WO2020025228A1

WO2020025228A1 - EUKARYOTIC TRANSLATION INITIATION FACTORS (EIFs) AS NOVEL BIOMARKERS IN PANCREATIC CANCER

Info

Publication number: WO2020025228A1
Application number: PCT/EP2019/067207
Authority: WO
Inventors: Johannes HAYBÄCK; Nicole GOLOB-SCHWARZL; Philip PUCHAS; Wilko Weichert; Benjamin GÖPPERT
Original assignee: Otto-Von-Guericke-Universität Magdeburg; Technische Universität München
Priority date: 2018-07-31
Filing date: 2019-06-27
Publication date: 2020-02-06

Abstract

The present invention relates to a method of diagnosing pancreatic cancer in an individual. Further, the present invention relates to a method of grading pancreatic cancer in an individual. Furthermore, the present invention relates to a method of differentiating between low-grade pancreatic cancer and high-grade pancreatic cancer in an individual. In addition, the present invention relates to a method for providing a survival prognosis to an individual suffering from pancreatic cancer. Moreover, the present invention relates to kits for performing the above-mentioned methods.

Description

EUKARYOTIC TRANSLATION INITIATION FACTORS (EIFs) AS NOVEL

BIOMARKERS IN PANCREATIC CANCER

The present invention relates to a method of diagnosing pancreatic cancer in an individual. Further, the present invention relates to a method of staging pancreatic cancer in an individual. Furthermore, the present invention relates to a method of differentiating between low-grade pancreatic cancer and high-grade pancreatic cancer in an individual. In addition, the present invention relates to a method for providing a survival prognosis to an individual suffering from pancreatic cancer. Moreover, the present invention relates to kits for performing the above-mentioned methods.

BACKGROUND OF THE INVENTION

Pancreatic cancer remains a devastating disease associated with extremely poor overall prognosis. The success rate for the treatment of pancreatic cancer, which is still one of the most fatal forms of cancer usually affecting the elderly with a 5-year survival rate of ~5%, has not improved during the past 20 years¹. Its major histological subtype - accounting for approximately 80% of cases - is pancreatic ductal adenocarcinoma (PD AC) originating from the exocrine portion of the gland¹. Pathogenetically, the disease mainly arises due to sporadic somatic mutations (in contrast to inherited germline mutations being associated with its occurence), and major progress in understanding the development of the disease on a molecular basis has been made by identifying subsets of activated oncogenes or inactivated tumor suppressor genes as well as by investigating mutational landscapes^1-3. Yet, the cellular alterations promoting carcinogenesis still need to be further elucidated as implementation of new diagnostic markers, prognostic tools and novel therapeutic targetis is of utmost importance.

Translation is the process of protein synthesis crucially needed for cellular homeostasis and is subdivided into four steps: initiation, elongation, termination and ribosome recycling. Initiation constitutes the rate-limiting step and is concerted by a plethora of eukaryotic translation initiation factors (elFs), acting in manifold ways to generate the fully functional ribosomal apparatus with its associated interaction partners such as the formyl-methionine- tRNA and the consecutively translated messenger RNA (mRNA)⁴. Briefly, translation initiation commences with the formation of the 43S-preinitiation complex, consisting of the 40S ribosomal subunit, e!Fl, e!FlA, eIF3 and the ternary complex composed by the initiating methionyl-tRNA bound to eIF2-GTP. This assembly consecutively scans the mRNA for the first start codon (AUG) in the 5' untranslated region (UTR), leading to codon-anticodon interaction assisted by elFl and elFlA. The scanning process is further assisted by the heterotrimeric eIF4F complex (eIF4A, eIF4E and eIF4G). Binding to the matching start codon leads to eIF2-GTP hydrolysis and dissociation of the respective eIF2-GDP. Ribosomal subunit joining is mediated through eIF5B, forming the fully functional ribosome⁴.

elFs have been described to be involved in tumor development and have, therefore, been proposed as potential drug targets, with some substances currently under preclinical and early clinical investigation. However, dysregulation of translation initiation, as well as its interaction with various signaling pathways, is just starting to be understood in pancreatic cancer such as PD AC. As pancreatic cancer is a devastating disease associated with extremely poor overall prognosis, there is a special need for the identification of new diagnostic biomarkers of pancreatic cancer.

The present inventors found that elFs represent crossroads for carcinogenesis in pancreatic cancer. They identified with elFl, eIF2a, eIF3C, and eIF6 new diagnostic bio markers for pancreatic cancer. These new diagnostic bio markers allow the diagnosis of pancreatic cancer, the staging of pancreatic cancer and the differentiation between low-grade and high-grade pancreatic cancer. In particular, the present inventors analyzed the expression of a subset of elFs in 174 cases of pancreatic ductal adenocarcinoma (PDAC) compared to non neoplastic pancreatic tissue (healthy control) via immunohistochemistry. This revealed significant downregulation of elFl, eIF2a, eIF3C and eIF6. They further investigated 28 frozen tissue samples of PDAC and 28 non-neoplastic tissue (NTT) samples as controls via immunoblot. Significant downregulation of elFl , eIF2a, eIF3C, and eIF6 was thereby confirmed. In addition, the present inventors found that elFl also acts as novel prognostic marker for the overall survival of PDAC patients. Thus, the methods of the present invention allow quick and accurate clinical diagnostics.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a method of diagnosing pancreatic cancer in an individual comprising the step of:

determining the level of at least one eukaryotic Initiation Factor (elF) in a biological sample from an individual, wherein the at least one elF is selected from the group consisting of eIF2a, elFl, eIF3C, and eIF6.

In a second aspect, the present invention relates to a method of staging/grading pancreatic cancer in an individual comprising the step of:

determining the level of at least one eukaryotic Initiation Factor (elF) in a biological sample from an individual,

wherein the at least one elF is selected from the group consisting of eIF2a, elFl, eIF3C, and eIF6.

In a third aspect, the present invention relates to a method of differentiating between low-grade pancreatic cancer and high-grade pancreatic cancer in an individual comprising the step of:

determining the level of at least one eukaryotic Initiation Factor (elF) in a sample from an individual,

In a fourth aspect, the present invention relates to a method of providing a survival prognosis to an individual suffering from pancreatic cancer comprising the step of:

determining the level of eukaryotic Initiation Factor 1 (elFl) in a biological sample from an individual suffering from pancreatic cancer.

In a fifth aspect, the present invention relates to the use of at least one elF for diagnosing pancreatic cancer in an individual, staging/grading pancreatic cancer in an individual, or differentiating between low-grade pancreatic cancer and high-grade pancreatic cancer in an individual,

wherein the at least one elF is selected from the group consisting of elFl, eIF2a, eIF3C, and eIF6.

In a sixth aspect, the present invention relates to the use of elFl for providing a survival prognosis to an individual suffering from pancreatic cancer.

In a seventh aspect, the present invention relates to a kit for diagnosing pancreatic cancer in an individual, staging/grading pancreatic cancer in an individual, or differentiating between low-grade pancreatic cancer and high-grade pancreatic cancer in an individual, wherein said kit comprises

means for determining the level of at least one elF in a sample from an individual,

wherein the at least one elF is selected from the group consisting of elFl, eIF2a, eIF3C, and eIF6. In an eighth aspect, the present invention relates to a kit for providing a survival prognosis to an individual suffering from pancreatic cancer, wherein said kit comprises means for determining the level of elFl in a biological sample from an individual suffering from pancreatic cancer.

This summary of the invention does not necessarily describe all features of the present invention. Other embodiments will become apparent from a review of the ensuing detailed description.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in“A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank Accession Number sequence submissions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

The term“comprise” or variations such as“comprises” or“comprising” according to the present invention means the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. The term“consisting essentially of’ according to the present invention means the inclusion of a stated integer or group of integers, while excluding modifications or other integers which would materially affect or alter the stated integer. The term“consisting of’ or variations such as“consists of’ according to the present invention means the inclusion of a stated integer or group of integers and the exclusion of any other integer or group of integers.

The terms“a” and“an” and“the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

The term“pancreatic cancer”, as used herein, refers to a disease arising when cells in the pancreas, a glandular organ behind the stomach, begin to multiply out of control and form a mass. These cancerous cells have the ability to invade other parts of the body. There are many types of pancreatic cancer known. The many types of pancreatic cancer can be divided into two general groups. The vast majority of cases (about 99%) occur in the part of the pancreas which produces digestive enzymes, known as the exocrine component (exocrine pancreatic cancer). The exocrine pancreatic cancer includes pancreatic ductal adenocarcinoma (PD AC). There are several sub-types of exocrine pancreatic cancers, but their diagnosis and treatment have much in common. The small minority of cancers that arise in the hormone-producing (endocrine) tissue of the pancreas have different clinical characteristics and are called pancreatic neuroendocrine tumors (neuroendocrine pancreatic cancer). Both groups occur mainly (but not exclusively) in people over 40, and are slightly more common in men, but some rare sub-types mainly occur in women or children. Thus, in one embodiment, the pancreatic cancer is exocrine pancreatic cancer such as PD AC. In one another embodiment, the pancreatic cancer neuroendocrine pancreatic cancer. Preferably, the pancreatic cancer is PD AC.

