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Late onset and high incidence of colon cancer of the mutator phenotype with hypermethylated hMLH1 gene in women
598
CORRESPONDENCE
GASTROENTEROLOGY Vol. 119, No. 2
Late Onset and High Incidence of Colon Cancer of the Mutator Phenotype With Hypermethylated hMLH1 Gene in Women Dear Sir: The presence of hundreds of thousands of clonal somatic mutations in simple repeated sequences characterizes cancer of the microsatellite mutator phenotype (MMP).1 Mutations impairing the DNA mismatch repair (MMR) lead to accumulation of mutations in cancer genes and to development of cancer of the MMP pathway.2 The MMP is a landmark of most hereditary nonpolyposis colorectal cancers as well as other cancers of different tissue origins without documented family history (sporadic cancer). Epigenetic changes may also lead to inactivation of the MMR system. The hMLH1 gene promoter is frequently methylated in MMP tumors, associated with gene silencing.3,4 We now report that, in the MMP pathway, colorectal tumors with methylated hMLH1 are distinct from the rest by a delayed onset and association with females. From a panel of 511 unselected colorectal carcinomas, 62 manifested the MMP (12%). Methylation of the hMLH1 gene promoter was detected in 26 of 56 (46.4%) MMP-positive (MMP⫹) tumors (Table 1). MMP⫹ tumors with methylated hMLH1 gene promoter occurred in patients approximately 18 years older than those without (average age of tumor onset, 70.8 years vs. 52.9 years, respectively; P ⬍ 0.0001, Student t test). These MMP⫹ tumors with hMLH1 methylation were also about twice as frequent in women than in men (P ⬍ 0.0002, Fisher exact test). This finding may explain previous observations of a higher incidence of MMP⫹ tumors in older women,5,6 because it establishes a link between the female gender and methylation of the hMLH1 mutator gene. This link can be explained by nongenetic or genetic gender-specific factors (for instance, a chromosome X–linked gene). Because there is no obvious reason to assume that stable epigenetic alterations need to be caused by epigenetic, rather than genetic events, we favor the second possibility. The late onset of colon cancer of the MMP in women could thus be explained by the additional genetic and epigenetic steps (not immediately affecting cell growth or survival) that appear to be involved in this particular pathway for tumorigenesis.
SERGEI R. MALKHOSYAN, Ph.D. HIROYUKI YAMAMOTO, M.D., Ph.D. ZHE PIAO, M.D. MANUEL PERUCHO, Ph.D. The Burnham Institute La Jolla, California 1. Ionov Y, Peinado MA, Malkhosyan S, et al. Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 1993;363:558 –561. 2. Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell 1996;87:159 –170. 3. Kane MF, Loda M, Gaida GM, et al. Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair– defective human tumor cell lines. Cancer Res 1997;57:808 – 811. 4. Toyota M, Ohe-Toyota M, Ahuja N, et al. Distinct genetic profiles in colorectal tumors with or without the CpG island methylator phenotype. Proc Natl Acad Sci U S A 1999;96:8681– 8686. 5. Breivik J, Lothe RA, Meling GI, et al. Different genetic pathways to proximal and distal colorectal cancer influenced by sex-related factors. Int J Cancer 1997;74:664 – 669. 6. Thibodeau S, French AJ, Cunningham JM, et al. Microsatellite instability in colorectal cancer: different mutator phenotypes and the principal involvement of hMLH1. Cancer Res 1998;58:1713–1718. 7. Malkhosyan S, Rampino N, Yamamoto H, et al. Frameshift mutator mutations. Nature 1996;382:499 –500. 8. Yamamoto H, Perez-Piteira J, Yoshida T, et al. Gastric cancers of the microsatellite mutator phenotype display characteristic genetic and clinical features. Gastroenterology 1999;166:1348 – 1357. 9. Schwartz S, Yamamoto H, Navarro M, et al. Frameshift mutations at mononucleotide repeats in caspase-5 and other target genes in endometrial and gastrointestinal cancer of the microsatellite mutator phenotype. Cancer Res 1999;59:2995–3002. 10. Herman JG, Graff JR, Myohanen S, et al. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 1996;93:9821–9826. doi:10.1053/gast.2000.16154
Table 1. Gender and Age of Patients With Colon Cancer Average age (yr)
MMP⫺ MMP⫹b (hMLH1 methylation⫺) (hMLH1 methylation⫹)c a
No. of casesa
Total
Men
Women
No. of men
No. of women
