Long-term Tyrosine Kinase Inhibitors Treatment Research Articles

Understanding the dynamics of TKI-induced changes in the tumor immune microenvironment for improved therapeutic effect

BackgroundThe dynamic interplay between tyrosine kinase inhibitors (TKIs) and the tumor immune microenvironment (TME) plays a crucial role in the therapeutic trajectory of non-small cell lung cancer (NSCLC). Understanding the...

Synergistic lethality in chronic myeloid leukemia – targeting oxidative phosphorylation and unfolded protein response effectively complements tyrosine kinase inhibitor treatment

Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), targeting the BCR::ABL1 oncoprotein. Still, resistance to therapy, relapse after treatment discontinuation, and side effects remain significant issues of long-term TKI treatment. Preliminary studies have shown that targeting oxidative phosphorylation (oxPhos) and the unfolded protein response (UPR) are promising therapeutic approaches to complement CML treatment. Here, we tested the efficacy of different TKIs, combined with the ATP synthase inhibitor oligomycin and the ER stress inducer thapsigargin in the CML cell lines K562, BV173, and KU812 and found a significant increase in cell death. Both, oligomycin and thapsigargin, triggered the upregulation of the UPR proteins ATF4 and CHOP, which was inhibited by imatinib. We observed comparable effects on cell death when combining TKIs with the ATP synthase inhibitor 8-chloroadenosine (8-Cl-Ado) as a potentially clinically applicable therapeutic agent. Stress-related apoptosis was triggered via a caspase cascade including the cleavage of caspase 3 and the inactivation of poly ADP ribose polymerase 1 (PARP1). The inhibition of PARP by olaparib also increased CML death in combination with TKIs. Our findings suggest a rationale for combining TKIs with 8-Cl-Ado or olaparib for future clinical studies in CML.

Improvement in Dasatinib-Induced Proteinuria after Switching to Nilotinib: A Case Report

Tyrosine kinase inhibitors (TKIs), such as imatinib, dasatinib, and nilotinib, have been used to treat chronic myelogenous leukemia (CML). The adverse effects of these TKIs vary according to the site of signaling pathway inhibition. Here, we report a case of dasatinib- induced proteinuria. A 56-year-old Korean woman was diagnosed with CML and treated with dasatinib. However, 3 years later, the patient developed hypertension and microalbuminuria. Losartan was ineffective, so a kidney biopsy was performed, which revealed dasatinib-associated glomerular changes. Subsequently, dasatinib was switched to nilotinib. After 1 month, the spot urine protein/creatinine ratio decreased from 2,985.0 mg/g to 237.8 mg/g. This case of heavy proteinuria developed after long-term TKI treatment and improved rapidly after switching to another TKI. The proposed strategy is important because it eliminates the need to discontinue the medication or use immunosuppressive drugs to treat proteinuria.

Chronic myeloid leukemia: where do we stand, where can we go?

The introduction of BCR-ABL tyrosine kinase inhibitors to the treatment of chronic myeloid leukemia (CML) has significantly changed the long term therapy results. After an initial 12 months of therapy with tyrosine kinase inhibitor (TKI), a 3-log reduction of the BCR-ABL copies number on an international scale is possible in 22–46% of patients, depending on the TKI used. In TKI-responsive patients, long-term TKI treatment results are even better, with the BCR-ABL transcript level decreasing over time, even to the point of becoming undetectable. Therefore, an operational cure can be diagnosed in CML patients with an optimal response to 1 st -line TKI treatment, atherapy duration of longer than 5–8 years, and BCR-ABL transcript level below MR4.0–MR4.5 for a period of more than two years. The latter has been the basis of multiple concepts of permanent or periodic discontinuation of TKI treatment [treatment-free remission (TFR)]. Initial TKI discontinuation clinical trials resulted in satisfactory results, with a disease recurrence rate of c.40–60% after 2–3 years. The mechanism of disease recurrence was then studied , with detailed characterization of the CML stem cells (CML SCs) immunophenotype and the mechanisms of survival and self-renewal under TKI selective pressure. A better understanding of the biology of CML allowed the formulation of new therapy concepts of CML SCs eradication, and new criteria for successful TFR qualification.

Tyrosine kinase inhibitor treatment discontinuation in chronic myeloid leukemia: patient views

Patients with chronic myeloid leukemia (CML) in deep molecular remission may discontinue tyrosine kinase inhibitor (TKI) treatment without relapse. The present study aims to gain insight into the views of CML patients on TKI treatment discontinuation and identify factors that are associated with their willingness to discontinue treatment. A cross-sectional study, among adult Dutch CML patients was conducted to assess willingness and their views on benefits of and concerns about discontinuation. A total of 185 patients participated of whom 76% were willing to discontinue TKI-treatment. Patients considered the absence of side effects the most important benefit whereas fear of disease recurrence was their most prominent concern. Adequate monitoring was the most important prerequisite for TKI-treatment discontinuation. However, ambiguity with respect to perquisites indicate that patients on long-term TKI treatment should be adequately informed both on the possibility to discontinue treatment and on its benefits, risks, and measures that address risks.

