GATA2 is required for lymphatic vessel valve development and maintenance.

Heterozygous germline mutations in the zinc finger transcription factor GATA2 have recently been shown to underlie a range of clinical phenotypes, including Emberger syndrome, a disorder characterized by lymphedema and predisposition to myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Despite well-defined roles in hematopoiesis, the functions of GATA2 in the lymphatic vasculature and the mechanisms by which GATA2 mutations result in lymphedema have not been characterized. Here, we have provided a molecular explanation for lymphedema predisposition in a subset of patients with germline GATA2 mutations. Specifically, we demonstrated that Emberger-associated GATA2 missense mutations result in complete loss of GATA2 function, with respect to the capacity to regulate the transcription of genes that are important for lymphatic vessel valve development. We identified a putative enhancer element upstream of the key lymphatic transcriptional regulator PROX1 that is bound by GATA2, and the transcription factors FOXC2 and NFATC1. Emberger GATA2 missense mutants had a profoundly reduced capacity to bind this element. Conditional Gata2 deletion in mice revealed that GATA2 is required for both development and maintenance of lymphovenous and lymphatic vessel valves. Together, our data unveil essential roles for GATA2 in the lymphatic vasculature and explain why a select catalogue of human GATA2 mutations results in lymphedema.

Genetic Variation in the TP53 Pathway and Bladder Cancer Risk. A Comprehensive Analysis

Introduction: Germline variants in TP63 have been consistently associated with several tumors, including bladder cancer, indicating the importance of TP53 pathway in cancer genetic susceptibility. However, variants in other related genes, including TP53 rs1042522 (Arg72Pro), still present controversial results. We carried out an in depth assessment of associations between common germline variants in the TP53 pathway and bladder cancer risk. Material and Methods: We investigated 184 tagSNPs from 18 genes in 1,058 cases and 1,138 controls from the Spanish Bladder Cancer/EPICURO Study. Cases were newly-diagnosed bladder cancer patients during 1998–2001. Hospital controls were age-gender, and area matched to cases. SNPs were genotyped in blood DNA using Illumina Golden Gate and TaqMan assays. Cases were subphenotyped according to stage/grade and tumor p53 expression. We applied classical tests to assess individual SNP associations and the Least Absolute Shrinkage and Selection Operator (LASSO)-penalized logistic regression analysis to assess multiple SNPs simultaneously. Results: Based on classical analyses, SNPs in BAK1 (1), IGF1R (5), P53AIP1 (1), PMAIP1 (2), SERINPB5 (3), TP63 (3), and TP73 (1) showed significant associations at p-value#0.05. However, no evidence of association, either with overall risk or with specific disease subtypes, was observed after correction for multiple testing (p-value$0.8). LASSO selected the SNP rs6567355 in SERPINB5 with 83% of reproducibility. This SNP provided an OR = 1.21, 95%CI 1.05–1.38, p-value = 0.006, and a corrected p-value = 0.5 when controlling for over-estimation. Discussion: We found no strong evidence that common variants in the TP53 pathway are associated with bladder cancer susceptibility. Our study suggests that it is unlikely that TP53 Arg72Pro is implicated in the UCB in white Europeans. SERPINB5 and TP63 variation deserve further exploration in extended studies.

Targeting cancer cell metabolism in pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is expected to become the second leading cause of cancer death by 2030. Current therapeutic options are limited, warranting an urgent need to explore innovative treatment strategies. Due to specific microenvironment constraints including an extensive desmoplastic stroma reaction, PDAC faces major metabolic challenges, principally hypoxia and nutrient deprivation. Their connection with oncogenic alterations such as KRAS mutations has brought metabolic reprogramming to the forefront of PDAC therapeutic research. The Warburg effect, glutamine addiction, and autophagy stand as the most important adaptive metabolic mechanisms of cancer cells themselves, however metabolic reprogramming is also an important feature of the tumor microenvironment, having a major impact on epigenetic reprogramming and tumor cell interactions with its complex stroma. We present a comprehensive overview of the main metabolic adaptations contributing to PDAC development and progression. A review of current and future therapies targeting this range of metabolic pathways is provided.

