**2. Recent status of pre-operative biomarkers to determine a response to neoadjuvant chemoradiation**

Neoadjuvant chemoradiation for the management of patients with stage II and III rectal cancer results in a clinical complete response (cCR), the absence of detectable rectal tumor with diagnostic modalities [i.e. endorectal ultrasound (EUS), magnetic resonance imaging (MRI), digital rectal exam (DRE), or proctoscopy], in 10-40% of patients [2].

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The fundamental pre-clinical and clinical question is to determine whether there are markers that can detect tumors that will respond well to neoadjuvant treatment such that these patients could be potential candidates for observation without operative intervention. Conversely, if a patient is not likely to respond to neoadjuvant chemoradiation, they should submit to surgical intervention sooner. Therefore, a myriad of pathways and molecules ranging from DNA-repair molecules to molecules that mediate cell cycle dynamics to apoptotic mediators as well as hypoxic mechanisms have been investigated with a wide range of results, which are summar‐ ized by Ramzan et al [3]. Currently, there is no unifying pathway that can reliably predict responses to chemoradiation in patients with rectal cancer.

#### **2.1. DNA repair molecules**

One of three pathways is responsible for the repair of DNA double-strand breaks (DSB) induced by ionizing radiation: homologous recombination (HR), non-homologous end-joining (NHEJ) pathway, or an alternate NHEJ pathway [3]. Of these, the non-homologous end-joining (NHEJ) pathway is fundamental for DSB repair. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an integral part of the NHEJ pathway. This mechanism can be broadly classified into three steps: (1) Ku 70/80 heterodimer identifies DSB and stick to DNA broken ends, facilitates the activation and recruitment of DNA-PKcs; (2) enzymatic processing of the DNA ends; and (3) ligation by DNA ligase IV. Ku 70/80 proteins play a fundamental role as they recruit DNA-PKcs which then set the cascade of DNA repair. Recent data dem‐ onstrate that DNA-PKcs and Ku proteins may have a central role in radiation induced cell death and might predict the response to radiation. However this is an area that is still under investigation.

#### **2.2. Apoptosis**

In the central mediators of apoptosis pathway in response to ionizing radiation, the initial response begins with an up-regulation of p53 (Figure 1). p53 then directly activates the cyclin dependent kinase inhibitors (such as p21). Cell cycle progression stops until the cell repairs the damage induced by ionizing radiation. If the cell is unable to repair itself, it undergoes apoptosis. The anti-apoptotic Bcl-2 inhibits p53, while p53 inhibits the inhibitor of apoptosis (survivin). All of these molecules have been investigated to determine if their up-regulation or down-regulation could predict a response to ionizing radiation. Mutations and manner of detection have to be considered in these studies. Additionally, contrary to expectations, p21 or Bax deficient cells lead to a more radiosensitive rather than a more radioresistant phenotype [4]. Thus, analyses of these molecules in predicting a response to ionizing radiation have been largely unyielding [5].

A recent systematic review analyzing the role of p53 as a predictor of a response to ionizing radiation included 30 studies of which 25 used p53 protein status, 7 used gene analysis detection and two used both. The results revealed that patients that demonstrated a p53 wildtype (and/or low expression) had a good response risk of 1.3 [CI=1.14 to 1.49], complete response RR was 1.65 [CI=1.19 to 2.30] and poor response RR 0.85 [CI = 0.75 to 0.96] [6].