A Real-World Assessment of Hepatic Dysfunction Among Chronic Phase Chronic Myeloid Leukemia (CP-CML) Patients Receiving First-Line (1L) Tyrosine Kinase Inhibitors (TKIs) in the United States

Introduction:Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the unregulated proliferation of myeloid cells in the bone marrow and their accumulation in the blood. The approval of imatinib (IM) and second-generation TKIs have substantially improved survival for patients with CML. Despite the effectiveness of these TKIs in controlling CML, continuous therapy is necessary for most patients, and may be associated with the development of unfavorable adverse events such as hepatotoxicity. Although patients with elevated liver function tests are excluded from key clinical trials, case reports and small single-center studies have described cases of TKI-associated hepatotoxicity among patients with CML. This study sought to describe the prevalence and incidence of hepatotoxicity in patients with newly diagnosed Philadelphia chromosome-positive chronic-phase (Ph+ CP) CML treated with 1L TKIs in community oncology practices. Methods:This retrospective, observational cohort study identified adult patients newly diagnosed with Ph+ CP CML and treated with 1L dasatinib (DAS), IM, or nilotinib (NIL) within the US Oncology Network (USON) between 1 January 2008 and 30 November 2018. Patients were required to have at least one comprehensive metabolic panel (CMP) within 90 days of treatment start, and ≥ 1 CMP during follow-up. Additionally, patients had to have at least two visits within the USON, no enrollment in any clinical trial during the study period, and no documentation of other primary cancer diagnoses. Descriptive statistics were generated for baseline patient characteristics and prevalence and incidence of hepatotoxicity, and were stratified by 1L TKI treatment initiation. Grades for hepatotoxicity were derived from laboratory data using the Common Terminology Criteria for Adverse Events version 5. Results:A total of 2743 patients with CP-CML were identified. Of those, 730 (26.6%), 1455 (53.0%), and 558 (20.3%) patients were treated with 1L DAS, IM, or NIL, respectively. With the exception of age and region, patient demographic characteristics were well balanced across cohorts. However, baseline hepatic functioning differed across TKIs (Table 1). Prevalence of any-grade liver enzyme elevation was observed in almost one-third (31.9%) of patients, including 246 (33.7%) patients treated with DAS, 416 (28.6%) patients treated with IM, and 213 (38.2%) patients treated with NIL. Among all patients with normal baseline hepatic function, 48.9% developed any-grade hepatotoxicity while on 1L therapy. Across TKI cohorts, significantly more patients treated with NIL developed any-grade hepatotoxicity (70.1%) compared to patients treated with DAS (45.5%) or IM (43.5%) (P< 0.0001). Among all patients receiving 1L treatment, 1.5% to 3.8% of patients experienced grade 3-4 hepatotoxicity. Significant differences in the proportion of patients that developed grade 3-4 hepatotoxicity were observed across treatments and were most common among patients treated with NIL (Table 2). Conclusions:Findings from this analysis suggest that hepatic dysfunction may be common at baseline among patients with CP-CML treated in real-world community oncology settings. Among patients with normal baseline hepatic function, more patients treated with NIL experienced any-grade hepatotoxicity. Patients treated with NIL were also more likely to develop grade 3-4 hepatotoxicity compared to patients treated with DAS or IM. These findings may provide insight into the effects of long-term TKI treatment on hepatic functioning and help to inform treatment choices for patients. Disclosures Kolibaba: AbbVie:Research Funding;Compass Oncology:Ended employment in the past 24 months;Janssen:Research Funding;TG Therapeutics:Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding;McKesson Life Sciences:Consultancy;Sumitomo Dainippon Pharma Oncology:Consultancy, Other: travel, accommodations, expenses, ;Gilead:Research Funding;Genentech:Research Funding;Cell Therapeutics:Research Funding;Celgene:Research Funding;Acerta:Research Funding;Atara Biotech:Membership on an entity's Board of Directors or advisory committees;Verastem:Honoraria;Seattle Genetics:Research Funding;Novartis:Research Funding;Pharmacyclics:Research Funding.Zhou:McKesson Corporation:Current Employment.Keating:Bristol Myers Squibb:Current Employment.Clark:McKesson Life Sciences:Current Employment, Current equity holder in publicly-traded company.Brokars:Bristol Myers Squibb:Current Employment.Kee:Bristol Myers Squibb:Current Employment.Copher:Bristol Myers Squibb:Current Employment.Stwalley:Bristol Myers Squibb:Current Employment, Current equity holder in publicly-traded company.Jabbour:Adaptive Biotechnologies:Other: Advisory role, Research Funding;Genentech:Other: Advisory role, Research Funding;BMS:Other: Advisory role, Research Funding;Amgen:Other: Advisory role, Research Funding;Pfizer:Other: Advisory role, Research Funding;Takeda:Other: Advisory role, Research Funding;AbbVie:Other: Advisory role, Research Funding.

