Next Generation Sequencing in AML-On the Way to Becoming a New Standard for Treatment Initiation and/or Modulation?

Acute myeloid leukemia (AML) is a clonal disease caused by genetic abberations occurring predominantly in the elderly. Next generation sequencing (NGS) analysis has led to a deeper genetic understanding of the pathogenesis and the role of recently discovered genetic precursor lesions (clonal hematopoiesis of indeterminate/oncogenic potential (CHIP/CHOP)) in the evolution of AML. These advances are reflected by the inclusion of certain mutations in the updated World Health Organization (WHO) 2016 classification and current treatment guidelines by the European Leukemia Net (ELN) and National Comprehensive Cancer Network (NCCN) and results of mutational testing are already influencing the choice and timing of (targeted) treatment. Genetic profiling and stratification of patients into molecularly defined subgroups are expected to gain ever more weight in daily clinical practice. Our aim is to provide a concise summary of current evidence regarding the relevance of NGS for the diagnosis, risk stratification, treatment planning and response assessment in AML, including minimal residual disease (MRD) guided approaches. We also summarize recently approved drugs targeting genetically defined patient populations with risk adapted- and individualized treatment strategies.

Durable remissions with venetoclax monotherapy in secondary AML refractory to hypomethylating agents and high expression of BCL-2 and/or BIM.

Acute myeloid leukemia (AML) is a disease of the elderly population and survival remains poor after failure of hypomethylating agents (HMA). The BCL-2 inhibitor venetoclax demonstrated activity as monotherapy and in combination with chemotherapy or HMA in AML. In this case series, patients with secondary AML (sAML) not eligible for intensive chemotherapy and refractory to HMA were treated with venetoclax within a named patient program at our tertiary cancer center in Salzburg, Austria. Between April 2017 and September 2018, seven patients with sAML received venetoclax therapy. Two out of seven patients achieved a complete remission upon venetoclax initiation with a PFS of 505 days and 352 days and another patient achieved complete peripheral blood blast clearing within nine days after start of venetoclax. Among the venetoclax responders, primary refractory disease to prior HMA therapy was documented, 2 patients harbored IDH1/IDH2 mutations and one patient had an antecedent myeloproliferative neoplasm. High BCL-2 and/or BIM expression in myeloblasts was found in venetoclax responders and response was significantly associated with overall survival (responders: 364 days versus non-responders: 24 days, P = 0.018). Venetoclax monotherapy is safe and is able to induce durable responses in elderly patients with secondary AML after treatment failure with HMA.

BIRC3 Expression Predicts CLL Progression and Defines Treatment Sensitivity via Enhanced NF-κB Nuclear Translocation.

PURPOSE: Chronic lymphocytic leukemia (CLL) pathophysiology is characterized by a complex crosstalk of tumor cells with the microenvironment. In this regard, NF-κB signaling is considered as important signaling axis, with a variety of key molecules aberrantly expressed or genetically altered in patients with CLL. One of these molecules is BIRC3 (cIAP2), a central regulator of noncanonical NF-κB signaling that serves as pathway brake in the absence of microenvironmental signals. However, the contribution of BIRC3 expression to CLL progression and potential therapeutic implications is unknown.Experimental Design: We analyzed the role of BIRC3 mRNA expression in primary CLL samples in correlation to clinical datasets and used ex vivo assays to investigate functional consequences on the level of NF-κB signaling and downstream target gene regulation. For proof-of-principle experiments, we used genetically modified cell lines. RESULTS: We demonstrate that patients with CLL with low BIRC3 expression experience a more rapid disease progression, which coincides with an enhanced activation of canonical NF-κB target genes evidenced by an increased p65/Rel-B nuclear translocation ratio. As a consequence of enhanced canonical NF-κB target gene activation, both anti- and proapoptotic Bcl-2 family members were upregulated in BIRC3low primary CLL cells, which was associated with higher sensitivity to venetoclax treatment in vitro. CONCLUSIONS: Here we show the impact of BIRC3 expression in CLL disease progression in the absence of BIRC3 mutations and show altered canonical NF-κB target gene activation with therapeutic implications.

