Ilona Seferynska

Status of minimal residual disease after induction predicts outcome in both standard and high-risk Ph-negative adult acute lymphoblastic leukaemia. The Polish Adult Leukemia Group ALL 4-2002 MRD Study

The treatment of adults with Philadelphia‐negative acute lymphoblastic leukaemia (ALL) depends on the presence of risk factors including age, white blood cell count, immunophenotype and time to complete remission. In recent years, status of minimal residual disease (MRD) has been postulated as an additional risk criterion. This study prospectively evaluated the significance of MRD. Patients were treated with a uniform Polish Adult Leukemia Group (PALG) 4‐2002 protocol. MRD status was assessed after induction and consolidation by multiparametric flow cytometry. Out of 132 patients included (age, 17–60 years), 116 patients were suitable for analysis. MRD level ≥0·1% of bone marrow cells after induction was found to be a strong and independent predictor for relapse in the whole study population (P < 0·0001), as well as in the standard risk (SR, P = 0·0003) and high‐risk (P = 0·008) groups. The impact of MRD after consolidation on outcome was not significant. The combination of MRD status with conventional risk stratification system identified a subgroup of patients allocated to the SR group with MRD <0·1% after induction who had a very low risk of relapse of 9% at 3 years as opposed to 71% in the remaining subjects (P = 0·001). We conclude that MRD evaluation after induction should be considered with conventional risk criteria for treatment decisions in adult ALL.

Genomic instability may originate from imatinib-refractory chronic myeloid leukemia stem cells.

Genomic instability is a hallmark of chronic myeloid leukemia in chronic phase (CML-CP) resulting in BCR-ABL1 mutations encoding resistance to tyrosine kinase inhibitors (TKIs) and/or additional chromosomal aberrations leading to disease relapse and/or malignant progression. TKI-naive and TKI-treated leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) accumulate high levels of reactive oxygen species (ROS) and oxidative DNA damage. To determine the role of TKI-refractory LSCs in genomic instability, we used a murine model of CML-CP where ROS-induced oxidative DNA damage was elevated in LSCs, including quiescent LSCs, but not in LPCs. ROS-induced oxidative DNA damage in LSCs caused clinically relevant genomic instability in CML-CP-like mice, such as TKI-resistant BCR-ABL1 mutations (E255K, T315I, H396P), deletions in Ikzf1 and Trp53, and additions in Zfp423 and Idh1. Despite inhibition of BCR-ABL1 kinase, imatinib did not downregulate ROS and oxidative DNA damage in TKI-refractory LSCs to the levels detected in normal cells, and CML-CP-like mice treated with imatinib continued to accumulate clinically relevant genetic aberrations. Inhibition of class I p21-activated protein kinases by IPA3 downregulated ROS in TKI-naive and TKI-treated LSCs. Altogether, we postulate that genomic instability may originate in the most primitive TKI-refractory LSCs in TKI-naive and TKI-treated patients.

ATR-Chk1 Axis Protects BCR/ABL Leukemia Cells from the Lethal Effect of DNA Double-Strand Breaks

BCR/ABL-positive leukemia cells accumulated more replication-dependent DNA double-strand breaks (DSBs) than normal counterparts after treatment with cisplatin and MMC, as assessed by pulse field gel electrophoresis (PFGE) and neutral comet assay. In addition, leukemia cells could repair these lesions more efficiently than normal cells and eventually survive genotoxic treatment. Elevated levels of drug-induced DSBs in leukemia cells were associated with higher activity of ATR kinase, and enhanced phosphorylation of histone H2AX on serine 139 (γ-H2AX). γ-H2AX eventually started to disappear in BCR/ABL cells, while continued to increase in parental cells. In addition, the expression and ATR-mediated phosphorylation of Chk1 kinase on serine 345 were often more abundant in BCR/ABL-positive leukemia cells than normal counterparts after genotoxic treatment. Inhibition of ATR kinase by caffeine but not Chk1 kinase by indolocarbazole inhibitor, SB218078 sensitized BCR/ABL leukemia cells to MMC in a short-term survival assay. Nevertheless, both caffeine and SB218078 enhanced the genotoxic effect of MMC in a long-term clonogenic assay. This effect was associated with the abrogation of transient accumulation of leukemia cells in S and G2/M cell cycle phases after drug treatment. In conclusion, ATR - Chk1 axis was strongly activated in BCR/ABL-positive cells and contributed to the resistance to DNA cross-linking agents causing numerous replication-dependent DSBs.

