MCAM+CD161- Th17 Subset Expressing CD83 Enhances Tc17 Response in Psoriasis.

Recent studies have highlighted the pathogenic roles of IL-17-producing CD8+ T cells (T-cytotoxic 17 [Tc17]) in psoriasis. However, the underlying mechanisms of Tc17 induction remain unclear. In this study, we focused on the pathogenic subsets of Th17 and their mechanism of promotion of Tc17 responses. We determined that the pathogenic Th17-enriched fraction expressed melanoma cell adhesion molecule (MCAM) and CCR6, but not CD161, because this subset produced IL-17A abundantly and the presence of these cells in the peripheral blood of patients has been correlated with the severity of psoriasis. Intriguingly, the serial analysis of gene expression revealed that CCR6+MCAM+CD161-CD4+ T cells displayed the gene profile for adaptive immune responses, including CD83, which is an activator for CD8+ T cells. Coculture assay with or without intercellular contact between CD4+ and CD8+ T cells showed that CCR6+MCAM+CD161-CD4+ T cells induced the proliferation of CD8+ T cells in a CD83-dependent manner. However, the production of IL-17A by CD8+ T cells required exogenous IL-17A, suggesting that intercellular contact via CD83 and the production of IL-17A from activated CD4+ T cells elicit Tc17 responses. Intriguingly, the CD83 expression was enhanced in the presence of IL-15, and CD83+ cells stimulated with IL-1ß, IL-23, IL-15, and IL-15Rα did not express FOXP3. Furthermore, CCR6+MCAM+CD161-CD4+ T cells expressing CD83 were increased in the peripheral blood of patients, and the CD83+ Th17-type cells accumulated in the lesional skin of psoriasis. In conclusion, pathogenic MCAM+CD161- Th17 cells may be involved in the Tc17 responses via IL-17A and CD83 in psoriasis.

Status and prognostic value of immunological biomarkers of breast cancer.

The immune response to cancer serves an important role in disease progression and patient prognosis. For triple-negative breast cancer showing aggressive behavior, immunotherapy has a good efficacy because of the potent immunogenicity of this type of cancer. However, the dominant subtype, luminal human epidermal growth factor receptor-2 (HER2)-negative breast cancer, is less immunogenic. To determine whether luminal HER2-negative cancer reacts to the anticancer immune response, the present study analyzed the status and prognostic value of the principal immunological biomarkers of breast cancer, including tumor-infiltrating lymphocytes (TILs), CD8+ T lymphocytes, the major histocompatibility complex and programmed cell death ligand-1 (PD-L1). The biomarkers were compared between patients with luminal HER2-negative breast cancer and those with immunogenic subtypes including triple-negative and HER2-overexpressed breast cancer. A total of 71 patients with primary breast cancer were classified into the immunogenic non-luminal (n=23) and less immunogenic luminal HER2-negative groups (n=48) based on immunogenicity. In the luminal HER2-negative group, compared with patients with low TIL levels, those with high TIL levels were at an advanced stage of cancer (P=0.024) and showed worse relapse-free survival (P=0.057); however, the remaining biomarkers exhibited no association with cancer progression or prognosis. In the non-luminal group, patients with high TIL levels showed significantly better RFS than those with low TIL levels (P=0.014). Compared with non-luminal patients negative for PD-L1, those positive for PD-L1 exhibited better overall survival (P=0.064). Notably, TIL status was found to exhibit contrasting prognostic predictions based on immunogenicity. In conclusion, TILs are a strong candidate for prognostic prediction in breast cancer, regardless of the subtype. PD-L1 is a potential candidate for prognostic prediction in immunogenic breast cancers, but not in the luminal HER2-negative subtype.

Involvement of a Transcription factor, Nfe2, in Breast Cancer Metastasis to Bone.