511 62 30 26
63.1 ⫾ 13.1 61.8 ⫾ 15.2 52.9 ⫾ 14.8 70.8 ⫾ 10.4
62.5 ⫾ 13.4 58.4 ⫾ 14.9 53.3 ⫾ 14.2 71.5 ⫾ 10.9
64.2 ⫾ 12.6 65.0 ⫾ 14.7 52.0 ⫾ 16.0 70.6 ⫾ 10.2
322 29 18 7
189 30 9 19
Unselected colon carcinomas without information on family history.1,7,8 The MMP was determined using 2 mononucleotide repeats and 1 dinucleotide repeat, as described previously.8,9 Tumors with dinucleotide but without mononucleotide repeat alterations were classified as MMP⫺.8,9 c Methylation of the hMLH1 gene promoter was detected in the MMP⫹ tumors as described previously.8,9 First, methylation was detected by digestion with HpaII restriction endonuclease of the 4 HpaII sites located between ⫺570 and ⫺337 nucleotides from the initiation codon.3 Second, methylation-specific polymerase chain reaction (MSP)10 was used to detect methylation of the 2 CpG sites at positions ⫺250 and ⫺214. Both methods yielded similar results. MMP⫹ tumors with methylated hMLH1 gene promoter were more frequent in women (19) than men (7). This difference was statistically significant compared with the rest of the tumors (19 and 198 women vs. 7 and 340 men; P ⫽ 0.0002, Fisher exact test) or with the MMP⫹ tumors without hMLH1 methylation (19 and 9 women vs. 7 and 18 men; P ⫽ 0.0040, Fisher exact test). MMP⫹ tumors without methylated hMLH1 were more frequent in men than women (18 vs. 9, respectively), but this was not significant compared with our control MMP⫺ tumors (9 and 189 women vs. 18 and 322 men; P ⫽ 0.85). The high male/female ratio of our tumor sample is similar to the incidence of colorectal cancer in the white U.S. population (58.7 men vs. 39.9 women per 100,000 people). Subjects with MMP⫹ tumors without hMLH1 methylation had a significantly earlier onset (52.9 years) than MMP⫺ cancer control patients (63.1 years; P ⬍ 0.0001, Student t test). The early cancer onset of these MMP⫹ tumors without methylated hMLH1 gene promoter could be explained as some of these tumors being hereditary because they carried germline mutations in one of the MMR genes, hMLH1, hMSH2, or hMSH6 (data not shown). b
CORRESPONDENCE
GASTROENTEROLOGY Vol. 119, No. 2
Late Onset and High Incidence of Colon Cancer of the Mutator Phenotype With Hypermethylated hMLH1 Gene in Women Dear Sir: The presence of hundreds of thousands of clonal somatic mutations in simple repeated sequences characterizes cancer of the microsatellite mutator phenotype (MMP).1 Mutations impairing the DNA mismatch repair (MMR) lead to accumulation of mutations in cancer genes and to development of cancer of the MMP pathway.2 The MMP is a landmark of most hereditary nonpolyposis colorectal cancers as well as other cancers of different tissue origins without documented family history (sporadic cancer). Epigenetic changes may also lead to inactivation of the MMR system. The hMLH1 gene promoter is frequently methylated in MMP tumors, associated with gene silencing.3,4 We now report that, in the MMP pathway, colorectal tumors with methylated hMLH1 are distinct from the rest by a delayed onset and association with females. From a panel of 511 unselected colorectal carcinomas, 62 manifested the MMP (12%). Methylation of the hMLH1 gene promoter was detected in 26 of 56 (46.4%) MMP-positive (MMP⫹) tumors (Table 1). MMP⫹ tumors with methylated hMLH1 gene promoter occurred in patients approximately 18 years older than those without (average age of tumor onset, 70.8 years vs. 52.9 years, respectively; P ⬍ 0.0001, Student t test). These MMP⫹ tumors with hMLH1 methylation were also about twice as frequent in women than in men (P ⬍ 0.0002, Fisher exact test). This finding may explain previous observations of a higher incidence of MMP⫹ tumors in older women,5,6 because it establishes a link between the female gender and methylation of the hMLH1 mutator gene. This link can be explained by nongenetic or genetic gender-specific factors (for instance, a chromosome X–linked gene). Because there is no obvious reason to assume that stable epigenetic alterations need to be caused by epigenetic, rather than genetic events, we favor the second possibility. The late onset of colon cancer of the MMP in women could thus be explained by the additional genetic and epigenetic steps (not immediately affecting cell growth or survival) that appear to be involved in this particular pathway for tumorigenesis.
