2. Análisis e interpretación de resultados
2.8 Conclusiones y Recomendaciones
Gastric cancer specimens and/or biopsies were evaluated from 22 patients (15 families) that presented with intestinal-type gastric cancer before the age of 40 (n=5) or fulfilled the following criteria2: (1) at least two first/second degree relatives affected by intestinal gastric cancer, one diagnosed before the age of 50 (n=5); or (2) three or more relatives with intestinal gastric cancer at any age (n=12). In ten specimens MSI was examined and MSI due to hypermethylation of the MLH1 promoter was detected in two patients. A history of H. pylori gastritis in this category was not increased compared to the other patients with a familial history (only one patient was diagnosed with H. pylori).
DISCUSSION
In the current study we present for the first time a comparison of the histological characteristics of patients with gastric cancer and a germline mutation in CDH1, Lynch syndrome or early-onset or familial intestinal or diffuse-type gastric cancer without a known germline mutation. Noteworthy, the majority of CDH1 mutation related gastric cancer cases did not present with predominant signet- ring cell cancer. Precursor lesions in the surrounding ‘normal’ gastric tissue, which were not reported in original pathology reports, could have led to the suspicion or diagnosis of a germline CDH1 mutation in most gastrectomies. Therefore in daily practice expertise is needed and attention should be paid to
5 precursor lesions in the evaluation of all diffuse type gastrectomy specimens.
Importantly, most CDH1-related gastric cancer cases show retained but aberrant immunohistochemical expression of E-cadherin protein. The majority of gastric cancer cases in Lynch syndrome can be defined by MMR-deficiency. Besides the presence of precursor lesions, there were no histological features of gastric cancer, that could distinguish between CDH1, Lynch syndrome or early-onset or familial gastric cancer without a known mutation.
Caldas et al. described 13 germline CDH1 mutation-related gastric cancer cases, 11 were of the diffuse-type and two were of the mixed-type (with both diffuse
and intestinal/glandular components).2 In the present study we also describe
(micro-)glandular components in five patients (22%) with a germline CDH1 mutation. It is important that (micro-)glandular growth does not exclude a diagnosis of CDH1-related gastric cancer.
We examined the ability of four different E-cadherin antibodies (recognizing different epitopes) to predict that gastric cancer arises in the context of a germline CDH1 mutation. Biallelic CDH1 gene inactivation does not invariably lead to complete loss of immunohistochemical E-cadherin protein expression which confirms previous reports.7-11 Although the international HDGC guideline does not recommend E-cadherin immunohistochemistry as a prescreening method to select patients for CDH1 mutation analysis,6 in daily practice there are laboratories that erroneously use E-cadherin immunohistochemistry on gastric cancer samples to predict and prescreen tissue for clinical genetic CDH1 testing. In literature various scoring criteria for the assessment of E-cadherin expression in DGC are used and often membranous expression was not assessed on irregularity. Distinguishing between normal and abnormal expression is very difficult, since even in normal gastric glands E-cadherin often is only expressed at the basolateral membrane and reduced or absent at the luminal membrane. The question remains how normal E-cadherin expression is defined in gastric cancer, since even though E-cadherin is expressed it might be a mutant protein. This stresses the difficulty of scoring E-cadherin expression in diffuse-type gastric cancer. In the current study, E-cadherin staining was defined as absent (0% expression), as aberrant (any degree of abnormal cytoplasmic, irregular or diminished membranous expression) or ‘normal’ (>75% crisp membranous expression with the same intensity as surrounding normal glands). Using this scoring system, we did not encounter ‘normal’ expression, however, we found >75% irregular membranous expression in seven cases (three without and four
with a germline CDH1 mutation). In daily practice, it is likely that this would be interpreted as ‘positive/ retained/ normal’ E-cadherin staining (see also supplementary files 3 and 4). There was little variability between the different E-cadherin antibodies. Total absence or focal (<10%) staining of E-cadherin was seen in only six of 20 cases (30%) with a CDH1 mutation and in 3/13 cases without a CDH1 mutation (23%). Overall, the E-cadherin antibody clone that was used did not influence staining results, although clone N-20 showed a lot of background staining and was difficult to interpret. E-cadherin staining was highly variable between individuals with the same CDH1 germline mutation and in the same individual between different gastric cancer lesions suggesting that the second inactivating hit of the wild-type allele is decisive for E-cadherin expression. Oliveira et al. found in 80% of HDGC families somatic CDH1 epigenetic and genetic alterations. Next to a somatic mutation, the second hit in HDGC lesions can also be promoter hypermethylation of CpG islands (50%), loss
of heterozygosity (LOH) (12.5%) or a combination of both (18.8%).10 Somatic
mutations affecting exon 8 and 9 are considered to be a hotspot in CDH1.12-14 So
depending on the E-cadherin antibody clone used, truncated protein might be missed by immunohistochemistry since its epitope may have been altered. In
2002, Becker et al.34 showed a DGC case with a somatic mutation resulting in an
in-frame exon 8 deletion, which similar to our results in one case did not show any immunoreactivity by using anti-E-cadherin clone HECD-1 while other anti-E- cadherin clones did show retained immunoreactivity. In our case somatic mutation analysis was not possible due to insufficient amount of DNA of the tumor. We have also examined the characteristics of gastric cancer in Lynch syndrome. The frequency of gastric cancer in patients with Lynch syndrome differs among
populations, gender and the gene that is mutated, varying from 0.2-30%.15,
16 Most reports in Western countries show a cumulative GC risk by age 70 in
the range of 2-6%.4, 17-22 There seems to be no clustering of gastric cancer in
the majority of families with Lynch syndrome. Three studies showed that the majority (around 75%) of GC associated with Lynch syndrome is of the intestinal- type and are characterized by microsatellite instability,4, 23, 24 however in our study a significant proportion (40%) had diffuse type histology. Four cases did not show MSI or loss of immunohistochemical expression of mismatch repair proteins. Therefore it is very well possible that the origin of GC is not related to MMR deficiency. For family risk and counseling it is important to confirm that Lynch syndrome patients with GC have MMR deficiency in their tumor.
5 Germline mutations in CDH1 were initially reported in 30-50% of HDGC families,
however recent publications of larger cohorts found germline CDH1 mutations in a minority of cases (<25%).3, 25, 26 The molecular basis for familial aggregation or early-onset of gastric cancer remains largely unknown. In patients without a CDH1 germline mutation, we did not find specific histopathological characteristics in GCs of 100 diffuse-type and 22 intestinal-type cases. In conclusion, important indications for gastric cancer related to a germline CDH1 mutation are the presence of precursor lesions. Only the minority is predominantly of the signet- ring cell type. Importantly, E-cadherin staining does not exclude the presence of a germline mutation in the CDH1 gene.
Box 1. Clinicopathologic characteristics of hereditary gastric cancer
CDH1 mutation -Multiple mucosal SRC carcinomas (pT1a); -Foci of in-situ or pagetoid spreading of SRCs;
-Poorly cohesive carcinoma with (a small subset of typical) SRCs;
-May contain areas of (micro-)glandular growth, mucinous lakes and cords of tumor cells;
-E-cadherin expression or absence does not discriminate between hereditary and sporadic diffuse type GC Lynch syndrome -Intestinal or diffuse histology
-History of a Lynch syndrome associated malignancy -Mismatch repair deficiency
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