Patients with triple negative breast cancer (TNBC) is frequently complicated by bone metastasis, which deteriorates the life expectancy of this patient cohort. In order to develop a novel type of therapy for bone metastasis, we established 4T1.3 clone with a high capacity to metastasize to bone after orthotopic injection, from a murine TNBC cell line, 4T1.0. To elucidate the molecular mechanism underlying a high growth ability of 4T1.3 in a bone cavity, we searched for a novel candidate molecule with a focus on a transcription factor whose expression was selectively enhanced in a bone cavity. Comprehensive gene expression analysis detected enhanced Nfe2 mRNA expression in 4T1.3 grown in a bone cavity, compared with in vitro culture conditions. Moreover, Nfe2 gene transduction into 4T1.0 cells enhanced their capability to form intraosseous tumors. Moreover, Nfe2 shRNA treatment reduced tumor formation arising from intraosseous injection of 4T1.3 clone as well as another mouse TNBC-derived TS/A.3 clone with an augmented intraosseous tumor formation ability. Furthermore, NFE2 expression was associated with in vitro growth advantages of these TNBC cell lines under hypoxic condition, which mimics the bone microenvironment, as well as Wnt pathway activation. These observations suggest that NFE2 can potentially contribute to breast cancer cell survival in the bone microenvironment.

Differentiation capacities of PS-clusters, adult pituitary stem/progenitor cell clusters located in the parenchymal-niche, of the rat anterior lobe.

Pituitary endocrine cells are supplied by Sox2-expressing stem/progenitor cells in the anterior lobe of the adult pituitary. In relation to their microenvironment ("niche"), SOX2-positive cells exist in two types of niches; the marginal cell layer-niche and the parenchymal-niche. Recently, we isolated dense stem/progenitor cell clusters from the parenchymal-niche as parenchymal stem/progenitor cell (PS)-clusters. We classified these PS-clusters into three subtypes based on differences in S100ß-expression (S100ß-positive, -negative, and -mixed type), and reported that S100ß-positive PS-clusters exhibited the capacity for differentiation into endocrine cells under 3-dimensional cultivation system. In the present study, we further characterized S100ß-positive PS-clusters using an in vitro 2-dimensional cultivation system. The results demonstrated that S100ß-positive PS-clusters in the 2-dimensional cultivation system proliferated more actively than S100ß-negative clusters. Moreover, in 2-dimensional cultivation conditions, S100ß-positive PS-clusters showed differentiation capacity into non-endocrine cells (Myogenin-, αSMA-, NG2-, or SOX17-positive cells) but not into endocrine cells, whereas S100ß-negative PS-clusters did not. Collectively, PS-clusters were heterogeneous, exhibiting different proliferation and differentiation properties based on the difference in S100ß-expression. Specifically, a part of SOX2-positive cells in the parenchymal-niche had capacities for differentiation into non-endocrine cells, and S100ß-positive PS-clusters may be in more progressive stages toward differentiation than S100ß-negative clusters.

Candidate biomarkers predictive of anthracycline and taxane efficacy against breast cancer.

BACKGROUND: Since breast cancer shows diversity in clinical behaviors, a standard therapy does not always lead to favorable outcomes. MATERIALS AND METHODS: The expression statuses of candidate markers, including topoisomerase-II alpha (TOP2A), beta-tubulin (B-tub), and tissue inhibitor of metalloprotease-1 (TIMP-1), were immunohistochemically evaluated in 70 breast cancer tissues from 68 patients with advanced breast cancers receiving chemotherapy. RESULTS: The response rates to anthracycline and taxane were 70.5% and 67.2%, respectively. Overall, 25.1% ± 29.7%, 8.32% ± 10.1%, and 16.37% ±17.5% of cancer cells in the tumors studied were positive for B-tub, TOP2A, and TIMP-1 expressions, respectively. However, positive molecule expression did not differ between patients who did and did not exhibit clinical responses to treatment. The proportion of TOP2A-positive cancer cells was significantly higher among anthracycline responders than among nonresponders in HR-negative cancer (15.4% ±17.5% vs. 2.0% ± 2.4%, respectively, P = 0.048), whereas TOP2A and TIMP-1 expression statuses did not differ in HR-positive cancer. When patients were stratified according to B-tub, TOP2A, or TIMP-1 expression statuses (B-tub ≥10% vs. <10%, TOP2A ≥5% vs. <5%, TIMP-1 ≤20% vs. >20%, respectively), the proportion of patients with ≥10% B-tub-positive cancer cells was significantly higher in taxane responders than in nonresponders (72.4% vs. 37.5%, respectively, P = 0.016). Anthracycline responders showed a trend to have a higher proportion of patients with either ≥5% TOP2A-positive cancer cells or ≤20% TIMP-1-positive cancer cells compared to nonresponders (86.7% vs. 61.5%, respectively, P = 0.066). CONCLUSION: Immunohistochemical TOP2A, TIMP-1, and B-tub expression analyses are expected to be useful for predicting tumor responses to chemotherapy.

