Early Changes in Alpha-Fetoprotein and Des-γ-Carboxy Prothrombin Are Useful Predictors of Antitumor Response to Durvalumab Plus Tremelimumab Therapy for Advanced Hepatocellular Carcinoma

The relationship between antitumor response and tumor marker changes was evaluated in patients with advanced hepatocellular carcinoma treated with durvalumab plus tremelimumab (Dur/Tre). Forty patients were enrolled in this retrospective evaluation of treatment outcomes. According to the Response Evaluation Criteria for Solid Tumors version 1.1 at 8 weeks, the objective response (OR) rate was 25% and the disease control (DC) rate was 57.5%. The median alpha-fetoprotein (AFP) ratio at 4 weeks was 0.39 in patients who achieved OR at 8 weeks (8W-OR group), significantly lower than the 1.08 in the non-8W-OR group (p = 0.0068); however, it was 1.22 in patients who did not achieve DC at 8 weeks (non-8W-DC group), significantly higher than the 0.53 in the 8W-DC group (p = 0.0006). Similarly, the median des-γ-carboxy-prothrombin (DCP) ratio at 4 weeks was 0.15 in the 8W-OR group, significantly lower than the 1.46 in the non-8W-OR group (p < 0.0001); however, it was 1.23 in the non-8W-DC group, significantly higher than the 0.49 in the 8W-DC group (p = 0.0215). Early changes in tumor markers after Dur/Tre initiation were associated with antitumor response. In particular, changes in AFP and DCP at 4 weeks may offer useful biomarkers for early prediction of both response and progressive disease following Dur/Tre.


Introduction
In the Phase III HIMALAYA trial [1], the combination of durvalumab (an anti-programmed cell death ligand-1 antibody) and tremelimumab (an anti-cytotoxic T-lymphocyteassociated protein 4 [CTLA-4] antibody) demonstrated an overall survival (OS) benefit over sorafenib in patients with advanced hepatocellular carcinoma (HCC).Based on these positive results, the combination of durvalumab and tremelimumab (Dur/Tre) is now recommended as a first-line systemic therapy for advanced HCC, as is atezolizumab plus bevacizumab (Atz/Bev) [2,3].However, no reports have detailed Dur/Tre's outcomes in actual clinical practice, and whether Dur/Tre will have the same efficacy and safety profile as in clinical trials has remained unclear.
Measuring concentrations of tumor markers for HCC, namely alpha-fetoprotein (AFP) and des-gamma-carboxyprotein (DCP), is less invasive and more common than tumor biopsy or imaging, and is widely used not only for diagnosing HCC, but also as an adjunctive diagnosis to determine treatment efficacy for HCC [4,5].Several studies have reported associations between changes in AFP and DCP and treatment response and prognosis following treatment with molecularly targeted agents (MTAs) such as sorafenib [6][7][8] and lenvatinib [9][10][11] and in combined immunotherapy with Atz/Bev [12][13][14][15].During the initial phase of using Dur/Tre at our institution, several cases were encountered in which patients with significant decreases in AFP and DCP early after the initiation of Dur/Tre showed good antitumor responses, whereas patients showing rapid increases in these markers showed poor antitumor responses.In the HIMALAYA study, changes in tumor markers during Dur/Tre were not reported in detail, and the relationship between changes in these tumor markers and the therapeutic effects of Dur/Tre have remained unclear.
In this study, we investigated changes in AFP, DCP, and the lens culinaris agglutininreactive fraction of alpha-fetoprotein (AFP-L3) in the early period after initiating Dur/Tre.We also evaluated the correlation between changes in these tumor markers and antitumor response to Dur/Tre.Finally, we also analyzed factors contributing to progression-free survival (PFS).

