KEAP1/NFE2L2 Pathway Signature Outperforms KEAP1/NFE2L2 Mutation Status and Reveals Alternative Pathway-Activating Mutations in NSCLC.

INTRODUCTION: Activation of the antioxidant KEAP1/NFE2L2 (NRF2) pathway leads to increased glutamine dependence and an aggressive phenotype in NSCLC. Because this pathway has been explored as a clinical target, we developed a transcriptomic signature for identifying KEAP1/NFE2L2-activated tumors. METHODS: A total of 971 NSCLC samples were used to train an expression signature (K1N2-score) to predict KEAP1/NFE2L2 mutations. There were 348 in-house NSCLCs that were analyzed using a NanoString expression panel for validation. RESULTS: The 46-gene K1N2 score robustly predicted KEAP1/NFE2L2 mutations in the validation set irrespective of histology and mutation (area under the curve: 89.5, sensitivity: 90.2%), suggesting that approximately 90% of KEAP1/NFE2L2 mutations are pathway-activating. The K1N2-score outperformed KEAP1/NFE2L2 mutational status when predicting patient survival (score p = 0.047; mutation p = 0.215). In K1N2 score-positive but KEAP1/NFE2L2 wild-type samples, enrichment testing identified SMARCA4/BRG1 and CUL3 mutations as mimics of KEAP1/NFE2L2 mutations. CONCLUSIONS: The K1N2-score identified KEAP1/NFE2L2-activated NSCLC by robustly detecting KEAP1/NFE2L2mut cases and discovering alternative genomic activators. It is a potential means for selecting patients with a constitutively active KEAP1/NFE2L2 pathway.

MET-FISH Evaluation Algorithm: Proposal of a Simplified Method.

MET amplifications (METamp) occur in 5% of NSCLC and represent in most case mechanisms of resistance to ALK and/or EGFR-targeted therapies. METamp detection can be performed using different techniques, although Fluorescence In-Situ Hybridization (FISH) remains the gold-standard, especially in the context of subclonality. To date current evaluation algorithms of MET amplifications are time consuming. Aim of the study was to identify a faster, equally reliable diagnostic algorithm for the detection of METamp, which is currently classified in negativity and low/intermediate/high-level amplification. N=497 NSCLC cases with available MET-FISH data had been selected. The results based on the first evaluated 20 cells had been re-calculated and compared with the definitive results based on 60 cells. For n=464 (93.4%) identical results had been obtained when counting 20 cells instead of 60 cells. Thirty-three cases (5.6%) showed a discrepancy, leading to an incorrect upgrade to a higher diagnostic category (n=25) and to an incorrect downgrade (n=8). We propose a simplified, yet equally reliable MET FISH-algorithm: after accurate screening of the whole tumor slide, twenty tumor cells have to be evaluated and results calculated: If the result is negative, or if all criteria of high-level METamp are fulfilled, the case can be signed out as such. All other cases should be considered as equivocal and additional 40 cells have to be counted. Given that, reliable results can be obtained by counting 20 cells only and an "equivocal" category for cases that need further investigation have been clearly defined.

Prevalence and potential biological role of TERT amplifications in ALK translocated adenocarcinoma of the lung.

AIMS: The advent of specific ALK-targeting drugs has radically changed the outcome of patients with ALK translocated non-small-cell lung cancer (NSCLC). However, emerging resistance to treatment with ALK inhibitors in these patients remains a major concern. In previous studies, we analysed two ALK+ patient cohorts (TP53 wild-type/TP53 mutated) in terms of copy number alterations. All patients belonging to the TP53 wild-type group had mainly genetically stable genomes, with one exception showing chromosomal instability and amplifications of several gene loci, including TERT. Here, we aimed to determine the prevalence of TERT amplifications in these ALK+ lung cancer patients by analysing an independent cohort of 109 ALK translocated cases. We further analysed the copy numbers of numerous cancer-relevant genes and other genetic aberrations. METHODS AND RESULTS: The prevalence of TERT amplifications was determined by means of FISH analyses. Copy numbers of 87 cancer-relevant genes were determined by NanoString nCounter® technology, FoundationOne® and lung-specific NGS panels in some of these TERT-amplified samples, and clinical data on patients with TERT-amplified tumours were collected. Our data revealed that five (4.6%) of all 109 analysed ALK+ patients harboured amplification of TERT and that these patients had genetically unstable genomes. CONCLUSIONS: Our preliminary study shows that ALK+ adenocarcinomas should be evaluated in the context of their genomic background in order to more clearly understand and predict patients' individual course of disease.

