Keiji Tanese

G-Protein-Coupled Receptor GPR49 is Up-regulated in Basal Cell Carcinoma and Promotes Cell Proliferation and Tumor Formation

The significance of Hedgehog (HH) signaling in the development of basal cell carcinoma (BCC) has been established. Although several target genes of HH signaling have been described previously, their precise role in tumorigenesis and cell proliferation is not yet known. To identify genes responsible for tumor formation in BCC, we screened a DNA microarray database of human BCC cases; the orphan G-protein-coupled receptor GPR49 was found to be up-regulated in all cases. GPR49 is a novel gene reported to be a marker of follicular and other tissue stem cells. Using real-time quantitative RT-PCR analysis, significant expression of GPR49 mRNA was observed in 19 of 20 BCC cases (95%) compared with controls. Up-regulation of GPR49 was confirmed by in situ hybridization. Moreover, knockdown of mouse Gpr49 showed suppression of cell proliferation in a mouse BCC cell line, and overexpression of GPR49 in human immortalized keratinocyte HaCaT cells induced proliferation. Furthermore, HaCaT cells overexpressing GPR49 showed tumor formation when transplanted into immunodeficient mice. In addition, inhibition of the HH signaling pathway in a mouse BCC cell line down-regulated endogenous Gpr49, whereas activation of HH signaling in mouse NIH3T3 cells up-regulated endogenous GPR49. These results suggest that GPR49 is expressed downstream of HH signaling and promotes cell proliferation and tumor formation in cases of BCC.

Identification of mutations in the prostaglandin transporter gene SLCO2A1 and its phenotype–genotype correlation in Japanese patients with pachydermoperiostosis

BACKGROUND Pachydermoperiostosis (PDP) is a rare genetic disorder characterized by 3 major symptoms: pachydermia including cutis verticis gyrata (CVG), periostosis, and finger clubbing. Recently, a homozygous mutation in the gene HPGD, which encodes 15-hydroxyprostaglandin dehydrogenase (15-PGDH), was found to be associated with PDP. However, mutations in HPGD have not been identified in Japanese PDP patients. OBJECTIVE We aimed to identify a novel responsible gene for PDP using whole exome sequencing by next-generation DNA sequencer (NGS). METHODS Five patients, including 2 patient-parent trios were enrolled in this study. Entire coding regions were sequenced by NGS to identify candidate mutations associated with PDP. The candidate mutations were subsequently sequenced using the Sanger method. To determine clinical characteristics, we analyzed histological samples, as well as serum and urinary prostaglandin E2 (PGE2) levels for each of the 5 PDP patients, and 1 additional patient with idiopathic CVG. RESULTS From initial analyses of whole exome sequencing data, we identified mutations in the solute carrier organic anion transporter family, member 2A1 (SLCO2A1) gene, encoding prostaglandin transporter, in 3 of the PDP patients. Follow-up Sanger sequencing showed 5 different SLCO2A1 mutations (c.940+1G>A, p.E427_P430del, p.G104*, p.T347I, p.Q556H) in 4 unrelated PDP patients. In addition, the splice-site mutation c.940+1G>A identified in 3 of 4 PDP patients was determined to be a founder mutation in the Japanese population. Furthermore, it is likely that the combination of these SLCO2A1 mutations in PDP patients is also associated with disease severity. CONCLUSION We found that SLCO2A1 is a novel gene responsible for PDP. Although the SLCO2A1 gene is only the second gene discovered to be associated with PDP, it is likely to be a major cause of PDP in the Japanese population.

Nivolumab for advanced melanoma: pretreatment prognostic factors and early outcome markers during therapy

Background An anti-programmed cell death protein 1 monoclonal antibody, nivolumab, is one of the most effective drugs for advanced melanoma. Tumor cell-derived or immune cell-derived markers and clinical predictors such as serum lactate dehydrogenase (LDH) and cutaneous adverse events, have already been described as prognostic factors for advanced melanoma treated with nivolumab. We sought to identify further clinical predictors that can be determined in routine clinical practice. Methods We retrospectively analyzed clinical findings of 98 consecutive patients with unresectable stage III or IV melanoma treated with nivolumab, at the National Cancer Center Hospital or at Keio University Hospital, in Tokyo, Japan, between July 2014 and July 2016. These patients had been administered nivolumab at a dose of 2mg/kg every 3 weeks. Results As for pretreatment prognostic factors, ECOG performance status (PS) ≥1, maximum tumor diameters of ≥30mm, elevated LDH and elevated C-reactive protein were significantly associated with poor overall survival (OS) (hazard ratio [HR] 0.29 [P<0.001], HR 0.40 [p=0.003], HR 0.29 [P<0.001], HR 0.42 [P=0.004], respectively) on univariate analysis. Among these factors, PS and LDH were identified as independent variables by multivariate analysis. As for early markers examined during therapy, patients with absolute lymphocyte count (ALC) ≥ 1000/μl (Week3: HR 0.40 [P=0.004], Week6: HR 0.33 [P=0.001]) and absolute neutrophil count (ANC) <4000/μl (Week3: HR 0.46 [P=0.014], Week6: HR 0.51 [P=0.046]) had significantly better OS. Conclusion ALC≥1000/μl and ANC<4000/μl during treatment appear to be early markers associated with OS. Nivolumab might have minimal efficacy in patients with a massive tumor burden.

