TAK1 mRNA expression in the tumor tissue of locally advanced head and neck cancer patients

Abstract

Resistance to radio and chemotherapy is one of the major drawbacks in the progression of head and neck squamous cell cancer (HNSCC) patients, evidencing the importance of finding optimum molecular prognosis markers to develop personalized treatment schedules. TGF-beta effector TAK1 activity has been related to a greater aggressiveness in several types of cancer (Kondo et al. 1998; Edlund et al. 2003; Kaur et al. 2005) and, although there has been described no significant implication of TAK1 in HNSCC development, we have further examined the role of its mRNA expression as a marker of prognosis in HNSCC. Fifty-nine advanced HNSCC patients were recruited for the study. The tumor expression of TAK1 mRNA was analyzed with RT-PCR using Taqman technology and its relationship with the clinical outcome of the patients studied. TAK1 mRNA expression was lower in patients that relapsed than in those that did not, but the difference was only significant between the patients that showed response to treatment (p < 0.001). ROC curve analyses pointed a 0.5 expression ratio TAK1/B2M value as an optimum cut-off point for relapse and response. Our data suggest the TAK1 mRNA analysis by Taqman RT-PCR can predict the risk of relapse in HNSCC patients.

Keywords: TAK1; head and neck cancer; molecular markers; prognosis; real-time PCR.

Figure 1

Using the control RNA as a “Calibrator Sample”, we built two standard curves or lines, one for the housekeeping gene, B2M, and one for the gene in study, TAK1. These standards enabled the control of the efficiency of the reaction in each experiment performed and, at the same time, they served as a third level of normalization for the data obtained from Real Time PCR. In particular, we used them to calculate in each sample the concentration of each mRNA, the housekeeping gene’s and TAK1 gene’s relative to the concentration of these mRNAs in the Calibrator sample. Calculating the ratio between these two Relative Concentrations we obtained a dimensionless measure directly proportional to the TAK1 mRNA amount expressed in each tissue sample tested. In the example exposed in the figure we calculated for a sample a relative expression value of TAK1 of 0.59 extrapolating the mean Ct values obtained in the Real Time PCR measurement in the standard curves/lines built with consecutive dilutions of the Calibrator mRNA. Once obtained a value of Ln Calibrator relative dilution for each gene, we raised e to that value and calculate the dimensionless ratio we were searching.

Figure 2

Contrast analysis of the interaction RESPONSE-RELAPSE regarding TAK1 mRNA tumor expression. In the columns situated in the left of the graphic, it can be observed that the expression levels difference, between those patients that suffered relapse and those that did not, is statistically significant among the patients that achieved some response to treatment.

Figure 3

ROC analysis for the previous contrast results. The curve on the left hand side analyzes the tumour TAK1 mRNA expression from the patients with response to treatment searching for a cut point for the presence of relapse; the second curve (right side) analyzes the differences in the patients that although relapsed locally showed some response to treatment and those that did not respond. Both curves present a high exactitude (Area Under the Curve approximately 0.9) and both curves point to the same TAK1 mRNA expression level in the tumour tissue as a cut off point between bad and good prognosis in these patients: a TAK1/B2M expression ratio of 0.5.

Figure 4

The table shows the patients’ distribution depending on their TAK1/B2M expression ratio related to the ROC curves’ designated cut off point of 0.5. The graphic represents the patient’s distribution according to their response pattern, relapse development pattern and the TAK1/B2M expression ratio. It can be observed that the former ROC curves have successfully found a TAK1 expression ratio that discriminates patients according to their clinical outcome, 90.0% of the ones with better prognosis, no relapse and response, having TAK1 mRNA expression ratios over 0.5, whereas most of the patients that present relapse, no response to treatment or both express TAK1 mRNA in the tumour tissue at lower levels than this designated ROC curve cut off point.