TCR Repertoire Analysis Reveals Mobilization of Novel CD8+ T Cell Clones Into the Cancer-Immunity Cycle Following Anti-CD4 Antibody Administration.

Depletion of CD4+ cells using an anti-CD4 monoclonal antibody (anti-CD4 mAb) induces the expansion of tumor-reactive CD8+ T cells and strong antitumor effects in several murine tumor models. However, it is not known whether the anti-CD4 mAb treatment activates a particular or a broad spectrum of tumor-reactive CD8+ T cell clones. To investigate the changes in the TCR repertoire induced by the anti-CD4 mAb treatment, we performed unbiased high-throughput TCR sequencing in a B16F10 mouse subcutaneous melanoma model. By Inter-Organ Clone Tracking analysis, we demonstrated that anti-CD4 mAb treatment increased the diversity and combined frequency of CD8+ T cell clones that overlapped among the tumor, draining lymph node (dLN), and peripheral blood repertoires. Interestingly, the anti-CD4 mAb treatment-induced expansion of overlapping clones occurred mainly in the dLN rather than in the tumor. Overall, the Inter-Organ Clone Tracking analysis revealed that anti-CD4 mAb treatment enhances the mobilization of a wide variety of tumor-reactive CD8+ T cell clones into the Cancer-Immunity Cycle and thus induces a robust antitumor immune response in mice.

Mumps Virus Induces Protein-Kinase-R-Dependent Stress Granules, Partly Suppressing Type III Interferon Production.

Stress granules (SGs) are cytoplasmic granular aggregations that are induced by cellular stress, including viral infection. SGs have opposing antiviral and proviral roles, which depend on virus species. The exact function of SGs during viral infection is not fully understood. Here, we showed that mumps virus (MuV) induced SGs depending on activation of protein kinase R (PKR). MuV infection strongly induced interferon (IFN)-λ1, 2 and 3, and IFN-ß through activation of IFN regulatory factor 3 (IRF3) via retinoic acid inducible gene-I (RIG-I) and the mitochondrial antiviral signaling (MAVS) pathway. MuV-induced IFNs were strongly upregulated in PKR-knockdown cells. MuV-induced SG formation was suppressed by knockdown of PKR and SG marker proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and T-cell-restricted intracellular antigen-1, and significantly increased the levels of MuV-induced IFN-λ1. However, viral titer was not altered by suppression of SG formation. PKR was required for induction of SGs by MuV infection and regulated type III IFN (IFN-λ1) mRNA stability. MuV-induced SGs partly suppressed type III IFN production by MuV; however, the limited suppression was not sufficient to inhibit MuV replication in cell culture. Our results provide insight into the relationship between SGs and IFN production induced by MuV infection.

Indication of metronomic chemotherapy for metastatic breast cancer: Clinical outcomes and responsive subtypes.

The survival of patients with metastatic breast cancer (MBC) has not improved, despite recent advances in therapeutic strategies. This is mainly due to the fact that cytotoxic agents cannot be administered over a long period, even if they exhibit favorable activity, due to treatment-related side effects or acquisition of tumor resistance to the administered agents. Thus, the development of therapeutic strategies that may be used over a long time period is required to improve survival. We assessed the availability and clinical outcomes of metronomic chemotherapy, which is defined as continuous or frequent treatment with low doses of cytotoxic drugs. A total of 80 patients with MBC received chemotherapy in the metastatic setting, and the clinicopathological factors and clinical outcomes were retrospectively compared between 52 patients who received metronomic regimens and 28 patients who received other cytotoxic regimens. As regards clinical outcomes, the median time-to-treatment failure (TTF) and overall survival (OS) were significantly longer in the metronomic group compared with those in the non-metronomic group (TTF, 15 vs. 4 months, P=0.0001; and OS, 53 vs. 28 months P=0.0012, respectively). In the metronomic group, none of the 18 patients who responded to the regimen had triple-negative (TN) cancer (17 had luminal-type tumors and 1 had a human epidermal factor receptor 2-type tumor). Furthermore, TTF and OS were significantly longer in patients with non-TN cancer compared with those in patients with TN cancer in the metronomic group (TTF, 16 vs. 7 months, P=0.0014; and OS, 108 vs. 20 months, P=0.000007, respectively). The proportion of patients who experienced treatment-related adverse events was significantly lower in the metronomic group compared with that in the non-metronomic group (36.5 vs. 61.5%, respectively; P=0.038). In conclusion, metronomic chemotherapy is a viable option for luminal-type MBC in terms of effectiveness and minimal toxicity, regardless of metastatic sites or prior treatment. However, an alternative treatment is required for TN cancer.

