Rechallenge of afatinib for EGFR-mutated non-small cell lung cancer previously treated with osimertinib: a multicenter phase II trial protocol (REAL study).

Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of advanced non-small cell lung cancer (NSCLC) and contributed to the development of precision medicine. Osimertinib is a standard first-line (1L) treatment for EGFR-mutated NSCLC and has demonstrated superior survival benefits over previous-generation TKIs. However, resistance to osimertinib is nearly inevitable, and subsequent treatment strategies remain unmet medical needs in this setting. Afatinib, a second-generation EGFR-TKI, exhibits activity against certain uncommon EGFR mutation types in the 1L setting. There are a few case reports on the efficacy of afatinib against EGFR-dependent resistance after osimertinib treatment, although these have not been prospectively investigated. Methods: The present phase II, single-arm multicenter trial aims to verify the efficacy and safety of afatinib rechallenge after 1L osimertinib resistance. Patients (aged ≥20 years) with advanced or recurrent non-squamous NSCLC harboring drug-sensitive EGFR mutations (deletion of exon 19 or L858R) who were previously treated with 1L osimertinib and second-line chemotherapy other than TKIs are considered eligible. Undergoing next-generation sequence-based comprehensive genomic profiling is one of the key inclusion criteria. The primary endpoint is the objective response rate; the secondary endpoints are progression-free survival, overall survival, and tolerability. Thirty patients will be recruited in December 2023. Discussion: The results of this study may promote incorporating afatinib rechallenge into the treatment sequence after 1L osimertinib resistance, a setting in which concrete evidence has not been yet established. Registration: UMIN Clinical Trial Registry: UMIN000049225.

Association between lower air pressure and the onset of ischemic colitis: a case-control study.

OBJECTIVE: Ischemic colitis (IC) often affects the elderly. Proarteriosclerotic factors, such as hypertension and smoking, and cardiovascular disease are considered major contributors to IC. Although a possible link between certain cerebrocardiovascular disorders and meteorological phenomena has been reported, the relationship between IC onset and weather changes remains uninvestigated. This study examined whether specific meteorological factors were associated with the occurrence of IC. PATIENTS AND METHODS: We retrospectively enrolled 303 patients who had been diagnosed with IC between January 2003 and June 2010 at Suwa Red Cross Hospital in Nagano Prefecture, Japan. The meteorological data of the days on which IC patients visited the hospital (IC+ days) were compared with those of the days on which IC patients did not (IC- days). RESULTS: Univariate analysis indicated that IC+ days had significantly lower air pressure (P<0.001), depressed air pressure from the previous day (P<0.001), and fewer daylight hours (P<0.001), as well as higher air temperature (P=0.017), air humidity (P=0.004), wind velocity (P<0.001), and rainfall (P=0.012) compared with IC- days. Multivariate logistic regression analysis of the meteorological data showed that air pressure (odds ratio: 0.935, P<0.001) and change in air pressure from the previous day (odds ratio: 0.934, P<0.001) were related to onset of IC. CONCLUSION: Lower air pressure and decrease in air pressure from the previous day are possible novel factors associated with the development of IC.

Low level light effects on inflammatory cytokine production by rheumatoid arthritis synoviocytes.

BACKGROUND AND OBJECTIVE: Low level light therapy (LLLT) is being evaluated for treating chronic and acute pain associated with rheumatoid arthritis (RA) and other inflammatory diseases. The mechanisms underlying the effectiveness of LLLT for pain relief in RA are not clear. The objectives of this study were to determine whether LLLT decreased production of pro-inflammatory cytokines by cells from RA joints, and, if so, to identify cellular mechanisms. STUDY DESIGN/MATERIALS AND METHODS: Synoviocytes from RA patients were treated with 810 nm radiation before or after addition of tumor necrosis factor-alpha (TNF-alpha). mRNA for TNF-alpha, interleukin (IL)-1beta, IL-6, and IL-8 was measured after 30, 60, and 180 minutes using RT-PCR. Intracellular and extracellular protein levels for 12 cytokines/chemokines were measured at 4, 8, and 24 hours using multiplexed ELISA. NF-kappaB activation was detected using Western blotting to follow degradation of IkappaBalpha and nuclear localization of the p65 subunit of NF-kappaB. RESULTS: Radiation at 810 nm (5 J/cm(2)) given before or after TNF-alpha decreases the mRNA level of TNF-alpha and IL-1beta in RA synoviocytes. This treatment using 25 J/cm(2) also decreases the intracellular levels of TNF-alpha, IL-1beta, and IL-8 protein but did not affect the levels of seven other cytokines/chemokines. TNF-alpha-induced activation of NF-kappaB is not altered by 810 nm radiation using 25 J/cm(2). CONCLUSIONS: The mechanism for relieving joint pain in RA by LLLT may involve reducing the level of pro-inflammatory cytokines/chemokines produced by synoviocytes. This mechanism may be more general and underlie the beneficial effects of LLLT on other inflammatory conditions.