Patients
Between March 2023 and May 2024, 40 consecutive patients with advanced unresectable HCC started Dur/Tre therapy at our institution.Eligibility criteria included the following: (1) HCC diagnosis by imaging or biopsy, (2) Barcelona Clinic Liver Cancer (BCLC) stage C or B ineligible for surgery or locoregional therapy, (3) Eastern Cooperative Oncology Group performance status (ECOG-PS) of 0 or 1, and (4) Child-Pugh score of 7 or less.In addition, three patients were included: one with multinodular fused BCLC A and a maximum tumor diameter of 100 mm and two with Child-Pugh scores of 8 and 9 due to large tumor volume despite chronic hepatitis.All 40 patients were included in this study for retrospective evaluation of treatment outcomes.

Dur/Tre Treatment, Evaluation of Adverse Events, and Changes in Liver Function
All patients received durvalumab 1500 mg and tremelimumab 300 mg intravenously on the first administration.Thereafter, durvalumab 1500 mg was administered every 4 weeks.The treatment line refers to the sequence of systemic therapy with MTA or immune checkpoint inhibitors (ICIs).Adverse events (AEs) were evaluated according to the Common Terminology Criteria for Adverse Events (CT-CAE), 5th edition [16].In the event of a drug-related AE, durvalumab was temporarily interrupted until symptoms improved to grade 1 or 2, according to guidelines provided by the manufacturer.Dur/Tre treatment was continued until a potentially fatal AE occurred or the tumor progressed clinically.To assess changes in liver function, albumin-bilirubin (ALBI) scores [17] were examined at baseline and at weeks 1, 2, 4, and 8 after Dur/Tre initiation.

Determination of Antitumor Response
Evaluation of antitumor response to Dur/Tre was performed according to the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1) [18] and modified RECIST (mRECIST) [19].Dynamic computed tomography (CT) was performed at baseline, 8 weeks after initiation of Dur/Tre, and every 4-12 weeks thereafter according to a predefined schedule.

Evaluation of Changes in AFP, DCP, and AFP-L3
Serum AFP and DCP were measured as tumor markers for HCC at baseline and after 2, 4, and 8 weeks of Dur/Tre.AFP-L3 was measured at baseline and after 4 and 8 weeks of Dur/Tre.For each patient, the baseline concentration of each tumor marker was set to 1, and the ratio of each tumor marker to baseline was calculated at weeks 2, 4, and 8 of treatment.Analysis of tumor marker changes was performed in patients with AFP ≥ 10 ng/mL, DCP ≥ 40 mAU/mL, and AFP-L3 ≥ 0.5%, excluding those in the normal range of tumor markers at baseline.

