Clinical and Molecular Biologic Characteristics of Early-onset Versus Late-onset
Colorectal Carcinoma in Filipinos Gemma B. Uy, M.D., 1 Leoncio L. Kaw, M.D., 1 Corazon K. Punzalan, M.D., 2 R. Ireneo Luis C. Querol, M.D., 1 Elena V. Koustova, Ph.D., 1 Mark W. Bowyer, M.D., 1 Christine M. Hobbs, M.D., 3 Leslie H. Sobin, M.D., 3 David C. Wherry, M.D. 1 1 Department of Surgery, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, 29814-4799 Bethesda, Maryland, USA 2 Division of Colorectal Surgery, University of the PhilippinesPhilippine General Hospital, Taft Avenue, 1100 Manila, Philippines 3 Gastrointestinal Section, Armed Forces Institute of Pathology, 20307 Bethesda, Maryland, USA Published Online: January 8, 2004 Abstract. A case-control study of Filipino patients who underwent surgical resection for colorectal cancer (CRC) during a 1-year period was under- taken. Thirty-five patients under age 40 years were identified. Paraffin blocks of these and 35 randomly selected patients over age 40 underwent histologic and immunohistochemical evaluation. Markers chosen for evaluation included the apoptosis-associated gene products (p53 and bcl- 2), a tumor proliferation activity-related factor (Ki-67), and the markers (MLH1 and MSH2) of DNA microsatellite instability (MSI). Results were correlated with age and the stage and location of the tumor. The average age of the early-onset group was 30.7 years compared to the late-onset group at 67.0 years; and the male/female ratio was equivalent. The younger patients had a significantly higher Dukes stage, the tumors were more poorly differentiated, and they were more frequently of the mucinous and signet ring cell histopathologic type. Expression of p53 was higher in the younger patients (p < 0.001) and was independent of the degree of differ- entiation or the stage of the tumor. No differences of expression were noted for the other markers measured. The increased frequency of CRC in Fili- pino patients less than 40 years of age offers a unique opportunity to gain a better understanding of carcinogenesis, which might be exploited during diagnosis and management. The differences noted between the early- and late-onset CRC are provocative and provide an impetus for increased screening in Filipinos. Colorectal cancer (CRC) remains a major concern throughout the world. Despite advances in medicine, there has been no significant decline in the incidence and mortality rates over the last few de- cades. In the United States, the colon and rectumare the third most common sites of newcancers, and CRCis the third leading cause of cancer deaths for both men and women, with an estimated 148,300 new cases and 56,600 deaths from this disease between 1979 and 1998 [1]. Although less commonly encountered in Asia than in Western countries, CRC remains a challenge in the Philippines. Statistics from 1998 showed that it ranked fourth in cancer inci- dence and fifth as a leading cause of cancer death [2]. Colorectal cancer is primarily a disease of older age groups. Younger patients are considered an interesting subset, as their can- cers generally behave differently from those in older patients and are commonly linked to a familial predisposition. In Western popu- lations, CRC in the young (< 40 years old) comprise around 2% to 8%of all the diagnosed cases [3]. In young Filipinos, several reports have shown a significantly higher frequency, in the range of 17%to 36% [46]. A recent 7-year review by Kaw et al. [5] of 1277 CRC patients showed that 17% were under the age of 40. In addition, it was found that these patients present at more advanced stages of the disease. The onset of CRC involves an interplay of genetic and environ- mental factors, which explains the wide variability of incidence among races. Known predisposing factors include adenomatous polyps, inflammatory bowel disease (ulcerative colitis and Crohns disease), and inherited disorders such as familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC). HNPCC has been well studied and involves a distinct subset of patients. They tend to be younger, have more right-sided tumors, and display a higher incidence of synchronous and meta- chronous CRC[7]. HNPCCis thought to involve an alternate path- way of tumorigenesis that is associated with microsatellite instabil- ity and is found in 4% to 6% of the population [8]. Interestingly, documented HNPCC cases are rare in the Philippines. So far, only one case has been reported [9]. Perhaps this low number is due to the lack of family histories in Filipino medical records or because cases do not meet the Amsterdam HNPCC criteria. The rare oc- currence of polyps