The term“diagnosing pancreatic cancer”, as used herein, means determining whether an individual shows signs of or suffers from pancreatic cancer.

The term“grading pancreatic cancer”, as used herein, means determining the stage of pancreatic cancer in an individual suffering from pancreatic cancer.

The term“differentiating between low-grade and high-grade pancreatic cancer” means determining whether an individual suffers from low-grade pancreatic cancer or high-grade pancreatic cancer.

The term“low-grade pancreatic cancer”, as used herein, means that the cancer cells are well differentiated. They look almost like normal cells. Lower grade cancer cells tend to grow slow and are less likely to spread. Pancreatic cancer of grades 0, 1, and 2 belongs to low-grade pancreatic cancer (see also staging below).

The term“high-grade pancreatic cancer”, as used herein, means that the cancer cells are poorly differentiated or undifferentiated. They look less normal, or more abnormal. Higher grade cancer cells tend to grow more quickly than low-grade cancer cells. Pancreatic cancer of grades 3 and 4 belongs to high-grade pancreatic cancer (see also staging below).

Knowing the grade of pancreatic cancer allows to determine how quickly the cancer may be growing and how likely it is to spread. The grade can also help to predict future outcomes (prognosis) and how the cancer might respond to treatment.

According to the TNM staging (System for the Classification of Malignant Tumors), pancreatic cancer has five stages - stage 0 followed by stages I to IV. The different stages of pancreatic cancer can be described as follows:

Stage 0 (or carcinoma in situ): The cancer cells are found only in the lining of the pancreas. Stage 1 : The tumor is 4 cm or smaller. For stage 1A, the tumor is 2 cm or smaller. For stage 1B, the tumor is larger than 2 cm, but not larger than 4 cm.

Stage 2: The tumor is larger than 4 cm (stage 2A). Or the tumor is any size and the cancer has spread to 1 to 3 nearby lymph nodes (stage 2B).

Stage 3: The cancer has spread to 4 or more nearby lymph nodes. Or the tumor has grown into large blood vessels outside of the pancreas and the cancer may have spread to nearby lymph nodes.

Stage 4: The cancer has spread to other parts of the body, such as the liver, lungs or abdominal cavity. This is also called metastatic pancreatic cancer.

The terms“stage” and“grade” are interchangeably used herein.

The term“providing a (survival) prognosis to an individual suffering from pancreatic cancer”, as used herein, refers to the prediction of the likelihood/probability of death of an individual suffering from pancreatic cancer. In particular, the term“providing a (survival) prognosis to an individual suffering from pancreatic cancer”, as used herein, means determining whether an individual has a good prognosis (low probability of death) or a poor prognosis (high probability of death) with respect to pancreatic cancer.

An individual suffering from pancreatic cancer may be considered to have a“good prognosis (low probability of death)” where, for example, the survival rate associated with pancreatic cancer is greater compared to the survival rate of (control) subjects suffering from the same disease. Preferably, an individual suffering from pancreatic cancer may be considered to have a“good prognosis” where, the survival rate associated with pancreatic cancer is greater compared to the survival rate of (control) subjects suffering from the same disease and showing another (expression) level of one or more prognostic bio markers. The prognostic bio marker of the present invention is the eukaryotic Initiation Factor 1 (e!Fl). In certain embodiments, a“good prognosis” indicates at least an increased expected survival time. A“good prognosis” preferably indicates a greater than 1%, more preferably a greater than 10%, 20%, 30%, or 40%, even more preferably a greater than 50%, 60%, or 70%, or most preferably a greater than 80%, 90% or 95% chance that the individual will survive at least to a specified time point (such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 month(s) or even 1, 2, 3, 4, or 5 year(s)) or a specific time period (such as at least a ¼-year, ½-year, l-year, 2-year, 3- year, 4-year, or 5-year period).

An individual suffering from pancreatic cancer may be considered to have a“poor prognosis (high probability of death)” where, for example, the survival rate associated with pancreatic cancer is lower compared to the survival rate of (control) subjects suffering from the same disease. Preferably, an individual suffering from pancreatic cancer may be considered to have a“poor prognosis” where, the survival rate associated with pancreatic cancer is lower compared to the survival rate of (control) subjects suffering from the same disease and showing another (expression) level of one or more prognostic markers. The prognostic biomarker of the present invention is the eukaryotic Initiation Factor 1 (elFl).

In certain embodiments, a“poor prognosis” indicates at least a decreased expected survival time.

A“poor prognosis” preferably indicates a lower than 1%, more preferably a lower than 10%, 20%, 30%, or 40%, even more preferably a lower than 50%, 60%, or 70%, or most preferably a lower than 80%, 90% or 95% chance that the individual will survive at least to a specified time point (such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 month(s) or even 1, 2, 3, 4, or 5 year(s)) or a specific time period (such as at least a ¼-year, ½-year, l-year, 2-year, 3-year, 4- year, or 5 -year period).

The prognostic method can be used clinically to determine the survival prognosis/survival probability of an individual suffering from pancreatic cancer. In particular, the method of providing a survival prognosis to an individual suffering from pancreatic cancer is a valuable tool in predicting whether overall (or long-term) survival of the individual, e.g. following therapy, is likely. The prognostic method can also be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular individual suffering from pancreatic cancer. In particular, the method of providing a survival prognosis to an individual suffering from pancreatic cancer is a valuable tool in predicting if an individual is likely to respond favorably to a treatment regimen, such as drug therapy and/or surgical intervention. The term“overall survival”, as used herein, refers to an individual’s survival suffering from pancreatic cancer for at least ¼-year, ½-year, 1 year, 2 years, at least 5 years, at least 8 years, or at least 10 years, e.g. following treatment such as drug therapy, radiotherapy, chemotherapy, and/or surgical intervention.

The term“individual”, as used herein, refers to any subject for whom it is desired to know whether she or he suffers from pancreatic cancer. In particular, the term“individual”, as used herein, refers to a subject suspected to be affected by pancreatic cancer. The individual may be diagnosed to be affected by pancreatic cancer, i.e. diseased, or may be diagnosed to be not affected by pancreatic cancer, i.e. healthy. The term“individual”, as used herein, also refers to a subject which is affected by pancreatic cancer, i.e. diseased. The patient may be retested for pancreatic cancer and may be diagnosed to be still affected by pancreatic cancer, i.e. diseased, or not affected by pancreatic cancer anymore, i.e. healthy, for example after therapeutic intervention. The individual may also be retested for pancreatic cancer and may be diagnosed as having developed an advanced form of pancreatic cancer (e.g. from pancreatic cancer of stages 1 or 2 to pancreatic cancer of stages 3 or 4, or from a low-grade pancreatic cancer to a high-grade pancreatic cancer). The individual suffering from pancreatic cancer may alternatively be tested in order to determine the individual’s survival prognosis.

It should be noted that an individual that is diagnosed as being healthy, i.e. not suffering from pancreatic cancer, may possibly suffer from another disease or condition not tested/known. The individual may be any mammal, including both a human and another mammal, e.g. an animal such as a rabbit, mouse, rat, or monkey. Human individuals are particularly preferred.

The term“(control) patient”, as used herein, refers to a subject known to be affected by pancreatic cancer, i.e. diseased. Said (control) patient may have developed an advanced form of pancreatic cancer. For example, the (control) patient is a (control) patient with pancreatic cancer of a specific grade (e.g. grade I/II or grade III). The (control) patient may also be a (control) patient suffering from low-grade pancreatic cancer or high-grade pancreatic cancer. The (control) patient may be any mammal, including both a human and another mammal, e.g. an animal such as a rabbit, mouse, rat, or monkey. Human (control) patients are particularly preferred.

The term“healthy (control) individual”, as used herein, refers to a subject known to be not affected by pancreatic cancer (negative control), i.e. healthy.

It should be noted that an individual which is known to be healthy, i.e. not suffering from pancreatic cancer, may possibly suffer from another disease or condition not tested/known. The healthy individual may be any mammal, including both a human and another mammal, e.g. an animal such as a rabbit, mouse, rat, or monkey. Human healthy individuals are particularly preferred.

