Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the present invention relates to the field of thrombotic diseases or disorders. More specifically, it relates to markers and methods for determining whether a subject, particularly a human subject, suffering or under the suspicion of suffering from cancer, is at risk of developing thromboembolic diseases or disorders, developing a thromboembolic event, having a thromboembolic disease or disorder, or is or will be experiencing a complication of a thromboembolic disease.
• VTE venous thromboembolism
• other thromboembolic diseases e.g. for 18% of all cases of incident VTE 1 .
• the risk for VTE is elevated 7-fold as compared to non- cancer patients, and in certain malignancies, the risk for VTE may be increased up to 17- fold 2 .
• VTE is discovered in a fifth of all cancer patients and as many as half of cancer postmortem examinations 3 .
• the incidence of cancer-associated VTE is particularly high during the first months after diagnosis, if distant metastases are present, and after initiating chemotherapy 2,4,5 .
• VTE cancer-associated VTE bears several clinical and economic implications, including increased hospitalization rates and the potential for delays in cancer therapy 6 .
• VTE is the second leading cause of death; in fact, of every seven patients with cancer who die while hospitalised, one will die of pulmonary embolism 1 , which is a typical direct consequence of VTE.
• pulmonary embolism 1 is a typical direct consequence of VTE.
• complications such as recurrent VTE, post-thrombotic syndrome, and chronic thromboembolic pulmonary hypertension are common, costly, and have a profound impact on the patient ' s quality of life 1 .
• Table 1 shows the currently known thrombotic risk factors identified in patients suffering from cancer-associated VTE. Those risk factors can be divided in demographic, cancer related, treatment related, and other risk factors. Table 1.
• P-selectin In the following, additional information is provided on both cancer- and treatment related risk factors.
• cancer related risk factors describe the incidence of thrombotic events, diseases or disorder, in particular of VTE, according to tumour type and stage.
• the rate of e.g. VTE is consistently higher in patients with cancer of the pancreas, stomach, brain, kidney, uterus, lung, prostate, leukemia or ovary 9 .
• haematological malignancies, lymphoma, leukemia and myeloma disease were reported to have the highest rates of VTE 10 ' 11 .
• VTE is the most common cause of death 30 days after surgery in cancer patients (46%), followed by cancer progression (12%) 14 .
• Non-surgical anticancer treatment strategies are also associated with a high incidence of thrombotic events, diseases or disorder, in particular of VTE.
• Active treatments including chemotherapy, adjuvant chemotherapy, hormonal therapy, antiangiogenic agents, and combination regimens all have a prothrombotic effect in cancer patients.
• Chemotherapy either as primary or adjuvant therapy, significantly increases the risk of thrombotic events, diseases or disorder, in particular of VTE complications in patients with cancer.
• a prospective study of nearly 4500 patients receiving outpatient chemotherapy reported a 2.7-fold increase in arterial thrombosis, and a 47-fold increase in the mortality rate from VTE compared with the general population 15 .
• Hormone therapy in combination with chemotherapy enhances the incidence of VTE in women with breast cancer. Studies have reported that women who received the selective estrogen receptor modulator tamoxifen had a 1.5- to 7.1-fold increase in the risk of developing symptomatic VTE, compared with placebo or no treatment.
• New therapeutic agents that inhibit angiogenesis are being developed as treatments for various solid tumours such as non-small-cell lung cancer, breast cancer, and colon cancer.
• Targeted antiangiogenic agents such as bevacizumab, the monoclonal antibody to vascular endothelial growth factor, have shown efficacy in improving survival rates among patients with advanced disease.
• bevacizumab the monoclonal antibody to vascular endothelial growth factor
• EPO erythropoietin
• haematopoietic growth factors such as granulocyte-macrophage colony-stimulating factor and granulocyte-colony stimulating factor
• CVC central venous catheters
• cancer site very high risk: stomach and pancreas; high risk: lung, lymphoma, gynaecologic, bladder, and testicular;
• body mass index >35kg/m 2 high risk.
• risk assessment can be conducted based on the above validated risk assessment tool developed by Khorana 16 .
• thrombotic events, diseases or disorder, in particular of VTE in c ⁇ er patients is of vital importance in light of the difficulties associated with the treatment of thrombotic events, diseases or disorder, in particular of VTE in these patients, as they are also prone to greater recurrence rates and a higher incidence of bleeding complications 17 .
• Thromboprophylaxis has been shown to decrease deep venous thrombosis (DVT) specifically in high-risk hospitalized patients.
• LMWH low molecular weight heparin
• Novel oral anticoagulants have emerged that can rapidly change the therapeutic scenario in patients with/without cancer. These oral anticoagulants that achieve rapid inhibition of activated factor X or thrombin may offer an easier solution than LMWH but studies focusing on treatment of cancer-associated thrombosis with these agents are lacking. To date, some of these new agents have shown comparable efficacy and safety compared with traditional anticoagulants in randomized trials that included primarily patients without cancer.
• Cancer patients are at high risk for recurrent thrombotic events, diseases or disorder, in particular of VTE.
• 20.7% of cancer patients developed recurrent VTE compared with 6.8% of patients without cancer.
• Cancer patients experience recurrence even within the first six month of anticoagulation.
• this risk varies depending on several parameters such as cancer site, cancer histology, or cancer stage.
