Months Of Tyrosine Kinase Inhibitor Treatment Research Articles

Therapy in Patients with Chronic Myeloid Leukemia Outside of Clinical Trials: Results of the German CML-Registry (CML-VI)

Introduction: Tyrosine kinase inhibitors (TKI) have significantly improved treatment outcomes for patients with newly diagnosed chronic myelogenous leukemia (CML) in chronic phase (CP). With the introduction of 2nd and 3rd generation TKI, the options in the management of CML have increased. In clinical trials, 25%-50% of patients do not remain on initial treatment due to intolerance or inefficacy. Information outside of clinical trials is limited. Aim: The aim of this study is to analyze the use of different TKI in first-line treatment and the frequency of TKI switches in CML in real world. Methods: Patients with newly diagnosed CML between 1.1.2013 and 31.12.2019 were registered and followed annually up to 31.12.2021. Primary endpoint was the time to and reason for switching of any first-line treatment including treatment free remission (TFR)attempt. Further endpoints were progression-free survival (PFS), overall survival (OS), cytogenetic and molecular response. Analyses were performed using Kaplan-Meier estimates with SPSS. Results: 540 adult patients have been registered from 91 centers, 512 were evaluable. 281 patients (55%), were treated by resident oncologists, 231 patients (45%) in hospitals including 82 patients (16%) in university hospitals. 271 patients (53%) were male. At diagnosis, median age was 59 (range 16-100) years. The median follow-up was 67 (3-108) months. Information on first-line TKI treatment was available for 480 patients: 203 patients (42%) were treated with imatinib, 137 (29%) with nilotinib and 140 (29%) with dasatinib. TKI switch was reported in 155 cases (31%) after a median of 11 (0-68) months. Reasons for switching was failure in 49 patients (32%), intolerance in 88 patients (57%), and 18 patients (11%) switched TKI because of other reasons (study inclusion, desire to have children, patient request, unknown). 33 patients (7%) switched TKI more than once. 13 (8,4%) of the 155 patients with TKI-switch died during follow-up, whereas only 16 of 325 (4,9%) patients without TKI-switch. OS of patients with TKI-switch was 90%, of patients without TKI-switch 94% after 6 years. TKI stop for more than one month has been reported in 118 patients (24%) due to different reasons (attempt for TFR, intolerance, patient wish, pregnancy). 61 patients (12%) started a TFR attempt after a median of 46 (10-96) months of TKI treatment. Discontinuation criteria were not always met. Only 10 patients (6,5%) with TKI-switch started a TFR attempt compared to 51 patients (16%) who had only been treated with one TKI. 501 patients were in CP at diagnosis, 11 patients in accelerated phase (AP) and 2 patients in blast crisis (BC). Progression occurred in 9 patients (BC n=6, AP n=3) after a median time of 12 (9-22) months. Overall, AP and BC were diagnosed in 22 patients (2,3%). Of the total cohort, 29 patients died, OS was 93% after 6 years. An evaluable cytogenetic examination was carried out in 291 patients (57%) at diagnosis or in the course of disease. No statements can be made about achieving of complete cytogenetic remission as follow up results were only available in 15% and monitoring was carried out using RQ-PCR in most patients. A major molecular response (MMR, BCR::ABL1 <0.1%) was reached by 422 patients (85%), a deep molecular response (defined as BCR::ABL1 ≤0.01) by 365 patients (74%) within the follow-up period. Cumulative incidence calculation and study of patients who lost their MMR are ongoing. There is no information about the molecular status of 18 patients. Conclusion: Results of the CML VI-registry provided information on treatment but also on baseline characteristics and diagnostic parameters of CML patients in real world. The median age of the patients at diagnosis was higher than in other clinical trials. In routine care, data demonstrated high switching rates of first-line treatment and frequent TFR attempts without meeting the guideline criteria. OS rates were as high as in clinical trials and confirm normal life expectation of CML patients. MMR was reached in 85% and DMR in 74% in this population. Progression is rare. In conclusion, the CML-registry offers importing data confirming a good outcome of response and survival for CML patients outside of clinical trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

In Search of Drivers of CD34+/CD38-/CD26+ Leukemia Stem Cells Persistence in CML Patients

