PTEN, ATM, IDH1 mutations and MAPK pathway activation as modulators of PFS and OS in patients treated by first line EGFR TKI, an ancillary study of the French Cooperative Thoracic Intergroup (IFCT) Biomarkers France project.

OBJECTIVES: Tumor mutation screening is standard of care for patients with stage IV NSCLC. Since a couple of years, widespread NGS approaches used in routine diagnostics to detect driver mutations such as EGFR, KRAS, BRAF or MET allows the identification of other alterations that could modulated the intensity or duration of response to targeted therapies. The prevalence of co-occurring alterations that could affect response or prognosis as not been largely analyzed in clinical settings and large cohorts of patients. Thanks to the IFCT program "Biomarkers France", a collection of samples and data at a nation-wide level was available to test the impact of co-mutations on first line EGFR TKI in patients with EGFR mutated cancers. MATERIALS AND METHODS: Targeted NGS was assessed on available (n = 208) samples using the Ion AmpliSeq™ Cancer Hotspot Panel v2 to screen for mutations in 50 different cancer genes. RESULTS: This study showed that PTEN inactivating mutations, ATM alterations, IDH1 mutations and complex EGFR mutations were predictors of short PFS in patients with a stage 4 lung adenocarcinoma receiving first line EGFR TKI and that PTEN, ATM, IDH1 and KRAS mutations as well as alterations in the MAPK pathway were related to shorter OS. CONCLUSION: These findings may lead to new treatment options in patients with unfavorable genotypes to optimize first line responses.

Rare EGFR exon 18 and exon 20 mutations in non-small-cell lung cancer on 10 117 patients: a multicentre observational study by the French ERMETIC-IFCT network.

BACKGROUND: There is scarce data available about epidermal growth factor receptor (EGFR) mutations other than common exon 19 deletions and exon 21 (L858R) mutations. PATIENTS AND METHODS: EGFR exon 18 and/or exon 20 mutations were collected from 10 117 non-small-cell lung cancer (NSCLC) samples analysed at 15 French National Cancer Institute (INCa)-platforms of the ERMETIC-IFCT network. RESULTS: Between 2008 and 2011, 1047 (10%) samples were EGFR-mutated, 102 (10%) with rare mutations: 41 (4%) in exon 18, 49 (5%) in exon 20, and 12 (1%) with other EGFR mutations. Exon 20 mutations were related to never-smoker status, when compared with exon 18 mutations (P < 0.001). Median overall survival (OS) of metastatic disease was 21 months [95% confidence interval (CI) 12-24], worse in smokers than in non-smoker patients with exon 20 mutations (12 versus 21 months; hazard ratio [HR] for death 0.27, 95% CI 0.08-0.87, P = 0.03). Under EGFR-tyrosine kinase inhibitors (TKIs), median OS was 14 months (95% CI 6-21); disease control rate was better for complex mutations (6 of 7, 86%) than for single mutations (16 of 40, 40%) (P = 0.03). CONCLUSIONS: Rare EGFR-mutated NSCLCs are heterogeneous, with resistance of distal exon 20 insertions and better sensitivity of exon 18 or complex mutations to EGFR-TKIs, probably requiring individual assessment.

[Feasibility of assessing EGFR mutation and others using samples obtained by EBUS transbronchial needle aspiration]. / Faisabilité de la recherche de mutations EGFR et KRAS sur des prélèvements obtenus par EBUS-PTBA.

INTRODUCTION: Assessment of mutation status in patients with non-small cell lung cancer (NSCLC) is often required. The aim of this study was to confirm the feasibility of molecular mutation analysis in cytologic specimens obtained by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA). METHODS: Patients with an EBUS-TBNA positive for adenocarcinoma or NSCLC (squamous cell carcinoma excluded) were included retrospectively from January to December 2010, and prospectively from January to August 2011. Specimens were collected on liquid based preparation and processed on paraffin-embedded cell blocks after ThinPrepÒ procedure. Molecular analysis was performed by a SnaPshot assay for EGFR and by pyrosequencing for KRAS on suitable samples (>5% tumor cells). RESULTS: Eighty-two patients were included (63 adenocarcinoma). Molecular analysis for EGFR was feasible in 80 (97.6%) patients and for KRAS in 78 (95.1%) patients. Molecular analysis identified EGFR and KRAS mutations in tumor samples from four (5%) and 18 (23%) patients respectively. All EGFR mutations were found in women. CONCLUSIONS: Molecular analysis mutations can be performed routinely in cytologic specimens obtained by EBUS-TBNA.

