Data_Sheet_1.pdf

<p>The TP53 gene is the most commonly mutated gene in human cancers and mutations in TP53 have been shown to have either gain-of-function or loss-of-function effects. Using the data generated by The Cancer Genome Atlas, we sought to define the spectrum of TP53 mutations in hepatocellular carcinomas (HCCs) and their association with clinicopathologic features, and to determine the oncogenic and mutational signatures in TP53-mutant HCCs. Compared to other cancer types, HCCs harbored distinctive mutation hotspots at V157 and R249, whereas common mutation hotspots in other cancer types, R175 and R273, were extremely rare in HCCs. In terms of clinicopathologic features, in addition to the associations with chronic viral infection and high Edmondson grade, we found that TP53 somatic mutations were less frequent in HCCs with cholestasis or tumor infiltrating lymphocytes, but were more frequent in HCCs displaying necrotic areas. An analysis of the oncogenic signatures based on the genetic alterations found in genes recurrently altered in HCCs identified four distinct TP53-mutant subsets, three of which were defined by CTNNB1 mutations, 1q amplifications or 8q24 amplifications, respectively, that co-occurred with TP53 mutations. We also found that mutational signature 12, a liver cancer-specific signature characterized by T>C substitutions, was prevalent in HCCs with wild-type TP53 or with missense TP53 mutations, but not in HCCs with deleterious TP53 mutations. Finally, whereas patients with HCCs harboring deleterious TP53 mutations had worse overall and disease-free survival than patients with TP53-wild-type HCCs, patients with HCCs harboring missense TP53 mutations did not have worse prognosis. In conclusion, our results highlight the importance to consider the genetic heterogeneity among TP53-mutant HCCs in studies of biomarkers and molecular characterization of HCCs.</p

Vascular endothelial growth factor A amplification in colorectal cancer is associated with reduced M1 and M2 macrophages and diminished PD-1-expressing lymphocytes

<div><p>VEGFA is an angiogenic factor secreted by tumors, in particular those with <i>VEGFA</i> amplification, as well as by macrophages and lymphocytes in the tumor microenvironment. Here we sought to define the presence of M1/M2 macrophages, PD-1-positive lymphocytes and PD-L1 tumoral and stromal expression in colorectal cancers harboring <i>VEGFA</i> amplification or chromosome 6 polysomy. 38 CRCs of which 13 harbored <i>VEGFA</i> amplification, 6 with Chr6 polysomy and 19 with neutral <i>VEGFA</i> copy number were assessed by immunohistochemistry for CD68 (marker for M1/M2 macrophages), CD163 (M2 macrophages), programmed death 1(PD-1)- tumor infiltrating and stromal lymphocytes as well as tumoral and stromal PD-1 ligand (PD-L1) expression. CRCs with <i>VEGFA</i> amplification or Chr6 polysomy were associated with decreased M1/M2 macrophages, reduced PD-1-expressing lymphocyte infiltration, as well as reduced stromal expression of PD-L1 at the tumor front. Compared to intermediate-grade CRCs, high-grade CRCs were associated with increased M1/M2 macrophages and increased tumoral expression of PD-L1. Our results suggest that <i>VEGFA</i> amplification or Chr6 polysomy is associated with an altered tumor immune microenvironment.</p></div

<i>VEGFA</i> copy number status in colorectal cancers measured by fluorescent <i>in situ</i> hybridization.

<p>Representative micrographs of (A) diploid, (B) polysomic and (C) amplified <i>VEGFA</i> using fluorescent <i>in situ</i> hybridization (FISH). FISH analysis was performed using two-color probes for <i>VEGFA</i> (red) and internal control (green). Scale bar 20 μm.</p

Number of macrophages in colorectal cancers, using CD68 and CD163 markers.

<p>Boxplots depict the number of (A, C) CD68+ and (B, D) CD163+ cells (A, B) in CRCs with and without <i>VEGFA</i> amplification/polysomy and (C, D) in CRCs of intermediate and high grade.</p

PD-1-positive tumor infiltrating and stromal lymphocytes and PD-L1 tumoral and stromal expression in colorectal cancers.

<p>Barplots depict the number of samples with high, low and negative expression of (A, B) PD-1 and (C, D) PD-L1 in (A, C) tumoral and (B, D) stromal areas of CRCs.</p

Distribution of macrophages in colorectal cancers, using CD68 and CD163 markers.

<p>Representative micrographs of (A, C) CD68+ cells and (B, D) CD163+ cells (A, B) at the invasive tumor front (T) and (C, D) in the surrounding tumor tissue (S). Magnification 40X. Scale bar 20 μm.</p