Targeted Therapy for Patients with BRAF-Mutant Lung Cancer: Results from the European EURAF Cohort.

INTRODUCTION: Approximately 2% of lung adenocarcinomas have BRAF (v-Raf murine sarcoma viral oncogene homolog B) mutations, including V600E and other types. Vemurafenib, dabrafenib, and sorafenib as BRAF inhibitors are currently tested in clinical trials, but access for patients is limited. The aim of this study was to document the clinical course of patients treated outside of clinical trials. METHODS: We conducted a retrospective multicenter cohort study in Europe of patients with advanced BRAF-mutant lung cancer treated with known BRAF inhibitors. Data were anonymized and centrally assessed for age, gender, smoking, histology, stage, local molecular diagnostic results, systemic therapies, and survival. Best response was assessed locally by RECIST1.1. RESULTS: We documented 35 patients treated in 17 centers with vemurafenib, dabrafenib, or sorafenib. Median age was 63 years (range 42-85); gender was balanced; 14 (40%) were never smokers; all (100%) had adenocarcinoma; 29 (83%) had V600E; 6 (17%) had other mutations; one of them had a concomitant KRAS mutation. Thirty (86%) patients had chemotherapy in the first line. Overall survival with first-line therapy was 25.3 months for V600E and 11.8 months for non-V600E. Thirty-one patients received one BRAF inhibitor, and four received a second inhibitor. Overall response rate with BRAF therapy was 53%, and disease control rate was 85%. Median progression-free survival with BRAF therapy was 5.0 months, and overall survival was 10.8 months. CONCLUSIONS: These results confirm the activity of targeted therapy in patients with BRAF-mutant lung adenocarcinoma. Further trials are warranted to study combination therapies and drug resistance mechanisms.

Phenotypic and genotypic characteristics of cryopyrin-associated periodic syndrome: a series of 136 patients from the Eurofever Registry.

OBJECTIVE: To evaluate genetic, demographic and clinical features in patients with cryopyrin-associated periodic syndrome (CAPS) from the Eurofever Registry, with a focus on genotype-phenotype correlations and predictive disease severity markers. METHODS: A web-based registry retrospectively collected data on patients with CAPS. Experts in the disease independently validated all cases. Patients carrying NLRP3 variants and germline-mutation-negative patients were included. RESULTS: 136 patients were analysed. The median age at disease onset was 9 months, and the median duration of follow-up was 15 years. Skin rash, musculoskeletal involvement and fever were the most prevalent features. Neurological involvement (including severe complications) was noted in 40% and 12% of the patients, respectively, with ophthalmological involvement in 71%, and neurosensory hearing loss in 42%. 133 patients carried a heterozygous, germline mutation, and 3 patients were mutation-negative (despite complete NLRP3 gene screening). Thirty-one different NLRP3 mutations were recorded; 7 accounted for 78% of the patients, whereas 24 rare variants were found in 27 cases. The latter were significantly associated with early disease onset, neurological complications (including severe complications) and severe musculoskeletal involvement. The T348M variant was associated with early disease onset, chronic course and hearing loss. Neurological involvement was less strongly associated with V198M, E311 K and A439 V alleles. Early onset was predictive of severe neurological complications and hearing loss. CONCLUSIONS: Patients carrying rare NLRP3 variants are at risk of severe CAPS; onset before the age of 6 months is associated with more severe neurological involvement and hearing loss. These findings may have an impact on treatment decisions.

