Recent Advances in the Molecular Diagnosis and Prognosis of Colorectal Cancer

Recent Advances in the Molecular Diagnosis and Prognosis of Colorectal Cancer

Abstract and Introduction

Abstract

Colon cancer remains a leading cause of mortality worldwide despite the well-characterized molecular events in the adenoma-to-carcinoma sequence. There has been a strong emphasis on early detection of colon cancer, and fecal DNA-based methods have been developed to assist with early screening. Tissue-based assays have been utilized for many years to assess tumor aggressiveness and to determine prognosis and response to chemotherapeutic interventions. The most widely used serum marker for colon cancer (carcinoembryonic antigen) remains a useful modality to assess for occult disease following curative resection. Identification of tumor mutations in circulating tumor cells and microarray analysis holds a great deal of promise in the diagnosis and prognosis of patients with colorectal cancer. The inhibitors of apoptosis may be important markers to determine resistance to radiation cytotoxicity in rectal cancer. This report presents a summary of the current status of the molecular markers of colorectal cancer to establish a diagnosis, determine prognosis and chemoradiotherapeutic interventions, and assess relapse following curative surgery.

Introduction

One of the best characterized models for development of carcinogenesis involves the multistep genetic modifications that occur in the process of colorectal neoplasia (Figure 1).[1,2] Colorectal cancer (CRC), however, remains a leading cause of mortality, affecting nearly 782,000 individuals worldwide each year. In the USA, CRC is the second most common cause of cancer-related deaths among men and women, with an estimated incidence of 153,760 and a mortality of 52,180 in 2007.[3] An understanding of the molecular aspects of CRC has led to the development of novel chemotherapeutic agents, which, when combined with effective wide screening practices, resulted in a 5% decrease in mortality in spite of a 6% increase in its incidence over the past 3 years (Figure 2).[3-8]

Figure 1.Somatic mutations that cause sporadic colorectal neoplasia. The accumulation of several mutations increases the risk of tumor formation from adenoma to carcinoma to metastatic disease.

 
 
Figure 2.Incidence and mortality of colorectal carcinoma in men and women in the USA over the past 6 years. Over the last 3 years there has been an increase in the incidence of colorectal carcinoma. Fortunately, a decrease in mortality has been observed over the same period of time.

The molecular diagnosis of CRC holds its best promise on fecal DNA-based tests. Serum tests such as CEA have poor sensitivity for detection of early CRC and, even when standardized among laboratories, may not be as sensitive as fecal specimens. Further sophistication in the current techniques utilized to detect mutations on fecal specimens, combined with a reduction of cost and amount of specimen needed, may allow fecal DNA-based tests to be evaluated in large cohorts of patients at risk of CRC and determine their clinical utility.

Fecal DNA Tests

Cells shed by the colonic mucosa in the feces may be examined in order to determine whether they harbor mutations in the adenoma-to-carcinoma sequence, thus enabling patient screening for CRC. A panel of DNA markers is more useful than a single marker, as the combination increases the sensitivity of the test. The genes most commonly assayed include APC (mutated in up to 70% of specimens), K-ras (mutated in up to 60% of specimens) and p53 (mutated in up to 60% of specimens).[13-15] The specificity of a panel of fecal DNA tests is 95%. However, its sensitivity is much more variable, ranging between 60 and 90%. These tests are presently expensive and are not currently useful as a screening tool for CRC because of their low sensitivity. Although the guaiac fecal occult blood test is less sensitive and less specific than fecal DNA testing, the low cost and its noninvasive nature make this test superior to fecal DNA testing for screening purposes and clinical applicability today.[15]

Carcinoembryonic Antigen

CEA is a family of related glycoproteins initially found in embryonic tissue and colon malignancies. The half-life of CEA is approximately 2 weeks. Plasma levels of CEA can be determined readily by radioimmunoassay. However, its usefulness in colon carcinogenesis screening is limited because of its high level in the plasma of a patient's malignancies originating from other sites such as breast, pancreas, stomach and lung.[16] Furthermore, CEA plasma levels may also be increased in smokers or patients with chronic diseases, such as inflammatory bowel disease, bronchitis and alcoholic liver disease.[17] The clinical usefulness of CEA for screening purposes is further limited by the fact that its increase in level typically occurs only when the tumor penetrates through the serosa. Thus, early lesions may not be detected by serum CEA level changes. CEA is typically elevated with metastatic liver disease, but rarely with peritoneal involvement.[17] Thus, while the specificity for CEA to identify occult CRCs is high, the sensitivity is low in most studies for screening.[17] Accordingly, CEA is not a useful test for the screening of CRC.[11,12]

