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Originally published as JCO Early Release 10.1200/JCO.2006.06.9781 on January 16 2007 © 2007 American Society of Clinical Oncology. Clinicopathologic and Pedigree Differences in Amsterdam I–Positive Hereditary Nonpolyposis Colorectal Cancer Families According to Tumor Microsatellite Instability Status
From the Familial Cancer Unit and the Human Genetics Group, Human Cancer Genetics Program, Spanish National Cancer Centre; Tumor Bank Unit, Molecular Pathology Program, Spanish National Cancer Centre, Madrid; Surgery Department, Hospital de Segovia, Segovia; Molecular Genetics Unit, Centro de Bioquímica y Genética Clínica, Hospital Virgen de la Arrixaca, Murcia, Spain Address reprint requests to Laura Valle, PhD, Familial Cancer Unit, Spanish National Cancer Centre, Melchor Fernández Almagro, 3, E-28029, Madrid, Spain; e-mail: lvalle{at}cnio.es
Purpose To establish the clinicopathologic and familial differences within Amsterdam I–positive families, showing either tumor microsatellite instability (MSI) or microsatellite stability (MSS) in order to confirm or deny the existence of hereditary nonpolyposis colorectal cancer (HNPCC) without defects in the mismatch repair system. Patients and Methods Sixty-four Amsterdam I–positive families were included in the study for which full, three-generation, family medical histories and colorectal paraffin-embedded tumors were obtained. Both personal and clinicopathologic information of patients were collected. In all cases, both the MSI status and the mismatch repair (MMR) protein expression were analyzed. MMR genetic testing was performed on the MSI families. Results Of the Amsterdam I–positive families, 59.4% were tumor MSI, and 40.6% were tumor MSS. When comparing both groups, the statistical differences were observed in the age of onset (MSI, 41 years; MSS, 53 years); in the colorectal tumor location, more frequently proximal in MSI cases; in fewer mucinous tumors in MSS; and loss of MMR protein expression in the MSI tumors. Regarding the individual and familial cancer history, we observed a predominance of individuals with multiple primary tumors in MSI pedigrees, as well as differences in the type of tumors developed within the family. Conclusion Our findings support the suspicion of another hereditary colorectal syndrome different from HNPCC and characterized by MSS, the normal MMR immunohistochemical expression, the presence of only colorectal tumors, and the absence of individuals with multiple primary tumors. All these circumstances suggest the existence of a non-MMR gene being responsible for this new syndrome.
Hereditary nonpolyposis colorectal cancer (HNPCC) is a dominantly inherited syndrome characterized by an increased risk for colorectal cancer (CRC), with a penetrance of around 80% and diagnosis at early ages. CRCs are more commonly right-sided, and have a mucinous and poorly differentiated histology.1 There is also an increased risk for cancers of the endometrium, stomach, ovary, kidney, ureter, small intestine, and hepatobiliary tract.2,3 CRC's molecular basis is a hereditary DNA mismatch repair (MMR) gene deficiency, which leads to changes in the length of DNA repetitive sequences, known as microsatellite instability (MSI). To define and select families for molecular studies, different guidelines have been proposed through the years. The most stringent, the Amsterdam I criteria (AC-I),4 includes the following conditions: (1) at least three relatives must have histologically verified CRC, one of the relatives must be a first-degree relative of the other two, familial adenomatous polyposis excluded; (2) at least two generations must be affected; and (3) at least one case must be diagnosed before patient age 50 years. These guidelines could exclude small families or those presenting HNPCC-associated tumors. In response to its stringency, the extended AC-II criteria were developed.5 The use of the Amsterdam criteria achieved the original purpose of classifying a family as having HNPCC, but its limited sensibility hampered decisions of which families should undergo genetic testing. For this reason, the Bethesda and the revised Bethesda guidelines were outlined.6,7 Tumor MSI is a phenotypic indicator of defective DNA MMR. It has been reported that more than 90% of the HNPCC-related cancers exhibit MSI.8-11 However, it has recently been suggested that another entity of HNPCC, not showing MSI, could exist.12-15 Some distinctive clinicopathologic features of HNPCC without MSI have been observed: microsatellite stable (MSS) HNPCC patients were older than those patients showing MSI; their tumors were less commonly on the right-hand-side of the colon, less often poorly differentiated and mucinous, and more often DNA aneuploid; they did not present multiple cancers; and had lesser penetrance than HNPCC with MMR deficiency.14-16 Lindor et al14 suggested the term "familial CRC type X," for those families who have a clustering of CRC, but in whose tumors no DNA MMR gene alteration was evident, to differentiate them from families with a hereditary DNA MMR deficiency. To avoid confusion with X chromosomal inheritance, the term "familial CRC of undetermined type" has been recently proposed.17 In our study, we have established the familial and clinicopathologic differences within the AC-I group, between those patients showing tumor MSI (independently of the presence or not of an identified germline MMR mutation), and with those patients with tumor MSS. The results obtained support the existence of a differential entity from the group of Lynch syndrome families, characterized by MSS, normal expression of MMR proteins, presence of exclusively CRC in family members, and absence of multiple primary tumors in the same individual.
