Immune suppression and resistance mediated by constitutive activation of Wnt/ß-catenin signaling in human melanoma cells.

Cancer-induced immunosuppression is a major problem reducing antitumor effects of immunotherapies, but its molecular mechanism has not been well understood. We evaluated immunosuppressive roles of activated Wnt/ß-catenin pathways in human melanoma for dendritic cells (DCs) and CTLs. IL-10 expression was associated with ß-catenin accumulation in human melanoma cell lines and tissues and was induced by direct ß-catenin/TCF binding to the IL-10 promoter. Culture supernatants from ß-catenin-accumulated melanoma have activities to impair DC maturation and to induce possible regulatory DCs. Those immunosuppressive culture supernatant activities were reduced by knocking down ß-catenin in melanoma cells, partly owing to downregulation of IL-10. Murine splenic and tumor-infiltrating DCs obtained from nude mice implanted with human mutant ß-catenin-overexpressed melanoma cells had less ability to activate T cells than did DCs from mice with control melanoma cells, showing in vivo suppression of DCs by activated Wnt/ß-catenin signaling in human melanoma. This in vivo DC suppression was restored by the administration of a ß-catenin inhibitor, PKF115-584. ß-catenin-overexpressed melanoma inhibited IFN-γ production by melanoma-specific CTLs in an IL-10-independent manner and is more resistant to CTL lysis in vitro and in vivo. These results indicate that Wnt/ß-catenin pathways in human melanoma may be involved in immunosuppression and immunoresistance in both induction and effector phases of antitumor immunoresponses partly through IL-10 production, and they may be attractive targets for restoring immunocompetence in patients with Wnt/ß-catenin-activated melanoma.

Operative treatment for scaphoid osteonecrosis (Preiser disease): salvage or excision? Report on two cases and a review of the literature.

Idiopathic avascular necrosis of the scaphoid, or Preiser disease, is a rare condition. Both the etiology and the pathology are still not well understood. We here present 2 cases with this disease and discuss the recommended treatment according to the stage of disease progression and by reference to previous studies in the literature. Similar to Kienböck disease, the recommended treatment should be selected according to the degree of disease progression. We believe that vascularized bone graft should be restricted to Herbert stage I or II cases with no evidence of radiocarpal arthritis or carpal instability. Our first case showed excellent revascularization following vascularized bone graft, as revealed by magnetic resonance image findings. The presence of arthrosis of the radiocarpal or mid-carpal joints is an indication for scaphoid excision with midcarpal fusion or proximal row carpectomy.

Isolated unilateral absence of the pulmonary artery (IUAPA) presenting with progressive interstitial shadows: A case report.

Unilateral absence of the pulmonary artery (UAPA) is a rare congenital condition. When UAPA occurs without associated congenital heart disease, it is referred to as isolated unilateral absence of the pulmonary artery (IUAPA). IUAPA is frequently not diagnosed until adulthood. A 61-year-old female patient presented with right chest pain and a worsening interstitial shadow over 9 years. Contrast-enhanced computed tomography revealed the absence of blood flow from the pulmonary artery in the right lung, leading to the diagnosis of IUAPA. In this case, the diagnosis of UAPA was established after approximately 40 years. This case underscores the importance of identifying vascular abnormalities to differentiate this condition in patients presenting with nonspecific symptoms and interstitial shadows.

Anaphylaxis and prolonged allergic reactions caused by intra-articular injection of diclofenac etalhyaluronate in knee osteoarthritis: A case report.

Osteoarthritis (OA) is a common condition. The treatment of knee OA aims to improve the quality of life and clinical symptoms, mainly knee pain. To develop a new treatment option, diclofenac etalhyaluronate (DF-HA;ONO-5704/SI-613; trade name: JOYCLU) was developed. Its anti-inflammatory and pain-relieving effects have been demonstrated. Conversely, adverse events have also been reported. Here, we report the case of a 71-year-old female who presented with anaphylaxis and prolonged allergic reactions after an intra-articular injection of JOYCLU. A basophil activation test (BAT) was performed to determine the cause of the anaphylaxis. BAT showed no positive findings. As the incidence of knee OA increases, the number of cases of JOYCLU use is expected to grow; therefore, careful administration is required. In this case, the cause was unknown by BAT. Thus, further development of appropriate testing methods is necessary. Intra-articular allergic reactions in knee OA may be modified and worsened besides normal allergic reactions; therefore, caution is required.

