EP3630187A1

EP3630187A1 - Predicting gastric cancer treatment outcome

Info

Publication number: EP3630187A1
Application number: EP18809143.3A
Authority: EP
Inventors: Todd Hembrough; Fabiola CECCHI; Sarit SCHWARTZ; Christina Yau
Original assignee: Expression Pathology Inc
Current assignee: Expression Pathology Inc
Priority date: 2017-06-02
Filing date: 2018-06-04
Publication date: 2020-04-08
Also published as: KR20200012895A; CA3065333A1; AU2018275156A1; CN110678203A; JP2020522500A; WO2018223112A1

Abstract

Methods are provided for identifying whether a cancer patient, and especially a gastric cancer patient, will be responsive to treatment with a sequential therapeutic strategy comprising a first administration of the FOLFIRI regimen (irinotecan/5-fluoruracil/folinic acid) followed by a separate sequential administration of the combination of the chemotherapy agents docetaxel and cisplatin (FOLFIRI + docetaxel/cisplatin). Specified TUBB3 and TYMP fragment peptides are precisely detected and quantitated by SRM-mass spectrometry directly in tumor cells, and especially gastric 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 sequential combination treatment of FOLFIRI + docetaxel/cisplatin.

Description

Predicting Gastric Cancer Treatment Outcome

Introduction

Improved methods are provided for treating cancer patients, and especially gastric cancer (GI) patients, by assaying tumor tissue surgically -removed from patients and identifying those patients most likely to respond to treatment with the standard chemotherapy strategy (treatment 1) comprising administration of 5-fluoruracil/folinic acid (DeGramont) versus a regimen (treatment 2) comprising a first administration of the FOLFIRI regimen

(irinotecan/5-fluoruracil/folinic acid) followed by a sequential administration of combination docetaxel/cisplatin. Treatment 1 comprises 5FU/LV while Treatment 2 comprises FOLFIRI + docetaxel/cisplatin.

More specifically, SRJV1 mass spectrometry is used to measure TUBB3 and TYMP proteins in tumor cells derived from patient tumor tissue and these measurements are then used to identify cancer patients, and in particular gastric cancer patients, most likely to respond to treatment with the sequential administration of FOLFIRI followed by combination docetaxel/cisplatin chemotherapy agents.

TUBB3, also known as class III β-tubulin, is a critical component of the cell microtubule assembly andis also actively involved in regulating ligand binding. Proteomic analysis has revealed that many factors bound to these cysteine residues are involved in the oxidative stress and glucose deprivation response. TUBB3 has been investigated as both a prognostic biomarker and an indicator of resistance to docetaxel and other similar taxane compounds where reports implicate high quantitative levels of TUBB3 as a biomarker of poor outcome. Accordingly, determining quantitative expression levels of the TUBB3 protein in patient cancer cells could help determine how cancer cells will respond to treatment with docetaxel.

TYMP, also known as thymidine phosphorylase, is a protein that synthesizes dTMP from thymine and is part of the dTMP biosynthesis salvage pathway, which is itself part of pyrimidine metabolism. TYMP is known as an angiogenic factor which promotes angiogenesis in vivo and stimulates the in vitro growth of a variety of endothelial cells.

TYMP normally has a highly restricted target cell specificity, acting only on endothelial cells. In some cases, however, TYMP can be abnormally expressed at high levels in tumor cells. TYMP functions to convert 5-dFUR to 5FU and 5FU to FdUM,P which promotes the functionality of 5FU to inhibit the TS protein, thereby blocking the production of DNA and inducing tumor cell apoptosis.

TUBB3 and TYMP are prognostic predictors of therapeutic outcome in cancer patients and as such can provide information about chemotherapy treatment strategies of cancer. The presence and/or quantitative levels of TUBB3 and TYMP protein expression in patient tumor cells procured from patient tumor tissue is determined by quantitating a specified peptide derived from subsequences of each of the TUBB3 and TYMP full-length proteins using the methodology of SRM mass spectrometry. Specific quantitative levels of the TUBB3 protein as detected by SRM mass spectrometry in cancer cells present within a cancer patient indicates that the patient is either more likely or less likely to respond in a positive manner to a chemotherapy regimen containing docetaxel. Specific quantitative levels of the TYMP protein as detected in cancer cells within a cancer patient by SRM mass spectrometry indicates that the patient is more likely or less likely to respond in a positive manner to a chemotherapy regimen that includes 5FU.

Irinotecan, also known as camptosar, is a cancer chemotherapy agent that interacts with DNA by intercalation, thereby inhibiting the progression of the enzyme topoisomerase I (TOPOl) which relaxes supercoils in DNA for transcription. Doxorubicin stabilizes the TOPOl complex after it has broken the DNA chain for replication, preventing the DNA double helix from being resealed and stopping the process of replication. This prevents cancer cells from synthesizing DNA and stops cancer cell division and tumor growth.