The role of melanoma tumor-derived nitric oxide in the tumor inflammatory microenvironment: Its impact on the chemokine expression profile, including suppression of CXCL10

Melanoma appears to be heterogeneous in terms of its molecular biology, etiology and epidemiology. We previously reported that the expression of inducible nitric‐oxide synthase (iNOS) in melanoma tumor cells is strongly correlated with poor patient survival. Therefore, we hypothesized that nitric oxide (NO) produced by iNOS promotes the melanoma inflammatory tumor microenvironment associated with poor outcome. To understand the role of NO and iNOS in the melanoma inflammatory tumor microenvironment, polymerase chain reaction arrays of inflammatory and autoimmunity genes were performed on a series of stage III melanoma lymph node metastasis samples to compare the gene expression profiles of iNOS‐expressing and nonexpressing tumor samples. The results indicate that expression of CXC chemokine ligand 10 (CXCL10) was inversely correlated with iNOS expression, and the high CXCL10‐expressing cases had more favorable prognoses than the low CXCL10‐expressing cases. Functional studies revealed that treating iNOS‐negative/CXCL10‐positive melanoma cell lines with a NO donor suppressed the expression of CXCL10. Furthermore, scavenging NO from iNOS‐expressing cell lines significantly affected the chemokine expression profile. Culture supernatants from NO scavenger‐treated melanoma cells promoted the migration of plasmacytoid dendritic cells, which was diminished when the cells were treated with a CXCL10‐neutralizing antibody. CXCL10 has been reported to be an antitumorigenic chemokine. Our study suggests that the production of NO by iNOS inhibits the expression of CXCL10 in melanoma cells and leads to a protumorigenic tumor microenvironment. Inhibiting NO induces an antitumorigenic environment, and thus, iNOS should be considered to be an important therapeutic target in melanoma.

Leucine-rich repeat-containing G protein-coupled receptor 5 regulates epithelial cell phenotype and survival of hepatocellular carcinoma cells.

The leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), also known as GPR49, is a seven-transmembrane receptor that is expressed in stem cells of the intestinal crypts and hair follicles of mice. LGR5 is overexpressed in some types of human cancer, and is one of the target genes of the Wnt signaling pathway. To explore the function of LGR5 in cancer cells, stable hepatocellular carcinoma (HCC) cell lines expressing FLAG-tagged LGR5 were established. Overexpression of LGR5 resulted in changes in cell shape from an extended flat (mesenchymal) phenotype to a round aggregated (stem cell-like) phenotype. Cells transfected with LGR5 showed higher colony forming activity, and were more resistant to a cytotoxic drug than cells transfected with empty vector. Overexpression of LGR5 inhibited cell motility. LGR5-transfected cells formed nodule type tumors in the livers of immunodeficient mice, whereas empty vector-transfected cells formed more invasive tumors. Down-regulation of LGR5 changed the morphology of HCC cells from the aggregated phenotype to an extended spindle phenotype, and cell motility was increased. This is the first study reporting the functional role of LGR5 in the biology of HCC cells, and the results suggest that aberrant expression of LGR5 regulates epithelial cell phenotype and survival.

Malignant eccrine spiradenoma: case report and review of the literature, including 15 Japanese cases.

Malignant eccrine spiradenoma (MES) is an extremely rare cutaneous malignant tumour. An 86‐year‐old man presented at our hospital with an enlarging tumour on the dorsum of the left hand. An excisional biopsy was taken and histological examination showed a solid island of cells of two distinct types: cells with abundant cytoplasm and oval vesicular nuclei, and small round cells with less cytoplasm and hyperchromatic nuclei with a high frequency of mitosis. We diagnosed this tumour as MES. Although we did not perform further treatment because of the patient’s age, there was no sign of recurrence or metastasis in the 2 years of follow‐up after excisional biopsy. We reviewed cases of malignant eccrine spiradenoma in the English and Japanese literature and found that ‘sarcomatous’ or ‘squamous’ change in histopathology was significantly correlated with a poorer prognosis. It is therefore important for treatment planning to evaluate the entire specimen histologically.

