Hiromi Sakashita

Pivotal Role of CXCR3 in Melanoma Cell Metastasis to Lymph Nodes

Chemokines and their receptors play key roles in leukocyte trafficking and are also implicated in cancer metastasis to specific organs. Here we show that mouse B16F10 melanoma cells constitutively express chemokine receptor CXCR3, and that its ligands CXCL9/Mig, CXCL10/IP-10, and CXCL11/I-TAC induce cellular responses in vitro, such as actin polymerization, migration, invasion, and cell survival. To determine whether CXCR3 could play a role in metastasis to lymph nodes (LNs), we constructed B16F10 cells with reduced CXCR3 expression by antisense RNA and investigated their metastatic activities after s.c. inoculations to syngeneic hosts, C57BL/6 mice. The metastatic frequency of these cells to LNs was markedly reduced to ∼15% (P < 0.05) compared with the parental or empty vector-transduced cells. On the other hand, pretreatment of mice with complete Freund’s adjuvant increased the levels of CXCL9 and CXCL10 in the draining LNs, which caused 2.5–3.0-fold increase (P < 0.05) in the metastatic frequency of B16F10 cells to the nodes with much larger foci. Importantly, such a stimulation of metastasis was largely suppressed when CXCR3 expression in B16F10 cells was reduced by antisense RNA or when mice were treated with specific antibodies against CXCL9 and CXCL10. We also demonstrate that CXCR3 is expressed on several human melanoma cell lines as well as primary human melanoma tissues (5 of 9 samples tested). These results suggest that CXCR3 inhibitors may be promising therapeutic agents for treatment of LN metastasis, including that of melanoma.

Chemokine receptor CXCR3 promotes colon cancer metastasis to lymph nodes.

Chemokines and their receptors are essential for leukocyte trafficking, and also implicated in cancer metastasis to specific organs. We have recently demonstrated that CXCR3 plays a critical role in metastasis of mouse melanoma cells to lymph nodes. Here, we show that some human colon cancer cell lines express CXCR3 constitutively. We constructed cells that expressed CXCR3 cDNA (‘DLD-1-CXCR3’), and compared with nonexpressing controls by rectal transplantation in nude mice. Although both cell lines disseminated to lymph nodes at similar frequencies at 2 weeks, DLD-1-CXCR3 expanded more rapidly than the control in 4 weeks. In 6 weeks, 59% of mice inoculated with DLD1-CXCR3 showed macroscopic metastasis in para-aortic lymph nodes, whereas only 14% of those with the control (P<0.05). In contrast, metastasis to the liver or lung was rare, and unaffected by CXCR3 expression. In clinical colon cancer samples, we found expression of CXCR3 in 34% cases, most of which had lymph node metastasis. Importantly, patients with CXCR3-positive cancer showed significantly poorer prognosis than those without CXCR3, or those expressing CXCR4 or CCR7. These results indicate that activation of CXCR3 with its ligands stimulates colon cancer metastasis preferentially to the draining lymph nodes with poorer prognosis.

Hexokinase II and VEGF expression in liver tumors: correlation with hypoxia-inducible factor-1α and its significance

BACKGROUND/AIMS We analyzed the expressions of hexokinase II (HK II), a key enzyme in glycolysis, and VEGF in hepatocellular carcinoma (HCC) and metastatic liver cancer in relation to tumor vascularity, and the participation of hypoxia-inducible factor-1 (HIF-1) was studied. METHODS A real-time quantitative reverse transcription-polymerase chain reaction was performed to examine the HK II and VEGF mRNA expression. Expression of HIF-1 alpha and HK II protein, and microvessel density (MVD) were examined immunohistochemically. RESULTS MVD was significantly higher in HCCs than in metastatic liver cancers, and VEGF mRNA expression was positively correlated only with MVD of HCCs. HK II mRNA expression was significantly higher in metastatic liver cancers, however, some cases of HCC pretreated with transcatheter arterial embolization (TAE) showed marked HK II mRNA expression. Both HIF-1 alpha and HK II protein expressions were co-localized in the cancer cells near necrosis, and the intensity of HIF-1 alpha protein expression was significantly correlated with HK II mRNA expression in both tumors. CONCLUSIONS These results suggest that, in metastatic liver cancers, glycolysis induced by HIF-1 is the predominant energy source under the hypoxic environment and, at least in some TAE-pretreated HCC cases, cancer cells obtain energy for growth by switching the metabolic profile to glycolysis through HIF-1.

