tissue samples from 13 patients ( untreated , without any neoadjuvant therapy ) with histologically confirmed diffuse gastric cancer who had undergone laparotomy were analyzed . in 8 of 13 patients , peritoneal carcinosis was histologically confirmed ( 5 female and 3 male patients ; average age 58 , age range 27 - 78 ). in 5 of 13 patients , no peritoneal carcinosis was confirmed ( 4 female and 1 male patient ; average age 70 , age range 60 - 76 ). all of the tumor samples were collected by the department of general surgery , gi tract and endocrine surgery , and transplantation surgery , university of tübingen , germany . the samples were quick - frozen in liquid nitrogen and stored at − 80 ° c . prior to use . each tumor sample was cryosectioned , hematoxylin and eosin - stained , classified by two experienced pathologists , and then reevaluated by an experienced surgical pathologist . the rna was extracted using the nucleo spin rna ii kit ( macherey - nagel , düren , germany ). rna quality and quantity were confirmed using the agilent 2100 bioanalyzer ( agilent technologies , palo alto , usa ) and the nanodrop spectrophotometer ( nanodrop technologies , wilmington , del ., usa ). microarray data were collected using oligonucleotide microarrays ( 65mer ) manufactured at the max planck institut , tübingen , germany . each of the arrays contained oligonucleotides for approximately 900 transcripts , with each oligonucleotide being placed on the array twice . in this assay , primary tumor samples from patients with gastric cancer with and without the development of peritoneal carcinoses were compared ( n = 6 ; 3 patients who developed peritoneal carcinosis were compared to 3 patients who did not develop this disorder ). in order to minimize variability because of the two dyes , 9 microarrays of 6 samples were conducted . further details on the array may be obtained from the national center for biotechnology information &# 39 ; s gene expression omnibus ( on the world - wide - web at ncbi . nlm . nih . gov / geo /) under access no . gpl5676 . amplification of the rna samples was carried out using ambion &# 39 ; s amino allyl messageamp ™ ii rna amplification kit ( ambion inc ., austin , usa ). dye coupling reactions were carried out using the amersham cydye postlabelling reactive dye pack ( ge healthcare , buckinghamshire , uk ). after rna fragmentation using ambion fragmentation reagents , hybridization was carried out at 48 ° c . for 14 hours . the sample rna was transcribed using the transcriptor first strand cdna synthesis kit ( roche , mannheim , germany ). selected cdnas were quantified by real time pcr on a lightcycler ® instrument ( roche , mannheim , germany ). sybr green jump start taq readymix ( sigma , taufkirchen , germany ) was used according to the manufacturer &# 39 ; s instructions for pcr - based amplification . the specificity of the pcr conditions was confirmed by determining the melting point and sequencing the product . quantification of the level of gene expression was carried out by means of a benchmark run on an external standard curve together with the samples of interest . the primer sequences used are shown in table 1 . raw data were collected using an imagene v . 5 . 0 . further statistical and bioinformatic analyses were carried out using the r language ( www . r - project . org ) and the limma package of the bioconductor project ( on the world - wide - web at bioconductor . org /). as the first step in signal extraction , the mean value of pixel distribution for each channel was used as the foreground signal , and the mean value for the background of each spot was used as an estimation value for the raw signal values . all of the spots were considered , regardless of their flag status . the test was carried out twice , with 9 arrays of 6 samples . the data were normalized by means of loess normalization , specifically using expression values and corrected with respect to the normexp background , followed by dye swap normalization and proportional normalization with respect to space arrays . both the loess and proportional normalization methods were used , as provided for in the limma package . the log ratio m of the samples with peritoneal carcinosis vs . the samples without peritoneal carcinosis for each spot was recorded as the result on the computer . based on the recorded m values , the differentially expressed genes were detected by using the welch one - sample t test , as implemented in r . using gene silencing experiments the influence of pgk1 on the regulation of cxcr4 / cxcl12 / beta - catenin / vegf on the mrna level and the protein level was determined . to this end , sirna was used . the post - transcriptional gene silencing was performed with the lipid - based transcription reagent lipofectamine rnaimax ( invitrogen , pasiley , uk ) according the manufacturer &# 39 ; s instructions in mkn - 45 cells ( gastric carcinoma cell line ). the used sirna ( sigenome on - targetplus smart - pool , dharmacon , chicago , usa ) consisted of four different target sirna &# 39 ; s for one gene , in order to achieve a higher silencing efficiency . the sequences of the sirna are shown in table 4 . as a control , mkn - 45 cells were transfected simultaneously with control sirna , which had no effect on the gene expression . the course of the changed gene expression was analyzed several times after transfection for the respective assays . gene