Impact of the COVID-19 pandemic on COPD exacerbations in Japanese patients: a retrospective study.

Various infection control measures implemented during the coronavirus disease (COVID-19) pandemic have reduced the number of respiratory infections, which are the most common cause of chronic obstructive pulmonary disease (COPD) exacerbations. Here, we investigated whether infectious disease prevention during the COVID-19 pandemic reduced COPD exacerbations and the characteristics of patients exhibiting exacerbations before and during the COVID-19 pandemic. We included outpatients and inpatients with moderate or severe COPD exacerbations who required systemic steroids between April 1, 2018 and March 31, 2022. Their medical records were retrospectively compared and analyzed in 2-year intervals (before and during the COVID-19 pandemic). During the 4-year observation period, 70,847 outpatients and 2,772 inpatients were enrolled; 55 COPD exacerbations were recorded. The number of COPD exacerbations decreased from 36 before to 19 during the COVID-19 pandemic. Regarding the characteristics of patients with exacerbations, the % forced expiratory volume in one second (52.3% vs. 38.6%, P = 0.0224) and body mass index (BMI) (22.5 vs. 19.3, P = 0.0127) were significantly lower during the COVID-19 pandemic than before the pandemic. The number of COPD exacerbations during the pandemic decreased. Additionally, the tendency for a reduction in COPD exacerbation was greatest in patients with preserved lung function or above-standard BMI patients.

[Susceptibilities of bacteria isolated from patients with lower respiratory infectious diseases to antibiotics (2006)].

From October 2006 to September 2007, we collected the specimen from 356 patients with lower respiratory tract infections in 14 institutions in Japan, and investigated the susceptibilities of isolated bacteria to various antibacterial agents and patients' characteristics. Of 414 strains that were isolated from specimen (mainly from sputum) and assumed to be bacteria causing in infection, 407 strains were examined. The isolated bacteria were: Staphylococcus aureus 64, Streptococcus pneumoniae 96, Haemophilus influenzae 87, Pseudomonas aeruginosa (non-mucoid) 52, P. aeruginosa (mucoid) 11, Klebsiella pneumoniae 20, and Moraxella catarrhalis 44. Of 64 S. aureus strains, those with 2 microg/ml or less of MIC of oxacillin (methicillin-susceptible S. aureus: MSSA) and those with 4 microg/ml or more of MIC of oxacillin (methicillin-resistant S. aureus: MRSA) were 27 (42.2%) and 37 (57.8%) strains, respectively. Against MSSA, imipenem had the most potent antibacterial activity and inhibited the growth of all strains at 0.063 microg/ml or less. Against MRSA, vancomycin and linezolid showed the most potent activity and inhibited the growth of all the strains at 1 microg/ml. Carbapenems showed the most potent activities against S. pneumoniae and in particular, panipenem inhibited the growth of all the strains at 0.063 microg/ml or less. Imipenem and faropenem also had a preferable activity and inhibited the growth of all the strains at 0.125 and 0.5 microg/ml, respectively. In contrast, there were high-resistant strains (MIC: over 128 microg/ml) for erythromycin (45.8%) and clindamycin (20.8%). Against H. influenzae, levofloxacin showed the most potent activity and its MIC90 was 0.063 microg/ml or less. Meropenem showed the most potent activity against P. aeruginosa (mucoid) and its MIC90 was 0.5 microg/ml. Against P. aeruginosa (non-mucoid), tobramycin had the most potent activity and its MIC90 was 2 microg/ml. Against K. pneumoniae, cefozopran was the most potent activity and inhibited the growth of all the strains at 0.063 microg/ml or less. Also, all the antibacterial agents except ampicillin generally showed a potent activity against M. catarrhalis and the MIC90 of them were 2 microg/ml or less. The approximately half the number (50.6%) of the patients with respiratory infection were aged 70 years or older. Bacterial pneumonia and chronic bronchitis accounted for 49.2% and 28.1% of all the respiratory infections, respectively. The bacteria frequently isolated from the patients with bacterial pneumonia were S. pneumoniae (29.2%), S. aureus (20.8%), and H. influenzae (12.9%). H. influenzae (25.0%) and P. aeruginosa (21.7%) also were frequently isolated from the patients with chronic bronchitis. Before the antibacterial agent administration, the bacteria frequently isolated from the patients were S. pneumoniae (27.5%) and H. influenzae (22.5%). The bacteria frequently isolated from the patients treated with macrolides was P. aeruginosa, and its isolation frequently was 39.4%.

