Single Tyrosine Kinase Inhibitors Research Articles

Peak study: A phase 3, randomized, open-label multicenter clinical study of bezuclastinib (CGT9486) and sunitinib in combination versus sunitinib in patients with gastrointestinal stromal tumors (GIST).

TPS766 Background: Imatinib is the worldwide standard for first-line therapy of advanced KIT-mutant GIST. However, secondary resistance mutations in the KIT ATP-binding domain (exons 13, 14), activation loop (exons 17, 18), or both develop and result in loss of imatinib-sensitivity. While no single tyrosine kinase inhibitor (TKI) inhibits all KIT mutations, the combination of bezuclastinib + sunitinib targets commonly occurring primary (exons 9, 11) and secondary (exons 13, 14, 17, and 18) KIT mutations. In a Phase 1/2 clinical study, pts with relapsed and/or refractory GIST and a median of 3 prior lines of therapy treated with bezuclastinib + sunitinib (n=15) experienced clinical benefit and an acceptable safety profile, warranting further evaluation in a randomized trial. Methods: Peak (NCT05208047) is a global, randomized, open-label, multi-part Phase 3 study evaluating the efficacy and safety of bezuclastinib + sunitinib versus sunitinib as second-line treatment in adult pts who were intolerant to imatinib or whose tumors had imatinib-resistance. The lead-in portion, to test a new formulation of bezuclastinib, (Part 1) has completed enrollment. Based upon PK and safety, a dose of bezuclastinib 600 mg QD + sunitinib 37.5 mg QD has been determined for Part 2 of the Peak study. Part 2 will enroll ~388 pts to be randomized (1:1) to bezuclastinib 600 mg QD + sunitinib 37.5 mg QD or sunitinib 37.5 mg QD alone. Key inclusion: >1 measurable lesion per modified Response Evaluation Criteria in Solid Tumors (mRECIST) v1.1, Eastern Cooperative Oncology Group Performance Status 0-2, adequate organ function, and prior imatinib therapy (no other prior therapy). Key exclusion: PDGFR mutations or succinate dehydrogenase deficiency, clinically significant cardiac disease, and use of strong CYP3A4 inhibitors or inducers. The primary endpoint is progression-free survival (PFS) confirmed by blinded independent central review per mRECIST v1.1. Additional efficacy (including overall survival and objective response rate) and safety endpoints will be evaluated. Clinical trial information: NCT05208047 .

Abstract 5489: Synergistic activity of SCD1 blockade in combination with tyrosine kinase inhibitors lenvatinib and cabozantinib in hepatocellular carcinoma (HCC)

Abstract Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Tyrosine kinase inhibitors (TKIs) are approved as a first-line treatment for unresectable HCC. Lenvatinib and cabozantinib are two of the most used TKIs, but the therapeutic duration is limited due to the development of drug resistance. Therefore, understanding the mechanisms of resistance and combining TKIs with other drugs antagonizing resistance should lead to antitumor synergy, eliminating drug resistance. It turns out that the simultaneous use of TKIs together with an inhibitor of stearoyl-CoA desaturase 1 (SCD1) prevents the development of drug resistance, leading to a durable response. SCD1 is the enzyme responsible for de novo fatty acid (FAs) synthesis, converting saturated fatty acids (SFAs) into monounsaturated fatty acids (MUFAs). MUFAs are an alternative energy source to glucose, integral to cellular membranes, and prevent endoplasmic reticulum (ER) stress and other signaling pathways. In our laboratory, we developed a novel, highly specific SCD1 inhibitor - SSI-4. We have tested the biological activity of SSI-4 against different HCC cell lines and patient-derived xenografts (PDX) mouse models. Of the twelve tested HCC cell lines, four were highly sensitive to SSI-4 (IC50 1-50 nM). Other cell lines showed moderate or no sensitivity to SSI-4. We tested the concomitant use of SSI-4 with lenvatinib and cabozantinib, tyrosine kinase inhibitors (TKIs) FDA-approved for HCC, in HCC cell lines in vitro and using HCC PDX in vivo mouse models. Our studies showed that the combination of the SCD1 inhibitor with both lenvatinib and cabozantinib showed a highly synergistic effect and no development of drug resistance i.e. durable response versus single TKI therapy. Ongoing mechanistic studies are examining whether known molecular targets of tested TKI’s such as VEGFR1, 2, and 3, PDGFRα, FGFR, KIT, and RET dominate in HCC drug resistance to lenvatinib and cabozantinib, and how the use of SSI-4 overcomes the phenomenon of resistance. Citation Format: Justyna J. Gleba, Aylin Alasonyalilar-Demirer, Matthew L. Pawlush, Ahmet Bilgili, Peyton G. Hickman, Kabir Mody, Lewis R. Roberts, Steven R. Alberts, Mark J. Truty, Tushar C. Patel, Han W. Tun, John A. Copland. Synergistic activity of SCD1 blockade in combination with tyrosine kinase inhibitors lenvatinib and cabozantinib in hepatocellular carcinoma (HCC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5489.