The term“treatment”, in particular“therapeutic treatment”, as used herein, refers to any therapy which improves the health status and/or prolongs (increases) the lifespan of an individual suffering from a disease or condition, in particular a tumor. Said therapy may eliminate the disease or condition in an individual, arrest or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, decrease the frequency or severity of symptoms in an individual, and/or decrease the recurrence in an individual who currently has or who previously has had a disease. The treatment of pancreatic cancer described herein includes, but is not limited to, administration of a drug, surgery, chemotherapy, and/or radiotherapy.

The term“level”, as used herein, refers to an amount (measured for example in grams, mole, or ion counts) or concentration (e.g. absolute or relative concentration) of the at least one elF claimed herein. The term“level”, as used herein, also comprises scaled, normalized, or scaled and normalized amounts or values. The level may also be a cut-off level. In one embodiment, the level is an expression level.

The term“eukaryotic Initiation Factor (elF)”, as used herein, refers to molecules which are involved in the initiation phase of eukaryotic translation. These factors help to stabilize the formation of the functional ribosome around the start codon and also provide regulatory mechanisms in translation initiation. The term“eukaryotic Initiation Factor (elF)”, as used herein, covers elF RNA transcripts (RNA transcript variants) such as mRNAs including splice variants of these transcripts and elF proteins encoded thereby. Thus, the level of the elFs may be determined by measuring mRNA or protein levels. The term“eukaryotic Initiation Factor (elF)”, as used herein, also covers elF isoforms. These elF isoforms are members of a set of highly similar molecules, in particular proteins, that perform the same or similar biological role. Described herein are the elFs: eIF2a (Gene: EIF2S1; Gene ID (GenBank): 1965), elFl (Gene: EIF1AD, Gene ID (GenBank): 84285), eIF3C (Gene: EIF3C; Gene ID (GenBank): 8663), and eIF6 (Gene: eIF6; Gene ID (GenBank): 3692).

The term“biological sample”, as used herein, refers to any biological sample from an individual or (control) patient comprising at least one of the elFs claimed herein. The biological sample may be a body fluid sample, e.g. a blood sample or urine sample, or a tissue sample. Biological samples may be mixed or pooled, e.g. a sample may be a mixture of a blood sample and a urine sample. Said biological samples may be provided by removing a body fluid from an individual or (control) patient, but may also be provided by using a previously isolated sample. For example, a blood sample may be taken from an individual or (control) patient by conventional blood collection techniques. The biological sample, e.g. urine sample or blood sample, may be obtained from an individual or (control) patient prior to the initiation of a therapeutic treatment, during the therapeutic treatment, and/or after the therapeutic treatment. If the sample, in particular the biological sample, is obtained from at least one (control) patient or healthy (control) individual, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, or 1.000 (control) patient(s) or healthy (control) individual(s), it is designated as a“reference biological sample”. Preferably, the reference biological sample is from the same source than the biological sample of the individual to be tested, e.g. both are blood samples or urine samples. It is further preferred that both are from the same species, e.g. from a human. It is also (alternatively or additionally) preferred that the measurements of the reference biological sample and the biological sample of the individual to be tested are identical, e.g. both have an identical volume. It is particularly preferred that the reference biological sample and the biological sample are from individuals/(control) patients of the same sex and similar age, e.g. no more than 2 years apart from each other.

The term“body fluid sample”, as used herein, refers to any liquid sample derived from the body of an individual or (control) patient containing at least one of the elFs claimed herein. Said body fluid sample may be a urine sample, blood sample, sputum sample, breast milk sample, cerebrospinal fluid (CSF) sample, cerumen (earwax) sample, gastric juice sample, mucus sample, lymph sample, endolymph fluid sample, perilymph fluid sample, peritoneal fluid sample, pleural fluid sample, saliva sample, sebum (skin oil) sample, semen sample, sweat sample, tears sample, cheek swab, vaginal secretion sample, liquid biopsy, or vomit sample including components or fractions thereof. The term“body fluid sample” also encompasses body fluid fractions, e.g. blood fractions, urine fractions or sputum fractions. Body fluid samples may be mixed or pooled. Thus, a body fluid sample may be a mixture of a blood and a urine sample or a mixture of a blood and cerebrospinal fluid sample. Said body fluid sample may be provided by removing a body liquid from an individual or (control) patient, but may also be provided by using previously isolated body fluid sample material. The body fluid sample allows for a non-invasive analysis of an individual. It is further preferred that the body fluid sample has a volume of between 0.01 and 20 ml, more preferably of between 0.1 and 10 ml, even more preferably of between 0.5 and 8 ml, and most preferably of between 1 and 5 ml.

The term“blood sample”, as used herein, encompasses a whole blood sample or a blood fraction sample such as a blood serum or blood plasma sample. It is preferred that the blood serum or plasma sample has a volume of between 0.01 and 20 ml, more preferably of between 0.1 and 10 ml, even more preferably of between 0.5 and 8 ml and most preferably of between 1 and 5 ml.

In the context of the present invention, the term“kit of parts (in short: kit)” is understood to be any combination of at least some of the components identified herein, which are combined, coexisting spatially, to a functional unit, and which can contain further components. Said kit may allow point-of-care testing (POCT).

The term“point-of-care testing (POCT)”, as used herein, refers to a medical diagnostic testing at or near the point of care that is the time and place of individual care. This contrasts with the historical pattern in which testing was wholly or mostly confined to the medical laboratory, which entailed sending off specimens away from the point of care and then waiting hours or days to learn the results, during which time care must continue without the desired information. Point-of-care tests are simple medical tests that can be performed at the bedside. The driving notion behind POCT is to bring the test conveniently and immediately to the individual to be tested. This increases the likelihood that the individual, physician, and care team will receive the results quicker, which allows for immediate clinical management decisions to be made. POCT is often accomplished through the use of transportable, portable, and handheld instruments and test kits. Small bench analyzers or fixed equipment can also be used when a handheld device is not available - the goal is to collect the specimen and obtain the results in a very short period of time at or near the location of the individual so that the treatment plan can be adjusted as necessary before the individual leaves the hospital.

Embodiments of the invention

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous, unless clearly indicated to the contrary.

The present inventors found that elFs represent crossroads for carcinogenesis in pancreatic cancer. They identified with elFl, eIF2a, eIF3C and eIF6 new diagnostic biomarkers for pancreatic cancer. These new diagnostic biomarkers allow the diagnosis of pancreatic cancer. Thus, in a first aspect, the present invention relates to a (an in vitro) method of diagnosing pancreatic cancer in an individual (suspected of suffering from pancreatic cancer) comprising the step of:

determining the level of at least one eukaryotic Initiation Factor (elF), e.g. 1, 2, 3, or 4 eIF(s), in a biological sample from an individual,

wherein the at least one elF is selected from the group consisting of eIF2a (Gene: EIF2S1; Gene ID (GenBank): 1965), elFl (Gene: EIF1AD, Gene ID (GenBank): 84285), eIF3C (Gene: EIF3C; Gene ID (GenBank): 8663), and eIF6 (Gene: eIF6; Gene ID (GenBank): 3692).

In particular, said individual is suspected of suffering from pancreatic cancer.

For example, the level(s) of at least 1, at least 2, at least 3 eIF(s), or of all of the elFs mentioned above is (are) determined.

Preferably, the at least one elF is (i) eIF2a, (i) elFl, (iii) eIF3C, (iv) eIF6, (v) eIF2a and elFl, (vi) eIF2a and eIF3C, (vii) eIF2a and eIF6, (viii) elFl and eIF3C, (ix) elFl and eIF6, (x) eIF3C and eIF6, (xi) eIF2a, elFl, and eIF3C, (xii) eIF2a, elFl, and eIF6, (xiii) eIF2a, eIF3C, and eIF6, (xiv) elFl, eIF3C, and eIF6, or (xv) eIF2a, elFl, eIF3C, and eIF6.

In one embodiment, the level of the at least one elF is compared to a reference level of said at least one elF (e.g. to at least 1, 2, 3 reference level(s), or 4 reference levels). Thus, in one particular embodiment, the present invention relates to a method of diagnosing pancreatic cancer in an individual (suspected of suffering from pancreatic cancer) comprising the steps of:

(i) determining the level of at least one eukaryotic Initiation Factor (elF), e.g. 1, 2, 3, or 4 eIF(s), in a biological sample from an individual, and

(ii) comparing the level of the at least one elF to a reference level of said at least one elF, wherein the at least one elF is selected from the group consisting of eIF2a, elFl, eIF3C, and eIF6.

The above comparison allows to diagnose pancreatic cancer in an individual, in particular in an individual suspected of having pancreatic cancer. The individual may be diagnosed as suffering from pancreatic cancer, i.e. being diseased, or as not suffering from pancreatic cancer, i.e. being healthy.

