WO2011149943A1 - Procédé de différenciation de carcinome pulmonaire à grandes cellules - Google Patents

Procédé de différenciation de carcinome pulmonaire à grandes cellules Download PDF

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WO2011149943A1
WO2011149943A1 PCT/US2011/037750 US2011037750W WO2011149943A1 WO 2011149943 A1 WO2011149943 A1 WO 2011149943A1 US 2011037750 W US2011037750 W US 2011037750W WO 2011149943 A1 WO2011149943 A1 WO 2011149943A1
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amount
score
subject
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lung carcinoma
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Fabien Gaire
Shalini Singh
Noemi Sebastiao
Maria R. Fischette
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Ventana Midical Systems, Inc.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung

Definitions

  • 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.
  • 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.
  • lung cancer 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.
  • SCLC small cell lung carcinoma
  • NSCLC non-small cell lung carcinoma
  • SQCC squamous cell carcinoma
  • ADC adenocarcinoma
  • 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.
  • NSCLC squamous cell carcinoma
  • SQCC 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).
  • ADC adenocarcinoma
  • 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
  • NSCLC histological subtype for example, squamous cell carcinoma or non-squamous cell carcinoma
  • squamous cell carcinoma may predict prognosis or treatment outcome for at least some chemotherapy drugs
  • a differential diagnosis is made by a pathologist based on observation of cellular morphology.
  • 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.
  • 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,
  • a squamous cell type lung carcinoma such as adenocarcinoma, large cell carcinoma, or undifferentiated carcinoma
  • an adenosquamous type lung carcinoma in the subject.
  • 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.
  • 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).
  • 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.
  • the first amount, second amount and fifth amount such as a combined first, second, and fifth amount
  • 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.
  • 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).
  • the disclosed methods include detecting the amount of cytokeratin 5 and/or cytokeratin 6, TTF1, and napsin A in an NSCLC sample.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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
  • 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.
  • AP/Fast Red alkaline phosphatase/Fast Red
  • 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.
  • HRP/DAB horseradish peroxidase/diaminobenzidine tetrahydrochloride
  • FIG. 2B shows background staining in a negative tissue control in the absence of serpin B13, CK 5/6, TTF1, or napsin A proteins.
  • 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.
  • AP/Fast Red alkaline phosphatase/Fast Red
  • ADC adenocarcinoma of lung
  • ASC adenosquamous lung carcinoma
  • CK5/6 cytokeratin 5 and/or 6
  • NSCLC non-small cell lung carcinoma
  • TTF1 thyroid transcription factor- 1 (NKX2-1)
  • NKX2-1 thyroid transcription factor- 1
  • ADC Adenocarcinoma
  • 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 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.
  • 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.
  • VH region and VL region are responsible for binding the antigen recognized by the antibody.
  • 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,
  • DAB diaminobenzidine
  • pNPP 4- nitrophenylphospate
  • BCIP bromochloroindolyl phosphate
  • NBT bromochloroindolyl phosphate
  • NBT nitro blue tetrazolium
  • BCIP/NBT nitro
  • TMB tetramethylbenzidine
  • ABTS 2,2'-azino-di-[3-ethylbenzothiazoline sulphonate]
  • ABTS 2,2'-azino-di-[3-ethylbenzothiazoline sulphonate]
  • ABTS 2,2'-azino-di-[3-ethylbenzothiazoline sulphonate]
  • ABTS 2,2'-azino-di-[3-ethylbenzothiazoline sulphonate]
  • ABTS 2,2'-azino-di-[3-ethylbenzothiazoline sulphonate]
  • ABTS 2,2'-azino-di-[3-ethylbenzothiazoline sulphonate]
  • ABTS 2,2'-azino-di-[3-ethylbenzothiazoline sulphonate]
  • ABTS 2,2
  • 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.
  • cytokeratin 5/6 (CK5/6) refers to cytokeratin 5 and/or 6.
  • Nucleic acid and protein sequences for CK5 and CK6 are publicly available.
  • GENBANK® Accession No. NM_000424 discloses an exemplary human CK5 nucleic acid sequence
  • GENBANK® Accession No. NP_000415 discloses an exemplary human CK5 protein sequence, both of which are
  • GENBANK® Accession Nos. NM_005554 (KRT6A), NM_005555 (KRT6B), and NM_173086 (KRT6C) disclose exemplary human CK6 nucleic acid sequences
  • 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
  • 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.
  • 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
  • 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).
  • 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 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
  • a sample including an 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).
  • 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.
  • NSCLC Non-small cell lung carcinoma
  • SQCC squamous cell carcinoma
  • ADC adenocarcinoma
  • ADC large cell 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.
  • 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.
  • samples 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.
  • a sample includes a control sample, such as a non-NSCLC cell or tissue sample.
  • Serpin B13 Serpin peptidase inhibitor, clade B, member 13; also known as proteinase inhibitor 13, HaCaT UV-repressible serpin (hurpin), or headpin
  • Serpin B13 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).
  • serpins are protease inhibitors that use a conformational change to inhibit target enzymes, mainly serine proteases.
  • serpin B13 was reported to inhibit cathepsin K and L (Weiss et ah, Biochemistry 42:7381-7389, 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
  • 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 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
  • lung carcinoma such as non-small cell lung carcinoma
  • TTF1 Thyroid transcription factor-1
  • NKX2-1 Thyroid transcription factor-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.
  • TTF1 controls expression of genes such as thyroglobulin and thyrotropin receptor.
