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A gene signature in histologically normal surgical margins is predictive of oral carcinoma recurrence.

Reis PP, Waldron L, Perez-Ordonez B, Pintilie M, Galloni NN, Xuan Y, Cervigne NK, Warner GC, Makitie AA, Simpson C, Goldstein D, Brown D, Gilbert R, Gullane P, Irish J, Jurisica I, Kamel-Reid S - BMC Cancer (2011)

Bottom Line: We identified 138 significantly over-expressed genes (> 2-fold, false discovery rate of 0.01) in OSCC.This signature comprised the invasion-related genes MMP1, COL4A1, P4HA2, and THBS2.Gene expression alterations occur in histologically normal margins in OSCC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Div, of Applied Molecular Oncology, Princess Margaret Hospital, Ontario Cancer Institute, University Health Network, Toronto, ON, Canada. juris@ai.utoronto.ca.

ABSTRACT

Background: Oral Squamous Cell Carcinoma (OSCC) is a major cause of cancer death worldwide, which is mainly due to recurrence leading to treatment failure and patient death. Histological status of surgical margins is a currently available assessment for recurrence risk in OSCC; however histological status does not predict recurrence, even in patients with histologically negative margins. Therefore, molecular analysis of histologically normal resection margins and the corresponding OSCC may aid in identifying a gene signature predictive of recurrence.

Methods: We used a meta-analysis of 199 samples (OSCCs and normal oral tissues) from five public microarray datasets, in addition to our microarray analysis of 96 OSCCs and histologically normal margins from 24 patients, to train a gene signature for recurrence. Validation was performed by quantitative real-time PCR using 136 samples from an independent cohort of 30 patients.

Results: We identified 138 significantly over-expressed genes (> 2-fold, false discovery rate of 0.01) in OSCC. By penalized likelihood Cox regression, we identified a 4-gene signature with prognostic value for recurrence in our training set. This signature comprised the invasion-related genes MMP1, COL4A1, P4HA2, and THBS2. Over-expression of this 4-gene signature in histologically normal margins was associated with recurrence in our training cohort (p = 0.0003, logrank test) and in our independent validation cohort (p = 0.04, HR = 6.8, logrank test).

Conclusion: Gene expression alterations occur in histologically normal margins in OSCC. Over-expression of the 4-gene signature in histologically normal surgical margins was validated and highly predictive of recurrence in an independent patient cohort. Our findings may be applied to develop a molecular test, which would be clinically useful to help predict which patients are at a higher risk of local recurrence.

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Heatmap of 138 genes up-regulated in OSCC. Expression values for each row (gene) are scaled to z-scores for visualization. Margins and tumors annotated with darker colors above the heatmap are from patients who experienced recurrence.
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Figure 1: Heatmap of 138 genes up-regulated in OSCC. Expression values for each row (gene) are scaled to z-scores for visualization. Margins and tumors annotated with darker colors above the heatmap are from patients who experienced recurrence.

Mentions: Meta-analysis of the five public data sets (Table 3) identified 667 up-regulated genes in OSCC; results are provided in Additional file 2, Table S1. Requiring two-fold up-regulation in tumors in both the meta-analysis of public datasets and our microarray experiment, with FDR < 0.01, identified 138 over-expressed genes (Additional file 3, Table S2). The expression patterns of these genes in tumors, margins, and normal oral tissue samples from healthy individuals are shown as a heatmap in Figure 1. As seen in the hierarchical clustering, the 138 genes accurately discriminate between the tumors and margins of the in-house study, as well as normal oral tissues from a publicly available study (as described in Methods). We performed LASSO penalized regression of these 138 genes and selected a total of six genes. Of these six genes, we retained four genes with the largest coefficients (MMP1, COL4A1, P4HA2 and THBS2), and eliminated two genes with very small coefficients (PXDN and PMEPA1), which together contributed less than 5% of the magnitude of the risk score. These four genes constituted a 4-gene signature of recurrence. In the heatmap, Gene cluster "B" contains three of the four genes in the signature (COL4A1, P4HA2 and THBS2), and it shows frequent up-regulation in the surgical margins compared to the normal oral tissues. Strikingly, MMP1, found in gene cluster "A", shows less frequent over-expression in the margins, but has extreme differential expression between margins and OSCCs (400-fold up-regulation as detected by microarrays, and 800-fold up-regulation, validated by RQ-PCR). The proteins encoded by the 138 genes are also shown in a protein interaction network that highlights the most highly inter-connected proteins (Figure 2 and Additional file 1, Methods S1). In the heatmap, the main features of clusters A and B are the large number of interacting MMP proteins in cluster A, which contains MMP1, and collagens plus TGFB1 in cluster B, which also contains P4HA2, THBS2 and COL4A1 genes of the signature. The large number of MMPs and collagen proteins are closely connected; in particular, MMP9 interacts with both THBS2 and COL4A1, and indirectly with MMP1.