• the model had a negative predictive value (probability of no VTE in those designated low risk) of 98.5%, a very low positive predictive value (probability of VTE in those designated high risk) of 7.1%, a low sensitivity (probability of high risk in those experiencing VTE) of 40.0%, and a specificity (probability of low risk in those not experiencing VTE) of 88% in the derivation cohort.
• the model had a negative predictive value of 98.5%, a very low positive predictive value of 6.7%, a low sensitivity of 35.7%, and a specificity of 89.6%.
• the invention provides a method which is suitable to overcome the limitations of the methods used nowadays to estimate the thromboembolism risk and/or to diagnose the thromboembolic events for a subject suffering from cancer.
• preferred aspects are described:
• a method for the thromboembolic event risk assessment in a subject suffering from cancer comprising the steps of determining in a sample isolated from said subject the presence of at least one allele of rs2232698, rs5985, rs6025 and rs4524, namely Serpin A10 (protein Z inhibitor) Arg67Stop (rs2232698), , factor XIII Val34Leu
• thromboembolic event vein thrombosis, deep vein thrombosis and/or pulmonary embolism
• a recurrent thromboembolic event vein thrombosis, deep vein thrombosis and/or pulmonary embolism
• a method for the diagnosis of developing or suffering a thromboembolic disease or event or a recurrent thromboembolic event in a subject suffering from cancer comprising the steps of determining a sample isolated from said subject the presence of at least one allele of rs2232698, rs5985, rs6025 and rs4524, namel" Serpin A10 (protein Z inhibitor) Arg67Stop (rs2232698), factor XIII Val34Leu (rs5985), factor V Leiden Arg506Gln (rs6025), and factor V K858R (rs4524) whereby said presence is indicative of the risk of suffering a thromboembolic event (vein thrombosis, deep vein thrombosis and/or pulmonary embolism) which is better than the risk assessment done by the methods nowadays in use, which is indicative of a risk of having a thromboembolic event and/or a recurrent thromboembolic event.
• thromboembolic disease is selected from the group of fatal or non-fatal myocardial infarction, stroke, transient, ischemic attacks, peripheral arterial disease, vein thrombosis, deep vein thrombosis, pulmonary embolism or a combination thereof, preferably from venous thromboembolism, deep vein thrombosis and pulmonary embolism.
• a method for identifying a subject suffering from cancer in need of anticoagulant and/or antithrombotic therapy or in need of prophylactic antithrombotic and/or anticoagulant therapy comprising the steps of determining in a sample isolated from said subject the presence in at least one allele of polymorphisms rs2232698, rs5985, rs6025 and rs4524, namely Serpin A10 (protein Z inhibitor) Arg67Stop (rs2232698), factor XIII Val34Leu (rs5985), factor V Leiden Arg506Gln (rs6025), and factor V K858R (rs4524), whereby said presence is indicative of having a decreased response to an antithrombotic and/or anticoagulant therapy or of being in need of early and aggressive antithrombotic and/or anticoagulant therapy or in need of prophylactic antithrombotic and/or anticoagulant treatment.
• the method as defined in any of items 1 to 4 further comprising determining one or more risk factor selected from the group consisting of body mass index (BMI), primary site of the tumor, tumor stage, family history of VTE, or additionally personal history of VTE, previous surgery, use of a central or peripheral catheter, chemotherapy, D-dimer, soluble p-selectin or additionally age, race, sex, smoking status, systolic blood pressure, diastolic blood pressure, hospitalization, plaster case, immobilization, surgery, trauma, hormonal contraceptive or hormone therapy, pregnancy, prolonged travel (>2 hours), collagen vascular diseases, heart failure, further medications, nephrotic syndrome, diabetes mellitus, low density
• LDL lipoprotein
• HDL high density lipoprotein
• cholesterol level cholesterol level
• triglyceride levels pregnancy, or Khorana score.
• the method according to any of the items 1 to 5 wherein the assistance in the diagnosis of a VTE or prediction of a first VTE or of a recurrent VTE or of the need to receive thromboprophyasis or the need to prolong the thromboprophylasis is made in a subject during the procedure to identify whether the patient is suffering from cancer and/or during the procedure to characterize the TNM stage of the cancer.
• the method according to any of the items 1 to 5 wherein the assistance in the diagnosis of a VTE or prediction of a first VTE or of a recurrent VTE or of the need to receive thromboprophyaxis or the need to prolong the thromboprophylaxis is made in a subject at any time from the diagnosis of the cancer.
• the method according to any of the items 1 to 7 wherein the assistance in the diagnosis of a VTE or prediction of a first VTE or of a recurrent VTE or of the need to receive thromboprophyaxis or the need to prolong the thromboprophylaxis is made in a subject suffering from cancer and treated in an out-patient setting.
• the sample is an oral tissue sample, scraping or wash or a biological fluid sample, preferably saliva, urine or blood, or buccal cells.
• a biological fluid sample preferably saliva, urine or blood, or buccal cells.
• the presence or absence of the genetic variant is identified by amplifying or failing to amplify an amplification product from the sample, wherein the amplification product is preferably digested with a restriction enzyme before analysis and/or wherein the genetic variant is identified by hybridizing the nucleic acid sample with a primer label which is a detectable moiety.