Essential roles of the interaction between cancer cell-derived chemokine, CCL4, and intra-bone CCR5-expressing fibroblasts in breast cancer bone metastasis.

From a murine breast cancer cell line, 4T1, we established a subclone, 4T1.3, which consistently metastasizes to bone upon its injection into the mammary fat pad. 4T1.3 clone exhibited similar proliferation rate and migration capacity as the parental clone. However, the intra-bone injection of 4T1.3 clone caused larger tumors than that of the parental cells, accompanied with increases in fibroblast, but not osteoclast or osteoblast numbers. 4T1.3 clone displayed an enhanced expression of a chemokine, CCL4, but not its specific receptor, CCR5. CCL4 shRNA-transfection of 4T1.3 clone had few effects on its in vitro properties, but reduced the tumorigenicity arising from the intra-bone injection. Moreover, intra-bone injection of 4T1.3 clone caused smaller tumors in mice deficient in CCR5 or those receiving CCR5 antagonist than in wild-type mice. The reduced tumor formation was associated with attenuated accumulation of CCR5-positive fibroblasts expressing connective tissue growth factor (CTGF)/CCN2. Tumor cell-derived CCL4 could induce fibroblasts to express CTGF/CCN2, which could support 4T1.3 clone proliferation under hypoxic culture conditions. Thus, the CCL4-CCR5 axis can contribute to breast cancer metastasis to bone by mediating the interaction between cancer cells and fibroblasts in bone cavity.

Measles Virus Genotype D Wild Strains Suppress Interferon-Stimulated Gene Expression More Potently than Laboratory Strains in SiHa Cells.

Changes in interferon (IFN)-stimulated gene (ISG) expression in cells infected with measles virus (MeV), four wild strains (belonging to different genotypes), and the laboratory strain Edmonston were examined. ISGs [MxA, 2'-5'-oligoadenylate synthetase, and interferon regulatory factor-1] were upregulated in an MeV-infection-induced manner and in an IFN-induced manner. In MeV-infected SiHa cell lines, the MeV infection-induced expression levels were in the order of A>H1>D8>D5>D3. On the other hand, all infected cell lines abolished type I and III IFN-induced ISG expression. However, partial type II IFN-mediated induction was observed in the MeV-infected cells. The wild strain of genotype D3 was the most potent inhibitor of MeV infection-induced and IFN-induced ISG expression and generated the highest titer of infectious viral particles. Edmonston triggered the highest levels of MeV infection-induced ISG expression in SiHa cells and produced the lowest titer of infectious particles. Expression of the viral C protein was associated with suppression of MeV infection-induced and type II IFN-induced ISG expression.

Reduced supply of monocyte-derived macrophages leads to a transition from nodular to diffuse lesions and tissue cell activation in silica-induced pulmonary fibrosis in mice.

Pulmonary fibrosis (PF) is an intractable disorder with a poor prognosis. Lung macrophages have been reported to regulate both progression and remission of bleomycin-induced diffuse PF. However, it remains unclear how macrophages contribute to silica-induced progressive nodular PF and the associated tissue cell responses in vivo. We found that lack of monocyte-derived macrophages results in the formation of diffuse PF after silica instillation. We found that the proportion and the number of monocyte-derived macrophages were persistently higher in silica-induced progressive PF compared with bleomycin-induced PF. Surprisingly, in Ccr2(-/-) mice, in which monocyte-derived macrophage infiltration is impaired, silica administration induced diffuse PF with loose nodule formation and greater activation of tissue cells. In the diffuse lesions, the distribution of epithelial cells, distribution of myofibroblasts, and architecture of the basement membrane were disrupted. Consistent with the development of diffuse lesions, genes that were differentially expressed in CD45(-) tissue cells from the lung of wild-type and Ccr2(-/-) mice were highly enriched in human diffuse, progressive PF. In gene ontology network analyses, many of these genes were associated with tissue remodeling and included genes not previously associated with PF, such as Mmp14, Thbs2, and Fgfr4. Overall, these results indicate that monocyte-derived macrophages prevent transition from nodular to diffuse silica-induced PF, potentially by regulating tissue cell responses.

