A Potential Predictive Marker for Advanced Hepatocellular Carcinoma PD-1 Inhibitors Combined with Radiation: Expression of PD-L1 on Circulating Tumor Cells

DOI: https://doi.org/10.21203/rs.3.rs-2277477/v1

Abstract

Purpose

In the treatment of advanced hepatocellular carcinoma (HCC), programmed death-1 (PD-1) immune checkpoint inhibitors has recently been shown to be highly effective when combined with radiotherapy. Furthermore, they have become the hotspot in cancer diagnosis and treatment for the detection of programmed death-ligand 1 (PD-L1) in circulating tumor cells (CTCs). However, their predictive effect is not well established. Therefore, this study examined whether PD-L1 expression in CTCs can be used as a marker to predict treatment response in patients with advanced HCC.

Methods

Patients treated with both PD-1 inhibitors and intensity-modulated radiation therapy (IMRT) were enrolled in the study. After radiation therapy, PD-1 inhibitor treatment was administered every 3 weeks until disease progression. Peripheral blood (2 mL) was collected from patients before and after treatment, and CTC PD-L1 was detected using the Watson Biotechnology reagent (China).

Results

A total of 28 patients with HCC were enrolled in this study. The disease control rate (DCR) was significantly higher in patients with PD-L1(+) CTC enrichment at baseline than in controls (92.3% and 50%, respectively). Before treatment, patients with PD-L1(+) CTCs ≥ 2 had a higher median progression-free survival (mPFS) than those with PD-L1(+) CTCs ≤ 1 (3.50 vs. 3.35 months). After treatment, CTCs with PD-L1(+) ≤ 1 were significantly associated with longer mPFS than CTCs with PD-L1(+) ≥ 2 (4.20 vs. 1.90 months, P < 0.01).

Conclusions

The presence of CTCs expressing PD-L1(+) might predict efficacy and prognosis in HCC patients treated with PD-1 inhibitors and radiotherapy.

Retrospectively registered

The study has been registered with the Chinese Clinical Trials Registry (registration number: ChiCTR2100044198).

Introduction

Over the past decades, the incidence of liver cancer and the mortality associated with the disease has been increasing year by year (Petrick et al. 2016), and currently, the disease ranks sixth in incidence and third in mortality globally (Sung et al. 2021; Siegel et al. 2019). Over 1 million people are expected to die from liver cancer by 2030 according to the World Health Organization (Villanueva 2019). Some noninvasive tests commonly used in clinical practice lack specificity and sensitivity for the diagnosis of HCC and do not provide a good response to the current treatment effect and prognosis of HCC.

In recent years, there has been a boom in immune checkpoint inhibitor research for HCC (Greten and Sangro 2017; Greten et al. 2006). Several studies have suggested that blocking the PD-1 and PD-L1 pathways improves the prognosis of patients with advanced HCC (Cha et al. 2019; Finn et al. 2020; Zhu et al. 2018). Furthermore, radiotherapy can have a huge impact on the killing of tumor cells in the body, rapidly release tumor-associated antigens, effectively activate the immune response, and exert excellent tumor-killing effects when costimulated with anti-PD-1 and anti-PD-L1 antibodies. By combining these two therapeutic measures, tumor immunomodulation is enhanced and therapeutic efficacy is significantly improved (Schumacher and Schreiber 2015; Brooks and Chang 2019; Postow et al. 2012).

Although PD-1 inhibitor immunotherapy has shown promising efficacy in HCC, the response rate of patients receiving this treatment is < 20% (Greten and Sangro 2017). Liquid biopsies, which enable repeat sampling, reflect the characteristics and dynamics of the tumor (Van et al. 2011; Alix-Panabieres and Pantel 2012; Crowley et al. 2013). Recently, CTCs and their surface PD-L1 expression has become a research hotspot in HCC. As key cells in tumor recurrence and metastasis, CTCs are the tumor cells that enter the circulation away from the primary or metastatic lesion (Follain et al. 2020). Although PD-L1(+) CTCs have been shown in some studies to be a beneficial prognostic marker in many types of cancer, including HCC (Winograd et al. 2020; Bergmann et al. 2020), some controversies remain (Ikeda et al. 2021; Strati et al. 2017; Kallergi et al. 2018; Khattak et al. 2020). This study aimed to examine the association between PD-L1 expression in CTCs and the therapeutic effect of PD-1 inhibitors combined with radiotherapy in patients with advanced HCC.

