining a fourth amount of napsin A protein in an NSCLC sample. In some embodiments, the methods further include determining a fifth amount of p63 protein in an NSCLC sample. In other embodiments, the methods include determining a first amount of cytokeratin 5 and/or cytokeratin 6 protein in the sample, determining a second amount of thyroid transcription factor- 1 protein in the sample, and determining a third amount of napsin A protein in an NSCLC sample.

Description

METHOD FOR DIFFERENTIATION OF NON-SMALL CELL LUNG

CARCINOMA

CROSS REFERENCE TO RELATED APPLICATION

This claims the benefit of U.S. Provisional Application No. 61/396,268, filed

May 24, 2010, which is incorporated by reference herein in its entirety.

FIELD

This disclosure provides methods for differentiating squamous cell and non- squamous cell subtypes of non- small cell lung carcinoma, particularly by detecting serpin B13, cytokeratin 5/6, thyroid transcription factor-1, napsin A and/or p63 proteins in a non-small cell lung carcinoma sample.

BACKGROUND

Cancer remains a major public health challenge despite progress in detection and therapy. Among the various types of cancer, lung cancer is a frequent cancer in the Western world and among the most frequent causes of cancer-related mortality. It is predominantly a disease of the elderly; incidence increases with age, affecting 482/100,000 men >65 years, and peaks at age 75, affecting about 502/100,000 men. A man aged 65 has a 50 times greater risk of developing lung cancer than a man aged 25, and has a 3 to 4 times greater risk than men aged 45 to 64.

About 90% of lung cancer cases in men and 80% in women are attributable to cigarette smoking. The risk of lung cancer is related to the total years of smoking, which exposes smokers to carcinogens and cancer-promoting agents. From initial exposure to cigarette smoke to clinical presentation, lung cancer probably has a 15 to 20 year natural history.

The majority of lung cancers can be divided into small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). Non-small cell lung carcinoma includes three major histological tumor types, squamous cell carcinoma (SQCC), adenocarcinoma (ADC), and large cell carcinoma. SCLC accounts for about 20-25% of all lung cancer cases. SCLC is an aggressive neuroendocrine type of lung cancer and has a very poor prognosis even if it is detected in early stages. SCLC is rarely amenable to curative treatment by resection. Because of the speed with which the disease progresses, SCLC is generally categorized using only two stages, limited and extensive disease.

About 75-80% of lung cancer diagnoses are grouped into the class of NSCLC, including squamous cell carcinoma (SQCC) and non-squamous cell carcinoma. Non-squamous cell carcinoma can be further subdivided into adenocarcinoma (ADC; including the subclasses of acinar carcinoma, papillary carcinoma, broncho alveolar carcinoma, solid tumor, and mixed subtypes), and large cell carcinoma (including the subclasses of giant cell tumors, clear cell carcinoma, adenosquamous carcinoma, and undifferentiated carcinoma). NSCLC, if detected at late stages, has a very poor prognosis. Surgical resection of the primary tumor is widely accepted as the treatment of choice for early stage NSCLC. Surgery is rarely, if ever, used in the management of late stage NSCLC. The earlier lung cancer is diagnosed the better the chances of long term survival.

Chemotherapy drugs and treatment regimens are being developed for NSCLC. Recent studies suggest that NSCLC histological subtype (for example, squamous cell carcinoma or non-squamous cell carcinoma) may predict prognosis or treatment outcome for at least some chemotherapy drugs (Hirsch et al, J Thome.

Oncol. 3: 1468-1481, 2008; Rossi et al, Int. J. Surg. Pathol. 17:206-218, 2009). For example, some drugs, such as pemetrexed {e.g., ALIMTA®) and bevacizumab {e.g., AVASTIN®) are approved for treatment of specific lung cancer subtypes, and thus accurate diagnosis is important for providing optimal treatment.

Currently, a differential diagnosis is made by a pathologist based on observation of cellular morphology. In some cases, the pathologist may use immunohistochemical techniques to help in making a diagnosis, such as the use of antibodies to thyroid transcription factor- 1, cytokeratin 5 and/or cytokeratin 6, napsin A, serpin B13, or p63. For example, studies have associated serpin B13 and cytokeratins (including CK5/6) with squamous cell carcinoma (see, e.g., Smith et al, Oncogene 22:8677-8687, 2003; de Koning et al, Int. J. Cancer 125: 1542-1550, 2009; Blobel et al, Virch. Arch. Cell Pathol. 45:407-429, 1984; Khayyata et al,

Diagn. Cytopathol. 37: 178-183, 2009; Kargi et al., Appl. Immunohistochem. Mol.

Morphol. 15:415-420, 2007; Jagirdar, Arch. Pathol. Lab. Med. 132:384-396, 2008).

Additional studies have associated napsin A and thyroid transcription factor- 1 with adenocarcinoma (see, .e.g., Perner et al., J. Pathol. 217:65-72, 2009; Anagnostou et al., J. Clin. Oncol. 27:271-278, 2009; Boggaram, Clin. Sci. 116:27-35, 2009;

Khayyatta et al., Diagn. Cytopathol. 37: 178-183, 2009; Chuman et al., FEBS Lett.

462: 129-134, 1999; Suzuki et al., Pathol. Res. Practice 201:579-586, 2005; Hirano et al., Lung Cancer 41: 155-162, 2003; Dejmek et al., Diagn. Cytopathol. 35:493- 497, 2007; Ueno et al., Br. J. Cancer 88: 1229-1233, 2003; Kargi et al., Appl.

Immunohistochem. Mol. Morphol. 15:415-420, 2007; Jagirdar, Arch. Pathol. Lab.

Med. 132:384-396, 2008; Yang and Nonaka, Mod. Pathol. 23:654-661, 2010).

Further studies have associated p63 with squamous cell carcinoma (see, e.g., Kargi et al., Appl. Immunohistochem. Mol. Morphol. 15:415-420, 2007; Jagirdar, Arch. Pathol. Lab. Med. 132:384-396, 2008; Khayyata et al, Diagn. Cytopathol. 37: 178-

186, 2009; Rossi et al., Int. J. Surg. Pathol. 17:206-218, 2009). However, site to site variability remains a challenge in diagnostic reproducibility, which can be further complicated by limited materials for testing as found in, for example, small sized biopsy samples.

SUMMARY

Disclosed herein are methods for diagnosing lung carcinoma type in a subject by differentiating subtypes of NSCLC, for example, diagnosing a squamous cell type lung carcinoma, a non- squamous cell type lung carcinoma (such as adenocarcinoma, large cell carcinoma, or undifferentiated carcinoma), or an adenosquamous type lung carcinoma in the subject.

In some embodiments, the disclosed methods include detecting the amount of at least four proteins (including serpin B13, cytokeratin 5 and/or 6, thyroid transcription factor- 1, and napsin A) in an NSCLC sample. In additional embodiments, the methods further include detecting the amount of p63 protein in an NSCLC sample. In some examples, the methods include detecting the amount of each of serpin B13, cytokeratin 5 and/or 6, thyroid transcription factor-1, and napsin A proteins in an NSCLC sample. In other examples, the methods include detecting the amount of each of serpin B13, cytokeratin 5 and/or 6, thyroid transcription factor-1, napsin A, and p63 proteins in an NSCLC sample.

In some embodiments, the methods include detecting a first amount of serpin

B13 protein in the sample, detecting a second amount of cytokeratin 5 and/or cytokeratin 6 (CK5/6) protein in the sample, detecting a third amount of thyroid transcription factor-1 (TTF1, also known as NXK2-1) protein in the sample, detecting a fourth amount of napsin A protein in the sample, and comparing the first amount and the second amount (such as a combined first and second amounts) with the third amount and the fourth amount (such as a combined third and fourth amounts), wherein the subject is diagnosed with squamous cell type lung carcinoma if the first amount and second amount is greater than the third amount and fourth amount, and the subject is diagnosed with non-squamous cell type lung carcinoma if the first amount and second amount is less than the third amount and fourth amount. In some examples, the subject is diagnosed with adenosquamous type lung carcinoma if the first amount and the second amount (such as a combined first and second amount) is approximately equal to the third amount and the fourth amount (such as a combined third and fourth amount).

In other embodiments, the methods further include detecting a fifth amount of p63 protein in the sample, wherein the subject is diagnosed with squamous cell type lung carcinoma if the first amount, second amount and fifth amount (such as a combined first, second, and fifth amount) is greater than the third amount and the fourth amount (such as a combined third and fourth amounts), and the subject is diagnosed with non-squamous cell type lung carcinoma if the first amount, second amount, and fifth amount is less than the third amount and the fourth amount. In still other embodiments, the methods further include detecting a fifth amount of p63 protein in the sample, wherein the subject is diagnosed with squamous cell type carcinoma if the fifth amount is positive (for example, detectable expression of p63 protein) and wherein the subject is diagnosed with non-squamous cell type lung carcinoma if the fifth amount is negative (for example, no detectable expression of p63 protein).

In additional embodiments, the disclosed methods include detecting the amount of cytokeratin 5 and/or cytokeratin 6, TTF1, and napsin A in an NSCLC sample. In some embodiments, the methods include detecting a first amount of cytokeratin 5 and/or cytokeratin 6 (CK5/6) protein in the sample, detecting a second amount of TTF1 protein in the sample, detecting a third amount of napsin A protein in the sample, and comparing the first amount with the second amount and the third amount (such as a combined second and third amounts), wherein the subject is diagnosed with squamous cell type lung carcinoma if the first amount is greater than the second amount and third amount, and the subject is diagnosed with non- squamous cell type lung carcinoma if the first amount is less than the second amount and third amount. In some embodiments, the subject is diagnosed with

adenosquamous type lung carcinoma if the first amount is approximately equal to the second amount and the third amount.

The amount of each of the serpin B13, CK5/6, TTF1, napsin A, and/or p63proteins in the sample can be detected by immunoassay methods, for example, immunohistochemistry (IHC), Western blotting, or ELISA. The amount of the proteins can be detected by a qualitative method (for example, visual observation), a semi-quantitative method (for example, an H score), or a quantitative method (for example, utilizing an automated imaging system).

In some embodiments, the amount of each of serpin B13, CK5/6, TTF1, napsin A, and/or p63 proteins is detected by determining an H score for each protein (or a combination of two or more thereof). In other embodiments, the amount of each of serpin B13, CK5/6, TTF1, napsin A, and/or p63 proteins (or a combination of two or more thereof) is detected by visual observation (for example, by observation of staining intensity and/or color).

The NSCLC sample can be any biological sample that includes NSCLC cells, for example, a tissue biopsy, fine needle aspirate, bronchoalveolar lavage, pleural fluid, sputum, or blood from a subject having or suspected to have NSCLC. In some examples, the sample is fixed and embedded in an embedding medium (such as paraffin). The sample can include a tissue section, for example, a section of a tissue biopsy.

