滋賀医科大学 分子診断病理学部門

Research theme and publications

① Lineage analysis of early and advanced gastric cancers

The cell lineage is discernible as very stable cellular characteristics, such as genomic DNA alteration patterns and DNA methylation patterns, which inherit from cell to cell. Genomic DNA changes are basically irreversible, not affected by environmental changes and more stable than epigenetic changes. Phenotypic cell lineages are maintained epigenetically in the normal cellular differentiation, whereas genetic lineages are discernible in normal lymphocytes after DNA rearrangements and tumors that are associated with genomic DNA alterations, such as sequence changes (mutations) and copy-number changes. Phenotypic expression in tumors thus reflects not only genomic DNA alterations but also environmental and epigenetic changes. In the stomach, for example, intestinal metaplasia accumulates time-dependently, and we should be careful in the assessment of intestinal expression as a lineage marker. More useful phenotypic information for lineage analysis in stomach tumors may be the retention or disappearance of gastric expression.

To clarify the independence of each tumor lineage, our studies cover the total natural history from earliest to advanced stages. Different lineages should start from different origins. From this point of view, gastric carcinomas (GCs) have been classified into intestinal type and diffuse type by the presence and absence, respectively, of close link to intestinal metaplasia in the background mucosa of an incipient tumor growth. Well differentiated gastric carcinoma had thus been used as the synonym of intestinal type. However, the discovery of gastric-type well differentiated adenocarcinoma as a common GC gave a great impact on this situation, disclosing that the majority of well differentiated adenocarcinoma arises not from intestinalized glands but from proper gastric glands. It also cast a question whether gastric adenomas, which are mostly intestinal type, are the precursor of well differentiated adenocarcinoma. We have been studying this problem from both phenotypic and genotypic aspects (Subtheme 1).

In Japan, early and advanced GCs are defined morphologically as a growth confined to the mucosa/submucosa and that involving the muscularis propria or deeper, respectively, irrespective of lymph node metastasis. Early GCs are not always the precursor of advanced GC but may be an indolent subtype that remains at an early stage for a long time. It has thus been a problem whether lineages are continuous or discontinuous between early and advanced GCs. Regarding the histotype, GCs have also been classified morphologically into differentiated type (DGC) and undifferentiated type (UGC), which develop well to moderately differentiated tubules and poorly differentiated sheets/nests/cord or isolated cells, respectively. But there is a problem whether UGC, including signet ring cell carcinoma (SIG), derives from dedifferentiation of DGC or has arisen de novo.

In this regard, another important discovery in GC is the identification of germline mutation of E-cadherin as the cause of the familial SIG. This indicates that SIG is not secondary cancer deriving from DGC by the secondary loss of E-cadherin expression in DGC. The discovery of familial SIGs enabled us to approach the earliest phase of tumor development; the background mucosa of the familial cases shows multiple occurrences of, some times, hundreds of minute SIGs. It was found from such stomachs, single-gland cancers were identified and that these cancers closely resemble the incipient canine SIGs that were induced by administration of carcinogen and described by us in 1991. The incipient phase of development of SIG is characterized by the formation of a layered structure (LS), in which the normal pattern of cell proliferation and differentiation is retained. LS is scarcely seen in secondary UGC dedifferentiated from DGC. The presence of LS may thus be a primary SIG. As far as LS is retained, cell proliferation is confined to the middle zone of the mucosal layer and the tumor never invades deeper. Nevertheless, UGC with LS often comes to have LS− parts and progresses to an advanced stage (Subtheme 2).

At present, it was accepted that UGC consists of two genetic lineages: one deriving from de-differentiated DGC and the other arising de novo. Recently we have confirmed that these two lineages are discernible as the different profiles of genomic DNA copy-number changes (Subtheme 3).

1. Development of tubular adenocarcinoma: viewed from de novo carcinogenesis vs adenoma-carcinoma sequence

1) Phnenotypic lineages in small intramucosal cancers and adenomas
Common de novo carcinogenesis in gastric lineage (Kushima et al, 1993; Tsukashita et al., 2001; 2003)
Common de novo carcinogenesis and infrequent adenoma-carcinoma sequence in gastric lineage (Nishimura et al., 2013)

2) Genotypic lineages in small intramucosal cancers and adenomas
→ APC-linked 5q LOH was common in low-grade adenoma and a small fraction (of pure intestinal phenotype) of DGC but not in most of DGC that had gastric phenotype (Wu et al., 1998; Tanaka et al., 2008).

