Biomedical subjects
Publications and source records attributed to N Hatta.
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To examine the distribution of occult micrometastases that could be potential sources of recurrence, complete maps of microscopic and submicroscopic metastases in entire inguinal lymph node basins were generated in 13 melanoma patients who had undergone elective or therapeutic lymphadenectomy. Occult micrometastases were analysed immunohistochemically for the pigment cell-specific antigen HMB-45 in all 155 nodes and using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect tyrosinase mRNA in 35 nodes. Five patients were determined to be node-negative by routine histopathology; three of these subjects were also negative by RT-PCR and/or immunohistochemistry. However, the remaining two patients had occult metastases, which were confined to a possibly sentinel node in one and were detected in multiple nodes in the other. Eight patients had histological evidence of lymph node metastasis. Three of these patients had no additional detectable submicroscopic disease, and one had occult metastasis in one node adjacent to the histologically positive node. In contrast, the other four patients had occult micrometastases in multiple non-sentinel, higher level nodes. The two patients who relapsed belonged to this group. The results show considerable variation in the distribution pattern of occult metastases in the regional lymph nodes, and have significant implications for the role of regional lymph node dissection, including sentinel node mapping with selective lymphadenectomy, in the management of melanoma patients.
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To examine for the genetic basis of metastatic progression in cutaneous melanoma, we have compared loss of heterozygosity (LOH) of several selected chromosome regions that are implicated in the initiation and progression of melanoma, and alterations of the p16INK4a gene in 14 pairs of primary tumor and synchronous or asynchronous metastasis excised from the same patients. The most frequent genetic alteration during metastatic progression detected was the loss of p16INK4a protein expression (four of 14 cases), whereas no somatic p16INK4a gene mutations were found in any primary or metastatic tumors. LOH analyses showed that most of the chromosome losses including 6q, 8p, 9p, 9q, and 18q were shared between primary tumors and their metastases. Nevertheless, LOH of 6q and 11q and LOH of 7q not detected in primary tumors were, respectively, observed in two lymph node metastases. These results suggest that loss of p16INK4a protein expression (but not p16INK4a gene mutation) and the losses of chromosome arms 6q, 7q, and 11q play an important role in the acquisition of metastatic potential in sporadic melanoma. Furthermore, comparison of genetic profiles between the primary tumor and its metastasis revealed in several cases that heterogenous tumor cell populations might already exist at the early stage of tumorigenesis and evolve independently in the primary tumor and its metastasis, strongly suggesting that metastatic progression of sporadic melanoma is not accounted for by a linear progression model.
AIMS: To evaluate immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR) for melanoma associated antigens (MAA) in detecting occult melanoma cells in lymph nodes which were missed on routine pathology. METHODS: Occult melanoma cells were sought in 436 lymph nodes from 32 patients with cutaneous melanoma of the lower extremities by immunohistochemistry using the melanoma specific antibody HMB-45. The detection sensitivity of routine histology, immunohistochemistry, and RT-PCR was also compared in 23 lymph node samples from six patients. RESULTS: Immunohistochemistry showed that 15 of 24 patients (62.5%) who had no detectable metastasis by routine histology had at least one lymph node containing HMB-45 positive cells, mostly seen singly in the medullary sinus. No difference was found in known clinicopathological prognostic factors and recurrence rates between the two groups of patients with and without occult HMB-45 positive cells. RT-PCR analyses showed that the nested PCR for tyrosinase was more sensitive than a combination of single round PCR for five different MAA, including tyrosinase, MART-1/Melan A, Pmel-17, TRP-1, and TRP-2, detecting tyrosinase mRNA in six nodes which were negative by HMB-45 immunohistochemistry. CONCLUSIONS: Immunohistochemistry + RT-PCR is more sensitive than routine histology in detecting occult melanoma cells in lymph nodes. The nested PCR for tyrosinase should be used in future studies investigating the prognostic significance of such lymph node micrometastases.
