Volume 117, Issue 14 p. 3112-3117
Original Article
Free Access

Germline mutations of the E-cadherin gene in families with inherited invasive lobular breast carcinoma but no diffuse gastric cancer

Ze Ming Xie MD, PhD

Ze Ming Xie MD, PhD

Department of Oncogenetics, Centre Jean Perrin, Clermont-Ferrand, France

Department of Breast Oncology, Sun Yat-Sen University Cancer Center, Guangzhou, People's Republic of China

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Lai Sheng Li MSc

Lai Sheng Li MSc

Department of Oncogenetics, Centre Jean Perrin, Clermont-Ferrand, France

Department of Breast Oncology, Sun Yat-Sen University Cancer Center, Guangzhou, People's Republic of China

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Claire Laquet BS

Claire Laquet BS

Department of Oncogenetics, Centre Jean Perrin, Clermont-Ferrand, France

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Frédérique Penault-Llorca MD, PhD

Frédérique Penault-Llorca MD, PhD

Department of Pathology, Centre Jean Perrin, Clermont-Ferrand cedex, France

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Nancy Uhrhammer PhD

Nancy Uhrhammer PhD

Department of Oncogenetics, Centre Jean Perrin, Clermont-Ferrand, France

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Xiao Ming Xie MD, PhD

Xiao Ming Xie MD, PhD

Department of Breast Oncology, Sun Yat-Sen University Cancer Center, Guangzhou, People's Republic of China

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Yves Jean Bignon MD, PhD

Corresponding Author

Yves Jean Bignon MD, PhD

Department of Oncogenetics, Centre Jean Perrin, Clermont-Ferrand, France

Fax: (011) 33-473-27-80-50

Department of Oncogenetics, Centre Jean Perrin, 58 rue Montalembert, Clermont-Ferrand 63011, France===Search for more papers by this author
First published: 26 January 2011
Citations: 57

Abstract

BACKGROUND:

Present data are highly suggestive but do not unequivocally prove the cosegregation of germ-line CDH1 mutations with inherited invasive lobular breast cancer (ILBC).

METHODS:

Two Caucasian families with 6 pathologically confirmed ILBC cases but no diffuse gastric cancer (DGC) were identified in our oncogenetics consultations. Screening for mutations of CDH1, BRCA1, and BRCA2 genes was performed on blood samples. When available, loss of heterozygosity (LOH) and immunohistochemistry (IHC) analyses were performed on tumor samples.

RESULTS:

No BRCA1 or BRCA2 mutation was found. Deleterious CDH1 germ-line mutations c.283C>T and c.1582del were found in all the 4 living women with ILBC in family 1 and family 2, respectively. The mutation c.283C>T was also present in a healthy 71-year-old male and 2 obligate carriers in family 1. No DGC was observed in the 2 families. Loss of the wild-type CDH1 allele in 1 of the breast tumors was confirmed by LOH and IHC studies, in accordance with the “2-hit” model of tumor suppressor genes.

CONCLUSIONS:

Germline CDH1 mutation can be cosegregated with ILBC in the absence of DGC. Present data do not support recommendation of prophylactic gastrectomy in CDH1 germline mutation carriers with ILBC. Cancer 2011. © 2011 American Cancer Society.

Germline CDH1 mutations have been demonstrated to underlie disease in hereditary diffuse gastric cancer (HDGC) families. The first description was made in 1998 by Pary Guilford on 3 Maori families with autosomal dominant HDGC.1 Carriers of deleterious mutations have an estimated cumulative risk of gastric cancer of 40% to 67% for men and 63% to 83 % for women.2, 3 As in diffuse gastric cancer (DGC), the absence of E-cadherin protein expression is characteristic of the majority of invasive lobular breast cancer (ILBC),4 and somatic truncating mutations of CDH1 are frequently detected in this subtype of breast cancer,5 all suggesting the etiological involvement of this gene in ILBC oncogenesis according to Knudson's “2 hit” model.

