Age at Onset Should Be a Major Criterion for Subclassification of Colorectal Cancer

CM 201 E 4J Pr M og D ra m

The Journal of Molecular Diagnostics, Vol. 16, No. 1, January 2014

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Age at Onset Should Be a Major Criterion for Subclassification of Colorectal Cancer José Perea,* Daniel Rueda,y Alicia Canal,y Yolanda Rodríguez,z Edurne Álvaro,* Irene Osorio,* Cristina Alegre,* Bárbara Rivera,x Joaquín Martínez,y Javier Benítez,x{ and Miguel Uriostex{ From the Departments of Surgery* and Pathologyz and the Molecular Biology Laboratory,y 12 de Octubre University Hospital, Madrid; the Human Genetics Group,x Human Cancer Genetics Program, Spanish National Cancer Centre, Madrid; and the Center for Biomedical Network Research on Rare Diseases,{ Carlos III Health Institute, Madrid, Spain CME Accreditation Statement: This activity (“JMD 2014 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“JMD 2014 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s)
. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.

Accepted for publication July 31, 2013. Address correspondence to José Perea, M.D., Ph.D., Department of Surgery, Hospital Universitario 12 de Octubre, C/ Rosas de Aravaca, 82A, 1 Dcha, E-28023, Madrid, Spain. E-mail: josepereag@ hotmail.com.

An important proportion of early-onset colorectal cancer (CRC) does not show a hereditary component with limited knowledge about its molecular basis and features. We analyzed a subset of patients with early-onset CRC and compared them with patients with late-onset CRC. We analyzed the microsatellite instability and CpG island methylator phenotype (CIMP) in both populations and classified them into four molecular subtypes. We analyzed the differential features between groups. Only 12 of 81 earlyonset cases (15%) showed microsatellite instability, 10 of which (83%) were Lynch syndrome cases; microsatellite instability cases in elderly patients were sporadic. Early-onset microsatellite-stable cases showed different tumor locations and more family history of cancer than the elderly. Microsatellite instability/CIMP-high early-onset CRC was associated with Lynch syndrome, whereas the elderly cases were associated with BRAF mutations. Early-onset microsatellite-stable/CIMP-high CRCs were more frequently mucinous and right sided than elderly cases, with a high incidence of Lynch syndrome neoplasms; early-onset microsatellite stable/CIMP-low/0 differed from elderly cases in location, stages, incidence of multiple primary neoplasms, and the familial component. The clinical and familial differences observed between early- and late-onset CRC when considering the different carcinogenetic pathways underline that the age at onset criterion should be considered when classifying CRC. (J Mol Diagn 2014, 16: 116e126; http://dx.doi.org/10.1016/j.jmoldx.2013.07.010)

The importance of colorectal cancer (CRC) is undeniable and is underscored by its increasing prevalence in the past years. Early-onset CRC has an incidence of 2% to 8% of all CRCs, and between 1992 and 2005 it increased in the United States at a rate of 1.5% per year in men and 1.6% per year in women.1,2 The classic description of colorectal carcinogenesis, the adenoma-carcinoma sequence,3 has considerably evolved, and CRC is increasingly classified into specific phenotypes on the basis of different carcinogenetic pathways. Most Copyright ª 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2013.07.010

sporadic cases (60%) show chromosomal instability (CIN), with chromosomal translocations and aneuploidy.4e6 They are also called microsatellite-stable (MSS) tumors. On the other hand, there are tumors with microsatellite instability (MSI), whose carcinogenetic pathway is also known as mutator phenotype7,8 and that accounts for 15% of CRCs; they include not only Lynch syndrome (LS) cases but also Supported by project PI10/0683 from the Spanish Ministry of Health and Consumer Affairs (J.P.).

Age at Onset in Colorectal Cancer more frequently sporadic cases in which the MLH1 DNA mismatch repair (MMR) gene is epigenetically silenced.9,10 These tumors are characterized by proximal location, mucinous histologic features, poor differentiation, and lymphocytic infiltration. The analysis of methylation of CpG islands as a mechanism of silencing genes in colon tumors has resulted in the identification of a third main pathway [CpG island methylator phenotype (CIMP)], which accounts for almost 40% of CRCs.11,12 CIMP-High tumors have a distinct clinical, pathologic, and molecular profile, such as an association with the proximal location in the colon, female sex, poor differentiation, MSI, and BRAF mutations.13 MSI and CIN are mutually exclusive pathways, whereas CIMP overlaps with the other two pathways, although most CIMP-high tumors have an MSI phenotype. It is important to classify CRCs according to global genomic or epigenomic status and to study correlation with molecular events in CRC or patient outcomes, as well as individualized therapies. For this reason, a molecular classification has been gaining strength that is based on three global cellular events (CIN, MSI, and CIMP), reflects underlying mechanisms of carcinogenesis, and correlates with some phenotypic features. Globally, sporadic CRC should be classified into four major subtypes: two MSI statuses (MSI-high versus MSI-low/MSS) times two CIMP statuses (CIMP-high versus CIMP-low/0), with distinct molecular correlates (BRAF, KRAS ) and pathologic features.13 As a whole, there are some controversial aspects about the natural history and prognosis of early-onset CRC, and there are also some clinical and pathologic differences compared with CRC in the elderly.14 Early onset of cancer is a marker of a potential hereditary component, with LS being the most frequent form of hereditary CRC. LS is characterized by earlier development of CRC than sporadic forms, the location of the tumor mostly in the right colon, and frequently poorly differentiated and mucinous tumors, and it has a tendency to develop synchronous and metachronous CRC.14e17 LS is caused by a germline mutation in one of the DNA MMR genes, which leads to MSI.18e20 Nevertheless, the proportion of MSI tumors found in early-onset CRC varies from 19.7% to 41%, depending on age at onset.14,19,21e24 Early age at onset, together with other features, has been proposed as an effective indicator of LS.25,26 However, MSI does not explain most of the earlyonset CRC cases because there still remain 60% of earlyonset CRCs that exhibit MSS. These early-onset MSS tumors are poorly characterized, and differences with MSS CRC in the elderly have not been defined. There are studies that have identified several molecular markers in MSS early-onset CRCs (modified expression of the APC, b-cateni, and p53 genes), but these molecular features have also been described in sporadic CRC.22 On the other hand, early-onset CRC has been shown to be occasionally familial after excluding cases owing to an MMR mutation or adenomatous polyposis. Specifically, Mourra et al27 suggest that chromosome 14 may harbor a gene playing an important role in this group of CRCs that is