Introduction: In Chronic Myeloid Leukemia (CML) patients, we confirmed that the expression of CD26 discriminates CML Leukemic Stem Cells (LSCs) from normal Hematopoietic Stem Cells (HSCs) or from LSCs of other myeloid neoplasms. CD34+CD38-CD26+ cells are easily measurable in Peripheral Blood (PB) at diagnosis, and circulating residual CD26+LSCs persist in most patients during treatment with Tyrosine Kinase Inhibitors (TKIs) and after successful TKI discontinuation. The reason for LCSs persistence may be correlated to mechanisms helping their survival, such as resistance to apoptosis (via BCL-2 or CD47 expression) or immune checkpoint escaping (via PD-L1). Moreover, few is still known about the "activity state" or "quiescence" of CML LSCs. Aim of the study: to unravel phenotypic characteristics that could help CML LSCs persistence, evaluating by flow-cytometry the expression level of BCL-2 protein, PD-L1, CD47 and Ki67 on CD26+ LSCs population. Methods: Flow-cytometry assays were performed on PB from newly diagnosed CML patients by using lyophilized reagents (CD34-FITC, CD26-PE, CD38-APC, CD45-V500) and liquid reagents (CD47-V450, PD-L1-Cy7, BCL-2-FITC, Ki67-PerCP-Cy 5.5). Analysis have been done using a BD FACSLyric cytometer. Results: 47 newly diagnosed CML patients entered this study. With regards to PD-L1 expression on CD26+LSCs, 22/47 (47%) scored negative while 25/47 (53%) resulted positive, albeit with a quite variable percentage of expression (median 28.5 % of CD26+ cells, range 12-82.8 %). BCL-2 protein was expressed in 47/47 (100%) CD26+ LSCs, and the level of expression evaluated comparing the Mean Fluorescent Index (MFI) was found much lower on CD26+ LSCs than lymphocytes (median 5.98, range 1.90-32.14; median 17.20, range 7-76, respectively, p value <0.05). Regarding CD47 expression, 47/47 (100%) patients scored positive, with an MFI that is double on lymphocytes compared to CD26+ LSCs (median 26.58, range 5.70-87.09; median 13.22, range 5.64-33.22, respectively). Lastly, all 47/47 (100%) CD26+ LSCs were found Ki-67 positive. Conclusions: These preliminary results show that in newly diagnosed CML patients, Ki67 is highly expressed on circulating CD26+ LSCs, suggesting an unexpected "active proliferative state" of CD26+ LSCs, rather than a "quiescent" status. The latter may explain the rapid reduction of the bulk of CD26+LSCs we observe already at 3 months of TKI treatment. Both BCL-2 protein and CD47 are expressed at a higher level on lymphocytes than on CD26+ CML LSCs, indicating that the "don't eat me signal" that blocks apoptosis may not be relevant for CML LSCs persistence. On the other hand, CD26+/PD-L1+ LSCs (found in about 53% of CML patients) could theoretically escape the immune checkpoint controls, an aspect that could be taken in consideration for a future Treatment Free Survival (TFR) approach in CML patients as it may lead to a loss of molecular response compared to patients with CD26+ LSCs scoring negative for PD-L1. Additional studies need to be performed on a larger cohort of patients with a longer follow-up to support these hypotheses; however, these preliminary data may start to pave the way to understand the relationship between CD26+ LSC and immunity of CML patients.

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.

SVCAM-1, and TGFβ1 in chronic phase, chronic myeloid leukemia patients treated with tyrosine kinase inhibitors

The outcome for chronic phase (CP) chronic myelogenous leukemia (CML) patients has changed dramatically since the introduction of tyrosine kinase inhibitor (TKI) therapy. We examined the characteristics of CML patients during TKI therapy by determining the plasma concentrations of soluble vascular cell adhesion molecule 1 (sVCAM-1), and transforming growth factor (TGFβ1) biomarkers. The plasma levels of sVCAM-1 and TGFβ1 were measured by ELISA at baseline and after 3 months of TKI treatment. The levels of sVCAM-1, and TGFβ1 were significantly elevated in patients with CML (P< 0.01). Dasatinib treatment was associated with a significant reduction in the levels of these biomarkers (P< 0.01). In conclusion, plasma levels of sVCAM-1 and TGFβ1 could have a role in the pathogenesis of CML and may be used as predictors of hematological and molecular responses to TKIs. A favorable outcome for Dasatinib therapy was observed.

Impact of the app-based and nurse-led supportive care program AKO@dom on dose intensity of oral-targeted therapies in patients with metastatic renal cell cancer: a multicentric observational retrospective study.

Tyrosine kinase inhibitors (TKIs) remain a cornerstone of metastatic kidney cancer (mRCC). Adverse events (AEs) may lead to dose downregulation, and optimal management of AEs is needed to maintain an efficient dose intensity (DI). The aim of our study was to evaluate the impact of an app-based and nurse-led supportive-care program on DI in mRCC patients. This multicenter (n = 3), retrospective study evaluated all consecutive mRCC patients who participated in the AKO@dom program, which consisted of an app-based and nurse-led weekly patient evaluation at home during the first 3months of TKI intake. Treatment patterns and modifications were described, and the mean DI (mDI) was calculated at the end of AKO@dom. Eighty-nine patients were included: 12 had sunitinib, 18 pazopanib, 12 axitinib, and 47 cabozantinib. Median age was 69years (60-76). TKIs were mainly initiated at standard doses except for cabozantinib (53% started at 40mg/day); 71% had prior systemic treatment. Nine patients discontinued permanent treatment during the program. Thirty-two patients required ≥ 1 dose interruption, and 29% experienced ≥ 1 grade 3 AE of any type. The mDI (in mg/day) at 3months was 34.4 ± 17.7 for sunitinib, 672.8 ± 144 for pazopanib, 8.6 ± 2.6 for axitinib, and 40 (36-48) for cabozantinib. Fifty-five patients [68.75% (95% CI: 57-78%)] had a mDI ≥ than reported in the literature. Overall survival at 12months was 64.2% (CI 95%: 55-75%). The AKO@dom program allowed 68.75% of patients to maintain a high dose intensity after 3months of TKI treatment. The impact on survival outcomes needs to be evaluated in randomized clinical trials.

Immune checkpoint inhibitors further aggravate proteinuria in patients with metastatic renal cell carcinoma after long-term targeted therapy.

BackgroundIncreasing number of patients with metastatic renal cell carcinoma (mRCC) are receiving subsequent programmed cell death protein-1 (PD-1) inhibitor combination therapy following tyrosine-kinase inhibitor (TKI) resistance. To explore whether PD-1 inhibitor would further deteriorate proteinuria and renal function, we observed their proteinuria’s and renal function’s condition since the administration of PD-1 inhibitor.MethodsTo assess the change in proteinuria and renal function, the data of 141 patients with mRCC treated with TKI were collected, 66 of whom were further prescribed PD-1 inhibitor. Proteinuria and estimated glomerular filtration rate (eGFR) were measured and analyzed. Logistic regression models were established to identify the predictors of proteinuria deterioration and significant eGFR decline (≥15%).ResultsOf the 141 patients, 74 (52%) had an increase in proteinuria level after an average of 22.98 months of TKI treatment. In multivariate analysis, longer duration of TKI (>12 months) and administration of PD-1 inhibitor were independent predictors for proteinuria deterioration. The median eGFR decreased from 81.56 mL/min/1.73 m2 to 66.75 mL/min/1.73 m2 after TKI treatment. Logistic regression identified older age (>60 years old) and longer duration of TKI (>12 months) as independent predictors for significant eGFR decline. Finally, of the 66 patients who received subsequent PD-1 inhibitor, 34 had sufficient proteinuria and eGFR data at follow-up. The level of proteinuria increased further after the administration of PD-1 inhibitor, although the decrease in eGFR was not statistically significant (P=0.182). Log-rank analysis identified proteinuria deterioration and eGFR decline were both significantly associated with patent’s survival (P<0.001).ConclusionsTargeted therapy was associated with an increase in proteinuria level and a decrease in eGFR in patients with mRCC. The administration of PD-1 inhibitor contributed to exacerbation in proteinuria, but no significant difference in a decrease of eGFR was observed.

