Review: Field Effect in Cancer–An Update

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Annals of Clinical & Laboratory Science, vol. 39, no. 4, 2009 331 Review: Field Effect in Cancer–An Update

Hong Chai and Robert E. Brown Department of Pathology and Laboratory Medicine, University of Texas Health Science Center–Medical School at Houston, Houston, Texas

Abstract. The concept “field effect in cancer” originated in 1953 from the histopathological observations of Slaughter and colleagues [1] regarding the occurrence of multiple primary oral squamous cell carcinomas and their local recurrences. The development of modern molecular technologies has extended the field effect concept by exploring the molecular abnormalities in tissues that appear histologically normal. To date, such field effect biomarkers have been reported in several sites and organs, eg, head and neck, colon and rectum, prostate, breast, lung, esophagus, stomach, and skin. Two popular hypotheses have been proposed. One hypothesis implicates genetic alterations that occur in a stepwise fashion (initiation, promotion, and progression); a clone gains growth advantage and acquires more genetic alterations, which eventually result in cancer. A second hypothesis focuses on epigenetic alterations, which include hypermethylation of the DNA promoter of certain tumor suppressor genes, leading to down-regulation of these genes. In this update, we discuss in detail the evidence that supports these two hypotheses. In addition, we attempt to provide a comprehensive overview of the field effect in carcinogenesis and its possible mechanisms in various organs. Moreover, we discuss the potential utilization of field effect biomarkers in cancer prevention, surgical considerations, and clinical prognosis.

Keywords: field effect, carcinogenesis, field cancerization, multiple primary cancers, DNA methylation

Introduction benign contiguous tissue. Based on these findings, they suggested field cancerization to be an The concept of the field effect in cancer, also known important factor in recurrence of oral cancer after as field defect or field cancerization, was introduced therapy. This interesting concept is considered to by Slaughter et al in 1953 [1]. In this classic paper, underlie the multicentricity of cancer in many, if the authors studied oral squamous cell carcinomas not all, patients who have multiple tumors in the in 783 patients from the gross and microscopic same organ but no apparent familial predisposition standpoint. They found 88 instances of independent to those tumors. In recent years, the development multiple tumors, which was far beyond the of modern molecular technologies has extended the statistical probability of their chance occurrence field effect concept by exploring the molecular [1]. They also observed microscopic abnormalities abnormalities in tissues that appear histologically (hyperplastic or atypical epithelium) in grossly normal. To date, such field effect biomarkers have been reported in various sites and organs, including Address correspondence to Robert E. Brown, M.D., Depart- the head and neck [2], lung [3], colon and rectum ment of Pathology and Laboratory Medicine, University of Texas Medical School-Houston, 6431 Fannin Street MSB [4,5], breast [6], stomach [7], prostate [8,9], and 2.286; Houston, TX 77030, USA; tel 713 500 5332; fax bladder [10]. 713,500 0695; e-mail: [email protected].

