Angiogenesis in SDH and VHL-related PH/PGL.

<p>(A) CD34 immunohistochemistry was performed to evaluate angiogenesis in all samples. Diaminobenzidin was used as a chromogen for detection (brown labeling). Calibration bar: 200 µm. (B) Quantification of vascular density showing an increased number of blood vessels in SDH, and VHL tissues. Data are means±SEM. *p<0.05, **p<0.01, ***p<0.001. (C) Correlation between vascular density and SCCR enzymatic values for individual patients.</p

Microarray analysis of oxidative phosphorylation in hereditary PH/PGL.

<p>(A) Unsupervised hierarchical clustering analysis of the 68 samples according to the expression of 200 genes. Expression profiles are shown as a heat map indicating high (red) and low (blue) expression according to a log2-transformed scale. The higher bipartition allows to distinguish VHL (white) and SDH (grey) patients from RET and NF1 (black) patients. (B) Principal component analysis of the 68 samples according to the expression of 200 genes. Three groups are focused on, corresponding to the SDH (red), VHL (green) and RET/NF1 (blue) patients. PC1: principal component; PC2: principal component 2; PC3: principal component 3. (C) Mean values for genes expression between SDH, VHL, RET and NF1 tumors. Data are means±SEM, represented as relative to NF1 expression values. **p<0.01, ***p<0.001.</p

Decreased oxidative phosphorylation in SDH and VHL-related PH/PGL.

<p>(A) The abundance of proteins of mitochondrial complexes I (20 kDa subunit), II (SDHB and SDHA), III (Core 2) and IV (Cox II) is lower in PH/PGL from SDH and most VHL than from RET and tumor tissues. (B) SDHB immunohistochemistry performed on the adrenal adjacent to a RET-related PH and in RET, VHL and SDHD-mutated PH reveals a strong labeling in the adrenal compared to tumor cells (asterisks). In VHL PH/PGL tumor, expression of SDHB was reduced when compared to RET-related PH while it is absent in SDHD-related tumor. Note that vascular immunostaining was present in all samples (arrows). Calibration bar: 50 µm. (C) Individual values of SCCR activity reveal that low complex II+III enzymatic activity is associated with low protein abundance. (D–F) Mean values for mitochondrial complexes II+III, III and IV reveal a generalized decrease in respiration in SDH and VHL PH/PGL. Data are means±SEM. **p<0.01, ***p<0.001.</p

Microarray analysis of glycolysis in hereditary PH/PGL.

<p>(A) Unsupervised hierarchical clustering analysis of the 68 samples according to the expression of 38 genes. Expression profiles are shown as a heat map indicating high (red) and low (blue) expression according to a log2-transformed scale. The different mutations are localized in three distinct clusters: SDH (grey), VHL (white) and RET/NF1 (black). (B) Mean values for genes expression between SDH, VHL, RET and NF1 tumors. Data are means±SEM, represented as relative to NF1 expression values. **p<0.01, ***p<0.001.</p

Pseudohypoxia in SDH and VHL-related PH/PGL.

<p>(A) HIF-1α and HIF-2α immunohistochemistry were performed to evaluate activation of the hypoxic pathway in all samples. Histogreen was used as a chromogen for detection (blue labeling). Calibration bar: 100 µm. Microarray evaluation of HIF-1α (B) and HIF-2α (C) expression between SDH, VHL, RET and NF1 tumors. Data are means±SEM. ***p<0.001.</p

Gene Expression Classification of Colon Cancer into Molecular Subtypes: Characterization, Validation, and Prognostic Value

<div><p>Background</p><p>Colon cancer (CC) pathological staging fails to accurately predict recurrence, and to date, no gene expression signature has proven reliable for prognosis stratification in clinical practice, perhaps because CC is a heterogeneous disease. The aim of this study was to establish a comprehensive molecular classification of CC based on mRNA expression profile analyses.</p><p>Methods and Findings</p><p>Fresh-frozen primary tumor samples from a large multicenter cohort of 750 patients with stage I to IV CC who underwent surgery between 1987 and 2007 in seven centers were characterized for common DNA alterations, including <i>BRAF</i>, <i>KRAS</i>, and <i>TP53</i> mutations, CpG island methylator phenotype, mismatch repair status, and chromosomal instability status, and were screened with whole genome and transcriptome arrays. 566 samples fulfilled RNA quality requirements. Unsupervised consensus hierarchical clustering applied to gene expression data from a discovery subset of 443 CC samples identified six molecular subtypes. These subtypes were associated with distinct clinicopathological characteristics, molecular alterations, specific enrichments of supervised gene expression signatures (stem cell phenotype–like, normal-like, serrated CC phenotype–like), and deregulated signaling pathways. Based on their main biological characteristics, we distinguished a deficient mismatch repair subtype, a <i>KRAS</i> mutant subtype, a cancer stem cell subtype, and three chromosomal instability subtypes, including one associated with down-regulated immune pathways, one with up-regulation of the Wnt pathway, and one displaying a normal-like gene expression profile. The classification was validated in the remaining 123 samples plus an independent set of 1,058 CC samples, including eight public datasets. Furthermore, prognosis was analyzed in the subset of stage II–III CC samples. The subtypes C4 and C6, but not the subtypes C1, C2, C3, and C5, were independently associated with shorter relapse-free survival, even after adjusting for age, sex, stage, and the emerging prognostic classifier Oncotype DX Colon Cancer Assay recurrence score (hazard ratio 1.5, 95% CI 1.1–2.1, <i>p</i> = 0.0097). However, a limitation of this study is that information on tumor grade and number of nodes examined was not available.</p><p>Conclusions</p><p>We describe the first, to our knowledge, robust transcriptome-based classification of CC that improves the current disease stratification based on clinicopathological variables and common DNA markers. The biological relevance of these subtypes is illustrated by significant differences in prognosis. This analysis provides possibilities for improving prognostic models and therapeutic strategies. In conclusion, we report a new classification of CC into six molecular subtypes that arise through distinct biological pathways.</p><p><i>Please see later in the article for the Editors' Summary</i></p></div