  • 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.
  • TTF1 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.
  • 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.
  • 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.
  • the methods further include detecting the amount of p63 protein in an NSCLC sample.
  • NSCLC for example, squamous cell or non-squamous cell carcinoma
  • 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
  • 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.
  • the first amount, second amount and fifth amount such as a combined first, second, and fifth amounts
  • 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.
  • 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).
  • the disclosed methods include detecting the amount of CK5/6, TTFl, and napsin A in an NSCLC sample.
  • 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.
  • 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 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.
  • the amounts of serpin B13, CK5/6, TTFl, napsin A, and p63 are detected in the same NSCLC sample from the subject.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the amount of TTF1 protein is the amount of TTF1 protein that is localized to the nucleus in the NSCLC sample.
  • the amount of napsin A protein is the amount of napsin A protein that has a cytoplasmic localization in the NSCLC sample.
  • the amount of p63 protein is the amount of p63 protein that is localized to the nucleus in the NSCLC sample.
  • 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.
  • the disclosed methods further include detecting a fifth amount of p63 protein in an NSCLC sample.
  • 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.
  • 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
  • the subject is diagnosed with adenosquamous carcinoma.
  • 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
  • Determining (or detecting) the amount of a protein includes measuring the quantity or concentration of a protein present in a sample.
  • the amount is measured by a semi-quantitative method (for example, by determining an H score) or a quantitative method.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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).
  • 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:
  • 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:
  • 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).
  • SQLCC squamous NSCLC
  • non-squamous NSCLC for example, ADC, large cell carcinoma, or undifferentiated carcinoma
  • 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:
  • 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:
  • 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).
  • SQLCC squamous NSCLC
  • non-squamous NSCLC for example, ADC, large cell carcinoma, or undifferentiated carcinoma
  • 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:
  • 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
  • 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).
  • SQLCC squamous NSCLC
  • non-squamous NSCLC for example, ADC, large cell carcinoma, or undifferentiated carcinoma
  • 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%,
  • 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
  • the subject is diagnosed with adenosquamous type lung carcinoma if the first intensity is approximately equal to the second intensity.
  • 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).
  • 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.
  • 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).
  • 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.
  • the subject is diagnosed with adenosquamous type lung carcinoma if the first intensity is approximately equal to the second intensity.
  • the first chromogen produces a red color and the second chromogen produces a brown color.
  • the first chromogen can be Fast Red and the second chromogen can be DAB.
  • 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).
  • 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.
  • 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.
  • comparing the intensities 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%).
  • 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.
  • 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.
  • NSCLC samples such as an NSCLC sample from a subject
  • NSCLC samples 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.
  • 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.
  • 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.
  • 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.
  • the NSCLC sample includes a tissue section (such as obtained by biopsy or fine-needle aspirate).
  • tissue samples are prepared using any method now known or hereafter developed in the art.
  • tissue samples are prepared by fixing and embedding the tissue in a medium.
  • 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.
  • fresh frozen (for example, unfixed) tissue sections may be used in the methods disclosed herein.
  • 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, OCTTM compound, agar, plastics, or acrylics.
  • 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.
  • 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
  • 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).
  • 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.
  • 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.
  • any standard immunoassay format (such as ELISA, Western blot, or RIA assay) can be used to detect protein amounts in a sample.
  • ELISA electrospray based assay
  • CK5/6 CK5/6
  • TTFl napsin A
  • p63 protein p63 protein
  • 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.
  • 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.
  • a target protein 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).
  • an enzyme for example, AP or HRP
  • 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).
  • DAB 3,3'-diaminobenzidine
  • 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.
  • an anti-p63 antibody a secondary antibody conjugated to alkaline phosphatase that can bind to the anti-p63 antibody
  • Fast Red 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.
  • 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 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).
  • a sample such as an NSCLC sample.
  • surface-enhanced laser desorption-ionization time-of-flight (SELDI-TOF) mass spectrometry is used to detect protein expression, for example by using the ProteinChipTM (Ciphergen Biosystems, Palo Alto, CA).
