EP2721055A1

EP2721055A1 - Diagnosis of cancer

Info

Publication number: EP2721055A1
Application number: EP12728843.9A
Authority: EP
Inventors: Theo Marten Luider; Johannes Nicolaas Maria IJZERMANS
Original assignee: Erasmus University Medical Center
Current assignee: Erasmus University Medical Center
Priority date: 2011-06-15
Filing date: 2012-06-11
Publication date: 2014-04-23
Also published as: WO2012173469A1

Abstract

The present invention is directed to diagnosis and/or prognosis of cancer. More specifically to the diagnosis and/or prognosis of cancer by determining the hydroxylation pattern of a peptide from a biological sample from an organism. The present invention also is directed to a method to monitor the progress of cancer or to monitor the treatment of cancer. Furthermore the present invention is related to cancer biomarkers and their use.

Description

Title: Diagnosis of cancer

The present invention is directed to diagnosis of cancer. More specifically to the diagnosis and/or prognosis of cancer by determining the hydroxylation pattern of a peptide from a biological sample from an organism.

Background

Cancer is a leading cause of death worldwide and accounted for 7.6 million deaths (around 13% of all deaths) in 2008. Although the five-year survival rate for cancer increases each year, it varies much among the different types of cancer going from less than 2% five-year survival rate to more than 95% five- year survival rate. For example pancreas, lung, oesophagus, stomach, brain, multiple myeloma, leukemia, kidney, rectum and colon cancer have a five-year survival rate of less than 50%. Whereas prostate, larynx, bladder, melanoma, Hodgkin's lymphoma, testis, breast, uterus and cervix cancer have a five-year survival rate of more than 50%.

Early detection of cancer is of critical importance in the treatment and survival of cancer. In addition, it is extremely important to diagnose precisely so that the right treatment can be given. Many cancer diagnostic methods exist but these are often specialized for a cancer type. In addition, many diagnosis methods are invasive, expensive, and/or not sensitive enough. Large scale screening of population for cancer has been shown to be effective to diagnose cancer early and thereby safe lives. Examples are population screening for breast cancer and cervix cancer.

Ideally a diagnostic method for cancer is easy to use, may be performed on a biological sample that is easily obtained from an organism, is not expensive, has a broad coverage of the types of cancer and is applicable in many labs.

The present invention has at least one of the above objectives. The present invention is directed to a method for diagnosing cancer by determining the hydroxylation pattern in a peptide from a biological sample from an organism. WO2010/031822 discloses diagnosis of kidney cell carcinoma by detecting at least 3 peptides markers in a urine sample wherein the peptide markers are selected from table 1 describing 517 peptides only characterized by their mass and migration time. Table 3 shows a long list of peptide sequences however does not indicate hydroxylation. There is nothing in WO2010/031822 that points to hydroxylation of the peptide markers let alone to the use of the hydroxylation pattern for detecting cancer.

Detailed description

The present invention is directed to a method for diagnosing cancer by determining the hydroxylation pattern in a peptide in a biological sample from an organism. It was found that for different cancer types, peptides showed different hydroxylation patterns. The present inventors realised, for the first time, that the simple detection of a hydroxylation pattern, i.e. whether a certain amino acid is hydroxylised or not, provides information on the type of cancer and also whether the cancer is primary or a secondary cancer. It is to be understood that the present invention is different then merely using a hydroxylised peptide as a biomarker. The present invention goes further. For the first time it is shown that the hydroxylation pattern, rather than a hydroxylised peptide, provides information on a cancer.

In the present invention, the term diagnosing includes determining whether an organism has cancer or not. It is to be understood that under diagnosis, in addition the indication of a stage or prognosis of a cancer in an organism is included. Prognosis relates to an opinion on how cancer will develop and/or how the cancer will influence other health condition and/or death/survival of the organism. Evidently, it is not always possible to diagnose with absolute certainty whether an organism has cancer by any method available presently, and this may also be true for the method of the invention. Therefore the term diagnosing in the present invention also includes determining diagnosis cancer by determining the hydroxylation pattern in a peptide in a biological sample with a certain specificity of at least 50% or at least 60%, more preferable with a higher specificity such as 62%, 65%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 91%, 93%, 95%, 96%, 97%, 98%, 99% and most preferably 100%.

