To Tyrosine Kinase Inhibitors Treatment Research Articles

Drug-Insensitive CML Stem/Progenitor Cells Highly Express Key Regulators of the Hedgehog Pathway and Inhibition of the Hedgehog Pathway Enhances Their Response to TKI Treatment

Chronic myeloid leukemia (CML) stem/progenitor cells are relatively insensitive to tyrosine kinase inhibitor (TKI) monotherapies, and may not exclusively rely on BCR-ABL activity for survival. This population rapidly generates therapy-resistant clones and is often responsible for relapse after TKI therapy discontinuation. It has been reported that Hedgehog (HH) signalling in hematopoietic stem/progenitor cells is essential for CML induction by BCR-ABL in a mouse model. Inhibition of Smoothened (SMO), a key regulator of the HH pathway, reduces the number of BCR-ABL+ stem cells in vitro and slows disease progression in vivo. However, it is not known if SMO and other key regulators are differentially expressed in stem/progenitor cells derived from imatinib (IM)-nonresponders and IM-responders. If so, they may serve as potential biomarkers and/or therapeutic targets. We have assessed gene expression changes in the HH pathway using RNA sequencing (RNA-seq) in 6 highly purified CD34+ stem/progenitor samples from newly-diagnosed chronic phase (CP)-CML patients. After IM therapy was initiated, 3 patients were classified retrospectively as IM-responders, and 3 as IM-nonresponders. This study identified 27 differentially expressed HH genes between healthy and CML patients (>1.5-fold). In particular, SMO and GLI2, key components of the HH pathway,were upregulated in CP-CML patients relative to healthy bone marrow (HBM) controls (n=3).In particular, GLI2 was highly overexpressed in CP-CML compared to HBM (48-fold upregulated in IM-responders and 166-fold upregulated in IM-nonresponders). To validate the RNA-seq results, expression of the principal HH genes (PTCH1, GLI1, GLI2, SMO) was assessed in 18 CD34+ CP-CML patient samples and 8 HBM samples using qRT-PCR. Interestingly, the expression levels correlated with patients’ TKI resistance status, with IM-nonresponders expressing significantly higher levels of SMO (p<0.01)and GLI2 (p<0.05) compared with IM-responders. GLI2 and SMO expression was then assessed in CD34-subpopulations in 5 IM-responders and 4 IM-nonresponder samples. GLI2 was exclusively and highly expressed in the most primitive population (lin-CD34+CD38-), while its expression was very low in more mature populations (lin-CD34+38+ and CD34-). This effect was amplified in IM-nonresponder samples, where GLI2 is greater than 40-fold upregulated in the lin-CD34+CD38- subpopulation compared with IM-responders. In contrast, SMO was more uniformly expressed in both stem and progenitor subpopulations, but was more highly expressed in IM-nonresponders than IM-responders.These results suggest that key regulators of the HH pathway are highly expressed in IM-nonresponder stem cells and that these cells may be more responsive to SMO inhibition than IM-responder cells.We then performed a viability and apoptosis analysis of 5 CD34+ CP-CML patient samples. IM-nonresponder samples were more sensitive to SMO inhibition using a highly selective SMO inhibitor (PF-04449913) compared with IM-responders, and this effect was enhanced in combination with the new TKI bosutinib (BOS). To investigate the effects of BOS and PF-04449913 on CML progenitor cells, colony forming cell assays were performed on CD34+ CP-CML cells from 4 IM-responders and 3 IM-nonresponders. Overall, combination treatment with BOS and PF-04449913 only modestly reduced colony forming ability compared to either agent alone. The inhibitory effects of combination therapy were, however, strikingly enhanced in a replating experiment, resulting in a greatly reduced replating efficiency compared to BOS treatment alone (15% vs. 30%), suggesting that the combination specifically targets primitive CP-CML cells. Taken together, we have demonstrated that both SMO and GLI2 are highly expressed in primitive CP-CML cells, and that the transcript levels of GLI2 are significantly increased in CP-CML stem cells from IM-nonresponders, which may serve as a new biomarker to predict patient TKI responsivity. We also found that IM-nonresponder stem/progenitor cells are more sensitive to SMO suppression in short- and long-term assays compared with IM-responders, suggesting that HH pathway activation may comprise a potential mechanism of resistance during TKI therapy, and that dual inhibition of the BCR-ABL and HH pathways may constitute a rational approach to abrogate drug resistance and disease progression in CML. DisclosuresWoolfson:Pfizer Inc: Employment.

Factors Associated With Tyrosine Kinase Inhibitor Initiation and Adherence Among Medicare Beneficiaries With Chronic Myeloid Leukemia.

Purpose There is substantial concern surrounding affordability of orally administered anticancer therapies, particularly for Medicare beneficiaries. We examined rates of initiation and adherence to tyrosine kinase inhibitors (TKIs) among Medicare beneficiaries with chronic myeloid leukemia (CML) with and without cost-sharing subsidies. We selected TKIs given their effectiveness and strong indication for use among patients diagnosed with CML. Patients and Methods Using SEER-Medicare data, we identified individuals diagnosed with CML from 2007 to 2011. We used Cox proportional hazards regression to assess time from diagnosis to TKI initiation. We used generalized estimating equations to examine treatment initiation within 180 days and TKI adherence among initiators. We defined adherence as at least 80% of days covered during the 6 months after TKI initiation. Results Among 393 individuals diagnosed with CML from 2007 to 2011, 68% initiated TKI treatment within 180 days after diagnosis. In multivariate analysis, individuals with cost-sharing subsidies, younger age, lower comorbidity, and later year of diagnosis were significantly more likely to initiate TKIs. Among TKI initiators, 61% were adherent; adherence was lower for individuals age 80 years or older versus 66 to 69 years. Conclusion Only 68% of Medicare beneficiaries with CML initiated TKI therapy within 6 months of diagnosis. Delayed initiation among individuals without cost-sharing subsidies suggests that out-of-pocket costs may be a barrier to timely initiation of therapy among individuals diagnosed with CML.