Endobronchial Watanabe Spigot Placement for Hepatic Abscess and Bronchobiliary Fistula Following Radiofrequency Ablation for Hepatocellular Carcinoma.

A bronchobiliary fistula (BBF) is an uncommon but severe complication after radiofrequency ablation (RFA). However, the definitive salvage methods are controversial. We herein report a patient with hepatocellular carcinoma with hepatic abscess and BBF following RFA. We also review previous reports of BBF after RFA. The patient was a man in his 70s who underwent RFA for recurrent hepatocellular carcinoma in the subphrenic area. Despite percutaneous transhepatic abscess drainage, bilioptysis persisted. Finally, the BBF was occluded with an endobronchial Watanabe spigot under fiber-optic bronchoscopy. Placing an endobronchial Watanabe spigot should be considered as a salvage therapy for refractory BBF following RFA.

Pathological activation of KIT in metastatic tumors of acral and mucosal melanomas.

Recent studies showed KIT gene aberrations in a substantial number of melanomas on acral skin and mucosa, suggesting the therapeutic benefit of tyrosine kinase inhibitors, such as imatinib. We therefore examined the expression and mutations of KIT in 4 primary and 24 metastatic acral and mucosal melanomas. Immunohistochemistry revealed moderate or strong KIT protein expression in 13 (48%) tumors. Sequence analysis revealed K642E and D820Y mutations in two metastases. Amplification of KIT was identified by real-time PCR in 4 tumors, including one that had K642E. Western blot analysis showed phosphorylation of the KIT receptor in 8 (62%) of 13 cryopreserved samples, indicating the frequent pathological activation of the receptor in vivo. Phosphorylation of KIT protein was detected in 2 tumors harboring KIT mutations, as well as in one tumor with KIT gene amplification. Furthermore, 5 tumors without detectable KIT gene aberrations showed phosphorylation of the KIT receptor. Expression of stem cell factor (SCF) in melanoma cells as well as stromal cells suggests SCF/KIT autocrine and paracrine activation in these tumors. Finally, we found significant growth suppressive effects of sunitinib in two acral melanoma cell lines; one harboring the D820Y mutation and one showing SCF-dependent KIT activation. These results show pathological activation of KIT in a substantial number of metastatic tumors of acral and mucosal melanomas, and suggest a potential therapeutic benefit of sunitinib for these melanomas.

Simultaneous suppression of MITF and BRAF V600E enhanced inhibition of melanoma cell proliferation.

Microphthalmia-associated transcription factor (MITF) is a master gene regulating differentiation of melanocytes, and a lineage survival oncogene mediating pro-proliferative function in malignant melanoma. However, high expression of MITF also has an anti-proliferative effect. To clarify the therapeutic implication of MITF as a molecular target for human melanoma, we evaluated the role of MITF in cell proliferation in a panel of human melanoma cell lines which express different levels of MITF. We found that both MITF depletion and forced expression of MITF significantly inhibited proliferation, suggesting that endogenous MITF is regulated at an appropriate level for melanoma cell proliferation, and could be a molecular target for melanoma. However, half of the melanoma cell lines in this study were relatively resistant to MITF depletion, indicating other treatment strategies are required for therapy. Our microarray analysis indicated that regulation of several cell growth-associated molecules may be independent of MITF and dependent on BRAF(V600E). Thus to enhance the anti-proliferative effect of MITF down-regulation, we combined shRNA-mediated MITF depletion with BRAF(V600E) inactivation, another known molecular target for melanoma. Indeed, simultaneous depletion of both MITF and BRAF(V600E) significantly inhibited melanoma growth even for the melanoma cell lines resistant to MITF depletion. These results suggest MITF may be an important molecular target for human melanoma and simultaneous inhibition of MITF and MAPK signaling may be an attractive strategy for melanoma treatment.

Multicenter study on finger systolic blood pressure test for diagnosis of vibration-induced white finger.

