Six patients presented with invasive lobular carcinoma and metastasis to the stomach, based on the original histological reports, immunohistochemical stains and clinical course. The median age at diagnosis of the primary breast cancer was 50 years (range 42 to 64) and of the gastric metastasis 56 years (range 47 to 71); all metastatic breast cancers lacked staining of HNF4A (Figure 1A-D and Table 2). In all patients, ER and BRST-2 expression was high in primary breast cancer and metastases (Table 2). In one of these six patients (patient 5) the pathology report of the gastric biopsy mentioned both metastases of the breast cancer and a signet ring cell carcinoma of the stomach. This was not supported by immunohistochemistry. HNF4A was completely negative and there was high expression of ER, PR and BRST-2 in various tumor samples available of stomach, breast and ovaries (gastric biopsy and autopsy material).
One patient was diagnosed with metastasis of diffuse gastric cancer to the breast, synchronously with the diagnosis of the gastric cancer (50 years of age, 2 months between both diagnoses). Both the primary tumor and the metastasis were HNF4A positive, while ER, PR, and BRST-2 did not show expression (Figure 1E-H and Table 2).
Thirteen patients presented with two primary carcinomas, one of the breast and the other of the stomach. According to initial pathology reports and review of cases, nine out of 13 patients had invasive ductal breast cancer, while the other
Table 1. HNF4A expression in gastric carcinomas (TMA validation set)
Histological type of gastric carcinoma Negative HNF4a staining Focal HNF4a staining Limited HNF4a staining Diffuse HNF4a staining Number of cases
Intestinal (tubular or papillary carcinoma)
1 4 14 43 62 Diffuse (poorly cohesive carcinoma,
incl. signet ring cell morphology)
0 0 3 18 21 Undifferentiated carcinoma 0 1 3 10 14 Mucinous carcinoma 0 0 1 7 8 Mixed carcinoma 0 0 0 2 2 Total gastric samples 1 5 21 80 107
A C E B D F
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Figure 1) A-I. H&E and HNF4A expression of breast and gastric carcinomas. A-D: patient (no 4) with
primary lobular breast carcinoma (A,B) with metastases to the stomach (C,D) without expression of HNF4A (B,D). Note nuclear expression of HNF4A in normal gastric glands (D). E-H: patient (no 7) with primary diffuse gastric carcinoma (E,F) with metastases to the breast (G,H). Both in the stomach (F) and in the breast (H) diffuse staining of HNF4A. I-L: patient (no 11) with both primary carcinomas of the breast (I,J) and stomach (K,L). There is no expression of HNF4A in the breast (J) but diffuse expression in signet ring cells of the stomach (L). Original magnification of all images 200x.
G I H J L K
four had invasive lobular breast cancer. The histology of gastric cancer showed 10 diffuse and three intestinal carcinomas. The median age at diagnosis was 49 years (range 33-79) for the first and 58 years (range 40-85) for the second tumor. The disease-free interval between diagnoses in patients with two primary carcinomas was approximately 4 years longer than in patients with metastatic disease. Four patients presented with gastric and 8 patients presented with breast cancer as the first primary tumor. One patient had a synchronous diagnosis of both gastric and breast cancer. In all patients, the primary tumors of the stomach stained positive for HNF4A, whereas the primary tumors of the breast were negative for HNF4A. In the primary gastric carcinomas there was no expression of ER, PR, and BRST-2. In the primary breast carcinomas there was expression of ER in 9/13, PR in 6/13 and BRST-2 in 11/13 cases (Figure 1I-L and Table 2).
Table 2. Results of immunohistochemical expression in patients with a metastatic breast carcinoma
(case 1-6), metastastatic gastric carcinoma (case 7), and 2 primary carcinomas of the breast and stomach (case 8-18)
Index No Gender Breast - histology HNF4A ER PR BRST-2 Stomach HNF4A ER PR BRST-2
1 F LBC 0 3 3 3 Metastasis LBC 0 3 1 2 2 F LBC 0 3 2 3 Metastasis LBC 0 3 1 2 3 F LBC NA NA NA NA Metastasis LBC 0 3 2 3 4 F LBC 0 3 3 3 Metastasis LBC 0 3 2 3 5 F LBC 0 3 2 3 Metastasis LBC 0 3 0 3 6 F LBC 0 3 0 3 Metastasis LBC 0 3 0 3 7 F Metastasis DGC 3 0 0 0 DGC 3 0 0 0 8 F LBC 0 3 0 3 DGC 3 0 0 0 9 F LBC 0 3 0 2 DGC 3 0 0 0 10 F LBC 0 3 2 2 DGC 3 0 0 0 11 F DBC 0 3 3 2 DGC 3 0 0 0 12 M DBC 0 3 3 1 DGC 3 0 0 0 13 F DBC 0 3 3 1 DGC 3 0 0 0 14 F DBC 0 0 0 3 DGC 3 0 0 0 15 F DBC 0 2 0 3 DGC 3 0 0 0 16 F DBC 0 0 0 0 IGC 3 0 0 0 17 F DBC 0 3 3 2 IGC 3 0 0 0 18 F DBC 0 0 0 0 DGC 3 0 0 0 19 F DBC 0 0 0 2 IGC 3 0 0 0 20 F LBC 0 3 2 2 DGC 3 0 0 0
LBC= invasive lobular breast carcinoma; DBC= invasive ductal breast carcinoma; IGC= intestinal- type gastric carcinoma; DGC= diffuse gastric carcinoma
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DISCUSSION
The present study shows that HNF4A is a highly useful marker in discriminating primary and metastatic breast from gastric carcinomas with a sensitivity of 99% and specificity of 100%, supporting the data of Koyama et al.16 We also confirmed that ER, PR, and BRST-2 staining could be helpful, since none of the primary gastric cancers did show expression of these markers. However, since a portion of primary breast carcinomas are negative for ER, PR, and BRST-2, absence of staining for these antibodies does not exclude breast cancer. Furthermore, in general a positive marker is more reliable than a negative one, because technical failure can never be excluded in case of absent staining without a positive internal control.