Materials And Methods

Design and participants of the study

Five inclusion criteria were considered for this study: (a) adults aged > 18 years; (b) biopsy/imaging confirmation of HCC; (c) diagnosis of stage B or C for the Barcelona Clinic Liver Cancer (BCLC); (d) availability of the complete clinical data of the patient; (e) after targeted drugs therapy, the patient progressed or could not tolerate it, and received radiotherapy combined with immunotherapy. The following were the exclusion criteria for this study: (a) the combination of other malignancies; (b) poor general health condition of the patient, not amenable to treatment with PD-1 inhibitor combined with radiotherapy; (c) peripheral blood collected > 6 h or of low quality.

Patients who received PD-1 inhibitors combined with radiotherapy from August 2020–2022 at our institution were included in this study. Meanwhile, the basic information and clinical data during the treatment were collected for these HCC patients. All patients voluntarily provided their signed informed consent form.

Circulating Tumor Cell Detection

The peripheral blood samples (2mL) were first collected from the patients within 6h and processed as quickly as possible. Phosphate-buffered solution (PBS) and the cell separation solution were added to the samples, and the density gradient centrifugation method was used to isolate the peripheral blood mononuclear cells (PBMCs). Next, the nano-microfluidic chip (i.e., CytoNanoChip chip) was coated with Anti-EpCMA Ab and Anti-CSV Ab antibodies, and the processed PBMCs were introduced into the package using The CytoSorter™ BioScanner System (Watson Biological Technology Company, China). The processed PBMCs were introduced into the nano-fluidic chip encapsulated with the captured antibodies through the SCx spiral sample chamber for further enrichment to CTC. Anti-CSV (FITC) Ab, Anti-PanCK (FITC) Ab, Anti-PD-L1 (Cy5) Ab, and Anti CD45(PE)Ab were used to identify CTCs and white blood cells (WBCs), and the cells were stained with Hoechst33342 for nuclear staining. PD-L1(+) CTC was defined as the phenotype of CSV&CK(+), PD-L1(+), CD45(-), and Nucleus(+) under fluorescence microscopy after cell staining.

Treatment Evaluation

The curative effect and prognostic index of the patients were mainly recorded. The effect of treatment was determined by comparing the patients' magnetic resonance imaging (MRI) scans before and after treatment. Based on the modified Response Evaluation Criteria in Solid Tumors (mRECIST) standard for evaluating the curative effects, the patients with HCC were classified as a disease-controlled (DC) when they underwent complete remission (CR), partial remission (PR), stable disease (SD), or progressive disease (PD). During PFS, the patients were evaluated from the time of initiation of treatment until the time of disease progression.

Statistical Analysis

Detailed processing and analyses of the data was performed with GraphPad Prism 8.0 and SPSS statistical software. For categorical variables in the study, Pearson’s Chi-square test and Fisher’s exact probability method were applied for specific analyses. We analyzed the survival data for PFS using the Kaplan–Meier method and compared the survival curves with the Mantel-Cox test. Statistically significant two-tailed p-values were considered at < 0.05.

Results

1. Basic characteristics of the patients with HCC

The study comprised 28 patients with HCC. All patients enrolled were men, their ages ranged from 28 to 72 years, had BCLC stage B or C, and Child-Pugh was either A or B. Triweekly PD-1 inhibitor treatments were administered to the patients after radiotherapy until disease progression occurred. The total dose of radiotherapy was 48Gy, with 3Gy per fraction. The predominant PD-1 inhibitors used were tirelizumab (18 cases), sindilizumab (3 cases), and karelizumab (7 cases). The use of the ROC curve to estimate the expected number of PD-L1(+) CTCs led to the determination of the critical value to be 2 PD-L1(+) CTCs in our previous work (Su et al. 2022).