and inflammatory bowel disease (IBD) in Fili- pinos and other unique characteristics of Filipino CRC patients make this population ideal for exploring possible variations in tu- mor biology. This study employed immunohistochemistry (IHC) to examine the differences between early-onset and late-onset CRC in Filipi- nos. Markers involved at various stages of tumor development were studied. Markers of apoptosis included p53 and bcl-2. Mutation of p53 is considered a relatively late event along the pathway and is a determinant of progression from adenoma to carcinoma, whereas bcl-2 is involved early on. The bcl-2 gene is a known inhibitor of Correspondence to: David C. Wherry, M.D. WORLD Journal of SURGERY 2004 by the Societe Internationale de Chirurgie World J. Surg. 28, 117123, 2004 DOI: 10.1007/s00268-003-7281-4 apoptosis and therefore allows accumulation of genetic alterations needed for neoplastic development [10]. The percentage of proliferating tumor cells was assessed using Ki-67 antibody. This antibody is directed against a nuclear antigen that is expressed only by proliferating cells [11]. Finally, alterations in mismatch repair (MMR) proteins were examined using antibod- ies to MLH1 and MHS2. As shown in several studies, IHCprovides a rapid, accurate means of identifying tumors with MSI [1214]. Materials and Methods Materials This study was approved by the Institutional Review Board of the Uniformed Services University of the Health Sciences (USUHS) in Bethesda, Maryland, USA. Surgical pathology reports from Janu- ary 1, 1999 to December 31, 1999 of the Department of Pathology at the Philippine General Hospital (PGH) were reviewed. A total of 35 paraffin blocks of resected adenocarcinoma of the colon or rectum from patients aged 40 years were identified. These pa- tients comprised the early-onset CRC group. Another 35 patients > 40 years of age were randomly selected and comprised the late- onset CRC group. Blocks from the carcinomas of each of these patients were also obtained. The data collected included patient age and sex, location of the primary tumor, depth of bowel wall invasion, and involvement of lymph nodes. Family history, pres- ence of distant metastasis, and outcome were not available fromthe records. The tumors were then staged using Dukes classification [15]. The location of the primary tumor was divided into three sites: right colon (from the cecum to the transverse colon), left colon (from the splenic flexure to the sigmoid colon), and rectum (recto- sigmoid junction, rectum, and anorectum excluding the anus). The blocks of formalin-fixed tissue fromthe subjects were coded and shipped to the USUHS for immunohistologic analysis by inves- tigators blinded to the patients data. These paraffin blocks were reembedded, sectioned, and mounted on slides (American His- tolabs, Gaithersburg, MD, USA) for immunohistochemistry and hematoxylin and eosin (H&E) staining. The H&E-stained slides were sent to the Division of Gastrointestinal Pathology at the Armed Forces Institute of Pathology (AFIP), Washington, DC for blinded histologic evaluation of the degree of differentiation, his- topathologic type, and depth of bowel wall invasion. Immunohistochemical Analysis The IHC analysis of p53, Ki-67, bcl-2, MLH1, and MSH2 was per- formed. Optimal concentrations of the various antibodies were de- termined in pilot experiments. The slides were deparaffinized in xylene and rehydrated in alcohol of graded concentrations and dis- tilled water. Antigen demasking was performed by heating the slides with Target Retrieval Solution (Dako, Carpinteria, CA, USA). Incubation with Peroxidase Suppressor (Pierce, Rockford, IL, USA) was done to quench any endogenous peroxidase activity. Primary antibodies were applied and incubated overnight at 4C at the following concentrations: p53, 1:500 (rabbit anti-p53 onco- protein polyclonal antibody; Chemicon, Temecula, CA, USA); Ki- 67, 1:500 (mouse anti-Ki-67 monoclonal antibody, Chemicon); bcl- 2, 1:500 (mouse anti-bcl-2a oncoprotein monoclonal antibody, Chemicon); MLH1, 1:500 [rabbit polyclonal immunoglobulin G (IgG), Oncogene Research Products, Boston, MA, USA); MSH2, 1:500 (rabbit polyclonal IgG, Oncogene Research Products). Horseradish peroxidase (HRP)-labeled secondary antibodies (Pierce) were then applied at a 1:2500 dilution for Ki-67, bcl-2, and MSH2 and at 1:5000 dilution for p53 and MSH1; they were incu- bated for 30 minutes at room temperature. TSA Biotin System (PerkinElmer Life Sciences, Boston, MA, USA) was used for signal amplification. Cells that reacted with the antibodies were visual- ized using the HRP chromogenic substrate Vector