WO2011129684A1 - Method for establishing and predicting resistance or responsiveness to chemotherapy using peptide mass profile - Google Patents

Abstract

Title: Method for establishing and predicting resistance or responsiveness to chemotherapy using peptide mass profile Abstract Method to predict responsiveness or resistance to chemotherapy in breast cancer comprising providing an optionally processed sample from a breast cancer patient as a test sample, wherein the sample comprise peptides and/or proteins; subjecting the test sample to MALDI- FT-ICR mass spectrometry to generate mass spectra for individual peptide mass in the sample and to quantify the amount of the individual peptide mass present in the test sample; and comparing the amount of an individual peptide mass present in the test sample with the amount of a corresponding peptide mass in a reference sample, wherein the peptide masses are selected from the peptide masses as identified in table 1 and 2

Description

Title: Method for establishing and predicting resistance or responsiveness to chemotherapy using peptide mass profile

The invention

FIELD OF THE INVENTION

The invention relates to the field of medical diagnostics, more specifically to the field of cancer diagnostics, especially breast cancer.

BACKGROUND OF THE INVENTION

Chemotherapy is a well-established treatment of breast cancer for patients for whom targeted therapies are not, or no longer, available. Once metastasis occurs, patients have a very poor survival, due to intrinsic or acquired resistance to drugs used. Ultimately, drug resistance of metastatic tumor cells is the main cause of death among breast cancer patients. At the moment there are no clinical markers that sufficiently predict the type of response to chemotherapeutic drugs. It is therefore of pivotal importance to identify new markers that can better predict the type of response or can act as new drugable targets for future therapy.

WO2008/085024 describes a method with which markers can be identified for a large number of diseases, thereby providing diagnostic tools for medical and veterinary diagnosis. It uses a combination of different MALDI techniques for marker detection.

WO 2008/133493 discloses a method for predicting the responsiveness to anti-estrogen treatment in breast cancer patients by assaying for one or more of proteins, which have found to be differentially expressed in responders and non- responders.

In March 2010 at the European Breast Cancer Conference in Spain researches found that an abnormality on chromosome 17, called CEP17, is a "highly significant indicator" that the tumor will respond to chemotherapy drugs called anthracyclines.

However, there is until now no protein marker for chemotherapy resistance in breast cancer. Protein markers have advantages over genetic markers. Protein markers may be used for clinical specimens that do not contain genetic information, such as serum. In addition, genetic markers may not always give the right information, as it is the expressed product, e.g. the protein that induces a certain effect. Often it is the level of expression of proteins that indicate whether a disease has a certain outcome or not, while at the DNA level there is no difference. In fact, it has been shown that RNA expression levels poorly correlate with protein levels [1, 2].

Therefore there is still a need for peptide biomarkers that are able to identify patients who will not respond to given therapy, and to select patients for various tailored treatments. This will enable doctors to stratify patients for the most suitable, tailored, treatment option, and avoid giving them toxic drugs that pose a heavy burden on the patient without being beneficial.

Surprisingly we have found a peptide mass profile that reliably predict the response to anthracycline based combination chemotherapy in breast cancer patients. SUMMARY OF THE INVENTION

The present invention relates to a method to predict responsiveness or resistance to chemotherapy in breast cancer comprising

(a) providing an optionally processed sample from a breast cancer patient as a test sample, wherein the sample comprise peptides and/or proteins;

(b) subjecting the test sample to MALDI- FT-ICR mass spectrometry to generate mass spectra for individual peptide mass in the sample and to quantify the amount of the individual peptide mass present in the test sample;

(c) comparing the amount of an individual peptide mass present in the test sample with the amount of a corresponding peptide mass in a reference sample, wherein the peptide masses are selected from the peptide masses as identified in table 1 and 2. DETAILED DESCRIPTION

Definitions

The term "breast cancer" as used herein refers to the erratic growth and proliferation of cells that originate in the breast tissue. A group of rapidly dividing cells may form a lump or mass of extra tissue. These masses are called tumors.

Tumors can either be cancerous (malignant) or non-cancerous (benign). Malignant tumors penetrate and destroy healthy body tissues. A group of cells within a tumor may also break away and spread to other parts of the body. Cells that spread from one region of the body into another are called metastases. In preferred embodiments of aspects of the invention the term breast cancer refers to a malignant tumor that has developed from cells in the breast. The teachings of the present invention could be extended to chemotherapy- and anthracycline resistance of other tumor types.

The term "predict" as used herein refers to identifying or forecasting whether a breast tumor will exhibit resistance or develop resistance against chemotherapy.

The term "resistance" as used herein with reference to resistance to treatment or therapy refers to the fact that the normal therapeutic efficacy of the drug is not attained and that, for instance, a tumor continues to grow. Preferably, a tumor is classified as being resistant if the patient from whom the tumor was derived is classified as being a non-responder according to, for instance, the criteria used or set forth by the European Organisation for Research and Treatment of Cancer (EORTC) or the Union International Contre le Cancer (UICC).

