Immunohistochemical pitfalls and the importance of glypican 3 and arginase in the diagnosis of scirrhous hepatocellular carcinoma.

Scirrhous hepatocellular carcinoma is a rare ill-defined morphological subtype of hepatocellular carcinoma characterized by marked stromal fibrosis. This variant can be difficult to distinguish from intrahepatic cholangiocarcinoma and metastatic adenocarcinoma, especially on needle biopsies. We performed immunohistochemistry for hepatocellular and adenocarcinoma-associated markers on 20 scirrhous hepatocellular carcinoma cases and compared the results with classical hepatocellular carcinoma and intrahepatic cholangiocarcinoma. Scirrhous hepatocellular carcinomas were significantly less likely to be HepPar-1 positive than classical hepatocellular carcinomas (26% and 74%, respectively; P<0.001) and were significantly more likely to express adenocarcinoma-associated markers such as epithelial cell adhesion molecule (63 vs 11%; P<0.001), cytokeratin 19 (26 vs 2%; P<0.001), and cytokeratin 7 (53 vs 2%; P<0.001). At least one of these adenocarcinoma-related markers was positive in 80% of scirrhous hepatocellular carcinoma cases. Glypican 3 and arginase were positive in 79% and 85% of cases of scirrhous hepatocellular carcinoma, respectively; the combined use of these two markers yielded 100% sensitivity for scirrhous hepatocellular carcinoma. In conclusion, the scirrhous morphology, absence of HepPar-1 staining, and frequent positivity with adenocarcinoma-related markers in scirrhous hepatocellular carcinoma can lead to an erroneous diagnosis of adenocarcinoma. Glypican 3 and arginase are the most reliable markers for identifying hepatocellular differentiation in this setting.

Oncogenic MAP2K1 mutations in human epithelial tumors.

The scirrhous subtype of gastric cancer is a highly infiltrative tumor with a poor outcome. To identify a transforming gene in this intractable disorder, we constructed a retroviral complementary DNA (cDNA) expression library from a cell line (OCUM-1) of scirrhous gastric cancer. A focus formation assay with the library and mouse 3T3 fibroblasts led to the discovery of a transforming cDNA, encoding for MAP2K1 with a glutamine-to-proline substitution at amino acid position 56. Interestingly, treatment with a MAP2K1-specific inhibitor clearly induced cell death of OCUM-1 but not of other two cells lines of scirrhous gastric cancer that do not carry MAP2K1 mutations, revealing the essential role of MAP2K1(Q56P) in the transformation mechanism of OCUM-1 cells. By using a next-generation sequencer, we further conducted deep sequencing of the MAP2K1 cDNA among 171 human cancer specimens or cell lines, resulting in the identification of one known (D67N) and four novel (R47Q, R49L, I204T and P306H) mutations within MAP2K1. The latter four changes were further shown to confer transforming potential to MAP2K1. In our experiments, a total of six (3.5%) activating mutations in MAP2K1 were thus identified among 172 of specimens or cell lines for human epithelial tumors. Given the addiction of cancer cells to the elevated MAP2K1 activity for proliferation, human cancers with such MAP2K1 mutations are suitable targets for the treatment with MAP2K1 inhibitors.

Clinical application of immunoreactivity of dihydropyrimidine dehydrogenase (DPD) in gastric scirrhous carcinoma treated with S-1, a new DPD inhibitory fluoropyrimidine.

BACKGROUND: A highly specific antibody against recombinant human dihydropyrimidine dehydrogenase (DPD) has been developed to immunohistochemically assess DPD expression in tumors. A new oral DPD inhibitory fluoropyrimidine (DIF), S-1, is reportedly effective against gastric scirrhous carcinoma. PATIENTS AND METHODS: In this study, the relationship between immunoreactivity to DPD in biopsy specimens and the effects of chemotherapy were investigated in 61 patients treated with first-line fluoropyrimidine-based chemotherapy (S-1:DIF, 5-FU:non-DIF) for gastric scirrhous carcinoma. RESULTS: The response rate was significantly higher in patients with DPD-positive tumors than in those with DPD-negative tumors in the S-1 group (45.5%, 10.0%: p < 0.05), as compared to the 5-FU group (0%, 5.6%: p = 0.398). According to the median survival time, there was no significant difference between patients with DPD-positive tumors (364 days) and those with DPD-negative tumors (406 days; p = 0.626) in either the S-1 group or the 5-FU group (181 days and 256 days, respectively; p = 0.543). CONCLUSION: This study indicates that S-1 may be effective even in gastric scirrhous carcinoma with a high level of DPD activity.

