Generation of induced pluripotent stem cell (iPSC) from NY-ESO-I-specific cytotoxic T cells isolated from the melanoma patient with minor HLAs: The practical pilot study for the adoptive immunotherapy for melanoma using iPSC technology.

Melanoma is one of the most severe skin cancers, derived from melanocytes. Among various therapies for melanoma, adoptive immunotherapy using tumor-infiltrating lymphocytes/chimeric antigen receptor-T cells (TCs) is advanced in recent years; however, the efficacy is still limited, and major challenges remain in terms of safety and cell supply. To solve the issues of adoptive immunotherapy, we utilized induced pluripotent stem cells (iPSCs), which have an unlimited proliferative ability and various differentiation capability. First, we monoclonally isolated CD8+ TCs specifically reactive with NY-ESO-1, one of tumor antigens, from the melanoma patient's monocytes after stimulated with NY-ESO-1 peptide by manual procedure, and cultured NY-ESO-1-specific TCs until proliferated and formed colonies. iPSCs were consequently generated from colony-forming TCs by exogenous expression of reprogramming factors using Sendai virus vector. After the RAG2 gene in TC-derived iPSCs (T-iPSCs) was knocked out for preventing T-cell receptor (TCR) rearrangement, T-iPSCs were re-differentiated into rejuvenated cytotoxic TCs. We confirmed that TCR of T-iPSC-derived TC was maintained as the same of original TCs. In conclusion, T-iPSCs have a potential to be an unlimited cell source for providing cytotoxic TCs. Our study could be a "touchstone" to develop iPSC-based adoptive immunotherapy for the treatment of melanoma for the future clinical use.

Role of interleukin-24 in the tumor-suppressive effects of interferon-ß on melanoma.

BACKGROUND: Type 1 interferons (IFNs), including IFN-ß, are widely used in adjuvant therapy for patients who undergo surgery for malignant melanoma to inhibit recurrence and in-transit metastasis. The precise mechanisms underlying the tumor-suppressive effects of IFN-ß on melanoma are not yet completely understood. OBJECTIVE: The purpose was to reveal the mechanisms underlying the tumor-suppressive effects of IFN-ß via interleukin (IL)-24. METHODS: Genome-wide oligonucleotide microarray, quantitative real-time reverse transcription-polymerase chain reaction (PCR), enzyme-linked immunosorbent assay and western blotting assay were performed using four melanoma cell lines (A375, RPMI-7951, SK-MEL-5 and SK-MEL-1) treated with natural-type IFN-ß to assess the expression of IL-24. Proliferation assay was performed using these melanoma cells and IL-24 knock-down melanoma cells. RESULTS: Genome-wide microarray analysis detected candidate genes upregulated in IFN-ß-sensitive cells after treatment with IFN-ß. We focused on IL-24 among the candidate genes encoding secretory proteins. Peak IL-24 mRNA expression completely correlated with the order of sensitivity of melanoma cells to IFN-ß. IFN-ß treatment induced extracellular IL-24 protein in IFN-ß-sensitive cells, but did not induce intracellular IL-24 protein. Knock-down of IL-24 changed melanoma cells into IFN-ß-resistant cells. The expression ratio of IL-22R1, one of the IL-24 receptors, correlated with the order of sensitivity of melanoma cells to IFN-ß. Treatment with recombinant human IL-24 did not have any effects on all the melanoma cell lines. CONCLUSION: Our data suggest that IFN-ß suppresses the proliferation of melanoma cells through extracellular IL-24 protein derived from melanoma cells.

Muir-Torre syndrome: sebaceous carcinoma concurrent with colon cancer in a kidney transplant recipient; a case report.

BACKGROUND: Sebaceous carcinoma is a rare but progressive malignant skin cancer, and the incidence is approximately five times higher in post-transplant patients than in people who have not received kidney transplants. Sebaceous carcinoma is sometimes found concurrently with visceral cancers and a genetic abnormality, Muir-Torre syndrome. We report the case of a female kidney transplant recipient with sebaceous carcinoma concurrent with colon cancer 10 years after transplantation. CASE PRESENTATION: A 43-year-old woman was admitted due to a rapidly progressive tumor on her head. Histologically, the tumor was diagnosed as sebaceous carcinoma. We diagnosed her with Muir-Torre syndrome based on the following evidence: 1) high prevalence of microsatellite instability in gene locus assay, 2) absence of mismatch repair proteins in the sebaceous carcinoma on immunohistochemical analysis, and 3) a genetic mutation of 1226_1227delAG in the MSH2 exon 7 in the lesion detected by DNA sequencing analysis. Several reports have shown an association between immunosuppressive agents and latent Muir-Torre syndrome progression. Therefore, the progression of colon cancer in this case originated from her genetic mutation for Muir-Torre syndrome and long-term use of immunosuppressive agents. CONCLUSION: This case report not only highlights the importance of adequate diagnosis and therapy for Muir-Torre syndrome, but also suggests the further prevention of the development of malignant tumors in kidney transplant recipients. Physicians should be mindful that sebaceous carcinoma in kidney transplant recipients is highly concurrent with Muir-Torre syndrome.

