Article
Camrelizumab plus apatinib for previously treated advanced adrenocortical carcinoma: a single-arm phase 2 trial
Received: 3 April 2024
Accepted: 16 November 2024
Published online: 29 November 2024 W ☒ Check for updates
Yu-Chun Zhu1,4, Zhi-Gong Wei2,4, Jing-Jing Wang2, Yi-Yan Pei2, Jing Jin2, Dong Li3, Zhi-Hui Li3, Zhe-Ran Liu2, Yu Min2, Rui-Dan Li2, Li Yang2, Ji-Yan Liu 2, Qiang Wei1 & Xing-Chen Peng 2
Adrenocortical carcinoma (ACC) is a rare, aggressive malignancy with a poor prognosis. Therapeutic options for patients with advanced ACC who have failed standard treatments are limited. Single-agent immunotherapy as a second-line treatment has shown unsatisfactory clinical outcomes. This phase II trial (NCT04318730) evaluated the efficacy and safety of the PD-1 inhibitor camrelizumab combined with the VEGFR inhibitor apatinib in previously treated advanced ACC. The primary endpoint was objective response rate (ORR). The secondary endpoints included progression-free survival (PFS), overall survival (OS), and safety. A total of 21 patients with advanced ACC received at least one dose of camrelizumab and apatinib. The ORR was 52% (95% CI, 30-74%), meeting the primary endpoint, and the disease control rate (DCR) was 95% (95% CI, 76-100%). The median PFS was 13.3 months (95% CI, 8.4-NE), and the median OS was 20.9 months (95% CI, 11.0-NE). The most common grade 3-4 treatment-related adverse events were alanine amino- transferase elevation, aspartate aminotransferase elevation, and lymphopenia. Predefined exploratory analyses indicated that patients with higher peripheral blood CXCR3 + CD8 + T cell abundance, lower immunosuppressive CD4 + T cell abundance, and higher overlap of clonotypes between tumor-infiltrating T cells and circulating T cells, were more likely to respond favorably to the combined therapy.
Adrenocortical carcinoma (ACC) is a rare, aggressive malignancy with a poor prognosis and limited treatment options, resulting in a 5-year survival of only 13% for stage IV disease1,2. Mitotane, a synthetic deri- vative of the pesticide dichlorodiphenyltrichloroethane, is the only approved drug for ACC. It exhibits adrenolytic activity but has limited efficacy, and is associated with serious toxicity3,4. Cytotoxic che- motherapy remains a cornerstone in the treatment of advanced ACC. The combination of etoposide, doxorubicin, and cisplatin (EDP) with mitotane is currently the standard first-line therapy based on the
results of the FIRM-ACT study. However, the response rate is only 23% and median duration of progression-free survival (PFS) is only 5.0 months5. For patients with advanced ACC whose disease progresses after Mitotane and/or chemotherapy, therapeutic options are severely limited.
Immune checkpoint inhibitors (ICIs), such as monoclonal anti- bodies targeting programmed cell death-1 (PD-1) or its ligand (PD-L1), have shown promise in the treatment of ACC6-9. However, the efficacy of anti-PD-1/PD-L1 monotherapy for advanced ACC remains
1Department of Urology, West China Hospital, Sichuan University, Chengdu, China. 2Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China. 3Department of Oncology, The General Hospital of Western Theater Command, Chengdu, China. 4These authors contributed equally: Yu-Chun Zhu, Zhi-Gong Wei. e-mail: pxx2014@163.com
22 participants assessed for eligibility
1 did not meet inclusion criteria
21 participants enrolled
21 allocated to and received intervention
13 discontinued intervention
7 had disease progression per RECIST 4 died with clinical progression
1 withdrew consent
1 had Grade 4 hepatotoxicity
1 finished intervention
7 still on treatment at data cutoff
unsatisfactory, with response rates ranging from 10% to 23%7,10,11. The potential for immunoresistance in ACC patients, due to both tumor- secreted endogenous glucocorticoids and glucocorticoid supple- mentation in those treated with mitotane, may reduce the effective- ness of single-agent immunotherapy12. Therefore, combining immune checkpoint inhibitors (ICIs) with other drugs might be more effective than immunotherapy alone.
Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) are highly expressed in ACC13. A phase II study demonstrated that anti-angiogenic therapy targeting VEGF/VEGFR has modest anti- tumor activity in patients with ACC14. Anti-angiogenesis therapy has been found to reprogram the tumor immune microenvironment in preclinical studies15. Recent clinical studies have also confirmed the synergistic anti-tumor effects of combining ICIs with anti-angiogenesis therapy in various advanced tumors16-20. Additionally, a retrospective case series reported that pembrolizumab, a PD-1 inhibitor, combined with lenvatinib, a multikinase inhibitor targeting VEGFR, achieved promising objective responses with manageable toxicity in ACC patients who had previously progressed through several lines of therapy21.
Here, we show that the combination of the PD-1 inhibitor cam- relizumab and the VEGFR2-targeted small molecular tyrosine kinase inhibitor (TKI) apatinib has promising activity and an acceptable safety profile in patients with previously treated advanced ACC. Moreover, we have explored relevant molecular biomarkers that may be associated with the clinical efficacy of the camrelizumab- apatinib combination therapy.