However, high expression levels of TOPOl enzyme in cancer cells can overcome the effects of irinotecan and cause resistance to the effects of irinotecan in the cancer cells, allowing them to synthesize DNA and promoting cellular division and tumor growth.

Fluorouracil (5-FU), also known as adrucil, is a chemotherapy agent that functions by blocking DNA, thereby inhibiting cell division and preventing tumor cells from dividing and growing. 5-FU acts in several ways, but acts principally as a thymidylate synthase (TS) inhibitor. Interrupting TS activity blocks synthesis of the pyrimidine thymidine, which is a nucleoside required for DNA replication. Thymidylate synthase methylates deoxyuridine monophosphate (dUMP) to form thymidine monophosphate (dTMP). Administration of 5-FU causes a scarcity in dTMP, and rapidly dividing cancerous cells undergo cell death due to this lack of thymine. High levels of thymidylate synthase can overcome the effects of 5-FU while high levels of the thymidine phosphorylase (TYMP) protein promotes the activity of 5-FU.

Docetaxel, also known as taxotere, is a semi-synthetic analogue of paclitaxel (Taxol). It is in the drug class of taxanes. Docetaxel binds to microtubules reversibly with high affinity, stabilizing microtubules and preventing depolymerization, thus killing dividing cells. This stabilization of the cellular microtubule assembly leads to a significant decrease in free tubulin (TUBB3), needed for microtubule formation, and results in inhibition of mitotic cell division between metaphase and anaphase, preventing further cancer cell division and tumor growth. High expression levels of the TUBB3 protein in cancer cells can overcome the effects of docetaxel and thus provide resistance to the effects of docetaxel in the cells, allowing them to depolymerize microtubule assembly and thus promote cellular division and tumor growth.

Cisplatin, also known as platinol, is a cancer chemotherapy agent that interferes with DNA replication thus preventing cells from dividing and leading to tumor cell death via apoptosis. Cisplatin irreversibly crosslinks DNA in several different ways, interfering with mitotic cell division. The damaged DNA elicits DNA repair mechanisms, which in turn activate apoptosis when repair proves impossible, thus killing the tumor cells.

Leucovorin calcium (LV), also known as folinic acid, does not have cancer-fighting properties but is a medication used to decrease the toxic side effects of chemotherapeutic agents. LV stabilizes binding of fluorodeoxy uridine monophosphate (FdUMP) to thymidylate synthase by increasing the intracellular pool of 5,10-methylene tetrahydrofolate (CH2THF), and thus enhancing thymidylate synthase (TS) inhibition). Leucovorin has almost no side effects of its own but when used in combination with fluorouracil it can increase the severity of side effects of that drug.

Brief Description of Figures

Figure 1 shows the Kaplan Meier overall survival (OS) curves using TUBB3 < 750amol^g and TUBB3 > 750amol^g cutoff for this gastric cancer cohort treated with treatment 1 regimen comprising 5FU/LV. Results indicate no statistically-significant predictive value for the overall survival of gastric cancer patients (n = 122) based on these TUBB3 protein levels using 2 different methods for calculating statistical significance (Mantel-Cox Test p = 0.4744; Gehan-Breslow-Wilcoxon Test p = 0.2179).

Figure 2 shows the Kaplan Meier overall survival (OS) curves using TYMP < 1335amol^g and TYMP > 1335amol^g cutoff for this gastric cancer cohort treated with treatment 1 regimen comprising 5FU/LV. Results indicate a small, yet statistically- significant, predictive value for the overall survival of gastric cancer patients (n = 122) based on the TYMP protein cutoff level of 1335amol^g using 2 different methods for calculating statistical significance (Mantel-Cox Test p = 0.0185; Gehan-Breslow-Wilcoxon Test p = 0.0378).

Figure 3 shows the Kaplan Meier overall survival (OS) curves using TUBB3 < 750amol^g and TUBB3 > 750amol^g cutoff for this gastric cancer cohort treated with treatment 2 regimen comprising FOLFIRI followed subsequently by administration of the combination docetaxel/cisplatin. Results indicate a small, yet statistically-significant, predictive value for the overall survival of gastric cancer patients (n = 125) based on these TUBB3 protein levels using 2 different methods for calculating statistical significance (Mantel-Cox Test p = 0.0382; Gehan-Breslow-Wilcoxon Test p = 0.0329).

