Method of determining acute myeloid leukemia response to treatment with farnesyltransferase inhibitors
The disclosed method rapidly identifies with desired accuracy AML patients, including elderly AML patients, likely to respond to treatment with a combination of a farnesyltransferase inhibitor and one or more of etoposide, teniposide, tamoxifen, sorafenib, paclitaxel, temozolomide, topotecan, trastuzumab and cisplatinum. In an embodiment, the improvements include the use of whole blood rather than the customary bone marrow sample, thus making the assay more accurate, rapid, less intrusive, less expensive as well as less painful. The method includes evaluation of a two-gene expression ratio (RASGRP1:APTX), which with a corresponding threshold, provides sufficient accuracy for predicting the response to the combination treatment. In the preferred embodiment the combination treatment combines tipifarnib (R115777, ZARNESTRA®) with etoposide. Further, the elderly AML patients identified as being likely responsive to the combination treatment with tipinifarb and etoposide have a complete recovery rate comparable to the best therapy available for younger patients.
1. A method of treating a myeloid disorder in a patient, the method of treating comprising:
administering a therapeutically effective amount of tipifarnib and etoposide to a patient responsive to tipifarnib and etoposide and having a ratio of RASGRP1 and APTX expression levels that exceeds a ΔΔCt threshold, wherein said ΔΔCt threshold has been determined using a specified sensitivity or specificity or a maximized sum of sensitivity and specificity in a ROC analysis in a test population comprising tipifarnib- and etoposide-treated subjects, and wherein, prior to administering tipifarnib and etoposide to said patient, the ratio of RASGRP1 and APTX expression has been determined experimentally in a sample obtained from the patient using the ΔΔCt method and at least one primer selected from the group consisting of
SEQ ID NO: 3
(i)
5′-CGCTTCCGATTGGGCTAC-3′
SEQ ID NO: 4
(ii)
5′-AGAATCAAAATCCTGGCTGATC-3′
SEQ ID NO: 5
(iii)
5′-CTGGACGATCTCATTGACAGC-3′
and
SEQ ID NO: 6
(iv)
5′-CTTGCAACAGTTGGTTACTTCG-3′.
2. The method of claim 1 wherein the ratio of RASGRP1 and APTX expression has been determined experimentally by using at least the following primers
SEQ ID NO: 3
(i)
5′-CGCTTCCGATTGGGCTAC-3′
SEQ ID NO: 4
(ii)
5′-AGAATCAAAATCCTGGCTGATC-3′
SEQ ID NO: 5
(iii)
5′-CTGGACGATCTCATTGACAGC-3′
and
SEQ ID NO: 6
(iv)
5′-CTTGCAACAGTTGGTTACTTCG-3′.
3. The method of claim 1 , wherein a level of expression of RASGRP1 is estimated relative to one or more genes selected from the group consisting of APTX, beta-actin, and HMBS.
4. The method of claim 1 , wherein the ΔΔCt threshold has been determined using an area under the curve of 70% or more.
5. The method of claim 1 , wherein the assay is performed in a single tube in a multiplex format.
6. The method of claim 2 , wherein the ratio of expression levels of RASGRP 1 relative to APTX has been determined using an external control.
7. The method of claim 6 , wherein the external control is selected from the group consisting of one or more reference cell lines, JY RNA, Universal RNA, a standardized RNA reference and a reference patient sample.
8. The method of claim 1 , wherein said ratio has been determined using a sample that comprises at least one of (i) a bone marrow sample; and (ii) a blood sample.
9. The method of claim 1 , wherein the disorder is AML.
10. A method for administering tipifarnib and etoposide to a patient diagnosed with Acute Myelogenous Leukemia, the method comprising:
administering a therapeutically effective amount of tipifarnib and etoposide to a patient responsive to tipifarnib and etoposide, wherein said administration is effected after the patient's ratio of RASGRP1 and APTX expression levels has been determined to exceed a ΔΔCt threshold, wherein said threshold has been determined using a sensitivity or specificity or a maximized sum of sensitivity and specificity in a ROC analysis in a test population comprising tipifarnib- and etoposide-treated subjects, and wherein, prior to administering tipifarnib and etoposide to said patient, the patient's ratio of RASGRP1 and APTX expression has been determined experimentally in a sample obtained from the patient using the ΔΔCt method and at least one primer selected from the group consisting of
SEQ ID NO: 3
(i)
5′-CGCTTCCGATTGGGCTAC-3′
SEQ ID NO: 4
(ii)
5′-AGAATCAAAATCCTGGCTGATC-3′
SEQ ID NO: 5
(iii)
5′-CTGGACGATCTCATTGACAGC-3′
and
SEQ ID NO: 6
(iv)
5′-CTTGCAACAGTTGGTTACTTCG-3′.
11. The method of claim 10 wherein the ratio of RASGRP1 and APTX expression has been determined experimentally by using the ΔΔCt method and at least the following primers
SEQ ID NO: 3
(i)
5′-CGCTTCCGATTGGGCTAC-3′
SEQ ID NO: 4
(ii)
5′-AGAATCAAAATCCTGGCTGATC-3′
SEQ ID NO: 5
(iii)
5′-CTGGACGATCTCATTGACAGC-3′
and
SEQ ID NO: 6
(iv)
5′-CTTGCAACAGTTGGTTACTTCG-3′.
12. The method of claim 10 wherein a level of expression of RASGRP1 is estimated relative to one or more of the group consisting of APTX, beta-actin and HMBS.
13. The method of claim 1 , wherein the ΔΔCt threshold has been determined using an area under the curve of 70% or more.
14. The method of claim 10 wherein the assay is performed in a single tube in a multiplex format.
15. The method of claim 11 wherein the ratio of expression levels of RASGRP1 relative to APTX has been determined using an external control.