SERGEI R. MALKHOSYAN, Ph.D. HIROYUKI YAMAMOTO, M.D., Ph.D. ZHE PIAO, M.D. MANUEL PERUCHO, Ph.D. The Burnham Institute La Jolla, California 1. Ionov Y, Peinado MA, Malkhosyan S, et al. Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 1993;363:558 –561. 2. Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell 1996;87:159 –170. 3. Kane MF, Loda M, Gaida GM, et al. Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair– defective human tumor cell lines. Cancer Res 1997;57:808 – 811. 4. Toyota M, Ohe-Toyota M, Ahuja N, et al. Distinct genetic profiles in colorectal tumors with or without the CpG island methylator phenotype. Proc Natl Acad Sci U S A 1999;96:8681– 8686. 5. Breivik J, Lothe RA, Meling GI, et al. Different genetic pathways to proximal and distal colorectal cancer influenced by sex-related factors. Int J Cancer 1997;74:664 – 669. 6. Thibodeau S, French AJ, Cunningham JM, et al. Microsatellite instability in colorectal cancer: different mutator phenotypes and the principal involvement of hMLH1. Cancer Res 1998;58:1713–1718. 7. Malkhosyan S, Rampino N, Yamamoto H, et al. Frameshift mutator mutations. Nature 1996;382:499 –500. 8. Yamamoto H, Perez-Piteira J, Yoshida T, et al. Gastric cancers of the microsatellite mutator phenotype display characteristic genetic and clinical features. Gastroenterology 1999;166:1348 – 1357. 9. Schwartz S, Yamamoto H, Navarro M, et al. Frameshift mutations at mononucleotide repeats in caspase-5 and other target genes in endometrial and gastrointestinal cancer of the microsatellite mutator phenotype. Cancer Res 1999;59:2995–3002. 10. Herman JG, Graff JR, Myohanen S, et al. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 1996;93:9821–9826. doi:10.1053/gast.2000.16154
Table 1. Gender and Age of Patients With Colon Cancer Average age (yr)
MMP⫺ MMP⫹b (hMLH1 methylation⫺) (hMLH1 methylation⫹)c a
No. of casesa
Total
Men
Women
No. of men
No. of women
511 62 30 26
63.1 ⫾ 13.1 61.8 ⫾ 15.2 52.9 ⫾ 14.8 70.8 ⫾ 10.4
62.5 ⫾ 13.4 58.4 ⫾ 14.9 53.3 ⫾ 14.2 71.5 ⫾ 10.9
64.2 ⫾ 12.6 65.0 ⫾ 14.7 52.0 ⫾ 16.0 70.6 ⫾ 10.2
322 29 18 7
189 30 9 19
Unselected colon carcinomas without information on family history.1,7,8 The MMP was determined using 2 mononucleotide repeats and 1 dinucleotide repeat, as described previously.8,9 Tumors with dinucleotide but without mononucleotide repeat alterations were classified as MMP⫺.8,9 c Methylation of the hMLH1 gene promoter was detected in the MMP⫹ tumors as described previously.8,9 First, methylation was detected by digestion with HpaII restriction endonuclease of the 4 HpaII sites located between ⫺570 and ⫺337 nucleotides from the initiation codon.3 Second, methylation-specific polymerase chain reaction (MSP)10 was used to detect methylation of the 2 CpG sites at positions ⫺250 and ⫺214. Both methods yielded similar results. MMP⫹ tumors with methylated hMLH1 gene promoter were more frequent in women (19) than men (7). This difference was statistically significant compared with the rest of the tumors (19 and 198 women vs. 7 and 340 men; P ⫽ 0.0002, Fisher exact test) or with the MMP⫹ tumors without hMLH1 methylation (19 and 9 women vs. 7 and 18 men; P ⫽ 0.0040, Fisher exact test). MMP⫹ tumors without methylated hMLH1 were more frequent in men than women (18 vs. 9, respectively), but this was not significant compared with our control MMP⫺ tumors (9 and 189 women vs. 18 and 322 men; P ⫽ 0.85). The high male/female ratio of our tumor sample is similar to the incidence of colorectal cancer in the white U.S. population (58.7 men vs. 39.9 women per 100,000 people). Subjects with MMP⫹ tumors without hMLH1 methylation had a significantly earlier onset (52.9 years) than MMP⫺ cancer control patients (63.1 years; P ⬍ 0.0001, Student t test). The early cancer onset of these MMP⫹ tumors without methylated hMLH1 gene promoter could be explained as some of these tumors being hereditary because they carried germline mutations in one of the MMR genes, hMLH1, hMSH2, or hMSH6 (data not shown). b