Assessment of estimated glomerular filtration rate in patients with chronic myeloid leukemia following discontinuation of tyrosine kinase inhibitors.

BCR-ABL1 tyrosine kinase inhibitors (TKIs) have dramatically improved survival outcomes in patients with chronic phase chronic myeloid leukemia (CML-CP) and are associated with a manageable safety profile. However, long-term TKI administration can lead to cardiovascular or renal adverse events. One goal in discontinuation of TKIs was reduction of adverse events, but it is unclear whether chronic toxicities are ameliorated as a result. In this study, we evaluated changes in estimated glomerular filtration rate (eGFR) in patients with CML-CP before and after TKI discontinuation. Long-term TKI treatment appears to induce renal toxicity, as eGFR at the time of TKI discontinuation correlated with the duration of TKI treatment (r = - 0.478, p = 0.005). Patients who received imatinib as first-line treatment exhibited lower eGFR levels than those treated with dasatinib or nilotinib, which may be correlated with long-term treatment (p = 0.027). After TKI discontinuation, no significant increases in eGFR were seen either in patients with treatment-free remission (66.8-71.2ml/min/1.73 m2) or molecular relapse (64.8-68.7ml/min/1.73 m2, p = 0.666). These data indicate that TKI-induced renal toxicities are associated with long-term TKI treatment, and may be irreversible even following treatment discontinuation.

Treatment-free remission in patients with chronic myeloid leukemia: literature review

Tyrosine kinase inhibitors have radically changed the course of chronic myeloid leukemia, significantly increasing survival and reducing the risk of disease progression. Nearly 50–70 % of patients achieve a consistently low or undetectable level of minimal residual disease – a deep molecular response. The long-term tyrosine kinase inhibitors treatment in about one-third of patients is accompanied by toxicity which impairs the quality of life. Therefore, the safe treatment discontinuation is relevant. The results of clinical trials have shown 40-60% possibility of maintaining treatment-free remission in patients with long-term deep molecular respons; however, all patients with molecular relapse regain molecular remission after the resumption of tyrosine kinase inhibitors therapy. Currently, clinical and biological factors associated with maintaining treatment-free remission are being studied. It is assumed that cessation of tyrosine kinase inhibitors therapy can improve the quality of life, but approximately 30 % of patients are reporting musculoskeletal pain – so called “withdrawal syndrome” – that begins or worsens after stopping tyrosine kinase inhibitors therapy. The mechanisms for the development of this phenomenon are currently unclear. Thus, many aspects concerning treatment-free remission require to be studied, which determines the importance of clinical trials in this area.

Renal dysfunction in patients with radioactive iodine-refractory thyroid cancer treated with tyrosine kinase inhibitors: A retrospective study.

In 2014/2015, tyrosine kinase inhibitors (TKIs) were introduced as a secondary treatment for refractory differentiated thyroid cancer (DTC) in Japan. While renal dysfunction is an adverse event of TKI, data on this adverse event in TKI-treated DTC remains insufficient. Here, we investigated renal function in patients undergoing TKI treatment for DTC and evaluated the efficacy of dose reduction/withdrawal for cases of renal dysfunction.A total of 73 cases of radioactive iodine-refractory DTC treated with sorafenib (n = 22) or lenvatinib (n = 51) were included. Patient data evaluated were TKI treatment period, estimated glomerular filtration rate (eGFR) before and after TKI therapy, incidence and degree (maximum value at time of TKI treatment) of proteinuria, and albumin levels before and after TKI therapy were compared.The mean ΔeGFR was −6.75% with lenvatinib and +5.90% with sorafenib. It was not significant (P = .15). The mean Δalbumin was −8.90% and −5.85% with lenvatinib and sorafenib, respectively; there was no significant difference between the lenvatinib and sorafenib groups (P = .77). According to our program of TKI dose reduction and withdrawal, all patients except 2 with diabetes were successfully continuing treatment.Overall, the present results demonstrated that renal function is negatively affected by long-term TKI treatment for RAI-refractory DTC. However, heightened proteinuria, decreased eGFR and albumin levels, and significant but apparently reversible renal dysfunction were more frequent with lenvatinib than sorafenib.

Vascular adverse events of ponatinib during treatment of Philadelphia chromosome-positive leukemia: a retrospective single-institution analysis

Ponatinib (PON) is a key drug for patients with second tyrosine kinase inhibitor (TKI)-resistant/intolerant Philadelphia chromosome-positive leukemia (Ph+ leukemia); however, the occurrence of vascular adverse events (VAEs) in patients treated with PON should be carefully monitored. A retrospective analysis involving seven patients treated with PON was conducted to elucidate the incidence rate and risk factor for the development of VAEs. In the present study, risk assessment and monitoring of VAEs were performed using SCORE Risk Chart and Suita Score (10-year risk for fatal cardiovascular event), respectively. Despite the prophylactic use of aspirin, cerebral infarction and unstable angina occurred in two patients. By contrast, deep vein thrombosis did not improve in a patient treated with edoxaban. Our data suggest that patients with Ph+ leukemia possessing risk factors, medical history of lifestyle diseases, and administration of long-term second TKI treatment require careful monitoring of VAEs and therapeutic intervention to lifestyle diseases.