The reference level may be any level which allows to determine whether an individual suffers from pancreatic cancer or not. It is preferred that the reference level is the level determined by measuring at least one reference biological sample, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference biological sample(s), from at least one healthy individual, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100,

150, 200, 250, 300, 400, 500, or 1.000 healthy individual(s). It is more preferred that the reference level is the level determined by measuring between 2 and 500 reference biological samples from between 2 and 500 healthy individuals. It is even more preferred that the reference level is determined by measuring between 50 and 500 reference biological samples from between 50 and 500 healthy individuals. It is most preferred that the reference level is determined by measuring between 100 and 500 reference biological samples from between 100 and 500 healthy individuals.

It is practicable to take one reference biological sample per individual for analysis. If additional reference biological samples are required, e.g. to determine the reference level in different reference biological samples, the same individual may be (re)tested. Said reference level may be an average reference level. It may be determined by measuring reference levels and calculating the“average” value (e.g. mean, median or modal value) thereof.

It is further preferred that the level of the at least one elF below the reference level indicates that the individual suffers from pancreatic cancer. Said at least one elF is selected from the group consisting of eIF2a, elFl, eIF3C, and eIF6.

Preferably, the level of the at least one elF is at least 0.6-fold or 0.7-fold, more preferably at least 0.8-fold or 0.9-fold, even more preferably at least 1.2-fold or 1.5-fold, and most preferably at least 2.0-fold or 3.0-fold below the reference level. For example, the level of the at least one elF is at least 0.6-fold, at least 0.7-fold, at least 0.8-fold, at least 0.9-fold, at least l .O-fold, at least l . l-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2.0-fold, at least 2.1- fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, or at least 3.0-fold below the reference level.

The present inventors further found out that the new diagnostic biomarkers for pancreatic cancer elFl, eIF2a, eIF3C and eIF6 allow grading of pancreatic cancer. Thus, in a second aspect, the present invention relates to a (an in vitro ) method of grading/staging pancreatic cancer in an individual (suffering from pancreatic cancer) comprising the step of: determining the level of at least one eukaryotic Initiation Factor (elF), e.g. 1 , 2, 3, or 4 eIF(s), in a biological sample from an individual,

In particular, said individual suffers from pancreatic cancer. For example, the level(s) of at least 1, at least 2, at least 3 eIF(s), or of all of the elFs mentioned above is (are) determined.

As to the Gene IDs of the elFs, it is referred to the first aspect of the present invention. In one embodiment, the level of the at least one elF is compared to a reference level of said at least one elF. Thus, in one particular embodiment, the present invention relates to a method of grading/staging pancreatic cancer in an individual (suffering from pancreatic cancer) comprising the steps of:

The above comparison allows to grade/stage pancreatic cancer in an individual, in particular in an individual suffering from pancreatic cancer. It may be determined that the individual suffers from pancreatic cancer of grade 0, 1, II, III, or IV, in particular of grade I/II or grade III.

Preferably, the above method allows to determine whether the individual suffers from pancreatic cancer of grade I/II or grade III.

The reference level may be any level which allows grading/staging of pancreatic cancer in an individual. It is preferred that the reference level is the level determined by measuring at least one reference biological sample, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference biological sample(s), from at least one healthy individual, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 healthy individual(s), from at least one patient with pancreatic cancer of grade I/II, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 patient(s) with pancreatic cancer of grade I/II, and/or from at least one patient with pancreatic cancer of grade III, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 patient(s) with pancreatic cancer of grade III. It is more preferred that the reference level is the level determined by measuring between 2 and 500 reference biological samples from between 2 and 500 healthy individuals, patients with pancreatic cancer of grade I/II, or patients with pancreatic cancer of grade III. It is even more preferred that the reference level is determined by measuring between 50 and 500 reference biological samples from between 50 and 500 healthy individuals, patients with pancreatic cancer of grade I/II, or patients with pancreatic cancer of grade III. It is most preferred that the reference level is determined by measuring between 100 and 500 reference biological samples from between 100 and 500 healthy individuals, patients with pancreatic cancer of grade I/II, or patients with pancreatic cancer of grade III.

It is further preferred that

(i) the reference level is the level determined by measuring at least one reference biological sample from at least one patient with pancreatic cancer of grade I/II,

wherein the level of the at least one elF above the reference level indicates that the individual has pancreatic cancer of grade III,

(ii) the reference level is the level determined by measuring at least one reference biological sample from at least one patient with pancreatic cancer of grade III,

wherein the level of the at least one elF below the reference level indicates that the individual has pancreatic cancer of grade I/II,

(iii) the reference level is the level determined by measuring at least one reference biological sample from at least one healthy individual and the reference level is the level determined by measuring at least one reference biological sample from at least one patient with pancreatic cancer of grade III,

wherein the level of the at least one elF below the reference level determined by measuring at least one reference biological sample from at least one healthy individual and below the reference level determined by measuring at least one reference biological sample from at least one patient with pancreatic cancer of grade III indicates that the individual has pancreatic cancer of grade I/II,

(iv) the reference level is the level determined by measuring at least one reference biological sample from at least one healthy individual and the reference level is the level determined by measuring at least one reference biological sample from at least one patient with pancreatic cancer of grade I/II,

wherein the level of the at least one elF below the reference level determined by measuring at least one reference biological sample from at least one healthy individual and above the reference level determined by measuring at least one reference biological sample from at least one patient with pancreatic cancer of grade I/II indicates that the individual has pancreatic cancer of grade III.

Preferably, the level of the at least one elF is at least 0.6-fold or 0.7-fold, more preferably at least 0.8-fold or 0.9-fold, even more preferably at least 1.2-fold or 1.5-fold, and most preferably at least 2.0-fold or 3.0-fold below/above the reference level. For example, the level of the at least one elF is at least 0.6-fold, at least 0.7-fold, at least 0.8-fold, at least 0.9-fold, at least l.O-fold, at least l.l-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5- fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2.0-fold, at least 2.l-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6- fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, or at least 3.0-fold below/above the reference level.

The present inventors further found out that the new diagnostic biomarkers for pancreatic cancer elFl, eIF2a, eIF3C and eIF6 allow differential diagnosis of pancreatic cancer. Thus, in a third aspect, the present invention relates to a (an in vitro) method of differentiating between low-grade pancreatic cancer and high-grade pancreatic cancer in an individual (suffering from pancreatic cancer) comprising the step of:

determining the level of at least one eukaryotic Initiation Factor (elF) , e.g. 1, 2, 3, or 4 eIF(s), in a biological sample from an individual,

In particular, said individual suffers from pancreatic cancer.

As to the Gene IDs of the elFs, it is referred to the first aspect of the present invention. In one embodiment, the level of the at least one elF is compared to a reference level of said at least one elF. Thus, in one particular embodiment, the present invention relates to a method of differentiating between low-grade pancreatic cancer and high-grade pancreatic cancer in an individual (suffering from pancreatic cancer) comprising the steps of:

The above comparison allows to differentiate between low- and high-grade pancreatic cancer in an individual. It may be determined that the individual suffers from low-grade pancreatic cancer or from high-grade pancreatic cancer.

Pancreatic cancer of grades 0, 1, and 2 belongs to low-grade pancreatic cancer. Pancreatic cancer of grades 3 and 4 belongs to high-grade pancreatic cancer.

Preferably, the above method allows to differentiate between pancreatic cancer of grade 1/2 (low grade pancreatic cancer) and pancreatic cancer of grade 3 (high grade pancreatic cancer).

The reference level may be any level which allows to differentiate between low-grade and high-grade pancreatic cancer in an individual. It is preferred that the reference level is the level determined by measuring at least one reference biological sample, e.g. at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference biological sample(s), from at least one healthy individual, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 healthy individual(s), from at least one patient with low- grade pancreatic cancer, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 patient(s) with low- grade pancreatic cancer, and/or from at least one patient with high-grade pancreatic cancer, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,

26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,

100, 150, 200, 250, 300, 400, 500, or 1.000 patient(s) with high-grade pancreatic cancer. It is more preferred that the reference level is the level determined by measuring between 2 and 500 reference biological samples from between 2 and 500 healthy individuals, patients with low- grade pancreatic cancer, or patients with high-grade pancreatic cancer. It is even more preferred that the reference level is determined by measuring between 50 and 500 reference biological samples from between 50 and 500 healthy individuals, patients with low-grade pancreatic cancer, or patients with high-grade pancreatic cancer. It is most preferred that the reference level is determined by measuring between 100 and 500 reference biological samples from between 100 and 500 healthy individuals, patients with low-grade pancreatic cancer, or patients with high-grade pancreatic cancer.