  • 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.
  • 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.
  • 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.
  • analytes bound to the surface can be desorbed and analyzed by any of several means, for example using mass spectrometry.
  • analytes bound to the surface such as serpin B 13, CK5/6, TTF1, napsin A, and/or p63
  • 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.
  • the analyte can be detectably labeled (for example with a fluorophore or radioactive isotope).
  • 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.
  • the chromatographic surface includes, consists essentially of, or consists of antibodies that specifically bind serpin B 13, CK5/6, TTF1, and napsin A.
  • the chromatographic surface includes, consists essentially of, or consists of, antibodies that specifically bind serpin B 13, CK5/6, TTF1, napsin A, and p63.
  • the chromatographic surface includes, consists essentially of, or consists of antibodies that specifically bind serpin B 13, CK5/6, TTF1, napsin A, and p63.
  • chromatographic surface includes, consists essentially of, or consists of antibodies that specifically bind CK5/6, TTF1, and napsin A.
  • the chromatographic surface includes antibodies that bind other molecules, such as housekeeping proteins (e.g., ⁇ -actin or myosin).
  • 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.
  • some or all of steps of the disclosed methods may be performed by automation, such as by an automated microscopy system.
  • 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
  • the analysis may be performed using the Ventana Image Analysis System (VIAS, Ventana Medical Systems, Arlington, AZ).
  • VIAS Ventana Image Analysis System
  • 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.
  • a therapy for a subject with lung carcinoma such as a NSCLC, for example a squamous cell carcinoma or a non- squamous cell carcinoma
  • a therapy is selected for the subject following diagnosis of the lung carcinoma type (for example, squamous or non-squamous type).
  • 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.
  • 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.
  • the selected therapy includes one or more
  • 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
  • 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 trastuzuma
  • monoclonal antibodies such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, and trastuzuma
  • 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.
  • the chemotherapeutic agent can include cisplatin or carboplatin, alone or in combination with one or more additional chemotherapeutic agent (such as docetaxel or etoposide).
  • the selected chemotherapeutic agent can include cisplatin or carboplatin, alone or in combination with one or more additional chemotherapeutic agent (such as docetaxel or etoposide).
  • chemotherapeutic agent can include one or more tyrosine kinase inhibitor, such as an epidermal growth factor receptor inhibitor (such as erlotinib, gefitinib, or lapatinib).
  • 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.
  • 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).
  • 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).
  • 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.
  • the disclosed methods include providing a therapeutically effective amount of the selected therapy (such as surgery, radiation therapy, and/or chemotherapeutics) to the subject.
  • a therapeutically effective amount of the selected therapy such as surgery, radiation therapy, and/or chemotherapeutics
  • agents which are administered at a therapeutically effective amount
  • treatments can be used alone or in
  • TMA tissue microarray
  • NSCLC non-small cell lung carcinoma
  • 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).
  • 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 ⁇ 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.
  • CCl Cell Conditioning 1
  • 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
  • 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 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.
  • ADC adenocarcinoma
  • SQCC squamous cell carcinoma
  • NSCC non-squamous cell carcinoma
  • ND not determined
  • NSCLC non-small cell lung cancer
  • BAC bronchoalveolar carcinoma
  • 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 ViewTM 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 ViewTM Alkaline Phosphatase Red, Ventana).
  • 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).
  • DAB brown color deposition
  • Fast Red red color deposition
  • 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 1 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, Arlington, AZ) using the Ultra ViewTM DAB and Ultra ViewTM 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).
  • 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 ⁇ g/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 ⁇ g/ml on the slide.
  • ADC adenocarcinoma
  • SQCC squamous cell carcinoma
  • ASC adenosquamous carcinoma
  • 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.
  • adenosquamous carcinomas showed distinct staining regions for each cell type. Besides clearly separated regions, there were also adeno- squamous variants with highly
  • 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.
  • lung carcinoma for example, non-squamous or squamous cell lung carcinoma
  • 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.
  • 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.
  • 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 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]
  • 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).
  • 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.
  • lung carcinoma for example, non-squamous or squamous cell lung carcinoma
  • 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.
  • 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.
  • results in patient samples 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.
  • DAB brown color deposition
  • Flust Red red color deposition
  • p63 red color
  • 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.
  • lung carcinoma for example, non-squamous or squamous cell lung carcinoma
  • 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.
  • results in patient samples 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.
  • DAB brown color deposition
  • Fast Red a dominant red color deposition indicative of expression of CK5/6