OBJECTIVE: A multicenter study (six Rosai hospitals around Japan) was performed to investigate the diagnostic value of changes in finger systolic blood pressure (FSBP) after segmental local cooling for vibration-induced white finger (VWF). METHODS: Subjects were 154 men without exposure to vibration and 135 men with occupational vibration exposure. They were classified into four groups: Group A, 154 unexposed control cases; Group B, 21 exposed cases without VWF; Group C, 31 cases with a history of VWF but without any signs of VWF within the last year; and Group D, 83 cases with active VWF within the last year. FSBP% measurements were taken at room temperatures of 23 +/- 1 and 21 +/- 1 degrees C, using a strain-gauge Digimatic 2000 plethysmograph (Medimatic). RESULTS: At a room temperature of 23 +/- 1 degrees C, there was a significant difference between Groups A and D, and B and D. At a room temperature of 21 +/- 1 degrees C, there was a significant difference between Groups A and C, A and D, and B and D. The values in Group D were the lowest at both room temperatures. Assuming a cut-off value of 75% at 23 +/- 1 degrees C, the sensitivity and specificity were 65.2 and 87.5%, respectively. Assuming the same cut-off value at 21 +/- 1 degrees C, the sensitivity and specificity were 73.9 and 82.5%, respectively. These values were not too high. Most of the subjects with WVF in this study were retired and had not used vibratory tools for many years. The situation of the subjects may affect the results of the FSBP test. Our data did not confirm a difference in diagnostic accuracy between room temperatures of 23 +/- 1 and 21 +/- 1 degrees C. CONCLUSIONS: Our study showed that the sensitivity and specificity of the FSBP test with a cut-off value of 75% at 23 +/- 1 degrees C, were 65.2 and 87.5%, respectively, and at 21 +/- 1 degrees C, they were 73.9 and 82.5%, respectively.

[Case of interstitial pneumonia induced by intravesical administration of Bacillus Calmette-Guerin].

An 81-year-old man with superficial bladder cancer, which was detected after he complained of hematuria and pain on urination, was treated 5 times with intravesical bacillus Calmette-Guerin (BCG) administration. Since he complained of a dry cough and dyspnea associated with severe hypoxemia, and had extensive bilateral pulmonary infiltrative shadows on his chest X-ray film, he was admitted to our hospital. He was treated with methylprednisolone under a diagnosis of interstitial pneumonia. Because BCG infection and/or bacterial pneumonia could not be denied, we administered antituberculosis agent and antibiotics. However, he died due to respiratory failure on 10th day after admission. Because a lymphocyte stimulation test for BCG was strongly positive, we established a diagnosis of interstitial pneumonia induced by intravesical administration of BCG. Because the mortality rate is high, the evaluation of interstitial pneumonia before administration of BCG is crucial.

A novel cancer testis antigen that is frequently expressed in pancreatic, lung, and endometrial cancers.

PURPOSE: To isolate cancer testis antigens that are expressed in pancreatic cancers and may be useful in clinical applications. EXPERIMENTAL DESIGN: To efficiently isolate cancer testis antigens, a testis cDNA library was immunoscreened (SEREX) with serum from a patient with pancreatic ductal adenocarcinoma. The expression of isolated antigens in various cancer cell lines and tissues was evaluated by reverse transcription-PCR and Northern blot analyses. The immunogenicity of the antigen in cancer patients was evaluated by detection of the IgG antibody in sera from patients with various cancers. RESULTS: Of the three clones isolated through screening of a total of 2 x 10(6) cDNA library clones, one clone (KU-CT-1) was found to be expressed in various cancers but only in testis among normal tissues, indicating that it was a novel cancer testis antigen. The KU-CT-1 gene is located on chromosome 10p12 and produces two splice variants, which encode proteins of 397 and 872 amino acids, respectively. KU-CT-1 was expressed in pancreatic cancer tissues (3 of 9, 33%), lung cancer tissues (9 of 24, 38%), and endometrial cancer tissues (7 of 11, 64%). Specific serum IgG antibodies were detected in 3 of 20 pancreatic cancer patients, 2 of 12 endometrial cancer patients, 1 of 18 colon cancer patients, and 1 of 10 prostate cancer patients but not detected in 30 healthy individuals. CONCLUSIONS: KU-CT-1 is a new cancer testis antigen that is expressed in pancreatic, lung, and endometrial cancers and may be useful for diagnosis and immunotherapy for patients with various cancers.

Expression of thymidylate synthase predicts clinical outcomes of S-1-based chemotherapy in squamous cell lung cancer.

