Cryptic BCR-ABL fusion gene as variant rearrangement in chronic myeloid leukemia: Molecular cytogenetic characterization and influence on TKIs therapy

At diagnosis, about 5% of Chronic Myeloid Leukemia (CML) patients lacks Philadelphia chromosome (Ph), despite the presence of the BCR/ABL rearrangement. Two mechanisms have been proposed about the occurrence of this rearrangement: the first one is a cryptic insertion between chromosomes 9 and 22; the second one involves two sequential translocations: a classic t(9;22) followed by a reverse translocation, which reconstitutes the normal morphology of the partner chromosomes. Out of 398 newly diagnosed CML patients, we selected 12 Ph-negative cases. Six Ph-negative patients treated with tyrosine kinase inhibitors (TKIs) were characterized, in order to study the mechanisms leading to the rearrangement and the eventual correlation with prognosis in treatment with TKIs. FISH analysis revealed cryptic insertion in 5 patients and classic translocation in the last one. In more detail, we observed 4 different patterns of rearrangement, suggesting high genetic heterogeneity of these patients. In our cases, the BCR/ABL rearrangement mapped more frequently on 9q34 region than on 22q11 region, in contrast to previous reports. Four patients, with low Sokal risk, achieved Complete Cytogenetic Response and/or Major Molecular Response after TKIs therapy. Therapy resistance was observed in one patient with duplication of BCR/ABL rearrangement and in another one with high risk. Even if the number patient is inevitably low, we can confirm that the rare Ph-negative CML patients do not constitute a "warning" category, meanwhile the presence of further cytogenetic abnormalities remains an adverse prognostic factor even in TKI era

Cytogenetic and molecular characterization in newly diagnosed chronic-phase chronic myeloid leukemia patients treated with tirosine kinase inhibitors: prognostic role.

L’applicazione della citogenetica convenzionale e molecolare può identificare: Ph-negatività, traslocazioni t(9;22) varianti e alterazioni citogenetiche addizionali (ACA) al cromsoma Ph in pazienti con LMC alla diagnosi. Prima dell’introduzione della terapia con Imatinib, esse mostravano un impatto prognostico negativo o non chiaro. Nel nostro studio, 6 casi di LMC Ph- erano trattati con Imatinib. La FISH identificava 4 casi con riarrangiamento BCR/ABL sul der(9q), 1 sul der(22q) e 1 su entrambi i derivativi. Quattro pazienti (66,7%) raggiungevano la RCgC, 2 fallivano il trattamento e 1 sottoposto a TMO. A causa dello scarso numero di casi, non era possibile nessuna correlazione con la prognosi. Nell’ambito di studi prospettici multicentrici del GIMEMA-WP, abbiamo valutato: traslocazioni varianti e ACA. Dei 559 pazienti arruolati, 30(5%) mostravano traslocazioni varianti, 24 valutabili in FISH: 18(75%) mostravano meccanismo 1-step, 4(16,7%) meccanismo 2-step e 2(8,3%) meccanismo complesso. Abbiamo confermato che le varianti non influenzano la risposta e la sopravvivenza dei pazienti trattati con Imatinib. Dei 378 pazienti valutabili alla diagnosi con citogenetica convenzionale, 21(5,6%) mostravano ACA: 9(43%) avevano la perdita del cromosoma Y, 3(14%) trisomia 8, 2(10%) trisomia 19, 6(28%) altre singole anomalie e 1 cariotipo complesso. La presenza di ACA influenzava la risposta: le RCgC e RMolM erano significativamente più basse rispetto al gruppo senza ACA e le curve di sopravvivenza EFS e FFS non erano significativamente diverse. Le curve di PFS e OS erano sovrapponibili nei due gruppi, per il basso numero di eventi avversi oppure perché alcuni raggiungevano la risposta con TKI di seconda generazione. Le anomalie “major route” mostravano decorso clinico peggiore, ma non è stato possibile determinare l’impatto prognostico in relazione al tipo di alterazione. Pertanto, le ACAs alla diagnosi rivestono un ruolo negativo nella prognosi dei pazienti trattati con Imatinib, che quindi rappresentano una categoria più a rischio per la risposta.At diagnosis in chronic myeloid leukemia (CML) patients, conventional cytogenetics and FISH analysis can identify: Ph- CML cases, variant t(9;22) translocations and additional chromosomal abnormalities (ACAs) in Ph-positive clone. Before the introduction of the Imatinib therapy, these characteristics showed a negative or not clear prognostic role. We analysed 6 Ph- cases treated with Imatinib. FISH analysis showed 4 cases with BCR/ABL rearrangement on der(9q), 1 on der(22q) and 1 on both derivatives chromosomes. Four patients (66,7%) reached CCgR, 2 failed treatment and 1 died after stem cell transplantation. Beacause of small of number of cases, we cannot stated that Ph-masked cases have a worse prognosis. Within the clinical trial of GIMEMA WP, we have analysed variant t(9;22) translocations and ACAs. Five-hundred-fifty-nine patients were enrolled; 30(5%) had variant translocations of which 24 could be evaluated by FISH: 18(75%) showed 1-step mechanism, 4(16,7%) 2-step mechanism and 2(8,3%) complex mechanism. We observed that variant translocations didn’t influence the responses and the outcome in patients treated with Imatinib. At diagnosis, 378 patients were evaluable by cytogenetics. Twenty-one (5,6%) showed ACAs: 9 (43%) had loss of Y chromosome, 3(14%) trisomy 8, 2(10%) trisomy 19, 6(28%) other isolated abnormalities and 1(15%) complex karyotype. We reported that the presence of ACAs influenced the responses: the CCgR and MMolR were significantly lower compared with those of the group without ACAs, but EFS and FFS were not significantly different. PFS and OS curves were overlapped, because of the low number of negative events or because some patients reached response with second generation TKI treatment. Cases with “major route” abnormalities showed worse outcome, but we cannot established the impact related to the kind of abnormality. However, ACAs at diagnosis had a negative role on the response to the therapy in CML patients, which constitute a “warning” category for Imatinib treatment

