The value of microsatellite instability in the detection of HNPCC families and of sporadic colorectal cancers with special biological features: An investigation on a series of 100 consecutive cases

Annals of Oncology, Aug 1998

Background: Microsatellite instability (MI) is a biological characteristic of most tumors involved in hereditary non-polyposis colorectal cancer (HNPCC). This disease appears to be caused by germline mutations in mismatch repair (MMR) genes, which are responsible for repairing single base-pair mismatches. At least five human genes participate in MMR. MI also occurs in 10%-15% of sporadic colorectal cancers. Because MI detection has been suggested as an alternative diagnostic tool for identification of HNPCC families, in this study we analyzed the MI pattern in 100 consecutive colorectal carcinomas in order to correlate them with the clinicopathologic features and family histories of the patients. Patients and methods: A series of 100 colorectal cancers was evaluated for MI with 10 polymerase chain reaction primer sets. Instability results were compared with family history and other clinical and biological characteristics. Results: MI was detected in 36 of 100 cancers, 27 of which showed low instability and nine a high instability. The low- and high-instability cases showed similar clinicopathological characteristics, and significantly positive associations were observed between MI and mucinous histological type (P = 0.0001) and MI and peritumoral lymphocytic infiltration (P = 0.01). A single HNPCC family was identified in the high-grade MI group, while two families belonged to the Mi-negative group. Conclusions: Our data suggest that MI screening is probably not an efficient strategy for identifying HNPCC cases. MI does, however, appear capable of defining a category of colorectal cancers with favourable prognostic features and should be investigated at least in all cases of mucinous adenocarcino-mas.

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The value of microsatellite instability in the detection of HNPCC families and of sporadic colorectal cancers with special biological features: An investigation on a series of 100 consecutive cases

Annals of Oncology D. Furlan 1 M. G. Tibiletti 0 1 M. Taborelli 1 L. Albarello 1 M. Cornaggia 0 1 C. Capella 1 0 Department ofPathology, Ospedale di Circolo , Varese , Italy 1 department of Clinical and Biological Sciences, University ofPavia at Varese The value of microsatellite instability in the detection of HNPCC families and of sporadic colorectal cancers with special biological features: An investigation on a series of 100 consecutive cases Summary Results: MI was detected in 36 of 100 cancers, 27 of which showed low instability and nine a high instability. The low- and Background: Microsatellite instability (MI) is a biological char- high-instability cases showed similar clinicopathological characteristic of most tumors involved in hereditary non-polyposis acteristics, and significantly positive associations were observed colorectal cancer (HNPCC). This disease appears to be caused between MI and mucinous histological type (P = 0.0001) and by germline mutations in mismatch repair (MMR) genes, MI and peritumoral lymphocytic infiltration (P = 0.01). A which are responsible for repairing single base-pair mis- single HNPCC family was identified in the high-grade MI matches. At least five human genes participate in MMR. MI group, while two families belonged to the Mi-negative group. also occurs in 10%-15% of sporadic colorectal cancers. Be- Conclusions: Our data suggest that MI screening is probcause MI detection has been suggested as an alternative diag- ably not an efficient strategy for identifying HNPCC cases. MI nostic tool for identification of HNPCC families, in this study does, however, appear capable of defining a category of colowe analyzed the MI pattern in 100 consecutive colorectal carci- rectal cancers with favourable prognostic features and should nomas in order to correlate them with the clinicopathologic be investigated at least in all cases of mucinous adenocarcinofeatures and family histories of the patients. mas. Patients and methods: A series of 100 colorectal cancers was evaluated for MI with 10 polymerase chain reaction primer sets. Instability results were compared with family history and Key words: colorectal cancers, histological type, HNPCC, other clinical and biological characteristics. RER phenotype Introduction Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant disease characterized by the early (i.e., in patients below the age of 45-50) occurrence of colorectal cancers. The disease appears to be due to germline mutations in the mismatch repair (MMR) genes which are responsible for repairing single basepair mismatches. The generally accepted mechanism for HNPCC tumorigenesis according to Knudson's model [1] involves inheritance of a single recessive MMR mutation with subsequent inactivation of its wild-type allele