Thus far, the exact underlying mechanisms of at the surgical margins [16]. This may partially the field effect in cancer are not fully understood. explain the high frequency of local recurrences and However, growing molecular evidence suggests a second primary tumors in HNSCC patients. Based carcinogenesis model in which the development of on these data, Tabor et al [16] proposed that a field with genetically altered cells plays a central accumulation of genetic alterations forms the basis role [2]. In addition, alterations of DNA methylation for the progression from a normal cell to a cancer patterns may contribute to field effects [11]. In this cell, referred to as the process of multistep review, we attempt to summarize the current carcinogenesis. This process involves at least three evidence regarding the cancer field effect in several steps. In the initial phase, a stem cell acquires well-studied organ systems. We discuss the findings genetic alterations and forms a “patch,” a clonal in each system and consider possible common unit of altered daughter cells. Such patches can be mechanisms for this phenomenon. We also review recognized on the basis of mutations in TP53 [17]. the possible role of field effect as a surrogate Next, a patch is converted into an expanding field. biomarker in cancer prevention and as a predictor Additional genetic alterations are required for this of carcinogenesis and clinical prognosis. step, and by virtue of its growth advantage, a proliferating field gradually replaces the normal Squamous Cell Carcinoma of Head and Neck mucosa. In the mucosa of the head and neck, as well as the esophagus, such fields have been detected Head and neck squamous cell carcinoma (HNSCC) with a diameter of >7 cm using molecular analysis, is one of the common malignancies in humans. whereas they are usually not detected by routine The average 5-yr survival rate is one of the lowest histopathological techniques [2]. Ultimately, clonal among aggressive cancers, and has not been divergence leads to the development of one or more significantly improved during the last two decades tumors within a contiguous field of pre-neoplastic [12]. Advances in surgical and nonsurgical therapy cells. An important clinical implication is that have led to increased local tumor control. However, fields often remain after surgery of the primary overall mortality rates have not improved, mostly tumor and may lead to new cancers, designated due to tumor recurrence at regional and distant presently by clinicians as “second primary tumor” sites [13]. Local recurrences and the development or “local recurrence,” depending on the exact site of second primary tumors occur in cases with and time interval. This hypothesis has also been advanced tumors, even with histopathologically postulated in other organ systems. tumor-free surgical margins [14]. A large proportion of these cases shows a common clonal origin, even Cancers of Colon and Rectum if the lesions are >7 cm apart [15]. Such observations support the hypothesis of a field effect in cancer. Colorectal cancer is currently the fourth most The term “field cancerization,” also known as common malignancy and the third most common “field effect,” was first used by Slaughter et al [1] in cause of death from cancer worldwide (WHO fact 1953. Originally, histological examinations were sheet, February 2009). It is typified by tumor performed on tumor-adjacent “normal tissue” and progression from adenoma to carcinoma, with surgical margins to assess the presence of a field mutations in the Adenomatous Polyposis Coli lesion. With the application of modern molecular (APC) gene believed to be one of the earliest genetic technologies, new findings support a carcinogenesis aberrations. In colorectal cancer, the field effect is model in which the development of a field with usually characterized by the simultaneous genetically altered cells plays a central role. occurrence of multiple but distinct tumors. In the Measuring the loss of heterozygosity (LOH) with multistep carcinogenesis model proposed by Fearon microsatellite analysis shows that a significant and Vogelstein [18], genetic alterations occur in a proportion of samples have tumor-associated stepwise fashion such that a clone with growth genetic alterations in biopsies from the histopath- advantage proliferates, acquires more genetic ologically normal mucosa adjacent to the tumor or alterations, and undergoes another selection for Field effect in cancer–an update 333 survival and growth, eventually resulting in cancer. in recent years suggest that there is synergy