Other serum tests have been investigated for the screening of CRC, but are not currently recommended by either the ASCO or EGTM (e.g., CA 19-9, CA 242 and TIMP-1).[11,12]

CRC prognosis

The prognosis of patients with CRCs and the selection of agents likely to result in a chemotherapeutic response have been largely evaluated by investigating molecular modifications of the tumor tissue compared with normal mucosa. All of these markers have been evaluated in detail by expert marker panels in the USA [11]and Europe.[12] None of them are currently recommended for clinical use. However, there has been a steady progression in the amount of literature produced on these tumor markers in the past few decades. As more standard techniques for their measurement become available and larger well-controlled studies are conducted, tissue-specific markers hold a significant promise in the prognosis and selection of therapeutic options in patients with CRC (table1). These markers are discussed in the following sections.

Chromosomal Deletions

Somatic mutations leading to chromosomal abnormalities occur with a frequency of 85% in sporadic forms of CRCs. These genetic abnormalities occur in the form of chromosomal deletions, translocation errors, gene amplifications and epigenic changes, such as gene silencing by hypermethylation. A number of such genetic abnormalities have been noted to occur on chromosome 18q. The genes most commonly implicated include DCC, DPC4 and genes that are part of the TGF-β1 pathway. These genes are mutated in up to 10% of patients with CRC. DCC is an adhesion molecule of the immunoglobulin family, responsible for homotypic binding between cells. However, the mechanism of action of DCC leading to CRC remains unclear. Both DCC and DPC4 are putative tumor-suppressor genes, affected as a result of 18q mutations.[18]

Loss of Heterozygosity at 18q

Loss of heterozygosity (LOH) can be defined as the manifestation that results from the loss of large genomic regions. Lack of amplification of a particular segment of DNA can be identified by PCR. Amplification of two to ten segments of interest is performed. Typically, the DCC locus is examined for 18qLOH. DCC protein loss can also be examined by immunohistochemistry.

Allelic loss at chromosome 18q [19]and loss of the region containing the DCC [20]gene are predictors for survival in patients with stage II cancers. A minimum loss of 18q containing at least two tumor-suppressor genes (i.e., DCC and DPC4) has been suggested to be a determinant of a malignant phenotype.[21] The 18qLOH was associated with a 24% decrease in overall survival in patients with stage III and high-risk stage II. Patients with a favorable outcome retained 18q and demonstrated microsatellite stability. Similarly, a 30% increase in overall survival occurred in microsatellite unstable tumors containing mutations of the type II TGF-β gene receptor.[22] The significance of the loss of 18q as a prognostic factor has been corroborated by systematic reviews.[11] Thus, chromosomal alterations of 18q are promising in predicting CRC patients at high risk for aggressiveness. Stage II tumors with 18qLOH should be considered as high risk and possible candidates for adjuvant chemotherapy.[23]

However, because 18qLOH has not been uniformly found to be a good predictor of survival in patients with CRC and because most studies that demonstrate prognostic significance are small and retrospective in nature,[24-26] the ASCO TMEP-2006 and the EGTM determined that it was too early to recommend 18qLOH as a clinical prognostic marker in colon cancer. Additionally, the panel concluded that DCC loss by immunohistochemistry is not recommended clinically for prognosis, therapeutic response predictability or for tumor operability assessment.[11,12]

p53 Status

DNA damage causes an intracellular increase in the protein levels of p53, which leads to cell-cycle arrest by upregulation of cyclin-dependent cell cycle inhibitor p21 Waf1/Cip1. This allows the cell to undergo DNA repair. However, if the cell is unable to repair the DNA injury, p53 leads to an increase in the levels of the proapoptotic protein BAX and the cell undergoes apoptosis.[27] p53 may also promote apoptosis by baseline suppression of the inhibitor of apoptosis (IAP), survivin, such that mutations of p53 give cells a survival advantage.[28,29]