**Figure 1.** Central mediators of apoptosis. Ionizing radiation (IR) leads to an increase of p53, which in turn activates p21 **Figure 1.** Central mediators of apoptosis. Ionizing radiation (IR) leads to an increase of p53, which in turn activates p21 and causes cell cycle arrest. P53 also activates Bax, which results in apoptosis

and causes cell cycle arrest. p53 also activates Bax, which

#### results in apoptosis. **2.3. Gene modifications and polymorphisms**

The fundamental pre-clinical and clinical question is to determine whether there are markers that can detect tumors that will respond well to neoadjuvant treatment such that these patients could be potential candidates for observation without operative intervention. Conversely, if a patient is not likely to respond to neoadjuvant chemoradiation, they should submit to surgical intervention sooner. Therefore, a myriad of pathways and molecules ranging from DNA-repair molecules to molecules that mediate cell cycle dynamics to apoptotic mediators as well as hypoxic mechanisms have been investigated with a wide range of results, which are summar‐ ized by Ramzan et al [3]. Currently, there is no unifying pathway that can reliably predict

One of three pathways is responsible for the repair of DNA double-strand breaks (DSB) induced by ionizing radiation: homologous recombination (HR), non-homologous end-joining (NHEJ) pathway, or an alternate NHEJ pathway [3]. Of these, the non-homologous end-joining (NHEJ) pathway is fundamental for DSB repair. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an integral part of the NHEJ pathway. This mechanism can be broadly classified into three steps: (1) Ku 70/80 heterodimer identifies DSB and stick to DNA broken ends, facilitates the activation and recruitment of DNA-PKcs; (2) enzymatic processing of the DNA ends; and (3) ligation by DNA ligase IV. Ku 70/80 proteins play a fundamental role as they recruit DNA-PKcs which then set the cascade of DNA repair. Recent data dem‐ onstrate that DNA-PKcs and Ku proteins may have a central role in radiation induced cell death and might predict the response to radiation. However this is an area that is still under

In the central mediators of apoptosis pathway in response to ionizing radiation, the initial response begins with an up-regulation of p53 (Figure 1). p53 then directly activates the cyclin dependent kinase inhibitors (such as p21). Cell cycle progression stops until the cell repairs the damage induced by ionizing radiation. If the cell is unable to repair itself, it undergoes apoptosis. The anti-apoptotic Bcl-2 inhibits p53, while p53 inhibits the inhibitor of apoptosis (survivin). All of these molecules have been investigated to determine if their up-regulation or down-regulation could predict a response to ionizing radiation. Mutations and manner of detection have to be considered in these studies. Additionally, contrary to expectations, p21 or Bax deficient cells lead to a more radiosensitive rather than a more radioresistant phenotype [4]. Thus, analyses of these molecules in predicting a response to ionizing radiation have been

A recent systematic review analyzing the role of p53 as a predictor of a response to ionizing radiation included 30 studies of which 25 used p53 protein status, 7 used gene analysis detection and two used both. The results revealed that patients that demonstrated a p53 wildtype (and/or low expression) had a good response risk of 1.3 [CI=1.14 to 1.49], complete response RR was 1.65 [CI=1.19 to 2.30] and poor response RR 0.85 [CI = 0.75 to 0.96] [6].

responses to chemoradiation in patients with rectal cancer.

**2.1. DNA repair molecules**

260 Updates on Cancer Treatment

investigation.

**2.2. Apoptosis**

largely unyielding [5].

It is possible that with standardized techniques, we might be able to utilize other molecules in the apoptotic pathway alone or in combination to better predict neoadjuvant chemotherapeutic responses in patient affected with rectal cancer. Another area that is gaining momentum is that of epigenetic changes. For instance methylation of the retinoic acid receptor gene (RARB) and the checkpoint with forkhead and ring finger gene (CHFR) discriminated between TI-2 *vs.* T2-3 un-irradiated tumors. RARB methylation was also associated with nodal metastasis and lymphovascular invasion (LVI). Methylation of other genes have also predicted nodal metastasis [7]. Thus, methylation can predict aggressiveness and in combination with the mutation status of other molecules, a predictive panel of a response to ionizing radiation can then be constructed. In a separate study, DNA analysis of biopsies of patients prior to radiation demonstrated a gene mutation and two gene polymorphisims to be associated with resistance to radiation as measured by pCR [8].