The foregoing and other features will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A to C is a series of digital images showing exemplary chromogenic immunohistochemistry on NSCLC samples from a tissue microarray. FIG. 1A shows brown chromogenic precipitate generated by anti-TTFl and anti-napsin A antibodies detected with a horseradish peroxidase/3,3 '-diaminobenzidine

tetrahydrochloride (HRP/DAB) detection system in a non-squamous NSCLC sample. FIG. IB shows background staining in a negative tissue control in the absence of serpin B13, CK5/6, TTF1, or napsin A proteins. FIG. 1C shows red chromogenic precipitate generated by anti-serpin B13 and anti-CK5/6 antibodies detected with an alkaline phosphatase/Fast Red (AP/Fast Red) detection system in a squamous cell NSCLC sample.

FIG. 2A to C is a series of digital images showing additional exemplary chromogenic immunohistochemistry on NSCLC samples from a tissue microarray. FIG. 2A shows brown chromogenic precipitate generated by anti-TTFl and anti- napsin A antibodies detected with a horseradish peroxidase/diaminobenzidine tetrahydrochloride (HRP/DAB) detection system in a non-squamous NSCLC sample. FIG. 2B shows background staining in a negative tissue control in the absence of serpin B13, CK 5/6, TTF1, or napsin A proteins. FIG. 2C shows red chromogenic precipitate generated by anti-serpin B13 and anti-CD5/6 antibodies detected with an alkaline phosphatase/Fast Red (AP/Fast Red) detection system in a squamous cell NSCLC sample. DETAILED DESCRIPTION

/. Abbreviations and Terms

ADC: adenocarcinoma of lung

AP: alkaline phosphatase

ASC: adenosquamous lung carcinoma

CK: cytokeratin

CK5/6: cytokeratin 5 and/or 6

DAB: 3,3'-diaminobenzidine

HRP: horseradish peroxidase

IHC: immunohistochemistry

NSCLC: non-small cell lung carcinoma

SQCC: squamous cell lung carcinoma

TTF1: thyroid transcription factor- 1 (NKX2-1) Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed invention belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. "Comprising" means "including." Hence "comprising A or B" means "including A" or "including B" or "including A and B."

Suitable methods and materials for the practice and/or testing of

embodiments of the disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which the disclosure pertains are described in various general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. All sequences associated with the GenBank Accession numbers mentioned herein are incorporated by reference in their entirety, to the extent permissible by applicable rules and/or law. In case of conflict, the present specification, including explanations of terms, will control.

In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

Adenocarcinoma (ADC): In the context of lung cancer, ADC is a type of lung cancer derived from glandular tissue (for example, surface alveolar epithelium or bronchial mucosal glands). ADC accounts for about 40% of all lung cancers and is the most common form of lung cancer among individuals who have never smoked. ADC is classified as a non-squamous cell type of NSCLC. Histologically, ADC shows gland formation, papillary structures, or solid growth with mucin production. ADC includes the subtypes acinar carcinoma, papillary carcinoma, broncho alveolar carcinoma, solid tumor, and mixed subtypes. ADC usually develops in peripheral portions of the lung and is generally slow-growing.

Adenosquamous lung carcinoma (ASC): A biphasic lung tumor including both adenocarcinoma lung carcinoma cells and squamous cell lung carcinoma cells. About 3-5% of NSCLC tumors are ASC tumors. In some examples, an ASC tumor includes at least 10% by volume of ADC and SCC cells.

Antibody: A polypeptide that includes at least a light chain or heavy chain immunoglobulin variable region and specifically binds an epitope of an antigen. Antibodies include monoclonal antibodies, polyclonal antibodies, or fragments of antibodies as well as others known in the art. In some examples, an antibody is labeled with a detectable label, such as an enzyme, hapten, or fluorophore.

Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. This includes intact immunoglobulins and the variants and portions of them well known in the art, such as Fab' fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes recombinant forms such as chimeric antibodies (for example, humanized murine antibodies) and heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of ordinary skill in the art, for instance by making hybrid antibody- forming cells from a fusion of myeloma cells with immune spleen cells. These fused cells and their progeny are termed "hybridomas." Monoclonal antibodies include humanized monoclonal antibodies.

Antigen: A molecule that stimulates an immune response. Antigens are usually proteins or polysaccharides. An epitope is an antigenic determinant, that is, particular chemical groups or peptide sequences on a molecule that elicit a specific immune response. An antibody binds a particular antigenic epitope. The binding of an antibody to a particular antigen or epitope of an antigen can be used to localize the position of the antigen for example in or on a biological sample, or determine if the particular antigen is present in a biological sample.

Chromogen: A substance capable of conversion to a colored product, such as a pigment or dye. Certain chromogens are electron donors that, when oxidized become a colored product. Production of a colored product, and the property of becoming insoluble upon chemical conversion, such as by oxidation, make chromogens useful for IHC and/or ISH. Particular examples of chromogenic compounds, without limitation, include diaminobenzidine (DAB), 4- nitrophenylphospate (pNPP), Fast Red, Fast Blue, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), BCIP/NBT, AP Orange, AP Blue,

tetramethylbenzidine (TMB), 2,2'-azino-di-[3-ethylbenzothiazoline sulphonate] (ABTS), o -dianisidine, 4-chloronaphthol (4-CN), nitrophenyl-P-D- galactopyranoside (ONPG), ophenylenediamine (OPD), 5-bromo-4-chloro-3- indolyl-P-galactopyranoside (X-Gal), methylumbelliferyl-P-D-galactopyranoside (MU-Gal), /7-nitrophenyl-a-D-galactopyranoside (PNP), 5-bromo-4-chloro-3- indolyl- β -D-glucuronide (X-Gluc), 3-amino-9-ethyl carbazol (AEC), New

Fuchsin, iodonitro tetrazolium (INT), tetrazolium blue and tetrazolium violet.

Cytokeratin 5 and 6: Members of the keratin gene family. Keratins are intermediate filament proteins that form a dense meshwork of filaments throughout the cytoplasm of epithelial cells. Keratins are generally expressed in particular pairs of type I and type II keratin proteins in a tissue-specific and cellular differentiation- specific manner. The keratin proteins of epithelial tissues are commonly known as "keratins" or are sometimes referred to as "epithelial keratins" or "cytokeratins."

Cytokeratins 5 (CK5; KRT5) and 6 (CK6; KRT6) are type II cytokeratins, which are basic or neutral proteins clustered in a region of chromosome 12ql2-ql3. CK5 is specifically expressed in the basal layer of the epidermis with family member KRT14. CK6 includes multiple genes (KRT6A, KRT6B, and KRT6C) that are highly conserved (>98 identity). CK6 is expressed with family members KRT16 and/or KRT17. In some examples, cytokeratin 5/6 (CK5/6) refers to cytokeratin 5 and/or 6.

Nucleic acid and protein sequences for CK5 and CK6 are publicly available. For example, GENBANK® Accession No. NM_000424 discloses an exemplary human CK5 nucleic acid sequence, and GENBANK® Accession No. NP_000415 discloses an exemplary human CK5 protein sequence, both of which are

incorporated by reference as provided by GENBANK® on May 24, 2010.

GENBANK® Accession Nos. NM_005554 (KRT6A), NM_005555 (KRT6B), and NM_173086 (KRT6C) disclose exemplary human CK6 nucleic acid sequences, and GENBANK® Accession Nos. NP_005545 (KRT6A), NP_005546 (KRT6B), and NP_775109 (KRT6C) disclose exemplary human CK6 protein sequences, all of which are incorporated by reference as provided by GENBANK® on May 24, 2010.

Detectable label: An agent capable of detection, for example by

immunohistochemistry, ELISA, Western blotting, spectrophotometry, flow cytometry, or microscopy. For example, a label can be attached to an antibody that specifically binds to a protein, thereby permitting detection (and in some examples, quantitation) of the protein. Examples of detectable labels include, but are not limited to, radioactive isotopes, enzyme substrates, co-factors, ligands,

chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, New York, 1989), Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), and Harlow and Lane (Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1999).

H score: A semi-quantitative value describing the amount of

immunoreactivity of a particular marker in a sample. An H score is determined by summing the products of the percentage of cells (0-100) staining at a given staining intensity in a sample (such as NSCLC sample, for example, an NSCLC tissue section) and the staining intensity (0-3 or 0-4), and if relevant can be rendered for one or more cell compartment (for example, cytoplasm, membrane, and/or nucleus). Staining intensity is rated by a slide reader on a numeric scale (for example, 0-3, where 0 indicates no staining relative to background, 1 indicates weak staining, 2 indicates moderate staining, and 3 indicates strong staining). For example, a sample with 10% of cells with staining intensity 3; 30% of cells staining with intensity 2; 20% of cells staining with intensity 1; and 40% of cells unstained (staining intensity 0) would have an H score of (3 x 10) + (2 x 30) + (1 x 20) + (0 x 40) = 110. An H score can also be determined utilizing a numeric scale of 0-4 for staining intensity. If a 0-3 staining intensity scale is used, the H score range is 0-300, while if a 0-4 staining intensity scale is used, the H score range is 0-400. See, e.g., McClelland et al, Cancer Res. 50:3545-3550, 1990. Immunohistochemistry (IHC): A method of determining the presence or distribution of an antigen (such as a protein) in a sample (such as an NSCLC sample, for example, a portion or section of tissue) by detecting interaction of the antigen with a specific binding agent, such as an antibody. A sample including an antigen (such as a target antigen) is incubated with an antibody under conditions permitting antibody-antigen binding. Antibody-antigen binding can be detected by means of a detectable label conjugated to the antibody (direct detection) or by means of a detectable label conjugated to a secondary antibody, which is raised against the primary antibody (e.g. , indirect detection). Exemplary detectable labels that can be used for IHC include, but are not limited to, radioactive isotopes, fluorochromes (such as fluorescein, fluorescein isothiocyanate, and rhodamine), haptens, enzymes (such as horseradish peroxidase or alkaline phosphatase), and chromogens (such as 3, 3 '-diaminobenzidine or Fast Red). In some examples, IHC is utilized to detect the presence of or determine the amount of one or more proteins in a sample, for example, an NSCLC sample.

Lung carcinoma: A neoplastic condition of lung tissue. The majority of lung carcinomas are non-small cell lung carcinoma (such as adenocarcinoma, squamous cell carcinoma, and large-cell lung cancer). Most other lung carcinomas are small-cell lung carcinomas. In particular examples, lung carcinoma includes non-small cell lung carcinoma.

Napsin A: An aspartic protease predominantly expressed in lung and kidney, also known as TA01/TA02. The pro-napsin A polypeptide includes a signal peptide, a pro-part region, the mature enzyme, and a C-terminal extension. The napsin A protease is involved in the N- and C-terminal processing of surfactant protein B in the lung.

Nucleic acid and protein sequences for napsin A are publicly available. For example, GENBANK® Accession No. NM_004851 discloses an exemplary human napsin A nucleic acid sequence, and GENBANK® Accession No. NP_004842 discloses an exemplary human napsin A protein sequence, both of which are incorporated by reference as provided by GENBANK® on May 24, 2010. Non-small cell lung carcinoma (NSCLC): Any type of lung cancer other than small cell lung carcinoma. NSCLC includes squamous cell carcinoma (SQCC), adenocarcinoma (ADC), and large cell carcinoma. Both ADC and large cell carcinoma are classified as non-squamous cell type carcinoma. ADC can be grouped into subclasses, including acinar carcinoma, papillary carcinoma, bronchoalveolar carcinoma (BAC), solid tumor, and mixed subtypes (2004 World Health Organization classification of lung tumors, Beasley et ah, Semin. Roentgenol. 40:90-97, 2004). Large cell carcinoma includes the subclasses giant cell tumors, clear cell carcinoma, adenosquamous carcinoma, and undifferentiated carcinoma.