Lung adenocarcinoma with EGFR 19Del and an ALK rearrangement benefits from alectinib instead of an EGFR-TKI: A case report

Rationale:A remarkable concurrence of an EGFR mutation and an EML4-ALK fusion (double positive) occasionally occurs within a narrow number of patients. Previous studies using targeted therapy on EGFR/ALK co-mutated patients have commonly focused on single tyrosine kinase inhibitors (TKIs) or on the sequential use of EGFR-TKIs and ALK-TKIs. At present, no consensus exists regarding the treatment of patients with double positive mutations. The effectiveness of precision therapy also remains unknown.Patient concerns:A 53-year-old female non-smoker who described recurrent coughing and blood in her sputum over a month-long interval was examined at a local hospital.Diagnosis:Using computed tomography (CT) and positron emission tomography CT (PET-CT), the patient was diagnosed with Stage IVb lung adenocarcinoma (T4N3M1).Interventions:The patient had a novel ALK-RAB10 rearrangement identified using DNA sequencing, which, at the transcript level, was actually a canonical ALK fusion that caused a response to alectinib therapy.Outcomes:The patient has achieved partial remission (PR), with a progression free survival (PFS) of 16 months, and continues to benefit.Lessons:Our results may indicate differential sensitivities to TKIs in patients harboring an EGFR mutation and an ALK rearrangement. Our patient’s response to alectinib, instead of to EGFR-TKIs, may lead to an expanded list of alectinib beneficiaries who have rare gene co-alterations in lung adenocarcinoma.

Health-Related Quality of Life of Patients with Chronic Myeloid Leukemia as Measured by Patient-Reported Outcomes: Current State and Future Directions.

Tyrosine kinase inhibitors (TKIs) allow many patients with chronic myeloid leukemia (CML) to live normal life spans but have the potential to impact patients' health-related quality of life (HRQOL). Patient-reported outcome (PRO) measures can provide valuable information to inform treatment decision-making. Here, we review pivotal studies that used PRO measures to evaluate HRQOL of patients with CML in the first-line and treatment-free remission (TFR), and identify areas for future research. PRO measures commonly studied in patients with CML include the SF-36, FACT-Leu, EORTC QLQ-CML24, and MDASI CML. Cohort or cross-sectional studies provide the most data on PRO measures in patients with CML, with less information available from randomized controlled trials (RCTs). Patients with CML taking TKIs have worse HRQOL compared to matched controls, with a few studies seeing a larger effect in younger patients (< 60years old). No single TKI consistently has better HRQOL compared to other agents. Fatigue is a predominant symptom associated with impaired HRQOL across many studies. Studies evaluating TFR show stable or improved HRQOL after TKI discontinuation. There are areas of HRQOL detrimental to patients with other types of cancer (e.g., cognition, sexuality) that warrant further evaluation in patients with CML. Understanding the HRQOL of patients with CML is increasingly important as patients live near-normal life expectancies. PRO measures have the potential to inform treatment decisions in this patient population. Future research opportunities include using PRO measures in RCTs and expanding the HRQOL topics studied in patients with CML.