Statistical Analysis
Statistical analysis was performed using Easy R (EZR) version 1.29 (Saitama Medical Center, Jichi Medical University) [20].Statistical analysis was performed using Fisher's direct probability test, the Mann-Whitney U test, Wilcoxon rank-sum test, Friedman test, Bonferroni method, etc., as appropriate.Cutoff values were determined using the receiver operating characteristic (ROC) curve and area under the curve (AUC).PFS was measured as the time from the start of Dur/Tre treatment to date of progression by RECIST 1.1 or date of death or last hospital visit.Overall survival (OS) was measured as the time from the start of Dur/Tre treatment to death or last hospital visit.PFS and OS were evaluated using the Kaplan-Meier method, and differences in survival were evaluated by the log-rank test.The Cox proportional hazards model was used to analyze factors contributing to PFS.For all analyses, the significance level was set at less than 0.05.
ROC curves were plotted using 8W-OR and 8W-DC as the state variables and AFP ratios at weeks 2 and 4 as the test variables.For 8W-OR, the AUCs at weeks 2 and 4 were 0.64 and 0.84, with an optimal week 4 cutoff of 0.53 (75% sensitivity, 89.5% specificity).For 8W-DC, the AUCs at weeks 2 and 4 were 0.78 and 0.89, with an optimal week 4 cutoff of 1.01 (86.7% sensitivity, 91.7% specificity).
ROC curves were plotted using 8W-OR and 8W-DC as the state variables and AFP ratios at weeks 2 and 4 as the test variables.For 8W-OR, the AUCs at weeks 2 and 4 were 0.64 and 0.84, with an optimal week 4 cutoff of 0.53 (75% sensitivity, 89.5% specificity).For 8W-DC, the AUCs at weeks 2 and 4 were 0.78 and 0.89, with an optimal week 4 cutoff of 1.01 (86.7% sensitivity, 91.7% specificity).Actual AFP levels at 0, 2, 4, and 8 weeks after Dur/Tre initiation, stratified according to 8W-RECIST 1.1, are shown in Supplementary Materials Table S2.Median actual AFP levels in the 8W-DC group at weeks 4 and 8 were significantly lower than those in the Non-8W-DC group (p = 0.0128 and p = 0.0063, respectively).4. Median DCP ratios at 2, 4, and 8 weeks were 0.46, 0.15, and 0.06 in patients with CR+PR (n = 10); 1.21, 1.52, and 1.26 in patients with SD (n = 11); and 1.13, 1.23, and 1.83 in patients with PD+NE (n = 16), respectively (Figure 4a).Median DCP ratios in the 8W-OR group at weeks 2, 4 and 8 were significantly lower than those in the Non-8W-OR group (p = 0.0049, p < 0.0001, and p < 0.0001, respectively) (Figure 4b).Median DCP ratios in the 8W-DC group at weeks 4 and 8 were significantly lower than those in the Non-8W-DC group p = 0.0215 and p = 0.0032, respectively) (Figure 4c).ROC curves were plotted using 8W-OR and 8W-DC as the state variables and DCP ratios at weeks 2 and 4 as the test variables.For 8W-OR, the AUCs at weeks 2 and 4 were 0.84 and 0.98, with an optimal week 4 cutoff of 0.48 (100% sensitivity, 92.6% specificity).For 8W-DC, the AUCs at weeks 2 and 4 were 0.63 and 0.72, with an optimal week 4 cutoff of 0.73 (61.9% sensitivity, 81.2% specificity).
Actual DCP levels at 0, 2, 4, and 8 weeks after Dur/Tre initiation, stratified according to 8W-RECIST 1.1, are shown in Supplementary Materials Table S3.Median actual DCP levels in the 8W-OR group at weeks 4 and 8 were significantly lower than those in the Non-8W-OR group (p = 0.0167 and p = 0.0001, respectively).Median actual DCP level in the 8W-DC group at week 8 was significantly lower than that in the Non-8W-DC group (p = 0.0048).5. Median AFP-L3 ratios at 4 and 8 weeks were 0.93 and 1.00 in 9 patients with CR+PR; 1.01 and 1.07 in 11 patients with SD; and 1.01 and 1.06 in 13 patients with PD+NE, respectively (Figure 5a).No significant differences in median AFP-L3 ratio were evident between the 8W-OR and Non-8W-OR groups and between the 8W-DC and Non-8W-DC groups (Figure 5b,c).ROC curves were plotted using 8W-OR and 8W-DC as state variables and the AFP-L3 ratio at week 4 as the test variable.The AUC was 0.60 for 8W-OR and 0.51 for 8W-DC, both indicating low correlation.

AFP-L3
Actual AFP-L3 levels at 0, 2, 4, and 8 weeks after Dur/Tre initiation, stratified according to 8W-RECIST 1.1, are shown in Supplementary Materials Table S4.The median actual AFP-L3 level in the 8W-DC group at week 8 was significantly lower than that in the Non-8W-DC group (p = 0.0390).

