Prognostic Value of Gene Signatures and Proliferation in Lymph-Node-Negative Breast Cancer

<div><p>Introduction</p><p>The overall survival rate is good for lymph-node-negative breast cancer patients, but they still suffer from serious over- and some undertreatments. Prognostic and predictive gene signatures for node-negative breast cancer have a high number of genes related to proliferation. The prognostic value of gene sets from commercial gene-expression assays were compared with proliferation markers.</p><p>Methods</p><p>Illumina WG6 mRNA microarray analysis was used to examine 94 fresh-frozen tumour samples from node-negative breast cancer patients. The patients were divided into low- and high-risk groups for distant metastasis based on the MammaPrint-related genes, and into low-, intermediate- and high-risk groups based on the recurrence score algorithm with genes included in Oncotype DX. These data were then compared to proliferation status, as measured by the mitotic activity index, the expressions of phosphohistone H3 (PPH3), and Ki67.</p><p>Results</p><p>Kaplan-Meier survival analysis for distant-metastasis-free survival revealed that patients with weak and strong PPH3 expressions had 14-year survival rates of 87% (<i>n</i> = 45), and 65% (<i>n</i> = 49, <i>p</i> = 0.014), respectively. Analysis of the MammaPrint classification resulted in 14-year survival rates of 80% (<i>n</i> = 45) and 71% (<i>n</i> = 49, <i>p</i> = 0.287) for patients with low and high risks of recurrence, respectively. The Oncotype DX categorization yielded 14-year survival rates of 83% (<i>n</i> = 18), 79% (<i>n</i> = 42) and 68% (<i>n</i> = 34) for those in the low-, intermediate- and high-risk groups, respectively (<i>p</i> = 0.52). Supervised hierarchical cluster analysis for distant-metastasis-free survival in the subgroup of patients with strong PPH3 expression revealed that the genes involved in Notch signalling and cell adhesion were expressed at higher levels in those patients with distant metastasis.</p><p>Conclusion</p><p>This pilot study indicates that proliferation has greater prognostic value than the expressions of either MammaPrint- or Oncotype-DX-related genes. Furthermore, in the subgroup of patients with high proliferation, Notch signalling pathway genes appear to be expressed at higher levels in patients who develop distant metastasis.</p></div

Long-term distant-metastasis-free survival curves according to PPH3 expression (A), classification by Oncotype DX RS (B), and classification by genes related to MammaPrint (C).

<p>Long-term distant-metastasis-free survival curves according to PPH3 expression (A), classification by Oncotype DX RS (B), and classification by genes related to MammaPrint (C).</p

Supervised hierarchical clustering for DMFS.

<p>Each row represents an mRNA and each column represents a patient sample. The mRNA clustering tree is shown on the left, and the sample clustering tree appears at the top. The colour scale shown at the bottom illustrates the relative expression level of an mRNA across all samples: red colour represents an expression level above mean, blue colour represents expression lower than the mean. Gray colour means that the specific mRNA has not been successfully detected with microarray. Numbers for clinicopathological features indicate the following: EOFUS (1 =  no distant metastasis, 2 =  distant metastasis), Tsize (Tumour size: 1≤2 cm, 2 >2 cm) and Nottgrade (Nottingham grade: 1 =  grade 1, 2 =  grade 2, 3 =  grade 3), Histologic type (1 =  Tubular, 2 =  Colloid, 3 =  Medullary, 4 =  Lobular, 5 =  Ductal, 6 =  mix Ductal/Lobular, 7 =  Others), MAI10 (1<10, 2≥10), PPH3_13 (0<13, 1≥13), HER2 (0 = 0 or 1+, 1 = 2+ or 3+), PR (1<1%, 2 = 1–9% positive tumour cells, 3≥10% positive tumour cells), ERα (1<1% positive tumour cells, 3≥1% positive tumour cells), TNP (0 =  positive for either ERα/PR/Her2, 1 =  negative for ERα and PR and HER2), and CK5/6 (0 =  no staining, 1 =  any percentage of positive tumour cells).</p

Distant-metastasis-free survival in lymph node negative breast cancer patient with Kaplan-Meier survival- and univariate analysis.

<p>Distant-metastasis-free survival in lymph node negative breast cancer patient with Kaplan-Meier survival- and univariate analysis.</p

List of gene-ontology terms related to 82-gene signatures and presence or not of distant metastases.

<p>List of gene-ontology terms related to 82-gene signatures and presence or not of distant metastases.</p

Supervised hierarchical clustering for ERα.

<p>Genes were filtered using analysis of variance, p-value ≤0.0001 and absolute correlation for ERα. The heat-map diagram shows the result of the two-way hierarchical clustering of miRNAs and samples. Good prognosis indicates patients with DMFS, while worse prognosis refers to patients with distant metastasis or who have died of distant metastasis. Each row represents a miRNA and each column represents a patient sample. The miRNA clustering tree is shown on the left, and the sample clustering tree appears at the top. The colour scale shown at the bottom illustrates the relative expression level of a miRNA across all samples: red colour represents an expression level above mean, blue colour represents expression lower than the mean. Gray colour means that the specific miRNA has not been successfully detected with qPCR. Numbers for clinicopathological features indicate the following: EOFUS (0 = no distant metastasis, 1 = distant metastasis), MAI10 (0<10, 1≥10), ERα (0<1% positive tumour cells, 1≥1% positive tumour cells), PPH3_13 (0<13, 1≥13), KP_Ki67 (0<10%, 1≥10%), Her2 (0 = 0 or 1+, 1 = 2+ or 3+), TNP (0 = positive for either ERα/PR/Her2, 1 = negative for ERα and PR and Her2), PR (0≤10% positive tumour cells, 1>10% positive tumour cells), CK5/6 (0 = no staining, 1 = any percentage of positive tumour cells), Tsize (Tumour size: 0≤2cm, 1>2 cm) and Nottgrade (Nottingham grade: 1 = grade 1, 2 = grade 2, 3 = grade 3).</p

Independent t-test between miRNAs and different clinical features for breast cancer.

<p>Proliferation includes MAI, PPH3 and Ki67. Star indicated that Ki67 did not give P-value under 0.05. The miRNAs with underscore/fat are inversely correlated to the different clinical features, meaning that low expression of let-7b is significantly associated to high proliferation.</p

An illustration of the miRNAs impact on ERα, based on literature and results presented in this paper.

<p>An illustration of the miRNAs impact on ERα, based on literature and results presented in this paper.</p

Kaplan-Meier survival analysis for Her2 and miR-106b.

<p>Kaplan-Meier survival analysis for Her2 and miR-106b.</p

Pathway analysis of the nine microRNAs by means of Diana microT 4.0 (beta version), PicTar and Targetscan 5.

<p>Only the most significant (from the top three) pathway that is common in all three software programmes is mentioned. Abbreviations: Diana 4.0 = Diana microT 4.0 (beta version), PT = PicTar, TS5 = Targetscan 5.</p