Effectiveness of tyrosine kinase inhibitors on uncommon E709X epidermal growth factor receptor mutations in non-small-cell lung cancer.

BackgroundClinical features of epidermal growth factor receptor (EGFR) mutations: L858R, deletions in exon 19, T790M, insertions in exon 20, G719X, and L861X in non-small-cell lung cancer (NSCLC) are well-known. The clinical significance of other uncommon EGFR mutations, such as E709X, is not well understood. This study aimed to improve the understanding of E709X, and the clinical response to tyrosine kinase inhibitors (TKIs) of NSCLC patients with such an uncommon mutation.MethodsSpecimens from 3,146 patients were tested for EGFR mutations. We surveyed the clinical data and the effectiveness of TKI treatment in NSCLC patients with EGFR mutations E709X.ResultsOf the 3,146 patients, 1,689 (53.7%) had EGFR mutations. This included 726 patients with deletions in exon 19, 733 patients with L858R, and 230 (13.6%) patients with other EGFR mutations. In the 230 patients who had mutations other than single deletion in exon 19 or single L858R in exon 21, 25 (1.5%) patients had the uncommon E709X mutations. Twenty patients had complex E709X mutations and five had single E709X mutation: delE709-T710insD. Of these 25 patients, 18 received either gefitinib or erlotinib treatment. The response rate of TKIs treatment was 50.0%, and the median progression-free survival was 6.2 months. All 5 patients who had delE709-T710insD were non-responders to TKI treatments.ConclusionE709X EGFR mutations constituted a small part of the whole group of EGFR mutations. Most patients had complex mutations. The mutation delE709-T710insD was a single mutation and was not associated with good response to TKI treatment.

Abstract 3964: Large scale noninvasive screening of EGFR mutations in advanced NSCLC patients: the Identify platform experience

Abstract Background: Activating mutations in the epidermal growth factor receptor gene (EGFR) confer sensitivity to tyrosine kinase inhibitors (TKIs) in patients with advanced non-small-cell lung cancer (NSCLC). Starting in June 2012, a nationwide platform (the Identify Blood Platform) was implemented in Spain for large-scale screening of EGFR mutations at presentation in the peripheral blood of unselected, advanced NSCLC patients. The Platform was restricted to patients with no tumor biopsy available or those with insufficient tumor tissue for genetic analyses. In May 2015, the Platform was extended to analyze blood of EGFR-mutated patients progressing to TKIs. Methods: Analysis of EGFR mutations was performed by a central laboratory using a sensitive multiplex TaqMan assay in the presence of a PNA clamp. Circulating free DNA (cfDNA) was isolated from both serum and plasma using an automatic extractor, and mutational analyses were performed in quadruplicates. Patients harbouring EGFR mutations were informed as eligible for TKI treatment. Results: From June 2012 to November 2015, 765 NSCLC patients from 102 institutions in Spain were prospectively screened at presentation for EGFR mutations in peripheral blood. In five cases (0.7%), blood collected was insufficient for molecular analysis. Clinical characteristics of the remaining 760 evaluable patients were as follows: prevalence of male (58.55%), former smokers (48.16%) and adenocarcinoma (79.34%). Sensitizing mutations were found in 76 of 760 patients (10%) and were prevalent in females (61.84%), never smokers (59.21%) and adenocarcinomas (80.26%). Of the 76 EGFR-mutated patients at presentation, 48 (63.16%) were positive in both serum and plasma, 19 (25%) only in plasma, and nine (11.84%) in serum alone. Regarding the type of mutation, 50 (65.8%) had exon 19 deletions, the most common being 15 bp deletions; 25 (32.9%) had L858R substitution, while L861Q mutation was detected in one patient (1.3%). Additionally, the T790M resistance mutation in exon 20 was analyzed in 223 pretreatment samples and not detected in any (0%). Finally, blood of 84 patients progressing to TKI treatment with no biopsy available was screened and T790M detected in 28 patients (33.33%). Conclusions: Our results demonstrate the feasibility of large-scale, nationwide screening of EGFR mutations in peripheral blood of NSCLC patients at presentation and after TKI progression. Mutational analysis in blood is useful to inform treatment decisions, particularly in patients with no biopsy available or after TKI progression Citation Format: Clara de la Caridad Mayo de las Casas, Nùria Jordana Ariza, Mónica Garzón Ibañez, Ariadna Balada Bel, Jordi Bertrán-Alamillo, Ana Pérez Rosado, Santiago Viteri Ramirez, Daniela Morales Espinosa, Juan Carlos Monasterio, Niki Karachaliou, Miguel Ángel Molina-Vila, Rafael Rosell. Large scale noninvasive screening of EGFR mutations in advanced NSCLC patients: the Identify platform experience. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3964.

Role of TP53 mutations in predicting response to TKI treatment in EGFR-mutated NSCLC patients.

9052Background: Patients with non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations are usually responsive to tyrosine kinase inhibitors (TKIs). However, ab...

Predictors of incipient dysfunction of all cardiac chambers after treatment of metastatic renal cell carcinoma by tyrosine kinase inhibitors.

