EMERGING MOLECULAR MARKERS OF CANCER

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1 EMERGING MOLECULAR MARKERS OF CANCER David Sidransky Alterations in gene sequences, expression levels and protein structure or function have been associated with every type of cancer. These molecular markers can be useful in detecting cancer, determining prognosis and monitoring disease progression or therapeutic response. But what is the best way to identify molecular markers and can they be easily incorporated into the clinical setting? The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross 818, Baltimore, Maryland 21205, USA. DOI: /nrc755 The histopathological progression from preneoplasia to cancer is accompanied by the accumulation of genetic alterations. These lead to altered expression patterns and modifications in protein structure and function 1. Changes that occur exclusively or more commonly in cancer cells compared with their normal tissue of origin can be detected by biopsy or in body fluids, and used as molecular markers of cancer (FIG. 1). These markers are useful in detecting cancer at early stages, assessing tumour burden, monitoring disease progression and determining response to therapy. There are many clinical situations in which molecular tumour markers are already being used (TABLE 1).For example, screening for prostate-specific antigen (PSA) a molecular marker of prostate cancer is now part of the physical examination of most male patients during yearly evaluations in the United States 2.However, about one-third of patients with an elevated PSA undergo further unnecessary medical procedures because they do not have a malignant form of prostate cancer. New markers would allow identification of patients who harbour the most dangerous tumours that require immediate treatment. Similarly, measurements of serum carcinoembryonic antigen (CEA) levels are now used to monitor disease progression and response to therapy in patients with colorectal cancer 3.However, only a proportion of colorectal cancers express elevated CEA levels at the time of diagnosis. More reliable serum markers are therefore needed to improve colorectal cancer diagnosis and to follow cancer progression. For most other types of cancer, there are no molecular markers available. For example, there are no molecular markers that can be used to detect lung cancer at early stages the time when it is most amenable to therapy. Imaging can be used to detect early tumours in patients with diseases such as breast cancer, but this approach is highly controversial 4. Metastasis-specific markers are also desperately needed to help to delineate the spread of the disease to neighbouring tissues, lymph nodes or distant parts of the body. Although many effective cancer therapies have recently been developed, there are only a few molecular markers that are available at present for determining treatment response. Breast cancer cells that express high levels of the tyrosine receptor kinase ERBB2 (also known as HER2/neu) are more likely to respond to trastuzumab (Herceptin, an antibody that targets this receptor), and assays have been developed to establish whether the patient s tumour actually expresses this protein before the drug is administered 5,6. Many of the new molecularly targeted therapeutics do not act by conventional cytotoxic mechanisms; instead, they disrupt other aspects of tumour growth such as angiogenesis or tissue invasion. It is difficult to monitor the clinical effect of these drugs, as they might not produce immediate or dramatic changes in tumour size 7, so other means of confirming drug activity are required. For example, if a drug is designed to inhibit a specific kinase, assays that measure phosphorylation of its target, or activation of downstream signalling proteins, can be used to determine drug activity 8. In addition, patients who do not seem to be responding to therapy could be tested to make sure that the drug produces the 210 MARCH 2002 VOLUME 2

2 Summary Alterations in gene sequences, expression levels, and protein structure or function can be used as molecular markers to detect cancers at an early stage, determine prognosis, and monitor disease progression or therapeutic response. DNA-based markers of cancer include mutations, loss-of-heterozygosity, microsatellite instability, DNA hypermethylation, mitochondrial DNA mutations and detection of viral DNA. There are various ways of detecting cancer cells by analysing RNA. Techniques to identify alterations in protein structure or function that are associated with cancer include antibody-based detection methods, measurement of enzymatic activity and two-dimensional gel analysis of samples. New approaches to discovering molecular markers include microarray analysis, serial analysis of gene expression and proteomic technologies. Clinical researchers should design future trials to incorporate plans for collecting and analysing molecular markers. Molecular biologists should take advantage of the number of tumour samples now available in tissue banks to identify new molecular markers and to more fully assess existing ones. mutations, MICROSATELLITE instability, hypermethylation of promoter regions and viral DNA 12. DNA can also be easily isolated from other body fluids such as saliva, urine and stools. Cancer-associated mutations. Proof of principle for this type of approach in solid tumours came in 1991 and 1992, when TP53 (the gene that encodes p53) mutations were identified in the urine of patients with bladder cancer 13 and RAS mutations in the stools of patients with colorectal cancer 14. This led researchers to search for other DNA mutations that could be used in cancer detection and diagnosis. But new assays were required to detect individual mutations in DNA samples that also contained large