Means and methods for molecular classification of breast cancer
The invention relates to a method of typing a sample from a breast cancer patient. More specifically, the invention relates to a method for classification of breast cancer according to the presence or absence of Estrogen Receptor (ER), Progesterone Receptor (PR) and Human Epidermal growth facto Receptor 2 (ERBB2; HER2). More specifically, the invention Provides methods and means to classify breast cancer as ER positive, triple negative (ER − , PR − and HER2 − ) and HER2+.
1. A method of assigning treatment to a patient suffering from breast cancer, comprising
(a) typing a sample from the patient according to a method comprising:
determining a level of RNA expression for ADM and CCDC74B, and, optionally for at least one gene selected from the group consisting of: BTD, HMGCL, ABAT, NPY1R, CHAD, ACADSB, HK3, IL6ST, SOX11, BECN1, BTRC, CDC25B, LILRB3, NUDT6, GREB1, TBC1D9, COQ7, GOLSYN, OCIAD1, DBNDD2, PREX1, TCTN1, HDAC11, TMEM101, PARD6B, KIAA1737, TMC4, SUSD3, UBXD3, TAPT1, RUNDC1, PPAPDC2, and TPRG1; and
determining a level of RNA expression for MSN and THSD4 and, optionally for at least one gene selected from the group consisting of: CHAD, ACADSB, DHRS2, GREB1, TBC1D9, SPEF1, PERLD1, ABCC11, PRR15, MAGED2, RTN4RL1; and
determining a level of RNA expression for PERLD1 and SYCP3 and optionally for ERBB2,
in the sample from the patient by microarray analysis with probes having SEQ ID NOs: 5, 34, 38, 43, 49 and 50,
whereby the sample from the patient comprises RNA expression products from a breast cancer cell of the patient, said breast cancer cell being obtained from the patient during or after surgery and/or biopsy;
converting said RNA expression products into a labelled sample comprising either complementary DNA (cDNA) or cRNA with a reverse-transcriptase enzyme and labelled nucleotides,
hybridizing the labelled sample against the probes that are spotted on an array,
washing the array to remove labelled sample that did not hybridize,
analyzing amount of labelled sample that remains on the array to determine a level of RNA expression of the at least six genes,
comparing said determined level of RNA expression of the at least six genes to the level of expression of the genes in a reference sample which reference sample comprises RNA expression products from breast cells; and
typing said sample from the patient based on the comparison of the determined levels of RNA expression, whereby downregulation of ADM and upregulation of CCDC74B in the sample from the patient, when compared to the reference sample, is indicative of ER+, downregulation of THSD4 and upregulation of MSN in the sample from the patient, when compared to the reference sample, is indicative of triple negative, and downregulation of SYCP3 and upregulation of PERLD1 in the sample from the patient, when compared to the reference sample, is indicative of HER2;
(b) classifying said sample from the patient as a ER+, triple negative, or HER2+;
(c) assigning treatment comprising a platinum agent and/or a taxane to an individual of which the sample from the patient is classified as triple negative and, assigning treatment comprising trastuzumab, lapatinib, an aromatase inhibitor and/or Tamoxifen to an individual of which the sample from the patient is classified as HER2+.
2. The method according to claim 1 , whereby at least one further gene is selected from SOX11, CDC25B and LILRB3 which are downregulated in an ER+ breast cancer, compared to the level of expression in the reference sample.
3. The method according to claim 1 , whereby at least one further gene is selected from PRR15, ABCC11, DHRS2, TBC1D9, GREB1, PERLD1, MAGED2, ACADSB, SPEF1, CHAD, and RTN4RL1 which are downregulated in a triple-negative breast cancer, compared to the level of expression in the reference sample.
4. The method according to claim 1 , whereby a level of RNA expression of at least three further genes from BTD, HMGCL, ABAT, NPY1R, CHAD, ACADSB, HK3, IL6ST, SOX11, BECN1, BTRC, CDC25B, LILRB3, NUDT6, GREB1, TBC1D9, COQ7, GOLSYN, OCIAD1, DBNDD2, PREX1, TCTN1, HDAC11, TMEM101, PARD6B, KIAA1737, TMC4, SUSD3, UBXD3, TAPT1, RUNDC1, PPAPDC2, and TPRG1 is determined.
5. The method according to claim 1 , further comprising determining a metastasizing potential of the sample from the patient.