VIP (Vector Laboratories, Burlingame, CA, USA). After each incubation, the slides were extensively washed with phosphate-buffered saline (PBS). Negative controls consisted of slides for which the primary antibody was omitted. Immunoreactivity was evaluated by counting the cells positive for the various antibodies using Image Pro Plus 4.1 (Media Cyber- netics, Silver Spring, MD, USA) at 100magnification for p53 and Ki-67 and at 400for bcl-2. For MLH1 and MSH2 staining, normal colorectal tissue adjacent to the tumor served as positive controls. Loss of expression of these proteins was recorded when positive nuclear immunostaining was observed in normal tissue or lympho- cytes but not in malignant cells [14, 16]. Statistical Analysis The clinicopathologic characteristics (sex, tumor location, degree of differentiation, Dukes stage) were compared between the early- and late-onset groups using the z test (Microsoft Excel 2000). Ex- pression of p53, bcl-2, and Ki-67 was stratified into five groups: 1% to 20%, 21%to 40%, 41%to 60%, 61%to 80%, and 81%to 100%. The distribution of cases between the two onset groups was ana- lyzed using Chi-Square Trend with Graphpad Prism Version 2.0 (Graphpad Software, San Diego, CA, USA). The 2 test or Fishers exact test, whichever was appropriate, was used to correlate antigen expression with the degree of differentiation and tumor stage (SPSS Program for Windows Version 11.0, Statistical Package for Social Sciences; SPSS, Chicago, IL, USA). Significance was defined as p < 0.05. Results One patient from the late-onset group was excluded because the paraffin block showed no tumor cells upon reviewat the AFIP. The mean age for the early-onset group was 30.7 years (range 1940 years), whereas that for the late-onset group was 67 years (range 5182 years). Of the 35 patients in the younger group 23 were men (65%), whereas only 19 of the 34 (55.9%) in the older group were of the male sex. In both groups, most of the cancers were in the rectum (57% and 56%); and although a higher percentage of CRCs was right-sided in the younger group (29% vs. 15%), the difference did not reach statistical significance. A statistically significant difference between the two groups was noted, with regard to the degree of differentiation. Younger pa- tients had a higher percentage of poorly differentiated CRCs (34.2%vs. 11.8%), whereas the older patients had a higher percent- age of moderately differentiated CRCs (64.7%vs. 28.6%). The mu- cinous type of adenocarcinoma was more common in the early- onset group, as was the presence of signet ring cells (p < 0.05). Coexistent granulomatous infections, such as schistosomiasis and tuberculous lymphadenitis, were noted in 2.9% and 7.2% of cases, respectively (Table 1). The depth of tumor invasion and degree of differentiation re- 118 World J. Surg. Vol. 28, No. 2, February 2004 ported in this study were based on the findings of the AFIP on their review of the tumor slides. The presence or absence of distant me- tastasis at the time of surgery was not part of the surgical pathology reports, so staging was limited to using Dukes systeminstead of the TNM classification. As shown in Table 1, the younger patients had pathologically more aggressive disease and a significantly higher percentage of stage C tumors than did the older group (p = 0.037). p53 Expression of p53 was graded based on the percentage of tumor cells that were immunoreactive with p53 antibody (Fig. 1) and cat- egorized into various groups using 20% range intervals. The younger group showed a higher percentage of p53-positive cells in their tumors than did the older group (p =0.0004). Interestingly, in most of the younger patients 61% to 80% of tumor cells expressed p53, whereas most of the older patients had a relatively equal dis- tribution of p53 expression over the 1% to 20%, 21% to 40%, and 41% to 60% ranges (Fig. 2). The degree of differentiation did not correlate with Dukes stage. bcl-2 The percentage of tumor cells that were bcl-2-positive was noted to be similar for the two groups (Fig. 3). Altogether, 63% of the pa- tients under age 40 and 65%of those over age 40 were in the 1%to 20%range, and 23%of the former and 32%of the latter were in the 21% to 40% range. No patient had a tumor with more than 60% bcl-2-positive cells (Fig. 4). As with p53, there was no correlation with the other clinicopathologic variables. Ki-67 The Ki-67 score was the percent of tumor cells counted that were Ki-67-positive (Fig. 5). There was substantial heterogeneity among the scores in both groups, with a mean Ki score of 36.06 23.28 for the early-onset group and 43.95 23.55 for the late-onset