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Molecular Characterization of Colorectal Neoplasia in Translational Research.

Molecular markers are being used extensively to characterize neoplastic processes. As we use these markers, the terms molecular markers and biomarkers refer to the evaluation of specific molecules in tissues as markers of either disease or induced changes in normal or diseased tissues. Molecular markers can be used in the descriptions of any disease process, but this manuscript will concentrate on molecular markers in neoplastic lesions of the colorectum. Much of the research using biomarkers translates the results of bench research for use in the diagnosis and treatment of human diseases and hence is designated translational research.

The use of molecular markers in translational research has expanded considerably during the last 3 decades, and this increased analysis of specific molecular changes has been associated with a concomitant decline in the use of more general and less specific histochemical stains and biochemical assays. Some of the applications for molecular markers include diagnosis, early detection, and prognosis. Also, specific molecular markers are used to study the biology of the disease,[1,2] to identify targets for novel therapies (eg, use of Herceptin), and to aid the selection of specific therapies.[3,4]

The utilization of molecular markers has advanced most rapidly in recent years. In the 1960s, pathologists relied on morphology, histochemical assays, and enzyme assays for diagnosis. To these tests, the modern pathologist and laboratorians have added immunohistochemical, ligand, and genotypic assays. Pathologists once relied on special histochemical stains to separate poorly differentiated tumors and achieve diagnosis; now, a wide range of specific and nonspecific molecular markers can aid in this process. Nevertheless, in substituting a single molecular marker for a more general histochemical test, which may identify many different molecular species, sensitivity is frequently lost and sometimes specificity is lost, especially when a specific marker is related only indirectly to the question being evaluated. For example, consider the use of either the periodic acid--Schiff (PAS) stain or the Alcian blue PAS (ABPAS) stain to identify mucins and thereby separate poorly differentiated adenocarcinomas from lymphoid or neuroendocrine malignancies of the colorectum. The PAS stain identifies a wide range of molecules, primarily sugar moieties that can be oxidized to produce reducing groups such as aldehydes. Aldehydes and other strong reducing groups reduce the dye of the PAS stain to produce an insoluble red-purple precipitate. The PAS stain can identify a specific molecule when that molecule is in a large group of similar molecules. This aggregation ensures that enough aldehydes are produced on oxidation of sugar moieties to produce a visually detectable amount of precipitated dye.

Several molecular markers are available for the characterization of colorectal adenocarcinomas (CRCs). These markers include tumor-associated glycoprotein 72 (TAG-72)[5-7] and carcinoembryonic antigen (CEA),[7,8] which identify and detect recurrence of adenocarcinomas. Other molecular markers can be used to diagnose specific metastatic tumors to the colorectum, such as prostate-specific antigen for metastatic prostate cancer or HMB-45 for metastatic melanomas.[9,10] To date, we are unaware of any molecular marker that is specific for the diagnosis of CRC. Of the markers for adenocarcinomas, our experience indicates that TAG-72 as recognized by the antibody CC49, may represent the most useful and sensitive of this group of molecular markers. The marker TAG-72, like CEA, Lewis X, or Lewis Y, is an oncofetal tumor antigen, that is usually not expressed in tissues that are "normal."[6]; however, like most oncofetal tumor antigens, both inflammatory and neoplastic events in the immediate vicinity of normal epithelial cells may induce the expression of TAG-72.[11] Just as TAG-72 and the PAS stain may be useful in separating specific subtypes of epithelial malignancies, other molecular markers, such as neuron-specific enolase[12] and leukocyte common antigen,[13] may be useful in identifying neuroendocrine and hematopoietic malignancies of the colorectum, respectively. Several schemes have developed to separate various broad categories of malignancies (Table 1).

[TABULAR DATA 1 NOT REPRODUCIBLE IN ASCII]

Recent molecular and immunophenotypic data have helped to correct earlier misclassifications of several types of malignancies. For instance, the studies of Ansell et al[14] and Wick et al[15] have demonstrated that malignant angioendotheliomatosis is a lymphoma and not a vascular tumor. Identification of this rare intravascular lymphoma and use of the correct specific therapy has resulted in a greatly improved clinical outcome for this previously fatal disease.[16] Similar molecular studies might promote an accurate understanding of certain tumor types, such as gastrointestinal stromal tumors; alveolar soft-part sarcomas; or solid, cystic, and papillary tumors of the pancreas, the histogenesis of which remain unclear.