Figure 4 shows the Kaplan Meier overall survival (OS) curves using TYMP <

1335amol^g and TYMP > 1335amol^g cutoff for this gastric cancer cohort treated with treatment 2 regimen comprising FOLFIRI followed subsequently by administration of the combination docetaxel/cisplatin. Results indicate no statistically-significant, predictive value for the overall survival of gastric cancer patients (n = 125) based on the TYMP protein cutoff level of 1335amol^g using 2 different methods for calculating statistical significance (Mantel-Cox Test p = 0.4200; Gehan-Breslow-Wilcoxon Test p = 0.3448).

Figure 5 shows the Kaplan Meier overall survival (OS) curves using TYMP < 2800amol^g and TYMP > 2800amol^g cutoff for this gastric cancer cohort treated with treatment 2 regimen comprising FOLFIRI followed subsequently by administration of the combination docetaxel/cisplatin. Results indicate a small, yet statistically-significant, predictive value for the overall survival of gastric cancer patients (n = 125) based on the TYMP protein cutoff level of 2800amol^g using 2 different methods for calculating statistical significance (Mantel-Cox Test p = 0.0344; Gehan-Breslow-Wilcoxon Test p = 0.0211).

Figure 6 shows the Kaplan Meier overall survival (OS) curves using the combination of TUBB3 cutoff of 750amol^g + TYMP cutoff of 1300amol^g for this gastric cancer cohort treated with treatment 2 regimen comprising FOLFIRI followed subsequently by administration of the combination docetaxel/cisplatin. Results indicate a slight predictive value, yet not statistically-significant, for the overall survival of gastric cancer patients (n = 58) using 2 different methods for calculating statistical significance (Mantel-Cox Test p = 0.0539; Gehan-Breslow-Wilcoxon Test p = 0.0561).

Figure 7 shows the Kaplan Meier overall survival (OS) curves using the combination of TUBB3 cutoff of 750amol^g + TYMP cutoff of 2800amol^g for this gastric cancer cohort treated with treatment 2 regimen comprising FOLFIRI followed subsequently by administration of the combination docetaxel/cisplatin. Results indicate highly statistically- significant, predictive value for the overall survival of gastric cancer patients (n = 32) using 2 different methods for calculating statistical significance (Mantel-Cox Test p = 0.0002;

Gehan-Breslow-Wilcoxon Test p = 0.0001).

Detailed Description

Methods are provided for determining if a cancer patient, and specifically a gastric patient, will clinically respond in a favorable manner to the therapeutic strategy comprising a first administration of the FOLFIRI regimen (irinotecan/5-fluoruracil/folinic acid) followed by a separate sequential administration of the combination of the chemotherapy agents docetaxel and cisplatin (FOLFIRI + docetaxel/cisplatin). Specifically, diagnostic methods for measuring the TUBB3 and TYMP proteins in a tumor sample or samples from the patient are provided. The sample is advantageously formalin-fixed. Using an SRM/MRM assay that simultaneously measures a specific TUBB3 peptide fragment and a specific TYMP peptide fragment, and particular characteristics about the peptide fragments, the amount of the

TUBB3 and TYMP proteins in cells derived from formalin fixed paraffin embedded (FFPE) tissue is determined. The peptide fragments derive from the full-length TUBB3 and TYMP proteins, wherein the peptide sequence for TUBB3 protein is SEQ ID NO: l

(ISVYYNEASSHK) while the peptide sequence for TYMP protein is SEQ ID NO:2

(DGPALSGPQSR). Surprisingly it has been found that these peptides can be reliably detected and quantitated simultaneously in digests prepared from FFPE samples of tumor tissue. See U.S. Pat. App. No. 13,993,045, the contents of which are hereby incorporated by reference in their entirety.

More specifically, this SRM/MRM assay can measure these peptides directly in complex protein lysate samples prepared from cells procured from patient tissue samples, such as formalin fixed cancer patient tissue. Methods of preparing protein samples from formalin-fixed tissue are described in U.S. Pat. No. 7,473,532, the contents of which are hereby incorporated by reference in their entirety. The methods described in U.S. Pat. No. 7,473,532 may conveniently be carried out using Liquid Tissue reagents and protocol available from Expression Pathology Inc. (Rockville, Md.).

The most widely and advantageously available form of tissue, and cancer tissue, from cancer patients is formalin fixed, paraffin embedded tissue. Formaldehyde/formalin fixation of surgically removed tissue is by far and away the most common method of preserving cancer tissue samples worldwide and is the accepted convention in standard pathology practice. Aqueous solutions of formaldehyde are referred to as formalin. "100%" formalin consists of a saturated solution of formaldehyde (this is about 40% by volume or 37% by mass) in water, with a small amount of stabilizer, usually methanol, to limit oxidation and degree of polymerization. The most common way in which tissue is preserved is to soak whole tissue for extended periods of time (8 hours to 48 hours) in aqueous formaldehyde, commonly termed 10% neutral buffered formalin, followed by embedding the fixed whole tissue in paraffin wax for long term storage at room temperature. Thus molecular analytical methods to analyze formalin fixed cancer tissue will be the most accepted and heavily utilized methods for analysis of cancer patient tissue.