Gene expression and mutation-guided synthetic lethality eradicates proliferating and quiescent leukemia cells

Quiescent and proliferating leukemia cells accumulate highly lethal DNA double-strand breaks that are repaired by 2 major mechanisms: BRCA-dependent homologous recombination and DNA-dependent protein kinase–mediated (DNA-PK–mediated) nonhomologous end-joining, whereas DNA repair pathways mediated by poly(ADP)ribose polymerase 1 (PARP1) serve as backups. Here we have designed a personalized medicine approach called gene expression and mutation analysis (GEMA) to identify BRCA- and DNA-PK–deficient leukemias either directly, using reverse transcription-quantitative PCR, microarrays, and flow cytometry, or indirectly, by the presence of oncogenes such as BCR-ABL1. DNA-PK–deficient quiescent leukemia cells and BRCA/DNA-PK–deficient proliferating leukemia cells were sensitive to PARP1 inhibitors that were administered alone or in combination with current antileukemic drugs. In conclusion, GEMA-guided targeting of PARP1 resulted in dual cellular synthetic lethality in quiescent and proliferating immature leukemia cells, and is thus a potential approach to eradicate leukemia stem and progenitor cells that are responsible for initiation and manifestation of the disease. Further, an analysis of The Cancer Genome Atlas database indicated that this personalized medicine approach could also be applied to treat numerous solid tumors from individual patients.

Clinical characteristics of patients with chronic eosinophilic leukaemia (CEL) harbouring FIP1L1-PDGFRA fusion transcript--results of Polish multicentre study.

A small subgroup of patients with hypereosinophilic syndrome (HES) demonstrates imatinib‐sensitive fusion transcript—the FIP1L1‐PDGFRA (F/P+). These cases are currently diagnosed as chronic eosinophilic leukaemia (CEL). In this paper, we screened 77 patients to estimate the frequency of FIP1L1‐PDGFRA transcript among patients with unexplained, long‐term hypereosinophilia exceeding 1.5 × 109/L and to analyse the clinical and serological features in F/P+ CEL population. The FIP1L1‐PDGFRA chimeric protein was detectable in 16 (14 males and 2 females) out of 77 examined HES patients (20%) by RT‐PCR. Two patients suffered from cough at diagnosis. Three out of 16 (18%) patients had no organ involvements, in 5‐one organ was affected and in the remaining eight cases—at least two. Eosinophilic organ damage/dysfunction identified splenomegaly in the majority of studied patients. We compared clinical and serological features between CEL F/P+ (n = 16) and HES (n = 61) patients. F/P+ cases had significantly increased WBC and absolute eosinophil count (AEC) at diagnosis (p = 0.008 and 0.02), whereas platelet count was decreased in this population (p = 0.03). Serum B12 and tryptase levels were increased (p = 0.002 and 0.004) in CEL F/P+ patients when compared to HES cases whereas serum IL‐5 levels were significantly increased in the latter group (p = 0.01). Male gender and splenomegaly occurred more frequent in CEL F/P+ population (p = 0.002 and 0.0007, respectively). Additionally, patients with F/P+ CEL (n = 16) were compared with F/P− CEL (n = 8). The latter group, was significantly older, had lower AEC and higher platelet count. In conclusion, significant clinical symptoms are infrequent present and splenomegaly remains the most common organ involvement in patients with CEL expressing F/P fusion transcript. Our study confirmed the long‐term remission on imatinib in this patient population. Copyright

Elevation of pulmonary artery pressure as a complication of nilotinib therapy for chronic myeloid leukemia

We present the case of a 72-year-old male with chronic phase myeloid leukemia. Elevation of the pulmonary artery pressure due to nilotinib therapy was noted. This effect on pulmonary artery pressure was nilotinib dose dependent.

