Pathogenic Germline BRCA1/2 Mutations and Familial Predisposition to Gastric Cancer

Most gastric cancers (GCs) are considered sporadic, however familial aggregation occurs in 10% of cases1, 2. Approximately 5% of GCs are caused by an autosomal dominant inherited trait, with carriers having a strongly increased risk of GC and other cancers3. Clinical criteria for this entity were defined by the International Gastric Cancer Linkage Consortium (IGCLC)4. Among these, hereditary diffuse gastric cancer (HDGC) is a well-known type of familial GC (FGC). About 40% of families fulfilling the clinical criteria for HDGC have germline CDH1 mutations5. A subset of the remaining families of HDGC, and ones fulfilling the criteria of other familial GC, harbor pathogenic germline mutation in other genes which associated with hereditary cancer predisposition syndromes4. 
 
Hereditary breast and ovarian cancer (HBOC) is one of the best-described inherited cancer predisposition syndromes, caused by pathogenic germline BRCA1 or BRCA2 (BRCA1/2) mutations6–9. The increased risks of cancers other than breast and ovarian cancers were observed in the carriers10. The association between germline BRCA1/2 mutation and increased risk of GC were demonstrated in previous studies for HBOC families11–14. Regarding FGC, a recent large-scale study demonstrated that germline BRCA2 mutations were identified in patients who had a family history which fulfilled the criteria of HDGC, but lacking CDH1 mutations15. Therefore, it is possible that germline BRCA1/2 mutations may cause familial predisposition to GC. 
 
Recent advances of comprehensive genomic analysis enable us to identify the genomic alterations in GC16. BRCA1/2 mutations were shown in the subset of GC tumor tissues, however the association between germline BRCA1/2 mutations and familial predisposition to GC were not fully understood. Previously we performed genomic sequencing of 207 Japanese GCs using 435-gene panel, and identified BRCA1/2 mutations in tumor17. In this study, we conducted BRCA1/2 genetic testing in seven Japanese GC patients whose tumor had BRCA1/2 mutations. We identified pathogenic germline BRCA1/2 mutations in three patients, who have a familial component of GC.


INTRODUCTION
Most gastric cancers (GCs) are considered sporadic; however, familial aggregation occurs in 10% of cases. 1,2 Approximately 5% of GCs are caused by an autosomal dominant inherited trait, with carriers having a strongly increased risk of GC and other cancers. 3 Clinical criteria for this entity were defined by the International Gastric Cancer Linkage Consortium. 4 Among these, hereditary diffuse gastric cancer (HDGC) is a well-known type of familial GC (FGC). Approximately 40% of families fulfilling the clinical criteria for HDGC have germline CDH1 mutations. 5 A subset of the remaining families of HDGC, and ones fulfilling the criteria of other familial GC, harbor pathogenic germline mutation in other genes that are associated with hereditary cancer predisposition syndromes. 4 Hereditary breast and ovarian cancer is one of the best-described inherited cancer predisposition syndromes, caused by pathogenic germline BRCA1 or BRCA2 (BRCA1/2) mutations. [6][7][8][9] The increased risks of cancers other than breast and ovarian cancers were observed in the carriers. 10 The association between germline BRCA1/2 mutation and increased risk of GC were demonstrated in previous studies for families with hereditary breast and ovarian cancer. [11][12][13][14] Regarding FGC, a recent large-scale study demonstrated that germline BRCA2 mutations were identified in patients who had a family history that fulfilled the criteria of HDGC but were lacking CDH1 mutations. 15 Therefore, it is possible that germline BRCA1/2 mutations may cause familial predisposition to GC.
Recent advances of comprehensive genomic analysis enable us to identify the genomic alterations in GC. 16 BRCA1/2 mutations were shown in the subset of GC tumor tissues; however, the association between germline BRCA1/2 mutations and familial predisposition to GC were not fully understood. Previously, we performed genomic sequencing of 207 Japanese GCs using a 435-gene panel and identified BRCA1/2 mutations in tumor. 17 In this study, we conducted BRCA1/2 genetic testing in seven Japanese patients with GC whose tumor had BRCA1/2 mutations. We identified pathogenic germline BRCA1/2 mutations in three patients who have a familial component of GC.

METHODS
Among 28 patients whose tumor had BRCA1/2 mutations in our previous study, seven patients who could provide written informed consent were enrolled. BRCA1/2 genetic testing was performed by Myriad Genetic Laboratories using genomic DNA extracted from peripheral blood samples. We assessed the family history using the criteria for referral for genetic risk assessment and FGC defined by International Gastric Cancer Linkage Consortium after the enrollment (Table 1). 4 Clinicopathological data are listed in Table 2. This study was approved by the institutional review boards at Niigata University and Niigata Cancer Center Hospital.

RESULTS
Family history of cases 1 to 4 met the criteria for referral to genetic risk assessment, and that of cases 1, 3, and 4 met the criteria for FGC ( Table 2). Pathogenic germline BRCA1 and/ or BRCA2 mutations were detected in cases 1 to 3. Cases 5 to 7 also had germline BRCA1 or BRCA2 mutations; however, pathogenicity was determined to be uncertain. Case 4 had no germline mutations in BRCA1/2 (Table 3). Somatic BRCA1/2 mutations detected in tumor by using a 435-gene panel are detailed in Table 3. Germline BRCA1/2 mutations in cases 1 to 3 were consistently detected in tumors.