NADPH oxidase 4 contributes to transformation phenotype of melanoma cells by regulating G2-M cell cycle progression.

Generation of reactive oxygen species (ROS) has been implicated in carcinogenic development of melanoma, but the underlying molecular mechanism has not been fully elucidated. We studied the expression and function of the superoxide-generating NADPH oxidase (Nox)4 in human melanoma cells. Nox4 was up-regulated in 13 of 20 melanoma cell lines tested. Silencing of Nox4 expression in melanoma MM-BP cells by small interfering RNAs decreased ROS production and thereby inhibited anchorage-independent cell growth and tumorigenecity in nude mice. Consistently, a general Nox inhibitor, diphenylene iodonium, and antioxidants vitamine E and pyrrolidine dithiocarbamate blocked cell proliferation of MM-BP cells. Flow cytometric analysis indicated that Nox4 small interfering RNAs and diphenylene iodonium induced G(2)-M cell cycle arrest, which was also observed with another melanoma cell line, 928mel. This was accompanied by induction of the Tyr-15 phosphorylated, inactive form of cyclin-dependent kinase 1 (a hallmark of G(2)-M checkpoint) and hyperphosphorylation of cdc25c leading to its increased binding to 14-3-3 proteins. Ectopic expression of catalase, a scavenger of ROS, also caused accumulation of cells in G(2)-M phase. Immunohistochemistry revealed that expression of Nox4 was detected in 31.0% of 13 melanoma patients samples, suggesting the association of Nox4 expression with some steps of melanoma development. The findings suggest that Nox4-generated ROS are required for transformation phenotype of melanoma cells and contribute to melanoma growth through regulation of G(2)-M cell cycle progression.

Adult onset X-linked chronic granulomatous disease in a woman patient caused by a de novo mutation in paternal-origin CYBB gene and skewed inactivation of normal maternal X chromosome.

We report a 28-year-old woman patient suffering from refractory subcutaneous abscess. Stimuli-induced microbicidal reactive oxygen metabolites formation test of the patient's neutrophils revealed that only 9.6% of the neutrophils produced H2O2. DNA analysis of the CYBB that encodes gp91(phox) demonstrated that she was heterozygous for a nonsense mutation, 206Trp(TGG)/stop(TGA) and therefore, a diagnosis of adult onset X-linked chronic granulomatous disease was made. Our molecular biological study revealed that her disease was caused by a de novo mutation in the CYBB gene on the paternal-origin X-chromosome and a skewed inactivation of the normal maternal X-chromosome.

A case of primary Epstein-Barr virus-associated cutaneous diffuse large B-cell lymphoma unassociated with iatrogenic or endogenous immune dysregulation.

Cutaneous Epstein-Barr virus (EBV)-associated B-cell lymphoma (EBVBL) in non-immunocompromised patients is very rare. Here, we report a case of cutaneous EBVBL in a 72-year-old Japanese woman without any signs of immunosuppression. She showed repeated high fever and skin eruptions on the face, limbs and palms. Histological diagnosis was diffuse large B-cell lymphoma. EBV infection was detected by in situ hybridization and Southern blotting. Immunostaining for viral proteins showed the patient to be positive for latent membrane protein 1 (LMP-1) and negative for Epstein-Barr virus nuclear antigen-1 (EBNA-2), indicating that a type II latency EBV infection pattern.

Establishment of a novel melanoma cell line SMYM-PRGP showing cytogenetic and biological characteristics of the radial growth phase of acral melanomas.

