Rationale: We have a limited understanding of the molecular underpinnings of early adenocarcinoma (ADC) progression. We hypothesized that the behavior of early ADC can be predicted based on genomic determinants.Objectives: To identify genomic alterations associated with resected indolent and aggressive early lung ADCs.Methods: DNA was extracted from 21 ADCs in situ (AISs), 27 minimally invasive ADCs (MIAs), and 54 fully invasive ADCs. This DNA was subjected to deep next-generation sequencing and tested against a custom panel of 347 cancer genes.Measurements and Main Results: Sequencing data was analyzed for associations among tumor mutation burden, frequency of mutations or copy number alterations, mutation signatures, intratumor heterogeneity, pathway alterations, histology, and overall survival. We found that deleterious mutation burden was significantly greater in invasive ADC, whereas more copy number loss was observed in AIS and MIA. Intratumor heterogeneity establishes early, as in AIS. Twenty-one significantly mutated genes were shared among the groups. Mutation signature profiling did not vary significantly, although the APOBEC signature was associated with ADC and poor survival. Subclonal KRAS mutations and a gene signature consisting of PIK3CG, ATM, EPPK1, EP300, or KMT2C mutations were also associated with poor survival. Mutations of KRAS, TP53, and NF1 were found to increase in frequency from AIS and MIA to ADC. A cancer progression model revealed selective early and late drivers.Conclusions: Our results reveal several genetic driver events, clonality, and mutational signatures associated with poor outcome in early lung ADC, with potential future implications for the detection and management of ADC.

Mutations in KRAS are among the most frequent RAS alterations in human cancers and the prevalent driver event in lung adenocarcinoma (LUAD). There are still no effective targeted therapies for KRAS-driven LUAD, although specific KRAS G12C inhibitors are showing very promising results in clinical trials. Small-molecule inhibitors of the MAPK pathway, one of the prominent downstream KRAS mediators, showed minimal clinical activity either as single agents or in combination with chemotherapy. We observed that loss of wild-type KRAS enhances tumor fitness in KRAS mutant cancer cells while concomitantly increasing sensitivity to MEK inhibition. Given the challenges of reanalyzing prior clinical trials, future clinical studies of targeted inhibitors should evaluate and/or stratify patients based on the relative expression of wild-type and mutant KRAS alleles to determine their correlation with treatment outcome. We also showed that dimerization/oligomerization between KRAS proteins is a key regulator for lung adenocarcinoma biology and determinant of treatment response. We generated an inducible system to force either wild-type/mutant or mutant/mutant KRAS dimerization, which showed that forced dimerization between wild-type/mutant KRAS resulted in impaired cell growth as compared to forced mutant/mutant KRAS dimerization. Summary: Loss of wild-type KRAS enhances tumor fitness in KRAS mutant cancer cells while concomitantly increasing sensitivity to MEK inhibition. Dimerization of wild-type KRAS with mutant KRAS results in growth inhibition and changes the therapeutic index for MEK inhibitors. Mutant-mutant KRAS dimerization is critical for the full oncogenic properties of mutant KRAS. Collectively these observations suggest that strategies designed to interfere with KRAS dimerization should be evaluated as a therapeutic approach in KRAS mutant cancers.

Beginning in 2006, the Urea Cycle Disorders Consortium (UCDC) has conducted a longitudinal study of eight inherited deficiencies of enzymes and transporters of the urea cycle, including 444 individuals with ornithine transcarbamylase deficiency (OTCD), of whom 300 (67 males, 233 females) received psychological evaluation. In a cross-sectional study (age range, 3-71years), analysis of covariance (ANCOVA) determined the association between outcomes in five cognitive domains (global intelligence, executive functions, memory, visuomotor integration, visual perception) and sex, age at testing and timing of disease onset defined as early onset (28days; EO), late onset (LO), or asymptomatic (AS). The dataset of 183 subjects with complete datasets (31 males, 152 females) revealed underrepresentation of EO subjects (2 males, 4 females), who were excluded from the ANCOVA. Although mean scores of LO and AS individuals were within 1 SD of the population norm, AS subjects attained significantly higher scores than LO subjects and males higher scores than females. Correlations between cognitive domains were high, particularly intelligence proved to be a distinguished indicator for cognitive functioning. Maximum plasma ammonium concentration and intelligence correlated significantly higher in EO (r=-0.47) than in LO subjects (r=0.04). Correlation between the number of hyperammonemic events and intelligence scores were similar for EO (r=-0.30) and LO (r=-0.26) individuals. The number of clinical symptoms was significantly associated with intelligence (r=-0.28) but not with scores in other domains. Results suggest that OTCD has a global impact on cognitive functioning rather than a specific effect on distinct cognitive domains.