Case 1
A 73-year-old man had undergone endoscopic submucosal dissection for multiple intramucosal Lauren intestinal-type GCs on two occasions. Two years later, advanced GC and hypopharyngeal cancer were synchronously detected by periodic endoscopy. He underwent curative gastrectomy with D2 lymphadenectomy for GC. Moderately differentiated tubular adenocarcinoma classified as Lauren intestinal type was shown with pathologic T4aN2M0 (Fig 1A). Helicobacter pylori (HP) was identified in non-neoplastic mucosa by reviewing the hematoxylin and eosin-stained sections ( Table 2). After that, he underwent concurrent chemoradiotherapy using cisplatin for hypopharyngeal cancer and achieved a complete response. He is alive with no evidence of recurrence 2 years after gastrectomy. He had a strong family history of cancers, with three GC and one breast cancer in first-or second-degree relatives (Fig 2A), which met the criteria for FGC (Table 1). Genetic testing detected a pathogenic frameshift germline mutation in BRCA2 c.89insA (p.L29fs), which was identical to the variant detected in tumor (Table 3).

Case 2
A 66-year-old woman underwent curative gastrectomy with D2 lymphadenectomy for GC. Mucinous and moderately differentiated adenocarcinoma classified as Lauren intestinal type was shown with pathologic T2N3bM0 (Fig 1B). HP was not identified in non-neoplastic mucosa ( Table 2). Although adjuvant chemotherapy with S-1, an oral fluoropyrimidine preparation combining tegafur, gimeracil, and oteracil potassium, was performed, she suffered para-aortic lymph node recurrence 6 months after gastrectomy. Cisplatin with fluorouracil and paclitaxel was administered, and she achieved partial response. After 2 years of treatment, mediastinal lymph node metastasis subsequently developed, and chemotherapy was converted to cisplatin with CPT-11. All of the metastatic sites were markedly reduced and were maintained in reduced state for 7 months. Although there was a recurrence in the mediastinal lymph node, she is alive and continuing chemotherapy with paclitaxel and ramucirumab 5 years after postoperative recurrence. She had a strong family history of cancers, with two GCs and one case each of breast, lung, and cervical cancers in first-or second-degree relatives, which did not fulfill the criteria for FGC (Fig 2B). Genetic testing detected double pathogenic nonsense germline mutations in BRCA1 c.188T>A (p.L63X) and in BRCA2 c.6922A>T (p.K2308X), which were identical to the variants detected in tumor (Table 3).

Case 3
A 56-year-old man underwent curative gastrectomy with D2 lymphadenectomy for GC. Poorly differentiated variant of tubular adenocarcinoma classified as Lauren indeterminate type was shown with pathologic T4bN1M0 (Fig 1C). HP was identified in non-neoplastic mucosa ( Table 2). Adjuvant chemotherapy was performed with S-1 for 1 year, and he is alive with no evidence of recurrence 2 years after gastrectomy. He had 2 ascopubs.org/journal/po JCO™ Precision Oncology   a strong family history of GC, with three cases in first-or second-degree relatives, which met the criteria for FGC (Fig 2C). Genetic testing detected a pathogenic nonsense germline mutation in BRCA2 c.9310A>T (p.K3104X), which was identical to the variant detected in tumor (Table 3).

DISCUSSION
Previous studies have shown that germline BRCA1/2 mutations increase the risk of GC. [11][12][13][14] However, there is a paucity of data regarding the relationship between germline BRCA1/2 mutations and familial predisposition to GC in Japan. In this report, we identified pathogenic germline BRCA1/2 mutations in three Japanese patients with GC with a familial component of GC. Among these, one patient (case 2) had double pathogenic germline mutations in BRCA1 and BRCA2, which were considered to be rare, as shown by a previous study for Ashkenazi Jewish double-founder mutations. 18 To the best of our knowledge, this is the first report of Japanese patients with FGC harboring BRCA1/2 mutations.
Multiple genetic and environmental factors influence the etiology of GC. The well-known environmental factors are high salt intake and HP infection, which cause chronic gastritis. 19 The development of GC stands out in the family history of our three cases, rather than breast and ovarian cancers (Fig 2). HP infection was detected in cases 1 and 3 and intestinal-type GC, which is most associated with chronic gastritis, was observed in cases 1 and 2 (Fig 1). These findings suggested that chronic gastritis, which is associated with HP infection and/or environmental factors, may cause the loss of BRCA1/2 function by the second-hit mutations in patients with the germline mutations. This synergistic exacerbatory effect confers the familial predisposition to GC in Japan, where a high incidence is observed.
BRCA1/2 mutation carriers might have a risk of multiple GCs. In this report, case 1, with a BRCA2 mutation, developed multiple metachronous GCs. BRCA1/2 mutations are considered risk factors for ipsilateral breast cancer recurrence. 20 Regarding HDGC caused by CDH1 germline mutation, multiple foci of signet ring cell carcinoma were detected in the stomach of individual patients. 21,22 Prophylactic total gastrectomy is recommended for CDH1 mutation carriers because of the high cumulative incidence and the difficulty of early detection of HDGC by endoscopy. 23 In this report, the intestinal type of GC, which is relatively easy to detect at an early stage by endoscopy, predominantly developed in the affected patients. Therefore, meticulous endoscopic surveillance may be recommended for BRCA1/2 mutation carriers in Asia if additional large-scale studies substantiate the findings in our limited study.