Clinical impact of PD-L1 and PD-1 expression in squamous cell cancer of the vulva.

PURPOSE: Squamous cell carcinoma of the vulva (SQCV) is the fifth most common cancer in women and accounts for about 5% of all genital cancers in women. The PD-L1 signaling pathway is activated in many malignant neoplasms and its blockade enhances anti-cancer immunity. The aim of our study was to examine the protein expression of PD-L1 and PD-1 in squamous cell cancer of the vulva, its correlations with clinicopathologic features and prognostic value. METHODS: Patients with SQCV treated in one institution were used for the analyses. PD-L1 immunohistochemistry was performed on 4 µm-thick section of the respective FFPE tissue blocks using the 28-8 antibody. PD-L1 scoring was performed separately for tumour cells (TC) and tumour associated immune cells. DNA was extracted to determine HPV status. Kaplan-Meier estimates for disease-free-survival and overall-survival were calculated and compared by log-rank test. RESULTS: PD-L1 expression in tumour cells could be observed in 32.9% of the patients. The expression of PD-L1 in peritumoural immune cells was confirmed in 91.4% of the patients. A significant correlation between PD-L1 expression in tumour cells and tumour stage was detected (p = 0.007). PD-L1 expression was independent from HPV status. Using the log-rank test we could not prove any significant differences in disease-free survival (p = 0.434) and overall survival (p = 0.858). Regression analysis showed that nodal status is a predictive factor of survival (p < 0.001). CONCLUSION: The present study showed that a relevant amount of patients with squamous cell cancer of the vulva express PD-L1 in both, tumour cells and tumour-associated immune cells. Furthermore, the significant correlation of PD-L1 expression in TCs with tumour stage indicated the clinical impact of PD-L1 expression during tumour development. These data indicate that SQCV might be amenable to immune checkpoint-inhibition and constitute a rational for the future clinical trials.

Comparison of the genomic background of MET-altered carcinomas of the lung: biological differences and analogies.

Although non-small-cell lung cancer is a leading cause of cancer-related deaths, the molecular characterization and classification of its genetic alterations has drastically changed treatment options and overall survival within the last few decades. In particular, tyrosine kinase inhibitors targeting specific molecular alterations, among other MET, have greatly improved the prognosis of non-small-cell lung cancer patients. Here, we compare the genomic background of a subset of non-small-cell lung cancer cases harboring either a MET high-level amplification (n = 24) or a MET exon 14 skipping mutation (n = 26), using next-generatison sequencing, fluorescence in situ hybridization, immunohistochemistry, and Nanostring nCounter® technology. We demonstrate that the MET-amplified cohort shows a higher genetic instability, compared with the mutant cohort (p < 0.001). Furthermore, MET mutations occur at high allele frequency and in the presence of co-occurring TP53 mutations (n = 7), as well as MDM2 (n = 7), CDK4 (n = 6), and HMGA2 (n = 5) co-amplifications. No other potential driver mutation has been detected. Conversely, in the MET-amplified group, we identify co-occurring pathogenic NRAS and KRAS mutations (n = 5) and a significantly higher number of TP53 mutations, compared with the MET-mutant cohort (p = 0.048). Of note, MET amplifications occur more frequently as subclonal events. Interestingly, despite the significantly (p = 0.00103) older age at diagnosis of stage IIIb/IV of MET-mutant patients (median 77 years), compared with MET high-level amplified patients (median 69 years), MET-mutant patients with advanced-stage tumors showed a significantly better prognosis at 12 months (p = 0.04). In conclusion, the two groups of MET genetic alterations differ, both clinically and genetically: our data strongly suggest that MET exon 14 skipping mutations represent an early driver mutation. In opposition, MET amplifications occur usually in the background of other strong genetic events and therefore MET amplifications should be interpreted in the context of each tumor's genetic background, rather than as an isolated driver event, especially when considering MET-specific treatment options.