expression on mrna level was determined by qrt - pcr . rna of the transfected cells was extracted using the rneasy mini kit ( qiagen , hilden , germany ) and subjected to qrt - pcr as described under c above . the primers used for the qrt - pcr are shown in table 1 . the pgk1 protein expression was analyzed by western blot analysis . to this end , the transfected cells were lysed in ripa buffer ( pierce , rockford , usa ) according to the instructions of the manufacturer and the whole proteins extracted . in the western blots , 50 μg of the whole protein of each sample was applied on a 10 % polyacrylamide gel and separated according to their size via gel electrophoresis . the proteins ware blotted on a pvdf - membrane ( millipore , billerica , usa ). apart from the primary antibodies for pgk1 ( abnova , paipei , taiwan ), 13 - actin ( affinity bio reagents , golden , usa ) was used as a control , also . the effects of pgk1 gene silencing on other proteins were analyzed by facs . the flow cytometric analysis of the harvested mkn45 cells was performed with each 5 × 10 5 cells with a vitality rate of at least 90 % per assay . intracellular staining was conducted with the intra prep permeabilization reagent ( beckman coulter , fullerton , usa ) according to the manufacturer &# 39 ; s instructions . a cxcr4 igg2b antibody ( r & amp ; d minneapolis , usa ) was used for the determination of protein expression , followed by labelling with a phycoerythrin conjugated secondary antibody ( bd , franklin lakes , usa ). protein analysis of beta - catenin was performed by using an alexa 488 conjugated beta - catenin antibody ( cell signaling , danvers , usa ). for each antibody , a respective isotype control was used . after antibody incubation , the fluorochrome labelled and washed mkn - 45 cells were analyzed in an epics xl mcl flow cytometer ( beckman coulter , fullerton usa ). prior to the analysis , the correct working of the apparatus was assured by using flow check beads fluorospheres ( beckman coulter , fullerton , usa ). evaluation was performed by observing positive cells at the indicated excitation wavelength , taking into account the zero compensation of the nonspecific fluorescence signal using mkn - 45 cells previously labelled with matching isotype control antibodies . for pgk1 overexpression the gene was cloned in plasmid vector pef - ires . by means of transfection reagent tfx - 50 ( promega , madison , usa ) the plasmid was transfected in mkn - 45 cells according to the manufacturer &# 39 ; s instructions . after 24 hr the transfected cells were cultivated in a selection medium ( rpmi 1640 , 20 % fcs and 5 μg puromycin per ml ). after one week , the cell selection was stopped and the positive clones stably overexpressing the plasmid vector and the resistance gene were persisting and were further cultivated . as soon as enough cells were on hand , whole rna was isolated using the rneasy mni kit ( qiagen , hilden , germany ); according to c :, the mrna expression of stably transfected mkn - 45 pef - ires cells was compared with control - transfected cells . gene expression on protein level was analyzed using western blots and facs analyses , which were performed as previously described under e . pgk1 overexpressing mkn - cells were analyzed in view of their invasiveness compared to non - transfected cells using a gelatine loaded migration assay in a boyden chamber . gene expression analysis was carried out using a specially prepared oligomicroarrays . several genes that were upregulated or downregulated compared to diffuse primary gastric tumor samples with and without peritoneal carcinoses were identified . after normalization , the t test was used to allow prognosis of differentially expressed genes in peritoneal carcinosis samples . at a level of significance of p & lt ; 0 . 05 , 57 genes were identified as showing differentiated regulation , of which 25 were upregulated in the peritoneal carcinosis samples and 32 were downregulated . in order to validate this result , 10 genes were selected for evaluation by quantitative real time pcr with pooled sample rna . for all of these genes , the expression observed on the microarrays was confirmed , with the exception of e cadherin and s100 . these results are shown in table 2 . a significant p value on the microarray and mrna overexpression on real time pcr ( pooled samples ) were observed for pgk1 ( p value = 0 . 015 ) on the microarray , with a result of 5 . 2 - fold in real time pcr in the primary tumor samples with known peritoneal carcinosis . based on this result , additional genes were investigated using quantitative real time pcr on individual samples ( not pooled ). these genes were pgk1 , hif - 1α , cxcr4 , cxcl12 , β - catenin , vegf , and il - 8 . specific mrna overexpression in diffuse primary tumors in stomach cancer with peritoneal carcinosis was detected for pgk1 ( 4 . 6 - fold ), hif - 1α ( 3 . 8 - fold ), cxcr4 ( 2 . 9 - fold ), cxcl12 ( 11 . 3 - fold ), and β - catenin ( 3 . 