Naohito Hatta

Extramammary Paget's disease : treatment, prognostic factors and outcome in 76 patients

Background  Extramammary Paget’s disease (EMPD) is a rare cutaneous carcinoma usually presenting as a genital erythematous lesion in the elderly. Although most EMPD tumours are in situ, invasive EMPD has a poor prognosis.

p16INK4a inactivation is not frequent in uncultured sporadic primary cutaneous melanoma.

In an attempt to examine whether the inactivation of p16INK4a is an important early event in the development of sporadic melanoma in vivo, we have systematically analysed 46 uncultured primary cutaneous melanomas. Loss of heterozygosity (LOH) of chromosome region 9p21-22 (where the p16INK4a resides) was detected in 11 tumours (24%) by PCR-based LOH analyses. Direct sequencing of all three exons of the p16INK4a gene in these 11 tumours revealed no somatic mutation although germline mutations which have not been reported previously as common polymorphisms were detected in two patients. Further sequencing analyses of the p16INK4a gene exon 2 in 19 additional tumours with no evidence of LOH on 9p21-22 identified only one heterozygous C – >T mutation at codon 81 altering a proline to a leucine. A sensitive methylation-specific PCR assay did not reveal de novo methylation of the 5′CpG island in exon 1 of the p16INK4a gene in any of the tumours showing 9p21-22 allelic loss or a heterozygous p16INK4a mutation. Complete loss of p16INK4a protein, most likely due to homozygous deletion of the p16INK4a gene, was observed in 6 (15%) out of 39 evaluable cases by immunohistochemical analyses on frozen sections using two different anti-p16INK4a antibodies. The results show that inactivation of p16INK4a is not as frequent in primary melanoma as has been reported in cell lines, and warrant further search for another tumour suppressor on 9p21-22. This study also emphasizes the importance of examining uncultured primary tumours rather than cell lines to define early events in tumorigenesis.

Glycine substitution mutations by different amino acids in the same codon of COL7A1 lead to heterogeneous clinical phenotypes of dominant dystrophic epidermolysis bullosa

Abstract Dystrophic epidermolysis bullosa (DEB), caused by mutations in the gene encoding type VII collagen (COL7A1), is known to show heterogeneous clinical phenotypes. Certain correlations between the nature or position of COL7A1 mutations and the resultant DEB phenotypes have been suggested, although such relationships may be more complex than initially thought. The purpose of the present study was to clarify the molecular basis of two different subtypes of dominant DEB (DDEB), EB pruriginosa and classical type. Interestingly, we found that both cases were caused by a missense glycine substitution mutation by different amino acids in the same codon of COL7A1 (G2028R and G2028A). These results further support the notion that different glycine substitution mutations in the same codon can lead to heterogeneous clinical phenotypes of DDEB, EB pruriginosa and classical type.

Molecular diagnosis of basal cell carcinoma and other basaloid cell neoplasms of the skin by the quantification of Gli1 transcript levels

Background:  Distinguishing basal cell carcinoma (BCC) from other benign and malignant skin tumors is sometimes a difficult task for the pathologists. Because the activation of hedgehog signals and the up‐regulation of its critical transcriptional factor Gli1 are well documented in BCC, a molecular technique measuring Gli1 transcripts may aide the diagnosis.

Atypical moles in a patient undergoing chemotherapy with oral 5-fluorouracil prodrug

We present a patient with multiple pigmented lesions on the palms, soles, oral mucosa and nails after chemotherapy with oral 5‐fluorouracil (5‐FU) prodrug. Dermoscopically, most of the macules showed similar features, with pigmentation present predominantly on the crista superficialis, while a large, dark macule also showed pigmentation along the sulcus superficialis with irregular hyperpigmentation and depigmentation, suggesting malignancy. However, histologically, both types of lesion showed basal hyperpigmentation and the presence of a small number of large atypical melanocytes. We diagnosed these lesions as pigment flecks induced by 5‐FU, and the pigmented lesions gradually diminished after the cessation of chemotherapy. Our findings suggested that immunosuppression and 5‐FU led to the development of the atypical pigmented lesions.

PRENATAL EXCLUSION OF PYLORIC ATRESIA-JUNCTIONAL EPIDERMOLYSIS BULLOSA SYNDROME

BACKGROUND The pyloric atresia--junctional epidermolysis bullosa (PA-JEB) syndrome is an autosomal recessive disorder with a poor prognosis. Electron microscopy of fetal skin has been the only reliable method for prenatal diagnosis. OBJECTIVE The purpose of this study was to make the prenatal diagnosis of PA-JEB syndrome with a more reliable method by means of immunocytochemical probes. METHODS Expression of a range of basement membrane antigens was examined in different types of JEB. On the basis of the results, a fetal skin biopsy specimen was obtained for prenatal diagnosis. RESULTS In PA-JEB syndrome (n = 2), GB3 antigen (BM600) was normally expressed; the 19-DEJ-1 antigen was completely absent. In fetal skin at risk for PA-JEB syndrome, the 19-DEJ-1 antigen was normally expressed, and no ultrastructural abnormality was found by electron microscopy. A normal male infant was delivered at 38 weeks of pregnancy. CONCLUSION 19-DEJ-1 monoclonal antibody serves as a useful probe for the prenatal diagnosis of PA-JEB syndrome.