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responsible for an inherited predisposition. Nevertheless, other alterations, such as epigenetic silencing of the tumor suppressor gene PTPRJ28 and some susceptibility genetic variants,29 have attracted the attention of researchers to clarify the important familial component of a subgroup of early-onset CRC. Even so, some studies suggest that there is a specific subset of MSS early-onset CRC without CIN and less frequent among sporadic cases; this is known as microsatellite- and chromosome-stable tumors and seems to be associated with a poor prognosis, an invasive phenotype, and early metastasis and is linked to familial clustering.30e32 In summary, knowledge about this probably clinically and genetically heterogeneous group in early-onset CRC remains limited, and further studies are essential. We analyzed early-onset CRC according to the three main different carcinogenetic pathways by characterizing clinical, pathologic, and familial features of each subset. We compared the results in early-onset tumors with CRC diagnosed at an older age (70 years old), when sporadic forms of CRC are more common, to identify differences that could support the idea that different molecular mechanisms may underlie CRCs with different ages at onset, apart from what is already known about the hereditary aspect.

Materials and Methods Families, Samples, and Data Collection A total of 88 consecutive individuals with CRC diagnosed at 45 years or younger were identified from our 12 de Octubre University Hospital. They were considered the index case of each family. Afterward, we identified 97 consecutive individuals who were diagnosed during the same period with CRC but at 70 years or older to compare with the early-onset CRC group. All the patients, or a first-degree relative in the case of death of the index case, provided written consent. This work was approved by the ethics committee of 12 de Octubre University Hospital. A full three-generation family cancer history and colorectal paraffin-embedded tumor tissue samples were obtained from each proband. Cancer diagnoses were verified by reviewing medical and pathologic reports. All the cases were reviewed by a common pathologist (Y.R.). Personal and clinicopathologic information was obtained, including age at onset, sex, location of the CRC (right or left colon or rectum), grade of cell differentiation (low, medium, or high), mucin production, the presence of signet ring cells, modified Astler-Coller stage, the existence of polyps during follow-up, type of polyps (adenomatous, hyperplastic, and mixed), the presence of synchronous or metachronous CRCs, and the presence of primary multiple neoplasms in the index case. Synchronous CRCs were defined as simultaneously diagnosed multiple invasive adenocarcinomas located in different anatomic segments of the large bowel. All the cases had a preoperatively complete colonoscopy. Metachronous CRCs were defined as a new CRC diagnosed as occurring >6

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Perea et al months after the first one, >1 cm from the previous anastomosis. To analyze the antecedents of cancer, families were classified into four groups: i) families fulfilling the Amsterdam II criteria for LS24; ii) families with mainly aggregation (one in first-degree or two in second-degree family members) of LSrelated neoplasms; iii) families with mainly aggregation of LS-unrelated neoplasms; and iv) cases without oncologic antecedents; these were considered sporadic cases. Families with the same number of relatives with LS-related and LSunrelated neoplasms were classified into group ii.

MSI and Mutational Analysis Tumoral and normal tissues of the paraffin-embedded samples of the index cases were selected by microscopic inspection. The proportion of tumor cells in the neoplastic material used for DNA extraction exceeded 70% in all cases. DNA was extracted using proteinase K digestion, phenolchloroform extraction, Phase Lock Gel Light (Eppendorf AG, Hamburg, Germany), and ethanol protocol precipitation. MSI analysis was performed using the Bethesda panel.33 The five microsatellite markers were PCR amplified, and fluorescently labeled fragments were evaluated using a 3100-Avant genetic analyzer (Applied Biosystems, Foster City, CA); data were analyzed using GeneMapper software version 3.5 (Applied Biosystems). Tumors were considered as MSI when showing high-frequency MSI (MSI-high) (two or more of the five markers showing instability), whereas the remaining tumors were classified as MSS. MSI cases were screened for germline mutations in the MMR genes MLH1, MSH2, and MSH6 by denaturing gradient gel electrophoresis using a DCode system (BioRad Laboratories, Hercules, CA), by denaturing highperformance liquid chromatography using a Varian ProStar system (Varian Australia Pty Ltd., Melbourne, Australia), or by high-resolution melting analysis using a LightCycler 480 real-time PCR system (Roche, Mannheim, Germany). Primers and denaturing and melting conditions were as previously reported, with slight modifications.25 When an anomalous band or pattern was observed by denaturing gradient gel electrophoresis, denaturing highperformance liquid chromatography, or high-resolution melting, the PCR product of the fragment was sequenced using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) and was analyzed using an ABI Prism 3130 genetic analyzer (Applied Biosystems).

