Microsatellite instability and mutation analysis among southern Italian patients with colorectal carcinoma: detection of different alterations accounting for MLH1 and MSH2 inactivation in familial cases

M. Colombino1,+, A. Cossu1,+, A. Arba1, A. Manca2, A. Curci3, A. Avallone3, G. Comella3, G. Botti3, F. Scintu4, M. Amoruso5, D. D’Abbicco5, M. R. d’Agnessa6, A. Spanu7, F. Tanda2 and G. Palmieri1,§

1 Istituto Chimica Biomolecolare-Sezione di Sassari, C.N.R., Tramariglio, Alghero; 2 Istituto di Anatomia Patologica, Università di Sassari, Sassari; 3 Istituto Nazionale Tumori ‘G. Pascale’, Napoli; 4 Chirurgia Generale II, Università di Cagliari, Cagliari; 5 Chirurgia Generale e Vascolare e Oncologia Clinica, Università di Bari, Bari; 6 Anatomia Patologica, Ospedali Riuniti, Foggia; 7 Medicina Nucleare, Università di Sassari, Viale San Pietro, Sassari, Italy

Received 12 February 2003; revised 7 April 2003; accepted 15 April 2003


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background:

Microsatellite instability (MSI) is due to defective DNA mismatch repair (MMR) and has been detected at various rates in colorectal carcinoma (CRC). The role of MSI in colorectal tumorigenesis was assessed further in this study by both microsatellite analysis of two CRC subsets [unselected patients (n = 215) and patients <50 years of age (n = 95)], and mutation screening of the two major MMR genes MLH1 and MSH2 among familial CRC cases.

Patients and methods:

PCR-based microsatellite analysis was performed on paraffin-embedded tissues. In CRC families, MLH1/MSH2 mutation analysis and MLH1/MSH2 immunostaining were performed on germline DNA and MSI+ tumour tissues, respectively.

Results:

The MSI+ phenotype was detected in 75 (24%) patients, with higher incidence in early-onset or proximally located tumours. Among 220 patients investigated for family cancer history, MSI frequency was markedly higher in familial [18/27 (67%)] than in sporadic [32/193 (17%)] cases. Three MLH1 and six MSH2 germline mutations were identified in 14 out of 36 (39%) CRC families. Prevalence of MLH1/MSH2 mutations in CRC families was significantly increased by the presence of: (i) fulfilled Amsterdam criteria; (ii) four or more CRCs; or (iii) one or more endometrial cancer. While MSH2 was found mostly mutated, almost all [8/9 (89%)] familial MSI+ cases with loss of the MLH1 protein were negative for MLH1 germline mutations.

Conclusions:

Both genetic (for MSH2) and gene-silencing (for MLH1) alterations seem to be involved in CRC pathogenesis.

Key words: colorectal cancer, gene inactivation, microsatellite instability, MLH1/MSH2 germline mutations


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Colorectal tumorigenesis is thought to occur by sequential accumulation of genetic mutations [1]. A multistep genetic model of tumorigenesis, based on specific genetic alterations in benign and primary malignant lesions, has been ascertained for colorectal carcinoma (CRC) [2, 3]. Genomic instability has been demonstrated to play a crucial role in the development of CRC tumours from normal epithelium (through onset of dysplastic lesions and adenomas, in a well defined pathogenetic sequence), and is mostly due to a defective replication fidelity [4].

Tumours with a non-functional DNA mismatch repair (MMR) display a genomic instability, as inferred by detection of ubiquitous somatic variation in the length of microsatellite sequences [2, 5]. Microsatellite instability (MSI) is characterized by small insertions or deletions within short tandem repeats in tumour DNA when compared with the corresponding normal DNA. MSI has been demonstrated in patients with hereditary non-polyposis colorectal carcinoma (HNPCC), an inherited cancer syndrome that also predisposes to endometrial cancer, which represents the most common extracolonic malignancy in HNPCC families [6]. Frequency of MSI has been reported ranging from 8% to 18% in unselected CRCs, and from 70% to ~90% in tumours from HNPCC patients [710]. Genetic instability seems to decrease moving from carcinomas originating in the proximal colon to those located in distal colon and rectum, where MSI frequency has been found at a very low rate (in some reports it was as little as ~2%) [11, 12].

