IRF4 Gene Expression on the Trail of Molecular Response: Looking at Chronic Myeloid Leukemia from Another Perspective.

INTRODUCTION: Interferon regulatory factor 4 (IRF4) is a transcriptional factor with a key role in the modulation of inflammation and immune surveillance. The IRF4 gene is downregulated in Philadelphia-negative myeloproliferative neoplasms, and its expression is associated with prognosis and response to treatment. METHODS: We evaluated the IRF4 expression kinetics during tyrosine kinase inhibitor (TKI) treatment in a cohort of 116 chronic myeloid leukemia (CML) patients to elucidate its role in the disease course. RESULTS: A relationship between the IRF4 expression and the disease burden was observed at various disease stages. A correlation analysis between the International Scale (IS) and IRF4 values confirmed this close association. A significant increase is detected after 3 months of TKI treatment. Patients achieving an early molecular response (EMR) had higher IRF4 values at both diagnosis and after 3 months of therapy as compared to those failing the EMR target. Patients achieving treatment-free remission did not show IRF4 fluctuations during monitoring, while a decreased IRF4 expression emerged at the time of molecular relapse. CONCLUSION: Our data seem to confirm the relevance of IRF4 in the pathogenesis of CML, suggesting a pivotal role at the disease onset and a predictive value during the CML course.

JAK2 V617f in chronic myeloid leukemia: driving force or passive bystander?

OBJECTIVES: BCR-ABL1 and JAK2 V617F coexistence in myeloproliferative neoplasms has been described as concomitant or sequential events. Despite this, we present a unique case of chronic myeloid leukemia (CML) not referable to either of the known scenarios. METHODS: BCR-ABL1 molecular monitoring was performed by real-time quantitative PCR (RQ-PCR). At the time of molecular relapse, a targeted next-generation sequencing analysis with a customized panel of 26 genes commonly mutated in myeloid diseases was performed. To investigate the kinetics of the JAK2 variant and its association with the BCR-ABL1 rearrangement, RQ-PCR was performed at different time points during the patient's follow-up. RESULTS: While negative at CML diagnosis, the JAK2 mutation was first detected 9 years later (VAF: 7.2%). The mutational burden of JAK2 remained stable in multiple determinations, with minor fluctuations independent of BCR-ABL1 kinetics. At the last available time point, the patient was in deep molecular response (MR4), the JAK2 mutational burden was 7%, and no clinical-laboratory findings of Ph-MPN were detectable. DISCUSSION: In the presented case, the JAK2variantoccurring during the course of the disease seems to stay in the shadows of CML, just as a bystander. The impact of this event (that may be considered suggestive of clonal hematopoiesis of indeterminate potential) on the disease outcome, even if seemingly irrelevant, has still to be explored.

Clonal Hematopoiesis at the Crossroads of Inflammatory Bowel Diseases and Hematological Malignancies: A Biological Link?

Inflammatory bowel diseases (IBDs) are a group of chronic conditions of the gastrointestinal tract in which nationwide studies have revealed a higher risk of hematological malignancies (HMs). Clonal hematopoiesis (CH) is a premalignant condition defined by the presence of an acquired somatic mutation characterized by a variant allele frequency (VAF) of ≥2%, in a gene frequently associated with HMs. A growing body of evidence suggests a correlation between inflammation and CH; its occurrence in the context of IBD has been previously demonstrated. With the aim to assess CH possible co-occurrence in patients with an IBD associated with HMs, we performed a targeted next-generation sequencing analysis in a cohort of thirteen patients who were referred to our center with IBD associated with HMs. Eleven (85%) patients showed one or more mutations in CH-associated genes; DNMT3A was the most frequently mutated gene, followed by ASXL1 and JAK2. These results may suggest that the mechanisms at the basis of the inflammatory environment could potentially select for the growth of hematopoietic clones harboring specific mutations. In this context, CH emergence may be boosted by the proinflammatory IBD environment, thus acting as a biological link between IBD and the HM onset. If these data are confirmed, IBD patients screened and positive for CH should undergo a hematologic follow-up to assess the risk of developing HM. Future study will clarify the relationship between these conditions.

Genomic BCR-ABL1 breakpoint characterization by a multi-strategy approach for "personalized monitoring" of residual disease in chronic myeloid leukemia patients.

