Abstract
Early-onset Colorectal Cancer (ECRC) represents a significant and increasing proportion of Colorectal Cancer (CRC), but it is a heterogeneous entity that probably encompasses specific subclasses. On the premise that the carcinogenetic mechanism and progression of CRC may differ with location, we analyzed molecular and clinical characteristics of ECRC according to tumor location in order to identify more homogeneous subgroups of CRC. Right-sided ECRC is a subset in which most Lynch Syndrome cases are found, with earlier stages at diagnosis and better prognosis. At this location the CpG Island Methylator Phenotype (CIMP) is predominant and Chromosomal Instability (CI) is rare. Left-sided ECRC appears as a transitional or intermediate location, except for CI tumors, that seem to predominate at this location. Finally, rectal ECRC shows Microsatellite Stability, CIMP low-0 and low CI - with recurrent altered chromosomal regions in common with left-sided ECRC-, possibly in relation with Microsatellite And Chromosomal Stable tumors, but with an unexpected familial component and worse prognosis. All this suggest that the molecular basis of ECRC varies with tumor location, which could affect the clinical management of patients.
Keywords: Early-onset colorectal cancer, microsatellite instability, Cpg island methylator phenotype, chromosomal instability, tumor location
Introduction
Early-onset CRC (ECRC) represents 11% of colon cancers and 18% of rectal cancers [1-4]. Currently, it is not generally considered as a subgroup of hereditary CRC, and Microsatellite Instability (MSI) does not explain the majority of cases [5-8]. Although ECRC could be a specific subgroup of CRC [6], it is a heterogeneous entity that probably encompasses particular subclasses of CRC [7,8]. Some studies have demonstrated that right- and left-sided CRCs exhibit different genetic, biological and demographical characteristics, suggesting that the carcinogenetic mechanism and progression of CRC may differ with tumor location [9-11]. Thus, the anatomic site of CRC could help to subclassify ECRC and to identify more homogeneous subgroups of CRC. To our knowledge, this should be the first analysis of molecular and clinical characteristics of ECRC according to tumor location.
Material and methods
We collected a total of 88 consecutive individuals with ECRC younger than 45 years old; six cases with Familial Adenomatous Polyposis were excluded. All patients, or a first degree relative when the proband was deceased, provided written consent. Clinicopathological, familial and follow-up characteristics of all cases and comparisons between location groups are shown in Table 1. Molecular analysis involves MSI analysis and screening for germline mutations in Mismatch Repair (MMR) genes [6]; CpG Island Methylator Phenotype (CIMP) characterization; and Chromosomal instability (CI) was evaluated by comparative genomic hybridization (CGH) array, and methods and calculation of the Genomic Instability Index (GII, defined as the fraction of altered genome) as recently reported [13]. For associations between tumor location and other discrete variables, statistical analyses were performed using Pearson’s Chi Square (X2) Test for parametric variables, and Fisher’s Exact Test for non-parametric variables. For continuous variables, Student’s t test was used.
Table 1.
