Serum alanine aminotransferase as an early marker of outcomes in patients receiving anti-PD-1 or anti-CTLA-4 antibody

Immune-oncology (IO) drug therapy is effective against various types of cancer. Although several, potential, clinical predictive markers have been identified, none so far have proven reliable. Herein we evaluated changes in serum alanine aminotransferase (ALT), which is upregulated by the accumulation of activated CD8+T cells in the liver, as a potentially reliable predictive marker. We retrospectively analyzed 265 patients with advanced malignancies at three institutions between 2016 and 2019. The patients received IO drug therapy. We defined the ALT ratio (ALR) as the serum ALT value at baseline / the highest serum ALT during IO drug therapy, then determined whether the ALR correlated with the objective response rate or progression-free survival. The median follow-up was 3.1 months. We observed objective responses in 65 patients. The ALR ranged from 0.19 to 32.2 (median 1.5), and a significant ALR increase was observed in responders (p < 0.001). In receiver operating characteristic analysis, ALR = 1.55 had the highest sensitivity and specificity. The patients with ALR < 1.55 had a significantly poorer PFS than those with ALR ≥ 1.55. A high ALR was associated with a tumor response and good PFS in patients with advanced malignancies. The ALR based on activated cytotoxic T lymphocyte dynamics is therefore a reliable predictive marker.

www.nature.com/scientificreports/ leukocyte count [31][32][33] , lactate dehydrogenase 33,34 , C-reactive protein 35 , and adverse events [36][37][38] , but as of yet, no reliable predictive marker has been found 39 . CTLs, which are activated by IO drugs, accumulate in the liver and undergo apoptosis 40,41 . Dong et al. showed a greater accumulation of CTLs in the liver of B7-H1-deficient mice than in wild-type mice 42 . In the present study, we evaluated changes in the serum alanine aminotransferase (ALT) level, which is a marker of liver injury, as a mechanism-based predictive marker for IO drug therapy.

Results
Patient characteristics. In total, 265 patients (134 with NSCLC (92 with adenocarcinoma and 42 with SCC); 38 with GC; 52 with RCC; 35 with UC; three with MM; and three with an unknown primary cancer) were enrolled ( Table 1). The median follow-up period was 3.1 months (0.2-46.3). The average age was 69.3 years , and the male: female ratio was 193 : 72. We observed objective responses in 65 patients (44 with NSCLC (26 with adenocarcinoma and 18 with SCC); four with GC, ten with RCC; five with UC; one with MM; and one with an unknown primary cancer).
ALR according to the response to immunotherapy. The ALR ranged from 0.19 to 32.2 (median 1.5).
We observed a significant increase in the ALR among responders (p < 0.001, Fig. 1). The ALR was able to predict the tumor response, as determined by ROC curve sensitivity (0.85) and specificity (0.70) (Fig. 2). We observed an increase in the ALR (≥ 1.55) in 52 of 65 responders (42 of 55 patients with a PR and in all ten patients with a CR) and 67 of 200 non-responders (27 of 55 patients with SD and 40 of 145 patients with PD) (odds ratio:9.7, p < 0.0001, Fig. 3). In the 67 non-responders with an elevated ALR, 12 experienced exacerbation or liver metastasis, nine experienced adverse events associated with immunotherapy (liver toxicity, etc.), and ten showed a mixed response, in which some tumors shrank, but PR was not achieved or a new metastasis occurred.