Requirement of Protocol Biopsy Before and After Complete Cessation of Immunosuppression After Liver Transplantation

Background. Operational tolerance is defined as long-term acceptance of a transplanted organ after complete cessation of immunosuppression (IS), but may not always protect against antigen-dependent changes in graft morphology. Method. IS free patients after living-donor liver transplantation (LDLT) underwent protocol biopsy (tolerance group [Gr-Tol]) and were evaluated for rejection and fibrosis. The degree of fibrosis was compared with those in the patients on maintenance IS group (Gr-IS) and the base line normal liver group (Gr-BS). When bridging fibrosis or progression of fibrosis was observed, IS was reintroduced or increased in Gr-Tol or in the patients in the weaning process. Results. Neither acute nor chronic rejection was observed. The degree of fibrosis, however, was significantly greater in Gr-Tol than those in Gr-IS and Gr-BS. In Gr-Tol, the number of graft infiltrating FOXP3+ cells was significantly increased, the interval between LDLT and biopsy plus the donor age was significantly longer, and recipient age at LDLT was significantly younger, compared with those in Gr-IS. However, none of these three parameters correlated with the degree of fibrosis. In 7 of 11 patients in whom IS was reintroduced or increased, the improvement of fibrosis was observed by the subsequent biopsy. Conclusion. Grafts of operationally tolerant patients after LDLT did not exhibit acute or chronic rejection, but they exhibited fibrosis. It remains elusive whether fibrosis observed in tolerant grafts is antigen dependent. The finding that after the reintroduction or the increase of IS fibrosis was improved supported the possibility that fibrosis in operationally tolerant patients was antigen dependent.

Significance of C4d staining in ABO-identical/ compatible liver transplantation

Complement degradation product C4d has become an important marker of humoral or antibody-mediated rejection in renal and heart allograft biopsies. Although there have been several reports on the detection of C4d in liver allografts, the significance of C4d in liver transplantation and its relationship with humoral rejection are still not clear. We investigated the frequency and pattern of C4d staining in liver allograft biopsies with reference to preoperative lymphocyte crossmatch tests, which detect donor-reactive lymphocyte antibody. Survival rates at 5 years were 77% for crossmatch-negative patients and 53% for crossmatch-positive patients (P=0.009). In crossmatch-negative patients, reproducible positive staining was obtained in 28 of 86 (33%) biopsies taken within 90 days after transplantation and 33 of 96 (34%) biopsies 90 days or after transplantation. Most C4d staining was observed in the portal areas, and no clear correlation was observed between C4d positivity and histological diagnosis. In crossmatch-positive patients, 9 of 11 (82%) biopsies showed positivity for C4d. C4d stained perivenular areas as well as portal areas. Histology of crossmatch-positive patients included acute rejection and cholangitis, but did not include periportal changes that were seen in humoral rejection in ABO-incompatible liver transplantation. In summary, focal C4d deposition was seen in various types of liver allograft injury and had little clinical impact on crossmatch-negative patients, but extensive C4d staining in crossmatch-positive patients may be associated with humoral rejection and poor graft survival.

Gastric foveolar metaplasia with dysplastic changes in Brunner gland hyperplasia: possible precursor lesions for Brunner gland adenocarcinoma.

Cases of adenocarcinomas developed in Brunner gland hyperplasia (BGH) have been sporadically reported. Herein, we report the morphologic spectrum of hyperplastic changes culminating into dysplasia and carcinoma in 722 cases of BGH listed in our files. Fifteen of these cases showed dysplastic changes, with 8 graded as low-grade dysplasia, 5 as high-grade dysplasia, 11 as atypical hyperplasia, and 2 as invasive carcinoma, although each frequently coexisted in the same tumor. In two carcinomas, one had high-grade dysplasia in the mucosa, and another had only atypical hyperplasia. Interestingly, hyperplastic glands around dysplastic foci were associated with gastric foveolar metaplasia and papillary configuration in 13 cases, 11 of which showed a gradual increase in nuclear atypism in the transition from metaplastic to dysplastic glands. All of the metaplastic gastric glands showed diffuse and strong immunopositivity for gastric foveolar mucin (MUC5AC). Immunohistochemical profiles also supported the concept of a continuous spectrum in carcinogenesis from gastric foveolar hyperplasia through atypical hyperplasia or dysplasia and eventually to frank adenocarcinoma. The results of our study suggest, therefore, that dysplastic and/or carcinomatous change does occur in BGH, that they form the continuous morphologic spectrum, and that papillary foveolar metaplasia may be a precursor lesion in the process of carcinogenesis with a background of BGH.