The term "reference sample" as used herein refers to a control sample that is a sample from a tumor which is resistant or that is a sample from a tumor that is not resistant to chemotherapy or that is a sample from a normal breast tissue, and which is used for comparison purpose with a test sample, that is, in order to classify the test sample— or more specifically the levels of certain proteins or peptides therein, as being indicative of a resistant or responsive tumor. In a preferred embodiment of a method according to the invention, the control sample is from a resistant tumor. The level of a protein or peptide of a control sample as described herein or an expression profile of proteins or peptides as described herein is compared to the level or profile in a suspected tumor sample. If there is no significant difference between the levels or profiles, then the suspected tumor sample is determined as having the same indication as that control sample. A control sample may be from a single individual or from multiple individuals.

The term "reference profile" as used herein refers to a collection of expression levels for a multitude of given proteins or peptides, which expression levels in combination and/or relative to each other provide specific information which can be used for the purpose of comparison with other profiles. The collection in aspects of this invention is indicative for a resistant or responsive phenotype of the tumor and can be used as a reference for comparison with the test profile. Preferably, such reference profile is based on samples of one or more non-resistant breast tumors. Alternatively, the reference profile may be based on samples of one or more resistant breast tumors. The skilled person is well aware of methods for comparing data collections such as test and reference profiles of gene expression data, and such methods can suitably be used in aspects of the present invention.

The term "chemotherapy-resistant tumor" as used herein refers to a tumor, including the individual cells therein, that is or becomes refractory to treatment by a chemotherapy or anthracycline based therapy such as FEC (= fluorouracil, epirubicine, cyclophosphamide), or FAC (= fluorouracil, doxorubicine,

cyclophosphamide). In specific embodiments, the chemotherapy-resistant tumor becomes resistant to chemo treatment after initiation of the treatment and may occur during the treatment. In further specific embodiments, the resistance to

chemotherapy manifests at about 2-24 months while the patient is receiving chemotherapy. In de novo resistance, the patient does not respond to initial therapy. Acquired resistance is where the patient develops metastatic disease during therapy.

Acquired resistance to chemotherapy is well-known in the art. In particular, breast cancer patients while undergoing treatment with chemotherapy have recurrence of the disease. In specific embodiments, the disease metastasizes during therapy with anthracycline based chemotherapy, which results in resistant metastases.

The term "chemotherapy or anthracycline -sensitive tumor" as used herein refers to a tumor, including the individual cells therein, that is treatable with chemotherapy or anthracycline. In specific embodiments, the chemotherapy or anthracycline -sensitive tumor remains sensitive during the treatment. In further specific embodiments, the chemotherapy or anthracycline -sensitive tumor is still sensitive up to at least about seven to ten years. Preferably, a tumor is classified as being sensitive if the patient from whom the tumor was derived is classified as being a responder as can be classified using EORTC or UICC criteria. Preferably, the responder is classified using EORTC or UICC criteria.

The term "different" as used herein with reference to the comparison of expression profiles or expression levels refers to a degree of difference which is statistically significantly increased or decreased compared to a reference or a control. The term "significantly" or "statistically significant" refers to statistical significance and generally means that values differ two standard deviations (SD). In preferred embodiments, the difference is classified as statistically significant if the expression level is at least a 20 percent increased or decreased compared to expression level of the same expression product in control individuals. Preferably, the increase or decrease is at least 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 or 250 percent. Most preferably, the increase or decrease is at least 100 percent (herein also referred to as "one fold").

The term 'level" as used herein refers to the measurable absolute level or a measurable relative level compared to the level of another protein. If the expression level is determined of a protein from more than one individual (as a control), usually the median or mean expression level of these individuals is used for comparison.

Some of the most common chemotherapy combinations used for breast cancer are CMF ( cyclophosphamide, methotrexate and fluorouracil), FEC (epirubicin, cyclophosphamide and fluorouracil), FAC (fluorouracil, doxorubicine,

cyclophosphamide), FEC-T (epirubicin, cyclophosphamide, fluorouracil and taxotere), E-CMF (epirubicin, followed by CMF), AC (doxorubicin (adriamycin) and

cyclophosphamide), MMM (methotrexate, mitozantrone and mitomycin), MM

(methotrexate and mitozantrone).

Drug resistance of metastatic tumor cells is the main cause of death among breast cancer patients. By comparing the mass spectra of tissues from patients with either breast cancer resistant to chemotherapy or breast cancer responsive to chemotherapy, the inventors have found that there are significant differences in the peptide masses profile. They found a list of peptide masses that were predominantly present in tissue from patients with breast cancer resistant to chemotherapy and found another list of peptide masses that were predominantly present in tissue from patients with breast cancer responsive to chemotherapy. The combination of peptide masses provide an unique profile that may be able to determine between a patient responsive to chemotherapy and a patient resistant to chemotherapy.

In a first aspect the present invention is directed to a method to predict responsiveness or resistance to chemotherapy in breast cancer comprising

(a) providing an optionally processed sample from a breast cancer patient as a test sample, wherein the sample comprise peptides and/or proteins;

(b) subjecting the test sample to MALDI- FT-ICR mass spectrometry to generate mass spectra for individual peptide mass in the sample and to quantify the amount of the individual peptide mass present in the test sample;

(c) comparing the amount of an individual peptide mass present in the test sample with the amount of a corresponding peptide mass in a reference sample, wherein the peptide masses are selected from the peptide masses as identified in table 1 and 2

Peptides masses listed in table 1 indicate a breast tumor responsive to chemotherapy. In contrast peptide masses listed in table 2 indicate breast tumor resistant to chemotherapy.