Safety and Changes in Liver Function
Table 7 shows the incidence of AEs occurring within 8 weeks of Dur/Tre initiation.The most common AEs in all patients were fever, diarrhea, anorexia, general fatigue, and pruritus.Twenty-five percent of patients (n = 10) experienced grade 3 or higher AEs, with diarrhea being the most common, in five patients (12.5%).Six patients (15.0%) required steroids as treatment for immune-related AEs.This included three patients with grade 3 enteritis, two patients with grade 2 skin rashes, and one patient with grade 3 elevated aspartate aminotransferase.The median duration of treatment with Dur/Tre was 6.2 months (95% CI: 1.9-8.4).Changes in ALBI scores within 8 weeks were assessed in 40 patients (Figure 7).The ALBI scores (median ± standard error (SE)) at baseline, weeks 1, 2, 4, and 8 were −2.26 ± 0.07, −2.19 ± 0.08, −2.25 ± 0.09, −2.12 ± 0.09, and −2.25 ± 0.08, respectively.There was no worsening of ALBI scores within 8 weeks.

Post-Progression Therapy
In 39 patients, excluding one patient who was transferred to another hospital within 8 weeks and whose post-transfer status was unknown, post-progression therapy (post-Figure 8. Flowchart on post-PD therapy.For patients with both Child-Pugh classification A and ECOG-PS 0 or 1 at time of PD confirmation, all 18 (100%) were able to progress to post-PD therapy, whereas only 3 of the remaining 7 patients (42.9%) were able to progress to post-PD therapy.PD, progressive disease; ECOG, Eastern Cooperative Oncology Group; PS, performance status; BSC, best supportive care.

Discussion
This is the first study to focus on the correlation between early changes in tumor marker levels and antitumor response after Dur/Tre initiation in patients with advanced HCC in clinical practice.In particular, we found that early changes in AFP and DCP at 4 weeks after Dur/Tre initiation were both significantly associated with OR and DC according to RECIST 1.1 at 8 weeks.Factors at the start of Dur/Tre that were associated with good PFS were 1st-line Dur/Tre treatment and NLR ≤ 3.00.