Clinical, pathological and familial features of the global group, and the comparison of the different location groups within early-onset CRC
| Early-onset CRC n (%) | Right colon Early-onset CRC n (%) | Left colon Early-onset CRC n (%) | Rectum Early-onset CRC n (%) | p (X2) | |
|---|---|---|---|---|---|
| Patients | 82 (100) | 20 (24) | 35 (43) | 27 (33) | |
| Mean age of onset (SD)1 | 39.6 (4.9) | 39.1 (6) | 39.3 (4.2) | 40.2 (4.9) | NS |
| Sex | |||||
| Male | 49 (59.8) | 10 (50) | 22 (63) | 17 (63) | |
| Female | 33 (40.2) | 10 (50) | 13 (37) | 10 (37) | NS |
| Tumour differentiation2 | |||||
| Poor | 8/60 (13.3) | 4/16 (25) | 3/24 (12.5) | 1/20 (5) | NS |
| Mucin production2 | 19/60 (32) | 6/16 (37.5) | 8/24 (33) | 5/20 (25) | NS |
| “Signet ring” cells2 | 4/60 (7) | 0/16 (0) | 2/24 (8) | 2/20 (10) | NS |
| Astler-Coller modified | |||||
| A | 23 (28) | 4 (20) | 12 (34) | 7 (26) | |
| B | 26 (32) | 12 (60) | 12 (34) | 2 (7) | |
| C | 14 (17) | 3 (15) | 3 (9) | 8 (30) | |
| D | 19 (23) | 1 (5) | 8 (23) | 10 (37) | 0.002 |
| Global Survival in months (SD)1 | 58.5 (34.8) | 81.6 (34.5) | 55.4 (32.6) | 44.6 (29.4) | <0.001 |
| Disease-Free Survival in months (SD)1 | 50.8 (39.9) | 72.4 (42.6) | 48.7 (37.2) | 36.7 (35.2) | 0.006 |
| Associated polyps | 46 (56) | 14 (70) | 21 (60) | 11 (41) | NS |
| Mean number (SD)1 | 1.8 (2.5) | 3 (3.2) | 1.6 (1.9) | 1.2 (2.3) | 0.046 |
| Type | |||||
| Adenomatous | 22 (48) | 5 (36) | 10 (48) | 7 (64) | NS |
| Hyperplastic | 8 (17) | 2 (14) | 4 (19) | 2 (18) | |
| Mixed | 16 (35) | 7 (50) | 7 (33) | 2 (18) | |
| Multiple primary neoplasms | 11 (13) | 5 (25) | 4 (11) | 2 (7) | NS |
| Synchronous or metachronous CRCs | 5 (6) | 4 (20) | 0 (0) | 1 (4) | 0.01 |
| Family history of cancer | 0.006 | ||||
| Amsterdam II-positive families | 15 (18) | 8 (40) | 6 (17) | 1 (4) | |
| Aggregation for Lynch-related neoplasms | 27 (33) | 7 (35) | 6 (17) | 14 (56) | |
| Aggregation for Lynch-unrelated neoplasms | 9 (11) | 1 (5) | 6 (17) | 2 (8) | |
| Sporadic cases | 31 (38) | 4 (20) | 17 (48) | 10 (37) |
Statistical analysis was carried out using Student’s t test.
Percentages shown are based on varying total numbers as some cases were excluded because only one biopsy was taken (stage D), or because tumors were severely dysplastic with “in situ” carcinoma, and it was not possible to study any other characteristic. SD: Standard Deviation.
NS: Not Significant. CRC: Colorectal Cancer.
Results
The earliest modified Astler-Coller tumor stages (A and B) were observed in 80% of the right-sided ECRCs, whereas the most advanced stages (C and D) predominated in rectal cases. The highest mean-number of associated polyps as well as the highest frequency of multiple, synchronous or metachronous, CRCs where associated with right location, decreasing progressively throughout the left colon and the rectum. Amsterdam II criteria were fulfilled by 40% of right-sided ECRC cases, and there was also an important rate of Lynch Syndrome (LS)-related tumors in rectum cases (56%). Prognosis worsened gradually from right colon cases, with best prognosis, to rectal cases, with worst prognosis, like Global Survival and Disease-Free Survival. All clinical data are shown in Table 1.
Eighty-one of the 82 early-onset cases were studied. Twelve were defined as MSI (15%) and the remaining 69 were defined as Microsatellite Stable (MSS) (Table 2). MSI was present in 30% of right-sided ECRC, in 17% of left-sided ECRC, and in 0% of rectum cases. Ten cases showed a pathogenic germline mutation in one of the MMR genes: two in MLH1 and four in MSH2 in right colon cases, and two in MLH1 and two in MSH6 in left colon cases. One left colon case showed hypermethylation of the MLH1 promoter as sporadic case, and none LS case appeared in rectal location.
Table 2.