Results Patients
Between November 2020 and April 2023, a total of 22 patients were assessed for eligibility. Of these, 21 patients received at least one dose of camrelizumab plus apatinib and were included in the modified intention-to-treat (mITT) population (Fig. 1). Among the 21 patients, eight (38%) had hormonally functioning tumors, with six producing cortisol and two producing both cortisol and androgens. All patients had previously received at least one type of anticancer therapy, with
90% (19/21) having received mitotane and 57% (12/21) having received chemotherapy (Table 1).
As of the data cutoff (December 1, 2023), seven patients were still undergoing treatment. One patient completed the two-year study intervention and achieved a durable response lasting 33 months. Thirteen patients discontinued treatment, seven due to radiographic disease progression, four due to death related to clinical progression, one due to withdrawal of consent after receiving 12 cycles of study drugs, and one due to grade 4 hepatotoxicity, leading to permanent discontinuation of all study drugs.
Anti-tumor activity
Of the 21 patients who received the treatment, 11 (52%; 95% confidence interval [CI], 30-74%) achieved objective responses, meeting the pri- mary endpoint. All these responses were partial responses (PRs), with no complete responses (CRs) observed. Nine additional patients (43%) exhibited stable disease (SD), and one patient (5%) had progressive disease (PD) as the best response at the end of follow-up (Fig. 2 and Supplementary Fig. 1). The disease control rate (DCR), defined as the proportion of patients achieving CR, PR, or SD, was 95% (95% CI, 76-100%). For the 11 patients who achieved PR, the median time to response was 2.8 months (range, 0.8-12.6), and median duration of response was not reached (Table 2).
For patients with non-hormonally functioning tumors, 69.2% (9/ 13) achieved PRs compared to 25% (2/8) for those with hormonally functioning tumors (p=0.08). Responses were observed across var- ious subgroups, irrespective of age, sex, Eastern Cooperative Oncol- ogy Group (ECOG) performance status, liver metastasis, lung metastasis, number of metastatic sites, prior chemotherapy, and prior mitotane treatment (Supplementary Fig. 2).
Survival outcomes were also included as secondary endpoints. After a median follow-up duration of 17.5 months (range, 2.1-37.1 months) for all patients in the mITT set, nine deaths were reported, all of which were cancer-specific. Among these patients, two had achieved a PR, six had SD, and one had PD as the best response. Notably, eight (72.3%) of the 11 patients who achieved PR were still alive without disease progression by the cutoff date (Fig. 3A). The median PFS was 13.3 months (95% CI, 8.4-NE) and the median overall survival (OS) was 20.9 months (95% CI, 11.0-NE). The 1-year PFS rate was 57.1%, and 1-year OS rate was 74.5% (Fig. 3B, C).
Safety
Treatment-related adverse events (TRAEs), as a secondary endpoint, were assessed in 21 patients, with grade 3-4 TRAEs occurring in nine patients (Table 3). The most common grade 3-4 TRAEs were alanine aminotransferase elevation, aspartate aminotransferase elevation, and lymphopenia. No grade 5 TRAEs leading to death were reported. The response rate among patients who experienced grade 3-4 TRAEs was 66.7% (6/9), compared to 41.7% (5/12) in those who did not; however, this difference was not statistically significant (p=0.387). Univariate Cox regression analysis showed no significant difference in the risk of death between patients who experienced grade 3-4 TRAEs and those who did not (HR 0.72, 95% CI 0.19-2.70; p= 0.62). Only one patient permanently discontinued all study drugs after receiving one treatment cycle because of severe hepatotoxicity. Despite this, the patient achieved a PR as the best response, and the disease has remained controlled off therapy for more than eight months without additional anti-tumor treatment. Moreover, nine patients experienced temporary discontinuation of camrelizumab and dose reduction of apatinib due to TRAEs. The administration of camrelizumab was delayed in seven patients due to COVID-19-related factors.
Exploratory analyses
Tumor immune microenvironment. Pretreatment archival tumor tis- sue samples were collected from 14 primary lesions and 2 recurrent/
| Characteristic | Patients (N= 21) | |
|---|---|---|
| Age, years | 48 | (35.5-55) |
| >50 | 9 | (43%) |
| ≤50 | 12 | (57%) |
| Sex | ||
| Female | 13 | (62%) |
| Male | 8 | (38%) |
| ECOG performance status | ||
| 0 | 14 | (67%) |
| 1 | 7 | (33%) |
| Smoking history | ||
| Never | 18 | (86%) |
| Current | 2 | (10%) |
| Former | 1 | (5%) |
| BMI, kg/m2 | 22 | (21-25) |
| ≥24 | 8 | (38%) |
| <24 | 13 | (62%) |
| Site of metastases | ||
| Lung | 14 | (67%) |
| Liver | 8 | (38%) |
| Tumor bed | 7 | (33%) |
| Others* | 10 | (48%) |
| Prior regional therapy | ||
| Adrenalectomy | 21 | (100%) |
| Radiotherapy | 3 | (14%) |
| Prior systemic therapy | ||
| Platinum-based therapy | 12 | (57%) |
| Mitotane | 19 | (90%) |
| Hormonally functioning tumor | ||
| Yes | 8 | (38%) |
| Cortisol | 6 | (28.6%) |
| Cortisol and androgens | 2 | (9.5%) |
| No | 13 | (62%) |
| Tumor mutation burdent | 1.9 | (0-4.5) |
| ≥10 | 2 | (10%) |
| <10 | 18 | (86%) |
| Not evaluable | 1 | (5%) |
| PD-L1 combined positive score | ||
| ≥1 | 2 | (10%) |
| <1 | 18 | (86%) |
| Not evaluable | 1 | (5%) |
| MSI status+ | ||
| MSI-H | 2 | (10%) |
| MSS | 18 | (86%) |
| Not evaluable | 1 | (5%) |
Data are n (%) or median (IQR), unless otherwise stated. Overall percentages might not add up to 100% due to rounding. * Others included abdominal and retroperitoneal lymph nodes, bone, and muscle. +Two participants were evaluated based on peripheral blood.