Invasive lobular breast carcinoma as a component of the tumor spectrum associated with germline CDH1 mutations was originally suggested by a gastric cancer family with a female family member who developed both lobular breast cancer and diffuse type gastric carcinoma.6 Additional studies have documented family members with breast cancer in HDGC families with CDH1 mutations or deletions.2, 3, 7-12. In all of these studies, only a minority of the breast cancer cases were pathologically confirmed to be ILBC, and not all the ILBC cases in each study were confirmed to carry the familial CDH1 mutations (Table 1). This may create an ascertainment bias, because the association between CDH1 and ILBC was not the subject of these studies. The data are supportive but still insufficient to prove a relation between lobular breast cancer and germline CDH1 mutations.11 Further demonstrations of cosegregation between these mutations with ILBC are still needed. To elucidate the role of constitutional CDH1 mutations in familial ILBC, we analyzed a large family (family 1) with 5 ILBC cases, including 2 bilateral cases, in 2 successive generations. An additional family (family 2) with CDH1 germ-line mutation and a familial member affected by ILBC was also identified in our oncogenetics consultation. It is noteworthy that no DGC was observed in either family, suggesting that gastric cancer is not an obligatory hallmark of families with CDH1 mutations.

Table 1. Summary of LBC in HDGC Families With CDH1 Mutations
Mutations Breast Cancer Cases (Confirmed LBC) CDH1 Mutations Confirmed in LBC Patients Reference No.
3G>C(start site) 1 (1) 1 9
49-2A>C 1 (0) 0 12
70G>T (E24X) 10 (2) 2 2, 7, 10
187 C >T (R63X) 4 (2) 0 2, 9
283 C.>T(Q95X) 2 (0) 0 8
372del 1 (1) 1 6
382del 2 (0) 0 11
IVS5(+1)G.A 1 (0) 0 11
832G>A 1 (1) 0 2
1003C>T (R335X) 1 (1) 1 7
1137G>A 2 (0) 0 3, 12
1212del 4 (0) 0 11
1391_1392del 1 (0) 0 12
1397_1398del 1 (0) 0 3
1588insC 1 (0) 0 10
1682-1683insA 1 (0) 0 3
IVS11(+5)GA 5 (1) 0 11
1792C>T (R598X) 3 (1) 1 7, 9
1901C>T (A634V) 1 (0) 0 3
2064-2065del 2 (0) 0 3
2195G>A (R732Q) 13 (4) 1 3, 7, 11
2276del 2 (1) 0 9
2310del 1 (0) 0 11
2343A>T(E781D) 1 (0) 0 3
2398del 16 (4) 0 3, 11
  • LBC indicates invasive lobular breast cancer; HDGC, hereditary diffuse gastric cancer.

MATERIALS AND METHODS

Both family 1 and family 2 are of French Caucasian origin. The living affected members and their offspring and siblings were recruited for genetic consultation, and informed consents for genetic testing were obtained from participating members. They were informed that all findings would be held in strict medical confidence and that their identities would be protected if the results of the study were published in the future. Individual cancer histories were confirmed by review of the original pathology reports and medical records. DNA was extracted from peripheral blood samples using the Talent kit (Euromedex, Souffelweyersheim, France) as directed. By using standard methods, complete screening of the CDH1, BRCA1, and BRCA2 genes was performed on cases III-1, III-6 of family 1 (Fig. 1) and the proband of family 2 (Fig. 2) by amplification and sequencing of all coding exons and intron-exon boundaries. Primer sequences and conditions are available on request. Mutations were confirmed by targeted testing of 2 independent DNA samples from each subject and participating relatives.

Details are in the caption following the image

Pedigree of family 1 is illustrated. bi-LBC indicates bilateral lobular breast cancer; C, colon cancer; E, esophageal cancer; P, pancreatic cancer; U, uterine cancer. affected family member with c.283C>T mutation; unaffected carriers; noncarriers.

Details are in the caption following the image

The pedigree of family 2 is shown. In the family with c.1582del mutation, the proband was diagnosed as ILBC at age 48 years (in 1995); her mother died of breast cancer (subtype unknown, age unknown).

Paraffin-preserved tumor samples were available for cases III-6 and IV-11 of family 1. Expression of E-cadherin was evaluated using standard immunohistochemical techniques. Frozen tumor samples were available for patient IV-11. DNA was extracted from microdissected tissues. Informative microsatellite loci flanking the CDH1 locus (D16S265, D16S3057) were amplified using standard reaction conditions and 1 fluorescently tagged primer per marker. Polymerase chain reaction (PCR) products were separated on an ABI 3130 sequencer (Life Technologies Applied Biosystems, Carlsbad, Calif), and allele peaks were quantified. Allelic loss was confirmed when allele ratios differed by greater than 40% in blood versus tumor DNA.13 The phase of microsatellite alleles was determined by segregation analysis.

RESULTS

Pedigrees

Figures 1 and 2 show the pedigrees of the 2 families that we studied. To protect the anonymity of the family members shown in the pedigrees, unaffected individuals are presented by sex-neutral diamond symbols. Information about affected individuals is fully displayed.