Molecular characteristics of early-onset CRC according to tumor location
| Right colon Early-onset CRC n (%) | Left colon Early-onset CRC n (%) | Rectum Early-onset CRC n (%) | p (X2) | |
|---|---|---|---|---|
| MSI | 6/20 (30) | 6/35 (17) | 0/26 (0) | 0.016 |
| MMR gene mutations | 6 (30) | 4 (11) | 0 | 0.009 |
| CIMP | 0.005 | |||
| CIMP-0 | 2 (12.5) | 14 (45) | 11 (52) | |
| CIMP-Low | 6 (37.5) | 14 (45) | 8 (38) | |
| CIMP-High | 8 (50) | 3 (10) | 2 (10) | |
| Molecular classification | 0.007 | |||
| MSI- CIMP High | 3 (19) | 1 (3) | 0 (0) | |
| MSI- CIMP Low-0 | 2 (12.5) | 4 (13) | 0 (0) | |
| MSS- CIMP High | 5 (31) | 2 (6.5) | 2 (9.5) | |
| MSS- CIMP Low-0 | 6 (37.5) | 24 (77.5) | 19 (90.5) | |
| GENOMIC INSTABILITY1 | ||||
| GII | ||||
| Gains | 0.048321 | 0.124064 | 0.115086 | NS |
| Losses | 0.020220 | 0.227445 | 0.018711 | 0.009 |
| Normal | 0.870251 | 0.692384 | 0.830362 | 0.05 |
| CNA per case (SD) | 91.5 (45) | 121 (128) | 61.6 (77) | 0.14 |
| Total gains per case (SD) | 43 (22) | 55 (67) | 34 (47) | NS |
| Total losses per case (SD) | 48.5 (22) | 66 (63) | 28 (32) | 0.03 |
| Mean of whole altered chromosomes (SD) | 1.5 (2) | 3 (3) | 3.5 (4) | 0.14 |
Statistical analysis was carried out using Student’s t test.
CIMP: CpG Island Methylator Phenotype. CNA: Copy Number Alterations. GII: Genomic Instability Index. MSI: Microsatellite Instability. SD: Standard Deviation. NS: Not Significant. MMR: Mismatch Repair. CRC: Colorectal Cancer.
CpG Island Methylator Phenotype (CIMP) characterization was carried out in 68 of the 82 early-onset tumours (Table 2). Right-sided CRCs showed an important component of CIMP (high in 50% and low in 37.5%). Left-sided and rectal CRCs demonstrated a low frequency of CIMP-High (10%).
We classified ECRC into four categories [12] based on CIMP status [6] and the results of the microsatellite study. Right-sided ECRCs showed the most even distribution of the four categories. Left-sided and rectal ECRCs were mainly MSS-CIMP low-0 (Table 2).
Quantitative analysis of CI in tumours according to location is shown in Table 2. The highest GII was observed in left-sided ECRCs, and the subgroup of Losses showed significant differences between the three tumour locations. Left-sided ECRCs also exhibited the highest number of copy number alterations (CNAs) per case, and the lowest number of CNAs per case was observed in rectal ECRCs. However, this latter group showed the highest mean number of whole chromosome aberrations.
Univariate and multivariate analysis performed with R Statistical Software® [13] were carried out in order to identify minimum recurrently lost or gained regions at the different locations (Supplementary Table 1). Recurrent gains and losses were more frequent in right-sided ECRCs. The only recurrent region that was observed in all three tumor locations was gain of 19p13.3-q13.24. Five regions were common to right- and left-sided ECRCs: losses at 1q12-q21.2, 5q13.2, 9p12-p13, 9p13.1, and 10q11.22. Other frequently altered regions were: gains at 7q22.1 and losses at 16p13.12-p12.3, in right-sided ECRCs; losses at 9q21.11 and 11q14.2-14.3, and gains at 20q11.21-q11.22, in left-sided ECRCs; and losses at 14q11.1-11.2, and gains at 17q21.31-q21.32, and 22q11.1 in rectal ECRCs. Based on the comparative analysis between tumor locations, two main groups of differentially altered chromosomal regions emerge: one predominantly associated with left-sided ECRCs, and another one common to left-sided and rectal ECRCs (Supplementary Table 2).