The tumor-suppressor gene p53 is absent or mutated in approximately 50% of all human colon cancers.[30] Some, but not all, mutations of the p53 gene promote tumor progression.[31] Furthermore, some mutations of the p53 gene do not assess its functional status. p53 status has been evaluated to determine:

  • Usefulness as a prognostic predictor
  • Aggressiveness for apparently localized tumors
  • Response to chemoradiotherapeutic interventions
  • Survival following curative resection

The results of several individual studies [32-34]as well as systematic reviews [35,36]of p53 abnormalities in patients with colon cancer have shown a high degree of variability in the methodologies employed to assess the status of p53, and the results have been inconsistent. The status of p53 in colorectal tumors has been investigated by a wide array of methods, including mutational analysis as well as immunohistochemical staining. Currently, the evidence of p53 as a tumor marker for prognosis of therapeutic response in patients with CRC is not uniformly in agreement to recommend p53 status in the clinical setting to assess prognosis or to determine the response to chemotherapeutic treatment.[11,12]

Microsatellite Instability

Genes that are involved in the surveillance and repair of DNA integrity during the S-phase of the cell cycle are called MMR (or caretaker genes). Therefore, MMR genes that undergo mutations or deletions accelerate carcinogenesis as the genome continues to accumulate replication errors. MMR genes include: hMLH1, hSMH2, hSMH3, hSMH6, hPMS1 and hPMS2. Germline mutations of hMHL1 and hSMH2 result in the development of the hereditary nonpolyposis syndrome (hereditary nonpolyposis CRC [HNPCC], Lynch syndrome), which is characterized by poorly differentiated and typically right-sided colonic neoplasia. Somatic mutations of MMR genes occur in up to 18% of sporadic forms of colon cancer.[37]

Microsatellites are repetitive sequences (1–5 nucleotides) of DNA that are present throughout the genome. Microsatellite instability (MSI), as defined by de la Chapelle, occurs when a germline microsatellite allele has gained or lost repeat units leading to a somatic change in length.[38] MSI is further categorized into high and low frequency, depending on the incidence of mutations and loci tested [11]in tumors compared with normal tissue (microsatellite stable tissue [MSS]).[39] In most studies, it is high-degree MSI (MSI-H) that is considered to be a poor prognostic factor for clinical outcome. Low-degree MSI (MSI-L) and microsatellite stable tumors are typically grouped together. A tumor is rated as MSI unstable by assessing mutations in a standard panel of five microsatellites, where none of them lie within a coding region. Immunohistochemical analysis of fresh-frozen or paraffin-embedded tissues with antibodies specific for protein products of the MMR genes can also be undertaken.

MSI is a widely studied marker for prognosis and response to chemotherapetutic interventions.[40] Some studies have shown that patients with stage II CRC and MSI-H had poorer clinical outcome compared with patients with MSI-L,[41,42] while others have found that CRC tumors with MSI-H had better clinical outcomes than MSI patients whose tumors were MSI-L/ MSS.[43-45] Others have shown that MSI-H was associated with worse survival,[22,46] had no association with clinical outcome [34,37,47]and was not a predictor for chemotherapeutic intervention.[42,48]

Based on the heterogeneity of the studies as well as the wide range within the findings, there are currently no recommendations for the clinical use of MSI as an independent predictor of prognosis or to assess chemotherapeutic interventions following surgery in early-stage CRC.[11,12]

Tumor DNA Ploidy

DNA ploidy is a test that measures the DNA content within tumor cells. Normal cells in the resting state contain a complete set of chromosomes, which is referred to as the diploid state. Cells that are ready to divide are in the tetraploid state (DNA index). Proliferation analysis determines the DNA content during DNA synthesis, which is referred to as the percentage S-phase. DNA measurements can be performed by microscopy or flow cytometry. Most studies assessing DNA content as a predictor to determine prognosis in CRC measure DNA ploidy by flow cytometry in tumor tissues. Flow cytometry typically reveals a large peak for the diploid state and a smaller peak for the tetraploid state. Tumor cells that are not dividing properly would reveal a different, third, smaller peak: the aneuploid state.

