A genetically defined signature of responsiveness to erlotinib in early-stage pancreatic cancer patients: Results from the CONKO-005 trial.

BACKGROUND: high recurrence rates of up to 75% within 2 years in pancreatic ductal adenocarcinoma (PDAC) patients resected for cure indicate a high medical need for clinical prediction tools and patient specific treatment approaches. Addition of the EGFR inhibitor erlotinib to adjuvant chemotherapy failed to improve outcome but its efficacy in some patients warrants predictors of responsiveness. PATIENTS AND METHODS: we analysed tumour samples from 293 R0-resected patients from the randomized, multicentre phase III CONKO-005 trial (gemcitabine ± erlotinib) with targeted sequencing, copy number, and RNA expression analyses. FINDINGS: a total of 1086 mutations and 4157 copy-number aberrations (CNAs) with a mean of 17.9 /tumour were identified. Main pathways affected by genetic aberrations were the MAPK-pathway (99%), cell cycle control (92%), TGFß signalling (77%), chromatin remodelling (71%), and the PI3K/AKT pathway (65%). Based on genetic signatures extracted with non-negative matrix factorization we could define five patient clusters, which differed in mutation patterns, gene expression profiles, and survival. In multivariable Cox regression analysis, SMAD4 aberrations were identified as a negative prognostic marker in the gemcitabine arm, an effect that was counteracted when treated with erlotinib (DFS: HR=1.59, p = 0.016, and OS: HR = 1.67, p = 0.014). Integration of differential gene expression analysis established SMAD4 alterations with low MAPK9 expression (n = 91) as a predictive biomarker for longer DFS (HR=0.49; test for interaction, p = 0.02) and OS (HR = 0.32; test for interaction, p = 0.001). INTERPRETATION: this study identified five biologically distinct patient clusters with different actionable lesions and unravelled a previously unappreciated association of SMAD4 alteration status with erlotinib effectiveness. Confirmatory studies and mechanistic experiments are warranted to challenge the hypothesis that SMAD4 status might guide addition of erlotinib treatment in early-stage PDAC patients.

EGR2 mutations define a new clinically aggressive subgroup of chronic lymphocytic leukemia.

Recurrent mutations within EGR2 were recently reported in advanced-stage chronic lymphocytic leukemia (CLL) patients and associated with a worse outcome. To study their prognostic impact, 2403 CLL patients were examined for mutations in the EGR2 hotspot region including a screening (n=1283) and two validation cohorts (UK CLL4 trial patients, n=366; CLL Research Consortium (CRC) patients, n=490). Targeted deep-sequencing of 27 known/postulated CLL driver genes was also performed in 38 EGR2-mutated patients to assess concurrent mutations. EGR2 mutations were detected in 91/2403 (3.8%) investigated cases, and associated with younger age at diagnosis, advanced clinical stage, high CD38 expression and unmutated IGHV genes. EGR2-mutated patients frequently carried ATM lesions (42%), TP53 aberrations (18%) and NOTCH1/FBXW7 mutations (16%). EGR2 mutations independently predicted shorter time-to-first-treatment (TTFT) and overall survival (OS) in the screening cohort; they were confirmed associated with reduced TTFT and OS in the CRC cohort and independently predicted short OS from randomization in the UK CLL4 cohort. A particularly dismal outcome was observed among EGR2-mutated patients who also carried TP53 aberrations. In summary, EGR2 mutations were independently associated with an unfavorable prognosis, comparable to CLL patients carrying TP53 aberrations, suggesting that EGR2-mutated patients represent a new patient subgroup with very poor outcome.

Spliceosome and other novel mutations in chronic lymphocytic leukemia and myeloid malignancies.

Spliceosome mutations represent a new generation of acquired genetic alterations that affect both myeloid and lymphoid malignancies. A substantial proportion of patients with myelodysplastic syndromes (MDSs) or chronic lymphocytic leukemia (CLL) harbor such mutations, which are often missense in type. Genotype-phenotype associations have been demonstrated for one of these mutations, SF3B1, with ring sideroblasts in MDS and 11q22 deletions in CLL. Spliceosome mutations might result in defective spliceosome assembly, deregulated global mRNA splicing, nuclear-cytoplasm export and altered expression of multiple genes. Such mutations are infrequent in other lymphomas, which instead display a separate group of novel mutations involving genes whose products are believed to affect histone acetylation and methylation and chromatin structure (for example, EZH2 and MLL2). On the other hand, some mutations (for example, NOTCH1) occur in both CLL and other immature and mature lymphoid malignancies. In the current review, we discuss potential mechanisms of cell transformation associated with spliceosome mutations, touch upon the increasing evidence regarding the clonal involvement of hematopoietic stem cells in some cases of otherwise mature lymphoid disorders and summarize recent information on recently described mutations in lymphomas.