Factors responsible for long-term survival in metastatic breast cancer.

BACKGROUND: Although survival of patients with metastatic breast cancer (MBC) has been significantly prolonged over the past decade due to improvement of anti-cancer therapeutics, only a few patients survive for more than 10 years. It has not been determined which patients can have long-term survival with treatment. METHODS: To determine prognostic factors responsible for long-term survival, we retrospectively compared clinicopathologic factors of patients with MBC who survived for 50 months or more after diagnosis with patients who did not. Of 70 patients with MBC who received chemotherapy between November 2005 and September 2011, 23 patients who survived for 50 months or more after diagnosis and 28 patients who died within 50 months after diagnosis were assessed for their clinicopathologic factors and outcomes. RESULTS: The proportion of patients with hormone receptor-positive (HR+) tumors was significantly higher and the proportion of patients with triple negative tumors (TN) was lower in long-term survivors than in non-long-term survivors (HR+: 87% versus 28.6%, P=0.000037; TN: 13.1% versus 53.6%, P=0.0028). Metastatic site, number of disease sites, prior chemotherapeutic regimens and human epidermal growth factor receptor-2 (HER2) status did not differ between the two groups. The proportion of patients who received metronomic regimens was significantly higher in long-term survivors than in non-long-term survivors (65.2% versus 35.7%, P=0.034) when the most effective regimen among regimens that were received in metastatic settings was compared between the two groups. Overall response rate was significantly higher (82.6% versus 17.9%, P<0.00001) and time to treatment failure after receiving the most effective regimen was longer in long-term survivors than in non-long-term survivors (26 versus 5 months, P=0.0001). The number of chemotherapeutic regimens for breast cancer and that for MBC did not differ between the two groups. CONCLUSIONS: Patients with luminal-type MBC who benefit at least once from chemotherapy including metronomic regimens, or patients who continued to receive the most effective regimen for more than two years can be expected to have long-term survival after diagnosis of MBC, regardless of the number of chemotherapeutic regimens they had received.

Lymph node stromal cells negatively regulate antigen-specific CD4+ T cell responses.

Lymph node (LN) stromal cells (LNSCs) form the functional structure of LNs and play an important role in lymphocyte survival and the maintenance of immune tolerance. Despite their broad spectrum of function, little is known about LNSC responses during microbial infection. In this study, we demonstrate that LNSC subsets display distinct kinetics following vaccinia virus infection. In particular, compared with the expansion of other LNSC subsets and the total LN cell population, the expansion of fibroblastic reticular cells (FRCs) was delayed and sustained by noncirculating progenitor cells. Notably, newly generated FRCs were preferentially located in perivascular areas. Viral clearance in reactive LNs preceded the onset of FRC expansion, raising the possibility that viral infection in LNs may have a negative impact on the differentiation of FRCs. We also found that MHC class II expression was upregulated in all LNSC subsets until day 10 postinfection. Genetic ablation of radioresistant stromal cell-mediated Ag presentation resulted in slower contraction of Ag-specific CD4(+) T cells. We propose that activated LNSCs acquire enhanced Ag-presentation capacity, serving as an extrinsic brake system for CD4(+) T cell responses. Disrupted function and homeostasis of LNSCs may contribute to immune deregulation in the context of chronic viral infection, autoimmunity, and graft-versus-host disease.

Qualitative rather than quantitative changes are hallmarks of fibroblasts in bleomycin-induced pulmonary fibrosis.

