Clinical features and mismatch repair genes analyses of Chinese suspected hereditary non-polyposis colorectal cancer: a cost-effective screening strategy proposal

Abstract

China has the largest numbers of hereditary non-polyposis colorectal cancer (HNPCC) patients based on its population of 1.4 billion. However, the clinical data and mismatch repair (MMR) gene analyses have been limited. Here we performed microsatellite instability (MSI) and immunohistochemistry (IHC) analyses on a series of patients with a high-risk for HNPCC: 61 patients with family histories fulfilling Amsterdam criteria II (ACII-HNPCC) or suspected HNPCC criteria (S-HNPCC), and 106 early onset colorectal cancer (CRC) patients. Sixty late-onset CRC patients were used as control. Methylation of the hMLH1 promoter was analyzed on tumors lacking hMLH1 expression. MMR germ-line mutations were screened on patients with tumors classified as MSI-H/L or negative for IHC. We identified 27 germ-line MMR variants in the 167 patients with a high-risk for HNPCC while only one germ-line mutation in hMSH6 was found in the late-onset CRC group. Of those, 23 were pathogenic mutations. The high incidence of gastric and hepatobiliary cancers coupled with the increasing number of small families in China reduces the sensitivity (43.5%, 30.4%) and positive predictive value (PPV) (45.5%, 17.9%) of the ACII- or S-HNPCC criteria. MSI or IHC testing are highly sensitive in detecting pathogenic mutations (sensitivities = 91.3% and 95.6%, respectively), but the PPVs are quite low (25.6% and 27.8%, respectively). Considering that all 12 tumors with pathogenic mutations in hMLH1 also showed promoter unmethylation, the sensitivity of IHC in conjunction with hMLH1 promoter methylation analysis is not reduced, but the PPV was increased from 27.8% to 61.1%, and the total cost was greatly reduced.

Figure 1

Results of microsatellite instability (MSI) status in different groups. MSI testing was performed on the genomic DNA from the paired tumor cells and peripheral‐blood lymphocytes or histologically normal cells. Five markers were analyzed using denaturing high‐performance liquid chromatography (DHPLC). The degree of instability in each tumor was scored as stable (MSS), low (MSI‐L), and high (MSI‐H) when 0, 1, and 2–5 markers were identified as unstable. ACII‐HNPCC, Amsterdam criteria II for hereditary non‐polyposis colorectal cancer; CRC, colorectal cancer; E‐CRC, early onset colorectal cancer; L‐CRC, late colorectal cancer; S‐HNPCC, suspected hereditary non‐polypsis colorectal cancer.

Figure 2

Representatives of the identified pathogenic germ‐line mutations. (1) Characterization of the nonsense mutation in the hMLH1 exon 18 of patient C30.1, c.2101C>T (predicted effect p.Q701X). Partial sequencing patterns of the hMLH1 exon 18 from the genomic DNA of index patient C30.1 and a healthy control are shown. The index patient is heterozygous for C/T in c. 2101, while the control is homozygous for C. (2) Characterization of the splicing donor site mutation in the hMSH2 exon 13 of patient E.C 29, c.2210 + 1G>A (predicted effect r.2006–2210del). Partial sequencing patterns of the boundary of exon 13 and intron 13 of index patient E.C 29 and a healthy control are shown. (3). Characterization of the small deletion in the hMLH1 exon 16 of patient C.11.2, c.1879–1882delTCTT (predicted effect p.Ser627_Leu628>TrpfsX9). Partial sequencing patterns of the hMLH1 exon 16 in genomic DNA of index patient C11.2 and a healthy control are shown. (4) Characterization of the large genomic deletion of hMSH2 exon 12 in patient E.C 62. (a) Schematic representation the germ‐line genomic deletion encompassing exon 12 of hMSH2. (b) The deletion mutation was confirmed by long‐range polymerase chain reaction (PCR) with genomic DNA from patient E.C 62, his daughter (Dau.), mother (Mo.), and a healthy control. Primer a (5′CACAACCTCCATTTCCTG3′) was located in intron 10 and and primer b (5′‐GGTGGTTCATGCCTGTAA‐3′) was complementary to intron 13. For the normal control, the PCR product is about 6.9 kb; for patient E.C 62, shortened PCR products were generated (1.9 kb). For the patient's daughter and mother, 6.9 kb PCR products were generated, indicting that they have no the pathogenic mutations. (c) The deletion was further confirmed by sequencing the PCR products. Sequencing the 1.9 kb and 6.9 kb PCR product indicted that the breakpoint region was located within intron 11 and intron 12 (deleted nucleotides 68176–73142, 4966 bp). The numbers refer to the nucleotide positions of Homo sapiens chromosome 2 and containing the whole sequence of hMSH2 (accession number NC_000002, GI:89161199).

Figure 3

Proposed strategy for hereditary non‐polypsis colorectal cancer (HNPCC) screening; IHC, immunohistochemical analysis; S‐HNPCC, suspected hereditary non‐polypsis colorectal cancer.