• M ⁇ ⁇ regression coefficient that expresses the risk (higher or lower) to present thrombosis associated with the value/presence of the predictor variable X n ,
• Pf.g regression coefficient that expresses the risk (higher or lower) to present thrombosis associated with the combined presence of the predictor variables Xf and x g>
• - X g value taken by the predictor variable X f in an individual
• - h i regression coefficient that expresses the risk (higher or lower) to present thrombosis associated with the combined presence of the predictor variables X and Xi
• a computer program or a cmputer-readable media containing means for carrying out a method as defined in any of items 1 to 11.
• a kit comprising reagents for detecting the identity of the nucleotide selected from the group of rs2232698, rs5985, rs6025 and rs4524, namely Serpin A10 (protein Z inhibitor) Arg67Stop (rs2232698), factor XIII Val34Leu (rs5985), factor V Leiden Arg506Gln (rs6025), and factor V K858R (rs4524).
• a kit comprising reagents, e.g. primer pairs, for detecting the identity of the nucleotide of risk within a nucleic acid sequence selected from the group of SEQ. ID NO: 11 and 12; 15 and 16; 21 and 22; and 23 and 24.
• the method provided according to the present invention overcomes the above limitations of known methods. It comprises the essential step of determining in a sample isolated from said subject the presence at least one of following genetic variants: rs6025, rs4524, rs2232698, and rs5985, which are indicative for the risk of suffering a thromboembolic event (in particular vein thrombosis, deep vein thrombosis and/or pulmonary embolism).
• a thromboembolic event in particular vein thrombosis, deep vein thrombosis and/or pulmonary embolism.
• the two following SNPs are additionally determined: factor V Cambridge Arg306Thr (118203906), factor V Hong Kong Arg306Gly (rsll8203905).
• the above genetic variants are further combined with additional predictive variables, which are in particular selected from one or more of the following:
• Body mass index (BMI) >25 is a risk factor
• the TNM tumor staging can be further categorized as follows:
• T2 - T3, T4 - refers to the size and/or extent of the main tumor. The higher the number after the T, the larger the tumor or the more is has grown into nearby tissues.
• Nl, N2, N3 refers to the number and location of lymph nodes that contain cancer. The higher the number after the N, the more lymph nodes that contain cancer.
• MO - Cancer has not spread to other parts of the body
• Ml - cancer has spread to other parts of the body.
• the invention relates to methods for establishing the probability of patient suffering from cancer of presenting a thromboembolic event based on the presence of one or more of the polymorphisms mentioned above, optionally in combination with one or more predictive variables, e.g. as described above.
• the invention relates to methods for establishing the probability of a patient suffering from cancer of presenting a recurrent thromboembolic event based on the presence of one or more of the polymorphisms mentioned above, optionally in combination with one or more predictive variables, e.g. as described above.
• the invention relates to methods for the assistance in the diagnosis in a patient suffering from cancer, of a thromboembolic event, based on the presence of one or more of the genetic variants mentioned above, optionally in combination with one or more predictive variables, e.g. as described above, optionally, to confirm an earlier tentative diagnosis of a thromboembolic event.
• the invention relates to methods for determining the need for preventive measurements in a patient suffering from cancer to prevent the development of a thromboembolic event and/or a recurrent thromboembolic event based on the prese ⁇ of one or more of the polymorphisms mentioned above, optionally in combination with one or more predictive variables, e.g. as described above.
• the invention relates to methods for the determination of a need for the prolonged treatment with anticoagulants in a patient suffering from cancer to prevent the development of a recurrent thromboembolic event based on the presence of one or more of the polymorphisms mentioned above, optionally in combination with one or more predictive variables, e.g. as described above.
• Thromboembolic event in the context of this application should be understood as the alteration of the hemostasis that leads to the development of a blood clot (thrombo) inside a vascular vessel (artery or vein).
• the thromboembolic event can also obstruct the vascular vessel completely and/or become detached and obstruct another vascular vessel.
• Thromboembolic event is meant to include in the present application among others the following conditions: arterial thrombosis, fatal- and non-fatal myocardial infarction, stroke, transient ischemic attacks, cerebral venous thrombosis, peripheral arteriopathy, deep vein thrombosis, vein thrombosis and pulmonary embolism.
• Recurrent thromboem bolic event in the context of this application should be understood as a thromboembolic event in a subject who has already developed one or more
• thromboembolic event in the context of this application is used interchangeably with “thromboembolism”.
• thromboembolic event in the context of this application is used interchangeably with “thrombosis”.
• Thromboembolic event in the context of this application is used interchangeably with “thromboembolic complication”.
• Thrombophilia in the context of this application should be understood as encompassing disorders of hemostasis that predispose to thrombosis. Included are heritable deficiencies of the natural anticoagulants antithrombin, protein C, and protein S and un-common and common genetic variants in the genes encoding clotting factors and acquired
• thrombophilias such as antiphospholipid antibodies. All these disorders and diseases are well known to the person of skill in the art.
• “Mutation” or “genetic variant” in the context of this application should be understood as the change of the sequence and/or structure of a gene, resulting in a variant form which may be transmitted to subsequent generations, caused by the alteration of single base units in the DNA, or the deletion, insertion, or rearrangement of larger sections of genes or chromosomes.