Multivariate analysis demonstrated that tumors in the diploid state were an independent predictor for better patient survival than tumors that were in the aneuploid state.[49,50] However, this finding has not been uniformly observed.[51-55] Based on the inconsistent observations in the results of the studies regarding DNA ploidy, the clinical utility of this parameter in patients with CRC is limited. There are currently no recommendations for the use of DNA ploidy as a predictor of outcome in patients with CRC.[11,12]

Thymidine Phosphorylase, Thymidine Synthase & Dihydropyrimidine Dehydrogenase

The enzymes thymidine phosphorylase, dihydropyrimidine dehydrogenase (DPD) and thymidine synthase (TS) play a crucial role in the metabolism of pyrimidines in human cells (Figure 3). TS is a rate-limiting enzyme in the DNA synthesis of the thymidine nucleotide. The chemotherapeutic first-line agent for most forms of CRC is 5-fluorouracil (5-FU). 5-FU is primarily metabolized in the liver by DPD. Because 5-FU inhibits TS, leading to DNA synthesis impairment, TS status has been investigated as a marker for prognosis and tumor response to chemotherapeutic interventions. Because of the heterogeneity of studies analyzed and because the method utilized by various studies has ranged from protein analysis by immunohistochemistry (e.g., TS activity in fresh-frozen specimens) to by reverse-transcriptase (RT)-PCR, there are currently no recommendations for the use of TS status as a predictor for survival or response to chemotherapeutic intervention in patients with CRC.[56] Currently, the only clinical application of DPD is as a predictor of 5-FU toxicity, because a lack of this enzyme would prevent the catabolism of 5-FU. DPD, however, is not used clinically to evaluate survival or tumor progression.[11] Current studies do not demonstrate consistent evidence to recommend the clinical use of thymidine phosphorylase as a marker for tumor response to chemotherapy or as a predictor of survival.[11,12]

Figure 3.Thymidine synthesis pathway. 5-FU binds and inhibits TS. 5-FU: 5-fluorouracil; DPD: Dyhyropyrimidine dehydrogenase; TP: Thymidine phosphorylase; TS: Thymidine synthase
 

Table 1. Current Molecular Markers for the Diagnosis, Prognosis and Surveillance of Patients With Colorectal Carcinoma


Method Diagnosis Prognosis Surveillance
Fecal marker Recommended:
FOBT screening
Not recommended:
DNA – tumor cells		    
Tumor tissue marker   Not recommended:
18qLOH (DCC, DPC4)
MSI
DNA (index)
% S phase
TP
DPD
TS
K-ras		  
Serum marker Not recommended:
CEA
CA 19.9		 Recommended:
CEA		 Recommended:
CEA
Genetic profiling Not recommended Not recommended Not recommended

 

The listed recommendations are based on the findings by the American Society for Clinical Oncology (ASCO)-2206 and the European Group on Tumor Markers (EGTM)-2007 with the exception of microarray analysis, which has not even made it to their evaluation for assessment as potential markers.

CEA: Carcinoembryonic antigen; DCC: Deleted in colorectal carcinoma; DPD: Dihydropyrimidine dehydrogenase; FOBT: Fecal occult blood test; LOH: Loss of heterozygosity; MSI: Microsatellite instability; TP: Thymidine phosphorylase; TS: Thymidine synthase.

K-ras

The K-ras oncogene is a well-described mutation in the adenoma-to-carcinoma sequence in CRC. Mutated forms of K-ras occur in up to 50% of CRC. Activation of the K-ras oncogene leads to increased cell proliferation and survival advantage in cancer cells. Several studies have investigated the potential role of K-ras as a predictor of aggressiveness in colon cancer. Most studies have shown that the presence of K-ras mutations in CRCs are a marker for poor clinical outcome.[57,58] Owing to the inconsistency of data from current studies and the high degree of variability in the data of individual studies, K-ras is not currently recommended as a marker for predicting tumor response or to assess chemotherapeutic response.[11,12]