In a preferred embodiment the reference sample is optionally processed sample from a breast cancer patient being responsive to chemotherapy or from a breast cancer patient being resistant to chemotherapy. Suitably the reference sample is a pooled sample of more than one breast cancer patient, suitably more than 10, more suitably more than 50, and even more than 100 cancer patients. In a preferred embodiment the reference sample is a combined pooled sample from more than two breast cancer patients, both from a patient responsive and from a patient resistant to chemotherapy. By comparing the peptide mass profile of a tissue of a patient with breast cancer, still unknown whether the cancer is responsive to chemotherapy or resistant, to the list of peptide masses of the present invention, a doctor can see whether the patient will be responsive to chemotherapy or not.

In another preferred embodiment the optionally processed sample is a body tissue sample processed by subjecting the sample to laser capture microdissection to provide collections of microdissected cells, the collections preferably amounting to about 200- 3,000 cells. Preferably the optionally processed samples are body tissue samples, body fluid samples, or collections of microdissected cells processed by subjection to protein digestion, preferably using trypsin, to provide processed samples comprising peptide fragments from the proteins in the samples. Suitably the body tissue is selected from the group consisting tissues of breast cancer tumor, breast cancer tumor stroma, and lymph node. Also so suitably the body fluid is selected from the group consisting of blood, serum, cerebrospinal fluid (CSF), urine, saliva and nipple aspirate. Preferred samples are body fluid samples, preferably a body fluid comprising about 0.05- 5 mg/ml of protein, and wherein in step (b) an amount of 1-10 μΐ of optionally processed body fluid is subjected to MALDI- FT- ICR mass

spectrometry. Another suitable test sample may be circulating tumor cells that are collected from blood or other fluids. Body fluids and circulating tumor cells are more easily obtained from a patient than tissue sample that often need local anaesthetic to obtain. Preferably the individual masses are in a mass range of 800 to 4,000 Da.

In preferred embodiment of the present invention the individual peptide masses present in the test sample are selected from the group consisting of peptide masses as identified in table 1 or 2 with a p value lower than 0.03, preferably lower than 0.01.

In another preferred embodiment it is determined whether a breast cancer may be responsive to chemotherapy. For this the amount of the individual peptide mass present in the test sample is determined. If the amount of the individual peptide mass selected from the group consisting of peptide masses as identified in table 1 is higher than the amount of peptide mass having a corresponding mass in the reference sample, it indicates a tumor responsive for chemotherapy. A preferred reference sample for determining responsiveness to chemotherapy is a reference sample that comprises one or more samples from one or more breast cancer patients being resistant to chemotherapy or a reference sample that comprises samples from both breast cancer patients resistant to chemotherapy and from breast cancer patients that are responsive to chemotherapy. The advantage of the present invention is that only the individual peptide masses as indicated in table 1, preferably the peptide masses with a p value lower than 0.03, more preferably lower than 0.01, are measured. This saves time, and provides a more reliable method for determining whether a patient is responsive to chemotherapy or not. In yet another preferred embodiment of the present invention it is determined whether a breast cancer is resistant to chemotherapy. For this the amount of the individual peptide mass present in the test sample is determined. If the amount of the individual peptide mass selected from the group consisting of peptide masses as identified in table 2 is higher than the amount of a peptide masses having a corresponding mass in the reference sample, this indicates a tumor resistant for chemotherapy. To determine chemotherapy resistance a preferred reference sample comprises one or more samples from one or more breast cancer patients being responsive to chemotherapy, or a reference sample that comprises samples from both breast cancer patients resistant to chemotherapy and from breast cancer patients that are responsive to chemotherapy is used. The advantage of the present invention is that only the individual peptide masses as indicated in table 2, preferably the peptide masses with a p value lower than 0.03, more preferably lower than 0.01, are measured. This saves time, and provides a more reliable method for determining whether a patient is responsive to chemotherapy or not.

Suitably in practice, a tissue from a breast cancer patient unknown whether to respond or being resistant to chemotherapy will be compared to two reference samples, one comprising tissue from breast cancer patients responsive to

chemotherapy, one comprising tissue from breast cancer patients resistant to chemotherapy. In a most preferred embodiment, the reference sample is a pooled combined sample from more than two patients, and from breast cancer patients responsive to chemotherapy, and from breast cancer patients resistant to

chemotherapy. In addition, suitably the peptide masses being indicative for responsiveness (peptide masses identified in table 1, preferably wit a p value lower than 0.03, more preferably lower than 0.01), together with the peptide masses being indicative for resistance (peptide masses identified in table 2, preferably with a p value lower than 0.03, more preferably lower than 0.01) are measured. This will give a differential peptide mass profile that will indicate whether a patient may be responsive to chemotherapy (having more peptide masses corresponding to peptide masses in table 1) or resistant to chemotherapy (having more peptide masses corresponding to peptide masses identified in table 2). Any suitable method to determine the amount of individual peptide mass may be used. Preferred methods are selected from the group consisting of mass spectrometry, peptide array, immuno-histochemical assay, ELISA, Protein array, Western Blot, and immunoaffinity chromatography.