Non-small cell lung cancer (NSCLC) patients with squamous cell carcinoma (SCC) histology have limited chemotherapeutic options. Treatment with S-1 combined with carboplatin (CBDCA) has been shown to provide a significant survival benefit in SCC patients compared with treatment with combined CBDCA and paclitaxel. The aim of the present study was to investigate the association between the expression of molecular markers related to the pharmacological action of S-1, including thymidylate synthase (TS), orotate phosphoribosyltransferase (OPRT) and dihydropyrimidine dehydrogenase (DPD), and the clinical efficacy of S-1-based chemotherapy in SCC patients. The immunohistochemical expression of TS, OPRT and DPD were retrospectively analyzed in tumor biopsy and resection specimens from patients with advanced SCC (n=32). Immunohistochemical H-scores were calculated and their association with S-1/CBDCA response was evaluated. Median progression-free survival time was significantly longer in patients with low TS H-scores than in those with high TS H-scores (162.5 vs. 97 days; P=0.004); by contrast, overall survival time was not observed to differ significantly between these groups (P=0.185). In the multivariate analysis, low TS expression was a significant positive factor for progression-free survival rate (hazard ratio, 0.40; P=0.021). A low TS H-score was also associated with an increased response to S-1-based chemotherapy compared with a high TS H-score (P=0.002). This indicates that SCC patients with low TS expression can benefit significantly from S-1-based chemotherapy, and that H-score measurement of intratumoral TS expression may represent a useful predictive biomarker for response to S-1-based chemotherapy by patients with SCC-type NSCLC.

Relation between distal motor latency delay and sensory action potential absence of the median nerve in carpal tunnel syndrome.

OBJECTIVE: To elucidate the cutoff value for distal motor latency (DML) at which sensory nerve action potentials (SNAPs) are absent in carpal tunnel syndrome (CTS) patients. METHOD: We examined 157 hands in 129 patients with CTS retrospectively. We classified the patients according to whether SNAPs were successfully obtained. Group A consisted of hands with SNAPs, while Group B consisted of hands without SNAPs. The cutoff value for DML was determined by receiver-operating characteristic curve analysis. We enrolled 130 hands with CTS for the analysis, because measurements were successful in 130 hands for compound muscle action potentials and in 82 hands for SNAPs from a total of 157 hands investigated. RESULTS: A significant correlation was observed between DML and SCV (P<0.0001, R*2=0.40). The most discriminative cutoff value for DML was 7.7ms, resulting in a sensitivity of 79.6% and specificity of 79.3%. CONCLUSION: Cases in which SNAPs are evoked despite a DML longer than 7.7ms should raise suspicion.

Diagnostic significance of cerebrospinal fluid EGFR mutation analysis for leptomeningeal metastasis in non-small-cell lung cancer patients harboring an active EGFR mutation following gefitinib therapy failure.

BACKGROUND: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been successfully used to treat patients with non-small cell lung cancer (NSCLC) harboring EGFR mutations. However, despite an initial excellent response, recurrence within one or two years is common. Diagnosis and treatment of leptomeningeal metastasis (LM), a form of NSCLC recurrence, remains particularly difficult. Here, we analyzed the EGFR mutation status of cerebrospinal fluid (CSF) directly using real-time polymerase chain reaction (PCR) and evaluated the efficacy of therapy with erlotinib, an EGFR TKI. PATIENTS AND METHODS: Seven NSCLC patients harboring activating EGFR mutations who had developed LM during or after therapy with gefitinib, an EGFR TKI, were retrospectively analyzed. CSF was obtained and subjected to cytological examination and EGFR mutation analysis, including detection of the resistance-associated T790M mutation, using real-time PCR. RESULTS: In all seven cases, the EGFR mutation detected in the CSF was the same as that detected in the primary tumor (sensitivity, 100%). Conversely, cytology results were positive in only two patients (sensitivity, 28.6%). No additional T790M mutations were detected. Erlotinib was efficacious in all cases, and improved performance status was achieved for five of the seven patients. The effect of erlotinib treatment was temporary, however, with time to treatment failure (TTF) ranging from 29 to 278 days (median, 65 days) and the interval between commencement of erlotinib treatment and death ranging from 45 to 347 days (median, 168 days). CONCLUSIONS: Analysis of EGFR mutations in CSF using a highly sensitive real-time PCR assay is a potentially powerful diagnostic method for LM.

Diagnosis of Severe Carpal Tunnel Syndrome Using Nerve Conduction Study and Ultrasonography.

