Cancer Hallmarks inferred from expression profiles correlate with breast laterality.

<p>In a different cohort of 25 breast tumors, Cancer Hallmarks were inferred from the expression profiles of 32 cancer related genes. CHs are represented in rows, tumors in columns. A color gradient from green to red is used to represent low to high values of CHs (from 5 to 52). By Unsupervised Hierarchical Cluster Analysis even though many clusters are formed, only two groups were established for bootstrapping 90–100% (shown by the red arm). By regression analyses a single association was found between clusters and the clinical variable BL.</p

CH1 and CH6 define two tumor clusters.

<p>Clustering of CH profiles of 51 tumors. (A) Tumors are represented in columns, CHs in rows. A color gradient from green to red is used to represent low to high values of CHs (from 0 to 49). Unsupervised Hierarchical Cluster Analysis was performed by the software MultiExperiment Viewer MeV v4.6. Even though many clusters are formed, only two groups were established for bootstrapping 90–100% (shown as red arm). (B) Principal component analysis performed by software InfoStat v.2014 shows that 92.7% of the grouping is predictable by CH1 and CH6.</p

Hallmarks of Cancer selected for the study.

<p>Five of the six cancer hallmarks proposed by Hanahan and Weinberg were selected for this study: i.e. Sustained proliferative signaling (CH1), Evasion of growth suppressors (CH2), Resistance to cell death (CH3), Induction of angiogenesis (CH4) and Activation of invasion and metastasis (CH6) The Hanahan-Weinberg hallmark “Enabled replicative immortality” is replaced in this study by “Genome instability” (CH5) as an alternative hallmark. The number of genes participating in the CH are shown each ellipse.</p

CpG location, genes and methylation frequencies in 113 breast tumors.

<p>CpG location, genes and methylation frequencies in 113 breast tumors.</p

Translation of Methylation profile to Cancer Hallmark profile.

<p>(A) The scheme describes how the MLPA-derived methylation data was converted into CH profiles, though a translation matrix. A multiplication operation was performed on two matrices: the Methylation Profile Matrix (MPM) and the Translation Matrix (TM). The MPM holds information of 51 CpGs located in 43 genes the MPM, for 51 tumors with complete clinical-pathological information. Green boxes represent the un-methylated status and red boxes the methylated status. The TM contains the <u>A</u>djusted <u>P</u>articipation <u>I</u>ndex (API) which expresses in a rank from 0 to 3, the influence of each of the 43 genes on the 6 studied Cancer Hallmarks (CH). This multiplication of both results in a CH Profile Matrix, which represents for each tumor, the values with which each CH is enhanced. The higher the values of the CH matrix, the more the methylation events have contributed to acquire specific CHs. (B) Results of the matrices multiplication operation performed for the 51 studied tumors. Each tumor (rows) presents a specific CH profile. The CH values are represented in a colored grey gradient (light grey are lower values; dark grey represent higher values).</p

Tumors from left-right breast sides are differentially predicted by cancer hallmarks.

<p>Regression analyses of CHs vs tumor stage in left and right sided tumors. (A) CHs of left sided lesions were better predictors of the tumor stage, revealing that CH1 increased with tumor stage while CH4, CH5 and CH6 decreased (adjusted R² = 0.76). (B) CHs of right sided tumors have no predictable value for tumor stages (adjusted R² = 0.00), supporting thereby the conclusion that the behavior of CHs differs in tumors of different sides.</p

Genes included in the expression analyses.

<p>Genes included in the expression analyses.</p

Patient and Tumor characteristics.

<p>Patient and Tumor characteristics.</p

Cancer Hallmark profiles are not randomly distributed in experimental tumors.

<p>Histograms presenting the distribution of CH values in experimental vs artificial (hypothetical) tumors. (A) By summing the values of the 6 CHs in each tumor, a Global CH value was obtained. Comparisons of the histograms representing the distribution of the Global CH values of experimental vs hypothetical tumors revealed a significant difference (Kolmogorov Smirnov test (KS)*, p<0.05). (B) By performing the comparison of the distribution of the single CH values of experimental vs hypothetical tumors, a significant difference was detected for CH1 and CH6 (Kolmogorov Smirnov test (KS*), p<0.05). These differences are suggesting that the CH profiles in experimental tumors are not generated by random accumulation of CpG methylations.</p