BCLC, Barcelona clinic liver cancer; AFP, alpha-fetoprotein; CTCs, circulating tumor cells; PD-L1, programmed death-ligand 1

2. Dynamic Changes In Pd-l1(+) Ctcs Before And After Treatment

As shown in Table 1, each of the 28 patients included in the study had CTCs in their peripheral blood. The total number of CTCs in the patients before the treatment varied from 2 to 10, and PD-L1(+) CTCs were detected in 26/28 patients before the treatment. Figure 1 shows the percentage of PD-L1(+) CTCs per patient at baseline. The total number of CTCs decreased in all 28 patients and the number of PD-L1(+) CTCs decreased in 19 patients after receiving PD-1 inhibitor along with radiotherapy.

3. Correlation Between Therapeutic Effect And Pd-l1(+) Ctc Expression

After treatment with PD-1 inhibitor combined with IMRT, 24 of the 28 patients with HCC had DC (SD13 + PR11) and 4 patients had PD. Patients with and without PD-L1(+) CTCs at baseline exhibited disease control rates of 92.3% and 50%, respectively, as shown in Fig. 2. The cotreatment of PD-1 inhibitor and radiotherapy was found to benefit patients with HCC in whom PD-L1(+) CTCs were detected at baseline. In the pre- and post-treatment comparisons, an overall reduction of CTCs was observed in 100% (24/24) of the DC patients and 50% (2/4) of the PD patients, as shown in Fig. 3A. PD-L1(+) CTCs were reduced in 75% (18/24) of the DC patients and 25%(1/4) of the PD patients in the analysis of PD-L1 expression, as shown in Fig. 3B.

4. Pre- and post-treatment correlation between PD-L1(+) CTCs and alpha-fetal protein (AFP) values in patients with HCC

When the AFP values recorded before and after the treatment were recorded, as shown in Fig. 4, patients with ≥ 2 PD-L1(+) CTCs before the treatment were found to show a significantly higher declining trend after the treatment than those with ≤ 1 PD-L1(+) CTCs. AFP values can, to a certain extent, determine the disease status of patients with HCC, and the dynamic variations in AFP reflect the better treatment outcomes of the patients if they had more PD-L1(+) CTCs before the treatment.

5. Pd-l1(+) Ctcs Indicate A Favorable Prognosis For Patients With Hcc Before And After Treatment

The patients’ PFS was monitored, which revealed that it was inversely correlated with the number of PD-L1(+) CTCs at baseline and after the treatment. At baseline, ≥ 2 PD-L1(+) CTCs were associated with an mPFS of 3.50 months, which was longer than that of ≤ 1 PD-L1(+) CTCs (3.35 months), as shown in Fig. 5A. After treatment, the mPFS of patients with ≤ 1 PD-L1(+) CTCs was 4.20 months, whereas that of patients with ≥ 2 PD-L1(+) CTCs was only 1.90 months; hence, the former was significantly better than the latter, as shown in Fig. 5B. The results demonstrated that both elevated PD-L1(+) CTCs before treatment and low PD-L1(+) CTCs after treatment can serve as prognostic indicators in patients with HCC.

Discussion

Researchers found that radiation therapy was an independent prognostic factor for good outcomes when taking PD-1/PD-L1 inhibitors and that the combined treatment had a synergistic effect, with patients exhibiting significantly higher PFS, overall survival, and response rates (Yamaguchi et al. 2019; Mauclet et al. 2019). However, not all patients with HCC benefit from immunotherapy.

PD-L1 CTC levels varied among patients with HCC. Evidence from previous studies suggests that anti-PD-1 and anti-PD-L1 therapy is more effective in patients who overexpress PD-L1 (Meng et al. 2015; Patel and Kurzrock 2015). A study found that in the treatment of HCC with anti-PD-1/PD-L1, a higher objective response rate was observed in the case of CTCs with PD-L1 expression than in those without PD-L1 expression (Winograd et al. 2020). According to our data, 92.3% of the patients with PD-L1(+) CTCs achieved DCR before treatment, which was much higher than the 50% of the patients without PD-L1(+) CTCs. It can be concluded that CTCs expressing PD-L1 can be clinically used as a potential prognostic marker for patients with HCC.