We aimed to explore the hypothesis that early subclinical cardiac chamber dysfunction secondary to tyrosine kinase inhibitors (TKIs) in patients with metastatic renal cell carcinoma could be signaled by abnormal cardiac mechanics demonstrated by velocity vector imaging. Echocardiographic images were acquired from the apical views in 23 metastatic renal cell carcinoma patients. All patients had baseline and at least a 3-month follow-up echocardiogram after receiving TKI therapy. Subendocardial borders of all the cardiac chambers were traced to obtain volumetric and deformation indices. Mean age was 67 ± 9 years with 92% men. The right ventricle peak systolic global longitudinal strain (GLɛ) and strain rate were significantly lower after TKIs (-23.49 ± 5.1 versus -19.81 ± 5.5, p = 0.002 and -1.52 ± 0.52 versus -1.24 ± 0.35 p = 0.02, respectively). LV GLɛ was not statistically different. Volumetric and deformation indices showed a minimal decrease of the right atrium reservoir and conduit functions, and no significant changes of left atrial function. The right heart exhibited greater strain changes than the left heart after TKI treatment. The implications of these findings and their potential significance warrant further work.

Mutation analysis of EGFR and its correlation with the HPV in Indian cervical cancer patients.

Cervical cancer is a major cause of morbidity and mortality particularly in developing countries. Somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene is associated with increased sensitivity to tyrosine kinase inhibitors (TKIs). In this study, the presence of EGFR mutations in cervical cancer and its correlation with HPV were identified. EGFR mutations were found in 31 out of 95 patients (32.63%). Results showed the presence of EGFR mutations in 5.263% of patients in exon 19. In exon 20, mutations were predominant in 25.26% patients. While in exon 21, 8.421% of patients had mutations. HPV, which is associated with cervical cancer development, was found in 95.78% (HPVL1), 92.63% (HPV16), and 3.15% (HPV18) of patients. No correlation was found between HPV16 and EGFR mutations (p = 0.0616). Overall, mutations like V742R, Q787Q, Q849H, E866E, T854A, L858R, E872Q, and E688Q were found. Next, impact of TKI inhibitor (gefitinib) was checked with respect to presence or absence of mutation considering Q787Q mutation in exon 20 (G/A genotype) which is present in 25.2% patients. Mutated cervical cancer cell lines showed higher sensitivity to gefitinib. Overall, this study suggests the importance of mutations in EGFR gene and indicates their relevance with respect to TKIs treatment in Indian cervical cancer patients.

Overall survival benefits of first-line EGFR tyrosine kinase inhibitors in EGFR-mutated non-small-cell lung cancers: a systematic review and meta-analysis.

Background:Accumulating data shows that exon 19 deletions and L858R, both activating epidermal growth factor receptor mutations in non-small-cell lung cancers (NSCLCs), are just two different entities in terms of prognosis and treatment response to tyrosine kinase inhibitors (TKIs).Methods:A systematic review and meta-analysis of randomized controlled trials comparing TKIs with conventional chemotherapy was performed. Eight trials of 1498 patients and five trials of 1279 patients with either exon 19 deletions or L858R were included in the meta-analysis.Results:TKI treatment demonstrated progression-free survival benefit in patients with exon 19 deletions (hazard ratio (HR): 0.27, 95% confidence interval (CI): 0.21–0.35) and L858R (HR: 0.45, 95% CI: 0.35–0.58). Patients with exon 19 deletions had significant overall survival (OS) benefit under TKI treatment (HR: 0.72, 95% CI: 0.60–0.88). Subgroup analyses showed that irreversible TKIs, but not reversible TKIs, had statistically significant OS benefit in these patients (irreversible TKIs, HR: 0.59, 95% CI: 0.47–0.73; reversible TKIs, HR: 0.84, 95% CI: 0.69–1.02). Patients with L858R demonstrated no OS benefit under first-line TKI use (HR: 1.15, 95% CI: 0.95–1.39).Conclusions:In patients with advanced NSCLC harbouring exon 19 deletions, TKIs are associated with better OS compared with conventional chemotherapy. Future clinical trials should take exon 19 deletions and L858R as distinct disease entities and evaluate the treatment efficacy separately.

Clinical and prognostic significance of 3q26.2 and other chromosome 3 abnormalities in CML in the era of tyrosine kinase inhibitors

AbstractChromosome 3q26.2 abnormalities in acute myeloid leukemia, including inv(3)/t(3;3) and t(3;21), have been studied and are associated with a poor prognosis. Their prevalence, response to tyrosine kinase inhibitor (TKI) treatment, and prognostic significance in chronic myelogenous leukemia (CML) are largely unknown. In this study, we explored these aspects using a cohort of 2013 patients with CML diagnosed in the era of TKI therapy. Chromosome 3 abnormalities were observed in 116 (5.8%) of 2013 cases. These cases were divided into 5 distinct groups: A, inv(3)(q21q26.2)/t(3;3)(q21;q26.2), 26%; B, t(3;21)(q26.2;q22), 17%; C, other 3q26.2 rearrangements, 7%; D, rearrangements involving chromosome 3 other than 3q26.2 locus, 32%; and E, gain or loss of partial or whole chromosome 3, 18%. In all, 3q26.2 rearrangements were the most common chromosome 3 abnormalities (50%, groups A-C). 3q26.2 rearrangements emerged at different leukemic phases. For cases with 3q26.2 rearrangements that initially emerged in chronic or accelerated phase, they had a high rate of transformation to blast phase. Patients with 3q26.2 abnormalities showed a marginal response to TKI treatment, and no patients achieved a long-term sustainable response at a cytogenetic or molecular level. Compared with other chromosomal abnormalities in CML, patients with 3q26.2 rearrangements had poorer overall survival. The presence or absence of other concurrent chromosomal abnormalities did not affect survival in these patients, reflecting the predominant role of 3q26.2 rearrangements in determining prognosis. Interestingly, although heterogeneous, chromosome 3 abnormalities involving non-3q26.2 loci (groups D, E) also conferred a worse prognosis compared with changes involving other chromosomes in this cohort.