Discussion
Early-onset CRC is being considered a specific and distinct subtype of CRC according to molecular, morphological and genetics features [8]. In order to better characterize carcinogenesis and its correlation with clinical, pathological and familial patterns, our group of research described the three main ways of CRC pathogenesis (microsatellite, chromosomal instability, and CpG island methylation) in the different tumour locations (right colon, left colon, and rectum) [5,6]. A molecular classification of CRC should be established in order to practical implications for diagnosis, prognosis and treatment, and, for that purpose and before a necessary comparison with late-onset CRC, an in-depth analysis of early-onset CRC must be done.
Right-sided ECRC shows, as a group in which most LS cases are found, some of its features, including earlier stages at diagnosis, more associated polyps, synchronous and metachronous CRC, and better prognosis. At this location CIMP-High is predominant and CI is rare, although recurrently altered chromosomal regions are observed (>40%) [9]. Left-sided ECRC appears as a transitional or intermediate location, except for CI tumors that seem to predominate at this location, as well as sporadic cases. Finally, rectal ECRC shows mainly MSS, CIMP low-0 and low CI, possibly in relation with Microsatellite and Chromosomal Stable (MACS) tumors [14,15], but with recurrent altered chromosomal regions in common with left-sided ECRC as well, and an unexpected familial component (Lynch-related tumors aggregation without diagnosis of Lynch Syndrome) and worse prognosis (not only explained by the advanced stage of diagnosis).
The most frequent common region, gain in 19p13.3-q13.42, has already been reported as more frequent in early-onset CRC population [16], in which only 20-50% cases showed LOH of this region, as well as in Peutz-Jeghers syndrome [17,18]. Regarding the most frequent regions commonly altered within right and left colon, only losses of 1q12-q21.2 have previously been reported in this subset of age [16], being the others rarely reported in association with sporadic CRC [19]. With respect to the most frequent regions for each group of location alone, only gain of 7q22.1 in right colon cancer (related with GAEC1) [20], and loss of 14q11.1-11.2 in rectal cancer (related with NDRG2) [21], have been previously associated with CRC; 11q13.3 region has been related to some cases of lymph node metastasis [22], and 2p24.3 showed evidence of linkage with cases of Familial CRC type X [23].
All these findings strongly suggest that molecular basis of ECRC varies with tumor location and may have a significant impact on the clinical management of patients. Next step should be the analysis and comparison of all these parameters within late-onset CRC regarding tumor location, in order to continue our differential characterization of ECRC, and also a complete definition and knowledge of the implications of the most important altered chromosome regions of each colon location.
Acknowledgements
This work was funded by Project PI10/0683 and PI13/0127 from the Spanish Ministry of Health and Consumer Affairs, and cofunded by the European Regional Development Fund, and approved by Project PI10/0683 from the Spanish Ministry of Health and Consumer Affairs, and approved by the Ethics Committee of our Institution. We thank the Tumor Registry of the Pathology Department of the 12 de Octubre University Hospital for providing us with the paraffin-embedded tissues, and Ron Hartong for his help with the English revision of this article.
Disclosure of conflict of interest
The authors declare that they have no conflicts of interest.