between According to this model, precancerous cells that genetic and epigenetic changes and that Knudson’s are in proximity to cancer cells should have some, two-hit hypothesis [23] may need to be revised: but not all, of the genetic alterations that are present instead of two possibilities (loss of heterozygosity in the fully developed cancer. At least three steps or homozygous deletion), a third possibility– are suggested in tumorigenesis: initiation, pro- transcriptional silencing by DNA methylation of motion, and progression [18]. Initiation starts when promoters–can disable tumor-suppressor genes a loss of specific chromosomal regions occurs, [11,24,25]. Shen et al [4] reported methylation of which is frequent in colorectal neoplasia. The the O6-methylguanine-DNA methyltransferase common region of loss on chromosome 17p in (MGMT) gene promoter in normal-appearing colorectal tumors has been identified and contains colorectal mucosa adjacent to colorectal cancer [4]. the p53 gene [19] (the TP53 gene in humans). p53 They demonstrated that normal appearing mucosa encodes a transcription factor that is important in located 1 cm from a colorectal cancer with MGMT multicellular organisms, where it regulates the cell promoter methylation is more likely to have hyper- cycle and functions as a tumor suppressor that is methylation than normal-appearing colorectal involved in preventing cancer [20]. The second mucosa located 10 cm from the same colorectal most common region of allelic loss is chromosome cancer, thus providing evidence for field effects due 18q, which includes another tumor suppressor to DNA methylation in normal-appearing colonic gene, Deleted in Colorectal Carcinoma (DCC), mucosa. Their data indicate thatMGMT promoter encoding a protein with significant homology to methylation may occur early in multistep carcino- the cell adhesion family of molecules [18]. Recent genesis, even before the emergence of morphologic studies suggest that DCC is a ligand-dependent changes in colorectal mucosa. They concluded that suppressor that is frequently epigenetically silenced some sporadic colorectal cancers may arise from a [21]. Other regions, including chromosomes 5q, field defect that is molecularly defined by epigenetic 1q, 4p, etc., may also be involved. When a stem cell inactivation of MGMT and an increased rate of acquires one or more genetic alteration, it forms a mutations in multiple genes, including KRAS. The patch with genetically altered daughter cells. As a causes of MGMT promoter methylation remain result of subsequent genetic alterations, the stem unclear. cell escapes normal growth control, gains growth Besides MGMT, other genes have also been advantage, and develops into an expanding clone reported with similar changes. Suzuki et al [26] of tumor initiating stem cells or tumor propagating showed that hypermethylation of four genes in the cells. The lesion gradually becomes a field, which secreted frizzled-related proteins (SFRP) family displaces the normal epithelium. As the lesion occurs frequently in colorectal cancer [26]. Their becomes larger, additional genetic hits give rise to initial report [26] indicated that APC mutations various subclones within the field and diverge at a are frequent throughout colorectal tumors with all certain time point [2]. combinations of hypermethylation of the SFRP However, the model of multistep carcinogenesis genes. A subsequent report [5] from the same group is unable to answer all the questions. Besides the showed that restoration of SFRP function in well-known genetic alterations, epigenetic changes colorectal cancer cells attenuates Wg (wingless) and are among the most common molecular alterations Int (WNT) signaling even in the presence of in human neoplasia [22]. Epigenetic denotes a trait downstream mutations. Suzuki et al [5] suggested that is heritable, yet not based upon a change in that the epigenetic loss of SFRP function occurs primary DNA sequence. Epigenetic changes differ early in colorectal cancer progression and may from genetic changes mainly in that they occur at provide constitutive WNT signaling that is required higher frequency, are reversible upon treatment to complement downstream mutations in the with pharmacological agents, and occur at defined evolution of colorectal cancer. Milicic et al [27] regions in a gene. DNA methylation is one of the reported persistent aberrant expression of placental well-known epigenetic mechanisms. Data obtained cadherin (P-cadherin) in sporadic colorectal cancer 334 Annals of Clinical & Laboratory Science, vol. 39, no. 4, 2009