For monitoring minimal residual disease (MRD) in chronic myeloid leukemia (CML) the most recommended method is quantitative RT-PCR (RT-qPCR) for measuring BCR-ABL1 transcripts. Several studies reported that a DNA-based assay enhances the sensitivity of detection of the BCR-ABL1 genomic rearrangement, even if its characterization results difficult. We developed a DNA-based method for detecting and quantifying residual BCR-ABL1 positive leukemic stem cells in CML patients. We propose two alternative approaches: the first one is a fluorescence in situ hybridization (FISH)-based step followed by Sanger sequencing; the second one employs MinION, a single molecule sequencer based on nanopore technology. Finally, after defining the BCR-ABL1 genomic junction, we performed the target CML patient-specific quantification, using droplet digital PCR (ddPCR). FISH and MinION steps, respectively, together with ddPCR analysis, greatly reduce the complexity that has impeded the use of "personalized monitoring" of CML in clinical practice. Our report suggests a feasible pipeline, in terms of costs and reproducibility, aimed at characterizing and quantifying the genomic BCR-ABL1 rearrangement during MRD monitoring in CML patients.

FLAG-Ida Regimen as Bridge Therapy to Allotransplantation in Refractory/Relapsed Acute Myeloid Leukemia Patients.

BACKGROUND: Patients with primary refractory or first relapse acute myeloid leukemia (AML) are considered to have worse clinical outcomes after treatment. For these patients, the achievement of complete remission appears crucial for them to be able to undergo allotransplantation, which might be the only possible treatment. PATIENTS AND METHODS: We used the FLAG-Ida (fludarabine, cytarabine [cytosine arabinoside], granulocyte colony-stimulating factor, idarubicin) regimen in patients with primary refractory/first relapse AML as a bridge to transplantation. We studied its efficacy in terms of overall response and overall survival to assess which variables (age, lactate dehydrogenase, bone marrow blast count, peripheral blood blast count, platelet count, white blood cell count, de novo or secondary AML, molecular-cytogenetic risk, duration of response, and relapsed or refractory disease) might have an effect on outcome. RESULTS: We analyzed the data from 108 consecutive adult patients (52 males, 66 females; median age, 49 years; range, 17-72 years) with newly diagnosed AML refractory to standard induction regimens or relapse after first complete remission, who had received the FLAG-Ida protocol as salvage therapy from January 2005 to December 2015. An overall response was achieved in 48 patients (44%). On multivariate analysis, the variables with a positive effect on the response rate were molecular-cytogenetic risk (P = .009), duration of first response in relapsed AML (P = .003), AML status (relapsed or refractory; P = .047), and peripheral blood blast count (P = .016). On multivariate analysis, overall survival was significantly associated with FLAG-Ida response (hazard ratio, 0.343; P = .001) and receipt of allotransplantation (hazard ratio, 0.277; P < .001). CONCLUSION: Our data seem to confirm the value of FLAG-Ida in this setting and might suggest its best usage as bridge therapy for patients awaiting allotransplantation.

Imatinib and polypharmacy in very old patients with chronic myeloid leukemia: effects on response rate, toxicity and outcome.

BACKGROUND: About 40% of all patients with chronic myeloid leukemia are currently old or very old. They are effectively treated with imatinib, even though underrepresented in clinical studies. Furthermore, as it happens in the general population, they often receive multiple drugs for associated chronic illnesses. Aim of this study was to assess whether or not in imatinib-treated patients aged >75 years the exposure to polypharmacy (5 drugs or more) had an impact on cytogenetic and molecular response rates, event-free and overall survival, as well as on hematological or extra-hematological toxicity. METHODS: 296 patients at 35 Italian hematological institutions were evaluated. RESULTS: Polypharmacy was reported in 107 patients (36.1%), and drugs more frequently used were antiplatelets, diuretics, proton pump inhibitors, ACE-inhibitors, beta-blockers, calcium channel blockers, angiotensin II receptors blockers, statins, oral hypoglycemic drugs and alpha blockers. Complete cytogenetic response was obtained in 174 patients (58.8%), 78 (26.4%) within 6 month, 63 (21.3%) between 7 and 12 months. Major molecular response was obtained in 153 patients (51.7%), 64 (21.6%) within the 12 month. One hundred and twenty-eight cases (43.2%) of hematological toxicity were recorded, together with 167 cases (56.4%) of extra-hematological toxicity. Comparing patients exposed to polypharmacy to those without, no difference was observed pertaining to the dosage of imatinib, cytogenetic and molecular responses and hematological and extra-hematological toxicity. CONCLUSION: Notwithstanding the several interactions reported in the literature between imatinib and some of the medications considered herewith, this fact does not seem to have a clinical impact on response rate and outcome.