A study observed that patients with high levels of PD-L1 expression on CTCs at baseline in gastrointestinal tumors tended to be more sensitive to anti-PD-1 and anti-PD-L1 therapy and had longer survival times (Tan et al. 2021). Our study analyzed the prognostic significance of PD-L1(+) CTCs by considering the patients’ PFS based on the numbers before and after treatment. At baseline, patients with ≥ 2 PD-L1(+) CTCs were more likely to have an extended median PFS (3.50 vs. 3.35 months). Meanwhile, those with ≤ 1 PD-L1(+) CTCs had significantly longer mPFS than those with ≥ 2 PD-L1(+) CTCs after the treatment (4.20 vs. 1.90 months, P < 0.01). Furthermore, the dynamic changes in AFP values signified, to some extent, that the patient's baseline CTC values can identify the immune-beneficiary population. Also, the post-treatment CTC counts reflected the benefit of PD-1 inhibitors combined with IMRT treatment.

CTC PD-L1 assay is convenient, noninvasive, real-time, and dynamic (Yue et al. 2018), and CTCs from patients were sorted using a cell sorter using the Multiplex Assessment of PD-L1 Expression™ BioScanner System. Several studies have demonstrated the sensitivity and specificity of this method in different solid cancers, with a sensitivity of 61.9–95.4% and specificity of 76.6–91.3% (Wei et al. 2019; JIN et al. 2020༛Xie et al. 2021; Gao et al. 2021). The assay helps clinicians select patients who are likely to benefit from PD-1 inhibitor therapy based on the expression of PD-L1 on their CTCs at baseline. Dynamic monitoring of the PD-L1 distribution on CTCs may provide more insights into the effectiveness of immunotherapy.

Numerous studies have demonstrated that the effectiveness of immunotherapy can be evaluated with the dynamic monitoring of PD-L1 expression on CTCs before and after treatment. However, owing to the limited number of cases collected, the results of multicenter randomized controlled trials are lacking. Therefore, CTC PD-L1 expression as an indicator of efficacy assessment in patients with HCC needs to be further validated in a prospective multicenter large sample.

Conclusions

This study has shown that patients with advanced HCC who have an overexpression of PD-L1(+) CTCs at baseline may be more likely to benefit from PD-1 inhibitors combined with radiotherapy. Detecting the dynamic changes in CTCs could help assess the prognosis in patients undergoing treatment.

Declarations

Acknowledgments

Special thanks to Hangzhou Watson Biotech, Inc.(Hangzhou, China)  for technical assistance. We also thank the patients and their families for their support of the study. Finally, we would like to thank all the reviewers who participated in the review, as well as MJEditor (www.mjeditor.com) for providing English editing services during the preparation of this manuscript.

Funding Information

This work was supported by a grant from Project of Science and Technology Department of Sichuan Province (2020JDTD0036).

Competing Interests

The authors declare no potential conflicts of interest. All authors endorse this manuscript.

Author contributions

JC and ZW analyzed the data and wrote the manuscript, KH, LG, and YH were involved in the conception and design of the study, MR, JZ, HL, KS, KX, TG, PW, HZ, and LH were responsible for collecting and organizing the data, All authors contributed to the article and approved the submitted version.

Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval

The protocol for this research project was approved by a suitably constituted Ethics Committee of the institution and it conforms to the provisions of the Declaration of Helsinki—The Committee of the Affiliated Hospital of Southwest Medical University (approval number: KY2021063). The study has been registered with the Chinese Clinical Trials Registry (registration number: ChiCTR2100044198). 

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Consent to publish

The authors confirm that human research participants provided informed consent for the publication of data relevant to this article.

