The impact of atezolizumab and bevacizumab in hepatocellular carcinoma with activated ß‐catenin signaling

Abstract Background To date, no biomarkers exist to predict response or resistance to immunotherapy in hepatocellular carcinoma (HCC). Recent approaches to classify HCC into different immunological states revealed a negative correlation between Wnt/ß‐catenin activation and immunogenicity and T‐cell infiltration. If these “cold” tumors with primary resistance to checkpoint inhibition (CPI) may benefit from dual treatment of CPI and anti‐angiogenic therapy has not been proved. Case Here, we describe the case of a male patient with metastatic HCC. After failure of standard of care treatment with lenvatinib, sorafenib and ramucirumab fourth‐line systemic therapy with atezolizumab and bevacizumab were applied leading to a phenomenal response. Immunohistochemical evaluations were compatible with Wnt/ß‐catenin pathway activation and accompanying low T‐cell infiltration as well as low PD‐L1 score. Conclusion Patients with Wnt/ß‐catenin activation may benefit from combination therapy with atezolizumab and bevacizumab regardless of potential predictive markers for immune checkpoint inhibition.


| INTRODUCTION
The hepatocellular carcinoma (HCC) is responsible for an increasing number of cancer-related deaths worldwide and is usually associated with liver cirrhosis. 1 Growing evidence indicates metabolic syndrome related to diabetes and obesity as mounting risk factors for HCC in the Western world. 2 While in patients with liver cirrhosis a multistep progression from cirrhotic nodules to HCC occurs, the mechanism is more heterogeneous in noncirrhotic patients. There, chronic infection with HBV, nonalcoholic steatohepatitis or the malignant transformation of hepatocellular adenoma are involved in cancer development.
Preclinically, two major molecular subtypes of HCC exist: a proliferation and nonproliferation class. The latter one is often characterized by an activation of the WNT signaling pathway based on the mutation of CTNNB1 (encoding for β-catenin). CTNNB1 and TERT mutations are frequently detected in liver adenomas and are marked by a particularly high rate of malignant progression. Besides tumor cell-specific alterations the tumor microenvironment (TME) has been increasingly recognized as modulator of tumor initiation and progression.  However, side effects and increasing deterioration of the nutritional status led to several treatment interruptions. After therapy with lenvatinib, sorafenib and ramucirumab (although AFP negative) the primary tumor in the liver was progressive (more than 25 cm in its largest extension) and a new lesion of the left adrenal gland, multiple suspicious abdominal lymph nodes and suspected diffuse small lung metastases occurred (timeline Figure 2). At this point we applied to the health insurance for permission to start a therapy with atezolizumab plus bevacizumab, not approved at that time. In parallel, we evaluated markers for response to immunotherapy of the liver biopsy made during first diagnosis. The immune checkpoint molecule PD-L1 (TPS = 0% and CPS = 1) was negative (Figure 3(A)). Similarly, there was no significant infiltration of T cells (Figure 3(B)) and the tumor presented a microsatellite stable situation (Figure 3(C)). During workup for immune markers, the tumor and matched normal liver tissue were reexamined. Based on a strong and homogeneous glutaminsynthetase (GS) expression ß-catenin was assessed and depicted a ubiquitous nuclear staining (Figure 1(B)). Given the literature to the pattern of GS and ß-catenin, a CTNNB1-exon-3-mutation can be

| DISCUSSION
From a scientific point of view there is a strong rationale for combining immunotherapy with VEGF inhibitors. Via complex mechanisms VEGF inhibition can modulate the immunosuppressive TME into an immunostimulatory micromilieu. 3 The results of the IMbrave study confirmed this preclinical hypothesis for HCC and thus may explain why checkpoint monotherapies have failed in the first-and second-line setting. Therapies with tyrosine kinase inhibitors (TKIs) such as lenvatinib (NCT03713593, LEAP-002 study) or cabozantinib (NCT03755791, COSMIC-312 study) may also be very promising in this context; however, the spectrum of side effects of TKIs has to be taken into account in patients with reduced performance status. Antiangiogenic therapy in combination with immunotherapy appears to be independent of the PD-L1 status. In addition, emerging evidence arises that activated Wnt/βcatenin signaling is associated with nonimmunogenic "cold" tumors and may be implicated in primary resistance to immunotherapy. 4 This hypothesis is supported by preclinical and clinical data. 5 Cells from the innate and adaptive immune response such as Treg cells F I G U R E 4 Representative computer tomographic images of the hepatocellular carcinoma in the liver in frontal and transverse views and polarized M2 macrophages secrete angiogenic factors to promote unrestrained angiogenesis and vascular immaturity. 6 It is precisely through this state that VEGF inhibition could have a crucial part in the effect of immunotherapy. Bevacizumab may normalize the aberrant vascular-immune crosstalk by reorganization of malformed tumor vessels to improve the infiltration of CD8+ T and CD4+ TH1 cells into the TME. Therefore, combination therapy with atezolizumab and bevacizumab should be investigated more extensively in the subgroups of HCC patients with CTNNB1 mutation as well as in noncirrhotic HCC.

| CONCLUSION
To the best of our knowledge, this is the first report showing an impressive response mediated by atezolizumab and bevacizumab in the fourth-line setting of a metastatic HCC in a patient with noncirrhotic liver and ß-catenin activation.

ACKNOWLEDGMENTS
Not applicable.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest. There was no financial support. Written consent has been obtained from the patient.

ETHICAL STATEMENT
Written informed consent for publication was obtained from the patient.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author.