amounts of wild-type DNA (BOX 1), which led to the development of highly sensitive polymerase chain reaction (PCR) assays 15,16. The limitations of PCR were quickly realized. Although PCR could be used to detect a specific single genetic mutation, cancer desired biological effect. So, what are the most effective molecular markers in use at present, and what techniques are being developed to identify new ones? a Organ Epithelium MICROSATELLITE DNA Repetitive stretches of short DNA sequences. Current cancer markers The earliest approaches to identifying cancer markers were based on preliminary clinical or pathological observations. Gold and Freedman 9 were the first to observe the overexpression of CEA a protein that is usually restricted to fetal development in colorectal cancer cells, and this led to its use as a sensitive serum-based marker for disease progression. Many other markers discovered in this manner, however, were found to be associated with only a small proportion of tumours, preventing their widespread use in diagnosis. The discovery of oncogenes led to the identification of genetic mutations that were associated with cancer phenotype and prognosis. The human genome sequence has further facilitated the discovery of new tumour-suppressor genes and proto-oncogenes. Nucleic-acid-based markers. Studies in human cancers and preneoplastic lesions led to the discovery of genetic modifications that occur at early stages of tumorigenesis, and which could be used to detect small tumours. The first genetic analyses were initially carried out on biopsy samples, but the discovery of free DNA in plasma opened new avenues in screening for cancer (FIG. 1). Free DNA was identified 50 years ago in the serum or plasma of patients with cancer, and elevated levels of DNA were found in the serum of patients with metastatic disease 10,11.The mechanism by which this DNA is released and survives in the circulation is not well understood. Serum or plasma DNA is not cancer specific elevated levels of DNA have also been observed in patients with severe infections and in those with autoimmune diseases. Plasma or serum from cancer patients is now analysed to detect tumour markers such as oncogene b Mitochondria DNA mutations Nucleus Tumour Cancer cells lost in body fluid (urine, blood) Chromosomes Aberrant copy number Translocations Deletions (LOH) Telomere extension DNA Point mutations Microsatellite alterations Promoter hypermethylation Viral sequences RNA Over/underexpression Point mutations Protein Structural alterations/modifcations Changes in enzymatic activity Mislocalization Altered expression Figure 1 Molecular marker detection. a As an epithelial tumour grows, cancer cells are sloughed off the organ epithelium into body fluids such as blood plasma, urine or saliva. This makes it possible to detect molecular markers such as DNA mutations, methylation patterns or microsatellite instability in these samples before they are symptomatic. b There are several different types of cancer marker that can be detected in serum, urine or saliva samples. DNA can be analysed for changes in gene copy number, chromosome translocations, deletions or loss of heterozygosity (LOH), telomere extension, microsatellite instability, promoter hypermethylation or point mutations. Mitochondrial DNA can also be analysed for mutations. RNA can be analysed for expression levels or point mutations, and proteins can be analysed for structural alterations, changes in enzymatic activity, localization or expression patterns. NATURE REVIEWS CANCER VOLUME 2 MARCH

3 Table 1 Selected molecular markers of cancer Cancer type Clinical sample DNA marker* RNA Protein marker Head and neck Saliva, serum TP53, microsatellite alterations, Cytokeratins SCC, CD44, presence of HPV and EBV DNA CYFRA, telomerase Lung Sputum/BAL, RAS and TP53 mutations, Cytokeratins, CEA, CA125, serum microsatellite alterations MAGE genes, telomerase, CEA CYFRA Breast Serum Microsatellite alterations Cytokeratins, CA15-3 (MS-1) hmam, MAGE CEA, CA125 genes,cea Colon Stool, serum RAS, APC and TP53 mutations Cytokeratins, CEA, CA19-9, CEA CA15-3, telomerase Pancreas Stool, serum RAS and TP53 mutations Cytokeratins, CA19-9 CEA Bladder Urine/wash, TP53 mutations, microsatellite Cytokeratins, CEA, CA125, serum alterations survivin, uroplakin CA19-9, telomerase, survivin, CD44 Prostate Urine, serum PSA, MAGE PSA, free PSA, genes, kallikrein telomerase, kallikrein *Promoter hypermethylation in DNA is listed separately in TABLE 2. Most protein markers in use are not specific enough for routine screening and are used predominantly to monitor response or disease progression. Virtually all genetic markers are still in early stages of development. Prostate-specific antigen (PSA) is widely used to screen men for prostate cancer. Cancer antigens include CA15-3, CA125, CA19-9 and CEA. Telomerase is a ribonucleoprotein and usually enzymatic activity is measured; some studies have used direct measurement of the RNA (htr) component. Most protein markers are measured in serum but other bodily fluids such as urine, saliva and nipple aspirates have been tested for the presence of aberrant proteins. APC, adenomatous polyposis coli; BAL, bronchoalveolar lavage; CYFRA, cytokeratin 19 fragment; EBV, Epstein Barr virus; hmam, mammaglobin; HPV, human papillomavirus; microsatellite alterations, loss of heterozygosity (LOH) and/or instability; SCC, squamous-cell carcinoma antigen. cells frequently contain several mutations in different oncogenes or tumour-suppressor genes. Simple PCR analysis is not useful in searching for groups of mutations that are associated with a given tumour type. More complex multiplex approaches involving initial amplification of the target gene fragment from DNA by PCR followed by mutation-specific ligation of