| dc.description.abstracteng | Background:
In Germany the standard treatment of locally advanced adenocarcinoma within the first 12cm of the rectum is a neoadjuvant radiochemotherapy (RCT) followed by a total mesorectal excision (TME) and adjuvant chemotherapy. Despite encouraging long-term results of this multimodality approach in reduction of local recurrence rate and improved disease-free survival (DFS) the patient`s response to preoperative RCT has remained very heterogeneous. It ranges between complete remission of rectal cancer to nearly complete resistance depending on the individual tumor biology. A good response to initial neoadjuvant treatment in form of a high tumor regression grade (TRG) is a positive prognostic factor.
Until yet predicting the tumor response to preoperative radiochemotherapy is not possible, but it would be neccessary in order to adapt multimodality treatment to the individual risk of cancer recurrence and to improve survival. Several clinicopathological parameters and so-called biomarkers offer the option for pre- or peritherapeutic prediction of response to multimodality therapy based on their involvement in tumor biology. In this context, the tumor suppressor protein p16 (also known as p16INK4a (INK4=inhibitor of CDK4), p16INK, INK4a; CDKN2 (cyclin-dependent-kinase-inhibitor 2); CDK4I (cyclin-dependent-kinase-4-inhibitor); TP16 (tumor suppressor protein 16) and MTS1 (multiple tumor suppressor 1)) seemed to be a promising biomarker for prediction of RCT-associated outcome. P16 mediates cell cycle arrest and inhibits proliferation within the retinoblastoma-pathway. Tumors with high expression of p16 could therefore have a prognostic advantage over tumors with low p16 expression levels.
Purpose:
In this study the expression of p16 was determined by immunohistochemistry (IHC) in cancer tissue of patients with and without RCT. The results were compared with several clinico-pathological parameters and survival outcome during standardized follow-up. All experiments and analyses were performed within the subproject 5a of the clinical research unit KFO-179-2 (www.kfo179.de).
Patients, materials and method:
A total of 261 patients with rectal cancer were included. For p16 expression analysis 40 pretherapeutically taken cancer biopsies of neoadjuvantly treated patients and tissue of rectal cancer specimens with (n=156) and without (n=98) preoperative RCT were investigated. Preoperative RCT consisted of radiotherapy (50.5 Gy) combined with 5-FU or 5-FU+Oxaliplatin followed by quality-controlled surgery and postoperative CTx with 5-FU+/-Oxaliplatin.
The p16 expression was assessed microscopically in normal mucosa (epithelium), in the adenoma region of the specimen and the rectal cancer itself as well as in stromal tissue. The p16 status was assessed by using paraffin-embedded tissue samples, which were cut into slices of 2μm. Using the specific anti-p16 (JC8: sc-56330) murine monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) a standardized IHC staining technique was performed on a Ventana Bench-Mark XT immunostainer (Ventana, Tucson, AZ). Furthermore a p16 score was established (including intensity of staining as well as quantity of stained cells) and heterogeneous or homogeneous staining of cancer cells was analysed. The results of the immunohistochemical analysis were compared with clinic-pathologic parameters (e.g. postsurgical residual (R-), tumor (T-), nodal (N-) and UICC-status according to the TNM-/UICC-classification system with special focus on RCT-induced tumor regression grading (TRG)) and survival (TTR, CSS).
Results:
The expression of p16 was very low in the normal epithelium of the rectum. In the normal mucosa there was no difference between the p16 expression of patients with or without RCT (n=192, p=0.4). Within the group of primarily operated tumors the p16 expression of the adenoma region and the carcinoma itself was higher than in the normal mucosa, but the p16 expression of adenomas and carcinomas did not differ significantly (p=0,857). Within the group of neoadjuvantly treated specimens the p16 expression of the carcinoma itself was significantly higher than the p16 expression of the adenoma region (p<0.001). In comparison between the patients with and without RCT, there was a significantly higher expression of p16 in the adenomas (n=56, p<0.001) as well as carcinomas (n=254, p<0.001) in the specimens of neoadjuvantly treated patients than in the specimens from patients without a preoperative therapy.
After RCT, there was a more homogeneous staining of residual cancer cell clusters or conglomerates (n=254, p<0,001). Furthermore the p16 expression of the specimens of neoadjuvantly treated patients correlated positively with the RCT-induced TRG quantitatively (in % amount, p=0.002) and qualitatively as measured in grading categories TRG-1 to TRG-4 (p<0.001). The expression of p16 in the pretherapeutic biopsies did not correlate with tumor regression (n=40, p=0.61). A correlation between age of patients and grade of stromal p16 staining was found (p=0.02) independently of preoperative RCT.
Despite these interesting results, neither the p16 expression of biopsies nor specimens correlated with time to recurrence (TTR) or cancer specific survival (CSS).
Conclusion: Further research with higher numbers of pretherapeutic biopsies should be considered to clarify the predictive value of p16 expression in order to support therapeutic decision making. With the positive correlation of high p16 expression with the amount and quality of RCT-induced tumor regression, p16 is a candidate to be included in a panel of biomarkers testing RCT-response prediction in ongoing as well as planned clinical trials with new drugs. The findings of this study and thesis could help in the future to stratify patients for more recurrence risk adapted strategies in pre- as well as in postoperative multimodal strategies. | de |