Abstract Background: Approximately 20% of patients with early ER+ breast cancer (BC) treated with adjuvant antiestrogen therapy eventually relapse with endocrine-resistant metastatic disease. We hypothesized that profiling newly diagnosed ER+ BC that persist following prolonged estradiol deprivation with letrozole would identify genomic alterations associated with endocrine resistance. Methods: We treated 57 postmenopausal women (median 77 years; range 60-86) with ER+/HER2– BC with neoadjuvant letrozole (median 7.5 months; range 3-36) followed by surgery and adjuvant endocrine therapy. Patients were followed with serial ultrasounds and defined as non-responders if they developed recurrent locally or metastatic disease, or had a preoperative endocrine prognostic index (PEPI) ≥4 (composite score of post-treatment ER, Ki67, T and N status). Post-treatment specimens were profiled by RNA-seq and targeted capture NGS of >300 cancer-related genes. We screened for variants with a high probability of disrupting protein function (GERP score >4) and excluded likely germline variants by filtering out every alteration not present in COSMIC, if the variant had an allele frequency >0.1% as per the ExAC dataset. Results: Ten patients (17.5%) had a PEPI 0 score, 31 (54%) were PEPI 1-3, and 16 (28%) were PEPI ≥4. After a median follow-up of 50 months (12-100), 9 patients (15.7%) had recurred with metastatic disease (4 with PEPI 1-3, 5 with PEPI ≥4). We identified 294 variants with a median coverage >250x (206 nonsynonymous, 21 nonsense, 58 indels, 8 splice site). Recurrent mutations included PIK3CA (38%), KMT2C (28%), CDH1 (15%), NF1 (12 %), TP53 (10%), MAP3K1 (7%), ERBB2 (7%) and ESR1 (5%). Recurrent amplifications were identified in MCL1 (31%), GNAS (19%), CCND1 (16%), FYN (14%), AURKA (12%), and ERBB2 (10%), while recurrent deletions were found in DUSP4 (12%), NCOR1 (8%) and NF1 (6%). Compared to alterations reported in untreated ER+ breast cancers in TCGA, we observed a significant increase in KMT2C, NF1, MCL1 and FYN alterations (FDR<0.05). MCL1, GNAS and FYN amplifications, DUSP4 deletions, MAP3K1 mutations, and ERBB2 and NF1 alterations were enriched in the non-responder group. Differential expression analysis of the RNA-seq data revealed an enrichment of E2F and MYC target genes, and genes involved in the G2/M checkpoint, TORC1 signaling, EMT and immunosuppression in non-responding tumors. PEPI 0 tumors were enriched with Luminal A subtype tumors, whereas PEPI 4 tumors were enriched with Luminal B, basal and HER2-enriched subtypes. Luminal A tumors exhibited improved disease free survival compared to other subtypes (HR 0.28, 95% CI 0.10-0.64). Gains in the proximal portion of chromosome 1q were associated with poor long-term outcomes as the relapse-free survival rate at 40 months for patients with 1q gains was 89% versus 41% for patients with no 1q gain (p=0.001). Conclusions: Genomic profiling of residual ER+ breast cancers treated with prolonged neoadjuvant letrozole revealed a different mutational landscape than primary untreated ER+ BC. These alterations may be associated with poor response to estrogen deprivation in early breast cancer and deserve further study. Citation Format: Guerrero AL, Stricker T, Hutchinson KE, Formisano L, Giltnane J, Fidalgo A, Schwarz LJ, Gavila J, Guillen V, Lluch A, Ruiz A, Arteaga CL. Genomic profiling of residual ER+ breast cancers treated with prolonged neoadjuvant letrozole reveals novel alterations in clinically resistant tumors [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-10-01.