small cdna strands were developed to identify multiple mutations. Recent studies report the use of this approach to detect common mutations in genes that encode RAS, adenomatous polyposis coli (APC) and p53 in stool samples of patients with colorectal cancer 17,18,19 (see also the Trial Watch article on page 159 in this issue). Mass spectroscopy has also been used to identify a panel of RAS mutations in the sputum of patients with lung cancer, and a specific TP53 mutation at codon 249 in the serum of patients with hepatocellular cancer 20,21. Similar panels of probes for other cancer-associated groups of mutations could be developed to detect different tumour types. Genetic mutation analysis is useful not only in detecting cancer in patients, but also in monitoring disease spread and in determining prognosis. In 1995, it was first reported 22 that TP53 mutations could be used to follow tumour spread into margins and draining lymph nodes of head and neck cancer patients. Approximately half of all head and neck tumours harbour TP53 mutations, and patients with mutant TP53-positive surgical-tissue margins were found to have a high risk of recurrence and poor overall survival. Further studies were carried out in patients with colorectal cancer, in which the presence of a tumour-specific KRAS or TP53 mutation in the lymph-node samples predicted poor outcome 23. Loss of heterozygosity and microsatellite instability. All cells contain two copies of autosomal genes one copy inherited from each parent. If a cell develops a mutation in one allele of a tumour-suppressor gene, loss of the remaining wild-type allele loss of heterozygosity (LOH) can initiate tumorigenesis. LOH is a hallmark of cancer that can be detected by various PCRbased approaches in most preneoplastic lesions and primary tumours. Microsatellite DNA markers are used as a tool to detect LOH in primary tumours. Because cancer cells are genetically unstable, microsatellites are often expanded or deleted in cancer cells. This characteristic known as microsatellite instability is another valuable clonal marker of cancer 24. In a study carried out in 1996, urine samples from 25 patients with suspicious bladder lesions that had been identified cystoscopically were analysed for LOH, and also by conventional cytology 25. Microsatellite alterations that matched those in the tumour were detected in the urine of 95% of the patients with bladder cancer, whereas urine cytology detected cancer cells in only 50% of the samples. These were the first results to indicate that microsatellite analysis could be a useful addition to current screening methods for detecting bladder cancer. Microsatellite analysis was also shown to be a useful technique for monitoring response to therapy. When urine samples from patients who had been treated for bladder cancer were analysed for 20 polymorphic microsatellite markers, recurrent lesions were detected in 10 out of 11 patients 26. The test correctly predicted the existence of a neoplastic-cell population in the urine of two patients, up to six months before cystoscopic evidence of the tumour. Furthermore, the assay was negative in all patients 212 MARCH 2002 VOLUME 2

4 Box 1 Sensitivity versus specificity One of the goals of molecular markers is to allow a physician to identify a small number of cancers in a tissue sample that contains many normal cells. Molecular tests usually begin with a preparation of a protein or DNA extract from a clinical sample. The ratio of neoplastic cells to normal cells varies considerably from one tissue sample to another. It is difficult to specifically isolate neoplastic cells for analysis from clinical samples, as samples are often composed primarily of cellular debris and free substrate (such as DNA, RNA and protein). So, clinical samples are frequently a heterogeneous mixture of normal and cancer-cell DNA and protein. Two of the most important factors in determining the efficiency of a molecularmarker assay are level of sensitivity (what is the minimal amount of the substrate that can be detected?) and specificity (what percentage of assays correctly distinguish normal from cancer-containing samples?). There is always a trade-off between sensitivity and specificity. High levels of sensitivity might reduce specificity, leading to the detection of the substrate when it not actually present (causing false-positive results). Alternatively, a highly specific probe might not be very sensitive, and might not always detect substrate when it is present (causing false-negative results). The sensitivity and specificity are initially established in well-controlled studies, using samples that contain a known amount of substrate. They are then formally assessed in clinical trials by comparing assay results to results of other, previously established tests. The bottom line is that more sensitive tests are not necessarily better. SINGLE-NUCLEOTIDE- POLYMORPHISM (SNP) ANALYSIS Analysis based on single basepair changes in DNA that differ among individuals. SNPs can be identified by various molecular means. CpG ISLANDS A region of DNA with a high density of cytosine phosphoguanine nucleotides, which are usually located in the promoter region or the first exons of a gene. CpG islands are involved in the regulation of transcription, because their methylation can lead to permanent silencing of the associated gene. BRONCHOALVEOLAR LAVAGE FLUID A fluid sample that is obtained by inserting a tube into the lung. NESTED PCR Amplification of a DNA sequence that entails one initial amplification with a set of primers, followed by a second amplification with a set of internal (nested) primers, to allow a more robust amplification. who had no evidence of cancer. Microsatellite analysis of urine therefore became a powerful clinical tool for the detection of