GGCCAAAA-30 . Each reaction mixture contained 3 ng of DNA, 0.3 mmol/L of each primer, 3 mmol/L MgCl2 plus LightCycler LC480 high-resolution melting master mix (Roche), and water to a final volume of 10 mL. PCR cycling conditions were as follows: 40 cycles at 95 C for 15 seconds, 67 C for 15 seconds, and 72 C for 15 seconds, followed by 5 minutes of extension at 72 C. Melting curve analysis was from 40 C to 93 C at a rate of 0.02 C per second in the same instrument. PCR products from samples with different melting patterns were treated with ExoSAP-IT reagent (GE Healthcare, Piscataway, NJ) and were sequenced using the BigDye XTerminator v3.1 cycle sequencing kit (Applied Biosystems) and an ABI3130 automated sequencer (Applied Biosystems). We studied BRAF mutations to assess the relationship between mutation status and CIMP phenotype and to identify possible MSI sporadic cases. The latter were also identified by determining the methylation status of the MLH1 gene promoter.

Analysis of the CIMP Panel The methylation status of promoter regions of CIMP panel genes CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS134,35 were studied by means of methylation-specific multiplex ligationedependent probe amplification36 using the SALSA MLPA kit (ME042-B1; MRC-Holland, Amsterdam, The Netherlands) following the manufacturer’s recommended procedure. The methylationspecific multiplex ligationedependent probe amplification products were resolved by capillary electrophoresis using a 3100-Avant genetic analyzer, and peaks were analyzed using GeneMapper software version 3.5. The methylation index at a specific CpG locus was calculated by dividing the normalized area of a given HhaI-digested probe sample and the undigested equivalent. CIMP-high was defined as the presence of at least six of eight methylated promoters, CIMPlow as one to five of eight methylated promoters, and CIMP0 as the absence (zero of eight) of methylated promoters.37

Molecular Classification According to the MSI and CIMP status, we classified both groups (early-onset and elderly CRC) into four categories, defined by Ogino and Goel13: MSI/CIMP-high, MSI/CIMPlow/0, MSS/CIMP-high, and MSS/CIMP-low/0.

Statistical Analysis

BRAF exon 15, including the hotspot V600E, was studied by high-resolution melting analysis as described previously herein. Primers were designed using NCBI Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast, last accessed November 2012): forward, 50 -AATGCTTGCTCTGATAGGAAAATGA-30 ; reverse, 50 -GCAGCATCTCAG-

Continuous variables are expressed as means  SD, and categorical variables are expressed as number (percentage) of cases. Differences were considered significant at P < 0.05. Associations between clinicopathologic and familial features and MSI/CIMP status in the early-onset CRC group were tested by the Pearson c2 test or the Fisher exact test when applicable. When those features were continuous variables, Student’s t test was used, as well as for some familial

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Determination of BRAF Mutational Status

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Age at Onset in Colorectal Cancer Table 1

Clinical, Pathologic, and Familial Features of the Global, MSS, and MSI Groups of Early- and Late-Onset CRCs Global

Feature

Early onset

MSI Late onset*

P value

Patients 82 (100) 97 (100) Age at onset (years), 39.56  4.9 77.9  5.7 means  SDz Sex Male 49 (59.8) 50 (51.5) Female 33 (40.2) 47 (48.5) Location Right colon 20 (24.4) 38 (39.2) Left colon 35 (42.7) 22 (22.7) Rectum 27 (32.9) 37 (38.1) 0.013 Poor tumor 8/60 (13.3) 4/90 (4.4) 0.036 differentiation{ 19/60 (31.7) 18/91 (19.8) 0.09 Mucin production{ Signet ring cells{ 4/60 (6.7) 2/91 (2.2) 0.22 Mlh1 expression (absent) 5 (6.2) 7 (7.2) 0.13 BRAF mutationjj 1/71 (1.4) 7 (7.2) 0.14 MMR gene mutations 10 (12.2) 1 (1.0) 0.003x Astler-Coller modified tumor stage A 23 (28.0) 5 (5.2) B 26 (31.7) 48 (49.5) C 14 (17.1) 25 (25.8) D 19 (23.2) 19 (19.6) <0.001x Associated polyps 46 (56.1) 62 (63.9) 0.22 Adenomatous 22 (47.8) 38 (61.3) 0.24 Hyperplastic 8 (17.4) 6 (9.7) Mixed 16 (34.8) 18 (29.0) Multiple primary 11 (13.4) 33 (34.0) 0.001x neoplasms Synchronous or 5 (6.1) 21 (21.6) 0.004 metachronous CRCs Family history of cancer <0.001x Amsterdam IIepositive 16 (19.5) 1 (1.0) families Aggregation for 21 (25.6) 12 (12.4) LS-related neoplasm Aggregation for 13 (15.9) 6 (6.2) LS-unrelated neoplasm Sporadic cases 32 (39.0) 78 (80.4)

MSS

Early onset

Late onset* P value Early onset

9 (9.3) 12 (14.8)y 34.33  5.2 75.8  5.7 9 (75.0) 3 (25.0)

1 (11.1) 8 (88.9)

6 (50.0) 6 (50.0) 0 2/11 (18.2)

7 (77.8) 2 (22.2) 0 1/9 (11.1)

6/11 (54.9) 2/11 (18.2) 5 (41.7) 0 10 (83.3)

1/9 (11.1) 0 7 (87.5) 4 (44.4) 1 (11.1)

1 (8.3) 9 (75.0) 2 (16.7) 0 8 (66.7) 2 (25.0) 2 (25.0) 4 (50.0) 2 (16.7)

1 (11.1) 7 (77.8) 1 (11.1) 0 7 (77.8) 3 (42.9) 1 (14.3) 3 (42.9) 4 (44.4)