Carcinoembryonic Antigen

Recent studies have shown CEA to provide prognostic information in the progression of patients with CRC.[59] CEA determined preoperatively may provide prognostic information if elevated following surgical intervention.[11,12]

    Summary of Tissue Markers for CRC Prognosis

    Although expert panels have not advocated 18qLOH, MSI, DNA index and K-ras in the clinical arena, the wealth of research is overwhelmingly promising and, together with ongoing prospective randomized clinical trials, this position is likely to change over the next few years. For instance, Chayvialle et al. are investigating p53 and MSI tumor status along with other pathological features to predict the efficacy of cetuximab and bevacizumab in patients with metastatic colonic liver lesions.[101] p53-mediated pathways are being evaluated to determine outcomes in patients with CRC receiving chemoradiation.[102] Liang et al. attempt to determine allocation of chemotherapeutic intervention based on tumor status of MSI, TS, DPD, microvessel density and EGF receptor in patients with metastatic colon cancer.[103] K-ras status is being evaluated in patients with metastatic colon cancer who have failed fluoropyrimidine- and oxaliplatin-based chemotherapy with bevacizumab to determine the efficacy of panitumumab in combination with irinotecan plus avastin (FOLFRI).[104] Similarly, K-ras mutations along with several other markers are being evaluated to determine clinical response to cetuximab and oxaliplatin/5-FU/leucovorin (FOLFOX4) versus FOLFOX4 alone in patients with initially unresectable colon cancers.[105]

    Indeed, the future of all these tumor markers in the assessment of prognosis and response to chemotherapeutic interventions shows a great deal of potential. Furthermore, tumor markers in combination with clinicopathological parameters, such as patients presenting with bleeding or perforated tumors (T4 tumors), can all be included in regression analysis models to provide strong independent predictors of outcomes in patients with CRC.

    CRC Surveillance

    A total of 80% of CRC recurrences will occur within the first 3 years following resection of the primary tumor, emphasizing the need for close surveillance during this period of time.[60] Tumor circulating cell antigens and microarray analysis (MA) are being developed to assist with the molecular aspects of surveillance in the management of CRC. Currently, however, the only serum marker clinically available to follow patients after surgical intervention for cure is CEA.[11,12] High CEA serum levels (> 15 ng/ml) are associated with poor prognosis independent of tumor stage.[16,61] Postoperatively, CEA levels should fall within 4 weeks. If CEA levels remain elevated, this is an indication of incomplete tumor resectability or occult metastasis. However, whether CEA levels are initially elevated or not, it is typically the first test to identify relapse in a cost-effective manner.[62] Additionally, The American Joint Committee on Cancer recommends CEA levels to be a part of the staging of CRC to further discriminate patients at risk of relapse.[63] The current recommendation for postoperative surveillance according to the National Comprehensive Cancer Network and ASCO guidelines is to obtain a baseline CEA every 3–6 months for 2 years, then every 6 months for the next 5 years for T2 lesion or higher in patients with CRC who are candidates for curative resection.[64]

    Novel Techniques

    Prior to seeding in a host organ, tumor cells must enter the circulation and be able to escape the immune system. In this early process, it is possible to detect tumor circulating cells in the serum of patients who underwent curative surgery to determine early relapse. The presence of tumor cells in peripheral blood can be detected by RT-PCR. The main advantage of molecular techniques for the detection of circulating cancer cells is its great sensitivity. Only 5 cells/ml of blood are required for the detection and amplification of a specific mRNA.

    CEA mRNA presence in peripheral blood samples of patients with CRC was an independent predictor for metastatic disease.[65,66] Other tumor markers present in tumor cells detected by RT-PCR in peripheral blood samples of patients with CRC include: hTERT, cytokeratin (CK)-19 and -20. In 72 CRC peripheral blood samples examined, mRNA amplification for hTERT, CK-19, CK-20 and CEA occurred in 70, 67, 53 and 72% of CRC patients, respectively. Amplification by RT-PCR of any of these genes did not occur in the peripheral blood derived from healthy subjects.[67] A noninvasive blood test membrane array, assaying hTERT, CK-19, CK-20 and CEA simultaneously determined that patients with all of these four tumor markers independently predicted relapse in patients with stage II CRC.[68] Along with pathological characteristics of the tumor, such as depth of tumor invasion and lymphovascular involvement, and the number of removed lymph nodes (which were also independent predictors by regression analysis), these markers could be used to select stage II patients for adjuvant chemotherapeutic intervention.[69]