group (Fig. 6). The mean Ki score was not statistically different between the two groups. The Ki score was found to be independent of dif- ferentiation, location, and stage of the tumor. MSH1 and MSH2 We used IHCto detect MMRdefects, and we considered failure of tumor cells to react with either MLH1 or MSH2 antibody as indica- tive of defective MMR (Table 2). In the early-onset group 8 of 35 tumors (22.9%) failed to react with one of the two antibodies, as did 7 of 34 tumors (20.6%) in the late-onset group. One tumor from a patient in the early-onset group did not react with either antibody. No statistical correlation was drawn with regard to age, degree of tumor differentiation, stage, or mucinous type between cases with defective or intact MMR. Discussion Colorectal cancer is a major health problemand remains one of the leading causes of cancer deaths worldwide. It is predominantly a Table 1. Demographic and clinicopathologic data of early- and late-onset colorectal cancer. Parameter Early onset Late onset p No. of patients 35 34 Age (years), mean 30.7 6.3 67 9.0 Male 23 (65.7%) 19 (55.9%) NS Female 12 (34.3%) 15 (44.1%) NS Location of tumor Right side 10 (29%) 5 (15%) NS Left side 5 (14%) 10 (29%) NS Rectum 20 (57%) 19 (56%) NS Differentiation Well 13 (37.1%) 8 (23.5%) NS Moderate 10 (28.6%) 22 (64.7%) 0.003 Poor 12 (34.2%) 4 (11.8%) 0.027 Dukes stage A 1 (2.9%) 3 (8.8%) NS B 8 (22.9%) 14 (41.2%) NS C 26 (74.3%) 17 (50%) 0.037 Other tumor characteristics Mucinous adenocarcinoma 10 (27.8%) 1 (2.9%) 0.004 Signet ring type 1 (2.9%) 0 With signet rings 8 (22.9%) 0 0.003 With Schistosoma eggs 2 (5.7%) 0 NS With tuberculous lymphadenitis 4 (11.4%) 1 (2.9%) NS Fig. 1. p53-positive cells (dark staining) in a patient with adenocarcinoma of the sigmoid colon. Fig. 2. Comparison of p53 staining for early- and late-onset colorectal car- cinoma groups. Squares: 40 years old; circles: > 40 years old. 119 Uy et al.: Colorectal Carcinoma in Filipinos disease of older age groups, with 90%of cases occurring in persons over 50 years of age and a peak incidence during the seventh decade of life. However, it can also occur at younger ages, especially in patients with a family history of the disease. Traditionally, CRC in the young has been considered to have a worse prognosis, partly because of its more aggressive character and partly due to delay in diagnosis. However, recent studies have shown a similar survival rate between the young and old groups when compared stage by stage [3]. Mitry et al. [17] showed better survival for patients less than age 45 using stage-stratified survival rates. This was explained by a significantly lower postoperative mortality rate and more fre- quent use of adjuvant therapy or palliative chemotherapy. Reviews by Talens et al. [6] and Kaw et al. [5] of CRC patients treated at the Philippine General Hospital showed a considerably higher frequency of CRC in the young (8% and 17%, respectively) than has been found in Western populations. In the Western litera- ture, patients younger than 40 years of age comprise only 2%to 8% of the total population [3]. The higher frequency of early-onset CRC in the Philippines provides an opportunity to compare and contrast age-specific genetic and molecular biologic characteris- tics. To this end, established markers of various tumorigenic prop- erties (i.e., p53, bcl-2, MLH1, MSH2, Ki-67) were studied in speci- mens obtained from patients under and over the age of 40. To our knowledge, this represents the first such attempt to quantify differ- ences between these two age groups. In our study, the younger patients had a higher frequency of Fig. 3. bcl-2-positive cells (dark staining) in a patient with adenocarcinoma of the colon. Fig. 4. Comparison of bcl-2 staining between early- and late-onset colorec- tal carcinoma groups. Squares: 40 years old; circles: > 40 years old. Fig. 5. Ki-67-positive cells (dark staining) in a patient with adenocarci- noma of the rectum. Fig. 6. Comparison of Ki-67 staining between early- and late-onset colo- rectal carcinoma groups. Squares: 40 years old; circles: > 40 years old. Table 2. MLH1 and MSH2 staining characteristics of early- versus late-onset colorectal cancer. MLH1/MSH2 staining Early onset Late onset +/+ 27 27 +/- 6 5 -/+ 1 2 -/- 1 0 Total 35 34 Defective MMR a 22.9% 20.6% Intact MMR b 77.1% 79.4% MMR: mismatch repair. a Failed to stain positively for either MLH1 or MSH2 antibody. b Stained positively for both MLH1 and MSH2 antibodies. 