Molecular markers may be used in diagnosis to describe subtypes of tumors, for example, the mucinous subtype of colorectal adenocarcinoma. Colorectal adenocarcinomas have been defined as tumors with a mucin composition of 50% or more. However, the results of our studies of phenotypic expression of molecular markers such as p53,[17] MUC1, MUC2,[18] and bcl-2[19,20] are more consistent with defining mucinous adenocarcinomas as tumors that have a mucinous component of 10% or more.[17]

The use of molecular markers in the early detection of neoplastic changes in the colorectum is in its infancy. Whereas preinvasive neoplastic changes that may lead to squamous cell carcinoma of the cervix have been characterized histopathologically, the preinvasive neoplastic changes that occur in the colorectum have not been characterized nearly as well. For example, it is only in the last 5 years that aberrant crypt foci have been identified as an important preinvasive neoplastic lesion in the human colorectal mucosa, even though these lesions were identified several years previously in a rat model of CRC.[21-23]

Table 2 lists the molecular features identified in aberrant crypt foci.[24] Also, recent studies have documented the reduced incidence of CRCs after the removal of colorectal polyps, supporting the neoplastic nature of adenomatous polyps.[25-27] Therefore, as our knowledge of preinvasive neoplastic lesions increases, pathologists are likely to be asked to identify relatively newly described preinvasive lesions based on molecular as well as histopathologic changes. Similarly, researchers will be challenged to develop schema that describe more numerous intermediate endpoints in the development of CRC. We believe that this characterization will be facilitated by the use of specific molecular markers to characterize specific preinvasive neoplastic lesions.

[TABULAR DATA 2 NOT REPRODUCIBLE IN ASCII]

Molecular characterization can also be used to clarify the biology of colorectal neoplasia. Studies of hereditary tumors of the colorectum have identified 2 major pathways by which CRCs develop--the suppressor gene pathway and the mutator phenotype pathway. The suppressor gene pathway was characterized from studies of hereditary CRC that developed in patients with familial adenomatous polyposis coli (Figure 1). In the suppressor gene pathway, sequential mutations occur in a series of suppressor genes, beginning with the suppressor gene, adenomatous polyposis coli (APC).[28-31] Mutation of both copies of this gene increases epithelial proliferation and hence the likelihood of additional mutations.[28,29] Subsequently, mutations develop in other suppressor genes such as p53, the mutated in colorectal cancer gene (MCC),[32] and the deleted in colorectal cancer (DCC) gene.[33] In addition, in the suppressor gene pathway, some proto-oncogenes such as K-ras[34] may also be activated by mutation. Patients with familial adenomatous polyposis coli inherit 1 mutated or deleted APC gene, but adenomatous polyps do not develop until somatic mutations develop in the originally nonmutated APC gene.[35] Tumor progression occurs as other suppressor genes (eg, p53) and proto-oncogenes (eg, K-ras) are subsequently somatically mutated in the proliferating cells carrying the 2 mutated copies of APC (Figure 1).

[Figure 1 ILLUSTRATION OMITTED]

In the mutator phenotype pathway, enzymes that normally act to repair inaccurately copied DNA no longer function or function only partially. When specific repair enzymes (eg, hMLH1, hMSH2, hMSH3, hPMS1, hPMS2, and hMSH6) can no longer repair DNA that has been copied inaccurately, errors accumulate in the DNA and ultimately cause an increase in the mutation rate (Figure 2). The repair enzymes that are affected are those enzymes that correct inaccurate copies of the DNA in areas of genes with multiple nucleotide repeats, usually mononucleotides, dinucleotides, trinucleotides, or tetranucleotides. Such repeats are called microsatellites. This pattern of tumor development was initially identified by studying CRCs that develop in patients from families with hereditary nonpolyposis colon cancer.[36,37] Typically, the affected family member inherits 1 copy of a mutated gene that codes for a specific repair enzyme. When the second, originally nonmutated copy develops a nonconservative, somatic mutation, functional molecules of the repair enzyme are no longer produced, and errors accumulate in multinucleotide repeats of the DNA. This accumulation of errors has been called microsatellite instability, ubiquitous somatic mutation, or replication error rate. Tumors are hypothesized to develop in the setting of MSI because microsatellites are found in the regulatory areas of several important genes, including receptor II for transforming growth factor [Beta][38,39] and the receptor for insulin-like growth factor 2[40]; abnormalities of this pathway are especially important in the development of colon cancers (Figure 2).

[Figure 2 ILLUSTRATION OMITTED]

Characterization of the molecular markers in sporadic CRCs has led to hypotheses that one, both, or neither of these pathways are involved in the development of specific sporadic CRCs. For example, both APC and p53 are mutated in the majority of sporadic CRCs; similarly, mutations in K-ras are common, but mutations in MCC and DCC are less frequent in sporadic CRCs. The temporal order in which these mutations develop in sporadic CRCs varies, although APC is probably the initial mutation in most cases. Molecular markers can be used to characterize early intermediate endpoints in the development of colorectal neoplasia; mutations in APC and K-ras are found in aberrant crypt foci and these mutations, along with mutations in p53 and DCC, can be found in large adenomatous polyps.[41-44]

Although the suppressor gene pathway and the mutator phenotype pathway can be described as part of the early development of some CRCs, much less effort has been devoted to describing molecular changes related to invasion and metastasis of CRCs.