References

  1. Alix-Panabières C, Pantel K. Circulating tumor cells: liquid biopsy of cancer. Clin Chem. 2013,59(1):110-118. doi:10.1373/clinchem.2012.194258
  2. Bergmann S, Coym A, Ott L, et al. Evaluation of PD-L1 expression on circulating tumor cells (CTCs) in patients with advanced urothelial carcinoma (UC). Oncoimmunology. 2020,9(1):1738798. Published 2020 Mar 23. doi:10.1080/2162402X.2020.1738798
  3. Brooks ED, Chang JY. Time to abandon single-site irradiation for inducing abscopal effects. Nat Rev Clin Oncol. 2019,16(2):123-135. doi:10.1038/s41571-018-0119-7
  4. Cha JH, Chan LC, Li CW, Hsu JL, Hung MC. Mechanisms Controlling PD-L1 Expression in Cancer. Mol Cell. 2019,76(3):359-370. doi:10.1016/j.molcel.2019.09.030
  5. Crowley E, Di Nicolantonio F, Loupakis F, Bardelli A. Liquid biopsy: monitoring cancer-genetics in the blood. Nat Rev Clin Oncol. 2013,10(8):472-484. doi:10.1038/nrclinonc.2013.110
  6. Finn RS, Qin S, Ikeda M, et al. Atezolizumab plus Bevacizumab in Unresectable Hepatocellular Carcinoma. N Engl J Med. 2020,382(20):1894-1905. doi:10.1056/NEJMoa1915745
  7. Follain G, Herrmann D, Harlepp S, et al. Fluids and their mechanics in tumour transit: shaping metastasis. Nat Rev Cancer. 2020,20(2):107-124. doi:10.1038/s41568-019-0221-x
  8. Gao Y, Fan WH, Song Z, Lou H, Kang X. Comparison of circulating tumor cell (CTC) detection rates with epithelial cell adhesion molecule (EpCAM) and cell surface vimentin (CSV) antibodies in different solid tumors: a retrospective study. PeerJ. 2021,9:e10777. Published 2021 Mar 2. doi:10.7717/peerj.10777
  9. Greten TF, Manns MP, Korangy F. Immunotherapy of hepatocellular carcinoma. J Hepatol. 2006,45(6):868-878. doi:10.1016/j.jhep.2006.09.004
  10. Greten TF, Sangro B. Targets for immunotherapy of liver cancer [published online ahead of print, 2017 Sep 18]. J Hepatol. 2017,S0168-8278(17)32287-0. doi:10.1016/j.jhep.2017.09.007
  11. Ikeda M, Koh Y, Teraoka S, et al. Longitudinal Evaluation of PD-L1 Expression on Circulating Tumor Cells in Non-Small Cell Lung Cancer Patients Treated with Nivolumab. Cancers (Basel). 2021,13(10):2290. Published 2021 May 11. doi:10.3390/cancers13102290
  12. Jin L, Zhao W, Zhang J, et al. Evaluation of the diagnostic value of circulating tumor cells with CytoSorter® CTC capture system in patients with breast cancer. Cancer Med. 2020,9(5):1638-1647. doi:10.1002/cam4.2825
  13. Kallergi G, Vetsika EK, Aggouraki D, et al. Evaluation of PD-L1/PD-1 on circulating tumor cells in patients with advanced non-small cell lung cancer. Ther Adv Med Oncol. 2018,10:1758834017750121. Published 2018 Jan 15. doi:10.1177/1758834017750121
  14. Khattak MA, Reid A, Freeman J, et al. PD-L1 Expression on Circulating Tumor Cells May Be Predictive of Response to Pembrolizumab in Advanced Melanoma: Results from a Pilot Study. Oncologist. 2020,25(3):e520-e527. doi:10.1634/theoncologist.2019-0557
  15. Mauclet C, Duplaquet F, Pirard L, et al. Complete tumor response of a locally advanced lung large-cell neuroendocrine carcinoma after palliative thoracic radiotherapy and immunotherapy with nivolumab. Lung Cancer. 2019,128:53-56. doi:10.1016/j.lungcan.2018.12.006
  16. Meng X, Huang Z, Teng F, Xing L, Yu J. Predictive biomarkers in PD-1/PD-L1 checkpoint blockade immunotherapy. Cancer Treat Rev. 2015,41(10):868-876. doi:10.1016/j.ctrv.2015.11.001
  17. Patel SP, Kurzrock R. PD-L1 Expression as a Predictive Biomarker in Cancer Immunotherapy. Mol Cancer Ther. 2015,14(4):847-856. doi:10.1158/1535-7163.MCT-14-0983