counterpart in a somatic cell, which is likely to occur in highly proliferating tissues [2]. Inactivation of MMR genes leads to genomic instability, characterized by either the expansion or contraction of short repeat DNA sequences [3, 4]. This form of instability, known as microsatellite instability (MI) or as replication error (RER), is thought to cause a rapid accumulation of somatic mutations in different oncogenes and tumor suppressor genes which play crucial roles in tumor initiation and progression [5-7]. At least five human genes participate in MMR. Germline mutations in these genes have been identified in HNPCC families, namely, hMSH2, hMLHl, hPMSl, hPMS2 and MSH6/GTBP [8-12]. More than 90% of mutations in HNPCC families are found in hMSH2 and hMLHl, while it appears that the hPMSl, hMSH6 and hPMS2 genes are only rarely mutated [2]. It has been demonstrated in verified HNPCC patients that MI is frequently associated with colorectal carcinomas [3, 13]. A population-based screening for MMR gene mutations to identify subjects at risk for HNPCC is time-consuming and extremely expensive because of the involvement of multiple genes and the heterogeneity of mutations in such patients [14, 15]. HNPCC is currently identified by pedigree reconstruction, using the so-called 'Amsterdam criteria' [16]. Although Jass et al. [17] recently suggested the use of RERs as an alternative diagnostic tool for this disease, several studies have demonstrated that the RER-positive phenotype is also detected in a considerable fraction (about 15%) of sporadic colorectal cancers and that by itself it is not an indicator of the presence of familiarity [13,18]. In this study we analyzed RER phenotypes at multiple loci in 100 consecutive colorectal carcinomas with the aim of: 1) correlating them with the clinicopathologic features and family histories of the patients; 2) comparing their value in identifying HNPCC cases to that of pedigree reconstruction. Patients and methods Patients and samples We investigated a consecutive series of 100 patients (98 unrelated and two belonging to the same family) who underwent surgery for colorectal cancer at the Varese Hospital from November 1995 to November 1996. In each case the patient's age and tumor site and size were recorded. The tumors located proximal to the splenic flexure were classified as right-sided and the tumors distal to the splenicflexureas left-sided In each case fresh tissue fragments from primary tumor, contiguous to the samples taken for conventional pathological investigation, and samples of non-neoplastic mucosa were stored at -80 C for molecular investigation. The presence of non-malignant 'contaminating' cells in the tumor specimens was evaluated on contiguous histological sections: in all samples the cut-off value of 50% for the proportion of malignant cells was used, but about 3/4 of the cells in most of the samples were malignant. Histological investigations The tissues were fixed in formalin and embedded in paraffin. Hematoxylin- and eosin-stained sections were used to categorize tumors according to the WHO Classification [19] The adenocarcinomas were graded into well- (Gl), moderately- (G2) and poorly (G3)-differentiated. Tumors were classified as mucinous when the amount of mucin was more than 50% of the examined areas. In all cases, the following histological parameters were evaluated, using a two-step scoring system (absent or minimal and present): pentumoral lymphocytic infiltration, necrosis, stromal fibrosis, vascular and neural invasion. The mitotic index was expressed as the number of mitoses per 10 high-power fields (HPF). The neoplasm extension was evaluated according to the TNM Classification [20]. Genomic DNA was isolated from frozen tissues using QIAamp tissue kits (Qiagen Germany). In 22 cases three different samples from the same tumor were used to evaluate tumor heterogeneity. DNA from paired normal and tumor tissues were PCR-amplified at 10 markers: D2S123 (2ql6), D2S119 (2pl6), D3S1611 (3p22-p24), APC, D7S481 (7pl5-pter), D7S517 (7pl5-pter), D13S175 (13qll), D17S787[17],TP53, DCC. The PCR mixture (50 vl) included 100-200 ng of genomic DNA, lOmM Tris, 50mM KC1, 1-2 mM MgCl2, 200 uM dNTPs, and 50 pmol of each primer, 2U Taq Polymerase (Perkin-Elmer, Italy). Samples were denatured at 94 C for five minutes followed by 30 cycles of denaturation (94 C, one minute), annealing (55 C, one minute), extension (72 C, one minute), and a final five-minute extension at 72 C in a Perkin-Elmer Gene Amp Thermal Cycler 480. PCR products were electrophoresed on 6% denaturing polyacrylamide sequencing gels for two hours at 1600 V, 40 mA, 50 C. Polyacrylamide gels,fixedon glass plates with methacryloxypropyltrimethoxysane, were silver-stained using DNA Silver Staining System (Promega, Italy). Silver-stained gels were reproduced on EDFfilms(Kodak, USA). Pedigree analysis Genetic counselling with psychological support was carried out at the first clinical follow-up of each patient. Using