In this study, we examined the effect of combination DFMO/sulindac treatment on rectal mucosal concentrations of polyamines and PGE2 as putative drug response biomarkers. Sulindac, an NSAID and nonselective COX inhibitor, has known inhibitory action on PGE2 as well as effects on polyamine transport5
. DFMO inhibits ODC with demonstrated effects on tissue polyamine levels, particularly putrescine and spermidine13
. We also explored the potential impact of self-reported low-dose aspirin use on biomarker levels, as aspirin is a commonly used NSAID with similar properties to sulindac.
Consistent with previous studies showing that DFMO treatment depletes putrescine and lowers Spd:Spm in rectal mucosa3,13
, we found a significant effect of DFMO/sulindac on rectal mucosal polyamines. In the treatment group, the decline in Spd:Spm was achieved by 12 months, with no evidence of additional change between 12 and 36 months. Putrescine levels showed a similar behavior to Spd:Spm, with change achieved by 12 months. However, putrescine subsequently increased between 12 and 36 months; consequently, putrescine levels at 36 months were no longer significantly different from those at baseline. A similar pattern was observed in a prior phase IIb biomarker trial investigating tissue polyamine responses in CRA patients after random assignment to 0.075 g/m2
DFMO, 0.2 g/m2
DFMO, 0.4 g/m2
DFMO, or placebo3
. In the two lower-dose DFMO groups (approximately 150 mg/day and 400 mg/day – i.e.
, lower than the 500 mg/day dose used in the present study), putrescine inhibition was observed at the earlier time point (6 months), but not the later time point (12 months). In contrast, putrescine levels remained suppressed at both 6 and 12 months in the highest DFMO group (approximately 800 mg/day). Effects of DFMO on putrescine levels at 36 months were not previously assessed. Our findings here in the 3-year trial strongly validate the results of the prior dose de-escalation study and support the selection of 500 mg/day as the lowest possible dose to achieve an effect on polyamine endpoints at the tissue level. Taken together, these findings support the potential for an adaptive tissue response to prolonged ODC suppression, resulting in increased cellular uptake of diet- or bacteria-derived putrescine from the colonic lumen13,14
. Polyamines are an important factor in colonic mucosa renewal15
; thus, prolonged inhibition of ODC with DFMO and increased export by sulindac may lead to compensatory uptake of luminal polyamines in the normal rectal mucosa.
In contrast with the polyamines, we found no measurable effect of treatment on PGE2 concentrations in rectal mucosa. Although we observed a clear difference in PGE2 levels at baseline between aspirin users and non-users that is consistent with PGE2 as a "drug-effect surrogate biomarker”11
, we found no evidence of an effect of DFMO/sulindac on PGE2 at 12 nor 36 months. The reason for the lack of treatment effect on tissue PGE2 levels is unknown and should be interpreted with caution given the sample size. The lack of evidence for a strong effect of treatment on PGE2 could alternatively suggest that treatment-associated reductions in colorectal tissue polyamine contents, as shown in this report, might be more important than previously appreciated for CRA prevention with combination DFMO/sulindac, as reported earlier1
. As noted, sulindac has been demonstrated to activate polyamine catabolism and export7
, possibly acting to complement the effects of DFMO to reduce tissue polyamine pools. This hypothesis needs to be tested in future clinical trials.
When we evaluated the role of biomarker change and response to intervention, we found no effect of biomarker response (i.e., percent change over time) on DFMO/sulindac efficacy for any of the three biomarkers investigated. We had hypothesized that individuals who achieved benefit from treatment would demonstrate greater reductions in their polyamine or PGE2 levels. It is notable that we observed a non significant greater benefit for participants who exhibited a ≥ 30% decrease in Spd:Spm (i.e., responders) compared with non-responders.
