We analyzed a cohort (n=295) of gastric adenocarcinoma published by the TCGA project for the mRNA expression levels of the 4 genes (SALL4, CLDN6, GPC3, and AFP) characterizing the primitive phenotype in our cohort.20 After excluding cases with no available expression data for any of the 4 genes, or cases with preoperative neoadjuvant therapy or palliative surgery, 210 cases were included for further analysis. On the basis of the z-scores for reads per kilobase per million mapped reads, expression levels of each gene were classified into 3 tiered categories: negative (z-score <1.0), low expression (z-score ≤1.0 to <10.0), and high expression (z-score ≥10.0), which correspond to the scoring system in the immunohistochemical analysis of our cohort (ie, negative, focal, and diffuse). On the basis of this, the 210 tumors were clustered into 3 groups: group 1 (primitive phenotype, n=25), low or high expression of at least one of the 3 genes (AFP, CLDN6, or GPC3) or high expression of SALL4; group 2 (SALL4 low, n=55), low expression of SALL4 but negative for the other 3 genes; group 3 (negative, n=130), negative expression of all 4 genes (Fig. 5A).
In terms of molecular subtypes defined by the TCGA project, namely chromosomal instability (CIN), genomically stable (GS), EBV-positive (EBV), and microsatellite instability (MSI), the majority of groups 1 (23/25=92%) and 2 (44/55=80%) were classified as CIN, whereas group 3 consisted of 4 types at nearly the same proportions. Frequent TP53 mutation was significant in groups 1 (17/25=68%, P=0.002) and 2 (37/53=70%, P<0.001) in comparison with group 3 (45/128=35%). Further, in an enrichment analysis employing the cBioPortal for Cancer Genomics, only TP53 mutation was identified as significantly enriched in tumors with SALL4, CLDN6, GPC3, or AFP expression (z-score≥1.0) compared with the other tumors (64.3% vs. 34.4%, q-value=0.0361 when calculated using the Benjamini-Hochberg procedure).20,22,23 Regarding histologic type, group 1 consisted essentially of intestinal-type tumors (23/25=92%), except for 2 tumors with mixed histology. Group 2 was also enriched with intestinal-type tumors (41/54=76%). The frequency of intestinal-type histology was significantly lower in group 3 (80/124=65%) compared with group 1 (P=0.008). Finally, group 1 patients showed significantly worse disease-free survival than group 3 patients (P=0.049), although the difference was not significant in terms of overall survival (Figs. 5B, C).
In comparison with ontogenesis, the expression patterns of primitive phenotype genes in group 1 tumors were comparable with those of gastrointestinal epithelium in early gestation.10,11,15–17 When compared with primitive germ cell tumors, which parallel embryonal development,25 expression patterns of group 1 tumors—positive for SALL4, CLDN6, GPC3, and AFP but negative for OCT4, NANOG, and LIN28—were close to those of yolk sac tumor or immature teratomas.11,26,27 Expression of OCT4 and NANOG is limited to the very early stages of embryogenesis, and is present only in the most primitive form of germ cell tumors, embryonal carcinoma and seminoma.26 On the basis of these findings, PEP gastric cancer in our study is likely to recapitulate fetal gut epithelium in early gestation, but is not as primitive as embryonic stem cells, which corresponds to embryonal carcinoma.25
The mechanism of primitive phenotypic gene activation remains an important question for future analysis. Recent studies have demonstrated that an epigenetic mechanism similar to cellular reprograming might activate embryonic stem cell-like gene expression signature in several tumors.7,28 For example, SALL4 re-expression induced by DNA demethylation was reported in hepatocellular carcinoma and hematopoietic malignancies.29–31 In line with these findings, a recent comprehensive analysis demonstrated that SALL4 was hypomethylated and overexpressed in 34% of 98% gastric cancers, with significant negative methylation–expression correlation (Pearson correlation coefficient=−0.655).32 In addition, the absence of TP53 function has been shown to enhance the efficiency of cellular reprogramming.33,34 Given these observations, we speculate that group 1 tumors in this study develop through primitive phenotypic transformation by an epigenetic mechanism, which is most likely to occur in CIN tumors with TP53 alterations.
