Incidence of gastric carcinoma
Until recently, gastric carcinoma was the second most common cancer worldwide, but now, with an estimated 934,000 new cases per year in 2002 (8.6% of new cases), it is in fourth place behind cancers of lung, breast and colon and rectum. It is the second most common cause of cancer death (700,000 deaths annually). Almost two-thirds of cases occur in developing countries and 42% in China alone. The geographic distribution of GC is characterized by wide international variations; high-risk areas include East Asia (China, Japan and Korea), Eastern Europe, and parts of Central and South America. Incidence rates are low (< 10 per 100,000 in men) in Southern Asia, North and East Africa, North America, Australia, New Zealand and Africa [17, 55].
In Europe, GC represents the fifth most common cancer, after colon and rectum, breast, lung and prostate, with an incidence of 149,000 per year and mortality rate of 116,000. The highest European risk area for GC is the Eastern zone with an incidence of 70,000 per year (Belarus area). Portugal and Italy represent one of the most high-risk European areas for stomach cancer, in particular, respectively, with incidence in 2008 of 41,100 and in Italy with incidence of 33,400 per year . In Italy, Tuscany and central regions present a GC mortality rate of 4,2/10,000 inhabitants (2002 ISTAT-The Italian National Institute of Statistics).
E-cadherin and diffuse gastric cancer
The CDH1 gene maps to chromosome 16q22.1 and consists of 16 exons that encode a 120-kDa protein called E-cadherin, which is a member of the transmembrane glycoproteins family. E-cadherin is expressed on epithelial tissues and is responsible for calcium-dependent cell-cell adhesion. Functionally, it is critical for establishing and maintaining polarized and differentiated epithelia through intercellular adhesion complexes. Human E-cadherin is considered an invasion suppressor, and under-expression of E-cadherin is correlated with the infiltrative and metastatic ability of the tumor .
In 1998 in New Zealand, a country that represents a low GC risk-area, Guilford and Colleagues identified firstly in Maori kindred the first CDH1 germline truncating mutation in cases with family history for diffuse gastric carcinoma . Subsequently, several international countries reported other CDH1 germline mutations in HDGC or in sporadic EODGC with different ethnicities [4, 54].
The definition of the HDGC syndrome was established in 1999 by the IGCLC that also delineated the criteria for the CDH1 genetic screening, as follows: 1) two or more documented cases of diffuse gastric cancer in 1st/2nd degree relatives, with at least one diagnosed before the age of 50, or 2) three or more cases of documented diffuse gastric cancer in 1st/2nd degree relatives, independently of age of onset . Subsequently, the last consensus conference of Cambridge in 2010 , modified the criteria as: a) two GC cases in family, one confirmed DGC < 50; b) three confirmed DGC cases in 1st or 2nd degree relatives independent of age; c) age < 40; Corso and Colleagues recently proposed the cut-off age of onset at ≤ 35 in sporadic EODGC ; d) personal or family history of DGC and lobular breast cancer, one < 50.
In this study, we reported a total of 122 CDH1 germline mutations, respectively 94 (77%) in group A (low-risk area) and 16 (13.1%) in group B (middle/high-risk area) (12-9.9% mutations were without ethnicity information).
Revising these 122 CDH1 mutations, we observed some main findings: 1) the overall mutation rate is lower in group B rather than in group A (13.1% vs. 77%; p < 0.001); 2) group A shows a higher frequency of non-missense mutations; moreover, CDH1 non-missense mutations are extremely lower in group B (5.7%), in which missense variants are predominant (32.4%) (Figure 2).
Considering the first point, we stated that CDH1 germline mutations in GC middle/high-risk area are rather rarely identified respecting to low-risk area; as reported, in middle/high-risk area the probability to perform a CDH1 genetic screening with negative result is high enough [10–12], in which maybe operates environmental factors.