2 (16.7)

3 (33.3)

10 (83.3)

0

69 (85.2)y 40.4  4.3

0.008

0.37 0.07 0.48 0.18 0.021 0.002

0.4

0.29

40 (58.0) 29 (42.0) 14 29 26 6/49

(20.3) (42.0) (37.7) (12.2)

13/49 (26.5) 2/49 (4.1) 0 1/60 (1.7) 0 22 17 12 18 37 20 6 11 7

(31.9) (24.6) (17.4) (26.1) (53.6) (54.1) (16.2) (29.7) (10.1)

Late onset*

P value

88 (90.7) 78.1 (5.7) 49 (55.7) 39 (44.3) 31 20 37 3/82

(35.2) (22.7) (42) (3.7)

0.02x 0.07

17/82 (20.7) 2/82 (2.4) 0 3 (3.4) 0 4 39 23 19 55 35 5 15 29

(4.5) (45.9) (27.1) (22.4) <0.001x (62.5) 0.25 (63.6) (9.1) (27.3) (33.0) 0.001x

3 (4.3)

18 (20.5)

6 (8.7)

1 (1.1)

2 (16.7)

2 (22.2)

18 (26.1)

10 (11.4)

0

1 (11.1)

13 (18.8)

5 (5.7)

0

6 (66.7)

32 (46.4)

72 (81.8)

0.004 0.0005x

Data are given as number (percentage) except where indicated otherwise. Only P Z 0.05/15 Z 0.003 has been used as the cutoff point for statistical significance (simple Bonferroni correction). *Age at onset older than 69 years. y Percentages are calculated for 81 tumors because we lacked material to analyze MSI in one case. z Statistical analysis was performed using Student’s t-test. x Statistically significant. { Percentages shown are based on varying total numbers because some cases were excluded as only one biopsy sample was collected (stage D) or tumors were severely dysplastic with in situ carcinoma, and it was not possible to study any other characteristic. jj Percentages shown are based on varying total numbers in the case of early-onset CRC because not all cases could be analyzed for BRAF mutations.

features, to compare the differences between groups. The same procedure was performed when comparing the categories of the molecular classification within and between early-onset CRC and elderly CRC. After all, we adjusted for multiple testing. In each table comparing molecular/clinical types (15 tests conducted), we used P Z 0.05/15 Z 0.003 as the cutoff point for statistical significance (simple Bonferroni correction). The SPSS for Windows statistical package version 11.5 (SPSS Inc., Chicago, IL) was used.

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Results Early-Onset CRC Global Group We studied 88 patients diagnosed as having CRC at age 45 years or younger. Six patients were diagnosed as having familial adenomatous polyposis and were excluded from further analysis. The clinicopathologic and familial characteristics of all the patients are shown in Table 1. The mean

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Perea et al age at onset was 39 years, and 60% of the patients were male. The most common tumor location was the left colon. Mucinous tumors and signet ring cell tumors represented 32% and 7%, respectively. The lowest Astler-Coller modified tumor stages (A and B) were identified in 60% of the cases. Other remarkable features were the appearance of polyps during follow-up (56%), and 48% of them were adenomatous. Amsterdam II criteria were fulfilled by 20% of the cases; 42% showed familial aggregation for neoplasms (26% for LS-related neoplasms and 16% for LS-unrelated ones); the remaining 39% were defined as sporadic cases, without familial antecedents of cancer. Left colon location; more poorly differentiated tumors but more reliable, earlier stages at diagnosis; and the familial component were differential characteristics of early-onset tumors compared with elderly CRCs. However, multiple primary neoplasms (including synchronous and metachronous CRCs) were less frequent in early-onset CRC.

MSI Analysis: LS and Sporadic Cases Eighty-one of the 82 early-onset cases were studied for MSI. Twelve cases were defined as MSI (15%), and the remaining 69 were defined as MSS. MSI correlated with lack of detectable expression of any of the MMR proteins in immunohistochemical analysis in all cases. Blood samples were collected from the MSI index cases to perform MLH1, MSH2, and MSH6 mutation screening. Ten of the 12 analyzed cases (83%) showed a pathogenic germline mutation in one of the MMR genes: four in MLH1, four in MSH2, and two in MSH6. One of the remaining two MSI cases showed lack of expression of Mlh1 and hypermethylation of its gene promoter; in the other case, no molecular alteration in MLH1, MSH2, and MSH6 was found, although the Msh6 protein was not expressed. We compared MSI and MSS cases in early-onset CRC (data not shown). The MSI group showed, as expected, LS features: earlier age at onset (34 versus 40 years old), higher frequency of right colon cancer (50% versus 20%), earlier stages of diagnosis (82% versus 57% for stage A and B, respectively), and more familial antecedents of cancer. We also performed microsatellite analysis in the elderly group. Nine tumors (9%) showed MSI. We compared the characteristics of MSI and MSS cases between the earlyonset and elderly groups (Table 1). Early-onset MSI CRCs were remarkably associated with MMR mutations (83%), whereas elderly MSI CRCs showed frequent BRAF mutations and appeared more commonly in females. Earlyonset MSS CRC cases, compared with elderly MSS CRCs, showed earlier stages, a lower proportion of multiple primary neoplasms, and an important familial aggregation for cancer.