Pro-Inflammatory and Pro-Oxidative Changes during Nilotinib Treatment in CML Patients: Results of a Prospective Multicenter Front-Line TKIs Study (KIARO Study)

Background: Tyrosine kinase inhibitors (TKI) may offer a normal life expectancy to Chronic Myeloid Leukemia (CML) patients. However, during treatment with nilotinib, a higher than expected incidence of arterial occlusive events (AOEs) was observed. We retrospectively showed an “inflammatory status” during nilotinib treatment that may explain this increased incidence of AOEs. Here, we report results of a prospective multicenter (KIARO) study including 186 CML patients (89 imatinib, 59 nilotinib, 38 dasatinib) in which pro/anti-inflammatory cytokines were measured at diagnosis and during treatment, with the aim to investigate potential changes in each patient's inflammatory status possibly favoring AOEs.Aims: The aims of this study are: 1) to analyze prospectively inflammation status during TKI treatment; 2) to record AOEs; 3) to calculate the SCORE and evaluate its predictive role for AOEs; 4) to analyze possible associations of AOEs with altered inflammation status.Methods: Inflammatory status was evaluated by measuring IL6, IL10, TNFα, oxLDL and hs-CRP plasma levels at diagnosis and during treatment (+1, +3, +6, +12 months); additionally, clinical and biochemical pro-atherothrombotic profiles and the 10-year SCORE chart were also evaluated.Results: 186 newly-diagnosed CML patients starting either imatinib, nilotinib or dasatinib treatment, entered this study. Regarding the inflammation status, we observed that TNFα and IL6 levels were high at diagnosis and decreased during the first 12 months of treatment regardless the type of TKI; instead, IL10 levels were comparable among the 3 TKI cohorts at baseline, but showed a remarkably different evolution during treatment. In fact, IL10 levels were significantly higher after 6 and 12 months of imatinib (p=0.012 and p=0.009, respectively) and dasatinib (p=0.032 and p=0.014, respectively) compared to nilotinib. Consequently, TNFα/IL10 ratio was significantly higher in nilotinib cohort at 6 and 12 months respect to imatinib (p=0.044 at 6 months and p=0.041 at 12 months) and dasatinib (p=0.040 at 6 months and p=0.044 at 12 months). As well, IL6/IL10 ratio was significantly higher in nilotinib cohort compared to imatinib and dasatinib both at 6 (p=0.042 and p=0.049, respectively) and 12 months (p=0.040 and p=0.041, respectively) (Figure 1). OxLDL levels were similar in the 3 groups for the first 6 months. At 12 months we detected a significant increase of oxLDL levels in the nilotinib cohort (p=0.041), respect to imatinib and dasatinib. We did not find significant differences in hs-CRP levels across the 3 TKIs, although a trend for higher levels was observed in nilotinib cohort. Overall, these results suggest a TKI-driven pro-inflammatory status in nilotinib treated patients.After a median follow-up of 23.3 months of TKI treatment, 10 patients (5.4%) suffered an AOE, specifically: 6 ACS, 2 PAOD, 1 TIA and 1 stroke. 5 events (50%) occurred in patients treated with nilotinib, either in first line (4 patients) or in third line (1 patient, after failure following brief treatment with imatinib and dasatinib). In this subgroup of 10 patients experiencing an AOE, we observed a trend of increased IL6 and TNFα median values both at diagnosis and at each time point, compared with the remaining no-AOE patients. IL10 and oxLDL had similar median concentrations in both AOE and no-AOE cohorts, except for oxLDL at 12 months which resulted higher in patients who experienced AOEs. Moreover, regarding AOEs, nilotinib treatment showed a 3.1 times increased risk compared to other TKIs (HR 3.1, 95% CI 2.6-4.4 p< 0.001), whereas 10-year SCORE was not predictive in the whole cohort (HR 0.6, 95% CI 0.33-0.94 p= 0.094) or in any subgroup (imatinib HR 0.8, 95% CI 0.49-1.03 p= 0.067; nilotinib HR 0.4, 95% CI 0.29-0.76 p= 0.112, dasatinib HR 0.6, 95% CI 0.37-0.92 p= 0.082).Conclusions: Our results showed a pro-inflammatory/oxidative milieu increasing along treatment with nilotinib compared with imatinib or dasatinib, as demonstrated by higher IL6/IL10 and TNFα/IL10 ratios, higher levels of oxLDL and a trend for higher hs-CRP only in nilotinib cohort. However, due to the low number of observed events, a formal statistical analysis for any association between AOEs and pro/anti-inflammatory cytokines levels was not possible. Therefore, a longer follow-up is needed to further confirm the active role of nilotinib in AOEs pathogenesis. [Display omitted] DisclosuresAbruzzese: Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Galimberti: Incyte: Speakers Bureau; AbbVie, Janssen: Honoraria, Other: Travel grants. Stagno: Pfizer: Consultancy, Honoraria, Other: Support for attending meetings and/or travel; InCyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Other: Support for attending meetings and/or travel, Research Funding.