Longitudinal Clinical Performance of the RNA-Based Aptima Human Papillomavirus (AHPV) Assay in Comparison to the DNA-Based Hybrid Capture 2 HPV Test in Two Consecutive Screening Rounds with a 6-Year Interval in Germany.

Longitudinal data on the E6/E7 mRNA-based Aptima human papillomavirus (AHPV) assay exceeding three years in comparison to the gold standard Digene Hybrid Capture 2 (HC2) test are not available. We previously reported the cross-sectional data of the German AHPV Screening Trial (GAST) in which 10,040 women were recruited and tested by liquid-based cytology, the HC2 assay, and the AHPV assay. Four hundred eleven test-positive women were followed for up to six years. In addition, 3,295 triple-negative women were screened after a median time of six years. Overall, 28 high-grade cervical intraepithelial neoplasia (CIN3) cases were detected. The absolute risk of developing high-risk HPV-positive CIN3+ over six years among those women that tested negative at baseline was 2.2 (95% confidence interval [95% CI], 1.0 to 4.9) and 3.1 (95% CI, 1.7 to 5.7) per 1,000 women screened by the HC2 and the AHPV tests; the additional risk for those with AHPV-negative compared with HC2-negative results was 0.9 (95% CI, -0.2 to 2.1) per 1,000. In comparison, the absolute risk following a negative LBC test was 9.3 (95% CI, 2.9 to 30.2). The relative sensitivity of AHPV compared to HC2 was 91.5% for CIN3+, and the negative predictive values were 99.8% (95% CI, 99.5 to 99.9%) for HC2 and 99.7% (95% CI, 99.4 to 99.8%) for AHPV. Our data show that the longitudinal performance of the AHPV test over six years is comparable to the performance of the HC2 test and that the absolute risk of CIN3+ over six years following a negative AHPV result in a screening population is low. (This study is registered at ClinicalTrials.gov under registration number NCT02634190.).

Genetic instability and recurrent MYC amplification in ALK-translocated NSCLC: a central role of TP53 mutations.

The anaplastic lymphoma kinase (ALK) rearrangement defines a distinct molecular subtype of non-small cell lung cancer (NSCLC). Despite the excellent initial efficacy of ALK inhibitors in patients with ALK+ lung cancer, resistance occurs almost inevitably. To date, there is no reliable biomarker allowing the identification of patients at higher risk of relapse. Here, we analysed a subset of 53 ALK+ tumours with and without TP53 mutation and ALK+ NSCLC cell lines by NanoString nCounter technology. We found that the co-occurrence of early TP53 mutations in ALK+ NSCLC can lead to chromosomal instability: 24% of TP53-mutated patients showed amplifications of known cancer genes such as MYC (14%), CCND1 (10%), TERT (5%), BIRC2 (5%), ORAOV1 (5%), and YAP1 (5%). MYC-overexpressing ALK+ TP53-mutated cells had a proliferative advantage compared to wild-type cells. ChIP-Seq data revealed MYC-binding sites within the promoter region of EML4, and MYC overexpression in ALK+ TP53-mutated cells resulted in an upregulation of EML4-ALK, indicating a potential MYC-dependent resistance mechanism in patients with increased MYC copy number. Our study reveals that ALK+ NSCLC represents a more heterogeneous subgroup of tumours than initially thought, and that TP53 mutations in that particular cancer type define a subset of tumours that harbour chromosomal instability, leading to the co-occurrence of pathogenic aberrations. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Comparison of Blood Collection Tubes from Three Different Manufacturers for the Collection of Cell-Free DNA for Liquid Biopsy Mutation Testing.