    Evaluation of all of these tumor markers assessed by membrane construct arrays was a good predictor for relapse in 157 subjects following curative surgery for CRC (stage I–III) with normal serum CEA levels. Concomitant evaluation of all markers predicted tumor invasion, lymph node metastasis, as well as CRC recurrence 6 months prior to elevation in serum CEA.[70] This combination method had a detection sensitivity of around 80% for circulating tumor cells.

    K-ras mutations have also been investigated by the generation of membrane arrays. The sensitivity and specificity of detecting K-ras mutations in circulating tumor cells in patients with CRC were 84 and 90%, respectively.[71] Perhaps a combination array of CEA, CK-19, CK-20 and hRTERT may increase the sensitivity and specificity of these tests to more accurately predict tumor relapse in patients with CRC submitting to curative surgery. Other tumor markers amplified by RT-PCR in the peripheral blood of patients with CRC include REG-4, uPA, and TIAM1, which are elevated in 80, 79 and 80% of the cases, respectively. A combination of up to six circulating tumor cell marker panels has demonstrated a sensitivity of 89%, specificity of 88% and accuracy of 88%.[67] Thus, the higher the number of markers analyzed in the peripheral blood, the better the accuracy. As more markers become available and the technique becomes more standardized and cost-effective, this technique can be further evaluated in larger well-controlled studies. At the present time, while the studies are promising, they still consist of small cohorts of patients, and more data are needed to further evaluate their clinical utility as a form of surveillance or to implement specific chemotherapeutic interventions.

    Microarray Analysis

    MA is a technique that permits the study of up to thousands of genes concurrently in a single experiment. On the GeneChip® oligonucleotide array, a given gene is represented by 15–20 different 25-mer oligonucleotides that serve as unique, sequence–sequence detectors. This requires RNA extraction, which can be obtained from tissue or from tumors or peripheral blood by standard laboratory techniques. Gene expression arrays may serve as molecular signatures to determine diagnosis and prognosis [72-75]as well as treatment strategies in patients with various malignancies.[76,77]

    In vitro MA was utilized to detect multiple genes responsible for resistance to cell death by cytotoxic challenge induced by cisplatin in metastatic colon cancer cells.[78] Gene array expression indentified 23 genes differentially expressed in patients with stage II colon cancer who developed recurrence within 3 years following surgical intervention.[79] Other studies have utilized two separate statistical analyses and indentified eight genes differentially expressed in stage II CRC at risk of relapse. Similarly, gene expression profiling of fresh-frozen samples from patients with stage III CRC indentified patients at risk of relapse.[80]

    Currently, there is a high degree of variability even in the same data sets examined, such that ten different unique signatures to differentiate colon tumor cells from normal cells can be derived from the same data set.[81] As a result of the high sensitivity of the MA, inherent noise in the data array may be the culprit for such variability. This results in a wide range in accuracy for this technique's ability (86–100%) to differentiate colon cancer cells from normal cells.[81] Newer methodologies have been shown to reduce the amount of noise and variability of microarrays for the evaluation of particular signatures in patients with colon cancer; however, they are still in the experimental stages.[82] As the technique develops in sophistication and noise is minimized, MA holds a great deal of promise in the molecular diagnosis and prognosis of CRC.

    MA is currently being investigated to predict the sensitivity of resistance to two first-line agents in patients with CRC.[106] Similarly, MA is being studied to determine the genetic profiles in patients with stage II and III rectal cancer before and after chemoradiation treatment.[107]

    Tissue Microarray

    Newer investigational techniques such as tissue microarray (TMA) may enable the processing of hundreds of markers within a single experiment. TMA, a novel technique developed by Kononen et al.,[83] allows for the study of as many as 1000 histological sections with a single paraffin block. These sections, dots, are then mounted onto slides for examination with any given specific antibody. Because of the ability to create multiple sections from the same paraffin-embedded block, a number of antibodies can be simultaneously assayed. This process accelerates the examination that would take much longer to be performed by immunohistochemistry of individual samples. Additionally, accuracy is much more reliable because the newly fabricated tissue dots are submitted to the same processing.