MSI has been associated with the presence of mutations in the two principal MMR genes, MLH1 and MSH2 (although MLH1 is altered most frequently) [13]. Patients with CRC tumours displaying MSI have a greater chance of presenting with a family history positive for colorectal cancer and/or synchronous CRCs [9, 10], strongly suggesting a hereditary cancer predisposition (MSI is considered a molecular feature particularly frequent in familial CRC) [12, 13].

The prognostic value of MSI in colorectal cancer is highly debated. Several studies have suggested a favourable outcome for patients with the MSI+ phenotype in colorectal and gastric cancer [10, 14, 15], whereas some recent results on MSI+ CRC patients have demonstrated a better survival only for cases with Dukes’ B and C disease stages, receiving adjuvant chemotherapy (survivals among untreated patients were found to be similar in the two groups with and without the MSI+ phenotype) [11, 16].

To investigate further the prevalence of MSI in CRC, as well as the existence of any clinicopathological difference in CRC patients with and without MSI that might be representative of distinctive tumour behaviour, a PCR-based microsatellite analysis was performed on archival tissues from patients with CRC at various stages of disease and originating from different regions of southern Italy. The incidence of CRC in this geographical area has been reported as being similar to that of other Western countries, with ~70 new cases per year per 100 000 inhabitants [17]. Statistical correlation between the presence of MSI and histopathological or clinical parameters was thus inferred. To evaluate the prevalence and spectrum of MLH1 and MSH2 germline mutations in this area, CRC families with at least three affected members underwent mutation analysis of both of these two MMR genes.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patient characteristics
Patients with a histologically proven diagnosis of CRC, whose primary tumour samples were available for molecular analysis, were included in the study. Thirty-six out of 346 enrolled patients were excluded from the series because of tissue DNA degradation. For the remaining 310 CRC patients, clinical stage was documented at the time of enrolment and followed up prospectively. All clinicopathological features, such as disease stage at diagnosis, type of surgical resection, therapy, relapse, disease-free survival and time of last control (for overall survival), were abstracted from the hospital record of each patient.

All patients for which family history was investigated were informed about the aims of the study; those on whom blood sampling was performed gave written informed consent.

Tissue samples and DNA extraction
Tissue samples from CRC patients were collected at the Departments and Institutes of Pathology participating in the study. Patients originated from both Sardinia and other geographical areas of southern Italy (in both cases, the incidence of CRC is similar to that found in other Western countries [17]). Family history of cancer was evaluated by questionnaire interviews with CRC patients after initial surgical treatment. Patients were classified as sporadic when no significant evidence of CRC tumours in first- and second-degree relatives was registered. Cases were classified as familial when at least three family members were affected by CRC. Presence of HNPCC syndrome was ascertained on the basis of the ‘Amsterdam criteria’, as follows: (i) three relatives with CRC, one of whom is a first-degree relative of the other two; (ii) CRC present in at least two generations; (iii) CRC diagnosed in at least one relative aged <50 years; and (iv) absence of familial adenomatous polyposis [9].

Genomic DNA was isolated from tumour samples and corresponding normal tissues, as described previously [18]. Tumour samples were estimated to contain at least 80% neoplastic cells by light microscopy.

Microsatellite analysis
Primers used to amplify simple sequence repeats at the five marker loci were as reported previously by a panel of experts [19]. The presence of MSI (referred to as MSI+) was defined by detection of at least two unstable (due to deletions or insertions) microsatellite markers in tumour DNA when compared with normal DNA. For each marker, PCR was carried out as described previously [18], using fluorescence-labelled primers. PCR products were analysed on a 377 Sequencer (Applied Biosystems, Foster City, CA).