Telomere shortening correlates with leukemic stem cell burden at diagnosis of chronic myeloid leukemia

AbstractTelomere length (TL) in peripheral blood (PB) cells of patients with chronic myeloid leukemia (CML) has been shown to correlate with disease stage, prognostic scores, response to therapy, and disease progression. However, due to considerable genetic interindividual variability, TL varies substantially between individuals, limiting its use as a robust prognostic marker in individual patients. Here, we compared TL of BCR-ABL−, nonleukemic CD34+CD38− hematopoietic stem cells (HSC) in the bone marrow of CML patients at diagnosis to their individual BCR-ABL+ leukemic stem cell (LSC) counterparts. We observed significantly accelerated telomere shortening in LSC compared with nonleukemic HSC. Interestingly, the degree of LSC telomere shortening was found to correlate significantly with the leukemic clone size. To validate the diagnostic value of nonleukemic cells as internal controls and to rule out effects of tyrosine kinase inhibitor (TKI) treatment on these nontarget cells, we prospectively assessed TL in 134 PB samples collected in deep molecular remission after TKI treatment within the EURO-SKI study (NCT01596114). Here, no significant telomere shortening was observed in granulocytes compared with an age-adjusted control cohort. In conclusion, this study provides proof of principle for accelerated telomere shortening in LSC as opposed to HSC in CML patients at diagnosis. The fact that the degree of telomere shortening correlates with leukemic clone's size supports the use of TL in leukemic cells as a prognostic parameter pending prospective validation. TL in nonleukemic myeloid cells seems unaffected even by long-term TKI treatment arguing against a reduction of telomere-mediated replicative reserve in normal hematopoiesis under TKI treatment.

Management of cardiovascular complications in CML patients treated with tyrosine kinase inhibitors

Tyrosine kinase inhibitors (TKIs) have significantly improved the clinical outcomes of patients with chronic myeloid leukemia (CML). However, patients with CML need to receive TKI therapy for several years. Hence, the safety of long-term TKI treatment warrants utmost attention. Among various adverse events caused by TKI therapy, cardiovascular events (CVEs), such as acute myocardial infarction, cerebral infarction, and pulmonary hypertension, are the most serious with high mortality. TKIs inhibit various off-target molecules involved in the occurrence of CVEs such as c-Kit, platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), and Tie-2/Tec. At present, nilotinib, dasatinib, and ponatinib, but not bosutinib, have been demonstrated to increase the risk of CVEs in patients with CML compared with imatinib. Conversely, patients with CML have also been shown to have high CVE risks regardless of the TKI treatment compared with non-cancer population. Hence, further analyses are required to deduce the influence of TKI treatment on CVEs. Whatever the cause may be, to prevent CVEs in patients with CML, we need to appropriately screen and monitor CVE risks such as hypertension, serum glucose, lipid levels, ankle-brachial index (ABI), Electrocardiography (ECG), and echocardiography before and during TKI treatment.

Comparative analysis of pulmonary hypertension in patients treated with imatinib, nilotinib and dasatinib.

Pulmonary hypertension (PH) is a rare, but life-threatening, adverse event in patients treated with tyrosine kinase inhibitors (TKIs), such as dasatinib, but has not been fully evaluated in patients treated with imatinib or nilotinib. We used echocardiography to noninvasively assess the incidence of PH in 105 patients with chronic myeloid leukaemia (CML) treated with imatinib (n=37), nilotinib (n=30) or dasatinib (n=38). The mean triscupid regurgitation peak gradient (TRPG), which reflects pulmonary arterial pressure, was 22·7mmHg in the imatinib group, 23·1mmHg in the nilotinib group and 23·4mmHg for dasatinib group. These values were not significantly different, but higher than those (19·0mmHg) in newly diagnosed CML patients. A TRPG>31mmHg, marking possible PH onset, was detected in 9 of 105 patients: one (2·7%) treated with imatinib, three (10·0%) with nilotinib and five (13·2%) with dasatinib. Only three patients complained of dyspnoea, whereas the other six were asymptomatic. In addition, there was a tendency toward correlation of TRPG value and age or TKI treatment duration. These results suggested that treatment with not only dasatinib, but also imatinib and nilotinib, can be associated with subclinical PH. Noninvasive echocardiography is useful for screening, especially in older patients with long-term TKI treatment.