120 World J. Surg. Vol. 28, No. 2, February 2004 poorly differentiated tumors, cancers with a mucinous component, cancers with signet ring cells, and cancers diagnosed at advanced stages. Poorly differentiated adenocarcinomas comprised 34.2%of the CRCs in the younger age group compared to only 11.8% in the older group. It has been shown that poorly differentiated adeno- carcinomas are associated with a poorer survival rate than are well differentiated tumors [18]. In our patient population, 74.3% of tu- mors in the younger age group were Dukes stage Cversus 50.0%in the older age group. This is important, as the prognosis is directly related to the tumor stage at operation. According to U.S. data, the 5-year survival rate for a localized tumor is an excellent 89.7%. This figure drops dramatically to 64.4% with spread to regional lymph nodes and to a dismal 8.3% with distant metastasis [1]. Because of the difficulties related to patient follow-up, there are no available local data that pertain to the survival rates of Filipino cancer pa- tients at different stages of the disease. The risk profile of Filipino CRCpatients is different fromthat of Western patients. Polyps are found in only 4.7% cases of resected CRC specimens from Filipino patients [5]. In addition, ulcerative colitis and Crohns disease are uncommon in Filipinos, as are re- ported cases of HNPCC and FAP [9]. Our study did reveal a high frequency of coexisting granulomatous infections, such as tubercu- lous lymphadenitis and schistosomiasis, especially in the younger age group. As suggested by Chen et al. [19], these infections could be involved in the multistep process of colorectal tumorigenesis by causing genetic alterations similar to those seen in ulcerative colitis. In colon cancers, as in others, there is progressive DNA damage involving a randomand multistep process that includes inactivation or deletion of tumor suppressor genes and activation of oncogenes [20]. CRCmay develop along one of several genetic pathways, with areas of overlap [21, 22]. This entire process may take several years, and during this time frame preventive interventions at various stages may significantly alter the progression to invasive cancer. New insights regarding the genetic basis of this disease will poten- tially have application in the clinical management of CRCpatients. It will be extremely valuable if molecular biologic factors that favor development along certain pathways can be identified for direct screening. Several prognostic markers may help us understand the subcellular mechanisms in colorectal tumorigenesis and identify those persons at increased risk of developing this type of cancer. The protein p53 was tested in this study, as it is the most commonly observed marker of a poor prognosis for patients with CRCs, oc- curring in approximately 75% [23]. This cell cycle regulatory pro- tein induces apoptosis in cases of DNA damage. It also has tumor suppressor activity. Mutations of p53 result in prolonged half-life and accumulation of the protein to levels detectable immunohisto- chemically in cancer cell nuclei. Overexpression of p53 reflects loss of normal growth regulation. Comparison of the percentages of p53-positive tumor cells between our two groups showed higher expression (p < 0.01) in the younger group than in the older one. This may provide an explanation for the more aggressive nature of CRCin young Filipinos. Indeed, as Schwandner et al. [24] found in their study, p53 was an independent predictor of recurrence and survival. In addition, Gallego et al. [25] noted a significantly shorter disease-free survival for their p53-positive group. In our study, p53 expression did not correlate with stage. This may be attributed to the central role of p53, as mutation occurs just before the tumor gains its invasive properties, so its presence is not stage-dependent. One important clinical implication of tumors with deficient p53 is that they show enhanced sensitivity to doxorubicin and resistance to 5-fluorouracil (5-FU) [26]. This may explain treatment failures when 5-FU is used as the first line of adjuvant chemotherapy. Another marker used in this study was bcl-2, an antiapoptotic protein that is thought to be involved early in the genetic pathway by acting synergistically with other genes to promote neoplastic changes. In a study by Sinirope et al. [10], bcl-2 positivity was local- ized to the basal epithelium in normal mucosa and hyperplastic crypts but was found superficially in dysplastic and malignant cells. An inverse correlation was found between bcl-2 and p53 positivity, but only in adenomas and not in carcinomas. This suggests down- regulation of bcl-2 by mutant p53 in premalignant polyps. In this as in other studies, no correlation was shown between p53 expression and