The molecular medicine of colorectal cancer

This report highlights some of the discussions at a Keystone Symposium entitled ‘The Molecular Medicine of Colorectal Cancer’ held at Taos, NM, USA, from 30 January to 4 February, 2001.     

Distinct molecular features of colorectal cancer in Ghana

Objectives: While colorectal cancer (CRC) is common, its incidence significantly varies around the globe. The incidence of CRC in West Africa is relatively low, but it has a distinctive clinical pattern and its molecular characteristics have not been studied. This study is one of the first attempts to analyze molecular, genetic, and pathological characteristics of colorectal cancer in Ghana. Methods: DNA was extracted from microdissected tumor and adjacent normal tissue of 90 paraffin blocks of CRC cases (1997–2007) collected at the University of Ghana. Microsatellite instability (MSI) was determined using fragment analysis of ten microsatellite markers. We analyzed expression of mismatch repair (MMR) proteins by immunohistochemistry and sequenced exons 2 and 3 of KRAS and exon 15 of BRAF. Results: MSI analysis showed 41% (29/70) MSI-High, 20% (14/70) MSI-Low, and 39% (27/70) microsatellite-stable (MSS) tumors. Sequencing of KRAS exons 2 and 3 identified activating mutations in 32% (24/75) of tumors, and sequencing of BRAF exon 15, the location of the common activating mutation (V600), did not show mutations at codons 599 and 600 in 88 tumors. Conclusions: Our study found a high frequency of MSI-High colorectal tumors (41%) in Ghana. While the frequency of KRAS mutations is comparable with other populations, absence of BRAF mutations is intriguing and would require further analysis of the molecular epidemiology of CRC in West Africa.     

Components of the Mediterranean Diet with chemopreventive activity toward colorectal cancer

Colorectal cancer is one of the leading causes of cancer incidence and death worldwide. For the past 30 years researchers have tried to find drugs suitable for the chemoprevention of the diseases. Epidemiological studies have demonstrated that adherence to the Mediterranean Diet (MD) is protective toward colon cancer development. Key components of this diet are olive oil (OO), fruits and vegetables, legumes and fish. Importantly, many bioactives present in these foods have shown anti-cancer activity in in vitro as well as in animal models of colon carcinogenesis, indicating a possible use as preventive agents. However, when translated to human trials, single bioactives have yielded conflicting results mostly due to irreproducibility of pre-clinical data. Since our preclinical models are usually designed to test single molecules at high concentrations and for very short time in order to provide significant effects, future studies should employ multiple bioactives that would resemble a more natural approach with possible significant synergisms that might be critical for cancer prevention. In this review we will discuss data obtained with components of the MD with a specific focus on OO, omega-3 polyunsaturated fatty acids and apples.     

Histological and molecular evaluation of patient-derived colorectal cancer explants

Mouse models have been developed to investigate colorectal cancer etiology and evaluate new anti-cancer therapies. While genetically engineered and carcinogen-induced mouse models have provided important information with regard to the mechanisms underlying the oncogenic process, tumor xenograft models remain the standard for the evaluation of new chemotherapy and targeted drug treatments for clinical use. However, it remains unclear to what extent explanted colorectal tumor tissues retain inherent pathological features over time. In this study, we have generated a panel of 27 patient-derived colorectal cancer explants (PDCCEs) by direct transplantation of human colorectal cancer tissues into NOD-SCID mice. Using this panel, we performed a comparison of histology, gene expression and mutation status between PDCCEs and the original human tissues from which they were derived. Our findings demonstrate that PDCCEs maintain key histological features, basic gene expression patterns and KRAS/BRAF mutation status through multiple passages. Altogether, these findings suggest that PDCCEs maintain similarity to the patient tumor from which they are derived and may have the potential to serve as a reliable preclinical model that can be incorporated into future strategies to optimize individual therapy for patients with colorectal cancer.     

Noninvasive molecular biomarkers for the detection of colorectal cancer

Colorectal cancer (CRC) is the third most common malignancy in the world. Because CRC develops slowly from removable precancerous lesions, detection of the disease at an early stage during regular health examinations can reduce both the incidence and mortality of the disease. Although sigmoidoscopy offers significant improvements in the detection rate of CRC, its diagnostic value is limited by its high costs and inconvenience. Therefore, there is a compelling need for the identification of noninvasive biomarkers that can enable earlier detection of CRC. Accordingly, many validation studies have been conducted to evaluate genetic, epigenetic or protein markers that can be detected in the stool or in serum. Currently, the fecal-occult blood test is the most widely used method of screening for CRC. However, advances in genomics and proteomics combined with developments in other relevant fields will lead to the discovery of novel non invasive biomarkers whose usefulness will be tested in larger validation studies. Here, noninvasive molecular biomarkers that are currently used in clinical settings and have the potential for use as CRC biomarkers are discussed.     