Age influences initial dose and compliance to imatinib in chronic myeloid leukemia elderly patients but concomitant comorbidities appear to influence overall and event-free survival.

We applied Charlson comorbidity index (CCI) stratification on a large cohort of chronic myeloid leukemia (CML) very elderly patients (>75 years) treated with imatinib, in order to observe the impact of concomitant diseases on both compliance and outcome. One hundred and eighty-one patients were recruited by 21 Italian centers. There were 95 males and 86 females, median age 78.6 years (range 75-93.6). According to Sokal score, 106 patients were classified as intermediate risk and 55 as high risk (not available in 20 patients). According to CCI stratification, 71 patients had score 0 and 110 a score ≥ 1. Imatinib standard dose was reduced at start of therapy (200-300 mg/day) in 68 patients independently from the evaluation of baseline comorbidities, but based only on physician judgement: 43.6% of these patients had score 0 compared to 34% of patients who had score ≥ 1. Significant differences were found in terms of subsequent dose reduction (39% of patients with score 0 compared to 53% of patients with score ≥ 1) and in terms of drug discontinuation due to toxicity (35% of patients with score 0 vs 65% of patients with score ≥ 1). We did not find significant differences as regards occurrence of hematologic side effects, probably as a consequence of the initial dose reduction: 39% of patients with score 0 experienced grade 3/4 hematologic toxicity (most commonly anemia) compared to 42% of patients with score ≥ 1. Independently from the initial dose, comorbidities again did not have an impact on development of grade 3/4 non-hematologic side effects (most commonly skin rash, muscle cramps and fluid retention): 62% of patients with score 0 compared to 52.5% of patients with score ≥ 1. Notwithstanding the reduced dose and the weight of comorbidities we did not find significant differences but only a trend in terms of efficacy: 66% of patients with score 0 achieved a CCyR compared to 54% of patients with score ≥ 1. Comorbidities appeared to have an impact on median OS (40.8 months for patients with score 0 vs 20.16 months for patients with score ≥ 1) on EFS and on non-CML death rate. Our results suggest that treatment of very elderly CML patients might be influenced by personal physician perception: evaluation at baseline of comorbidities according to CCI should improve initial decision-making in this subset of patients.

Gene expression profiling of chronic myeloid leukemia with variant t(9;22) reveals a different signature from cases with classic translocation.

BACKGROUND: The t(9;22)(q34;q11) generating the BCR/ABL1 fusion gene represents the cytogenetic hallmark of chronic myeloid leukemia (CML). About 5-10% of CML cases show variant translocations with the involvement of other chromosomes in addition to chromosomes 9 and 22. The molecular bases of biological differences between CML patients with classic and variant t(9;22) have never been clarified. FINDINGS: In this study, we performed gene expression microarray analysis to compare CML patients bearing variant rearrangements and those with classic t(9;22)(q34;q11). We identified 59 differentially expressed genes significantly associated with the two analyzed groups. The role of specific candidate genes such as TRIB1 (tribbles homolog 1), PTK2B (protein tyrosine kinase 2 beta), and C5AR1 (complement component 5a receptor 1) is discussed. CONCLUSIONS: Our results reveal that in CML cases with variant t(9;22) there is an enhancement of the MAPK pathway deregulation and show that kinases are a common target of molecular alterations in hematological disorders.

CD3+/Tregs ratio in donor grafts is linked to acute graft-versus-host disease and immunologic recovery after allogeneic peripheral blood stem cell transplantation.