Elevated cyclooxygenase-2 expression is associated with histological grade in invasive ductal breast carcinoma.

PURPOSE: The aim of this study was to evaluate the expression of cyclooxygenase-2 in human breast cancer using immunohistochemistry and to determine whether the expression of cyclooxygenase-2 is associated with clinicopathological factors in invasive ductal breast carcinoma. METHODS: Cyclooxygenase-2 expression was investigated by immunohistochemistry in 30 invasive ductal breast carcinoma specimens and relationships between cyclooxygenase-2 expression and age, histological grade, histological type, nodal status, and hormone receptor status were evaluated. RESULTS: Cyclooxygenase-2 expression was found in 56.7% of the tumor samples and was related to histological grade (P<0.01) and histological type (P<0.001). CONCLUSIONS: Our results suggest that cyclooxygenase-2 expression has an important role in tumor differentiation in invasive ductal breast carcinoma.

Differences between scirrhous and non-scirrhous human gastric carcinomas from the aspect of proMMP-2 activation regulated by TIMP-3.

Gastric carcinomas can be classified into scirrhous carcinomas (SC), i.e. 'linitis plastica' or Borrmann 4 gastric cancer, and non-scirrhous carcinomas (NSC). SC are characterized by diffuse invasive growth patterns with marked fibrosis, frequent peritoneal dissemination and lymph-node metastases and poor prognosis, while NSC show medullary growth patterns and common hematogenous metastases. To study the differences in local expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) between SC and NSC, we examined the expression of MMPs and TIMPs in human gastric carcinoma tissues by several methods including sandwich-enzyme immunoassay systems, gelatin zymography, reverse transcriptase-polymerase chain reaction (RT-PCR), real-time quantitative PCR, immunoblotting, immunohistochemistry and in situ zymography. Of the seven MMPs and two TIMPs tested, only proMMP-2 levels were remarkably higher in SC than in NSC (P < 0.01), and proMMP-2 activation ratio was significantly lower in SC than in NSC (P < 0.05). TIMP-3 mRNA levels were remarkably about 2-fold higher in SC than in NSC tissues (P < 0.01). TIMP-3 production in SC was confirmed by immunoblotting and TIMP-3 was immunolocalized to stromal fibroblasts in SC. TIMP-3 mRNA levels inversely correlated with proMMP-2 activation ratios, although the expression levels of MT1-MMP and MT2-MMP were not different in SC and NSC. By in situ zymography, gelatinolytic activity appeared to be weaker in SC than in NSC. All these data suggest that proMMP-2 activation is down-regulated by TIMP-3 expressed in scirrhous gastric carcinomas. Our findings may explain the differences in clinical behaviors of SC and NSC.

[Significance of tissue thymidine phosphorylase and dihydropyrimidine dehydrogenase activities in ovarian cancer].

Thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) are important enzymes in the pyrimidine salvage pathway. In the meantime, TP and DPD are a converting enzyme of 5'-DFUR to 5-FU and the major catabolic enzyme of 5-FU, respectively. Because little is known about their protein expressions in ovarian cancers, we investigated TP and DPD protein expressions quantitatively in 24 ovarian cancers and their normal counterparts by ELISA. Higher TP expression was observed in ovarian cancers than in normal ovaries. The higher expression was also correlated with the histological grade and clinical stage. No relation was observed between the expression of DPD and the clinical and pathological parameters. The higher TP/DPD ratio, which appears to be a predictor of 5-fluorouracil sensitivity, was observed in ovarian cancers than in normal counterparts. In univariate analysis, a higher TP/DPD ratio was found to be a predictor of progression-free survival in ovarian cancer patients. This would suggest that capecitabine and 5'-DFUR are potential candidates for ovarian cancer chemotherapy.