3) Epigenetic aspect
Microsatellite instability can be obtained during tumor progression (Ling et al., 2010)

2. Development of signet ring cell carcinoma: viewed from the aspect of a layered structure (LS)

1) How LS is formed and how regular pattern of cell proliferation and differentiation are retained in LS at an incipient stage of tumor development (from single gland cancers to intramucosal cancers)?
→ LS is a kind of gland formation (Sugihara et al., 1987).
→ LS is continuous to single-gland cancers (Sugihara et al., 1991).
→ Retention of LS causes intramucosal tumor spreading and eventually results in superficially spreading, large monoclonal lesions. The monoclonality was demonstrated by the HUMARA assay (Bamba et al., 1998).

2) How regular pattern of cell proliferation and differentiation is disturbed during progression of LS+ UGCs (from intramucosal cancers to submucosal or deeply invasive cancers)?
→ Cell dissociation and invasion are different phenomena; in UGC, cell-cell adhesion via E-cadherin-catenin complex is attenuated in intramucosal growth and in subserosal invasion but is enhanced in submucosal invasion (Nakamura et al., 2005).
→ Cellular dissociation due to mutant E-cadherin is correlated not with deep invasion but with superficially spreading growth of UGC (Bamba et al., 2008).
→ Occurrence of epithelial to mesenchymal transition (EMT) associated with deep invasion and dedifferentiation in UGC was demonstrated by double APAAP staining for integrin and cytokeratin and subsequent image analyses (Yanchenko et al., 2009; 2011).

3) Phenotypic and genotypic aspects:
→ Time-dependent accumulation of intestinal phenotype and progression-related loss of phenotypic differentiation in UGC (Bamba et al., 2001; Natsagdorj et al., 2008).
→ Gains of chromosome 20q, Xp and Xq and loss of 17p are related to the progression from signet ring cell carcinoma to poorly differentiated adenocarcinoma (Peng et al., 2003).
→ TP53 mutation was rare but could occur during tumor progression in signet ring cell carcinoma (Yoshimura et al., 2006).
Even LS+ intramucosal cancers showed aggressive signature in gene copy-number changes and inevitably become advanced.(Sonoda et al, 2013).

3. Genetic lineage and progression in gastric carcinomas: relationship between early and advanced cancers

UGC have two genetic lineages: LS+ UGC deriving from signet ring cell carcinoma and LS-/TC(a tubular component)+ UGC dedifferentiated from DGC (Peng et al., 2004; Yoshimura et al., 2006; Sonoda et al., 2013). These different lineages were separated by the cluster analysis of genomic DNA copy-number profiles (Sonoda et al., 2013).
In LS+ UGC, early and advanced cancers are continuous; additional chromosomal changes occur during inevitable progression from early to advanced stage in UGC (Peng et al., 2003; Sonoda et al., 2013).
In DGC (having two genetic lineages: dormant and aggressive subtypes), early and advanced cancers are often discontinuous;the dormant subtype accounts for the majority of early DGCs and can invade the submucosa but not deeper part, whereas the aggressive subtype is common in the deeper part of advanced DGCs (Nakayama et al., 2010).
The outcome of individual DGC is not stochastically determined but can be predicted from the genomic copy-number profile even at the non-invasive stage. Non-invasive neoplasms of the unstable clusters, which accounted for 21% of non-invasive neoplasms, may progress to invasive carcinomas, whereas those of stable cluster may not (Vo et al., 2015)

4. Methodology of genomic and epigenomic analyses

→ Southern blot analyses of early and advanced GCs revealed that the time course of oncogene amplification and the kinds of genes amplified may be different between DGC and UGC (Tsujimoto et al., 1997).
→ Our first paper on chromosomal CGH, using GC cell lines (Okada et al., 2000).
→ Establishment of combined DNA ploidy and CGH analyses after DOP-PCR amplification of genomic DNA (Kamitani et al., 2002).
→ Random primer labeling was recommended for chromosomal CGH as well as for array CGH, and the adverse effect of DOP-PCR amplification of the whole geneme on chromosomal and array CGH data was within acceptable levels (Tsubosa et al., 2005).
→ Optimization of comparative expressed sequence hybridization (CESH) (Ling et al., 2007).