In family 1, the female proband (III-6) was diagnosed with ILBC at the age of 60 years (in 1997). Her daughter (IV-11) was diagnosed with bilateral ILBC at age 42 years (in 2007). And, 1 of her sisters (III-9) died of ILBC diagnosed at age 53 years. Two of her cousins were also diagnosed with ILBC, at ages 63 years (III-1, bilateral, diagnosed in 1995) and 50 years (III-2). In family 2, the proband was diagnosed with ILBC at age 48 years (in 1995). Her mother died of breast cancer (subtype unknown), and her aunt died more than 50 years ago, of primary hepatocellular carcinoma at age 52, without hints of a gastric pathology. There was no DGC observed in either family. Other specific cancers and the age of cancer onset for the affected individuals are summarized in Figures 1 and 2.

Mutation Screening

No BRCA1 or BRCA2 mutation was found in either family. In family 1, a deleterious mutation of CDH1, c.283C>T, was found in the 3 living women with ILBC (III-1, III-6, and IV-11) and in a healthy 71-year-old male (III-7; Fig. 1). The mutation was also present in 2 obligate carriers (II-1 and II-2), 1 of whom developed pancreatic cancer at age 56 years, and the other died in an accident at age 75 years. Another deleterious mutation, c.1582del, was found in the proband of family 2.

Immunohistochemistry and Loss of Heterozygosity

Tumor samples from patients III-6 and IV-11 of family 1 were tested for the retention of wild-type CDH1. Immunohistochemistry showed that tumor cells in all the samples were negative for E-cadherin expression, whereas adjacent normal breast tissue was positive (Fig. 3), which is a common finding in lobular breast cancer.4 Analysis of loss of heterozygosity (LOH) on DNA microdissected from frozen tumor tissues from IV-11 showed clear deviation of peak ratios found in the corresponding peripheral blood DNA, suggesting that the wild-type CDH1 allele was lost in both tumor samples from this patient (Fig. 4).

Details are in the caption following the image

Pathology results are (A and C) hematoxylin and eosin staining of lobular breast tumor sections from patients III-6 and IV-11, respectively, and (B and D) E-cadherin staining of the corresponding samples where the epithelium of the normal ducts are E-cadherin positive whereas the tumor cells are E-cadherin negative.

Details are in the caption following the image

Loss of heterozygosity at the CDH1 locus in breast tumor tissue from IV-11 is shown. The arrow indicates the lost allele at D16S3057, which is known to represent the wild-type gene (additional markers showed similar results).

DISCUSSION

The CDH1 gene at 16q22.1 codes for E-cadherin, a calcium-dependent cell adhesion molecule crucial for the establishment and maintenance of epithelial polarity and structural integrity.14 The specific c.283C>T mutation observed in family 1 has already been described in HDGC families.3, 8 In a HDGC family case report with this mutation, 2 breast cancer cases were present in the pedigree, but no information was given on the histological type of breast cancer nor whether the patients carried the mutation.8 This same mutation was also observed in a HDGC family reported by Kaurah and colleagues, but was not associated with breast cancer.3

In the present study, there were 5 pathologically confirmed ILBC cases in family 1: the c.283C>T mutation was found in all 3 living women with ILBC from 2 successive generations, including 2 bilateral ILBC cases. No DGC was observed in this family. This family is probably not an isolated exception. We observed a second family with a germ-line CDH1 mutation (c.1582del) without any DGC cases in our oncogenetics consultation. The proband was affected by a pathologically confirmed ILBC at age 48 years. After 15 years of follow-up, there was no evidence of DGC in this woman or her relatives, although the family size was smaller. Masciari and colleagues also identified 1 woman with a CDH1 mutation (c.517insA) among 23 women with invasive lobular or mixed ductal and lobular breast cancer who had at least 1 close relative with breast cancer or had themselves been diagnosed before age 45 years, and, again, no DGC cases were observed in this family.15 All of these findings strongly suggest that when there is a cosegregation between germline CDH1 mutations with ILBC in the absence of DGC, gastric cancer is not an obligatory hallmark of families with CDH1 mutations.