Non-small cell lung cancer: the prevalence of oncogenic driver mutations in Switzerland

Background. Predictive molecular marker analyses are standard of care in order to select non-small cell lung cancer (NSCLC) patients for targeted therapies. The aim of this study was to determine the prevalence of targetable oncogenic driver mutations including EGFR, KRAS, BRAF, HER2, ALK and ROS1 in Switzerland. Methods. Eight Swiss pathology institutions provided retrospective and anonymized data on their predictive molecular marker results performed on NSCLC from January 2012 to December 2014. Clinico-pathological data were recorded including age, gender, histological NSCLC-subtype and specimen type (biopsy, conventional cytology and cell block, respectively) used for molecular analyses. The prevalence of oncogenic mutations were calculated and compared between the centres. Results. A total of 4187 NSCLC were included into the study. The median age was 67 years and 55% were male patients. The tumor specimens for molecular analysis were mostly derived from biopsies (69%), 26% were from conventional cytology specimens and only in 5% from cell blocks. The most prevalent gene mutation was KRAS with 30.6% (range: 27.3-33.9%), followed by EGFR, BRAF and HER2 mutations in 12.2% (range: 10.2-13.1%), 3.9% (range: 2.5-5.6%) and 1.1% (range: 0.9-4.0%), respectively, without significant differences between the eight centers. Concomitant EGFR and KRAS mutations were detected in only 3/2027 NSCLC. In contrast the prevalence of ALK (mean 6.5%, range: 2.8-11.7%) and ROS1 (mean 2.4%, range: 1.5-6.2%) rearrangements varied significantly between centers. Conclusions. The Prevalence of EGFR, KRAS, BRAF and HER2 mutations are well in line with data from other West European populations. Concomitant EGFR, KRAS, BRAF or HER2 mutations are exceptional. ALK FISH results vary significantly between the eight centres. Concomitant ALK FISH positive results in NSCLC harbouring other oncogenic driver mutation have only been observed in two smaller centres, highlighting the difficulty in ALK-FISH interpretation.

A mild form of SLC29A3 disorder: a frameshift deletion leads to the paradoxical translation of an otherwise noncoding mRNA splice variant

We investigated two siblings with granulomatous histiocytosis prominent in the nasal area, mimicking rhinoscleroma and Rosai-Dorfman syndrome. Genome-wide linkage analysis and whole-exome sequencing identified a homozygous frameshift deletion in SLC29A3, which encodes human equilibrative nucleoside transporter-3 (hENT3). Germline mutations in SLC29A3 have been reported in rare patients with a wide range of overlapping clinical features and inherited disorders including H syndrome, pigmented hypertrichosis with insulin-dependent diabetes, and Faisalabad histiocytosis. With the exception of insulin-dependent diabetes and mild finger and toe contractures in one sibling, the two patients with nasal granulomatous histiocytosis studied here displayed none of the many SLC29A3-associated phenotypes. This mild clinical phenotype probably results from a remarkable genetic mechanism. The SLC29A3 frameshift deletion prevents the expression of the normally coding transcripts. It instead leads to the translation, expression, and function of an otherwise noncoding, out-of-frame mRNA splice variant lacking exon 3 that is eliminated by nonsense-mediated mRNA decay (NMD) in healthy individuals. The mutated isoform differs from the wild-type hENT3 by the modification of 20 residues in exon 2 and the removal of another 28 amino acids in exon 3, which include the second transmembrane domain. As a result, this new isoform displays some functional activity. This mechanism probably accounts for the narrow and mild clinical phenotype of the patients. This study highlights the"rescue" role played by a normally noncoding mRNA splice variant of SLC29A3, uncovering a new mechanism by which frameshift mutations can be hypomorphic.

Further molecular profiling of tumors harboring therapeutic targets within non-small cell lung cancer