Pulmonary fibrosis is characterized by accumulation of activated fibroblasts that produce excessive amounts of extracellular matrix components such as collagen type I. However, the dynamics and activation signatures of fibroblasts during fibrogenesis remain poorly understood, especially in vivo. We examined changes in lung tissue cell populations and in the phenotype of activated fibroblasts after acute injury in a model of bleomycin-induced pulmonary fibrosis. Despite clustering of collagen type I-producing fibroblasts in fibrotic regions, flow cytometry-based quantitative analysis of whole lungs revealed that the number of fibroblasts in the lungs remained constant. At the peak of inflammation, fibroblast proliferation and apoptosis were both increased, suggesting that the clustering was not merely a result of proliferation, but also of fibroblast migration from nearby alveolar walls. Parabiosis experiments demonstrated that fibroblasts were not supplied from the circulation. Comprehensive gene expression analysis of freshly isolated fibroblasts revealed a detailed activation signature associated with fibrogenesis, including changes in genes responsible for migration and extracellular matrix construction. The Spp1 gene, which encodes osteopontin, was highly up-regulated and was an identifying characteristic of activated fibroblasts present at the sites of remodeling. Osteopontin may serve as a useful marker of profibrotic fibroblasts. These results provide insights into the cellular and molecular mechanisms underlying pulmonary fibrosis and provide a foundation for development of specific antifibrotic therapies.

Epidermal cyst of the breast treated by vacuum-assisted biopsy.

A 38-year-old woman presented for evaluation of a firm mass (measuring 20 × 20 mm) in the upper outer quadrant of her left breast. On the basis of the clinical and radiologic findings, we diagnosed a benign breast tumor and scheduled removal by a hand-held vacuum-assisted biopsy device (VABD) under ultrasonographic guidance. Because the first specimen removed from the tumor was white, flaky, and waxy material, we strongly suspected that the lesion was an epidermal cyst. We continued VABD treatment until the tumor was invisible on real-time ultrasonography. Histologic examination demonstrated that the tumor was composed of mature stratified squamous epithelium and laminated layers of keratin, findings consistent with a diagnosis of epidermal cyst. These cysts rarely occur in the breast and are sometimes difficult to distinguish from breast cancer. To our knowledge, this is the first report of an epidermal cyst treated by VABD.

Metronomic chemotherapy for metastatic breast cancer to prolong time to treatment failure to 12 months or more.

The objective of treatment for metastatic breast cancer (MBC) is to control the disease or disease-related symptoms. Prolonged survival has also often been achieved by chemotherapeutic regimens in this setting. Long-term administration of one therapeutic regimen is essential for prolonging survival as well as for maintaining quality of life in these patients. In this study, we focused on time to treatment failure (TTF) as a parameter that predicts patient survival and we retrospectively compared clinical outcomes of patients with MBC who showed TTF of ≥12 months (26 patients) and <12 months (29 patients). The proportion of hormone receptor-positive tumors and the number of prior chemotherapy regimens for MBC were significantly higher and tumor grade was lower in patients with TTF ≥12 months compared to those with TTF <12 months. With regard to clinical outcomes, the objective response rate (ORR) in patients with TTF ≥12 months was significantly higher and median time to progression (TTP) and overall survival (OS) were longer compared to those with TTF <12 months. Of note, the proportion of patients who received metronomic regimens was significantly higher in patients with TTF ≥12 months compared to those with TTF <12 months (80.8 vs. 24.1%, P=0.00003). To assess the clinical benefit of metronomic regimens, the efficacy in patients receiving metronomic and those receiving non-metronomic regimens was compared. Although there was no difference in ORR between the two groups, median TTP and OS were significantly longer in the metronomic compared to the non-metronomic group (TTP: 30 vs. 4 months, P=0.0017; OS: 68 vs. 28 months, P=0.0005). The results suggested that metronomic chemotherapy is useful for palliative care and also improved clinical outcomes as a regimen for which long-term administration may be expected.

Clinical usefulness of human epidermal growth factor receptor-2 extracellular domain as a biomarker for monitoring cancer status and predicting the therapeutic efficacy in breast cancer.