Only 19% of these CRCs (13 of 68) were CIMP-high. Amsterdam IIepositive families and those with LS-related tumors were more associated with CIMP-high cases. Right colon tumors were more frequent in these CIMP-high cases as well. CIMP analysis was also performed in 90 elderly CRCs. The most frequent phenotype in elderly CRC was CIMP0 (49%). MSI cases associated with BRAF mutations and absent Mlh1 protein expression due to MLH1 promoter hypermethylation mainly fell into the CIMP-high group (Supplemental Table S1). We compared early- and late-onset (elderly) tumors regarding their CIMP status. Results are shown in Table 2. Early-onset CIMP-high CRCs were associated with MMR gene germline mutations and more frequent mucinous tumors and exhibited a larger familial component. In contrast, elderly CIMP-high CRCs included most sporadic MSI cases. Earlyonset CIMP-low CRCs were more commonly located in the left colon, whereas elderly tumors with the same phenotype showed a tendency to develop other primary neoplasias, including CRCs. Finally, early-onset CIMP-0 cases were rarely in the right colon, had a high proportion of AstlerColler modified stage A tumors, and had an important familial component.

Molecular Classification of CRC

CIMP characterization was performed in 68 of the 88 earlyonset tumors. Results are shown in Supplemental Table S1.

Based on MSI and CIMP status, we defined four molecular groups, as described in Materials and Methods. Clinical, pathologic, and familial characteristics of the four groups in early-onset CRC are shown in Table 3. MSS/CIMP-low/0 tumors were the most frequent. Age at onset was younger for MSI/CIMP-high, with an average age at diagnosis of 29 years old. Locations of tumors differed: right-sided neoplasms were related to CIMP-high with and without MSI. Advanced stages at diagnosis were observed in MSS/CIMPlow/0 tumors. The highest familial cancer component was observed in MSI tumors where LS appeared, even after applying Bonferroni correction. However, aggregation for LS-related and LS-unrelated tumors was also substantial in MSS cases. Supplemental Table S2 shows the clinical, pathologic, and familial features of the four groups in elderly CRC. The comparison between early- and late-onset MSI tumors is not shown in the tables, and because of the small size of their subsets, differences should result in very cautious use. Although early-onset MSI/CIMP-high tumors were LS cases, all of them fulfilling Amsterdam II criteria, in the elderly group these tumors were associated with BRAF mutations, all of which were sporadic. Differences between both MSI/ CIMP-low/0 groups are observed regarding sex (earlyonset cases were mostly male, whereas elderly cases were all female) and the familial cancer component (early-onset CRC showed an important familial component related with LS, whereas all the elderly cases were sporadic). LS cases accounted for 50% in both early- and elderly-onset MSI/ CIMP-low/0 groups, and no BRAF mutations were found.

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CIMP Analysis

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Age at Onset in Colorectal Cancer Table 2

Comparison of Each CIMP Group between Early- and Late-Onset CRCs CIMP-high

CIMP-low

Feature

Early onset Late onset* P value Early onset

Patients Age at onset (years), means  (SD)y Sex Male Female Location Right colon Left colon Rectum Poor tumor differentiationz Mucin productionz Signet ring cellsz MSI Mlh1 expression (absent) BRAF mutation MMR gene mutations Astler-Coller modified tumor stage A B C D Associated polyps Adenomatous Hyperplastic Mixed Multiple primary neoplasms Synchronous or metachronous CRCs Family history of cancer Amsterdam IIepositive families Aggregation for LS-related neoplasm Aggregation for LS-unrelated neoplasms Sporadic cases

13 (19.1) 22 (24.4) 36.9  6.4 77.8  5 6 (46.2) 7 (53.8)

8 (36.4) 14 (63.6)

(30.8) (46.2) (15.4) (7.7) (76.9) (30.0) (10.0) (60.0) (30.8)

1 12 3 6 16 9 2 5 7

3 (23.1)

18 (64.3) 10 (35.7) 6 14 8 3/22 8/22 3/22 4 1 1 2

(4.5) (54.5) (13.6) (27.3) 0.14 (72.7) (56.3) (12.5) 0.43 (31.3) (31.8)

4 (18.2)

4 (30.8)

0

3 (23.1)

Late onset* P value Early onset

28 (41.2) 24 (26.7) 39.4  5 78.2  5.3

8 (61.5) 7 (31.8) 3 (23.1) 3 (13.6) 2 (15.4) 12 (54.5) 0.07 2/9 (22.2) 2 (10.0) 4/9 (44.4) 1/20 (5.0) 0.02x 0/9 0/20 4 (30.8) 7 (31.8) 2 (15.4) 6/10 (60.0) 0.08 0 6 (27.3) 0.07 4 (30.8) 0 0.014x 4 6 2 1 10 3 1 6 4

CIMP-0

7 9 7 5 14 8 2 4 3

1 (3.6) 0.08

27 (39.7) 44 (48.9) 40.15  4.5 77.6  6.4

15 (62.5) 9 (37.5)

18 (66.7) 9 (33.3)

(21.4) 12 (50.0) (50) 4 (16.7) (28.6) 8 (33.3) 0.026 (13.6) 0 0.18 (36.4) 5/21 (23.8) (13.6) 1/21 (4.8) (14.3) 1 (4.2) 0.3 (3.6) 0 (3.6) 1 (4.2) (7.1) 1 (4.2) (25.0) (32.1) (25.0) (17.9) (50.0) (57.1) (14.3) (28.6) (10.7)

2(8.3) 7 (29.2) 6 (25) 9 (37.5) 0.3 14 (58.3) 9 (64.3) 1 (7.1) 4 (28.6) 9 (37.5) 0.02 6 (25.0) 0.034

5 (17.9)

1 (4.2)

2 (9.1)

5 (17.9)

1 (7.7)

2 (9.1)

5 (38.5)

18 (81.8)

Late onset* P value

0.1

23 (52.3) 21 (47.7)