Monoubiquitinated Fanconi anemia D2 (FANCD2-Ub) is required for BCR-ABL1 kinase-induced leukemogenesis

Fanconi D2 (FANCD2) is monoubiquitinated on K561 (FANCD2-Ub) in response to DNA double-strand breaks (DSBs) to stimulate repair of these potentially lethal DNA lesions. FANCD2-Ub was upregulated in CD34+ chronic myeloid leukemia (CML) cells and in BCR-ABL1 kinase-positive cell lines in response to elevated levels of reactive oxygen species (ROS) and DNA cross-linking agent mitomycin C. Downregulation of FANCD2 and inhibition of FANCD2-Ub reduced the clonogenic potential of CD34+ CML cells and delayed BCR-ABL1 leukemogenesis in mice. Retarded proliferation of BCR-ABL1 positive FANCD2−/− leukemia cells could be rescued by FANCD2 expression. BCR-ABL1 positive FANCD2−/− cells accumulated more ROS-induced DSBs in comparison with BCR-ABL1 positive FANCD2+/+ cells. Antioxidants diminished the number of DSBs and enhanced proliferation of BCR-ABL1 positive FANCD2−/− cells. Expression of wild-type FANCD2 and FANCD2(S222A) phosphorylation-defective mutant (deficient in stimulation of intra-S phase checkpoint, but proficient in DSB repair), but not FANCD2(K561R) monoubiquitination-defective mutant (proficient in stimulation of intra-S phase checkpoint, but deficient in DSB repair) reduced the number of DSBs and facilitated proliferation of BCR-ABL1 positive FANCD2−/− cells. We hypothesize that FANCD2-Ub has an important role in BCR-ABL1 leukemogenesis because of its ability to facilitate the repair of numerous ROS-induced DSBs.

Karyotype changes during long-term targeted therapy of chronic myeloid leukemia with imatinib.

The main risk factors during imatinib therapy of chronic myeloid leukemia are still subject to discussion. A group of 39 patients was cytogenetically examined and monitored before and during long-term treatment with imatinib. The cytogenetic response was investigated using karyotype analysis and fluorescence in situ hybridisation method. Different therapy effects were shown for three subgroups distinguished before the start of treatment: patients with the sole translocation t(9;22) with a typical pattern of BCR/ABL fusion vs. patients with submicroscopic deletion in the fusion region ABL/BCR of the sole t(9;22) vs. patients with aberrations additional to t(9;22) and without submicroscopic deletion. Of the two group with sole t(9;22) the group with deletion in the ABL/BCL region suffered a poorer treatment outcome than the group without deletion. The risk of progression of cytogenetic changes in group with deletion was more than nine times higher than in patients with sole t(9;22) without deletion (statistically significant).

Differential expression of BIRC family genes in chronic myeloid leukaemia – BIRC3 and BIRC8 as potential new candidates to identify disease progression