The inventors of the present application have surprisingly found that the hydroxylation of certain amino acids of peptides that may be found in a biological sample of an organism are different for different cancers. It is stressed that in the present invention hydroxylation pattern is used to determine the presence of cancer and the type of cancer and not merely the presence of a certain hydroxylised peptide. It was even found that one was able to distinguish between a primary tumour and a secondary tumour. A secondary tumour is a tumour that originates from another tumour by e.g. metastasis. For example the present invention has shown to distinguish between a primary tumour in the liver, a hepatocellular carcinoma (HCC) and a metastatic tumour in the liver from colorectal cancer (CRLM). For the context of the present invention tumour and cancer are used interchangeably and refer both to uncontrolled growth of cells that may invade and intrude upon and destroy adjacent tissue and includes both malignant and benign tumours. Generally, benign tumours are tumours that lack the ability to metastasize, although some tumours like glioma do not metastasize but are considered to be malignant. Malignant tumours are able to metastasize and spread to other locations of the body via the lymph or blood system.

Hydroxylation is a process that introduces a hydroxyl group into an amino acid and is facilitated by enzymes called hydroxylases. The principal residue to be hydroxylated in proteins is proline. The hydroxylation occurs mostly at the γ-C atom, forming hydroxyproline (Hyp). In some cases, proline may be

hydroxylated instead on its 6-C atom. Lysine may also be hydroxylated on its δ-C atom, forming hydroxylysine (Hyl). These reactions may be catalyzed by multi-subunit enzymes prolyl 4-hydroxylase, prolyl 3-hydroxylase and lysyl 5- hydroxylase, respectively. Also cysteine, phenylalanine, tyrosine are examples of amino acids that may be hydroxylated.

Hydroxylation pattern of a peptide may be measured by e.g. mass

spectrometry wherein the mass of a hydroxylated peptide is different from a non-hydroxylated peptide. In addition the fragmentation of a hydroxylated peptide by e.g. MS-MS indentifies the position of the hydroxyl group, i.e. the hydroxylation pattern. Other suitable methods to determine the hydroxylation pattern are any method that measures the interaction of any of the

hydroxylated peptides, such as immuno assays, multiplex assays, competitive assays, beads, carrier chips, arrays, sticks, columns. A suitable method may be immunoassay, multiplex assay, competitive assay and selection reaction monitoring (SRM). The detection may be indicated by any suitable means available such as chemiluminence and/or fluorescence.

The present invention is suitable for determining cancer in an organism. For the context of the present invention an organism is a multicellular organism and preferably comprises a circulatory system such as a blood system and/or preferably comprises a digestive tract and/or preferably is motile. For the present invention, animals are included in the term organism. In a preferred embodiment an organism is preferably a vertebrate, and/or a mammal, and most preferably a human. A peptide is a polymer of amino acids linked by peptide bonds. They have the same peptide bonds as those in proteins, but are commonly shorter in length. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond. According to the invention a peptide may be up to 1000 amino acids long, e.g. between 10 and 500 amino acids, preferably between 12 and 450 amino acids, more preferably between 15 and 400 amino acids, more preferably between 17 and 375 amino acids, more preferably between 20 and 350 amino acids, more preferably between 22 and 300 amino acids, more preferably between 25 and 250 amino acids, more preferably between 27 and 225 amino acids, more preferably between 30 and 200 amino acids, more preferably between 33 and 175 amino acids, more preferably between 35 and 150 amino acids, more preferably between 37 and 150 amino acids, more preferably between 40 and 125 amino acids, more preferably between 45 and 100 amino acids, more preferably between 50 and 85 amino acids, more preferably between 55 and 75 amino acids and most preferably between 60 and 70 amino acids. Peptides have an amino end and a carboxyl end, unless they are cyclic peptides. It is to be understood that polypeptides, oligopeptides and even proteins are envisioned under the term peptides according to the present invention. In a preferred embodiment according to the invention and/or other

embodiments described herein the peptides of the present invention are naturally occurring peptides. Naturally occurring peptides are peptides that are formed by the organism itself, preferably formed without the addition of a pre-cursor to the organism. It is to be understood however that it is within the scope of the invention to add a precursor of the peptides of the invention prior to the method of the invention to increase the amount of peptides to be measured.