Results from the SRM/MRM assay can be used to correlate accurate and precise quantitative levels of the TUBB3 and TYMP proteins within the specific cancer of the patient from whom the tissue was collected and preserved, including gastric cancer tissue. This not only provides diagnostic/prognostic information about the cancer, but also permits a physician or other medical professional to determine appropriate therapy for the patient. In this case, utilizing these assays can provide information about specific levels of TUBB3 and TYMP protein expression simultaneously in cancer tissue and whether or not the patient from whom the cancer tissue was obtained will respond in a favorable way to the therapeutic strategy comprising administering the FOLFIRI regimen (irinotecan/5-fluoruracil/folinic acid) followed by a separate sequential administration of the combination of the

chemotherapy agents docetaxel and cisplatin (FOLFIRI + docetaxel/cisplatin).

Treating cancer patients with the FOLFIRI regimen is a common and effective strategy that has been shown to prolong the lives of cancer patients, especially gastric cancer patients. One of the agents in the combination therapy FOLFIRI regimen is 5-fluoruracil (5FU) which functions in multiple ways that involve blocking the action of thymidylate synthase and thus stopping the production of DNA as well as blocking the production of RNA and thus causing apoptosis. The TYMP protein is involved in converting 5-dFUR to active 5FU as well as converting 5FU to FUDR. Both of these conversions promote the activity of exogenously administered 5FU and thus enhance tumor cell killing by 5FU and higher levels of the TYMP protein are desirable when treating the cancer patient with a regimen that includes 5FU.

Treating cancer patients with docetaxel is also a common and effective strategy for prolonging the lives of the patients. The TUBB3 protein functions as a critical component of the cell microtubule assembly and is also actively involved in regulating ligand binding. Both functions are involved in helping cells to grow and divide. Docetaxel inhibits normal microtubule processes and ligand binding and thus prevent tumor cells from growing and dividing. It therefore is useful for a clinician to know quantitative levels of the TUBB3 protein in a patient's cancer cells because the therapeutic effects of docetaxel in those cells can be overcome simply be overexpression of the TUBB3 protein. If a clinician knows that a cancer patient's tumor cells express very high levels of TUBB3 protein he/she will likely not prescribe docetaxel to the patient because the patient is unlikely to respond favorably.

Likewise, if the patient's tumor cells express very low amounts of TUBB3 protein the clinician will be more likely to prescribe docetaxel as one of the chemotherapy agents in a combination therapy strategy because the drug will likely be able to inhibit normal microtubule function and ligand binding in the cancer cells and thus help to shut down growth of the tumor.

Presently the most widely-used and applied methodology to determine protein presence in cancer patient tissue, especially FFPE tissue, is immunohistochemistry (IHC). IHC methodology utilizes an antibody to detect the protein of interest. The results of an IHC test are most often interpreted by a pathologist or histotechnologist. This interpretation is subjective and does not provide quantitative data that are predictive of sensitivity to therapeutic agents that target specific oncoprotein targets, such as 5FU and docetaxel sensitivity in a TUBB3 and TYMP positive tumor cell population.

Research from other IHC assays, such as the Her2 IHC test suggest the results obtained from such tests may be wrong. This is probably because different laboratories use different rules for classifying positive and negative IHC status. Each pathologist running the tests also may use different criteria to decide whether the results are positive or negative. In most cases, this happens when the test results are borderline, meaning that the results are neither strongly positive nor strongly negative. In other cases, tissue from one area of cancer tissue can test positive while tissue from a different area of the cancer tests negative.

Inaccurate IHC test results may mean that patients diagnosed with cancer do not receive the best possible care. If all or part of a cancer is positive for a specific target oncoprotein but test results classify it as negative, physicians are unlikely to recommend the correct therapeutic treatment, even though the patient could potentially benefit from those agents. If a cancer is oncoprotein target negative but test results classify it as positive, physicians may recommend a specific therapeutic treatment, even though the patient is unlikely to get any benefits and is exposed to the agent's secondary risks. Thus there is great clinical value in the ability to correctly evaluate quantitative levels of the TUBB3 and TYMP proteins in tumors, especially gastric tumors, so that the patient will have the greatest chance of receiving the most optimal treatment.