Efficacy and Cardiovascular Adverse Events of Long-term Treatment with Tyrosine Kinase Inhibitors for Chronic Myeloid Leukemia: A Report from the Nagasaki CML Study Group.

Objective The standard treatment for chronic myeloid leukemia (CML) is the continuous use of tyrosine kinase inhibitors (TKIs), which results in a favorable prognosis for the majority of patients. Recent studies have identified cardiovascular diseases (CVDs) as late adverse events (AEs) related to TKIs. In this study, we evaluated the long-term efficacy and AEs of TKIs, focusing on CVDs. Methods We performed a retrospective survey of CML patients (diagnosed from 2001 to 2016) treated with TKIs in Nagasaki Prefecture. Clinical data were obtained from their medical records. We analyzed the survival, estimated cumulative incidence of CVDs, and risk factors for CVD among CML patients treated with TKIs. Results The overall survival rate of 264 CML patients treated with TKIs (median age 58 years old) was 89.6% [95% confidence interval (CI), 84.9-92.9%], and 80.5% (95% CI, 73.4-85.9%) at 5 and 10 years after the CML diagnosis, respectively. CVD events occurred in 26 patients (9.8%, median age 67.5 years old) with a median 65.5 months of TKI treatment. The cumulative incidences at 2 and 5 years was 2.4% (95% CI, 1.0-4.8%) and 5.2% (95% CI, 2.8-8.6%), respectively. Hypertension and a high SCORE chart risk at the diagnosis of CML were associated with CVD events during TKI treatment. Conclusion TKI treatment contributed to the long-term survival of CML patients in Nagasaki Prefecture in a “real-world” setting, but the incidence of CVDs seemed to be increased in these patients. A proper approach to managing risk factors for CVD is warranted to reduce CVD events during TKI treatment.

Assessment of soluble cytotoxic T lymphocyte-associated antigen-4, transforming growth factor β1, and platelet-derived microparticles during dasatinib therapy for patients with chronic myelogenous leukemia.

BackgroundThe outcome for chronic myelogenous leukemia (CML) patients presented in the chronic phase has changed dramatically since the introduction of tyrosine kinase inhibitor (TKI) therapy. Notably, an increased incidence of large granular lymphocytes (LGLs), which is related to immunological conditions, appears to be predictive of a favorable outcome for dasatinib therapy. We therefore examined the immunological characteristics of CML patients during dasatinib therapy by determining the plasma concentrations of five different biomarkers.MethodsThe plasma levels of biomarkers, specifically interleukin-6, platelet-derived microparticles (PDMPs), soluble vascular cell adhesion molecule 1 (sVCAM-1), transforming growth factor (TGF) β1, and soluble cytotoxic T lymphocyte-associated antigen-4 (sCTLA-4), were measured by ELISA at baseline and after 2 and 6 months of TKI treatment. The incidence of LGLs was estimated by microscopic examination.ResultsThe levels of PDMPs, sVCAM-1, and TGFβ1 were significantly elevated in patients with CML. Dasatinib treatment was associated with a significant reduction in the levels of these markers and with an increased incidence of LGLs compared with imatinib or nilotinib treatment. In addition, an increased incidence of LGLs was significantly correlated with a decreased sCTLA-4 level during dasatinib therapy.ConclusionThe assessment of the levels of specific biomarkers may be beneficial to understand the immunological conditions of patients with CML during dasatinib treatment.

Prevalence and Dynamics of BCR-ABL Independent Gene Mutations in Patients with Chronic Myeloid Leukemia

Living without treatment has become a realistic target for patients with chronic myeloid leukemia (CML) who achieved durable deep molecular remission under treatment with tyrosine kinase inhibitors (TKI). Recently, we have identified novel BCR-ABL-independent gene mutations in newly diagnosed CML patients that may function as important cofactors in the evolution of CML and persistence of the disease (Schmidt M, Rinke J, et al. Leukemia 2014; 28(12): 2292-9). Here, we sought to investigate the dynamics of such mutations during a 2-year treatment with the second generation TKI nilotinib. To check if BCR-ABL independent mutations are an age associated phenomenon we also screened a cohort of children and adolescents with CML. A total of 51 chronic phase CML patients were analyzed including 31 adults (male, n=23; median age 57 years, range 42 to 73 years) and 20 children/adolescents (male, n= 14; median age 14 years, range 10 months to 27 years). For each adult patient, samples were analyzed at diagnosis, in subsequent complete cytogenetic remission (CCyR) after 3 months and in deep molecular remission (≤ MMR) after 24 months of nilotinib therapy. Children/adolescents samples were investigated at diagnosis only. Targeted deep next-generation sequencing (NGS) was used to analyze a panel of 30 commonly mutated genes in myeloid disorders. Constitutional DNA obtained from buccal swabs at diagnosis was investigated to check whether mutations are of somatic origin. BCR-ABL independent gene mutations were found in 8/31 (26%) adult CML patients at diagnosis affecting the genes ASXL1 (n=3), DNMT3A (n=3), RUNX1 (n=1) and RUNX1 plus TET2 (n=1). No mutation was recognized in corresponding constitutional DNA specimens indicating that all mutations were somatically acquired. Analysis of individual hematopoietic colonies from three adult patients revealed that most mutations were part of the BCR-ABL-positive clone. NGS of subsequent samples obtained after three months of TKI therapy identified two DNMT3A mutations in BCR-ABL-negative cells that were also present in BCR-ABL-positive cells at diagnosis, implying that this mutation preceded the BCR-ABL rearrangement. After 24 months of TKI treatment, NGS revealed these two DNMT3A mutations again as well as a novel low-level EZH2 mutation in a patient with deep molecular remission (MR4.5) that was neither detectable at diagnosis nor after 3 months of treatment indicating the emergence of a BCR-ABL-independent subclone in BCR-ABL-negative cells during long-term nilotinib treatment. Within the children/adolescents cohort BCR-ABL independent gene mutations were identified in 5/20 (25%) subjects all affecting the ASXL1 gene. All mutations were stop codon or frameshift mutations implying loss of ASXL1 gene function. We conclude that BCR-ABL independent gene mutations can be found frequently in CML patients and affect predominately epigenetic modifier genes. The finding of frequent mutations also in children/adolescents implicates that BCR-ABL independent gene mutations are not just age-related events. Such molecular aberrations may play an important role in the evolution and persistence of the disease and may affect therapy in both children and adults with CML. These findings may provide important novel genetic information regarding CML biology and for the design and performance of discontinuation trials. DisclosuresHochhaus:Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding.