The novel SLCO2A1 heterozygous missense mutation p.E427K and nonsense mutation p.R603* in a female patient with pachydermoperiostosis with an atypical phenotype.

DEAR EDITOR, Pachydermoperiostosis (PDP), or primary hypertrophic osteoarthropathy (PHO: MIM 167100), is a rare genetic disease affecting the skin and bones. The major diagnostic criteria include finger clubbing, periostosis, pachydermia and cutis verticis gyrata (CVG). Additional symptoms, including sebaceous hyperplasia, hyperhidrosis and arthropathy, have been reported. Uppal et al. discovered that a homozygous mutation in HPGD, which encodes 15-hydroxyprostaglandin dehydrogenase (15-PGDH), causes PHO and PDP. However, PHO and PDP are genetically heterogeneous. Exome analysis of PDP in Japanese, Chinese, Caucasian and other races has revealed homozygous mutations in the solute carrier organic anion transporter family member 2A1 (SLCO2A1) gene, which encodes prostaglandin transporter (PGT). Increased levels of prostaglandin E2 (PGE2) resulting from defective degradation contribute to the pathogenesis of PHO and PDP. A genetic defect in either SLCO2A1 or HPGD can cause PHO and PDP. In this study, we describe the first observation of a SLCO2A1 mutation in a female patient. A 67-year-old woman was referred for SLCO2A1 mutation analysis. At the age of 43 years, she developed myelopathy of unknown aetiology. She received rehabilitation therapy without medication. A neurologist had examined her muscle weakness at the T-helper 7 level on the right side following a diagnosis of suspected multiple sclerosis. At the age of 64 years, she had multiple seronegative arthralgias but no serious problems. She was referred to Tohoku Kouseinenkin Hospital because of recurring arthralgia and was treated with methotrexate and prednisone. She responded favourably to the medication with alleviation of the pain and decreased serum levels of C-reactive protein. Physical examination revealed finger clubbing and swelling of the large joints, as seen in Figure 1b,c. No skin manifestations, including facial coarseness or greasiness, and no hyperhidrosis were observed. Marked thickening of the scalp (CVG) was not evident. Radiological examination showed the presence of periostosis of the diaphysis of the tibia and fibula (Fig. 1d). No hydrarthrosis was evident. A diagnosis of possible incomplete type of PDP or PHO was made because of minimal pachydermia. She had no history of peptic ulcers or anaemia. Diagnostic imaging and laboratory data revealed no evidence of secondary PDP. She has a healthy son and daughter. This study was approved by the ethics committee of the National Centre for Child Health and Development and Keio University School of Medicine. The participants provided written informed consent. All exons of HPGD and SLCO2A1 along with sequences adjacent to the exon–intron borders were amplified, sequenced and screened for mutations. Serum and urinary levels of PGE2 were measured with a commercial enzyme immunoassay kit (Cayman, Cayman Biochemical, Ann Arbor, MI, U.S.A.). We identified compound heterozygous novel mutations c.1279G>A/p.E427K and c.1807C>T/p.R603* in SLCO2A1 (Fig. 2). We also detected a heterozygous mutation c.1279G>A in her daughter, but she has not developed any triad of PDP. In her seventh decade, the patient’s atypical history showed minimal impact of pachydermia. Serum PGE2 was not detected and her urinary PGE2 was within normal limits (372 pg mL ). One of the mutations, c.1279G>A (p.E427K), is included within the region of a previously reported deletion, c.1279_1290del12 (p.E427_P430del). Another mutation, c.1807C>T/p.R603*, is detected close to the C-terminus of PGT, resulting in a shortened predicted protein. The loss of function in truncated PGT is consistent with the presence of the p.R603* mutation in another patient, who had the complete type of PDP (manuscript in preparation). This patient is the first woman with PDP who had an SLCO2A1 gene mutation. It is unlikely that the mild phenotype of P1 was due to the missense mutation p.E427K. A recent report on PDP in a Chinese family described a homozygous p.A286Qfs*35 frameshift mutation in a male proband who had PDP. Two of the proband’s sisters were also homozygous for p.A286Qfs*35, but at ages 42 years and 47 years, they had neither history nor findings suggestive of PDP. Diggle et al. reported that two women in two PDP families were homozygous for pathogenic SLCO2A1 mutations. One had mild finger clubbing but no musculoskeletal or skin symptoms at 34 years of age. The other was asymptomatic at 19 years of age. Taken together, we propose that PDP resulting from SLCO2A1 defects is a sex-dependent autosomal recessive disease, and women homozygous for pathogenic SLCO2A1 mutations may develop late-onset PDP symptoms. The explanation of mildand low-frequency disease in women remains unclear. Hatano et al. suggested that reactivity to prostaglandin was milder in women than in men. Ospina et al.