Sequential immunotherapy in a patient with primary refractory Hodgkin lymphoma and novel mutations.

Primary resistant Hodgkin lymphoma is an aggressive disease with few treatment options and short survival. Neoplastic cells of classical Hodgkin lymphoma are heavily dependent on microenvironmental stimuli, regularly express PD-L1, and a relevant proportion of relapsed patients is sensitive to blocking of the PD1/PD-L1 axis. However, response duration is limited and further treatment options are unknown but urgently needed. We report a case of a patient without relevant response to five subsequent chemotherapy regimens who immediately and dramatically responded to an anti-PD1 mab. During the following two years she responded to the anti-CTLA-4 mab ipilimumab, the Jak2 inhibitor ruxolitinib, and a combination of lenalidomide plus cyclophosphamide given in subsequent relapses. A thorough genomic analysis demonstrated seven genomic alterations with six of them not previously described in this disease (i.e. BRIP1 G212fs*62, KRAS L19F, KDM5A R1239W, MYC A59T, ARIDA1A E1683fs*15 and TP53 277Y). Three alterations were considered actionable and one of them drugable. The number of mutations increased over time and the BRIP1 mutation was found to be a germline mutation.

Characterization of two Austrian porcine reproductive and respiratory syndrome virus (PRRSV) field isolates reveals relationship to East Asian strains.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes major problems for the swine industry worldwide. Due to Austria's central location in Europe, a large number of animals are transported through the country. However, little is known about current PRRSV strains and epidemiology. We determined full-length genome sequences of two Austrian field isolates (AUT13-883 and AUT14-440) from recent PRRSV outbreaks and of a related German isolate (GER09-613). Phylogenetic analysis revealed that the strains belong to European genotype 1 subtype 1 and form a cluster together with a South Korean strain. Remarkably, AUT14-440 infected the simian cell line MARC-145 without prior adaptation. In addition, this isolate showed exceptional deletions in nonstructural protein 2, in the overlapping region of glycoprotein 3 and 4 and in the 3' untranslated region. Both Austrian isolates caused similar lung lesions but only pigs infected with AUT14-440 developed clear clinical signs of infection. Taken together, the genetic and biological characterization of two novel Austrian PRRSV field isolates revealed similarities to East Asian strains. This stresses the necessity for a more detailed analysis of current PRRSV strains in Europe beyond the determination of short ORF5 and ORF7 sequences.

Yno1p/Aim14p, a NADPH-oxidase ortholog, controls extramitochondrial reactive oxygen species generation, apoptosis, and actin cable formation in yeast.

The large protein superfamily of NADPH oxidases (NOX enzymes) is found in members of all eukaryotic kingdoms: animals, plants, fungi, and protists. The physiological functions of these NOX enzymes range from defense to specialized oxidative biosynthesis and to signaling. In filamentous fungi, NOX enzymes are involved in signaling cell differentiation, in particular in the formation of fruiting bodies. On the basis of bioinformatics analysis, until now it was believed that the genomes of unicellular fungi like Saccharomyces cerevisiae and Schizosaccharomyces pombe do not harbor genes coding for NOX enzymes. Nevertheless, the genome of S. cerevisiae contains nine ORFs showing sequence similarity to the catalytic subunits of mammalian NOX enzymes, only some of which have been functionally assigned as ferric reductases involved in iron ion transport. Here we show that one of the nine ORFs (YGL160W, AIM14) encodes a genuine NADPH oxidase, which is located in the endoplasmic reticulum (ER) and produces superoxide in a NADPH-dependent fashion. We renamed this ORF YNO1 (yeast NADPH oxidase 1). Overexpression of YNO1 causes YCA1-dependent apoptosis, whereas deletion of the gene makes cells less sensitive to apoptotic stimuli. Several independent lines of evidence point to regulation of the actin cytoskeleton by reactive oxygen species (ROS) produced by Yno1p.