ECOG Eastern Cooperative Oncology Group, MSI microsatellite instability, MSS microsatellite stability.
metastatic lesions to evaluate the tumor immune microenvironment. The expression levels of CD4 and CD8 were consistently low, with most samples exhibiting less than 5% CD4+ or CD8+ cells (Fig. 4A). Treat- ment response was not significantly associated with the percentages of CD4 +, CD8+, or CD56+ cells (Fig. 4B and Supplementary Fig. 3). Moreover, there was no significant difference in the percentages of
CD4 +, CD8+, or CD56+ cells between hormonally functioning and non-hormonally functioning tumors (Supplementary Fig. 4).
T-cell receptor (TCR) repertoires. The level of TCR diversity in per- ipheral blood T cells and the clonality of tumor-infiltrating T cells did not differ significantly between responders and nonresponders (Fig. 4C). However, correlation analysis revealed a significant correla- tion between TCR diversity and the best tumor size reduction (r = 0.49, p=0.02) (Supplementary Fig. 5). Responders also had a significantly higher overlap index (OLI) compared to nonresponders (p=0.04) (Fig. 4℃).
Systemic immunity. Flow cytometry analyses of pretreatment peripheral blood showed that patients with a higher baseline percen- tage of CD8+T cells were more likely to respond to combined therapy (p=0.002), while a trend towards a higher percentage of CD4 + T cells was observed in nonresponders (p = 0.055). The baseline CD4 +/CD8+T cell ratio was significantly lower in responders (p=0.002) (Fig. 4D). After two treatment cycles, nonresponders showed a tendency for a decline in CD4 +T cells (Supplementary Fig. 6), which was accompanied by a significant decrease in the CD4 + / CD8+ T cell ratio (p= 0.009) (Fig. 4E). CyTOF analysis was further used to investigate the impact of systemic immune response on treatment outcomes. Similar trends were observed in the baseline abundance of CD4+ and CD8+T cells, although these differences did not reach statistical significance (Supplementary Fig. 7). More specifically, responders showed increased expression of the chemokine receptor CXCR3 in CD8 + T cells, particularly in naïve CD8 + T cells (p=0.017) (Fig. 4F, G). CXCR3 is known for its role in regulating the migration, differentiation, and activation of immune cells. Additionally, the expression of TIM-3, an immunosuppressive molecular, was sig- nificantly decreased in CD4 + T cells from patients who responded to treatment (p=0.03) (Supplementary Fig. 8).
PD-L1 and MSI status. The level of PD-L1 expression was determined using the combined positive score (CPS). Only two patients (10%) were PD-L1 positive with a CPS ≥1; one patient had SD as the best response (CPS=2), while the other achieved a PR (CPS =5). The patient with PR also had both a high tumor mutational burden (TMB) and micro- satellite instability-high (MSI-H) status. Notably, only two patients had MSI-H tumors.
Genetic alterations and gut microbial diversity. Next-generation sequencing (NGS) results are detailed in Fig. 5A and Supplementary Fig. 9. The most frequently mutated genes were CKD4 (38.1%), KRAS (33.3%), PDGFRA (33.3%), KDR (28.6%), KIT (28.6%), and MDM2 (28.6%). No specific somatic alterations were associated with treatment response (Supplementary Table 1). We also analyzed the gut micro- biota composition and diversity from fecal samples collected prior to treatment (7 from responders and 4 from nonresponders). Microbiota a diversity, measured by the Shannon index and Ace index at the OTU level, was similar between responders and nonresponders (Fig. 5B). Moreover, ß diversity analysis assessed by PCoA revealed no sig- nificant association between gut microbiota composition and treat- ment response (Fig. 5C).
Discussion
This prospective study demonstrated that the combination of anti-PD- 1 and anti-VEGFR therapy provides promising clinical activity with an acceptable safety profile in advanced ACC patients who have pro- gressed after standard therapy. As a second-line therapy, the combi- nation of camrelizumab and apatinib achieved an objective response rate (ORR) of 52% and a durable survival benefit, which is higher than the published data for pembrolizumab (a PD-1 inhibitor) monotherapy
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(14-23%)7,11 and exceeds the response rates with EDP plus mitotane in the first-line setting (23%)5. Moreover, this study reported that higher peripheral blood CXCR3 + CD8 +T cell abundance, lower immuno- suppressive CD4 + T cell abundance, and higher overlap of clonotypes between tumor-infiltrating T cells and circulating T cells were poten- tially associated with a more favorable response to camrelizumab plus apatinib therapy.