Molecular genetic and cytogenetic determinants of primary resistance or loss of the response to treatment with tyrosine kinase inhibitors in patients with chronic myeloid leukemia.

Aim of this study was to analyze molecular genetic and cytogenetic reasons for disease resistance to tyrosine kinase inhibitors (TKI) imatinib (IM) and nilotinib (NI) in patients with chronic myeloid leukemia (CML). Material and methods. A group of 32 CML patients with primary or acquired resistance to TKI treatment was investigated. Cytogenetic response was determined by conventional karyotyping with differential banding. Presence of BCR-ABL kinase domain mutations was investigated by direct sequencing. Results and discussion. The frequency of mutations was 37% (12 patients) with prevailing occurrence of mutations with low sensitivity to nilotinib – E255K/V; Т315I; F359V; Y253H. In 47% of the cases (15 patients) additional chromosome aberrations (ACA) were revealed which could also be the reason for TKI resistance in patients without BCR/ABL mutations. Patients with detected mutations of BCR/ABL gene were either switched to nilotinib or treated with increased dose of IM. Cytogenetic response was achieved in only 2 patients with mutations and in 12 patients without them. Frequency of blast crisis development did not differ significantly in both groups. Conclusions. Among the investigated patients with CML resistant to IM BCR/ABL gene mutations were detected in more than third of the cases whereas ACA were found in almost half of the group. Taking into account revealed prevalence of mutations not sensitive to the 2nd generation TKI nilotinib, investigation of mutational status has to be obligatory in all patients for whom treatment correction is considered. Presence of ACA should also be taken into account in patients requiring administration of the second line TKI since they can adversely influence expected treatment response as well.

Clinical and prognostic significance of 3q26 rearrangements and other chromosome 3 abnormalities in chronic myelogenous leukemia in era of tyrosine kinase inhibitors

Chromosome 3q26.2 abnormalities in acute myeloid leukemia (AML), including inv(3)/t(3;3) and t(3;21), have been studied and are associated with a poor prognosis. Their prevalence, response to tyrosine kinase inhibitor (TKI) treatment and prognostic significance in chronic myelogenous leukemia (CML) are largely unknown. In this study we explored these aspects using a cohort of 2013 CML patients in the era of TKI therapy. Chromosome 3 abnormalities were observed in 116 of 2013 (5.8%) cases. These cases were divided into five distinct groups: A, inv(3)(q21q26.2)/t(3;3)(q21;q26.2), 26%; B, t(3;21)(q26.2;q22), 17%; C, other 3q26.2 rearrangements, 7%; D, rearrangements involving chromosome 3 other than 3q26.2 locus, 32%; and E, gain or loss of partial or whole chromosome 3, 18%. In all, 3q26.2 rearrangements were the most common chromosome 3 abnormalities (50%, groups A-C). 3q26.2 rearrangements emerged at different leukemic phases. For cases with 3q26.2 rearrangements that initially emerged in chronic or accelerated phase, they had a high rate of transformation to blast phase. Patients with 3q26.2 abnormalities showed a marginal response to TKIs treatment and no patients achieved a long-term sustainable response at cytogenetic or molecular level. Compared to other chromosomal abnormalities in CML, patients with 3q26.2 rearrangements had a poorer overall survival. The presence or absence of other concurrent chromosomal abnormalities did not affect survival in these patients, reflecting the predominant role of 3q26.2 rearrangements in determining prognosis. Interestingly, although heterogeneous, chromosome 3 abnormalities involving non-3q26.2 loci (groups D, E) also conferred a worse prognosis compared with changes involving other chromosomes in this cohort.

Does Molecular Genotype Provide Useful Information in the Management of Radioiodine Refractory Thyroid Cancers? Results of a Retrospective Study.

Whether mutation status should be used to guide therapy is an important issue in many cancers. We correlated mutation profile in radioiodine-refractory (RAIR) metastatic thyroid cancers (TCs) with patient outcome and response to tyrosine kinase inhibitors (TKIs), and discussed the results with other published data. Outcome in 82 consecutive patients with metastatic RAIR thyroid carcinoma prospectively tested for BRAF, RAS and PI3KCA mutations was retrospectively analyzed, including 55 patients treated with multikinase inhibitors. Papillary thyroid carcinomas (PTCs) were the most frequent histological subtype (54.9 %), followed by poorly differentiated thyroid carcinoma [PDTC] (30.5 %) and follicular thyroid carcinoma [FTC] (14.6 %). A genetic mutation was identified in 23 patients (28 %) and BRAF was the most frequently mutated gene (23 %). Median progression-free survival (PFS) on first-line TKI treatment was 14.6 months (95% CI 9.9-18.4). BRAF mutation positively influenced median PFS, both in the entire TKI-treated cohort (median PFS 34.7 months versus 11.6 months; hazard ratio [HR] 0.29; 95% CI 0.09-0.98; p = 0.03) and in the TKI-treated PTC cohort (n = 22) [log-rank p = 0.086; HR 2.95; 95 % CI 0.81-10.70). However, in TKI-treated patients, PDTC histologic subtype was the only independent prognostic factor for PFS identified in the multivariate analysis (HR 2.36; 95% CI 1.01-5.54; p = 0.048). Patients with BRAF-mutant PTC had a significantly longer PFS than BRAF wild-type when treated with TKIs. However, due to the small number of BRAF-mutant patients, further investigations are required, especially to understand the potential positive effect of BRAF mutations in RAIR TC patients while having a negative prognostic impact in RAI-sensitive PTC patients.