Cancer-elicited inflammation attenuates response and outcome in tyrosine kinase inhibitor naive patients with advanced NSCLC

ObjectiveCancer elicited inflammation is the main environmental cause leading to carcinogenesis and metastasis of non-small cell lung cancer (NSCLC). Roles of the inflammatory biomarker in predicting the clinical efficacy of tyrosine kinase inhibitor (TKI) and prognosis of naive patients with advanced NSCLC need to be determined, and the best inflammatory predicted biomarker remains unknown. MethodsA total of 178 eligible advanced NSCLC patients (124 and 54 cases within discovery and validation cohorts, respectively) who received first-line EGFR-TKI between July of 2014 and October of 2020 were enrolled in the present study. We detected circulating immune cell counting, albumin (Alb), pre-albumin (pAlb), ALP, AST, LDH, GGT, HDL-c, and fibrinogen (Fib) concentrations, and calculated 22 inflammatory ratios and scores. Logistic regression and Cox proportional hazards models were used to assess the impact of these ratios and scores on objective response and disease control rate (ORR and DCR) as well as progression-free survival (PFS) in these patients. ResultsTwenty-five percentage and 24.07% of NSCLC patients were observed objective response to the treatment of first-line EGFR-TKI in discovery and validation cohort, respectively. Univariate and multivariate Cox regression showed that high PLR, NPS, SII, SIS, mSIS, GLR and FPR as well as low PNI were significantly associated with poor PFS in discovery cohort. However, only high SII and FPR were found to be associated with unsatisfactory outcome in validation cohort. Time-dependent areas under ROC of FPR were 0.702 (0.517–0.888) in discovery cohort, and 0.767 (0.613–0.921) in validation cohort, which were extremely higher than the other biomarkers. The patients with FPR-SII combined score 2 harbored worse prognosis compared to the combined score 0 in discovery (plog-rank = 0.003, adjusted HR = 2.888, 95%CI = 1.500–5.560) and validation cohort (plog-rank = 0.001, adjusted HR = 3.769, 95%CI = 1.676–8.478) as well as overall population (plog-rank < 0.001, adjusted HR = 3.109, 95%CI = 1.878–5.147), and its time-dependent AUCs were 0.747 (0.594–0.900) and 0.815 (0.688–0.942) in the two cohorts, respectively, which were significantly higher than the single biomarker in the two cohorts. The patients with high FPR and FPR-SII score harbored worse DCR than the low patients in the two cohorts and overall population, respectively. Moreover, the similar poor survival was observed in advanced high-FPR NSCLC patients with different treatment options, however, the survival of low-FPR patients with treatment of single TKI, radiotherapy or chemotherapy or radio-chemotherapy combined TKI was good compared to the high-FPR patients with radio-chemotherapy combined TKI, and the survival differences were observed between TKI (plog-rank < 0.001) or radiotherapy combined TKI (plog-rank = 0.014) treated low-FPR patients and the high FPR patients. Additionally, FPR-SII combined score could monitor the progression of the disease in real-time, and the median month of the positive score appearance was significantly earlier than CT/MRI detection (p < 0.001 for 3 months vs. 13 months). ConclusionsHigh-grade cancer elicited inflammation could attenuates response and outcome in tyrosine kinase inhibitor naive patients with advanced NSCLC. FPR-SII combined score was the best inflammatory biomarker to monitor and predict the progression of advanced NSCLC patients with treatment of TKI.

Maintenance Tyrosine Kinase Inhibitors Following Allogeneic Hematopoietic Stem Cell Transplantation for Chronic Myelogenous Leukemia: A Center for International Blood and Marrow Transplant Research Study.

