The significance of early warning in chronic myeloid leukemia

We have read with great interest the manuscript by Eskazan and colleagues entitled \u201cCritical appraisal of European LeukemiaNet (ELN) 2013 recommendations for the management of chronic myeloid leukemia: is it early for a warning?\u201d. After a revision of the relatively limited literature, the Authors conclude that there are still no solid data to suggest a switch of therapy in patients with warning signs and that long-term survival remains a highly significant endpoint in CML patients. While we generally agree with these thoughts, we would like to stress a couple of additional points on the issue of ELN 2013 \u2013 defined \u201cwarning\u201d. The ELN recommendations defines warning as less than partial cytogenetic response (PCyR) and/or BCR-ABL1 >10% (according to the International Scale \u2013 IS) at 3 months, less than complete cytogenetic response (CCyR) and/or BCR-ABL1 >1%IS at 6 months, and BCR-ABL1 >0.1% IS, i.e. no major molecular response (MMR) at 12 months. So, at the first two time-points, conventionally considered as \u201cearly\u201d, both cytogenetic and molecular status define response, while at 12 months only BCR-ABL1 level >0.1 to 1%IS identifies warning patients, as anything less than CCyR is regarded as a failure. Our group analyzed the outcome of 216 CML patients treated with front-line standard dose (400 mg/day) imatinib with discordant cytogenetic and molecular responses at 3 and 6 months. Patients with even a single warning sign at 3 months (i.e. no PCyR or BCR-ABL1 >10%IS) had a significantly lower chance to obtain a subsequent CCyR (37% compared to 85% in patients with concordant optimal cytogenetic and molecular responses) and worse failure-free survival (FFS) (39% vs 81% at 48 months). Similarly, a warning sign at 6 months identified patients less prone to attain a MMR at 12 months (17% vs 82% in concordantly optimal patients) and with worse FFS (62% vs 88%). In our experience, most discordant patients had a \u201cmolecular warning\u201d, as 15/17 discordant at 3 months were in PCyR or better but with BCR-ABL1 transcript >10%IS and at 6 months 20/25 discordant were in CCyR with BCR-ABL1 >1%IS. This finding is an indirect confirmation of the importance of a BCR-ABL1 transcript level <10%IS at 3 months (now defined \u201cearly molecular response\u201d, EMR) as a positive predictor of long-term outcome, as reported by different studies. Despite EMR is gaining ground as a factor for an early switch of therapy, as suggested by NCCN guidelines, some reports indicate, in line with ELN recommendations, to consider also the 6-month cytogenetic or molecular status to assess a two-point evaluation of response to TKI therapy. The MDACC group analyzed the outcome of 453 CML patients treated with different TKIs, finding that 19 out of 44 patients (43%) not achieving major (i.e. optimal) cytogenetic response (MCyR) at 3 months obtained this response at 6 months and had an outcome comparable to patients achieving an earlier MCyR [8]. A Canadian study reviewed 320 patients receiving imatinib therapy with 3 and 6 month BCR-ABL1 transcript levels available, reporting that patients not achieving an EMR at 3 months but with BCR-ABL1 transcript <1% at 6 months (n=18) had similar FFS, progression-free survival (PFS) and overall survival (OS) compared to patients in EMR (n=184). Taken together, these data suggest that cytogenetic and molecular response at 6 months can identify a subgroup with favorable outcome among patients \u201cwarning\u201d at 3 months. However, considering patients with cytogenetic and/or molecular warning at 3 months in our series (n=41), only 2 had a subsequent optimal cytogenetic and molecular response at 6 months (unpublished). Moreover, we found that the rates of warning responses at 3 and 6 months were higher in cases with b2a2 BCR-ABL1 transcript type compared to those with b3a2 variant (32% vs 24% at 3 months and 31% vs 12% at 6 months, respectively). If there is still debate on the practical significance of a warning at 3 or 6 months, even less consensus and significantly less data are about the meaning of a late (i.e. at 12 months) warning. Starting from their database of 483 patients treated with four different TKI strategies, colleagues at MDACC found no benefit, in term of survival, in patients achieving MMR while in CCyR, even if their landmark analysis was performed at 18 and 24 months, and not at the 12-months timepoint. A landmark analysis of PFS and OS on the bases of molecular response at 12 months of imatinib performed in 128 patients from our database did not find any difference between patients in MMR or not (personal data, unpublished). Concordantly, a Spanish group showed that, in 198 patients treated with standard-dose imatinib and in CCyR without MMR at 12 months, a switch to a second-generation TKI was associated with a higher probability of subsequently major and deep molecular response, but no advantage in terms of PFS and OS and higher rates of discontinuation for adverse events, compared to patients continuing imatinib. Hopefully, more information on the therapeutic approach to \u201cwarning\u201d patients will come from an upcoming study of the GIMEMA Working Party on CML study aimed to evaluate efficacy of nilotinib frontline versus imatinib followed by switch to nilotinib in the case of absence of ELN-defined optimal response at 3, 6 or 12 months

