CN110678203A

CN110678203A - Prediction of therapeutic effect of gastric cancer

Info

Publication number: CN110678203A
Application number: CN201880035577.2A
Authority: CN
Inventors: 托德·哈姆布拉夫; 法比奥拉·切基; S·施瓦兹; C·邱
Original assignee: Expression Pathology Inc
Current assignee: Expression Pathology Inc
Priority date: 2017-06-02
Filing date: 2018-06-04
Publication date: 2020-01-10
Also published as: KR20200012895A; CA3065333A1; AU2018275156A1; EP3630187A1; JP2020522500A; WO2018223112A1

Abstract

The present invention provides a method for determining whether a cancer patient, particularly a gastric cancer patient, will respond to the use of a sequential treatment strategy comprising first administering a FOLFIRI regimen (irinotecan/5-fluorouracil/folinic acid) followed by separate sequential administration of the combination chemotherapeutic drugs docetaxel and cisplatin (FOLFIRI + docetaxel/cisplatin). Specific TUBB3 and TYMP fragment peptides were accurately detected and quantified directly in tumor cells, particularly gastric cancer tumor cells, collected from tumor tissues obtained from cancer patients by SRM mass spectrometry and compared to reference levels to determine whether the cancer patients responded positively to FOLFIRI + docetaxel/cisplatin sequential combination therapy.

Description

Prediction of therapeutic effect of gastric cancer

Introduction to the design reside in

Improved treatment is provided for cancer patients, particularly gastric cancer patients, by analyzing surgically resected tumor tissue from the patient and identifying those patients most likely to respond to standard chemotherapeutic strategies, including (treatment protocol 1) administration of 5-fluorouracil/folinic acid (degemont), and (treatment protocol 2) a protocol comprising first administration of the FOLFIRI protocol (irinotecan/5-fluorouracil/folinic acid), followed by sequential administration of a docetaxel/cisplatin combination. Treatment regimen 1 included 5FU/LV, while treatment regimen 2 included FOLFIRI + docetaxel/cisplatin.

More specifically, TUBB3 and TYMP proteins in tumor cells extracted from tumor tissue of a patient are measured using SRM mass spectrometry, and these measurements are then used to identify cancer patients, particularly gastric cancer patients, who are most likely to respond to treatment with sequential FOLFIRI administration followed by a combination docetaxel/cisplatin chemotherapeutic drug.

TUBB3, also known as class iii β -tubulin, is an important component of cellular microtubule assembly and is also involved in regulating ligand binding. Proteomic analysis shows that many factors binding to cysteine residues are involved in oxidative stress and glucose deprivation responses. TUBB3 has been studied as an indicator of prognostic biomarker and resistance to docetaxel and other similar taxanes, where TUBB3 with higher quantitative levels has been reported to be a poor prognostic biomarker. Thus, determining quantitative expression levels of TUBB3 protein in patient cancer cells helps to determine cancer cell response to docetaxel treatment.

TYMP, also known as thymidine phosphorylase, is a protein that synthesizes dTMP from thymine and is part of the dTMP biosynthetic repair pathway, itself part of pyrimidine metabolism. TYMP is an angiogenic factor that promotes angiogenesis in vivo and stimulates the growth of a variety of endothelial cells in vitro. Normally, TYMP has a highly restricted target cell specificity and acts only on endothelial cells. However, in some cases, TYMP may be abnormally highly expressed in tumor cells. TYMP has the functions of converting 5-dFUR into 5FU, converting 5FU into FdUM, and promoting 5FU to inhibit the function of TS protein, thereby blocking DNA generation and inducing tumor cell apoptosis.

TUBB3 and TYMP are prognostic predictors of treatment outcome in cancer patients and therefore can provide information about cancer chemotherapy treatment strategies. The presence and/or quantitative levels of TUBB3 and TYMP protein expression in patient tumor cells obtained from patient tumor tissue were determined by quantifying the specific peptides extracted from each subsequence of TUBB3 and TYMP full-length proteins using SRM mass spectrometry. The specific amount of TUBB3 protein levels detected in cancer cells present in cancer patients by SRM mass spectrometry indicates that patients are more or less likely to respond positively to docetaxel-containing chemotherapy regimens. The detection of a specific amount of a level of TYMP protein in cancer cells present in a cancer patient by SRM mass spectrometry indicates that the patient is more or less likely to respond positively to a chemotherapeutic regimen comprising 5 FU.