To further explore a potential mechanistic role for PGE2 and polyamines in DFMO/sulindac treatment for CRA prevention, we stratified the association between treatment and metachronous CRA by baseline biomarker levels. We investigated whether or not baseline status of the biomarker could act as an indicator of drug response. Though we lacked sufficient power to detect statistically significant differences, we found evidence suggesting that baseline PGE2 and Spd:Spm levels may modify responsiveness to DFMO/sulindac for CRA prevention. Our findings suggest that subjects with low baseline Spd:Spm were more responsive to the chemopreventive effects of DFMO/sulindac than individuals with high baseline Spd:Spm. Interestingly, the most responsive subgroup to treatment were subjects who entered the study with both high PGE2 and low Spd:Spm in their rectal mucosal biopsy. This subgroup experienced zero recurrences, compared with 39% observed in the corresponding placebo subgroup. Strikingly, the subgroup with the exact opposite characteristics, low PGE2 and high Spd:Spm, showed no significant treatment benefit. These findings suggest that high Spd:Spm levels in tissues, which may differ among individuals as a consequence of genetic variability (e.g., ODC polymorphisms) and/or exposures to dietary or gut flora polyamines, render subjects less pharmacologically responsive to DFMO/sulindac for CRA prevention. Further, the magnitude of risk reduction for CRA appears to be reduced in individuals who do not express a COX dependent, pro-PGE2 background in the target tissue. Importantly, among individuals with low baseline Spd:Spm levels, we observed significant risk reduction with DFMO/sulindac even in the absence of high baseline PGE2, though this subgroup experienced a few “breakthrough” CRAs. Possible COX2 negativity of these few CRAs should be considered. While highly speculative, we believe that these results show an important role for polyamine metabolism in CRA risk and suggest that the combination of DFMO/sulindac is most effective when the NSAID target (COX2) is present and the polyamine levels are in a treatable range (low). In a separate analysis, we found significant metachronous CRA risk reduction ascribed to DFMO/sulindac in patients consuming low-to-moderate levels of dietary polyamines, but there was no benefit among individuals in the highest quartile of polyamine intake (Raj KP et al., unpublished results; Abstract #279; Gastrointestinal Cancers Symposium; Orlando, Florida; Jan.22 24, 2010). Taken together, these data also suggest that the ability to overcome high polyamine background levels (e.g., dietary modification to reduce polyamine intakes or other drug targets in the pathway) may lead to even greater efficacy of drug combinations directed at the polyamine/COX2 or polyamine/”other” pathways. Although these results derive from subgroup analyses, they strongly suggest a role for baseline PGE2 and polyamine status as potential drug-response predictors and warrant further investigation.
There are a number of limitations in the current study. A strong overall effect of DFMO/sulindac treatment on the development of CRA and resulting small sample size from the early termination of the trial yielded inadequate statistical power. Thus, we were limited in our ability to fully study the relationships between biomarker levels and CRA development as originally planned. Additional limitations are inherent to the measurement of rectal mucosal levels of PGE2 and polyamines, which are prone to measurement error, and, as intermediate markers, they may not accurately reflect biological effects at the CRA level. This latter issue may be particularly true for normal rectal colonocytes, which, unlike colonocytes taken from neoplastic tissues, express low levels of the DFMO and sulindac drug targets, ODC16
, respectively. Further, greater dependence of normal rectal mucosa on exogenously derived luminal polyamines18
may explain the significant increase in putrescine between 12 and 36 months and our inability to associate biomarker change in the surrogate tissue with response to intervention.
In conclusion, future studies should consider the measurement of surrogate tissue sources of Spd:Spm and PGE2 as potential baseline predictors of drug responsiveness. Investigation of urinary measures, as suggested by Kawakita et al.19
, may prove more feasible in monitoring response to DFMO/NSAIDs in planned future trials than the collection of rectal mucosal biopsy specimens. Finally, although the number of metachronous CRAs in the DFMO/sulindac treatment group was low, additional study of the factors contributing to these “breakthrough” adenomas is needed.