16. The method of claim 15 , wherein the external control is selected from the group consisting of one or more reference cell lines, JY RNA, Universal RNA, a standardized RNA reference and a reference patient sample.
17. The method of claim 10 , wherein said ratio has been determined using a sample that comprises at least one of (i) a bone marrow sample; and (ii) a blood sample.
18. The method of claim 10 , wherein said patient diagnosed with Acute Myelogenous Leukemia is 55 years of age or older.
19. A method of treating a myeloid disorder in a patient, the method of treating comprising:
determining a ratio of RASGRP1 and APTX expression in a sample from the patient using the ΔΔCt method and at least one primer selected from the group consisting of
SEQ ID NO: 3
(i)
5′-CGCTTCCGATTGGGCTAC-3′
SEQ ID NO: 4
(ii)
5′-AGAATCAAAATCCTGGCTGATC-3′
SEQ ID NO: 5
(iii)
5′-CTGGACGATCTCATTGACAGC-3′
and
SEQ ID NO: 6
(iv)
5′-CTTGCAACAGTTGGTTACTTCG-3′,
and
subsequently administering a therapeutically effective amount of tipifarnib and etoposide to the patient having a ratio of RASGRP1 and APTX expression levels that exceeds a ΔΔCt threshold, wherein said ΔΔCt threshold has been determined using a specified sensitivity or specificity or a maximized sum of sensitivity and specificity in a ROC analysis in a test population comprising tipifarnib- and etoposide-treated subjects.
20. A method of treating a myeloid disorder in a patient having a ratio of RASGRP1 and APTX expression levels that exceeds a ΔΔCt threshold, the method of treating comprising:
administering a therapeutically effective amount of tipifarnib and etoposide to said patient,
wherein said ΔΔCt threshold has been determined using a specified sensitivity or specificity or a maximized sum of sensitivity and specificity in a ROC analysis in a test population,
wherein, prior to administering tipifarnib and etoposide to said patient, the ratio of RASGRP1 and APTX expression has been determined experimentally in a sample obtained from the patient using the ΔΔCt method and at least one primer selected from the group consisting of
SEQ ID NO: 3
(i)
5′-CGCTTCCGATTGGGCTAC-3′
SEQ ID NO: 4
(ii)
5′-AGAATCAAAATCCTGGCTGATC-3′
SEQ ID NO: 5
(iii)
5′-CTGGACGATCTCATTGACAGC-3′
and
SEQ ID NO: 6
(iv)
5′-CTTGCAACAGTTGGTTACTTCG-3′,
and
wherein the sample is a blood sample.
21. A method for administering tipifamib and etoposide to a patient diagnosed with Acute Myelogenous Leukemia, the method comprising:
administering a therapeutically effective amount of tipifamib and etoposide to said patient,
wherein said administration is effected after the patient's ratio of RASGRP1 and APTX expression levels has been determined to exceed a ΔΔCt threshold,
wherein said threshold has been determined using a sensitivity or specificity or a maximized sum of sensitivity and specificity in a ROC analysis in a test population,
wherein, prior to administering tipifarnib and etoposide to said patient, the patient's ratio of RASGRP1 and APTX expression has been determined experimentally in a sample obtained from the patient using the ΔΔCt method and at least one primer selected from the group consisting of
SEQ ID NO: 3
(i)
5′-CGCTTCCGATTGGGCTAC-3′
SEQ ID NO: 4
(ii)
5′-AGAATCAAAATCCTGGCTGATC-3′
SEQ ID NO: 5
(iii)
5′-CTGGACGATCTCATTGACAGC-3′
and
SEQ ID NO: 6
(iv)
5′-CTTGCAACAGTTGGTTACTTCG-3′,
and
wherein the sample is a blood sample.
22. A method of treating a myeloid disorder in a patient having a ratio of RASGRP1 and APTX expression levels that exceeds a ΔΔCt threshold, the method of treating comprising:
determining the ratio of RASGRP1 and APTX expression using the ΔΔCt method and at least one primer selected from the group consisting of
SEQ ID NO: 3
(i)
5′-CGCTTCCGATTGGGCTAC-3′
SEQ ID NO: 4
(ii)
5′-AGAATCAAAATCCTGGCTGATC-3′
SEQ ID NO: 5
(iii)
5′-CTGGACGATCTCATTGACAGC-3′
and
SEQ ID NO: 6
(iv)
5′-CTTGCAACAGTTGGTTACTTCG-3′,
and
subsequently administering a therapeutically effective amount of tipifarnib and etoposide to said patient,
wherein said ΔΔCt threshold has been determined using a specified sensitivity or specificity or a maximized sum of sensitivity and specificity in a ROC analysis in a test population, and
wherein the ratio of RASGRP1 and APTX expression levels is determined by using a blood sample of said patient.
23. The method of claim 1 wherein the ratio of RASGRP1 and APTX expression has been determined experimentally in a sample obtained from the patient using the ΔΔCt method according to equation 1:
RASGRP1:APTX ratio=2{circumflex over ( )}−(( A−B )−( C−D )) Equation 1,
where:
A: Sample RasGRP1 Ct value,
B: JY (or Universal) RNA (+) RASGRP1 Ct value,
C: Sample APTX Ct Value, and
D: JY (or Universal) RNA (+) APTX Ct Value.
24. The method of claim 1 , wherein treating comprises
administering the therapeutically effective amount of tipifarnib and etoposide to patients in a patient population responsive to tipifarnib and etoposide and having a ratio of RASGRP1 and APTX expression levels that exceeds a ΔΔCt threshold, and
detecting a response to the therapeutically effective amount of tipifarnib and etoposide in greater than 50% of the patients in the patient population.