recurrent bladder cancer. Microsatellite analysis has also been used to detect head and neck squamous-cell carcinoma (HNSCC) one of the cancer types that is desperately in need of molecular markers due to its rapid progression 27. Analysis of a panel of 23 microsatellite markers detected microsatellite instability in exfoliated oral mucosal cells: LOH or microsatellite instability of at least one marker was detected in 86% of primary tumours, and identical alterations were found in the saliva samples in 79% of cases. No microsatellite alterations were detected in samples from control subjects. In another study, 29% of HNSCC patients were found to have one or more microsatellite alteration in serum that precisely matched that in the primary tumours 28. Microsatellite alterations have also been observed in 76% of small-cell lung cancers (SCLC) and also in plasma samples 29. The lower frequency of LOH that is detected in the serum of HNSCC patients compared with the plasma of SCLC patients might reflect the increased propensity of SCLC to metastasize to distant sites. A role for measuring LOH in any of the above clinical situations awaits further trials in larger cohorts. LOH analysis is not particularly sensitive and therefore requires large quantities of cancer-cell DNA. In most cases, 50% of the total DNA in the sample has to be tumour-derived to reliably detect LOH. Microsatellite instability is easier to detect than LOH, and can identify approximately one cancer cell among 200 normal cells 24 (BOX 1). Some cancers, such as a subset of colon cancers, have widespread microsatellite instability due to deficiencies in mismatch-repair proteins. Direct comparison of molecular detection methods in paired fluids of patients with lung cancer, however, showed that PCR analysis of oncogene mutations was more sensitive than microsatellite analysis in detecting the presence of cancer cells 30. Microsatellite analysis is also difficult to carry out on a large number of clinical samples because many markers must be tested. Improved techniques have reduced the time required, but a large panel (15 20) of microsatellite markers is still required to reliably detect cancer 31. Analysis of SINGLE NUCLEOTIDE POLYMORPHISMS (SNPs) is also being developed as a means to detect cancer cells, although this approach requires an even larger number of markers approximately three SNPs are required to have the same efficiency as microsatellite detection. However, compared with microsatellite analysis, SNPs that are detected by hybridization with DNA probes or other approaches might yield more linear and reliable results 32. SNP assays have also been incorporated into high-throughput platforms in continuing efforts to identify cancer-associated polymorphic alleles. DNA methylation. Another encouraging type of marker is based on detecting hypermethylation of promoter regions of cancer-associated genes (TABLE 2). CpG ISLANDS in gene-promoter regions are methylated as part of gene regulation 33. A large amount of cytosine methylation in a gene promoter can lead to a complete block of transcription 34. Many types of cancer cell have been shown to use this mechanism to inactivate tumour-suppressor genes. PCR assays have been developed to rapidly and accurately identify methylated regions of DNA 35. In this procedure, DNA is treated with bisulphite, which converts unmethylated cytosines to uracil, whereas methylated cytosines are protected and not converted. The sensitivity of the assay is approximately one cancer cell among 1,000 normal cells, which is sensitive enough to detect tumour DNA in most body fluids. This approach has been used to detect cancer cells in the saliva of patients with oral cancer 36, in the sputum and BRONCHOALVEOLAR LAVAGE FLUID of patients with lung cancer 30,37,38, and in the serum of patients with lung 39, head and neck, and colorectal cancers 40,41. Groups of hypermethylated genes can be associated with different tumour types. Techniques that allow analysis of multiple molecular markers are important, as combinations of several markers are likely to provide a more accurate prognosis than analysis of one marker at a time. For example, methylation of genes that encode INK4A (also known as p16; a cyclin-dependent kinase inhibitor that is encoded by CDKN2A), DAPK (death-associated protein kinase, which is involved in resistance to apoptosis) and MGMT (methyl O-guanine methyltransferase, which is involved in DNA repair) has been associated with lung, and head and neck cancer. These genes were found to be methylated in serum samples of over 50% of lung, and head and neck cancer patients 39,40. Similarly, methylation of APC, which is involved in WNT signalling, was observed in the serum and plasma DNA of early-stage lung cancer and oesophageal cancer patients 42,43. The list of cancer-associated methylated genes is expanding as methylated targets have been catalogued in many primary cancers 44. Recent attempts to use NESTED PCR have increased sensitivity, but its use might also increase the number of false-positive results 38. NATURE REVIEWS CANCER VOLUME 2 MARCH