other clinicopathologic variables [10, 27]. The proliferative activity of the tumor cells was evaluated using antibody to Ki-67 protein. Our results concur with other studies that have shown the percentage of tumor cells expressing Ki-67 to be independent of age, stage, and differentiation in CRC [11, 28, 29]. The discovery of MSI and its association with HNPCC has given new insights into the tumor biology of CRC. It offers an alternate pathway to the established adenomacarcinoma sequence [30]. MSI is due to failure of the DNA mismatch repair system to repair errors during DNA replication, thereby leading to the accumula- tion of mutations [31, 32]. Knowledge of MSI has also broadened our approach to the clinical management of patients with heredi- tary susceptibility to development of CRC. However, it has been shown that MSI is not exclusive to either CRC or HNPCC [33]. Although MSI is found in more than 90% of HNPCC patients, it is also present in 10% to 15% of sporadic CRCs [34]. The rarity of HNPCCin the population (46%) explains why it accounts for only 16% of all cases of MSI. The other 84% of cases are sporadic. The critical implication of MSI status is that it is common in a unique subset of CRC patients whose tumors tend to be more proximal in location, poorly differentiated, mucinous, and associated with peri- tumoral lymphocytic infiltration and DNA diploidy. In addition, these patients have been shown to have a better prognosis than patients with non-MSI-related adenocarcinomas and are sensitive to 5-FU-based chemotherapy [3538]. Still, these patients are in need of long-term surveillance, as they are at increased risk for metachronous CRC as well as extracolonic tumors. Several studies have shown the usefulness of IHC using MLH1 and MSH2 antibodies to detect MMR defects. IHC is a rapid, sen- sitive, specific method for screening large numbers of patients. This contrasts with genetic analysis of MSI status, which is expensive, time-consuming, and requires specialized equipment [12]. IHChas shown a predictive value of 100%for detecting high-frequency MSI and 96.7% for detecting microsatellite stability and low-frequency MSI, as reported by Lindor et al. [13]. Stone et al. [14] showed 96% sensitivity using IHC for detecting MSI compared to molecular analysis with the polymerase chain reaction method using the five microsatellite loci defined by the National Cancer Institute guide- lines. Thus, IHC can be utilized to screen for patients needing fur- ther testing to confirm germ line mutations [39, 40]. Our study found a somewhat higher proportion of MSI cases in the younger group (22.9%) than in the older group (20.6%). In addition, loss of expression of MSH2 was more commonly seen than loss of expres- sion of MLH1 (75%vs. 25%). Lanza et al. [37] divided patients into two groups: those with or suspected of having HNPCC and those without a family history suggestive of HNPCC. This study showed a nearly equal frequency of loss of MLH1 and MSH2 in the former 121 Uy et al.: Colorectal Carcinoma in Filipinos group, whereas patients with sporadic CRCs more frequently lost MLH1 expression (87.5%). Hypermethylation of the MLH1 promoter region has been sug- gested as the primary mechanism in the development of sporadic CRC[26]. Our findings stimulate interest in further study of MMR defects in Filipino patients. For instance, one could investigate whether Filipinos have a unique type of mutation or some kind of familial clustering of CRCcases that does not meet established cri- teria of known syndromes. An association between MSI-positive tumors and certain unique clinicopathologic features was not found in this study. Loss of expression of one of the markers of defective MMR (MLH1 or MSH2) was the same for both the younger and older patients. Location of the tumor and the degree of differentiation did not differ significantly between intact and de- fective MMRcases. Although MLH1 and MSH2 are the most com- mon genes involved in this pathway, other genetic mutations may occur. Testing for all such mutations in future studies may aid in the further elucidation of CRC tumorigenesis. There is still much to learn about the molecular behavior of co- lorectal cancer. Other markers must be tested to lay out a clearer viewof the genetic alterations involved in the development of these cancers. The concept of de novo carcinoma is emerging and pro- vides an alternate pathway to the established adenomacarcinoma sequence [41]. Future studies comparing Filipinos with age- and stage-matched non-Filipinos may reveal divergent pathways of co- lorectal tumorigenesis. A survey of survival differences