Masamichi Bamba

Availability of CD10 Immunohistochemistry as a Marker of Breast Myoepithelial Cells on Paraffin Sections

CD10, also called common acute lymphoblastic leukemia antigen (CALLA), was recently found to be expressed in nonhematopoietic tissues. Although CD10 was also identified in human breast myoepithelial cells, its availability of immunohistochemistry on paraffin sections has not been examined so far. In the present study, we demonstrated CD10 immunohistochemically on paraffin sections of both normal and pathological breast tissues, comparing its staining patterns to those of smooth muscle actin (SMA), which is now commonly used to highlight myoepithelium. CD10 was consistently positive in normal breast myoepithelial cells. CD10 also clearly highlighted myoepithelial cells in intraductal papilloma, adenosis, ductal hyperplasia, fibroadenoma, and phyllodes tumor as well as SMA did. In atypical ductal hyperplasia and ductal carcinoma in situ, continuous, discontinuous, and totally negative stainings of both CD10 and SMA were noted, depending on foci of neoplastic cell nests. However, both stainings clearly demonstrated myoepithelial cells of cancerized acini, being useful in differentiating lobular cancerization from microinvasion. Because SMA was also positive in normal vessels and spindle-shaped stromal cells, CD10, which was negative in vessels, was useful in differentiating myoepithelial cells from thin vascular wall in intracystic lesions with delicate papillae. Although background staining of spindle-shaped stromal cells was also noted in CD10, the positive cell number was less, and the signal was weaker than that of SMA. The absence of myoepithelial cells in invasive ductal carcinomas was more clearly highlighted by CD10 than SMA. We concluded that CD10 could be another useful marker of breast myoepithelial cells on paraffin sections. Combination of CD10 and SMA will provide more sophisticated information about presence or absence of myoepithelial cells in confusing breast lesions.

MUC gene expression and histogenesis of adenocarcinoma of the stomach.

To elucidate the histogenesis of adenocarcinomas of the stomach, we examined MUC gene expression in gland‐forming intramucosal neoplastic lesions. Eighty tumors were histopathologically assigned to 1 of the following 3 groups based upon the Vienna classification: group A (low‐grade adenoma/dysplasia), group B (high‐grade adenoma/dysplasia) and group C (intramucosal carcinoma). Immunohistochemic staining was performed with monoclonal antibodies against MUC2 (goblet cell mucin), MUC5AC (gastric‐foveolar mucin), MUC6 (pyloric‐gland mucin) and CD10 (brush border). Ki‐67 staining was also carried out. An obvious difference existed in MUC gene expression between lesions in group A and those in groups B and C. The majority of group A lesions strongly expressed intestinal markers in which proliferating cell zones were formed but generally expressed no gastric markers, whereas more than 50% of groups B and C tumors expressed gastric markers. These findings suggest that group A lesions are of a stable intestinal phenotype, whereas those in groups B and C are phenotypically and genotypically unstable, indicating that the adenoma‐carcinoma sequence is not a major pathway, but instead that adenocarcinomas arise de novo.

NEOEXPRESSION OF N-CADHERIN IN E-CADHERIN POSITIVE COLON CANCERS

In our study, we aimed to investigate the expression of N‐cadherin and E‐cadherin and their dependency on epithelial‐mesenchymal transition regulators SNAI1, SIP1 and TWIST in human colon cancer. Expression of E‐cadherin and N‐cadherin was examined by immunohistochemistry in 80 colon carcinomas by using paraffin embedded and formalin fixed tissues. Those cases were partly analyzed for mRNA expression of N‐cadherin (42 cases), TWIST (18 cases), SNAI1 (25 cases) and SIP1 (25 cases) by real‐time quantitative RT‐PCR. Additionally, colon carcinomas that showed amplification of 20q13, the localization of the human SNAI1 gene, were examined. We found cytoplasmic and/or membrane‐associated immunoreactivity of N‐cadherin in 35/80 (44%) of the cases. However, there was no correlation to upregulated TWIST mRNA levels, as we have shown previously for diffuse‐type gastric cancers with abnormal N‐cadherin expression. Reduced and/or cytoplasmic E‐cadherin immunoreactivity was detected in 19% (15/80) of the cases. Expression of SNAI1 or SIP1 mRNA was not seen in any of the 25 cases analyzed. There was no correlation between amplification of 20q13 and SNAI1 mRNA expression. Remarkably, N‐cadherin was almost exclusively expressed in those cases showing normal E‐cadherin immunoreactivity, suggesting a mutual exclusion between abnormal E‐cadherin reduction and upregulation of N‐cadherin. For the first time, we postulate a role for N‐cadherin in primary colon cancer progression, which may be similar to the effect discovered by others in breast cancer cell lines, where coexpressed N‐cadherin can exert a dominant function over E‐cadherins adhesive function and thus promote tumor invasiveness.

Time-dependent expression of intestinal phenotype in signet ring cell carcinomas of the human stomach.

