HCC is considered the fifth most common malignancy worldwide, with the third-highest mortality [1]. An estimated that 80% of patients present with advanced stage tumor not amenable to curative therapy [1, 2]. Oral tyrosine kinase inhibitor (sorafenib) has been the frontline standard of care since 2007 for treatment of advanced HCC with preserved liver function [3]. Newer systemic treatments with immunotherapy agents are being investigated, such as Nivolumab and Pembrolizumab (anti–PD-1 mAb) which enhance immune function and cytotoxic T-lymphocyte (CTL)-mediated immune response against cancer cells [4–6].

Imaging assessment of HCC response to targeted therapies is challenging since reduction in size may not occur. Tumor stability is used as a marker of response without necessarily conferring improved outcomes [7–9]. MR Elastography (MRE) is a relatively novel technique, and has been shown to be superior to ultrasound-based transient elastography for assessment of liver fibrosis [10]. MRE may be used to distinguish malignant from benign liver tumors [11], which is thought be due to the abnormal cellular microenvironment of neoplastic conditions, including denser extracellular matrix, increase cellularity, vascularity, and interstitial pressure, causing increased stiffness.

Immunotherapy response decreases viable tumor cells, but increases immune content, and causes stromal and fibrosis flux due to effects on immune cell function. We hypothesize that such changes in tumor cellularity and stroma in patients treated with anti-PD-1 immunotherapy would effect MRE tumor stiffness. The purpose of our study was to determine if stiffness changes measured by magnetic resonance elastography (MRE) can be a predictor of immunotherapy response in patients with advanced HCC.

This was a prospective, Institutional Review Board approved study. A total of 15 patients were accrued through our Liver Center, with biopsy proven advanced HCC (not amenable to curative therapy), Child-Pugh Score A, who were treated with anti-PD-1, Pembrolizumab monotherapy. Clinical responses were determined by blinded independent review using RECIST 1.1 and mRECIST 1.1. All patients provided written informed consent. Eligible patients were over 18 years of age with radiographic disease progression on sorafenib or intolerance to sorafenib treatment, and ECOG 0 or 1. All patients underwent liver MRI with MR Elastography (MRE) and liver biopsy at baseline and at 6 weeks of therapy. Date of documented disease progression on patient follow-up and date of death were obtained from the patients’ electronic medical records.

MR Elastography (MRE) was performed on a 3 T whole body MRI scanner (Discovery 750 HD; GE Healthcare, Waukesha, WI) with a 32-channel phased-array torso coil. Acoustic waves at 60 Hz were generated by an active driver and transmitted to the liver through an external passive driver placed over the subject’s right upper quadrant (overlying the liver). Data was acquired using a 2D-echoplanar imaging (EPI) based MRE sequence with the following imaging parameters: TR/TE = 600 ms/Minimum Full; slice thickness/gap =7/2.5 mm; FOV 38-42 cm; acquisition matrix = 64 × 64; NEX = 2; 6 axial slices through widest cross-section of the liver including at least one slice through the tumor; parallel imaging factor = 2, and acquisition time = 16–19 s (one breath hold). Automated in-line post-processing was used to generate quantitative maps or “elastograms” of liver stiffness in units of kilopascals (kPa) [12–16], and a mask overlay to exclude pixels with low inversion processing confidence [12].

Of the total of 15 patients, 4 withdrew (2 died before the followup MRE scan could be performed, 1 patient decided on hospice care and declined further treatment; 1 patient was intolerant to treatment), 1 patient did not undergo MRE scan, and 1 patient had MRE exam failure. The remaining 9 patients included 6 men. Median age was 71 years (range, 54–78).

HCC was well-differentiated in 2 of 9 patients, moderately differentiated in 6 and poorly differentiated in 1. Median overall survival and time to progression were 44 weeks (range, 16–70) and 13 weeks (range, 9–48), respectively. Etiology of liver disease was HCV (n = 4), HBV (n = 1) and NASH (n = 4).

No correlation was found for stiffness of non-tumor liver and etiology of liver disease.

Average non-tumor liver stiffness was 3.2 kPa (range, 2.1–4.3), and did not significantly change at 6 weeks (p = 0.42). Baseline non-tumor liver stiffness did not significantly correlate with overall survival, (p = 0.056), Table 1.Tab1

Correlation of imaging and pathology with with overall survival; (R, Spearman correlation coefficient; kPa, kilopascals)

Seven of 9 HCC demonstrated > 50% enhancement at baseline, 1 demonstrated 20–50% and 1 < 25%. Decrease in HCC enhancement category was only seen in 2 of 9 patients at 6 weeks.