clinical reports or death certificates for validation of cancer cases, family medical histories of at least three generations were established. For HNPCC identification the so-called 'Amsterdam criteria' were adopted [16]. The families with one or two missing 'Amsterdam criteria' were considered suspect for HNPCC [21]. Statistical analysis Associations between variables were tested by Pearson's x2"test o r Fisher's exact test when appropriate. Results MI was detected in 36 of 100 colorectal carcinomas: 27 of them were unstable at 1 or 2 loci, while nine were unstable at three or more loci. Both microsatellite expansions (Figure 1) and contractions were identified independently of the number of altered loci. The loci more frequently altered were APC (11 cases), D2S123 (10 cases), and D3S1611, D7S481 and DCC (nine cases each). Tumors showing MI were divided into two groups according to the number of involved loci. Twenty-seven carcinomas showed low instability (MI at 1 or 2 loci) and nine high instability (MI at more than 2 loci). The unstable loci were different in the two groups, as shown in Figure 2. The D13S175 locus was exclusively involved in high-instability cases, while D3S1611 and DCC were more frequently involved in low-instability cancers. In the 22 cases in which multiple samples of the same tumor were analyzed we detected complete concordance among altered MI loci in all fragments of the same case. The relationships between MI, clinico-pathological data and family history are listed in Tables 1 and 2. The low- and high- instability cases showed similar clinicopathological characteristics. Among the various clinicopathologic parameters examined (Table 1) only two, the mucinous histological type (P - 0.0001) and the presence of peritumoral lymphocytic infiltration (P - 0.01), were significantly associated with both low- and highgrade MI. Clinicopathologic characteristics MI absent (64 cases) Low MIa (27 cases) High MIb (nine cases) Total (100 cases) P-value 64.4 14.7 23/41 19 Grade 1 2 Absent Present Present Present Absent It Family history HNPCC Suspected HNPCC Sporadic Lost to follow-up Number of relatives with malignant tumors of any site Number of relatives without malignant tumors Number of relatives with colorectal cancers Number of relatives without colorectal cancers Number of relatives with malignant tumors other than colorectal0 Number of relatives without malignant tumors other than colorectalc MI absent (64 cases, Low MIa (27 cases) 63 families) High MIb (nine cases) Total (100 cases, P-value 99 families) Cl inicopathologic characteristics MI absent (64 cases) MI present (36 cases) Total (100 cases) Histological type Mucinous Non-mucinous Lymph node metastases Absent Present Lymphocytic infiltration Minimal/absent Moderate/abundant Mitotic index =50/10HPF >50/HPF Number of relatives with colorectal cancers Number of relatives without colorectal cancers With respect to pedigree analysis, the average family size in both RER-positive and -negative cases was 14 members (range nine to 16). A single HNPCC family which met the Amsterdam criteria qualified for placement in the high-grade MI group, and two HNPCC families belonged to the Mi-negative group; MI thus showed a 33.3% rate of sensitivity for detecting HNPCC. In the only HNPCC family showing RER+ phenotype cancers no hMSH2 or hMLHl mutations were identified by the use of SSCP analysis (Dr. G. Guanti, University of Bari, Italy, personal communication). Suspected (one Amsterdam criteria missing) HNPCC families were observed in all three groups: 13 in the MI negative-, one in the low- and six in the high-MI groups, respectively. In the group of cases with low and high MI the pedigree analysis demonstrated a significantly (P - 0.012) higher number (60 of 238 and 31 of 86, respectively) of relatives affected by malignant tumors, mainly consisting of colorectal cancers, in comparison to the group of MInegative cases (145 of 754). Owing to the strict similarities in clinico-pathologic features of carcinomas with low and high MI, all RER+ cases were considered as a single group whose pathologic and clinical parameters were then compared with those of the group of RER-negative cases (Table 3). The RER-positive phenotype proved to be positively correlated with mucinous histological type (P = 0.00007), peritumoral lymphocytic infiltration (P = 0.008), absence of lymph node metastasis, low mitotic index (P 0.01) and a high number of relatives affected by colorectal cancer (P = 0.03). Discussion In this study the MI analysis, performed with 10 polymerase chain reaction primer sets on a consecutive series of colorectal cancers, was compared with family history, and based on genetic counselling of the patients and with other pathological and biological characteristics. Genomic instability was detected in 36% of the series of 100 consecutive primary colorectal cancers examined. We observed MI at multiple loci (more than 2 loci altered) in 9% and MI at 1-2 loci in 27% of