The improvement in sensitive techniques has allowed the detection of tumor-specific aberrations in circulating tumor (ct) DNA. Amplification-refractory mutation system PCR has been used for the analysis of ctDNA to detect resistance-causing mutations in the epidermal growth factor receptor gene (eg, EGFR T790M) in lung cancer patients. So far, Streck tubes have been widely used as standard blood collection tubes. Here, we compared blood collection tubes manufactured by Streck with tubes from Roche and Qiagen regarding their utility in stabilizing ctDNA in plasma samples. Venous blood from healthy donors was collected in tubes from Streck, Roche, and Qiagen. Samples were spiked with artificially fragmented EGFR T790M-mutated DNA and stored for different periods of time or spiked with different DNA amounts before plasma preparation. Extracted ctDNA was analyzed by amplification-refractory mutation system PCR. Mutant DNA, spiked into blood samples from healthy donors at quantities of 1 or 3 ng, was still reliably detectable in all samples after 7 days. EGFR T790M could be detected when spiking was performed with an amount of artificial ctDNA of 0.5 ng when tubes from Roche and Qiagen were used. Blood collection tubes from Roche and Qiagen are highly suitable for ctDNA stabilization and subsequent liquid biopsy testing. Even low ctDNA concentrations allow the detection of somatic mutations.

Study-based evaluation of the Abbott RealTime High Risk HPV test in comparison to the HC2 HR HPV test in women aged ≥30 years using residual LBC ThinPrep specimens.

BACKGROUND: High-risk human papillomavirus (HR HPV) testing is already part of cervical cancer screening programs in a number of countries. New tests need to be validated not only in clinical studies but also in routine screening settings with regard to their clinical performance. METHODS: The Abbott RealTime High Risk HPV Test (RT hrHPV test) was evaluated in a random sample of 1,456 patients from a German routine screening population of 13,372 women ≥30 years of age screened primarily by liquid-based cytology (LBC) that was complemented by 48 CIN3+ cases. Clinical sensitivities, relative specificities and positive predictive values (PPV) for both HPV tests were determined based on histologically confirmed high-grade cervical disease (CIN3+) as clinical outcome. RESULTS: HR HPV prevalence in residual LBC samples was found to be 5.4 % by the RT hrHPV test and 5.6 % by the HR HC2 test, respectively. The Kappa-value for overall agreement between the RT hrHPV test and the HC2 assay for detection of HR HPV was 0.87. Relative sensitivities for detection of CIN3+ in patients with abnormal cytology was 93.8 % for the RT hrHPV assay and 97.9 % for HC2 (p-value = 0.5). Relative specificities and PPVs were comparable for both tests. The highest PPV was calculated for the specific detection of HPV16 by the RT hrHPV test (84.2 %). The RT hrHPV test showed a reduced sensitivity for detection of HVP31-positive CIN3 + . CONCLUSION: The RT hrHPV assay is as sensitive and specific in detecting severe cervical lesions in women with abnormal cytology as the HC2 HR HPV test.

Head-to-Head Comparison of the RNA-Based Aptima Human Papillomavirus (HPV) Assay and the DNA-Based Hybrid Capture 2 HPV Test in a Routine Screening Population of Women Aged 30 to 60 Years in Germany.

Testing for E6/E7 mRNA in cells infected with high-risk (HR) human papillomavirus (HPV) might improve the specificity of HPV testing for the identification of cervical precancerous lesions. Here we compared the RNA-based Aptima HPV (AHPV) assay (Hologic) and the DNA-based Hybrid Capture 2 (HC2) HPV test (Qiagen) to liquid-based cytology (LBC) for women undergoing routine cervical screening. A total of 10,040 women, 30 to 60 years of age, were invited to participate in the study, 9,451 of whom were included in the analysis. Specimens were tested centrally by LBC, the AHPV test, and the HC2 test, and women who tested positive on any test were referred for colposcopy. Genotyping was performed on all HR-HPV-positive samples. Test characteristics were calculated based on histological review. As a result, we identified 90 women with cervical intraepithelial neoplasia grade 2+ (CIN2+), including 43 women with CIN3+. Sensitivity differences between the AHPV test and the HC2 test in detecting CIN2+ (P = 0.180) or CIN3+ (P = 0.0625) lesions were statistically nonsignificant. Of three CIN3 cases that were missed with the AHPV test, two cases presented lesion-free cones and one had a non-HR HPV67 infection. The specificity (

A population-based observational study comparing Cervista and Hybrid Capture 2 methods: improved relative specificity of the Cervista assay by increasing its cut-off.