  18. Petrick JL, Kelly SP, Altekruse SF, McGlynn KA, Rosenberg PS. Future of Hepatocellular Carcinoma Incidence in the United States Forecast Through 2030. J Clin Oncol. 2016,34(15):1787-1794. doi:10.1200/JCO.2015.64.7412
  19. Postow MA, Callahan MK, Barker CA, et al. Immunologic correlates of the abscopal effect in a patient with melanoma. N Engl J Med. 2012,366(10):925-931. doi:10.1056/NEJMoa1112824
  20. Schumacher TN, Schreiber RD. Neoantigens in cancer immunotherapy. Science. 2015,348(6230):69-74. doi:10.1126/science.aaa4971
  21. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin. 2019,69(1):7-34. doi:10.3322/caac.21551
  22. Strati A, Koutsodontis G, Papaxoinis G, et al. Prognostic significance of PD-L1 expression on circulating tumor cells in patients with head and neck squamous cell carcinoma. Ann Oncol. 2017,28(8):1923-1933. doi:10.1093/annonc/mdx206
  23. Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021,71(3):209-249. doi:10.3322/caac.21660
  24. Su K, Guo L, He K, et al. PD-L1 expression on circulating tumor cells can be a predictive biomarker to PD-1 inhibitors combined with radiotherapy and antiangiogenic therapy in advanced hepatocellular carcinoma. Front Oncol. 2022,12:873830. Published 2022 Aug 2. doi:10.3389/fonc.2022.873830
  25. Tan Z, Yue C, Ji S, et al. Assessment of PD-L1 Expression on Circulating Tumor Cells for Predicting Clinical Outcomes in Patients with Cancer Receiving PD-1/PD-L1 Blockade Therapies. Oncologist. 2021,26(12):e2227-e2238. doi:10.1002/onco.13981
  26. van de Stolpe A, Pantel K, Sleijfer S, Terstappen LW, den Toonder JM. Circulating tumor cell isolation and diagnostics: toward routine clinical use. Cancer Res. 2011,71(18):5955-5960. doi:10.1158/0008-5472.CAN-11-1254
  27. Villanueva A. Hepatocellular Carcinoma. N Engl J Med. 2019,380(15):1450-1462. doi:10.1056/NEJMra1713263
  28. Wei T, Zhang X, Zhang Q, et al. Vimentin-positive circulating tumor cells as a biomarker for diagnosis and treatment monitoring in patients with pancreatic cancer. Cancer Lett. 2019,452:237-243. doi:10.1016/j.canlet.2019.03.009
  29. Winograd P, Hou S, Court CM, et al. Hepatocellular Carcinoma-Circulating Tumor Cells Expressing PD-L1 Are Prognostic and Potentially Associated With Response to Checkpoint Inhibitors. Hepatol Commun. 2020,4(10):1527-1540. Published 2020 Aug 4. doi:10.1002/hep4.1577
  30. Xie X, Wang L, Wang X, et al. Evaluation of Cell Surface Vimentin Positive Circulating Tumor Cells as a Diagnostic Biomarker for Lung Cancer. Front Oncol. 2021,11:672687. Published 2021 May 14. doi:10.3389/fonc.2021.672687
  31. Yamaguchi O, Kaira K, Hashimoto K, et al. Radiotherapy is an independent prognostic marker of favorable prognosis in non-small cell lung cancer patients after treatment with the immune checkpoint inhibitor, nivolumab. Thorac Cancer. 2019,10(4):992-1000. doi:10.1111/1759-7714.13044
  32. Yue C, Jiang Y, Li P, et al. Dynamic change of PD-L1 expression on circulating tumor cells in advanced solid tumor patients undergoing PD-1 blockade therapy. Oncoimmunology. 2018,7(7):e1438111. Published 2018 Mar 6. doi:10.1080/2162402X.2018.1438111
  33. Zhu AX, Finn RS, Edeline J, et al. Pembrolizumab in patients with advanced hepatocellular carcinoma previously treated with sorafenib (KEYNOTE-224): a non-randomised, open-label phase 2 trial [published correction appears in Lancet Oncol. 2018 Sep,19(9):e440]. Lancet Oncol. 2018,19(7):940-952. doi:10.1016/S1470-2045(18)30351-6