Recent publications from many investigators have identified the importance of the MAPK pathway in melanoma (1
). Sixty to 80% of melanomas have activating BRAF
). In addition, activation of the AKT pathways, through loss of PTEN
, is seen in a proportion of melanoma tumors, implying that activation of this pathway may also drive tumor progression in many melanoma tumors (2
). These observations suggest that strategies to block both of these pathways in melanoma will be needed to interrupt melanoma growth. The observation that both mutated BRAF and AKT are hsp90 client proteins suggested that using 17-AAG to inhibit hsp90 could be an efficient strategy to block both the MAPK and AKT pathways in melanoma. Several phase I trials have been reported exploring a variety of 17-AAG treatment schedules (12
). To maximize feasibility, we elected to use a weekly schedule for which the maximum tolerated dose was identified as 450 mg/m2
weekly. Also, at this dose and schedule, 2 of 11 melanoma patients had shown prolonged stable disease in a previous trial (15
). In that study, tumors were biopsied after 24 hours in a subset of patients with a variety of tumor types treated at doses of 320 to 450 mg/m2
and compared with pretreatment biopsies. RAF-1 (CRAF) was decreased in 4 of 6 patients measured; CDK4 decreased in 8 of 9 patients biopsied. Here, we have conducted a phase II trial in melanoma at the maximum tolerated dose, stratifying for mutation in the BRAF
gene with tumor pharmacodynamic data on 13 patients. Although we observed no objective clinical responses, the pharmacodynamic results may provide an explanation. The effects of 17-AAG on tumors seem to be minimal and short-lived. At the time of the posttreatment biopsy (median 44 hours after dose 1), we could detect an increase in hsp70 levels and a decrease in cyclin D1 levels. These findings, together with the observation of changes in RAF-1 and CDK4 at 24 hours in an earlier study (15
), suggest that there may have been transient decreases in raf kinases, phospho-ERK, and CDK4 that had already recovered by the time of the posttreatment biopsy but that in several of the tumors, cyclin D1 levels were still depressed. This suggests that there was a biological effect of 17-AAG, but it was short-lived. Further, these changes in the components of the MAPK pathway were not sufficient to cause tumor shrinkage. Future trials in melanoma will require a more potent hsp90 inhibitor that can be administered chronically, resulting in more prolonged suppression of the MAPK pathway.
We tested whether the tumor BRAF
mutation status could be reliably assessed from plasma DNA. Previous investigators have shown that in colorectal and pancreas cancer patients, tumor DNA harboring KRAS
mutations is readily shed into the plasma and can be detected by PCR (22
). With this in mind, we analyzed plasma DNA in our patients for the presence of mutated BRAF
to test whether plasma could be used instead of tumor to determine BRAF
status. In 6 of the 7 patients tested who harbored a BRAF
mutation in their tumors, we were able to detect a BRAF
mutation in plasma-derived DNA. However, among the six BRAF
wild-type patients tested, we detected mutant BRAF
in plasma DNA in two of them. Although the number of patients tested is small, this experience indicates that the specificity of this approach may be too low to yield reliable information about the BRAF
status of the tumor. An alternative explanation is that direct Sanger sequencing of tumor DNA is insufficiently sensitive for detecting BRAF
mutations. However, when we analyzed tumor DNA by MS-PCR, the plasma PCR results still lacked specificity (data not shown).
Another issue raised by this trial is the feasibility of pretreatment and posttreatment tumor biopsies. Melanoma patients with easily biopsied tumors are not seen as often as many investigators assume. Thus, accrual to these trials is slow and is optimized using a multicenter design. Even in the best of hands, not all biopsies contain viable tumor sufficient for Western blotting, and it is difficult to obtain more than one posttreatment sample. Because the exact pharmacodynamic time course is rarely known beforehand, this makes it easy to miss the critical biological effects, which is what we believe happened in our trial. These issues must be taken into account in future trials studying drugs that target specific pathways.
This is the first 17-AAG phase II trial in melanoma. The lack of objective tumor responses is consistent with what has been reported in hormone-refractory prostate cancer (26
) and in renal cell carcinoma (27
) using lower doses of 17-AAG. However, tumor biopsies were not obtained in those trials and so we do not know if there were pharmacodynamic effects of 17-AAG.
Preclinical data indicate that among hsp90 client proteins, there is a range of sensitivity to hsp90 inhibition. One of the most sensitive client proteins is HER2 (28
) and consistent with this, recent data from a phase I/II trial showed multiple partial responses in HER2+
metastatic breast cancer patients using the same dose and schedule of 17-AAG used in this trial (29
). This indicates that inhibitors of hsp90 can be used to deplete client proteins critical for tumor survival and that antitumor effects can be observed in patients. However, the current formulation of 17-AAG seems to be suboptimal for inhibiting BRAF and RAF-1 and therefore for patients with melanoma. As a weekly infusion, the drug is inconvenient, associated with toxicity related to the DMSO diluent, and results in only transient inhibition of the MAPK pathway in melanoma. Depletion of less sensitive hsp90 client proteins, such as BRAF, will require novel hsp90 inhibitors or formulations that can more effectively suppress RAF kinase expression.