Tumor microsatellite instability in early onset gastric cancer

Abstract

Gastric cancer (GC) remains a leading cause of cancer mortality worldwide. Genetic factors are implicated, including DNA mismatch repair (MMR) deficiency manifested as tumor microsatellite instability (MSI). However, a standardized panel of markers and a definition of low-versus-high level MSI in GC are lacking. We examined a population-based cohort of early onset (<or=50 yrs) gastric cancer. We identified 211 cases of early onset gastric cancer in Central-East Ontario from 1989 to 1993, with archival material available for 139 cases. Testing included a six-mononucleotide marker panel and a three-MMR immunohistochemical panel. Overall, 30% (41 of 139) of GC were MSI+, with allelic shifts at one to eight markers. An unexpected discordance between the BAT-25, BAT-26, and BAT-40 markers was observed in the MSI+ cases. Six cases showing multiple loci instability (>or=3 markers MSI+/MSI-high) demonstrated MMR protein deficiency. Three novel hMLH1 mutations (two germline frameshift and one somatic nonsense) were also found. The only significant clinicopathological associations were increased tumor size in MSI+ cases (P=0.04) and Lauren histotype (P=0.006) and tumor grade (P=0.007) in MSI-high cases. Tumor size, location, depth, nodal status, and Ming subtype were significant prognostic variables. Therefore, we propose a new definition of high-level MSI based on unifying characteristics of instability of more than or equal to three of six mononucleotide markers and loss of MMR protein expression.

Figure 1

Case 4 is an MSI+/hMLH1-immunonegative case with six of eight loci instability. A: Microsatellite instability in all six mononucleotide loci (red arrows, extra alleles in tumor genomic DNA) and normal-sized alleles in both normal and tumor using both dinucleotide markers D5S346 and D17S25. Note allelic shifts in DNA from tumor (T) samples compared with DNA from corresponding normal (N) tissue. B through E: Photomicrographs taken at 40× magnification. B: H&E showing mixed-type gastric cancer using the Carneiro system; C: hMLH1 immunostaining; D: hMSH2 immunostaining; E: hMSH6 immunostaining. Note the lack of tumor nuclear staining by hMLH1 and normal hMSH2 and hMSH6 immunostaining. Internal positive control immunostaining is demonstrated in nuclear staining of lymphocytes.

Figure 2

Case 100 is an MSI+/hMLH1-immunonegative case with eight of eight marker instability. A through D: Photomicrographs taken at 40× magnification. A: H&E shows glandular type gastric cancer using the Carneiro system; B: hMLH1 immunostaining; C: hMSH2 immunostaining; D: hMSH6 immunostaining. Note the lack of tumor nuclear staining by hMLH1 and intact hMSH2 and hMSH6 immunostaining. Internal positive control immunostaining is demonstrated in nuclear staining of lymphocytes. E: Methylation-specific PCR for hMLH1 in case 100. Note the band in tumor DNA and the absence of PCR products in normal DNA using specific primers for the methylated (M) hMLH1 in lanes 4 and 2, respectively. Lanes 1 and 3: As expected, both normal and tumor DNA show unmethylated (U) hMLH1 PCR products.

Figure 3

hMLH1 exon 16 d-HPLC analyses and manual sequencing results. A: Representative chromatogram showing absorption/wave patterns of cases: 4N, 87N, 87T, 100N, 101T, 192N, and controls including normal (genomic DNA from a noncancer patient), positive (genomic DNA from a colon cancer patient with known hMLH1 exon 16 mutation), and negative control (hMLH1 exon 16 sample without a DNA template). Note the variable absorption patterns seen in mutation-positive samples 87N, 87T, 101T, and positive control relative to the wave pattern in the normal control. B: hMLH1 exon 16 manual sequencing results for samples 87N/T (asterisks) and normal control. C: hMLH1 exon 16 manual sequencing results for sample 101T (asterisk) and normal control. B and C were sequenced in the forward direction. Nucleotide and amino acid sequences are shown along the margins. All mutations were also confirmed in the reverse direction (not shown). N, normal/genomic DNA sample; T, tumor DNA sample.