Abstract

L'invention concerne des procédés de diagnostic de types de carcinome pulmonaire chez un patient par la différenciation de sous-types de carcinome pulmonaire à grandes cellules (NSCLC), par exemple par le diagnostic d'un carcinome pulmonaire de type à cellules squameuses ou d'un carcinome pulmonaire de type non squameux chez le patient. Dans certains modes de réalisation, les procédés comprennent la détermination d'une première quantité de protéine serpine B13 dans l'échantillon, la détermination d'une seconde quantité de protéine cytokératine 5 et/ou cytokératine 6 dans l'échantillon, la détermination d'une troisième quantité de protéine facteur 1 de transcription thyroïdien dans l'échantillon et la détermination d'une quatrième quantité de protéine napsine A dans un échantillon de NSCLC. Dans certains modes de réalisation, les procédés comprennent en outre la détermination d'une cinquième quantité de protéine p63 dans un échantillon de NSCLC. Dans encore d'autres modes de réalisation, les procédés comprennent la détermination d'une première quantité de protéine cytokératine 5 et/ou cytokératine 6 dans l'échantillon, la détermination d'une seconde quantité de protéine facteur 1 de transcription thyroïdien dans l'échantillon et la détermination d'une troisième quantité de protéine napsine A dans un échantillon de NSCLC.
PCT/US2011/037750 2010-05-24 2011-05-24 Procédé de différenciation de carcinome pulmonaire à grandes cellules WO2011149943A1 (fr)

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EP2971284A4 (fr) * 2013-03-15 2017-01-18 HTG Molecular Diagnostics, Inc. Sous-typage des cancers du poumon
WO2020243733A1 (fr) * 2019-05-30 2020-12-03 Sakura Finetek Usa, Inc. Détection d'antigènes dans un échantillon biologique à l'aide de réactifs à base d'anticorps
EP4010021A4 (fr) * 2019-08-06 2023-08-30 Regents of the University of Minnesota Compositions et méthodes de traitement de troubles liés à la serpine b13

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013173627A1 (fr) 2012-05-16 2013-11-21 Expression Pathology, Inc. Méthode srm/mrm pour sous-typage histologique des tumeurs pulmonaires
US9261506B2 (en) 2012-05-16 2016-02-16 Expression Pathology, Inc. SRM/MRM assay for subtyping lung histology
US9470687B2 (en) 2012-05-16 2016-10-18 Expression Pathology, Inc. SRM/MRM assay for subtyping lung histology
US9766246B2 (en) 2012-05-16 2017-09-19 Expression Pathology, Inc. SRM/MRM assay for subtyping lung histology
EP2971284A4 (fr) * 2013-03-15 2017-01-18 HTG Molecular Diagnostics, Inc. Sous-typage des cancers du poumon
WO2020243733A1 (fr) * 2019-05-30 2020-12-03 Sakura Finetek Usa, Inc. Détection d'antigènes dans un échantillon biologique à l'aide de réactifs à base d'anticorps
EP4010021A4 (fr) * 2019-08-06 2023-08-30 Regents of the University of Minnesota Compositions et méthodes de traitement de troubles liés à la serpine b13

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