NSCLC accounts for about 75-80% of lung carcinomas. Adenocarcinomas account for approximately 50% of all cases of NSCLC, squamous cell carcinomas account for approximately 30% of all cases of NSCLC, and large cell carcinomas account for about 10% of all NSCLC.

p63: Also known as tumor protein p63 or TP63. A member of the p53 family of transcription factors. p63 is involved in development and maintenance of epithelial tissues and mutations in p63 are associated with ectodermal dysplasia and cleft lip/palate syndrome, split-hand/foot malformation 4, ankyloblepharon- ectodermal defects-cleft lip/palate; ADULT syndrome, limb-mammary syndrome, Rap-Hodgkin syndrome, and orofacial cleft 8.

Nucleic acid and protein sequences for p63 are publicly available. For example, GENBANK® Accession Nos. NM_003722, NM_001114978,

NM_001114979, NM_001114980, NM_001114981, and NM_001114982 disclose exemplary human p63 nucleic acid sequences, and GENBANK® Accession Nos. NP_003713, NP_001108450, NP_001108451, NP_001108452, NP_001108453, and NP_001108454 disclose exemplary human p63 protein sequences, all of which are incorporated by reference as provided by GENBANK® on May 24, 2011.

Sample: A biological specimen containing genomic DNA, RNA (including mRNA), protein, or combinations thereof, obtained from a subject. Examples include a specimen containing at least one NSCLC cell (an "NSCLC sample"), for example, a tissue or tumor biopsy, fine needle aspirate, bronchoalveolar lavage, pleural fluid, sputum, surgical specimen, lymph node, an NSCLC metastasis, peripheral blood, or autopsy material. In other examples, a sample includes a control sample, such as a non-NSCLC cell or tissue sample.

Serpin B13: Serpin B13 (serpin peptidase inhibitor, clade B, member 13; also known as proteinase inhibitor 13, HaCaT UV-repressible serpin (hurpin), or headpin) is an approximately 44 kDa protein including 391 amino acids. An isoform of serpin B13 (approximately 38 kDa, 339 amino acids) which is produced by alternative splicing is reported in the literature. Serpin B13 belongs to the broadly distributed protein superfamily of serpins (serine protease inhibitors).

Generally, serpins are protease inhibitors that use a conformational change to inhibit target enzymes, mainly serine proteases. In particular, serpin B13 was reported to inhibit cathepsin K and L (Weiss et ah, Biochemistry 42:7381-7389, 2003;

Jayakumar et ah, Arch. Biochem. Biophys. 409:367-374, 2003).

Nucleic acid and protein sequences for serpin B13 are publicly available. For example, GENBANK® Accession Nos. NM_012397, AJ278717, and

AF216854 disclose exemplary human serpin B13 nucleic acid sequences, and

GENBANK® Accession Nos.: NP_036529, CAC03569, and AAF72879 disclose exemplary human serpin B13 protein sequences, all of which are incorporated by reference as provided by GENBANK® on May 24, 2010.

Sensitivity and specificity: Statistical measurements of the performance of a binary classification test. Sensitivity measures the proportion of actual positives which are correctly identified (e.g., the percentage of NSCLC tumors that are identified as being non- squamous cell carcinoma). Specificity measures the proportion of negatives which are correctly identified (e.g., the percentage of NSCLC tumors identified as not being non-squamous cell carcinoma).

Squamous cell lung carcinoma (SQCC): A cancer of the squamous epithelium of the lungs or bronchi. SQCC is a type of NSCLC that accounts for about 30% of all cases of lung cancer. It is strongly linked with a history of cigarette smoking. SQCC tumors are typically found in the central region of the lung, for example, in the proximal bronchi. Classic SQCC can be identified histologically based on areas of keratinization and associated inflammatory component. However, less differentiated forms of SQCC may lack keratinization and have smaller undifferentiated cells.

Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals.

Therapeutically effective amount: A dose sufficient to prevent

advancement, delay progression, or to cause regression of a disease, or which is capable of reducing symptoms caused by the disease, such as cancer, for example lung carcinoma (such as non-small cell lung carcinoma).

Thyroid transcription factor-1 (TTF1): Also known as NK2 homeobox 1 (NKX2-1). TTF1 is a homeodomain-containing transcription factor that regulates expression of genes in the thyroid, lung, and central nervous system and is involved in morphogenesis and differentiation of thyroid and lung. In the thyroid, TTF1 controls expression of genes such as thyroglobulin and thyrotropin receptor. In the lung, TTF1 controls expression of surfactant proteins A, B, and C, and Clara cell secretory protein. Multiple transcript variants encoding different isoforms of TTF1 have been identified.

Nucleic acid and protein sequences for TTF1 are publicly available. For example, GENBANK® Accession Nos. NM_001079668 and NM_003317, disclose exemplary human TTF1 nucleic acid sequences, and GENBANK® Accession Nos. NP_001073136 and NP_003308 disclose exemplary human TTF1 protein sequences, all of which are incorporated by reference as provided by GENBANK® on May 24, 2010.

//. Method for Diagnosing Lung Carcinoma Type

Disclosed herein are methods for diagnosing lung cancer type in a subject, for example, methods for differentiating the type of NSCLC (for example squamous cell or non-squamous cell carcinoma) in an NSCLC sample from the subject. In some embodiments, the methods include detecting the amount of at least four proteins (including serpin B13, cytokeratin 5 and/or 6, thyroid transcription factor-1, and napsin A) in an NSCLC sample. In some examples, the methods include detecting the amount of each of serpin B13, cytokeratin 5 and/or 6, thyroid transcription factor- 1, and napsin A in an NSCLC sample. In other examples, the methods further include detecting the amount of p63 protein in an NSCLC sample.

Also disclosed herein are methods for diagnosing lung cancer type in a subject, for example differentiating the type of NSCLC (for example, squamous cell or non-squamous cell carcinoma), including detecting the amount of CK5/6, TTF1, and napsin A in an NSCLC sample from the subject.

In some embodiments, the methods include detecting a first amount of serpin B13 protein in the sample, detecting a second amount of cytokeratin 5 and/or cytokeratin 6 (CK5/6) protein in the sample, detecting a third amount of thyroid transcription factor- 1 (TTF1, also known as NXK2-1) protein in the sample, detecting a fourth amount of napsin A protein in the sample, and comparing the first amount and the second amount (such as a combined first and second amounts) with the third amount and the fourth amount (such as a combined third and fourth amounts), wherein the subject is diagnosed with squamous cell type lung carcinoma if the first amount and second amount is greater than the third amount and fourth amount, and the subject is diagnosed with non-squamous cell type lung carcinoma if the first amount and second amount is less than the third amount and fourth amount. In some embodiments, the subject is diagnosed with adenosquamous type lung carcinoma if the first amount and the second amount (such as a combined first and second amounts) is approximately equal to the third amount and the fourth amount (such as a combined third and fourth amounts).

In other embodiments, the methods further include detecting a fifth amount of p63 protein in the sample, wherein the subject is diagnosed with squamous cell type lung carcinoma if the first amount, second amount and fifth amount (such as a combined first, second, and fifth amounts) is greater than the third amount and the fourth amount (such as a combined third and fourth amounts), and the subject is diagnosed with non-squamous cell type lung carcinoma if the first amount, second amount, and fifth amount is less than the third amount and the fourth amount. In still other embodiments, the methods further include detecting a fifth amount of p63 protein in the sample, wherein the subject is diagnosed with squamous cell type carcinoma if the fifth amount is positive (for example, detectable expression of p63 protein) and wherein the subject is diagnosed with non-squamous cell type lung carcinoma if the fifth amount is negative (for example, no detectable expression of p63 protein).

In additional embodiments, the disclosed methods include detecting the amount of CK5/6, TTFl, and napsin A in an NSCLC sample. In some

embodiments, the methods include detecting a first amount of CK5/6 protein in the sample, detecting a second amount of TTFl protein in the sample, detecting a third amount of napsin A protein in the sample, and comparing the first amount with the second amount and the third amount (such as a combined second and third amounts), wherein the subject is diagnosed with squamous cell type lung carcinoma if the first amount is greater than the second amount and third amount, and the subject is diagnosed with non-squamous cell type lung carcinoma if the first amount is less than the second amount and third amount. In some embodiments, the subject is diagnosed with adenosquamous type lung carcinoma if the first amount is approximately equal to the second amount and the third amount.

In some examples, the amounts of serpin B13, CK5/6, TTFl, napsin A, and/or p63 proteins are detected in the same NSCLC sample from the subject (such as the same tissue section). In some examples, the amounts of serpin B13, CK5/6, TTFl, and napsin A are detected in the same NSCLC sample from the subject. In other examples, the amounts of serpin B13, CK5/6, TTFl, and napsin A are detected in the same NSCLC sample from the subject and the amount of p63 is detected in a different NSCLC sample from the same subject. In another example, the amounts of serpin B13, CK5/6, TTFl, napsin A, and p63 are detected in the same NSCLC sample from the subject. In other examples, the amount of serpin B13, CK5/6, TTFl, napsin A, and/or p63 proteins are each detected in a separate NSCLC sample from the subject (such as individual tissue sections, for example, serial tissue sections). In further examples, the amounts of two or three of serpin B13, CK5/6, TTFl, napsin A, and p63 proteins are detected in the same NSCLC sample from the subject. In a particular example, serpin B13 and CK5/6 are detected in one sample from the subject and TTFl and napsin A are detected in another sample from the subject. In another example, the amounts of CK5/6, TTFl, and napsin A are detected in the same NSCLC sample from the subject. If the amounts of two or more of serpin B13, CK5/6, TTF1, napsin A, and p63 are detected in the same sample from a subject, the amount of one or more proteins can be detected as a combined amount (for example, if more than one protein is detected with the same label). For example, a combined amount of serpin B 13 and CK5/6 can be detected and/or a combined amount of TTF1 and napsin A can be detected.

In some embodiments, the amount of one or more proteins is detected in one or more particular cellular compartments, such as one or more of the nucleus, cytoplasm, or cell membrane. Exemplary patterns of cellular reactivity for TTF1, napsin A, CK5/6, serpin B13, and p63 proteins are provided in Table 1. In some examples, the amount of serpin B13 protein detected is the amount of serpin B13 protein that has a cytoplasmic localization (for example, fine granular cytoplasmic localization) in the NSCLC sample. In other examples, the amount of CK5/6 protein is the amount of CK5/6 protein that has a cytoplasmic localization (for example, granular cytoplasmic localization) in the NSCLC sample. In further examples, the amount of TTF1 protein is the amount of TTF1 protein that is localized to the nucleus in the NSCLC sample. In still further examples, the amount of napsin A protein is the amount of napsin A protein that has a cytoplasmic localization in the NSCLC sample. In additional examples, the amount of p63 protein is the amount of p63 protein that is localized to the nucleus in the NSCLC sample.

