Expression Pathology Incorporated

Nantomics, LLC

Seoul National University

The University of Chicago

West Virginia University

Expresssion Pathology, Inc.

Todd Hembrough

David B Krizman

Sheeno Thyparambil

Wei-Li Liao

Fabiola Cecchi

Eunkyung An

Adele Blackler

Dongyao Yan

Yung-Jue Bang

Manish Monga

Sarit Schwartz

Kerry Scott

Paolo Nuciforo

Daniel Catenacci

Do-Youn Oh

Jean-Charles Soria

Wei-Li Lao

Wei-Liao Liao

The current disclosure provides specific peptides, and derived ionization characteristics of the peptides from the estrogen receptor (ER), progesterone receptor (PR), and/or antigen Ki67 (Ki67) proteins that are particularly advantageous for quantifying the ER, PR, and/or Ki67 proteins directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring/Multiple Reaction Monitoring (SRM/MRM) mass spectrometry. Such biological samples are chemically preserved and fixed wherein the biological sample is selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from a biological sample using the Liquid Tissue&#x2122; reagents and protocol, and the ER, PR, and/or Ki67 proteins are quantitated in the Liquid Tissue&#x2122; sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the peptides described for one or more of the ER, PR, and/or Ki67 proteins. These peptides can be quantitated if they reside in a modified or in an unmodified form. An example of a modified form of an ER, PR, and/or Ki67 peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

Multiplex MRM assay for evaluation of cancer

Improved methods for treating cancer are provided herein by determining if a cancer patient, particularly a colon cancer patient or a gastric cancer patient, will clinically respond in a favorable manner to a therapeutic strategy comprising the FOLFOX regimen (fluorouracil, leucovorin, and oxaliplatin) or a combination of capecitabine and cisplatin. Diagnostic methods for measuring the OPRT, TYMP, and/or UCK2 proteins in a tissue sample, such as a tumor sample, from the patient are provided.

OPRT expression and cancer treatment outcome

Methods are provided herein for identifying whether a cancer patient, for example a colorectal cancer patient, will be responsive to treatment with a therapeutic strategy comprising administration of the FOLFOX regimen (5-fluorouracil, leucovorin, and oxaliplatin). Specified TYMP and UCK2 fragment peptides are precisely detected and quantitated by SRM-mass spectrometry directly in tumor cells, for example colorectal cancer tumor cells, that are collected from tumor tissue obtained from a cancer patient and compared to reference levels in order to determine if the cancer patients will positively respond to treatment with the combination treatment of FOLFOX (5-fluorouracil, leucovorin, and oxaliplatin).

Predicting optimal chemotherapy for CRC

Methods are provided for treating a cancer patient suffering from a glioblastoma (GBM) by administering to the patient an effective amount of temozolomide, wherein a mass spectrometry analysis of a protein digest of a formalin-fixed tumor sample from the patient evidences an amount of a MGMT fragment peptide less than or substantially equal to 150 amol/&#x3bc;g.

Quantifying MGMT protein for optimal cancer therapy of glioblastoma

The current disclosure provides for specific peptides, and derived ionization characteristics of the peptides, from the tyrosine-protein kinase receptor UFO protein (AXL) that are particularly advantageous for quantifying the AXL protein directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring (SRM) mass spectrometry, or what can also be termed as Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed wherein said biological sample is selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from said biological sample using the Liquid Tissue reagents and protocol and the AXL protein is quantitated in the Liquid Tissue sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the peptides described. These peptides can be quantitated if they reside in a modified or an unmodified form. An example of a modified form of an AXL peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

SRM/MRM assay for the tyrosine-protein kinase receptor UFO(AXL) protein

Methods are provided for identifying whether a lung tumor will be responsive to treatment with the combination of the therapeutic agents cisplatin and pemetrexed. Specified ERCC1, TS, p16, and FR&#x3b1; fragment peptides are precisely detected and quantitated by SRM-mass spectrometry directly in lung tumor cells collected from lung tumor tissue that was obtained from a cancer patient and compared to reference levels in order to determine if the lung cancer patient will positively respond to treatment with the combination of cisplatin and pemetrexed therapeutic agents.

Methods of treating lung cancer by predicting responders to cisplatin-pemetrexed combination therapy

Methods are provided for quantifying the cyclin-dependent kinase inhibitor 2A protein (p16) p16 protein directly in biological samples that have been fixed in formalin by SRM/MRM mass spectrometry. A protein sample is prepared from the biological sample using, for example, the Liquid Tissue reagents and protocol and the p16 protein is quantitated in the resulting sample by quantitating in the protein sample at least one fragment peptide from p16. Peptides can be quantitated in modified or unmodified form. An example of a modified form of a p16 peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

SRM/MRM assay for the cyclin-dependent kinase inhibitor 2A (p16) protein

Methods are provided for quantifying the fibroblast growth factor receptor 2 protein (FGFR2) directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring (SRM)/Multiple Reaction Monitoring (MRM) mass spectrometry. The biological sample may be selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells. A protein sample is prepared from the biological sample and the FGFR2 protein is quantitated in the sample using the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one fragment peptide derived from FGFR2.