It is further preferred that

(i) the reference level is the level determined by measuring at least one reference biological sample from at least one patient with low-grade, in particular grade I/II, pancreatic cancer,

wherein the level of the at least one elF above the reference level indicates that the individual has high-grade, in particular grade III, pancreatic cancer,

(ii) the reference level is the level determined by measuring at least one reference biological sample from at least one patient with high-grade, in particular grade III, pancreatic cancer,

wherein the level of the at least one elF below the reference level indicates that the individual has low-grade, in particular grade I/II, pancreatic cancer,

(iii) the reference level is the level determined by measuring at least one reference biological sample from at least one healthy individual and the reference level is the level determined by measuring at least one reference biological sample from at least one patient with high-grade, in particular grade III, pancreatic cancer,

wherein the level of the at least one elF below the reference level determined by measuring at least one reference biological sample from at least one healthy individual and below the reference level determined by measuring at least one reference biological sample from at least one patient with high-grade, in particular grade III, pancreatic cancer indicates that the individual has low-grade, in particular grade I/II, pancreatic cancer,

(iv) the reference level is the level determined by measuring at least one reference biological sample from at least one healthy individual and the reference level is the level determined by measuring at least one reference biological sample from at least one patient with low-grade, in particular grade I/II, pancreatic cancer,

wherein the level of the at least one elF below the reference level determined by measuring at least one reference biological sample from at least one healthy individual and above the reference level determined by measuring at least one reference biological sample from at least one patient with low-grade, in particular grade I/II, pancreatic cancer indicates that the individual has high-grade, in particular grade III, pancreatic cancer.

Preferably, the level of the at least one elF is at least 0.6-fold or 0.7-fold, more preferably at least 0.8-fold or 0.9-fold, even more preferably at least 1.2-fold or 1.5-fold, and most preferably at least 2.0-fold or 3.0-fold below/above the reference level. For example, the level of the at least one elF is at least 0.6-fold, at least 0.7-fold, at least 0.8-fold, at least 0.9-fold, at least l .O-fold, at least l . l-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5- fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2.0-fold, at least 2. l-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6- fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, or at least 3.0-fold below/above the reference level.

Preferably, the low-grade pancreatic cancer is pancreatic cancer of grade I/II and the high-grade pancreatic cancer is pancreatic cancer of grade III.

In a fourth aspect, the present invention relates to a (an in vitro) method of providing a survival prognosis to an individual suffering from pancreatic cancer comprising the step of: determining the level of eukaryotic Initiation Factor 1 (elFl) in a biological sample from an individual suffering from pancreatic cancer.

It is preferred that the method of providing a survival prognosis to an individual suffering from pancreatic cancer is carried out at the time of pancreatic cancer diagnosis, immediately after pancreatic cancer diagnosis, e.g. within 1 week, 2 week, 3 weeks, or 1 month after pancreatic cancer diagnosis, or within a time period after pancreatic cancer diagnosis, e.g. within a time period of no more than 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, or 4 years after pancreatic cancer diagnosis. The method of providing a survival prognosis to an individual suffering from pancreatic cancer may also be repeated several times during the disease/illness period in order to determine, whether the survival prognosis has changed, e.g. from a good survival prognosis to a low survival prognosis or from a low survival prognosis to a good survival prognosis.

It is also preferred that the method allows a at least ¼-year, ½-year, l-year, 2-year, 3- year, 4-year, 5-year , 6-year, 7-year, 8-year, 9-year, or lO-year survival prognosis (after diagnosis)/a survival prognosis of at least up to about ¼-year, ½-year, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years (after diagnosis). The prognostic method can be used clinically to determine the survival prognosis/survival probability of an individual suffering from pancreatic cancer. In particular, the method of providing a survival prognosis to an individual suffering from pancreatic cancer is a valuable tool in predicting whether overall (or long-term) survival of the individual, e.g. following therapy, is likely. The prognostic method can also be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular individual suffering from pancreatic cancer. In particular, the method of providing a survival prognosis to an individual suffering from pancreatic cancer is a valuable tool in predicting if an individual is likely to respond favorably to a treatment regimen, such as drug therapy, chemotherapy, radiotherapy, and/or surgical intervention.

It is further preferred that the level of elFl is compared to a reference level of said elFl . The above comparison allows to determine the survival prognosis/probability of the individual suffering from pancreatic cancer. The reference level may be any level which allows to determine the survival prognosis/probability of the individual suffering from pancreatic cancer. It may be obtained from a (control) subject (i.e. a subject different from the individual to be tested) or from the same individual. In the latter case, the survival prognosis/probability of the individual suffering from pancreatic cancer may be retested, e.g. in the form of a longitudinal monitoring. It may be determined that the individual has now (in the re-test) a good survival prognosis/high survival probability and not a poor survival prognosis/low survival probability anymore or that the individual has now a poor survival prognosis/low survival probability (in the re-test) and not a good survival prognosis/high survival probability anymore. The re-test may be carried out regularly, e.g. every year or two, three, or four times per year.

The reference level is preferably determined at the time of pancreatic cancer diagnosis, immediately after pancreatic cancer diagnosis, e.g. within 1 week, 2 weeks, 3 weeks, or 1 month after pancreatic cancer diagnosis, or within a time period after pancreatic cancer diagnosis, e.g. within a time period of no more than 1 week, 2 weeks, 3 weeks, or 1 month after pancreatic cancer diagnosis of the (control) subject. Preferably, the level and the reference level are determined at the same time points, e.g. at the time of pancreatic cancer diagnosis, or during the same time period.

The reference level may be the level determined by measuring at least one reference biological sample from at least one healthy subject and/or at least one subject suffering from pancreatic cancer.

More preferably, the reference level is the level determined by measuring at least one reference biological sample, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42,

43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference biological sample(s), from at least one subject suffering from pancreatic cancer, e.g. from at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,

31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250,

300, 400, 500, or 1.000 subject(s) suffering from pancreatic cancer.

Even more preferably,

the level of elFl comparable with or below the reference level indicates that the individual has a poor survival prognosis, or

the level of elFl above the reference level indicates that the individual has a good survival prognosis.

The level of elFl is preferably at least 0.6-fold or 0.7-fold, more preferably at least 0.8- fold or 0.9-fold, even more preferably at least 1.2-fold or 1.5-fold, and most preferably at least 2.0-fold or 3.0-fold above/below the reference level. For example, the level of elFl is at least 0.6-fold, at least 0.7-fold, at least 0.8-fold, at least 0.9-fold, at least l .O-fold, at least l . l-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7- fold, at least 1.8-fold, at least 1.9-fold, at least 2.0-fold, at least 2. l-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8- fold, at least 2.9-fold, or at least 3.0-fold above/below the reference level.

A level which is comparable with the reference level is preferably identical with the reference level.

The pancreatic cancer referred to in the methods of the first to fourth aspect of the present invention is preferably exocrine pancreatic cancer or neuroendocrine pancreatic cancer. More preferably, the pancreatic cancer is PD AC. PD AC is an exocrine pancreatic cancer.

In the methods of the first to fourth aspect of the present invention, it is preferred that the biological sample is a tissue sample, e.g. tumor tissue sample, or a body fluid sample. It is also preferred that the reference biological sample is a tissue sample, e.g. tumor tissue sample, or a body fluid sample. Preferably, the body fluid sample is selected from the group consisting of a blood sample, an urine sample, a lymph sample, a saliva sample and a combination thereof. More preferably, the blood sample is a whole blood sample or a blood fraction sample. Even more preferably, the blood fraction sample is a blood serum sample or a blood plasma sample.

Preferably, the aforementioned samples are pre-treated before they are used in the methods of the first to fourth aspect of the present invention. Said pre-treatment may include treatments required to separate the at least one e!F described herein, or to remove excessive material or waste. Furthermore, pre-treatments may aim at sterilizing samples and/or removing contaminants such as undesired cells, bacteria or viruses. Suitable techniques comprise centrifugation, extraction, fractioning, ultrafiltration, protein precipitation followed by filtration and purification and/or enrichment of compounds. Moreover, other pre-treatments are carried out in order to provide the at least one elF described herein in a form or concentration suitable for analysis.

In one preferred embodiment of the methods of the first to fourth aspect of the present invention, the biological sample used to determine the level of the at least one elF is a tissue sample, e.g. tumor tissue sample (obtainable e.g. by biopsy) or a body fluid sample. The elF markers of the present invention can be found in the tissue affected with the tumor and in body fluids like blood and blood components (e.g. plasma or serum).