Evaluation of a new panel of six mononucleotide repeat markers for the detection of DNA mismatch repair-deficient tumours.

BACKGROUND: Microsatellite instability (MSI) is a molecular phenotype due to defective DNA mismatch repair (MMR) system. It is used to predict outcome of colorectal tumours and to screen tumours for Lynch syndrome (LS). A pentaplex panel composed of five mononucleotide markers has been largely recommended for determination of the MSI status. However, its sensitivity may be taken in default in occasional situations. The aim of the study was to optimise this panel for the detection of MSI. METHODS: We developed an assay allowing co-amplification of six mononucleotide repeat markers (BAT25, BAT26, BAT40, NR21, NR22, NR27) and one polymorphic dinucleotide marker (D3S1260) in a single reaction. Performances of the new panel were evaluated on a cohort of patients suspected of LS. RESULTS: We demonstrate that our assay is technically as easy to use as the pentaplex assay. The hexaplex panel shows similar performances for the identification of colorectal and non-MSH6-deficient tumours. On the other hand, the hexaplex panel has higher sensitivity for the identification of MSH6-deficient tumours (94.7% vs 84.2%) and MMR-deficient tumours other than colorectal cancer (92.9% vs 85.7%). CONCLUSION: The hexaplex panel could thus be an attractive alternative to the pentaplex panel for the identification of patients with LS.

Rapp-Hodgkin and Hay-Wells ectodermal dysplasia syndromes represent a variable spectrum of the same genetic disorder.

BACKGROUND: Rapp-Hodgkin syndrome (RHS) and Hay-Wells [also known as ankyloblepharon-ectodermal defects-cleft lip/palate (AEC)] syndrome have been designated as distinct ectodermal dysplasia syndromes despite both disorders having overlapping clinical features and the same mutated gene, TP63. OBJECTIVES: To search for TP63 mutations in two unrelated cases of RHS and two of AEC syndrome and to review the TP63 mutation database and clinical descriptions of affected individuals, the goal being to refine genotype-phenotype correlation and to determine the clinical/molecular justification for RHS and AEC continuing to exist as separate entities. METHODS: Clinical examination of four affected cases and sequencing of genomic DNA using TP63-specific primers. Literature review of published clinical descriptions of RHS and AEC syndrome cases containing TP63 mutation data. RESULTS: Cases of RHS and AEC show considerable clinical overlap, particularly with regard to hypotrichosis and mid-face hypoplasia, and the clinical feature of ankyloblepharon in AEC is often subtle, transient and a poor distinguishing clinical sign. We identified two new and two recurrent heterozygous mutations in TP63: c.1456insA (p.Leu486fsX52), RHS; c.1537T>G (p.Phe513Val), RHS; c.1787delG (p.Gly596fsX68), AEC; and c.1682G>A (p.Gly561Asp), AEC. Including this study, 42 different mutations in TP63 in RHS and AEC have now been reported, three of which are exactly the same in both syndromes. CONCLUSIONS: Our clinicopathological and molecular findings indicate that there is no justification for the continued use of eponyms in referring to these particular ectodermal dysplasia syndromes. We support the view that the terms 'Hay-Wells' and 'Rapp-Hodgkin' should be abandoned in favour of the all-inclusive diagnosis 'AEC syndrome', notwithstanding the inconsistency or often transient nature of the ankyloblepharon.

An intermediate phenotype between Hay-Wells and Rapp-Hodgkin syndromes in a patient with a novel P63 mutation: confirmation of a variable phenotypic spectrum with a common aetiology.

The ankyloblepharon-ectodermal defects-cleft lip and palate (Hay-Wells or AEC) and the Rapp-Hodgkin syndrome (RHS) are rare autosomal dominant ectodermal dysplasias due to mutations in the transcription factor gene P63. Both are caused by mutations affecting SAM or TID domains of TP63 protein. The two disorders share common features and may represent different phenotypic expressions of the same clinical entity. To date more than 20 P63 mutations have been described associated with AEC and RHS, the majority of which are missense or nonsense mutations. Molecular heterogeneity cannot account for the clinical heterogeneity, because the same mutations were observed both in patient with RHS and with AEC syndrome. Here we report on a novel P63 mutation (the first repeat variation described in the gene) in a patient showing overlapping phenotype of AEC and RH syndromes.

