Patent Application
20110294136
The invention relates to a method for the diagnosis of pancreatic cancer (synonymous term: pancreatic carcinoma) (PaCa) and the precursor and/or concomitant illnesses thereof, particularly PDAC (pancreatic ductal adenocarcinoma), PanIN (pancreatic intraepithelial neoplasias), pancreatic lesions, CP (chronic pancreatitis), including endocrine tumors of the pancreas, where a determination is carried out using selected biomarkers. Furthermore, the invention relates to suitable combinations of biomarkers, particularly for in vitro diagnostics.
The 5-year-survival rate for pancreatic carcinoma of approx. 1% is the lowest of all cancer types (Parkin, D. M., F. Bray, et al. (2001). “Estimating the world cancer burden: Globocan 2000.” Int J Cancer 94(2): 153-6). Early diagnosis might increase the 5-year survival rate to 40% (Yeo, C. J. and J. L. Cameron (1998). “Prognostic factors in ductal pancreatic cancer.” Langenbecks Arch Surg 383(2): 129-33). Therefore, for diagnosis, the precursor diseases of pancreatic cancer need to be considered as well, such as PDAC (pancreatic ductal adenocarcinoma), PanIN (pancreatic intraepithelial neoplasias), pancreatic lesions, CP (chronic pancreatitis), including endocrine tumors of the pancreas. Especially PanID are associated with pancreatic lesions and differentiate them morphologically into PanIn 1A, 1B, 2, and 3 (Kern, S., R. Hruban, et al. (2001). “A white paper: the product of a pancreas cancer think tank.” Cancer Res 61(12): 4923-32). Pancreatic lesions have also been described for CP. Endocrine (benign or malignant) tumors of the pancreas, particularly neuroendocrine tumors, are relevant as well.
For the purpose of a useful therapy of pancreatic cancer or of precursor and/or concomitant illnesses thereof, particularly PDAC (pancreatic ductal adenocarcinoma), PanIN (pancreatic intraepithelial neoplasias), pancreatic lesions, CP (chronic pancreatitis), including endocrine tumors of the pancreas, there is a requirement of early diagnosis and differentiation in connection with the need for clinical decisions.
However, a drawback of the current diagnostic methods using the presently known markers is that the early and comprehensive identification of risk patients is unsuccessful, which is why diagnosis is incomplete or even too late.
An underlying objective of the invention is therefore to develop a method for diagnosis of pancreatic cancer or of precursor and/or concomitant illnesses thereof, enabling an improved early diagnosis and identification of risk patients as well as an improvement of the therapeutic success.
Another disadvantage is that often no sufficient sensitivity and/or specificity of the markers can be obtained in the art. For example, the early diagnosis of PDAC is associated with the significant problem of not having a specific biomarker. The most commonly used serum biomarker for pancreatic cancer is C-19-9, with a specificity of only 69-90%, since this marker can be detected in the blood in other diseases as well, particularly in chronic pancreatitis (Banfi et al. (1996) CA 19.9, CA 242 and CEA in the diagnosis and follow-up of pancreatic cancer, Int J Biol Markers, 77-81, Banfi et al (1993) Behavior of tumor markers CA19.9, CA195, CAM43, CA242, and TPS in the diagnosis and follow-up of pancreatic cancer, Clin Chem, 420-3).
