Serum Alanine Aminotransferase As An Early Marker of Outcomes in Patients Receiving Immuno-Oncology Drugs

Immune-oncology (IO) drug therapy is effective against various types of cancer. Although several, potential, clinical, predictive markers have been identied, 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 dened 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 signicant ALR increase was observed in responders (p < 0.001). In receiver operating characteristic analysis, ALR = 1.55 had the highest sensitivity and specicity. The patients with ALR < 1.55 had a signicantly 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 is a reliable predictive marker based on activated cytotoxic T lymphocyte dynamics.

Despite their e cacy, IO drugs bene t only a small number of patients. Therefore, reliable predictive markers allowing the identi cation of responders and non-responders is crucial to choosing the optimal treatment. Several studies have discussed predictive markers, such as PD-L1 expression 29 , overall mutational burden 30 , leukocyte count [31][32][33] , lactate dehydrogenase 33,34 , C-reactive protein 35 and adverse events 36-38 , but as yet, no reliable predictive marker has been found.
CTLs, which are activated by IO drugs, accumulate in the liver and undergo apoptosis 39,40 . Dong et al.
showed a greater accumulation of CTLs in the liver of B7-H1-de cient mice than in wild-type mice 41 . 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.

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) Figure   3). In 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.

Timing of the serum ALT increase in responders
We analyzed the timing of the serum ALT increase in 52 responders ( Figure 4A). The serum ALT increased in 28 (54%) and 42 (81%) patients within 30 and 60 days, respectively, following the initial drug infusion.
In all the responders, a serum ALT increase occurred within three months. Next, we evaluated the differences by drug. In patients receiving nivolumab, one (4%), 11 (40%), and 21 (75%) of 28 patients showed a serum ALT increase within 10, 30, and 60 days, respectively, following the initial drug infusion.
Progression-free survival estimated using the ALR Among patients with NSCLC, GC, RCC, and UC, an ALR<1.55 was associated with signi cantly poorer PFS than ALR≥1.55. The median PFS among patients with NSCLC with ALR<1.55 and ALR≥1.55 was 2.6 and 12 months, respectively (p<0.001, Figure 5A). Subgroup analysis performed for tumor histology demonstrated that elevated ALR remained a signi cant prognostic factor. The median PFS in patients with adenocarcinoma with an ALR<1.55 and ALR≥1.55 was 2.9 and 11.2 months, respectively (p=0.002, Figure 5B). The median PFS in patients with squamous cell carcinoma with an ALR<1.55 and ALR≥1.55 was 1.5 and 20.3 months, respectively (p<0.001, Figure 5C). Elevated ALR was a signi cant prognostic factor in patients with gastric cancer, RCC, and UC. The median PFS in patients with gastric cancer with an ALR<1.55 and ALR≥1.55 was 1.8 and 2.2 months, respectively (p=0.015, Figure 5D). The median PFS in patients with RCC with an ALR<1.55 and ALR≥1.55 was 4.1 months and not achieved, respectively (p=0.006, Figure 5E). The median PFS in patients with UC with an ALR<1.55 and ALR≥1.55 was 1.4 and 6.5 months, respectively (p<0.001, Figure 5F).

Discussion
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 eld hypothesis) 39,40 . In B7-H1 de cient mice, activated PD-1 + CD8 T cells accumulated in the liver 41 . 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 42 . After assessing both serum aspartate aminotransferase and ALT, we found that serum ALT was a better predictive marker (data not shown).
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 one to three 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 a nity of each IO drug to the PD-1 molecule. An antibody with a strong a nity might affect the immune system sooner than an antibody with a weaker a nity; pembrolizumab has stronger a nity to the immune system than nivolumab. Usually, serum ALT rises immediately prior to the nding of tumor shrinkage on a radiologic modality. These ndings 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 not a perfect predictive marker and presented certain some 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 a number of 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 su cient CTL activation to cause hepatopathy; in effect, there was insu cient injury to the liver to elevate the ALT level. Another reason for the minimal injury to the liver was very good liver function.
There are also a number of 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, despite the achievement of SD or PD according to the RECIST criteria, a portion of the tumors showed shrinkage. 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 response to IO drug therapy when transitioning to the next treatment.
The PFS duration was signi cantly 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 nonresponders. 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 tumor mutation burden 43 . 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. In contrast to those markers, some studies revealed that certain blood and clinical markers can be routinely used, such as the leukocyte count, lactate dehydrogenase, and adverse events 43 . ALR is also one such available blood markers. However, in this respect the ALR, based on CTL dynamics, is a superior marker of immunotherapy and has the potential to serve as a simple, reliable prognostic marker in risk strati cation 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. An increase in ALT levels during IO drug therapy is reliable because it is based on activated CTL dynamics, making the ALR an useful predictive marker. Further studies aimed at re ning the use of the ALR as a predictive marker based on the mechanism of IO drugs therapy are warranted. Treatment and response assessment Patients received nivolumab 3 mg/kg every two weeks, pembrolizumab 200 mg every three weeks or nivolumab 240 mg every three weeks plus ipilimumab 3 mg per kilogram every three weeks in four doses, followed by nivolumab 240 mg every two weeks.

Patients
All the patients underwent a blood test before starting IO drug therapy until the end of follow-up, which was performed at intervals of one to two weeks. We de ned 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 eight to 12 weeks 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 de ned a clinical bene t as a complete response (CR), partial response (PR) or stable disease (SD). We de ned progression-free survival (PFS) as the time from immunotherapy initiation to the date of progression.

Statistics
We performed statistical analyses using the JMP® software package, with p <0.05 indicating statistical signi cance. 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.

Declarations
Author Contributions TA designed the present study and critically revised the manuscript. TA, TT, YM, SN, TO, K.I. and M.T. performed data collection. TA analyzed the data. TA wrote the manuscript. TA, TT, YM, SN, TO, K.I. and M.T. revised the manuscript. All the authors read and approved the nal manuscript.

Con icts of Interest
All the authors state that they have no con icts of interest. Figure 1 ALR according to the response to immunotherapy (SD/PD, stable/progressive disease; PR/CR, partial/complete response) Figure 2 Receiver operating curve (ROC) analysis based on the ALR for the tumor response. In this model, the sensitivity was 85.0%, the speci city was 70.0%, and the AUC was 0.790. p<0.001. AUC, area under the curve.  Timing of the serum ALT increase in responders.