Irinotecan, also known as Camptosar, is a cancer chemotherapeutic drug that interacts with DNA through insertions, thereby inhibiting the progression of topoisomerase I (TOPO1), and TOPO1 releases the supercoiled portion of DNA for transcription. Doxorubicin stabilizes the TOPO1 complex after the DNA replication strand is disrupted, preventing the DNA duplex from being re-encapsulated, terminating the replication process. This prevents the cancer cells from synthesizing DNA, preventing cancer cell division and tumor growth. However, the TOPO1 enzyme, highly expressed in cancer cells, can overcome and tolerate the action of irinotecan, allowing it to synthesize DNA and promote cell division and tumor growth.

Fluorouracil (5-FU), also known as Adrucil, is a chemotherapeutic drug that acts by blocking DNA to inhibit cell division, prevent tumor cell division and growth. 5-FU has multiple effects, but mainly acts as a Thymidylate Synthase (TS) inhibitor. Blocking TS activity blocks the synthesis of thymine, which is a nucleoside essential for DNA replication. Thymidylate synthase methylates deoxyuridine monophosphate (dUMP) to form thymidine monophosphate (dTMP). Administration of 5-FU results in a deficiency of dTMP, and rapid dividing cancer cells undergo cell death due to the absence of thymine. High levels of thymidylate synthase overcome the effects of 5-FU, while high levels of thymidine phosphorylase (TYMP) protein promote the activity of 5-FU.

Docetaxel, also known as docetaxel (taxotere), is a semi-synthetic analog of paclitaxel (taxol). It belongs to taxane drugs. Docetaxel reversibly binds to microtubules with high affinity, stabilizes microtubules and prevents depolymerization, thereby killing dividing cells. This stability of cellular microtubule assembly results in a significant reduction in free tubulin (TUBB3) required for microtubule formation and in the inhibition of the metaphase and anaphase of mitotic cell division, thereby preventing further division of cancer cells and tumor growth. High expression levels of TUBB3 protein in cancer cells can overcome the effects of docetaxel, thereby allowing the cells to tolerate the effects of docetaxel and disaggregate microtubule assembly, thereby promoting cell division and tumor growth.

Cisplatin, also known as cisplatin, is a cancer chemotherapeutic drug that interferes with DNA replication, thereby preventing cell division and leading to tumor cell death by apoptosis. Cisplatin irreversibly crosslinks DNA in several different ways, interfering with mitotic cell division. The damaged DNA triggers DNA repair mechanisms that in turn activate apoptosis when repair becomes impossible, thereby killing the tumor cells.

Calcium Leucovorin (LV), also known as folinic acid, has no anticancer activity, but is a drug used to reduce the toxic side effects of chemotherapeutic drugs. LV stabilizes the binding of fluorodeoxyuridine monophosphate (FdUMP) to Thymidylate Synthase (TS) by increasing the intracellular pool of 5, 10-methylenetetrahydrofolate (CH2THF), thereby enhancing Thymidylate Synthase (TS) inhibition. Leucovorin itself has few side effects, but when used in combination with fluorouracil, it aggravates the side effects of the drug.

Drawings

FIG. 1 shows a Kaplan-Meier Total survival (OS) curve for a gastric cancer population receiving treatment with 5FU/LV in regimen 1 using a cut-off value (cutoff) of TUBB3 ≦ 750amol/μ g and TUBB3>750amol/μ g. The results show that there is no statistically significant predictive value for the overall survival of gastric cancer patients (n 122) based on these TUBB3 protein levels using two different statistical significance calculation methods (Mantel-Cox test p 0.4744; Gehan-Breslow-Wilcoxon test p 0.2179).

FIG. 2 shows a Kaplan-Meier Total survival (OS) curve for gastric cancer populations treated with treatment regimen 1 containing 5FU/LV using cut-offs of TYMP ≦ 1335amol/μ g and TYMP >1335amol/μ g. The results show that using two different statistical significance calculation methods (Mantel-Cox test p 0.0185; Gehan-Breslow-Wilcoxon test p 0.0378), a cutoff level of TYMP protein based on 1335amol/μ g has a small but statistically significant predictive value for overall survival in gastric cancer patients (n 122).

FIG. 3 shows a Kaplan-Meier Total survival (OS) curve for a gastric cancer population receiving treatment regimen 2, including FOLFIRI followed by administration of a docetaxel/cisplatin combination, using TUBB3 ≦ 750amol/μ g and TUBB3>750amol/μ g cut-off. The results show that using two different statistical significance calculation methods (Mantel-Cox test p-0.0382; Gehan-Breslow-Wilcoxon test p-0.0329), there is a small but statistically significant predictive value for overall survival in gastric cancer patients (n-125) based on these TUBB3 protein levels.