The National Institutes of Health TCGA project has highlighted drug-targetable pathways in each molecular subgroup. Relevant targetable pathways identified in CIN tumors were related to receptor tyrosine kinase gene amplifications, including HER2, EGFR, MET, FGFR, and VEGFA.20 Novel agents for these targets have been examined in clinical trials, but the results were largely disappointing, with the notable exception of trastuzumab targeting HER2.35 Because the primitive markers analyzed in this study are not expressed in normal adult tissues, they are ideal therapeutic targets, and in fact, several novel agents are under evaluation. Anti-CLDN6 monoclonal antibodies such as IMAB027 or 6PHU3 have been developed for cancer treatment, and the early phase of clinical trials of IMAB027 has been conducted involving patients with ovarian cancer.36,37 GPC3 is also expected to be a target for immunotherapy as well as monoclonal antibody therapy (Codrituzumab), currently under clinical trials.38–40 As for SALL4, a peptide that inhibits interaction of SALL4 with its downstream target was developed, which successfully reduced tumorigenesis of xenograft hepatocellular carcinoma in the NOD/SCID mouse.41,42 Given the aggressive nature of PEP gastric cancer, development of these novel agents is greatly anticipated.
1. Huntly BJ, Gilliland DG. Leukaemia stem cells and the evolution of cancer-stem-cell research. Nat Rev Cancer. 2005;5:311–321.
2. Ratajczak MZ. The embryonic rest hypothesis of cancer development—an old XIX century theory revisited in light of evidence showing that early development stem cells reside in dormant state in postnatal tissues. Int J Mol Med. 2015;36:S8.
3. Ben-Porath I, Thomson MW, Carey VJ, et al. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet. 2008;40:499–507.
4. Hassan KA, Chen G, Kalemkerian GP, et al. An embryonic stem cell-like signature identifies poorly differentiated lung adenocarcinoma but not squamous cell carcinoma. Clin Cancer Res. 2009;15:6386–6390.
5. Schoenhals M, Kassambara A, De Vos J, et al. Embryonic stem cell markers expression in cancers. Biochem Biophys Res Commun. 2009;383:157–162.
6. Zvelebil M, Oliemuller E, Gao Q, et al. Embryonic mammary signature subsets are activated in Brca1-/- and basal-like breast cancers. Breast Cancer Res. 2013;15:R25.
7. Chiang JH, Cheng WS, Hood L, et al. An epigenetic biomarker panel for glioblastoma multiforme personalized medicine through DNA methylation analysis of human embryonic stem cell-like signature. OMICS. 2014;18:310–323.
8. Matsunou H, Konishi F, Jalal RE, et al. Alpha-fetoprotein-producing gastric carcinoma with enteroblastic differentiation. Cancer. 1994;73:534–540.
9. Murakami T, Yao T, Mitomi H, et al. Clinicopathologic and immunohistochemical characteristics of gastric adenocarcinoma with enteroblastic differentiation: a study of 29 cases. Gastric Cancer
10. Ushiku T, Shinozaki A, Shibahara J, et al. SALL4
represents fetal gut differentiation of gastric cancer
, and is diagnostically useful in distinguishing hepatoid gastric carcinoma from hepatocellular carcinoma. Am J Surg Pathol. 2010;34:533–540.
11. Ushiku T, Shinozaki-Ushiku A, Maeda D, et al. Distinct expression pattern of claudin-6, a primitive phenotypic tight junction molecule, in germ cell tumours and visceral carcinomas. Histopathology. 2012;61:1043–1056.
12. Xu CY, Shen JG, Xie SD, et al. Positive expression of Lin28 is correlated with poor survival in gastric carcinoma. Med Oncol. 2013;30:32.
13. Kono K, Amemiya H, Sekikawa T, et al. Clinicopathologic features of gastric cancers producing alpha-fetoprotein. Dig Surg. 2002;19:359–365.
14. Zhang L, Xu Z, Xu X, et al. SALL4
, a novel marker for human gastric carcinogenesis and metastasis. Oncogene. 2014;33:5491–5500.
15. Gitlin D, Gitlin GM, Perricelli A. Synthesis of alpha-fetoprotein by liver, yolk sac, and gastrointestinal tract of human conceptus. Cancer Res. 1972;32:979–982.
16. Filmus J, Church JG, Buick RN. Isolation of a cDNA corresponding to a developmentally regulated transcript in rat intestine. Mol Cell Biol. 1988;8:4243–4249.
17. Ushiku T, Uozaki H, Shinozaki A, et al. Glypican 3-expressing gastric carcinoma: distinct subgroup unifying hepatoid, clear-cell, and alpha-fetoprotein-producing gastric carcinomas. Cancer Sci. 2009;100:626–632.
18. Sobin LH, Gospodarowicz MK, Wittekind C, et al. TNM Classification of Malignant Tumours. Hoboken, NJ: Wiley & Sons Inc.; 2009.
19. Lauren P. The two histological main types of gastric carcinoma, an attempt at a histoclinical classification. Acta Pathol Microbiol Scand. 1965;64:31–49.