A Randomized Phase II Trial of Interferon (IFN)/GM-CSF Versus K562/GM-CSF Vaccination in Chronic Phase CML Patients on Frontline Tyrosine Kinase Inhibitor (TKI) Therapy: Potential of IFN to Enhance Molecular Remissions

Background: Tyrosine kinase inhibitors (TKIs) have dramatically improved outcomes in chronic myeloid leukemia (CML), but most patients either never achieve a complete molecular remission (CMR) or recur upon TKI discontinuation due to the persistence of leukemic stem cells. The kinetics of the initial TKI response appear predictive of long term outcomes as front-line imatinib patients who achieve a major molecular response (MMR) at 12 months have a 72% chance of attaining a CMR versus just 5% for those who do not (median time on Imatinib (IM) prior to CMR 66 months) (Branford. Clin Cancer Res. 2007). Interestingly, patients who achieve and maintain a CMR for ≥2 years on single agent IM have been the subject of several TKI discontinuation trials demonstrating that 38-47% of such patients maintain treatment-free remissions (TFR) at 2 years (median time on IM prior to discontinuation of 50-70 months) (Mahon. Lancet Oncol. 2010 and Ross. Blood. 2013). Interferon-alpha (IFN) and K562/GM-CSF vaccines have been shown to enhance the frequency of deep molecular responses to TKIs (Preudhomme. N Engl J Med. 2010 and Smith. Clin Cancer Res. 2010). We have shown that IFN targets LSCs, which may be the mechanism of improved IM responses (Angstreich. Br J Haematol. 2005). We now report initial outcomes for patients randomized between these treatments as a means of achieving TFR.Methods: Patients with Ph+ CML in complete cytogenetic remission on first-line TKI treatment for ≥1 year were randomized to receive either K562/GM-CSF vaccination at 3 week intervals (vaccine) or daily interferon/GM-CSF (IFN/GM) in combination with their TKI. If CMR was achieved after a minimum of 6 months of adjuvant treatment, then all therapy was stopped, and monthly BCR-ABL PCR testing was performed to monitor for relapse. Based on the limits of detection of our assay, CMR is defined as at least MR4.3, which is IS <0.01. At disease recurrence, or if CMR was not achieved at 12 months of combination therapy, patients were allowed to cross-over. The treatment arms were defined as IFN alone (A), vaccine alone (B), IFN followed by vaccine (C), or vaccine followed by IFN (D).Results: 34 patients were enrolled with final treatment allocation shown in Table 1. Most patients were on IM (n=31), nilotinib (n=2) and dasatinib (n=1) with a median duration of TKI use at enrollment of 19 (range 12-80) months. Both arms of the trial were well-tolerated. The vaccine arm was notable for grade 1 and 2 injection site reactions in all patients. Predictably, the IFN arm produced grade 1 and 2 myalgias and flu-like symptoms in the majority of patients. Three patients had to discontinue treatment on the IFN arm due to grade 3 adverse events including a neurological event of unknown causality (1) and ALT elevation (2). Table 1 shows the responses by treatment arm with 62% of patients achieving CMR at a median of 28.6 (range 11-100) months of TKI therapy (compared to 66 months in IRIS). 15 patients (44%) discontinued TKI treatment per study plan after a median of 27.5 (range 19-56) months of TKI treatment (compared to median of 50-70 months of TKI in STIM/TWISTER), and 4 patients (27%) remain off therapy with TFRs of 64, 64, 85, and 92 months. Those with recurrence were off TKI for a median of 3 (range 1-5) months. Importantly, the long term responders who achieved CMR with adjuvant therapy all received IFN adjuvant, and all had relatively little prior TKI treatment (21, 27, 28, and 44 months) compared to published discontinuation studies. As expected in this group, all 4 had achieved a MMR at 12 months on TKI alone (p=0.05). What was unexpected was that 50% of patients treated with one of the IFN arms who had not achieved a MMR at 12 months on TKI alone went on to CMR after treatment on study compared to previous reports and just 15% of historical controls in our institutional database who achieved CMR on TKI alone (p=0.02)Conclusions: Adjuvant therapies may improve the likelihood of achieving CMR and allow for discontinuation of TKI in CML patients with minimal TKI exposure. Even patients predicted to not achieve CMR based on a lack of MMR at 12 months did so at a higher rate than expected following IFN treatment. IFN likely played a role in long term TFR in 4 exceptional responders who successfully discontinued IM sooner than in previously reported discontinuation trials, possibly owing to IFNs effect on primitive CML progenitors. [Display omitted] DisclosuresSmith:Actinium Pharmaceuticals, Inc.: Research Funding.