A gene signature in histologically normal surgical margins is predictive of oral carcinoma recurrence.

Reis PP, Waldron L, Perez-Ordonez B, Pintilie M, Galloni NN, Xuan Y, Cervigne NK, Warner GC, Makitie AA, Simpson C, Goldstein D, Brown D, Gilbert R, Gullane P, Irish J, Jurisica I, Kamel-Reid S - BMC Cancer (2011)

Heatmap of 138 genes up-regulated in OSCC. Expression values for each row (gene) are scaled to z-scores for visualization. Margins and tumors annotated with darker colors above the heatmap are from patients who experienced recurrence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198722&req=5

Figure 1: Heatmap of 138 genes up-regulated in OSCC. Expression values for each row (gene) are scaled to z-scores for visualization. Margins and tumors annotated with darker colors above the heatmap are from patients who experienced recurrence.
Mentions: Meta-analysis of the five public data sets (Table 3) identified 667 up-regulated genes in OSCC; results are provided in Additional file 2, Table S1. Requiring two-fold up-regulation in tumors in both the meta-analysis of public datasets and our microarray experiment, with FDR < 0.01, identified 138 over-expressed genes (Additional file 3, Table S2). The expression patterns of these genes in tumors, margins, and normal oral tissue samples from healthy individuals are shown as a heatmap in Figure 1. As seen in the hierarchical clustering, the 138 genes accurately discriminate between the tumors and margins of the in-house study, as well as normal oral tissues from a publicly available study (as described in Methods). We performed LASSO penalized regression of these 138 genes and selected a total of six genes. Of these six genes, we retained four genes with the largest coefficients (MMP1, COL4A1, P4HA2 and THBS2), and eliminated two genes with very small coefficients (PXDN and PMEPA1), which together contributed less than 5% of the magnitude of the risk score. These four genes constituted a 4-gene signature of recurrence. In the heatmap, Gene cluster "B" contains three of the four genes in the signature (COL4A1, P4HA2 and THBS2), and it shows frequent up-regulation in the surgical margins compared to the normal oral tissues. Strikingly, MMP1, found in gene cluster "A", shows less frequent over-expression in the margins, but has extreme differential expression between margins and OSCCs (400-fold up-regulation as detected by microarrays, and 800-fold up-regulation, validated by RQ-PCR). The proteins encoded by the 138 genes are also shown in a protein interaction network that highlights the most highly inter-connected proteins (Figure 2 and Additional file 1, Methods S1). In the heatmap, the main features of clusters A and B are the large number of interacting MMP proteins in cluster A, which contains MMP1, and collagens plus TGFB1 in cluster B, which also contains P4HA2, THBS2 and COL4A1 genes of the signature. The large number of MMPs and collagen proteins are closely connected; in particular, MMP9 interacts with both THBS2 and COL4A1, and indirectly with MMP1.

Bottom Line: We identified 138 significantly over-expressed genes (> 2-fold, false discovery rate of 0.01) in OSCC.This signature comprised the invasion-related genes MMP1, COL4A1, P4HA2, and THBS2.Gene expression alterations occur in histologically normal margins in OSCC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Div, of Applied Molecular Oncology, Princess Margaret Hospital, Ontario Cancer Institute, University Health Network, Toronto, ON, Canada. juris@ai.utoronto.ca.

ABSTRACT

Background: Oral Squamous Cell Carcinoma (OSCC) is a major cause of cancer death worldwide, which is mainly due to recurrence leading to treatment failure and patient death. Histological status of surgical margins is a currently available assessment for recurrence risk in OSCC; however histological status does not predict recurrence, even in patients with histologically negative margins. Therefore, molecular analysis of histologically normal resection margins and the corresponding OSCC may aid in identifying a gene signature predictive of recurrence.

Methods: We used a meta-analysis of 199 samples (OSCCs and normal oral tissues) from five public microarray datasets, in addition to our microarray analysis of 96 OSCCs and histologically normal margins from 24 patients, to train a gene signature for recurrence. Validation was performed by quantitative real-time PCR using 136 samples from an independent cohort of 30 patients.

Results: We identified 138 significantly over-expressed genes (> 2-fold, false discovery rate of 0.01) in OSCC. By penalized likelihood Cox regression, we identified a 4-gene signature with prognostic value for recurrence in our training set. This signature comprised the invasion-related genes MMP1, COL4A1, P4HA2, and THBS2. Over-expression of this 4-gene signature in histologically normal margins was associated with recurrence in our training cohort (p = 0.0003, logrank test) and in our independent validation cohort (p = 0.04, HR = 6.8, logrank test).

Conclusion: Gene expression alterations occur in histologically normal margins in OSCC. Over-expression of the 4-gene signature in histologically normal surgical margins was validated and highly predictive of recurrence in an independent patient cohort. Our findings may be applied to develop a molecular test, which would be clinically useful to help predict which patients are at a higher risk of local recurrence.

Show MeSH
Related in: MedlinePlus