The object is attained through a method for diagnosis of pancreatic cancer or precursor and/or concomitant illnesses thereof, whereby a determination of at least one polypeptide/proteins selected from the group
a.) Keratin 8 protein (SEQ ID No. 1), Vimentin (SEQ ID No. 2), Mitochondrial malate dehydrogenase (SEQ ID No. 3), Beta tropomyosin (SEQ ID No. 4), ACTG1 protein (SEQ ID No. 5), Thioredoxin delta 3 (SEQ ID No. 6), B Chain B Triosephosphate Isomerase (SEQ ID No. 7), Annexin A2 (SEQ ID No. 8), TPM4-ALK fusion oncoprotein type 2 (SEQ ID No. 9), Peptidylprolyl isomerase A (SEQ ID No. 10), Smooth muscle mysoin light chain (SEQ ID No. 11), Desmin (SEQ ID No. 12), Major vault protein 1 (SEQ ID No. 13), Heterogeneous nuclear ribonucleoprotein A1 (SEQ ID No. 14), S100A10 (SEQ ID No. 15), EF1a-like protein (SEQ ID No. 16), Regulatory myosin light chain long version (SEQ ID No. 17), Tropomyosin 1 alpha chain isoform 3 (SEQ ID No. 18), Tropomyosin 2 (beta) isoform 2 (SEQ ID No. 19), Myosin regulatory light chain MRCL3 (SEQ ID No. 20), Alpha-2-globin (SEQ ID No. 21), Tropomyosin 4 (SEQ ID No. 22), Transgelin (SEQ ID No. 23), Keratin 7 (SEQ ID No. 24), ACTB protein (SEQ ID No. 25), M2-type pyruvate kinase (SEQ ID No. 26), Actin related protein ⅔ complex subunit 5 (SEQ ID No. 27), Anterior gradient 2 homolog (AGR 2) (SEQ ID No. 28), Stratifin (14-3-3 sigma) (SEQ ID No. 29), Coactosin-like 1 (SEQ ID No. 30), Chaperonin heat shock 60 kD protein 1 (SEQ ID No. 31), Transgelin 2 (SEQ ID No. 32), Aldehyde dehydrogenase 1 (SEQ ID No. 33), Sarcomeric tropomyosin kappa (SEQ ID No. 34), Annexin A3 (SEQ ID No. 35), Delta-globin (SEQ ID No. 36), Serum albumin (SEQ ID No. 37), Protein PP4-X (Annexin A4) (SEQ ID No. 38), Crystallin (SEQ ID No. 39), Myosin regulatory light chain MRCL3 (SEQ ID No. 40)
or
group b.) aldehyde dehydrogenase 1 (SEQ ID No. 41), Aldehyde dehydrogenase 1A1 (SEQ ID No. 42), T-complex protein 1 subunit beta (SEQ ID No. 43), Apolipoprotein A4 (SEQ ID No. 44), Malate dehydrogenase mitochondrial precursor (SEQ ID No. 45), Voltage-dependent anion selective channel protein 1 (SEQ ID No. 46), glyceraldehydes-3-phosphate dehydrogenase (SEQ ID No. 47), uracil DNA glycosylase (SEQ ID No. 48), aging-associated-associated 9 protein (SEQ ID No. 49), Nipsnap homolog 3A (SEQ ID No. 50), peroxiredoxin 2 isoform b (SEQ ID No. 51), thiol-specific antioxidant protein (SEQ ID No. 52), enhancer protein (SEQ ID No. 53), Chromosome 17 open reading frame 25 (SEQ ID No. 54), hypothetical protein LOC51031 (SEQ ID No. 55), CGI-150 protein (SEQ ID No. 56), Gelsolin isoform a (SEQ ID No. 57), Gelsolin precursor (SEQ ID No. 58), ATP-specific succinyl-CoA synthetase beta subunit (SEQ ID No. 59), TAR DNA binding protein (SEQ ID No. 60), 2,4-dienoyl-CoA reductase mitochondrial precursor (SEQ ID No. 61), MDH2 (SEQ ID No. 62), heat shock protein beta-1 (SEQ ID No. 63), mitochondrial malate dehydrogenase precursor MDH-2 (SEQ ID No. 64), prostate and colon associated protein (SEQ ID No. 65), secretagogin (SEQ ID No. 66), TPD 52 (SEQ ID No. 67), tumor protein D52 (SEQ ID No. 68), N8 protein long isoform (Fragment) variant (SEQ ID No. 69), tumor protein D52 isoform 2 (SEQ ID No. 70), triosephosphate isomerase 1 (SEQ ID No. 71) or partial peptides or fragments thereof is carried out on a patient to be investigated (hereinafter referred to as method according to the invention”).
