Phase II trial of 17-allylamino-17-demethoxygeldanamycin in patients with metastatic melanoma.

PURPOSE: Activation of the mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3-kinase/AKT pathway seems to be critical for melanoma proliferation. Components of these pathways are client proteins of heat-shock protein 90 (hsp90), suggesting that inhibition of hsp90 could have significant antimelanoma effects. We conducted a phase II trial using the hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) in melanoma patients. The primary end points were clinical responses and whether treatment inhibited MAPK pathway activity. EXPERIMENTAL DESIGN: Melanoma patients with measurable disease were stratified on the basis of whether or not their tumor harbored a V600E BRAF mutation. The hsp90 inhibitor 17-AAG was administered i.v. once weekly x 6 weeks at 450 mg/m2. Tumor biopsies were obtained pretreatment and 18 to 50 hours after the first dose of 17-AAG, and were snap-frozen. RESULTS: Fifteen evaluable patients were treated; nine had BRAF mutations and six were wild-type. No objective responses were observed. Western blot analysis of tumor biopsies showed an increase in hsp70 and a decrease in cyclin D1 expression in the posttreatment biopsies but no significant effect on RAF kinases or phospho-extracellular signal-regulated kinase expression. Plasma analyzed by mutant-specific PCR for V600E BRAF showed 86% sensitivity and 67% specificity in predicting tumor DNA sequencing results. CONCLUSIONS: At this dose and schedule of 17-AAG, the effects of 17-AAG on RAF kinase expression were short-lived, and no objective antimelanoma responses were seen. Future trials in melanoma should focus on a more potent hsp90 inhibitor or a formulation that can be administered chronically for a more prolonged suppression of the MAPK pathway.

Detection of B-RAF and N-RAS mutations in human melanoma.

BACKGROUND: It is now known that activating point mutations in components of the mitogen-activated protein kinase pathway commonly occur in melanoma. We previously described a method to detect point mutations in heterogenous tissues containing both wild-type and mutant B-RAF and N-RAS genes by using site-directed mutagenesis to introduce new restrictions sites in the cDNA sequence when the specific point mutations are present. We modified this technique to improve sensitivity and used it to determine the incidence of B-RAF and N-RAS mutations in human melanoma. STUDY DESIGN: We screened 115 melanoma samples for the most common B-RAF and N-RAS mutations found in melanoma using a site-directed mutagenesis-based detection technique. Southern blotting was used to increase sensitivity of the basic system. We also tested this method of genetic mutation detection in fine-needle aspiration specimens and paraffin-embedded tissues. RESULTS: Sixty-eight samples (20 of 36 primaries, 18 of 27 regional metastases, 16 of 40 nodal metastases, and 9 of 12 distant metastases) harbored the V599E B-RAF mutation (59%), 17 contained a Q61R N-RAS mutation, and 4 contained a Q61K N-RAS mutation. We were able to detect the V599E mutation in genomic DNA from paraffin-embedded melanoma samples and could routinely detect this mutation in fine-needle aspirations of melanoma tumors. This method of detection was sensitive and specific with no false positives. CONCLUSIONS: Activating mutations of B-RAF and N-RAS were present in approximately 60% and 18%, respectively, of samples tested. The site-directed mutagenesis system of mutation detection was both sensitive and specific in detecting these mutations and will likely prove very clinically useful in future studies.

Detection of mutations in the mitogen-activated protein kinase pathway in human melanoma.

PURPOSE: Recent studies suggest that activating point mutations in B-RAF may commonly occur in melanoma. We devised a method to detect point mutations in heterogeneous tissues containing both wild-type and mutant B-RAF and N-RAS genes by using site-directed mutagenesis to introduce new restrictions sites in the cDNA sequence when the specific point mutations are present. We used this technique to determine the incidence of mitogen-activated protein kinase (MAPK) mutations in human melanoma. EXPERIMENTAL DESIGN: We screened 85 melanoma samples for the most common B-RAF and N-RAS mutations found in melanoma using a site-directed mutagenesis-based detection technique. Western blotting was used to evaluate downstream up-regulation of the mitogen-activated protein kinase pathway in these tissues. RESULTS: Thirty-three samples (7 of 25 primaries, 15 of 25 regional metastases, 5 of 25 nodal metastases, and 6 of 10 distant metastases) harbored the V599E B-RAF mutation (39%), 12 contained a Q61R N-RAS mutation and 5 a Q61K N-RAS mutation. Western blotting with antiphosphorylated extracellular signal-regulated kinase 1/2 antibodies demonstrated up-regulation of the MAPK pathway in samples containing activating B-RAF or N-RAS mutations compared with wild-type samples. This method of detection was sensitive and specific with no false positives. CONCLUSIONS: Activating mutations of the MAPK pathway were present in approximately 60% of samples tested and caused activation of this cellular pathway that appears to be important in the pathogenesis of melanoma.

Patterns of recurrence in patients with melanoma and histologically negative but RT-PCR-positive sentinel lymph nodes.

BACKGROUND: We studied the patterns of recurrence of patients with only reverse transcriptase-polymerase chain reaction (RT-PCR) evidence of regional nodal spread to see whether or not proposed treatment interventions are likely to be effective. STUDY DESIGN: One hundred seventy-five patients who underwent selective lymphadenectomy for clinical stage I and II melanomas were included in this analysis. We preserved a portion of each sentinel lymph node (SLN) in liquid nitrogen in the operating room and performed RT-PCR on the specimens to detect the melanoma/melanocyte-specific marker tyrosinase. We then compared the pattern of recurrence (regional dermal metastases, regional nodal recurrence, or distant metastatic spread) of the patients with histologically positive SLNs to that of patients who had histologically negative SLNs. RESULTS: The mean followup time of the 175 patients was 33.83 months (SD = 15.94, median = 34.17, maximum = 62.95, minimum = 6.21). Thirty-four patients had at least one histologically positive SLN, and 17 of these patients had a recurrence (50%). Of the 141 patients that had histologically negative SLNs, 73 had SLNs that were also negative for tyrosinase by RT-PCR, and none of these patients had a recurrence. Of the 68 patients that had histologically negative but RT-PCR-positive SLNs, 14 had a recurrence (20.6%). CONCLUSIONS: Because the pattern of recurrence of patients with only RT-PCR evidence of melanoma in SLNs was identical to that in patients who had histologically evident melanoma in the SLN and underwent subsequent completion lymphadenectomy, we conclude that completion lymphadenectomy might be ineffective in decreasing the recurrence rate of patients with only RT-PCR evidence of melanoma in SLNs.

PKD1 induces p21(waf1) and regulation of the cell cycle via direct activation of the JAK-STAT signaling pathway in a process requiring PKD2.

Autosomal dominant polycystic kidney disease is characterized by cyst formation in the kidney and other organs and results from mutations of PKD1 or PKD2. Previous studies suggest that their gene products have an important role in growth regulation. We now show that expression of polycystin-1 activates the JAK-STAT pathway, thereby upregulating p21(waf1) and inducing cell cycle arrest in G0/G1. This process requires polycystin-2, a channel protein, as an essential cofactor. Mutations that disrupt polycystin-1/2 binding prevent activation of the pathway. Mouse embryos lacking Pkd1 have defective STAT1 phosphorylation and p21(waf1) induction. These results suggest that one function of the polycystin-1/2 complex is to regulate the JAK/STAT pathway and explain how mutations of either gene can result in dysregulated growth.