Article Figures & Data
Figures
- Fig. 1.
Immunohistochemical images (with MLH1 antibody) and corresponding MSI plots (with BAT25 marker) for patients 8 (A) and 24 (B) with germ-line mutation in MLH1. In patient 8, simple hyperplasia (SH; top left), complex hyperplasia with atypia (CAH; middle), and endometrial carcinoma (EC; top right) lacked MLH1 protein (red arrows), whereas stromal cells showed normal expression of MLH1 (black arrows) as indicated by brown staining. In the same patient, BAT25 mononucleotide repeat was stable in simple hyperplasia and unstable in complex hyperplasia with atypia (CH) and endometrial carcinoma. Short vertical lines, constitutional peaks of 121 bp; arrowheads, size shifts indicative of MSI. In patient 24, simple hyperplasia expressed and complex hyperplasia without atypia did not express MLH1 protein, and both specimens were microsatellite-stable.
- Fig. 2.
Comparison of MS-MLPA results for individual tumor suppressor loci in the Lynch syndrome series (groups I and II combined) and reference series (group III). Loci that showed promoter methylation in at least one sample are included. Results for normal endometrium, proliferatory and secretory combined (A), simple hyperplasia (B), complex hyperplasia without atypia (C), complex hyperplasia with atypia (D), and endometrial carcinoma, group I (E).
- Fig. 3.
Examples of MS-MLPA plots representing normal endometrium (top), complex hyperplasia without atypia (middle), and complex hyperplasia with atypia (bottom). Twenty-four different tumor suppressor loci were interrogated with specific probe pairs (two pairs of probes were included for RASSF1), combined with a set of control probes (C). The fragment size (increasing from left to right) identifies each locus. An amplification product (that is, visible peak) is obtained only if a given tumor suppressor locus is methylated (in contrast, control loci, which do not include HhaI sites, yield peaks invariably). Arrows, methylation of particular tumor suppressor gene loci in complex hyperplasia without atypia and complex hyperplasia with atypia, whereas no methylation is observed in normal endometrium.
Tables
- Table 1.
Number of available samples and average ages (in years) in which specimens were taken for groups I to III
Histology Group I (mutation positive, endometrial carcinoma positive) Group II (mutation positive, endometrial carcinoma negative) Group III (sporadic, mutation negative, endometrial carcinoma negative) Total No. Age No. Age No. Age No. Age Normal 13 42.0 37 45.4 21 48.9 71 45.4 Simple hyperplasia 3 46.7 8 48.4 11 47.1 22 47.4 Complex hyperplasia without atypia 3 46.7 7 50.4 14 61.0 24 52.7 Complex hyperplasia with atypia 10 48.7 15 47.9 16 61.0 41 52.5 Endometrial carcinoma 14 50.3 — — — — 14 50.3 Total 43 46.9 67 48.0 62 54.5 172 49.9 - Table 2.
Frequencies of MSI, decreased MMR protein expression, and tumor suppressor promoter methylation in groups I to III
Group I Group II Groups I and II (Lynch syndrome) Group III (reference) Normal Simple hyperplasia Complex hyperplasia without atypia Complex hyperplasia with atypia Endometrial carcinoma Normal Simple hyperplasia Complex hyperplasia without atypia Complex hyperplasia with atypia Normal Simple hyperplasia Complex hyperplasia without atypia Complex hyperplasia with atypia Normal Simple hyperplasia Complex hyperplasia without atypia Complex hyperplasia with atypia “Core” series Proportion with unstable marker (%) 0 10/10 (100) 2/3 (67) 0/2 (0) 5/8 (62) 4/11 (36) 24/26 (92) 3/3 (100) 1/1 (100) 3/5 (60) 34/36 (94) 5/6 (83) 1/3 (33) 8/13 (61) 21/21 (100) N/A N/A N/A 1 0/10 (0) 1/3 (33) 0/2 (0) 0/8 (0) 4/11 (36) 2/26 (8) 0/3 (0) 0/1 (0) 1/5 (20) 2/36 (6) 1/6 (17) 0/3 (0) 1/13 (7) 0/21 (0) N/A N/A N/A 2-3 0/10 (0) 0/3 (0) 2/2 (100) 3/8 (38) 3/11 (28) 0/26 (0) 0/3 (0) 0/1 (0) 1/5 (20) 0/36 (0) 0/6 (0) 2/3 (67) 4/13 (32) 0/21 (0) N/A N/A N/A Frequency of MSI defined as at least one unstable marker (%) 0/10 (0) 1/3 (33) 2/2 (100) 3/8 (38) 7/11 (64) 2/26 (8) 0/3 (0) 0/1 (0) 2/5 (40) 2/36 (6) 1/6 (17) 2/3 (67) 5/13 (38) 0/21 (0) N/A N/A N/A Proportion (%) with decreased MMR protein corresponding to germ-line mutation 0/8 (0) 2/3 (67) 2/2 (100) 7/7 (100) 11/11 (100) 2/21 (10) 0/2 (0) 1/1 (100) 4/5 (80) 2/29 (7) 2/5 (40) 3/3 (100) 11/12 (92) N/A N/A N/A N/A “Extended” series Frequency of MSI defined as at least one unstable marker (%) 0/13 (0) 1/3 (33) 3/3 (100) 6/10 (60) 8/12 (67) 2/37 (5) 3/8 (38) 4/7 (57) 11/15 (73) 2/50 (4) 4/11 (36) 7/10 (70) 17/25 (68) Average no. tumor suppressor genes with promoter methylation (out of 24)/sample 0.3 1.0 4.0 2.1 2.1 1.5 2.3 2.8 2.6 1.1 2.0 3.1 2.4 0.3 0.6 2.4 2.7 NOTE: See Materials and Methods for “core” and “extended” series. N/A, not available.