FIG. 4 shows a Kaplan-Meier Total survival (OS) curve for a gastric cancer population receiving treatment regimen 2, including FOLFIRI followed by administration of a docetaxel/cisplatin combination, using TYMP ≦ 1335amol/μ g and TYMP >1335amol/μ g cut-off values. The results show that there is no statistically significant predictive value for the overall survival of gastric cancer patients (n 125) based on a cutoff level of TYMP protein of 1335 amol/. mu.g, using two different statistical significance calculation methods (Mantel-Cox test p 0.4200; Gehan-Breslow-Wilcoxon test p 0.3448).

FIG. 5 shows a Kaplan-Meier Total survival (OS) curve for a gastric cancer population receiving treatment regimen 2 including FOLFIRI followed by administration of a docetaxel/cisplatin combination, using TYMP ≦ 2800amol/μ g and TYMP >2800amol/μ g cut-off values. The results show that using two different statistical significance calculation methods (Mantel-Cox test p 0.0344; Gehan-Breslow-Wilcoxon test p 0.0211), there is a small but statistically significant predictive value for overall survival in gastric cancer patients (n 125) based on a threshold level of TYMP protein of 2800amol/μ g.

FIG. 6 shows a Kaplan-Meier Total survival (OS) curve for a gastric cancer population receiving treatment regimen 2, including FOLFIRI followed by administration of a docetaxel/cisplatin combination, in combination with a TUBB3 cut-off of 750 amol/. mu.g + TYMP cut-off of 1300 amol/. mu.g. The results show that the overall survival of gastric cancer patients (n-58) is slightly predictive of the overall survival using two different statistical significance calculations (Mantel-Cox test p-0.0539; Gehan-Breslow-Wilcoxon test p-0.0561), but not statistically significant.

FIG. 7 shows a Kaplan-Meier Total survival (OS) curve for a gastric cancer population receiving treatment regimen 2 including FOLFIRI followed by administration of a docetaxel/cisplatin combination, in combination with a TUBB3 cut-off of 750 amol/. mu.g + TYMP cut-off of 2800 amol/. mu.g. The results show that two different statistical significance calculation methods (Mantel-Cox test p ═ 0.0002; Gehan-Breslow-Wilcoxon test p ═ 0.0001) have a high predictive value of statistical significance for the overall survival of gastric cancer patients (n ═ 32).

Detailed Description

Methods are provided for determining whether a cancer patient, particularly a gastric cancer patient, has clinically good response to a treatment strategy comprising first administering a FOLFIRI regimen (irinotecan/5-fluorouracil/folinic acid) followed by separate, sequential administrations of the combination chemotherapeutic drugs docetaxel and cisplatin (FOLFIRI + docetaxel/cisplatin). In particular, diagnostic methods for measuring TUBB3 and TYMP protein in a tumor sample or a sample from a patient are provided. The sample is preferably fixed with formalin. The SRM/MRM method was used to simultaneously assay specific peptide fragments of TUBB3 and specific peptide fragments of TYMP and to analyze specific characteristics of these peptide fragments to determine the amount of TUBB3 and TYMP proteins in cells from formalin-fixed paraffin-embedded (FFPE) tissue. The peptide fragments were from full-length TUBB3 and TYMP proteins, where the peptide sequence of TUBB3 protein is SEQ ID NO:1(ISVYYNEASSHK) and the peptide sequence of TYMP protein is SEQ ID NO:2 (DGPALSGPQSR). Surprisingly, these peptides can be reliably detected and quantified simultaneously in lysates prepared from FFPE samples of tumor tissue. See U.S. patent application No. 13,993,045, which is incorporated by reference herein in its entirety.

More specifically, the SRM/MRM method described above can measure these peptides directly in complex protein lysate samples prepared from cells obtained from patient tissue samples (e.g., formalin-fixed cancer patient tissue). Methods for preparing protein samples from formalin-fixed tissue are described in U.S. Pat. No. 7,473,532, the entire contents of which are incorporated herein by reference. The method described in U.S. Pat. No. 7,473,532 can be conveniently performed using the Liquid Tissue reagent and protocol supplied by Expression Pathology, Inc. (Rockville, Md.).

The most widespread and convenient form of tissue from cancer patients and cancer tissue is formalin-fixed paraffin-embedded tissue. Formaldehyde/formalin fixation of surgically excised tissue is currently the most common method of preserving cancer tissue samples worldwide, and is also a accepted practice in standard pathological practice. An aqueous solution of formaldehyde is called formalin. "100%" formalin consists of a saturated aqueous solution of formaldehyde (about 40% by volume or about 37% by mass) and a small amount of a stabilizer (usually methanol) to limit oxidation and degree of polymerization. The most common method of preserving tissues is to soak whole tissues in aqueous formaldehyde (commonly referred to as 10% neutral buffered formalin) for extended periods of time (8 to 48 hours) and then embed the fixed whole tissues in paraffin for long term storage at room temperature. Therefore, formalin-fixed cancer tissue molecular analysis will be the most widely accepted and used method for analyzing cancer patient tissue.