Hereditary leukonychia, or porcelain nails, resulting from mutations in PLCD1.

Hereditary leukonychia (porcelain nails or white nails) is a rare nail disorder with an unknown genetic basis. To identify variants in a gene underlying this phenotype, we identified four families of Pakistani origin showing features of hereditary leukonychia. All 20 nails of each affected individual were chalky and white in appearance, consistent with total leukonychia, with no other cutaneous, appendageal, or systemic findings. By using Affymetrix 10K chip, we established linkage to chromosome 3p21.3-p22 with a LOD score (Z) of 5.1. We identified pathogenic mutations in PLCD1 in all four families, which encodes phosphoinositide-specific phospholipase C delta 1 subunit, a key enzyme in phosphoinositide metabolism. We then identified localization of PLCD1 in the nail matrix. It was recently shown that PLCD1 is a component of the human nail plate by proteomic analysis and is localized in the matrix of human nails. Furthermore, mutations detected in PLCD1 resulted in reduced enzymatic activity in vitro. Our data show that mutations in PLCD1 underlie hereditary leukonychia, revealing a gene involved in molecular control of nail growth.

Generation of keratinocytes from normal and recessive dystrophic epidermolysis bullosa-induced pluripotent stem cells.

Embryonic stem cells (ESCs) have an unlimited proliferative capacity and extensive differentiation capability. They are an alternative source for regenerative therapies with a potential role in the treatment of several human diseases. The clinical use of ESCs, however, has significant ethical and biological obstacles related to their derivation from embryos and potential for immunological rejection, respectively. These disadvantages can be circumvented by the alternative use of induced pluripotent stem cells (iPSCs), which are generated from an individual's (autologous) somatic cells by exogenous expression of defined transcription factors and have biological characteristics similar to ESCs. In recent years, patient-specific iPSCs have been generated to study disease mechanisms and develop iPSC-based therapies. The development of iPSC-based therapies for skin diseases requires successful differentiation of iPSCs into cellular components of the skin, including epidermal keratinocytes. Here, we succeeded in generating iPSCs not only from normal human fibroblasts but also from fibroblasts isolated from the skin of two patients with recessive dystrophic epidermolysis bullosa. Moreover, we differentiated both of these iPSCs into keratinocytes with high efficiency, and generated 3D skin equivalents using iPSC-derived keratinocytes, suggesting that they were fully functional. Our studies indicate that autologous iPSCs have the potential to provide a source of cells for regenerative therapies for specific skin diseases.

A pediatric case of multiple palisaded encapsulated neuromas of the palms and soles.

Palisaded encapsulated neuroma (PEN) usually presents as a solitary, skin-colored papule on the face in middle-aged adults. We present a rare pediatric case of multiple PEN of the palms and soles. DNA analysis of the RET proto-oncogene in our patient showed no mutations in exons 13, 15, and 16.

The medial plantar flap vascularized by the reverse flow lateral plantar artery: a novel variation through the case of aggressive digital papillary adenocarcinoma of the sole.

Aggressive digital papillary adenocarcinoma (ADPA) is a rare neoplasm of eccrine sweat gland origin that typically presents as a mass on the distal extremities. It is associated with high rates of local recurrence and distal metastasis. Presented here is the case of a 61-year-old male who developed ADPA on his distal sole just above the head of the first metatarsal bone. Wide excision of the tumor involving a 3-cm skin margin from previous surgical scar of biopsy was performed, and sentinel lymph node biopsies were taken from the popliteal fossa and inguinal regions. During this wide excision surgery, the pedicle for the reverse medial plantar flap had to be removed along with the tumor. Reconstructive surgery was performed with a medial plantar flap that was vascularized with a lateral plantar artery in a reverse fashion. This flap successfully covered the defect and the patient can walk without any problems. However, the pedicle crossed the donor site somewhat tightly and the flap became congested for a while. Therefore, it is important to ensure careful handling of the donor site when performing this procedure.