Background: Treatment of NSCLC has been revolutionized in recent years with the introduction of several targeted therapies for selected genetically altered subtypes of NSCLC. A better understanding of molecular characteristics of NSCLC, which features common drug targets, may identify new therapeutic options. Methods: Over 6,700 non-small cell lung cancer cases referred to Caris Life Sciences between 2009 and 2014. Diagnoses and history were collected from referring physicians. Specific testing was performed per physician request and included a combination of sequencing (Sanger, NGS or pyrosequencing), protein expression (IHC), gene amplification/rearrangement (CISH or FISH), and/or RNA fragment analysis. Results: Tumors profiles from patients with hormone receptor positive disease (HER2, ER, PR, or AR positive by IHC) (n=629), HER2 mutations (n=8) ALK rearrangements (n=55), ROS1 rearrangement (n=17), cMET amplification or mutation (n=126), and cKIT mutation (n=11) were included in this analysis and compared to the whole cohort. Tumors with ALK rearrangement overexpressed AR in 18% of cases, and 7% presented with concomitant KRAS mutation. Lower rates of PTEN loss, as assessed by IHC, were observed in ALK positive (20%), ROS1 positive (9%) and cKIT mutated tumors (25%) compared to the overall NSCLC population (58%). cMET was overexpressed in 66% of ROS1 translocated and 57% of HER2 mutated tumors. cKIT mutations were found co-existing with APC (20%) and EGFR (20%) mutations. Pathway analysis revealed that hormone receptor positive disease carried more mutations in the ERK pathway (32%) compared to 9% in the mTOR pathway. 25% of patients with HER2 mutations harbored a co-existing mutation in the mTOR pathway. Conclusions: Pathway profiling reveals that NSCLC tumors present more often than reported with several concomitant alterations affecting the ERK or AKT pathway. Additionally, they are also characterized by the expression of potential biological modifiers of the cell cycle like hormonal receptors, representing a rationale for dual inhibition strategies in selected patients. Further refining of the understanding of NSCLC biomarker profile will optimize research for new treatment strategies.

Factors Affecting Cancer Behavior with Special Reference to Lymphatic Vessels, Macrophages, EGFR, and Pim-1 in Colorectal Cancer

Syövän käyttäytymiseen ja ennusteeseen vaikuttavat monet tekijät, muun muassa muutokset syöpäsoluissa sekä kasvainta ympäröivässä mikroympäristössä. Tutkimuksen tavoitteena oli tutkia uusia prediktiivisiä ja prognostisia ennustetekijöitä syöpäsoluissa (EGFR geenikopiomäärä, EGFR, onkogeeni pim-1) sekä syöpäkasvaimen mikroympäristöön kuuluvissa imuteissä (CLEVER-1, podoplaniini), makrofageissa (CD68, CLEVER-1) ja T lymfosyyteissä (CD3) kolorektaalisyövässä. Lisäksi tutkittiin imuteiden molekulaarisia ominaisuuksia tarkemmin (CD73, LYVE-1, podoplaniini) kuten myös lymfosyyttien ja dendriittisolujen liikennöintiä imuteissä. Tutkimustulokset osoittavat että korkea Pim-1 ekspressiotaso, suuri peritumoraalinen CD68+ makrofagimäärä sekä varhaisen vaiheen taudissa suuri CLEVER-1+ peritumoraalinen makrofagimäärä ovat hyvän ennusteen tekijöitä kolorektaalisyövässä. Metastaattisessa taudissa sen sijaan suuri määrä CLEVER-1+ makrofageja, sekä intra- että peritumoraalisesti, liittyy huonoon tautiennusteeseen. EGFR geenikopiomäärä, EGFR proteiinipitoisuuden ohjaaman hopea in situ hybridisaatiomenetelmän avulla määritettynä, ennusti vastetta anti-EGFR hoidolle metastaattisessa kolorektaalisyövässä tarkemmin kuin nykyisin rutiinisti käytössä oleva KRAS määritys. Lisäksi havaittiin että imutiet ovat monimuotoisia imutiemarkkeri ekspressionsa suhteen sekä normaali- että syöpäkudoksissa. CD73 molekyylin funktio imuteissä poikkesi selvästi molekyylin funktiosta verisuonissa. Yhteenvetona voidaan todeta että kolorektaalisyövän ennusteeseen vaikuttavien tekijöiden merkitys vaihtelee taudin levinneisyysasteen sekä imuteiden että makrofagien sijainnin perusteella. Korkea Pim-1 ilmentyminen on yhteydessä hyvään kolorektaalisyöpäennusteeseen. Lisäksi EGFR geenikopiomäärä osoittautui lupaavaksi uudeksi prediktiiviseksi ennustetekijäksi KRAS villintyypin metastaattista kolorektaalisyöpää sairastavilla potilailla.