The results of the SRM/MRM analysis can be used to correlate accurate quantitative TUBB3 and TYMP protein levels in specific cancers of patients whose tissues are collected and preserved, including gastric cancer tissues. This not only provides diagnostic/prognostic information about the cancer, but also allows a physician or other medical professional to determine an appropriate treatment regimen for the patient. In this case, using these assays can provide information about the specific levels of both TUBB3 and TYMP proteins expressed in tumor tissue, and whether the patient obtaining the tumor tissue will respond favorably to a treatment strategy (FOLFIRI + docetaxel/cisplatin) that involves administration of FOLFIRI regimen (irinotecan/5-fluorouracil/folinic acid) followed by sequential administration of the combination chemotherapeutic drugs docetaxel and cisplatin, respectively.

Treatment of cancer patients with FOLFIRI regimens is a common and effective strategy that has been shown to prolong the life of cancer patients, particularly those with gastric cancer. One drug in the FOLFIRI combination regimen is 5-fluorouracil (5FU), which acts in a variety of ways, including blocking thymidylate synthase, thereby preventing DNA production, and blocking RNA production, thereby causing apoptosis. TYMP protein is involved in the conversion of 5-dFUR into active 5FU and in the conversion of 5FU into FUDR. Both of these transformations promote the activity of exogenously administered 5FU, thereby enhancing the killing of tumor cells by 5FU, and higher levels of TYMP protein are desirable when treating cancer patients with a regimen containing 5 FU.

Treatment of cancer patients with docetaxel is also a common and effective strategy to prolong the life of patients. The TUBB3 protein is an important component of cellular microtubule assembly and is also actively involved in regulating ligand binding. Both of these functions are associated with aiding cell growth and division. Docetaxel inhibits normal microtubule processes and ligand binding, thereby preventing tumor cell growth and division. Thus, it is useful for clinicians to understand the quantitative levels of TUBB3 protein in patient cancer cells, as the therapeutic effect of docetaxel in these cells can be overcome simply by overexpression of TUBB3 protein. If the clinician knows that the tumor cells of a cancer patient express a very high amount of TUBB3 protein, he/she may not be able to administer docetaxel to the patient because the patient is unlikely to respond well. Also, if the patient's tumor cells express very low TUBB3 protein, the clinician would be more likely to use docetaxel as one of the chemotherapeutic drugs in a combination therapy strategy, as this drug would likely inhibit normal microtubule function and ligand binding in cancer cells, thereby helping to stop tumor growth.

The most widely used method for detecting protein content in cancer patient tissue, particularly FFPE tissue, is Immunohistochemistry (IHC). The IHC method detects a protein of interest using an antibody. The results of IHC testing are typically interpreted by a pathologist or histotechnologist. This interpretation is subjective and does not provide quantitative data to predict the sensitivity of therapeutic drugs to specific tumor protein targets, such as 5FU and docetaxel sensitivity in TUBB3 and TYMP positive tumor cell populations.

Studies from other IHC assays, such as the Her2IHC assay, indicate that the results obtained from these assays may be erroneous. This may be because different laboratories use different classification rules for positive and negative status of IHC. Each pathologist performing the test may also use different criteria to decide whether the result is positive or negative. In most cases, this occurs when the test result is ambiguous, meaning that the test result is neither strongly positive nor strongly negative. In other cases, tissue from one region of cancerous tissue may detect positivity, while tissue from another region of cancerous tissue may detect negativity. Inaccurate IHC detection may mean that patients diagnosed with cancer are not best treated. If all or a portion of the cancer is positive for a particular target tumor protein, but the test results would classify it as negative, then the physician is unlikely to recommend the correct treatment, even though the patient may benefit from these drugs. If the tumor protein target is negative, but the test results would classify it as positive, the physician may recommend a particular treatment, even if the patient is unlikely to receive any benefit and would also be exposed to a secondary risk from the drug.

Therefore, accurate assessment of the quantitative levels of TUBB3 and TYMP proteins in tumors, particularly gastric tumors, is of significant clinical value, maximizing the chances of obtaining optimal treatment for the patient.