Summary of evidence: CDH1 mutations and geographic variability
The abovementioned clinical criteria represents an important approach in selecting probands for the CDH1 genetic screening. Currently, different modifications have been appointed at these criteria [2, 4, 32], evaluating clinical and molecular results along the international consensus conferences; so far, the risk area vs. incidence of gastric carcinoma has never been discussed in these scientific meetings. We stated that "GC risk area and incidence" can represent an important emerging factor that relates strongly to the prognosis and other important behaviors of GC patients; ethnicity of GC patients coming from low- or high-risk areas could be considered in these criteria in order to identify cases with high or low-risk mutation carriers in hereditary syndromes. Information about the ancestry, can improve the accuracy of proband selection for the CDH1 genetic screening; in particular, we can consider GC patients as high or low-risk mutation carriers respectively in low or high-risk GC areas whenever the abovementioned criteria is fulfilled. This data verifies that in high-risk areas, clinical criteria should be applied strictly to cases with a strong family history for GC in particular when it has identified very young members.
It is important to stress that geographical variability represents a novel risk factor for gastric carcinoma, clinico-pathological features and some molecular results can confirm it. These acknowledgments, like the GC risk-area, provide important information and can propose different approaches in clinical practice for GC prevention and treatment.
Molecular features and pathogenicity of E-cadherin missense mutations
The second consideration of this study is that GC high-risk area shows a higher frequency of CDH1 missense mutations; identification of a CDH1 missense alteration also implicated its functional assessment, because in vitro and in silico analysis with study of control population demonstrates that this mutation can be a novel polymorphic variant without pathogenic impact. We also revised the frequencies of CDH1 germline missense mutations with a pathogenic role that was assessed in 21 CDH1 missense alterations [4, 56]; 17 of these showed a deleterious impact and respectively 5 (29.4%) were coming from middle/high risk-areas and 12 (70.6%) from low risk-areas. The pathogenicity of the remaining 13 mutations is unknown. As reported, the probability of finding a novel pathogenic CDH1 missense mutation (including also non-missense mutations) is higher in GC low-risk areas. Very recently, we revised the role of the CDH1 germline mutation in pathogenesis of sporadic EODGC; a total of 19 mutations have been reported to date in this GC group but only six (2.3%) of them did in fact represent variants with a proven potentially deleterious effect. This predicted pathogenicity was based on the type of mutation (frameshift) or on the results obtained from in vitro functional analysis. Five of the six germline pathogenic CDH1 mutations were detected in EOGC patients from low or moderate incidence GC areas, whereas only one arose in a patient from Portugal, a high incidence GC area. Although CDH1 germline mutations have been searched for in EOGC patients from other areas where high incidence rates of GC are verified, namely in countries like China, Korea, Japan, and even Italy, no deleterious CDH1 germline variants have been identified .
These results demonstrated that functional assessment of CDH1 germline missense mutations is mandatory, in particular in GC high-risk cases that show a higher frequency of unpathogenic missense mutations.
Limitations of the current study
Certainly, this study presents some limitations that should be discussed, also for further consideration. First, the CDH1 germline mutation frequency could be affected by research activity, as publication bias or access to medical care, considering that some CDH1 variants could be unpublished in MEDLINE; this factor probably is due to the lack of a CDH1 recorded mutation database. However, MEDLINE is the unique search engine in which we can access easily and freely; unfortunately we do not have access in confidential and in unpublished data. However, this bias should be very small, because we carefully collected all CDH1 mutations recorded in MEDLINE; we also discussed this point in the "result" section. The second point is that we do not know precisely in what proportion of cases CDH1 mutations are identified in low vs. middle/high risk areas, and if the mutation frequency is related to the number of screened subjects; moreover, the accuracy of this study could be affected by the human migrations. The overall CDH1 mutation number identified to date is rather low and we are not able to also include this factor (as ratio between number of mutations vs. number of screened kindred, migration of population, etc.) risking to dissipate the statistical value, in the excess of data stratification. In the specific, we were not able to assess the quantitative genetic analysis due to the lack of information about the impact of environmental factor in DGC development and about migratory behavior. Further, if applicable and also considering that the clinical criteria are rather limited, in a larger family collection, this data could be considered. The third limitation is that we cannot analyse intestinal GC and other risk factors, such as environmental agents (i.e. specific diet habits); however, the aim of this study is only to assess the frequency of CDH1 germline mutation in DGC, in which environmental factors are probably less important than in intestinal hystotype. Moreover, the CDH1 germline or somatic mutations have not reported in intestinal gastric carcinoma, and for this reason, it was excluded from this systematic review.