Pancreatic enzyme elevation in chronic myeloid leukemia patients treated with nilotinib after imatinib failure

An increase in the serum concentration of pancreatic enzymes (amylase and lipase) was reported in a proportion of imatinib-resistant and/or intolerant Philadelphia-positive chronic myeloid leukemia patients treated with nilotinib. This report describes chronic myeloid leukemia patients who developed serum lipase/amylase elevation during treatment with nilotinib

FGFR1 and KAT6A rearrangements in patients with hematological malignancies and chromosome 8p11 abnormalities: biological and clinical features

No abstract available

Circulating calreticulin is increased in myelofibrosis: Correlation with interleukin-6 plasma levels, bone marrow fibrosis, and splenomegaly

Myelofibrosis (MF) is a clonal neoplasia of the hemopoietic stem/progenitor cells associated with genetic mutations in the Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR) genes. MF is also characterized by a state of chronic inflammation. Calreticulin (CRT), as a multifunctional protein, is involved in a spectrum of cellular processes including inflammation, autoimmunity, and cancer initiation/progression. Based on this background, we hypothesised that in MF circulating CRT might reflect the inflammatory process. In the present study we show that circulating CRT is increased in MF patients compared to healthy controls. Also, in MF, CRT levels highly correlate with bone marrow fibrosis, splenomegaly, and Interleukin-6 (IL-6) plasma levels. In turn, higher IL-6 levels also correlated with disease severity in terms of increased spleen size, bone marrow fibrosis, number of circulating CD34+ cells, and lower hemoglobin values. These results demonstrate that the circulating CRT takes part in the inflammatory network of MF and correlates with aggressiveness of the disease

Circulating Calreticulin Is Increased in Myelofibrosis: Correlation with Interleukin-6 Plasma Levels, Bone Marrow Fibrosis, and Splenomegaly

Myelofibrosis (MF) is a clonal neoplasia of the hemopoietic stem/progenitor cells associated with genetic mutations in the Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR) genes. MF is also characterized by a state of chronic inflammation. Calreticulin (CRT), as a multifunctional protein, is involved in a spectrum of cellular processes including inflammation, autoimmunity, and cancer initiation/progression. Based on this background, we hypothesised that in MF circulating CRT might reflect the inflammatory process. In the present study we show that circulating CRT is increased in MF patients compared to healthy controls. Also, in MF, CRT levels highly correlate with bone marrow fibrosis, splenomegaly, and Interleukin-6 (IL-6) plasma levels. In turn, higher IL-6 levels also correlated with disease severity in terms of increased spleen size, bone marrow fibrosis, number of circulating CD34+ cells, and lower hemoglobin values. These results demonstrate that the circulating CRT takes part in the inflammatory network of MF and correlates with aggressiveness of the disease

BCR-ABL1-Associated Reduction of Beta Catenin Antagonist Chibby1 in Chronic Myeloid Leukemia