Multiple Phenotypic Conversion in Malignant Melanoma: Obtainment of Granulocyte Colony-Stimulating Factor-Producing Ability during the Intermission of Nivolumab Therapy.

Malignant melanoma (MM) is one of the most aggressive, recalcitrant, and recurrence-prone skin neoplasms. Its feature is likely to be associated with phenotypic conversion due to tumor heterogeneity. The multidisciplinary assessment, including surgery, drug therapy using anticancer agents and immune checkpoint inhibitors, and radiotherapy, is needed for the treatment of advanced MM. Herein, we report a long-term follow-up MM, in which multiple phenotypic conversion occurred during several treatments. In particular, our case obtained granulocyte colony-stimulating factor-producing ability during the intermission of nivolumab therapy and it was successfully controlled by re-administration of nivolumab. Sharing the case having a varied clinical course is meaningful to increase the knowledge and decision branches for the treatment of melanoma.

Regression of CD30-positive large cell transformation arising on patch lesion of early mycosis fungoides.

CD30-positive large cell transformation that occurs in early mycosis fungoides potentially possesses characteristics of spontaneous regression as with CD30-positive lymphoproliferative disorders. Such transformation may not relate to poor prognosis.

Footprint-free gene mutation correction in induced pluripotent stem cell (iPSC) derived from recessive dystrophic epidermolysis bullosa (RDEB) using the CRISPR/Cas9 and piggyBac transposon system.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a monogenic skin blistering disorder caused by mutations in the type VII collagen gene. A combination of biological technologies, including induced pluripotent stem cells (iPSCs) and several gene-editing tools, allows us to develop gene and cell therapies for such inherited diseases. However, the methodologies for gene and cell therapies must be continuously innovated for safe clinical use. OBJECTIVE: In this study, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to correct the pathogenic mutation in RDEB-specific iPSCs, and the piggyBac transposon system so that no residual gene fragments remained in the genome of iPSCs after correcting the mutation. METHODS: For homologous recombination (HR)-based gene editing using CRISPR/Cas9, we designed guide RNA and template DNA including homologous sequences with drug-mediated selection cassette flanked by inverted repeat sequences of the transposon. HR reaction using CRISPR/Cas9 was induced in RDEB-specific iPSCs, and mutation-corrected iPSCs (MC-iPSCs) was obtained. Consequently, the selection cassette in the genome of MC-iPSCs was removed by transposase expression. RESULTS: After CRISPR/Cas9-induced gene editing, we confirmed that the pathogenic mutation in RDEB-specific iPSCs was properly corrected. In addition, MC-iPSCs had no genetic footprint after removing the selection cassette by transposon system, and maintained their "stemness". When differentiating MC-iPSCs into keratinocytes, the expression of type VII collagen was restored. CONCLUSIONS: Our study demonstrated one of the safer approaches to establish gene and cell therapies for skin hereditary disorders for future clinical use.

Approach for the Derivation of Melanocytes from Induced Pluripotent Stem Cells.

Induced pluripotent stem (iPS) cells have the ability to differentiate into multiple cell types in the body and have an unlimited growth potential. However, iPS cell-derived melanocytes produced by existing protocols have significant limitations in developing novel strategies for regenerative medicine and cell therapies of pigmentation disorders in humans because they involve culture in media containing fetal bovine serum and nonphysiological agents. In this study, we established an in vitro approach to generate iPS cell-derived human melanocytes that have higher proliferation rates and increased melanin production compared with melanocytes prepared by previously reported approaches. Importantly, our iPS cell-derived human melanocytes are prepared in fetal bovine serum-free culture conditions that do not contain any nonphysiological agents. We designed two original methods, transferring black colonies by pipette and recovering black cell pellets from centrifuged medium, and numerous human iPS cell-derived melanocytes proliferated in gelatinous dishes coated with Matrigel after 12 days. We also succeeded in inducing melanin pigmentation in the nude mouse skin in vivo using those human iPS cell-derived melanocytes. We propose that this method using iPS cells established from T cells in the blood of normal human volunteers could be applied clinically to develop regenerative medicine and cell therapies for various forms of human pigmentation disorders.

Successful treatment of disseminated superficial actinic porokeratosis with Q-switched ruby laser.