This study investigated the correlation between nerve conduction study and ultrasonographic findings for assessment of the usefulness of ultrasonography in determining carpal tunnel syndrome severity. Hands of adults with carpal tunnel syndrome were assessed using ultrasound and nerve conduction studies and grouped according to median nerve cross-sectional area (CSA). There were significant differences (p < 0.01) in mean median nerve CSA between controls, patients with median sensory nerve conduction velocity ≤40 m/s and patients with absent sensory nerve action potential and between controls, patients with median nerve distal motor latency ≥4.5 ms and patients with absent compound muscle action potentials of the abductor pollicis brevis. This is the first report to define median nerve CSA cutoff values (18 mm(2)) for determining carpal tunnel syndrome severity in patients with absent compound muscle action potentials of the abductor pollicis brevis. Median nerve CSA values below the cutoff values should prompt clinicians to consider other disorders, such as cervical compressive myelopathy.

The N-ERC index is a novel monitoring and prognostic marker for advanced malignant pleural mesothelioma.

BACKGROUND: Although N-ERC/mesothelin (N-ERC) is an attractive diagnostic and treatment monitoring biomarker for malignant pleural mesothelioma (MPM), its clinical utility for predicting the prognosis has not yet been clarified. The aim of this study is to investigate whether the serum N-ERC level can accurately predict the outcome in patients with MPM. METHODS: Twenty-six patients with MPM were enrolled. Serum N-ERC level was measured before and after chemotherapy. The N-ERC index was determined by the logarithm of the division of the N-ERC level after two courses of chemotherapy by the prior level. RESULTS: The median N-ERC index in the partial response (PR) group was significantly lower than that in patients with the stable disease (SD) plus the progressive disease (PD) group. The overall survival in the group whose median N-ERC index was lower than its median value was significantly longer than the group whose median N-ERC index was higher than its median value. CONCLUSIONS: The N-ERC index is therefore considered to be a useful biomarker for predicting not only the chemotherapeutic response, but also the prognosis in patients with advanced MPM.

Hydration with magnesium and mannitol without furosemide prevents the nephrotoxicity induced by cisplatin and pemetrexed in patients with advanced non-small cell lung cancer.

BACKGROUND: The aim of this study was to examine the effect of hydration with magnesium and mannitol without furosemide on the nephrotoxocity accompanying combination chemotherapy using cisplatin and pemetrexed in patients with advanced non-small cell lung cancer (NSCLC). METHODS: Fifty patients with NSCLC who received cisplatin plus pemetrexed, using either old hydration protocol including normal saline with mannitol and furosemide, or a new one including normal saline with magnesium and mannitol without furosemide were retrospectively analyzed. Nephrotoxicity was compared between patients treated using the old protocol and those treated with the new protocol. Univariate and multivariate analyses were performed to identify the independent factors associated with protection against nephrotoxicity in patients with NSCLC who received cisplatin plus pemetrexed. RESULTS: Thirty patients received the old hydration protocol, while 20 patients were treated using the new hydration protocol. The patients treated using the new hydration protocol showed a significantly greater increase in creatinine clearance (P=0.0004) and a decrease in the serum creatinine level (P=0.0148) after one course of chemotherapy compared with those treated using the old hydration protocol. There were no differences in the chemotherapeutic response or overall survival between the groups (P=0.572). The new hydration protocol with supplemented magnesium with mannitol without furosemide was an independent factor for the protection against nephrotoxicity induced by cisplatin and pemetrexed in patients with advanced NSCLC [HR 0.232 (95% CI: 0.055-0.986), P=0.039]. CONCLUSIONS: These results demonstrate that the new hydration protocol comprising supplementation with magnesium without furosemide could prevent the nephrotoxicity induced by cisplatin and pemetrexed without affecting the treatment outcome.

Polyclonality of BRAF mutations in acquired melanocytic nevi.

Melanocytic nevi are thought to be senescent clones of melanocytes that have acquired an oncogenic BRAF mutation. BRAF mutation is considered to be a crucial step in the initiation of melanocyte transformation. However, using immunomagnetic separation or laser-capture microdissection, we examined BRAF mutations in sets of approximately 50 single cells isolated from acquired melanocytic nevi from 13 patients and found a substantial number of nevus cells that contained wild-type BRAF mixed with nevus cells that contained BRAF(V600E). Furthermore, we simultaneously amplified BRAF exon 15 and a neighboring single nucleotide polymorphism (SNP), rs7801086, from nevus cell samples obtained from four patients who were heterozygous for this SNP. Subcloning and sequencing of the polymerase chain reaction products showed that both SNP alleles harbored the BRAF(V600E) mutation, indicating that the same BRAF(V600E) mutation originated from different cells. The polyclonality of BRAF mutations in acquired melanocytic nevi suggests that mutation of BRAF may not be an initial event in melanocyte transformation.