8 - fold ), while specific mrna suppression of vegf ( 5 - fold ) was detected in diffuse primary stomach cancer tumors with peritoneal carcinosis . for il - 8 , a wide range of differences in expression was observed among the patients ( see fig4 , outermost right plot in the bottom row ). nevertheless , both the array data and the quantitative real time pcr data from the pooled rna indicated twofold downregulation in primary tumors of gastric cancer tissue with peritoneal carcinosis compared to primary tumors without peritoneal carcinosis . in a comparison of primary tumors with and without peritoneal carcinoses , no differences in cadherin mrna were observed . the results of quantitative real time pcr for the individual samples are shown in table 3 . the gene silencing and overexpression experiments have shown that pgk1 has an important influence on the regulation of the signalling molecules cxcr4 , cxcl12 , beta - catenin and vegf , and vice versa . these results confirm the data generated on expression level , so that the influence of pgk1 on its signalling molecules could be further confirmed and a regulation mechanism on expression and protein level could be shown ( see fig4 a / b ). the results of the pgk1 overexpression ( transfection of pgk1 ) ( see fig5 ) show that pgk1 mrna as well as cdcr4 mrna was upregulated compared to controls without transfection ( see fig5 a ). further , the facs results of cxcr4 , 13 - catenin and cxcl12 on protein level show that these genes are upregulated in the cells with pgk1 transfection , again , compared to controls ( see fig5 b ). also , it could be shown , that pgk1 is increased on protein level in the transfected cells , also , compared to the controls ( see fig5 c ). the analyses regarding the invasiveness of the cells with and without pgk1 overexpression showed that from the gastric carcinoma cells mkn45 without pgk1 overexpression only two cells invaded the gel ( see fig6 , right ). this fiftyfold increased invasiveness of gastric carcinoma cells mediated by a pgk1 - overexpression compared to gastric carcinoma cells without pgk1 - overexpression shows the malignancy increasing influence of pgk1 and its signalling molecules in view of tumorgenesis and peritoneal carcinosis . in the tests described above , the differential expression of genes in samples from patients with diffuse gastric cancer with and without peritoneal carcinoses were investigated using oligonucleotide microarrays and quantitative real time pcr . in the view of the applicant , this constitutes the first study that shows highly specific overexpression of pgk1 mrna in diffuse primary gastric cancer with histologically confirmed peritoneal carcinosis . in addition to its function as a glycolytic enzyme , pgk1 has also been shown to play an important role in malignant tumors such as breast cancer , uterine cancer , and pancreatic cancer , but not yet in gastric cancer . the results of this study show that pgk1 overexpression could promote dissemination in the peritoneum . it is well - known that hif - 1α regulates the expression of pgk1 . the elimination of hif - 1α stops the induction of pgk1 , which confirms that pgk1 is a downstream target of hif - 1α . these results show overexpression of hif - 1α mrna in primary gastric cancer tumors with peritoneal carcinoses , a finding that shows a clear relation with the pgk1 expression values obtained . these results also showed a significant increase in cxcr4 and cxcl12 mrna , as well as strong suppression of vegf and il - 8 mrna in gastric cancer tissue with peritoneal carcinoses . accordingly , these results are also the first to show a strong link between pgk1 regulation and the cxcr4 -/ cxcl12 axis in diffuse primary gastric cancer with known peritoneal carcinoses . the present results also showed overexpression of β - catenin mrna in primary tumors with peritoneal carcinosis . these results are not consistent with reports in which prostate cancer cell lines were investigated and in which high levels of pgk1 lead to downregulated expression of β - catenin . to date there are no true diagnosis markers available that would allow prognosis of whether a primary gastric tumor will spread into the peritoneum . although there are a few quantitative prognosis markers that serve as guidelines in selecting treatment in order to achieve the maximum therapeutic effect , these cannot be used to predict whether spreading will occur . the highly clear differential expression of pgk1 , hif - 1α , cxcr4 , cxcl12 , β - catenin , and vegf is highly specific . for this reason , these molecules may be used as outstanding markers , either individually or in combination , in order to detect primary tumors that are highly likely to spread into the peritoneum and to develop metastases .
the present invention relates to a method for diagnosing peritoneal carcinoses or metastatic primary tumors in a subject , as well as to a method for providing a prognosis to a subject diagnosed with a primary tumor to develop metastases , in particular peritoneal carcinosis , comprising the step of determining the level of expression of at least phosphoglycerate kinase 1 gene . further , the invention relates to a diagnostic kit , comprising at least one substance for detection of the activity and / or expression of phosphoglycerate kinase 1 and / or β - catenin , either alone or in combination with the detection of cxcr4 and / or cxcl12 , for the diagnosis or prognosis of peritoneal carcinoses and / or metastatic primary tumors . also , a method for the preventive treatment of peritoneal carcinoses and / or metastatic primary tumors in a subject in need thereof is disclosed , wherein the method comprises the step of administering to the subject at least a pharmaceutically effective amount of a substance inhibiting the activity and / or expression of phosphoglycerate kinase 1 .