among Asian emigrants to the United States has already shown that Fili- pino immigrants were more likely to be diagnosed with advanced CRC and had worse survival rates than Chinese, Japanese, and non-Hispanic white immigrants [42]. Because access to structured medical care has been eliminated as a factor for this delay in diag- nosis, a genetic predisposition may explain the particular aggres- siveness of this disease in Filipinos. Our study was limited by its retrospective nature, lack of clinico- pathologic data, and poor patient follow-up. These issues are being addressed in an ongoing prospective study of the molecular biology of CRC in Filipinos. With most patients presenting at an advanced stage, the need for earlier diagnosis cannot be overstated. It is hoped that the knowledge that we gain fromthis and future studies of the molecular biology of CRC allows an earlier diagnosis and more targeted management of our patients. Re sume . Cette e tude cas-te moin concerne des patients philippins ayant eu une re section chirurgicale pour cancer colorectal pendant une pe riode dobservation dun an. Trente cinq patients de moins de 40 ans ont e te identifie s. Lexamen histologique et immunohistochimique apre `s fixation a` la paraffine a inte resse ces pie `ces de re sections et compare a` ceux de 35 autres patients se lectionne s au hasard. Les marqueurs choisis pour e valuation comprenaient les produits ge ne tiques associe s a` lapoptose (p53 et bcl-2), un facteur dactivite de prolife ration tumorale (Ki-67), et les marqueurs (MLH1 et MSH2) de linstabilite microsatellite dADN (MSI). Les re sultats ont e te corre le s avec lage, le stade et la localisation de la tumeur. Lage moyen du groupe porteur de cancer dapparition pre coce a e te de 30.7 ans compare a` celui du groupe dapparition tardive qui a e te de 67 ans; le sexe ratio a e te de 1. Les patients les plus jeunes avaient un stade de Dukes significativement plus avance , des cancers plus indiffe rencie s, et e taient plus souvent mucineux ou contenant des cellules a` bague de chaton. Lexpression P53 e tait plus forte chez les patients plus jeunes (p < 0.001), inde pendamment du degre de de diffe renciation ou du stade de la tumeur. On na retrouve aucune diffe rence dans lexpression des autres marqueurs tumoraux mesure s. Lincidence plus e leve e de cancer colorectal chez les patients philippins de moins de 40 ans est une situation unique pour pourvoir mieux comprendre la carcinogene `se et qui pourrait e tre exploite e pour ame liorer le diagnostic et la prise en charge. Les diffe rences note es entre les cancers dapparition pre coce et tardive sont provocatrices et nous incitent a` proposer un programme acce le re de de pistage. Resumen. Se realizo un estudio de control de casos de pacientes filipinos sometidos a reseccion por cancer colo-rectal (CCR) en un periodo de un ano. Treinta y cinco pacientes menores de 40 anos fueron identificados. Se hizo el estudio histologico e inmunohistoqu mico de e stos y de otros 40 pacientes mayores de 40 anos seleccionados al azar. Los marcadores escogidos para la evaluacion incluyeron los productos del gen asociado con la apoptosis (p53 y bel-2); un factor relacionado con la actividad tumoral (Ki-67); y los marcadores (MlH1) y MSH2) de inestabilidad microsatelital de ADN (MSI). Los resultados fueron correlacionados con la edad, el estado y la ubicacion del tumor. La edad promedio de los pacientes con presentacion temprana de la enfermedad fue 30.7 anos, en tanto que la edad promedio del grupo con presentacion tard a fue 67 anos; la relacion masculino a femenino fue equivalente. Los pacientes mas jovenes mostraron un estado de Duke mas alto, sus tumores eran mas pobremente diferenciados y mas comunmente del tipo histopatolo gico mucinoso y en anillo de sello. La expresio n de P53 aparecio mas alta en el grupo de pacientes mas jovenes (p <0.001) e independiente del grado de diferenciacion del tumor. No se encontraron diferencias en la expresio n de los otros marcadores. La mayor frecuencia de CCR en los pacientes filipinos menores de 40 anos ofrece una oportunidad unica para lograr una mejor comprension del proceso de carcinogenesis que podr a ser explotada tanto en el diagnostico como en el manejo de la neoplasia. Las diferencias observadas entre los grupos de menor y mayor edad en cuanto a la presentacion del CCRson provocativas y dan fundamento a incrementar el tamizaje en los filipinos. References 1. Colorectal Cancer and Early Detection. Cancer Facts and Figures 2002, Surveillance Research, Atlanta, American Cancer Society, 2002:2027 2. 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