According to another preferred embodiment of the methods of the first to fourth aspect of the present invention, the level of the at least one elF is determined by measuring mRNA or protein levels. The levels of the elFs in the methods of the first to fourth aspect of the present invention can be determined either by measuring mRNA molecules encoding said elFs or the elFs as such in form of proteins. Methods to determine mRNA levels and protein levels in a sample are well known. mRNA expression levels are usually measured by polymerase chain reaction (PCR), in particular by reverse transcription quantitative polymerase chain reaction (RT-PCR and qPCR) or real-time PCR. RT-PCR is used to create a cDNA from the mRNA. The cDNA may be used in a qPCR assay to produce fluorescence as the DNA amplification process progresses. This fluorescence is proportional to the original mRNA amount in the samples. Other methods to be used include Northern blots, Fluorescence in situ hybridization (FISH), microarrays, and RT-PCR combined with capillary electrophoresis. Protein levels of elFs are preferably determined using immunoassays. Such methods are based on the binding of an antibody, a derivative or a fragment thereof to its corresponding target (i.e. elF). Polyclonal and monoclonal antibodies can be used in such methods. Derivatives or fragments of antibodies include Fab fragments, F(ab')₂ fragments, Fv fragments, single chain antibodies and single domain antibodies. Preferred immunoassays include Western blot, Immunohistochemistry, ELISA (enzyme- linked immunosorbent assay), radioimmunoassays, fluorescence resonance energy transfer (FRET) or time resolved-FRET (TR-FRET). Immunoassays detection is possible in lymphoma and HCC. It is particularly preferred to use antibodies and derivatives or fragments of antibodies which have been obtained from a non-human source. These antigen binding molecules can be of porcine, rabbit, murine, camel or rat origin. Of course, it is also possible to use antibodies and derivatives or fragments thereof which are recombinantly produced in plants or cell cultures, in particular microbial cell cultures (e.g. bacteria, yeast).

In a fifth aspect, the present invention relates to the (in vitro) use of at least one elF, e.g. 1, 2, 3, or 4 eIF(s), for diagnosing pancreatic cancer in an individual (suspected of suffering from pancreatic cancer), grading pancreatic cancer in an individual (suffering from pancreatic cancer), or differentiating between low-grade pancreatic cancer and high-grade pancreatic cancer in an individual (suffering from pancreatic cancer),

As to the Gene IDs of the elFs, it is referred to the first aspect of the present invention.

The pancreatic cancer may be pancreatic cancer of grade 0, 1, II, III, or IV, in particular of grade I/II or grade III. Pancreatic cancer of grades 0, 1, and 2 belongs to low-grade pancreatic cancer. Pancreatic cancer of grades 3 and 4 belongs to high-grade pancreatic cancer. Preferably, the low-grade pancreatic cancer is pancreatic cancer of grade I/II and the high-grade pancreatic cancer is pancreatic cancer of grade III.

The pancreatic cancer is preferably exocrine pancreatic cancer or neuroendocrine pancreatic cancer. More preferably, the pancreatic cancer is PD AC. PD AC is an exocrine pancreatic cancer.

For the above-mentioned use, the level of the above-mentioned elFs is determined in a biological sample from an individual to be tested. It is preferred that the biological sample is a tissue sample, e.g. tumor tissue sample, or body fluid sample. Preferably, the body fluid sample is selected from the group consisting of a blood sample, a urine sample, and a combination thereof. More preferably, the blood sample is a whole blood sample or a blood fraction sample. Even more preferably, the blood fraction sample is a blood serum sample or a blood plasma sample.

As to further preferred embodiments, it is referred to the first to third aspect of the present invention.

In a sixth aspect, the present invention relates to the (in vitro) use of elFl for providing a survival prognosis to an individual suffering from pancreatic cancer.

As to the Gene ID of elFl, it is referred to the first aspect of the present invention. The pancreatic cancer is preferably exocrine pancreatic cancer or neuroendocrine pancreatic cancer. More preferably, the pancreatic cancer is PD AC. PD AC is an exocrine pancreatic cancer.

For the above-mentioned use, the level of elFl is determined in a biological sample from an individual to be tested. It is preferred that the biological sample is a tissue sample, e.g. tumor tissue sample, or body fluid sample. Preferably, the body fluid sample is selected from the group consisting of a blood sample, a urine sample, and a combination thereof. More preferably, the blood sample is a whole blood sample or a blood fraction sample. Even more preferably, the blood fraction sample is a blood serum sample or a blood plasma sample.

As to further preferred embodiments, it is referred to the fourth aspect of the present invention.

In a seventh aspect, the present invention relates to a kit for diagnosing pancreatic cancer in an individual (suspected of suffering from pancreatic cancer), grading pancreatic cancer in an individual (suffering from pancreatic cancer), or differentiating between low-grade pancreatic cancer and high-grade pancreatic cancer in an individual (suffering from pancreatic cancer), wherein said kit

comprises means for determining the level of at least one elF, e.g. 1, 2, 3, or 4 eIF(s), in a biological sample from an individual,

For example, the means are for determining the level(s) of at least 1, at least 2, at least 3 eIF(s), or of all of the elFs mentioned above is (are) determined.

Said means may be primers or primer pairs allowing the detecting of the above- mentioned elFs on the R A transcript, e.g. mR A, level and/or antibodies, antibody derivatives or fragments of antibodies allowing the detection of the above-mentioned elFs on the protein level.

In addition, said means encompass dipstrips or dipsticks, e.g. urine or blood dipstrips or dipsticks. Said means are tools used to determine changes in individual’s urine or blood. A dipstrip or dipstick comprises different chemical pads or reagents which react (e.g. change color, in particular by applying an immune assay) when immersed in (e.g. blood or urine), and then removed from the biological sample (e.g. urine or blood sample). The result can be read after a few minutes, preferably after a few seconds.

It is preferred that the kit is useful for conducting the methods of the first to third aspect of the present invention.

In an eighth aspect, the present invention relates to a kit for providing a survival prognosis to an individual suffering from pancreatic cancer, wherein said kit comprises means for determining the level of elFl in a biological sample from an individual suffering from pancreatic cancer.

As to the Gene ID of elFl, it is referred to the first aspect of the present invention.

Said means may be primers or primer pairs allowing the detection of elFl on the RNA transcript, e.g. mRNA, level and/or antibodies, antibody derivatives or fragments of antibodies allowing the detection of elFl on the protein level.

It is preferred that the kit is useful for conducting the method of the fourth aspect of the present invention.

It is further preferred that the above-mentioned kits comprise

(i) a container, and/or

(ii) a data carrier.

Said data carrier may be a non-electronical data carrier, e.g. a graphical data carrier such as an information leaflet, an information sheet, a bar code or an access code, or an electronical data carrier such as a floppy disk, a compact disk (CD), a digital versatile disk (DVD), a microchip or another semiconductor-based electronical data carrier. The access code may allow the access to a database, e.g. an internet database, a centralized, or a decentralized database. The access code may also allow access to an application software that causes a computer to perform tasks for computer users or a mobile app which is a software designed to run on smartphones and other mobile devices.

Said data carrier may further comprise a reference level of the at least one elF referred to herein. In case that the data carrier comprises an access code which allows the access to a database, said reference level is deposited in this database.

In addition, the data carrier may comprise information or instructions on how to carry out the methods of the first to fourth aspect of the present invention.

Said kit may also comprise materials desirable from a commercial and user standpoint including a buffer(s), a reagent(s) and/or a diluent(s) for determining the level mentioned above.

The individual tested in the methods of the first to fourth aspect of the present invention and referred to in the fifth to eighth aspect of the present invention may be a mammal. Preferably, the mammal is a human.

The present invention is summarized as follows:

1. A method of diagnosing pancreatic cancer in an individual comprising the step of: determining the level of at least one eukaryotic Initiation Factor (elF) in a biological sample from an individual,

2. The method of item 1 , wherein the level of the at least one elF is compared to a reference level of said at least one elF.

3. The method of item 2, wherein the reference level is the level determined by measuring at least one reference biological sample from at least one healthy individual.

4. The method of items 2 or 3, wherein the level of the at least one elF below the reference level indicates that the individual suffers from pancreatic cancer.

5. A method of grading pancreatic cancer in an individual comprising the step of:

wherein the at least one elF is selected from the group consisting of eIF2a, elFl, eIF3C, and eIF6. The method of item 5, wherein the level of the at least one elF is compared to at least one reference level of said at least one elF.

The method of item 6, wherein the at least one reference level is the level determined by measuring at least one reference biological sample

from at least one healthy individual,

from at least one patient with pancreatic cancer of grade I/II, and/or

from at least one patient with pancreatic cancer of grade III.

The method of items 6 or 7, wherein

(i) the reference level is the level determined by measuring at least one reference biological sample from at least one patient with pancreatic cancer of grade I/II, wherein the level of the at least one elF above the reference level indicates that the individual has pancreatic cancer of grade III,

(ii) the reference level is the level determined by measuring at least one reference biological sample from at least one patient with pancreatic cancer of grade III, wherein the level of the at least one elF below the reference level indicates that the individual has pancreatic cancer of grade I/II,

wherein the level of the at least one elF below the reference level determined by measuring at least one reference biological sample from at least one healthy individual and above the reference level determined by measuring at least one reference biological sample from at least one patient with pancreatic cancer of grade I/II indicates that the individual has pancreatic cancer of grade III. A method of differentiating between low-grade pancreatic cancer and high-grade pancreatic cancer in an individual comprising the step of:

The method of item 9, wherein the level of the at least one elF is compared to at least one reference level of said at least one elF.