Families, Samples, and Data Collection A total of 64 families fulfilling the AC-I criteria were collected from the set of families suspected to have HNPCC, as assessed through the Familial Cancer Unit of the Spanish National Cancer Centre (Madrid, Spain). All patients provided written consent. A full three-generation family medical history and a colorectal paraffin-embedded tumor were obtained from each family (if possible, from the family member diagnosed at the earliest age). Efforts were made to verify reported cancer diagnoses, primarily by reviewing medical and pathologic reports, and when this was not possible, by interviewing multiple family members. The patient whose paraffin-embedded tumor was MSI analyzed was considered as the index case. Personal and tumor clinicopathologic information was collected taking into account the age of onset, the patient's sex, the location of the CRC (right/left colon or rectum), the cell differentiation (low, medium, or high), the mucin production, the modified Astler-Coller stage, the existence of polyps, and the presence of synchronous or metachronous CRCs (Table 1).
To analyze the familial cancer history of each group, families were classified depending on the presence of the distinctive tumors: (1) families with CRC alone; (2) families with HNPCC-related neoplasms, CRC included; and (3) families with non–HNPCC-related tumors, other than CRC and other Lynch-associated tumors (Table 2). For these evaluations, the reference triad of each pedigree was included, as well as an evaluation of the presence of multiple primary tumors. To study the presence of HNPCC-related tumors in both groups' family members, the reference triad of each pedigree was excluded and the remaining affected first- and second-degree family members were considered (Table 3).
DNA Extraction As mentioned herein, a tissue specimen was obtained from an affected individual of each family. Before the DNA extraction, tumor and normal areas of the paraffin-embedded samples were selected via microscopic inspection. The proportion of tumor cells in the material used for DNA extraction exceeded 70% in all cases. DNA was extracted using a proteinase K, phenol–chloroform, phase Lock Gel Light (Eppendorf AG, Germany) and ethanol protocol.
Microsatellite Instability and MMR Immunohistochemistry Analyses BAT26 was polymerase chain reaction (PCR) amplified, and fragments were evaluated by using an ABI automated sequencer and the GeneScan software (Perkin Elmer, Wellesley, MA). For the analysis of the Bethesda panel, we used the HNPCC Microsatellite Instability Test (Roche, Mannheim, Germany). The PCR products were run on an ABI Prism 3700 automated sequencer (PE Applied Biosystems, Foster City, CA) using fragment software (GeneScan 3.5, Perkin Elmer, Wellesley, MA). The immunohistochemical staining of MLH1, MSH2, MSH6, and PMS2 was performed using the clones: G168-15 from Pharmigen (San Diego, CA); FE11 from Oncogene Research Products (Cambridge, MA); clone 44 from BD Transduction Laboratories (San Diego, CA); and C20 from Santa Cruz Biotechnology (Santa Cruz, CA), respectively, as previously described.22,23 The scoring of tumor staining was carried out without any knowledge of the patients' family history or results of mutation analyses. The protein expression was considered absent when there was no staining of the nuclei of tumor cells in the presence of nuclear staining in nearby stromal lymphocytes and/or in normal epithelial cells.