Simultaneous inhibition of the VEGF-VEGFR2 and PD-1-PD-L1 path- ways results in a synergistic anti-tumor effect by promoting tumor vas- cular normalization and enhancing immune cell infiltration22. Apatinib, a small molecule inhibitor of VEGFR-2, combined with the PD-1 inhibitor camrelizumab, has demonstrated superior efficacy across several malignancies in clinical trials17,20,23,24, but there have been no reported experiences in patients with ACC to date. Our prospective study shows
the promising activity of camrelizumab plus apatinib as a second-line or later therapy for this rare and aggressive disease, with a manageable toxicity profile. In our study, all participants had progressed on at least one prior treatment. To date, only two of the eleven responding patients have died, indicating a sustained survival benefit. Our findings suggested that this combined regimen could serve as an alternative salvage therapy following the failure of standard therapy. Additionally, patients with non- hormonally functioning tumors may be more likely to benefit from camrelizumab plus apatinib. Overcoming potential inherent resistance to immunotherapy due to excess glucocorticoid production by the tumors remains a challenge for the treatment of ACC25.
The observed safety profile of camrelizumab plus apatinib was consistent with the known individual toxicities of each drug, as well as with findings from previous studies19,20,26. The most commonly repor- ted grade 3-4 adverse events were related to hepatotoxicity, and no
| Patients (N = 21) | ||
|---|---|---|
| Complete response | 0 | |
| Partial response | 11 | (52%) |
| Stable disease | 9 | (43%) |
| Progressive disease | 1 | (5%) |
| Objective response rate | 52% | (30%-74%) |
| Disease control rate | 95% | (76%-100%) |
| Median duration of response, months | Not reached | |
| Median progression-free survival, months | 13.3 | (95% CI: 8.4-NE) |
| Median overall survival, months | 20.9 | (95% CI: 11.0-NE) |
Data are n (%) or % (95% CI), unless otherwise stated. All objective responses in this study were classified as partial responses by RECIST v1.1.
treatment-related deaths occurred. The incidence of grade 3-4 adverse events was lower in patients receiving this combination compared to those treated with mitotane plus chemotherapy (43% vs. 58%)5. Only one patient permanently discontinued treatment due to uncontrollable transaminase elevation. Notably, this patient achieved a PR despite receiving only one cycle of camrelizumab, with remission lasting more than eight months. Previous studies have suggested that adverse events are more likely to occur in patients who respond to ICI treatment, both in ACC7,9 and other cancers27. However, our study did not find evidence that TRAEs were associated with a favorable response or improved clinical outcomes.
Our exploratory analyses aimed to identify potential response- related biomarkers for camrelizumab plus apatinib in ACC from mul- tiple perspectives. Due to variations in cutoff values, reported rates of positive PD-L1 expression in ACC range from 10.7% to 29% (10.7%28; 21%7; 29%6), compared to 10% in our study, as determined by CPS. With only two patients testing positive for PD-L1, we were unable to analyze the correlation between PD-L1 expression and treatment efficacy. A similar limitation applies to MSI/MMR analysis. Importantly, the majority of patients who lacked positive tumor PD-L1 expression or MSI-H status still responded well to the combination therapy of cam- relizumab and apatinib. This suggests that the relationship between these biomarkers and treatment response in ACC requires further investigation in the context of combined therapy.
The efficacy of immunotherapy in cancer patients has been linked to the immune composition within the tumor microenvironment29, as well as the immune landscape in the blood circulation system30. We analyzed pre-treatment tumor tissues to assess the immune micro- environment and found relatively sparse immune infiltration in ACC, which was consistent with previous reports31. ACC is traditionally characterized as an immune-depleted tumor, a condition believed to be influenced by adrenal glucocorticoid production32. It has been reported that CD4+ tumor-infiltrating lymphocytes negatively corre- late with glucocorticoid levels in ACC patients33. In our study, CD4 +
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| TRAEs | Overall (N= 21) | |||
|---|---|---|---|---|
| Grade 1 | Grade 2 | Grade 3 | Grade 4 | |
| Fatigue | 6 (28.6) | 6 (28.6) | 1 (4.8) | 0 |
| Aspartate aminotransfer- ase elevation | 5 (23.8) | 4 (19) | 3 (14.3) | 2 (9.5) |
| Alanine aminotransferase elevation | 2 (9.5) | 4 (19) | 5 (23.8) | 1 (4.8) |
| Hypertriglyceridemia | 6 (28.6) | 0 | 0 | 0 |
| Lymphopenia | 0 | 5 (23.8) | 4 (19) | 1 (4.8) |
| Hypercholesterolemia | 5 (23.8) | 0 | 0 | 0 |
| Hypertension | 3 (14.3) | 2 (9.5) | 0 | 0 |
| Reactive cutaneous capil- lary endothelial proliferation | 4 (19) | 0 | 0 | 0 |
| BNP elevation | 4 (19) | 0 | 0 | 0 |
| Leukocytopenia | 2 (9.5) | 2 (9.5) | 0 | 0 |
| Nausea | 2 (9.5) | 2 (9.5) | 0 | 0 |
| Hypothyroidism | 2 (9.5) | 2 (9.5) | 0 | 0 |
| Anorexia | 2 (9.5) | 2 (9.5) | 0 | 0 |
| Hypoalbuminema | 3 (14.3) | 0 | 0 | 0 |
| Hyponatremia | 3 (14.3) | 0 | 0 | 0 |
| Hypokalaemia | 2 (9.5) | 1 (4.8) | 0 | 0 |