Detection of the peptides and determination of the quantitative levels of the specific TUBB3 and TYMP fragment peptides were determined by the SRM/MRM method in a mass spectrometer, in which the SRM/MRM characteristic chromatographic peak area of each peptide was determined in a complex peptide mixture present in a Liquid Tissue lysate (see U.S. Pat. No. 7,473,532, described above). The SRM/MRM characteristic chromatographic peak areas of individual specific peptides from TUBB3 and TYMP proteins in one biological sample were then compared to known amounts of internal standard "spiked" for each individual specific TUBB3 and TYMP fragment peptide, and the quantitative levels of TUBB3 and TYMP proteins were determined by the SRM/MRM method. In one embodiment, the internal standard is a synthetic version of the same TUBB3 and TYMP fragment peptide, wherein the synthetic peptide comprises one or more amino acid residues labeled with one or more heavy isotopes. Such isotopically labeled internal standards are synthesized so that mass spectrometry analysis produces a predictable and consistent characteristic chromatographic peak for SRM/MRM that is distinct and distinct from the characteristic chromatographic peaks for native TUBB3 and TYMP fragment peptides and can be used as a control peak. Thus, when a known amount of labeled internal standard is added to a protein or peptide prepared from a biological sample and analyzed by mass spectrometry, the SRM/MRM characteristic chromatographic peak area of the native peptide is compared to the SRM/MRM characteristic chromatographic peak area of the internal standard peptide, and this comparison of values indicates the absolute molar concentration and/or absolute weight of the native peptide present in the original protein preparation from the biological sample. Quantitative data for fragment peptides were characterized by the amount of protein analyzed for each sample.

To establish the SRM/MRM analysis method of TUBB3 and TYMP fragment peptides, the mass spectrometer can utilize additional information beyond simple peptide sequences. This additional information is important for guiding and operating mass spectrometers (e.g., triple quadrupole mass spectrometers) for the correct and focused analysis of specific TUBB3 and TYMP fragment peptides. An important consideration when performing SRM/MRM analysis is that such analysis can be performed efficiently on a triple quadrupole mass spectrometer. This type of mass spectrometer may be considered the most suitable tool for analyzing a single isolated peptide of interest in a very complex protein lysate, which may contain hundreds of thousands to millions of individual peptides from all proteins within a cell. The additional information provides a correct indication for triple quadrupole mass spectrometers, allowing analysis of a single isolated peptide of interest in very complex protein lysates that may contain hundreds of thousands to millions of individual peptides from all proteins within a cell.

While SRM/MRM analysis can be performed and performed on any type of mass spectrometer, including MALDI, ion traps, hybrid ion trap/quadrupole, or triple quadrupole, the instrument platform that is currently most advantageous for SRM/MRM analysis is generally considered to be the triple quadrupole instrument platform. General additional information about the target peptide, in particular about the specific TUBB3 and TYMP fragment peptides, may include one or more of the monoisotopic mass of each peptide, its precursor charge state, the precursor m/z value, the m/z transition ion, and the ion type of each transition ion. The peptide sequences of the specific TUBB3 and TYMP fragment peptides are shown in table 1.

TABLE 1

SEQ ID	Protein	Peptide sequences
			SEQ ID NO:1	TUBB3	ISVYYNEASSHK
SEQ ID NO:2	TYMP	DGPALSGPQSR

To determine appropriate reference levels for TUBB3 and TYMP quantification, tumor samples were obtained from a group of cancer patients (e.g., gastric cancer). Tumor samples were formalin fixed by standard methods and the levels of TUBB3 and TYMP in the samples were determined by the method described above. Tissue samples can also be detected using methods well known in the art, IHC and FISH. Patients in this group were treated in combination with the following two regimens: 1) the chemotherapy regimen (treatment 1) included administration of 5-fluorouracil/folinic acid (degemont); or 2) the chemotherapy regimen (treatment regimen 2) comprised administration of the FOLFIRI regimen (irinotecan/5-fluorouracil/folinic acid) first, followed by sequential administration of the docetaxel/cisplatin combination. Treatment regimen 1 included 5FU/LV, while treatment regimen 2 included FOLFIRI + docetaxel/cisplatin.

Patient response is measured using methods well known in the art, such as recording the overall survival time of the patient at intervals following treatment. Suitable reference levels may be determined using statistical methods known in the art, for example by determining the lowest p-value of a log-rank test. Once the reference level is determined, it can be used to identify those patients whose expression levels of TUBB3 and TYMP protein indicate that they are likely to benefit from a combination of regimen 1 or regimen 2. One skilled in the art will appreciate that the FOLFIRI regimen, which includes irinotecan + 5-fluorouracil + folinic acid, is a common treatment regimen for patients with gastric cancer. The levels of TUBB3 and TYMP protein in patient tumor samples are usually expressed in amol/μ g, but other units may be used. Those skilled in the art will recognize that the reference level may be expressed as a range around a central value, e.g., +/-250, 150, 100, 50 or 25amol/μ g.