5 Table 2 Examples of genes that are hypermethylated in cancer Tumour type Primary tumour* Body fluid Colon CDKN2A, MGMT, Serum (MLH1, CDKN2A) MLH1, DAPK, TIMP-3, APC Breast CDKN2A, BRCA1, GSTP1, CDH1, TIMP-3, RASSF1A Nipple aspirate (CCND2, RARβ) Lung CDKN2A, MGMT, DAPK, TIMP-3, APC, RASSF1A Serum and sputum/bal (CDKN2A, MGMT) Head and neck CDKN2A, MGMT, DAPK, RASSFIA Serum and saliva (CDKN2A, MGMT, DAPK) Bladder APC, RASSF1A, CDH1, CDH3, FHIT, RARβ Urine (RASSFIA, RARβ) Pancreas CDKN2A, MGMT, APC None Prostate CDKN2A, GSTP1, ER, CH1, CD44, EDNRB Serum and urine (GSTP1, CD44) *Genes found to be methylated in more than 10% of primary tumours or tested in DNA isolated from a body fluid. APC, adenomatous polyposis coli; BAL, bronchoalveolar lavage; BRCA1, breast and ovarian cancer-1; CCND2, the gene that encodes for cyclin D2; CD44, cluster designation 44; CDH1, E-cadherin-1; CDH3, E-cadherin-3; CDKN2A, cyclin-dependent kinase inhibitor-2a; DAPK, deathassociated protein kinase; EDNRB, endothelin receptor B; ER, oestrogen receptor; FHIT, fragile histidine triad; GSTP1, glutathione S- transferase protein 1; MLH1, Mut L homologue 1; MGMT, methylguanine-dna methyltransferase; RARβ, retinoic acid receptor-β; RASSFIA, human RAS association domain family 1A; TIMP-3, tissue inhibitor of metalloproteinases-3. REAL-TIME PCR The ability to monitor the linear phase of PCR during the actual amplification stages, usually by means of a fluorogenic compound and a laser to detect the accumulation of amplification products. MITOCHONDRIAL D-LOOP A section of the mitochondrial genome that is thought to be involved in replication, and contains short poly-pyrimidine tracts. HOMOPLASMIC The presence of identical genomes within all of the mitochondrial organelles in a cell. A recent innovation in promoter hypermethylation detection has been the development of quantitative assays. REAL-TIME-PCR-based approaches which involve amplification by DNA polymerases with monitoring of fluorescent signals during the actual amplification process, such as Taqman can be used to quantify the number of methylated alleles (in a single region) among wild-type DNA. DNA that is isolated from tumour biopsies harbours high levels of specific gene methylation, whereas cell samples that are taken from benign tumours show no methylation or very low levels. These assays have been used to detect DNA with cancer-cellspecific methylation patterns in the serum of patients with oesophageal cancer and lung cancer 42,43. The ability to quantify methylation represents an important innovation in molecular-marker analysis. In anecdotal cases, methylation levels were quantified and monitored in patients during the course of therapy 43. Methylation levels of the gene that encodes the detoxification protein glutathione-s-transferase placental enzyme 1 (GSTP1) were found to be elevated in prostate cancer cells compared with normal tissue 45. This study was unique in that it associated a specific level of DNA methylation with cancer. GSTP1 methylation analysis was also used to correctly distinguish between cancer and non-cancer patients, when results were compared with those that are obtained by biopsy 45. Assays that measure GSTP1 methylation therefore have the potential to be developed as quantitative prostate cancer markers. Mitochondrial DNA mutations. Mitochondrial DNA mutations have also been associated with various tumour types. Mitochondria dysfunction was proposed to be involved in cancer over 50 years ago 46, although mutations in mitochondrial DNA were thought to be rare in tumour cells. Mitochondria are believed to be more susceptible to exogenous mutagens and also have less efficient DNA-repair mechanisms. Investigators described tumour-specific mutations in various regions of the mitochondrial genomes of cancer cells that were taken from patients with colorectal cancer 47. This work was quickly expanded to identify mitochondrial mutations in more than 50% of tumour types, including lung, head and neck, bladder and breast cancers 48. Although mitochondrial mutation analysis suffers from some of the same limitations as oncogene-mutation analysis in that mutations are spread throughout the genome the mitochondrial genome is significantly smaller than the nuclear genome, at slightly over 16,000 base pairs in size. Moreover, frameshift mutations in the MITOCHONDRIAL D- LOOP a region that seems to be a hot spot for alterations were found to occur at a high frequency (up to 25%) in many common tumour types 49. A unique feature of mitochondrial mutations is that they seem to be HOMOPLASMIC in cancer cells. This aids in their detection, as a given cell might harbour several hundred copies of the mitochondrial genome. Mitochondrial mutations are therefore easier to detect than nuclear DNA mutations. In patients with lung cancer, bronchoalveolar lavage samples were found to harbour almost 200-fold more mitochondrial mutations than nuclear TP53 mutations 48. High-throughput methods need to be developed in order to carry out mitochondrial DNA analysis of clinical samples on a regular basis. Viral DNA. Detection of viral DNA is also being developed as a molecular marker for virus-associated cancers. For example, the human papillomavirus (HPV) has been associated with cervical carcinoma, so testing for the presence of HPV DNA is a useful approach for identifying women who are at risk of developing this cancer. An assay used to measure HPV DNA was shown to detect cervical carcinoma in 100% of women whose cancer was detected by biopsy, and it also detected 75% of patients with high-grade squamous intraepithelial lesions, as well as 62% of patients with low-grade squamous intraepithelial lesions 50. HPV has been implicated as an aetiological factor in a subset of head and neck squamous-cell carcinoma, and HPV DNA was detected in the serum of patients with HPVpositive head and neck tumours, but not in those with HPV-negative tumours MARCH 2002 VOLUME 2