In a preferred embodiment the amount of the individual peptide mass in the test sample is at least 20% higher than the amount of the corresponding peptide mass in the reference sample. Preferably at least 10 of the individual peptide mass are present in a higher amount in the test sample than the corresponding peptide mass in the reference sample. Suitably, at least 2, more suitably at least 5, most suitably at least 10, or even at least 15 peptide masses selected from the group consisting of peptide masses from table 1 and 2 with a p value lower than 0.03 are present in an higher amount in the test sample than the corresponding peptide mass in the reference sample. Most suitably, at least 2, more suitably at least 5, most suitably at least 10, peptide masses selected from the group consisting of peptide masses from table 1 and 2 with a p value lower than 0.01 are present in an higher amount in the test sample than the corresponding peptide mass in the reference sample.

The present invention is suitable to detect responsiveness or resistance to chemotherapy in breast cancer. More suitably the chemotherapy is anthracycline based preferably selected from the group consisting of FEC, FAC.

Yet another aspect of the invention is directed to a method to identify a compound that target breast cancer tumor that is resistant to chemotherapy comprising the steps

a) providing a test sample from a breast cancer patient that is resistant to chemotherapy

b) subjecting the test sample to MALDI- FT-ICR mass spectrometry to generate mass spectra for individual peptide masses in the test sample and to quantify the amount of the individual peptide masses present in the test sample; c) contact the test sample with a compound to provide a treated sample d) subjecting the treated sample to MALDI- FT-ICR mass spectrometry to generate mass spectra for individual peptide masses in the treated sample and to quantify the amount of the individual peptide masses present in the treated sample; e) comparing the amount of an individual peptide masses present in the treated sample with the amount of a corresponding peptide mass in the test sample, wherein the peptide masses are selected from the peptide masses as identified in table 1, preferably with a p value lower than 0.03, more preferably lower than 0.01

f) identifying the compound wherein the treated sample has at least 2, more preferably at least 5, most preferably at least 10 peptide masses selected from the group consisting of peptide masses as identified in table 1, preferably with a p value lower than 0.03, more preferably lower than 0.01.

The peptide mass profile as identified in table 1 indicates tumor being responsive to chemotherapy. This information may be used to identify compounds that may be able to treat the breast tumor that is resistant to chemotherapy.

Exposing a tumor sample resistant to chemotherapy to a compound, determining the peptide profile of the treated sample may identify the compound that causes especially the peptide masses in table 1 to be enhanced. This compound may be a promising compound to treat breast cancer patients that are resistant to

chemotherapy.

METHODS

Experimental procedures

Reagents

PEN membrane (1 mm) covered glass slides and PALM caps were purchased from PALM laboratories (Carl Zeiss Microimaging, GmbH, Munich, Germany), PCR clean LoBind 0.5ml tubes were from Eppendorf AG (Hamburg, Germany), trypsin gold mass spectrometry grade was obtained from Promega (Promega Benelux B.V., Leiden, Netherlands), RapiGest SF surfactant and LC glass vials were from Waters

Corporation (Milford, MA, USA), MTP AnchorChip™ 600/384 T F target plate, 2,5- dihydroxybenzoic acid (DHB), and peptide calibration standard (containing angiotensin I and II, substance P, bombesin, rennin substrate, ACTH clip 1-17, ACTH clip 18-39, and somatostatin 28) were obtained from Bruker (Bruker Daltonik GmbH, Bremen, Germany), HPLC grade water and acetonitrile (ACN) were purchased from Fluka analytical (Sigma-Aldrich Corporation, St. Louis, MO, USA), and trifluoroacetic acid (TFA) was purchased from Thermo Fischer Scientific Inc. (Rockford, IL, USA).

Patients and tumor tissues

For the discovery phase of this study, we have used 50 snap frozen primary breast tumor tissues present in our N2 bio bank, of which long-term clinical follow-up was available. Tissues were selected from patients that received anthracyclin-based chemotherapy; 13 FEC (5-fluorouracil + epirubicin + cyclofosfamide) / 37 FAC (5- fluorouracil + doxorubicin [Adriamycin®] + cyclofosfamide). Of these patients, 22 showed objective response (OR), 5 stable disease (SD) >6 months, 21 progressive disease (PD), and 5 SD <6 months upon treatment. Furthermore, 31 tumors were estrogen receptor (ER) negative, and 19 were ER positive, as assessed by ligand binding assay or enzyme-linked immuno sorbent assay (>=10 fmol/mg cytosolic protein). Clinical response was defined by standards of the International Union against Cancer criteria of tumor response [3] .

This study was approved by the Medical Ethics Committee of the Erasmus MC Rotterdam, The Netherlands (MEC 02.953), and was performed in accordance to the Code of Conduct of the Federation of Medical Scientific Societies in the Netherlands. Where ever possible we adhered to the Reporting Recommendations for Tumor Marker Prognostic Studies REMARK [4].

Laser Capture Microdissection (LCM)

LCM was performed on 10 μπι tissue cryosections that were fixed in ice-cold 70% ethanol and stained with hematoxylin as previously described [5] . Laser

microdissection and pressure catapulting (LMPC) was performed directly after staining. Tumor epithelial and stromal cells were separately collected, using a P.A.L.M. LMPC device, type P-MB (Carl Zeiss Microimaging, GmbH, Munich, Germany). From each cryosection, an area of ~400,000 μπι² that corresponds to ~4,000 cells (area x slide thickness / 1000 μπι³ cell volume) was collected in P.A.L.M. tube caps containing 10 μΐ of 0.1% RapiGest, and then spun down into 0.5 ml Eppendorf Protein LoBind tubes. Collected cells were stored at— 80°C until further processing. Since we used small numbers of microdissected cells in this study, protein

concentration was typically below the detection limit of any protein assay. Hence, protein concentration for samples undergoing MS analysis was estimated based on microdissected tissue area and extrapolations from protein assays performed on whole tissue lysates (i.e., ~4,000 cells corresponds to ~400 ng of total protein) as described previously [6].