Management of Chronic Myeloid Leukemia in Advanced Phase

Management of chronic myeloid leukemia (CML) in advanced phases remains a challenge also in the era of tyrosine kinase inhibitors (TKIs) treatment. Cytogenetic clonal evolution and development of resistant mutations represent crucial events that limit the benefit of subsequent therapies in these patients. CML is diagnosed in accelerated (AP) or blast phase (BP) in &lt;5% of patients, and the availability of effective treatments for chronic phase (CP) has dramatically reduced progressions on therapy. Due to smaller number of patients, few randomized studies are available in this setting and evidences are limited. Nevertheless, three main scenarios may be drawn: (a) patients diagnosed in AP are at higher risk of failure as compared to CP patients, but if they achieve optimal responses with frontline TKI treatment their outcome may be similarly favorable; (b) patients diagnosed in BP may be treated with TKI alone or with TKI together with conventional chemotherapy regimens, and subsequent transplant decisions should rely on kinetics of response and individual transplant risk; (c) patients in CP progressing under TKI treatment represent the most challenging population and they should be treated with alternative TKI according to the mutational profile, optional chemotherapy in BP patients, and transplant should be considered in suitable cases after return to second CP. Due to lack of validated and reliable markers to predict blast crisis and the still unsatisfactory results of treatments in this setting, prevention of progression by careful selection of frontline treatment in CP and early treatment intensification in non-optimal responders remains the main goal. Personalized evaluation of response kinetics could help in identifying patients at risk for progression

Current Strategies and Future Directions to Achieve Deep Molecular Response and Treatment-Free Remission in Chronic Myeloid Leukemia

The treatment of chronic myeloid leukemia (CML) has been radically changed by the approval of tyrosine kinase inhibitors (TKIs), which target BCR-ABL1 kinase activity. CML is now managed as a chronic disease requiring long-term treatment and close molecular monitoring. It has been shown that in a substantial number of patients who have achieved a stable deep molecular response (DMR), TKI treatment can be safely discontinued without loss of response. Therefore, treatment-free remission (TFR), through the achievement of a DMR, is increasingly regarded as a feasible treatment goal in many CML patients. However, only nilotinib has approval in this setting and a number of controversial aspects remain regarding treatment choices and timings, predictive factors, patient communication, and optimal strategies to achieve successful TFR. This narrative review aims to provide a comprehensive overview on how to optimize the path to DMR and TFR in patients with CML, and discusses recent data and future directions

Macrophages may promote cancer growth via a GM-CSF/HB-EGF paracrine loop that is enhanced by CXCL12

<p>Abstract</p> <p>Background</p> <p>Increased numbers of tumour-associated macrophages correlate with shortened survival in some cancers. The molecular bases of this correlation are not thoroughly understood. Events triggered by CXCL12 may play a part, as CXCL12 drives the migration of both CXCR4-positive cancer cells and macrophages and may promote a molecular crosstalk between them.</p> <p>Results</p> <p>Samples of HER1-positive colon cancer metastases in liver, a tissue with high expression of CXCL12, were analysed by immunohistochemistry. In all of the patient biopsies, CD68-positive tumour-associated macrophages presented a mixed CXCL10 (M1)/CD163 (M2) pattern, expressed CXCR4, GM-CSF and HB-EGF, and some stained positive for CXCL12. Cancer cells stained positive for CXCR4, CXCL12, HER1, HER4 and GM-CSF. Regulatory interactions among these proteins were validated <it>via </it>experiments <it>in vitro </it>involving crosstalk between human mononuclear phagocytes and the cell lines DLD-1 (human colon adenocarcinoma) and HeLa (human cervical carcinoma), which express the above-mentioned ligand/receptor repertoire. CXCL12 induced mononuclear phagocytes to release HB-EGF, which activated HER1 and triggered anti-apoptotic and proliferative signals in cancer cells. The cancer cells then proliferated and released GM-CSF, which in turn activated mononuclear phagocytes and induced them to release more HB-EGF. Blockade of GM-CSF with neutralising antibodies or siRNA suppressed this loop.</p> <p>Conclusions</p> <p>CXCL12-driven stimulation of cancer cells and macrophages may elicit and reinforce a GM-CSF/HB-EGF paracrine loop, whereby macrophages contribute to cancer survival and expansion. The involvement of mixed M1/M2 GM-CSF-stimulated macrophages in a tumour-promoting loop may challenge the paradigm of tumour-favouring macrophages as polarized M2 mononuclear phagocytes.</p