6 Epstein Barr virus (EBV) DNA has been detected in the plasma and serum of patients with nasopharyngeal carcinoma (NPC), which indicates an association between this virus and this form of cancer. EBV DNA concentrations were measured by PCR in the serum from patients with NPC and correlated with disease burden 52. Moreover, in patients with NPC, those with recurrence or metastasis within the first year after treatment had a higher median plasma EBV DNA concentration than those without. EBV DNA has been shown to be an independent prognostic indicator for early clinical events 53.Moreover,EBV serum DNA levels in NPC correlated well with the response to therapy and disease recurrence 53. RNA-based approaches. Genetic alterations lead to marked changes in the expression of many genes at the mrna level. Several mrna-based approaches have been developed to detect cancer cells in clinical samples. Isolation of intact RNA from bodily fluids and tissue samples is also possible, but generally requires cumbersome efforts to neutralize ubiquitous RNase enzymes. New methods have also been developed to extract degraded RNA molecules from paraffin tissues. One of the most common approaches that is used to identify and quantify mrna levels in tissue samples is reverse transcriptase PCR This assay can be used to quantify mrna levels of markers such as tyrosinase, which is only expressed in benign moles and melanoma cells. Its presence in serum can be a sign of cancer Cytokeratin mrna is a common marker of epithelial cells, so detection of cytokeratin transcripts in blood or lymph nodes can also indicate the presence of cancer cells. Although pathologists already test for the expression of cytokeratin protein by immunohistochemical analysis, many studies have attempted to detect cytokeratin mrna by reverse transcriptase PCR in the lymph nodes of patients with cancer to determine the spread of disease. Theoretically, patients with cytokeratin-positive lymph nodes might benefit from more aggressive therapies. Reverse transcriptase PCR can also be used to detect other markers, such as elevations in CEA levels that indicate colorectal cancer, or elevations in PSA that indicate prostate cancer. Reverse transcriptase PCR has also been used to detect these transcripts in lymph-node, bone-marrow or peripheral-blood samples, which indicate tumour metastasis 54,57. The presence of PSA mrna in bone-marrow aspirates from patients with prostate cancer has been used to confirm tumour spread 61. It is important to remember, however, that altered expression of some of these genes has also been reported in normal cells, which leads to false-positive results. More quantitative analyses might eventually determine a cut-off level for differentiating between cancer and normal cells, which would be similar to the approach that is used in quantitative methylation. A recent study 62 used a quantitative assay to detect a multimarker mrna panel in the serum of patients with breast cancer. Quantitative RNA analysis can also be used to monitor patient response to anticancer therapies. Levels of mrnas that encode dihydropyrimidine dehydrogenase, thymidylate synthase and thymidine phosphorylase in colon tumour samples have been correlated with patient response to 5-fluorouracil therapy 63,64. Protein markers. Several protein-based assays have also been developed to detect cancer cells. Most of these are antibody-based assays, although many new approaches are being developed. Protein-based assays typically detect proteins that are overexpressed or structurally altered in cancer cells, compared with normal cells. These approaches are generally used in the research setting, and are not yet feasible for larger clinical studies. One of the most common protein-based screens for cancer is to measure serum levels of PSA, as high serum levels of this protein are associated with prostate cancer. PSA levels, however, cannot be used as a sole indicator of cancer, and results must be considered in the context of other factors, such as age, prostate size, the presence of a prostate nodule and, ultimately, a tissue biopsy. At least a third of patients with high PSA levels do not have prostate cancer, and biopsies can miss small nests of cancer cells, so new tests for malignant prostate cancer are needed. One interesting approach will be to combine assays, such as the GSTP methylation analysis (described above), with the PSA test, to identify the patients that have cancer and need immediate therapy. After cancer is diagnosed, expression levels of certain protein markers can be followed to assess therapeutic response and to signal disease recurrence. Traditionally, increased expression of oncofetal proteins such as the human glycoprotein hormone (β-hcg) and α-fetoprotein (AFP) have been used to monitor testicular cancer and hepatocellular cancer, whereas expression levels of CEA are commonly used to monitor colon cancer progression. Older biomarkers, such as PSA for prostate cancer and cancer antigen (CA)125 for ovarian cancer, are used extensively to determine the response to therapy. CA125 has a positive-predictive value of less than 10% as a single marker for detecting cancer, but the addition of other tests, such as ultrasound screening, has improved its predictive value to about 20%. Newer markers such as CA15-3 for breast cancer and CA19-9 for pancreatic cancer are found in the serum of only a fraction of affected patients and do not always correlate with therapeutic response Additionally, expression levels of all these protein markers are elevated in the serum of patients with other cancers, and are occasionally elevated in patients without disease, precluding their use as individual agents in cancer screening. Immunohistochemical studies have also shown that protein markers can be used to predict patients in whom cancer is most likely to recur after therapy and, perhaps, those most likely to benefit from adjuvant therapy 70,71. Telomerase a ribonucleoprotein that is involved in telomere maintenance represents a promising molecular marker for cancer. This enzyme is required NATURE REVIEWS CANCER VOLUME 2 MARCH

