-deletions occur recurrently in acute lymphoblastic leukemia, especially in the
-rearranged subtype. The
-deletion was shown to positively impact prognosis of patients with
-deletion and its presence ...precludes assignment into
group, a novel high-risk category on AIEOP-BFM ALL trials. We analyzed the impact of different methods on
-deletion detection rate, evaluated
-deletion as a potential marker for
-rearranged leukemia, studied its associations with molecular and clinical characteristics within this leukemia subtype, and analyzed its clonality. Using single-nucleotide-polymorphism array, genomic polymerase chain reaction (PCR) and amplicon-sequencing we found
-deletion in 34% (16 of 47), 66% (33 of 50) and 78% (39 of 50) of
-rearranged leukemia, respectively. False negativity of
-deletion by single-nucleotide-polymorphism array caused
misclassification in 5 patients. No
-deletion was found outside the
-rearranged cases. Within
-rearranged leukemia, the
-deletion was associated with higher total number of copy-number aberrations, and, importantly, the
-deletion positivity by PCR was associated with better outcome 5-year event-free survival (EFS),
-deletion-positive 93%
-deletion-negative 68%,
=0.022; 5-year overall survival (OS),
-deletion-positive 97%
-deletion-negative 75%,
=0.029. Ultra-deep amplicon-sequencing revealed distinct co-existing
-deletions in 22 of 24 patients. In conclusion, our data demonstrate inadequate sensitivity of single-nucleotide-polymorphism array for
-deletion detection, unacceptable for proper
classification. Even using more sensitive methods (PCR/amplicon-sequencing) for its detection,
-deletion is absent in 22-34% of
-rearranged leukemia and does not represent an adequately sensitive marker of this leukemia subtype. Importantly, the
-deletion potentially stratifies the
-rearranged leukemia into biologically/clinically distinct subsets. Frequent polyclonal pattern of
-deletions shows that late origin of this lesion is more common than has been previously described.
Intragenic ERG deletions occur in 3-5% of B-cell precursor acute lymphoblastic leukemia, specifically in B-other subtype lacking the classifying genetic lesions. They represent the only genetic ...lesion described so far present in the majority of cases clustering into a subgroup of B-other subtype characterized by a unique gene expression profile, probably sharing a common, however, not yet fully described, biological background. We aimed to elucidate whether ERG deletions could drive the specific biology of this ERG-related leukemia subgroup through expression of aberrant or decreased expression of wild type ERG isoforms. We showed that leukemic cells with endogenous ERG deletion express an aberrant transcript translated into two proteins in transfected cell lines and that one of these proteins colocalizes with wild type ERG. However, we did not confirm expression of the proteins in acute lymphoblastic leukemia cases with endogenous ERG deletion. ERG deletions resulted in significantly lower expression of wild type ERG transcripts compared to B-other cases without ERG deletion. However, cases with subclonal ERG deletion, clustering to the same ERG deletion associated subgroup, presented similar levels of wild type ERG as cases without ERG deletion. In conclusion, our data suggest that neither the expression of aberrant proteins from internally deleted allele nor the reduced expression of wild type ERG seem to provide a plausible explanation of the specific biology of ERG -related leukemia subgroup.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recent studies have demonstrated that several chelators possess marked potential as potent anti-neoplastic drugs and as agents that can ameliorate some of the adverse effects associated with standard ...chemotherapy. Anti-cancer treatment employs combinations of several drugs that have different mechanisms of action. However, data regarding the potential interactions between iron chelators and established chemotherapeutics are lacking. Using estrogen receptor-positive MCF-7 breast cancer cells, we explored the combined anti-proliferative potential of four iron chelators, namely: desferrioxamine (DFO), salicylaldehyde isonicotinoyl hydrazone (SIH), (E)-N'-1-(2-hydroxy-5-nitrophenyl)ethyliden isonicotinoyl hydrazone (NHAPI), and di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), plus six selected anti-neoplastic drugs. These six agents are used for breast cancer treatment and include: paclitaxel, 5-fluorouracil, doxorubicin, methotrexate, tamoxifen and 4-hydroperoxycyclophosphamide (an active metabolite of cyclophosphamide). Our quantitative chelator-drug analyses were designed according to the Chou-Talalay method for drug combination assessment. All combinations of these agents yielded concentration-dependent, anti-proliferative effects. The hydrophilic siderophore, DFO, imposed antagonism when used in combination with all six anti-tumor agents and this antagonistic effect increased with increasing dose. Conversely, synergistic interactions were observed with combinations of the lipophilic chelators, NHAPI or Dp44mT, with doxorubicin and also the combinations of SIH, NHAPI or Dp44mT with tamoxifen. The combination of Dp44mT with anti-neoplastic agents was further enhanced following formation of its redox-active iron and especially copper complexes. The most potent combinations of Dp44mT and NHAPI with tamoxifen were confirmed as synergistic using another estrogen receptor-expressing breast cancer cell line, T47D, but not estrogen receptor-negative MDA-MB-231 cells. Furthermore, the synergy of NHAPI and tamoxifen was confirmed using MCF-7 cells by electrical impedance data, a mitochondrial inner membrane potential assay and cell cycle analyses. This is the first systematic investigation to quantitatively assess interactions between Fe chelators and standard chemotherapies using breast cancer cells. These studies are vital for their future clinical development.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We used the genomic breakpoint between BCR and ABL1 genes for the DNA-based monitoring of minimal residual disease (MRD) in 48 patients with childhood acute lymphoblastic leukemia (ALL). Comparing ...the results with standard MRD monitoring based on immunoglobulin/T-cell receptor (Ig/TCR) gene rearrangements and with quantification of IKZF1 deletion, we observed very good correlation for the methods in a majority of patients; however, >20% of children (25% 8/32 with minor and 12.5% 1/8 with major-BCR-ABL1 variants in the consecutive cohorts) had significantly (>1 log) higher levels of BCR-ABL1 fusion than Ig/TCR rearrangements and/or IKZF1 deletion. We performed cell sorting of the diagnostic material and assessed the frequency of BCR-ABL1-positive cells in various hematopoietic subpopulations; 12% to 83% of non–ALL B lymphocytes, T cells, and/or myeloid cells harbored the BCR-ABL1 fusion in patients with discrepant MRD results. The multilineage involvement of the BCR-ABL1-positive clone demonstrates that in some patients diagnosed with BCR-ABL1-positive ALL, a multipotent hematopoietic progenitor is affected by the BCR-ABL1 fusion. These patients have BCR-ABL1-positive clonal hematopoiesis resembling a chronic myeloid leukemia (CML)–like disease manifesting in “lymphoid blast crisis.” The biological heterogeneity of BCR-ABL1-positive ALL may impact the patient outcomes and optimal treatment (early stem cell transplantation vs long-term administration of tyrosine-kinase inhibitors) as well as on MRD testing. Therefore, we recommend further investigations on CML-like BCR-ABL1-positive ALL.