A case of pigmented dermatofibrosarcoma protuberans (Bednár tumor) occurring in a Japanese infant is reported. A 6-month-old girl developed a 16 x 10 mm erythematous tumor with a slight elevation on the lower back at 1 month of age. Histological examination revealed proliferation of spindle-shaped, fibroblast-like cells in the myxoid interstitium. Subsequently the tumor grew gradually to a red-purplish tumor measuring 45 x 36 mm. The second biopsy specimen presented hypercellular proliferation composed of spindle-shaped cells showing slight nuclear atypia and a characteristic storiform pattern, with scattered slender dendritic cells containing a large amount of brown pigment. Immunohistochemical studies of the second biopsy specimen showed that the spindle-shaped tumor cells were positive for vimentin and CD34 and negative for factor XIIIa. The number of CD34-reactive spindle-shaped tumor cells increased in the second biopsy specimen compared with the first biopsy.
The neurofibromatosis type 1 (NF1) gene has been considered to be a tumor suppressor gene, since the NF1 gene product downregulates the ras oncogene product (p21(ras)). In addition, children with NF1 show an increased incidence of myelogenous leukemia, including juvenile chronic myelogenous leukemia (JCML). We studied 8 Japanese JCML patients without NF1 for mutations in exons 21-36 of the NF1 gene by using polymerase chain reaction/single-strand conformation polymorphism analysis. This region was chosen because it includes two main hotspots in the NF1 gene as well as the functionally important domain, GTPase-activating protein-related domain, which mediates the downregulation of ras activity. One of the 8 JCML patients exhibited a G to T transversion at the third nucleotide of the codon GAG for Glu at amino acid residue 1699, which results in the heterozygous conversion to Asp (E1699D). This variation was detected neither in 65 healthy volunteers nor 50 NF1 patients. No other variations were detected in our JCML patients. We suggest that NF1 gene mutation does not occur frequently in JCML without NF1.
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.
Extramammary Paget's disease is a particular form of skin cancer of unknown histogenesis. To look for the genetic defects underlying the pathogenesis of this tumour, we have examined loss of heterozygosity (LOH), p53 and human papillomavirus (HPV) status, and the expression of c-erbB-2 and bcl-2 proteins in 14 cases. Unexpectedly, no LOH was detected at several loci commonly lost in other human cancers (namely 3p, 9p, 9q, 13q, 16q, 17p, and 17q) in 12 tumours examined. Altered p53 protein expression was entirely or mostly negative in all 14 cases. Direct sequencing of exons 5-8 of the p53 gene in eight cases revealed no mutation. Polymerase chain reaction amplification of the L1 gene of human papillomavirus (HPV) did not detect the virus that could inactivate p53 and retinoblastoma tumour-suppressor gene products. As expected, c-erbB-2 proto-oncogene protein was overexpressed in six cases. The expression of bcl-2 was negative in all cases. The results presented in this study suggest that molecular events underlying extramammary Paget's disease differ from those of other common epithelial malignancies and that tumour-suppressor genes located in chromosome regions not examined in this study may be important.
Serum 5-S-cysteinyl dopa (5-S-CD), circulating intercellular adhesion molecule-1 (cICAM-1) and soluble interleukin-2 receptor (sIL-2R) have each been reported as useful markers for melanoma progression. To assess the clinical relevance of these three markers, we simultaneously assayed their serum levels in 30 Japanese melanoma patients. Pre-surgical serum levels of 5-S-CD, cICAM-1 and sIL-2R were elevated in six, 13 and five patients respectively. These abnormal values returned to normal after curative surgery in most of the patients, suggesting a direct relationship to the presence of the primary tumour. Pre-surgical values of these three markers, either individually or in combination, did not predict the development of subsequent metastases. The sequential measurements of the three markers in eight patients who had relapse after surgery showed that serum 5-S-CD is the most useful marker for disease progression, although it is dependent on the melanin-producing ability of individual recurrent tumours. sIL-2R seemed to reflect in vivo activation of the host immune system and was a good indicator for predicting occult metastasis in selected cases. Circulating ICAM-1 levels were less relevant to the clinical disease course in our cases, although they tended to increase strikingly after liver metastasis. Our results in this limited number of cases show that the significance of the three markers varied with the individual and suggest that the simultaneous measurement of these markers may facilitate the early detection of metastases and proper therapeutic intervention.
