Introduction: Hepatocellular carcinoma (HCC) is the 2nd most common cause of cancer-related death worldwide, and a major cause of cirrhosis-related mortality. Most patients present with advanced disease requiring systemic therapy¹. While oral tyrosine kinase inhibitor (sorafenib) is the frontline standard-of-care treatment for advanced HCC, drug intolerance due to side-effects and drug resistance are common. Promising newer systemic treatments with immune checkpoint blockade (ICB), such as anti-PD-1 antibodies (e.g. pembrolizumab and nivolumab), reduce tumor-mediated suppression of cytotoxic T-cells². However, a major challenge of ICB treatment in HCC is the lack of predictive biomarkers of outcome³. MR Elastography (MRE) employs propagating mechanical shear waves, which are imaged using a modified phase contrast pulse sequence with cyclic motion encoding gradients⁴. While MRE is used clinically to measure liver stiffness (kPa) as a surrogate for liver fibrosis, HCC shear stiffness has been reported to be lower after loco-regional ablation compared to untreated tumors ^{5, 6}. In this work we evaluated whether HCC stiffness change on Magnetic Resonance Elastography (MRE) and tumor enhancement are associated with response to ICB.

Methods: This was a prospective, Institutional Review Board approved study. We enrolled 38 patients with biopsy proven HCC and Child-Pugh Score A (non-surgical n=15; pre-surgical n=23) through our Liver Center. Data from the non-surgical patients has been previously published⁷. All patients provided written informed consent. Eligible patients were over 18 years of age with ECOG 0 or 1. All patients had contrast enhanced liver MRI/MRE and HCC biopsy at baseline, and repeat MRI/MRE with biopsy or resection after 6 weeks immunotherapy. Treatment response was defined as overall survival >1 year for non-surgical patients (treated with anti-PD-1 antibody pembrolizumab), and <50% viable tumor on resection for surgical patients (treated with anti-PD-1 antibody nivolumab +/- anti-CTL4 ipilimumab).
MRI and MRE was performed on a 3T whole body MRI scanner with a 32‐channel phased‐array torso coil. Liver MRI included the following: axial T2-weighted FRFSE, coronal SSFSE, axial IDEAL-IQ; T2* mapping for iron and fat quantification; axial DWI (b-values 50, 400, 800); axial LAVA pre- and post-dynamic contrast enhancement with gadoxetate and 20 minute delay (hepatobiliary phase) imaging. Liver MRE exam was performed with acoustic waves at 60 Hz ³. Data was acquired using a 2D-echoplanar imaging (EPI) based MRE sequence with the following imaging parameters: TR/TE = 600ms/min; slice thickness/gap =7/2.5mm; FOV 38-42cm; matrix = 64x64; NEX = 2; motion encoding gradient along the z-direction, 6 axial slices through widest cross-section of the liver including the bulk of the tumor; parallel imaging factor = 2, and acquisition time = 16-19 seconds. Automated in-line post-processing was used to generate quantitative maps or “elastograms” of liver shear stiffness in units of kilopascals (kPa) ^3-5.
Images were reviewed by 2 radiologists blinded to data on treatment response. Percent HCC enhancement was categorized on arterial phase images using a 5-point scale (0-4): none=0, <25%=1, 25-50%=2, >50 to 75%=3, >75% =4. HCC size was measured on 20 minute delay contrast-enhanced images (hepatobiliary phase). MRE-derived average stiffness measurements (kPa) were measured by placing regions-of-interest (ROIs) on the MRE elastograms (stiffness maps) as previously described ^{5, 6}. Average HCC stiffness was measured by drawing ROIs on the elastograms covering as much of the tumor as possible on adjacent slices (minimum of 2 slices). In the case of multiple tumors, the largest tumor was selected. HCC apparent diffusion coefficient (ADC) was measured by placing ROIs to include the darkest regions of the tumor on the ADC map.
Spearman rank correlation was estimated between imaging measurements (baseline and change in both HCC size and stiffness, ADC value and percent arterial phase enhancement) at the 2 time-points and treatment response. Wilcoxon rank-sum test or Fisher’s Exact test was used for comparison of HCC stiffness and change in stiffness between non-responders and responders; p-value <0.05 was considered statistically significant. Statistical analyses was performed using SAS version 9.3 and JMP version 14.0 (SAS Institute, Cary, NC).

Results: 25 patients were evaluable. Median age 67 years (32, 78). Etiology of liver disease: NASH (n=8), HCV (n=8), HBV (n=2) and unknown (n=7). Treatment response occurred in 11/25 (44%) patients: 5/9 (56%) of non-surgical and 6/16 (38%) of surgical cases.
Median HCC size and change in size were 4.7 cm (1.2, 14.0) and –0.32 cm, respectively. Median baseline HCC stiffness and change in stiffness were 5 kPa (2.2, 12.4) and –0.1 kPa (–2.2, 1.5), respectively. Median change in HCC size for responders and non-responders was –1.2 cm (–4.8, 0.4) and 0 cm (–1.5, 1.1), respectively (p=0.02).
Treatment response was significantly correlated with increase in HCC stiffness at 6 weeks (p< 0.001), Figures 1 and 2. No significant correlation was seen between treatment response and change in HCC size, ADC value, or percent HCC enhancement.

Discussion: Immunotherapy response correlated with an increase in MRE stiffness. Immune checkpoint blockade has been associated with HCC immune cell infiltration⁸. Our observation of an increase in HCC stiffness of responding patients could reflect an increase in tumor lymphocyte infiltration and associated alterations in the tumor matrix.

Conclusion: MRE stiffness change may be useful biomarkers of immune cell activated response to ICB therapy.

Acknowledgements

No acknowledgement found.