BACKGROUND: High-risk human papillomavirus (HR HPV) testing has been shown to be a valuable tool in cervical cancer screening for the detection of cervical pre-cancer and cancer. METHODS: We report a purely observational study evaluating HR HPV prevalences in residual liquid-based cytology (LBC) samples using both the Cervista™ HPV HR Test and the Digene Hybrid Capture 2 High-Risk HPV DNA Test (HC2) in a sample of 1,741 women aged ≥30 years of a German routine screening population of 13,372 women. Test characteristics were calculated and a novel method for measuring test performances was applied by calculating ratios of sensitivity or specificity. RESULTS: The overall agreement of both tests for detection of HR HPV was excellent (κ = 0.8). Relative sensitivities for the detection of histologically confirmed severe cervical intraepithelial dysplasia (CIN3+) were similar for both HPV-tests, which was confirmed by the ratio analysis. However, discrepancy analysis between the Cervista HPV HR test and HC2 revealed a high false positive rate of the Cervista HPV HR test in the cytology normal category. CONCLUSIONS: Performance of the Cervista HPV test in cervical specimens with abnormal cytology is comparable to HC2 as both tests were highly sensitive and specific for the detection of high grade cervical disease. We also demonstrate evidence that modification of the cut-off values drastically reduces the false positive rate in the cytology normal category without affecting the detection of CIN3+, which ultimately improved specificity of the Cervista HPV HR assay.

Prevalence of low-risk and high-risk types of human papillomavirus and other risk factors for HPV infection in Germany within different age groups in women up to 30 years of age: an epidemiological observational study.

Human papillomavirus (HPV) infection is frequent in young women and persistent infection may lead to cervical cancer. Therefore, vaccination against HPV is recommended for young women in the age group from 12-17 years in Germany. However, epidemiological data on the prevalence of HPV types and risk factors for infection for younger women in Germany is scarce. To address this, an observational study was performed in Germany including 1,692 women aged 10-30 years. After a routine Pap smear, cervical swabs were tested for high-risk and low-risk HPV, respectively, using the Hybrid Capture 2 (HC2) test, and genotyped using the PCR-based tests SPF(10)/LiPA(25) and PapilloCheck®. In addition, the women were interviewed regarding their medical history and lifestyle factors. Three hundred seventy-seven (22.28%) women had positive HC2 results. The proportion of HPV positive women was highest in the 20-22 age group with 28.3%. Predominant HPV types were HPV 16, 42, 51 and HPV 16, 51, 31 as defined by PapilloCheck® and SPF(10)/LiPA(25), respectively. 95.8% of women did not show signs of any cervical lesion. Adjusted analysis identified number of sexual partners (OR:1.105; 95% CI:[1.069-1.142]), smoking (OR:1.508; [1.155-1.968]), and vaccination against HPV (OR:0.589; [0.398-0.872]) rather than increasing age as risk associated with HPV infection. Comparison of the genotyping assays showed that they correspond well regarding the high-risk HPV types but less well for low-risk HPV types. This epidemiological study shows that high-risk HPV infection is common in young women in Germany. According to our data, vaccination of young women could have a potential impact on the prevention of HPV infection and cervical disease.

Evaluation of the performance of the novel PapilloCheck HPV genotyping test by comparison with two other genotyping systems and the HC2 test.