Mapping of SOMU1 and M1 epitopes on the apomucin encoded by the 5' end of the MUC5AC gene.

We have developed 11 monoclonal antibodies (MAbs) against human gastric mucin, (1-13M1, 2-11M1, 2-12M1, 9-13M1, 58M1, 19M1, 21M1, 45M1, 463M, 589M, 62M1), which specifically stained by immunohistochemisty both the human gastric surface mucosa and colon adenoma. Among them, five (19M1, 21M1, 463M, 589M, 62M1) immunoreacted with the peptide encoded by the 3' region of the MUC5AC gene (Nollet et al: Int J Cancer 2002;99:336-343). In this study, we identified in the 5' region of this gene the nucleotide fragments encoding peptides immunoreacting with three other anti-M1 MAbs (1-13M1, 2-11M1 and 9-13M1), as well as the SOMU1 MAb (Sotozono et al: J Immunol Methods 1996;192:187-196). 1-13M1 MAb immunoreacts with peptides, including the Cys 2 and Cys 4 domains. The SOMU1 MAb recognized the Cys 5 domain, and the MAbs 2-11M1 and 9-13M1 the globular D1/D2 and D3 domains, respectively. Using serial sections of the mucosae adjacent to colon adenocarcinomas and colon adenomas, we observed that the anti-M1 and anti-SOMU1 MAbs displayed the same immunostaining patterns. The three anti-M1 MAbs (2-12M1, 58M1, and 45M1) did not react with the products of the MUC5AC gene tested until now. The MUC5AC apomucin is now well characterized by MAbs immunoreacting against seven different epitopes belonging to the different main cystein globular domains of this macromolecule. Such antibodies are useful tools for studying the biosynthesis, polymerization, and degradation of mucin.

Normal respiratory mucosa, precursor lesions and lung carcinomas: differential expression of human mucin genes.

Mucins are glycoproteins synthesized by epithelial cells and thought to promote tumor-cell invasion. Eight human mucin genes have been well characterized: MUC2, MUC5AC, MUC5B, MUC6 map to 11p15.5 and encode secretory gel forming mucins while MUC1, MUC3, MUC4, MUC7 are scattered on different chromosomes and encode membrane-bound or secreted mucins. The expression pattern of the mucin genes is complex in normal airways involving six genes, mainly MUC5AC and MUC5B in mucus-producing cells and MUC4 in a wide array of epithelial cells. MUC5AC overexpression in metaplasia, dysplasia and normal epithelium adjacent to squamous cell carcinoma provides additional arguments for a mucous cell origin of preneoplastic squamous lesions. MUC5AC and MUC5B expression is related to mucus formation in adenocarcinomas. Mucinous bronchioloalveolar carcinoma (BAC) has a particular pattern of mucin gene expression indicating that it has sustained a well-differentiated phenotype similar to the goblet cell, correlated with distinctive features i.e. a noninvasive pattern and a better prognosis than nonBACs. MUC4 is the earlier mucin gene expressed in the foregut, before epithelial differentiation and is expressed independently of mucus secretion both in normal adult airways and carcinomas. These findings are in favor the histogenetic theory of non-small-cell carcinoma originating from a pluripotent mucous cell.

Structural organization and classification of the human mucin genes.

The cells of living organisms in contact with the external environment are constantly attacked by different kinds of substances such as micro-organisms, toxins, and pollutants. With evolution, defense mechanisms, such as the secretion of mucus has been developed. Mucins are the main components of mucus. They are synthesized and secreted by specialized cells of the epithelium and in some case, by non mucin-secreting cells. Little was known about the structure of mucins until a decade ago. This is principally due to heavy glycosylation of mucins, which complicated their analysis. With the application of molecular biological methods, structures of the mucin core peptides (apomucins) are beginning to be elucidated. A total of eleven human mucin (MUC) genes have been identified and numbered in chronological order of their description: MUC1-4, MUC5AC, MUC5B, MUC6-8, and MUC11-12. Of these, the complete cDNA sequence are published only for six mucins MUC1, MUC2, MUC4, MUC5B, MUC5AC, and MUC7. Human mucin genes, in general, show three common features: I) a nucleotide tandem repeat domain; II) a predicted peptide domain containing a high percentage of serines and threonines; III) complex RNA expression. The tandem repeats in mucins make up the majority of the backbone. Related to their structure, mucins can be classified in three distinct sub-families: gel-forming, soluble, and membrane-bound. Each member from one family possesses common characteristics and probably specific functions. For a long time, they were thought to have the unique function of protecting and lubricating the epithelial surfaces. The study of the mucins structure as well as the relationship between structure and function show that mucins also possess other important functions, such as growth, direct implication in the fetal development, the epithelial renewal and differentiation, the epithelial integrity, carcinogenesis, and metastasis. This review presents the actual knowledge on the mucins structure and the best-characterized function related to their structure.