Although the therapy of chronic myeloid leukaemia (CML) with tyrosine kinase inhibitors (TKI) is considered a major advance in oncology, a significant group of patients still develops drug resistance and does not benefit from targeted therapy (Marin et al, 2013). These patients are at high risk of progression from chronic phase (CML-CP) to almost inevitably fatal blastic phase (CML-BP) (or blast crisis). Therefore, there is a need to identify suboptimal responders to imatinib who should be switched to secondor third-generation TKI as early as possible (Marin et al, 2013). Although the Sokal and Hasford risk scores remain the most used prognostic indicators at diagnosis, reliable markers of suboptimal response and/or progression of the disease are needed. BCR-ABL1 transcript level monitoring is used to evaluate response to the therapy, and its predictive value at 3 and/or 6 months after starting TKI therapy has recently been shown (Neelakantan et al, 2013). Among other possible candidates, high levels of cancerous inhibitor of PP2A (CIP2A) may be an independent determinant of progression to blast crisis (Lucas et al, 2011). Searching for potential candidates of disease progression we have focused on the BIRC (baculoviral IAP repeat-containing; BIRC) gene family expression in various stages of CML. To date, there is no comprehensive analysis of the whole family of BIRC genes in CML. The BIRC family comprises eight functionallyand structurally-related members. Their common structural feature, a motif termed the baculovirus IAP repeat, is required for their cytoprotective function, hence the majority of BIRC serve as endogenous inhibitors of apoptosis (Deveraux & Reed, 1999). While the first human BIRC gene (originally termed BIRC1, now NAIP, encoding the NLR family, apoptosis inhibitory protein) was described in the neurodegenerative disorder, spinal muscular atrophy (Roy et al, 1995), most recent studies focus on the role of BIRC genes in various types of neoplasms. Overexpression of BIRC genes has been associated with cancer progression, multidrug resistance, poor prognosis and short survival in several cancers including haematological malignancies. One of the most extensively studied members of BIRC family – BIRC5 (BIRC5, also known as survivin) is upregulated in haematological malignancies and solid tumours, and was also shown to be overexpressed in CML-BP in comparison to CML-CP (Hernandez-Boluda et al, 2005). Higher expression of BIRC5 was also linked to higher Sokal score and was positively correlated with P-glycoprotein expression in late CML-CP (Reis et al, 2011). Recently, the disruption of BIRC3, leading to truncated protein expression has been implicated in resistance to fludarabine in chronic lymphocytic leukaemia (CLL) (Rossi et al, 2012). To elucidate the potential role of the BIRC family of genes in CML we investigated the relative expression of all eight known BIRC family members. We analysed sequential samples of cDNA from peripheral blood obtained from CML patients at various stages of the disease. Blood samples were taken after informed consent was obtained and reverse transcription (RT)-quantitative real time polymerase chain reaction (qPCR) experiments were performed according to MIQE (Minimum Information for Publication of qPCR Experiments) guidelines (Bustin et al, 2009). Initially we looked at samples from CML-CP patients at diagnosis and after development of TKI resistance (confirmed as a loss of cytogenetic response, n = 5). Four patients developed resistance to imatinib (one had V299L mutation); the fifth was resistant to imatinib, dasatinib and nilotinib (no mutation detected) (Fig. 1A). We then analysed sequential samples from patients with CML who progressed to either accelerated phase (CMLAccPh) (n = 2) or to CML-BP (n = 4). Contrary to the general view of the role of BIRC genes in tumour progression, we found a significant decrease in BIRC3 and BIRC8 expression in CML-CP after development of TKI resistance (Fig. 1A) and also in progression to CML-AccPh/BP (Fig. 1B). We also observed a marked increase in BIRC5 expression after progression to CML-AccPh/BP (as previously shown by Hernandez-Boluda et al, 2005) (Fig. 1B) but not in CML-CP samples obtained after development of TKI resistance (Fig. 1A). Expression of other BIRC genes, namely: NAIP (BIRC1), BIRC2, XIAP (BIRC4), BIRC6 and BIRC7 was comparable in all the studied stages of the disease (data not shown). To confirm these results analysis of a larger group of patients was performed. Samples were obtained from patients in either CML-CP at diagnosis prior to any treatment (n = 15) or in CML-BP (n = 11) (Fig. 2). To compare BIRC family genes expression in CML with that of normal haematopoietic cells, cDNA from healthy blood donors was also used (n = 10). In accordance with paired samples analysis, we observed downregulation of BIRC3 and BIRC8 expression in CML-BP, while BIRC5 was upregulated in CML-BP patients (as compared to CML-CP and healthy blood donors) (Fig. 2). There was no significant difference in the relative expression of other BIRC family members (data not shown). correspondence

Immune thrombocytopenia in patients with chronic lymphocytic leukemia treated with cladribine-based regiments or chlorambucil – follow-up of PALG-CLL randomized trials

The relationship between treatments of chronic lymphocytic leukemia (CLL) with cladribine (2‐CdA) or chlorambucil and immune thrombocytopenia (IT) has not been yet determined.