| Fever | 2 (9.5) | 1 (4.8) | 0 | 0 |
| Anemia | 2 (9.5) | 0 | 1 (4.8) | 0 |
| Vomiting | 1 (4.8) | 2 (9.5) | 0 | 0 |
| Proteinuria | 0 | 3 (14.3) | 0 | 0 |
| Blood bilirubin elevation | 2 (9.5) | 0 | 0 | 0 |
| Hyperuricemia | 2 (9.5) | 0 | 0 | 0 |
| Hand-foot syndrome | 2 (9.5) | 0 | 0 | 0 |
| Constipation | 1 (4.8) | 1 (4.8) | 0 | 0 |
| Neutropenia | 1 (4.8) | 1 (4.8) | 0 | 0 |
| Diarrhea | 0 | 2 (9.5) | 0 | 0 |
| Oral mucositis | 0 | 2 (9.5) | 0 | 0 |
| Rash | 0 | 2 (9.5) | 0 | 0 |
| Anasarca | 0 | 2 (9.5) | 0 | 0 |
| Gastrointestinal bleeding | 0 | 2 (9.5) | 0 | 0 |
| Creatinine elevation | 1 (4.8) | 0 | 0 | 0 |
| Thrombocytopenia | 1 (4.8) | 0 | 0 | 0 |
| Pruritus | 1 (4.8) | 0 | 0 | 0 |
Data are n (%). No grade 5 treatment-related adverse events were reported. Patients could report more than one adverse event. If a patient experienced multiple occurrences of the same adverse event, they were counted once at the maximum recorded grade. TRAE treatment-related adverse event, BNP brain natriuretic peptide.
T cells, CD8 + T cells, and NK cells did not show a significant correlation with treatment response or with whether the tumor was hormonally functional. Given the confirmed variability in immune infiltration between primary and recurrent/metastatic tumors33, and considering that the tissues analyzed in our study were predominantly from pri- mary tumors previously surgically resected, it is crucial to further investigate whether the findings in the context of salvage therapy truly reflect the correlation between the immune microenvironment and the efficacy of combined treatment, as well as glucocorticoids.
We also reported the impact of circulating immune cells on the efficacy of the combined therapy in ACC. Patients who responded to camrelizumab plus apatinib had higher peripheral blood CD8 + T cell abundance prior to treatment. CD8+ T cells, also known as cytotoxic T lymphocytes, are well recognized for their anti-tumor functions34. Notably, naïve CD8 +T cells with higher CXCR3 expression likely played an important role in the treatment response. CXCR3+ naïve
CD8 + T cells, considered young memory T cells, are more likely to differentiate and acquire effector functions, enabling timely immune responses against foreign antigens35-37. The CXCR3 axis is involved in the migration, differentiation, and activation of immune cells, con- tributing to tumor suppression38. CXCR3 expression in peripheral T cells has also been proposed as a predictor of immunotherapy effi- cacy, with enhanced CXCR3 signaling potentially aiding PD-1 inhibitors in suppressing tumor growth39. In addition, tumor response was associated with low pre-treatment circulating CD4 + T cell abundance. However, due to the limited sample size, we lack sufficient evidence to distinguish between different CD4 + T cell subtypes. Interestingly, we observed higher TIM-3 expression in CD4 + T cells in nonresponders. TIM-3 is a known co-inhibitory molecule that contributes to an immunosuppressive tumor environment40. Given the generally lower expression level of TIM-3 in CD4 + T cells, this finding requires further validation.
In addition, consistent with this study, a lower CD4 + /CD8 + T cell ratio was significantly associated with a superior objective response to anti-PD-1 immunotherapy in patients with gastric, esophageal, and colorectal cancers41,42. It has been reported that the proportions of CD4 + T cells and CD4 +/CD8 + T cell ratio significantly increased after immunotherapy in the peripheral blood of gastric cancer patients who responded to treatment43. In the present study, although changes in peripheral blood CD4+ or CD8 + T cells were not significant in ACC patients who responded to treatment, we observed a significant decrease in the CD4 +/CD8 + T cell ratio after treatment in patients who did not respond, largely attributed to a decrease in CD4 + T cells. Different CD4 + T cell subtypes may have opposing roles in tumor immunity; however, due to the lack of an analysis of CD4 + T cell subtypes after treatment, we cannot specify which subtypes of CD4 + T cells are declining. In addition, an analysis of tumor-infiltrating lymphocyte subtypes before and after treatment would also help to better explain the specific changes in lymphocyte subtypes in the peripheral blood.
Investigations into the impact of TCR diversity on the treatment response to immunotherapy have resulted in varied and sometimes conflicting findings. For instance, Postow et al. examined the effect of the initial TCR repertoire on melanoma patients undergoing immu- notherapy and found that high TCR diversity in peripheral blood was associated with better survival outcomes44. Conversely, Hogan et al. reported that reduced TCR diversity predicted superior responses to PD-1 inhibitors and longer PFS45. In this study, we also observed that TCR diversity in peripheral blood was inversely correlated with tumor regression in ACC. Moreover, the presence of overlapping clones between the blood and tumor prior to immunotherapy provided more informative insights into clinical outcome. Specifically, a higher fre- quency of shared clones was associated with a more favorable treat- ment response46. Our findings confirmed this association, indicating that ACC patients with a higher overlap index were more likely to benefit from the combined therapy.