7 Analysed by algorithm to generate a cancer-associated pattern Proteins Patient serum sample collected Protein spectra Fractionation Analysed by mass spectroscopy Peptides Digested with proteolytic enzymes Separated by chromatography Figure 2 Proteomic detection of cancer. Protein-pattern analysis, or proteomics, can be used to detect cancer cells in patient samples such as blood. Proteins are isolated from samples, fractionated and digested with proteolytic enzymes. The resulting peptides are further separated by chromatography, and this pattern is analysed by technologies such as mass spectroscopy. Bioinformatic technologies are then used to identify cancer-specific proteomic patterns, and to detect cancer in other samples, based on similarities between these patterns. for cellular immortalization, and is therefore expressed by almost every cancer type. There are several assays for measuring telomerase activity, including the telomerase repeat amplification protocol (TRAP) assay, which measures the ability of cellular extracts to add telomere repeats to a substrate 72. Other assays involve detection of the specific RNA component (htr) in clinical samples 73. Telomerase has been detected in the urine of patients with bladder cancer and in the saliva of patients with oral cancer One of the main limitations of this approach has been the discovery that various lymphocyte subtypes also express telomerase at early stages in development. These cells are found in the circulation, lymph nodes and even primary tissues, and low levels of telomerase have been detected in non-cancerous samples, which leads to a notable number of false-positive results in telomerase activity assays More quantitative telomerase assays that can determine precise levels of activity might be more useful in determining the presence of cancer cells in clinical samples. Some protein markers have been discovered through the presence of tumour-antigen-specific antibodies in the serum of cancer patients. Cancer cells overexpress proteins that are recognized by the immune system as antigens, due to their aberrant expression patterns (for example, normally expressed only during fetal development), post-translational modification or structural changes. Antibodies to p53 and its family member p63 have been described in the serum of patients with various types of cancers 77,78. In a case report, production of anti-p53 antibodies proceeded the clinical detection of lung cancer by two years 79. Molecular marker discovery Recently, high-throughput screening approaches that can simultaneously analyse expression patterns of several genes and proteins have been used to search for cancer-associated molecules 80,81. These comprehensive approaches are yielding a plethora of new molecular markers. Microarray analysis is now one of the most common ways to detect changes in gene expression in cancer and normal cells, and is quickly replacing differential PCR-based approaches. This approach allows for rapid surveillance of the expression of tens of thousands of genes in one experiment, and can be used to identify changes in gene-expression patterns in normal and cancer cells, or in different types of cancers 80,81. Serial analysis of gene expression (SAGE), alternatively, is a comprehensive cloning and sequencing method that can be used to identify and quantify expression of new genes as well as that of known genes 82. This technique is especially useful for analysing expression patterns of low-copy-number genes. A SAGE approach has recently been used to identify mesothelin a membrane-bound protein as a new marker for pancreatic cancer 83. Microchip-based approaches are also being developed to identify methylated regions of gene promoters, and gene-specific chips are available that can be used to screen a large number of samples for mutations in oncogenes such as TP53 (REF. 84). Although genomics has been the recent trend powered by tremendous investments from genomics companies and government projects racing to clone the human genome attention has turned to proteomics for the next wave of innovation. This approach is important, because post-translational protein modifications, some of which are cancer-cellspecific, cannot be determined from genomic information. Proteomic approaches have begun to provide interesting new cancer markers. Instead of searching for a single molecular marker of cancer, proteomics like genomics allows unbiased quantitative analysis of a large number of proteins in normal and malignant tissues, and in cell populations. Traditionally, proteomic methods have involved analysis of two-dimensional gels that separate proteins on the basis of size and charge. These have been refined to allow simultaneous analysis of a large pattern of proteins 85. Recently, investigators compared the proteomic profile of cancer cells with normal cells, and using this information were able to correctly identify unique proteins in the serum of patients with prostate cancer and ovarian cancer 86,87. Virtually every pharmaceutical company is now investing in high-throughput proteomic approaches to identify potential drug targets. High-throughput mass spectroscopy can now be used to compare protein profiles between cancerous and normal tissue samples, and to also detect cancer in patient samples (FIG. 2). Technologies such as matrix-assisted laser-desorption-ionization time-offlight (MALDI-TOF) mass spectroscopy or liquidchromotography ion-spray tandem mass spectroscopy (LC-MS/MS) have revolutionized protein analysis. In these approaches, protein samples, such as those from a patient s serum, are fractionated and 216 MARCH 2002 VOLUME 2