Successful Preservation of Native BCR::ABL1 in Chronic Myeloid Leukemia Primary Leukocytes Reveals a Reduced Kinase Activity

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the acquisition of t(9;22) generating the fusion tyrosine kinase BCR::ABL1. However, despite the crucial role of this protein in the dysregulation of numerous signal transduction pathways, a direct measure of BCR::ABL1 kinase activity in chronic phase (CP) CML was never accomplished due to intense degradative activity present in mature leukocytes. Therefore, we developed a procedure suitable to preserve BCR::ABL1 protein under non-denaturing, neutral pH conditions in primary, chronic phase (CP)-CML samples. As a result, specific kinase activity was detected utilizing a biotinylated peptide substrate highly selective for c-ABL1. Furthermore, through this approach, BCR::ABL1 kinase activity was barely detectable in CP-CML compared to Ph+ acute lymphoblastic leukemia primary samples, where kinase activity is comparable to those measured in Ph+ cell lines. These in vitro findings provide the first direct measure of BCR::ABL1 kinase activity in primary CP-CML and reveal the presence of a still uncharacterized inhibitory mechanism that maintains BCR::ABL1 in a low activity state in CP-CML despite its overexpression

Regulative Loop between \u3b2-catenin and Protein Tyrosine Receptor Type \u3b3 in Chronic Myeloid Leukemia

Protein tyrosine phosphatase receptor type \u3b3 (PTPRG) is a tumor suppressor gene, down-regulated in Chronic Myeloid Leukemia (CML) cells by the hypermethylation of its promoter region. \u3b2-catenin (CTNNB1) is a critical regulator of Leukemic Stem Cells (LSC) maintenance and CML proliferation. This study aims to demonstrate the antagonistic regulation between \u3b2-catenin and PTPRG in CML cells. The specific inhibition of PTPRG increases the activation state of BCR-ABL1 and modulates the expression of the BCR-ABL1- downstream gene \u3b2-Catenin. PTPRG was found to be capable of dephosphorylating \u3b2-catenin, eventually causing its cytosolic destabilization and degradation in cells expressing PTPRG. Furthermore, we demonstrated that the increased expression of \u3b2-catenin in PTPRG-negative CML cell lines correlates with DNA (cytosine-5)-methyl transferase 1 (DNMT1) over-expression, which is responsible for PTPRG promoter hypermethylation, while its inhibition or down-regulation correlates with PTPRG re-expression. We finally confirmed the role of PTPRG in regulating BCR-ABL1 and \u3b2-catenin phosphorylation in primary human CML samples. We describe here, for the first time, the existence of a regulative loop occurring between PTPRG and \u3b2-catenin, whose reciprocal imbalance affects the proliferation kinetics of CML cells

Pro-apoptotic activity of α-bisabolol in preclinical models of primary human acute leukemia cells

<p>Abstract</p> <p>Background</p> <p>We previously demonstrated that the plant-derived agent α-bisabolol enters cells <it>via </it>lipid rafts, binds to the pro-apoptotic Bcl-2 family protein BID, and may induce apoptosis. Here we studied the activity of α-bisabolol in acute leukemia cells.</p> <p>Methods</p> <p>We tested <it>ex vivo </it>blasts from 42 acute leukemias (14 Philadelphia-negative and 14 Philadelphia-positive B acute lymphoid leukemias, Ph^-/Ph⁺B-ALL; 14 acute myeloid leukemias, AML) for their sensitivity to α-bisabolol in 24-hour dose-response assays. Concentrations and time were chosen based on CD34⁺, CD33⁺my and normal peripheral blood cell sensitivity to increasing α-bisabolol concentrations for up to 120 hours.</p> <p>Results</p> <p>A clustering analysis of the sensitivity over 24 hours identified three clusters. Cluster 1 (14 ± 5 μM α-bisabolol IC₅₀) included mainly Ph^-B-ALL cells. AML cells were split into cluster 2 and 3 (45 ± 7 and 65 ± 5 μM IC₅₀). Ph⁺B-ALL cells were scattered, but mainly grouped into cluster 2. All leukemias, including 3 imatinib-resistant cases, were eventually responsive, but a subset of B-ALL cells was fairly sensitive to low α-bisabolol concentrations. α-bisabolol acted as a pro-apoptotic agent <it>via </it>a direct damage to mitochondrial integrity, which was responsible for the decrease in NADH-supported state 3 respiration and the disruption of the mitochondrial membrane potential.</p> <p>Conclusion</p> <p>Our study provides the first evidence that α-bisabolol is a pro-apoptotic agent for primary human acute leukemia cells.</p