• polymorphism and “single nucleotide polymorphism” (SNP) are used herein interchangeably and relate to a genetic variant occurring when a single nucleotide in the genome or another shared sequence differs between members of species or between paired chromosomes in an individual.
• a SNP can also be designated as a mutation with low allele frequency greater than about 1% in a defined population.
• Single nucleotide polymorphisms may fall within coding sequences of genes, non-coding regions of genes or the intronic regions between genes.
• SNPs in high linkage disequilibrium are encompassed.
• SNPs in high linkage disequilibrium are those with more than 70%, more preferred more than 80 %, even more preferred more than 95 %, linkage disequilibrium.
• Linkage disequilibrium is well known to the person skilled in the art who will understand how to determine further SNPs in linkage disequilibrium to a given SNP.
• a strong linkage disequilibrium is one with an r 2 value of more than 0.7, wherein r 2 is defined as follows:
• p a f is the frequency of baplotypes having aide a at locus 1 and allele b at locus 2
• the respective information can also be acquired in a straight-forward manner from public genome browsers like http://www.ensembl.org.
• sample refers to any sample from a biological source and includes, without limitation, cell cultures or extracts thereof, biopsied material obtained from a mammal or extracts thereof, and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof.
• a preferred sample in the present context is blood or saliva, even more preferred buccal cells.
• Sociodemographic and clinical characteristics in the context of this application should be understood as age, gender, diabetes mellitus, smoking, family history of thromboembolic event, personal history of thromboembolic event, pregnancy, and body mass index.
• the invention relates to a computer program or a computer-readable media containing means for carrying out any of the methods of the invention, in particular means which allow for the determination of the above preferred selection of genetic variants, and/or means for the determination of the above preferred additional variables.
• the invention relates to a kit comprising reagents for detecting genetic variants rs2232698, rs5985, rs6025, and rs4524 (i.e. Serpin A10 (protein Z inhibitor) Arg67Stop (rs2232698), factor XIII Val34Leu (rs5985), factor V Leiden Arg506Gln (rs6025), and factor V K858R (rs4524)).
• the kit comprises those reagents which are suitable for detecting the preferred or more preferred combination of genetic variants, as listed above.
• the kit additionally comprises reagents which are suitable for the determination of the additional variables, in particular, the combination of additional variables, as also mentioned above.
• the authors of the present invention have solved the problems identified above in the methods in use nowadays for the calculation of the risk in a subject to develop a thromboembolic event a nd/or recurrent thromboembolic event, as those terms have been defined above.
• the authors of the present invention have identified a series of genetic variants which are associated with a particularly high risk of presenting a thromboembolic event and/or a recurrent thromboembolic event. These genetic variants show improved predictive and diagnostic value.
• the present application solves the above-described limitations of the methods used nowadays to calculate a thromboembolic event risk and/or a recurrent thromboem bolic event risk and/or to diagnose a thromboembolic event, in particular of VTE, and in particular in cancer patients.
• This is achieved, if a particular combination (as described above) of genetic markers is used, which has been selected and evaluated by the inventors after a complex and genuine analysis of thousands of possible markers.
• GRS genetic risk score
• the inventors have determined a particular preferred combination and could show, as proven in the examples below, that it provided the best possible results.
• the accumulated number of risk alleles from those SNPs listed in table 3 below that are present in each individual is considered.
• every individual can have 0, 1 or 2 alleles of risk.
• the inventors have additionally generated new algorithms for the thromboembolic risk estimation which further assist the determination of a risk and the actual diagnosis, which in turn will lead to an indication to the person of skill in the art of whether or not treatment or preventive measures should be ta ken to prevent and/or treat a thromboembolic event, in particular VTE.
• predictive risks may be categorized by using risk cut-off threshholds.
• nucleotides present according to the method of the invention in an individual's nucleic acid can be done by any method or technique capable of determining nucleotides present in a polymorphic site.
• the nucleotides present in the polymorphic markers can be determined from either nucleic acid strand or from both strands.
• a biological sample from a subject e.g., a bodily fluid, such as urine, saliva, plasma, serum, or a tissue sample, such as a buccal tissue sample or a buccal cell, preferably a buccal cell
• a tissue sample such as a buccal tissue sample or a buccal cell, preferably a buccal cell
• blood plasma or serum
• saliva or buccal cells the detection of a sequence variation or allelic variant SNP is typically undertaken.
• a biological sample from a subject e.g., a bodily fluid, such as urine, saliva, plasma, serum, or a tissue sample, such as a buccal tissue sample or a buccal cell, preferably a buccal cell
• blood plasma or serum
• any of a variety of methods that exist for detecting sequence variations may be used in the methods of the invention.
• the particular method used is not important in the estimation of cardiovascular risk or treatment selection.
• Other examples of possible commercially available methods exist for high throughput SNP identification not using direct sequencing technologies, for example, lllumina's Veracode Technology, Taqman ® SNP Genotyping Chemistry and KASPar SNP genotyping Chemistry.
• a variation on the direct sequence determination method is the Gene ChimpTM method available from Affymetrix ® .
• Perkin Elmer adapted its TAQjnan AssayTM to detect sequence variation.
• Orchid Biosciences has a method called SNP- ITTM (SNP-ldentification technology) that uses primer extension with labeled nucleotide analogs to determine which nucleotide occurs at the position immediately 3' of an oligonucleotide probe, the extended base is then identified using direct fluorescence, an indirect colorimetric assay, mass spectrometry, or fluorescence polarization.