Abstract 5462: Extracellular ATP promotes cancer cell drug resistance to tyrosine kinase inhibitors by competing for the ATP-binding site of tyrosine kinase

Abstract ATP plays important roles in cellular processes in cancer cells, which are a heterogeneous population in tumors regarding their genetic background and oxygen and energy supplies. The fact that cancer cells undergo rapid proliferation suggests that they must possess mechanisms, which may not be present in normal cells, to meet their high energy needs. Studies by others revealed that extracellular ATP levels of various cancer types are 10^3 to 10^4 times higher than those in their corresponding normal tissues (1). We recently reported that extracellular ATP can be taken up directly by cancer cells by macropinocytosis (2). Based on these results, we hypothesized that the internalized extracellular ATP not only promotes cancer cell growth and survival, but also drug resistance. In this study, human lung cancer A549 and breast cancer MCF7 cells were used as cell models to determine the effects of extracellular ATP on drug resistance to tyrosine kinase inhibitors (TKIs) in cancer cells. Cancer cell viability and intracellular ATP measurement results showed that extracellular ATP substantially increased intracellular ATP levels and promoted cancer cell drug resistance to TKIs. Viability of TKI treated cancer cells was significantly increased in the presence of extracellular ATP, compared to cancer cells with TKI treatment alone. Administration of macropinocytosis inhibitors significantly reduced the drug resistance induced by extracellular ATP, suggesting that extracellular ATP promoted drug resistance by contributing to the intracellular ATP pool. Protein and protein phosphorylation analyses results suggested that the increased intracellular ATP reversed the inhibition of TKIs by competing with the inhibitor for the ATP-binding site of platelet-derived growth factor receptor (PDGFR) and enhancing Akt and MAPK signaling pathways. These findings identify, for the first time, new biological functions of extracellular ATP in tumors and may lead to novel anti-cancer strategies combating drug resistance in cancers. 1. Pellegatti P, et al. PLoS ONE 2008;3:e2599. 2. Qian Y, et al. Cancer Lett 2014;351:242-51. Citation Format: Xuan Wang, Yanrong Qian, Yunsheng Li, Xiaozhuo Chen. Extracellular ATP promotes cancer cell drug resistance to tyrosine kinase inhibitors by competing for the ATP-binding site of tyrosine kinase. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5462. doi:10.1158/1538-7445.AM2015-5462

Tyrosine kinase inhibitors in patients with chronic myelogeneous leukemia: defining the role of social risk factors and non-adherence to treatment.

To assess the role of social risk factors on adherence to tyrosine kinase inhibitors (TKI) therapy in chronic myeloid leukemia (CML) patients. This is a retrospective study and eligible patients were adults with CML on TKI treatment. Cases of no adherence to treatment were confirmed during pharmacists' consultation (patient-reported adherence). Baseline characteristics between groups were compared between cases and controls groups. Risk factors identified in bivariate analysis (p<0.2) were included in multivariate model. A qualitative investigation assessed whether such predictors of non-adherence had causal relationship. Of 151 patients with CML consulted by pharmacists, 21% had adherence problems. Despite patients with secondary school (p=0.03), most of investigated social risk factors did not differ between groups. However, by using a qualitative approach, patients' level of education could not explain low adherence rates behavior. Social determinants of health, herein investigated, were unlikely to predict adherence to treatment. Regression techniques may lead to untrue statements, so future researches should consider investigating the causes, not only the statistical estimates.

Response-related predictors of survival in CML

The assessment of response to tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) does not only reflect tumor burden at a given time but has been shown to be linked to long-term survival outcomes as well. Therefore, the quantification of molecular or cytogenetic response as early as 3 months on treatment allows a prognostic stratification of a patient's individual risk. With competing TKI regimens available, a timely switch of treatment can be considered if unfavorable outcome has to be expected due to early response failure. Numerous studies have demonstrated the association of long-term outcome with early response for first-line treatment with imatinib, with second-generation TKI and for second-line TKI treatment as well.

The scaffolding protein NHERF1 sensitizes EGFR-dependent tumor growth, motility and invadopodia function to gefitinib treatment in breast cancer cells.

Triple negative breast cancer (TNBC) patients cannot be treated with endocrine therapy or targeted therapies due to lack of related receptors. These patients overexpress the epidermal growth factor receptor (EGFR), but are resistant to tyrosine kinase inhibitors (TKIs) and anti-EGFR therapies. Mechanisms suggested for resistance to TKIs include EGFR independence, mutations and alterations in EGFR and in its downstream signalling pathways. Ligand-induced endocytosis and degradation of EGFR play important roles in the downregulation of the EGFR signal suggesting that its activity could be regulated by targeting its trafficking. Evidence in normal cells showing that the scaffolding protein Na+/H+ exchanger regulatory factor 1 (NHERF1) can associate with EGFR to regulate its trafficking, led us to hypothesize that NHERF1 expression levels could regulate EGFR trafficking and functional expression in TNBC cells and, in this way, modulate its role in progression and response to treatment. We investigated the subcellular localization of NHERF1 and its interaction with EGFR in a metastatic basal like TNBC cell model, MDA-MB‑231, and the role of forced NHERF1 overexpression and/or stimulation with EGF on the sensitivity to EGFR specific TKI treatment with gefitinib. Stimulation with EGF induces an interaction of NHERF1 with EGFR to regulate its localization, degradation and function. NHERF1 overexpression is sufficient to drive its interaction with EGFR in non-stimulated conditions, inhibits EGFR degradation and increases its retention time in the plasma membrane. Importantly, NHERF1 overexpression strongly sensitized the cell to the pharmacological inhibition by gefitinib of EGFR-driven growth, motility and invadopodia-dependent ECM proteolysis. The further determination of how the NHERF1‑EGFR interaction is regulated may improve our understanding of TNBC resistance to the action of existing anticancer drugs.

