Successful Treatment of Hereditary Folate Malabsorption With Intramuscular Folinic Acid.

BACKGROUND: Hereditary folate malabsorption is a multisystem disease owing to biallelic variants in the gene encoding the proton-coupled folate transporter. Hereditary folate malabsorption is treated with folinic acid, aimed to restore blood and cerebrospinal fluid folate levels. Little is known as to whether oral or intramuscular supplementation of folinic acid is most effective. METHODS: Here we describe a one-year-old boy with hereditary folate malabsorption presenting with the typical features including failure to thrive, aphthous stomatitis, macrocytic anemia along with severe developmental impairment and epilepsy, as well as a magnetic resonance imaging of the brain showing bilateral occipital, cortical calcifications characteristic of hereditary folate malabsorption. We compared the effect of treatment with oral folinic acid versus intramuscular folinic acid supplementation by measuring plasma and cerebrospinal fluid folate levels. RESULTS: Compared with oral administration, intramuscular treatment resulted in higher folate levels in blood and, most importantly, normalization of folate levels in cerebrospinal fluid. Clinically, nearly all systemic and neurological symptoms resolved. CONCLUSION: Normal cerebrospinal fluid folate levels can be achieved in individuals with hereditary folate malabsorption with intramuscular (but not with oral) administration of folinic acid.

Comprehensive DNA methylation study identifies novel progression-related and prognostic markers for cutaneous melanoma.

BACKGROUND: Cutaneous melanoma is the deadliest skin cancer, with an increasing incidence and mortality rate. Currently, staging of patients with primary melanoma is performed using histological biomarkers such as tumor thickness and ulceration. As disruption of the epigenomic landscape is recognized as a widespread feature inherent in tumor development and progression, we aimed to identify novel biomarkers providing additional clinical information over current factors using unbiased genome-wide DNA methylation analyses. METHODS: We performed a comprehensive DNA methylation analysis during all progression stages of melanoma using Infinium HumanMethylation450 BeadChips on a discovery cohort of benign nevi (n = 14) and malignant melanoma from both primary (n = 33) and metastatic (n = 28) sites, integrating the DNA methylome with gene expression data. We validated the discovered biomarkers in three independent validation cohorts by pyrosequencing and immunohistochemistry. RESULTS: We identified and validated biomarkers for, and pathways involved in, melanoma development (e.g., HOXA9 DNA methylation) and tumor progression (e.g., TBC1D16 DNA methylation). In addition, we determined a prognostic signature with potential clinical applicability and validated PON3 DNA methylation and OVOL1 protein expression as biomarkers with prognostic information independent of tumor thickness and ulceration. CONCLUSIONS: Our data underscores the importance of epigenomic regulation in triggering metastatic dissemination through the inactivation of central cancer-related pathways. Inactivation of cell-adhesion and differentiation unleashes dissemination, and subsequent activation of inflammatory and immune system programs impairs anti-tumoral defense pathways. Moreover, we identify several markers of tumor development and progression previously unrelated to melanoma, and determined a prognostic signature with potential clinical utility.

Effectiveness and safety of antihistamines up to fourfold or higher in treatment of chronic spontaneous urticaria.

BACKGROUND: Treatment with second-generation antihistamines is recommended in patients with chronic spontaneous urticaria (CSU). Some patients remain unresponsive even after up-dosing up to fourfold. Many third line treatment options have limited availability and/or give rise to significant side effects. We investigated effectiveness and safety of antihistamine treatment with dosages up to fourfold and higher. METHODS: This retrospective analysis of patients' records was performed in adult CSU patients suffering wheals and/or angioedema (AE). Demographic, clinical, and therapeutic data was extracted from their medical records. We recorded the type, maximum prescribed dosage, effectiveness, and reported side effects of antihistamine treatment. RESULTS: Of 200 screened patients, 178 were included. Treatment was commenced with a once daily dose of antihistamines. Persisting symptoms meant that up-dosing up to fourfold occurred in 138 (78%) of patients, yielding sufficient response in 41 (23%). Up-dosing antihistamines was necessary in 110 (80%) patient with weals alone or weals with angioedema and 28 (64%) with AE only (p = 0.039). Of the remaining 97 patients with insufficient response, 59 were treated with dosages higher than fourfold (median dosage 8, range 5-12). This was sufficient in 29 patients (49%). Side effects were reported in 36 patients (20%), whereof 30 (17%) experienced somnolence. Side effects after up-dosing higher than fourfold were reported in six out of 59 patients (10%). CONCLUSION: Up-dosing antihistamines higher than fourfold dosage seems a feasible therapeutic option with regards to effectiveness and safety. The need for third line therapies could be decreased by 49%, with a very limited increase of reported side effects.

High-throughput oncogene mutation profiling shows demographic differences in BRAF mutation rates among melanoma patients.