Supporting Information
References
- 1.Boyle P, Ferlay J. Cancer incidence and mortality in Europe, 2004. Ann Oncol. 2005;16:481–488. doi: 10.1093/annonc/mdi098. [DOI] [PubMed] [Google Scholar]
- 2.Siegel RL, Jemal A, Ward EM. Increase in incidence of colorectal cancer among young men and women in the United States. Cancer Epidemiol Biomarkers Prev. 2009;18:1695–8. doi: 10.1158/1055-9965.EPI-09-0186. [DOI] [PubMed] [Google Scholar]
- 3.Sia CS, Paul E, Wale RJ, Lynch AC, Heriot AG, Warrier SK. No increase in colorectal cancer in patients under 50 years of age: a Victorian experience from the last decade. Colorectal Dis. 2014;16:690–95. doi: 10.1111/codi.12648. [DOI] [PubMed] [Google Scholar]
- 4.Ahnen DJ, Wade SW, Jones WF, Sifri R, Mendoza Silveiras J, Greenamyer J, Guiffre S, Axilbund J, Spiegel A, You YN. The increasing incidende of young-onset colorectal cancer: A call to action. Mayo Clin Proc. 2014;89:216–24. doi: 10.1016/j.mayocp.2013.09.006. [DOI] [PubMed] [Google Scholar]
- 5.Perea J, Álvaro E, Rodríguez Y, Gravalos C, Sánchez-Tomé E, Rivera B, Colina F, Carbonell P, González-Sarmiento R, Hidalgo M, Urioste M. Approach to the early-onset colorectal cancer: Clinicopathological, familial, molecular and immunohistochemical characteristics. World J Gastroenterol. 2010;16:3697–3703. doi: 10.3748/wjg.v16.i29.3697. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Perea J, Rueda D, Canal A, Rodríguez Y, Álvaro E, Osorio I, Alegre C, Rivera B, Martínez J, Benítez J, Urioste M. Age at onset should be a major criterion for subclassification Colorectal Cancer. J Mol Diagn. 2014;16:116–26. doi: 10.1016/j.jmoldx.2013.07.010. [DOI] [PubMed] [Google Scholar]
- 7.Boardman LA, Johnson RA, Viker KB, Hafner KA, Jenkins RB, Riegert-Johnson DL, Smyrk TC, Litzelman K, Seo S, Gangnon RE, Engelman CD, Rider DN, Vanderboom RJ, Thibodeau SN, Petersen GM, Skinner HG. Correlation of chromosomal instability, telomere length ant telomere maintenance in microsatellite stable rectal cancer: a molecular subclass of rectal cancer. PLoS One. 2013;8:e80015. doi: 10.1371/journal.pone.0080015. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Kirzin S, Marisa L, Guimbaud R, De Reynies A, Legrain M, Laurent-Puig P, Cordelier P, Pradère B, Bonnet D, Meggetto F, Portier G, Brousset P, Selves J. Sporadic Early-onset colorrectal cancer is a specific subtype of cancer: a morphological, molecular and genetics study. PLoS One. 2014;9:e103159. doi: 10.1371/journal.pone.0103159. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Iacopetta B. Are there two sides to colorectal cancer? Int J Cancer. 2002:403–8. doi: 10.1002/ijc.10635. [DOI] [PubMed] [Google Scholar]
- 10.Glebov OK, Rodriguez LM, Nakahara K, Jenkins J, Cliatt J, Humbyrd CJ, DeNobile J, Soballe P, Simon R, Wright G, Lynch P, Patterson S, Lynch H, Gallinger S, Buchbinder A, Gordon G, Hawk E, Kirsch IR. Distinguishing right from left colon by the pattern of gene expression. Cancer Epidemiol Biomarkers Prev. 2003;12:755–62. [PubMed] [Google Scholar]
- 11.Snaebjornsson P, Jonasson L, Jonsson T, Möller PH, Theodors A, Jonasson JG. Colon cancer in Iceland- a nationwide comparative study on various pathology parameters with respect to right and left tumor location and patients’ age. Int J Cancer. 2010;127:2645–53. doi: 10.1002/ijc.25258. [DOI] [PubMed] [Google Scholar]