Table 1. Exemplary cellular reactivity for ADC/SCC markers

Normal ADC Cells SCC Cells

Serpin PC VII negative Negative Fine granular B13 Bronchi(ol)i basal cytoplasmic,

Cytoplasmic and nuclear Gl/12 positive

p63 PC VII negative Negative Nuclear

Bronchi(ol)i basal

Nuclear positive

In some examples, the disclosed methods include detecting a first amount of serpin B 13 protein in an NSCLC sample, detecting a second amount of CK5/6 protein in the sample, detecting a third amount of TTF1 protein in the sample, detecting a fourth amount of napsin A protein in the sample, calculating a first sum of the first amount and the second amount, and calculating a second sum of the third amount and the fourth amount. If the first sum is greater than the second sum, the subject is diagnosed with squamous cell type lung carcinoma, whereas if the first sum is less than the second sum, the subject is diagnosed with a non-squamous cell type lung carcinoma (such as adenocarcinoma or large cell carcinoma). In some examples, if the first sum and the second sum are approximately equal, the subject is diagnosed with an adenosquamous carcinoma.

In other examples, the disclosed methods further include detecting a fifth amount of p63 protein in an NSCLC sample. For example, the methods include detecting a first amount of serpin B 13 protein in an NSCLC sample, detecting a second amount of CK5/6 protein in the sample, detecting a third amount of TTF1 protein in the sample, detecting a fourth amount of napsin A protein in the sample, detecting a fifth amount of p63 protein in the sample, calculating a first sum of the first amount, the second amount, and the fifth amount, and calculating a second sum of the third amount and the fourth amount. If the first sum is greater than the second sum, the subject is diagnosed with squamous cell type lung carcinoma, whereas if the first sum is less than the second sum, the subject is diagnosed with a non- squamous cell type lung carcinoma (such as adenocarcinoma or large cell carcinoma). In some examples, if the first sum and the second sum are

approximately equal, the subject is diagnosed with adenosquamous carcinoma. In further examples, the disclosed methods include detecting a first amount of CK5/6 protein in an NSCLC sample, detecting a second amount of TTFl protein in the sample, detecting a third amount of napsin A protein in the sample, and calculating a sum of the second amount and the third amount. If the first amount is greater than the sum of the second amount and the third amount, the subject is diagnosed with squamous cell carcinoma, whereas if the first amount is less than the sum of the second amount and the third amount, the subject is diagnosed with a non- squamous cell type lung carcinoma (such as adenocarcinoma or large cell carcinoma). In some examples, if the first amount is approximately equal to the sum of the second amount and the third amount, the subject is diagnosed with

adenosquamous carcinoma.

Determining (or detecting) the amount of a protein (such as serpin B13, CK5/6, TTFl, napsin A, or p63) includes measuring the quantity or concentration of a protein present in a sample. In some embodiments, the amount is measured by a semi-quantitative method (for example, by determining an H score) or a quantitative method. In other embodiments, the amount is measured qualitatively, for example by visually assessing the presence or intensity of staining or the color present in a sample (for example utilizing colorimetric detection methods). In some examples, determining the amount of a protein includes determining whether a protein is present or absent in a sample (for example, the sample is positive or negative for the protein).

The amount of each of the serpin B13, CK5/6, TTFl, napsin A, and/or p63 proteins in the sample can be detected by immunoassay methods, for example, immunohistochemistry (IHC), Western blotting, or ELISA. In some embodiments, the amount of each protein is detected by determining an H score (for example, determined by IHC) for each protein. The H score is a standard semi-quantitative value that describes the amount of immunoreactivity of a particular marker in a sample (e.g., an NSCLC sample). See, e.g., McClelland et al, Cancer Res.

50:3545-3550, 1990. An H score is determined by summing the products of the percentage of cells staining at a given staining intensity (0-100) in a sample (such as NSCLC sample, for example, an NSCLC tissue section) and the staining intensity (e.g., 0-3 or 0-4). Staining intensity is rated by a slide reader on a numeric scale (for example, a scale of 0-3, where 0 indicates no staining relative to background, 1 indicates weak staining, 2 indicates moderate staining, and 3 indicates strong staining; or a scale of 0-4 where 0 indicates no staining relative to background, 1 indicates weak staining, 2 indicates moderate staining, 3 indicates strong staining, and 4 indicates very strong staining).

In some examples, the methods include detecting an amount of each of serpin B 13, CK5/6, TTFl, and napsin A proteins in an NSCLC sample from a subject by determining an H score for serpin B 13 protein, determining an H score for CK5/6 protein, determining an H score for TTFl protein, and determining an H score for napsin A protein in the sample. A value is then calculated according to the following formula:

[serpin B 13 H score + CK5/6 H score] - [TTFl H score + napsin A H score]

In additional examples, the H score for TTFl is the H score for nuclear staining for TTFl and the H scores for serpin B 13, CK5/6 and napsin A are the H scores for cytoplasmic staining for each protein. A value is then calculated according to the following formula:

[serpin B 13 cytoplasmic H score + CK5/6 cytoplasmic H score] - [TTFl nucleus H score + napsin A cytoplasmic H score]

In some examples, if the value is greater than one, the subject is diagnosed with a squamous NSCLC (SQCC) and if the value is less than one, the subject is diagnosed with a non-squamous NSCLC (for example, ADC, large cell carcinoma, or undifferentiated carcinoma).

In other embodiments, the methods include detecting an amount of each of serpin B 13, CK5/6, TTFl, napsin A, and p63 proteins in an NSCLC sample from a subject by determining an H score for serpin B 13 protein, determining an H score for CK5/6 protein, determining an H score for p63 protein, determining an H score for TTFl protein, and determining an H score for napsin A protein in the sample. A value is then calculated according to the following formula:

[serpin B13 H score + CK5/6 H score + p63 H score] - [TTFl H score + napsin A H score]

In some examples, the H scores for TTFl and p63 are the H scores for nuclear staining for each protein and the H scores for serpin B13, CK5/6 and napsin A are the H scores for cytoplasmic staining for each protein. A value is then calculated according to the following formula:

[serpin B13 cytoplasmic H score + CK5/6 cytoplasmic H score + p63 nuclear H score] - [TTFl nucleus H score + napsin A cytoplasmic H score]

In some examples, if the calculated value is greater than one, the subject is diagnosed with a squamous NSCLC (SQCC) and if the value is less than one, the subject is diagnosed with a non-squamous NSCLC (for example, ADC, large cell carcinoma, or undifferentiated carcinoma).

In other embodiments, the methods include detecting an amount of each of CK5/6, TTFl, and napsin A proteins in an NSCLC sample from a subject by determining an H score for CK5/6 protein, determining an H score for TTFl protein, and determining an H score for napsin A protein in the sample. A value is then calculated according to the following formula:

[CK5/6 H score] - [TTFl H score + napsin A H score]

In some examples, the H score for TTFl is the H score for nuclear staining for TTFl protein and the H scores for CK5/6 and napsin A are the H scores for cytoplasmic staining for each protein. A value is then calculated according to the following formula: [CK5/6 cytoplasmic H score] - [TTFl nucleus H score + napsin A

cytoplasmic H score]

In some examples, if the calculated value is greater than one, the subject is diagnosed with a squamous NSCLC (SQCC) and if the value is less than one, the subject is diagnosed with a non-squamous NSCLC (for example, ADC, large cell carcinoma, or undifferentiated carcinoma).

In some examples, the calculated value (such as the value calculated by determining a quantity for each of serpin B13, CK5/6, TTFl, napsin A, and/or p63 protein, such as an H score for each protein) can predict with a sensitivity of at least

80% and a specificity of at least 80% for a non-squamous cell carcinoma, such as a sensitivity of at least 85%, at least 90%, and at least 95% (for example, 80%, 81%,

82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,

96%, 97%, 98%, 99% or 100%) and a specificity of at least of at least 80%, at least 85%, at least 90%, and at least 95% (for example, 81%, 82%, 83%, 84%, 85%, 86%,

87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or

100%).

In other embodiments, the amounts of serpin B13 protein and CK5/6 protein are detected by determining a first intensity of a first chromogen and the amounts of TTFl protein and napsin A protein are detected by determining a second intensity of a second chromogen. The first intensity and the second intensity are compared, and the subject is diagnosed with squamous cell type lung carcinoma if the first intensity is greater than the second intensity and the subject is diagnosed with non-squamous cell type lung carcinoma (for example ADC, large cell carcinoma, or

undifferentiated carcinoma) if the first intensity is less than the second intensity. In some examples, the subject is diagnosed with adenosquamous type lung carcinoma if the first intensity is approximately equal to the second intensity.

In further embodiments, determining the first intensity of the first chromogen further includes detecting the amount of p63 protein (in the same or a separate NSCLC sample from the subject). In some examples, the subject is diagnosed with squamous cell type lung carcinoma if the first intensity is greater than the second intensity and the subject is diagnosed with non-squamous cell type lung carcinoma if the first intensity is less than the second intensity. In other examples (such as when the amount of p63 protein is detected in a separate sample from the subject), the subject is diagnosed with squamous cell type lung carcinoma if the intensity of the first chromogen for p63 is positive (for example, detectable p63 expression) and the subject is diagnosed with non-squamous cell type lung carcinoma if the intensity of the first chromogen for p63 is negative (for example, no detectable p63 expression).

In other embodiments, the amount of CK5/6 protein is detected by determining a first intensity of a first chromogen and the amounts of TTF1 protein and napsin A protein are detected by determining a second intensity of a second chromogen. The first intensity and the second intensity are compared, and the subject is diagnosed with squamous cell type lung carcinoma if the first intensity is greater than the second intensity and the subject is diagnosed with non-squamous cell type lung carcinoma (for example, ADC or large cell carcinoma) if the first intensity is less than the second intensity. In some examples, the subject is diagnosed with adenosquamous type lung carcinoma if the first intensity is approximately equal to the second intensity.

In particular examples, the first chromogen produces a red color and the second chromogen produces a brown color. For example, the first chromogen can be Fast Red and the second chromogen can be DAB. In a particular example, IHC is performed according to standard methods, wherein serpin B13 and CK5/6 proteins are detected utilizing Fast Red chromogen and alkaline phosphatase (AP) and TTF1 and napsin A proteins are detected utilizing DAB chromogen and horseradish peroxidase (HRP). In other examples, IHC is performed according to standard methods, wherein serpin B13, CK5/6, and p63 proteins are detected using Fast Red chromogen and AP, and TTF1 and napsin A proteins are detected utilizing DAB chromogen and HRP. In further examples, IHC is performed according to standard methods, wherein CK5/6 protein is detected using Fast Red chromogen and AP, and TTF1 and napsin A proteins are detected utilizing DAB chromogen and HRP.

In some examples, comparing the intensities (such as comparing the first intensity and second intensity and determining if the first intensity is greater than the second intensity) can predict with a sensitivity of at least 80% and a specificity of at least 80% for a non- squamous cell carcinoma, such as a sensitivity of at least 85%, at least 90%, and at least 95% (for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) and a specificity of at least of at least 80%, at least 85%, at least 90%, and at least 95% (for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%).