A 37-kb restriction map of the human immunoglobulin lambda variable locus, VB cluster, harboring four functional genes and two non-coding Vl sequences

The human immunoglobulin lambda variable locus (IGLV) is mapped at chromosome 22 band q11.1-q11.2. The 30 functional germline v-lambda genes sequenced untill now have been subgrouped into 10 families (Vl1 to Vl10). The number of Vl genes has been estimated at approximately 70. This locus is formed by three gene clusters (VA, VB and VC) that encompass the variable coding genes (V) responsible for the synthesis of lambda-type Ig light chains, and the Jl-Cl cluster with the joining segments and the constant genes. Recently the entire variable lambda gene locus was mapped by contig methodology and its one- megabase DNA totally sequenced. All the known functional V-lambda genes and pseudogenes were located. We screened a human genomic DNA cosmid library and isolated a clone with an insert of 37 kb (cosmid 8.3) encompassing four functional genes (IGLV7S1, IGLV1S1, IGLV1S2 and IGLV5a), a pseudogene (VlA) and a vestigial sequence (vg1) to study in detail the positions of the restriction sites surrounding the Vl genes. We generated a high resolution restriction map, locating 31 restriction sites in 37 kb of the VB cluster, a region rich in functional Vl genes. This mapping information opens the perspective for further RFLP studies and sequencing

Utilization of microsatellites for the analysis of genomic alterations in colorectal cancers in Brazil

Two different pathogenetic mechanisms are proposed for colorectal cancers. One, the so-called "classic pathway", is the most common and depends on multiple additive mutational events (germline and/or somatic) in tumor suppressor genes and oncogenes, frequently involving chromosomal deletions in key genomic regions. Methodologically this pathway is recognizable by the phenomenon of loss of heterozygosity. On the other hand, the "mutator pathway" depends on early mutational loss of the mismatch repair system (germline and/or somatic) leading to accelerated accumulation of gene mutations in critical target genes and progression to malignancy. Methodologically this second pathway is recognizable by the phenomenon of microsatellite instability. The distinction between these pathways seems to be more than academic since there is evidence that the tumors emerging from the mutator pathway have a better prognosis. We report here a very simple methodology based on a set of tri-, tetra- and pentanucleotide repeat microsatellites allowing the simultaneous study of microsatellite instability and loss of heterozygosity which could allocate 70% of the colorectal tumors to the classic or the mutator pathway. The ease of execution of the methodology makes it suitable for routine clinical typing

Nasopharyngeal colonization by pathogenic bacteria: effect of polymorphisms in innate immune genes of young children

Nasopharyngeal bacteria can asymptomatically colonize the nasopharynx of infants and young children but are also associated with the development of respiratory infections and diseases. Such nasopharyngeal bacteria include Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae and Staphylococcus aureus. The host defense against invading pathogens is largely relies germline-encoded pattern recognition receptors (PRR), which are expressed on the cells of innate immunity, and different cytokines. These include toll-like receptors (TLR), mannose-binding lectin (MBL) and different cytokines such as IL-17A. Single nucleotide polymorphisms (SNP) in these receptors and cytokines have been reported. The aim of this study was to investigate genetic polymorphisms in the genes for TLR2, 3 and 4, MBL as well as for IL-17A and their associations with nasopharyngeal pathogenic bacterial colonization during a two-year follow-up. The study revealed that polymorphisms in TLRs, MBL2 and IL17A are associated with the nasopharyngeal bacterial colonization in young children. Healthy young (2.6 months of age) children with variant types of MBL2, TLR2 R753Q or TLR4 D299G had an increased risk to be colonized by S. pneumonia, S. aureus or M. catarrhalis, respectively. Moreover, variant types of MBL2 in healthy children with might facilitate human rhinovirus (HRV)-induced S. pneumoniae colonization at 2.6 months of age. The polymorphism of TLR4 D299G was shown to be associated with M. catarrhalis colonization throughout the whole two-year follow-up (2.6, 13 and 24 months of age) and also with the bacterial load of this pathogen. Also, the polymorphism of IL17A G152A was shown to be associated with increased risk to be colonized by S. pneumoniae at 13 and 24 months of age. Furthermore, the results suggest that IL17A G152A has an effect on production of serum IL-17A already at young age. In conclusion, the results of this study indicate that polymorphisms in the key PRRs and IL17A seem to play an important role to colonization of S. pneumoniae, M. catarrhalis, and S. aureus in healthy young Finnish children. The nasopharyngeal colonization by these pathogenic bacteria may further promote the development of respiratory infections and may be related to development of asthma and allergy in the later life of children. These findings offer a possible explanation why some children have more respiratory infections than other children and provide a rational basis for future studies in this field.