2 (7.4) 17 (38.6) 14 (51.9) 14 (31.8) 11 (40.7) 13 (29.5) 0 2 (4.7) 5/24 (20.8) 8/43 (18.6) 0/24 1/43 (2.3) 2 (7.4) 1 (2.3) 0 1/27 (3.7) 0 0 2 (7.4) 0 7 10 4 6 14 8 4 2 5

(25.9) (37) (14.8) (22.2) (52) (57.1) (28.6) (14.3) (18.5)

4 (14.8)

1 26 13 4 29 18 3 8 15

(2.3) (59.1) (29.4) (9.2) (65.9) (62.1) (10.3) (27.6) (34.1)

0.011

0.003x 0.32 0.3 0.18

10 (22.7)

5 (18.5)

0

3 (12.5)

8 (29.6)

6 (13.6)

6 (21.4)

2 (8.3)

4 (14.8)

2 (4.5)

12 (42.9)

18 (75)

10 (37)

0.32

<0.001x

36 (81.8)

Data are given as number (percentage) except where indicated otherwise. Only P Z 0.05/15 Z 0.003 has been used as the cutoff point for statistical significance (simple Bonferroni correction). *Age at onset older than 69 years. y Statistical analysis was performed using Student’s t-test. z Percentages shown are based on varying total numbers because some cases were excluded as only one biopsy sample was collected (stage D) or tumors were severely dysplastic with in situ carcinoma, and it was not possible to study any other characteristic. x Statistically significant.

Results of comparisons in the MSS groups are shown in Table 4. Early-onset MSS/CIMP-high tumors show earlier stages at diagnosis and are mainly right sided. Familial cancer history was still important in this group, mainly aggregation for LS-related neoplasms. Elderly cases were mainly sporadic (93%). Finally, comparison of MSS/CIMP-Low/0 tumors in both populations showed differential features: early-onset tumors were mainly located in the left colon and rectum, were homogeneously distributed among all stages, and exhibited a

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more important familial component. On the other hand, elderly MSS/CIMP-low/0 tumors were more frequently right sided, were intermediate stages, were frequently associated with multiple primary neoplasms, and were mainly sporadic (79%).

Discussion The incidence of early-onset CRC seems to be increasing, and its effect on the population is consequently important.

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Perea et al Table 3 Comparison of the Clinical, Pathologic, and Familial Characteristics between the Different Molecular Categories in Early-Onset CRC According to MSI and CIMP Status Characteristic

MSI/CIMP-high

MSI/CIMP-low/0

MSS/CIMP-high

MSS/CIMP-low/0

P value

Patients Age at onset (years), means  SD* Sex Male Female Location Right colon Left colon Rectum Poor tumor differentiationz Mucinous tumorsz Signet ring cellsz Mlh1 expression (absent) BRAF mutation MMR gene mutations Astler-Coller modified tumor stage A B C D Associated polyps Adenomatous Hyperplastic Mixed Synchronous or metachronous CRCs Multiple primary neoplasms Family history of cancer Amsterdam IIepositive families Aggregation for LS-related neoplasms Aggregation for LS-unrelated neoplasms Sporadic cases

4 (5.9) 29.25  3.3

6 (8.8) 37.17  3.3

9 (13.2) 40.3  1.3

49 (72.1) 40.1  4.8

<0.001y

2 (50.0) 2 (50.0)

5 (83.3) 1 (16.7)

4 (44.4) 5 (55.6)

31 (63.3) 18 (36.7)

2 (33.3) 4 (66.7) 0 0 4/6 (66.7) 1/6 (16.7) 1 (16.7) 0 3 (50.0)

5 (55.6) 2 (22.2) 2 (22.2) 2/6 (33.3) 3/6 (50.0) 0/6 0 0 0

6 (12.2) 24 (49.0) 19 (38.8) 3/40 (7.5) 9/40 (22.5) 2/40 (5.0) 0 1 (2.0) 0

3 (75.0) 1 (25.0) 0 0 1/3 (33.3) 0/3 2 (50.0) 0 4 (100.0) 1 (25.0) 3 (75.0) 0 0 4 (100.0) 2 (50.0) 0 2 (50.0) 2 (50.0) 2 (50.0)

0 6 (100.0) 0 0 4 (66.7) 0 2 (50.0) 2 (50.0) 0 0

3 3 2 1 6 1 1 4 1 2

(33.3) (33.3) (22.2) (11.1) (66.7) (16.7) (16.7) (66.7) (11.1) (22.2)

14 13 11 11 24 16 4 4 5 8

(28.6) (26.5) (22.4) (22.4) (49.0) (66.7) (16.7) (16.7) (10.2) (16.3)

4 (100.0) 0 0 0

5 (83.3) 1 (16.7) 0 0

0 3 (33.3) 1 (11.1) 5 (55.6)

5 12 10 22

(10.2) (24.5) (20.4) (44.9)

0.004 0.13 0.08 0.0015y <0.001y

0.011 0.21 0.018 0.12 0.2 <0.001y

Data are given as number (percentage) except where indicated otherwise. Only P Z 0.05/15 Z 0.003 has been used as the cutoff point for statistical significance (simple Bonferroni correction). *Statistical analysis was performed using Student’s t-test. y Statistically significant. z Percentages shown are based on varying total numbers because some cases were excluded as only one biopsy sample was collected (stage D) or tumors were severely dysplastic with in situ carcinoma, and it was not possible to study any other characteristic.