The novel PapilloCheck genotyping test was compared with SPF10 PCR LiPav1 and PGMY09/11 on hybrid capture 2 (HC2)-pretested samples. From results of 826 cervical samples detection rates and kappa values for the tests were calculated using a HPV type consensus definition. With PapilloCheck HPV types 53, 56, and 33 were found with a sensitivity of 100%. The lowest detection rate was observed for HPV 35 (72.2%). The SPF10 PCR LiPav1 was found to be 100% positive for HPV 18, 31, 53, 56, and 35 and lowest for HPV 59 (81%). The PGMY09/11 system detected only HPV 59 at 100% detection rate and showed lowest sensitivity for HPV 56 (40.5%). Multiple infection rates ranged from 25.8% (PGMY09/11 PCR-LBA), over 39.5% (PapilloCheck) to 55.9% (SPF10 PCR LiPav1). In samples with higher viral DNA load detection rates and concordance between the genotyping tests increases. The kappa values in comparison to the HPV consensus type ranged from k = 0.21 to k = 0.82 for comparing SPF10 PCR with the HPV consensus type, while values for PGMY09/11 PCR ranged from k = 0 to k = 0.96 and were best for the PapilloCheck (k = 0.49-0.98). Detection rates for the identification of high-grade cervical intraepithelial neoplasia (CIN2+) ranged from 93.7% (PGMY09/11 PCR) to 98.4% (PapilloCheck, SPF10 PCR, HC2). In conclusion, this study shows that the PapilloCheck give comparable results to established PCR methods. However, these results also show a necessity for the standardization of genotype-specific HPV detection assays.

Comparison of the performance of different HPV genotyping methods for detecting genital HPV types.

Classification of high-risk HPV types for cervical cancer screening depends on epidemiological studies defining HPV type-specific risk. The genotyping tests that are used, are however, not uniform with regard to type-specific detection rates making comparisons between different studies difficult. To overcome the lack of a "gold standard" four tests were evaluated crosswise using 824 cervical smears pretested by HC2. The tests evaluated were the L1-PCR-based assays PGMY09/11 LBA, HPV DNA Chip and SPF LiPA and an E1 consensus PCR followed by cycle sequencing (E1-PCR). A subset of 265 samples was tested in addition with the GP5+/6+ reverse line blot assay. Differences were noted in the sensitivity and range for specific HPV types, e.g. with detection rates for HPV53 ranging from 2.3% to 11.6%. HPV16 was the most prevalent type detected by all tests except for the SPF-10 LiPa, which detected HPV31 more often. Kappa values calculated ranged from poor (k=0.20) to intermediate (k=0.54) for HPV positivity, but were higher for high-risk type positivity (k=0.31-0.61) and best for recognition of HPV16 (k=0.53-0.72). The analytical sensitivity of the tests ranged between 15% and 97% for individual types and specificity was highly dependent on which test system was used as "gold standard" for the analysis. The results of histology were used for calculation of clinical sensitivity and specificity. E1-PCR, PGMY09/11 LBA and SPF-10 LiPA had a high clinical sensitivity (>95%) for the detection of cervical intraepithelial neoplasia 2 or higher, whereas the HPV DNA Chip reached only 84.1%.

Delta6-desaturase (FADS2) deficiency unveils the role of omega3- and omega6-polyunsaturated fatty acids.

Mammalian cell viability is dependent on the supply of the essential fatty acids (EFAs) linoleic and alpha-linolenic acid. EFAs are converted into omega3- and omega6-polyunsaturated fatty acids (PUFAs), which are essential constituents of membrane phospholipids and precursors of eicosanoids, anandamide and docosanoids. Whether EFAs, PUFAs and eicosanoids are essential for cell viability has remained elusive. Here, we show that deletion of delta6-fatty acid desaturase (FADS2) gene expression in the mouse abolishes the initial step in the enzymatic cascade of PUFA synthesis. The lack of PUFAs and eicosanoids does not impair the normal viability and lifespan of male and female fads2 -/- mice, but causes sterility. We further provide the molecular evidence for a pivotal role of PUFA-substituted membrane phospholipids in Sertoli cell polarity and blood-testis barrier, and the gap junction network between granulosa cells of ovarian follicles. The fads2 -/- mouse is an auxotrophic mutant. It is anticipated that FADS2 will become a major focus in membrane, haemostasis, inflammation and atherosclerosis research.

Characterizing potential exposure to the public from low-level radioactive waste transportation by truck.