Abstract Hypothesis: Endocrine therapy (ET) is an effective treatment for estrogen-receptor positive (ER+) breast cancer; however, more than 80% of women will develop endocrine resistance. ER ChIP-seq studies in clinical samples show tumors likely to relapse have a unique set of ER binding sites that correlate with gene signatures predicting clinical outcome. Understanding how several factors, including transcriptional co-activator-complexes, influence ER binding location is crucial to improving treatment. MLL3, which is recurrently mutated in ~7% of ER+ cases, is a member of one such complex that interacts with ER. It is a histone methyltransferase that marks active enhancers. We hypothesized that mutation of MLL3 may change the genomic landscape of enhancers and thus change binding patterns of ER, altering response to ET. Methods: To elucidate transcriptional consequences of MLL3 mutation in ER+ breast cancers, we leveraged TCGA ER+ breast cancer RNA-seq data. We used a gene-by-gene multivariate linear model to identify differentially expressed genes (DEGs) between MLL3-mutant and wild-type (WT) samples. This data was compared to DEG found with in-house RNA-seq data for MLL3-mutant MCF7 cells, MLL3-WT ZR751 cells, and MLL3-knockdown (KD) ZR751 cells to identify a testable MLL3-mutant signature. ChIP-seq data for ER and H3K4me1, the histone mark made by MLL3, was then produced using these cell lines in order to pinpoint genes with changes in both H3K4me1 and ER-binding upon MLL3 mutation/KD. This gene list was compared to DEG found through RNA-Seq. To illuminate inherent differences between MLL3-WT and mutant/KD cells in response to ET, Cell-Titer Blue, Cell-Titer Glo, and Caspase-Glo assays were performed using DMSO, 4-OHT, and Fulvestrant. Results: TCGA RNA-seq data revealed MLL3-mutant ER+ breast cancers have differentially expressed transcriptional regulators, including ER itself. iRegulon analysis showed enrichment for DEG-regulated by ER-tethering factor SP1, a phenomenon suggested to be associated with more aggressive behavior. ChIP-seq data revealed substantial shifts in both ER-binding locations and H3K4me1 marks upon MLL3 mutation/KD. Interestingly, annotated peaks for MLL3-KD ZR751 showed overlap with those of MLL3-mutant MCF7, and GREAT analysis of new ER binding sites in MLL3-KD ZR751 showed an up-regulation of signatures associated with endocrine resistance. Indeed, MLL3-KD ZR751 cells were more resistant to 4-OHT/Fulvestrant than MLL3-WT ZR751. Conclusions: Loss of MLL3 function leads to a massive shift in H3K4me1, indicating a shift in the genomic landscape of enhancers. This shift is associated with a shift in ER binding as well as alterations in gene expression. Pathway analysis of these genes suggest that loss of MLL3 function may increase endocrine resistance, and indeed, cell lines with loss of MLL3 function are more resistant to ER inhibition than MLL3-WT cell lines. This work demonstrates that loss of MLL3 function leads to shifts in the enhancer landscape, alterations in ER binding and regulation of gene expression, and contributes to more aggressive tumor behavior. Citation Format: Stauffer K, Stricker T. MLL3 mutation in ER+ breast cancers leads to dramatic shift in ER binding locations and consequentially alters transcriptional programs [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-10.

A male patient, born in 1999, was diagnosed with ornithine transcarbamylase deficiency as neonate and was managed with a strict low-protein diet supplemented with essential amino acids, L-citrulline, and L-arginine as well as sodium benzoate. He had an extensive history of hospitalizations for hyperammonemic crises throughout childhood and early adolescence, which continued after the addition of sodium phenylbutyrate in 2009. In December 2013 he was switched to glycerol phenylbutyrate, and his metabolic stability was greatly improved over the following 7 months prior to liver transplant.