Sample preparation

Microdissected cells were lysed by sonication directly in RapiGest solution in a cup horn sonifier bath, using an Ultrasonic Disrupter Sonifier II (Model W-250/W-450, Bransons Utrasonics, Danbury, CT, USA) for 1 min at 60% amplitude. Proteins were subsequently equilibrated for 2 min at 37°C, and denatured at 99°C for 5 min, and processed for overnight trypsin digestion at a 1:20 v/v ratio, as previously described [5] and according to the instructions of the RapiGest manufacturer. Peptides were lyophilized and stored in— 80°C until further analysis. Prior to FTICR MS analysis, samples were reconstituted in 6 μΐ 50% ACN, 0.1% TFA. In order to fully cleave RapiGest in the samples, 0.6 μΐ 500mM HC1 was added, shortly mixed, incubated at 37°C for 45 minutes, and centrifuged for 10 minutes at 10,600g to pellet any contaminating particulate material. MALDI-FTICR MS

For MALDI-FTICR MS analysis, a matrix solution was prepared from 10 mg/ml DHB in 0.1% TFA in MilliQ water. The matrix solution was vortexed for 1 min prior to use. 0.5μ1 DHB matrix was spotted on an AnchorChip™ target plate, after which 0.5 μΐ prepared sample was added and mixed on the spot. Peptide calibration standard was spotted on distinct calibration positions together with DHB matrix. Samples were spotted in duplicate and left on the AnchorChip™ for drying prior to MALDI-FTICR analysis on the Apex IV Qe instrument with a 9.6 tesla magnet equipped with a 20 Hz nitrogen laser (Bruker Daltonik). For each measurement, 100 scans of 10 shots each at 60% laser power were accumulated, and spectra were acquired in the mass range of 800-4,000 m/z using XMass software v7.0.8 (Bruker). MALDI-FTICR data were acquired at 512K and further processed with a Gaussian filter and two zero fillings. For each sample, mass spectra were recorded from duplicate spots and accumulated in to one final spectrum. Prior to sample analysis, external calibration was performed on the peptide calibration standard using a quadratic equation. In addition, a post- acquisition internal calibration step was performed in DataAnalysis software, version 3.3, build 139 (Bruker) using auto-lytic trypsin MH+ peptide masses (842.50940, 1045.56370, 2211.10400 m/z), and ubiquitous keratin (1179.60100 m/z), actin

(1198.70545, 1790.89186 m/z) and histone MH+ peptide masses (976.44824,

1515.74913, 2343.16486, 3183.61423 m/z), so that accuracy of <lppm could be acquired. Spectra in which less than 5 calibration peaks were present, were excluded from further data analysis because of poor quality, which was the case for 1 OR tumor, 1 PD tumor, 4 OR stroma, and 3 PD stroma spectra. Data analysis

All isotopic peaks with a signal-to-noise ratio >4 were annotated using DataAnalysis software package, version 3.3, build 139 (Bruker Daltonik). Peak lists were saved in general text format and imported into the home made script in the R-program, Peptrix v2.4.1. The Peptrix package was used to compare mass spectra for

identification of differentially abundant peaks by label free quantitation using MS peak intensity. A matrix file was generated indicating presence, absence, and intensities of peaks in different samples. Peak masses present in less than 4 samples were omitted from the matrix. Within Peptrix, a univariate Wilcoxon-Mann-Whitney rank sum test was performed for pair wise comparisons between tumor and stromal cells and between OR and PD patients. Peaks were considered to be differentially abundant if p-values were <0.05. Differentially abundant peak masses (annotated as doubly and triply charged peptide masses) were subjected to targeted MS/MS analysis for peptide identification purposes.

LC-MS/MS

Identification of differential peptide masses was performed by nLC-MS/MS analysis on an Orbitrap (Thermo Fischer Schientific, Germany) XL mass spectrometer, using an inclusion list of target peptide masses. Tryptic digests of whole tumor tissue lysates were subjected to nLC separation, using a 15 cm x 75 μπι inner diameter C18 reversed phase column. Peptides were eluted from the column by the following binary gradient: from 100% solvent A (0.1% formic acid in water) to 75% solvent A/ 25% solvent B (80% acetonitrile and 0.08% formic acid in water) in 120 minutes, followed by 25-50% solvent B for a further 60 minutes, with a column flow rate of 300 nl/min. A data dependent acquisition was performed in the high resolution Orbitrap with a survey scan from 400-1800 Th. Based on this survey scan, up to 5 ions corresponding to the masses in the inclusion list were fragmented if present. If no target ions were present, the 5 most intensive ions were selected for collision- activated dissociation fragmentation, after which these masses were excluded for further MS/MS analysis for 3 minutes.

