Methods for the detection and prognosis of breast tumors
View Patent ↗This invention relates to the production of specific antibodies for the detection of human tumor associated urokinase-type plasminogen activator receptor (uPAR) deletion variants in biological samples as well as to their diagnostic and therapeutic application.
1. A method for the detection of a breast tumors associated with the generation of at least one uPAR deletion variant comprising the steps of:
(a) obtaining a cDNA containing sample from a breast tumor to be investigated, and
(b) analyzing the cDNA of said sample to determine whether alternative splicing of uPAR has occurred and at least one uPAR deletion variant has been generated, wherein the at least one uPAR deletion variant is del4+5, wherein the tumor sample is obtained from a human patient, and
wherein the presence of the at least one uPAR deletion variant in said tumor indicates the presence of a tumor associated with the generation of at least one uPAR deletion variant.
2. The method according to claim 1 , wherein the step of analyzing the cDNA of said sample comprises: amplifying the cDNA to obtain a quantified control amplification product comprising the nucleic acid coding for wild type uPAR and a quantified amplification product comprising the nucleic acid coding for the at least one uPAR deletion variant, and determining the ratio of wild type uPAR:uPAR deletion variant by comparing the quantified amplification products.
3. The method according to claim 2 , wherein the amplification of the cDNA is done by real-time RT-PCR amplification.
4. The method according to claim 2 , wherein the ratio of wild type uPAR:uPAR deletion variant is greater than 0.043.
5. The method according to claim 1 , wherein the cDNA is amplified with a first set of primers complementary to sequences contained in regions bridging the deleted sequences in uPAR deletion variants to obtain the amplification product comprising the nucleic acid coding for the at least one uPAR deletion variant, and wherein the cDNA is amplified with a second set of primers complementary to sequences contained in the wild type uPAR to obtain the control amplification product comprising the nucleic acid coding for wild type uPAR.
6. The method according to claim 1 , wherein the method provides a prognosis for the tumor associated with the generation of at least one uPAR deletion variant.
7. The method according to claim 6 , wherein the prognosis comprises characterization of the tumor, said characterization comprising indicating whether the tumor is more invasive, is more likely to have metastasis, is more likely to have a shorter disease free survival period or is more likely to have a higher rate of recurrence than a tumor not associated with the generation of uPAR deletion variants.
8. A method of providing a prognosis for a breast tumor-bearing patient comprising the steps of:
(a) obtaining a cDNA containing sample from a breast tumor of said patient,
(b) analyzing the cDNA of said sample to determine whether alternative splicing of uPAR has occurred and at least one uPAR deletion variant has been generated, wherein the at least one uPAR deletion variant is del4+5, and
(c) correlating the presence of the at least one uPAR deletion variant in the tumor of the patient with a prognosis of the patient.
9. The method according to claim 8 , wherein the step of analyzing the cDNA of said sample comprises: amplifying the cDNA to obtain a quantified control amplification product comprising the nucleic acid coding for wild type uPAR and a quantified amplification product comprising the nucleic acid coding for the at least one uPAR deletion variant, and determining the ratio of wild type uPAR:uPAR deletion variant by comparing the quantified amplification products.
10. The method according to claim 9 , wherein the amplification of the cDNA is done by real-time RT-PCR amplification.
11. The method according to claim 8 , wherein the cDNA is amplified with a first set of primers complementary to sequences contained in regions bridging the deleted sequences in uPAR deletion variants to obtain the amplification product comprising the nucleic acid coding for the at least one uPAR deletion variant, and wherein the cDNA is amplified with a second set of primers complementary to sequences contained in the wild type uPAR to obtain the control amplification product comprising the nucleic acid coding for wild type uPAR.