The proteins according to the invention are identified as potential biomarkers by means of a differential proteome analysis from ill pancreatic ductal tissue—five progression phases—in comparison to normal (healthy) pancreatic ductal tissue. Hereto, appropriate tissue samples were taken from ill patients. The samples were homogenized with lysis buffer in a hand-held homogenizer and removed from DNA and other cell material resulting in a protein concentrate.
The proteins were labeled with a dye and subject to a 2D gel electrophoresis with an isoelectric focusing in the first dimension and a SDS gel electrophoresis in the second dimension. The differential illustration (ill/healthy) is presented in tables (1 to 3), examples and figures showing different characteristic expressions (up- and down-regulated and read out by using the spots).
Further examination was carried out by means of LC-ESI-MS(/MS) (Liquid-Chromatographie-Electrospray-Ionization-Mass Spectrometry). In a first instance the proteins were fragmented in specific peptide fragments by means of trypsin within the gel, afore the samples were separated. Those were each other separated by means of reversed-phase HPLC and examined with mass spectrometry in order to identify each protein. It should be understood that other methods of mass spectrometry are also suitable like MALDI-TOF-MS.
The proteins in accordance with the invention (biomarkers) are identified as follows:
The invention refers also to such amino acid sequences of SEQ ID No. 1 to SEQ ID No. 71 (polypeptide, proteins), having a sequence identity or homology of 70% and more, preferably 80% and more, most preferably 90-95%.
Likewise are included such analogous amino acid sequences having although due to a replacement of one or more amino acid(s) the desired function of a biomarker for diagnosis of pancreatic cancer. Expressly included according to the invention are in particular partial peptides or fragments of SEQ ID No. 1 to SEQ ID No. 71.
In a further preferred embodiment of the invention combinations of biomarkers according to the invention are advantageously (Sub-combinations of the above entirety of all biomarkers according to the invention) for diagnosis. Particularly preferred are such combinations within the group
a.) comprising at least Stratifin (14-3-3 sigma) (SEQ ID No. 29) and/or Vimentin (SEQ ID No. 2) and/or Major vault protein 1 (SEQ ID No. 13) and/or Anterior gradient 2 homolog (AGR 2) (SEQ ID No. 28), and/or S100A10 (SEQ ID No. 15) and/or EF1a-like protein (SEQ ID No. 16) and/or Annexin A2 (SEQ ID No. 8) and/or Annexin A4 (SEQ ID No. 38).
The term “pancreatic cancer” in accordance with the invention encompasses also precursor and/or concomitant illnesses thereof, in particular PDAC (Pancreatic ductal adenocarcinoma), PanIN (pancreatic intraepithelial neoplasias), pancreatic lesions, CP (chronic pancreatitis), including endocrine pancreatic tumors, particularly pancreatic tumors und pancreatic neoplasm.
The invention therefore further relates to the identification of patients with increased risk and/or unfavorable prognosis of pancreatic cancer, particularly by symptomatic and/or asymptomatic patients.
The method according to the invention thus allows clinical decisions resulting in rapid therapeutic success and avoidance of mortalities. Such clinical decisions also include further treatment with medicaments for treatment or therapy of pancreas cancer. Clinical decisions of this type likewise include further treatment by means of pharmaceuticals for the treatment or therapy of pancreatic cancer.
Therefore, the invention relates also to a method for diagnosis of patients having pancreatic cancer for carrying out clinical decisions, like further treatment and therapy by means of medicaments.
In one further preferred embodiment of the method according to the invention, diagnosis is carried out for prognosis, differential diagnostic early detection and identification, severity assessment, and prognostic assessment in conjunction with therapy.
In one further preferred embodiment, the invention relates to a method for diagnostics for early or differential diagnosis or prognosis of pancreatic cancer or a precursor illness, wherein the biomarker is determined on a patient to be examined.
In one embodiment of the method according to the invention, tissue samples or bodily fluid (blood, plasma pancreatic secretion) is withdrawn from the patient to be examined, and the diagnosis is made in vitro/ex vivo, i.e. outside the human or animal body. As a result of the determination of the marker according to the invention high sensitivity and specificity for pancreatic cancer or precursor and/or concomitant illnesses thereof are achieved and diagnosis may be performed based on the quantity present or its shifting (level: increase/decrease) in at least one patient sample.