The method of item 10, wherein the at least one reference level is the level determined by measuring at least one reference biological sample from

from at least one healthy individual,

at least one patient with low-grade pancreatic cancer , and/or

at least one patient with high-grade pancreatic cancer.

The method of items 10 or 11, wherein

(i) the reference level is the level determined by measuring at least one reference biological sample from at least one patient with low-grade pancreatic cancer, wherein the level of the at least one elF above the reference level indicates that the individual has high-grade pancreatic cancer,

(ii) the reference level is the level determined by measuring at least one reference biological sample from at least one patient with high-grade pancreatic cancer, wherein the level of the at least one elF below the reference level indicates that the individual has low-grade pancreatic cancer,

(iii) the reference level is the level determined by measuring at least one reference biological sample from at least one healthy individual and the reference level is the level determined by measuring at least one reference biological sample from at least one patient with high-grade pancreatic cancer,

wherein the level of the at least one elF below the reference level determined by measuring at least one reference biological sample from at least one healthy individual and below the reference level determined by measuring at least one reference biological sample from at least one patient with high-grade pancreatic cancer indicates that the individual has low-grade pancreatic cancer,

(iv) the reference level is the level determined by measuring at least one reference biological sample from at least one healthy individual and the reference level is the level determined by measuring at least one reference biological sample from at least one patient with low-grade pancreatic cancer,

wherein the level of the at least one elF below the reference level determined by measuring at least one reference biological sample from at least one healthy individual and above the reference level determined by measuring at least one reference biological sample from at least one patient with low-grade pancreatic cancer indicates that the individual has high-grade pancreatic cancer.

The method of any one of items 9 to 12, wherein the low-grade pancreatic cancer is pancreatic cancer of grade I/II and wherein the high-grade pancreatic cancer is pancreatic cancer of grade III.

A method of providing a survival prognosis to an individual suffering from pancreatic cancer comprising the step of:

The method of item 14, wherein the level of elFl is compared to a reference level of said elFl.

The method of item 15, wherein the reference level is the level determined by measuring at least one reference biological sample from at least one subject suffering from pancreatic cancer.

The method of items 15 or 16, wherein

The method of any one of items 1 to 17, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PD AC).

The method of any one of items 1 to 18, wherein the biological sample is a tissue sample or a body fluid sample.

The method of item 19, wherein the body fluid sample is blood, lymph, or saliva. The method of item 20, wherein the blood is whole blood or a blood fraction, preferably serum or plasma.

The method of any one of items 1 to 21, wherein the level of the at least one elF is determined by measuring mR A or protein levels. 23. Use of at least one elF for diagnosing pancreatic cancer in an individual, grading pancreatic cancer in an individual, or differentiating between low-grade pancreatic cancer and high-grade pancreatic cancer in an individual,

24. Use of elFl for providing a survival prognosis to an individual suffering from pancreatic cancer.

25. A kit for diagnosing pancreatic cancer in an individual, grading pancreatic cancer in an individual, or differentiating between low-grade pancreatic cancer and high-grade pancreatic cancer in an individual, wherein said kit comprises

means for determining the level of at least one elF in a biological sample from an individual,

26. The kit of item 25, wherein the kit is useful for conducting the methods according to any one of items 1 to 13 or 18 to 22.

27. A kit for providing a survival prognosis to an individual suffering from pancreatic cancer, wherein said kit comprises

means for determining the level of elFl in a biological sample from an individual suffering from pancreatic cancer.

28. The kit of item 27, wherein the kit is useful for conducting the method according to any one of items 14 to 22.

29. The kit of items 25 to 28, wherein the kit further comprises

(i) a container, and/or

(ii) a data carrier.

Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope of invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art in the relevant fields are intended to be covered by the present invention.

The present invention is further illustrated by the following examples, however, without being restricted thereto. BRIEF DESCRIPTION OF THE FIGURES

Figure 1: elF expression in subgrouped PDAC samples (well and poorly differentiated) versus non-neoplastic pancreatic tissue. (A) elFl expression (B) eIF2a expression (C) eIF3C expression (D) eIF6 expression. *p<0.05 **p<0.0l ***p<0.00l ****p<0.000l, case numbers are indicated in the graphs.

Figure 2: Immunohistochemistry of non-neoplastic tissue (NNT) (left) and tumor tissue (right) stained for elFl, eIF2a, eIF3C and eIF6. A & B: elFl expression in NNT (A) and tumor tissue (B). C & D: eIF2a expression in NNT (C) and tumor tissue (D). E & F: eIF3C expression in NNT (E) and tumor tissue (F). G & H: eIF6 expression in NNT (G) and tumor tissue (H). Scale bar represents 400pm. (I) Densitometric analyses of immunohistochemical stainings of elFl in tumor tissue (PDAC) compared to non-neoplastic tissues (NNT). (J) Densitometric analyses of immunohistochemical stainings of eIF2a in tumor tissue (PDAC) compared to non neoplastic tissues (NNT). (K) Densitometric analyses of immunohistochemical stainings of eIF3C in tumor tissue (PDAC) compared to NNT. (L) Densitometric analyses of immunohistochemical stainings of eIF6 in tumor tissue (PDAC) compared to NNT.

Figure 3: (A) elFl expression in tumor tissue (PDAC) samples (n=28) versus non neoplastic pancreatic tissue (n=28). (B) eIF2a expression in tumor tissue (PDAC) samples (n=28) versus non-neoplastic pancreatic tissue (n=28) . *p<0.05 **p<0.01 ***p<0.001

Figure 4: (A) eIF3C expression in tumor tissue (PDAC) samples (n=28) versus non neoplastic pancreatic tissue (n=28). (B) eIF6 expression in tumor tissue (PDAC) samples (n=56) versus non-neoplastic pancreatic tissue (n=56) . *p<0.05 **p<0.01 ***p<0.00l

Figure 5: elFl is a prognostic marker for PDAC. Kaplan-Meier curves reflect the effect of elFl expression on overall survival for PDAC. Cases are divided into low or high expression according to whether expression is below or above median, and survival is compared using the log-rank test. EXAMPLES

Methods

Samples

A total of 174 PD AC cases on tissue microarrays (TMAs) were examined. Each case was represented by three cores on the TMA slides. Patient data and clinicopathological classification are summarized in Table 1. As a control, non-neoplastic pancreatic tissue was collected at the Institute of Pathology at the Medical University of Graz, Graz, Austria under approval of the local ethics committee (EK-Nr.: 28-294 ex 15/16). A total of 9 formalin- fixed, paraffin- embedded (FFPE) control samples classified by two board-certified pathologists (J.H. and I.B.) were collected.

Table 1: Patient data and clinicopathological parameters, n=l74.

All tumor tissue samples were acquired during surgery under approval of both the ethics committee at the Otto-von-Guericke-University Magdeburg, Germany (Nr. 08/18) and the ethics committee of the Medical University of Graz, Austria (EK-Nr.: 28-294 ex 15/16, Amendment February 2018) and were immediately frozen in liquid nitrogen and stored at - 80°C. A total of 28 tumor samples and 28 non-neoplastic pancreatic tissue samples were analyzed.

Immunohistochemistry

TMAs and non-neoplastic FFPE samples were stained for expression of elFl, eIF2a, eIF3A, eIF3C, eIF4E, eIF4G, eIF5 and eIF6. Slices of 4um thickness were attached to adhesive-coated glass slides and fixed at 65°C for one hour. The slides were stained using a Ventana Immunostainer XT (Ventana Medical Systems, Tucson, USA) with an ultra- VIEW universal DAB Detection Kit (Ventana Medical Systems, Tucson, USA) and cell-conditioning solution for 30 minutes via heat-induced epitope retrieval (HIER). The primary antibodies and dilutions used are listed in Table 2. Antibodies were incubated for 30 minutes.

Table 2: Antibodies used for immunohistochemical staining.

Expression evaluation/scoring

Staining was evaluated for its intensity and the amount of postively stained tumor cells (and normal glandular duct epithelium, respectively). Staining intensity was classified using an intensity score ranging from 0-3 (0: no staining, 1 : weak staining, 2: moderate staining, 3: strong staining), whereas the amount of cells was quantified using a proportion score ranging from 0- 100% (0%: 0, 1-10%: 1, 11-49%: 2, 50-79%: 3, 80-100%: 4). An overall tissue intensity score (TIS) taking both parameters into account was calculated by multiplication ranging from 0-12; final expression was classified as follows: 0 no expression, 1-4 weak expression, 5-8 moderate expression, 9-12 strong expression.