6. The method according to claim 5 , whereby the metastasizing potential is determined by a 70 gene profile.
7. The method according to claim 1 , whereby at least one further gene is selected from TMEM101, BTRC, ABAT, UBXD3, DBNDD2, TMC4, ACADSB, GOLSYN, PREX1, PARD6B, TBC1D9, GREB1, CHAD, SUSD3, TPRG1, NPY1R, OCIAD1, NUDT6, BECN1, COQ7, HMGCL, HDAC11, HK3, PPAPDC2, TAPT1, TCTN1, KIAA1737, RUNDC1, IL6ST, and BTD, which are upregulated in a ER+ breast cancer, compared to the level of expression in the reference sample.
8. A method of assigning treatment to a patient suffering from breast cancer, comprising
(a) typing a sample from the patient according to a method comprising:
determining a level of RNA expression for ADM and CCDC74B, and, optionally for at least one gene selected from the group consisting of: BTD, HMGCL, ABAT, NPY1R, CHAD, ACADSB, HK3, IL6ST, SOX11, BECN1, BTRC, CDC25B, LILRB3, NUDT6, GREB1, TBC1D9, COQ7, GOLSYN, OCIAD1, DBNDD2, PREX1, TCTN1, HDAC11, TMEM101, PARD6B, KIAA1737, TMC4, SUSD3, UBXD3, TAPT1, RUNDC1, PPAPDC2, and TPRG1; and
determining a level of RNA expression for MSN and THSD4, and, optionally for at least one gene selected from the group consisting of: CHAD, ACADSB, DHRS2, GREB1, TBC1D9, SPEF1, PERLD1, ABCC11, PRR15, MAGED2, RTN4RL1; and
determining a level of RNA expression for PERLD1 and SYCP3, and optionally for ERBB2, in the sample from the patient, by microarray analysis with probes having SEQ ID NOs: 5, 34, 38, 43, 49 and 50,
whereby the sample from the patient comprises RNA expression products from a breast cancer cell of the patient, said breast cancer cell being obtained from the patient during or after surgery and/or biopsy;
converting said RNA expression products into a labelled sample comprising either complementary DNA (cDNA) or cRNA with a reverse-transcriptase enzyme and labelled nucleotides,
hybridizing the labelled sample against the probes that are spotted on an array,
washing the array to remove labelled sample that did not hybridize,
analyzing the amount of labelled sample that remains on the array to determine a level of RNA expression of the at least six genes,
comparing said determined level of RNA expression of the at least six genes to the level of expression of the genes in a reference sample which reference sample comprises RNA expression products from breast cells; and
typing said sample from the patient based on the comparison of the determined levels of RNA expression, whereby downregulation of ADM and upregulation of CCDC74B in the sample from the patient, when compared to the reference sample, is indicative of ER+, downregulation of THSD4 and upregulation of MSN in the sample from the patient, when compared to the reference sample, is indicative of triple negative, and downregulation of SYCP3 and upregulation of PERLD1 in the sample from the patient, when compared to the reference sample, is indicative of HER2;
(b) classifying said sample as a ER+, triple negative, or HER2+;
(c) assigning hormone therapy comprising an aromatase inhibitor, Tamoxifen and/or a lutenizing hormone releasing hormone (LHRH) blocker to an individual if the sample is classified as a ER+.
9. The method according to claim 8 , whereby at least one further gene is selected from SOX11, CDC25B and LILRB3, which are downregulated in a ER+ breast cancer, compared to the level of expression in the reference sample.
10. The method according to claim 8 , whereby at least one further gene is selected from PRR15, ABCC11, DHRS2, TBC1D9, GREB1, PERLD1, MAGED2, ACADSB, SPEF1, CHAD, and RTN4RL1, which are downregulated in a triple-negative breast cancer, compared to the level of expression in the reference sample.
11. The method according to claim 8 , whereby a level of RNA expression of at least five genes from SEQ ID NOs: 1-35 is determined.
12. The method according to claim 8 , further comprising determining a metastasizing potential of the sample from the patient.
13. The method according to claim 12 , whereby the metastasizing potential is determined by a 70 gene profile.
14. The method according to claim 8 , whereby at least one further gene is selected from TMEM101, BTRC, ABAT, UBXD3, DBNDD2, TMC4, ACADSB, GOLSYN, PREX1, PARD6B, TBC1D9, GREB1, CHAD, SUSD3, TPRG1, NPY1R OCIAD1, NUDT6, BECN1, COQ7, HMGCL, HDAC11, HK3, PPAPDC2, TAPT1, TCTN1, KIAA1737, RUNDC1, IL6ST, and BTD, which are upregulated in a triple negative breast cancer, compared to the level of expression in the reference sample.