• Sequenom ® uses a hybridization capture technology plus MALDI-TOF (Matrix Assisted Laser Desorption/ionization-Time-of Flight mass spectrometry) to detect SNP genotypes with their MassARRAYTM system.
• MALDI-TOF Microx Assisted Laser Desorption/ionization-Time-of Flight mass spectrometry
• Promega ® provides the READITTM SNP/Genotyping System (U.S. Pat. No 6,159,693).
• DNA or RNA probes are hybridized to target nucleic acid sequences. Probes that are complementary to the target sequence at each base are depolymerized with a proprietary mixture of enzymes, while probes which differ from the target at the interrogation position remain intact.
• the method uses pyrophosphorylation chemistry in combination with luciferase detection to provide a highly sensitive and adaptable SNP scoring system.
• Third Wave Technologies ® has the Invader OSTM method that uses proprietary Cleavage enzymes, which recognize and cut only the specific structure formed during the Invader process.
• Invader OS ® relies on linear amplification of the signal generated by the Invader process, rather than on the exponential amplification of the target.
• the Invader OS assay does not utilize PCR in any part of the assay.
• RFLPs restriction fragment length polymorphisms
• the presence or absence of the SNPs is identified by amplifying or failing to amplify an amplification product from the sample
• Polynucleotide amplifications are typically template-dependent. Such amplifications generally rely on the existence of a template strand to make additional copies of the template.
• Primers are short nucleic acids that are capable of priming the synthesis of a nascent nucleic acid in a template-dependent process, which hybridize to the template strand. Typically, primers are from ten to thirty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded and/or single-stranded form, although the single-stranded form generally is preferred.
• pairs of primers are designed to selectively hybridize to distinct regions of a template nucleic acid and are contacted with the template DNA under conditions that permit selective hybridization.
• high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers.
• hybridization may occur under reduced stringency to allow for amplification of nucleic acids containing one or more mismatches with the primer sequences.
• a number of template dependent processes are available to amplify the oligonucleotide sequences present in a given template sample.
• One of the best known amplification methods is the polymerase chain reaction.
• PCR pairs of primers that selectively hybridize to nucleic acids are used under conditions that permit selective hybridization.
• the term "primer”, as used herein, encompasses any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Primers may be provided in double- stranded or single-stranded form, although the single-stranded form is preferred. Primers are used in any one of a number of template dependent processes to amplify the target gene sequences present in a given template sample.
• PCR One of the best known amplification methods is PCR, which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,156, each incorporated herein by reference.
• two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target gene(s) sequence.
• the primers will hybridize to form a nucleic acid primer complete if the target- gene(s) sequence is present in a sample.
• An excess of deoxribonucleoside triphosphates is added to a reaction mixture along with a DNA polymerase, e.g. Taq polymerase that facilitates template-dependent nucleic acid synthesis.
• a DNA polymerase e.g. Taq polymerase that facilitates template-dependent nucleic acid synthesis.
• the polymerase will cause the primers to be extended along the target-gene(s) sequence by adding on nucleotides.
• the extended primers will dissociate from the target-gene(s) to form reaction products, excess primers will bind to the target-gene(s) and to the reaction products and the process is repeated.
• cycles are conducted until a sufficient amount of amplification product is produced.
• the amplification product may be digested with a restriction enzyme before analysis.
• the presence or absence of the SNP is identified by hybridizing the nucleic acid sample with primer labeled with a detectable moiety.
• the detectable moiety is detected in an enzymatic assay, radio assay, immunoassay or by detecting fluorescence.
• the primer is labeled with a detecta ble dye (e.g., SYBR Green 1, YO-PRO-1, thiazole orange, Hex, pico green, edans, fluorescein, FAM, or TET).
• the primers are located on a chip.
• the primers for amplification are specific for said SNPs.
• LCR ligase chain reaction
• LAMP loop-mediated isothermal amplification
• Strand Displacement Amplification is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.
• a similar method, called Repair Chain Reaction (RCR) involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection.
• nucleic acid amplification procedures include transcription-based amplification systems, including nucleic acid sequence based amplification.
• nucleic acid sequence based amplification the nucleic acids are prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and mini-spin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
• amplification techniques involve annealing a primer, which has target specific sequences.
• DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization.
• the double-stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6.
• a polymerase such as T7 or SP6.
• the RNA's are revers transcribed into double stranded DNA, and transcribed once against with a polymerase such as T7 or SP6.
• the resulting products whether truncated or complete, indicate target specific sequences.
• modified primers are used in a PCR-like template and enzyme dependent synthesis.
• the primers may be modified by labelling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).
• a capture moiety e.g., biotin
• a detector moiety e.g., enzyme
• a nucleic acid amplification process involves cyclically synthesizing single-stranded RNA (“ssRNA”), SSDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention.
• the ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA- dependent DNA polymerase).
• RNA- dependent DNA polymerase reverse transcriptase
• the RNA is then removed from the resulting DNA-RNA duplex by the action of ribonuclease H (RNase H, and RNase specific RNA in duplex with either DNA or RNA).
• the resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template.