Prevalence and Risk Predictive Factors of Comorbid Malignancies in Patients with Chronic Myeloid Leukemia

Introduction: As chronic myeloid leukemia (CML) patents are generally diagnosed at old age and live longer by active use of BCR-ABL1 tyrosine kinase inhibitors (TKIs), the occurrence of other malignancy (OM) is becoming a critical issue as a long-term comorbidity. An increased rate of OM has been reported in myeloproliferative disorders and long-term TKI treatment may induce OM in CML. To explore exact prevalence and characteristics of OM, we reviewed medical records of CML patients and compared with those of age-matched Korean population.Methods: The medical records of 1,469 CML patients who diagnosed between January 2000 and December 2014 were reviewed using Korean data-set of Asia CML Registry (ACR). With a cut-off date of July 2016, age-standardized prevalence rates (A-SPR) of OM (except benign tumors and other leukemias) were analyzed and compared with that of general population in Korea Central Cancer Registry (KCCR). In addition, we analyzed cumulative incidence rate of OM and various risk factors.Results: The median duration of follow-up was 84 (1-197) months, and 96 CML patients had at least one OM. Forty three patients had a history of OM before a median 69 (1-161) months of CML diagnosis and 53 patients developed OM after a median 53 (range; 0.2-172) months of CML diagnosis. The OM included 32 thyroid cancers, 19 colorectal cancers, 16 stomach cancers, 9 breast cancers, 4 gynecological cancers (3 cervical cancers and 1 uterine endometrial cancers), 3 lymphoma (2 non-Hodgkin’s lymphoma and 1 Hodgkin’s lymphoma), 3 biliary cancer, 3 skin cancers, 3 prostate cancers, 2 lung cancer, 2 tongue cancer, 2 liver cancer, 2 esophageal cancer, 1 pancreatic cancer, and 1 bladder cancer.A-SPR of OM was 1.7 times higher in CML patients. Hodgkin’s lymphoma (8.7 times), thyroid cancer (2.6 times), biliary cancer (2.6 times), colorectal cancer (2 times), non-Hodgkin’s lymphoma (1.8 times), cervical cancer (1.8 times), and breast cancer (1.6 times) had a higher A-SPR. On the other hands, skin cancer (3.3 times), lung cancer (2 times), and liver cancer (2 times) were lower than that of general population. With 53 patients who had OM after CML diagnosis, we analyzed the cumulative incidence. The risk of OM was increased over the follow-up period (2.7% at 7 years) Univariate analysis revealed that patients who were more than 37 years old at CML diagnosis (4.3% vs. 0.4%, p<0.001) and who had family history of cancer (8.2% vs. 2.3%, p=0.002) were associated with a higher OM. After adjusting for factors, multivariate analysis showed that older age (HR of 4.19, P<0.001) and family history (HR of 3.17, P=0.001) were independently associated with increased risk. There was no difference in 7-year overall survival (OS) between patients with OM (n=96) and without OM (n=1,373) (84.9% vs. 86.9%, p=0.573). However advanced cancer stages (stage 3 and 4) of OM significantly affected poor OS ( 88.3% vs. 65.6% P=0.0406).Conclusion: Although comorbid malignancies did not significantly affect CML survival, poor survival in advanced stages and the high risk of other cancers warn the need of systematic screening in long-term CML survivors. In addition, the specific cancer types with a significantly higher A-SPR should be considered for further studies including genetic mechanisms. DisclosuresKim:ILYANG: Consultancy, Honoraria, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Long-Term Treatment with Imatinib Is Associated with Decreased Estimated Glomerular Filtration Rate and Hemoglobin Level in Patients with Chronic Myelogenous Leukemia

