Alexander Kohlmann

Selumetinib Plus Docetaxel Compared With Docetaxel Alone and Progression-Free Survival in Patients With KRAS-Mutant Advanced Non-Small Cell Lung Cancer: The SELECT-1 Randomized Clinical Trial.

Importance There are no specifically approved targeted therapies for the most common genomically defined subset of non–small cell lung cancer (NSCLC), KRAS-mutant lung cancer. Objective To compare efficacy of the mitogen-activated protein kinase kinase (MEK) inhibitor selumetinib + docetaxel with docetaxel alone as a second-line therapy for advanced KRAS-mutant NSCLC. Design, Setting, and Participants Multinational, randomized clinical trial conducted at 202 sites across 25 countries from October 2013 through January 2016. Of 3323 patients with advanced NSCLC and disease progression following first-line anticancer therapy tested for a KRAS mutation, 866 were enrolled and 510 randomized. Primary reason for exclusion was ineligibility. The data cutoff date for analysis was June 7, 2016. Interventions Patients were randomized 1:1; 254 to receive selumetinib + docetaxel and 256 to receive placebo + docetaxel. Main Outcomes and Measures Primary end point was investigator assessed progression-free survival. Secondary end points included overall survival, objective response rate, duration of response, effects on disease-related symptoms, safety, and tolerability. Results Of 510 randomized patients (mean age, 61.4 years [SD, 8.3]; women, 207 [41%]), 505 patients (99%) received treatment and completed the study (251 received selumetinib + docetaxel; 254 received placebo + docetaxel). At the time of data cutoff, 447 patients (88%) had experienced a progression event and 346 deaths (68%) had occurred. Median progression-free survival was 3.9 months (interquartile range [IQR], 1.5-5.9) with selumetinib + docetaxel and 2.8 months (IQR, 1.4-5.5) with placebo + docetaxel (difference, 1.1 months; hazard ratio [HR], 0.93 [95% CI, 0.77-1.12]; P = .44). Median overall survival was 8.7 months (IQR, 3.6-16.8) with selumetinib + docetaxel and 7.9 months (IQR, 3.8-20.1) with placebo + docetaxel (difference, 0.9 months; HR, 1.05 [95% CI, 0.85-1.30]; P = .64). Objective response rate was 20.1% with selumetinib + docetaxel and 13.7% with placebo + docetaxel (difference, 6.4%; odds ratio, 1.61 [95% CI, 1.00-2.62]; P = .05). Median duration of response was 2.9 months (IQR, 1.7-4.8; 95% CI, 2.7-4.1) with selumetinib + docetaxel and 4.5 months (IQR, 2.3-7.3; 95% CI, 2.8-5.6) with placebo + docetaxel. Adverse events of grade 3 or higher were more frequent with selumetinib + docetaxel (169 adverse events [67%] for selumetinib + docetaxel vs 115 adverse events [45%] for placebo + docetaxel; difference, 22%). Conclusions and Relevance Among patients with previously treated advanced KRAS-mutant non–small cell lung cancer, addition of selumetinib to docetaxel did not improve progression-free survival compared with docetaxel alone. Trial Registration clinicaltrials.gov: NCT01933932

Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC)

Introduction Non-invasive mutation testing using circulating tumour DNA (ctDNA) is an attractive premise. This could enable patients without available tumour sample to access more treatment options. Materials & Methods Peripheral blood and matched tumours were analysed from 45 NSCLC patients. We investigated the impact of pre-analytical variables on DNA yield and/or KRAS mutation detection: sample collection tube type, incubation time, centrifugation steps, plasma input volume and DNA extraction kits. Results 2 hr incubation time and double plasma centrifugation (2000 x g) reduced overall DNA yield resulting in lowered levels of contaminating genomic DNA (gDNA). Reduced “contamination” and increased KRAS mutation detection was observed using cell-free DNA Blood Collection Tubes (cfDNA BCT) (Streck), after 72 hrs following blood draw compared to EDTA tubes. Plasma input volume and use of different DNA extraction kits impacted DNA yield. Conclusion This study demonstrated that successful ctDNA recovery for mutation detection in NSCLC is dependent on pre-analytical steps. Development of standardised methods for the detection of KRAS mutations from ctDNA specimens is recommended to minimise the impact of pre-analytical steps on mutation detection rates. Where rapid sample processing is not possible the use of cfDNA BCT tubes would be advantageous.