[27]. They showed that ectopic expression of P- benign tissue adjacent to prostate cancer [30,31]. cadherin in the colon is not limited to the neoplastic As summarized by Nonn et al [9], multiple region but extends into the adjacent, phenotypically biomarkers have been identified that support the normal, colonic mucosa. The P-cadherin promoter existence of a field effect in prostate carcinogenesis was hypomethylated in colonic aberrant crypt foci, [9]. These biomarkers identify cytomorphologic, colorectal cancer, and occasionally in normal genetic, epigenetic, and gene/protein expression epithelium adjacent to the cancer, demonstrating a abnormalities in putatively normal prostatic tissue. potential field effect of cancerization. Nonn et al [9] discussed different approaches to Two types of methylation pattern have been select the ‘normal’ prostate tissues (microscopically reported in colorectal cancer: type A for aging- negative area on the cancer sample, tissue from specific methylation and type C for cancer-specific non-cancer prostate, and tissue from ‘nested’ case- methylation [28,29]. Type A is characterized by a control studies of negative biopsies) and suggested high incidence of CpG island methylation in that the latter approach offers unique advantages. tumors and is accompanied by a slight incidence in Mehrota et al [8] used core biopsy samples to detect normal colon mucosa. By contrast, type C epigenetic changes in the primary prostate tumor methylation occurs exclusively in a subset of and surrounding tissues. They checked the colorectal cancers and at lower frequency. Type A methylation ratio of GSTP1, APC, RARb2, and methylation is thought to increase the predisposition RASSF1A using quantitative methylation-specific of aging cells to become transformed, whereas type PCR analysis in 159 cores from 37 prostatectomy C methylation may contribute to neoplastic samples. Based on these data, combined with progression in a subset of cases [28]. Type C histopathological observations, they claimed that methylation in colorectal cancer is observed for no field effect, defined as an absence of epigenetically INK4a/ARF, thrombospondin-1 (THBS1), a p53- transformed cells, was observed for GSTP1, whereas inducible angiogenesis inhibitor, and the mismatch APC, RARb2, and RASSF1A showed a field effect repair gene hMLH1 [28,29]. This raises the question up to 3 mm from the malignant core in 3 prostat- whether age is a determining factor in the ectomy samples [8]. Similar results were reported development of the field effect in cancer. This could by Hanson et al [32], who examined 5 prostate be a subject for future investigation. samples obtained by laser capture microdissection and 5 samples by expression microdissection. Prostate Cancer The progress in identifying the field effect in prostate cancer has been slower than in HNSCC Prostate cancer develops most frequently in men and colorectal cancers. This may be due in part to >50 yr old and is one of the most prevalent types of the fact that transrectal ultrasound (TRUS)-guided cancer in men. Many patients who develop prostate needle biopsy is the method of choice for early cancer never have symptoms, undergo no therapy, detection of prostate cancer. The limited amount of and eventually die of other causes. Due to tissue obtained by such biopsy makes the evaluation widespread use of a serum marker, prostate-specific of field cancerization sites or pre-cancerous lesions antigen (PSA), and the lack of any practical means relatively difficult. for imaging lesions, prostate cancer is the only solid tumor that is routinely detected by indirect tissue Breast Cancer sampling without visualization of a suspicious lesion [9]. Breast cancer is the second most common type of Consistent with the cancer field effect theory, cancer and the fifth most common cause of cancer step-wise sectioning of radical prostatectomy and death (WHO fact sheet, February 2009). Fackler autopsy specimens has shown that prostate cancer et al [33] reported tumor-specific promoter hyper- is almost always multi-focal [30]. Studies of nuclear methylation of RASSF1A, TWIST, Cyclin D2, and morphology using digital image analysis have HIN-1 using a quantitative multiplex methylation- identified subtle changes in the histologically specific PCR method in breast ductal carcinoma in Field effect in cancer–an update 335 situ (DCIS). Evaluation of the cumulative cervical disease. Of the positive women, 50% had hypermethylation of the four genes within each peri-anal disease or extensive cervical disease. None sample revealed high sensitivity (84%) and of the negative women had these conditions [39]. specificity (89%) of detection of methylation. The A cross-sectional study of 396 HIV-positive and data showed the advantage of a cumulative scoring 395 HIV-negative women confirmed these findings. system of promoter hypermethylation among The prevalence of lower genital tract condyloma multiple genes in distinguishing normal and benign was higher in the HIV-positive vs HIV-negative from malignant tissues. Similar results have been women. High-grade vulvar dysplasia (VIN) was reported by other groups [34,35]. As in colon present in two of the positive women vs none of the cancer, Vasilatos et al [36] reported that specific negative women, and HPV growths were far more CpG island promoter methylation events are likely to demonstrate the field effect in the women associated with early mammary carcinogenesis. with HIV [40]. Other studies suggest connections Their study provides evidence that the combination between smoking [41] or occupational exposure to of RARB M4, INK4a/ARF, PRB, and HIN-1 CpG polychlorinated biphenyls [42] and gene changes island promoter methylation may predict non– that have been linked to cancers. Taken together, BRCA1/2-associated mammary carcinogenesis and these data suggest that common viral and chemical tumor progression, which comprise the majority carcinogens may serve as initiators of multifocal (95%) of breast cancers. and multicentric neoplastic alterations. In a study of histologically normal tissue from multiple geographic zones adjacent to primary Summary and Conclusions breast tumors, Yan et al [6] reported methylation changes of extending as far as 4 cm from primary The field effect in carcinogenesis, in which a tumors [6]. As part of the breast conservation morphologically normal epithelium develops into a strategy, lumpectomy is now the preferred surgical tumor, is the result of expansion of a genetically procedure in patients with small and early tumors abnormal clone. The term, “field effect,” remains [37]. Thus far, pathologic examination by frozen controversial even after half a century. According section is the only means to ensure that the surgical to this theory, precancerous cells that are in margins are cancer-free. Since the amplicons used proximity to tumors have some of the genetic by Yan et al [6] were prepared from small amounts fingerprints that are present in the fully developed of DNA, sufficient DNA samples could be obtained tumors. Such phenomena have been documented from cytology touch imprints made by gently in some cancers of various sites and organs [2-10]. pressing the corresponding surgical margin to the Development of an expanding pre-neoplastic glass slide. This raises the possibility of using field appears to be a critical step in epithelial methylation profiling to decide whether patients carcinogenesis and has serious clinical consequences. should receive radiation therapy or other treatment Mutational theories predict that neoplasms have a to prevent recurrence. monoclonal composition, whereas aberrant differentiation processes or field effects might give Other Possible Mechanisms rise to neoplasms with polyclonal compositions. The somatic alterations hypothesis suggests that in As previously noted, the initial event or cause of the the initial phase, a stem cell acquires genetic field effect is still undetermined. It is possible that alterations and forms a patch, a clonal unit of viral and chemical stimuli play important roles in altered daughter cells. Later, clonal divergence leads the early stages. A study of human papilloma virus to the development of one or more tumors within a (HPV) disease in HIV-infected women demon- contiguous field of preneoplastic cells. An important strated that warts and other HPV-related growths clinical implication is that the field often remains frequently show a field effect in the genital tract after surgery of the primary tumor and may lead to [38]. Another report compared cases of HIV- new cancers. Thus the diagnosis and treatment of positive and HIV-negative patients with preinvasive epithelial cancers should be focused not only on 336 Annals of Clinical & Laboratory Science, vol. 39, no. 4, 2009 the tumor but also on the field from which it novel mechanism for field carcinogenesis. J Clin Invest developed [18]. 1997;100:2133-2137. 4. 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