Detection of peptides and determining quantitative levels of specified TUBB3 and TYMP fragment peptides are determined in a mass spectrometer by the SRM/MRM methodology, whereby the SRM/MRM signature chromatographic peak area of each peptide is determined within a complex peptide mixture present in a Liquid Tissue lysate (see U. S. Pat. No. 7,473,532, as described above). Quantitative levels of the TUBB3 and TYMP proteins are then determined by the SRM/MRM methodology whereby the SRM/MRM signature chromatographic peak area of an individual specified peptide from each of the TUBB3 and TYMP proteins in one biological sample is compared to the SRM/MRM signature chromatographic peak area of a known amount of a "spiked" internal standard for each of the individual specified TUBB3 and TYMP fragment peptides. In one embodiment, the internal standard is a synthetic version of the same exact TUBB3 and TYMP fragment peptides where the synthetic peptides contain one or more amino acid residues labeled with one or more heavy isotopes. Such isotope labeled internal standards are synthesized so that mass spectrometry analysis generates a predictable and consistent SRM/MRM signature chromatographic peak that is different and distinct from the native TUBB3 and TYMP fragment peptide chromatographic signature peaks and which can be used as comparator peaks. Thus when the internal standard is spiked in known amounts into a protein or peptide preparation from a biological sample and analyzed by mass spectrometry, the SRM/MRM signature chromatographic peak area of the native peptide is compared to the SRM/MRM signature chromatographic peak area of the internal standard peptide, and this numerical comparison indicates either the absolute molarity and/or absolute weight of the native peptide present in the original protein preparation from the biological sample. Quantitative data for fragment peptides are displayed according to the amount of protein analyzed per sample.

In order to develop the SRM/MRM assay for the TUBB3 and TYMP fragment peptides additional information beyond simply the peptide sequence may be utilized by the mass spectrometer. That additional information is important in directing and instructing the mass spectrometer, (e.g., a triple quadrupole mass spectrometer) to perform the correct and focused analysis of the specified TUBB3 and TYMP fragment peptides. An important consideration when conducting an SRM/MRM assay is that such an assay may be effectively performed on a triple quadrupole mass spectrometer. That type of a mass spectrometer may be considered to be the most suitable instrument for analyzing a single isolated target peptide within a very complex protein lysate that may consist of hundreds of thousands to millions of individual peptides from all the proteins contained within a cell. The additional information provides the triple quadrupole mass spectrometer with the correct directives to allow analysis of a single isolated target peptide within a very complex protein lysate that may consist of hundreds of thousands to millions of individual peptides from all the proteins contained within a cell.

Although SRM/MRM assays can be developed and performed on any type of mass spectrometer, including a MALDI, ion trap, ion trap/quadrupole hybrid, or triple quadrupole, presently the most advantageous instrument platform for SRM/MRM assay is often considered to be a triple quadrupole instrument platform. The additional information about target peptides in general, and in particular about the specified TUBB3 and TYMP fragment peptides, may include one or more of the mono isotopic mass of each peptide, its precursor charge state, the precursor m/z value, the m/z transition ions, and the ion type of each transition ion. The peptide sequences of the specified TUBB3 and TYMP fragment peptides are shown in Table 1.

Table 1

To determine an appropriate reference level for TUBB3 and TYMP quantitation, tumor samples are obtained from a cohort of patients suffering from cancer, for example gastric cancer. The tumor samples are formalin-fixed using standard methods and the level of TUBB3 and TYMP in the samples is measured using the methods as described above. The tissue samples may also be examined using IHC and FISH using methods that are well known in the art. The patients in the cohort are treated with the combination of either: 1) the chemotherapy strategy (treatment 1) comprising administration of 5-fluoruracil/folinic acid (DeGramont); or 2) the chemotherapy treatment strategy (treatment 2) comprising a first administration of the FOLFIRI regimen (irinotecan/5-fluoruracil/folinic acid) followed by a sequential administration of combination docetaxel/cisplatin. Treatment 1 comprises 5FU/LV while Treatment 2 comprises FOLFIRI + docetaxel/cisplatin. Patient response is measured using methods that are well known in the art, for example by recording the overall survival of the patients at time intervals after treatment. A suitable reference level can be determined using statistical methods that are well known in the art, for example by determining the lowest p value of a log rank test. Once a reference level has been determined it can be used to identify those patients whose TUBB3 and TYMP protein expression levels indicate that they may likely benefit from the combination of treatment regimen 1 or treatment regimen 2. The skilled artisan will recognize that the FOLFIRI regimen comprising irinotecan + 5-fluoruracil + folinic acid is a common treatment regimen for gastric cancer patients. Levels of TUBB3 and TYMP proteins in patient tumor samples typically are expressed in amol^g, although other units can be used. The skilled artisan will recognize that a reference level can be expressed as a range around a central value, for example, +/- 250, 150, 100, 50 or 25 amol/ μg.