Plasma proteomics in CML patients before and after initiation of tyrosine kinase inhibitor therapy reveals induced Th1 immunity and loss of angiogenic stimuli

Background and aimsThe simultaneous measurement of many proteins is now possible using multiplex assays. In this pilot study we investigated a total of 124 proteins in plasma from chronic myeloid leukemia (CML) patients with the purpose of identifying proteins that are differently expressed at diagnosis and after tyrosine kinase inhibitor (TKI) treatment initiation. MethodsSamples were taken from 14 CML patients at diagnosis and after three months of TKI treatment (imatinib or dasatinib). Samples were analyzed by Mesoscale Discovery, Myriad RBM MAP technology and Olink Proseek. ResultsMultiple plasma proteins were differentially expressed before and after initiation of TKI therapy. Protein patterns demonstrated a possible shift towards Th1-immunity and reduced angiogenic stimuli. Further, some plasma proteins were identified that can be of potential interest to study further for biologic, prognostic or therapeutic significance such as E-selectin, uPAR, growth hormone and carbonic anhydrase IX. ConclusionsPlasma proteomics seems feasible and useful in CML patients, both for studying patterns of protein expression and for identifying single proteins differentially expressed before and after treatment. Plasma proteomics may be useful to map disease activity and biological processes. Hence, plasma proteomics can be used to understand drug mechanisms and treatment responses in CML.

Molecularly Defined Clonal Evolution in Patients with Chronic Myeloid Leukemia Independent of the BCR-ABL Status

Molecularly defined clonal evolution has been identified as a key phenomenon in the biology of acute myeloid leukemia. Molecular aberrations may be markers of individual subclones of the disease, but also responsible for treatment resistance. In most patients with chronic myeloid leukemia (CML), treatment with tyrosine kinase inhibitors (TKI) induces complete cytogenetic remissions (CCyR) characterized by polyclonal hematopoiesis. However, some CML patients show emergence of clonal cytogenetic abnormalities (CCA) in Philadelphia (Ph) negative cells during treatment indicating clonal hematopoiesis. We searched for BCR-ABL independent gene mutations in both Ph-negative and Ph-positive clones in 29 chronic phase CML patients (male, n=16; median age 58 years, range 29-73 years) using targeted deep next-generation sequencing of 25 genes frequently mutated in myeloid disorders: ASXL1, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, LNK, MPL, NPM1, NRAS, RUNX1, SF3B1, SRSF2, TP53, TET2, U2AF1, UTX, WT, and ZRSR2. Ph-negative clones were analyzed in 14 CML patients who developed CCA in Ph-negative cells (trisomy 8, n=10; nonrecurrent reciprocal translocations, n=2; monosomy 7, n=2). At time of analysis, patients were in CCyR (n=7), partial CyR (n=6), or minimal CyR (n=1) after a median of 21 months (range 7-114 months) of TKI treatment with imatinib, and/or nilotinib, and/or dasatinib. After exclusion of known polymorphisms, mutations were detected in 6/14 patients (43%) affecting the genes DNMT3A, EZH2, RUNX1, TET2, TP53, U2AF1, and ZRSR2. These patients were in CCyR (n=2), partial CyR (n=3), or minimal CyR (n=1), respectively, indicating BCR-ABL independent mutations in both Ph-negative and Ph-positive subclones. In two patients, the mutations were also found in corresponding diagnostic samples at higher or lower mutation level. To further investigate BCR-ABL independent gene mutations in Ph-positive clones, 15 randomly selected CML patients at diagnosis were analyzed. Mutations additional to BCR-ABL were found in 5/15 CML patients (33%) affecting ASXL1, DNMT3A, RUNX1, and TET2. None of the mutations were recognized in corresponding constitutional DNA specimens indicating that all mutations had been somatically acquired. Deep-sequencing of subsequent samples obtained in early CCyR after three months of TKI treatment revealed one DNMT3A mutation in Ph-negative cells which was also present in Ph-positive cells at diagnosis. Follow-up investigation showed that the mutation persisted in Ph-negative cells throughout CCyR and deep molecular remission (MR4.5) up to month 36 thereby not significantly changing its mutation level of approximately 15% implying a clonal hematopoiesis before the acquisition of the BCR-ABL rearrangement. In summary, BCR-ABL independent gene mutations were frequently found in Ph-negative and Ph-positive clones of CML patients and may be considered as important cofactors in the evolution of CML. Additional mutations acquired in the Ph-positive clone may impact on response to TKI treatment. Mutations preexisting to the occurrence of the BCR-ABL rearrangement may predispose patients to secondary hematological neoplasms. Our findings provide novel genetic information regarding CML biology and warrant further studies and impact on the design and performance of discontinuation trials. DisclosuresSchnittger:MLL Munich Leukemia Laboratory: Other. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; ARIAD: Honoraria, Research Funding; Pfizer: Consultancy, Research Funding.

Human MICL Is An Early Marker In Myeloid Differentiation and Identifies a Subgroup Of CML Patients With Expanded Granulocyte-Macrophage Progenitor Populations At Diagnosis

