Ratio based biomarkers and methods of use thereof

A method of determining cancer prognosis for a subject with lung cancer, comprising:

• i) identifying at least two cancer associated proteins in a lung tumor sample from the subject;
• ii) quantifying the at least two cancer associated proteins in the sample, comprising:
• a) transferring proteins from the lung tumor sample to a stack of membranes;
• b) probing the stack of membranes with primary antibodies for detection of the at least two cancer associated proteins on the membranes;
• c) detecting with fluorescent secondary antibodies the primary antibodies bound to the at least two cancer associated proteins on the membranes;
• d) quantifying the signal intensity of the secondary antibodies to determine the quantity of the at least two cancer associated proteins;
• e) biotinylating total cellular proteins present in the membranes;
• f) incubating the membranes with a fluorescent secondary probe;
• g) detecting fluorescence of the secondary probe;
• and
• h) quantifying signal intensity of the fluorescence, thereby quantifying the total cellular proteins;
• iii) normalizing the at least two cancer associated proteins in the sample against the total cellular proteins to obtain a normalized value for each cancer associated protein in the sample;
• iv) comparing the normalized value of the first cancer associated protein with the normalized value of the second cancer associated protein to obtain a biomarker indicator wherein the biomarker indicator comprises a ratio of the normalized value of the first cancer associated protein to the normalized value of the second cancer associated protein;
• and
• v) correlating the biomarker indicator with prognosis of the subject with lung cancer;
• wherein the at least two cancer associated proteins comprise p-mTOR and p-AKT, and wherein the first cancer associated protein is p-mTOR and the second cancer associated protein is p-AKT,
• wherein the prognosis of the subject with lung cancer is improved when the biomarker indicator is higher than a subject with a lower level of the biomarker indicator.