② Duodenal contents reflux and gastro-esophageal carcinogenesis: in preparation


③ Epigenetic and genetic aspects in esophageal carcinogenesis

For trials of the application of new technologies, esophageal squamous cell carcinomas (ESCCs) have given us suitable materials because tumor samples with high purity are easier to take from ESCCs that show a sheet-like pattern of growth. Especially, for the application of chromosomal and array-based comparative genomic hybridization (CGH) and methylation analysis to carcinomas of the stomach and the gastroesophageal junction, the foregoing experiences using ESCC have been precious.

→ Application of the HUMARA assay to female ESCCs disclosed the presence of multiclonal incipient growths before the appearance of single dominant clone (Tamura et al., 2001).
→ Modes of silencing of p16 gene either by promoter methylation or by genomic loss were mapped onto ESCC lesions and the surrounding mucosa, and were found to be concordant between the carcinoma and the surrounding mucosa. The modes of silencing are thus determined in each patient before occurrence of ESCC (Tokugawa et al., 2002).
→ Dendrogram analysis based on combined DNA ploidy and CGH analyses has disclosed that genomic duplication occurs repeatedly as late events in tumor progression (Shiomi et al., 2003).
→ Establishment of combined DNA ploidy and CGH analyses after DOP-PCR amplification of genomic DNA in esophageal squamous cell carcinoma (Kamitani et al., 2002).