Although several tumor markers are present in the blood (such as CA-19-9 LASA-p antimalignin)[45] or present within tumors (such as DNA ploidy markers of proliferation, mucins, or mucin antigens), serum carcinoembryonic antigen (CEA) is the only molecular marker that has been recommended for the detection of recurrent colorectal cancer in patients by both the American Society of Clinical Oncology and the College of American Pathologists.[46]

An important application for molecular markers is their use for the identification of subsets of cancers that are more or less aggressive. We believe that within the next decade, clinicians will no longer consider the routine histopathologic diagnosis of specific neoplastic lesions alone to be adequate. In addition to the histopathologic diagnosis, the medical care team will require the results from a panel of molecular markers that characterize the prognosis of specific tumors.

Our laboratory is currently involved in identifying prognostic molecular markers in several epithelial tumors. We have described patterns for 3 molecular markers--p53 nuclear accumulation and phenotypic expression of bcl-2 and MUC1--that aid in identifying aggressive subsets of CRCs. Specifically, we have noted that immunohistochemical demonstration of nuclear accumulation of p53 in proximal tumors of Caucasian patients indicates a poor prognosis (Figure 3, A).[1] We do not find p53 to be prognostically useful for distal colorectal tumors in Caucasians, or for proximal or distal colorectal tumors in African Americans (Figure 3, B through D). MUC1 is also prognostically useful in Caucasians, but not in African Americans; however, its usefulness as a prognostic marker does not vary with anatomic location[18] (Figure 4). In contrast, strong phenotypic expression of bcl-2 is associated with less aggressive tumors of the distal colorectum in both Caucasians and African Americans (Figure 5) 20; in addition, p53 and bcl-2 combine to become an important aggregate prognostic factor for colorectal cancers in Caucasian patients (Figure 6, A),[19,20] as do p53 and MUC1 (Figure 6, B).[18] Another molecular marker with prognostic significance for patients with tumors of the colorectum is p27.[47,48]

[Figure 3-6 ILLUSTRATION OMITTED]

Another closely related application is the use of biomarkers for selecting or excluding specific types of novel therapies. For example, in immunotherapy, selected targets such as [p185.sup.erbB-2] must be expressed on the surface of neoplastic cells for the immunotherapy to be effective.[49,50] For the immunoglobulin to reach its target, the marker must be expressed on the external membrane, unless the immunoglobulin is attached to a ligand that has a cell-surface receptor, which allows the immunoglobulin to enter the cell and reach its internal target. One of the better examples of effective immunotherapy is the treatment of ductal carcinoma of the breast with Herceptin, a monoclonal antibody that recognizes and binds to the external domain of the [p185.sup.erbB-2] proto-oncogene.[51] Herceptin interacts with the [p185.sup.erbB-2] proto-oncogene, which is strongly expressed on the cell membrane, and causes cellular toxicity.[52] Other antibodies that are toxic to specific cells simply as a result of their interaction with specific receptors include some that have been developed against the epidermal growth factor receptor.[53] Several institutions, including the University of Alabama at Birmingham, are investigating the use of antibodies to these receptors for the treatment of CRC. Alternatively, death of malignant cells can be induced by antibodies that are coupled with a radionuclide, for the delivery of local radiotherapy, or coupled with a toxin, to deliver localized toxic chemotherapy.[54] Some newer methods include using antibodies to specific tumor-cell targets, such as TAG-72, that are coupled with biotin; attachment of the antiTAG-72 antibodybiotin complex to TAG-72 on the tumor cells is followed by a chemotherapeutic or toxic agent attached to streptavidin.[55] The therapeutic concept is that by using such directed, local radiotherapy or chemotherapy, high doses of an agent that cannot be tolerated by the whole body can be selectively delivered to the tumor Immunotherapeutic antibodies may not only have direct cytotoxic actions or deliver agents that produce toxicity, but their interaction with cell-surface targets may also make cells more susceptible to conventional therapies (eg, radiation or chemotherapy).

In addition to immunotherapy directed at targets identified by molecular markers, specific forms of gene therapy may sometimes require the demonstration that either a molecular pathway is abnormal or that a specific target molecule is expressed. For example, p53 mutations could be demonstrated before delivering native p53 to tumor cells to correct the apoptosis pathway. Similarly, if a particular target receptor is shown to be expressed on the surface of tumor cells, a ligand for the receptor may be used as an entry point into the cell for the transfection of the gene of interest.[56-57] For example, cells may be transfected with cytosine deaminase; treatment with the nontoxic compound 5-fluorocytosine then results in the local production of the toxic metabolite 5-fluorouracil.[58]

Molecular markers can also be used to identify cellular features, which may indicate that a specific therapy may be more or less effective. For example, certain chemotherapeutic agents such as 5-fluorouracil are less effective in cells with mutations in the p53 gene. Similarly, breast tumors, which express [p185.sup.erbB-2], are less sensitive to specific chemotherapeutic regimens.[59,60] Thus, in the future, laboratorians may be asked to provide the immunophenotype of specific tumors to guide the selection of a patient's therapeutic regimen.