Protein identification and quantitation

Bioworks 3.2 software package (Thermo Fischer Scientific, Germany) was used for peak picking and MS/MS identification, and corresponding SEQUEST features, using HUPO criteria, were obtained from the UniProt and SwissProt databases. The number of allowed missed cleavages was set to 1, mass tolerance for precursor ions was 10 ppm, and for fragment ions 0.5 Da. The cut-off for matching identified peptides with theoretical mass was 2 ppm.

Precursor MS scan peptide mass intensities were used for peptide and protein quantitation. Results

Primary breast cancer tissues from patients that received anthracycline-based chemotherapy in advanced stage of disease were subjected to global proteome analysis. For the identification of proteins that associate with chemotherapy resistance, we compared proteomes of tissues from patients responsive (OR + SD >6 months) to therapy with tissues from therapy-resistant (PD + SD <6 months) patients. Patient characteristics are summarized in Table 1. Tumor tissues were subjected to LCM, and, whenever possible, ~4000 tumor and surrounding stromal cells were collected from each tissue. Tumor cells were procured from 20 different OR, 10 SD, and 16 PD tissues, and stromal cells were collected from 18 OR, 10 SD, and 18 different PD tissues. Tryptic digests were prepared and analyzed in duplicate by MALDI-FTICR MS.

Comparative proteomics of breast cancer tissues

Mass spectra of sufficient quality were obtained for 19 OR tumor, 14 OR stroma, 15 PD tumor, 15 PD stroma, and all SD samples.... Using Peptrix software, OR and PD tumor, OR and PD stroma, and all tumor versus all stroma mass sprectra were compared based on peak intensity and peak count. All detected peaks in all mass spectra were populated into a matrix file, which was than used for further statistical analysis. In total, 3,948 peaks were detected in all tumor samples, taking all isotopic peaks into account. In stromal samples, a total of 4,715 peaks were detected.

Wilcoxon-Mann- Whitney rank sum analysis revealed the presence of 135 differential peak masses (p<0.05) between OR and PD tumor cells, of which 28 peak masses had a p-value <0.01 (supplemental table Si). Furthermore, 231 differential peaks were observed between OR and PD stromal cells (p<0.05), of which 17 had a p-value <0.01. Comparison of tumor and stromal samples revealed a very large difference in sample composition.

Identification of differentially abundant proteins

For the identification of peptide sequences corresponding to differentially abundant peak masses, a mass search was performed. Differential peak masses were matched to our list of identified proteins obtained through nLC-MS/MS analysis. From the 135 differential peak masses between OR and PD tumor cells, 70 were matched with peptide sequences, representing 65 different proteins (see tables 1-4). 11 differential stromal peak masses were matched to peptides and their corresponding proteins. Based on this putative peptide profile, chemotherapy responding breast cancer patients can be distinguished from chemotherapy resistant patients (fig 1). In a principal components analysis, it was shown that chemotherapy-resistant breast cancer patients (light dots) can be separated from chemotherapy responsive breast cancer patients (dark dots).

Figure 1. Principal Components Analysis of breast cancer patients. Groups can be separated from each other based on the differentiating peptide profile.