Amino acids as used herein are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. Twenty-two amino acids are naturally incorporated into polypeptides and are called proteinogenic or natural amino acids. Of these, 20 are encoded by the universal genetic code. The remaining 2, selenocysteine (Sec, U) and pyrrolysine (Pyl, O), are incorporated into proteins by unique synthetic mechanisms. The natural amino acids include Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic acid (Asp, D), Cysteine (Cys, C), Glutamic acid (Glu, E), Glutamine (Gin, Q), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), Valine (Val, V). The three and one letter code for the amino acids is given in parenthesis.

Aside from the 22 standard amino acids, there are many other amino acids that are called non-proteinogenic or non-standard. Those either are not found in proteins (for example carnitine, GABA), or are not produced directly and in isolation by standard cellular machinery (for example, hydroxyproline and selenomethionine) .

Non-standard amino acids that are found in proteins and/or peptides may be formed by post-translational modification, which is modification after translation during protein synthesis, e.g. hydroxylation.

In a preferred embodiment according to the invention and/or other

embodiments described herein, the peptide is a collagen derived peptide. Collagen is the most abundant protein in humans and is an important component of the extracellular matrix (ECM). So far, 29 types of collagen have been described. It is to be understood that the present invention encompasses peptides of all 29 types of collagen. Preferred peptides are derived from collagen chosen from the group consisting of collagen type I, collagen type II, collagen type III, collagen type IV and collagen type V. Most preferred collagens are collagen type I, collagen type III.

In a preferred embodiment according to the invention and/or other

embodiments described herein, the peptide has a mass of less than 20 kDa. More preferably the peptide has a mass of between 0.5 and 18 kDa, more preferably between 1 -15kDa, more preferably between 1.5-12 kDa, more preferably between 1.7 and 10, more preferably beteeen 2 and 8 and most preferably between 2.2 and 6 kDa.

According to a preferred embodiment according to the invention and/or other embodiments described herein, the hydroxylation is on proline or lysine residues, most preferably on proline.

In a preferred embodiment according to the invention and/or other

embodiments described herein the hydroxylation pattern of at least 1 peptide is determined. More preferably the hydroxylation pattern of at least 2, more preferably of from 1-50, even more preferably from 3-40, more preferably from 4-30, more preferably from 5-25, more preferably from 6 to 20, more preferably from 7 to 15, more preferably from 8 to 12, and most preferably from 9-11 peptides the hydroxylation pattern is determined.

Suitably in the embodiment according to the invention and/or other

embodiments described herein the hydroxylation of 2-15, or 3-10, or 4-8 peptides is determined.

In a preferred embodiment according to the invention and/or other

embodiments described herein, the biological sample is selected from the group consisting of whole blood, plasma, serum, urine, sputum, saliva, nipple aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, cerebrospinal fluid, sweat, pericrevicular fluid, semen, prostatic fluid, faeces, cell lysate, tissue sample, biopsy, tissue lysate and tears. More preferably the biological sample is selected from the group consisting of whole blood, plasma, serum, urine, sputum, saliva, nipple aspirate, vaginal fluid, gastric fluid, cerebrospinal fluid, semen, prostatic fluid, faeces, cell lysate, tissue sample, biopsy, and tissue lysate. More preferably the biological sample is selected from the group consisting of whole blood, plasma, serum, urine, sputum, saliva, cerebrospinal fluid, cell lysate, tissue sample, biopsy, and tissue lysate. Most preferably, the biological sample is selected from the group consisting of whole blood, plasma, serum, urine, tissue sample or tissue lysate. Most suitable are urine, sputum, saliva, and faeces because no invasive procedure is needed to obtain such biological sample. Most suitable biological sample is urine. Preferably the biological sample is obtained from the organism. More preferably the method of the invention is conducted on the biological sample outside the organism.