Molecular markers are also used to identify cellular changes that are produced by chemotherapeutic or chemopreventive agents. Identification of changes in specific molecular markers in response to these agents may be an approach for monitoring the effectiveness of preventive and other therapies. When molecular markers are used in this approach, they are referred to as surrogate endpoint biomarkers. Changes in specific molecular markers may indicate that a therapeutic or preventive agent is having a clinical effect; ideally, such a marker would be on the direct pathway of the neoplastic development and/or progression. Alterations in this marker could therefore indicate that an agent is ultimately either going to prevent the development of clinically significant cancers or is preventing the progression of such tumors. The use of markers of proliferation in assessing response to radiation therapy in rectal cancer is a good illustration. Even though neither proliferating cell nuclear antigen (PCNA) nor Ki-67/MIB-1 is a prognostic indicator (because proliferation is not associated with clinical outcome in CRC), Willett et al[61,62] demonstrated that small rectal tumors with high PCNA staining in preoperative biopsies responded better to radiation therapy than tumors with low proliferating indices; furthermore, they reported a significant decrease in PCNA staining in postirradiation biopsies.

Although we and others have demonstrated that molecular markers can mirror the response of tumors to therapeutic interventions in animal models, their use as surrogate endpoint biomarkers in chemoprevention studies has not been demonstrated. Also, several concerns surround the use of alterations in cell-cycle regulators, protooncogenes, and tumor suppressor genes as molecular markers in chemoprevention trials. The usefulness of biomarkers as surrogate endpoint biomarkers in chemoprevention trials may depend on several factors, such as the ability to reproducibly identify activation of specific protooncogenes; the extent of phenotypic expression of specific biomarkers in tumors; and the stage of tumor development at which specific molecular changes occur. The complexity of genetic and epigenetic changes in particular types of tumors also make it difficult to use molecular markers as biomarkers; furthermore, the association between a single molecular change and the changes in different stages of tumor development caused by chemopreventive agents is not always clear. However, there are several frequently activated proto-oncogenes, such as the ras oncogene, which is mutationally activated during the early development of a variety of tumors, including preinvasive neoplastic lesions that lead to colorectal cancers. The effect of sequential biopsies on adjacent tissues is yet another problem. We have noted a pronounced activation of the prostate (increased proliferation index and increased expression of tumor growth factor-[Alpha], epidermal growth factor receptor, and [p185.sup.erbB-2]) following an initial biopsy. This has been designated as a "biopsy effect".[63] The extent of biopsy-induced changes in specific molecular markers and the temporal pattern of biopsy effects in various tissues remain to be determined.

In summary, the characterization of molecular markers can aid in the diagnosis and early detection of colorectal neoplasia. Molecular markers enable better definition of subtypes of colorectal neoplasia (eg, mucinous adenocarcinomas) as well as more complete molecular descriptions of intermediate endpoints in the progression of colorectal neoplasia (eg, aberrant crypt foci). Similarly, molecular markers have been useful in clarifying the biology of colorectal neoplasia and in identifying aggressive subsets (immunophenotypes) of tumors. In the near future, molecular characterization will not only be important in the selection and evaluation of specific therapeutic approaches, including chemopreventive, chemotherapeutic, and novel therapies (eg, immunotherapy and gene therapy), it will also be helpful in histologic classification of colorectal tumors.

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Accepted for publication August 15, 2000.

From the Department of Pathology, Comprehensive Cancer Center, Bio-Statistics Unit, University of Alabama at Birmingham, Birmingham, Ala.

Presented at the Ninth Annual William Beaumont Hospital DNA Technology Symposium, DNA Technology in the Clinical Laboratory, Roayl Oak, Mich, April 13-15, 2000.

Reprints: William E. Grizzle, MD, PhD, Department of Pathology, University of Alabama at Birmingham, Zeigler REsearch Building, Room 422, 703 South 19th St, Birmingham, AL 35233-0007 (e-mail: grizzle@path.uab.edu).
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Author:Grizzle, William E.; Manne, Upender; Jhala, Nirag C.; Weiss, Heidi L.
Publication:Archives of Pathology & Laboratory Medicine
Geographic Code:1USA
Date:Jan 1, 2001
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SUBJECTS ENROLLED IN PRESEPT COLORECTAL CANCER SCREENING.
Potential new target to stop colorectal cancer's spread identified.
Aspirin 'cuts colorectal cancer death risk'.

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