Table 1: peptide masses that are present in OR:PD positive patients

Peptide mass

Da p-value OR:PD Tissue

2305.072724 9.80E-04 + stroma

2063.929124 0.002111308 + stroma

3085.446824 0.002507105 + stroma

2009.058124 0.002714834 + tumor

1834.914924 0.003742871 + tumor

1800.868124 0.005462092 + stroma

2220.950824 0.005804654 + stroma

2347.075324 0.005946875 + stroma

1325.626924 0.006096424 + stroma

1697.815724 0.006096424 + stroma

2265.126724 0.006096424 + stroma

2306.073824 0.006096424 + stroma

1240.552924 0.006501702 + stroma

794.515324 0.007192917 + stroma

842.425424 0.007192917 + stroma

2462.088524 0.007215417 + stroma

3341.762124 0.007955847 + tumor

1514.729524 0.008980671 + stroma

1703.789324 0.008980671 + stroma

1969.067724 0.009611013 + tumor

2182.005724 0.009882035 + stroma

2643.261824 0.009882035 + stroma

3330.552924 0.01426209 + tumor

3634.960924 0.01426209 + tumor

1848.806724 0.01607209 + tumor

2722.284024 0.01798863 + tumor

1038.006024 0.0202379 + tumor

3637.950724 0.02531842 + tumor

1803.933324 0.0255138 + tumor 1944.878924 0.0255138 + tumor

2922.352824 0.02760192 + tumor

3340.755324 0.02794024 + tumor

3350.576624 0.02933639 + tumor

1029.591224 0.03002501 + tumor

976.062824 0.03010091 + tumor

2138.049324 0.03010091 + tumor

3076.644624 0.03010091 + tumor

2419.249624 0.03105303 + tumor

886.061224 0.03131214 + tumor

1404.810024 0.03131214 + tumor

1426.766724 0.03131214 + tumor

1611.843424 0.03131214 + tumor

1743.791024 0.03131214 + tumor

1793.878324 0.03131214 + tumor

2279.186824 0.03131214 + tumor

2567.243124 0.03131214 + tumor

2662.385424 0.03131214 + tumor

2768.326324 0.03131214 + tumor

2779.313424 0.03131214 + tumor

2854.397024 0.03131214 + tumor

2960.334724 0.03131214 + tumor

2961.318724 0.03131214 + tumor

3342.747024 0.03131214 + tumor

3351.589024 0.03131214 + tumor

3795.858424 0.03131214 + tumor

954.080624 0.036303 + tumor

2674.262824 0.036303 + tumor

2794.297324 0.036303 + tumor

3332.559124 0.0363532 + tumor

2312.185924 0.04253066 + tumor

2896.487524 0.04253066 + tumor

3348.565024 0.04253066 + tumor

2008.053424 0.04533272 + tumor

2663.384224 0.04627936 + tumor

1026.078324 0.04795378 + tumor

2918.517924 0.04795378 + tumor

1987.076124 0.04878663 + tumor Table 2: peptide masses that are present in OR:PD negative patients

Peptide mass

Da p-value OR:PD Tissue

1991.891624 0.000499394 - tumor

2745.290324 0.001232227 - tumor

1940.080824 0.001334505 - tumor

1921.064524 0.003801999 - tumor

1985.063324 0.004223652 - tumor

1613.900624 0.005804654 - stroma

1763.799724 0.006732545 - stroma

2183.209624 0.007411747 - tumor

2822.293524 0.007411747 - tumor

3113.604424 0.007411747 - tumor

2420.241624 0.008041432 - stroma

1938.071424 0.00845795 - tumor

2824.307324 0.008658083 - stroma

1516.749224 0.008980671 - stroma

3085.435724 0.008980671 - stroma

3030.592824 0.009611013 - tumor

993.497324 0.009882035 - stroma

882.208824 0.01031402 - tumor

1780.794624 0.01188119 - tumor

2263.117124 0.01188119 - tumor

2531.373624 0.01200892 - tumor

842.497524 0.01888901 - tumor

1138.500424 0.01888901 - tumor

1640.742324 0.01888901 - tumor

1686.973124 0.01888901 - tumor

2368.252624 0.01888901 - tumor

2799.343024 0.01888901 - tumor

2904.371124 0.01888901 - tumor

3330.643724 0.01888901 - tumor

801.879724 0.02324039 - tumor

1420.662424 0.02324039 - tumor

1639.908824 0.02324039 - tumor

1642.804324 0.02324039 - tumor

2694.208924 0.02324039 - tumor

2736.260824 0.02324039 - tumor

2854.388424 0.02324039 - tumor

3029.589024 0.02324039 - tumor

1038.001424 0.02606067 - tumor

1614.907424 0.02606067 - tumor

1934.008924 0.02606067 - tumor

1787.927524 0.03010091 - tumor

1984.058524 0.03010091 - tumor

2166.059224 0.03010091 - tumor

2181.199824 0.03105303 - tumor

2182.204524 0.03105303 - tumor 2312.181824 0.03105303 - tumor

2436.231424 0.03105303 - tumor

2245.199024 0.03415649 - tumor

1044.557424 0.036303 - tumor

2367.252324 0.04101963 - tumor

795.466524 0.04533272 - tumor

1610.890724 0.04533272 - tumor

1709.903924 0.04533272 - tumor

1769.997824 0.04533272 - tumor

1920.070624 0.04533272 - tumor

2038.020624 0.04533272 - tumor

2417.273624 0.04533272 - tumor

2709.285624 0.04533272 - tumor

2732.304124 0.04533272 - tumor

2842.301024 0.04533272 - tumor

References

[1] Anderson, L., Seilhamer, J., A comparison of selected mRNA and protein abundances in human liver. Electrophoresis 1997, 18, 533-537.

[2] Washburn, M. P., Roller, A., Oshiro, G., Ulaszek, R. R., Plouffe, D., Deciu, C, Winzeler, E., Yates, J. R., 3rd, Protein pathway and complex clustering of correlated mRNA and protein expression analyses in Saccharomyces cerevisiae. Proc. Natl. Acad. Set. U. S. A. 2003, 100, 3107-3112.

[3] Hayward, J. L., Carbone, P. P., Heuson, J. C, Kumaoka, S., Segaloff, A., Rubens, R. D., Assessment of response to therapy in advanced breast cancer: a project of the Programme on Clinical Oncology of the International Union Against Cancer, Geneva, Switzerland. Cancer 1977, 39, 1289-1294.

[4] McShane, L. M., Altman, D. G., Sauerbrei, W., Taube, S. E., Gion, M., Clark, G. M., Statistics Subcommittee of the, N. C. I. E. W. G. o. C. D., Reporting

recommendations for tumor marker prognostic studies (REMARK). J Natl Cancer Inst 2005, 97, 1180- 1184.

[5] Umar, A., Kang, H., Timmermans, A. M., Look, M. P., Meijer-van Gelder, M. E., den Bakker, M. A., Jaitly, N., Martens, J. W., Luider, T. M., Foekens, J. A., Pasa- Tolic, L., Identification of a putative protein profile associated with tamoxifen therapy resistance in breast cancer. Mol Cell Proteomics 2009, 8, 1278- 1294.

[6] Umar, A., Dalebout, J. C, Timmermans, A. M., Foekens, J. A., Luider, T. M.,

Method optimisation for peptide profiling of microdissected breast carcinoma tissue by matrix-assisted laser desorption/ionisation-time of flight and matrix- assisted laser desorption/ionisation-time of flight/ time of flight-mass spectrometry. Proteomics 2005, 5, 2680-2688.