In some examples, a subject known to have an NSCLC is pre-selected for diagnosis of lung cancer type using the methods disclosed herein. Methods for identifying a subject having an NSCLC are well known to one of skill in the art. For example, NSCLC can be identified based on morphologic evaluation of a lung cancer sample such as a histological section (for example, a section stained with hematoxylin and eosin). See, e.g., Travis et ah, WHO Classification of Tumours: Pathology and Genetics of Tumours of the Lung, Pleura, Thymus, and Heart, I ARC Press, Lyon, France, 2004.

///. Methods for Detecting Proteins

Samples, for example, NSCLC samples (such as an NSCLC sample from a subject) used in the methods described herein, such as a tissue or other biological sample, can be prepared using any method known in the art. NSCLC samples include any solid or fluid sample containing at least one NSCLC cell obtained from, excreted by or secreted by a subject. For example, an NSCLC sample can be a biological fluid obtained from, for example, blood, plasma, serum, saliva, sputum, bronchoalveolar lavage, pleural fluid, or any bodily secretion containing NSCLC cells. In some examples, an NSCLC sample includes a blood sample, for example, blood containing circulating tumor cells. An NSCLC sample can also be a tissue sample obtained from an NSCLC tumor (including a biopsy, fine needle aspirate, or autopsy specimen, such as a tumor biopsy or tumor core), a lymph node including NSCLC cells, or an NSCLC tumor metastasis. In other examples, an NSCLC sample is a tissue microarray (TMA) that includes two or more tissue samples (such as tumor cores) from NSCLC biopsies. Methods for making and using TMAs are well known in the art. See, e.g., Battifora, Lab. Invest. 55:244-248, 1986; Battifora and Mehta, Lab. Invest. 63:722-724, 1990; Kononen et al, Nature Med. 4:844-847, 1998. In particular embodiments, the NSCLC sample includes a tissue section (such as obtained by biopsy or fine-needle aspirate).

The samples described herein can be prepared using any method now known or hereafter developed in the art. Generally, tissue samples are prepared by fixing and embedding the tissue in a medium. In other examples, samples include a cell suspension which is prepared as a monolayer on a solid support (such as a glass slide) for example by smearing or centrifuging cells onto the solid support. In further examples, fresh frozen (for example, unfixed) tissue sections may be used in the methods disclosed herein.

In some examples an embedding medium is used. An embedding medium is an inert material in which tissues and/or cells are embedded to help preserve them for future analysis. Embedding also enables tissue samples to be sliced into thin sections. Embedding media include paraffin, celloidin, OCT™ compound, agar, plastics, or acrylics.

Many embedding media are hydrophobic; therefore, the inert material may need to be removed prior to histological or cytological analysis, which utilizes primarily hydrophilic reagents. The term deparaffinization or dewaxing is broadly used herein to refer to the partial or complete removal of any type of embedding medium from a biological sample. For example, paraffin-embedded tissue sections are dewaxed by passage through organic solvents, such as toluene, xylene, limonene, or other suitable solvents.

The process of fixing a sample can vary. Fixing a tissue sample preserves cells and tissue constituents in as close to a life-like state as possible and allows them to undergo preparative procedures without significant change. Fixation arrests the autolysis and bacterial decomposition processes that begin upon cell death, and stabilizes the cellular and tissue constituents so that they withstand the subsequent stages of tissue processing, such as for IHC.

Tissues can be fixed by any suitable process, including perfusion or by submersion in a fixative. Fixatives can be classified as cross-linking agents (such as aldehydes, e.g., formaldehyde, paraformaldehyde, and glutaraldehyde, as well as non-aldehyde cross-linking agents), oxidizing agents (e.g., metallic ions and complexes, such as osmium tetroxide and chromic acid), protein-denaturing agents (e.g., acetic acid, methanol, and ethanol), fixatives of unknown mechanism (e.g., mercuric chloride, acetone, and picric acid), combination reagents (e.g., Carnoy's fixative, methacarn, Bouin's fluid, B5 fixative, Rossman's fluid, and Gendre's fluid), microwaves, and miscellaneous fixatives (e.g., excluded volume fixation and vapor fixation). Additives may also be included in the fixative, such as buffers, detergents, tannic acid, phenol, metal salts (such as zinc chloride, zinc sulfate, and lithium salts), and lanthanum. The most commonly used fixative in preparing samples for IHC is formaldehyde, generally in the form of a formalin solution (4% formaldehyde in a buffer solution, referred to as 10% buffered formalin). In one example, the fixative is 10% neutral buffered formalin.

Antibodies specific for serpin B13, CK5/6, TTF1, napsin A, or p63 can be used for detection of each protein in an NSCLC sample by one of a number of immunoassay methods that are well known in the art, such as those presented in Harlow and Lane (Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York, 1988). Methods of constructing such antibodies are known in the art. In addition, such antibodies may be commercially available. Exemplary commercially available antibodies include those shown in Table 2. In another example, the serpin B13 antibody is a polyclonal serpin B13 antibody, such as the antibody disclosed in International Patent Publication WO 10/099923, incorporated herein by reference.

Table 2. Exemplary commercially available antibodies

Protein Source Catalog Number(s)

Ventana Medical Systems (Tucson, AZ) 790-4554; 760-4253

TTFl Abeam ab40880; ab76013;

ab82743; ab72876

Cell Marque 343M

Santa Cruz Biotechnology sc-25331; sc-8761; sc- 8762

Ventana Medical Systems 790-4398

Napsin A Abeam ab73021

Cell Marque 352A

Santa Cruz Biotechnology sc-50125

p63 Santa Cruz Biotechnology sc-8431

Abeam ab735

Ventana Medical Systems 790-4509

Any standard immunoassay format (such as ELISA, Western blot, or RIA assay) can be used to detect protein amounts in a sample. Thus, in one example, the presence and/or amount of serpin B13, CK5/6, TTFl, napsin A, and/or p63 protein in an NSCLC sample can readily be evaluated using these methods.

Immunohistochemical techniques can also be utilized for protein detection and/or quantification. General guidance regarding such techniques can be found in Bancroft and Stevens (Theory and Practice of Histological Techniques, Churchill Livingstone, 1982) and Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998). In some examples, serpin B13, CK5/6, TTFl, napsin A, and/or p63 proteins are detected by JJTC and an H score is determined for each protein.

In some examples, a sample is contacted with an antibody to a target protein (a "primary" antibody), such as an antibody specific for serpin B13, CK5/6, TTFl, napsin A, or p63. The primary antibody is then contacted with a secondary antibody raised against the primary antibody, such as a secondary antibody conjugated to an enzyme (for example, AP or HRP). The presence of the target protein is detected by contacting the antibody-enzyme conjugate with a chromogen and/or a substrate composition. In a particular example, the sample is contacted with an anti-serpin B13 antibody and an anti-CK5/6 antibody, a secondary antibody conjugated to alkaline phosphatase that can bind to both the anti-serpin B13 and the anti-CK5/6 antibodies, and Fast Red. The same sample or a separate sample from the same subject is contacted with an anti-TTFl antibody and an anti-napsin A antibody, a secondary antibody conjugated to horseradish peroxidase that can bind to both the anti-TTFl and anti-napsin A antibodies, and 3,3'-diaminobenzidine (DAB). In additional examples, the same sample or a separate sample from the same subject is also contacted with an anti-p63 antibody, a secondary antibody conjugated to alkaline phosphatase that can bind to the anti-p63 antibody, and Fast Red. In some examples, the intensity of the red (Fast Red; serpin B13, CK5/6, and/or p63) and brown (DAB; TTF1 and napsin A) signals are assessed qualitatively. In other examples, the red (Fast Red; serpin B13, CK5/6, and/or p63) and brown (DAB; TTF1 and napsin A) signals are assessed semi-quantitatively, for example, by determining an H score.

Quantitative spectroscopic methods, such as SELDI, can also be used to analyze serpin B13, CK5/6, TTF1, napsin A, and/or p63 protein expression in a sample (such as an NSCLC sample). In one example, surface-enhanced laser desorption-ionization time-of-flight (SELDI-TOF) mass spectrometry is used to detect protein expression, for example by using the ProteinChip™ (Ciphergen Biosystems, Palo Alto, CA). Such methods are well known in the art (for example see U.S. Pat. No. 5,719,060; U.S. Pat. No. 6,897,072; and U.S. Pat. No. 6,881,586). SELDI is a solid phase method for desorption in which the analyte (such as a protein) is presented to the energy stream on a surface that enhances analyte capture or desorption.

Briefly, one version of SELDI uses a chromatographic surface with a chemistry that selectively captures analytes of interest, such as serpin B13, CK5/6, TTF1, napsin A, and/or p63. Chromatographic surfaces can be composed of hydrophobic, hydrophilic, ion exchange, immobilized metal, or other chemistries. For example, the surface chemistry can include binding functionalities based on oxygen-dependent, carbon-dependent, sulfur-dependent, and/or nitrogen-dependent means of covalent or noncovalent immobilization of analytes. The activated surfaces are used to covalently immobilize specific "bait" molecules such as antibodies or other molecules that bind the analyte(s) of interest. The surface chemistry allows the bound analytes to be retained and unbound materials to be washed away. Subsequently, analytes bound to the surface (such as serpin B 13, CK5/6, TTF1, napsin A, and/or p63) can be desorbed and analyzed by any of several means, for example using mass spectrometry. When the analyte is ionized in the process of desorption, such as in laser desorption/ionization mass spectrometry, the detector can be an ion detector. Mass spectrometers generally include means for determining the time-of-flight of desorbed ions. This information is converted to mass. However, one need not determine the mass of desorbed ions to resolve and detect them: the fact that ionized analytes strike the detector at different times provides detection and resolution of them. Alternatively, the analyte can be detectably labeled (for example with a fluorophore or radioactive isotope). In these cases, the detector can be a fluorescence or radioactivity detector. A plurality of detection means can be implemented in series to fully interrogate the analyte components and function associated with retained molecules at each location in the array.

Therefore, in a particular example, the chromatographic surface includes, consists essentially of, or consists of antibodies that specifically bind serpin B 13, CK5/6, TTF1, and napsin A. In other examples, the chromatographic surface includes, consists essentially of, or consists of, antibodies that specifically bind serpin B 13, CK5/6, TTF1, napsin A, and p63. In further examples, the

chromatographic surface includes, consists essentially of, or consists of antibodies that specifically bind CK5/6, TTF1, and napsin A. In some examples, the chromatographic surface includes antibodies that bind other molecules, such as housekeeping proteins (e.g., β-actin or myosin).