• This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E coli DNA polymerase I), resulting in a double-stranded ONA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
• This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
• amplification products are separated by agarose, agarose acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al., 1989, see infra). Separated amplification products may be cut out and eluted from the gel for further manipulation . Using low melting point agarose gels, the separated band may be removed by heating the gel, followed by extraction of the nucleic acid. Separation of nucleic acids may also be effected by chromatographic techniques known in the art.
• the amplification products are visualized.
• a typical visualization method involves staining of a gel with ethidium bromide and visualization of brands under UV light.
• the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the separated amplification products can be exposed to X-ray film or visualized with light exhibiting the appropriate excitatory spectra.
• the presence of the polymorphic positions according to the methods of the invention can be determined by hybridization or lack of hybridization with a suitable nucleic acid probe specific for a polymorphic nucleic acid but not with the non-mutated nucleic acid.
• hybridize is meant a pair to form a double-stranded molecule between complementary polynucleotide sequences, or portions thereof, under various conditions of stringency.
• stringent salt concentration will ordinarily be less than about 750 mM NaCI and 75 mM trisodium citrate, preferably less than about 500 mM NaCI and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCI and 25 mM trisodium citrate.
• Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least a bout 50% formamide.
• Stringent temperature conditions will ordinarily include temperatures of at least about 30°C, more preferably of at least about 37°C, and most preferably of at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are all well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. I n a preferred em bodiment, hybridization will occur at 30°C in 750 mM NaCI, 75 mM trisodium citrate, and 1% SDS.
• SDS sodium dodecyl sulfate
• hybridization will occur at 37°C in 500 mM NaCI, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ⁇ g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42°C in 250 mM NaCI, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 ⁇ g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
• wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
• stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCI and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCI and 1.5 mM trisodium citrate.
• Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25°C, more preferably of at least about 42°C, and even more preferably of at least about 68°C.
• wash steps will occur at 25°C in 30 mM NaCI, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42°C in 15 mM NaCI, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68°C in 15 mM NaCI, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well-known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad.
• Nucleic acid molecules useful for hybridization in the methods of the invention include any nucleic acid molecule which exhibits substantial identity so as to be able to specifically hybridize with the target nucleic acids.
• Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.
• substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence or nucleic molecule exhibiting at least 50% identity to a reference amino acid sequence or nucleic acid to the sequence used for comparison.
• Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical are similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine, tyrosine. I n an exemplary approach to determining the degree of identity, a BLAST program may be used with a probability score between e ⁇ "3> and e ⁇ "100> indicating a closely related sequence.
• sequence analysis software for example, Sequence Analysis Software Package of the Genetics Computer Group,
• a detection system may be used to measure the absence, presence and amount of hybridization for all of the distinct sequences simultaneously.
• a scanner is used to determine the levels and patterns of fluorescence.
• Another object of the present invention is the development of an algorithm - in close connection and relationship to the SNPs (and optionally further variables) to be determined - to estimate the risk of a patient suffering from cancer to develop and/or to being suffering a thromboembolic event or a recurrent thromboembolic event.
• the algorithm is shown as function 1.
• the individual estimation of the risk of thromboembolic event is based on a logistic regression model.
• the aim of this model is to calculate the probability that a person has of presenting thromboembolic event and/or a recurrent event according to his/her genetic, sociodemographic and clinical characteristics. To calculate this probability we use the following equation:
• Probability 1 I xi. Xn probability of presenting a thromboembolic event in a particular cancer patient with concrete a nd measureable characteristics in a number of variables 1, n. This probability could range between 0 and 1 (This is the probability considering all the variables included in the algorithm and it can be expressed as 0 - 1 or alternatively as a percentage from 0-100%. "0" means low probability and 1 (or 100%) means a high probability.
• the GRS score is one of the variables included and the GRS can range from 0 to 22,a s described above);
• - ⁇ 0 coefficient that defines the risk (the probability) of thromboembolic event non-related with the variables 1 to n. This coefficient can take a value from - ⁇ to + ⁇ and is calculated as the natural logarithm of the incidence of thromboembolic event in the population:
• - ⁇ regression coefficient that expresses the risk (higher or lower) to present thromboembolic event associated with the value/presence of the predictor variable Xi. This coefficient can take a value from - ⁇ to + ⁇ .
• - ⁇ ⁇ regression coefficient that expresses the risk (higher or lower) to present a thromboembolic eventAssociated with the value/presence of the predictor variable X n .
• This coefficient can take a value from - ⁇ to + ⁇ ;
• - X n value taken by the predictor variable Xn in an individual.
• the range of possible values depends on the variable.
• the model includes the effect of the combination of some variables in terms of interaction or modification of the effect. That is, the effect size (regression coefficient) of a single variable (x f ) can be Pf but if this variable is present in combination with another variable (X g ) the effect size may vary (increase or decrease). To consider the effect size of the variable X f it is therefore necessary to consider not only the Pf but also a second regression coefficient Pf. g by adding the Pf and the Pf. g .
• Pf. g second regression coefficient
• This coefficient can take a value from - ⁇ to + ⁇ ;
• This coefficient can take a value from - ⁇ to + ⁇ ;
• GRS genetic risk score
• “Stage” is the Stage of the TNM tumor staging system described above. This staging system is well accepted and can be used for the present tumor stage definition. "Lower” and “upper” are defined as the estimated lowest possible value and the estimated highest posible value according to the present data and regression coefficient
• KhHR means High risk based on the Khorana score (i.e. +1), and "KhVHR” means very high risk based on the Khorana Score (i.e. +2).