▪Background: Among tyrosine kinase inhibitors (TKIs), imatinib mesylate was the first TKI successfully used for the treatment of chronic myelogenous leukemia (CML) in chronic phase (CP), and a majority of patients still remains on its long-term treatment. Although imatinib has been well tolerated in clinical practice, and the side effect profile has usually been mild to moderate, there are limited data available regarding the long-term TKIs treatment on kidney function and associated complications such as anemia. This study aimed to evaluate the effect of long-term imatinib treatment on estimated glomerular filtration rate (eGFR) and hemoglobin (Hb) level in patients with CML in CP.Patients and methods: By using the institutional database, patients with CML in CP who had been on imatinib as the first and the only TKI for over 5 years at Keio University Hospital (Tokyo, Japan) were selected and retrospectively analyzed. Estimated GFR was calculated by the Modification of Diet in Renal Disease equation for Japanese defined by the Japanese Society of Nephrology. All statistical analyses were performed with EZR, which is a graphical user interface for R.Results: Eighty-two patients were evaluable. The median age at initiating imatinib was 49.5 years (range, 19-76). The median duration of imatinib treatment was 105 months (range, 60-170). During this study period, imatinib was given at a dose of 400mg/day. The dose reduction was indicted in 8 patients but not because of progressing renal impairment. The mean eGFR was 77 ml/min/1.73m2 (range, 38-120), and the value was below 60 ml/min/1.73m2 in 12 patients before initiating imatinib. The mean value significantly decreased to 62 ml/min/1.73m2 (range, 34-98) over the 5 years after imatinib treatment (P<0.001), and the values reached <60 ml/min/1.73m2 in 43 of 82 patients (P<0.001). In an univariate analysis of patients excluding 12 patients with below 60 ml/min/1.73m2, older age and lower eGFR value at the initiation of imatinib were associated with later development of chronic kidney disease (<60 ml/min/1.73m2) (P<0.001 and 0.002, respectively). Mean Hb level at 5 years after starting imatinib was significantly lower as compared with that before (12.9+1.7 g/dl vs. 12.4+1.3 g/dl, P<0.01). The declining rate of eGFR was negatively correlated with those of Hb levels (correlation coefficient -0.249, P<0.05). In 20 patients with low Hb level (9.7+1.2 g/dl) and renal dysfunction, median serum erythropoietin (EPO) level was 31.9 mIU/ml (range, 9.1-119). Furthermore, 11 patients with eGFR<60 ml/min/1.73m2 achieving a durable molecular remission took part in an institutional TKI discontinuation trial. At 1 year after discontinuing imatinib, their mean eGFR values significantly improved (50.0 + 6.5 to 56.0 + 10.2 ml/min/1.73m2, P<0.05) as well as Hb level (12.0 + 1.7 to 14.0 + 1.6 g/dl, P<0.01).Conclusion: Our findings indicated that long-term use of imatinib is frequently associated with reversible but continuous decline in eGFR level, which could lead to anemia partly due to inadequate production of EPO. Although the degree of nephrotoxicity is usually mild, close monitoring of renal function is recommended particularly in older patients with pre-existing renal dysfunction. DisclosuresSakurai:Celgene: Honoraria. Karigane:Celgene: Honoraria. Matsuki:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Nippon Shinyaku: Honoraria. Kikuchi:Celgene: Honoraria; Takeda Pharmaceutical Company: Honoraria; Kyowa Hakko Kirin: Honoraria. Okamoto:Nippon Shinyaku Co., Ltd.: Research Funding; Alexion Pharmaceuticals, Inc.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Asahi Kasei Pharma Corp.: Research Funding; Teijin Pharma Limited: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Shionogi & Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Pfizer Inc.: Honoraria, Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding.

Modulation of Leukotriene Signaling Affecting Cell Growth in Chronic Myeloid Leukemia: A Key Pathway to Cure?

▪Background: Recent data suggest that tyrosine kinase inhibitor (TKI)-insensitive leukemic stem cells often prevail in chronic myeloid leukemia (CML) patients subjected to long-term TKI treatment. To achieve cure additional aberrant pathways in these patients, such as leukotriene (LT) signaling, may need to be targeted. We have previously shown that human CML cells have an increased capacity to produce LT and that LT can stimulate normal myelopoiesis (Tornhamre ExpHem 2003). Chen later noted a striking up-regulation of ALOX5/5LO (catalyzing an initial step in LT formation) in CML mice, and that inhibition of this enzyme improved survival of these animals by a magnitude similar to that induced by imatinib (Chen NatureGen 2009). Here wexve assessed the effect of LT modulating agents on the growth of human CML cells in vitro.Materials and methods: Human CML cells, derived from the three cell lines K562, Kcl22 and KU812, were grown in microtitre plate cultures, in the presence of RPMI 1640. The MTT technique was used to evaluate the number of viable cells at 72 hours. Protein expression was assessed by Western blot and immunocytochemistry.Results: Several specific modulators of LT-signaling were capable of inducing dose-dependent growth inhibitory effects on CML cells. Thus, the cysteinyl-LT receptor antagonist montelukast, a drug with approved clinical use in human asthma, was shown to reduce the growth of all three tested CML cell lines. In Fig. 1 this is examplified by K562 cells, where also additive effects between montelukast and imatinib are indicated. All cell lines expressed the cysLT1-receptor. Furthermore, the LTB4 receptor antagonist etalocib, as well as the 5-LO activating protein (FLAP) inhibitors licofelone and Bay-X-1005, also executed inhibitory effects at concentrations considered as physiological. As expected, addition of the TKIs imatinib, nilotinib or dasatinib to the cultures also generated dose-dependent inhibitory effects on the growth of all tested CML cell lines. An LD50 of approximately 0.5 µM, 10 nM and 0.1 nM was noted for imatinib, nilotinib and dasatinib, respectively, for K562 cells at 72 hours.Conclusions: We demonstrate that several compounds, known to specifically inhibit leukotriene signaling by different mechanisms, were at clinically relevant concentrations capable of significantly suppressing the growth of human CML cells. Some of these compounds are already in clinical practice for non-hematological disorders. They could provide an additional therapeutic possibility in the quest to cure CML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CD93 Is a Novel Biomarker of Leukemia Stem Cells in Chronic Myeloid Leukemia