Urea cycle disorders (UCDs) are common inborn errors of metabolism, with an incidence of one in 30,000 births. They are caused by deficiencies in any of six enzymes and two carrier proteins, the most common being Ornithine Transcarbamylase Deficiency (OTCD). OTCD results in impairment to excrete nitrogen, causing toxic buildup of ammonia with resultant encephalopathy. Hyperammonemia (HA) induces the conversion of glutamate to glutamine in the brain. Excess glutamine in the brain causes osmotic changes, cerebral edema, changes in astrocyte morphology, and cell death. Acute symptoms of HA include vomiting, hyperventilation, seizures, and irritability. Long-term neurological effects include deficits in working memory and executive function. To date, there are no predictors of prognosis of infants with neonatal onset OTCD outside of the plasma ammonia level at presentation and duration of a hyperammonemic coma. We provide a comprehensive analysis of a 16-year-old male with neonatal onset of OTCD as an example of how brain biomarkers may be useful to monitor disease course and outcome. This male presented at 8 days of life with plasma ammonia and glutamine of 677 and 4024 micromol/L respectively, and was found to have a missense mutation in Exon 4 (p. R129H). Treatment included protein restriction, sodium benzoate, and citrulline, arginine, and iron. Despite compliance, he suffered recurrent acute hyperammonemic episodes triggered by infections or catabolic stressors. We discuss the long-term effects of the hyperammonemic episodes by following MRI-based disease biomarkers.

BACKGROUND:Acute liver failure (ALF) has been reported in ornithine transcarbamylase deficiency (OTCD) and other urea cycle disorders (UCD). The frequency of ALF in OTCD is not well-defined and the pathogenesis is not known.AIM:To evaluate the prevalence of ALF in OTCD, we analyzed the Swiss patient cohort. Laboratory data from 37 individuals, 27 females and 10 males, diagnosed between 12/1991 and 03/2015, were reviewed for evidence of ALF. In parallel, we performed cell culture studies using human primary hepatocytes from a single patient treated with ammonium chloride in order to investigate the inhibitory potential of ammonia on hepatic protein synthesis.RESULTS:More than 50% of Swiss patients with OTCD had liver involvement with ALF at least once in the course of disease. Elevated levels of ammonia often correlated with (laboratory) coagulopathy as reflected by increased values for international normalized ratio (INR) and low levels of hepatic coagulation factors which did not respond to vitamin K. In contrast, liver transaminases remained normal in several cases despite massive hyperammonemia and liver involvement as assessed by pathological INR values. In our in vitro studies, treatment of human primary hepatocytes with ammonium chloride for 48 hours resulted in a reduction of albumin synthesis and secretion by approximately 40%.CONCLUSION:In conclusion, ALF is a common complication of OTCD, which may not always lead to severe symptoms and may therefore be underdiagnosed. Cell culture experiments suggest an ammonia-induced inhibition of hepatic protein synthesis, thus providing a possible pathophysiological explanation for hyperammonemia-associated ALF.

Urea cycle disorders (UCDs) are inherited disorders of ammonia detoxification often regarded as mainly of relevance to pediatricians. Based on an increasing number of case studies it has become obvious that a significant number of UCD patients are affected by their disease in a non-classical way: presenting outside the newborn period, following a mild course, presenting with unusual clinical features, or asymptomatic patients with only biochemical signs of a UCD. These patients are surviving into adolescence and adulthood, rendering this group of diseases clinically relevant to adult physicians as well as pediatricians. In preparation for an international workshop we collected data on all patients with non-classical UCDs treated by the participants in 20 European metabolic centres. Information was collected on a cohort of 208 patients 50% of which were ≥ 16 years old. The largest subgroup (121 patients) had X-linked ornithine transcarbamylase deficiency (OTCD) of whom 83 were female and 29% of these were asymptomatic. In index patients, there was a mean delay from first symptoms to diagnosis of 1.6 years. Cognitive impairment was present in 36% of all patients including female OTCD patients (in 31%) and those 41 patients identified presymptomatically following positive newborn screening (in 12%). In conclusion, UCD patients with non-classical clinical presentations require the interest and care of adult physicians and have a high risk of neurological complications. To improve the outcome of UCDs, a greater awareness by health professionals of the importance of hyperammonemia and UCDs, and ultimately avoidance of the still long delay to correctly diagnose the patients, is crucial.

