Locally advanced rectal cancer: which treatment after complete response after neoadjuvant therapy? Combined treatment for locally advanced rectal cancer achieves a 5-year overall survival close to90% \[15\]. This multimodal approach can be also applied to elderly patients with stage III rectal cancer obtaining improved oncological outcomes \[16\]. Some patients with rectal cancer who receive neoadjuvant chemoradiotherapy (nCRT) achieve a pathologic complete response (pCR) and may be eligible for less radical surgery or non-operative management \[17\]. Moreover, complete regression of the rectal cancer after neoadjuvant chemoradiation therapy have been confirmed by clinical and radiographic evaluation-this is known as complete clinical response (cCR). The "watch and wait" approach was first proposed by Dr. Angelita Habr-Gama in Brazil in 2009. Those patients with cCR are followed with rigorousphysical, endoscopical, and imaging surveillance \[18\]. Several studies aimed to identify variables that predict pCR after nCRT for rectal cancer. A large retrospective review of the NCDB performed from 2006 to 2011, including a total of 23,747 patients identified the clinical factors associated with pCR. They were lower tumor grade, lower clinical T and N stage, higher radiation dose, and delaying surgery by more than 6-8weeks after the end of radiation, while lack of health insurance was linked with a lower likelihood of pCR \[17\]. Another study identified predictive factors in the models included tumor length, tumor circumferential extent, age, and ApoA1 \[19\]. A different study showed that responders after neoadjuvant chemoradiation had a lower incidence of cytokeratin 20 positive circulating tumor cells compared to non-responders, which might be a result of effective systemic and local treatment prior to surgery \[20\]. Finally, the role of immune microenvironment was investigated analyzing Foxp3(+), CD3(+), CD4(+), CD8(+) and IL-17(+) cell density in post-CRT surgical samples from 128 patients with rectal cancer. Stromal Foxp3(+) cell density was strongly associated with tumor regression grade. A low stromal Foxp3(+) cell density was observed in 84% of patients who had a pathologic complete response (pCR) compared to41% of patients who did not. Low stromal Foxp3(+) cell density was also associated with improved recurrence-free survival. Regulatory T cells in the tumor microenvironment may inhibit response to neoadjuvant CRT and may represent a therapeutic target in rectal cancer \[21\].

Hypotheses and Specific aims The main hypothesis underlying our research is that aggressiveness of rectal cancer is determined by the complex interactions between the malignant cells and the local immune microenvironment. Our hypothesis is that the local immune activation may involve the "healthy" rectal mucosa surrounding the cancer and the sampling of this mucosa may provide useful information about rectal cancer behavior. In fact, a combination of different factors, including molecular signaling networks (i.e. checkpoint genes) within different cell population, presence of soluble chemical factors and quantity/quality of immune cells infiltrate will decide rectal cancer behavior and its response to neoadjuvant chemoradiation. Moreover, tumor cells actively interact with the microenvironment secreting and degrading extracellular matrix components, and the release of soluble molecules (e.g. miRNA) can significantly influence the inflammatory and immune responses of the "healthy" rectal mucosa surrounding the cancer. By using state of the art histological/molecular/immunological markers and bio-statistical analysis tools we aim to identify clinically relevant molecular targets to design effective therapeutic strategies for rectal cancer patients.

Therefore, the aim of the study is to identify a prognostic test that accurately predicts rectal cancer behavior even in case when the tumor samples are scarce (due to small size of early rectal cancer) or absent (in case of complete response to therapy). This prognostic test will be constructed on the combinatory analysis of the transcriptome, immune cells activation and checkpoints, epithelial cells activity as antigen presenting cells and miRNA expression in normal tissue specimens of prospectively collected rectal cancers. The question is how to identify, among locally advanced rectal cancer, those with immediate and sustained complete response to neoadjuvant chemo/radiotherapy. These patients might safely avoid a low rectal resection with all the consequences for their quality of life. In patients with locally advanced rectal cancer (N+) an ideal biomarker should be able to identify the cohort of patients that will have a complete response to neoadjuvant therapy and will not require further treatments.

Study design Tissue samples will be obtained from cancer tissue (if possible) and from normal rectal mucosa adjacent to the cancer at surgery. The very large number of patients need to answer the two questions imply a multicentric design. Complete medical record and follow-up will be collected from each center. The analysis will be centralized mostly in Azienda Ospedaliera di Padova. The study will be articulated in two parts each of them aiming to answer to one of the above described questions. Each part of the study will be articulated in a retrospective and exploratory step (A) and in a prospective validation step (B).