Since both nucleic acids and proteins can be analyzed from the same Liquid Tissue biomolecule preparation, additional information on disease diagnosis and drug treatment decisions can be obtained from nucleic acids in the same sample of the analyzed proteins. For example, if TUBB3 and TYMP proteins are expressed at higher levels in certain cells, these data can provide information about the cell state and its potential for uncontrolled growth, selection of optimal treatment, and potential drug resistance when tested with SRM. Meanwhile, information about the status of genes and/or nucleic acids and their encoded proteins (e.g., mRNA molecules and their expression levels or splicing mutations) can be obtained from nucleic acids present in the same Liquid Tissue biomolecule preparation. Nucleic acid evaluation can be performed simultaneously with SRM analysis of proteins, including TUBB3 and TYMP proteins. In one embodiment, information about the TUBB3 and TYMP proteins and/or one, two, three, four, or more additional proteins can be assessed by examining the nucleic acids encoding these proteins. For example, these nucleic acids can be detected by one or more, two or more, or three or more of the following means: sequencing methods, polymerase chain reaction methods, restriction fragment polymorphism analysis, insertions, deletion identification, and/or determination 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 correlation of protein levels to OS in a univariate Cox model for treatment regimen 1 including 5 FU/LV. In addition to analysis using continuous TUBB3 data, bisection of patients based on TUBB3 levels using the Cox proportional model and Gehan-Breslow-Wilcoxon significance test led to cutoff values. Half of the patients were randomly selected, balancing the number of OS events and groups (first and second groups) as a training set. Each possible value between the 25 th and 75 th percentiles is taken as a potential threshold for dividing the training set into a "high" group and a "low" group; and a series of univariate Cox models of OS were fitted over the binary TUBB3 variables. The threshold of best Cox model fit obtained in the training set is then applied to the test set, and Cox model fitness is evaluated in a dichotomy test case. This process was repeated 1000 times; the likelihood ratio test (LR) p values for 1000 test sets were combined using the logit method. The threshold is selected at least 10 times and the threshold with the lowest combined p-value is selected. A Kaplan-Meier curve of a binary TUBB3 population layered composition dataset was constructed for visualization. Using the above procedure, a threshold of 750 amol/. mu.g was chosen to test the likelihood of segregation in the patient population. As a continuous variable, there was essentially no associated trend between TUBB3 and OS, and no predictive value for treatment outcome, although there was a slight separation between the two patient populations, with lower TUBB3 levels not associated with better or worse OS. TUBB3 levels were <750amol/μ g for 93 patients and TUBB3 levels were >750amol/μ g for 29 patients. The Kaplan-Meier Overall Survival (OS) curves for gastric cancer patients using TUBB3 ≦ 750amol/μ g and TUBB3>750amol/μ g cut-offs show that statistical significance was calculated using two different methods based on these TUBB3 protein levels (Mantel-Cox test p ═ 0.4744; Gehan-Breslow-Wilcoxon test p ═ 0.2179), and that the overall survival of gastric cancer patients (n ═ 122) had no predictive value for statistical significance. The median OS for the TUBB3<750 amol/. mu.g group was 1325 days, and the median OS for the TUBB3>750 amol/. mu.g group was 1991 days. The results are shown in FIG. 1.

The same analytical method as described above for TUBB3 was used to assess the relationship between TYMP and OS in the same patient population treated with treatment regimen 1 comprising 5 FU/LV. 1335 amol/. mu.g was chosen as the threshold according to the above procedure. TYMP has a slight significant correlation with OS as a continuous variable (Cox test p 0.0185; Gehan-Breslow-Wilcoxon test p 0.0378), and therefore has some predictive value for treatment outcome. TYMP levels in 57 patients are ≤ 1335 amol/. mu.g, and TYMP levels in 65 patients are >1335 amol/. mu.g. Patients with TYMP >1335 amol/. mu.g had significantly longer OS than patients with TYMP ≦ 1335 amol/. mu.g. A Kaplan-Meier-OS curve of the union cohort dichotomy at a TYMP level of 1335 amol/. mu.g is shown. The median OS of the group TYMP ≦ 1335 amol/. mu.g was 1062 days, while the median OS of the group TYMP >1335 amol/. mu.g was 2362 days. The results are shown in FIG. 2.