A person of ordinary skill in the art will appreciate that embodiments of the methods disclosed herein for detection of one or more molecules, such as by immunohistochemistry, can be automated. Ventana Medical Systems, Inc. is the assignee of a number of United States patents disclosing systems and methods for performing automated analyses, including U.S. Patent Nos. 5,650,327; 5,654,200; 6,296,809; 6,352,861 ; 6,827,901 ; and 6,943,029, and U.S. Pat. Publication Nos. 2003/0211630 and 2004/0052685. In some embodiments, some or all of steps of the disclosed methods may be performed by automation, such as by an automated microscopy system. In particular examples, an automated method may include automatically imaging label(s) bound to proteins (such as by immunohistochemistry), automatically analyzing the image for the distribution and/or intensity of the label(s), and providing a result of the analysis (such as an H score). Such methods are known in the art, e.g., U.S. Pat. Publication Nos. 2003/0170703, 2006/0188140, and

2008/0213769; Cregger et al, Arch. Pathol. Lab. Med. 130: 1026-1030, 2006;

Stevens et ah, J. Mol. Diagn. 9: 144-150, 2007. In one example, the analysis may be performed using the Ventana Image Analysis System (VIAS, Ventana Medical Systems, Tucson, AZ).

IV. Treatment

In some embodiments, the methods disclosed herein include selecting a therapy for a subject with lung carcinoma (such as a NSCLC, for example a squamous cell carcinoma or a non- squamous cell carcinoma) and administering a therapeutically effective amount of the selected therapy to the subject.

In some examples of the methods disclosed herein, a therapy is selected for the subject following diagnosis of the lung carcinoma type (for example, squamous or non-squamous type). Examples of therapies include, but are not limited to, surgical treatment for removal or reduction of the tumor (such as surgical resection, cryotherapy, or chemoembolization), radiation therapy, and/or anti-tumor pharmaceutical treatments which can include radiotherapeutic agents, anti-neoplastic chemotherapeutic agents, antibiotics, alkylating agents and antioxidants, kinase inhibitors, and other agents.

After the lung carcinoma type (e.g., squamous or non-squamous) is determined, the selection of appropriate treatment for the specific cancer depends on a complex array of variables, including tumor site, tumor stage, tumor grade, patient treatment history, relative morbidity of various treatment options, patient performance and nutritional status, concomitant health problems, social and logistic factors, previous primary tumors, and patient preference. Treatment planning generally requires a multidisciplinary approach involving specialist surgeons and medical and radiation oncologists.

In some examples, the selected therapy includes one or more

chemo therapeutic agents. Chemo therapeutic agents include, but are not limited to alkylating agents, such as nitrogen mustards (for example, chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (for example, carmustine, fotemustine, lomustine, and strep tozocin), platinum compounds (for example, carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites, such as folic acid (for example, methotrexate, pemetrexed, and raltitrexed), purine (for example, cladribine, clofarabine, fludarabine, mercaptopurine, and tioguanine), pyrimidine (for example,

capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids, such as podophyllum (for example, etoposide, and teniposide), taxane (for example, docetaxel and paclitaxel), vinca (for example, vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic/antitumor antibiotics, such as anthracycline family members (for example, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, hydroxyurea, and mitomycin; topoisomerase inhibitors, such as topotecan and irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, and trastuzumab; photosensitizers, such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfin; and other agents , such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, bexarotene, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, pentostatin, masoprocol, mitotane, pegaspargase, and tretinoin.

Chemotherapeutic agents can be administered individually, or in combination. Selection and therapeutic dosages of such agents are known to those skilled in the art, and can be determined by a skilled clinician.

In a particular example, if the subject has (or had) an NSCLC tumor of squamous cell carcinoma type, the chemotherapeutic agent can include cisplatin or carboplatin, alone or in combination with one or more additional chemotherapeutic agent (such as docetaxel or etoposide). In other examples, the selected

chemotherapeutic agent can include one or more tyrosine kinase inhibitor, such as an epidermal growth factor receptor inhibitor (such as erlotinib, gefitinib, or lapatinib). See, e.g., Hirsch et al., J Thorac. Oncol. 3:1468-1481, 2008 and Rossi et al, Int. J. Surg. Pathol. 17:206-218, 2009.

In another example, if the subject has (or had) an NSCLC tumor of non- squamous type (such as adenocarcinoma, large cell carcinoma, or undifferentiated carcinoma), the chemotherapeutic agent can include cisplatin or carboplatin, alone or in combination with one or more additional chemotherapeutic agent (such as gemcitabine, etoposide, vincristine, platinum, taxane, epirubicin, cyclophosphamide, or methotrexate). In other examples, the selected chemotherapeutic agent can include one or more tyrosine kinase inhibitor, such as an epidermal growth factor receptor inhibitor (such as erlotinib, gefitinib, or lapatinib). In further examples, the chemotherapeutic agent can include bevacizumab or pemetrexed. See e.g., Hirsch et al., J Thorac. Oncol. 3: 1468-1481, 2008 and Rossi et al., Int. J. Surg. Pathol.

17:206-218, 2009.

In some examples, the disclosed methods include providing a therapeutically effective amount of the selected therapy (such as surgery, radiation therapy, and/or chemotherapeutics) to the subject. These agents (which are administered at a therapeutically effective amount) and treatments can be used alone or in

combination. Methods and therapeutic dosages of such agents and treatments are known to those skilled in the art, and can be determined by a skilled clinician.

The present disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

Example 1

Differentiation of NSCLC Using Multiplex Antibody Assay

Three tissue microarray (TMA) blocks were created using a TMArrayer™ semi-automated tissue arrayer (Pathology Devices, Inc., Westminster, MD).

Twenty-one adenocarcinoma non-small cell lung carcinoma (NSCLC) tissue cores were obtained for TMA1, twenty squamous cell carcinoma NSCLC cores were obtained for TMA2, and nineteen undifferentiated NSCLC cores were obtained for TMA3. Each core had a 1.5 mm diameter and was obtained from different areas of primary tumor from each human patient.

Immunohistochemical staining was performed on the Ventana BenchMark®

XT Automated Platform (Ventana Medical Systems, Tucson, AZ) using the

Ultra View™ DAB and Ultra View™ Universal Alkaline Phosphatase Red detection kits (Ventana Medical Systems). Antibodies utilized for target detection for IHC were anti-TTFl (clone 8G7G3/1; Cell Marque, Rocklin, CA), anti-CK5/6 (Clones D5 and 16B4; Cell Marque), anti-serpin B13 (Roche Diagnostics; International Patent Publication WO 10/099923), and anti-napsin A (clone KCG 1.1; Abeam, Cambridge, MA). The antibodies were combined into two cocktails; cocktail #1 contained anti-TTFl used as the diluent for anti-napsin A in a 1:200 dilution and cocktail #2 contained anti-CK5/6 used as the diluent for anti-serpin B 13 in a 1:2000 dilution. All analyses were performed on formalin fixed, paraffin embedded tissues, where serial 4 μιη thick tissue sections were cut from the TMA blocks. The tissue containing slides were heated overnight at 55°C and deparaffinized. Antigen retrieval was performed using Cell Conditioning 1 (CCl) solution (Ventana Medical Systems) for one hour. The antibody cocktails were applied to the treated TMA slides, both cocktails on the same slide, and the slides were incubated at 37°C for 32 minutes, followed by slide staining for target detection. Cocktail #1 was detected by DAB staining and Cocktail #2 was detected on the same slide by Alkaline

Phosphatase Red staining.

A pathologist assessed and scored each tissue. Results included H scoring for nucleus, cytoplasm and membranous staining when applicable (Table 3).

Cytoplasmic staining was scored for serpin B13, napsin A and CK5/6; nuclear staining was scored for and TTF1. The scoring algorithm for determining NSCLC type (non-squamous versus squamous) was: IF ([serpin B13 cytoplasmic H score] + [CK5/6 cytoplasmic H score]) - ([TTFl nuclear H score] + [napsin A cytoplasmic H score]) <1, THEN "non- squamous" ELSE "squamous" H&E differential diagnosis, which is the gold standard, was determined by a board of certified pathologists based on World Health Organization accepted classification/descriptions.

Table 3. NSCLC tumor type using H&E and Multiplex Methods

Serpin MPX

Tissue H&E H&E CK5/6 TTFl Napsin A

B13 value; Perf. No. Score Outcome H score H score H score

H score outcome

1 ADC -3, ADC 0 0 150 134 -284, NSCC True

2 ADC -3, ADC 0 0 145 130 -275, NSCC True

3 ADC -3, ADC 0 0 16 10 -26, NSCC True

4 ADC -3, ADC 0 0 0 0 0, NSCC True

5 ADC -3, ADC 0 0 145 180 -325, NSCC True

6 ADC -3, ADC 0 0 150 0 -150, NSCC True

7 ND 0, ND 5 0 35 0 -30

8 ND 0, ND 0 0 134 140 -274

9 ND 0, ND 0 0 124 40 -164

10 ADC -3, ADC 0 18 0 0 18, SQCC False

11 ADC -3, ADC 0 0 0 35 -35, NSCC True

12 ADC -3, ADC 5 0 134 200 -329, NSCC True

13 ADC -3, ADC 0 0 102 200 -302, NSCC True

14 ADC -3, ADC 0 0 135 210 -345, NSCC True

15 ND 0, ND 0 0 12 10 -22

16 ADC -3, ADC 20 0 130 170 -280, NSCC True

Insufficient

17 0 0 0 0 0 0 NA tumor

18 ADC -3, ADC 0 0 0 0 0, NSCC True

19 ADC -3, ADC 76 0 115 0 -39, NSCC True

20 ADC -3, ADC 5 0 0 0 5, SQCC False

21 SQCC 3, SQCC 15 195 0 0 210, SQCC True

22 No tumor 0 0 0 0 0 0 NA

23 SQCC 3, SQCC 35 145 0 0 180, SQCC True

24 SQCC 3, SQCC 0 0 0 0 0, NSCC False

25 SQCC 3, SQCC 35 252 4 0 283, SQCC True

26 SQCC 3, SQCC 25 290 7 0 308, SQCC True

27 ND 0, ND 40 300 7 0 333

28 SQCC 3, SQCC 120 0 0 0 120, SQCC True

29 SQCC 3, SQCC 80 17 0 0 97, SQCC True

30 ND 0, ND 0 293 6 0 287

31 SQCC 3, SQCC 35 10 25 0 20, SQCC True

32 SQCC 3, SQCC 75 135 0 0 210, SQCC True

33 SQCC 3, SQCC 125 250 0 0 375, SQCC True

34 SQCC 3, SQCC 45 267 0 0 312, SQCC True Serpin MPX

Tissue H&E H&E CK5/6 TTF1 Napsin A

B13 value; Perf. No. Score Outcome H score H score H score

H score outcome

35 SQCC 3, SQCC 130 197 0 0 327, SQCC True

36 ND 0, ND 40 0 95 0 -55

37 ND 0, ND 85 18 0 0 103

38 SQCC 3, SQCC 50 268 0 0 318, SQCC True

39 SQCC 3, SQCC 120 257 0 0 377, SQCC True

40 SQCC 3, SQCC 200 290 0 0 490, SQCC True

Poorly

41 differen. 0 0 0 0 0 0

NSCLC

Poorly

42 differen. 0 25 12 0 0 37

NSCLC

Poorly

43 differen. 1, SQCC 100 210 0 0 310, SQCC True SQCC

44 BAC 4, other 0 0 150 70 -220, NSCC

Spindle cell

tumor

45 mesothel4, other 17 0 0 0 17

ioma,

undifferen.

