Determining argininosuccinate synthetase (ASS) expression in patients with melanoma treated with arginine depleting therapy.

Abstract

10627 Background: In our phase II trial in advanced melanoma (ASCO 2010) with arginine depleting therapy using ADI-PEG20 (Polaris Pharmaceuticals), we found that response to therapy correlated with tumor expression of enzyme, ASS. Normal cells which possess ASS can recycle citrulline to arginine and hence evade cell death, whereas melanoma tumors lacking ASS do not. Thus, 11 of 17 patients (pts) with ASS(-) tumors (by immunohistochemistry or real-time PCR) had evidence of antitumor activity versus 1 of 10 pts with ASS (+) tumors, (P value = 0.01). To study why not all ASS(-) melanoma pts respond to ADI-PEG20, we have developed a sensitive method to detect small amounts of mRNA by PCR. Methods: Total RNA was prepared from primary culture or cell lines and used for cDNA synthesis. Real-time PCR of ASS was performed using specific primers. The reaction was based on SYBR Green and performed in a Bio-Rad iCycler PCR machine equipped with a MyiQ module. ΔΔCt method was used to calculate the relative ASS mRNA level. Levels of ASS mRNA were corrected with that of GAPDH and normalized with that of BJ-1 normal skin fibroblast, the value of which was set as 1. Results: Real-time PCR was performed in a panel of 15 primary cultures derived from patients’ melanoma samples. The relative mRNA expression ranged from 0.0005 to 4.9. 5 primary cultures were treated with ADI-PEG for three days and assayed for ASS expression. In 2/5 with ASS mRNA <0.003, ASS expression was not inducible. The ASSmRNA of less than 0.001 exhibit higher sensitivity to ADI-PEG20 treatment (ID50 <0.1 ug/ml). However, in one cell line with baseline ASS mRNA of 0.003, it increased to 0.06 upon arginine deprivation. In a panel of melanoma cell lines we have also found that immunocytochemistry cannot detect ASS when their mRNA is <0.09. Thus, it is possible that the ASS(-) melanoma pts who do not respond to ADI-PEG20 have low detectable levels of mRNA which can be induced upon arginine deprivation. Conclusions: While the assay for ASS mRNA is cumbersome, it may better define melanoma pts who will respond to arginine depleting therapy with ADI-PEG20. Other factors which regulate ASS expression such as HIF-1alpha and cMyc are also being investigated. Supported by 1R01CA109578.