Although chronic myeloid leukemia (CML) originates from a primitive hematopoietic stem cell (HSC), it is the more differentiated progenitor cells that drive the expansion of the malignant clone. In addition, previous studies of chronic phase CML have shown that, despite the marked leukocytosis observed here, megakaryocyte-erythroid progenitors dominate the progenitor fraction. We sought to elucidate this by employing the new marker for leukemic stem cells, the human myeloid inhibitory C-type lectin-like receptor (hMICL), in the study of progenitor cell expansion in CML.Bone marrow or peripheral blood stem cells were acquired from 11 normal donors and 31 CML patients at diagnosis in chronic phase and/or after 3-119 months of tyrosine kinase inhibitor (TKI) treatment. Cells were stained with fluorescent monoclonal antibodies and FACS sorted into HSCs (CD34+CD38-), hMICL+ progenitors (MpP; CD34+CD38+hMICL+), and hMICL- progenitors (MnP; CD34+CD38+hMICL-). Sorted cell subsets were subjected to growth in a 14-day methylcellulose assay and analyzed quantitatively for expression of the BCR-ABL fusion transcript.In normal donors, hMICL expression reproducibly identified a well-defined subpopulation of the CD34+CD38+ cells (fig 1A). The MpPs were highly enriched for cells of granulocyte-macrophage progenitors (GMP) phenotype compared to the MnPs (p=0.012) (fig 1B). Sorted MpPs produced almost exclusively granulocyte and/or macrophage (CFU-GM) colonies (median: 92% of colonies), while colonies from MnPs were dominated by BFU-Es (91%) and, as opposed to the MpPs, also contained CFU-GEMM colonies (0.64%) (fig 1C). Thus, hMICL seems to be a useful marker for the GMP population. [Display omitted] In CML at diagnosis we found decreased numbers of MpPs (mean 23% of CD34+CD38+ cells (range: 6.3-48%)), compared to the normal donors (33% (18-48%), p=0.030) (fig 2A). Extraordinarily, the MpP fraction varied considerably in size among CML patients, and 12/23 patients had MpP fractions within the 90% reference range (RR) of normal donors (MpPHIGH patients) and thus distinctly higher than the remaining patients (MpPLOW patients) (fig 2B-C). High MpP fractions significantly correlated with high WBC (Spearman's r = 0.47, p=0.049) (fig 2D), high neutrophil counts (r = 0.55, p=0.043), large spleen size (r = 0.66, p=0.0069), and low hemoglobin at the time of diagnosis (r = -0.58, p=0.014). Within the progenitor compartment, high ratio of BCR-ABL in the MpP to BCR-ABL in the MnP significantly correlated with large MpP fractions (r = 0.54, p=0.021). [Display omitted] During the first 6 months of TKI treatment differing developments in MpP fraction size were observed for MpPLOW and MpPHIGH patients. While MpPLOW patients showed increasing MpP fractions during the first 6 months of treatment (fig 2E), 4/4 and 2/4 MpPHIGH patients displayed a decrease in MpPs at 3 and 6 months, respectively (fig 2F). Thus, in these patients, the majority of the Ph+ progenitor cells being cleared seemed to be GMPs.In conclusion, our data demonstrate that hMICL is an early marker of granulocyte-macrophage differentiation, and provides a readily accessible approach to assessing the GMP population during TKI therapy in CML. Using the present approach we have uncovered a higher degree of variability in the composition of the progenitor compartment at diagnosis than previously reported, and shown that a significant proportion of the patients have expanded GMP populations. Ongoing studies are aimed at determining whether these patients may represent patients with a more advanced form of disease at the time of diagnosis. Disclosures:Stentoft:Novartis: Consultancy, Financial support for relevant congress participation Other, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers-Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Danish Regions: Membership on an entity's Board of Directors or advisory committees. Hokland:Novartis: Research Funding.

Molecular analysis of the BCR-ABL1 kinase domain in chronic-phase chronic myelogenous leukemia treated with tyrosine kinase inhibitors in practice: Study by the Nagasaki CML Study Group

An appropriate trigger for BCR-ABL1 mutation analysis has not yet been established in unselected cohorts of chronic-phase chronic myelogenous leukemia patients. We examined 92 patients after 12 months of tyrosine kinase inhibitor (TKI) treatment in Nagasaki Prefecture, Japan. Univariate analysis revealed that significant factors associated with not attaining a major molecular response (MMR) were the presence of the minor BCR-ABL1 fusion gene, a low daily dose of TKI, and the emergence of BCR-ABL1 kinase domain mutations conferring resistance to imatinib. Factors associated with the loss of sustained MMR were a low daily dose of TKI and the emergence of alternatively spliced BCR-ABL1 mRNA with a 35-nucleotide insertion. Taken together, our results suggest that the search for BCR-ABL1 mutations should be initiated if patients have not achieved MMR following 12 months of TKI treatment.

Predictive value of molecular response after treatment with tyrosine kinase inhibitor for 3 months in patients with chronic myeloid leukemia

To understand the predictive value of early monitoring BCR-ABL transcripts in patients with chronic myeloid leukemia (CML) after treatment with tyrosine kinase inhibitor (TKI), and to provides the information for early assessment of prognosis and treatment options. BCR-ABL transcripts of 53 CML patients before and after TKI treatment were detected by using real-time quantitative RT-PCR. The relationship between BCR-ABL transcripts level after TKI treatment for 3 months and the later molecular response, progression and mutation was analyzed. The median values of BCR-ABL transcripts in peripheral blood samples from 30 newly diagnosed patients were 43.99%, which was used as a baseline of BCR-ABL transcripts for molecular response evaluation. Of 53 patients, 31 (58.49%) had a BCR-ABL mRNA ≤ 4.40% (reduced more than 1 log) and 22 (41.51%) greater than 4.40% (reduced to less than 1 log) after 3 months of TKI treatment. The former 31 patients had a significantly higher 18-months cumulative incidence of major molecular response (MMR) (90.32% vs 18.18%, P=0.000) and 3-year cumulative incidence of complete molecular response (CMR) (48.39% vs 0, P=0.000) compared with the latter 22 patients. The lower BCR-ABL level was, the earlier MMR reached. The proportion of patients with a mutation in group of BCR-ABL mRNA>4.40% was significantly higher than that of BCR-ABL mRNA ≤ 4.40% (22.73% vs 0, P=0.021). The incidence of progression increased in group of BCR-ABL mRNA>4.40%, but the difference was not statistically significant (P=0.052). It is important for the prognosis evaluation of the patients to monitor the level of BCR-ABL transcripts at 3 months after TKI treatment, which might help to early optimization of treatment and to improve curative effect of CML patients.