The same analysis as above was used to assess the relationship between TUBB3 and OS in a population of gastric cancer patients receiving treatment regimen 2 comprising administration of docetaxel/cisplatin combination following FOLFIRI (irinotecan/5-fluorouracil/folinic acid). 750 amol/. mu.g was chosen as threshold according to the procedure described above. TUBB3 was significantly correlated with OS as a continuous variable (Cox test p 0.0382; Gehan-Breslow-Wilcoxon test p 0.0329). The TUBB3 levels in 100 patients were <750 amol/. mu.g, and the TUBB3 levels in 25 patients were >750 amol/. mu.g. Patients with TUBB3 levels <750 amol/. mu.g had significantly longer OS than patients with TUBB3 levels >750 amol/. mu.g. A Kaplan-Meier-OS curve of the joint cohort dichotomy at a TUBB3 level of 750 amol/. mu.g is shown. The median OS of the group TUBB3 ≤ 750amol/μ g was 1563 days, and the median OS of the group TUBB3>750amol/μ g was 886 days. The results are shown in FIG. 3.

Figure 4 shows the relationship between TYMP and OS in a population of gastric cancer patients treated with treatment regimen 2 comprising administration of a docetaxel/cisplatin combination following administration of FOLFIRI (irinotecan/5-fluorouracil/folinic acid). The previously derived cut-off value of 1335 amol/. mu.g was selected for comparison with the previous analysis. As a continuous variable TUBB3 has no significant correlation with OS (Cox test p ═ 0.420; Gehan-Breslow-Wilcoxon test p ═ 0.3448). TYMP levels in 69 patients were ≤ 1335 amol/. mu.g, and TYMP levels in 56 patients were >1335 amol/. mu.g. OS in patients with TYMP levels >1335 amol/. mu.g was not longer than in patients with TYMP levels <1335 amol/. mu.g. A Kaplan-Meier-OS curve of the union cohort dichotomy at a TYMP level of 1335 amol/. mu.g is shown. The median OS of the TYMP ≦ 1335amol/μ g group was 1379 days, while the median OS of the TYMP >1335amol/μ g group was 1146 days.

To obtain an unbiased correlation of TYMP expression and OS, the same analysis method described above was used to assess the relationship between TYMP and OS in a population of gastric cancer patients receiving treatment regimen 2, which included administration of FOLFIRI (irinotecan/5-fluorouracil/folinic acid) followed by administration of a docetaxel/cisplatin combination. 2800 amol/. mu.g was chosen as threshold according to the procedure described above. TYMP is significantly related to OS as a continuous variable (Cox test p ═ 0.0344; Gehan-Breslow-Wilcoxon test p ═ 0.0211). TYMP levels of <2800 amol/. mu.g in 94 patients and >2800 amol/. mu.g in 31 patients. OS was significantly longer in patients with TYMP >2800 amol/. mu.g than in patients with TYMP <2800 amol/. mu.g. A Kaplan-Meier-OS curve of the union cohort dichotomy at a TYMP level of 2800 amol/. mu.g is shown. The median OS of TYMP ≦ 2800 amol/. mu.g was 1146 days, whereas the median OS of the TYMP >2800 amol/. mu.g group could not be determined, since this patient group did not reach the median OS (>2800 days). The results are shown in FIG. 5.

When hierarchical analysis of the combined population based on a combination of TUBB3 and TUMP under selected threshold conditions (TUBB3<750amol/μ g; TUMP >1335amol/μ g) was performed, 51 patients were TUBB3<750amol/μ g and TUMP >1335amol/μ g, 7 patients were TUBB3>750amol/μ g and TOPO2A <1335amol/μ g. The Kaplan-Meier-OS curves were layered into 2 different TUBB3/TYMP sets. As a continuous variable, patients with TUBB3<750amol/μ g and TYMP >1335amol/μ g had longer OS but no statistical significance (Cox test p ═ 0.0539; Gehan-Breslow-Wilcoxon test p ═ 0.0561). Median OS for the patient group of TUBB3<750amol/μ g and TYMP >1335amol/μ g was 1566 days, while median OS for the patient group of TUBB3>750amol/μ g and TYMP <1335amol/μ g was 464 days. The results are shown in FIG. 6.

When hierarchical analysis of the combined population was performed based on the combination of TUBB3 and TYMP under different selection thresholds (TUBB3<750amol/μ g; TYMP >2800amol/μ g), TUBB3<750amol/μ g and TYMP >2800amol/μ g in 19 patients, TUBB3>750amol/μ g and TYMP <2800amol/μ g in 13 patients. The Kaplan-Meier-OS curves were layered into 2 different TUBB3/TYMP sets. As a continuous variable, patients with TUBB3<750amol/μ g and TYMP >2800amol/μ g had longer OS with significant statistical significance (Cox test p ═ 0.0002; Gehan-Breslow-Wilcoxon test p ═ 0.0001). Median OS could not be determined for TUBB3<750amol/μ g and TYMP >2800amol/μ g patient group because this patient group did not reach median OS (>2800 days); whereas the median OS in the TUBB3>750 amol/. mu.g and TYMP <2800 amol/. mu.g patient groups was 408 days. The results are shown in FIG. 7.

