Laboratory methods of monitoring disease response after allogeneic haematopoietic stem cell transplantation for myelofibrosis.

The era of molecular prognostication in myelofibrosis has allowed comprehensive assessment of disease risk and informed decisions regarding allogeneic haematopoietic stem cell transplantation (HSCT). However, monitoring disease response after transplantation is difficult, and limited by disease and sample-related factors. The emergence of laboratory techniques sensitive enough to monitor measurable residual disease is promising in predicting molecular and haematological relapse and guiding management. This paper summarises the existing literature regarding methods for detecting and monitoring disease response after HSCT in myelofibrosis and explores the therapeutic use of measurable residual disease (MRD) assays in transplant recipients. Laboratory assessment of disease response in myelofibrosis post-allogeneic transplant is limited by disease and treatment characteristics and by the sensitivity of available conventional molecular assays. The identification of MRD has prognostic implications and may allow early intervention to prevent relapse. Further applicability is limited by mutation-specific assay variability, a lack of standardisation and sample considerations.

High incidence of minor and micro breakpoints in Chronic Myeloid Leukaemia with additional cytogenetic abnormalities at diagnosis - the Western Australian series.

Introduction and objective: Chronic Myeloid Leukaemia (CML) is defined by the presence of the Philadelphia chromosome, a balanced translocation between chromosomes 9 and 22 that results in the constitutively active tyrosine kinase, BCR-ABL1. Additional chromosomal abnormalities (ACAs) at diagnosis occur in 5-10% of CML patients, and are important for prognosis. They are classified as major or minor route. The purpose of our study was to determine the frequency and type of ACAs in 193 newly diagnosed CML patients, and to evaluate patient characteristics, treatment response, and survival. Methods: Medical records, in conjunction with data from the PathWest cytogenetics and molecular laboratories, were analysed. Results: ACAs were present in 14 (7.3%) of patients at diagnosis. Seven patients had major-route abnormalities, with additional chromosome 8 (+8) the most common. All patients were treated with tyrosine kinase inhibitors (TKIs). Three patients presented in blast crisis; two patients have died. Of note, there was a high incidence of the rare minor and micro BCR-ABL1 fusion transcripts. Conclusions: Frequency of ACAs at diagnosis was similar to that of previous reports. These patients consist a higher-risk cohort, and require individualised treatment, with consideration of frontline and secondary TKIs, adjunct chemotherapy, novel agents, and allogeneic stem cell transplant.

Clonal cytopenia of undetermined significance and atypical Behçet's: the importance of zinc.

Atypical Behçet's is recognised in myelodysplastic syndrome (MDS) cases and is associated with trisomy 8. Clonal cytopenia of undetermined significance (CCUS) is recognised as a precursor to MDS. Our case describes the presentation of atypical Behçet's, in association with CCUS, post a Streptococcal infection. A mutation of a zinc finger RNA spliceosome, ZRSR2, is also described. Our patient initially presented with macrocytic anaemia, together with neutropenia and lymphocytopenia on routine monitoring. Later gastrointestinal symptoms together with oral and anal ulcerations developed. He was treated with oral zinc therapy and had resolution of recurrent oral ulcerations and significant reduction in severity of anal ulcerations. The functional impact of ZRSR2 mutation on spliceosome assembly is yet to be defined, but has been previously reported in CCUS with a clinical phenotype of macrocytic anaemia.

Laboratory quality assessment of candidate gene panel testing for acute myeloid leukaemia: a joint ALLG / RCPAQAP initiative.

Accurate classification of acute myeloid leukaemia (AML) has become increasingly reliant on molecular characterisation of this blood cancer. Throughout Australia and New Zealand massively parallel sequencing (MPS) is being adopted by diagnostic laboratories for the routine evaluation of patients with AML. This technology enables the surveying of many genes simultaneously, with many technical advantages over single gene testing approaches. However, there are many variations in wet and dry lab MPS procedures, which raises the prospect of discordant results between laboratories. This study compared the results obtained from MPS testing of ten diagnostic AML bone marrow aspirate samples sent to eight participating laboratories across Australasia. A reassuringly high concordance of 94% was observed with regard to variant detection and characterisation of pathogenicity. The level of discordance observed, although low, demonstrates the need for ongoing assessment of concordance between diagnostic testing laboratories through quality assurance programs.

Control of nuclear-cytoplasmic shuttling of Ankrd54 by PKCδ.

AIM: To identify and characterize the effect of phosphorylation on the subcellular localization of Ankrd54. METHODS: HEK293T cells were treated with calyculin A, staurosporin or phorbol 12-myristate 13-acetate (PMA). Cells were transfected with eGFP-tagged Ankrd54 with or without Lyn tyrosine kinase (wild-type, Y397F mutant, or Y508F mutant). The subcellular localization was assessed by immunofluorescence imaging of cells, immunoblotting of subcellular fractionations. The phosphorylation of Ankrd54 was monitored using Phos-tagTM gel retardation. Phosphorylated peptides were analysed by multiple-reaction-monitoring (MRM) proteomic analysis. RESULTS: Activation of PKC kinases using PMA promoted nuclear export of Ankrd54 and correlated with increased Ankrd54 phosphorylation, assayed using Phos-tagTM gel retardation. Co-expression of an active form of the PKCδ isoform specifically promoted both phosphorylation and cytoplasmic localization of Ankrd54, while PKCδ, Akt and PKA did not. Alanine mutation of several serine residues in the amino-terminal region of Ankrd54 (Ser14, Ser17, Ser18, Ser19) reduced both PMA induced cytoplasmic localization and phosphorylation of Ankrd54. Using MRM proteomic analysis, phosphorylation of the Ser18 residue of Ankrd54 was readily detectable in response to PMA stimulation. PMA stimulation of cells co-expressing Ankrd54 and Lyn tyrosine kinase displayed increased co-immunoprecipitation and enhanced co-localization in the cytoplasm. CONCLUSION: We identify phosphorylation by PKCδ as a major regulator of nuclear-cytoplasmic shuttling of Ankrd54, and its interaction with the tyrosine kinase Lyn.