20. Cancer Genome Atlas Research Network. Comprehensive molecular characterization of gastric adenocarcinoma. Nature. 2014;513:202–209.
21. Lauwers GY, Franceschi S, Carneiro F, et alBosman FT. World Health Organization, International Agency for Research on Cancer. WHO classification of tumours of the digestive system. World Health Organization Classification of Tumours. Lyon: International Agency for Research on Cancer; 2010:48–58.
22. Cerami E, Gao J, Dogrusoz U, et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2012;2:401–404.
23. Gao J, Aksoy BA, Dogrusoz U, et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal. 2013;6:pl1.
24. Ahn S, Lee SJ, Kim Y, et al. High-throughput protein and mRNA expression-based classification of gastric cancers can identify clinically distinct subtypes, concordant with recent molecular classifications. Am J Surg Pathol. 2017;41:106–115.
25. Skotheim RI, Lind GE, Monni O, et al. Differentiation of human embryonal carcinomas in vitro and in vivo reveals expression profiles relevant to normal development. Cancer Res. 2005;65:5588–5598.
26. Santagata S, Ligon KL, Hornick JL. Embryonic stem cell transcription factor signatures in the diagnosis of primary and metastatic germ cell tumors. Am J Surg Pathol. 2007;31:836–845.
27. Gillis AJ, Stoop H, Biermann K, et al. Expression and interdependencies of pluripotency factors LIN28, OCT3/4, NANOG and SOX2 in human testicular germ cells and tumours of the testis. Int J Androl. 2011;34:e160–e174.
28. Zheng YW, Nie YZ, Taniguchi H. Cellular reprogramming and hepatocellular carcinoma development. World J Gastroentero. 2013;19:8850–8860.
29. Lin J, Qian J, Yao DM, et al. Aberrant hypomethylation of SALL4
gene in patients with myelodysplastic syndrome. Leuk Res. 2013;37:71–75.
30. Ma JC, Qian J, Lin J, et al. Aberrant hypomethylation of SALL4
gene is associated with intermediate and poor karyotypes in acute myeloid leukemia. Clin Biochem. 2013;46:304–307.
31. Fan H, Cui Z, Zhang H, et al. DNA demethylation induces SALL4
gene re-expression in subgroups of hepatocellular carcinoma associated with Hepatitis B or C virus infection. Oncogene. 2016. doi: 10.1038/onc.2016.399.
32. Wang K, Yuen ST, Xu J, et al. Whole-genome sequencing and comprehensive molecular profiling identify new driver mutations in gastric cancer
. Nat Genet. 2014;46:573–582.
33. Kawamura T, Suzuki J, Wang YV, et al. Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature. 2009;460:1140–1144.
34. Yi L, Lu C, Hu W, et al. Multiple roles of p53-related pathways in somatic cell reprogramming and stem cell differentiation. Cancer Res. 2012;72:5635–5645.
35. Fontana E, Smyth EC. Novel targets in the treatment of advanced gastric cancer
: a perspective review. Ther Adv Med Oncol. 2016;8:113–125.
36. Sahin U, Jaeger D, Marme F, et al. First-in-human phase I/II dose-escalation study of IMAB027 in patients with recurrent advanced ovarian cancer (OVAR): preliminary data of phase I part [abstr 5537]. J Clin Oncol. 2015;33(suppl):5537.
37. Stadler CR, Bahr-Mahmud H, Plum LM, et al. Characterization of the first-in-class T-cell-engaging bispecific single-chain antibody for targeted immunotherapy of solid tumors expressing the oncofetal protein claudin 6. Oncoimmunology. 2016;5:e1091555.
38. Abou-Alfa GK, Puig O, Daniele B, et al. Randomized phase II placebo controlled study of codrituzumab in previously treated patients with advanced hepatocellular carcinoma. J Hepatol. 2016;65:289–295.
39. Sawada Y, Yoshikawa T, Ofuji K, et al. Phase II study of the GPC3
-derived peptide vaccine as an adjuvant therapy for hepatocellular carcinoma patients. Oncoimmunology. 2016;5:e1129483.
40. Suzuki S, Sakata J, Utsumi F, et al. Efficacy of glypican-3-derived peptide vaccine therapy on the survival of patients with refractory ovarian clear cell carcinoma. Oncoimmunology. 2016;5:e1238542.
41. Yong KJ, Gao C, Lim JS, et al. Oncofetal gene SALL4
in aggressive hepatocellular carcinoma. N Engl J Med. 2013;368:2266–2276.
42. Gao C, Dimitrov T, Yong KJ, et al. Targeting transcription factor SALL4
in acute myeloid leukemia by interrupting its interaction with an epigenetic complex. Blood. 2013;121:1413–1421.