Because of advances in targeted therapies, the clinical evaluation of cutaneous melanoma is increasingly based on a combination of traditional histopathology and molecular pathology. Therefore, it is necessary to expand our knowledge of the molecular events that accompany the development and progression of melanoma to optimize clinical management. The central objective of this study was to increase our knowledge of the mutational events that complement melanoma progression. High-throughput genotyping was adapted to query 159 known single nucleotide mutations in 33 cancer-related genes across two melanoma cohorts from Ireland (n=94) and Belgium (n=60). Results were correlated with various clinicopathological characteristics. A total of 23 mutations in 12 genes were identified, that is--BRAF, NRAS, MET, PHLPP2, PIK3R1, IDH1, KIT, STK11, CTNNB1, JAK2, ALK, and GNAS. Unexpectedly, we discovered significant differences in BRAF, MET, and PIK3R1 mutations between the cohorts. That is, cases from Ireland showed significantly lower (P<0.001) BRAF(V600E) mutation rates (19%) compared with the mutation frequency observed in Belgian patients (43%). Moreover, MET mutations were detected in 12% of Irish cases, whereas none of the Belgian patients harbored these mutations, and Irish patients significantly more often (P=0.027) had PIK3R1-mutant (33%) melanoma versus 17% of Belgian cases. The low incidence of BRAF(V600E)(-) mutant melanoma among Irish patients was confirmed in five independent Irish cohorts, and in total, only 165 of 689 (24%) Irish cases carried mutant BRAF(V600E). Together, our data show that melanoma-driving mutations vary by demographic area, which has important implications for the clinical management of this disease.

BRAFV600E immunopositive melanomas show low frequency of heterogeneity and association with epithelioid tumor cells: a STROBE-compliant article.

Treatment of BRAFV600E-mutant melanoma by small molecule inhibitors that target BRAFV600E or MEK kinases is increasingly used in clinical practice and significantly improve patient outcome. However, patients eventually become resistant and therapeutic improvement is required. Molecular diversity within individual tumors (intratumor heterogeneity) and between tumors within a single patient (intrapatient heterogeneity) poses a significant challenge to precision medicine. Using immunohistochemistry, we determined the extent of BRAFV600E intratumor and intrapatient heterogeneity and the influence of morphological heterogeneity in a large series of 171 melanomas of 81 patients. The BRAFV600E mutation rate found in our melanoma series is 44%, with none of 22 (0%) melanoma in situ, 23 of 56 (41%) primary tumors, 28 of 59 (48%) regional metastases, and 24 of 34 (71%) distant metastases harboring the mutation. In general, a diffuse homogeneous immunostaining was seen, even in tumors consisting of more than one cell type, that is, epithelioid, spindle, and/or small cell types. Nevertheless, BRAFV600E-mutant melanomas more often had a purely epithelioid cell population (P=0.063), that is more evident among distant metastases (P=0.014). Only two of 75 (3%) mutated specimens (one primary and one metastasis) displayed heterogeneous BRAFV600E expression. The primary tumor was also morphologically heterogeneous and exclusively displayed BRAFV600E in the epithelioid component, confirming an association between BRAFV600E and epithelioid cells. Twenty-eight of 30 patients (93%) had concordant BRAFV600E mutation status between their tumors. Taken together, BRAFV600E intratumor and intrapatient heterogeneity in melanoma is diminutive, nevertheless, the identified exceptions will have important implications for the clinical management of this disease.

Promoter CpG island hypermethylation in dysplastic nevus and melanoma: CLDN11 as an epigenetic biomarker for malignancy.

Dysplastic nevi are melanocytic lesions that represent an intermediate stage between common nevus and melanoma. Histopathological distinction of dysplastic nevus from melanoma can be challenging and there is a requirement for molecular diagnostic markers. In this study, we examined promoter CpG island methylation of a selected panel of genes, identified in a genome-wide methylation screen, across a spectrum of 405 melanocytic neoplasms. Promoter methylation analysis in common nevi, dysplastic nevi, primary melanomas, and metastatic melanomas demonstrated progressive epigenetic deregulation. Dysplastic nevi were affected by promoter methylation of genes that are frequently methylated in melanoma but not in common nevi. We assessed the diagnostic value of the methylation status of five genes in distinguishing primary melanoma from dysplastic nevus. In particular, CLDN11 promoter methylation was specific for melanoma, as it occurred in 50% of primary melanomas but in only 3% of dysplastic nevi. A diagnostic algorithm that incorporates methylation of the CLDN11, CDH11, PPP1R3C, MAPK13, and GNMT genes was validated in an independent sample set and helped distinguish melanoma from dysplastic nevus (area under the curve 0.81). Melanoma-specific methylation of these genes supports the utility as epigenetic biomarkers and could point to their significance in melanoma development.

Loss of microRNA-200a and c, and microRNA-203 expression at the invasive front of primary cutaneous melanoma is associated with increased thickness and disease progression.