To the best of our knowledge this study describes the largest validation series of HNF4A staining to discriminate primary breast and primary stomach cancers thus far presenting data of 107 gastric and 105 breast cancer samples. Our data are supported by the study of Tanaka et al., in which HNF4A immunohistochemistry in the stomach was performed with a sensitivity and specificity of 100% in 14 gastric cancer cases.18 In another study of Takano et al., expression was shown in all differentiated-type early gastric carcinoma samples of 39 patients.20 In our series, one out of 107 gastric carcinomas tested negative, which could not be explained by technical failure as the surrounding normal gastric mucosa was positive.
Histological differentiation between primary or metastatic breast and gastric carcinoma can be difficult. It has been suggested that univacuolated signet ring cells are a feature of metastastic lobular breast carcinomas, rather than primary gastric carcinoma.22 However, metastatic breast and primary diffuse gastric carcinoma often contain both uni- and multivacuolated signet ring cells.16, 22 All metastatic breast carcinomas in our series were lobular breast carcinomas, while most patients with two primary carcinomas had an invasive ductal breast carcinoma.
Currently, for the differential diagnosis between gastric and breast carcinomas a panel that includes breast-associated immunomarkers ER, PR, BRST-2, and/or mammaglobin and the stomach-associated immunomarkers CDX2 and CK20 are often used. Breast carcinomas never show expression of CDX2 and almost never CK20 (approximately 1%).16,18,22-28 The disadvantage of these markers is that they are positive in only about 50% of gastric carcinomas (Table 3). With expression in 99% of gastric cancers and 0% of breast cancers HNF4A outperforms both CDX2 and CK20 as a positive predictor for gastric cancer.
Table 3. Literature of immunohistochemical expression in breast and gastric carcinoma
Antibody* Primary GC** Primary BC** Metastatic BC in GI-tract** p-value†
ER 16,22-25,27 0 81 (75-89) 74 (72-76) <,001 PR 16,23 0 71 (69-75) 33 (33) <,001 BRST-2 16,23,27 0 56 (33-75) 69 (62-78) <,001 CK7 22-25,27 59 (35-92) 92 (81-100) 90 (90) <,001 CK20 16,22-25,27,28 52 (18-100) 1 (0-5) 0 <,001 CDX2 16,22,23,25-27 57 (18-100) 0 0 <,001 HNF4A 16,18 100 0 0 <,001 MUC1 22,25,29,30 32 (17-67) 100 NA‡ <,001 MUC2 22,23,25,29,30 54 (42-92) 27 (3-100) 24 (24) <,001 MUC5AC 16,22,23,29,30 63 (38-85) 5 (0-17) 5 (5-6) <,001 MUC6 16,23,25,29 35 (27-58) 27 (8-50) 0 0,36
*Studies are included when immunohistochemistry was performed in both primary GC and BC **data are given as percentage (range) of expression in cases
‡NA= not available
† Primary gastric adenocarcinoma versus primary breast carcinoma
In conclusion, our data validate HNF4A as a good marker and can be added to an immunohistochemical panel to differentiate between primary gastric cancer and breast cancer derived metastasis in the stomach.
Acknowledgements: The authors would like to thank Monique Link (technical assistance).
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REFERENCES
1. Fitzgerald RC, et al. Hereditary diffuse gastric cancer: updated consensus guidelines for clinical management and directions for future research. J Med Genet 2010; 47:436-44.
2. Pharoah PD, et al. Incidence of gastric cancer and breast cancer in CDH1 (E-cadherin) mutation carriers from hereditary diffuse gastric cancer families. Gastroenterology 2001; 121:1348-53. 3. Benusiglio PR, et al. CDH1 germline mutations and the hereditary diffuse gastric and lobular
breast cancer syndrome: a multicentre study. J Med Genet 2013; 50:486-9.