Detection of Mutations and Large Deletions Multiplex ligation-dependent probe amplification (SALSA P003 MLH1/MSH2, MRC-Holland, Amsterdam, Holland) was used to search for large deletions in MLH1 and MSH2.26
Statistical Analyses
From a total of 64 families fulfilling the AC-I criteria, 26 families (40.6%) resulted as MSS, and 38 families (59.4%) as MSI. Within the MSI group, 65.7% of families (23 of 35) carried a germline mutation (point mutation or large deletion) in either MLH1 (15 of 23) or MSH2 (8 of 23); and 34% of them (12 of 35) did not. The MSH6 gene was also analyzed but no sequence alterations were identified. Within this group of MSI-positive MMR-negative patients, a lack of tumor MMR protein expression was observed in six families: two families showed a lack of MLH1 expression, one family showed a lack of MSH2 expression, one family lacked MLH1 and PMS2, one family lacked both MSH2 and MSH6, and finally, another family showed a lack of expression of MLH1 and MSH6. Of the three families for whom the analysis of the genes was not performed because of a lack of peripheral blood of a cancer-affected individual, two families showed a normal expression of the three MMR proteins analyzed, and one family showed a lack of MSH2 and MSH6. In all, taking into account the presence of MMR mutations and/or the absence of MLH1/MSH2/MSH6/PMS2 expression, in 79% (30 of 38) of all MSI families an MMR defect was identified. In contrast, within the MSS family group, MMR immunohistochemistry revealed that 20 of 26 families showed expression of the four MMR proteins analyzed, and the other six could not be evaluated due to technical reasons. Clinicopathologic characteristics of all cases included in the study, and in particular, of the two defined groups (MSS and MSI), as well as the P values obtained from the comparison of those two tumor groups, are listed in Table 2. Statistically significant differences were observed for age of onset, which was earlier in the MSI tumors; for CRC location, which was more frequently proximal in MSI cases; for mucin production, which was infrequent in MSS tumors; and for MMR protein expression, which was frequently lost in the MSI cases. No differences were found when comparing the other clinicopathologic variables that were analyzed. However, we consider it important to underline that 36% of the MSS cases presented associated adenomatous polyps compared with 17.6% of the MSI cases, and that MSI index cases more frequently developed other colorectal tumors, either synchronous or methachronous. Table 2 shows the familial cancer history information and results obtained from comparing the MSS and MSI families studied. Fifty-eight percent of all the MSS families, compared with 13.2% of the MSI ones, were characterized by the presence of CRC as the sole neoplasm developed by their members. In addition, MSI tumors were more likely to appear in families with other HNPCC-associated tumors, and in families with multiple primary tumors. The incidence of different tumor types in first- and second-degree relatives varied between the MSS and MSI groups (Table 3). In summary, MSI families had a greater number of relatives affected with colorectal and endometrial cancers. Although no statistical analyses were performed (because of the low frequency of tumors different from CRC, endometrial, ovarian, and gastric cancers), in general, the MSI families showed a greater number of cancer-affected relatives with other HNPCC-associated tumors.
HNPCC was first defined by family cancer history, and later was defined by some common differential characteristics associated with the disease.1,27 Germline mutations in the MMR genes, mainly in MLH1 and MSH2, and to a lesser extent in MSH6, PMS1, and PMS2, have been identified as the alterations responsible for this syndrome.28 Nevertheless, mutations are not found in all cases defined as HNPCC, according to the clinical criteria. In fact, these mutations are identified in 50% to 70% of the families who meet the AC-I criteria. This could be explained, at least in part, by the fact that mutations in these genes are of different nature, and maybe we are not able to detect them with current mutation-detection techniques.29 In fact, it has been reported that when using more sophisticated techniques, mutation detection can be significantly improved.30 Therefore, in our series, four MSI cases showed a lack of MLH1 protein expression in the tumor, but no germline mutation in the gene was identified. Apart from problems in mutation detection or in the immunohistochemical performance or evaluation, this could be explained by somatic silencing of MLH1 by promoter hypermethylation. However, this phenomenon has been reported to be rare in familial cases, as were those included in our study (AC-I), whereas it has only been observed in sporadic MSI colorectal tumors.31,32 However, all those explanations (technical problems or somatic silencing of MLH1) could be valid for patients presenting with features of MMR gene mutations, such as MSI or a lack of MMR expression, but the remaining proportion still remains high. This suggests that in those patients, other pathways of carcinogenesis, different from the MMR gene deficiency, could be involved. We chose MSI as a marker of MMR system malfunction and performed the study in AC-I families in order to avoid the introduction of cases that were not clearly hereditary due to the high incidence of CRC familial aggregation in developed populations. Mutation and immunohistochemical results confirmed that the MSI and MSS groups were well-characterized and defined by the MMR defect and the normal MMR function, respectively. Little is known about the group of families with HNPCC who have tumor MSS, and no genes have been identified to be altered in the germline in this group, which comprises a high proportion of AC-I families, as was observed in our series (40.6%). When comparing MSS and MSI AC-I families, we observed that MSI CRCs, compared with MSS, develop at an earlier age (mean age of onset, 41 years), are frequently located in the right colon (70.6%), and a high proportion of tumors produce mucin (40%). In contrast, MSS tumors develop at a later age (mean age of onset, 