The method according to the invention and/or embodiments described herein are directed to diagnosing of cancer. There are many types of cancer such as, breast cancer, skin cancer, bone cancer, prostate cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, gallbladder cancer, pancreas cancer, rectum cancer, parathyroid cancer, thyroid cancer, adrenal cancer, neural tissue cancer, head and neck cancer, colon cancer, stomach cancer, bronchi cancer, kidney cancer, basal cell carcinoma, squamous cell carcinoma, metastatic skin carcinoma, osteo sarcoma, Ewin's sarcoma, vesticulum cell sarcoma, myeloma, giant cell tumor, small cell lung tumor, non-small-cell lung carcinoma, gallstones, islet cell tumor, primary brain tumor, acute and chronic lymphatic, and granulocytic tumors, hairy cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neurons, intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilm's tumor, seminoma, ovarian, tumor, leiomyomater tumor, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, sof t tissue sarcoma, malignant carcinoid topical skin lesion, mycosis fungoide,

rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera,

adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignant melanoma's epidermoid carcinomas, and other carcinomas and sarcomas. Preferably the cancer is selected from the group consisting of colorectal liver metastases, hepatocellular carcinoma, adenoma, glioma and prostrate carcinoma.

In a preferred embodiment according to the invention and/or other

embodiments described herein to determine the difference between primary and secondary tumors. It has surprisingly been found that a primary tumor (HCC) and a secondary tumor (e.g. CRLM) have different hydroxylation pattern of peptides. In a preferred embodiment according to the invention and/or other

embodiments described herein the peptide is a peptide as depicted in table 1-3. More preferably the peptide is a peptide selected from the group consisting of SEQ ID 1-16. Also preferred are peptides with the mass as indicated in table 1- 3. In addition, preferred peptides are those peptides as depicted in table 1-3 which are 5-25% longer or shorter than the peptides as depicted in table 1-3, preferably 10-15% longer or shorted than the peptides as depicted in table 1-3. With longer and shorter is meant having 5-25%, 10-25% more or less amino acids. The method according to the invention and/or other embodiments described herein is also suitable to monitor progress of cancer or to monitor effect of treatment of cancer. Following the hydroxylation pattern over time may provide an indication whether the cancer is developing stronger or is becoming less. In a preferred embodiment, a biological sample obtained at time 1 and a biological sample obtained at time 2 are measured and the hydroxylation pattern of a peptide is compared. Preferably the peptide is a collagen peptide, and/or preferably the peptide is less than 20 kDa, and/or preferably the peptide is a peptide as depicted in table 1-3 and/or selected from the group consisting of SEQ ID 1-16. In another aspect the invention is also directed to a biomarker selected from the group consisting of SEQ ID 1-16. In a preferred embodiment the biomarker is used to detect or diagnose cancer, to monitor the progress of cancer or to monitor the effect of a treatment of cancer. In a preferred embodiment, a biological sample obtained at time 1 and a biological sample obtained at time 2 are measured and the hydroxylation pattern of a peptide is compared.

Experimental

Methods

To obtain a "threshold set" for the identification of markers specific for metastatic liver disease, urine samples of 11 patients with diagnosed colorectal liver metastases (CRLM), 10 patients with hepatocellular carcinoma (HCC), 10 patients with benign liver adenomas, and 12 healthy controls were collected and stored at -85°C until workup. Prior to analysis by an Orbitrap mass spectrometer, 1 ml urine aliquots were filtered to remove masses higher than 20 kDa and subsequently desalted on a PD-10 column, lyophilized and dissolved in HPLC-grade water. Each sample was fractionated using nano-LC chromatography with a 90 minute gradient online coupled to a mass

spectrometer (Orbitrap). This approach allowed us to identify and sequence natural occurring peptides in urine. Hydroxylation and position of

hydroxylation was measured by MS-MS. A second set consisting of 48 samples (13 CRLM, 10 HCC, 12 adenomas, 13 and healthy individuals) was used to verify the results obtained. Results

MS spectra obtained from the threshold set were aligned using the vector- based Progenesis software package (Non-Linear Dynamics, New Castle UK). A total of 28380 different masses were detected and out of these masses, 2480 unique peptides could be identified in the Swissprot protein database using the Mascot search engine. Of these 2480 peptides, 1386 (55%) belonged to 26 different collagens. The second (verification) set provided 58545 masses with 3442 unique peptides identified. Out of these 3443 peptides, 1304 belonged to 34 different collagens. Univariate analysis of the threshold set revealed 43 collagen peptides that were significantly different (pO.01). Out of these 43, 7 were also significantly different in the verification set. Independent, multivariate testing of these 7 peptides provided a 64% sensitivity, 100% specificity and 85% sensitivity, 92% specificity in the threshold set and verification set, respectively. To confirm the usefulness of the peptide set that overlaps between the threshold set and the verification set, regression was used to construct a statistically relevant model. Nine collagen peptides were identified that could build a model with 92% sensitivity and 100% specificity. Three of these peptides were already identified using a univariate analysis.