BACKGROUND:Oral cavity and in particular oral tongue cancers occur with a rising incidence in younger patients often lacking the typical risk factors of tobacco use, alcohol use, and human papilloma virus (HPV) infection. Their prognosis when treated with chemoradiation has not been well studied and responsible risk factors remain elusive. A viral etiology (other than HPV) has been hypothesized. METHODS:First we analyzed outcomes from 748 head and neck cancer patients with locoregionally advanced stage tumors treated with curative-intent chemoradiation by anatomic site. Second, we analyzed seven oral tongue (OT) tumors from young, non-smokers/non-drinkers for the presence of viral mRNA using short-read massively-parallel sequencing (RNA-Seq) in combination with a newly-developed digital subtraction method followed by viral screening and discovery algorithms. For positive controls we used an HPV16-positive HNC cell line, a cervical cancer, and an EBV-LMP2A transgene lymphoma. RESULTS:Younger patients with oral cavity tumors had worse outcomes compared to non-oral cavity patients. Surprisingly none of the seven oral tongue cancers showed significant presence of viral transcripts. In positive controls the expected viral material was identified. CONCLUSION:Oral cavity tumors in younger patients have a poor prognosis and do not appear to be caused by a transcriptionally active oncovirus.

The Urea Cycle Disorders Consortium (UCDC) was created as part of a larger network established by the National Institutes of Health to study rare diseases. This paper reviews the UCDC’s accomplishments over the first 6 years, including how the Consortium was developed and organized, clinical research studies initiated, and the importance of creating partnerships with patient advocacy groups, philanthropic foundations and biotech and pharmaceutical companies.

Non-small cell lung cancer (NSCLC) is a common cancer with a poor prognosis. The aim of this study was to screen Finnish NSCLC tumor samples for common cancer-related mutations by targeted next generation sequencing and to determine their concurrences and associations with clinical features.Sequencing libraries were prepared from DNA isolated from formalin-fixed, paraffin-embedded tumor material of 425 patients using the AmpliSeq Colon and Lung panel covering mutational hot spot regions of 22 cancer genes. Sequencing was performed with the Ion Torrent Personal Genome Machine (PGM).Data analysis of the hot spot mutations revealed mutations in 77% of the patients, with 7% having 3 or more mutations reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Two of the most frequently mutated genes were TP53 (46%) and KRAS (25%). KRAS codon 12 mutations were the most recurrently occurring mutations. EGFR mutations were significantly associated with adenocarcinoma, female gender and never/light-smoking history; CTNNB1 mutations with light ex-smokers, PIK3CA and TP53 mutations with squamous cell carcinoma, and KRAS with adenocarcinoma. TP53 mutations were most prevalent in current smokers and ERBB2, ERBB4, PIK3CA, NRAS, NOTCH1, FBWX7, PTEN and STK11 mutations occurred exclusively in a group of ever-smokers, however the association was not statistically significant. No mutation was found that associated with asbestos exposure.Finnish NSCLC patients have a similar mutation profile as other Western patients, however with a higher frequency of BRAF mutations but a lower frequency of STK11 and ERBB2 mutations. Moreover, TP53 mutations occurred frequently with other gene mutations, most commonly with KRAS, MET, EGFR and PIK3CA mutations.