Csk-binding protein controls red blood cell development via regulation of Lyn tyrosine kinase activity.

Erythropoiesis is controlled principally through erythropoietin (Epo) receptor signaling, which involves Janus kinase 2 (JAK2) and Lyn tyrosine kinase, both of which are important for regulating red blood cell (RBC) development. Negative regulation of Lyn involves C-Src kinase (Csk)-mediated phosphorylation of its C-terminal tyrosine, which is facilitated by the transmembrane adaptor Csk-binding protein (Cbp). Although Cbp has significant functions in controlling Lyn levels and activity in erythroid cells in vitro, its importance to primary erythroid cell development and signaling has remained unclear. To address this, we assessed the consequence of loss of Cbp on the erythroid compartment in vivo and whether Epo-responsive cells isolated from Cbp-knockout mice exhibited altered signaling. Our data show that male Cbp-/- mice display a modest but significant alteration to late erythroid development in bone marrow with evidence of increased erythrocytes in the spleen, whereas female Cbp-/- mice exhibit a moderate elevation in early erythroid progenitors (not seen in male mice) that does not influence the later steps in RBC development. In isolated primary erythroid cells and cell lines generated from Cbp-/- mice, survival signaling through Lyn/Akt/FoxO3 was elevated, resulting in sustained viability during differentiation. The high Akt activity disrupted GAB2/SHP-2 feedback inhibition of Lyn; however, the elevated Lyn activity also increased inhibitory signaling via SHP-1 to restrict the Erk1/2 pathway. Interestingly, whereas loss of Cbp led to mild changes to late RBC development in male mice, this was not apparent in female Cbp-/- mice, possibly due to their elevated estrogen, which is known to facilitate early progenitor self-renewal.

Loss of PRDM11 promotes MYC-driven lymphomagenesis.

The PR-domain (PRDM) family of genes encodes transcriptional regulators, several of which are deregulated in cancer. By using a functional screening approach, we sought to identify novel tumor suppressors among the PRDMs. Here we demonstrate oncogenic collaboration between depletion of the previously uncharacterized PR-domain family member Prdm11 and overexpression of MYC. Overexpression of PRDM11 inhibits proliferation and induces apoptosis. Prdm11 knockout mice are viable, and loss of Prdm11 accelerates MYC-driven lymphomagenesis in the Eµ-Myc mouse model. Moreover, we show that patients with PRDM11-deficient diffuse large B-cell lymphomas (DLBCLs) have poorer overall survival and belong to the nongerminal center B-cell-like subtype. Mechanistically, genome-wide mapping of PRDM11 binding sites coupled with transcriptome sequencing in human DLBCL cells evidenced that PRDM11 associates with transcriptional start sites of target genes and regulates important oncogenes such as FOS and JUN. Hence, we characterize PRDM11 as a putative novel tumor suppressor that controls the expression of key oncogenes, and we add new mechanistic insight into B-cell lymphomagenesis.

Lyn kinase plays important roles in erythroid expansion, maturation and erythropoietin receptor signalling by regulating inhibitory signalling pathways that control survival.

Erythroid homoeostasis is primarily controlled by Epo (erythropoietin) receptor signalling; however, the Lyn tyrosine kinase plays an important subsidiary role in regulating the erythroid compartment. Nonetheless, specific erythroid pathways that require Lyn activity and their biological significance remain unclear. To address this, we asked what consequence loss of Lyn had on the ex vivo expansion and maturation of splenic erythroid progenitors and Epo receptor signalling. Pharmacological inhibition of Lyn with PP2 inhibited the survival of terminally differentiated erythroblasts. Less committed erythroid progenitors expanded well, whereas early splenic Lyn(-/-) erythroblasts had attenuated ex vivo expansion, and late stage Lyn(-/-) erythroblasts were retarded in completing morphological maturation ex vivo. Furthermore, immortalized Lyn(-/-) erythroblasts were slower growing, less viable and inhibited in their differentiation. Signalling studies showed that Lyn was required for both positive GAB2/Akt/FoxO3 (forkhead box O3) survival signals as well as negative feedback of JAK2 (Janus kinase 2)/STAT5 (signal transducer and activator of transcription 5) and ERK1/2 (extracellular-signal-regulated kinase 1/2) signals via SHP-1 (Src homology 2 domain-containing protein tyrosine phosphatase 1). During differentiation, Lyn controls survival and cell cycle exit as demonstrated by reduced STAT5 and FoxO3/GSKα/ß (glycogen synthase kinase α/ß) phosphorylation and diminished p27(Kip1) induction in Lyn-deficient erythroblasts. Lyn deficiency alters the balance of pro- and anti-apoptotic molecules (BAD and BclXL), thereby reducing survival and preventing cell cycle exit. Consequently, Lyn facilitates normal erythrocyte production by influencing different stages of erythroid progenitor expansion, and mature cell development and survival signalling.