Mutation analysis of the MLH1/MSH2 genes
Mutation analysis was performed on DNA from peripheral blood samples of familial CRC cases. Primers sequences for MLH1/MSH2 genes were as reported previously [20]. PCR products corresponding to all exons and intron–exon boundaries of these two MMR genes were analysed by denaturing high-performance liquid chromatography (DHPLC), as described previously [20]. All PCR products with abnormal DHPLC profiles were sequenced on the ABI3100 Automated Sequencer (Applied Biosystems) [20].

To assess whether MLH1/MSH2 germline variants detected by sequencing were mutations or polymorphisms, 53 unrelated normal individuals (corresponding to 106 chromosomes), originating from the same geographical area and with no family history for cancer, were used as controls.

Expression analysis of the MLH1/MSH2 genes
Expression of the MLH1/MSH2 proteins was detected by immunohistochemistry (IHC) on 2-µm sections of formalin-fixed, paraffin-embedded tissues, following previously described protocols [21]. Staining was evaluated semiquantitatively, using normal epithelial cells or the centres of lymphoid follicles as internal controls. IHC scoring was performed by at least two investigators.

Statistical analysis
Differences in dichotomous variables were evaluated using Pearson’s {chi}2 test or by Fisher’s exact test. The exact coefficient for sample proportion analysis was performed to determine all significant parameters (below the P = 0.05 level). All analyses were performed using the statistical package SPSS/7.5 for Windows.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Microsatellite analysis of archival tissues
Paired normal and tumour paraffin-embedded tissues from 310 patients with CRC were analysed for genetic instability by PCR-based microsatellite analysis. As shown in Table 1, 95 patients (collected during 1989–1997) presented at diagnosis aged <=50 years, and 215 patients (consecutively collected from 1998 to 2000) were unselected cases. Altogether, an approximately equal proportion of males and females was included into the study; the median age was 54 years (range 22–90 years). The majority of patients [245 (79%)] presented with an intermediate disease stage (Dukes’ B and C stages) and a localization of the primary tumour in the distal portions of the large bowels [including sigma with or without rectum (196/310, 63%)] (Table 1). Among 220 patients investigated for family cancer history, 27 (12%) patients with familial recurrence of CRCs (the presence of at least three affected members in the family, referred to as familial cases) were identified (Table 1).


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Table 1. Patient characteristics (cases are grouped according to selection criteria)
 
Comparison of amplified DNA from tumour and corresponding normal tissues allowed identification of the genetic instability as an electrophoretic mobility shift due to changes in microsatellite length. For each marker, typical examples of MSI are shown in Figure 1. Tumours were classified as MSI+ when at least two markers displayed evidence of mutant alleles in tumour DNA compared with corresponding normal tissue DNA.



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Figure 1. Representative examples of microsatellite instability at the five locus markers. Peaks visualized on the ABI377 sequencer and corresponding to normal (N) and tumour (T) DNA are labelled N and T, respectively. (A) and (B), examples of stable and unstable CRC cases, respectively.

 
Among the 310 CRC patients analysed, 75 (24%) exhibited a MSI+ phenotype; the remaining 235 (76%) (including 29 tumours showing only one unstable marker) were classified as exhibiting a microsatellite stable (MSI–) phenotype (Table 2). The frequency of MSI was detected at a similar rate among the different disease stages (varying from 28% at Dukes’ stage A to 22% at Dukes’ stage D), whereas a preponderance of instability was observed in tumours from proximal colon [19/50 (38%) compared with 38/196 (19%) from the distal tract; P = 0.03] or from patients with earlier CRC onset [33/86 (38%) cases aged <=45 years at diagnosis compared with 21/150 (14%) cases aged >=55 years; P <0.01)] (see Table 2).


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Table 2. Frequencies of microsatellite instability (MSI) and correlation between number of unstable (MSI+) CRC cases and several clinicopathological features
 
Considering the familial recurrence of CRC, 18 (67%) out of 27 familial cases (three or more affected members) presented with the MSI+ phenotype compared with 32 (17%) out of 193 patients with no significant evidence of tumours in first- and second-degree relatives (in this group, we also included the 26 patients with only one another additional CRC case in the family) (Table 2). Fifteen (7%) out of 220 patients investigated for family cancer history fulfilled the Amsterdam criteria, indicating the presence of HNPCC syndrome; among them, 11 (73%) presented with a MSI+ tumour.