DNA aptamers as molecular probes for colorectal cancer study

Background

Understanding the molecular features of specific tumors can increase our knowledge about the mechanism(s) underlying disease development and progression. This is particularly significant for colorectal cancer, which is a heterogeneous complex of diseases developed in a sequential manner through a multistep carcinogenic process. As such, it is likely that tumors with similar characteristics might originate in the same manner and have a similar molecular behavior. Therefore, specific mapping of the molecular features can be potentially useful for both tumor classification and the development of appropriate therapeutic regimens. However, this can only be accomplished by developing high-affinity molecular probes with the ability to recognize specific markers associated with different tumors. Aptamers can most easily meet this challenge based on their target diversity, flexible manipulation and ease of development.

Methodology and Results

Using a method known as cell-based Systematic Evolution of Ligands by Exponential enrichment (cell-SELEX) and colorectal cancer cultured cell lines DLD-1 and HCT 116, we selected a panel of target-specific aptamers. Binding studies by flow cytometry and confocal microscopy showed that these aptamers have high affinity and selectivity. Our data further show that these aptamers neither recognize normal colon cells (cultured and fresh), nor do they recognize most other cancer cell lines tested.

Conclusion/Significance

The selected aptamers can identify specific biomarkers associated with colorectal cancers. We believe that these probes could be further developed for early disease detection, as well as prognostic markers, of colorectal cancers.     

Reconciliation of classification systems defining molecular subtypes of colorectal cancer

Recently we published two independent studies describing novel gene expression-based classifications of colorectal cancer (CRC). Notably, each study stratified CRC into a different number of subtypes: one reported 3 subtypes, whereas the second highlighted 5. Given that each ascribed clinical significance, distinctive biology, and therapeutic prognosis to the different subtypes, we sought to reconcile this apparent incongruity in subtype stratification of CRC, and to interrelate the results. To do so, we each evaluated the other’s data sets and analytical methods and discovered that the subtypes and their classifiers are, in fact, clearly related to each other; indeed, the 5 subtype outcomes can be coalesced into the same three. In addition to presenting this clarification, we briefly discuss how both classification methods can be viewed within the broader literature on CRC subtypes, and potentially applied.     

Novel molecular classification of colorectal cancer and correlation with survival

IntroductionColorectal cancer (CRC) is one of the most common cancers worldwide. This study was designed to evaluate biological patterns, explore molecular classification and correlate with survival outcome in treatment naïve CRC patients.MethodsOver 11 years consecutive series of 435 CRC patients were operated on as primary surgical therapy. A total of 201 CRC patients were included, whose complete set of clinical information was available, and their good quality tumour blocks were retrieved. Immunohistochemistry was used for tumour analysis, and partitional clustering was performed using R software for cluster analysis.ResultsThe median age was 43 (range 10–85) years; adenocarcinoma was the most commonly seen histological type. The great majority had positive CK20, CEA, E-Cadherin, Ki67, CDX2, and p53 expression. There were four distinct molecular classes found, whereas Ki67, CDX2, and p53 play the main role in partitioning. Younger age negatively impacted survival; overall and disease-specific survival was 26 months only with 50 months’ longest survival.ConclusionColorectal cancer is a biologically heterogeneous disease with at least four distinct molecular patterns, where cell proliferation and gene repair mechanisms appear to play the key role.     

Rho GTPase signaling in the development of colorectal cancer

The involvement of Rho GTPases in major aspects of cancer development, such as cell proliferation, apoptosis, cell polarity, adhesion, migration, and invasion, have recently been attracting increasing attention. In this review, we have summarized the current findings in the literature, and we discuss the participation of the Rho GTPase members RhoA, Rac1, and Cdc42 in the development of colorectal cancer, the second most lethal neoplasia worldwide. First, we present an overview of the mechanisms of Rho GTPase regulation and the impact that regulator proteins exert on GTPase signaling. Second, we focus on the participation of Rho GTPases as modulators of colorectal cancer development. Third, we emphasize the involvement of activation and expression alterations of Rho GTPases in events associated with cancer progression, such as loss of cell-cell adhesion, proliferation, migration, and invasion. Finally, we highlight the potential use of novel anticancer drugs targeting specific components of the Rho GTPase signaling pathway with antineoplastic activity in this cancer type.     