The evaluation of vulvovaginitis, which is common in pediatric practice, depends on the pubertal development of the patient, keeping the possibility of sexual abuse in mind. Prepubescent girls are especially susceptible to vulvovaginitis because of anatomic and hormonal factors and because of their tendency to have poor local hygiene. If symptoms persist despite hygienic measures vaginal secretions should be investigated microbiologically and specific antimicrobial treatment prescribed accordingly. When the major complaint is of perineal pruritus, especially at night, empirical treatment with Mebendazole can be considered. In adolescents, who usually present with vaginal discharge, pruritus or dysuria, the pH of vaginal secretions should be tested and the secretions should be examined under the light microscope and sent for microbiological investigations. Physiologic leukorrhea is a common cause of vaginal discharge in adolescents. In the sexually active adolescent a complete pelvic examination with speculum should be performed including evaluation of endocervical specimen for sexually transmitted pathogens. Treatment is then directed at the specific cause. The diagnosis of one sexually transmitted disease necessitates investigation for others and treatment of the partner.

Background Previous data in infants at risk of the development of atopy demonstrate that neutral prebiotic oligosaccharides (OS) (short chain galacto-OS and long chain fructo-OS, ratio 9 : 1, Immunofortis) reduce the incidence of atopic dermatitis (AD). The aim of this study was to assess the effect of immunologically active OS-supplemented formula feeding on the incidence of early AD in infants at low risk of developing atopy. Methods In this randomised controlled double blind European multicentre trial (seven centres in five countries) 1187 infants without a family history of atopy were recruited. 1130 infants remained in the full analysis set for the intention-to-treat analysis (new prebiotic group: 414; control: 416, breastfeeding reference: 300). AD was diagnosed according to the criteria recommended by the European Task Force on Atopic Dermatitis. Results The cumulative incidence of AD at age 16 weeks was 3.6%, 4.8% and 1.3%, at 24 weeks 4.6%, 7.7% and 3.3% in the new prebiotic, control and reference groups, respectively. At 52 weeks, the cumulative AD incidence was significantly lower in the new prebiotic group (5.6%) than in the control group (9.4%; p = 0.0469), but similar to the reference group (7.0%; ns). AD occurred significantly earlier in the control group than in the new prebiotic group (p = 0.0411). Conclusion This trial indicates a preventive effect of immunoactive OS-supplemented formula feeding on the incidence of early AD in infants at low risk of atopy development. The finding is particularly intriguing because most children with AD in a general population come from low atopy risk strata.

1. Tamar Stricker, MD* 2. Ulrich Lips, MD* 3. Felix H. Sennhauser, MD, Prof.* 1. *University Children's Hospital, Zurich, Switzerland An 8-month-old girl who has a history of atopic dermatitis diagnosed at 2½ months of age presents to the clinic with the complaint of worsening skin lesions. She had been seen by her pediatrician 10 days ago and was prescribed oral amoxicillin-clavulanic acid and oral betamethasone for a suspected bacterial superinfection of atopic dermatitis. However, soon after starting the medications, her skin lesions increased rapidly. Physical examination reveals an ill-appearing but afebrile infant. Height and weight are normal for age. There is a marked cutaneous eruption on the face and neck (Fig. 1), characterized by large areas of red, denuded skin with occasional crusting, oozing, and bleeding on her cheeks interspersed with elevated, fluid-containing lesions. There are crops of small, round, punched-out lesions that have raised edges on her forehead and upper chest. Examination of the dorsal and palmar aspects of both hands reveals crops of punched-out and fluid-containing elevated lesions, with some appearing hemorrhagic (Fig. 2). The remainder of the physical examination findings are normal. Figure 1. Facial areas of denuded skin with crusting, oozing, and bleeding and fluid-containing and punched-out lesions on the forehead. Figure 2. Crops of punched-out lesions with raised edges on the hand. A complete blood count demonstrates a hematocrit of 36% (0.36) and a white blood cell count of 11.6×103/mcL (11.6×109/L) with 58% neutrophils, 27% lymphocytes, and 14% monocytes. Platelet count is 471×103/mcL (471×109/L). A serum C-reactive protein measurement is 33 mg/L. A culture of a skin lesion confirms the diagnosis. ### Eczema Herpeticum A viral culture of fluid obtained from a skin vesicle grows herpes simplex virus (HSV) that is confirmed by fluorescent antibody staining, which detected HSV …