Loss of E-cadherin expression in melanoma correlates with increased tumor thickness and reduced disease-free survival. The molecular mechanisms underpinning its differential expression in melanoma tissue remain elusive. MicroRNAs (miRNAs) have been implicated in tumor progression and regulation of E-cadherin expression. Here, we demonstrate a significant correlation between tumor thickness and loss of expression of miR-200a, miR-200c, and miR-203 in a series of 23 frozen primary melanomas, where it was confirmed in two subsequent validation series (series 1: six nevi, 15 primary melanomas, and 16 metastases; series 2: 11 matched pairs of primary melanomas and metastases). Decreased levels of miR-200a, miR-200c, and miR-203 correlated with increasing thickness in the combined validation series (P = 0.024, 0.033, and 0.031, respectively). In addition, progressive loss of miR-200a expression with disease progression was observed in series 1 (P < 0.001) and in series 2 (P = 0.029). MiR-200 in situ hybridization and E-cadherin immunohistochemistry demonstrated reduced expression of both at the deep invasive margin of the tumor. Furthermore, a functional validation study using an anti-miR200 strategy demonstrated that loss of miR-200 expression in melanoma cell lines reduced E-cadherin expression. Collectively, our data point towards an important role for miR-200 and miR203 expression in regulating E-cadherin during melanoma progression.

Genetics and epigenetics of cutaneous malignant melanoma: a concert out of tune.

Cutaneous malignant melanoma (CMM) is the most life-threatening neoplasm of the skin and is considered a major health problem as both incidence and mortality rates continue to rise. Once CMM has metastasized it becomes therapy-resistant and is an inevitably deadly disease. Understanding the molecular mechanisms that are involved in the initiation and progression of CMM is crucial for overcoming the commonly observed drug resistance as well as developing novel targeted treatment strategies. This molecular knowledge may further lead to the identification of clinically relevant biomarkers for early CMM detection, risk stratification, or prediction of response to therapy, altogether improving the clinical management of this disease. In this review we summarize the currently identified genetic and epigenetic alterations in CMM development. Although the genetic components underlying CMM are clearly emerging, a complete picture of the epigenetic alterations on DNA (DNA methylation), RNA (non-coding RNAs), and protein level (histone modifications, Polycomb group proteins, and chromatin remodeling) and the combinatorial interactions between these events is lacking. More detailed knowledge, however, is accumulating for genetic and epigenetic interactions in the aberrant regulation of the INK4b-ARF-INK4a and microphthalmia-associated transcription factor (MITF) loci. Importantly, we point out that it is this interplay of genetics and epigenetics that effectively leads to distorted gene expression patterns in CMM.

Epigenetic changes in Basal Cell Carcinoma affect SHH and WNT signaling components.

BACKGROUND: The genetic background of Basal Cell Carcinoma (BCC) has been studied extensively, while its epigenetic makeup has received comparatively little attention. Epigenetic alterations such as promoter hypermethylation silence tumor suppressor genes (TSG) in several malignancies. OBJECTIVE: We sought to analyze the promoter methylation status of ten putative (tumor suppressor) genes that are associated with Sonic Hedgehog (SHH), WNT signaling and (hair follicle) tumors in a large series of 112 BCC and 124 healthy control samples by methylation-specific PCR. RESULTS: Gene promoters of SHH (P = 0.016), adenomatous polyposis coli (APC) (P = 0.003), secreted frizzled-related protein 5 (SFRP5) (P = 0.004) and Ras association domain family 1A (RASSF1A) (P = 0.023) showed significantly more methylation in BCC versus normal skin. mRNA levels of these four genes were reduced for APC and SFRP5 in BCC (n = 6) vs normal skin (n = 6). Down regulation of SHH, APC and RASSF1A could be confirmed on protein level as well (P<0.001 for all genes) by immunohistochemical staining. Increased canonical WNT activity was visualized by ß-catenin staining, showing nuclear ß-catenin in only 28/101 (27.7%) of BCC. Absence of nuclear ß-catenin in some samples may be due to high levels of membranous E-cadherin (in 94.1% of the samples). CONCLUSIONS: We provide evidence that promoter hypermethylation of key players within the SHH and WNT pathways is frequent in BCC, consistent with their known constitutive activation in BCC. Epigenetic gene silencing putatively contributes to BCC tumorigenesis, indicating new venues for treatment.

TLR2 activation is essential to induce a Th1 shift in human peripheral blood mononuclear cells by plant stanols and plant sterols.

Plant sterols may induce a Th1 shift in humans. However, whether plant stanols have similar effects as well as the underlying mechanism are unknown. We have now shown that (like sitosterol) sitostanol, both 4-desmethylsterols, induces a Th1 shift when added in vitro at physiological concentrations to human PBMCs. This conclusion was based on a higher IFNgamma production, with no change in the production of IL-4 and IL-10. alpha-Amyrin, a 4.4-dimethylsterol, had comparable effects. Because 4.4-dimethylsterols cannot activate transcription factor LXR, this finding indicates that LXR activation was not involved. Sitosterol and sitostanol did not alter the production of IL-12 and IL-18 in PBMCs as well as in monocyte-derived U937 cells, suggesting that plant sterols directly affect T-helper cells, without activating APCs. However, in PBMCs treated with a TLR2 blocker (T2.5), IFNgamma production was completely inhibited, whereas blocking TLR4 with HTA125 had no such effect. To confirm these findings, PBMCs from TLR2(-/-) mice were cultured in the presence of sitosterol and sitostanol. In these cells, no Th1 shift was observed. Our results, therefore, indicate that TLR2 activation is essential to induce a Th1 shift in human PBMCs by plant stanols and plant sterols.