Expression Pathology Incorporated

The United States of America, As Represented by the Secretary, Department of Health and Human Services

USA As Represented by Secretary of the Navy

Nantomics, LLC

Expresssion Pathology, Inc.

Paul D. Smith

Lance A. Liotta

Seth R. Goldstein

Tom Pohida

W. Marston Linehan

Rodrigo Chuaqui

John I. Peterson

Michael Emmert-buck

Jeffrey M. Trent

David B. Krizman

David B Krizman

Todd Hembrough

Sheeno Thyparambil

Wei-Li Liao

Marlene M Darfler

Eunkyung An

Lance A Liotta

Paul D Smith

Michael R Emmert-Buck

Robert F Bonner

Seth R Goldstein

John I Peterson

W Marston Linehan

Rodrigo F Chuaqui

Jeffry M Trent

Bradley R Ringeisen

Adele Blackler

Wei-Liao Liao

R Andrew McGill

Douglas B Chrisey

Alberto Pique

Peter K Wu

Jason Barron

Wei-Li Lao

Michael Emmert-Buck

Jeffrey M Trent

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

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 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

This disclosure provides ten (10) specific peptides, and particular peptide characteristics, from the cell membrane-bound Her2 protein and a diagnostic assay useful for determining the presence and amount of full length and truncated versions of the full-length Her2 protein in cells derived from formalin fixed paraffin embedded tissue.

Truncated Her2 SRM/MRM assay

Methods are provided for directly converting histopathologically processed biological samples, tissues, and cells into a multiuse biomolecule lysate. This method allows for simultaneous extraction, isolation, solubilization, and storage of all biomolecules contained within the histopathologically processed biological sample, thereby forming a representative library of said sample. This multi-use biomolecule lysate is dilutable, soluble, capable of being fractionated, and used in biochemical assays.

Liquid tissue preparation from histopathologically processed biological samples, tissues and cells

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

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

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.

SRM/MRM assay for the ephrin type-A receptor 2 protein

Peptides from the tyrosine-protein kinase receptor UFO protein (AXL) are provided 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)/Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed and include 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 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.

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

Methods are provided for quantifying the Androgen receptor protein (AR) protein directly in biological samples that have been fixed in formalin, using Selected Reaction Monitoring (SRM)/Multiple Reaction Monitoring (MRM) mass spectrometry. The biological samples are chemically preserved and fixed and can be, for example, tissues 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 sample is prepared from said biological sample using, for example, the Liquid Tissue protocol and the AR 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.

SRM/MRM assay for the androgen receptor (AR) protein

Methods are provided for detecting and quantifying the Mesothelin protein (MSLN) in biological samples that have been fixed in formalin using Selected Reaction Monitoring (SRM)/Multiple Reaction Monitoring (MRM) mass spectrometry. The biological sample may be, for example, 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 and the MSLN protein is quantitated by SRM/MRM mass spectrometry by quantitating one or more MSLN fragment peptides in the protein sample.

SRM/MRM assay for the mesothelin (MSLN) protein

The current disclosure provides methods for detecting and quantitating the 6-O-methylguanine-DNA methyltransferase protein (MGMT) 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 with formaldehyde containing agents/fixatives and may include 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 and the MGMT protein is quantitated in the sample using SRM/MRM mass spectrometry by quantitating one or more fragment peptides.

SRM/MRM assay for the 6-O-methylguanine-DNA methyltransferase (MGMT) protein

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Gaithersburg, MD, United States of America

David B Krizman