- 12.Ogino S, Goel A. Molecular classification and correlates in colorectal cancer. J Mol Diagn. 2008;10:13–27. doi: 10.2353/jmoldx.2008.070082. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Arriba M, García JL, Inglada-Pérez LS, Rueda D, Osorio I, Rodríguez Y, Álvaro E, Sánchez R, Fernánde-Miguel T, Pérez J, Hernández JM, Benítez J, González-Sarmiento R, Urioste M, Perea J. Array-based Comparative Genomic Hybridation defines age at onset as a factor for differential Chromosomal instability within Colorectal Cancer. Mol Carcinog. 2015 doi: 10.1002/mc.22315. [Epub ahead of print] [DOI] [PubMed] [Google Scholar]
- 14.Tang R, Changchien CR, Wu MC, Fan CW, Liu KW, Chen JS, Chien HT, Hsieh LL. Colorectal cancer without high microsatellite instability and chromosomal instability-an alternative genetic pathway to human colorectal cancer. Carcinogenesis. 2004;25:841–6. doi: 10.1093/carcin/bgh074. [DOI] [PubMed] [Google Scholar]
- 15.Sinicrope FA, Rego RL, Halling KC, Foster N, Sargent DJ, La Plant B, French AJ, Laurie JA, Goldberg RM, Thibodeau SN, Witzig TE. Prognostic impact of microsatellite instability and DNA ploidy in human colon carcinoma patients. Gastroenterology. 2006;131:729–37. doi: 10.1053/j.gastro.2006.06.005. [DOI] [PubMed] [Google Scholar]
- 16.Berg M, Ågesen TH, Thiis-Evensen E INFACstudy group. Merok MA, Teixeira MR, Vatn MH, Nesbakken A, Skotheim RI, Lothe RA. Distinct high resolution genome profiles of early-onset and late-onset colorectal cancer integrated with gene expression data identify candidate susceptibility loci. Mol Cancer. 2010;9:100. doi: 10.1186/1476-4598-9-100. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Trojan K, Brieger A, Raedle J, Esteller M, Zeuzem S. 5’-CpG island methylation of the LKB1/STK11 promoter and allelic loss at chromosome 19p13.3 in sporadic colorectal cancer. Gut. 2000;47:272–76. doi: 10.1136/gut.47.2.272. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Entius MM, Keller JJ, Westerman AM, Van Rees BP, van Velthuysen ML, de Goeij AF, Wilson JH, Giardiello FM, Offerhaus GJ. Molecular genetic alterations in hamartomatous polyps and carcinomas of patients of Peutz-Jeghers syndrome. J Clin Pathol. 2001;54:126–31. doi: 10.1136/jcp.54.2.126. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Dyrsø T, Li J, Wang K, Lindebjerg J, Kølvraa S, Bolund L, Jakobsen A, Bruun-Petersen G, Li S, Crüger DG. Identification of chromosome aberrations in sporadic microsatellite stable and unstable colorectal cancers using array comparative genomic hybrization. Cancer Genet. 2011;204:84–95. doi: 10.1016/j.cancergencyto.2010.08.019. [DOI] [PubMed] [Google Scholar]
- 20.Gopalan V, Smith RA, Nassiri MR, Yasuda K, Salajegheh A, Kim SY, Ho YH, Weinstein S, Tang JC, Lam AK. GAEC1 and colorectal cancer: a study of the relationships between a novel oncogene and clinicopathological features. Hum Pathol. 2010;41:1009–15. doi: 10.1016/j.humpath.2009.11.014. [DOI] [PubMed] [Google Scholar]
- 21.Shen L, Qu X, Ma Y, Zheng J, Chu D, Liu B, Li X, Wang M, Xu C, Liu N, Yao L, Zhang J. Tumor supressor NDGR2 tips the balance of oncogenic TGF-ß via EMT inhibition in colorectal cancer. Oncogenesis. 2014;3:e86. doi: 10.1038/oncsis.2013.48. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Nakao M, Kawauchi S, Furuya T, Uchiyama T, Adachi J, Okada T, Ikemoto K, Oga A, Sasaki K. Identification of DNA copy number aberrations associated with metastases of colorectal cancer using array CGH profiles. Cancer Genet Cytogenet. 2009;188:70–6. doi: 10.1016/j.cancergencyto.2008.09.013. [DOI] [PubMed] [Google Scholar]
- 23.Sanchez-Tomé E, Rivera B, Perea J, Pita G, Rueda D, Mercadillo F, Canal A, Gonzalez-Neira A, Benitez J, Urioste M. Genome-wide linkage analysis and tumoral characterization reveal heterogeneity in familial colorectal cancer type X. J Gastroenterol. 2015;50:657–66. doi: 10.1007/s00535-014-1009-0. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.