Melanoma,

46 4, other 1 0 115 165 -279

possible

Neuro¬

47 endocrine, 4, other 0 0 0 0 0

carcinoid

48 No tumor 0 0 0 0 0 0

SQCC,

49 moderately 2, SQCC 60 0 42 0 18, SQCC True differen.

SQCC,

50 poorly 1, SQCC 20 6 0 0 26, SQCC True differen.

51 No tumor 0 0 0 0 0 0

Mesothel¬

52 ioma or 4, other 0 287 0 0 287

melanoma

SQCC,

53 poorly 1, SQCC 3 266 0 0 269, SQCC True differen.

Poor tissue

quality, too

54 many 4, other 10 0 3 0 7

plasma

cells

55 Melanoma 4, other 35 0 132 0 -97

SQCC,

56 poorly 1, SQCC 0 16 0 0 16, SQCC True differen.

57 Undifferen. 0 15 0 7 0 8

58 Undifferen. 0 10 0 0 0 10 Serpin MPX

Tissue H&E H&E CK5/6 TTF1 Napsin A

B13 value; Perf. No. Score Outcome H score H score H score

H score outcome

59 Undifferen. 0 0 0 0 0 0

60 Undifferen. 0 0 0 0 0 0

ADC, adenocarcinoma; SQCC, squamous cell carcinoma; NSCC, non-squamous cell carcinoma; ND, not determined; NSCLC, non-small cell lung cancer; BAC, bronchoalveolar carcinoma

Of the 35 samples that were scored as ADC, SQCC or poorly differentiated SQCC by H&E staining, only three did not correlate when comparing the H&E staining to the scoring algorithm as described.

Example 2

Colorimetric Methods for Visual NSCLC Differentiation on Tissue

Microarrays

Assays were performed on TMAs as described in Example 1 to provide a visual means of differentiating squamous from non-squamous NSCLC. Assays were performed on a BenchMark® XT as previously described, except the

concentrations of the antibodies and incubation of the antibodies on the tissues were varied to provide for maximal detection by the detection assays utilized. Anti-TTFl and anti-napsin A antibodies were mixed and applied to fixed tissue samples. The two antibodies were detected utilizing an HRP/DAB enzymatic detection system (ultra View™ DAB, Ventana Medical Systems). On the same sample, a cocktail of anti-CK 5/6 and anti-Serpin B13 was subsequently applied and antibodies detected using an alkaline phosphatase/naphthol/Fast Red colorimetric assay (ultra View™ Alkaline Phosphatase Red, Ventana).

A number of different colorimetric detection systems and their combination with the assay antibodies were evaluated. However, the detection system

combination utilized in the present Example was shown to provide a reliable visual representation for differentiation of NSCLC which did not exist with the other combinations. For example, if the detection assays (HRP and AP) were switched, or different detection colors were utilized, background was a significant problem and the reliability of the assay was decreased. As a result, the developed assay system provided a dominant brown color deposition (DAB) for expression of TTF1 and napsin A that correlated to non- squamous cell carcinoma (FIGS 1A and 2A), while a dominant red color deposition (Fast Red) correlated to a squamous cell carcinoma (FIGS. 1C and 2C). FIGS. IB and 2B represent tissue samples that served as negative controls. This assay method thereby provides a fast and easy confirmation of a morphological diagnosis of either squamous or non- squamous NSCLC.

Example 3

NSCLC Differentiation: Multiplex Assay Plus p63

The utility of the multiplex assay described in Example 1 with the addition of p63 staining (AdenoSquamous 4+1 panel) to correctly diagnose the origin of NSCLC as either adenocarcinoma or squamous cell carcinoma was tested.

A lung TMA (LUC1503, Pantomics, Inc., Richmond, CA) consisting of 70 independent cases of various lung cancers with normal lung and other control tissues was utilized. The TMA was stained with either the multiplex panel (serpin B13, CK5/6, TTF1, and napsin A), p63, or H&E. Immunohistochemical staining was performed on the Ventana BenchMark® XT Automated Platform (Ventana Medical Systems, Tucson, AZ) using the Ultra View™ DAB and Ultra View™ Universal Alkaline Phosphatase Red detection kits (Ventana Medical Systems). Antibodies utilized for target detection for IHC were anti-TTFl (clone 8G7G3/1; Ventana Medical Systems, catalog number 790-4398), anti-CK5/6 (Clones D5 and 12eB4; Ventana Medical Systems, catalog number 790-4554), anti-serpin B13 (Roche Diagnostics; International Patent Publication WO 10/099923), anti-napsin A (clone KCG 1.1; Abeam, Cambridge, MA, catalog number ab73021), and anti-p63 (clone 4A4, Ventana Medical Systems, catalog number 790-4509). The antibodies were combined into two cocktails; cocktail #1 contained anti-TTFl used as the diluent for anti-napsin A in a 1: 1000 dilution and cocktail #2 contained anti-CK5/6 used as the diluent for anti-serpin B13 in a 1:2000 dilution. The anti-p63 antibody (0.140 μg/ml) was not diluted prior to use. One drop of the anti-p63 antibody was applied to the slide following the rinse step, resulting in an antibody concentration of about 0.035 μg/ml on the slide.

All analyses were performed on formalin fixed, paraffin embedded tissues, where serial 4 μιη thick tissue sections were cut from the TMA blocks. The tissue containing slides were heated overnight at 55°C and deparaffinized. Antigen retrieval was performed using Cell Conditioning 1 (CCl) solution (Ventana Medical Systems) for one hour. Cocktail #1 was applied to the treated TMA slides and incubated at 37°C for 32 minutes, followed by DAB staining for target detection. Cocktail #2 was then applied to the same slides, incubated at 37 °C for 32 minutes and detected by Alkaline Phosphatase Red staining. p63 staining was carried out on separate slides, incubated at 37 °C for 32 minutes and detected by Alkaline

Phosphatase Red staining. All slides were counterstained with Hematoxylin II for 4 minutes followed by Bluing for 4 minutes. Two pathologists independently scored the cases based on visual assessment as either adenocarcinoma (ADC), squamous cell carcinoma (SQCC) or adenosquamous carcinoma (ASC) using the multiplex assay plus p63, and compared the results to the H&E staining results.

Using the AdenoSquamous 4 +1 panel, well-differentiated cases were easily identified. Adenocarcinoma and squamous cell carcinoma were clearly detected by reactivity with TTFl and napsin A or serpin B13, CK5/6, and p63, and visualized by DAB (brown) or AP (red) detection, respectively. In addition, adenosquamous carcinomas showed distinct staining regions for each cell type. Besides clearly separated regions, there were also adeno- squamous variants with highly

intermingled cell populations, which would have been difficult to discriminate without the present IHC panel.

Several cases were reclassified based upon their staining patterns with the full panel of antibodies. After comparing the dual color multiplex (serpin B13, CK5/6, TTFl, and napsin A) and p63 results to the external diagnosis of ADC, SQCC or ASC based upon H&E staining, 37% of the cases were reclassified including ADC to SQCC or ASC, SQCC to ASC, and ASC to ADC (Table 4). The percent of cases reclassified was reduced to 13% when the data were compared to internal H&E scoring (Table 5). Staining with p63 was important for correct classification. Two ADC and two SQCC cases which were reclassified by p63 did not react with CK5/6 or serpin B13 (Tables 4 and 5). The loss of serpin B13 reactivity might be the result of its down-regulation, which has been reported for advanced squamous head and neck cancers.

Table 4. Reclassification of externally diagnosed NSCLCs (n=68)

'Detected by p63 only Table 5. Reclassification of internally diagnosed NSCLCs (n=68)

'Detected by p63 only

Example 4

Diagnosis of Lung Carcinoma Type

This example describes particular methods that can be used to diagnose the type of lung carcinoma (for example, non-squamous or squamous cell lung carcinoma) in a subject with NSCLC. However, one skilled in the art will appreciate that methods that deviate from these specific methods can also be used to successfully provide the diagnosis of non-squamous or squamous cell lung carcinoma in a subject with NSCLC.

An NSCLC sample (such as a tumor biopsy) is obtained from the mammalian subject, such as a human. Tissue samples are prepared for IHC, including deparaffinization and antigen retrieval and/or protease digestion. In one example, the sample, such as a tissue or cell sample present on a substrate (such as a microscope slide) is incubated with an anti-serpin B13 antibody, an anti-cytokeratin 5/6 antibody, an anti-thyroid transcription factor- 1 antibody, and an anti-napsin A antibody. The antibodies can be applied to the same sample (for example, a multiplex assay) or each antibody can be applied different samples from the same subject (for example, serial sections of a tumor sample). Binding of the antibodies to the sample is detected, for example, using microscopy.

A quantity for each protein (serpin B13, CK5/6, TTFl, and napsin A) is determined by calculating the H score for each protein. The H scores for serpin B13and TTFl are each calculated for nuclear staining with the respective antibodies. The H scores for CK5/6 and napsin A are each calculated for cytoplasmic staining with the respective antibodies. A value is calculated according to the following formula: [serpin B13 nuclear H score + CK5/6 cytoplasmic H score] - [TTFl nuclear H score + napsin A cytoplasmic H score]

In some examples, if the calculated value is greater than 0 (serpin B13 + CK5/6 is greater than TTFl + napsin A), the subject is diagnosed with squamous cell carcinoma and if the calculated value is less than 0 (serpin B13 + CK5/6 is less than TTFl + napsin A), the subject is diagnosed with non-squamous carcinoma (such as adenocarcinoma). In other examples, if the calculated value is greater than or equal to 1 the subject is diagnosed with squamous cell carcinoma, and if the calculated value is less than 1 the subject is diagnosed with non-squamous carcinoma (such as adenocarcinoma).

Example 5

Colorimetric Methods for Visual NSCLC Differentiation on Biopsy Specimens

This example describes particular methods that can be used to diagnose the type of lung carcinoma (for example, non-squamous or squamous cell lung carcinoma) in a subject with NSCLC. However, one skilled in the art will appreciate that methods that deviate from these specific methods can also be used to successfully provide the diagnosis of non-squamous or squamous cell lung carcinoma in a subject with NSCLC.

Assays can be performed on human or animal tissue biopsy specimens, cell blocks, etc. as described in Example 2, thereby providing a visual means of differentiating and diagnosing squamous from non-squamous NSCLC in direct surgical tissue samples.

Assays are performed, for example, on a BenchMark® XT as previously described, wherein the concentrations of the antibodies and incubation of the antibodies on the tissues are varied to provide for maximal detection by the detection assays utilized. Anti-TTFl and anti-nap sin A antibodies are mixed and applied to the fixed tissue samples. The two antibodies are detected utilizing an HRP/DAB enzymatic detection system. On the same sample, a cocktail of anti-CK 5/6 and anti-Serpin B13 is subsequently applied and antibodies detected using an alkaline phosphatase/Fast Red colorimetric assay. An anti-p63 antibody can optionally be applied to the same or a separate tissue sample and detected using an alkaline phosphatase/Fast Red detection system.