SEQUENCE LISTING

<110> Acotion spectacular Pathology research company (Expression Pathology, Inc.)

<120> prediction of therapeutic effect on gastric cancer

<130>P60338LRH

<140>

<141>06-01-2018

<150>62/514,364

<151>06-02-2017

<160>2

<170>PatentIn version 3.5

<210>1

<211>12

<212>PRT

<213>Homo sapiens

<400>1

Ile Ser Val Tyr Tyr Asn Glu Ala Ser Ser His Lys

1 5 10

<210>2

<211>11

<212>PRT

<213>Homo sapiens

<400>2

Asp Gly Pro Ala Leu Ser Gly Pro Gln Ser Arg

1 5 10

Claims

1. A method of treating a cancer patient comprising:

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

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

(c) when the level of TUBB3 fragment peptide is below the TUBB3 reference level, the patient is treated with a treatment regimen comprising first administering a FOLFIRI regimen (irinotecan/5-fluorouracil/folinic acid) followed by separate sequential administrations of the combination chemotherapeutic drug docetaxel and cisplatin (FOLFIRI + docetaxel/cisplatin), or

(d) When the level of TUBB3 fragment peptide is above the TUBB3 reference level, the patient is treated with a different treatment regimen comprising different therapeutic agents than the treatment strategy comprising first administering the FOLFIRI regimen (irinotecan/5-fluorouracil/folinic acid) followed by the sequential administration of the combination chemotherapeutic agents docetaxel and cisplatin alone (FOLFIRI + docetaxel/cisplatin), or

(e) When the level of TYMP fragment peptide is above the TYMP reference level, the patient is treated with a treatment regimen comprising first administering a FOLFIRI regimen (irinotecan/5-fluorouracil/folinic acid) followed by separate sequential administrations of the combination chemotherapeutic drugs docetaxel and cisplatin (FOLFIRI + docetaxel/cisplatin), or

(f) When the level of TUBB3 fragment peptide is below the TYMP reference level, the patient is treated with a different treatment regimen comprising different therapeutic drugs, which is different from the FOLFIRI regimen (irinotecan/5-fluorouracil/folinic acid) administered first, followed by the sequential administration of the combination chemotherapeutic drugs docetaxel and cisplatin alone (FOLFIRI + docetaxel/cisplatin).

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

3. The method of claim 1 or 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 of the preceding claims, wherein the specific TUBB3 peptide has the amino acid sequence set forth in SEQ ID No. 1.

8. The method of any one of the preceding claims, wherein the specified TYMP peptide has the amino acid sequence of SEQ ID NO 2.

9. The method of any of the preceding claims, wherein the tumor sample is a cell, a 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 of the above claims, wherein the quantifying a particular TUBB3 fragment peptide comprises determining the amount of TUBB3 peptide in the sample by comparison to a known amount of a tagged internal standard peptide, wherein both the native peptide and the internal standard peptide in the biological sample correspond to the same amino acid sequence of the TUBB3 fragment peptide as set forth in SEQ ID No. 1.

13. The method of any of the preceding claims, wherein quantifying the specified TYMP fragment peptide comprises determining the amount of TYMP peptide in the sample by comparison to a known amount of a spiked internal standard peptide, wherein the native peptide and the internal standard peptide in the biological sample both correspond to the same amino acid sequence of the TYMP fragment peptide as set forth in SEQ ID NO 2.

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

15. The method of any one of the preceding claims, wherein the isotopically labeled internal standard peptide comprises a peptide selected from the group consisting of¹⁸O、¹⁷O、¹⁵N、¹³C、²Heavy stable isotopes of one or more of H or combinations thereof。

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

17. The method of claim 1 wherein the specific level of TYMP peptide fragment is about 2800amol/μ g of protein analyzed.

18. The method of claim 12, wherein detecting and quantifying a particular TUBB3 fragment peptide is combined with detecting and quantifying other peptides from other proteins in a multiplex format such that a therapeutic decision of which drug to use for treatment is based on a particular level of a particular TUBB3 fragment peptide in combination with other peptides/proteins in a biological sample.

19. The method of claim 13, wherein said detecting and quantifying a specific TYMP fragment peptide is combined with detecting and quantifying other peptides from other proteins in a multiplex format such that therapeutic decisions on which drugs to use for therapy are based on specific levels of the specific TYMP fragment peptide in combination with other peptides/proteins in the biological sample.