It remains unknown whether the administration of tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 after allogeneic hematopoietic cell transplantation (HCT) is associated with improved outcomes for patients with chronic myelogenous leukemia (CML). In this registry study, we analyzed clinical outcomes of 390 adult patients with CML who underwent transplantation between 2007 and 2014 and received maintenance TKI following HCT (n = 89) compared with no TKI maintenance (n = 301), as reported to the Center for International Blood and Marrow Transplant Research. All patients received TKI therapy before HCT. The majority of patients had a disease status of first chronic phase at HCT (n = 240; 62%). The study was conducted as a landmark analysis, excluding patients who died, relapsed, had chronic graft-versus-host disease, or were censored before day +100 following HCT. Of the 89 patients who received TKI maintenance, 77 (87%) received a single TKI and the other 12 (13%) received multiple sequential TKIs. The most common TKIs used for maintenance were dasatinib (n = 50), imatinib (n = 27), and nilotinib (n = 27). As measured from day +100, the adjusted estimates for 5-year relapse (maintenance, 35% versus no maintenance, 26%; P = .11), leukemia-free survival (maintenance, 42% versus no maintenance, 44%; P = .65), or overall survival (maintenance, 61% versus no maintenance, 57%; P = .61) did not differ significantly between patients receiving TKI maintenance or no maintenance. These results remained unchanged in multivariate analysis and were not modified by disease status before transplantation. In conclusion, our data from this day +100 landmark analysis do not demonstrate a significant impact of maintenance TKI therapy on clinical outcomes. The optimal approach to TKI administration in the post-transplantation setting in patients with CML remains undetermined.

P2.14-34 Tyrosine-Kinase Inhibitors (TKI) in First-Line Treatment of 470 Patients with Non-Small Cell Lung Cancer (NSCLC) from the Czech Republic

From October 2013 there is a possibility to treat patients with NSCLC and with activated epidermal growth factor receptor (EGFR) mutations with three TKI in the Czech Republic. We have tried to find differences among patient groups treated with single TKI in 1st line of treatment. The TULUNG registry was used as a data source for this analysis. This clinical registry is focused on the collection of epidemiological and clinical data of patients with NSCLC treated with target therapy in the Czech Republic. A total of 959 patients treated with TKI inhibitors was enrolled in registry until December 31, 2018. Only patients with EGFR mutation and in which 1st line treatment started in October 2013 and later were included in analysis. With respect to defined inclusion criteria we analysed 470 patients. With gefitinib were treated 234, with afatinib 180 and with erlotinib 56 patients. Descriptive statistics and frequency tables were used to characterize the sample data set. Statistical significance of differences among three TKI inhibitors subgroups was assessed using the Fisher’s exact test or Kruskal-Wallis test for continuous variables. OS and PFS were estimated using Kaplan-Meier method and all point estimates include 95% confidence intervals (95% CI). Statistical significance of differences in survival between subgroups was assessed using the log-rank test. All statistical tests were performed at a significance level of α=0.05. There was statistically significant difference according to age (<0.001) and PS (<0.001), patients treated with gefitinib were statistically significant older and had significantly significant higher PS than patients treated with afatinib and erlotinib There was statistically significant difference in the occurrence of adverse events (p<0.001). Patients treated with gefitinib had a significantly lower incidence of adverse events than patients treated with afatinib and erlotinib. Between these three groups of patients there was no statistically significant difference in sex (p=0.863), in smoking habits (p=0.463, in type of EGFR mutation (p=0.103), in adenocarcinoma proportion (p=0.183). There was no statistically significant difference according to disease control (p=0.183), in response to treatment (p = 0.804), in OS and (p=0.053, in PFS (p=0.06). Between these three groups of patients we found a statistically significant difference in age, PS segmentation and in occurrence of adverse events. We have not found any important statistically difference in sex, smoking, in histology type of EGFR mutation, difference in response to treatment, in disease control and OS.

Abstract 3037: Stromal cell activation of MAPK signaling pathways mediates resistance to MERTK inhibition in acute myeloid leukemia