To address public concern about potential exposure to gamma radiation from legal-weight low-level radioactive waste truck shipments to the Nevada Test Site, a stationary, automated array of four pressurized ion chambers was established for trucks to pass through. Data were collected from 1,012 of the 2,260 trucks that transported low-level radioactive waste to the Nevada Test Site from February through December 2003. To avoid perception of biasing a potential exposure low, the maximum reading (muR per hour; muR h(-1)) from the array was assigned as the gross measurement value for each truck. [In this article, exposure measurements are reported as Roentgen (R), as this unit is consistent with the data readings of the measurement instruments and has been historically presented to public stakeholders. Subsequently, dose measurements are reported as Roentgen Equivalent Man (rem).] To calculate the "net exposure" for each truck, the average and standard deviation of the maximum background values during the corresponding 12-h period when the truck arrived were subtracted from the gross value. For 483 trucks (47.7%), calculated net exposure values were equal to or less than zero, indicating that the exposure from the truck was indistinguishable from background. An additional 206 trucks (20.4%) had calculated net exposure values ranging between 0.0 and 1.0 muR h(-1). Cumulative exposure scenarios appropriate for rural transportation routes to the Nevada Test Site were developed; however, these scenarios assumed the unlikely case that the same individual was exposed to all of the trucks on that route. Cumulative exposure values were dominated by a small percentage of the trucks with comparatively high values. In communities along transportation routes, the probability of an individual receiving a potential exposure from a single truck may be a more meaningful perspective.

Neutral sphingomyelinase (SMPD3) deficiency causes a novel form of chondrodysplasia and dwarfism that is rescued by Col2A1-driven smpd3 transgene expression.

Neutral sphingomyelinase SMPD3 (nSMase2), a sphingomyelin phosphodiesterase, resides in the Golgi apparatus and is ubiquitously expressed. Gene ablation of smpd3 causes a generalized prolongation of the cell cycle that leads to late embryonic and juvenile hypoplasia because of the SMPD3 deficiency in hypothalamic neurosecretory neurons. We show here that this novel form of combined pituitary hormone deficiency is characterized by the perturbation of the hypothalamus-pituitary growth axis, associated with retarded chondrocyte development and enchondral ossification in the epiphyseal growth plate. To study the contribution by combined pituitary hormone deficiency and by the local SMPD3 deficiency in the epiphyseal growth plate to the skeletal phenotype, we introduced the full-length smpd3 cDNA transgene under the control of the chondrocyte-specific promoter Col2a1. A complete rescue of the smpd3(-/-) mouse from severe short-limbed skeletal dysplasia was achieved. The smpd3(-/-) mouse shares its dwarf and chondrodysplasia phenotype with the most common form of human achondrodysplasia, linked to the fibroblast-growth-factor receptor 3 locus, not linked to deficits in the hypothalamic-pituitary epiphyseal growth plate axis. The rescue of smpd3 in vivo has implications for future research into dwarfism and, particularly, growth and development of the skeletal system and for current screening and future treatment of combined dwarfism and chondrodysplasia.

Obesity resistance of the stearoyl-CoA desaturase-deficient (scd1-/-) mouse results from disruption of the epidermal lipid barrier and adaptive thermoregulation.

Targeted deletion of the stearoyl-CoA desaturase 1 gene (scd1) in mouse causes obesity resistance and a severe skin phenotype. Here, we demonstrate that SCD1 deficiency disrupts the epidermal lipid barrier and leads to uncontrolled transepidermal water loss, breakdown of adaptive thermoregulation and cold resistance, as well as a metabolic wasting syndrome. The loss of omega-hydroxylated very long-chain fatty acids (VLCFA) and ceramides substituted with omega-hydroxylated VLCFA covalently linked to corneocyte surface proteins leads to the disruption of the epidermal lipid barrier in scd1-/- mutants. Artificial occlusion of the skin by topical lipid application largely reconstituted the epidermal barrier and also reversed dysregulation of thermogenesis and cold resistance, as well as the metabolic disturbances. Interestingly, SCD1 deficiency abolished expression of the key transcription factor Lef1, which is essential for interfollicular epidermis, sebaceous glands, and hair follicle development. Finally, the occurrence of SCD1 and a newly described hSCD5 (ACOD4) gene in humans suggests that the scd1-/- mouse mutant might be a valuable animal model for the study of human skin diseases associated with epidermal barrier defects.