investigated cancers. Genomic instability in colorectal cancers has been reported in percentages ranging from 15% to 28% [3, 18, 22, 23] and in all studies the frequency of MI was higher in familial than in sporadic cases. The high percentage of MI instability detected in our study over those in other studies [3,18, 22 and 23] may be explained by the high number and the location of loci investigated to determine MI, and this technical approach may increase the percentage of Mi-positive cases. We found a different involvement of loci between the group with high (MI at more than 2 loci) and the one with low (MI at 1 or 2 loci) instability. In the first group microsatellites mapping (D2S123, D2S119, D3S1611, D7S481 and D7S517) near the HNPCC genes and near or within (D13S175, APC, TP53 and D17S787) the genes involved in cell-cycle or cell adhesion control were more often involved. By contrast, in the low-instability group, the more frequently unstable loci were D3S1611 and DCC, suggesting the presence of a different type of instability for this group of cases. For this reason, the instability degree in this group of cancers could be underestimated. However, the locus or allelic heterogeneity may cause a variable degree of instability without detectable clinico-pathological differences. In our study the RER phenotype was correlated with different clinico-pathological characteristics, including age and sex of the patients, and location, size, histological type, grade, stage, lymph node metastasis, peritumoral lymphocytic infiltration, tumor necrosis, stromal fibrosis, vascular invasion, neural invasion and mitotic index of the tumors. No significant differences between RER-positive and RER-negative cases were observed for the majority of variables considered. A highly significant correlation, however, was found between the mucinous type and RER-positive phenotype of colorectal cancers, indicating that genomic instability is involved in the tumorigenesis of mucinous adenocarcinomas. In addition to the mucinous aspect, the RER+ cases in our study were characterized by intense lymphoid peritumoral infiltrates, a relatively low mitotic index and a low incidence of lymph node metastasis. Our findings are in agreement with data previously reported by other authors [24, 25] and confirm that tumors with replication errors comprise a distinct biological entity within the colorectal cancers. In particular, the mucinous aspect, the low proliferation rate, as determined by analysis of S phase fraction and AgNOR content [24] and the marked peritumoral inflammatory infiltrates, probably representing host response [25], indicate that RER-positive tumors are less aggressive than RER-negative tumors. This is also confirmed by studies suggesting that patients with RER-positive sporadic cancers have a better prognosis than those with RER-negative cancers [22, 23, 26]. With respect to family history, the RER+ phenotype is significantly associated with familial clustering of colorectal cancers but alone is not always suggestive of the typical HNPCC. In fact, we identified one HNPCC family in the RER+ group, and two HNPCC families in the RER- group. Also the 'suspected' HNPCCs (one Amsterdam criterion missing) were more frequent in RER- than in RER+ cancer groups. Also noteworthy was the fact that the mean ages of patients with RER+ and RER- cancers were not significantly different. These results are unexpected and clearly indicate that the HNPCC cancers are not caused by MMR-defective genes only, but that other genes may also play a role. On the other hand, these results correlate well with other reports from the literature indicating the presence of HNPCC families meeting Amsterdam criteria and lacking MMR gene mutations [28, 29]. In conclusion, our data demonstrate that RER screening is probably not an efficient strategy for identifying HNPCC cases, despite various relevant considerations [17]. RER detection is less efficient and more expensive than pedigree analysis. Although MI in human colon cancers is not a useful clinical indicator of HNPCC, it appears to indicate a category of colorectal cancers with favourable prognostic features and should be investigated at least in all cases of mucinous colorectal adenocarcinoma. Acknowledgement This research was supported by grants from the Italian MURST and the AIRC (progetto speciale tumori del colon ereditari). References 1. Knudson AJ Jr. Heredity and cancer in man . Progr Med Genet 1973 ; 9 : 113 - 58 . 2. Eshleman JR , Markowitz SD . 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Received 5 March 1998; accepted 27 May 1998 .


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D. Furlan, M. G. Tibiletti, M. Taborelli, L. Albarello, M. Cornaggia, C. Capella. The value of microsatellite instability in the detection of HNPCC families and of sporadic colorectal cancers with special biological features: An investigation on a series of 100 consecutive cases, Annals of Oncology, 1998, 901-906,