CXCL12 and [N33A]CXCL12 in 5637 and HeLa Cells: Regulating HER1 Phosphorylation via Calmodulin/Calcineurin

In the human neoplastic cell lines 5637 and HeLa, recombinant CXCL12 elicited, as expected, downstream signals via both G-protein-dependent and \u3b2-arrestin-dependent pathways responsible for inducing a rapid and a late wave, respectively, of ERK1/2 phosphorylation. In contrast, the structural variant [N33A]CXCL12 triggered no \u3b2-arrestin-dependent phosphorylation of ERK1/2, and signaled via G protein-dependent pathways alone. Both CXCL12 and [N33A]CXCL12, however, generated signals that transinhibited HER1 phosphorylation via intracellular pathways. 1) Prestimulation of CXCR4/HER1-positive 5637 or HeLa cells with CXCL12 modified the HB-EGF-dependent activation of HER1 by delaying the peak phosphorylation of tyrosine 1068 or 1173. 2) Prestimulation with the synthetic variant [N33A]CXCL12, while preserving CXCR4-related chemotaxis and CXCR4 internalization, abolished HER1 phosphorylation. 3) In cells knockdown of \u3b2-arrestin 2, CXCL12 induced a full inhibition of HER1 like [N33A]CXCL12 in non-silenced cells. 4) HER1 phosphorylation was restored as usual by inhibiting PCK, calmodulin or calcineurin, whereas the inhibition of CaMKII had no discernable effect. We conclude that both recombinant CXCL12 and its structural variant [N33A]CXCL12 may transinhibit HER1 via G-proteins/calmodulin/calcineurin, but [N33A]CXCL12 does not activate \u3b2-arrestin-dependent ERK1/2 phosphorylation and retains a stronger inhibitory effect. Therefore, we demonstrated that CXCL12 may influence the magnitude and the persistence of signaling downstream of HER1 in turn involved in the proliferative potential of numerous epithelial cancer. In addition, we recognized that [N33A]CXCL12 activates preferentially G-protein-dependent pathways and is an inhibitor of HER1

Multiple large osteolytic lesions in a patient with systemic mastocytosis: a challenging diagnosis

Patients with advanced variants of Systemic Mastocytosis may develop destructive bone lesions when massive mast cell (MC) infiltrates are present. Finding of large osteolyses in indolent systemic mastocytosis, typically characterized by low MC burden, should prompt investigations for an alternative explanation

Benefit-risk profile of cytoreductive drugs along with antiplatelet and antithrombotic therapy after transient ischemic attack or ischemic stroke in myeloproliferative neoplasms

We analyzed 597 patients with myeloproliferative neoplasms (MPN) who presented transient ischemic attacks (TIA, n = 270) or ischemic stroke (IS, n = 327). Treatment included aspirin, oral anticoagulants, and cytoreductive drugs. The composite incidence of recurrent TIA and IS, acute myocardial infarction (AMI), and cardiovascular (CV) death was 4.21 and 19.2%, respectively at one and five years after the index event, an estimate unexpectedly lower than reported in the general population. Patients tended to replicate the first clinical manifestation (hazard ratio, HR: 2.41 and 4.41 for recurrent TIA and IS, respectively); additional factors for recurrent TIA were previous TIA (HR: 3.40) and microvascular disturbances (HR: 2.30); for recurrent IS arterial hypertension (HR: 4.24) and IS occurrence after MPN diagnosis (HR: 4.47). CV mortality was predicted by age over 60 years (HR: 3.98), an index IS (HR: 3.61), and the occurrence of index events after MPN diagnosis (HR: 2.62). Cytoreductive therapy was a strong protective factor (HR: 0.24). The rate of major bleeding was similar to the general population (0.90 per 100 patient-years). In conclusion, the long-term clinical outcome after TIA and IS in MPN appears even more favorable than in the general population, suggesting an advantageous benefit-risk profile of antithrombotic and cytoreductive treatment