Familial benign hypercalcemia--from clinical description to molecular genetics.

Familial benign hypercalcemia (or familial hypocalciuric hypercalcemia), a syndrome of lifelong hypercalcemia inherited as an autosomal dominant trait, is distinct from the multiple endocrine neoplasia syndromes and other forms of inherited parathyroid disease. Familial benign hypercalcemia results from the inappropriate secretion of parathyroid hormone despite hypercalcemia, enhanced renal tubular reabsorption of calcium (independent of parathyroid hormone), and apparent tissue resistance to adverse effects of hypercalcemia. Heterozygosity for the familial hypercalcemia trait is benign, although homozygosity for the trait may lead to severe neonatal primary hyperparathyroidism. Genetic linkage studies show that most persons affected with familial hypercalcemia have a mutation on the long arm of chromosome 3 (3cen-q21), although one phenotypically indistinguishable family appears to have a mutation on the short arm of chromosome 19 (19p), and another family has neither 3q nor 19p mutations. One group has recently shown mutations in a putative parathyroid cell-surface calcium receptor that are plausible causes for the chromosome 3q variant of the familial hypercalcemia syndrome. Perhaps the other genes for this syndrome encode proteins representing hitherto-unknown regulators of systemic calcium metabolism independent of parathyroid cell calcium sensing or proteins involved in signal transduction from the calcium receptor.     

Mendel's genes: toward a full molecular characterization

The discipline of classical genetics is founded on the hereditary behavior of the seven genes studied by Gregor Mendel. The advent of molecular techniques has unveiled much about the identity of these genes. To date, four genes have been sequenced: A (flower color), LE (stem length), I (cotyledon color), and R (seed shape). Two of the other three genes, GP (pod color) and FA (fasciation), are amenable to candidate gene approaches on the basis of their function, linkage relationships, and synteny between the pea and Medicago genomes. However, even the gene (locus) identity is not known for certain for the seventh character, the pod form, although it is probably V. While the nature of the mutations used by Mendel cannot be determined with certainty, on the basis of the varieties available in Europe in the 1850s, we can speculate on their nature. It turns out that these mutations are attributable to a range of causes-from simple base substitutions and changes to splice sites to the insertion of a transposon-like element. These findings provide a fascinating connection between Mendelian genetics and molecular biology that can be used very effectively in teaching new generations of geneticists. Mendel's characters also provide novel insights into the nature of the genes responsible for characteristics of agronomic and consumer importance.     

Construction of preferential cDNA microarray specialized for human colorectal carcinoma: molecular sketch of colorectal cancer

cDNA microarray analyses can be used to identify candidate genes that play important roles in human carcinogenesis. To gain insight into the molecular sketch of colorectal cancer, we have constructed cDNA microarrays specialized for colorectal cancer, which we named "Colonochip" by selecting genes that are expressed in colorectal cancer, normal colonic mucosa, and liver metastatic cancer tissues. This microarray contained 4608 nonredundant cDNA clones from over 30,000 cDNA clones derived from the three types of human cDNA libraries, as well as clones from 170 additional conventional major genes suspected to be involved in colorectal carcinogenesis, according to literatures. Using this "Colonochip," we were able to identify 59 genes showing twofold or more differential expression between primary cancer and normal colonic mucosa, potent candidates for diagnosis, and therapy of colorectal cancer for further studies.     

Vaccines for colorectal cancer.

Despite recent advances in the treatment of colorectal cancer, the overall survival rate for those patients with advanced locoregional disease remains less than 50%. Although adjuvant systemic chemotherapy has improved survival of these patients, more effective therapies are needed. Immunotherapy is an approach that could have a particular role in the adjuvant therapy of colorectal cancer. There is now convincing evidence that the immune system can specifically recognize and destroy malignant cells. Although both antibody- and T-cell-mediated anti-tumor responses have been documented, the cellular immune response with its direct cytotoxic mechanisms is felt to be the principal anti-tumor arm of the immune system. Analysis of the T cells that recognize tumors has led to the identification and characterization of many tumor-associated antigens including several colorectal antigens. Current approaches to developing a vaccine for colorectal cancer use our expanded understanding of these tumor-associated antigens and the conditions that allow development of an effective cellular immune response to them.