Until recently, series regarding this CRC group showed important variations concerning tumor location, the presence of mucinous or signet ring cell neoplasms, etc.15,21 Only when focus was shifted to the MSI component did the characteristics of LS CRC become clear, and early age at onset was considered to be a consequence of the higher proportion of LS.14,22 The present results confirm that MSI in early-onset tumors is mostly related to LS and is rarely due to somatic inactivation of MLH1,22 with 83% of MSI cases explained by a mutation in one of the MMR genes. These findings support previous results pointing out that the criterion of early age at onset, together with other clinicopathologic and familial features, should be helpful in identifying LS cases more easily.25 However, note that most early-onset CRCs (85%) involve MSS tumors, and there are few studies that evaluate the clinicopathologic features of this subset of early-onset CRC.

First, the proportions of MSS tumors in previous studies with early-onset CRC, with the same age at onset as in the present series, are slightly smaller but reach 80%.22 On the other hand, Chang et al38 found a striking predilection of sporadic MSS early-onset CRC for the distal colon, particularly the sigmoid colon and rectum, and observed that these tumors more frequently showed adverse histologic features. In the present series, early-onset MSS tumors are remarkably different from those in late-onset MSS CRC. Left colon location, low frequency of other primary neoplasms, and an important familial component are significant features of early-onset MSS CRC. These aspects are more reliable when we perform a more in-depth molecular classification by including the CIMP phenotype in the analysis. CIMP has rarely been evaluated in early-onset CRC. Previous large series, without using age at onset as a differential criterion, showed that LS cases were mainly

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Age at Onset in Colorectal Cancer Table 4

Comparison of the MSS/CIMP-High and MSS/CIMP-Low/0 Molecular Categories between Early- and Late-Onset CRC MSS/CIMP-high

MSS/CIMP-low/0

Feature

Early onset

Late onset*

Patients Age at onset (years), means  SDy Sex Male Female Location Right colon Left colon Rectum Poor tumor differentiationx Mucin productionx Signet ring cellsx Mlh1 expression (absent) BRAF mutation MMR gene mutations Astler-Coller modified tumor stage A B C D Associated polyps Adenomatous Hyperplastic Mixed Multiple primary neoplasms Synchronous or metachronous CRCs Family history of cancer Amsterdam IIepositive families Aggregation for LS-related neoplasms Aggregation for LS-unrelated neoplasm Sporadic cases

9 (13.2) 40.3  1.3

15 (16.7) 79.1  4.9

P value

Early onset

Late onset*

49 (72.1) 40.1  4.8

66 (73.3) 77.8  6

4 (44.4) 5 (55.6)

7 (46.7) 8 (53.3)

31 (63.3) 18 (36.7)

38 (57.6) 28 (42.4)

5 (55.6) 2 (22.2) 2 (22.2) 2/6 (33.3) 3/6 (50.0) 0/6 0 0 0

2 (13.3) 1 (6.7) 12 (80.0) 1 (7.7) 0/13 0/13 0 2 (13.3) 0

6 (12.2) 24 (49.0) 19 (38.8) 3/40 (7.5) 9/40 (22.5) 2/40 (5.0) 0 1 (2.0) 0

27 (40.9) 18 (27.3) 21 (31.8) 2 (3.2) 13/62 (21.0) 2/62 (3.2) 0 1 (1.5) 0

3 3 2 1 6 1 1 4 2 1

(33.3) (33.3) (22.2) (11.1) (66.7) (16.7) (16.7) (66.7) (22.2) (11.1)

0 3 (33.3) 1 (11.1) 5 (55.6)

0 7 2 6 9 6 1 2 3 1

(46.7) (13.3) (40.0) (60) (66.7) (11.1) (22.2) (20) (6.7)

0 0 1 (6.7) 14 (93.3)

0.021 0.18 0.02

0.007 0.16

0.02

14 13 11 11 24 16 4 4 8 5

(28.6) (26.5) (22.4) (22.4) (49) (66.7) (16.7) (16.7) (16.3) (10.2)

3 29 19 12 44 28 4 12 24 16

(4.8) (46.0) (30.2) (19.0) (66.7) (63.6) (9.1) (27.3) (36.4) (24.2)

5 12 10 22

(10.2) (24.5) (20.4) (44.9)

1 9 4 52

(1.5) (13.6) (6.1) (78.8)

P value

0.002z

0.003z 0.09

0.004 0.08 <0.001z

Data are given as number (percentage) except where indicated otherwise. Only P Z 0.05/15 Z 0.003 has been used as the cutoff point for statistical significance (simple Bonferroni correction). *Age at onset older than 69 years. y Statistical analysis was performed using Student’s t-test. z Statistically significant. x Percentages shown are based on varying total numbers because some cases were excluded as only one biopsy sample was collected (stage D) or tumors were severely dysplastic with in situ carcinoma, and it was not possible to study any other characteristic.

included in the CIMP-low group, whereas sporadic MSI cases were more likely to be CIMP-high, with high rates of BRAF mutations.13 The present results confirm these features only for the elderly CRC group, whereas in early-onset CRC, LS primarily falls in the CIMP-high group, and the only two cases we found with MSI and no MMR mutation fell in the CIMP-low category. This could be the consequence, among others, of low BRAF mutations and hypermethylation of the MLH1 promoter in early-onset CRC, observed by us and others.26,39 It is known that the difference between CIMP-low and CIMP-0 is unclear when considering CRC in general. In the early-onset population, a variety of differences compared with the elderly group draw the attention: a progressive disappearance of right colon tumors and an increase in age at onset as CIMP decreases; a higher rate of CIMP-low cases; and an important familial