8 digested with proteolytic enzymes, and then separated by chromatographic methods 88. The partially resolved sample is then analysed by mass spectroscopy. Tumour-specific proteomic spectra are then determined by a combination of peptide mass analysis and partial peptide sequencing, in conjunction with database searches 89,90. Proteomic profiles can contain thousands of data points, and require sophisticated analytical tools. Recently, an algorithm has been created to analyse serum protein-mass spectra and to correctly identify ovarian cancer cases including subjects with stage-i disease with a sensitivity of 100% and a specificity of 95% (REF. 91, and see the In Brief article on page 155 in this issue). Proteomics can also be used to detect the targets of tumour-specific auto-antibodies that are commonly found in the serum of cancer patients. Antibody-containing patient sera can be used to probe two-dimensional blots of protein extracts extracted from the patient s own tumours to identify antigenic tumour proteins 92,93. Assays of circulating levels of such antigenic proteins, or their corresponding antibodies, might have some use in the early diagnosis of cancer. Using this approach, investigators identified several proteins, including annexin and RS/DJ-1, as immunological targets. After screening a larger number of sera from cancer patients, the authors confirmed that these proteins were expressed at significantly higher levels in breast and colon cancer samples than in controls. These newly discovered tumour antigens might also be developed as targets for immune therapy. Other investigators have used various molecular approaches to identify proteins that are secreted by cancer cells, as these are also likely to be elevated in serum. A wide range of new cancer-cell protein markers and/or protein sets are likely to be discovered using proteomic approaches. It is also likely that proteomics will be integrated into the clinical setting sometime in the future, and used to identify patients with cancer and to monitor disease progression. Comprehensive whole-genome approaches for marker detection will continue to flourish. The platforms on which they are based rely on similar concepts, but have significant differences in technical layout. Together, all of these techniques are useful in identifying new markers and cancer-associated expression or proteomic profiles. But the sheer number of markers that are identified by these technologies is overwhelming, and new bioinformatic approaches are still needed to distinguish true, clinically relevant markers 94,95. Future directions Although thousands of papers are published each year describing genetic mutations or alterations in expression levels that are associated with various types of cancers, very few of these are developed into reliable molecular markers that can be used routinely in the clinical setting. Some molecular markers are used in combination with other techniques, such as imaging or histological studies, to detect, diagnose and monitor cancer. However, as assays become more sensitive and quantitative, a more thorough assessment of a patient s cancer status will be based on molecular markers alone. The sensitivity and specificity of a molecular marker cannot be fully realized until careful testing is carried out in large numbers of tumour specimens and compared with normal controls to identify markers that are truly over-represented or qualitatively different in the neoplastic cells compared with normal cells. Incorporation of molecular-marker analysis into clinical trials is therefore a crucial part of marker development. Future clinical trials should be designed to incorporate assays to both identify molecular markers and to correlate known markers with patient outcome. Early-phase clinical trials can be anticipated to record not only a patient s therapeutic response, but also a gene-expression or proteomic profile. As a drug moves into advanced testing, specific molecular-marker profiles could be correlated with response. This data could be used in predicting other patients responses to the treatment, or to determine if a certain marker profile is associated with a specific type of cancer or pattern of cancer progression. The procurement of patient samples and subsequent testing in preneoplastic lesions to identify the timing of their appearance in tumour progression is of great importance. For many reasons, including initial analytical validation in cancers and controls, biological samples are often difficult to acquire. So, access to, and further development of, tissue banks which pair tissue samples with clinical data is of great importance. There are already many patient samples in tumour and tissue banks that can be used to identify markers, and have been analysed for markers that are associated with patient outcome. Unfortunately, not all samples have been preserved adequately for molecular analysis, and consent issues must be addressed before some of these stored samples can be used. Efforts to discover markers use large amounts of primary tumour samples or cell lines to facilitate initial analysis, and clinical samples of some tumour types are scarce. Several high-throughput strategies, such as capillary separation systems and hybridization platforms, have been miniaturized to reduce the amount of clinical sample that is required for analysis. Further development of molecular markers depends on cooperation between molecular biologists and clinical researchers. Molecular biologists must take advantage of the numerous biopsy samples and paired controls that are already preserved in tissue banks, and clinical researchers must incorporate marker analysis into their trials. It has been difficult to translate the large-scale use of molecular markers to the clinic, owing to the difficulty and expense that is involved in developing the necessary platforms and technology to use these assays on a large number of samples. So, new high-throughput techniques are necessary not only to identify new markers, but also to analyse known markers in a cost-effective fashion. NATURE REVIEWS CANCER VOLUME 2 MARCH

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