Graft-versus-host disease (GVHD), mediated by mature T cells present in the donor graft, remains a major complication after allogeneic peripheral blood stem cell transplantation (PBSCT). Regulatory T cells (Tregs) (CD4(+)CD25(high)Foxp3(+)) are believed to maintain tolerance and to inhibit GVHD after allogeneic PBSCT (allo-PBSCT). In this study, we analyzed the graft CD3(+)/Tregs ratio (gCD3/Tregs R) and evaluated its impact on acute GVHD (aGVHD) and immunologic recovery after myeloablative allo-PBSCT. We analyzed 65 consecutive patients who underwent transplantation with unmanipulated peripheral blood stem cells from an HLA-identical related donor (n = 45) or an HLA-identical unrelated donor (n = 20). The median CD3(+) and Tregs doses administered were 256 × 10(6)/kg of body weight (range, 67-550 × 10(6)/kg) and 12 × 10(6)/kg (range, 2-21 × 10(6)/kg), respectively; the median gCD3/Tregs R value was 18 (range, 8-250). Patients were subdivided into a high gCD3/Tregs R (≥36) group (HR; n = 26) and a low gCD3/Tregs R (<36) group (LR; n = 39). The incidence of aGVHD (grade II-IV) was lower in the LR group compared with the HR group (8/39 [20%] versus 22/26 [84%]; P < .001). Median cytomegalovirus-specific CD8(+) T lymphocytes were significantly higher in the LR group than in the HR group at 1 month (2 cells/µL versus 0 cells/µL; P < .001), 2 months (6 cells/µL versus 1 cell/µL; P < .001), and 3 months (15 cells/µL versus 3 cells/µL; P < .001) months. Moreover, cytomegalovirus infection/disease was observed in 15% of patients in the LR group versus 69% of patients in the HR group (P < .001). At multivariate logistic regression, gCD3/Tregs R was correlated both with aGVHD (odds ratio, 2.50; 95% confidence interval, 1.30-4.50; P = .05) and with cytomegalovirus infection/disease (odds ratio, 2.35; 95% confidence interval, 0.9-5.00; P = .05). Taken together, our data may suggest that the balance in favor of graft Tregs content is able to mediate protective effects against aGVHD and to maintain an optimal microenviroment for the reconstitution of functional immunity.

Health-related quality of life in chronic myeloid leukemia patients receiving long-term therapy with imatinib compared with the general population.

The main objective of this study was to investigate whether patients with chronic myeloid leukemia (CML) in treatment with long-term therapy imatinib have a different health-related quality-of-life (HRQOL) profile compared with the general population. In total, 448 CML patients were enrolled, and the SF-36 Health Survey was used to compare generic HRQOL profiles. Symptoms were also assessed. HRQOL comparisons were adjusted for key possible confounders. The median age of patients was 57 years and the median time of imatinib treatment was 5 years (range 3-9 years). The largest HRQOL differences were found in younger patients. In particular, patients aged between 18 and 39 years had marked impairments in role limitations because of physical and emotional problems, respectively: -22.6 (P < .001), -22.3 (P < .001). Patients with CML age 60 or older had a HRQOL profile very similar to that reported by the general population. Women had a worse profile than men when each were compared with their peers in the general population. Fatigue was the most frequently reported symptom. The HRQOL of CML patients is comparable with that of population norms in many areas, however, younger and female patients seem to report the major limitations.

Dasatinib is safe and effective in unselected chronic myeloid leukaemia elderly patients resistant/intolerant to imatinib.

To highlight dasatinib role in the elderly, 125 unselected patients with CP-CML aged >60 years resistant/intolerant to imatinib were retrospectively evaluated. Grade 3-4 haematological and extra-haematological toxicities were reported in 39 (31.2%) and 34 (27.2%) patients; grade 3-4 haematological toxicity was higher in patients with 140 mg starting dose (50.0% vs 19.6%, p=0.001). Grade 3-4 pleuro-pericardial effusions occurred in 10 patients (8.0%). Dose reductions were more common in patients with 140 mg (88.4% vs 26.7%, p<0.001). Of 122 evaluable patients, 72 (59.1%) had cytogenetic response [12 (9.8%) partial, 60 (49.3%) complete]. Overall, 38/60 patients in complete CyR also achieved a molecular response. Cumulative OS at 24 and 48 months were 93.1% (95% CI 88.4-97.8) and 84.2% (95% CI 74.6-93.7). Dasatinib, at the recommended dose of 100mg/day, is effective and safe also in unselected elderly subjects.

Recovery of CMV-specific CD8+ T cells and Tregs after allogeneic peripheral blood stem cell transplantation.