Molecular analysis of MLH1/MSH2 genes in familial CRC cases
DNA from 44 CRC patients as probands among 36 families with at least three affected members (including 16 families from the aforementioned population screening and 20 newly selected families originating from the same geographical area) was analysed for germline mutations of the MLH1 and MSH2 genes. All coding regions and splice boundaries of these two genes were screened by DHPLC analysis; PCR products with abnormal denaturing profiles compared with normal controls were sequenced using an automated approach.

As shown in Table 3, three MLH1 and six MSH2 germline mutations were detected in our CRC families (one family proband presented with two MSH2 germline mutations: Val342Ile and IVS6+1G->T). With the exception of the MSH2-Gly322Asp, previously described as a low penetrance variant [22], none of the remaining mutations has been reported previously in the database (missense variants present unknown functional significance due to the lack of any information about the effects on protein function). Altogether, 14 (39%) families were found to be positive for germline mutations in these two MMR genes [four (11%) in MLH1 and 10 (28%) in MSH2]. In CRC families, positivity to the Amsterdam criteria, or the presence of at least four CRC cases or association with endometrial cancer (at least one family member with it) were all factors significantly increasing the prevalence of MLH1/MSH2 germline mutations (Table 4).


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Table 3. MLH1 and MSH2 gene variants identified by screening of the 36 colorectal cancer families
 

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Table 4. Correlation between family features and the presence of MLH1/MSH2 germline mutations among CRC families
 
Expression of the MLH1/MSH2 proteins has been evaluated by IHC analysis in MSI+ tumours from patients with familial recurrence of CRC (data not shown). Altogether, a negative nuclear immunostaining for the MLH1 and MSH2 proteins was observed in nine (43%) and two (10%) out of 21 MSI+ tumours tested, respectively. Conversely, all 20 additional MSI– cases used as negative controls presented with normal expression of both MLH1 and MSH2 proteins. The two CRC cases with negative MSH2 immunostaining were found to carry a germline mutation into the corresponding gene (MSH2-34insG and MSH2-1781insCT), whereas only one (11%) out of nine patients with loss of the MLH1 protein presented a MLH1 germline mutation (Leu749Pro). Interestingly, two first-degree relatives from the same family presenting the Leu749Pro mutation were both negative for MLH1 immunostaining, suggesting that this missense variant might be considered as a disease-causing mutation. In other words, none of the 19 familial CRCs negative for MSH2 germline mutations presented a somatic lack of MSH2 protein expression, whereas eight out of 20 (40%) family probands negative for MLH1 germline mutations presented loss of the MLH1 protein into the tumour.


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
In this study, we performed a microsatellite analysis on patients with CRC in order to investigate better the prevalence and the role of genetic instability in such a disease, as well as the existence of differences in MSI incidence among two groups of patients (selected for diagnosis age <=50 years, and collected consecutively, thus unselected for any clinico-pathological parameter).

Using a reference subset of five polymorphic markers (two mononucleotide repeats and three dinucleotide repeats), DNA from paraffin-embedded tissues of 310 CRC patients was investigated for genome-wide MSI. As recommended by a panel of experts [19], alterations in two or more locus markers were needed to identify tumours with high MSI (also referred to as MSI+ phenotype). Altogether, the presence of MSI was detected in 75 (24%) CRC cases.

The frequency of this genetic alteration in the CRC population in southern Italy is slightly higher than that reported by others for unselected CRCs (usually percentages range from <10% to 18% [7, 11, 12]). However, our findings are strongly consistent with the previously reported data regarding a higher MSI frequency in CRC patients with earlier onset age (<=45 years) or proximal tumour location [2, 4]. Indeed, there is increasing evidence that proximal and distal colon tumours comprise distinct diseases, with marked differences in the expression of tumour-associated proteins as well as in the level of genetic alterations [23]. Conversely, no significant correlation between MSI and stage of disease was observed, suggesting that such genetic alteration may occur before tumour dissemination, and may also play a role in the first stages of CRC development.