SRM/MRM assay for the fibroblast growth factor receptor 2 (FGFR2) protein

Methods are provided for quantifying specific proteins directly in biological samples that have been fixed in formalin by SRM/MRM assay. Such biological samples are chemically preserved and fixed wherein said biological sample is selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks. A protein digest is prepared from the biological sample using, for example, the Liquid Tissue reagents and protocol and a designated protein is quantitated in the digest sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the described peptides. The proteins that can be detected and/or quantitated are CD3D, B7H3, B7-2, STAT1, GBP1, GPNMB, CD27, CD3E, and CD8.

SRM/MRM assays for cancer

Methods of treating breast cancer are provided where a quantitative Her2 assay is used to identify whether a breast tumor will be responsive to treatment with anti-Her2 therapeutic agents such as lapatinib and trastuzumab, followed by selection of a suitable treatment regimen and administration of the regimen. A specific Her2 fragment peptide is precisely quantitated by SRM-mass spectrometry directly in breast tumor cells collected from breast tumor tissue that was obtained from a cancer patient and compared to a reference level in order to determine if the breast cancer patient will positively respond to treatment with a therapeutic agent that specifically targets the Her2 protein.

Methods for treating Her2-positive breast cancer

Specific peptides are provided, and derived ionization characteristics of those peptides, from the Hepatocyte Growth Factor Receptor (cMET) protein. The peptides are particularly and surprisingly advantageous for quantifying by the method of Selected Reaction Monitoring (SRM) mass spectrometry the cMET protein directly in biological samples that have been fixed in formalin, or what can also be termed as Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed where the biological sample is selected from tissues and cells treated with formaldehyde containing agents/fixatives including: formalin-fixed tissue/cells; formalin-fixed/paraffin embedded (FFPE) tissue/cells; FFPE tissue blocks and cells from those blocks; and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from the biological sample using the Liquid Tissue&#x2122; reagents and protocol and the cMET protein is quantitated in the Liquid Tissue&#x2122; sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the peptides described. These peptides can be quantitated if they reside in a modified or an unmodified form. An example of a modified form of a cMET peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

CMET protein SRM/MRM assay

Methods are provided for treating a gastric cancer patient. A specific Met fragment peptide is precisely quantitated by SRM-mass spectrometry directly in gastric tumor cells collected from gastric tumor tissue that was obtained from the cancer patient and compared to a reference level. If the Met peptide is below the reference level a second therapeutic regimen is used to treat the patient whereas if the Met peptide is above the reference level then a first therapeutic regimen combining, for example, the second regimen with one or more Met inhibitor therapeutic agents may be used to treat the patient.

Quantifying met protein for cancer treatment

Specific peptides, and derived ionization characteristics of those peptides, from the Bcl-2-like protein 11 (BIM) are provided that are particularly advantageous for quantifying the BIM protein directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring (SRM) mass spectrometry, or what can also be termed as Multiple Reaction Monitoring (MRM). Such biological samples are chemically preserved and fixed where the biological sample is selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from the biological sample using the Liquid Tissue&#x2122; reagents and protocol, and the BIM protein is quantitated in the Liquid Tissue&#x2122; sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the peptides described. These peptides can be quantitated if they reside in a modified or an unmodified form. An example of a modified form of a BIM peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

Bcl-2-like protein 11 SRM/MRM assay

Improved methods of treatment are provided for patients suffering from cancer. The methods identify whether a tumor will be responsive to treatment with a therapeutic regime that includes anti-Her2 therapeutic agents. A specific Her2 fragment peptide is precisely quantitated by SRM-mass spectrometry directly in tumor cells collected from tumor tissue that was obtained from a cancer patient and compared to a reference level in order to determine if the cancer patient will positively respond to treatment with a therapeutic agent that specifically targets the Her2 protein.

Quantifying Her2 protein for optimal cancer therapy

Specific peptides, and derived ionization characteristics of the peptides, from the Ephrin Type-A Receptor 2 (EPHA2) protein are provided that are particularly advantageous for quantifying the EPHA2 protein directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring (SRM) mass spectrometry, or what can also be termed as Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed and are selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from said biological sample using the Liquid Tissue&#x2122; reagents and protocol and the EPHA2 protein is quantitated in the Liquid Tissue&#x2122; sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the peptides described. These peptides can be quantitated if they reside in a modified or an unmodified form. An example of a modified form of an EPHA2 peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

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Gaithersburg, MD, United States of America

Todd Hembrough