To investigate the risk of DGC after the diagnosis of LBC, we reviewed the relevant literatures. Keywords CDH1, germ-line mutation, lobular breast cancer, and diffuse gastric cancer were used for online searches of Pubmed (US National Center for Biotechnology Information [NCBI] at the National Library of Medicine [NLM], Bethesda, Md) and Google Scholar (Google, Mountain View, Calif). Cases with pathologically confirmed LBC and informative follow-up data reported in the literature were identified for comparison. Five patients with both ILBC and DGC have been reported, all from HDGC families, with ILBC occurring either synchronously or before DGC.3, 6, 7, 9 The lapse from the diagnosis of ILBC to the diagnosis of DGC was 4-9 years in nonsynchronous cases,3, 6, 7 whereas 3 of the 4 living ILBC cases in the present families had ILBC diagnosed more than 12 years ago (12-15 years, Table 2). Two ILBC cases identified in the literature had been followed for 10 and 26 years, respectively.7 No evidence of DGC was reported, although they are from HDGC families and both were confirmed to be CDH1 germ-line mutation carriers. No follow-up data for other ILBC cases with CDH1 mutations was available in the literature, making it difficult to systematically evaluate the risk of DGC in such carriers. Whether the present criteria for recommending prophylactic gastrectomy in HDGC families should be applied to hereditary ILBC families with CDH1 mutations remains controversial. Furthermore, in the present families, 3 of the 5 living carriers are older than 70 years, so the risk of morbidity and mortality from prophylactic gastrectomy should also be considered. Prophylactic gastrectomy was indeed discussed with the CDH1-positive patients of our families, although none of them chose this option, considering their age and adverse effects of surgery.

Table 2. Age at Diagnosis of LBC and DGC
Source Age at LBC, y Age at DGC, y Lapse/ Follow-Up, y
Ref 9 73 73 0
Ref 3 68 72 4
Ref 6 49 58 9
Ref 7 58 66 8
40 49 9
39 na 26
58 na 10
Present family 1 63 na 14
60 na 12
42 na 1
50 na 10a
53 na 4a
Family 2 48 na 15
  • LBC indicates invasive lobular breast cancer; DGC, diffuse gastric cancer; y indicates years; Ref, reference number in reference section; na, not applicable (the patients have not developed DGC).
  • a These 2 patients died of invasive lobular breast cancer (ILBC) at ages 60 years and 57 years, respectively.

There is currently no reliable screening method available for detecting occult DGC in CDH1 mutation carriers. Computed tomography (CT) and positron emission tomography (PET) has been shown to have poor sensitivity for screening.16 Preoperative endoscopic screening has been reported to miss more than 90% of DGC cases in mutation carriers undergoing prophylactic gastrectomy.16, 17 Thus, prophylactic gastrectomy remains the only reliable preventive intervention in CDH1 mutation carriers from HDGC families. But for individuals not wishing to have prophylactic surgery, regular surveillance may be helpful to detect clinically relevant small foci,18 and the International Gastric Cancer Consortium also recommends that annual endoscopic surveillance should be offered by experienced teams to mutation carriers in whom gastrectomy is not currently pursued.19 This consensus may also be applicable to the present families at a time when prophylactic gastrectomy is not warranted.

Other nongastric malignancies also were observed in the present families. There was 1 case each of colon cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, and uterine cancer. With the exception of pancreatic cancer, which occurred in an obligate carrier, it is unknown whether other cases with nongastric malignancies are mutation carrier or not, because no DNA was available for them. It also remains unclear whether pancreatic cancer is attributable to germline CDH1 mutations or not, because it is not significantly over-represented in the present family or other HDGC families, although the E-cadherin protein was demonstrated to be involved in the transition from adenoma to carcinoma in a transgenic mouse model of pancreatic β-cell carcinogenesis.20

The underlying molecular mechanism responsible for the phenotypic divergence of families with CDH1 mutations is unknown. Although 1 group has suggested that LBC-associated mutations may tend to cluster in the 3′ half of the gene compared with those associated with DGC, the association was weak.21 Such a position effect cannot explain the present family 1, because the same mutation has also been described in DGC-only and mixed DGC/LBC families, strongly suggesting the existence of modifier genes.

In conclusion, our report clearly demonstrates that germline CDH1 mutation can cosegregate with invasive lobular breast cancer in the absence of diffuse gastric cancer. Present data do not support recommendation of prophylactic gastrectomy in CDH1 germline mutation carriers with ILBC. The risk of DGC in these families and the underlying molecular mechanisms leading to this phenotypic divergence, such as the epistatic effects of other genes, merit further study.

CONFLICT OF INTEREST DISCLOSURES

This work was supported by a grant from the FEDER (Fonds Européens de Développement en Région), convention nb 31,355-2008 and Clermont Communauté.