Because both nucleic acids and protein can be analyzed from the same Liquid Tissue biomolecular preparation it is possible to generate additional information about disease diagnosis and drug treatment decisions from the nucleic acids in the same sample in which proteins were analyzed. For example, if the TUBB3 and TYMP proteins are expressed by certain cells at increased levels, when assayed by SRM the data can provide information about the state of the cells and their potential for uncontrolled growth, choice of optimal therapy, and potential drug resistance. At the same time, information about the status of genes and/or the nucleic acids and proteins they encode (e.g., mRNA molecules and their expression levels or splice variations) can be obtained from nucleic acids present in the same Liquid Tissue biomolecular preparation. Nucleic acids can be assessed simultaneously with the SRM analysis of proteins, including the TUBB3 and TYMP proteins. In one

embodiment, information about the TUBB3 and TYMP proteins and/or one, two, three, four or more additional proteins may be assessed by examining the nucleic acids encoding those proteins. Those nucleic acids can be examined, for example, by one or more, two or more, or three or more of: sequencing methods, polymerase chain reaction methods, restriction fragment polymorphism analysis, identification of deletions, insertions, and/or determinations of the presence of mutations, including but not limited to, single base pair polymorphisms, transitions, transversions, or combinations thereof.

Examples

TUBB3 was assessed by determining the association between its protein levels and OS in a univariate Cox model for treatment 1 regimen comprising, 5FU/LV. In addition to the analysis using the continuous TUBB3 data, a cutoff threshold was derived for dichotomizing patients by TUBB3 levels, using Cox proportional modeling and Gehan- Breslow-Wilcoxon significance test. Half the patients were randomly selected, balancing for the number of OS events and cohort (first vs. second), as the training set. Every possible value between the 25th and 75th percentile was evaluated as a potential threshold to dichotomize the training set into 'High' vs. 'Low' groups; and fit a series of univariate Cox models of OS on the binary TUBB3 variable. The threshold that gave the best Cox model fit in the training set was then applied to the test set; and the Cox model fit was assessed in the dichotomized test cases. The procedure was repeated 1000 times; and the likelihood ratio test (LR) p values across the 1000 test sets were combined using the logit method. The threshold selected at least 10 times and the lowest combined p value was selected. Kaplan Meier curves of the combined dataset stratified by binary TUBB3 groups were constructed for visualization. Using the procedure described above, a threshold of 750amol^g was selected to test the possibility of patient population separation. As a continuous variable, while there was slight separation of the 2 patient populations there was essentially no trend for association between TUBB3 and OS, nor was there predictive value for treatment outcome, where lower TUBB3 levels were not associated with better or worse OS. 93 patients show TUBB3 levels < 750amol^g and 29 patients show TUBB3 levels > 750amol^g. Kaplan Meier overall survival (OS) curves using TUBB3 < 750amol^g and TUBB3 > 750amol^g cutoff for this gastric cancer cohort demonstrate no statistically-significant predictive value for the overall survival of gastric cancer patients (n = 122) based on these TUBB3 protein levels using 2 different methods for calculating statistical significance (Mantel-Cox Test p = 0.4744; Gehan-Breslow-Wilcoxon Test p = 0.2179). The median OS of the TUBB3 < 750amol^g group is 1325 days; while the median OS of the TUBB3 > 750amol^g group is 1991 days. The results are shown in Figure 1.

The same analytical approach as described above for TUBB3 was used to assess associations between TYMP and OS in the same patient population treated with treatment regimen 1 comprising 5FU/LV. Following the procedure described above, a threshold of 1335amol^g was selected. As a continuous variable, TYMP was slightly significantly associated with OS (Cox test p = 0.0185; Gehan-Breslow-WilcoxonTest p = 0.0378) thus providing some predictive value for treatment outcome. 57 patients had TYMP levels < 1335amol^g and 65 patients had TYMP levels >1335amol^g. Patients with TYMP levels > 1335amol^g had significantly better OS than those with TYMP levels < 1335amol^g.

Kaplan Meier OS curves of the combined cohort dichotomized at TYMP levels = 1335amol^g are shown. The median OS of the TYMP < 1335amol^g was 1062 days; while the median OS of the TYMP > 1335amol^g group was 2362 days. These results are shown in Figure 2.

The same analytical approach as described above was used to assess associations between TUBB3 and OS in the gastric cancer patient population treated with treatment regimen 2 comprising FOLFIRI (irinotecan/5-fluoruracil/folinic acid) followed subsequently by administration of the combination docetaxel/cisplatin. Following the procedure described above, a threshold of 750amol^g was selected. As a continuous variable, TUBB3 was significantly associated with OS (Cox test p = 0.0382; Gehan-Breslow-WilcoxonTest p = 0.0329). 100 patients had TUBB3 levels≤ 750amol^g and 25 patients have TUBB3 levels > 750amol^g. Patients with TUBB3 levels < 750 amol^g hd significantly better OS than those with TUBB3 levels > 750amol^g. Kaplan Meier OS curves of the combined cohort dichotomized at TUBB3 levels = 750amol^g was shown. The median OS of the TUBB3 < 750amol^g was 1563 days; while the median OS of the TUBB3 > 750amol^g group was 886 days. These results are shown in Figure 3.