Claims

Claims

1. Method to predict responsiveness or resistance to chemotherapy in breast cancer comprising

(a) providing an optionally processed sample from a breast cancer patient as a test sample, wherein the sample comprise peptides and/or proteins;

(b) subjecting the test sample to MALDI- FT-ICR mass spectrometry to generate mass spectra for individual peptide mass in the sample and to quantify the amount of the individual peptide mass present in the test sample;

(c) comparing the amount of an individual peptide mass present in the test sample with the amount of a corresponding peptide mass in a reference sample, wherein the peptide masses are selected from the peptide masses as identified in table 1 and 2

2. Method according to claim 1 wherein the reference sample is optionally processed sample from a breast cancer patient being responsive to chemotherapy or from a breast cancer patient being resistant to chemotherapy.

3. Method according to claim 1 or 2, wherein the optionally processed sample is a body tissue sample processed by subjecting the sample to laser capture microdissection to provide collections of microdissected cells, the collections preferably amounting to about 200- 3,000 cells.

4. Method according to any one of the preceding claims, wherein the optionally processed samples are body tissue samples, body fluid samples, or collections of microdissected cells processed by subjection to protein digestion, preferably using trypsin, to provide processed samples comprising peptide fragments from the proteins in the samples.

5. Method according to any one of the preceding claims, wherein the body tissue is selected from the group consisting tissues of breast cancer tumor, breast cancer tumor stroma, and lymph node.

6. Method according to any one of the preceding claims, wherein the body fluid is selected from the group consisting of blood, serum, cerebrospinal fluid (CSF), urine, saliva and nipple aspirate.

7. Method according to any one of the preceding claims, wherein the samples are body fluid samples, preferably a body fluid comprising about 0.05- 5 mg/ml of protein, and wherein in step (b) an amount of 1-10 μΐ of optionally processed body fluid is subjected to MALDI- FT-ICR mass spectrometry.

8. Method according to any of the preceding claims wherein the sample is a sample comprising circulating tumor cells that are collected from blood or other fluids.

9. Method according to any one of the preceding claims, wherein in step (b) the individual masses are in a mass range of 800 to 4,000 Da.

10. Method according to any one of the preceding claims, wherein the individual peptide masses present in the test sample are selected from the group consisting of peptide masses as identified in table 1 or 2 with a p value lower than 0.03, preferably lower than 0.01.

11. Method according to any one of the preceding claims, wherein the amount of the individual peptide mass present in the test sample is higher than the amount of peptide mass having a corresponding mass in the reference sample indicates a tumor responsive for chemotherapy, wherein the individual peptide mass from the test sample is selected from the group consisting of peptide masses as identified in table 1 preferably with a p value lower than 0.03, more preferably lower than 0.01

12. Method according to any one of the preceding claims, wherein the amount of the individual peptide masses present in the test sample is higher than the amount of a peptide masses having a corresponding mass in the reference sample indicates a tumor resistant for chemotherapy, wherein the individual peptide masses from the test sample is selected from the group consisting of peptide masses as identified in table 2, preferably with a p value lower than 0.03, more preferably lower than 0.01.

13. Method according to any of the preceding claims wherein the amount of peptide or protein is determined by a method selected from the group consisting of mass spectrometry, peptide array, immuno-histochemical assay, ELISA, Protein array, Western Blot, and immunoaffinity chromatography.

14. Method according to any one of the preceding claims wherein the amount of the individual peptide mass in the test sample is at least 20% higher than the amount of the corresponding peptide mass in the reference sample, and wherein the individual peptide mass from the test sample is selected from the group consisting of peptides as identified in table 1 and 2, preferably with a p value lower than 0.03, more preferably with a p value lower than 0.01.

15. Method according to any one of the preceding claims wherein at least 10 of the individual peptide mass are present in a higher amount in the test sample than the corresponding peptide mass in the reference sample, and wherein the individual peptide mass from the test sample is selected from the group consisting of peptides as identified in table 1 and 2, preferably with a p value lower than 0.03, more preferably with a p value lower than 0.01.

16. Method according to any one of preceding claims wherein the chemotherapy is anthracycline based preferably selected from the group consisting of FEC and FAC.

17. Method to identify a compound that target breast cancer tumor that is resistant to chemotherapy comprising the steps

a) providing a test sample from a breast cancer patient that is resistant to chemotherapy

b) subjecting the test sample to MALDI- FT-ICR mass spectrometry to generate mass spectra for individual peptide masses in the test sample and to quantify the amount of the individual peptide masses present in the test sample; c) contact the test sample with a compound to provide a treated sample d) subjecting the treated sample to MALDI- FT-ICR mass spectrometry to generate mass spectra for individual peptide masses in the treated sample and to quantify the amount of the individual peptide masses present in the treated sample; e) comparing the amount of an individual peptide masses present in the treated sample with the amount of a corresponding peptide mass in the test sample, wherein the peptide masses are selected from the peptide masses as identified in table 1, preferably with a p value lower than 0.03, more preferably lower than 0.01

f) identifying the compound wherein the treated sample has at least 2, more preferably at least 5, most preferably at least 10 peptide masses selected from the group consisting of peptide masses as identified in table 1.