Discussion
This is the first study to focus on the correlation between early changes in tumor marker levels and antitumor response after Dur/Tre initiation in patients with advanced HCC in clinical practice.In particular, we found that early changes in AFP and DCP at 4 weeks after Dur/Tre initiation were both significantly associated with OR and DC according to RECIST 1.1 at 8 weeks.Factors at the start of Dur/Tre that were associated with good PFS were 1st-line Dur/Tre treatment and NLR ≤ 3.00.
Regarding the antitumor response of Dur/Tre according to RECIST 1.1, the HI-MALAYA study reported an ORR of 20.1% and a DCR of 60.1% [1].In the present study, antitumor response according to RECIST 1.1 at 8 weeks was similar, with an 8W-ORR of 25.0% and an 8W-DCR of 57.5%.An updated analysis of the HIMALAYA trial reported a 3-year survival rate of 44.6% and a 4-year survival rate of 36.2% for patients who achieved DC with the best response (60.1%) [21].Dur/Tre therapy is called the STRIDE regimen, and the anti-CTLA-4 antibody tremelimumab is administered only once at the first time.In the course of systemic therapy for HCC, an initial single dose of a CTLA-4 inhibitor can provide long-term prognosis for some patients.Although the follow-up period in the current study was too short to draw conclusions, no deaths were seen among patients who achieved DC (CR+PR+SD group) with Dur/Tre treatment, and if DC can be achieved as in this clinical trial, good prognosis can be expected.
Several reports have examined the relationship between changes in AFP levels and antitumor response and prognosis in patients with advanced HCC treated with MTA and Atz/Bev [6][7][8][9][10][11][12][13][14][15].Patients with decreased AFP levels during treatment reportedly achieved better antitumor response and prognosis, whereas patients with increased AFP levels showed worse antitumor response and prognosis.In the current study, the median AFP ratio was significantly lower in the 8W-OR group than in the non-8W-OR group at 4 and 8 weeks after starting Dur/Tre treatment.On the other hand, the median AFP ratio of the non-8W-DC group was significantly higher than that of the 8W-DC group at weeks 2, 4, and 8.The optimal cutoff values for the AFP ratio at week 4 predicting 8W-OR and 8W-DC were 0.53 and 1.01, respectively.These results suggest that in Dur/Tre therapy, as in other previously reported systemic therapy regimens, changes in AFP in the early treatment period may provide a useful predictor of antitumor response for both responsive cases and PD cases.
Several studies have also evaluated the relationship between changes in DCP after treatment initiation and antitumor efficacy and prognosis.Reports on MTA treatment have shown that, unlike AFP, early changes in DCP are not a useful predictor of antitumor efficacy or prognosis [6,22].This is because some responders and SD patients who are considered to have achieved an antitumor effect also have elevated DCP, indistinguishable from the elevated DCP in PD.Tumor hypoxia due to anti-angiogenic therapy may lead to increased DCP production by the tumor itself [23].In our own study, even with Atz/Bev treatment, early changes in DCP were not associated with antitumor efficacy [12].In the present study, median DCP ratios among 8W-OR patients at weeks 2, 4, and 8 were significantly lower than those of non-8W-OR patients.DCP ratios at weeks 4 and 8 in the non-8W-DC group were also significantly higher than those in the 8W-DC group, suggesting that Dur/Tre treatment, unlike Atz/Bev treatment, does not involve VEGF inhibition and no mechanism may be needed for producing DCP from within the tumor due to hypoxia from inhibited angiogenesis.The optimal cutoff values for the DCP ratio at week 4 predicting 8W-OR and 8W-DC were 0.48 and 0.73, respectively.Therefore, in Dur/Tre treatment, changes in DCP and AFP changes may be associated with antitumor effects (response and PD cases).
Few reports have examined the relationship between changes in AFP-L3 and the efficacy of systemic therapy [13].In the present study, AFP-L3 was unchanged in most patients after 4 weeks of treatment.After 8 weeks, a trend was seen toward a decrease in the 8W-OR group compared to the non-8W-OR group.With the advent of ICI therapy, including Atz/Bev, a small number of patients have been able to achieve clinical CR.In these patients, AFP-L3 eventually declines to normal levels.If a subsequent decrease in AFP-L3 is obtained, a favorable antitumor response can therefore be expected.However, based on the results of this study, AFP-L3, unlike AFP and DCP, may not offer a useful early predictor of antitumor response.
Combining multiple tumor markers or integrating markers with age and tumor factors has been reported as a useful indicator for predicting antitumor efficacy and prognosis [24][25][26][27].Therefore, in Dur/Tre treatment, the use of a combination of these factors may be a more effective biomarker for predicting treatment outcome.
In this study, significant predictive factors at Dur/Tre initiation associated with good PFS were first-line Dur/Tre treatment and NLR ≤ 3.00.Although the number of cases in this study was small and the ability to draw conclusions is limited, the results suggest that, at least when Dur/Tre is introduced as a first-line treatment, we could expect similar results to those achieved in the HIMALAYA study.As for NLR, low NLR levels have been reported as predictive of good antitumor efficacy and favorable OS in ICI therapies, including Atz/Bev therapy for HCC [28][29][30][31][32][33].Dur/Tre therapy is a combination of only two immune checkpoint inhibitors and, as with ICI therapy for other cancers, low NLR

Figure 8 .
Figure 8. Flowchart on post-PD therapy.For patients with both Child-Pugh classification A and ECOG-PS 0 or 1 at time of PD confirmation, all 18 (100%) were able to progress to post-PD therapy, whereas only 3 of the remaining 7 patients (42.9%) were able to progress to post-PD therapy.PD, progressive disease; ECOG, Eastern Cooperative Oncology Group; PS, performance status; BSC, best supportive care.

Table 1 .
Baseline characteristics at initiation of durvalumab plus tremelimumab.

Table 6 .
Uni-and multivariate survival analyses of factors at initiation of durvalumab plus tremelimumab associated with good PFS.

Table 6 .
Uni-and multivariate survival analyses of factors at initiation of durvalumab plus tremelimumab associated with good PFS.