The introduction of BCR-ABL tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of chronic myeloid leukemia (CML). However, although the majority of patients with chronic phase (CP)-CML obtain durable complete cytogenetic and major molecular responses, there is low level disease persistence postulated to be due to a population of TKI-insensitive leukemia stem cells (LSC). The aims of this study were (1) to fully characterize differences in gene expression between normal hematopoietic stem cells (HSC) and CP-CML LSC and (2) identify potential novel therapeutic targets specific to CML LSC.Lin-CD34+CD38- CD45RA-CD90+ normal HSC (n=3) and CP-CML LSC (n=6 patients at diagnosis), populations were isolated using a FACSAria and applied to Affymetrix HuGene 1.0ST arrays. The raw data (.CEL files) was imported into Partek Genomics Suite and Ingenuity Pathway Analysis software and principal component analysis and gene ontology ANOVA performed. A total of 1217 genes were significantly deregulated between normal HSC and CP-CML LSC. The most significantly deregulated genes and pathways were involved with the molecular and cellular functions of cell cycle, cell assembly and organisation, cellular movement, cell death and DNA replication, recombination and repair. These results suggested that CML LSC were less quiescent than normal HSC. Importantly, complimentary functional studies indicated that CML LSC have significantly increased proliferation (14 fold expansion; P<0.001) compared to normal HSC (no expansion) after 5 days in vitro culture. In addition, equivalent numbers of CML LSC produce ~4-fold more colonies in colony forming cell (CFC) assays than normal HSC (329±56 versus 86±17 per 2,000 cells, respectively; P<0.05). Fluorescence in situ hybridisation (FISH) demonstrated that >90% of lin- CD34+ CD38- CD45RA- CD90+ CML LSC from all patient samples were BCR-ABL positive (+).In addition to these deregulated intracellular pathways, we sought to assess if there were differences in expression of cell surface molecules that may be amenable to therapeutic manipulation. Of particular interest, our microarray studies demonstrated that CD93 was highly upregulated in CP-CML LSC (6 fold, p = 2.5x10-6). Increased CD93 expression was validated by Fluidigm digital PCR (6 fold increase, p = 0.02; n=6). Furthermore, using flow cytometry, we demonstrated significant upregulation of CD93 protein expression on lin-CD34+ CD38- CD45RA- CD90+ CML LSC from peripheral blood and bone marrow of CP-CML patients (n= 17; mean = 63.8% CD93+) compared to normal HSC from healthy peripheral blood stem cell donors (n=7; mean = 0.8% CD93+) and bone marrow donors (n=4; mean = 0.2% CD93+; p < 0.0001). FISH confirmed that 100% of lin-CD34+ CD38- CD90+ CD93+ CML cells were BCR-ABL+ in all samples assessed.CD93 (also known as C1qRp) is a C-type lectin-like domain (CTLD)-containing glycoprotein which regulates phagocytosis, with roles in cell adhesion and leukocyte migration. It is normally expressed on endothelial cells, hematopoietic precursors and mature cells including neutrophils, monocytes and platelets. Previous studies have shown CD93 to be upregulated in a proportion of AML patients (Saito et al, Sci Transl Med, 2010. 2(17): p. 17ra9).Short term (24h) in vitro exposure of lin-CD34+CD38- CD45RA- CD90+ CML LSC to TKIs (Imatinib or Dasatinib; n=3) reduced, but did not fully eliminate CD93 expression (Imatinib, 48.5% to 22.9%; Dasatinib, 47.7% to 9.2%). Importantly, following long-term TKI treatment of patients, lin-CD34+CD38-CD45RA-CD90+ cells from CP-CML patient bone marrow samples (n=2) taken in major molecular response demonstrated a small, but persistent population of CD93+ LSC which were BCR-ABL+ by FISH. Furthermore, in xenograft transplantation experiments (n = 5), after 16 weeks, CD34+CD93+ CML LSC engrafted lethally irradiated NOD/SCID/IL-2Rg-/- (NSG) mice with BCR-ABL+ cells, whereas CD34+CD93- cells from the same patient samples failed to engraft to significant levels (3.5-30 fold increase in engraftment with CD34+CD93- cells; p < 0.03). FISH confirmed that engrafted human cells were BCR-ABL+.Taken together, our results identify CD93 as a potential novel biomarker of CML LSC, which may also be helpful in assessing minimal residual disease at the LSC level. Further studies are ongoing to assess the therapeutic potential of inhibiting CD93 in CML LSC. DisclosuresCopland:Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Long-term imatinib treatment does not cause testicular toxicity in male adolescents with chronic myeloid leukemia and in a juvenile rat model.