Homozygosity for killer immunoglobin-like receptor haplotype A predicts complete molecular response to treatment with tyrosine kinase inhibitors in chronic myeloid leukemia patients

Several recent reports suggest a possible role for killer immunoglobulin-like receptors (KIR) in the onset of chronic myeloid leukemia (CML) and response to therapy with tyrosine kinase inhibitors (TKIs). To explore this hypothesis, we studied KIRs and their human leukocyte antigen class I ligands in 59 consecutive patients with chronic-phase CML (mean age, 53 years; range, 23-81 years) and a group of 121 healthy control participants belonging to the same ethnic group as the patients. The 2-year cumulative incidence of complete molecular response, obtained after a median of 27 months (range, 4-52 months), was 51.2%. An increased frequency of the activating receptor KIR2DS1 (pm = 0.05) and a reduced frequency of the KIR-ligand combination KIR2DS2/2DL2 absent/C1 present (pm = 0.001) were significantly associated with CML. Moreover, KIR repertoires in patients appeared to influence response to TKI therapy. Homozygosity for KIR haplotype A (pm = 0.01), a decreased frequency of the inhibitory KIR gene KIR2DL2 (pm = 0.02), and low numbers of inhibitory KIR genes (pm = 0.05) were all significantly associated with achievement of complete molecular remission. These data suggest that a decrease in properly stimulated and activated NK cells might contribute to the occurrence of CML and indicate homozygosity for KIR haplotype A as a promising immunogenetic marker of complete molecular response that could help clinicians decide whether to withdraw treatment in patients with CML.

Sensitivity to Imatinib In BCR-ABL1-Positive Chronic Myeloid Leukemia Cells Depends on the Presence of Normal ABL1.

AbstractAbstract 3405 Background:BCR-ABL1 encoding for oncogenic tyrosine kinase results from t(9;22)(q34;q11) reciprocal translocation or variants generating the Philadelphia chromosome (Ph), which initiates chronic myeloid leukemia in chronic phase (CML-CP). The second (wild-type) ABL1 and BCR alleles in CML-CP cells remain intact on the non-rearranged homologues of chromosome 9 and 22, respectively. Accordingly, CML-CP cells at early stages express both forms of the ABL1 kinase, oncogenic BCR-ABL1 and normal ABL1. ABL1 tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib and nilotinib revolutionized the treatment of CML-CP, but they do not eradicate leukemia. Patients who do achieve complete cytogenetic remission (CCyR) may eventually stop responding and acquire resistance to TKIs, which may lead to disease relapse and malignant progression. Mutations in the sequence encoding BCR-ABL1 kinase have been detected in approximately 50% of CML-CP patients resistant to TKIs, but other factors contributing to this phenomenon are poorly characterized. Results:Here, we identified a novel mechanism of TKI resistance: loss of the remaining normal ABL1 allele resulting from cryptic deletion in the 9q34 region in the normal chromosome 9 [del(9)(q34)]. Using bacterial artificial chromosome probes (BACs) for dual color/dual probe fluorescent in situ hybridization (D-FISH), and oligonucleotide array comparative genomic hybridization (aCGH) we show that genomic deletion of ABL1 allele in non-translocated chromosome 9 acquired during TKI therapy in CML-CP patients was associated with resistance to imatinib and dasatinib. del(9)(q34) was detected in approximately 10% of the patients who initially failed to achieve CCyR within 12 months of TKI treatment. Moreover, BCR-ABL1-positive Abl1-/- murine leukemia cells were refractory to imatinib in comparison to BCR-ABL1–positive Abl1+/+ counterparts as indicated by persistent BCR-ABL1 –mediated tyrosine phosphorylation, lack of BCR-ABL1 protein degradation, increased cell survival and clonogenic activity. Expression of exogenous ABL1 kinase in BCR-ABL1–positive Abl1-/- cells restored their sensitivity to imatinib. These results provide direct evidence that Abl1 plays a crucial role in regulation of the sensitivity of BCR-ABL1-positive leukemia cells to imatinib. ABL1 is regarded as a cell cycle regulatory and pro-apoptotic protein, thus antagonistic to BCR-ABL1. The cell cycle inhibitory activity is independent of ABL1 kinase, whereas the pro-apoptotic function is dependent on its kinase activity. However, acquired resistance to TKIs caused by a loss of the wild-type ABL1 kinase does not appear to depend directly on the lack of ABL1-induced cell cycle arrest and/or apoptosis. In contrast to BCR-ABL1 –positive Abl1+/+ leukemia cells, imatinib exerted only modest effect on BCR-ABL1 kinase-dependent tyrosine phosphorylation and did not downregulate BCR-ABL1 protein in Abl1-/- leukemia cells, suggesting the role of Abl1 in cellular uptake of the drug and/or BCR-ABL1 degradation. Expression of imatinib cellular importer Oct-1 and cellular exporters Abcb1 and Abcg2 does not appear to favor the resistance to TKI in BCR-ABL1-positive Abl1-/- cells, but the impact of ABL1 on intracellular metabolism of imatinib cannot be excluded. In addition, expression of chaperone protein Hsp90, which protects BCR-ABL1 from proteasomal degradation, is not affected by Abl1. However, >3-fold downregulation of cathepsin B may be responsible for lack of degradation of BCR-ABL1 protein in imatinib-treated Abl1-/- cells. Conclusions:Altogether, it can be postulated that loss of expression of ABL1 kinase plays an important role in TKI resistance in CML. It can be achieved by interstitial deletion in chromosome 9 [del(9)(q34)] causing loss of normal ABL1 allele, which could be eventually combined with epigenetic silencing of the alternative ABL1 promoter retained in the CpG island of the BCR-ABL1 gene in t(9;22)(q34:q11). Detection of the del(9)(q34) is beyond the resolution of conventional karyotyping currently used to monitor TKI treatment response. In contrast, D-FISH using commercially available probes can identify such loss in both quiescent and dividing cells. In summary, downregulation of ABL1 caused by del(9)(q34) may serve as an important prognostic factor and have a significant impact on CML treatment. Disclosures:No relevant conflicts of interest to declare.