Collagen biosynthesis in gastric cancer: immunohistochemical analysis of prolyl 4-hydroxylase.

BACKGROUND AND OBJECTIVES: The mechanism of the desmoplastic response in gastric carcinoma tissues is largely unknown. The objective of this study is to determine the localization of prolyl 4-hydroxylase (PH), an enzyme that plays a crucial role in collagen biosynthesis. METHODS: Freshly prepared gastric carcinoma tissues from 51 cases, including 13 of the scirrhous type (diffusely infiltrative type), were immunostained by using monoclonal antibodies to human placental PH. RESULTS: Although cytoplasmic staining for PH was observed in both fibroblasts and carcinoma cells, there was increased expression of the alpha-subunit in fibroblasts and no difference in expression between the scirrhous and non-scirrhous type gastric carcinomas. In scirrhous type samples, there was increased PH expression in fibroblasts located in the tumor periphery when compared with fibroblasts in the tumor center. These findings suggested that maintenance of a balance between production and degradation of collagen in gastric carcinoma tissues might be important for stroma formation. CONCLUSIONS: It is speculated that activated fibroblasts participate in collagen biosynthesis at the tumor periphery rather than in the tumor center and that increased collagen biosynthesis at the tumor periphery in scirrhous gastric carcinoma may assist further invasion of tumor cells.

[Activity of the enzymes of DNA metabolism in the blood of patients with breast cancer]. / Aktivnost' fermentov metabolizma DNK v syvorotke krovi bol'nykh rakom molochnoi zhelezy.

Age-related changes in the activity of thymidine- and adenosine-metabolizing enzymes were studied in healthy females and those with breast cancer aged 46-70 years. A significant increase in activity of thymidine kinase, adenosine deaminase and 5'-nucleotidase and a decrease in that of thymidine phosphorylase were registered in blood serum of breast cancer patients of all age brackets. Adenosine deaminase activity in blood serum and lymphocytes of breast cancer patients was found to significantly change after surgery. A direct correlation was established between pretreatment thymidine phosphorylase activity and histological type of tumor, on the one hand and results of chemotherapy, on the other. The applicability of enzyme level assay for evaluating response to pre- and postoperative medication was studied.

[The role of thymidylate synthetase in sequential dose of MTX and 5-FU in the advanced scirrhous type gastric cancer].

The biochemical rationale for the potentiation of the effects of 5-FU by MTX is based on an increased PRPP level or MTX polyglutamate produced by MTX. The cytotoxic action of MTX results not only from inhibition of DHFR but also depends upon thymidylate synthetase (TS), a key enzyme in DNA synthesis. We obtained a monoclonal antibody to TS using a hydrophilic peptide consisting of 20 amino acids in the TS amino acid sequence and demonstrated by PAP that TS was detectable in poorly differentiated adenocarcinoma cells but not in well differentiated adenocarcinoma cells. Upon clinical application of sequential doses of MTX and 5-FU, the median survival durations were 318 days and 156 days for scirrhous-type gastric cancer patients and non-scirrhous-type gastric cancer patients respectively. These results suggest that immunohistochemistry with TS antibody is available as an indicator of the effect of this drug regimen.

Mechanisms of calcitonin induction of prolonged activation of adenylate cyclase in human cancer cells.

Four human cancer cell lines (MCF 7, T 47D, ZR 75 and BEN), each of which possess calcitonin-responsive adenylate cyclase, have been investigated. Pre-treatment of cells with calcitonin resulted in two effects on their adenylate cyclase. First, the ability of adenylate cyclase to respond to further stimulation by calcitonin was reduced. Secondly, membranes from calcitonin-preincubated cells had increased activity of adenylate cyclase compared with control membranes, the activated state not only surviving washing and cell-disrupting steps but persisting for some hours after the removal of hormone from the incubation medium. This phenomenon has therefore been termed "persistent activation" and may be peculiar to the action of calcitonin. It is dose dependent and is seen with calcitonins of various origins (sCT, hCT, pCT and [Asu1-7] eCT). When pre-bound hormone was removed from the cell surface by acid washing, adenylate cyclase activities in preincubated membranes fell towards control activities and responsiveness of the enzyme to a second challenge with calcitonin returned. Inhibitors of protein synthesis prevented full recovery from desensitization but had no effect on the return towards control of residual enzyme activity in preincubated membranes. These results are consistent with continued receptor occupancy leading to a persistent activation of adenylate cyclase until internalization of the receptor has occurred.