Abstract MERTK is a TAM family (TYRO-3, AXL, MERTK) receptor tyrosine kinase that is aberrantly expressed in &amp;gt;80% of primary acute myeloid leukemia (AML) samples. MERTK inhibition mediated by the small molecule tyrosine kinase inhibitor (TKI), MRX-2843, induced apoptosis in leukemia cell cultures and prolonged survival in mouse xenograft models of AML, but was not curative. In these models, treatment efficiently reduced peripheral disease burden but was less effective in the bone marrow, suggesting a role for the bone marrow microenvironment in therapeutic resistance. To evaluate the role of the bone marrow stromal niche in resistance to MERTK inhibition by MRX-2843, Kasumi-1 and OCI-AML5 AML cell lines were cultured with the fibroblast-like Hs27 cell line and induction of apoptosis and cell death was measured by flow cytometry after treatment with MRX-2843 or vehicle. Co-culture with Hs27 cells significantly reduced AML cell death in response to MRX-2843 compared to leukemia cells alone (e.g. Kasumi-1 31.9% vs 61.2%, p&amp;lt;0.05). To identify parallel signaling pathways that may be activated under cytokine rich bone marrow stromal conditions and mediate resistance to MRX-2843, a unbiased phospho-kinase array was performed to identify targets differentially activated by co-culture and/or MRX-2843. Kasumi-1 cells were treated with MRX-2843 in the presence or absence of stromal co-culture, then cell lysates were incubated with human phospho-kinase arrays and proteins were quantitated by densitometry. Five kinases were differentially inhibited by &amp;gt;1.5-fold in the presence or absence of stromal cells, including numerous MAPK pathway components - p38α, ERK1/2, EGFR, TOR, and HSP27. Changes in ERK1/2 phosphorylation (pERK) were confirmed by immunoblot in Kasumi-1 and OCI-AML5 cell cultures. In the absence of co-culture MRX-2843 potently inhibited pERK at doses as low as 100nM, while in co-culture even a 3-fold higher dose did not fully inhibit pERK, consistent with a potential role in resistance. To determine whether the observed upregulation and persistent activation of ERK1/2 has functional significance, Kasumi-1 cells were treated with MRX-2843 and the MEK1/2 TKI pimasertib in the presence or absence of stromal co-culture. Treatment with MRX-2843 or pimasertib alone did not significantly affect survival of AML cells in co-culture (31.3% MRX-2843, 28.1% pimasertib vs 22.7% vehicle, p=ns), but treatment with a combination of MRX-2843 and pimasertib resulted in significant induction of apoptosis compared to vehicle or single TKIs (56.7%, p&amp;lt;0.05). Similar results were observed in OCI-AML5 cell cultures and in combination with the MEK1/2 inhibitor PD0325901. Together these results indicate a critical role for stromal cell-mediated activation of ERK1/2 in resistance to MRX-2843 and demonstrate the utility of translational strategies targeting both MERTK and MAPK pathways to overcome resistance. Citation Format: Katherine A. Minson, Eleana Vasileiadi, Madeline G. Huey, Xiadong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. Stromal cell activation of MAPK signaling pathways mediates resistance to MERTK inhibition in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3037.

Concomitant resistance mechanisms to multiple tyrosine kinase inhibitors in ALK-positive non-small cell lung cancer

ObjectivesALK tyrosine kinase inhibitors (TKIs), including crizotinib and several next generation TKIs, have demonstrated beneficial clinical outcomes in ALK-positive non-small cell lung cancer (NSCLC). However, resistance mechanisms following multiple TKI treatments in ALK-positive NSCLC are not fully elucidated. Materials and methodsMutation profiles of 422 cancer-relevant genes in 52 patients with post-TKI biopsy samples were analyzed using next-generation sequencing (NGS), and compared between patients receiving crizotinib alone (n = 35) and multi-TKIs (n = 17). ResultsEML4-ALK variant 3 is the most frequent ALK variants in this cohort, followed by EML4-ALK variant 1. Half of the patients harbored ALK activating mutations upon progression on crizotinib treatment. After multi-TKIs treatment, 59% of the cases developed resistant ALK mutations, and concomitant ALK activating mutations were more commonly observed in this cohort (P = 0.031). Specifically, ALK G1269 A, L1196 M, and C1156Y substitutions were more common in crizotinib-alone samples, while ALK G1202R was significantly more enriched post-multi-TKIs (P = 0.009). Activated bypass signaling tended to be more prevalent in patients post-multi-TKIs. Furthermore, dual activation of ALK and bypass signaling was more frequently found in the multi-TKIs group (5/17, 29%) in contrast to crizotinib-alone (2/35, 6%) (P = 0.031). Additionally, concurrent TP53 mutation demonstrated significantly shorter progression-free survival (PFS) compared with TP53 wildtype in crizotinib-alone group (median PFS: 8 vs 13 months, Hazard Ratio = 1.494, P = 0.019). ConclusionConcurrent ALK activating mutations and/or upregulated bypass signaling are more enriched in patients undergoing multiple ALK TKI treatments compared to crizotinib alone. Concomitant TP53 mutation correlated to unfavorable survival when receiving a single TKI crizotinib.

