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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 validation data and Kaplan-Meier plot of disease recurrence. (A) Unsupervised hierarchical clustering of the quantitative real-time PCR (validation data) showing the maximum expression levels of MMP1, P4HA2, THBS2 and COL4A1 in margins from patients with and without recurrence and with a follow-up time ≥ 12 months. Margins annotated with darker green above the heatmap are from patients who experienced recurrence. Margins from patients with locally recurrent tumors show increased expression levels of the four-gene signature compared to patients who did not recur. (B) Kaplan-Meier plot of quantitative real-time PCR data for patients in the validation set. Patients are assigned to high or low risk based on their four-gene signature risk score. As seen in the Kaplan-Meier plot, patients with over-expression of the 4-gene signature are at high risk for disease recurrence; all patients who experienced recurrence in the validation set are in the high risk group, suggesting that over-expression of this signature was highly predictive of recurrence in the validation set.
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Figure 3: Heatmap of validation data and Kaplan-Meier plot of disease recurrence. (A) Unsupervised hierarchical clustering of the quantitative real-time PCR (validation data) showing the maximum expression levels of MMP1, P4HA2, THBS2 and COL4A1 in margins from patients with and without recurrence and with a follow-up time ≥ 12 months. Margins annotated with darker green above the heatmap are from patients who experienced recurrence. Margins from patients with locally recurrent tumors show increased expression levels of the four-gene signature compared to patients who did not recur. (B) Kaplan-Meier plot of quantitative real-time PCR data for patients in the validation set. Patients are assigned to high or low risk based on their four-gene signature risk score. As seen in the Kaplan-Meier plot, patients with over-expression of the 4-gene signature are at high risk for disease recurrence; all patients who experienced recurrence in the validation set are in the high risk group, suggesting that over-expression of this signature was highly predictive of recurrence in the validation set.

Mentions: We performed quantitative PCR validation of the 4-gene signature (MMP1, COL4A1, P4HA2 and THBS2) in a separate patient cohort (Table 2). This validation analysis confirmed that all four genes were up-regulated in margins and OSCC samples from patients with disease recurrence compared to margins and OSCCs from patients who did not recur (Figure 3A). The risk score was calculated using the maximum expression in any margin of each patient, and was dichotomized at the median score. These risk groups were predictive of recurrence in an independent test cohort assayed by RQ-PCR (136 samples; N = 30 patients) (HR = 6.8, p = 0.04, logrank test) (Figure 3B). In multivariate Cox analysis, correction for T (tumor size) and N (nodal status) resulted only in a slight increase in significance level of the four-gene risk score (p = 0.06, likelihood ratio test). Clinical variables, alone or in combination, were not predictive of recurrence in either training or validation cohorts. The coefficients of the 4-gene risk score, for use with Z-score scaled expression values, are summarized in Table 4.


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 validation data and Kaplan-Meier plot of disease recurrence. (A) Unsupervised hierarchical clustering of the quantitative real-time PCR (validation data) showing the maximum expression levels of MMP1, P4HA2, THBS2 and COL4A1 in margins from patients with and without recurrence and with a follow-up time ≥ 12 months. Margins annotated with darker green above the heatmap are from patients who experienced recurrence. Margins from patients with locally recurrent tumors show increased expression levels of the four-gene signature compared to patients who did not recur. (B) Kaplan-Meier plot of quantitative real-time PCR data for patients in the validation set. Patients are assigned to high or low risk based on their four-gene signature risk score. As seen in the Kaplan-Meier plot, patients with over-expression of the 4-gene signature are at high risk for disease recurrence; all patients who experienced recurrence in the validation set are in the high risk group, suggesting that over-expression of this signature was highly predictive of recurrence in the validation set.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Heatmap of validation data and Kaplan-Meier plot of disease recurrence. (A) Unsupervised hierarchical clustering of the quantitative real-time PCR (validation data) showing the maximum expression levels of MMP1, P4HA2, THBS2 and COL4A1 in margins from patients with and without recurrence and with a follow-up time ≥ 12 months. Margins annotated with darker green above the heatmap are from patients who experienced recurrence. Margins from patients with locally recurrent tumors show increased expression levels of the four-gene signature compared to patients who did not recur. (B) Kaplan-Meier plot of quantitative real-time PCR data for patients in the validation set. Patients are assigned to high or low risk based on their four-gene signature risk score. As seen in the Kaplan-Meier plot, patients with over-expression of the 4-gene signature are at high risk for disease recurrence; all patients who experienced recurrence in the validation set are in the high risk group, suggesting that over-expression of this signature was highly predictive of recurrence in the validation set.
Mentions: We performed quantitative PCR validation of the 4-gene signature (MMP1, COL4A1, P4HA2 and THBS2) in a separate patient cohort (Table 2). This validation analysis confirmed that all four genes were up-regulated in margins and OSCC samples from patients with disease recurrence compared to margins and OSCCs from patients who did not recur (Figure 3A). The risk score was calculated using the maximum expression in any margin of each patient, and was dichotomized at the median score. These risk groups were predictive of recurrence in an independent test cohort assayed by RQ-PCR (136 samples; N = 30 patients) (HR = 6.8, p = 0.04, logrank test) (Figure 3B). In multivariate Cox analysis, correction for T (tumor size) and N (nodal status) resulted only in a slight increase in significance level of the four-gene risk score (p = 0.06, likelihood ratio test). Clinical variables, alone or in combination, were not predictive of recurrence in either training or validation cohorts. The coefficients of the 4-gene risk score, for use with Z-score scaled expression values, are summarized in Table 4.

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