References

Authors Title Journal Vol pages Year
Bamba M, Sugihara H, Becker KF, Becker I, H?fler H, Hattori T. A case of multiple diffuse gastric carcinoma with regional expression of mutant E-cadherin. Virchows Arch 452 581-583 2008
Bamba M, Sugihara H, Kushima R, Okada K, Tsukashita S, Horinouchi M, Hattori T Time-dependent expression of intestinal phenotype in signet ring cell carcinomas of the human stomach Virchows Arch 438 49-56 2001
Kamitani S, Sugihara H, Shiomi H, Tani T, Hattori T. Intratumoral regional variations in copy number of the chromosomal part revealed by microdissection and combined ploidy and comparative genomic hybridization analyses in esophageal squamous cell carcinoma. Cancer Genet Cytogenet 132 30-35 2002
Kushima R, Hattori T. Histogenesis and characteristics of gastric-type adenocarcinomas in the stomach. J Cancer Res Clin Oncol. 120 103-111 1993
Ling ZQ, Sugihara H, Tatsuta T, Mukaisho K, Hattori T Optimization of comparative expressed sequence hybridization for genome-wide expression profiling at chromosome level. Cancer Genet Cytogenet 175 144-153 2007
Ling ZQ, Tanaka A, Li P, Nakayama T, Fujiyama Y, Hattori T, Sugihara H Microsatellite instability with promoter methylation and silencing of hMLH1 can regionally occur during progression of gastric carcinoma. Cancer Letters 297 244-251 2010
Nakamura E, Sugihara H, Bamba M, Hattori T. Dynamic alteration of the E-cadherin/catenin complex during cell differentiation and invasion of undifferentiated-type gastric carcinomas. J Pathol 205 349-358 2005
Nakayama T, Ling ZQ, Mukaisho K, Hattori T, Sugihara H Lineage analysis of early and advanced tubular adenocarcinomas of the stomach: continuous or discontinuous? BMC Cancer 10 311 2010
Natsagdorj L, Sugihara H, Bamba M, Hattori T. Intratumoural heterogeneity of intestinal expression reflects environmental induction and progression-related loss of induction in undifferentiated-type gastric carcinomas. Histopathology 53 685-697 2008
Nishimura R, Mukaisho KI, Yamamoto H, Sonoda A, Andoh A, Fujiyama Y, Hattori T, Sugihara H. Precursor-derived versus de-novo carcinogenesis depends on lineage-specific mucin phenotypes of intramucosal gland-forming gastric neoplasms. Histopathology 63 616-629 2013
Okada K, Sugihara H, Bamba M, Bamba T, Hattori T Sequential numerical changes of chromosomes 7 and 18 in diffuse-type stomach cancer cell lines: Combined comparative genomic hybridization, fluorescence in situ hybridization and ploidy analyses. Cancer Genet Cytogenet 118 99-107 2000
Peng DF, Sugihara H, Mukaisho K, Ling ZQ, Hattori T. Genetic lineage of poorly differentiated gastric carcinoma with a tubular component analysed by comparative genomic hybridization. J Pathol 203 884-895 2004
Peng DF, Sugihara H, Mukaisho K, Tsubosa Y, Hattori T. Alterations of chromosomal copy number during progression of diffuse-type gastric carcinomas: metaphase- and array-based comparative genomic hybridization analyses of multiple samples from individual tumours. J Pathol 201 439-450 2003
Shiomi H, Sugihara H, Kamitani S, Tokugawa T, Tsubosa Y, Okada K, Tamura H, Tani T, Kodama M, Hattori T. Cytogenetic heterogeneity and progression of esophageal squamous cell carcinoma. Cancer Genet Cytogenet 147 50-61 2003
Sonoda A, Mukaisho K, Nakayama T, Diem VT, Hattori T, Andoh A, Fujiyama Y, Sugihara H. Genetic lineages of undifferentiated-type gastric carcinomas analysed by unsupervised clustering of genomic DNA microarray data. BMC Med Genomics 6 25 2013
Sugihara H, Hattori T, Imamura Y, Noriki S, Fukuda M, Katsura K, Tsuchihashi Y, Fujita S Morphalogy and modes of cell proliferation in earliest signet-ring-carcinomas induced in canine stomachs by N-ethyl-N’-nitro-N-nitrosoguanidine J Cancer Res Clin Oncol. 117 197-204 1991
Tamura H, Sugihara H, Bamba M, Tani T, Hosokawa M, Kodama M, Hattori T Clonal analysis of esophageal squamous cell carcinoma with intraepithelial components. Pathobiology 69 289-296 2001
Tanaka A, Takemura-Tsukashita S, Kushima R, Sugihara H, Fujiyama Y, Hattori T Low-grade gastric adenomas/dysplasias: Phenotypic expression, DNA ploidy pattern, and LOH at microsatellites linked to the APC gene. Pathol Res Pract. 204 1-9 2008
Tokugawa T, Sugihara H, Tani T, Hattori T. Modes of silencing of p16 in development of esophageal squamous cell carcinoma Cancer Res 62 4938-4944 2002
Tsubosa Y, Sugihara H, Mukaisho K, Kamitani S, Peng DF, Ling ZQ, Tani T, Hattori T. Effects of degenerate oligonucleotide-primed polymerase chain reaction amplification and labeling methods on the sensitivity and specificity of metaphase- and array-based comparative genomic hybridization. Cancer Genet Cytogenet 158 156-166 2005
Tsujimoto H, Sugihara H, Hagiwara A, Hattori T Amplification of growth factor receptor genes and DNA ploidy pattern in the progression of gastric cancer. Virchows Arch 431 388-389 1997
Tsujimoto H, Sugihara H, Hattori T, Hagiwara A Microsatellite instability, DNA ploidy, and mutation of transforming growth factor-β receptor type II gene in sporadic gastric cancer. Cancer Journal 10 211-216 1997
Tsukashita S, Kushima R, Bamba M, Nakamura E, Mukaisho K, Sugihara H, Hattori T. Beta-catenin expression in intramucosal neoplastic lesions of the stomach. Comparative analysis of adenoma/dysplasia, adenocarcinoma and signet-ring cell carcinoma. Oncology 64 251-258 2003
Tsukashita S, Kushima R, Bamba M, Sugihara H, Hattori T MUC gene expression and histogenesis of adenocarcinoma of the stomach. Int J Cancer 94 166-170 2001
Vo DT, Nakayama T, Yamamaoto H, Mukaisho K, Hattori T, Sugihara H. Progression risk assessments of individual non-invasive gastric neoplasms by genomic copy-number profile and mucin phenotype BMC Med Genomics 8 60 2015
Wu LB, Kushima R, Borchard F, Molsberger G, Hattori T ntramucosal carcinomas of the stomach: phenotypic expression and loss of heterozygosity at microsatellites linked to the APC gene. Pathol Res Pract. 194 405-411 1998
Yanchenko N, Sugihara H, Hattori T. Application of a novel method of double APAAP staining with subsequent quantitative image analysis to the examination of integrin expression in undifferentiated-type gastric carcinomas. J Histochem Cytochem 57 1183-1193 2009
Yoshimura A, Sugihara H, Ling ZQ, Peng DF, Mukaisho K, Fujiyama Y, Hattori T. How wild-type TP53 is inactivated in undifferentiated-type gastric carcinomas: analyses of intratumoral heterogeneity in deletion and mutation of TP53. Pathobiology 73 40-49 2006