Discussion
Reliable predictive markers are needed to distinguish responders from non-responders. Although several candidate markers have already been identified, they have some limitations [29][30][31][32][33][34][35][36][37][38] . Our study showed that ALR may serve as a novel predictive marker based on the dynamics of activated CTLs, and its utility may be improved by further basic research. Crispe et al. revealed that CTLs activated by an antigen were deleted in the liver, suggesting either a preferential accumulation in the liver of activated CTLs undergoing apoptosis (the graveyard hypothesis) or a trap for activated CTLs in the liver for subsequent killing (the killing field hypothesis) 40,41 . In B7-H1 deficient mice, activated PD-1+ CD8 T cells accumulated in the liver 42 . We surmised that the accumulation and deletion of activated CTLs in the liver might impose a burden on the liver, which could induce hepatopathy and elevate serum liver enzymes 43 . After assessing both serum aspartate aminotransferase and ALT, we found that serum ALT was a better predictive marker ( Supplementary Fig. 1).
The timing of the serum ALT increase varies for each IO drug. In most patients receiving pembrolizumab or a combination of nivolumab and ipilimumab, the timing of serum ALT elevation was 1-3 weeks following the initial drug infusion. However, the timing for nivolumab was different, varying from soon after the initial infusion to later in some cases. This difference may depend on the affinity of each IO drug to the PD-1 molecule. An antibody with a strong affinity might affect the immune system sooner than an antibody with a weaker affinity; pembrolizumab has a stronger affinity than nivolumab to PD-1 expressing T cells 44 . Usually, serum ALT rises immediately prior to the finding of tumor shrinkage on a radiologic modality. These findings suggested that ALR might serve as a predictive marker of IO drug therapy.
The present study has the limitations inherent in any retrospective analysis. The patient population varied widely. The ALR was an effective predictive marker, but presented certain problems, such as the occurrence of false negative and false positive cases. We observed no increase in the ALR in 11 (17.0%) responders while we found an increase in 67 (33.5%) non-responders.
There are several possible explanations for the false negatives. First, our timing of ALT analysis was inadequate in that we failed to perform the analysis when the ALT had reached its highest value. Second, because injury to the liver was small, we did not observe elevations in the serum ALT levels. There are two reasons for the low level of injury to the liver. First, the shrunken tumor volume was too small to induce sufficient CTL activation to cause hepatopathy; in effect, there was insufficient injury to the liver to elevate the ALT level. Another reason for the minimal injury to the liver was exceptional liver function. There are also several possible explanations for the false positive results. In twelve patients, a new liver metastasis or the progression of an existing liver metastasis induced a serum ALT increase. In nine patients, other reasons, for example, autoimmune hepatitis, autoimmune cholangitis, septic shock, other drug-induced hepatopathy, etc., accounted for the elevated ALT. In ten patients, We were able to identify other, clinical causes of elevated ALT as well; after excluding 21 cases of elevated ALT due to other causes from the cohort and adding ten cases showing some tumor shrinkage to the responder group, the odds ratio rose from 9.7 to 16.8. In clinical practice, the ALR may serve as a very promising predictor of the  www.nature.com/scientificreports/ response to IO drug therapy when transitioning to the next treatment. These circumstances should be taken into consideration, and ALR should be used carefully. The PFS duration was significantly longer in the high ALR group than in the low ALR group for four types of cancer, suggesting that the ALR can help differentiate between long and short SD among non-responders. The ALR is useful for patients receiving cancer immunotherapy because it facilitates judging whether immunotherapy should be continued or switched to an alternative treatment. Previous studies have reported several, reliable predictive markers for IO drug therapy, including PD-L1 expression and the tumor mutation burden 39 . However, these factors have limitations due to cost-related concerns; these markers require additional analysis, the involvement of specialists or the use of expensive equipment. Some studies revealed that in contrast to these markers, certain blood and clinical markers, such as the leukocyte count, lactate dehydrogenase, and adverse events, can be routinely used 39 . ALR is also one such available blood marker. However, in this respect the ALR, www.nature.com/scientificreports/ based on CTL dynamics, is an effective marker of immunotherapy and has the potential to serve as a simple, reliable prognostic marker in risk stratification and provide better treatment allocation in cancer patients. A high ALR was associated with the tumor response and PFS in patients with various types of cancer. The accumulation and deletion of activated CTLs in the liver might impose a burden on the liver, which induce the elevation of ALR. Thus, an increase in ALT levels during IO drug therapy is reliable because the ALR is associated with CTL dynamics.
Further studies aimed at refining the use of the ALR as a predictive marker based on the mechanism of IO drug therapy are warranted.

Materials and methods
Patients. Between 2016 and 2019, we reviewed the medical records of 265 patients with advanced malignancies at Tokyo Metropolitan Tama Medical Center, Kanto Rosai Hospital, and Tomishiro-Chuo Hospital who received single-agent nivolumab, pembrolizumab or a combination nivolumab and ipilimumab treatment.
The present study was approved by the ethical review board of Tama Medical Center, Kanto Rosai Hospital, and Tomishiro Central Hospital and was conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice Guidelines. Comprehensive iInformed consent was obtained from all the participants.
Treatment and response assessment. Patients received nivolumab 3 mg/kg every 2 weeks, pembrolizumab 200 mg every 3 weeks or nivolumab 240 mg every 3 weeks plus ipilimumab 3 mg per kilogram every 2 weeks in four doses, followed by nivolumab 240 mg every 2 weeks.
All the patients underwent blood testing from before the commencement of IO drug therapy to the end of follow-up at intervals of 1-2 weeks. We defined ALR as the serum ALT value at baseline / the highest serum ALT during IO drug therapy.
Computed tomography or magnetic resonance imaging was performed at baseline and repeated at 8-to 12-week intervals. The clinical tumor response during treatment was assessed using the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 based on the patients' medical records. We defined a clinical benefit as a complete response (CR), partial response (PR) or stable disease (SD). We defined progression-free survival (PFS) as the time from the commencement of immunotherapy to the date of progression.
Statistics. Statistical analyses were performed using the JMP® software package, with p < 0.05 indicating statistical significance. We compared continuous variables using the two-tailed unpaired Student's t-test. To predict the response to, and prognosis of, IO drug therapy, we determined the optimal cutoff value of the ALR based on the receiver operating characteristic (ROC) curve and the area under the ROC curve (AUC). We constructed the distribution of the recurrence-free survival (RFS) rate using the Kaplan-Meier method. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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