    It is possible that with continued use of this technique, multiple tissue markers can be assayed simultaneously. For instance, Bonavida's group has demonstrated by TMA that the transcription factor Ying-Yang-1 predicted relapse in patients with prostate cancer, independently of prostate-specific antigen levels.[84] These same principles can be applied to CRC.

    Thus, while other markers under current investigation such as DCC, DPC4 and others have not been endorsed for clinical use by expert panels, it is important to keep them in close proximity while awaiting the implementation of newer technological advances.

    Rectal Cancer

    Rectal cancer accounts for up to 28% of all CRC cases and more than 70% of patients with the disease present stages II–IV (involvement beyond the colonic mucosa).[85] Even though colon and rectal cancers are typically grouped and staged together, the pelvic location of the rectum and its proximity to the anal sphincter and bladder, as well as to sympathetic and parasympathetic nerves, results in a substantially more challenging inability to obtain clear radial margins. As a result, morbidity (including sexual and bladder dysfunction as well as pelvic recurrence) remains high. In light of this, neoadjuvant modalities have been developed to downstage tumors to aid with surgical intervention and decrease the rate of local recurrence. Despite current aggressive multimodality treatments, there is still a substantially high rate of recurrence (5–15%).[86-90] The expected 5-year survival rate of these patients is a dismal 9%.[91] Efforts to improve local control and survival in rectal cancer are the focus of multiple current clinical and research efforts.

    One important difference in the management of rectal cancer compared with colon cancer is the use of ionizing radiation either in the neoadjuvant setting or as an adjuvant modality. However, pre-, intra- or postoperative radiation therapy, either alone or in combination with chemotherapy, results in a tremendously wide clinical response, with nearly 10% of patients achieving complete pathological response and up to 9% being nonresponders.[91] The wide heterogeneity in tumor response is the result of tumor size, differentiation and cellular phenotype of rectal cancer cells. The relapse rate of up to 15% in early rectal cancers and the 90% mortality in patients with recurrence mandates a refinement of surgical techniques and an optimization of the therapeutic ratios of radiotherapy and chemotherapy. An understanding of the mechanisms leading to tumor cell radiation resistance will result in optimal operative intervention, reduced rates of recurrence, lower mortality, and decreased adverse effects of radiation therapy by appropriate radiosensitizing interventions. Ionizing radiation exerts its therapeutic response by causing single- and double-stand breaks in DNA leading to apoptosis.

    Within the multiple pathways of apoptosis, the NF-κB-mediated pathway of apoptosis leading to the elevation in the levels of the IAPs, particularly survivin, has emerged as a potential mechanism resulting in a radio-resistant phenotype. Elevated levels of the IAP, survivin, resulted in resistance to radiation-induced cell death, and silencing of this gene by small-interference RNA studies radiosensitized CRC cells to radiotoxic challenge.[92] The IAP family consists of eight members, of which survivin has been the only one studied in radiation resistance-mediated apoptosis. Nonetheless, XIAP is the most potent of all IAPs. Preliminary studies from our laboratory demonstrate a more substantial increase in XIAP compared with survivin in radio-resistant CRC cells.[93]

    It is possible that TMA of multiple IAPs, including survivin, may lead to the identification of genes resistant to radiation resistance such that therapy can be implemented in this group of patients. Similarly, patients with tumor samples at the time of diagnosis with elevated levels of IAPs by TMA may lead to identification of radio-resistant tumors such that aggressive surgical intervention can be implemented.