•Combination of Ig/TCR and BCR-ABL1 genomic approach for MRD monitoring in childhood ALL reveals patients with CML-like disease.•Monitoring ALL using BCR-ABL1 genomic breakpoint is feasible and enables the most specific and sensitive MRD quantification.
Transient myeloproliferative disorder (TMD) is a hematopoietic disease, characterized by a clonal proliferation of immature megakaryoblasts in the neonatal period occurring in approximately 10% of ...newborns with Down syndrome (DS). Rarely, TMD occurs in non-DS newborns but then it is associated with somatic trisomy 21 (tri21). Tri21 together with in-utero gained mutations in the GATA1 gene encoding a myeloid transcription factor are thus considered essential in TMD.
Recently, we have identified a TMD with a typical manifestation and course in a newborn without DS/somatic tri21, which admits that tri21 is dispensable for TMD development. To elucidate the alternative TMD pathogenesis, we performed comprehensive genomic/transcriptomic profiling of this TMD case. We utilized high-density SNP array and whole exome and transcriptome sequencing (WES/RNAseq) to detect copy number changes, mutations and fusion genes. We did not find any aberrations on chromosome 21 and any fusion genes. Two focal intronic losses, likely representing benign germline variants, were found on chromosome X. In addition to 6 missense mutations affecting genes without established roles in hematopoietic disorders, we found in-frame deletions in the GATA1 and JAK1 genes. Both mutations are novel. The GATA1 D65_C228del mutation is predicted to result in an internally truncated protein - GATA1aber. Unlike GATA1s (resulting from GATA1 mutations in DS-TMD) which lacks the transactivation domain (TAD) but retains both Zinc fingers (ZF), GATA1aber lacks part of TAD and the N-terminal ZF. Nevertheless, we hypothesize that GATA1aber substitutes the pathogenetic role of GATA1s. The JAK1 gene encodes a non-receptor tyrosine-kinase engaged in the JAK/STAT signaling pathway. The identified mutation results in the loss of phenylalanine 636 (F636del), which is located in the pseudokinase domain and belongs to a conserved amino acid triad (F636-F575-V658) that is believed to mediate a structural switch controlling the JAK1 catalytic activity (Toms, Nat Struct Mol Biol, 2013). JAK1 mutations are implicated in various hematological malignancies including acute megakaryocytic leukemia, and we hypothesize that JAK1 F636del co-operates with GATA1aber on TMD pathogenesis via deregulation of cytokine/growth factor signaling.
We cloned the coding sequences of GATA1aber and JAK1 F636del and transfected them into a model cell line in which we confirmed the expression of both in-silico predicted proteins. Their subcellular trafficking was analogous to that of their wild type counterparts; GATA1aber was found in the nucleus and JAK1 F636del in both the nucleus and cytoplasm. Next, we assessed the kinase activity of JAK1 F636del. To distinguish auto- from trans-phosphorylation, we utilized the JAK1 F636del construct harboring an inactivating mutation of an ATP-binding site (K908G). The JAK1 F636del (but not JAK1 F636del + K908G) was autophosphorylated on Y1034/Y1035 and induced STATs phosphorylation both under steady-state conditions and following non-specific stimulation with PMA. However, at all studied time points all phosphorylation levels were lower compared to wild-type JAK1. Moreover, unlike constitutively active JAK1 V658I, JAK1 F636del did not confer IL3-independent growth to the murine B-cell progenitor cell line BAF3. Interestingly, the transforming potential of double-mutated JAK1 (JAK1 V658I + F636del) was enforced compared to JAK1 V658I. These data show that F636del does not lead to constitutive activation, but in the same time it is not functionally neutral. As the impact of F636del on JAK1 function may vary depending on upstream signaling, we are currently assessing JAK1 F636 kinase activity/transforming potential in BAF3 cells stably expressing the IL6 receptor, which (unlike the IL3 receptor) directly activates JAK1 upon ligand binding. In the future, we plan to study the impact of JAK1 F636del on GATA1s induced deregulation of erythroid/megakaryocytic lineage development and to demonstrate “GATA1s-like” function of GATA1aber.
To conclude, we identified two novel mutations affecting GATA1 and JAK1 as likely drivers in an alternative tri21-independent TMD pathogenesis. As the pathogenetic role of tri21 has been poorly understood so far, we believe that by clarifying an alternative mechanism of TMD development, we could improve our understanding of this intriguing disease in general.
Support: GAUK 86218
No relevant conflicts of interest to declare.
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