Erratum: Cell Surface CD74-MIF Interactions Drive Melanoma Survival in Response to Interferon-γ

While melanoma is believed to be a highly immunogenic tumor and recent developments in immunotherapies are promising. Interferon-γ (IFN-γ) produced by immune cells plays a crucial role in tumor immune surveillance; however, it has also been reported to be pro-tumorigenic. In the current study, we found that IFN-γ enhances the expression of CD74, which interacts with its ligand, macrophage migration inhibitory factor (MIF), and thereby activates the PI3K/AKT pathway in melanoma, promoting tumor survival. IFN-γ increased phosphorylation of AKT Ser473 and upregulated total and cell surface expression of CD74 in human melanoma cell lines tested. CD74 was highly expressed in melanoma tissues. Moreover, the expression of CD74 on tumor cells correlated with plasma IFN-γ levels in melanoma patient samples. In our analysis of melanoma cell lines, all produced MIF constitutively. Blockade of CD74-MIF interaction reduced AKT phosphorylation and expression of pro-tumorigenic molecules, including interleukin-6, interleukin-8 and BCL-2. Inhibition of CD74-MIF interaction significantly suppressed tumor growth in the presence of IFN-γ in our xenograft mouse model. Thus, we conclude that IFN-γ promotes melanoma cell survival by regulating CD74-MIF signaling, suggesting that targeting the CD74-MIF interaction under IFN-γ–stimulatory conditions would be an effective therapeutic approach for melanoma.

Epstein-Barr Virus-Positive Hodgkin Lymphoma-Like Earlobe Lymphoid Infiltrate: Case Report

Hodgkin lymphoma (HL) is a malignancy of the lymph system usually occurring on the lymph nodes. A 57-year-old Japanese woman presented with the chief complaint of an enlarging tumor of the left ear. An excisional biopsy was taken, and histological examination showed a mixed infiltration of cells, including Reed-Sternberg cells and their mononuclear forms against a background of small lymphocytes. Reed-Sternberg cells were CD15, CD20, CD30, Ki-67, MUM-1, CD45, EMA, and Epstein-Barr virus-encoded small RNA was detected by in situ hybridization. We diagnosed this tumor as a skin infiltration with a lymphocyte-rich classical HL pattern. Skin involvement of HL is most often a secondary phenomenon representing a rare late manifestation of disease dissemination; however, we could not detect any evidence of systemic lesion for 6 months after the initial presentation. A case of HL only involving the skin was reported by several past reports, which termed it primary cutaneous HL. But, it is still controversial whether HL initially occurs on the skin because a diagnostic gray zone exists between HL, some non-HL entities, and nonneoplastic lymphoid infiltrates. Clinical and histological features of this case suggest that the skin will become a primary site of HL.

Pathological characterization of pachydermia in pachydermoperiostosis

Pachydermoperiostosis is a rare hereditary disease, which presents with the cutaneous manifestations of pachydermia and cutis verticis gyrata. Histological findings in pachydermia frequently include dermal edema, mucin deposition, elastic fiber degeneration, dermal fibrosis and adnexal hyperplasia. However, the severity of these findings varies between clinical reports, and a systematic multiple‐case clinicopathological correlative analysis has not been performed to date. In the present study, we reviewed the skin biopsy specimens obtained from the pachydermia of six pachydermoperiostosis patients. The severity of the characteristic histological features was semiquantitatively evaluated and correlated with the grade of pachydermia. Dermal edema, mucin deposition and elastic fiber degeneration were observed in all cases. Patients with severe pachydermia had sebaceous gland hyperplasia and fibrosis. These results suggest that the triad of mucin deposition, dermal edema and elastic fiber degeneration are found from very early stage pachydermia, and could be considered diagnostic findings. To ensure an earlier diagnosis of pachydermoperiostosis, a biopsy should be taken when a patient has grade 1 pachydermia to determine the presence of this histological triad.