Protein extraction and immunoblot

Frozen tissue samples were homogenized with a MagNA Lyser homogenizer (Roche Diagnostics, Risch-Rotkreuz, Switzerland) and lysed in NP-40 Lysis buffer (0.05 M Tris-HCl, 5 mM NaCl, 0.5% NP-40, 0.1 mM Pefabloc, 1 mM DTT, complete Mini, PhosSTOP). The protein concentration was determined using Bradford protein assay (Biorad Protein Assay Dye Reagent, 500-0006; BioRad Laboratories GmbH, Munich, Germany). Equal amounts of 30 pg protein were loaded onto SDS-PAGE gels (30% Acrylamid / Bisacrylamid solution; ROTH, Karlsruhe, Germany), subjected to electrophoresis in Mini-vertical electrophoresis units (Hoefer Inc, Richmond, USA) and blotted onto PVDF membranes (Immobilin-P Transfer Membrane; Millipore, Massachusetts, USA) using a Semi Dry Blotting Unit (SCIE-PLAS; Cambridge, England). The membranes were blocked in TBS tween (TBST) with 5% non-fat milk (AppliChem; Darmstadt, Germany) for lh at room temperature. The primary antibodies were diluted in TBST, 5% BSA and applied overnight at 4°C. The membranes were washed with TBST, followed by incubation with a horseradish peroxidase conjugated secondary antibody (anti-mouse 1 :3000 and anti-rabbit 1 :5000; GE Healthcare Life Sciences, Buckinghamshire, England). Proteins were visualized using a chemiluminescence ECL kit (GE Healthcare Life Sciences), followed by exposure on the Image Quant LAS 500 (GE Healthcare, Little Chalfont, UK). The signal was normalized using GAPDH antibody (mAb dilution 1 : 1000, Sigma- Aldrich, Missouri, USA).

Statistical analysis

Graphical analysis and statistical testing were done using GraphPad Prism 4.03 software (GraphPad software Inc., La Jolla, CA, USA). All data are presented as means ± standard of the mean (SEM). Significance level was set as p<0.05; all data were analyzed with descriptive statistics and Mann-Whitney-U-test.

The Cancer Genome Atlas (TCGA) public dataset, including 125 PDCA subjects, was analyzed to identify the association between gene expression stratified by the median survival. Kaplan- Meier curves were generated using the survival R package. The log rank test was applied to test for an association of survival and gene expressioa All results were expressed as mean ± standard deviation (SD). Differences between groups were assessed using Students t-test or Mann- Whitney U test based on data distribution. A p <0.05 was considered as statistically significant. Statistical analysis and graph generation were performed using GraphPad PRISM version 5.0 (GraphPad software Inc., La Jolla, CA, USA).

Results

Immunohistochemistry - downregulation of elFl, eIF2a, eIF3C and eIF6

The expression of four elF subunits (elFl, eIF2a, eIF3C and eIF6) was found to be downregulated in the PD AC samples compared to non-neoplastic pancreatic tissue (healthy control, Fig. 1 and 2). elFl demonstrated downregulation in well and poorly differentiated samples, p<0.000l. Downregulation of eIF2a was detected at the same significance level, p<0.000l, whilst significant differences were present between poorly and well differentiated samples (p=0.0l 19). Further, expression of eIF3C was mitigated in tumor samples, p<0.000l. Finally, eIF6 was downregulated compared to healthy tissue, p=0.0083 for well-differentiated samples and p=0.0252 for poorly differentiated samples.

Immunoblot - downregulation of elFl, eIF2a and eIF3C

Protein expression of three elF subunits was found to be downregulated in the PD AC samples compared to non-neoplastic pancreatic tissue, namely elFl, eIF2a, eIF3C, and eIF6 (Fig. 3 and Fig. 4). elFl demonstrated downregulation in tumor samples (PD AC), p<0.0l. A significant downregulation of eIF2a could be detected, p<0.0l. Further, lower expression of eIF3C was observed in tumor samples (PD AC), p<0.0l. eIF6 was also downregulated in tumor samples (PDAC). p<0.00l. In this respect, please note that * means p<0.05, ** means p<0.0l, and *** means p<0.00l.

Fligh elFl expression correlates with better overall survival in PDAC patients

Based on the results shown above, the correlation between the expresssion of elFl and patients’ overall survival was tested. An in silico analysis including 125 patients of The Cancer Genome Atlas (TCGA) database was performed. To determine the statistical significance, the log rank test was carried out. Kaplan-Meier curves were generated to assess a potential association of elFl expression with overall survival in PDAC patients. Lower expression of elFl in PDAC indicated a significantly poorer overall survival as compared to high elFl (p= 0.048) expression (see Fig. 5). Thus, elFl also acts as novel prognostic marker for the overall survival of PDAC patients. Discussion

Eukaryotic translation initiation factors demonstrate alterations in PD AC, indicating involvement and contribution to pancreatic carcinogenesis. The data revealed downregulation of various elF subunits, pointing to their applicability as biomarkers. Immunohistochemically assessing the expression of elFl, eIF2a, eIF3C and eIF6 might be regarded as a future diagnostic parameter for identification of PDAC - findings that were further undermined through Immunoblot analysis for elFl, eIF2a, eIF3C, and eIF6. Protein synthesis is commonly upregulated in tumor cells due to their high proliferation rates and the hereby encompassed need for proteins. The finding that these translation-enhancing factors are downregulated - in contrast to logical expectations - is very surprising. In addition, elFl also acts as novel prognostic marker for the overall survival of PDAC patients.

REFERENCES

1. Ducreux, M. et al. Cancer of the pancreas: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann. Oncol. 26, v56-v68 (2015).

2. Yeo, T. P. Demographics, epidemiology, and inheritance of pancreatic ductal

adenocarcinoma. Semin. Oncol. 42, 8-18 (2015).

3. Waddell, N. et al. Whole genomes redefine the mutational landscape of pancreatic cancer. Nature 518, 495-501 (2015).

4. Jackson, R. J., Hellen, C. U. T. & Pestova, T. V. The mechanism of eukaryotic

translation initiation and principles of its regulation. Nat. Rev. Mol. Cell Biol. 11, 113— 27 (2010).

Claims

2. The method of claim 1, wherein the level of the at least one elF is compared to a reference level of said at least one elF.

3. The method of claim 2, wherein the reference level is the level determined by measuring at least one reference biological sample from at least one healthy individual.

4. The method of claims 2 or 3, wherein the level of the at least one elF below the reference level indicates that the individual suffers from pancreatic cancer.

6. The method of claim 5, wherein the level of the at least one elF is compared to at least one reference level of said at least one elF.

7. The method of claim 6, wherein the at least one reference level is the level determined by measuring at least one reference biological sample

from at least one healthy individual,

from at least one patient with pancreatic cancer of grade I/II, and/or

from at least one patient with pancreatic cancer of grade III.

8. The method of claims 6 or 7, wherein (i) the reference level is the level determined by measuring at least one reference biological sample from at least one patient with pancreatic cancer of grade I/II, wherein the level of the at least one elF above the reference level indicates that the individual has pancreatic cancer of grade III,

9. A method of differentiating between low-grade pancreatic cancer and high-grade pancreatic cancer in an individual comprising the step of:

10. The method of claim 9, wherein the level of the at least one elF is compared to at least one reference level of said at least one elF.

11. The method of claim 10, wherein the at least one reference level is the level determined by measuring at least one reference biological sample from

from at least one healthy individual,

at least one patient with low-grade pancreatic cancer , and/or

at least one patient with high-grade pancreatic cancer.

12. The method of claims 10 or 11, wherein

13. The method of any one of claims 9 to 12, wherein the low-grade pancreatic cancer is pancreatic cancer of grade I/II and wherein the high-grade pancreatic cancer is pancreatic cancer of grade III.

14. A method of providing a survival prognosis to an individual suffering from pancreatic cancer comprising the step of:

15. The method of claim 14, wherein the level of elFl is compared to a reference level of said elFl .

16. The method of claim 15, wherein the reference level is the level determined by measuring at least one reference biological sample from at least one subject suffering from pancreatic cancer.

17. The method of claims 15 or 16, wherein

18. Use of at least one elF for diagnosing pancreatic cancer in an individual, grading pancreatic cancer in an individual, or differentiating between low-grade pancreatic cancer and high-grade pancreatic cancer in an individual,

19. Use of elFl for providing a survival prognosis to an individual suffering from pancreatic cancer.

20. A kit for diagnosing pancreatic cancer in an individual, grading pancreatic cancer in an individual, or differentiating between low-grade pancreatic cancer and high-grade pancreatic cancer in an individual, wherein said kit comprises

21. A kit for providing a survival prognosis to an individual suffering from pancreatic cancer, wherein said kit comprises