Immunofluorescence studies of junctional epidermolysis bullosa with pyloric atresia (JEB-PA) have suggested abnormalities in the expression of the alpha6 beta4 integrin, an integral component of hemidesmosomes. In this study, we examined a family with two affected individuals with JEB-PA for mutations in the ITGA6 and ITGB4 genes which encode the alpha6 and beta4 integrin polypeptides, respectively. Mutation detection strategy based on PCR amplification of genomic DNA, followed by heteroduplex analysis and direct nucleotide sequencing, did not reveal sequence variants in ITGA6. Putative pathogenic mutations, however, were identified in both ITGB4 alleles. Specifically, the proband was a compound heterozygote for a 1-bp maternal deletion, 3434delT, and an 8-bp paternal deletion, 4050de18. Both mutations result in a frameshift and premature termination codon downstream from the deletion. At the protein level, immunofluorescence of the skin of the proband revealed negative staining for the integrin alpha6 and markedly reduced staining for the beta4 subunit. Thus, the results support the notion of close association of the alpha6 beta4 integrin subunits and further attest to the critical role of this integrin in providing physiologic stability to the dermal-epidermal junction.
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We describe a patient with subacute cutaneous lupus erythematosus, widespread pruritic papulosquamous plaques, and hypocomplementemia. Skin biopsy specimens revealed liquefaction degeneration and colloid bodies and dyskeratotic cells in basal and suprabasal layers. An immunofluorescence study revealed deposits of IgG, IgM, and C3 at the dermalepidermal junction in a bandlike pattern, and particulate IgG deposition in the basal and suprabasal layers. Treatment with prednisolone (15 mg/day), chloroquine phosphate (200 mg/ day), cyclosporine (5 mg/kg daily), and gold (10 mg/day) failed to reduce pruritic plaque formation, and pulse methylprednisolone therapy led to only a transient remission. Clinical exacerbations correlated with a decrease in complement levels. The disease was successfully controlled with dapsone (75 mg/day) and prednisolone (25 mg/day).
To evaluate the role of the OKT4 epitope in human herpesvirus 7 (HHV-7) infection, we studied the susceptibility to HHV-7 infection of CD4+ T cells isolated from two individuals with OKT4 epitope deficiency. HHV-7-infected OKT4-Leu3a+ T cells exhibited the characteristic cytopathic effect, reactivity with HHV-7-seropositive serum by immunofluorescence and down-modulation of surface CD4 in a manner similar to HHV-7-infected OKT4+Leu3a+ T cells. A semiquantitative PCR revealed that the amounts of HHV-7 replicated in OKT4+Leu3a+ T cells and OKT4-Leu3a+ T cells were not significantly different. Although it has been reported that OKT4 monoclonal antibody efficiently inhibits HHV-7 infection, the present study demonstrated that the interaction of HHV-7 with CD4+ T cells does not require participation of the epitope defined by OKT4 monoclonal antibody.