Detection of BCR-ABL1 Compound and Polyclonal Mutants in Chronic Myeloid Leukemia Patients Using a Novel Next Generation Sequencing Approach That Minimises PCR and Sequencing Errors

BackgroundBCR-ABL1 kinase domain (KD) mutations are the most common known cause of resistance to tyrosine kinase inhibitors (TKIs) in CML. Mutation analysis is critical for selection of subsequent TKI therapy after treatment failure. Low level and compound mutants (>1 KD mutation in the same molecule) may also lead to therapy failure. However, compound and multiple polyclonal mutants cannot be distinguished by conventional methods as they determine the average genotype of all molecules. Next generation sequencing (NGS) has the potential to sensitively detect these mutants, however sequencing and PCR errors confound the detection of true, low level mutants using current approaches. Indeed, we demonstrated that the reported frequency of BCR-ABL1 compound mutants may be over estimated due to PCR recombination artifacts that mimic compound mutations (Parker Blood 2014). More reliable methods are needed to appropriately assess the impact of various mutations on patient (pt) outcome.AimTo develop a clinically applicable NGS assay that can robustly distinguish BCR-ABL1 compound and polyclonal mutants.MethodWe have developed a novel NGS assay termed Single Molecule Consensus Sequencing (SMCS) that involves tagging individual BCR-ABL1 cDNA molecules before library amplification, enabling identification and elimination of most PCR and sequencing errors. NGS was performed on the Illumina MiSeq, 2 x 300 bp; aa 244 - 407 of the KD was examined. Reads derived from an initial BCR-ABL1 molecule are identified bioinformatically by virtue of sharing the same tag sequence. The consensus sequence of reads with the same tag is determined using automated variant calling and filtering algorithms. The consensus sequence represents the sequence of the initial BCR-ABL1 cDNA molecule (Fig A).ResultsTo test the validity of SMCS, we examined 10 samples lacking KD mutations and 5 mock samples created by mixing compound mutant plasmids or pt samples. Examination of raw sequencing reads revealed a complex spectrum of mutants, similar to previous clinical reports. SMCS enabled bioinformatic filtering of these artifacts, largely eliminating PCR and sequencing error, and exclusively reported the compound and polyclonal mutants known to be present in the mock samples. We estimated the background error rate to be ~2x10-5 per base. The error spectrum was consistent with DNA damage causing first round PCR errors.SMCS was used to retrospectively examine samples of 46 pts (36 CP, 2 AP, 8 BP) who were resistant to ≤4 TKIs (1st and 2nd generation). 71 mutations were previously detected by Sanger sequencing in these samples, collected before starting next line TKI. Within the region examined using SMCS, there was 100% detection concordance with Sanger sequencing.We compared the results of SMCS with an amplicon NGS method performed at another centre for 24/46 pts (Ion Torrent, depth ~10000). Ion Torrent detected 34 compound mutants in 24 pts. Of the 30/34 that were within the region examined by SMCS we only detected 8. Based on observations in Parker Blood 2014, 14 of the 22 compound mutants not detected by SMCS were likely to be PCR recombination artifacts. The other 8/22 were low level (1 - 4%) and most (6/8) involved mutations rarely/never reported in TKI resistant pts so may also be artifacts (Fig B). We detected 3 additional compound mutants in these 24 pts, plus 5 in the remaining 22/46 pts. The compound mutants detected by SMCS were consistent with the pts’ TKI treatment history.ConclusionWe demonstrated detection of BCR-ABL1 compound and polyclonal mutants in pt samples using a novel NGS assay that has the potential to overcome technical artifacts generated with other published methods. Whilst there is no gold standard method that can accurately detect low level compound mutations, SMCS has correctly identified sequencing and PCR recombination artifacts using mock samples. The accuracy and clinical utility of SMCS for sensitive compound and polyclonal mutant detection is currently being validated in another group of 200 imatinib resistant pts. The frequency of compound mutants detected in pts with >1 mutation by SMCS in the current analysis (35%) is approximately half of that reported previously, which suggests the published frequency may have been overestimated. Our novel assay takes an important step towards enabling a more concrete understanding of the mutation spectra in pts and their association with resistance. [Display omitted] DisclosuresYeung:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Lustgarten:ARIAD Pharmaceuticals Inc: Employment, Equity Ownership. Hodgson:ARIAD Pharmaceuticals, Inc.: Employment, Equity Ownership. Rivera:ARIAD Pharmaceuticals Inc: Employment, Equity Ownership. Hughes:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria, Research Funding. Branford:Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding.

Integrin-Linked Kinase As a Key Mediator of Stromal Cell-Enhanced Resistance of Primitive CML Cells to Tyrosine Kinase Inhibitors