- Table 3.
Molecular alterations in endometrial samples taken at different time points from carriers of MMR gene germ-line mutations (groups I and II)
Patient ID Germ-line mutation Endometrial histology Time before last sampling Protein expression by immunohistochemistry No. unstable markers out of 3 Methylated genes out of 24 MLH1 MSH2 MSH6 Group I 1 MLH1 del ex16 Normal 13 y + + cytopl.+ 0 None Normal 11 y + + (+) 0 None Normal 6 y + + + 0 None Endometrial carcinoma 0 − + + 2 RASSF1, CDH13 2 MLH1 del ex16 Normal 3 y + + + 0 APC, CDH13 + Complex hyperplasia with atypia − (+) + Complex hyperplasia with atypia 1 mo − + + 0 None +Normal + + + Endometrial carcinoma 0 − + + 0 CDH13 3 MLH1 del ex16 Normal 3 y + + + 0 NA Simple hyperplasia 3 mo + + + 0 GSTP1 Endometrial carcinoma 0 − + + 0 None 4 MLH1 del ex16 Normal 3 y + + + 0 RASSF1 + Complex hyperplasia with atypia − + + Endometrial carcinoma 0 − + + 0 RASSF1, GSTP1, CDH13 5 MSH6 c3938-3941 dupGTTA Normal 12 y + (+) + 0 CHD13 Normal 2 y + + + 0 None Endometrial carcinoma 0 + − NA 1 CDKN2B, RASSF1, CDH13 7 MLH1 R659X Simple hyperplasia 0.5 mo − + + 1 NA Complex hyperplasia with atypia 0.5 mo +/− + NA 1 NA + Simple hyperplasia +/− + NA Normal 0 + + + 0 NA Complex hyperplasia with atypia 0 − + + 2 NA Endometrial carcinoma 0 − +/− + 3 PTEN, TP73, CDH13, GSTP1 8 MLH1 del ex16 Normal 4 y + + + 0 NA Normal 3 y + + (+) 0 NA Simple hyperplasia 2 y − + + 0 NA Complex hyperplasia with atypia 0 − + (+) 3 RASSF1, CDH13 Endometrial carcinoma 0 − + + 2 CDKN2A Group II 13 MLH1 R659P Endometritis chronica 8 y +/− + + 0 CDKN2B, CDH13, GSTP1 Complex hyperplasia with atypia 3 mo − + + 2 TIMP3, CD44, RASSF1, CDH13 Complex hyperplasia with atypia 0 − − − 1 TIMP3, CDKN2A, PTEN, RASSF1, CDH13, GSTP1 + Normal + + + 14 MLH1 R659P Simple hyperplasia 1 y + + + 0 TIMP3, GSTP1 Normal 7 mo + + + 0 CDKN2B, CDH13 Normal 0 NA NA NA 0 None 15 MLH1 454-1 g>a Normal 2 NA NA NA 0 CDH13 Simple hyperplasia 0 + + + 0 ATM, CDKN2B, CDKN1B 20 MLH1 del ex16 Normal 5 y NA NA NA 0 CDH13, GSTP1 Complex hyperplasia with atypia 0.5 mo + + + 0 APC, RASSF1, CDH13, GSTP1 Normal 0 − + + 0 None + Complex hyperplasia with atypia − + + 24 MLH1 del ex16 Normal 6 y + + + 0 RARB, PTEN, ESR1, RASSF1 Simple hyperplasia 0 + + + 0 RASSF1, CDH13 Complex hyperplasia without atypia 0 − + + 0 RASSF1, CDH13 109 MLH1 Normal 2 mo 0 ND Normal 0 0 None Complex hyperplasia without atypia 2 mo 1 FHIT, CDH13, GSTP1 Complex hyperplasia with atypia 0 0 APC, CDH13 Abbreviations: +, positive expression; −, negative expression; (+), weak positive expression; +/−, positive and negative expression.
Volume 15, Issue 18