Prevalence of BRCA1 and BRCA2 gene mutations in families with medium and high risk of breast and ovarian cancer in Brazil

Of all malignant neoplasias affecting women, breast cancer has the highest incidence rate in Brazil. The objective of the present study was to determine the frequency of genetic modifications in families with medium and high risk for breast and ovarian cancer from different regions of Brazil. An exploratory, descriptive study was carried out on the prevalence of the BRCA1 and BRCA2 mutations in case series of high-risk families for breast and/or ovarian cancer. After heredogram construction, a blood sample was taken and DNA extraction was performed in all index cases. The protein truncation test was used to screen for truncated mutations in exon 11 of the BRCA1 gene and in exons 10 and 11 of the BRCA2 gene. Of the 612 individuals submitted to genetic testing, 21 (3.4%), 19 women and 2 men, had mutations in the BRCA1 or BRCA2 genes. Of the 19 BRCA1 mutations found in the 18 participants, 7 consisted of ins6kb mutations, 4 were 5382insC, 3 were 2156delGinsCC, 2 were 185delAG, 1 was C1201G, 1 was C3522T, and 1 was 3450del4. With respect to the BRCA2 gene, 3 mutations were found: 5878del10, 5036delA and 4232insA (one case each). The prevalence of germline mutations in the BRCA1 and BRCA2 genes found in the present study was lower than reported by other studies on high-risk Brazilian populations. The inclusion of individuals with medium risk may have contributed to the lower prevalence observed.

Cytogenetic characterization and evaluation of c-MYC gene amplification in PG100, a new Brazilian gastric cancer cell line

Gastric cancer is the fourth most frequent type of cancer and the second cause of cancer mortality worldwide. The genetic alterations described so far for gastric carcinomas include amplifications and mutations of the c-ERBB2, KRAS, MET, TP53, and c-MYC genes. Chromosomal instability described for gastric cancer includes gains and losses of whole chromosomes or parts of them and these events might lead to oncogene overexpression, showing the need for a better understanding of the cytogenetic aspects of this neoplasia. Very few gastric carcinoma cell lines have been isolated. The establishment and characterization of the biological properties of gastric cancer cell lines is a powerful tool to gather information about the evolution of this malignancy, and also to test new therapeutic approaches. The present study characterized cytogenetically PG-100, the first commercially available gastric cancer cell line derived from a Brazilian patient who had a gastric adenocarcinoma, using GTG banding and fluorescent in situ hybridization to determine MYC amplification. Twenty metaphases were karyotyped; 19 (95%) of them presented chromosome 8 trisomy, where the MYC gene is located, and 17 (85%) presented a deletion in the 17p region, where the TP53 is located. These are common findings for gastric carcinomas, validating PG100 as an experimental model for this neoplasia. Eighty-six percent of 200 cells analyzed by fluorescent in situ hybridization presented MYC overexpression. Less frequent findings, such as 5p deletions and trisomy 16, open new perspectives for the study of this tumor.