Figure 4 shows the association between TYMP and OS in the gastric cancer patient population treated with treatment regimen 2 comprising FOLFIRI (irinotecan/5- fluoruracil/folinic acid) followed subsequently by administration of the combination docetaxel/cisplatin. The previously derived cutoff of 1335amol^g was selected for comparison to the earlier analysis. As a continuous variable, TUBB3 was not significantly associated with OS (Cox test p = 0.420; Gehan-Breslow-WilcoxonTest p = 0.3448). 69 patients have TYMP levels < 1335amol^g and 56 patients had TYMP levels > 1335amol^g. Patients with TYMP levels > 1335amol^g hadno greater OS than those with TYMP levels < 1335amol^g. Kaplan Meier OS curves of the combined cohort dichotomized at TYMP levels = 1335amol^g are shown. The median OS of the TYMP < 1335amol^g was 1379 days; while the median OS of the TYMP > 1335amol^g group was 1 146 days.

To derive an unbiased association of TYMP expression and OS, the same analytical approach as described above was used to assess associations between TYMP and OS in the gastric cancer patient population treated with treatment regimen 2 comprising FOLFIRI (irinotecan/5-fluoruracil/folinic acid) followed subsequently by administration of the combination docetaxel/cisplatin. Following the procedure described above, a threshold of 2800amol^g was selected. As a continuous variable, TYMP was significantly associated with OS (Cox test p = 0.0344; Gehan-Breslow-WilcoxonTest p = 0.021 1). 94 patients had TYMP levels≤ 2800amol^g and 31 patients had TYMP levels > 2800amol^g. Patients with TYMP levels > 2800amol^g had significantly better OS than those with TYMP levels < 2800amol^g. Kaplan Meier OS curves of the combined cohort dichotomized at TYMP levels = 2800amol^g are shown. The median OS of the TYMP < 2800amol/ng was 1146 days; while the median OS of the TYMP > 2800amol^g group could not be assigned because the median OS had not been reached by this patient group (>2800 days). These results are shown in Figure 5.

When the combined cohort was stratified by using the combination of TUBB3 and TYMP at selected thresholds (TUBB3 < 750amol^g; TYMP > 1335amol^g), there were 51 patients with TUBB3 < 750amol^g and TYMP > 1335amol^g, 7 patients with TUBB3 > 750amol^g and TOP02A < 1335amol^g. The Kaplan Meier OS curves stratified into 2 distinct TUBB3/TYMP groups. As continuous variables, patients with TUBB3 <

750amol^g and TYMP > 1335amol^g had greater OS but were not considered statistically significant (Cox test p = 0.0539; Gehan-Breslow-WilcoxonTest p = 0.0561). The median OS of the TUBB3 < 750amol^g and TYMP > 1335amol^g patient group was 1566 days; while the median OS of the TUBB3 > 750amol^g and TYMP < 1335amol^g patient group was 464 days. These results are shown in Figure 6.

When the combined cohort was stratified by using the combination of TUBB3 and TYMP at different selected thresholds (TUBB3 < 750amol^g; TYMP > 2800amol^g), there were 19 patients with TUBB3 < 750amol^g and TYMP > 2800amol^g, 13 patients with TUBB3 > 750amol^g and TYMP < 2800amol^g. The Kaplan Meier OS curves stratified into 2 distinct TUBB3/TYMP groups. As continuous variables, patients with TUBB3 < 750amol^g and TYMP > 2800amol^g had much greater OS that was extremely statistically significant (Cox test p = 0.0002; Gehan-Breslow-WilcoxonTest p = 0.0001). The median OS of the TUBB3 < 750amol^g and TYMP > 2800amol^g patient group could not be assigned because the median OS had not been reached by this patient group (> 2800 days); while the median OS of the TUBB3 > 750amol^g and TYMP < 2800amol^g patient group was 408 days. These results are shown in Figure 7.