Human mucin gene MUC5AC: organization of its 5'-region and central repetitive region.

Human mucin gene MUC5AC is clustered with MUC2, MUC5B and MUC6 on chromosome 11p15.5. We report here the full length cDNA sequence upstream of the repetitive region of human MUC5AC. We have also determined the sequence of its large central tandem repeat array. The 5'-region reveals high degree of sequence similarity with MUC2 and MUC5B and codes for 1336 amino acids organized into a signal peptide, four pro-von Willebrand factor-like D domains (D1, D2, D' and D3) and a short domain which connects to the central repetitive region. In the central region, 17 major domains have been identified. Nine code for cysteine-rich domains (Cys-domains 1-9) and exhibit high sequence similarity to the cysteine-rich domains described in the central region of MUC2 and MUC5B. Cys-domains 1-5 are interspersed by domains enriched with serine, threonine, and proline residues. Cys-domains 1-9 are interspersed by four domains (TR1-TR4) composed of various numbers of MUC5AC-type repeats. Southern-blot analyses reveal allelic variations both in length and nucleotide sequence. The length polymorphism which is due to variable numbers of tandem repeats is located in TR1 and TR4, whereas a mutation polymorphism detected with TaqI is located in Cys-domain 6. In this study, the organization of MUC5AC has been entirely elucidated showing extensive similarity to the other chromosome 11p15 MUC genes, particularly MUC5B, and providing additional arguments for common evolution from a single ancestral gene.

Alternative splicing generates a family of putative secreted and membrane-associated MUC4 mucins.

The MUC4 mucin gene encodes a putative membrane-anchored mucin with predicted size of 930 kDa, for its 26.5-kb allele. It is composed of two regions, the 850-kDa mucin-type subunit MUC4alpha and the 80-kDa membrane-associated subunit MUC4beta. In this study, we cloned and characterized unique MUC4 cDNA sequences that differ from the originally published sequence. Eight alternative splice events located downstream of the central large tandem repeat array generated eight new, distinct cDNAs. The deduced sequences of these MUC4 cDNAs (sv1-MUC4 to sv8-MUC4, the full length cDNA being called sv0-MUC4) provided seven distinct variants, five secreted forms and two membrane-associated forms. Furthermore, two other alternative splicing events located on both sides of the tandem repeat array created two variants, MUC4/Y and MUC4/X, both lacking the central tandem repeat. Therefore, MUC4 can be expressed in three distinct forms, one membrane-bound, one secreted, and one lacking the hallmark feature of mucin, the tandem repeat array. Although no specific function has yet been discovered for the family of proteins putatively produced from the unique MUC4 gene, we suspect that the MUC4 proteins may be implicated in the integrity and renewal of the epithelium.

Expression of p-glycoprotein in multidrug-resistant human leukemia k562 cells during erythroid-differentiation.

Expression of P-glycoprotein (P-gp), the multidrug resistance gene product, has previously been shown to be downregulated during differentiation of normal haematopoietic cells. In order to determine whether such a regulation also occurs in leukemic cells, we have investigated the relevance of differentiation levels to P-gp expression in multidrug-resistant leukemia K562R/7 cells and in parental drug-sensitive K562 cells. These leukemic cells were exposed to hemin and sodium butyrate, two known inducers of erythroid differentiation. Analysis of hemoglobin-synthesizing cells indicated that hemin induced both K562R/7 and K562 cells to differentiate into erythroid cells while sodium butyrate led to hemoglobin synthesis in only K562 cells. Northern blotting and immunolabelling experiments revealed elevated levels of MDR1 mRNAs and P-gp in both untreated and hemin-treated K562R/7 cells while P-gp expression was not detected in both uninduced and hemin-induced K562 cells. Flow cytometric analysis of cellular doxorubicin retention demonstrated that K562R/7 cells poorly accumulated the anticancer drug regarless their level of differentiation. These results therefore suggest that erythroid differentiation of leukemic drug-resistant K562R/7 cells in response to hemin treatment did not result in major alteration of P-gp expression and activity.