We assessed the clinical usefulness of human epidermal growth factor receptor-2 extracellular domain (HER2ECD) as a biomarker for detecting cancer and monitoring disease status and for predicting the efficacy of anticancer treatment in breast cancer. Five-hundred and eighty serum samples from 252 patients with breast cancer were examined for the concentration of HER2ECD to compare with conventional tumor markers (CEA, CA15-3, NCC-ST439 and BCA225). Also, in 19 patients with HER2-overexpressed advanced or recurrent breast cancer who were treated with trastuzumab, clinical outcomes were evaluated retrospectively to determine whether their serum HER2ECD levels predict clinical responses. The proportion of patients with elevated HER2ECD levels was 15.1%, which was compatible with those with elevated conventional marker levels. In patients with HER2-overexpressed breast cancer, the positive rate of HER2ECD was significantly higher (24.1%) than those of conventional markers (7.4-12.9%), suggesting the usefulness of HER2ECD for detecting cancer in this population. HER2-overexpressed patients responding to trastuzumab (12 of 19 patients) showed significantly higher serum HER2ECD level (p = 0.033) and longer time to progression (TTP) (p = 0.039) and overall survival (OS) (p = 0.031) than did patients not responding (seven patients). Furthermore, higher response rates were observed in patients with elevated HER2ECD levels than in patients without elevated HER2ECD levels (91.3% vs. 14.3%, p = 0.032), whereas there was no difference in survival between the two groups. The results suggest that HER2ECD is a useful biomarker not only for detecting breast cancer recurrence but also for predicting tumor responses to trastuzumab.

Genome-wide profiling of DNA methylation in human cancer cells.

Global changes in DNA methylation correlate with altered gene expression and genomic instability in cancer. We have developed a methylation-specific digital sequencing (MSDS) method that can assess DNA methylation on a genomic scale. MSDS is a simple, low-cost method that combines the use of methylation-sensitive restriction enzymes with second generation sequencing technology. DNA methylation in two colon cancer cell lines, HT29 and HCT116, was measured using MSDS. When methylation levels were compared between the two cell lines, many differentially methylated regions (DMRs) were identified in CpG island shore regions (located within 2kb of a CpG island), gene body regions and intergenic regions. The number of DMRs in the vicinity of gene transcription start sites correlated with the level of expression of TACC1, CLDN1, and PLEKHC1 (FERMT2) genes, which have been linked to carcinogenesis. The MSDS method has the potential to provide novel insight into the functional complexity of the human genome.

Comprehensive gene expression analysis of 5'-end of mRNA identified novel intronic transcripts associated with hepatocellular carcinoma.

To elucidate the molecular feature of human hepatocellular carcinoma (HCC), we performed 5'-end serial analysis of gene expression (5'SAGE), which allows genome-wide identification of transcription start sites in addition to quantification of mRNA transcripts. Three 5'SAGE libraries were generated from normal human liver (NL), non-B, non-C HCC tumor (T), and background non-tumor tissues (NT). We obtained 226,834 tags from these libraries and mapped them to the genomic sequences of a total of 8,410 genes using RefSeq database. We identified several novel transcripts specifically expressed in HCC including those mapped to the intronic regions. Among them, we confirmed the transcripts initiated from the introns of a gene encoding acyl-coenzyme A oxidase 2 (ACOX2). The expression of these transcript variants were up-regulated in HCC and showed a different pattern compared with that of ordinary ACOX2 mRNA. The present results indicate that the transcription initiation of a subset of genes may be distinctively altered in HCC, which may suggest the utility of intronic RNAs as surrogate tumor markers.

Massive transcriptional start site analysis of human genes in hypoxia cells.

Combining our full-length cDNA method and the massively parallel sequencing technology, we developed a simple method to collect precise positional information of transcriptional start sites (TSSs) together with digital information of the gene-expression levels in a high throughput manner. We applied this method to observe gene-expression changes in a colon cancer cell line cultured in normoxic and hypoxic conditions. We generated more than 100 million 36-base TSS-tag sequences and revealed comprehensive features of hypoxia responsive alterations in the transcriptional landscape of the human genome. The features include presence of inducible 'hot regions' in 54 genomic regions, 220 novel hypoxia inducible promoters that may drive non-protein-coding transcripts, 191 hypoxia responsive alternative promoters and detailed views of 120 novel as well as known hypoxia responsive genes. We further analyzed hypoxic response of different cells using additional 60 million TSS-tags and found that the degree of the gene-expression changes were different among cell lines, possibly reflecting cellular robustness against hypoxia. The novel dynamic figure of the human gene transcriptome will deepen our understanding of the transcriptional program of the human genome as well as bringing new insights into the biology of cancer cells in hypoxia.