• the GRS (i.e. genetic risk score) can be formed by the genetic variants included in table 5.
• the combination of genetic variants in particular in the combination described above as preferred (those in table 5), and even more preferred in combination with clinical variables included in the present invention (e.g. as set forth in table 4) have proved to be capable to determining the risk of a patient with cancer to develop a thromboempolic disease or event or a recurrent thromboembolic disease or event with a higher accuracy than that obtained using the methods nowadays in use or published functions including genetic information.
• the additional use of the above described function further assists these methods. This is particularly applicable to patients which have not yet undergone chemotherapy.
• the preferred combination of genetic variants in particular in combination with clinical variables included in the present invention and further preferably using the function described in function 1 have proved to be capable to assist in the diagnosis of a thromboembolic disease or event in a patient suffering from cancer with a higher accuracy that that obtained using the methods nowadays in use or published functions including genetic information.
• a personalized risk for a patient with cancer is obtained for the development of a thromboem bolic event or a recurrent thromboem bolic event, in particular fatal- and non-fatal-myocardium infarction, stroke, transient ischemia attack, peripheral ateriopathy, deep vein thrombosis, pulmonary embolism or a combination thereof.
• a personalized risk for a patient with cancer is obtained for the development of a thromboembolic event or a recurrent thromboembolic event, in particular fatal- and non-fatal-myocardium infarction, stroke, transient ischemia attack, peripheral ateriopathy, deep vein thrombosis, pulmonary embolism or a combination thereof.
• the VTE risk estimation can be performed at the same time the characterization of the cancer process is being performed by the pysician even before the consideration of a chemotherapy is established. This is very relevant as close to half of the VTE events in cancer patients are occuring even before chemotherapy is initiated.
• the aim of this study was to evaluate the capability of a new genetic risk score and a new clinical-genetic algorithm (the so called "TiC-Onco") for the identification of cancer patients in outpatient setting at risk of developing VTE in comparison to the standard risk assessment method.
• TiC-Onco a new clinical-genetic algorithm
• the study protocol was approved by the participant hospitals' institutional review boards. Signed, informed consent was obtained from each patient.
• This study - the ONCOTHRO MB 12-01 study - is a multicentric observational cohort study involving an 18 month monitoring period with analysis at 6, 12 and 18 months. This example presents the results for the first 6 months.
• the initial study subjects were 406 out-patients, all over 18 years of age, with a recent diagnosis (following standard procedures) of colorectal, oesophago-gastric, lung, or pancreatic cancer. All had an ECOG/WHO/Zubrod score (One of several well known scores for the classification of the performance of Cancer patients: The Eastern Cooperative Oncology Group (ECOG) score, also called the WHO or Zubrod score (after C. Gordon Zubrod), runs from 0 to 5, with 0 denoting perfect health and 5 death:
• ECOG Eastern Cooperative Oncology Group
• WHO Zubrod score
• Deep vein thrombosis in the lower limbs was diagnosed by ultrasound or ascending venography. Pulmonary embolism was diagnosed by ventilation-perfusion lung scanning, pulmonary angiography, or spiral computed tomography. Visceral vein thrombosis was diagnosed by ultrasound, spiral computed tomography or magnetic resonance imaging. Intracranial venous thrombosis was diagnosed by magnetic resonance imaging.
• the TiC-Onco risk score tool was developed in three steps:
• multivariate logistic regression analysis was performed to determine the weight of each genetic variable in the appearance of a VTE event.
• the final genetic risk score was determined using the genetic variants associated with an increased risk of VTE in the multivariate model ( ⁇ 0 ⁇ 25).
• Table 6a shows the tested further predictive variables.
• Esof or esofagus is meant to be “oesophagus”.
• the genetic risk score and the clinical variables selected were subjected to multivariate logistic regression analysis using an AlC-based backward selection process.
• the risk prediction capacity of the Khorana and TiC-Onco risk score was evaluated using the c-statistic, which represents the area under the receiver operating characteristic (ROC) curve (larger values indicate better discrimination) 23 .
• Standard measures of sensitivity, specificity, positive and negative predictive value (PPV and NPV), diagnostic odds ratio (DOR), and positive and negative likelihood ratios (PLR and NLR) 24 were determined for specific cut-off points.
• the cut-off for high risk as determined using the Khorana score was set at >3 (the normal cut-off value), and for the TiC-Onco score either as the point on the ROC curve giving the same specificity as provided by the Khorana score, or the point providing the best Youden Index.
• Table 6 shows the clinical and demographic characteristics of the 391 patients at the start of the study. For each variable, the number and percentage of patients who experienced a VTE, or not, at some point in the 6 month study period, are shown. The overall incidence of VTE was 18.16%. Patients suffering from pancreatic cancer experienced VTE at a significantly higher frequency (40.28%) than patients with other type of cancers (p ⁇ 0.001) (Table 6).
• Table 7 shows the genetic and clinical markers that were significantly associated by multivariate analysis with a VTE event, and thus selected for inclusion in the TiC-Onco risk score model.