BACKGROUND AND DESIGN: The expression of basement membrane-related antigens was surveyed in 2 Japanese siblings who died of pyloric atresia-junctional epidermolysis bullosa syndrome in early infancy. RESULTS: The skin specimens of both patients demonstrated complete absence of detectable alpha 6 integrin and markedly reduced amounts of beta 4 integrin. All the other subtypes of epidermolysis bullosa used as controls demonstrated normal intensity of expression of alpha 6 and beta 4 integrin. In contrast to the negative immunoreactivity of monoclonal antibody GB3 in gravis-Herlitz junctional epidermolysis bullosa (n = 4), a bright linear pattern along the epidermal basement, membrane was demonstrated in the skin of both siblings with pyloric atresia-junctional epidermolysis bullosa syndrome. Based on these data, a monoclonal antibody against alpha 6 integrin was successfully used as a prenatal diagnostic probe for a skin biopsy specimen from a fetus at risk for pyloric atresia-junctional epidermolysis bullosa syndrome in this family. CONCLUSION: The absence of detectable alpha 6 integrin, but not beta 4 integrin, in these cases raises the possibility that alpha 6 integrin or its ligands are responsible for the pyloric atresia-junctional epidermolysis bullosa syndrome phenotype seen in this family.
Neurofibromatosis 1 (NF1) is an autosomal dominant disease characterized by abnormalities in multiple tissues derived from the neural crest. We analysed 50 unrelated Japanese patients for NF1 mutations by using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis for exons 28 to 36. Here, we demonstrate a single base pair (bp) insertional mutations in exon 31 in one patient (5843insA/5844insA/5845insA/5846insA) and a single adenine to guanine transitional mutation 4 bp upstream from the 3' end of intron 31 in two unrelated cases. The insertional mutation in exon 31 was novel and resulted in premature termination of the transcript. The other intron 31 mutations resulted in 4 bp insertions of cDNA between exon 31 and exon 32 with premature termination of the transcript, indicating that those transitions of intron 31 caused aberrant splice acceptor sites upstream from the 5' end of exon 32. However, as the same mutation of intron 31 has been reported previously in two cases of unrelated Caucasians, the splice junction mutation of intron 31 is thought to be common among different ethnic groups.
Recently, a peculiar self-healing neonatal blistering disease has been reported, which is characterized by perinuclear stellate inclusions within basilar keratinocytes, representing abnormal retention of type VII collagen. We report a Japanese patient with this condition, in whom we studied the expression of a variety of basement membrane zone (BMZ)-related antigens. Skin biopsy specimens at 3 days of age showed abundant accumulation of both the NC-1 domain and the collagenous part of type VII collagen within the basal and suprabasal keratinocytes, in addition to patchy and weak staining along the BMZ. In contrast, at 4 years of age, when the disease activity was markedly attenuated, a second biopsy showed complete linear staining of type VII collagen along the BMZ, with no detectable intracytoplasmic deposits. Expression of other BMZ-related antigens, including laminin 5, alpha 6 and beta 4 integrins, bullous pemphigoid antigens 1 and 2, and type IV collagen, was normal in both the biopsy specimens. Our observations further confirm that the perinuclear stellate bodies seen in this peculiar condition are composed of both collagenous and non-collagenous domains of type VII collagen retained within the epidermis, and that these bodies disappear when the disease activity remits.
We have investigated the effects of interleukin-4 (IL-4) on the proliferation of chronic myelomonocytic leukemia (CMMoL) cells in the chronic and leukemic transformation phases in vitro. CMMoL cells formed colonies spontaneously in both phases. IL-4 suppressed the spontaneous growth in the chronic phase, but on the other hand, stimulated colony formation in the leukemic transformation phase. Anti-IL-6 antibody inhibited spontaneous colony formation in both phases. CMMoL cells in both phases produced high levels of IL-6, compared with those produced by acute myelogenous leukemia (AML) cells showing myelomonocytic differentiation and normal monocytes. IL-4 suppressed the IL-6 production by CMMoL cells in both phases. None of anti-IL-6, anti-macrophage colony-stimulating factor (M-CSF), anti-granulocyte-macrophage colony-stimulating factor (GM-CSF), anti-tumor necrosis factor-alpha (TNF-alpha) and anti-IL-1-beta antibodies inhibited IL-4-stimulated colony formation. These results suggest that IL-4 directly stimulates the growth of CMMoL cells once leukemic transformation has occurred and that the therapeutic use of IL-4 for CMMoL should be viewed with caution, especially in the leukemic transformation phase.