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component, a result of LS-related, but also LS-unrelated, cancer history, in the case of CIMP-low early-onset CRC, whereas in CIMP-0 tumors, an important number of LSrelated neoplasias is observed. Jass40 was the first to propose a classification of CRC based on the correlation of the main molecular carcinogenetic pathways and clinical and molecular features; this classification was later modified by Ogino and Goel.13 These latter authors pointed out that i) MSI/CIMP-high tumors (10%) commonly show, among others, MLH1 methylation and BRAF mutations, poor differentiation, lymphocytic reactions, and mucinous or signet ring cell features; clinically, this is generally known as sporadic MSI-high and is associated with elderly females and tumor location in the proximal colon; ii) MSI/CIMP-low/0 tumors (5%) include LS and also show tumor location in the proximal colon, lymphocytic reactions,

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Perea et al and mucinous features, but not with poor differentiation or signet ring cell features; iii) MSS/CIMP-high tumors (5% to 10%) commonly show BRAF mutations, poor differentiation, and signet ring cell features and are associated with elderly females and tumor location in the right colon; and iv) MSS/ CIMP-low/0 tumors (75% to 80%), with a heterogeneity of features, are mainly associated with male sex and predominant location of tumor in the distal colon. Familial cancer history was not studied in this series. We also performed an analysis of MSI status and CIMP in this series of early- and late-onset CRCs to evaluate the molecular classification according to age at onset. In the present series, MSI/CIMP-high tumors are mainly right sided in both age groups. However, all early-onset MSI/CIMP-high tumors (6%) were LS tumors, without sex preference, whereas elderly MSI/CIMP-high tumors (8%) were associated with BRAF mutations in female patients. Half of the early-onset MSI/CIMP-low/0 cases were LS tumors with a mucinous component, but 67% of tumors were left sided. From these results we can deduce that features of the MSI groups (regardless of the CIMP) from the corresponding categories of the Ogino and Goel classification are comparable with the elderly CRC consecutive categories but that they are remarkably different from the early-onset population. The early-onset criterion seems to make a change in the MSI groups of Ogino and Goel’s molecular classification, particularly in MSI/CIMP-high tumors, because they contain an important proportion of LS in early-onset MSI CRC. However, as mentioned in Results, because of the small size of these subsets (MSI groups, regardless of their CIMP status), differences should result in very cautious use. The early-onset MSS/CIMP-high group represented 13% of early-onset tumors. They were right sided, mucinous, at earlier stages at diagnosis, and without sex preference or identified BRAF mutations; they differed from the equivalent elderly category (17%), with BRAF mutations in 13% and a predominant left colon/rectum location. Consequently, in the MSS/CIMP-high category, there are significantly more similarities with the correlative group in the Ogino and Goel molecular classification.13 Furthermore, there is also an important familial cancer component in this early-onset subset of tumors, mainly LS-related neoplasm aggregation. In these groups of MSS/CIMP-high tumors, differences should be taken on account with care because none reach statistical significance after applying Bonferroni correction. Last, the most common phenotype was MSS/CIMP-low/0 CRC in the early-onset group (72%) comparable with what was observed in the late-onset group (73%). When combined, they are heterogeneous according to the Ogino and Goel classification,13 but when divided according to age at onset, each consecutive group tends to homogenize. There are some clinical and histopathologic features that are similar in both groups (mainly male sex and mucinous tumors), but there are many more differences. Compared with elderly MSS/CIMP-low/0 tumors, early-onset tumors are rarely in

the right colon (12%), are less associated with polyps during follow-up, show an association with other primary neoplasms, and contain an important familial cancer component. A multivariate analysis was performed to discover any independent predictor in the study, but because of the small size of some categories, it revealed no statistical significance. Only early-onset CRC groups with MSI showed a better prognosis, related to global and disease-free survival. Familial antecedents are different in early-onset CRC than in elderly CRC, as early age at onset is one of the characteristics of hereditary forms of cancer. The MSS/ CIMP-low/0 group probably includes familial CRC type X. This type of familial CRC is probably one of the reasons for the different features of early-onset MSS/CIMP-low/0 CRC compared with elderly CRC. On the other hand, microsatellite- and chromosome-stable tumors would be included in MSS groups in early-onset and elderly CRCs and would be responsible for some differences in these groups. To date, microsatellite- and chromosome-stable tumors seem to be more frequent in early-onset CRC and show a predominantly distal location and some familial aggregation for LS neoplasms31; although some studies indicate that CIMP does not seem to play a key role in this type of CRC,41 others propose that microsatellite- and chromosome-stable tumors have distinct clinicopathologic features and can be distinguished from other CRCs by a specific set of methylation loci.42 Recently, LINE-1 hypomethylation has been shown to be a feature of young age at onset of CRC43,44; LINE-1 hypomethylated CRCs are also associated with family history of CRC.45,46 According to the molecular classification of CRC based on MSI and CIMP status, both CIMP-high and MSI-high are inversely associated with LINE-1 hypomethylation, suggesting that CIMP/MSI and genomic hypomethylation may represent different pathways to CRC. In addition, in noneMSI-high tumors, CIN is correlated with LINE-1 hypomethylation, supporting the possible link between genome-wide hypomethylation and CIN. This molecular feature of LINE-1 hypomethylation related to early-onset MSS CRC suggests that CIMP/MSI and genomic hypomethylation may represent different pathways to CRC.43 The importance of the differential features arising in MSS early-onset CRC according to CIMP status led us to analyze these two groups in more detail not only with respect to their molecular basis but also including their important familial component.

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Acknowledgments We thank the Tumor Registry of the Pathology Department of the 12 de Octubre University Hospital for providing the paraffin-embedded tissues, Ron Hartong for proofreading the manuscript, and Lucia Inglada-Pérez for helping with the statistical analysis.

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Supplemental Material Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.jmoldx.2013.07.010.

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