We report herein the proof-of-principle demonstration that tissue platinum level can be reliably measured using FAAS in resected NSCLC specimens from patients who received neoadjuvant platinum-based chemotherapy. Furthermore, using this approach, we report the novel findings that tissue platinum concentration is significantly associated with tumor response and survival in NSCLC, supporting reduced drug accumulation as a significant mechanism of platinum resistance in clinical tumor specimens.
Tumor platinum concentrations were similar in patients treated with carboplatin versus cisplatin despite platinum doses being considerably higher with carboplatin. This is in keeping with cellular uptake being slower for carboplatin than for cisplatin.18
The weak correlation between time from last treatment and platinum concentration is in keeping with the long half-life previously noted for platinum in human tissues,13
including dorsal root ganglion,19
and kidney cortex,21
and in earlier studies of human tumor platinum concentrations.22,23
The weak correlation between the number of cycles of platinum therapy and tumor platinum concentrations is similar to previous observations with respect to human autopsy kidney cortex platinum concentrations after cisplatin, where tissue concentrations varied with dose given in the first cycle but correlated only weakly with cumulative dose.20,21
This suggests that for kidney and tumor, there may be adaptive response/resistance-inducing transport factors that limit further net platinum accumulation after initial exposure. In this regard, uptake transporters, such as the copper transporters CTR1 and CTR2, and various efflux transporters, such as the multidrug resistance protein (MRP), ABCB1, and ATP7B, are potential contributors.4
Interestingly, more is known about the function of efflux transporters than uptake transporters. MRP expression is associated with decreased cellular drug accumulation of cisplatin.24
In autopsy NSCLC tumor tissues, mRNA expression levels of MRP325
were significantly higher in patients who were exposed to platinum drugs compared with patients who had not received platinum drugs. However, it is not certain whether tumor MRP expression correlates with clinical outcome in NSCLC.4
Conversely, the role of ABCB1 in transport of platinum drugs seems to be less significant, because its expression in NSCLC cell lines did not correlate significantly with sensitivity to cisplatin or intracellular platinum accumulation.9,27
Likewise, in NSCLC tissues, ABCB1 expression by immunohistochemistry did not correlate with response to cisplatin.28–30
The copper transporter ATP7B is also thought to play a role in efflux of platinum drugs. Human tumor cells transfected with ATP7B acquired significant resistance to cisplatin, mainly as a result of increased cisplatin efflux.31
Furthermore, ATP7B mRNA and immunohistochemistry expression significantly correlated with cisplatin resistance in NSCLC xenografts.32
However, in clinical NSCLC specimens, only the copper uptake transporter CTR1, but not ATP7A or ATP7B, predicted clinical outcome after platinum-based chemotherapy.33
In addition, the observation that NSCLC dose-response curves flatten at higher platinum dose-intensities34
would be more in keeping with resistance being related to reduced uptake rather than increased efflux.35
Validating functions of transport factors in NSCLC and incorporating them as biomarkers for platinum sensitivity into future clinical trials would be of significant clinical value. Furthermore, designing a novel platinum complex that is not subject to reduced platinum accumulation in resistant cells could be another potential strategy to overcome platinum resistance.
There are factors other than transporters that may modulate intratumoral platinum concentration. A low extracellular pH favors uptake of weak acids such as aquated cisplatin and enhances cytotoxicity.36
Serum levels of lactate dehydrogenase, which converts pyruvate to lactate in tumors,37
did not correlate with tissue platinum concentration. Contrary to the potential importance of low pH on platinum uptake, we observed a positive correlation between hemoglobin level and tumor platinum concentration, despite the fact that higher hemoglobin level would have been expected to be associated with higher tissue pH. Because pretreatment hemoglobin level was used for correlation, chemotherapy-induced anemia does not explain this observation. It is possible that anemia contributes to tumor hypoxia affecting drug delivery of platinum agents. This would be consistent with a previous report in which prevention of anemia using darbepoetin alfa (an erythropoiesis-stimulating protein) resulted in reduced tumor hypoxia, higher intracellular platinum concentration, and greater tumor response in a murine model of Lewis lung carcinoma.38
ERCC1 may also play a role in modulating intratumoral platinum concentration. Platinum-DNA adducts can be removed by the nucleotide excision repair pathway in which ERCC1 plays an important role.39,40
It is unclear what happens once adducts get removed from DNA by nucleotide excision repair, but they could potentially leave the cell, thereby contributing to reduced intracellular platinum concentration. Against ERCC1 being the major factor driving tumor platinum concentrations is the weak correlation with cumulative drug dose. If an active resistance factor such as ERCC1 (or an efflux pump) were responsible, one might expect little drug accumulation at lower drug doses and then a relatively steep increase in drug concentration with increasing dose as one saturated the capacity of the resistance-inducing factor at higher doses.35
Our sample size was relatively small because only a small proportion of all patients with NSCLC are candidates for neoadjuvant chemotherapy and because we were limited by the availability of sufficient quantities of archived fresh-frozen tumor to permit analysis in this study. Despite these small patient numbers, we achieved adequate statistical power to establish a correlation between tumor platinum concentration and therapeutic efficacy that has proven our hypothesis to be correct. Nevertheless, it is still appropriate to stress that this is a single-institution, retrospective study that, therefore, will require independent validation with a larger number of patients.
Once platinum gets into tumor cells and accumulates to adequate levels, platinum-DNA adducts are generated.1
We attempted to measure DNA adduct levels in our specimens, but the analytic sensitivity proved limiting, and it was impractical to increase the amount of tissue for extracting an adequate amount of DNA for reliable detection of adducts by FAAS. Furthermore, there is ample evidence that the amount of adduct formation is significantly associated with intracellular platinum accumulation.41
However, we believe that total intratumoral platinum concentration is still the most accurate variable that can be used to validate reduced drug accumulation as a significant mechanism of platinum resistance.
Another limitation of our study is the potential influence of second agents (mostly taxanes) that were given concurrently with platinum agents. The factors that confer resistance to one agent may render tumors resistant to several other agents.4
Alternating multiple agents with different mechanisms of action, including cisplatin and paclitaxel, does not improve clinical outcome in NSCLC.42
However, there were a few outlying patients in our study whose tumors demonstrated clinically significant shrinkage despite low detectable levels of intratumoral platinum (A). This shrinkage may have been a result of the concurrent taxane, although this remains uncertain. Additionally, number of cycles was associated with OS. However, the clinical significance of number of cycles cannot be definitely answered using our data.
In conclusion, to our knowledge, this is the first study evaluating impact of tissue platinum concentration on NSCLC response, recurrence, and survival. Our data strongly support reduced drug accumulation as a significant mechanism of platinum resistance. Because transport-related mechanisms of resistance are likely to persist or are acquired after platinum treatment in a variety of cancer tissue types,1,4
we anticipate that our novel approach of measuring platinum concentration in resected specimens after neoadjuvant chemotherapy by FAAS could potentially be used to investigate the relationship between intratumoral platinum concentration and tumor response in the metastatic setting or in other tumor types, such as advanced ovarian carcinoma, in which neoadjuvant platinum-based chemotherapy could be considered before debulking surgery.43
Finally, enhanced understanding of the molecular mechanism of platinum accumulation by tumor cells will be necessary to identify surrogate biomarkers for platinum accumulation that could be developed prospectively for individualizing therapy.