Refractory anemia with ring sideroblasts and marked thrombocytosis cases harbor mutations in SF3B1 or other spliceosome genes accompanied by JAK2V617F and ASXL1 mutations

Refractory anemia with ring sideroblasts and marked thrombocytosis (RARS-T) is a rare entity with characteristics of both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN).[1][1] Patients have been shown to be frequently JAK2 V617F and SF3B1 , and less commonly MPL W515, mutated

Chromothripsis Is a Recurrent Genomic Abnormality in High-Risk Myelodysplastic Syndromes

To explore novel genetic abnormalities occurring in myelodysplastic syndromes (MDS) through an integrative study combining array-based comparative genomic hybridization (aCGH) and next-generation sequencing (NGS) in a series of MDS and MDS/myeloproliferative neoplasms (MPN) patients. 301 patients diagnosed with MDS (n = 240) or MDS/MPN (n = 61) were studied at the time of diagnosis. A genome-wide analysis of DNA copy number abnormalities was performed. In addition, a mutational analysis of DNMT3A, TET2, RUNX1, TP53 and BCOR genes was performed by NGS in selected cases. 285 abnormalities were identified in 71 patients (23.6%). Three high-risk MDS cases (1.2%) displayed chromothripsis involving exclusively chromosome 13 and affecting some cancer genes: FLT3, BRCA2 and RB1. All three cases carried TP53 mutations as revealed by NGS. Moreover, in the whole series, the integrative analysis of aCGH and NGS enabled the identification of cryptic recurrent deletions in 2p23.3 (DNMT3A; n = 2.8%), 4q24 (TET2; n = 10%) 17p13 (TP53; n = 8.5%), 21q22 (RUNX1; n = 7%), and Xp11.4 (BCOR; n = 2.8%), while mutations in the non-deleted allele where found only in DNMT3A (n = 1), TET2 (n = 3), and TP53 (n = 4). These cryptic abnormalities were detected mainly in patients with normal (45%) or non-informative (15%) karyotype by conventional cytogenetics, except for those with TP53 deletion and mutation (15%), which had a complex karyotype. In addition to well-known copy number defects, the presence of chromothripsis involving chromosome 13 was a novel recurrent change in high-risk MDS patients. Array CGH analysis revealed the presence of cryptic abnormalities in genomic regions where MDS-related genes, such as TET2, DNMT3A, RUNX1 and BCOR, are located.

Gene expression profiling for the diagnosis of acute leukaemia

An optimised diagnostic setting in acute leukaemias combines cytomorphology and cytochemistry, multiparameter immunophenotyping, cytogenetics, fluorescence in situ hybridisation, and polymerase chain reaction (PCR)-based assays. This allows classification and definition of biologically defined and prognostically relevant subtypes, and allows directed treatment in some subentities. Over the last years the microarray technology has helped to quantify simultaneously the expression status of ten thousands of genes in single experiments. This novel approach will hopefully become an essential tool for the molecular classification of acute leukaemias in the near future. It can be anticipated that new biologically defined and clinically relevant subtypes of leukaemia will be identified based on their unique gene expression profiles. This method may therefore guide therapeutic decisions and should be investigated in a diagnostic setting in parallel to established standard methods.

Next-generation sequencing and FISH studies reveal the appearance of gene mutations and chromosomal abnormalities in hematopoietic progenitors in chronic lymphocytic leukemia

BackgroundChronic lymphocytic leukemia (CLL) is a highly genetically heterogeneous disease. Although CLL has been traditionally considered as a mature B cell leukemia, few independent studies have shown that the genetic alterations may appear in CD34+ hematopoietic progenitors. However, the presence of both chromosomal aberrations and gene mutations in CD34+ cells from the same patients has not been explored.MethodsAmplicon-based deep next-generation sequencing (NGS) studies were carried out in magnetically activated-cell-sorting separated CD19+ mature B lymphocytes and CD34+ hematopoietic progenitors (n = 56) to study the mutational status of TP53, NOTCH1, SF3B1, FBXW7, MYD88, and XPO1 genes. In addition, ultra-deep NGS was performed in a subset of seven patients to determine the presence of mutations in flow-sorted CD34+CD19− early hematopoietic progenitors. Fluorescence in situ hybridization (FISH) studies were performed in the CD34+ cells from nine patients of the cohort to examine the presence of cytogenetic abnormalities.ResultsNGS studies revealed a total of 28 mutations in 24 CLL patients. Interestingly, 15 of them also showed the same mutations in their corresponding whole population of CD34+ progenitors. The majority of NOTCH1 (7/9) and XPO1 (4/4) mutations presented a similar mutational burden in both cell fractions; by contrast, mutations of TP53 (2/2), FBXW7 (2/2), and SF3B1 (3/4) showed lower mutational allele frequencies, or even none, in the CD34+ cells compared with the CD19+ population. Ultra-deep NGS confirmed the presence of FBXW7, MYD88, NOTCH1, and XPO1 mutations in the subpopulation of CD34+CD19− early hematopoietic progenitors (6/7). Furthermore, FISH studies showed the presence of 11q and 13q deletions (2/2 and 3/5, respectively) in CD34+ progenitors but the absence of IGH cytogenetic alterations (0/2) in the CD34+ cells. Combining all the results from NGS and FISH, a model of the appearance and expansion of genetic alterations in CLL was derived, suggesting that most of the genetic events appear on the hematopoietic progenitors, although these mutations could induce the beginning of tumoral cell expansion at different stage of B cell differentiation.ConclusionsOur study showed the presence of both gene mutations and chromosomal abnormalities in early hematopoietic progenitor cells from CLL patients.