Abstract. Signet ring cell carcinomas of the stomach are thought to arise from the proper gastric mucosa without intestinal metaplasia. It was recently reported that intestinal phenotypes appear along with tumor progression. In this study, we performed several experiments to reconsider the significance of this intestinalization in the growth of signet ring cell carcinoma. We applied mucin histochemistry with monoclonal antibodies MUC2 (Ccp58) and M1 (45M1), and paradoxical concanavalin A staining for class III mucin [PCS(III)] reaction to 29 intramucosal and 25 deeply invasive carcinomas of this type and correlated the phenotypic expression with the size of the mucosal spread and the depth of tumor invasion. It was found that the larger the size of the mucosal lesion, the more frequently the intestinal phenotypes were demonstrated. There was no significant increase in the expression of the intestinal phenotype as the tumor invaded the deeper part of the mucosa or as the intestinal metaplasia increased in the background mucosa. The intestinal expression appeared to be suppressed in the earlier phase of deep invasion. In the mucosal part of the tumor, the intestinal phenotype was often expressed regionally and incompletely, coexisting with gastric phenotypes at the cellular and the tissue levels. These findings indicate that the expression of the intestinal phenotype is a time-dependent and unstable phenomenon probably based on the accumulation of genetic changes and plays a neutral role in progression of signet ring cell carcinomas.

'Pyloric gland-type adenoma' arising in heterotopic gastric mucosa of the duodenum, with dysplastic progression of the gastric type

Abstract ’Pyloric gland-type adenoma’ is a recently described and very rare entity. We report a case of a pedunculated polyp of the duodenal bulb showing the features of pyloric gland-type adenoma. Heterotopic gastric mucosa was found adjacent to the tumour. Immunohistochemically, the tumour cells at the surface of the polyp showed foveolar-type mucin (M1) while most other tumour cells showed deep gastric mucin (M2), displaying a pattern of differentiation similar to the normal gastric mucosa. The polyp also showed villous or papillary structures with disorganization of gastric differentiation and marked increase of proliferating in foci cells. This is the first case of pyloric gland-type adenoma found to arise in heterotopic gastric mucosa of the duodenum, showing dysplastic progression of the gastric type.

Myoepithelial cells in solid variant of intraductal papillary carcinoma of the breast: a potential diagnostic pitfall and a proposal of an immunohistochemical panel in the differential diagnosis with intraductal papilloma with usual ductal hyperplasia.

We examined myoepithelial status in intraductal papillary carcinoma (IPC) along with the expression of high-molecular weight cytokeratin (HMWK) and neuroendocrine markers, with special reference to the differential diagnosis of solid intraductal papillary carcinoma(SIPC) and intraductal papilloma with usual ductal hyperplasia (IP-UDH). Twenty-six (93%) of the twenty-eight intraductal papillomas (IP) had myoepithelial cells in >70% of the epithelial–stromal interface of the intraluminal proliferating component. Six (29%) of twenty-one SIPC had almost complete myoepithelial layer like IP-UDH at the epithelial–stromal interface. HMWK (34βE-12) was diffusely positive in 14 (93%) of 15 IP-UDH, but 16 (76%) of 21 SIPC were completely negative for HMWK. Neuroendocrine markers were positive in 14 (67%) of SIPC, but all 28 IPs were completely negative. If only the presence of myoepithelial cells is emphasized as a benign hallmark, about 30% of SIPCs may be underdiagnosed as IP-UDH. However, by using a combination of myoepithelial markers, HMWK, and neuroendocrine markers, all of the 36 solid intraductal papillary lesions were properly classified as benign and malignant. Solid intraductal papillary lesions meeting at least two of the following criteria are highly likely to be malignant: (1) absence of myoepithelial cells(<10% of epithelial–stromal interface of intraluminal proliferating component), (2) negative HMWK(<10%), (3) positive neuroendocrine markers (>10%).

Highly anaplastic extraventricular ependymoma arising in an adult, mimicking metastatic adenocarcinoma with heavy stromal inflammation and emperiporesis

We report a case of extraventricular ependymoma arising in a 50‐year‐old woman that took an aggressive clinical course with recurrence three times. The initial tumor was a well‐circumscribed nodule in the right temporal white matter measuring 2 cm in diameter. It showed variegated histological findings mimicking metastatic adenocarcinoma: an epithelioid arrangement of highly pleomorphic cells with pseudopapillary structures and perivascular pseudorosettes, and bizarre multinucleated giant cells with occasional emperiporesis surrounded by abundant mononuclear inflammatory cells, as well as a focal small area of conventional ependymoma. Emperiporesis and abundant mononuclear cell infiltration were not previously described in an ependymoma. The recurrent tumors predominantly showed an epithelioid pattern with frequent formation of astroblastoma‐like pseudopapillary structures. Neoplastic cells were markedly atypical and had characteristic intracytoplasmic eosinophilic inclusion bodies. Much of the cells in both the initial and recurrent tumors showed a positive immunostaining for glial fibrillary acidic protein (GFAP) with accentuation to the cytoplasmic processes of the pseudorosettes and pseudopapillary structures. Epithelial membrane antigen (EMA) highlighted the epithelial differentiation of the tumor cells, while cytokeratin was completely negative. Although this tumor might be classified to at least WHO grade III from the histology and aggressive behavior, the exact grading is still controversial because of the rarity of such cases.