53 years), most of the tumors (79%) are localized in the left colon and rectum, and in general, the tumors do not produce mucin. Apart from these features, there seems to be a higher association with polyps in this group of patients, although the differences do not reach statistical significance. The first report addressing this issue observed that MSI HNPCC patients showed an earlier age of onset, more frequently presented with multiple cancers, and more frequently presented with tumors that were right-sided, poorly differentiated, and mucinous.16 The older age at diagnosis of MSS patients has been confirmed in other studies, and a lower incidence of CRC and endometrial tumors has also been observed.14,33 Recently, Llor et al15 compared the clinicopathologic characteristics within a group of 25 AC-I/II families (AC-I, 17 families; MSI, nine families; MSS, eight families). Despite their small sample size, the authors observed important differences between both groups, coinciding with the differences observed between MSI and MSS sporadic or unselected CRCs. Compared with the studies performed previously, we have limited the study to AC-I families, and the number of families included (n = 64) is, by far, larger and, therefore, obtains more consistent results. If we do not consider MSS families within classical HNPCC (with MMR deficiency), the characteristics of the Lynch syndrome are even more marked and defined, especially regarding tumor location, mucin production, and age of onset. Regarding familial cancer history, we observe a clear predominance of individuals with multiple primary tumors (all type and HNPCC-related cancers) in the MSI pedigrees, as well as differences in the type of tumors developed within the family. In the MSS group, more than half of the families showed only CRCs in their pedigrees, whereas in the MSI group, the vast majority showed CRC together with other HNPCC-associated neoplasias or with other tumors that were not included in the defined Lynch-related group. This observation suggests that the cancer causing gene or genes defining the AC-I MSS families could be implicated only in colorectal tumorigenesis, discarding the implication of genes implying a more generalized loss of control, which happens when an MMR gene is mutated. The high percentage of MSS AC-I families, as well as the familial and clinicopathologic differences observed between this and the MSI AC-I group, firmly support the presence of a different entity or syndrome within HNPCC; the so-called "familial CRC type X" or "familial CRC of undetermined type."14,17 These observations and previous studies suggest that the cancer screening guidelines defined for HNPCC, including annual colonoscopy, endometrial cancer screening, and transvaginal ultrasound or endometrial aspirate, do not seem to be appropriate for familial CRC of undetermined type families, making it necessary to redefine the surveillance measures for this group. It must be taken into account that this subgroup of families would be included in the Lynch syndrome I category, which is characterized by an increased CRC risk and a lack of associated extracolonic cancers, a term defined before knowledge about both MMR mutations and their casual significance in the syndrome.34,35 Little or nothing has been elucidated about the mechanisms of the carcinogenesis implicated in this MSS-hereditary form of CRC or about the germline cancer-causing alterations occurring in these families. In fact, it is not known whether this cancer is an entity by itself or if it comprises several MSS HNPCC subclasses. Classically, two forms of genomic instability have been defined, MSI and chromosomal instability (CIN), and CRCs have been classified as MSS/CIN-positive or MSI/CIN.36,37 Recently, Chan et al38 reported on a group of CRCs with near diploid DNA, few chromosome imbalances, and MSS. The authors observed that the proportion of these kinds of tumors was significantly greater in early-onset versus late-onset tumors, and some of the patients had a positive family cancer history. In 2005, the existence of novel pathways to colorectal carcinogenesis for MMR gene mutation–negative cancers was suggested by Abdel-Rahman et al39: one group of CRC characterized by MSS, no CIN, infrequent p53 mutations, a membranous ß-catenin expression, a right location of the tumor, and a younger age of onset; and another group (minority) with a nuclear ß-catenin expression, a predominance of left CRCs, later age of onset, and molecular features similar to classical MSS/CIN-positive sporadic CRCs. In summary, our findings support the existence of an MSS HNPCC group of families that differs from the MSI HNPCC group in clinicopathologic and familial features. The correct understanding and definition of this entity becomes essential in order to establish appropriate surveillance guidelines and to characterize the molecular mechanisms and pathways involved in the carcinogenic process.
The authors indicated no potential conflicts of interest.
Conception and design: Laura Valle, Jose Perea, Javier Benitez, Miguel Urioste Provision of study materials or patients: Jose Perea, Pablo Carbonell, Victoria Fernandez, Javier Benitez, Miguel Urioste Collection and assembly of data: Laura Valle, Jose Perea, Pablo Carbonell, Ana M. Dotor, Miguel Urioste Data analysis and interpretation: Laura Valle, Jose Perea, Miguel Urioste Manuscript writing: Laura Valle, Jose Perea, Javier Benitez, Miguel Urioste Final approval of manuscript: Laura Valle, Jose Perea, Pablo Carbonell, Victoria Fernandez, Ana M. Dotor, Javier Benitez, Miguel Urioste
This work was funded by the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo (FIS-PI04/2237). We thank the Spanish National Tumor Bank Network for providing us with the paraffin-embedded tissues, and Luz Alvarez and Roger Milne for their assistance.
published online ahead of print at www.jco.org on January 16, 2007. L.V. and J.P. contributed equally to this article. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test for parametric variables and Fisher's exact test for nonparametric variables. The t test was used for features that were continuum variables, as well as for some familial features, as listed in 