<div><p>Beta Catenin signaling is critical for the self-renewal of leukemic stem cells in chronic myeloid leukemia. It is driven by multiple events, enhancing beta catenin stability and promoting its transcriptional co-activating function. We investigated the impact of BCR-ABL1 on Chibby1, a beta catenin antagonist involved in cell differentiation and transformation. Relative proximity of the Chibby1 encoding gene (C22<i>orf</i>2) on chromosome 22q12 to the BCR breakpoint (22q11) lets assume its involvement in beta catenin activation in chronic myeloid leukemia as a consequence of deletions of distal BCR sequences encompassing one C22<i>orf</i>2 allele. Forty patients with chronic myeloid leukemia in chronic phase were analyzed for C22<i>orf</i>2 relocation and Chibby1 expression. Fluorescent in situ hybridization analyses established that the entire C22<i>orf</i>2 follows BCR regardless of chromosomes involved in the translocation. In differentiated hematopoietic progenitors (bone marrow mononuclear cell fractions) of 30/40 patients, the expression of Chibby1 protein was reduced below 50% of the reference value (peripheral blood mononuclear cell fractions of healthy persons). In such cell context, Chibby1 protein reduction is not dependent on C22<i>orf</i>2 transcriptional downmodulation; however, it is strictly dependent upon BCR-ABL1 expression because it was not observed at the moment of major molecular response under tyrosine kinase inhibitor therapy. Moreover, it was not correlated with the disease prognosis or response to therapy. Most importantly, a remarkable Chibby1 reduction was apparent in a putative BCR-ABL1+ leukemic stem cell compartment identified by a CD34+ phenotype compared to more differentiated hematopoietic progenitors. In CD34+ cells, Chibby1 reduction arises from transcriptional events and is driven by C22<i>orf</i>2 promoter hypermethylation. These results advance low Chibby1 expression associated with BCR-ABL1 as a component of beta catenin signaling in leukemic stem cells.</p></div

Cby1 reduced transcription in CD34+ cells is driven by DNA hypermethylation of C22<i>orf</i>2 promoter.

<p>PCR amplification of methylated DNA lets detect a significant increment of 5<i>orf</i>2 promoter encompassing nucleotides −85 to +120 in CD34+ cells of HP and 4 CML-CP patients compared to MCF (p<0.05 or less). The DNA input after the isolation of 5 mC-enriched DNA was used as an internal control for PCR.</p

Cby1 reduced expression in MCF of CML-CP patients is restricted to the leukemic clone.

<p>At the moment of MMR Cby1 protein (<b>A</b>) and transcript (<b>B</b>) were significantly increased in all cases (p<0.05 or less) and approached the HP levels in 3 cases (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081425#pone.0081425.s007" target="_blank">Table S3</a> for signal intensity details and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081425#pone.0081425.s002" target="_blank">Figure S2</a> for blots). The levels of Cby1 protein and transcript were evaluated in MCF from bone marrow samples of 5 CML-CP patients at diagnosis (black columns) and at the moment of MMR under TK inhibitor therapy (white columns). At this instance, a 3-log reduction in BCR-ABL1 transcripts compared to diagnosis was seen (data not shown). Cby1 expression was expressed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081425#pone-0081425-g002" target="_blank">Figure 2</a>, with HP signal intensities used as reference ( = 1).</p

<i>C22orf2</i>, the Cby1-encoding gene, follows BCR sequences and relocates to the der(9q) or third chromosome involved in variant translocations.

<p><b>A-</b> Location of BCR and Cby1 genes on chromosome 22 are shown with red marks on chromosome 22 ideogram using NCBI Map Viewer (<a href="http://www.ncbi.nlm.nih.gov/mapview/" target="_blank">http://www.ncbi.nlm.nih.gov/mapview/</a>). <b>B- panel a:</b> typical FISH pattern of BCR-ABL1 rearrangement with one fusion signal at der(22q), one green signal at the non-rearranged 22q, and one red signal at 9q in the metaphases of a CP-CML patient with t(9;22) translocation; <b>panel b:</b> one pair of Cby1 signals, with the green one labeling the promoter origin and the red one the end of the gene, was translocated to der (9q) in metaphases of a CML-CP patient with t(9;22) translocation; <b>panel c:</b> the green signal corresponding to BCR was relocated to chromosome 1 in one patient exhibiting the t(1;9;22) variant translocation (#9 of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081425#pone.0081425.s005" target="_blank">Table S1</a>); <b>panel d:</b> in the last case, Cby1 signals were relocated at the third chromosome involved in translocation. The results shown in panel b were confirmed in CML-CP patients with t(9;22) translocation included in the study, and those shown in panel d were confirmed in two CML-CP patients with variant translocations encompassing chromosomes 1 and 7 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081425#pone.0081425.s002" target="_blank">Figure S2</a>). All images were acquired using a 100× objective. FISH analyses were performed according to procedures illustrated in the Materials and Methods section. At least 20 metaphases were analyzed for the detection of BCR-ABL1 and C22orf2 signals.</p