4. Jakubowska A, et al. A high frequency of BRCA2 gene mutations in Polish families with ovarian and stomach cancer. Eur J Hum Genet 2003; 11:955-8.
5. Friedenson B. BRCA1 and BRCA2 pathways and the risk of cancers other than breast or ovarian.
MedGenMed 2005; 7:60.
6. Moran A, et al. Risk of cancer other than breast or ovarian in individuals with BRCA1 and BRCA2 mutations. Fam Cancer 2012; 11:235-42.
7. Graham WP, GASTRO-INTESTINAL METASTASES FROM CARCINOMA OF THE BREAST. Ann
Surg 1964; 159:477-80.
8. Choi SH, Sheehan FR, Pickren JW. METASTATIC INVOLVEMENT OF THE STOMACH BY BREAST CANCER. Cancer 1964; 17:791-7.
9. Cifuentes N, Pickren JW. Metastases from carcinoma of mammary gland: an autopsy study. J
Surg Oncol 1979; 11:193-205.
10. Reiman T, Butts CA. Upper gastrointestinal bleeding as a metastatic manifestation of breast cancer: a case report and review of the literature. Can J Gastroenterol 2001; 15:67-71.
11. Almubarak MM, et al. Gastric metastasis of breast cancer: a single centre retrospective study. Dig
Liver Dis 2011; 43:823-7.
12. Taal BG, et al. The spectrum of gastrointestinal metastases of breast carcinoma: I. Stomach.
Gastrointest Endosc 1992; 38:130-5.
13. Sato T, et al. Metastatic breast cancer from gastric and ovarian cancer, mimicking inflammatory breast cancer: report of two cases. Breast Cancer 2008; 15:315-20.
14. Hamby LS, et al. Gastric carcinoma metastatic to the breast. J Surg Oncol 1991; 48:117-21. 15. Boutis AL, et al. Gastric signet-ring adenocarcinoma presenting with breast metastasis. World J
Gastroenterol 2006; 12:2958-61.
16. Koyama T, et al. Hepatocyte nuclear factor 4A expression discriminates gastric involvement by metastatic breast carcinomas from primary gastric adenocarcinomas. Hum Pathol 2011; 42:1777-84. 17. Kojima K, et al. The expression of hepatocyte nuclear factor-4alpha, a developmental regulator of visceral endoderm, correlates with the intestinal phenotype of gastric adenocarcinomas.
Pathology 2006; 38:548-54.
18. Tanaka T, et al. Dysregulated expression of P1 and P2 promoter-driven hepatocyte nuclear factor-4alpha in the pathogenesis of human cancer. J Pathol 2006; 208:662-72.
19. Sladek FM, et al. Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily. Genes Dev 1990; 4:2353-65.
20. Takano K, et al. Immunohistochemical staining for P1 and P2 promoter-driven hepatocyte nuclear factor-4alpha may complement mucin phenotype of differentiated-type early gastric carcinoma. Pathol Int 2009; 59:462-70.
21. Sugai M, et al. Expression of hepatocyte nuclear factor 4 alpha in primary ovarian mucinous tumors. Pathol Int 2008; 58:681-6.
22. Chu PG and Weiss LM. Immunohistochemical characterization of signet-ring cell carcinomas of the stomach, breast, and colon. Am J Clin Pathol 2004; 121:884-92.
23. O’Connell FP, Wang HH, Odze RD. Utility of immunohistochemistry in distinguishing primary adenocarcinomas from metastatic breast carcinomas in the gastrointestinal tract. Arch Pathol
Lab Med 2005; 129:338-47.
24 Tot T. The role of cytokeratins 20 and 7 and estrogen receptor analysis in separation of metastatic lobular carcinoma of the breast and metastatic signet ring cell carcinoma of the gastrointestinal tract. APMIS 2000; 108:467-72.
25 Chu PG et al. Determining the site of origin of mucinous adenocarcinoma: an immunohistochemical study of 175 cases. Am J Surg Pathol 2011; 35:1830-6.
26. Werling RW et al. CDX2, a highly sensitive and specific marker of adenocarcinomas of intestinal origin: an immunohistochemical survey of 476 primary and metastatic carcinomas. Am J Surg Pathol 2003; 27:303-10.
27. Dennis JL et al. Markers of adenocarcinoma characteristic of the site of origin: development of a diagnostic algorithm. Clin Cancer Res 2005; 11:3766-72.
28. Moll R et al. Cytokeratin 20 in human carcinomas. A new histodiagnostic marker detected by monoclonal antibodies. Am J Pathol 1992; 140:427-47.
29. Nguyen MD, et al. Mucin profiles in signet-ring cell carcinoma. Arch Pathol Lab Med 2006; 130:799-804.
30. Lau SK, Weiss LM, Chu PG. Differential expression of MUC1, MUC2, and MUC5AC in carcinomas of various sites: an immunohistochemical study. Am J Clin Pathol 2004; 122:61-9.
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