    Pathways in Colorectal Cancer

    The development of newer diagnostic techniques may allow for the investigation of pathways in the molecular prognosis and response to chemotherapeutic interventions in patients with CRC. The pathways in colorectal neoplasia can be broadly divided into:

    • Proliferation
    • Apoptosis
    • Angiogenesis
    • MMR genes

    In colon cancer, two well-characterized pathways resulting in cell proliferation are the Wingless (Wnt) and the Hedeghog (Hg) pathways.[18] Activation of the Hg pathway requires the collaboration of multiple circulating ligands and participation of smoothened proto-oncognene, which leads to activation of cytoplasmic elements culminating in transcriptional factor modifications and cell proliferation. Activation of the Wnt pathway may result from mutations of the APC gene, which destabilizes β-catenin leading to the activation or expression of cell proliferation genes, such as c-MYC and cyclin D.[18] Novel techniques that enable the detection of multiple genes or proteins with the same mRNA or same tissue block, such as MA and TMA, may allow for the detection of various genes simultaneously, which along with other markers of proliferation, such as K-ras, may be used in patients with CRC to predict tumor aggressiveness or relapse.

    Chemotherapeutic response is likely to be determined by the investigation of multiple proteins in the apoptotic pathway. Because most chemotherapeutic interventions elicit an upregulation of proapoptotic genes with a concomitant downregulation of antiapoptotic mediators leading to tumor cell resistance to apoptosis, this pathway is the most likely to yield usefulness in chemotherapeutic response prediction. Within the pathway of apoptosis, the intrinsic (mitochondrial) pathway is activated by elevation of p53, which then stimulates BAX, leading to cytochrome c release and activation of the caspases.[27] Examination of these mediators by MA or TMA is likely to result in predictive information regarding chemotherapeutic interventions. Investigation of these apoptotic mediators along with analysis of the thymidine synthesis pathway is likely to specifically predict patient's response to 5-FU.

    Examination of the targeted pathways including VEGF and EGF receptors is likely to provide predictive information for metastatic potential or response to bevacizumab or cetuximab. Both of these targeted pathways can be examined by either MA or TMA. Similarly, multiple MMR genes examined simultaneously by MA or TMA may increase the sensitivity of screening for patients with HNPCC.

    Expert Commentary & Five-year View

    The greatest promise for the analysis of CRC diagnosis and prognosis rests with molecular analysis of CRC pathways. As new approaches become available and the cost of current technologies is reduced, the molecular diagnosis of CRC will become more widely available to undertake well-randomized controlled studies. Similarly, standardization of techniques for currently available tumor markers for the prognosis of CRC in tissue specimens, such as 18qLOH (DCC, DPC4), MSI, DNA (index), percentage S-phase, thymidine phosphorylase, DPD, TS and K-ras, will eanble more uniform data collection to provide more consistent results such that these can be included in clinical practice to determine tumor aggressiveness or response to chemotherapeutic interventions. Implementation of MA and TMA techniques may facilitate the reliable use of multiple tumor markers in prognosis and therapeutics of CRCs.

    The current techniques that hold the greatest promise in the next 5 years for the diagnosis and prognosis of colon cancer are fecal serial analysis of gene expression (BAT-26, L-DNA, APC, K-ras and p53 mutations) and serial analysis of tumor circulating antigens, MA and TMA. More sophisticated and standard techniques in the detection of fecal-based DNA assays are likely to be developed over the next 5 years, and hold a great deal of promise in the early identification of cancers and to serve as a screening tool. Tumor-circulating cell markers are most promising in providing diagnostic information in patients following curative surgery, and for assessing relapse and determining specific forms of chemotherapeutic modalities. MA and TMA assays may have promise in facilitating the evaluation of specific signatures in tumor tissues to determine prognosis. Thus, in the next 5 years, the utility of MA in the diagnosis, prognosis and surveillance of CRC is promising.

    The inhibitors of apoptosis including survivin and XIAP are important mediators of radiation resistance in rectal cancer. It is possible that TMA techniques can be implemented to determine radio-resistant rectal tumors or rectal tumors at risk of relapse,

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    Krishan Maggon

    Make your own contribution please

    There is no point in copy and paste efforts. It is bad for both knol and you as well as readers.

    As a student of medicine you can give your own understanding and insight of the disease. It is unethical to steal someone work and ideas without citation.



    Aug 18, 2009 10:01 AM
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    micheal miller
    micheal miller
    student in medicine
    ALGERIA
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