Adenosine triphosphatases as histochemical markers for the cell of origin in experimental mammary carcinoma.

Different adenosine triphosphatase (ATPase) activities were detected at an ultrastructural level in order to differentiate epithelial and myoepithelial cells in normal and neoplastic mouse mammary tissues. Mg2+ dependent and Na+-K+-dependent ATPase activities were studied in: BALB/c mouse mammary gland; a BALB/c carcinoma from a transplantable D2 hyperplastic nodule; a stable cell line, MCF-8, derived from the BALB/c carcinoma; and a BALB/c scirrhous-like carcinoma induced by MCF-8 cell inoculation. Mg2+-dependent ATPase was detected in the plasma membranes of the normal mouse mammary epithelial cells, the epithelial component of the BALB/c carcinoma, the MCF-8 cells in culture, and the atypical epithelial component of the scirrhous-like carcinoma. Na+-K+-dependent and Mg2+-dependent ATPase were localized in the plasma membranes of the myoepithelial cells of the normal mammary gland and the BALB/c carcinoma. The results from these histochemical studies established that the cell of origin in both the BALB/c carcinoma and the scirrhous-like carcinoma was the mammary epithelial rather than the myoepithelial cells. Furthermore, these results indicated that the MCF-8 cell line was derived from the epithelial component of the primary BALB/c carcinoma. These conclusions, which were based on histochemical study, were supported by the presence of intracisternal type A viral particles in the epithelial cells of the primary BALB/c carcinoma, the MCF-8 cells in culture, and the epithelial cells of the scirrhous-like carcinoma. Thus, the enzymatic markers were specific for cell type and remained unchanged by the process of cell transformation.

Histological grading, histochemistry, and electron microscopy of scirrhous carcinoma of the breast.

Twenty-six scirrhous carcinomas of the breast were divided into three histological grades of malignancy; low (Grade I), intermediate (Grade II), and high (Grade III), and the correlation of the grades with histochemical and electron microscopic findings in both tumor cells and host tissues was examined. The tumor cells contained increased amounts of lysosomal acid phosphatase and beta-glucuronidase. This increase was most marked in Grade I and II tumors and the increase was consistent with lysosome-like fine structures. Both intracytoplasmic lumina and microvilli against stroma were characteristic findings of carcinoma cells and they were mostly found in Grade I and II tumors. Segments of intact basal laminae and myoepithelial cells were also found in Grade I and II carcinomas. The stroma contained moderately increased amounts of intracellular acid phosphatase and beta-glucuronidase, independent of tumor grade. The stroma also contained a large amount of acid mucopolysaccharide ground substance, irrespective of the three grades. There was a striking difference in the ultrastructural organization of the stroma between normal and neoplastic tissues. Although fragmented elastic fibers and increased amount of acid mucopolysaccharide granules, and macrophages rich in phagolysosomes were prominent fine structures of the stroma of carcinomas, there was no apparent difference in them among the three grades of malignancy.

[Acid phosphatase activity in breast dysplasias and in carcinomas of the breast]. / L'attività della fosfatasi acida nelle displasie mammarie e nei carcinomi della mammella

The histochemical reactions of acid phosphatase were studied in tissues deriving from normal breasts, from mammary dysplasia, and from breast carcinoma. Acid phosphatase was demonstrated in all cases with more intense activity in breast carcinoma where activity was particularly accentuated in the stroma. In benign tumours and fibroadenosis, phosphatase was prevalently localized in the periductal cells and within the ducts. Exceptions were the cases of fibroadenosis with acute or chronic collateral inflammation where phosphatase granules were located in the stroma also, even though they were always quantitatively inferior to the corresponding localization of cases of breast carcinomas.