Exploring outcomes based on genomic characteristics and gut microbiome aimed to identify additional biomarkers of response. However, no significant association was found between the treatment response and TMB, somatic alterations, or microbial diversity.
As an early-phase exploratory trial, this study had several inherent limitations, including a small sample size and the lack of a control cohort. These factors may limit the ability to provide statistically meaningful information, particularly in the exploratory analysis of bio- markers. Meanwhile, the absence of post-treatment tumor tissue restricts the assessment of changes in the tumor microenvironment attributed to camrelizumab and apatinib treatment. Given the rarity of ACC, these constraints reflect the challenges of conducting clinical research in rare conditions. Furthermore, while mitotane has been shown to be safe when administered concurrently with immunotherapy6,9, its role as a potent CYP3A4 inducer and its long
A
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Percentage of CD4+ cells (%)
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p=0.63
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0
Responders
Nonresponders
0.0
Responders
Nonresponders
0.0
Responders
Nonresponders
D
E
Percentage of CD4+ T cells (%)
80
Percentage of CD8+ T cells (%)
60
p=0.002
4.0
2.5
p=0.19
4.0
p=0.055
CD4+/ CD8+ T cell ratio
p=0.002
CD4+ / CD8+ T cell ratio
p=0.009
60
3.0
2.0
CD4+/ CD8+ T cell ratio
3.0
40
1.5
40
2.0
2.0
1.0
20
20
1.0
0.5
1.0
0
Responders
Nonresponders
0
Responders
Nonresponders
0.0
Responders
Nonresponders
0.0
0.0
Pre
Post
Pre
Post
Responders
Nonresponders
F
CXCR3
G
Responders
Nonesponders
1.5
2.0
CXCR3 in CD8+ T cells
p=0.051
CXCR3 in naïve CD8+ T cells
p=0.017
Effector memory CD8+ T
1.5
Value
1.0
TSNE2
3.0
2.0
1.0
1.0
Naïve CD8+
0.0
0.5
0.5
Effector CD8+ T
0.0
0.0
TSNE1
Responders
Nonresponders
Responders
Nonresponders
half-life create pharmacokinetic interactions with other drugs, particu- larly TKIs47,48. In this study, although all patients who had previously received mitotane discontinued its use for at least four weeks before starting the study treatment, we did not monitor and record the mito- tane blood level at enrollment and throughout the study period, which
limits our ability to accurately assess its potential impact on the study medications.
In conclusion, the combination of camrelizumab and apatinib demonstrated promising activity and an acceptable safety profile in previously treated advanced ACC patients. Pre-treatment peripheral
A
B
OS (Months)
35
350
p=0.26
4.0
p=0.27
Ace index of OTU level
Shannon index of OTU level
300
O
O
0
Response
3.5
Hormonally functioning tumor
250
O
O
PD-L1
o
o
MSI
3.0
TMB
200
OS status
CDK4
Alive
150
2.5
PDGFRA
KRAS
Dead
KDR
Response
100
O
2.0
KIT
PR
Nonresponders
Responders
Nonresponders
Responders
MDM2
SD
MAF
CDKN2B
PD
TP53
Hormonally functioning
CDKN2A
tumor
RB1
Yes
C
CHEK2
PCoA on OTU level
MEN1
No
0.3
NF1
PD-L1
Nonresponders
R = - 0.03
SMARCB1
Positive
Responders
p = 0.52
PDCD1LG2
Negative
0.2
DAXX
FGFR3
Not evaluated
EP300
MSI
BRCA2
MSI-H
PC2 (13.38%)
0.1
MYC
MSS
JAK2
ATRX
Not evaluated
0
CDK6
MET
FGFR2
TMB
Alternations
CD274
40
missense
-0.1
CBFB
30
nonsense
GNAS
20
copy number gain
-0.2
CTNNB1
NF2
10
copy number loss
EGFR
0
frameshift
KEAP1
splice
-0.3
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
PC1 (23.11%)
blood immune cell subsets may serve as potential predictors of treatment efficacy. Given these encouraging results, evaluation of camrelizumab in combination with apatinib in future research is warranted.
Methods Study design and patients
This was an investigator-initiated, prospective, single-arm, open-label, phase 2 trial conducted at a single medical center, West China Hospital of Sichuan University. The study was approved and monitored by the Ethics Committee and the Clinical Trial Center of West China Hospital, Sichuan University. This study was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmo- nization Good Clinical Practice guidelines. Written informed consent was obtained from all patients prior to study enrollment. This study was registered on ClinicalTrials.gov under the identifier NCT04318730, with the registration submitted on March 20, 2020.
Patients were eligible for inclusion if they were at least 18 years old; had a pathological diagnosis of recurrent or metastatic ACC; had failed one or more lines of therapy; had an ECOG performance status of 0 or 1; had at least one measurable lesion; and had adequate organ function. Key exclusion criteria included a history of treatment with immunotherapy, anti-angiogenic small molecule TKIs, or anti- angiogenic monoclonal antibodies; a history of uncontrolled hyper- tension; and an autoimmune condition requiring systemic therapy. The full study protocol is available in the Supplementary Information
file. The first patient was enrolled on November 6, 2020, and the final patient was recruited on April 24, 2023.