In a further embodiment of the invention, for an in vitro diagnosis the method according to the invention may be carried out by means of parallel or simultaneous determinations of the markers (for example, using multititer plates containing 96 or more cavities), wherein the determinations are carried out for at least one patient sample.
In a further embodiment, the method according to the invention may be carried out by means of 2D-elektrophoresis, wherein in a first dimension an isoelectric focusing and in the second dimension a SDS gel electrophoresis are conducted (This is understood in the broadest sense as proteome research (“proteomics”)).
In a further embodiment, the method according to the invention and determinations therefor may be carried out using a rapid test (for example, a lateral flow test) in either single- or multi-parameter determinations.
In a further embodiment, the method according to the invention may be carried out in-vivo, wherein the biomarkers are detected with a probe, particularly with an antibody, having a marked contrast agent and which are detectable with an image making suitable detector (“Molecular Imaging”) (Ralph Weissleder, Molecular Imaging in Cancer, Science, Vol. 312, 1168 (2006)).
The invention further relates to the use of the biomarker according to the invention for diagnosis and/or prognosis and/or for early or differential diagnosis of myocardial infarction of pancreatic cancer or precursor and/or concomitant illness thereof.
A further object is to provide a corresponding diagnostic device for carrying out the methods according to the invention.
Within the scope of the invention, such a diagnostic device, in particular an array or assay (for example, immunoassay, ELISA, etc.), is understood in the broadest sense as a device for carrying out the methods according to the invention, particularly a protein biochip (U.S. Pat. No. 6,346,413B1. US20050014292). The invention further relates to a kit for carrying out the methods according to the invention, particularly containing detection reagents and further adjuvants. Such detection reagents include antibodies, for example.
The detection and the quantification of the biomarkers according to the invention may also be performed with the aid of further protein diagnostic methods known to those skilled in the art, in particular employing radioactive or fluorescence-marked antibodies. In particular, bioanalytical methods suitable for this purpose are to be cited here, such as immunohistochemistry, antibody arrays, luminex, ELISA, immunofluorescence, and radio immunoassays as well as further bioanalytical methods suitable for this purpose, such as mass-spectrometry methods, e.g., MRM (multi-reaction monitoring) or AQUA (absolute quantification), with the aid of which the biomarkers may be quantitatively measured.
The following examples and figures are used for a more detailed explanation of the invention, but do not limit the invention to said examples and figures.
The tissue samples were obtained from surgical patients of the General Surgery Department of the University Hospital Schleswig-Holstein, Campus Kiel (German). Tumor tissues from ductal pancreatic cancer and peritumoral parenchyma were shock frozen at −80° C. immediately postsurgically and stored thereafter. For visualization of normal pancreatic ducts and PanINs, 5 μm thick frozen sections were prepared of the peritumoral pancreas parenchyma, briefly fixed in ethanol (Merck, Darmstadt, Germany), stained with hematoxylin-eosin and subsequently evaluated by a pathologist. The PanINs were classified according to accepted criteria (Hruban, R. H., N. V. Adsay, et al. (2001). “Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions.” Am J Surg Pathol 25(5): 579-86). Serial tissue block sections (10 μm) containing the required PanIN lesions were obtained. For the 2-D electrophoresis, the tissue sections were stained only with hematoxylin and immediately stored at −20° C. The PanIN lesions were microdissected under a microscope (BH2, Olympus, Wetzlar, Germany) using a sterile injection needle (size 0.65×25 mm, Braun company, Melsungen, Germany). Primarily medium sized interlobular ducts were selected, in order to avoid contamination with periductal mesenchymal and acinar tissue. The microdissected cells were taken up in 100 μL lysis buffer (Tris-Cl 30 mM; thiourea 2M; urea 7M; CHAPS 4%, pH 8.0) and treated on ice in an ultrasonic bath immediately after microdissection (6×10 s pulses; ultrasonic cleaner, VWR Darmstadt, Darmstadt).
