## 3 Discussion  
We identified a panel of predictive markers for nCRT response in rectal carcinomas with ICPL. ICPL was recently described as powerful tool to identify thousands of proteins in extracts of body fluids or tissue. The technique is characterized by a highly accurate and reproducible quantification method for proteins [10; 11]. ICPL is especially useful to identify regulated proteins in different samples. It is an innovative technology, which showed its value for marker screening in human tissue samples in our study. It was possible to validate randomly selected proteins by immunohistochemistry of tumor biopsies. This is a further indicator for the reliability of the identified proteins. One crucial point is the amount of tissue needed for proteome analysis. We harvested one tumor biopsy for proteomics which is generally also needed for histological diagnosis under clinical routine conditions. No patient suffered from any complication during sample harvesting. From our biopsies sufficient material was accumulated after LCM to perform ICPL. Thus ICPL not only provides reliable results but is also a valuable tool for proteome studies of human specimens which are harvested under clinical routine conditions. The patients who underwent nCRT were all included in a clinical trial (CAO/ARO/AIO-04) [12]. This is important because not only standardized tissue sampling and preparation but also standardized patient care and treatment increases the value of molecular predictive marker evaluation. In our cohort two different types of chemotherapy were added to radiation. It is not surprising that several proteins were regulated only in relation to nCRT I or II. These markers may reflect the response to the different chemotherapy regiments. However 14 markers were uniformly regulated in response to radiation. Chemotherapy regiments added to nCRT change more frequently than radiation itself. Therefore these proteins and especially the very highly expressed ones, which indicate good radiation response, may be the most valuable for further validation in the future, because a highly expressed marker such as a stable protein can easily be identified e.g., by immunohistochemistry in a routinely sampled tumor biopsy under clinical conditions. PKM2, which was one of these markers, was recently described as highly expressed in colorectal cancer and correlated with later stage, lymph node metastases and oxaliplatin metabolism [13; 14]. It is a cytosolic enzyme involved in nucleic acid, phospholipid- and amino acid synthesis, which provides critical cell building materials for highly proliferating cells, such as tumor cells. A high expression of Transketolase was already described in colorectal and urothelial  
Figure 1: Immunohistochemical validation of protein expression in colorectal carcinoma biopsy tissue identified by isotope coded protein label (ICPL); (**A**) Picetin-1; (**B**) Transketolase; (**C**) Transgelin; (**D**) HADHA Trifunctional enzyme subunit \(\alpha\), mitochondrial precursor. cancer and was associated with poor prognosis. In breast cancer Transketolase was identified as a potential target against tumor growth [15]. It is involved in the non-oxidative part of the pentose phosphate pathway which is important during cell metabolism. A decrease in Transketolase expression levels in tumor cells was assumed to delay tumor growth. These findings are indicators that several of the markers identified in this study are related to cancer. We classified our patients for good and poor nCRT response referring to the regression grading of Dworak [7,16]. This histological method is based on the stroma/tumor cell relation within the tumor after nCRT. It is a commonly used method to quantify tumor response after neoadjuvant treatment under clinical routine. Unfortunately it is of no value for identification of response to CRT prior to CRT application.