Claims

1. A method of treating a patient suffering from cancer, comprising:

(a) quantifying the level of a specified TUBB3 fragment peptide and quantifying the level of a specified ΤΎΜΡ fragment peptide in a protein digest prepared from a tumor tissue sample obtained from the patient and calculating the level of the TUBB3 and TYMP peptides in said sample by selected reaction monitoring using mass spectrometry;

(b) comparing the level of said TUBB3 fragment peptide to a TUBB3 reference level and said TYMP fragment peptides to a TYMP reference level, and

(c) treating the patient with a therapeutic regimen comprising a first administration of the FOLFIRI regimen (irinotecan/5-fluoruracil/folinic acid) followed by a separate sequential administration of the combination of the chemotherapy agents docetaxel and cisplatin (FOLFIRI + docetaxel/cisplatin) when the level of the TUBB3 fragment peptide is below said TUBB3 reference level, or

(d) treating the patient with a different therapeutic regimen comprising different therapeutic agents other than the therapeutic strategy comprising a first administration of the FOLFIRI regimen (irinotecan/5-fluoruracil/folinic acid) followed by a separate sequential administration of the combination of the chemotherapy agents docetaxel and cisplatin (FOLFIRI + docetaxel/cisplatin) when the level of the TUBB3 fragment peptide is above said TUBB3 reference level, or

(e) treating the patient with a therapeutic regimen comprising a first administration of the FOLFIRI regimen (irinotecan/5-fluoruracil/folinic acid) followed by a separate sequential administration of the combination of the chemotherapy agents docetaxel and cisplatin (FOLFIRI + docetaxel/cisplatin) when the level of the TYMP fragment peptide is above said TYMP reference level, or

(f) treating the patient with a different therapeutic regimen comprising different therapeutic agents other than a first administration of the FOLFIRI regimen (irinotecan/5- fluoruracil/folinic acid) followed by a separate sequential administration of the combination of the chemotherapy agents docetaxel and cisplatin (FOLFIRI + docetaxel/cisplatin) when the level of the TUBB3 fragment peptide is below said TYMP reference level

2. The method of claim 1, wherein said cancer is gastric cancer.

3. The method of claim 1 or claim 2, wherein said protein digest comprises a protease digest.

4. The method of claim 3, wherein said protein digest comprises a trypsin digest.

5. The method of any preceding claim, wherein mass spectrometry comprises tandem mass spectrometry, ion trap mass spectrometry, triple quadrupole mass spectrometry, MALDI-TOF mass spectrometry, MALDI mass spectrometry, hybrid ion trap/quadrupole mass spectrometry and/or time of flight mass spectrometry.

6. The method of claim 5, wherein the mode of mass spectrometry used is Selected Reaction Monitoring (SRM), Multiple Reaction Monitoring (MRM), Parallel Reaction Monitoring (PRM), intelligent Selected Reaction Monitoring (iSRM), and/or multiple Selected Reaction Monitoring (mSRM).

7. The method of any preceding claim, wherein the specified TUBB3 peptide has the amino acid sequence as set forth as SEQ ID NO: 1.

8. The method of any preceding claim, wherein the specified TYMP peptide has the amino acid sequence as set forth as SEQ ID NO:2.

9. The method of any preceding claim, wherein the tumor sample is a cell, collection of cells, or a solid tissue.

10. The method of claim 9, wherein the tumor sample is formalin fixed solid tissue.

11. The method of claim 10, wherein the tissue is paraffin embedded tissue.

12. The method of any preceding claim, wherein quantifying the specified TUBB3 fragment peptide comprises determining the amount of the TUBB3 peptide in said sample by comparing to a spiked internal standard peptide of known amount, wherein both the native peptide in the biological sample and the internal standard peptide corresponds to the same amino acid sequence of the TUBB3 fragment peptide as shown in SEQ ID NO: l .

13. The method of any preceding claim, wherein quantifying the specified TYMP fragment peptide comprises determining the amount of the TYMP peptide in said sample by comparing to a spiked internal standard peptide of known amount, wherein both the native peptide in the biological sample and the internal standard peptide corresponds to the same amino acid sequence of the TYMP fragment peptide as shown in SEQ ID NO:2.

14. The method of any preceding claim, wherein the internal standard peptide is an isotopically labeled peptide.

15. The method of any preceding claim, wherein the isotopically labeled internal standard peptide comprises one or more heavy stable isotopes selected from ¹⁸0, ¹⁷0, ¹⁵N, ¹ C, ²H or combinations thereof.

16. The method of claim 1, wherein the specified level of the TUBB3 peptide fragment is about 750 amol^g protein analyzed.

17. The method of claim 1 , wherein the specified level of the TYMP peptide fragment is about 2800 amol^g protein analyzed.

18. The method of claim 12, wherein detecting and quantitating the specified TUBB3 fragment peptide is combined with detecting and quantitating other peptides from other proteins in a multiplex format so that the treatment decision about which agent used for treatment is based upon specific levels of the specified TUBB3 fragment peptide in combination with other peptides/proteins in the biological sample.

19. The method of claim 13, wherein detecting and quantitating the specified TYMP fragment peptide is combined with detecting and quantitating other peptides from other proteins in multiplex so that the treatment decision about which agent used for treatment is based upon specific levels of the specified TYMP fragment peptide in combination with other peptides/proteins in the biological sample.