A Population-Based Study of Chronic Myeloid Leukemia Treated with Imatinib in First Line

Background. Chronic myeloid leukemia (CML) treatment is based on company-sponsored and academic trials testing different tyrosine kinase inhibitors (TKIs) as first-line therapies. However these clinical trials included patients selected according to different inclusion and exclusion criteria, particularly age and comorbidities, and with specific treatment obligations. In daily clinical practice (real life), no inclusion and exclusion criteria do exist, and treatment outcomes depend not only on choice of the first-line TKI, but also on doctor- and patient-choice of second- and third-line TKIs. Aims. To investigate the response and the outcome on first-line imatinib, with switch to 2nd generation TKIs in case of failure or toxicity, in BCR-ABL1+ CML patients enrolled in a population-based registry (real-life setting). Methods. We report here the results from a prospective study, enrolling all newly diagnosed patients (N = 236, median age 60 years) living in two Italian regions, that were registered according to population-based criteria and treated front-line with imatinib. The decision to switch to second-line treatment was up to the Local Investigator (no predefined criteria) Definitions: major molecular response (MMR or MR3.0): BCR-ABL1IS ratio 10,000 ABL1 copies; failure and suboptimal response: according to 2009 European LeukemiaNet (ELN) criteria; progression: transformation to advanced phases according to ELN criteria; leukemia-related death (LRD): death after progression. Results. Median age was 60 years at diagnosis and 64 years at last contact, with a median follow up of 48 months (range 2-86 months). High risk patients according to Sokal score and new EUTOS long-term survival (ELTS) score were 23% and 18%, respectively. A switch from imatinib to a second generation TKI was reported in 14% of patients for side-effects, and in 24% for failure or suboptimal response, with an improvement of molecular response in 57% of them. The median time to switch was 8 months for toxicity and 20 months for failure. At 12 months, 55% of all evaluable patients were in MR3.0 or better. At last contact, 75% of all evaluable patients were in MR3.0 or better and 48% were in MR4.0 or better. The molecular response of patients switching for failure or suboptimal response was evaluable in 61/63 patients: improved in 57%, not modified in 38%, worsened by at least 1 log in 5% of patients. Overall, the treatment switch was associated with a remarkable increase of MR3.0 rate: from 10% at the time of switching to 52% at last qPCR. Overall, 33% achieved a stable MR4.0, of whom 22% with imatinib alone, and 11% after switching to a second generation TKI (Table 1). The 5-year overall survival (OS) and leukemia-related survival (LRS) were 85% and 93%, respectively. Twenty-seven patients (11%) died, of whom 13 (6%) of leukemia, after disease progression and 14 (6%) of "other" causes, without any evidence of progression. Cardiovascular complications were reported in 4% of patients treated with imatinib alone and in 6% of patients who received nilotinib in second-line. OS was not affected by age in the interval between 18 and 69 years (5-year OS 92%, ranging from 89% to 95 % in the different decades), but 5-year OS probabilities of elderly (70-79 years) and very elderly patients (≥ 80 years) were significantly lower (78%, P = 0.003, and 34%, P Conclusions. The results of this prospective population-based study emphasize the importance of second-line treatment in the clinical practice: the CML management with the 3 available TKIs in the period 2008-2012, according to current treatment recommendations for switching and without any predefined obligation, resulted in response rates and outcomes, that are in the high range of prospective interventional studies investigating the efficacy and the safety of a single TKI in selected patients. This study provides a robust dataset on the results of CML treatment with TKI in clinical practice, that will be important for interpreting and evaluating the results of the next prospective studies, arguably limited to selected patients. Disclosures Castagnetti:Bristol-Myers Squibb: Consultancy, Honoraria; ARIAD Pharmaceuticals: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Gugliotta:Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Marasca:Janssen: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees; Pfizer: Honoraria; Roche: Honoraria. Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Ariad: Consultancy. Martinelli:Genentech: Consultancy; ARIAD: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy. Cavo:Bristol-Myers Squibb: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Rosti:Novartis: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding, Speakers Bureau; Incyte: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau. Baccarani:Ariad: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau.