• the TiC-Onco score showed an area under the ROC curve of 0.734 (0.673-0.794), a sensitivity of 49.30%, and a specificity of 81.25%. Its PPV was 36.84%, NPV 87.84%, PLR 2.63, NLR 0.62, and DOR 4.21 (Table 8).
• the Khorana score showed a significantly lower capacity to distinguish between patients who experienced/did not experience a VTE event (0.734 vs. 0.580; ⁇ 0 ⁇ 001).
• the sensitivity of the TiC-Onco score was significantly higher than that of the Khorana (49.30% vs. 22.54%; ⁇ 0 ⁇ 002), while the specificities of both scores were similar (81.25% vs.
• TiC-Onco 1 and 2 refer to two differnt points in the same AUC curve.
• the cutt-off point for TiC-Onco 1 is selected by a mathematical model selecting the best combination of sensitivity and specificity (the point providing the best Youden Index .
• the cut-off for TiC-Onco 2 is selected to have a specificity similar to that of Khorana.
• Tables 9 and 10 show the distribution of patients deemed likely/not likely to experience a VTE event according to the Khorana score, and for the TiC-Onco score (with the cut-off set at the same specificity as the former score).
• the great majority of patients who suffered a VTE event (77.46%) were identified by the Khorana score as being at low or moderate risk (values 0, 1 and 2).
• 17 (31%) were detected as high risk patients by the TiC-Onco score.
• the cut-off for high risk was taken as the best Youden Index, the TiC-Onco score returned significantly better predictions of risk than the Khorana score, especially in terms of sensitivity (85.92% vs.
• the Khorana-score is as follows:
• Positive Predictive Value PPV
• Negative Predictive Value NPV
• the positive and negative predictive values are the proportions of positive and negative results in statistics and diagnostic tests that are true positive and true negative results, respectively, (Fletcher, Robert H. Fletcher ; Suzanne W. (2005). Clinical epidemiology : the essentials (4th ed.). Baltimore, Md.: Lippincott Williams & Wilkins. p. 45. ISBN 0-7817- 5215-9.)
• the PPV and NPV describe the performance of a diagnostic test or other statistical measure. A high result can be interpreted as indicating the accuracy of such a statistic.
• the PPV and NPV are not intrinsic to the test; they depend also on the prevalence.
• LR Likelihood Ratio
• CI is the confidence interval, again well known to the person skilled in the art, i.e. a confidence interval (CI) is a type of interval estimate of a population parameter. It is an observed interval (i.e., it is calculated from the observations), in principle different from sample to sample, that potentially includes the unobservable true parameter of interest.
• DOR is the diagnostic odds ratio, again well known to the person skilled in the art, i.e. In medical testing with binary classification, the diagnostic odds ratio is a measure of the effectiveness of a diagnostic test. It is defined as the ratio of the odds of the test being positive if the subject has a disease relative to the odds of the test being positive if the subject does not have the disease.
• VTE The incidence of VTE in the studied population at six months of follow up is 18.16% (71 VTE cases in 391 patients).
• VTE is a multifactorial, complex disease that results from a combination of genetic and acquired risk factors.
• the heritability of VTE has been estimated to be at about 60%.
• the genetic factors underlying the risk of VTE include some well-established genetic variants such as factor V Leiden and prothrombin variant G20210A and new variants coming out from GWAS studies.
• cancer associated VTE is a multifactorial entity where clinical and genetic factors are acting
• a multivariate approach of feature selection allowing us to identify a subset or a combination of informative genetic variants and non-genetic variables that underlies the risk of developing VTE. That approach is capable of capturing the multifactorial characteristics of VTE.
• TiC-Onco for the identification of patients at high risk of developing a cancer associated VTE. As the risk of cancer associated VTE is high even 6 months before cancer diagnosis and the peak of the incidence is from 0 to 6 months post diagnosis, it should be recommended to use TiC-Onco at the moment a cancer is suspected in a patient.
• the present study is a multi-location study, with a large portion of patients in advanced stages of TNM and that we have performed a sub-analysis in post-chemotherapy VTE cases. It is also a relevant strength that significantly better results are obtained based on TiC-Onco in comparison to Khorana, especially when analysing those classification functions such as sensitivity, specificity, positive and negative likelihood ratios more strongly associated with the capacity of the test than with the specific characteristics of the population in which it is tested.
• TiC-Onco has high sensitivity to identify most (70-79%) of the cancer patients who will develop VTE.
• TiC-Onco has high specificity to identify most (69-73%) of the cancer patients who will NOT develop VTE.
• TiC-Onco is superior to Khorana both to confirm the existence of a high risk to develop VTE as well as to confirm the absence of risk to develop VTE.
• VTE thromboembolism
• Falanga A The Incidence and Risk of Venous Thromboembolism Associated With Cancer and Nonsurgical Cancer Treatment. Cancer Invest . 2009;27(1): 105- 115.
• Thromboembolism is a leading cause of death in cancer patients receiving outpatient chemotherapy. J Thromb Haemost. 2007;5(3):632-634. doi: 10.1 11 1/j .1538- 7836.2007.02374.x.
• VTE venous thromboembolism
• Falanga A Russo L. Epidemiology, risk and outcomes of venous thromboembolism in cancer. Hamostaseologie. 2012;32(2):1 15-125. doi:10.5482/ha-l 170.

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