Background: Hepatocellular carcinoma (HCC) typically occurs in patients with chronic liver disease. Such diseases create a demand for liver regeneration due to injury, which in turn promotes upregulation of c-Met, a receptor for hepatocyte growth factor (HGF) involved in wound healing, leading to increased cellular proliferation, survival, and mobilization. c-Met is the product of the proto-oncogene MET, so increased c-Met activity not only effects wound healing but can also contribute to the development and progression of HCC. Aberrant c-Met signaling is associated with rapid tumor growth and aggressively invasive disease in HCC, resulting in poor patient prognosis. We examined c-Met expression and MET gene amplification in 69 procured HCC samples and c-Met expression in adjacent non-tumor tissue. Methods: c-Met expression was assessed using MET IHC MSC2156119J pharmDx anti-c-MET (clone D1C2) rabbit mAb. c-Met expression was scored semiquantitatively on a scale of 0-3+ based on the staining intensity in ≥50% of cells [Koeppen et al. Proc USCAP 2012 (abstract 2001)]; tumors scoring 2+ or 3+ were judged positive. MET gene copy number (GCN) was assessed using the MET IQFISH Kit-111480 with a probe covering a 269 kb segment on chromosome 7q31.2 containing the MET gene. Tumors were considered to be FISH positive if scored cells had a mean MET/CEN7 ratio ≥2.0 or ≥50% of cells contained ≥5 MET signals. Results: All cases showed typical HCC morphology and were classified as low to high grade trabecular, pseudoglandular, or solid with common cytoplasmic features. The tumor cells and cells of the adjacent cirrhotic/non-cirrhotic liver tissue (bile duct epithelia, hepatocytes endothelial cells) showed cytoplasmic and membrane staining of varying extent and intensity. c-Met staining in tumor was heterogeneous and present in 68/68 cases. Semiquantitative analysis showed that 11/67 (16%) tumors were c-Met-positive (3+, 3 cases; 2+, 8 cases). Six out of 69 (8.7%) were amplified by ratio and 3 (4.3%) others had ≥5 MET gene copies in ≥50 % of cells, giving an overall frequency of FISH positivity of 13%. Most interestingly, amplification and/or increased gene copy number was homogeneous throughout the tumor in all cases, in contrast to heterogeneous c-Met staining observed in some of the same tumors with IHC. Five of the 11 c-Met-positive (IHC 2+/3+) tumors were MET amplified, and all 3 of the IHC 3+ tumors were amplified. Conversely, 4 of the 9 FISH-positive tumors were c-Met-negative. Conclusion: These data provide insight into the frequency of c-Met/MET abnormalities in HCC and show there is discordance between c-Met protein expression and MET GCN alterations. This may be of importance for selection of patients with HCC for clinical trials of c-Met inhibitors. Citation Format: Christian Ihling, Sienna Yoast, Matthew DeNicola, Josef Straub, Klaus Dücker, Karsten Nielson, Russell A. Baldocchi, Aaron R. Ellison, Holly Yamada. Analysis of c-Met protein expression by immunohistochemistry and MET gene copy number in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4668. doi:10.1158/1538-7445.AM2017-4668
Still-emerging evidence suggests that HCC is at least moderately responsive to therapeutic agents that target tumor immune suppression pathways, specifically the programmed death 1/programmed death ligand 1 (PD-L1) pathway. Prior studies raise the possibility that PD-L1 is a prognostic biomarker in HCC, where its expression levels have been reported to correlate with tumor aggressiveness and recurrence following surgical resection. The purpose of this study was to evaluate the expression of PD-L1 in non-cirrhotic liver tissue adjacent to HCC and HCC itself in 68 procured tissue samples, as well as elucidate the immune cell composition of the HCC tumor microenvironment. All cases showed the typical morphology of HCC and were classified as low- to high-grade trabecular, pseudoglandular, or solid with the common cytoplasmic features. Immunohistochemical (IHC) staining for PD-L1 in tumor-free liver (TFL) revealed PD-L1 staining in sinusoidal lining cells (macrophages [Mϕ] and endothelial cells [EC], as confirmed by double labeling for CD68 and CD31), although there was considerable heterogeneity in the extent of PD-L1 staining. More specifically, in TFL, some but not all CD68+ Mϕ were also PD-L1+, whereas most CD31+ EC were also PD-L1+. Furthermore, we evaluated PD-L1 staining qualitatively and semiquantitatively in HCC. Only few tumor cells displayed membranous PD-L1 staining, and 62/68 of the cases (91.2%) were categorized as PD-L1- vs 6/68 (8.8%) PD-L1+ (≥1% and <25% = PD-L1+); instead, PD-L1 expression within the tumor microenvironment predominantly emanated from immune cell infiltrates (as confirmed by PD-L1/pan-cytokeratin double labeling). Then we assessed the immune milieu in HCC tissue specimens using quantitative IHC. We found considerable interspecimen variation in the number of CD68+ Mϕ, CD8+ cytotoxic T lymphocytes, and FoxP3+ regulatory T lymphocytes. Comparing the number of immune cells in different tumor compartments showed that the prevalence of CD68+ cells (p=.0005), CD8+ cells (p=.0004), and FoxP3+ cells (p=.05) was significantly higher within the invasive margin vs the center of the tumor. Quantitative analysis of the immune cell content also showed that there was no correlation between T lymphocyte infiltration (CD8+/FoxP3+) and Mϕ infiltration in any compartment. Taken together, our findings confirm the intertumoral heterogeneity of the immune cell microenvironment in HCC; however, future studies are needed to correlate these findings to the clinical setting with immunooncologic treatments. Citation Format: Christian Ihling, Sienna Yoast, Yue Zhang, Bartholomew Naughton, Miriam Urban, P. Alexander Rolfe, Eveline Frick-Krieger, Isabelle Dussault. Characterization of PD-L1 expression and the immune cell microenvironment in hepatocellular carcinoma (HCC) and non-cirrhotic liver tissue adjacent to HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 624. doi:10.1158/1538-7445.AM2017-624
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