Gene expression profiling in biopsied tumor tissues
The invention concerns sensitive methods to measure mRNA levels in biopsied tumor tissues, including archived paraffin-embedded biopsy material. The invention also concerns breast cancer gene sets important in the diagnosis and treatment of breast cancer, and methods for assigning the most optimal treatment options to breast cancer patient based upon knowledge derived from gene expression studies.
1. A method of determining likelihood of survival without breast cancer recurrence for a human breast cancer patient, the method comprising:
providing a tissue sample obtained from a primary ductal or lobular breast tumor of the patient following surgical removal of the primary tumor;
extracting RNA from the tissue sample;
reverse transcribing RNA transcripts from the extracted RNA to produce cDNAs, wherein the RNA transcripts include transcripts of each of GRB7, HER2 (ERBB2), CCNB1, Ki67, STK15, and BIRC5;
amplifying the cDNAs to produce amplicons from the cDNAs, wherein amplification of GRB7 utilizes primers comprising SEQ ID NOs: 133 and 134, and wherein amplification of HER2 (ERBB2) utilizes primers comprising SEQ ID NOs: 115 and 116;
quantitatively assaying levels of the amplicons;
normalizing said levels of the amplicons against a level of an amplicon from a cDNA of at least one reference RNA transcript in the extracted RNA from the tissue sample to provide normalized amplicon levels;
comparing the normalized RNA amplicon levels to data based on normalized RNA amplicon levels of the same transcripts obtained from reference breast cancer samples, wherein the reference breast cancer samples yield a distribution of normalized expression levels of the RNA transcripts assayed and wherein increased normalized amplicon levels of each of CCNB1, Ki-67, STK15, and BIRC5 in comparison to reference breast cancer sample data from patients who survived without breast cancer recurrence correlates with decreased likelihood of survival without breast cancer recurrence; and
determining the patient's likelihood of survival without breast cancer recurrence based on the patient's normalized amplicon levels of CCNB1, Ki67, STK15, and BIRC5 compared to the reference breast cancer sample data.
2. The method of claim 1 , wherein the patient is an invasive breast cancer patient.
3. The method of claim 1 , wherein the patient is estrogen receptor (ER) positive.
4. The method of claim 1 , further comprising a step of creating a report based upon the normalized amplicon levels of GRB7, HER2 (ERBB2), CCNB1, Ki67, STK15, and BIRC5.
5. The method of claim 1 , wherein the reverse transcribing is done as part of reverse transcriptase polymerase chain reaction (RT-PCR).
6. The method of claim 5 , wherein the PCR is quantitative real-time polymerase chain reaction (qPCR).
7. The method of claim 1 , wherein the RNA transcripts include transcripts for each of GRB7, HER2 (ERBB2), CCNB1, Ki67, STK15, BIRC5, GSTM1, and BAG1, wherein increased normalized amplicon levels of each of CCNB1, Ki-67, STK15, and BIRC5 and decreased normalized amplicon levels of each of GSTM1 and BAG1 in comparison to reference breast cancer sample data from patients who survived without breast cancer recurrence correlate with decreased likelihood of survival without breast cancer recurrence.
8. A method of treating breast cancer in a human breast cancer patient, the method comprising:
providing a tissue sample obtained from a primary ductal or lobular breast tumor of the human breast cancer patient following surgical removal of the primary tumor;
extracting RNA from the tissue sample;
reverse transcribing RNA transcripts from the extracted RNA, wherein the RNA transcripts include transcripts for each of GRB7, HER2 (ERBB2), CCNB1, Ki-67, STK15, and BIRC5, to produce cDNAs;
amplifying the cDNAs to produce amplicons from the cDNAs, wherein amplification of GRB7 utilizes primers comprising SEQ ID NOs: 133 and 134, and wherein amplification of HER2 (ERBB2) utilizes primers comprising SEQ ID NOs: 115 and 116;
quantitatively assaying levels of the amplicons;
normalizing said levels of the amplicons against a level of an amplicon from a cDNA of at least one reference RNA transcript in the extracted RNA from the tissue sample to provide normalized amplicon levels;
comparing the normalized RNA amplicon levels to data based on normalized RNA amplicon levels of the same transcripts obtained from reference breast cancer samples, wherein the reference breast cancer samples yield a distribution of normalized expression levels of the RNA transcripts assayed and wherein increased normalized amplicon levels of each of CCNB1, Ki-67, STK15, and BIRC5 in comparison to reference breast cancer sample data from patients who survived without breast cancer recurrence correlates with decreased likelihood of survival without breast cancer recurrence;
determining that the patient has a decreased likelihood of survival without breast cancer recurrence based on the patient's normalized amplicon levels of CCNB1, Ki-67, STK15, and BIRC5 compared to the reference breast cancer sample data; and
administering an effective amount of adjuvant chemotherapy to the patient.
9. The method of claim 8 , wherein the patient is an invasive breast cancer patient.
10. The method of claim 8 , wherein the patient is estrogen receptor (ER) positive.
11. The method of claim 8 , further comprising a step of creating a report based upon the normalized amplicon levels of GRB7, HER2 (ERBB2), CCNB1, Ki-67, STK15, and BIRC5.
12. The method of claim 8 , wherein the reverse transcribing is done as part of reverse transcriptase polymerase chain reaction (RT-PCR).
13. The method of claim 12 , wherein the PCR is quantitative real-time polymerase chain reaction (qPCR).
14. The method of claim 8 , wherein the RNA transcripts include transcripts for each of GRB7, HER2 (ERBB2), CCNB1, Ki67, STK15, BIRC5, GSTM1, and BAG1, wherein increased normalized amplicon levels of each of CCNB1, Ki-67, STK15, and BIRC5 and decreased normalized amplicon levels of each of GSTM1 and BAG1 in comparison to reference breast cancer sample data from patients who survived without breast cancer recurrence correlate with decreased likelihood of survival without breast cancer recurrence.
15. The method of claim 1 , wherein RNA transcripts of at least 15 genes are reverse transcribed.
16. The method of claim 15 , further comprising a step of creating a report based upon the normalized amplicon levels of the at least 15 genes.
17. The method of claim 8 , wherein RNA transcripts of at least 15 genes are reverse transcribed.
18. The method of claim 17 , further comprising a step of creating a report based upon the normalized amplicon levels of the at least 15 genes.