A Meta-Analysis and Meta-Regression of the Efficacy of Front-Line Treatment Combinations with Ponatinib Versus 1st- and 2nd-Generation Tyrosine Kinase Inhibitors for Ph+ Acute Lymphoblastic Leukemia

▪Background: The current standard induction therapy for de novo Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) is a tyrosine kinase inhibitor (TKI) in combination with chemotherapy or corticosteroids. Three generations of TKIs are currently available for the treatment of Ph+ ALL. Ponatinib, a third generation multi-targeted TKI, is a more potent inhibitor of BCR/ABL than previous generations. It also is effective in cases where there is a threonine-to-isoleucine substitution at position 315 (T315I mutation), which confers resistance to all first- and second-generation TKIs (i.e., imatinib, dasatinib, and nilotinib). However, evidence on the comparative effectiveness of front-line combination therapy with ponatinib relative to other TKIs in de novo Ph+ ALL has not been well-established.Aims: The purpose of this study is to evaluate the effectiveness, as measured by complete molecular response (CMR) and 2- and 3-year overall survival (OS), of front-line treatment combinations with ponatinib versus first- and second-generation TKIs in de novo Ph+ ALL.Methods: Twenty-six studies of front-line Ph+ ALL treatment with TKI in combination with chemotherapy or corticosteroids (18 studies of imatinib, 5 of dasatinib, 2 of nilotinib, and 1 of ponatinib) were identified from published targeted literature reviews and recently published trials. These consisted of 25 phase 2 through 4 trials and 1 retrospective analysis. Study arms in which patients received chemotherapy or corticosteroids only, a single TKI agent, or in which they received autologous stem cell transplant exclusively were excluded. Data on aggregated patient characteristics were extracted and summarized using the median (range). The proportions of patients achieving CMR (following induction or consolidation therapy) and 2- and 3-year OS were also extracted from all study arms and summarized by TKI group (ponatinib versus all other first and second generation TKIs) using pooled estimates with 95% confidence intervals (CIs) from a random-effects meta-analysis. A binomial distribution was assumed to calculate the 95% CIs for the ponatinib trial. Multivariate logistic meta-regressions were conducted to examine the association between each TKI treatment group and CMR rates, 2-year OS, and 3-year OS, separately, adjusting for age and gender.Results: A total of 32 TKI treatment arms were included in the analysis. The median (range) of age across trial arms was 46 years (36-69 years) and proportion of male patients was 53% (42-67%). The pooled proportion of patients achieving CMR with ponatinib was higher than that with first and second generation TKIs (79% versus 34%). The pooled 2- and 3-year OS with ponatinib were also higher than those with other TKIs (2-year: 83% versus 58%; 3-year: 79% versus 50%). Relative to other TKIs, ponatinib was associated with a statistically significant 6.09-fold increase in the odds of achieving CMR (N=25) [odds ratio=6.09 (95% CI: 1.16-31.90), p=0.034]. While ponatinib was not significantly associated with an increase in the odds of 2-year OS (N=27) [odds ratio=3.70 (95% CI: 0.93-14.73), p=0.062], it was associated with a statistically significant 4.49-fold increase in the odds of 3-year OS (N=19) [odds ratio=4.49 (95% CI: 1.00-20.13), p=0.050] compared to earlier generation TKIs.Conclusion: Frontline treatment with ponatinib in combination with chemotherapy or corticosteroids was associated with significantly better odds of CMR and 3-year OS in patients newly diagnosed with Ph+ ALL than combination therapy with earlier generations of TKIs. In particular, ponatinib was associated with a 6-fold increase in CMR, the most important factor in predicting long-term survival outcomes. The improved efficacy of ponatinib in Ph+ ALL may be particularly important for patients who are ineligible to undergo stem cell transplantation due to lack of suitably matched donors, advanced age, or significant comorbidities. In such cases, combination therapy with TKI may be a suitable alternative. Though the number of studies included was small and few covariates could be used to adjust for heterogeneity across trials, the results suggest that ponatinib in combination with chemotherapy may represent an effective front-line treatment option for patients with Ph+ ALL. Prospective head-to-head clinical trials are needed to confirm these results.Sponsor: ARIAD Pharmaceuticals, Inc. [Display omitted] DisclosuresJabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. DerSarkissian:ARIAD: Research Funding. Duh:Allergan: Consultancy; Abbvie: Consultancy; Novartis: Research Funding; GSK: Research Funding; Bayer: Research Funding; Janssen: Research Funding; Eisai: Research Funding; Pfizer: Research Funding; Medtronic: Research Funding; Takeda: Research Funding; Novo Nordisk: Research Funding; Sanofi: Research Funding; Ariad: Research Funding. McCormick:ARIAD: Research Funding. Cheng:ARIAD: Research Funding. McGarry:ARIAD: Employment, Equity Ownership. Souroutzidis:ARIAD: Research Funding. Huang:ARIAD: Employment, Equity Ownership. Kantarjian:Bristol-Myers Squibb: Research Funding; ARIAD: Research Funding; Amgen: Research Funding; Pfizer Inc: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding.