Prognostic implications and molecular associations of NADH dehydrogenase subunit 4 (ND4) mutations in acute myeloid leukemia.

To study the prevalence and prognostic importance of mutations in NADH dehydrogenase subunit 4 (ND4), a mitochondrial encoded transmembrane component of the electron transport chain respiratory Complex I, 452 AML patients were examined for ND4 mutations by direct sequencing. The prognostic impact of ND4 mutations was evaluated in the context of other clinical prognostic markers and genetic risk factors. In all, 29 of 452 patients (6.4%) had either somatic (n=12) or germline (n=17) ND4 mutations predicted to affect translation. Somatic mutations were more likely to be heteroplasmic (P<0.001), to occur in predicted transmembrane domains (P<0.001) and were predicted to have damaging effects upon translation (P<0.001). Patients with somatically acquired ND4 mutations had significantly longer relapse-free survival (P=0.017) and overall survival (OS) (P=0.021) than ND4(wildtype) patients. Multivariate analysis also demonstrated a tendency for increased survival in patients with somatic ND4 mutations (RFS: hazard ratio (HR) 0.25, confidence interval (CI) 0.06-1.01, P=0.052; OS: HR 0.29, CI 0.74-1.20, P=0.089). Somatic ND4(mutated) patients had a higher prevalence of concomitant DNMT3A mutations (P=0.023) and a higher percentage of the NPM1/FLT3-ITD low-risk genotype (P=0.021). Germline affected cases showed higher BAALC (P=0.036) and MLL5 (P=0.051) expression levels. Further studies are warranted to validate the favorable prognostic influence of acquired ND4 mutations in AML.

Prevalence and prognostic value of IDH1 and IDH2 mutations in childhood AML: a study of the AML-BFM and DCOG study groups.

Mutations in the NADP(+)-dependent isocitrate dehydrogenase genes 1 and 2 (IDH1 and IDH2) have recently been found in adult acute myeloid leukemia (AML) patients with a prevalence rising up to 33%. To investigate the frequency of IDH1/2 mutations in pediatric AML, we characterized the mutational hotspot (exon 4) of these genes in diagnostic samples from 460 pediatric AML patients. Our analysis identified somatic IDH1/2 mutations in 4% of cases (IDH1 R132 n=8; IDH2 R140 n=10) and the minor allele of single-nucleotide polymorphism (SNP) rs11554137 in 47 children (10.2%). IDH mutations were associated with an intermediate age (P=0.008), FAB M1/M2 (P=0.013) and nucleophosmin1 mutations (P=0.001). In univariate analysis, IDH(mutated) compared with IDH(wildtype) patients showed a significantly improved overall survival (OS; P=0.032) but not event-free survival (EFS; P=0.14). However, multivariate analysis did not show independent prognostic significance. Children with at least one minor allele of IDH1 SNP rs11554137 had similar EFS (P=0.27) and OS (P=0.62) compared with major allele patients. Gene expression profiles of 12 IDH(mutated) were compared with 201 IDH(wildtype) patients to identify differentially expressed genes and pathways. Although only a small number of discriminating genes were identified, analysis revealed a deregulated tryptophan metabolism, and a significant downregulation of KYNU expression in IDH(mutated) cases.

Reduced expression of C1q-mRNA in monocytes from patients with systemic lupus erythematosus.

Inherited C1q deficiency is associated strongly with the development of systemic lupus erythematosus (SLE). The aim of our study was to evaluate the ability of monocytes from SLE patients without inherited C1q deficiency to up-regulate C1q-mRNA upon stimulation. Furthermore, we wanted to elucidate the physiological stimulus for up-regulation of C1q-mRNA. Peripheral blood mononuclear cell (PBMC)-derived monocytes from 10 SLE patients, 10 patients with rheumatoid arthritis (RA) and 10 healthy controls (HC) were stimulated with dexamethasone (DXM), interferon-gamma or both. Additionally, purified monocytes from HC were stimulated with interleukin (IL)-10. C1q-mRNA expression was measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR). C1q protein was detected using the standard alkaline phosphatase/anti-alkaline phosphatase (APAAP) technique. SLE monocytes were significantly less able to up-regulate C1q-mRNA when compared to RA or HC. IL-10 was identified as an important stimulus for C1q synthesis. In SLE patients there is a significant functional impairment of monocytes to synthesize C1q upon stimulation. As C1q is linked to the process of recognition and removal of apoptotic cells, this relative C1q deficiency is likely to contribute to the reduced phagocytosis of apoptotic material observed in SLE and thereby might be a central pathogenetic factor.