The results in patient samples (for example, surgical biopsy tissues) would be used to diagnose squamous versus non-squamous NSCLC, in that a dominant brown color deposition (DAB) indicative of expression of TTF1 and napsin-A is correlated to diagnosis of non-squamous cell carcinoma, while a dominant red color deposition (Fast Red) indicative of expression of serpin B13 and CK5/6 is correlated to diagnosis of squamous cell carcinoma. Expression of p63 (red color) is also correlated to diagnosis of squamous cell carcinoma.

Example 6

Three Marker Colorimetric Methods for Visual NSCLC Differentiation on

Biopsy Specimens

This example describes particular methods that can be used to diagnose the type of lung carcinoma (for example, non-squamous or squamous cell lung carcinoma) in a subject with NSCLC. However, one skilled in the art will appreciate that methods that deviate from these specific methods can also be used to successfully provide the diagnosis of non-squamous or squamous cell lung carcinoma in a subject with NSCLC.

Assays can be performed on human or animal tissue biopsy specimens, cell blocks, etc. as described in Example 2, thereby providing a visual means of differentiating and diagnosing squamous from non-squamous NSCLC in direct surgical tissue samples.

Assays are performed, for example, on a BenchMark® XT as previously described, wherein the concentrations of the antibodies and incubation of the antibodies on the tissues are varied to provide for maximal detection by the detection assays utilized. Anti-TTFl and anti-nap sin A antibodies are mixed and applied to the fixed tissue samples. The two antibodies are detected utilizing an HRP/DAB enzymatic detection system. On the same sample, anti-CK 5/6 is subsequently applied and detected using an alkaline phosphatase/Fast Red colorimetric assay.

The results in patient samples (for example, surgical biopsy tissues) would be used to diagnose squamous versus non-squamous NSCLC, in that a dominant brown color deposition (DAB) indicative of expression of TTF1 and napsin-A is correlated to diagnosis of non-squamous cell carcinoma, while a dominant red color deposition (Fast Red) indicative of expression of CK5/6 is correlated to diagnosis of squamous cell carcinoma.

In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A method for diagnosing lung carcinoma type in a non-small cell lung carcinoma sample from a subject, comprising:

detecting a first amount of serpin B13 protein in the sample;

detecting a second amount of cytokeratin 5 and/or 6 protein in the sample; detecting a third amount of thyroid transcription factor 1 protein in the sample;

detecting a fourth amount of nap sin A protein in the sample; and

comparing the first amount and the second amount with the third amount and the fourth amount, wherein the subject is diagnosed with squamous cell type lung carcinoma if the first amount and the second amount is greater than the third amount and the fourth amount, and the subject is diagnosed with non-squamous cell type lung carcinoma if the first amount and the second amount is less than the third amount and the fourth amount.

2. The method of claim 1, wherein detecting the first amount, detecting the second amount, detecting the third amount, and detecting the fourth amount comprises immunohistochemistry, Western blotting, or ELISA.

3. The method of claim 1 or claim 2, wherein detecting the first amount, detecting the second amount, detecting the third amount, and detecting the fourth amount are performed by an automated imaging system.

4. The method of any one of claims 1 to 3, wherein detecting the first amount comprises determining a first H score, detecting the second amount comprises determining a second H score, detecting the third amount comprises determining a third H score, and detecting the fourth amount comprises determining a fourth H score.

5. The method of claim 4, further comprising calculating a first sum of the first H score and the second H score and calculating a second sum of the third H score and the fourth H score, wherein the subject is diagnosed with squamous cell type lung carcinoma if the first sum is greater than the second sum and the subject is diagnosed with non-squamous cell type lung carcinoma if the first sum is less than the second sum.

6. The method of claim 5, further comprising subtracting the second sum from the first sum, thereby generating a value, wherein the subject is diagnosed with squamous cell type lung carcinoma if the value is greater than one and the subject is diagnosed with non-squamous cell type lung carcinoma if the value is less than one.

7. The method of any one of claims 1 to 3, wherein detecting the first amount and detecting the second amount comprises determining a first intensity of a first chromogen and detecting the third amount and detecting the fourth amount comprises determining a second intensity of a second chromogen, wherein the subject is diagnosed with squamous cell type lung carcinoma if the first intensity is greater than the second intensity and the subject is diagnosed with non-squamous cell type lung carcinoma if the first intensity is less than the second intensity.

8. The method of claim 7, wherein the first chromogen produces a red color and the second chromogen produces a brown color.

9. The method of claim 8, wherein the first chromogen is Fast Red and the second chromogen is 3,3'-diaminobenzidine.

10. The method of any one of claims 1 to 9, further comprising determining a fifth amount of p63 protein in the sample, wherein the subject is diagnosed with squamous cell type carcinoma if the fifth amount is positive, and the subject is diagnosed with non-squamous cell carcinoma if the fifth amount is negative.

11. The method of any one of claims 1 to 9, further comprising:

determining a fifth amount of p63 protein in the sample; and

comparing the first amount, the second amount, and the fifth amount with the third amount and the fourth amount, wherein the subject is diagnosed with squamous cell type lung carcinoma if the first amount, the second amount, and the fifth amount is greater than the third amount and the fourth amount, and the subject is diagnosed with non-squamous cell type lung carcinoma if the first amount, the second amount, and the fifth amount is less than the third amount and the fourth amount.

12. The method of claim 10 or claim 11, wherein determining the fifth amount comprises determining a fifth H score.

13. The method of claim 12, further comprising calculating a first sum of the first H score, the second H score, and the fifth H score, and calculating a second sum of the third H score and the fourth H score, wherein the subject is diagnosed with squamous cell type lung carcinoma if the first sum is greater than the second sum and the subject is diagnosed with non-squamous cell type lung carcinoma if the first sum is less than the second sum.

14. The method of claim 7, wherein determining the first intensity of the first chromogen further comprises determining a fifth amount of p63 protein in the sample, wherein the subject is diagnosed with squamous cell type lung carcinoma if the first intensity is greater than the second intensity and the subject is diagnosed with non-squamous cell type lung carcinoma if the first intensity is less than the second intensity.

15. A method for diagnosing lung carcinoma type in a non-small cell lung carcinoma sample from a subject, comprising:

determining a first H score for serpin B13 protein in the sample by immunohistochemistry with an anti- serpin B13 antibody; determining a second H score for cytokeratin 5 and/or 6 protein in the sample by immunohistochemistry with an anti-CK5/6 antibody;

determining a third H score for thyroid transcription factor 1 protein in the sample by immunohistochemistry with an anti-TTFl antibody;

determining a fourth H score for napsin A protein in the sample by immunohistochemistry with an anti-napsin A antibody;

calculating a first sum of the first H score and the second H score and calculating a second sum of the third H score and the fourth H score; and

subtracting the second sum from the first sum, thereby generating a value, wherein the subject is diagnosed with squamous cell type lung carcinoma if the value is greater than one and the subject is diagnosed with non-squamous cell type lung carcinoma if the value is less than one.

16. The method of claim 15, wherein the first H score is a cytoplasmic serpin B13 H score, the second H score is a cytoplasmic CK5/6 H score, the third H score is a nuclear TTFl H score, and the fourth H score is a cytoplasmic napsin A H score.

17. The method of claim 15 or claim 16, further comprising:

determining a fifth H score for p63 protein by immunohistochemistry with an anti-p63 antibody;

calculating a first sum of the first H score, the second H score, and the fifth H score and calculating a second sum of the third H score and the fourth H score; and

subtracting the second sum from the first sum, thereby generating a value, wherein the subject is diagnosed with squamous cell type lung carcinoma if the value is greater than one and the subject is diagnosed with non-squamous cell type lung carcinoma if the value is less than one.

18. A method for diagnosing lung carcinoma type in a non-small cell lung carcinoma sample from a subject, comprising: determining a first intensity of a first chromogen for serpin B13 protein and cytokeratin 5/6 protein in the sample, wherein the first chromogen is Fast Red; determining a second intensity of a second chromogen for TTF1 protein and napsin A protein in the sample, wherein the second chromogen is 3,3'- diaminobenzidine; and

comparing the first intensity and the second intensity, wherein the subject is diagnosed with squamous cell type lung carcinoma if the first intensity is greater than the second intensity and the subject is diagnosed with non-squamous cell type lung carcinoma if the first intensity is less than the second intensity.

19. The method of claim 18, wherein determining the first intensity further comprises determining the intensity of the first chromogen for p63 protein in the sample.

20. A method for diagnosing lung carcinoma type in a non-small cell lung carcinoma sample from a subject, comprising:

detecting a first amount of cytokeratin 5 and/or 6 protein in the sample; detecting a second amount of thyroid transcription factor 1 protein in the sample;

detecting a third amount of napsin A protein in the sample; and

comparing the first amount with the second amount and the third amount, wherein the subject is diagnosed with squamous cell type lung carcinoma if the first amount is greater than the second amount and the third amount, and the subject is diagnosed with non-squamous cell type lung carcinoma if the first amount is less than the second amount and the third amount.

21. The method of claim 20, wherein detecting the first amount, detecting the second amount, and detecting the third amount comprises

immunohistochemistry, Western blotting, or ELISA.

22. The method of claim 20 or claim 21, wherein detecting the first amount, detecting the second amount, and detecting the third amount are performed by an automated imaging system.

23. The method of any one of claims 20 to 22, wherein detecting the first amount comprises determining a first H score, detecting the second amount comprises determining a second H score, and detecting the third amount comprises determining a third H score.

24. The method of claim 23, further comprising calculating a sum of the second H score and the third H score, wherein the subject is diagnosed with squamous cell type lung carcinoma if the first H score is greater than the sum and the subject is diagnosed with non-squamous cell type lung carcinoma if the first H score is less than the sum.

25. The method of claim 24, further comprising subtracting the sum from the first H score, thereby generating a value, wherein the subject is diagnosed with squamous cell type lung carcinoma if the value is greater than one and the subject is diagnosed with non-squamous cell type lung carcinoma if the value is less than one.

26. The method of any one of claims 20 to 25, wherein detecting the first amount comprises determining a first intensity of a first chromogen and detecting the second amount and detecting the third amount comprises determining a second intensity of a second chromogen, wherein the subject is diagnosed with squamous cell type lung carcinoma if the first intensity is greater than the second intensity and the subject is diagnosed with non-squamous cell type lung carcinoma if the first intensity is less than the second intensity.

27. The method of claim 26, wherein the first chromogen produces a red color and the second chromogen produces a brown color.

28. The method of claim 27, wherein the first chromogen is Fast Red and the second chromogen is 3,3'-diaminobenzidine.

29. The method of any one of claims 1 to 28, wherein the non- squamous cell type lung carcinoma comprises adenocarcinoma or large cell carcinoma.

30. The method of any one of claims 1 to 29, wherein the sample comprises a tissue biopsy, fine needle aspirate, bronchoalveolar lavage, pleural fluid, or sputum.

31. The method of claim 30, wherein the tissue biopsy is a tissue section.

32. The method of any one of claims 1 to 31, wherein the sample is fixed and embedded in paraffin.

33. The method of any one of claims 1 to 32, further comprising selecting a therapy for the subject and administering a therapeutically effective amount of the therapy to the subject.