Mutations of TP53 gene in adult acute lymphoblastic leukemia at diagnosis do not affect the achievement of hematologic response but correlate with early relapse and very poor survival

In adult acute lymphoblastic leukemia (ALL) the evaluation of clinical and biological conventional risk criteria at diagnosis is important but not sufficient to predict clinical outcome. The impact of TP53 mutations has been investigated in a limited number of studies and has still not been defined

A Low Frequency of Losses in 11q Chromosome Is Associated with Better Outcome and Lower Rate of Genomic Mutations in Patients with Chronic Lymphocytic Leukemia

To analyze the impact of the 11q deleted (11q-) cells in CLL patients on the time to first therapy (TFT) and overall survival (OS), 2,493 patients with CLL were studied. 242 patients (9.7%) had 11q-. Fluorescence in situ hybridization (FISH) studies showed a threshold of 40% of deleted cells to be optimal for showing that clinical differences in terms of TFT and OS within 11q- CLLs. In patients with ≥40% of losses in 11q (11q-H) (74%), the median TFT was 19 months compared with 44 months in CLL patients with <40% del(11q) (11q-L) (P<0.0001). In the multivariate analysis, only the presence of 11q-L, mutated IGHV status, early Binet stage and absence of extended lymphadenopathy were associated with longer TFT. Patients with 11q-H had an OS of 90 months, while in the 11q-L group the OS was not reached (P = 0.008). The absence of splenomegaly (P = 0.02), low LDH (P = 0.018) or β2M (P = 0.006), and the presence of 11q-L (P = 0.003) were associated with a longer OS. In addition, to detect the presence of mutations in the ATM, TP53, NOTCH1, SF3B1, MYD88, FBXW7, XPO1 and BIRC3 genes, a select cohort of CLL patients with losses in 11q was sequenced by next-generation sequencing of amplicons. Eighty % of CLLs with 11q- showed mutations and fewer patients with low frequencies of 11q- had mutations among genes examined (50% vs 94.1%, P = 0.023). In summary, CLL patients with <40% of 11q- had a long TFT and OS that could be associated with the presence of fewer mutated genes.

TET2 Overexpression in Chronic Lymphocytic Leukemia Is Unrelated to the Presence of TET2 Variations

TET2 is involved in a variety of hematopoietic malignancies, mainly in myeloid malignancies. Most mutations of TET2 have been identified in myeloid disorders, but some have also recently been described in mature lymphoid neoplasms. In contrast to the large amount of data about mutations of TET2, some data are available for gene expression. Moreover, the role of TET2 in chronic lymphocytic leukemia (CLL) is unknown. This study analyzes both TET2 expression and mutations in 48 CLL patients. TET2 expression was analyzed by exon arrays and quantitative real-time polymerase chain reaction (qRT-PCR). Next-generation sequencing (NGS) technology was applied to investigate the presence of TET2 variations. Overexpression of TET2 was observed in B-cell lymphocytes from CLL patients compared with healthy donors (P = 0.004). In addition, in CLL patients, an overexpression of TET2 was also observed in the clonal B cells compared with the nontumoral cells (P = 0.002). However, no novel mutations were observed. Therefore, overexpression of TET2 in CLL seems to be unrelated to the presence of genomic TET2 variations.

Current status and trends in the diagnostics of AML and MDS

Diagnostics of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) have recently been experiencing extensive modifications regarding the incorporation of next-generation sequencing (NGS) strategies into established diagnostic algorithms, classification and risk stratification systems, and minimal residual disease (MRD) detection. Considering the increasing arsenal of targeted therapies (e.g. FLT3 or IDH1/IDH2 inhibitors) for AML, timely and comprehensive molecular mutation screening has arrived in daily practice. Next-generation flow strategies allow for immunophenotypic minimal residual disease (MRD) monitoring with very high sensitivity. At the same time, standard diagnostic tools such as cytomorphology or conventional cytogenetics remain cornerstones for the diagnostic workup of myeloid malignancies. Herein, we summarize the most recent advances and new trends for the diagnostics of AML and MDS, discuss the difficulties, which accompany the integration of these new methods and their results into daily routine, and aim to define the role hemato-oncologists may play in this new diagnostic era.