The human bone marrow (BM) compartment consists of a heterogeneous, multi-functional network of cells and extracellular matrix that interact with hematopoietic stem cells. Growing evidence suggests that this microenvironment also likely adds to the resistance of chronic myeloid leukemia (CML) stem cells to tyrosine kinase inhibitor (TKI) therapy in vivo. Integrin-linked kinase (ILK) is a serine/threonine kinase that is an important constituent of focal adhesions, a regulator of spindle formation during mitosis, and a key mediator of multiple signaling pathways. However, its potential role in the regulation of primitive CML cells and their response to TKIs is unknown.Our RNA-seq analysis revealed that ILK expression and its downstream targets, such as AKT, are highly upregulated in pre-treatment CD34+ CML stem/progenitor cells (n=6) compared to normal BM controls (n=3, p<0.05). We confirmed this finding by qRT-PCR analysis of CD34+ cells obtained from 28 CML patients (including some who subsequently became clinically resistant to imatinib mesylate (IM) treatment) compared to CD34+ BM cells from 9 normal adults (p<0.05). Elevated expression of ILK protein in CD34+ CML cells was also demonstrated by Western blot analysis (threefold, n=4, p<0.05). Interestingly, we also found ILK transcript levels to be much higher in the more primitive and rarer CD38- CML stem cell-enriched subset of the lin-CD34+ population as compared to the more mature and prevalent CD38+ subset or the terminally differentiated lin+CD34- cells (p<0.01). Both ILK transcripts and intracellular protein levels were elevated in CD34+ CML cells, but not in CD34- differentiated cells, in response to IM treatment in vitro, especially in the presence of expanded BM stromal cells, suggesting differential regulation of ILK expression in primitive CML cells and their more mature counterparts.We next asked whether ILK influences primitive CML cell responses to TKIs by using a clinically validated and selective ILK inhibitor (QLT0267) to suppress ILK activity in colony assays of CD34+ CML cells. QLT0267 plus TKIs significantly reduced the yield of colonies obtained compared to any single agent or combination of TKIs and this enhanced cell killing was most pronounced on cells from IM non-responders (n=6; p<0.01). Analysis of the lineages affected showed that the combination of IM or dasatinib (DA) with QLT0267 had a more pronounced effect on myeloid colony formation (CFU-GM), at concentrations where either or both TKIs had little effect (i.e. 85-90% inhibition vs. 30-45%). Moreover, the simultaneous suppression of ILK and BCR-ABL activities also effectively inhibited the growth of more primitive CML cells (from IM non-responder patients) when these were co-cultured with stromal cells in 6-week long-term cultures (LTC), in contrast to the lack of these effects in the presence of single TKIs or TKI combinations.Mechanistically, we found that the combination of QLT0267 with a TKI enhanced the induction of apoptosis of CD34+ CML cells in suspension cultures within the first three days (from 10-15% to 30-45% apoptotic cells relative to untreated controls) and CFSE (carboxy-fluorescein diacetate succinimidyl diester) tracking of cell division showed that QLT0267 specifically targets quiescent CML stem cells from IM-resistant patients (n=4; p<0.05). In addition, treatment with the ILK inhibitor alone or in combination with a TKI enhanced apoptosis of primitive CML cells in vitro by abolishing the protective effect of BM stromal cells observed under TKI treatment alone (25-30% apoptotic cells vs. 3-8%) and combination treatments confirmed strong synergy between DA and QLT0267 (CI value <0.3). Importantly, QLT0267 (up to 10 μM) was not toxic to normal CD34+BM cells in either short- or long-term culture systems (with and without stromal cells).Together, these findings indicate that ILK is a critical player in the regulation of TKI response/resistance of primitive CML cells. The enhanced and selective effect obtained by dual inhibition of both targets, particularly in the presence of protective stromal cells to mimic their response within the BM microenvironment, may offer an important new therapeutic possibility. DisclosuresNo relevant conflicts of interest to declare.

Peptides from Nme2 Are Preferentially Expressed By Chronic Phase CML Cells, Recognized By T Cells and Restricted By the Most Frequent HLA-Antigens

Nme2 (non metastasis protein 2) is a nucleoside diphosphate kinase that plays multiple roles in signalling and metabolism. We previously identified Nme2 as a tumor antigen in chronic myeloid leukaemia (CML) and found Nme2 protein over-expression to be a universal and specific feature of Bcr/Abl+ cells in chronic-phase of the disease. Nme2 up-regulation in Bcr/Abl+ cells is post-transcriptional and is reversed by TKI treatment. Furthermore, the IL-3 independence induced by Bcr/Abl transfection of Ba/F3 cells is also accompanied by Nme2 over-expression and is reversed by specific miRNA-mediated down-regulation of Nme2, suggesting that Nme2 contributes to Bcr/Abl driven leukemogenesis. Here, we have investigated the potential of Nme2 as a target for CML specific immunotherapy by assessing it’s antigenicity in common HLA backgrounds.Peripheral blood mononuclear cells (PBMCs) were obtained from 40 patients with CML and 24 healthy donors who carried the common HLA-A02 (n=21/7); A03 (n=12/8) and A24 (n=8/9) antigens. Together, these represent the most common HLA types in the Caucasian population. Samples were obtained before (n = 13) and after (n = 36) hematopoietic stem cell transplantation (HCT) and Nme2 specific cytotoxic T lymphocytes (CTL) were generated using artificial antigen presenting cells (aAPC) engineered to express both Nme2 and the appropriate HLA-A antigen. PBMC were primed with aAPC and then re-stimulated weekly. Following CD8+ T-cell selection on day 14, IFNγ production in response to Nme2 expressing stimulator cells was evaluated in Elispot assays. In addition Nme2 reactivity was investigated in peripheral blood cells of CML patients by Elispot assays at various time points up to 10 years after HCT.The frequency of Nme2 specific T cells was significantly higher in CML patients pre HCT (39.3 specific T cells/105 CD8+ cells, p=0.004) than in healthy donors (2.17 specific T cells/105 CD8+ cells). A further rise in specific T cell response was detected in CML patients post HCT (142 specific T cells/105 CD8+ cells, p=0.0006), with a significant increase being evident by day +50 after HCT. The subsequent activity varies, with a weakly pronounced second peak occurring around 3 years post-transplant. However, Nme2 specific T cells persisted over the entire observation period with a significant Nme2 specific T cell response still being detectable nearly 10 years post transplant. The highest Nme2 specific T cell activity was found in HLA-A03 positive patients (187 T cells/105 CD8+ cells) and the lowest in the HLA-A02 background (102 T cells/105 CD8+ cells, p=0.00001). Restriction by HLA-A24 was associated with an intermediate activity (141 Nme2 specific T cells/105 CD8+ cells). We identified 8 patients in which an increase of Bcr-Abl transcript level from 0% Bcr-Abl/Abl to a detectable amount (0.28%, range 0.001 to 5.017% Bcr-Abl/Abl) was observed. In all cases, a transient increase in Nme2 reactive T cells (2.7 fold, p=0.007) was associated with re-entry into molecular remission. Furthermore, Nme2 specific T cells were lower before relapse (88 T cells/105 CD8+ cells) than in patients with long lasting molecular remission (186 T cells/105 CD8+ cells, p=0.026).Finally, we successfully generated Nme2 specific CTLs from healthy donors (4/5) as well as from patients with CML who had received HCT (10/10). CTLs showed a mean expansion rate of 23 (healthy donors) to 30 fold (CML patients) and HLA class I restricted Nme2 specific recognition. Partially HLA matched, primary CML cells were targeted by these Nme2 specific CTLs (6/6), regardless of the presence of the T315I Bcr/Abl mutation that bestows resistance to tyrosine kinase inhibitors (TKI).In summary, we show here that Nme2 is a strong inducer of immune response in transplanted CML patients carrying common HLA class I alleles, implying that the immune response to Nme2 may be involved in the GvL effect post HCT. Our results suggest that Nme2 specific CTLs may be of therapeutic relevance in eradicating the residual leukemic cells after therapy with TKI or HCT. DisclosuresNo relevant conflicts of interest to declare.