Correlation of overall survival with baseline and change in HCC size and stiffness are shown in Table 1. Average HCC size and change in size were 4 cm (range, 1.5–8.5) and - 0.32 (range, [− 2.2] - 0.4), respectively. There was no significant correlation between overall survival and baseline HCC size (p = 0.4).

Average baseline HCC stiffness and change in stiffness were 5 kPa (range, 2.4–9.1) and 0.12 kPa (range, [− 2.1] – 2.8), respectively. There was no significant correlation between overall survival and baseline HCC stiffness (p = 0.055), Table 1. Increase in HCC stiffness on follow-up imaging (Fig. 1a and b) was seen in 5 patients, decrease in 3 patients and no change in 1 patient (Table 2). Increase in HCC stiffness at 6 weeks correlated significantly with overall survival (R = 0.81, p = 0.008), Fig. 2a, and with survival of more than 52 weeks from start of therapy (p = 0.02), Fig. 2b. Increase in HCC stiffness at 6 weeks also correlated significantly with time to progression (R = 0.88, p = 0.009), Fig. 2c.Fig1Fig. 1

Elastogram color map. a gray scale b at baseline showing HCC (arrow). HCC stiffness increased on as indicated by increased red color of the tumor (c and d). Liver is demarcted by hashed lines (----)

Imaging predictors of immunotherapy response in HCC could aid the identification of patients more likely to benefit from the treatment and represent an important unmet need. Our preliminary data suggests that an early increase in HCC stiffness may be an indicator of early immunotherapy (anti–PD-1) response.

There is limited data on MRE assessment of HCC. Thompson et al. [17], reported a trend toward increased HCC stiffness in well-moderately differentiated compared to poorly differentiated HCC but no correlation with size (n = 21). Our findings are concordant with this study since we also did not observe a significant correlation between tumor stiffness and tumor grade. However, this could have been due to there only being 1 patient with poorly differentiated HCC in our study. We observed a correlation between HCC stiffness and HCC size (p = 0.036), which is discordant with Thompson et al. [17]. This may have been related to differences in HCC stiffness measurement technique. Thompson et al. [17], only included solid portions of the tumor, but we included as much of the tumor as possible. Furthermore, it is possible that measurements of smaller tumors are assocated with partial volume averaging from the adjacent liver. Larger studies stratifying stiffness measurements with respect to tumor size are needed to understand the relationship between thesee observations.

To our knowledge, there are no prior studies evaluating HCC stiffness changes using MRE in patients treated with immunotherapy. Interestingly, increase in HCC stiffness at 6 weeks was correlated with improved outcome but not change in HCC size or enhancement. Our findings suggest effects related to immune cell infiltration and alteration in tumor stroma (including fibrosis and angiogenesis), can result in early increased tumor stiffness as an independant biomarker of response.

We found a significant correlation between HCC stiffness on MRE and the number of lymphocytes on tumor biopsy. This is supportive of the theory that anti-PD-1 therapy causes increased T lymphocyte activation in the immune-mediated response to tumor. However, further larger studies are necessary to better understand the underlying mechanisms. If our findings are confirmed, MRE would have an important clinical impact on the response assessment of advanced HCC treatment with checkpoint inhibitors, enabling early identification of treatment response.

Our study has some limitations. First, this is a pilot study that is meant to be hypothesis-generating, and the number of patients is small. Nevertheless, our study showed a strong correlation between early increase in tumor stiffness on MRE and overall survival. Further larger studies are necessary to validate our initial promising observations. Second, HCC was sampled with image-guide core biopsies. Given the heterogeneity of HCC, the biopsy sample may not have been representative of the whole tumor. However, we did observe a correlation between HCC stiffness and tumor T lymphocytes. Third, since patients were being treated with pembrolizumab, they had previously failed treatment or were intolerant to sorafenib. Prior treatment may have affected patient outcomes. However, anti-PD-1 therapy was used as a second-line treatment for all the subjects. Our study focused on change in HCC stiffness on serial MRE, and showed all patients that had an increase in tumor stiffness had better outcomes irrespective of baseline HCC stiffness. Fourth, in our exploratory study HCC stiffness measurements were made by a single independent radiologist placing region-of-interest on the tumor. Future larger studies could benefit having more than 1 radiologist measure tumor stiffness to allow assessment of interobserver agreement.

In summary, our preliminary data demonstrated that HCC stiffness increase on MRE in patients treated with immunotherapy significantly correlated with overall survival and time to progression. MRE has the potential to be a useful tool in the assessment of anti-PD-1 therapy in advanced HCC and may be beneficial to the many active immunotherapy trials.

Our pilot MRE data suggests early change in tumor stiffness may be an indicator of immunotherapy response in patients with advanced HCC.