We established a novel melanoma cell line, SMYM-PRGP, which was non-tumorigenic in vivo, from an acral melanoma in radial growth phase under a low-oxygen environment. SMYM-PRGP was wild-type for known mutation sites in the BRAF and NRAS genes, and showed focal amplification of the human telomerase reverse transcriptase and cyclin D1 genes as well as the fibroblast growth factor-3 and fibroblast growth factor-4 genes. Neither mutation nor copy number loss of the CDKN2A gene was observed. The p16(INK4A) protein was expressed at a level equal to that in normal melanocytes. Among the various melanocyte growth factors added to the culture of SMYM-PRGP cells, endothelin-1 was the strongest growth stimulator, the effect of which was significantly augmented by the addition of calcium chloride. The growth stimulatory effect of endothelin-1 was shown to be mediated via the endothelin B receptor. The protein level of cyclin D1 in SMYM-PRGP cells was approximately 10 times higher than that in normal melanocytes. Although the stimulation with endothelin-1 plus calcium chloride increased cyclin D1 protein levels after 4-6 h, the level of phosphorylated retinoblastoma protein did not increase, suggesting that overexpression of cyclin D1 protein may have little effect on cell cycle progression but rather act as a pro-survival factor. SMYM-PRGP is an excellent tool for investigating the development and progression of acral melanoma.

Specific dermoscopy patterns and amplifications of the cyclin D1 gene to define histopathologically unrecognizable early lesions of acral melanoma in situ.

OBJECTIVE: To define early lesions of acral melanoma in situ that cannot be recognized histopathologically. DESIGN: A retrospective review of the clinical, dermoscopic, and histopathological findings. SETTING: University department of dermatology. PATIENTS: Thirty-three patients with melanocytic lesions on acral volar skin that were clinically suspected of being early melanomas. MAIN OUTCOME MEASURES: Fluorescent in situ hybridization studies to detect the cyclin D1 gene amplification in proliferating melanocytes, which is a characteristic genetic aberration recently found in acral melanoma. RESULTS: Seventeen of 33 lesions were histopathologically diagnosed as either melanoma in situ (8 lesions) or benign melanocytic nevi (9 lesions). Amplification of the cyclin D1 gene was observed in 2 (25%) of the 8 melanomas in situ. None of the 9 nevi showed the amplification. The remaining 16 lesions were, however, difficult to classify histopathologically because most of them showed only a slight increase of nonatypical melanocytes in the basal cell layer of the epidermis. On dermoscopic examination, 9 of these 16 lesions exhibited the parallel ridge pattern that has been reported to be highly specific to melanoma in situ, and 4 (44%) of them had amplifications of the cyclin D1 gene. Amplifications were not found in any of the remaining 7 lesions that showed dermoscopic patterns suggestive of melanocytic nevi. CONCLUSIONS: Cyclin D1 gene amplification detected by fluorescent in situ hybridization identified a very early progression phase of acral melanoma that precedes histopathologically defined melanoma in situ. The present study also indicates the specificity of the parallel ridge pattern on dermoscopy to detect melanomas on acral volar skin at such a very early developmental phase.

Constitutive activation of the mitogen-activated protein kinase signaling pathway in acral melanomas.

One of the most attractive clinical targets for melanoma is the mitogen-activated protein kinase (MAPK) signaling pathway. In this study, we examined MAPK signaling activation in a total of 28 acral melanoma samples, consisting of 13 primary tumors and 15 metastases. In line with the previous reports, NRAS/BRAF mutations were rare; only one metastatic tumor had an NRAS E61R mutation, and one primary tumor and two metastases harbored BRAF V599E mutations. Western blot analyses, however, revealed phosphorylated extracellular signal-regulated kinase (ERK)1/2 proteins in 11 of 14 (78.5%) of the acral melanoma tumors. Furthermore, fluorescence in situ hybridization analyses revealed the prominent amplification of the cyclin D1 (CCND1) gene, which is an important down-stream effecter of the MAPK pathway, in 5 of 21 (23.8%) tumors examined. Interestingly, two of three tumors that were negative for phosphorylated ERK proteins according to western blot harbored CCND1 amplifications, suggesting that the increased gene dosage of CCND1 may exert effects similar to phosphorylated ERK proteins in cell growth. We conclude that, despite the low frequency of BRAF/NRAS mutations, the MAPK signaling pathway is constitutively activated in the majority of acral melanomas. This provides a rational basis to include acral melanomas into the clinical trials with MAPK inhibitors.