Recovery of cytomegalovirus (CMV)-specific CD8+ T cells after allogeneic stem cell transplantation (SCT) is critical for protection against CMV infection and disease. Moreover, Foxp3+CD4+CD25(high) regulatory T cells (Tregs) are a major regulator of adaptive immunity, preventing graft-versus-host disease (GVHD) and so promoting timely and complete immune recovery. The aim of our study was to evaluate the recovery of circulating tetramer-based CMV-specific CD8+ T cells and T regs in 46 patients after allogeneic peripheral blood SCT (PBSCT). CMV infection and/or disease was observed in 7% and 94% of patients with or without CMV-specific CD8+ T cells recovery (P < .001), and in 77% and 4% of patients with or without acute GVHD (aGVHD) (P < .001), respectively. T regs values were higher in patients without than with CMV infection and/or disease at 2 (P < .001) and 3 months (P < .001) after allogeneic PBSCT, respectively. Moreover, we observed a positive correlation between T regs and the recovery of CMV-specific CD8+ T cells at 2 (r = .61, P < .0001) and 3 (r = .72, P < .00001) months, respectively. Tregs were higher in patients without than with aGVHD at 1, 2 (P < .001) and 3 months (P < .0001), respectively. At multivariate logistic regression, aGVHD (odds ratio [OR]: 2.60, 95% confidence interval [CI] [1.3-5.0], P = .0006) and CMV-specific CD8+ T cells recovery (OR:2.25, 95% CI [1.2-4.8], P = .05) were correlated with CMV infection and/or disease, whereas no correlation was found for Tregs, absolute neutrophil count, patients' and donors' age, disease status pretransplantation, type of disease, and CMV serology. Taken together, our data may suggest the existence of a correlation between Tregs and the recovery of CMV-specific CD8+ T cells; Tregs may preserve an optimal microenvironment for the reconstitution of functional immunity and mediate protective effects against aGVHD.

Non random distribution of genomic features in breakpoint regions involved in chronic myeloid leukemia cases with variant t(9;22) or additional chromosomal rearrangements.

BACKGROUND: The t(9;22)(q34;q11), generating the Philadelphia (Ph) chromosome, is found in more than 90% of patients with chronic myeloid leukemia (CML). As a result of the translocation, the 3' portion of the ABL1 oncogene is transposed from 9q34 to the 5' portion of the BCR gene on chromosome 22 to form the BCR/ABL1 fusion gene. At diagnosis, in 5-10% of CML patients the Ph chromosome is derived from variant translocations other than the standard t(9;22). RESULTS: We report a molecular cytogenetic study of 452 consecutive CML patients at diagnosis, that revealed 50 cases identifying three main subgroups: i) cases with variant chromosomal rearrangements other than the classic t(9;22)(q34;q11) (9.5%); ii) cases with cryptic insertions of ABL1 into BCR, or vice versa (1.3%); iii) cases bearing additional chromosomal rearrangements concomitant to the t(9;22) (1.1%). For each cytogenetic group, the mechanism at the basis of the rearrangement is discussed.All breakpoints on other chromosomes involved in variant t(9;22) and in additional rearrangements have been characterized for the first time by Fluorescence In Situ Hybridization (FISH) experiments and bioinformatic analyses. This study revealed a high content of Alu repeats, genes density, GC frequency, and miRNAs in the great majority of the analyzed breakpoints. CONCLUSIONS: Taken together with literature data about CML with variant t(9;22), our findings identified several new cytogenetic breakpoints as hotspots for recombination, demonstrating that the involvement of chromosomes other than 9 and 22 is not a random event but could depend on specific genomic features. The presence of several genes and/or miRNAs at the identified breakpoints suggests their potential involvement in the CML pathogenesis.

In patients with myelodysplastic syndromes response to rHuEPO and G-CSF treatment is related to an increase of cytogenetically normal CD34 cells.

The in vivo effect of recombinant human erythropoietin (rHuEpo) and granulocyte colony-stimulating factor (G-CSF) combined treatment on CD34(+) cells was evaluated by fluorescence in situ hybridization (FISH) in 13 myelodysplastic syndrome (MDS) patients with known cytogenetic abnormalities. After treatment, responsive patients presented a significantly lower proportion of FISH abnormal CD34(+) cells than before treatment (P = 0.003), and in comparison with unresponsive cases (P = 0.007). Response to treatment was associated with a reduced degree of apoptosis in CD34(+) cells (P = 0.021): however, no difference in telomere length was observed in responsive patients after growth factor administration. Although the number of patients analysed was relatively small, the present data suggest that, in MDS patients, response to rHuEpo and G-CSF may be related to the proliferation of karyotypically normal but potentially defective CD34(+) progenitor cells.