Applicability of radiocolloids, blue dyes and fluorescent indocyanine green to sentinel node biopsy in melanoma

Patients with primary cutaneous melanoma underwent sentinel node (SN) mapping and biopsy at 25 facilities in Japan by the combination of radiocolloid with gamma probe and dye. Technetium‐99m (99mTc)‐tin colloid, 99mTc‐phytate, 2% patent blue violet (PBV) and 0.4% indigo carmine were used as tracers. In some hospitals, 0.5% fluorescent indocyanine green, which allows visualization of the SN with an infrared camera, was concomitantly used and examined. A total of 673 patients were enrolled, and 562 cases were eligible. The detection rates of SN were 95.5% (147/154) with the combination of tin colloid and PBV, 98.9% (368/372) with the combination of phytate and PBV, and 97.2% (35/36) with the combination of tin colloid or phytate and indigo carmine. SN was not detected in 12 cases by the combination method, and the primary tumor was in the head and neck in six of those 12 cases. In eight of 526 cases (1.5%), SN was detected by PBV but not by radiocolloid. There were 13 cases (2.5%) in which SN was detected by radiocolloid but not by PBV. In 18 of 36 cases (50%), SN was detected by radiocolloid but not by indigo carmine. Concomitantly used fluorescent indocyanine green detected SN in all of 67 cases. Interference with transcutaneous oximetry by PVB was observed in some cases, although it caused no clinical trouble. Allergic reactions were not reported with any of the tracers. 99mTc‐tin colloid, 99mTc‐phytate, PBV and indocyanine green are useful tracers for SN mapping.

A novel p63 sterile alpha motif (SAM) domain mutation in a Japanese patient with ankyloblepharon, ectodermal defects and cleft lip and palate (AEC) syndrome without ankyloblepharon.

Summary We present a sporadic case of a Japanese female infant with ectodermal dysplasia, complete cleft lip and palate, severe skin erosions at birth and recurrent scalp infection. She had typical clinical features of ankyloblepharon, ectodermal defects and cleft lip and palate (AEC) syndrome without ankyloblepharon. Histological and immunohistochemical analyses showed reduced granular cell layers and aberrant expression of the p63 protein in the suprabasal keratinocytes. Mutation analysis of the exon 13 of p63 gene revealed a heterozygous in‐frame 3‐bp insert (c. 538–539 ins TTC) encoding additional amino acid residues (F). This is the first report of sterile alpha motif domain mutation except for single nucleotide transitions.

Expression of Structurally Unaltered Androgen Receptor in Extramammary Paget's Disease

Extramammary Pagets disease (EMPD) is an uncommon neoplasm of the skin that shows differentiation to an apocrine sweat gland. Although we previously showed that erbB-2 overexpression may play a part in the progression of EMPD, molecular genetic defects underlying the development of EMPD are poorly understood. In the study described here, we examined androgen receptor expression and gene alterations in 30 cases of EMPD without internal malignancy. Immunohistochemistry revealed that 24 of 30 (80%) cases of EMPD variably expressed nuclear androgen receptor. Semi-quantitation of receptor content by scoring immunostained sections showed no difference between in situ (n = 17) and invasive (n = 13) EMPD tumors. Androgen receptor expression was also observed in four of six lymph node metastases. In these lymph nodes, expression of androgen receptor mRNA was confirmed by reverse transcriptase-polymerase chain reaction. Direct sequencing of exon 2 through exon 8, which encodes DNA- and hormone-binding domains of the androgen receptor gene, revealed no mutation in any of the 10 advanced stage tumors. Neither amplification nor deletion of the androgen receptor gene locus was detected by dual color fluorescence in situ hybridization analysis in 14 tumors. The present findings showing frequent expression of structurally unaltered androgen receptor in an advanced stage of EMPD may provide a rational basis for hormone therapy, which is widely used in the treatment of metastatic prostate cancer and androgen receptor-positive breast cancer recurrence.

Tape stripping induces marked epidermal proliferation and altered TGF-α expression in non-lesional psoriatic skin

Psoriasis is a chronic inflammatory disorder characterized by epidermal hyperproliferation. Although recent evidence suggests that T cell activation is a primary trigger for psoriasis lesions, there may be alterations in the keratinocyte growth regulatory pathways which induce epidermal hyperproliferation in psoriatic patients. To test this hypothesis, we investigated the proliferative activity of epidermal keratinocytes 48 h after tape stripping, one of the standard mechanical ways to stimulate the epidermis, in 20 psoriasis patients and in 18 controls. Epidermal cell kinetics were assessed with DNA flow cytometry, the mitotic index, bromodeoxyuridine incorporation, Ki-67 antigen expression and DNA polymerase alpha expression. The expression of TGF-alpha and EGF receptors, critical mediators of keratinocyte proliferation, were also investigated immunohistochemically. The results of multiparameter assays showed that the baseline proliferative activity in uninvolved skin was the same in psoriasis patients and normal controls. After tape stripping, although both psoriasis patients and the normal controls showed significant increases in epidermal cell proliferation, the values of all the parameters investigated were significantly greater in the psoriasis patients than in the normal controls. EGF receptors were overexpressed in basal and suprabasal keratinocytes after tape stripping in both the psoriasis patients and the normal controls. In contrast, overexpression of TGF-alpha was only observed in the patients with psoriasis, which may explain their increased proliferative response to trauma.