Preparation of the Reference Proteome
For generation of the reference proteome, 100 mg adenocarcinoma tissue was homogenized in 148 μL lysis buffer (Tris-Cl 30 mM; thiourea 2M; urea 7M; CHAPS 4%, pH 8). Then the samples were sonicated (6×10 pulses, on ice) and centrifuged (12.000×g for 5 min). Protein determination was performed using a protein assay (Bio-Rad).
The samples, each with 1000 microdissected cells in 100 μL lysis buffer, were reduced by addition of 2 nmoles TCEP, and were then incubated at 37° C. for 1 h in the dark. The saturation dyes Cy3 and Cy5 were first diluted with DMF (2 nmol/μL; Sigma) and were then added to the reduced samples in a concentration of 4 nmoles. The incubation took place at 37° C. for 30 min in the dark. To stop the labeling reaction, 10 μL DTT (1.08 g/mL; Bio-Rad) was added. Then, 10 μL Ampholine 2-4 (GE Healthcare) was added to each sample.
For separation of the proteins in the first dimension, carrier ampholyte-based IEF (slab gels 20 cm×1.5 mm) was conducted according to Klose and Kobalz (Klose, J. and U. Kobalz (1995). “Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome.” Electrophoresis 16(6): 1034-59). After completion of a voltage program with 21.25 hrs, the ejected cylindrical gels were incubated in equilibration buffer (125 mM Tris, 40% (w/v) glycerin, 3% (w/v) SDS, 65 mM DTT, pH 6.8) for 10 min. The second dimension was obtained in an Desaphor VA 300 system with polyacrylamide gels (15.2% acrylamide (total), 1.3% bisacrylamide) (Klose and Kobalz 1995 (supra)). The cylindrical gels were applied to the polyacrylamide gels (20 cm×30 cm×1.5 mm) and fixed with 1% agarose containing 0.01% (w/v) bromophenole blue dye (Riedel deHaen, Seelze, Deutschland). The gel system used for protein identification (IEF: 20 cm×1.5 mm, SDS-PAGE: 20 cm×30 cm×1.5 mm) was processed under equal conditions. For this procedure, the MS-compatible silver staining protocol according to Nesterenko et al. was used (Nesterenko, M. V., M. Tilley, et al. (1994). “A simple modification of Blum's silver stain method allows for minutes detection of proteins in polyacrylamide gels.” J Biochem Biophys Methods 28(3): 239-42).
For image acquisition with the Typhoon 9400 fluorescence scanner (Amersham Biosciences/GE Healthcare) the gels remained between the glass plates. The excitation wave length and the emission filters were selected specifically for the respective fluorescence dyes according to the manual. Prior to the image analysis with the DeCyder software (Amersham Biosciences/GE Healthcare) the images were cropped using the ImageQuant™ software (Amersham Biosciences/GE Healthcare). Intra-gel spot detection and quantification took place using the Differential In-gel Analysis (DIA) mode of the DeCyder software. The estimated spot number was set to 3000. As an exclusion filter, an increase of the spot slope of more than 1.6 was selected. For determination of the reference proteome the matching rates between microdissected PDAC cells, a pancreatic cell lines pool, and PDAC tumor tissue were determined for various gel areas.
The spots were punched out manually from a preparative gel. In order to determine the position of the spots in the gel, a true to scale gel print was placed underneath the gel after image acquisition. Then, the spots were digested in the gel with trypsin (Promega, Mannheim, Germany), and the peptides were extracted as described in Schaefer et al. (Schaefer, H., J. P. Chervet, et al. (2004). “A peptide preconcentration approach for nano-high-performance liquid chromatography to diminish memory effects.” Proteomics 4(9): 2541-4; Schaefer, H., K. Marcus, et al. (2003). “Identification of phosphorylation and acetylation sites in alphaA-crystallin of the eye lens (mus musculus) after two-dimensional gel electrophoresis.” Anal Bioanal Chem 376(7): 966-72). For peptide analytics, a system consisting of FAMOS™ (automatic sampler), Switchos™ (loading pump and switch valves), and Ultimate™ (separation pump and UV detector) (LC Packings Dionex, Amsterdam, Niederlande), coupled on-line with an ion-trap mass spectrometer LCQ Deca XP (Thermo Electron, San Jose, Calif., USA) and equipped with a nanoelectrospray ion source (PicoView™100, New Objective Inc., Woburn, Mass., USA), and SilicaTips™ (FS360-20-10-D, New Objective Inc.) were used.