Microsatellite instability has been documented at various rate in many tumour types, suggesting that the presence of MSI might be a marker for a tendency for replication errors in human cancers [24, 25]. In our series, the probability of detecting MSI seems to be increased by the presence of familial recurrence of CRC (17% among patients with less than three affected members versus 67% among patients with three or more CRC cases in a family). Considering the Amsterdam criteria, 11/15 (73%) HNPCC cases tested showed MSI (this is consistent with previously reported rates, varying from ~70% to >90% [710]).

A large proportion of CRC families have been demonstrated to harbor both germline mutations of the MLH1/MSH2 genes (together accounting for 80–85% of all mutations in HNPCC families [26]) and altered somatic immunoexpression of these two MMR genes (loss of the MLH1 protein has been described in ~75% of the MSI+ tumours [8, 21]). In our series, percentages were markedly lower: 12/23 (52%) HNPCC families and 11/21 (52%) MSI+ tumours presented germline mutations and lack of expression of the MLH1/MSH2 genes, respectively. Even taking into consideration some limitations of the screening strategy (e.g. DHPLC analysis may not detect large deletions), the frequency of MLH1/MSH2 germline mutations in our CRC families was still lower than expected.

The detection of a much higher fraction of MSI+ familial CRCs presenting loss of MLH1 protein compared with those harboring a MLH1 mutation [nine (43%) versus one (5%) out of the 21 MSI+ tumours tested, respectively] strongly suggests that either genetic alterations not affecting the coding portion (i.e. within the regulatory regions) or epigenetic modifications may account for the MLH1 inactivation. In this regard, additional data from our group indicated that about one-fifth of MSI+ cases with tumour loss of the MLH1 protein and absence of MLH1 germline mutations presented MLH1 promoter hypermethylation at the somatic level using a methylation-specific PCR assay [27].

Our findings seem to support the criticisms of the general idea that inactivation of MLH1 in colorectal cancer is prevalently due to gene mutations in familial MSI+ cases and to epigenetic changes (mostly promoter hypermethylation) in sporadic MSI+ cases [25, 28]. Conversely, inactivation of the MSH2 gene seems to be strictly correlated with the presence of genetic mutations in its coding regions. Finally, the remaining familial cases presenting neither MLH1 nor MSH2 inactivations may have alterations in other known MMR genes (such as MSH6 or PMS2) or new genes, which await identification.

Considering the correlation with clinical parameters, preliminary data seem to indicate a favourable outcome in patients with MSI compared with those without MSI (not shown). Previous investigations have demonstrated a predictive value as prognostic factor for MSI in colorectal cancer, although in some cases a significant survival advantage has been observed only in MSI+ patients receiving chemotherapy [16, 29]. In this regard, an adequately powered, prospective trial should be performed to understand better the clinical behaviour of MSI+ tumours, as well as to define the real effect of MSI on prognosis.


    Acknowledgements
 
The authors are grateful to Dr Domenico Milano and the Centro clinico di Ricerca Malattie Genetiche (CRMG), as well as to all other members of this cooperative group: S. Astolfi, M.R. Bona, G.M. Bonomo, G. Casula, A. Criscuolo, G. Lucarelli, A. Margari, C. Natale, D. Piscitelli, G. Posillico and F. Tricarico. This work was funded by the Regione Autonoma della Sardegna and Asso-ciazione Italiana Ricerca sul Cancro.


    Footnotes
 
+ M. Colombino and A. Cossu contributed equally to this work. Back

§ Correspondence to: Dr G. Palmieri, Istituto Chimica Biomolecolare-Sezione di Sassari, C.N.R., Località Tramariglio-Alghero, 07040 Santa Maria La Palma, Sassari, Italy. Tel: +39-079-946706; Fax: +39-079-3961036; E-mail: gpalmieri{at}yahoo.com Back


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