Molecular Response to Tyrosine Kinase Inhibitors (TKIs) for Chronic Myeloid Leukemia (CML). an Update on Experience at Tawam Hospital, Abudhabi, UAE in the Light of ELN Guidelines

BackgroundChronic Myeloid Leukemia (CML) is a clonal myeloproliferative disorder characterized by the presence of a balanced reciprocal translocation involving the long arms of chromosomes 9 and 22. The fusion gene that is created by this translocation (BCR-ABL1) encodes for a constitutively active protein tyrosine kinase that is primarily responsible for the leukemic phenotype.Targeted therapy with Tyrosine Kinase Inhibitors (TKIs) has become the recommended first-line treatment for patients with CML. Commercially available TKIs include Imatinib, Nilotinib, Dasatinib, Ponatinib and Bosutinib. The first three drugs are approved for first line therapy while the later 2 are for resistant disease. Monitoring of the response to therapy is done with quantification of the BCR-ABL transcripts by RQ-PCR–based molecular technique.MethodsRetrospective review of charts of patients diagnosed with CML between January 2010 and June 2014. Data points were measured at 3, 6, 12, and 24 months post initiation of therapy and results were documented as per ELN guidelines.Results56 patients were diagnosed with CML. The median age was 33 years (range 19-73 years). Male to female ratio was 4:1. Sokal score was calculated for 53 patients (data for 3 patients was incomplete) 21 were low risk, 24 were intermediate risk and 8 were high risk.7 patients were lost from follow up after diagnosis and are excluded from analysis. 1 patient has not reached the 3 month mark and is also excluded from analysis. 13 patients were started on initial treatment with Imatinib 400 mg while 35 with second generation TKIs (Dasatinib 100 mg daily or Nilotinib 300 mg BID) daily as upfront therapy.With a median follow up of 27 months (range 3 month to 51 months) 4/13 (30%) patients on Imatinib as first line therapy were able to achieve optimal repsone (one at a dose of 600 mg daily). 8 patients were switched to second line therapy and 7/8 patients have achieved MMR (1 patient remains in full cytogenetic relapse due to non-compliance). 35 patients were treated with 2ndgeneration TKIs as first line therapy. 22 out of 35 (62 %) patients have achieved optimal results within 18 months of front line therapy . Second line therapy was initiated in patients achieving suboptimal results or intolerance in 11 patients. 5 of these patients subsequently achieved MMR while 5 remain with suboptimal results. 1 patient had disease progression while one was lost to follow-up after achieving a partial cytogenetic response at 6 months.Interestingly 12 (25%) patients achieved MMR/CMR within 6 months of starting TKIs. Of these only 1 was on Imatinib while the rest were on 2ndgeneration TKIs. MMR 4.5 or undetectable levels of bcr-abl transcript have been documented in 10 patients (20 %) for the whole cohort.4 patients had disease progression on TKI (treatment failure rate of 8 %). One patient had disease progression to accelerated and then blast crisis (lymphoid and myeloid) while the other 3 patients are alive with only CHR with one resistant to all TKIs, one with intolerance of all TKIs and one secondary to noncompliance.ConclusionWe have previously reported on our experience with CML. We have now updated our report with a larger cohort of patients. The median age remains much lower as compared to Western countries, just reflecting differences in the age distribution of the population in the UAE with 80% being below the age of 65 years.MMR report from Enestnd trial is 67-71% in favor of Nilotinib as compared to Imatinib 44%, while the Dasision trial reported a MMR of 44 % in favor of Dasatinib with faster rate to response. Our results mirror the results of these phase 3 randomized trial with optimal response of 62 % with second generation TKIs% as compared to 30 % with Imatinib as front line therapy.Expatriates accounts for approximately 80% of the population in the UAE and many are temporary employed, having limited health care coverage, limited financial means as well as limited possibilities to attend regular follow-ups. This leads to compliance problems, loss from follow-up and suboptimal management and monitoring of their disease. These factors also complicate any chance of treatment free period for patients who achieve undetectable levels of bcr-abl transcripts. Additionally absence of technology for mutational analysis makes it impossible to determine underlying reasons for suboptimal response with accuracy. DisclosuresAlam:BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Hussain:BMS: Honoraria; Novartis: Honoraria. Lal:BMS: Honoraria; novartis: Honoraria.