For protein identification, the MS/MS spectra were searched against the NCBI protein sequence sub-database (human) (http://www.ncbi.nlm.nih.gov) using the SEQUEST™ algorithm and accounting for the following search parameters: mass tolerance ±1.5 Da for parent and fragment ions. Cy3 modification of all cysteins. One overread trypsin cutting site. Proteins with a SequestMetaScore (Proteinscape™) larger than 10 with 3 or more peptides were considered as identified.
For normal pancreatic ducts as well as for PanINs, one 1.5 mm thick tissue cylinder (two for ductal adenocarcinomas) was punched out of each representative area and embedded in paraffin reception blocks, so 300 cylinders with pancreatic tissues (in altogether 6 tissue arrays) as well as two control cylinders each with healthy tonsil tissue were processed in total. Processing took place using an MTA1 tissue arrayer instrument (Beecher Instruments, Sun Prairie, Wis., USA). Normal pancreatic ducts and the PanIN ducts were derived from 12 pancreases of healthy suicide victims that had been autopsied at the Pathology Department of the Semmelweis University in Budapest, Hungary (approval number: 140-1/1996), and from 81 pancreases that had been removed by surgical resection of gastrointestinal and pancreatic tumors in surgical departments at the university hospitals in Kiel and Dresden, Germany. For the tissue arrays of the pancreatic cancer, tissue blocks of 48 pancreases were used that had been removed in the surgical university clinic, Kiel, Germany.
All investigations were conducted on formalin-fixed paraffin-embedded tissue. 3 μm thin sections were deparaffinized and rehydrated. Then, immunohistochemical stainings were performed according to the established method. Prior to the application of the primary antibody a 20 min. serum block was performed. The murine anti-14-3-3-sigma antibody (Acris, 1.N.6., 2.5 μg/μL, 1:40), the anti-LRP/MVP antibody (Kamiya Biomedical Company, 1032, 0.5 μg/μL, 1:400) and the rabbit anti-AGR2 antibody (Imgenex, 10 μg/μL, 1:50) were used as primary antibodies. The development of the signal was conducted using a mouse or rabbit staining kit (Vectastain Elite Peroxidase kit, PK-6102, Vector Laboratories, Burmingame, USA). As a negative control, the primary antibody was omitted.
The intensity of the staining was classified into mild, moderate und strong (with a score of 1, 2, or 3, respectively). The stained areas were estimated in percent in terms of pancreatic ducts or tumor regions, and also classified into scores (<10%=1, 10-50%=2, 51-80%03, >80%=4). The final score was determined from the product of the staining intensity and the percentage of positively stained cells (minimum 0, maximum 12) (Remmele, Hildebrand et al. 1986).
Average values of the immunohistochemically determined scores of the normal pancreatic ducts, the various PanIN lesions as well as the ductal adenocarcinoma were compared using the Mann-Whitney U and Kruskal-Wallis H tests. A level of significance of 0.05 was applied to all statistical tests that were conducted. For multiple comparisons, the p-value was modified according to Bonferroni. All statistical calculations were performed using the SPSS 10.1 software. For identification of the biomarker candidates for pancreatic tumor progression, a differential proteome analysis of microdissected cells from PanIN lesions, PDAC and normal pancreatic ducts was performed. For this approach, tumors from 9 pancreas cancer patients, each providing 4-9 samples per lesion, were examined. The identified differential biomarkers were immunohistochemically validated with samples (tissue arrays) from 130 patients.
In the differential proteome analysis via 2-D electrophoresis, 86 different protein spots showing differential expression were detected in total. Among these, 19 spots in the PanIN 1A lesion, 37 in the PanIN 1B lesion, 40 in the PanIN 2 lesion, 39 in the PanIN 3 lesion, and 32 in PDAC were regulated differentially compared to normal pancreatic ducts (p<0.05, regulation factor >1.6).