Study treatment
All study patients received camrelizumab 200 mg intravenously on the first day of each 3-week cycle, combined with apatinib 250 mg orally once daily, until disease progression, intolerable toxicity, or with- drawal of consent. Dose modification of camrelizumab was not per- mitted. If adverse events occurred that were not alleviated by supportive care, dose adjustments and interruptions of apatinib were allowed. The initial dose reduction involved administering apatinib 250 mg once daily on a two days on, one day off schedule. Further reduction to 250 mg every other day was permitted if the adverse events persisted. Once the apatinib dose was reduced, it could not be escalated. If the adverse event did not improve to grade 1 or lower after the 4-week interruption, apatinib was permanently discontinued.
Study assessments
All patients underwent regular evaluations for response and safety. These included history and physical examination, adverse event assessment, hematological, biochemical, and endocrinological tests upon study entry and before each treatment cycle. Detailed endocri- nological test information is provided in Supplementary Table 2. Baseline tumor response assessments were performed within 14 days before treatment using contrast-enhanced computed tomography or magnetic resonance imaging of the brain, chest, abdomen, pelvis, and
any other known sites of disease. Subsequent regular assessments were conducted every two cycles, following the same methodology as the baseline evaluations. Patients with clinically unstable disease could be evaluated at any time with unplanned imaging assessments.
The primary endpoint was the objective response rate (ORR), defined as the proportion of participants achieving a CR or PR per RECIST version 1.1. Both CR and PR had to be confirmed at least 4 weeks after the initial response. The best overall response was the best tumor response recorded at any point after treatment initiation. Secondary endpoints included PFS, defined as the time from the initiation of treatment to disease progression or death, whichever occurred first; OS, defined as the time from the treatment initiation to the date of death or the end of follow-up, whichever occurred first; and safety, defined as the incidence of TRAEs per NCI-CTCAE version 5.0.
Exploratory endpoints included molecular biomarker analyses and their association with treatment response. PD-L1 expression was asses- sed using the CPS with the PD-L1 22C3 pharmDx assay (concentration: 3 µg/ml; Dako, Agilent Technologies, Carpinteria, USA). Multiplex immunofluorescence was used to detect the percentages of CD4+ (clone 458G4A1, ready-to-use, Abcarta), CD8+ (clone 815R4B2, ready-to- use, Abcarta), and CD56+ (clone 682F4C5, ready-to-use, Abcarta) cells within the tumor-related area of pre-treatment tumor tissues.
TCR diversity in peripheral blood mononuclear cells (PBMCs) and clonality of tumor-infiltrating T cells in pre-treatment tissues were analyzed. Genomic DNA from PBMCs and tumor tissues was extracted using commercial kits (Qiagen, Duesseldorf, Germany) and sequenced for the variable CDR3 region of the TCR ß-chain using the immunoSEQ assay, as previously described49. From the CDR30 data, T-cell diversity in PBMCs and clonality of tumor-infiltrating T cells were assessed. The overlap index (OLI) was calculated from the detection data of the TCR immunogroup bank, based on the number and abundance of shared clones between tumor-infiltrating T cells and circulating T cells.
The analysis of immune cell subsets was conducted on PBMCs obtained from pre-treatment blood samples using flow cytometry and mass cytometry (CyTOF) method with an immune-related panel con- sisting of 41 markers (Supplementary Table 3). Cells were initially stained with cisplatin to exclude dead cells, followed by staining with a cocktail of surface antibodies. After fixation with an intercalation solution (Maxpar Fix and Perm Buffer containing 250 nM 191/193Ir, Fluidigm), cells were stained with an intracellular antibody cocktail. The cells were then washed, resuspended, and mixed with 20% EQ beads before being acquired on a mass cytometer (Helios, Fluidigm). Data for each sample were debarcoded from raw data using a doublet- filtering scheme50, normalized through bead normalization51, and manually gated to exclude debris and dead cells using FlowJo software (FlowJo, Oregon, USA). The Phenograph algorithm was employed for clustering based on marker expression, with clusters annotated according to marker patterns52. The t-distributed stochastic neighbor embedding (t-SNE) dimension reduction technique was used to visualize the clusters and illustrate marker expression53.
Tumor tissue samples, along with self-blood negative controls, were sequenced using a comprehensive 503 cancer-related panel. Genomic alterations and gene fusions/rearrangements, were identified using GATK, MuTect (version 1.1.4) (http://www.broadinstitute.org/ cancer/cga/mutect), and BreakDancer54, respectively. MSI status was assessed using MSIsensor (version 0.2)55. Tumors with an MSI score of 10 or higher were classified as MSI-H. TMB was calculated based on the number of all somatic nonsynonymous mutations, insertions, and deletions per megabase of coding regions sequenced.
Microbiota diversity of the gut prior to treatment was assessed using 16S rRNA sequencing. Data analysis of the gut microbiota was performed on the Majorbio Cloud platform (https://cloud.majorbio. com). Based on the OTUs information, a diversity indices, including ACE and Shannon indices, were calculated using Mothur v1.30.156.
The similarity among microbial communities in different samples was determined by principal coordinate analysis (PCoA) using the Vegan v2.5-3 package (https://vegandevs.github.io/vegan/).
