An immunogenomic exome landscape of triple positive primary antiphospholipid patients.

Primary antiphospholipid syndrome is characterized by thrombosis and autoantibodies directed against phospholipids or associated proteins. The genetic etiology of PAPS remains unknown. We enrolled 21 patients with thromboembolic events associated to lupus anticoagulant, anticardiolipin and anti ß2 glycoprotein1 autoantibodies. We performed whole exome sequencing and a systematic variant-based analysis in genes associated with thrombosis, in candidate genes previously associated with APS or inborn errors of immunity. Data were compared to public databases and to a control cohort of 873 non-autoimmune patients. Variants were identified following a state-of-the-art pipeline. Enrichment analysis was performed by comparing with the control cohort. We found an absence of significant HLA bias and genetic heterogeneity in these patients, including when testing combinations of rare variants in genes encoding for proteins involved in thrombosis and of variants in genes linked with inborn errors of immunity. These results provide evidence of genetic heterogeneity in PAPS, even in a homogenous series of triple positive patients. At the individual scale, a combination of variants may participate to the breakdown of B cell tolerance and to the vessel damage.

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.

Detection of K-Ras mutations in tumour samples of patients with non-small cell lung cancer using PNA-mediated PCR clamping.

Non-small cell lung cancers (NSCLC), in particular adenocarcinoma, are often mixed with normal cells. Therefore, low sensitivity of direct sequencing used for K-Ras mutation analysis could be inadequate in some cases. Our study focused on the possibility to increase the detection of K-Ras mutations in cases of low tumour cellularity. Besides direct sequencing, we used wild-type hybridisation probes and peptide-nucleic-acid (PNA)-mediated PCR clamping to detect mutations at codons 12 and 13, in 114 routine consecutive NSCLC frozen surgical tumours untreated by targeted drugs. The sensitivity of the analysis without or with PNA was 10 and 1% of tumour DNA, respectively. Direct sequencing revealed K-Ras mutations in 11 out of 114 tumours (10%). Using PNA-mediated PCR clamping, 10 additional cases of K-Ras mutations were detected (21 out of 114, 18%, P<0.005), among which five in samples with low tumour cellularity. In adenocarcinoma, K-Ras mutation frequency increased from 7 out of 55 (13%) by direct sequencing to 15 out of 55 (27%) by clamped-PCR (P<0.005). K-Ras mutations detected by these sensitive techniques lost its prognostic value. In conclusion, a rapid and sensitive PCR-clamping test avoiding macro or micro dissection could be proposed in routine analysis especially for NSCLC samples with low percentage of tumour cells such as bronchial biopsies or after neoadjuvant chemotherapy.

EGFR-TKI and lung adenocarcinoma with CNS relapse: interest of molecular follow-up.

The epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) erlotinib improves survival of lung cancer as second- or third-line therapy. However, after an initial response, most patients will recur, particularly within the central nervous system. The present study reports the case of a 27-yr-old nonsmoking male presenting with a metastatic lung adenocarcinoma with EGFR exon 19 deletion, associated with sensitivity to EGFR-TKI. Gefitinib, followed by chemotherapy and finally erlotinib resulted in prolonged disease control, until multiple liver metastases were detected. After stopping EGFR-TKI, brain metastases with carcinomatous meningitis were diagnosed. A secondary T790M mutation, associated with resistance to EGFR-TKI, was found on the liver biopsy but not in the cerebrospinal fluid. Erlotinib was reintroduced and allowed a quick neurological improvement, even though the extra-cranial disease remained resistant to erlotinib. The present report underscores the interest of molecular monitoring in lung cancer. Persistent cerebral tyrosine kinase inhibitor sensitivity should be considered in patients presenting with an early central nervous system relapse after stopping epidermal growth factor receptor tyrosine kinase inhibitor, even with a T790M-resistant mutation in noncerebral metastases. Questions remain concerning the selection of sub-clones during epidermal growth factor receptor tyrosine kinase inhibitor therapy, which could differ according to metastatic sites, especially in the central nervous system.

Genetic alterations in primary osteosarcoma from 54 children and adolescents by targeted allelotyping.

At present, the only recognised prognostic factor for primary osteosarcoma is the histological response to preoperative chemotherapy. Our study was designed to identify new diagnostic markers that could eventually have a prognostic value. A total of 54 patients under 20 years of age with primary osteosarcomas were studied while under treatment by the French Society of Paediatric Oncology OS 94 protocol. Paired normal and biopsy samples were collected. In addition, surgical resection specimens, following preoperative chemotherapy, were obtained in 13 cases. After genomic DNA extraction, an allelotyping analysis targeting microsatellites linked to Rb and p53 genes, and 9p21, 7q31 and 5q21 regions was performed. In all, 94% of the samples at diagnosis showed allelic imbalance and the biopsies were highly rearranged except for the microsatellite targeting 7q31. The same panel was highly informative at surgical resection. Microsatellites investigating Rb, p53 and the 9p21 region were particularly altered without a significant correlation with prognosis. On the other hand, the alteration of the 7q31 locus at diagnosis was significantly correlated with a worse prognosis and a new frequently altered locus, 5q21, was described. In conclusion, this panel allowed us to characterise paediatric osteosarcomas. Correlation of prognosis with the altered 7q31 region could be a useful tool and further studies are required to confirm the importance of 5q21.

Modification of topoisomerase genes copy number in newly diagnosed childhood acute lymphoblastic leukemia.

Topoisomerase genes were analyzed at both DNA and RNA levels in 25 cases of newly diagnosed childhood acute lymphoblastic leukemia (ALL). The results of molecular analysis were compared to risk group classification of children in order to identify molecular characteristics associated with response to therapy. At diagnosis, allelic imbalance at topo-isomerase IIalpha (TOP2A) gene locus was found in 75% of informative cases whereas topoisomerase I and IIbeta gene loci are altered in none or only one case, respectively. By semi-quantitative Polymerase chain reaction, we found a 2.5 to 8-fold TOP2A gene amplification in 72% of the children, which was correlated to gene overexpression in every case. These results show that TOP2A gene amplification is a frequent event in ALL at diagnosis. Interestingly, we also identified a small population of children that do not present TOP2A gene amplification or gene overexpression and who are significantly associated with very high risk classified patients showing glucocorticoid resistance. In conclusion, characterization of TOP2A gene status in childhood ALL at diagnosis provides useful complementary information for risk assessment.