For identification of the differential protein spots, the reference proteome of the pancreatic tumor tissue was used, the proteome pattern of which being highly consistent with the proteome of the microdissected material (>91%). Using LC-ESI-MS/MS, 38 non-redundant proteins in total could be identified (Table 1).
In order to be able to select proteins for immunohistochemical validation, their respective expression profiles during tumor progression were considered. Therefore, the differential protein spots were divided into 3 groups: 1) protein spots showing early regulation in the PanIN 1A and PanIN 1B lesions; 2) consistently modified protein spots throughout tumor progression; 3) protein spots with differential expression in an advanced tumor stage (PanIN 2 to PDAC) (see Table 1). Furthermore, the potential role of the proteins in tumor biology was taken as another criterion for immunohistochemical validation. Initially, among the 38 non-redundant proteins, seven were selected for validation in 130 patients: AGR2, MVP, stratifin, annexin A2, EFla-like protein, annexin A4 and S100A10. The proteome data could be confirmed for six of these proteins. The comparison of the proteome data and the validation is illustrated for three proteins: 14-3-3 sigma, MVP, and AGR2 (
The MVP antibody stainings revealed an intra-cytoplasmic staining reaction. The average scores for MVP staining were as follows: normal ducts 3.70 (standard deviation 3.0, range 0-9); PanIN-1a 4.60 (standard deviation 3.2, range 0-12); PanIN-1b 7.82 (standard deviation 3.2, range 0-12); PanIN-2 7.93 (standard deviation 3.8, range 2-12); PanIN-3 10.00 (standard deviation 2.8, range 3-12) as well as ductal adenocarcinomas 8.32 (standard deviation 3.0, range 1-12) (
Staining of the tissue arrays with the 14-3-3-sigma antibody displayed a primarily intra-cytoplasmic and less membrane-based staining reaction. The average scores for the 14-3-3 sigma staining were as follows: normal pancreatic ducts 2.04 (standard deviation 3.1, range 0-12); PanIN-1A 2.80 (standard deviation 2.6, range 0-8); PanIN-1B 5.30 (standard deviation 3.8, range 0-12); PanIN-2 8.34 (standard deviation 3.1, range 2-12); PanIN-3 10.61 (standard deviation 1.9, range 6-12), and PDAC 9.61 (standard deviation 2.8, range 2-12) (
Staining of the tissue arrays with the AGR2 antibody displayed a primarily intra-cytoplasmic and less membrane-based expression pattern. The average scores for the AGR2 staining were as follows: normal pancreatic ducts 7.59 (standard deviation 3.5, range 2-12); PanIN-1A 10.97 (standard deviation 2.0, range 6-12); PanIN-1B 10.16 (standard deviation 2.6, range 3-12); PanIN-2 8.96 (standard deviation 2.9, range 3-12); PanIN-3 8.47 (standard deviation 3.3, range 3-12), and PDAC 6.53 (standard deviation 2.6, range 1-12) (
For the proteins AGR2, 14-3-3 sigma and MVP, the present study revealed increased expression during progression of PDAC by proteome investigation and by immunohistochemical analysis. In order to evaluate the application of these proteins to differentiate between pancreatic cancer and pancreatitis, their expression was also studied in tissue arrays of 40 pancreatitis patients. In contrast to pancreatic cancer patients, there was no or little detectable concentration in pancreatitis patients. The expression level of these proteins in the tissue of the pancreatitis patients is comparable to the level of expression in healthy tissue. Thus, AGR2, 14-3-3 sigma and MVP show a high potential for being used as non-invasive or in vivo biomarkers to differentiate (differential diagnosis) between PDAC or pancreatic cancer and pancreatitis (see
The sequences according to the invention (SEQ ID No. 1-71) are as follows:
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2006-056-784.6 | Dec 2006 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/DE2007/002174 | 12/3/2007 | WO | 00 | 1/15/2010 |