Gastric carcinogenesis by duodenal reflux through gut regenerative cell lineage.

To elucidate the histogenesis of gastric stump cancer, we performed an operation in rats to make all duodenal contents flow back into the glandular stomach. The subjects were 41 rats, and sequential morphological changes of the duodenogastric stoma and the incidence of stump cancers were studied. Serial sections around the stoma were studied with mucin stains such as paradoxical concanavalin A (Con A), galactose oxidase Schiff (GOS), and high-iron diamine–Alcian blue (HID-AB). An immunohistochemical study on cell proliferation with bromodeoxyuridine (BrdU) was also done. At week 30, pyloric gland type cells positive for Con A first appeared at the base of the intestinal crypts and the fundic glands adjacent to the anastomosis. These glands became large with time, resulting in formation of cystically dilated glands. These gland cells were partially stained with GOS, and then they retained a proliferative activity. These changes seemed to resemble “gastritis cystica profunda” in human remnant stomachs. At 50 and 80 weeks, adenocarcinomas were observed in 4 of 10 rats (40.0%) and in 16 of 21 rats (76.2%), respectively. We have noted that the early change of cystic proliferation of mucous glands resembled the so-called “ulcer associated cell lineage (UACL)” described by others, but our characteristic finding was not only pyloric but also foveolar metaplasia. This pyloric–foveolar metaplasia subsequently led to development of glands with intestinal-type goblet cells, which looked like incomplete intestinal metaplasia. This sequence was different from UACL, and very recently, we proposed a concept of “gut regenerative cell lineage (GRCL)semi; from pyloric–foveolar to with goblet cell metaplasia in regeneration,” common to all parts of the gut, and the stump cancer appeared to arise from GRCL.

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

Sequential changes of chromosomal copy number were analyzed retrospectively in five diffuse-type gastric cancer cell lines by comparative genomic hybridization (CGH), DNA cytometry, and fluorescence in situ hybridization (FISH) with centromeric and painting probes. By CGH, we found loss of 18q21 in all of the cell lines and gains of 7p11-q31, 20q, and 22 in four of the five cell lines. Actual copy numbers of chromosomes 7 and 18 were determined by FISH: disomy 18 with (partial) loss of 18q in the two DNA-diploid cell lines (AGS and MKN-45), trisomy 7 in MKN-45, disomy 18 and tetrasomy 7 with one-copy loss of 7p and one-copy gain of 7q tip in DNA-triploid HSC-39/40A, and trisomy 18 and hexasomy 7 with one-copy loss of 7q in DNA-tetraploid KATO-III. Because the DNA aneuploidy is thought to result through tetraploidization, and the duplicated chromosomal changes in DNA aneuploid tumors seem to precede tetraploidization, the duplicated gain of chromosome 7 and one-copy loss of 7q in KATO-III were inferred to have occurred before and after tetraploidization, respectively. Similarly, HSC-39/40A were inferred to be preceded by the DNA-diploid stage with disomy 7 and monosomy 18. As the loss of 18q21 and the gain of 7p11-q31 were inferred to have occurred already in the DNA diploid stage in at least four and two of the cell lines, respectively, the 18q21 loss may be more important than the 7q gain as an earlier event in the genesis of diffuse-type stomach cancer. The combined CGH, FISH, and ploidy analyses thus give us a clue to extract important earlier events from the chromosomal changes that were screened by CGH alone.

Dynamic alteration of the E‐cadherin/catenin complex during cell differentiation and invasion of undifferentiated‐type gastric carcinomas

To examine qualitative alterations of the E‐cadherin/catenin complex (CCC) during cell differentiation and invasion of undifferentiated‐type gastric carcinoma, immunoreactivity for the intracytoplasmic domain and the extracellular domain (ECD) of E‐cadherin, and that of β‐catenin, was analysed in the mucosal, submucosal, and deepest invasive parts of 20 early and 20 advanced cancers that had a component of intramucosal signet ring cell carcinoma. Histological subtype affected the mode of E‐CCC alteration. The tumours with a tubular component and without organized differentiation of signet ring cells in a layered structure were associated with nuclear expression of β‐catenin and may derive from tubular adenocarcinomas through de‐differentiation and de‐regulation of the Wnt pathway. These tumours were characterized by relatively stable ECD expression throughout the course of tumour progression. On the other hand, the tumours with a layered structure, which may derive from signet ring cell carcinoma by de novo abnormality of E‐cadherin, were characterized by dynamic alteration of ECD expression during cell differentiation and tumour progression; intramucosal spread (with a layered structure) as well as deep invasion (beyond the submucosa) commonly showed cellular dissociation with downregulation of ECD, whereas submucosal invasion and lymph node metastasis often showed cellular cohesion and retention (or ‘reappearance’) of ECD. Thus, cellular dissociation did not always reflect enhanced invasive activity but may be reversibly regulated during tumour progression. Copyright