Statistical analyses
A Simon’s two-stage design was used to determine the sample size for the study57,58. Historical data indicated an ORR of 14% for single-agent pembrolizumab as salvage therapy in ACC11. To detect an approximate 26% improvement (reaching an ORR of 40%), with 80% power and a one-sided alpha of 0.05, a sample size of 19 patients was required. In stage I, nine patients were enrolled; if at least one of the nine patients exhibited a PR or CR, the study would proceed to stage II to enroll an additional 10 patients. The combination treatment would be con- sidered worthwhile if six or more objective responses were observed among the 19 patients. Considering an anticipated 10% dropout rate, the study needed to enroll a total of 21 patients.
Analyses for all primary and secondary endpoints were conducted in the mITT population, including all patients who received at least one dose of the study drug. Continuous variables were presented as medians with interquartile range (IQR), while categorical variables were shown as frequency with corresponding percentage. The ORR was presented with its corresponding 95% CI, calculated using the Clopper-Pearson method. Median follow-up time was calculated using the reverse Kaplan-Meier method59. The median duration of response, median PFS, and median OS were estimated using the Kaplan-Meier method; corresponding 95% CIs were estimated using the Brookmeyer-Crowley method. Safety data was described using descriptive statistics.
Pearson’s chi-square test or Fisher’s exact test was used to assess the association of clinicopathological characteristics and genetic mutations with ORR. The normality of the distribution of continuous variables was confirmed using the Shapiro-Wilk normality test. If the data passed the normality test, Student’s t test was used to examine the association of immune cell proportions in blood or tumor tissue, TCR diversity, or microbial diversity index with treatment response. If the distribution was not normal, the Mann-Whitney U test was applied. A paired student’s t test was used to compare immune cell proportions before and after treatment. Pearson correlation coefficient was cal- culated to assess the correlations between the best tumor change and immune cell proportion or TCR diversity. Statistical analysis was per- formed using GraphPad Prism software (version 9.4.1, San Diego, California) and R software (version 3.6.1, Vienna, Austria).
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
All raw sequencing data generated in this study have been deposited in the National Genomics Data Center (NGDC) under the accession code HRA008803, HRA008789, and HRA008669. The raw sequencing data contain information unique to individuals and are available under con- trolled access. Access to the data can be requested by completing the application form via GSA-Human System and is granted by the corresponding Data Access Committee. Additional guidance can be found at the GSA-Human System website [https://ngdc.cncb.ac.cn/ gsa-human/document/GSA-Human_Request_Guide_for_Users_us.pdf]. The raw patient data are protected and not publicly available due to data privacy laws. The de-identified individual patient data will be available upon reasonable request for academic research purposes by contacting the corresponding author (pxx2014@163.com) for 10 years. The study protocol is available in the Supplementary Information file. The remaining data are available within the Article, Supplementary Infor- mation, and Source Data file. Source data are provided with this paper.
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Acknowledgements
This work was supported by the National Key Research and Develop- ment Program of China (2021YFE0206600, X.P.), the Clinical Research Incubation Project of West China Hospital (2020HXFH031, 20HXFH037, X.P.), the Sichuan Science and Technology Program (2022YFSY0012, 2021ZYCD011, 2021YFSY0008, 2021CDDZ-25, 2021CDZG-24, X.P.), the Chengdu International Science and Technology Cooperation Program (2022-GH03-00004-HZ, X.P.), the West China Nursing Discipline Development Special Fund Project (HXHL21008, X.P.), the Post-Doctor
Research Project, West China Hospital, Sichuan University (2020HXBH119, X.P.), the Translational Medicine Fund of West China Hospital (CGZH19002, X.P.), and the China Medical Board (Grant 22- 482, X.P.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We sin- cerely thank the patients and their families for their participation in this study. We also extend our gratitude to the study coordinators and nurses for their dedication and enthusiasm. We appreciate the support from Jiangsu Hengrui Pharmaceuticals. We also thank Zhejiang Puluoting Health Technology Co., Ltd. for their work in performing CyTOF. Addi- tionally, we thank Yan Wang, Jian Yang, Xiang-Yi Ren, Cong Li, and Jing- Yao Zhang from the Core Facilities, West China Hospital of Sichuan University for their assistance with sample processing.
Author contributions
Conception and design: Xing-Chen Peng, Yu-Chun Zhu, Qiang Wei, Ji- Yan Liu, Zhi-Gong Wei, Jing-Jing Wang, Zhe-Ran Liu, Yi-Yan Pei, Yu Min, Rui-Dan Li, Li Yang Provision of study materials or patients: Xing-Chen Peng, Yu-Chun Zhu, Qiang Wei, Ji-Yan Liu Collection and assembly of data: Yu-Chun Zhu, Zhi-Gong Wei, Jing-Jing Wang, Dong Li, Zhi-Hui Li, Zhe-Ran Liu, Yi-Yan Pei, Jin Jing, Yu Min, Rui-Dan Li, Li Yang Data analysis and interpretation: Yu-Chun Zhu, Zhi-Gong Wei, Jing-Jing Wang, Xing- Chen Peng. Manuscript writing: All authors. Final approval of manu- script: All authors. Accountable for all aspects of the work: All authors.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-024-54661-9.
Correspondence and requests for materials should be addressed to Xing-Chen Peng.
Peer review information Nature Communications thanks Barbara Altieri, Sara Bedrose, Martin Fassnacht and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.
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