The impact of exposure to the tyrosine kinase inhibitor (TKI) imatinib (IMA) on the male reproductive endocrine system is still discussed controversially. We therefore investigated testosterone (Testo) and inhibin B (InB) in blood serum from male adolescents with chronic myeloid leukemia (CML) under long-term TKI treatment. Also long-term exposure to TKIs was studied in a juvenile rat model. Serum was collected at 3 months intervals from 13 boys (age: 7.8-18.9 years, median: 12.8 years) with CML receiving TKI treatment over 3-58 months (median: 18 months). 4 weeks (w) old male rats were exposed, either chronically or intermittently, via the drinking water to a standard (SD) and a high dose (=2-fold SD) of IMA, dasatinib (DASA), or bosutinib (BOSU) over a 10 w period. Controls received water only. Animals were sacrificed after 2 w (prepubertal), 4 w (pubertal), and 10 w (postpubertal) of exposure. Testo and InB serum levels were measured by ELISA. Boys exhibited Testo and InB levels within normal age-related reference ranges and no pattern of rising or falling levels during TKI treatment could be observed. In rats, Testo levels under IMA exposure tended to be non-significantly lowered at postpubertal age compared to controls while no significant differences were found under DASA and BOSU exposure. Animals' InB levels did not significantly differ from controls for all TKIs, at all doses, and by all application schemes tested. With the limitation that the number of individuals tested was rather small, testicular toxicity due to TKI seems unlikely as no alterations of Testo and InB blood levels neither in male adolescent patients nor in rats under long-term TKI exposure was observed.

Skeletal Effects of the Tyrosine Kinase Inhibitors Imatinib, Dasatinib, and Bosutinib in Young Rats

Abstract 4429 Background:In patients with chronic myeloid leukemia (CML) tyrosine kinase inhibitors (TKIs) such as imatinib (IMA), dasatinib (DASA), and bosutinib (BOSU) are used to selectively inhibit the oncogenic BCR-ABL tyrosine kinase. However, these TKIs also exhibit off-target effects on other kinases (e.g. PDGFR, c-FMS) involved in the regulation of bone metabolism. In adult patients with CML, disturbances in bone structure and metabolism are observed during IMA therapy, while in pediatric patients longitudinal growth retardation represents a major concern. Here, we compared the skeletal effects of chronically administered IMA, DASA, and BOSU in young, growing rats. Methods:From age 4 weeks to age 14 weeks, groups (n=10 each) of male Wistar rats were chronically exposed via the drinking water to concentrations representing i) standard dose and ii) two-fold dose of the standard dose of IMA (i: 1 mM and ii: 2 mM), DASA (i: 50 μM and ii: 100 μM), or BOSU (i: 50 μM and ii: 100 μM). Additional groups received the high doses of the TKIs tested in an intermittent fashion over 3 consecutive days per week. Controls were left untreated. Three to four rats of each group were sacrificed after 2 weeks (prepubertal), 4 weeks (pubertal), or 10 weeks (postpubertal) of exposure, respectively. Biochemical bone markers were analyzed in serum. Bone length of femora and tibiae was determined with calipers. Trabecular (trab) and cortical (cort) bone mineral density (BMD) and cortical thickness were assessed by pQCT. Bone strength of femora was measured by a 3-point bending test. Results:Continuous exposure to IMA and DASA led to dose dependent reductions in femoral and tibial length. No such effect was observed under BOSU exposure. Intermitted exposure of all 3 TKIs tested reduced effects on femoral and tibial length. IMA and DASA lowered femoral and tibial trab BMD in prepubertal rats. At pubertal age, rats receiving high dose IMA showed reduced femoral and tibial trab BMD compared to controls, while no such effect was observed for DASA and BOSU. Postpubertally, femoral and tibial trab BMD did not differ from controls in all groups. Moreover, TKI exposure did not affect femoral or tibial cortical BMD and cortical thickness at any dose and age tested. The bone resorption marker tartrate resistant acidic phosphatase (TRACP) was reduced in rats receiving continuously high dose of IMA at all time points tested, and also in postpubertal rats receiving 50 μM and 100 μM of BOSU. The bone formation marker osteocalcin was reduced in all groups and at all time points under IMA exposure, whereas no such effect was observed for DASA and BOSU. Femoral mechanical properties did not differ from controls pre- and pubertally for all 3 TKIs tested. However, high dose IMA reduced femoral breaking strength in postpubertal rats (Fig.1). DASA or BOSU did not cause changes in biomechanical bone strength. Conclusion:The juvenile rat model is an appropriate model to examine side effects of long-term exposure of TKIs on growing bones. Compared with IMA and DASA, BOSU seems to exert a less negative effect on the bone parameters tested. Given the limited pediatric experience with TKI, regular monitoring of skeletal side effects under long-term TKI treatment is warranted. Moreover, skeletal status and fracture rates should be systematically analyzed in patients under IMA treatment. Acknowledgment:Supported by grant DFG SU122-3/1 to MS, DFG HO 1875/10-1 to LCH, and FWF I 734 to RGE. [Display omitted] Disclosures:No relevant conflicts of interest to declare.