Table 1: Sequences and hydroxylation for colorectal liver metastases

Sequence Hydroxylat Mass # P, K SE ion pattern R Q

ID NO

ADGQPGAKGEpGDAGAKGDAGPpGP 11(P) 23(P) 2206.0096 5P 2K 1

4(P) 10(P) 6P IK 2

AGPpGEAGKpGEQGVpGDLGApGP 16(P) 2(P) 2176.0331

7(P) 10(P) 3P 3 K 3

KGNSGEpGApGSKGDTGAKGEpGPVG 22(P) 2356.1145

NVGAPGAkGARGSAGPpGATGFpGAAGRVGpPG 8(K) 17(P) IK 2R 4 P 23(P) 31(P) 2974.5056 7P

3(P) 6(P) 7 P IK 5 14(P) 18(P) I R

GFpGSpGAKGEVGpAGSpGSNGApGQRGEpGP 24(P) 30(P) 2926.3466

DkGETGEQGDRGIkGHRG 2(K) 14(K) 1928.9258 2K 2R 6

GPpGPpGKNGDDGEAGKPG 3(P) 6(P) 1735.7971 5P 2K 7

3(P) 15(P) 7 P 2K 8

GPpGKNGDDGEAGKpGRpGERGpPGP 18(P) 23(P) 2517.1891 2R

5(P) 8(P) 8P IK 9 9(P) 15(P)

GLPGpAGppGEAGKpGEQGVpGDLGApGP 21(P) 27(P) 2629.2644

8(P) 11(P) IR 4P 10 14(K) 23(P) 2K

ERGEAGIpGVpGAkGEDGKDGSpGEpGA 26(P) 2671.2301

3(P) 9(P) 7P 2K 11 12(K) 15(P)

GPpGADGQpGAkGEpGDAGAKGDAGpPGPA 26(P) 2633.1933 Table 2: Sequences and hydroxylation for HCC

Table 3: Sequences and hydroxylation pattern for adenoma

Hydroxylation pattern: number corresponds with the amino acid in the peptide sequence and are indicated in either p (hydroxyproline) or k (hydroxylysine).

Claims

1. Method for diagnosing cancer by determining the hydroxylation pattern in a peptide in a biological sample from an organism.

2. Method according to claim 1 wherein the peptide is a collagen derived peptide.

3. Method according to claim 1 or 2 wherein the peptide has a mass of less than 20 kDA.

4. Method according to any of claims 1 to 3 wherein the peptide has a mass of between 0.5 and 15 kDa.

5. Method according to any of claim 1 to 4 wherein the hydroxylation is on proline or lysine residues.

6. Method according to any of claim 1 to 5 wherein the biological sample is selected from the group consisting of blood, plasma, serum, urine, sputum, saliva, tissue sample, biopsy, tissue lysate.

7. Method according to any of claim 1 to 6 wherein the peptide is a peptide derived from collagen selected from the group consisting of collagen type I, collagen type III, collagen type II, collagen type IV, collagen type V.

8. Method according to any of the claims 1 to 7 wherein the peptide is a peptide as depicted in table 1-3.

9. Method according to any of the claims 1-8 wherein the peptide is a peptide selected from the group consisting of SEQ ID 1-16.

10. Method according to any of the claims 1 to 9 wherein the

hydroxylation pattern of at least 2 peptides is determined.

11. Method according to any of the claims 1 to 10 wherein the cancer is selected from the group consisting of colorectal liver metastases, hepatocellular carcinoma, adenoma, glioma and prostate carcinoma.

12. Method according to any of the claims 1-11 to determine the difference between primary and secondary tumours.

13. Method according to any of the claims 1-11 to monitor progress of cancer.

14. Method according to any of the claims 1-11 to monitor effect of treatment of cancer.

15 Biomarker for the detection of cancer selected from the group consisting of SEQ ID 1-16.

16. Use of a biomarker according to claim 15 to diagnose cancer, to monitor the progress of cancer or to monitor the effect of treatment of cancer.