Suppression of Colonic Polyposis by Homeoprotein CDX2 through its Nontranscriptional Function that Stabilizes p27Kip1

Caudal-related homeoprotein CDX2 is expressed in intestinal epithelial cells, in which it is essential for their development and differentiation. A tumor suppressor function is suggested by evidence that CDX2 levels are decreased in human colon cancer specimens and that an inactivating mutation of Cdx2 in Apc(Δ716) mice markedly increases the incidence of colonic polyps. In this study, we investigated roles for transcriptional and nontranscriptional functions of CDX2 in suppression of colonic tumorigenesis. Mutagenic analysis of CDX2 revealed that loss of function stabilizes CDK inhibitor p27Kip1 by a nontranscriptional but homeodomain-dependent mechanism that inhibits cyclin E-CDK2 activity and blocks G0/G1-S progression in colon cancer cells. p27Kip1 stabilization was mediated by an inhibition of ubiquitylation-dependent proteolysis associated with decreased phosphorylation of Thr187 in p27Kip1. siRNA-mediated knockdown of p27Kip1 relieved the decrease in cyclin E-CDK2 activity and S-phase cell fraction elicited by CDX2 expression. Together, these results implicate a nontranscriptional function of CDX2 in tumor suppression mediated by p27Kip1 stabilization. Up to approximately 75% of low-CDX2 human colon cancer lesions show reduced levels of p27Kip1, whereas approximately 68% of high-CDX2 lesions retain expression of p27Kip1. These results show that low levels of CDX2 accelerate colon tumorigenesis by reducing p27Kip1 levels.

Coexistence of Epstein–Barr virus-associated lymphoproliferative disorder and marginal zone B-cell lymphoma of the thymus in a patient with rheumatoid arthritis treated with methotrexate

that DLBCLs arising in these sites show decreased or absent expression of MHC-II, allowing tumour cells to escape from immune attack. By extrapolation, it is distinctly possible that lack of MHC-II on the EATL type II in this case allowed it to home-in on immuneprivileged sites, probably explaining the clinical presentation of vitreous involvement and imaging-confirmed brain involvement. Once in these sites, the lymphoma could expand unchecked, probably explaining the rapid neurological deterioration of the patient. Finally, very little is known of MHC-II down-regulation in T-cell lymphomas of all subtypes in general. In the light of the observations made in this case report, a lack of MHC-II on T-cell lymphomas may be relevant in the prediction of likely tumour behaviour, and alert the clinician to keep a close watch on immune-privileged sites (i.e. brain, eye, and testis) for post-treatment recurrences.

Pseudodysplastic regenerative mucosa associated with congenital ileal atresia

A male neonate was clinically diagnosed with congenital intestinal atresia. Surgical operation was performed and the ileum including the atretic portion was resected. Grossly, there was a plaque‐like elevation of mucosa at the proximal side of the ileal atresia. Microscopic examination of this lesion revealed proliferation of severely atypical glands. Although dysplasia was a serious diagnostic concern, we concluded that ischemia due to the intestinal atresia induced this benign pseudodysplastic regenerative mucosa, judging from the pattern of coexisting inflammation and the literature review.

Acute Epstein–Barr virus infection presenting as severe gastroenteritis without infectious mononucleosis-like manifestations

Primary Epstein–Barr virus (EBV) infection is usually a self-limiting disease. Although it is sometimes accompanied by severe complications such as thrombocytopenia, hemolytic anemia, and splenic rupture, predominantly gastrointestinal complications are rarely reported. We studied an unusual case of primary EBV infection associated with severe hemorrhagic gastroenteritis. EBV infection was confirmed in the biopsy specimen by demonstrating the presence of EBV DNA by polymerase chain reaction, and of EBV-encoded small RNA (EBER)-positive cells by in-situ hybridization. Our patient was suspected of having primary EBV infection from the serological findings—EBV-viral capsid antigen IgM (+) and EBV nuclear antigen (−)—but he did not show typical clinical features of infectious mononucleosis such as lymph node swelling, pharyngitis, liver dysfunction, and splenomegaly. A definite diagnosis of primary EBV infection was made using biopsy specimens by demonstrating the presence of EBV DNA and EBER-positive cells.