Porokeratosis, a cutaneous disorder that is characterized histologically by the presence of a "cornoid lamella", is a progressive disease with limited therapeutic modalities. We report a case of a 61-year-old man suffering from disseminated superficial actinic porokeratosis, one of the clinical types of porokeratosis, treated with Q-switched ruby laser, commonly used for the treatment of pigmented skin diseases. The laser therapy provided striking improvement and no clinical recurrence was noted.

Integration-free T cell-derived human induced pluripotent stem cells (iPSCs) from a patient with lymphedema-distichiasis syndrome (LDS) carrying an insertion-deletion complex mutation in the FOXC2 gene.

Expanded human T cells from a Japanese male with lymphedema-distichiasis syndrome (LDS) were used to generate integration-free induced pluripotent stem cells (iPSCs) by exogenous expression of four reprogramming factors, OCT3/4, SOX2, cMYC, KLF4, using Sendai virus vector (SeVdp). The authenticity of established iPSC line, LDS-iPSC8, was confirmed by the expression of stem cell markers and the differentiation capability into three germ layers. LDS-iPSC8 may be a useful cell resource for the establishment of in vitro LDS modeling and the study for vascular and lymph vessel development.

Integration-free T cell-derived human induced pluripotent stem cells (iPSCs) from a healthy individual: WT-iPSC2.

Expanded human T cells from a Japanese healthy male were used to generate integration-free induced pluripotent stem cells (iPSCs) by exogenous expression of four reprogramming factors, OCT3/4, SOX2, cMYC, KLF4, using Sendai virus vector (SeVdp). The authenticity of established iPSC line, WT-iPSC2, was confirmed by the expressions of stem cell markers and the differentiation capability into three germ layer. WT-iPSC2 may be a useful cell resource as a normal control for the comparative study using disease-specific iPSCs.

Integration-free T cell-derived human induced pluripotent stem cells (iPSCs) from a healthy individual: WT-iPSC4.

Expanded human T cells from a Japanese healthy male were used to generate integration-free induced pluripotent stem cells (iPSCs) by exogenous expression of four reprogramming factors, OCT3/4, SOX2, cMYC, KLF4, using Sendai virus vector (SeVdp). The authenticity of established iPSC line, WT-iPSC4, was confirmed by the expressions of stem cell markers and the differentiation capability into three germ layer. WT-iPSC4 may be a useful cell resource as a normal control for the comparative study using disease-specific iPSCs.

Integration-free T cell-derived human induced pluripotent stem cells (iPSCs) from a healthy individual: WT-iPSC1.

Expanded human T cells from a Japanese healthy male were used to generate integration-free induced pluripotent stem cells (iPSCs) by exogenous expression of four reprogramming factors, OCT3/4, SOX2, cMYC, KLF4, using Sendai virus vector (SeVdp). The authenticity of established iPSC line, WT-iPSC1, was confirmed by the expressions of stem cell markers and the differentiation capability into three germ layers. WT-iPSC1 may be a useful cell resource as a normal control for the comparative study using disease-specific iPSCs.

A Case of Advanced Extramammary Paget's Disease Successfully Controlled by Monthly but Not Weekly Docetaxel Chemotherapy.

Extramammary Paget's disease (EMPD) is an uncommon cutaneous adenocarcinoma arising from the apocrine glands within the epidermis or underlying skin appendages in the anogenital and axillary regions. Surgical excision is basically performed as a treatment for EMPD. However, therapeutic options for EMPD in an advanced stage are limited. Herein, we report the case of a Japanese woman with advanced EMPD successfully controlled by monthly but not weekly docetaxel therapy. We also demonstrate the possibility that a monthly regimen of docetaxel is a more effective and optimal schedule than a weekly one through this case report.

Integration-free T cell-derived human induced pluripotent stem cells (iPSCs) from a patient with recessive dystrophic epidermolysis bullosa (RDEB) carrying two compound heterozygous mutations in the COL7A1 gene.

Expanded human T cells from a Japanese female with recessive dystrophic epidermolysis bullosa (RDBE) were used to generate integration-free induced pluripotent stem cells (iPSCs) by exogenous expression of four reprogramming factors, OCT3/4, SOX2, cMYC, KLF4, using Sendai virus vector (SeVdp). The authenticity of established iPSC line, RDEB-iPSC26, was confirmed by the expressions of stem cell markers and the differentiation capability into three germ layer. RDEB-iPSC26 may be a useful cell resource for the establishment of in vitro RDEB modeling and the study for developing gene and cell therapy.