A clinical study of interstitial lung disease induced by TKI concurrent radiotherapy

Objective To investigate the influence of radiotherapy with concurrent oral epidermal growth factor receptor (EGFR) and tyrosine kinase inhibitor (TKI) for advanced non-small cell lung cancer (NSCLC) on the incidence of interstitial lung disease (ILD), and to analyze the risk factors for the pathogenesis of ILD. Methods The incidence of ILD in 72 patients with NSCLC who were admitted to Department of Radiotherapy Oncology of Lu'an Hospital of Traditional Chinese Medicine and treated with radiotherapy with concurrent oral EGFR-TKI from January 2010 to January 2014 was analyzed retrospectively, and some patients were selected as controls to compare the difference in the incidence of ILD between the TKI concurrent radiotherapy group and the other groups. The univariate analysis using chi-square test and multivariate logistic regression analysis were applied to analyze the risk factors for the incidence of ILD in the patients receiving TKI concurrent radiotherapy. Results The TKI concurrent radiotherapy group had a significantly higher incidence rate of ILD than the palliative chemotherapy group, single TKI group, and concurrent chemoradiotherapy group (19.4% vs. 2.5%, 12%, and 7.5%, P=0.045). The multivariate regression analysis showed that male sex, long-term smoking, underlying lung disease, tumor volume>5 cm, and application of conventional radiotherapy were the risk factors for the pathogenesis of ILD in the TKI concurrent radiotherapy group. Conclusions Radiotherapy with concurrent TKI for advanced NSCLC may increase the incidence of ILD. Key words: Tyrosine kinase inhibitors; Interstitial lung disease; Lung neoplasms/radiotherapy; Lung neoplasms/chemotherapy

A Novel Compound Inhibits Chronic Myeloid Leukemia Via Upregulating Apoptosis

Chronic myeloid leukemia (CML) is a myeloid proliferative disorder mainly result from chimeric protein BCR-ABL1 encoded by a fusion gene at the t(9;22) (q34;q11) chromosomal translocation. Intrinsically, this recombined protein results in an increased tyrosine kinase (TK) activity that directly related to hematopoietic stem cell malignant proliferation. Consequently, the drugs derived from tyrosine kinase inhibitors (TKI) have been developed as an infective therapy, and greatly improved patients survival in clinic. Unfortunately, single TKI administration led to toxicities or tolerance in long-term treated CML patients. Even worse is, about 5% CML patients were not caused by bcr-abl gene mutation. Thus better medicines are badly needed to compensate CML therapy. Herein, we investigated the undefined function of a biscoumarins. The new synthesized compound exhibited a null toxicity on HUVECs but intensive toxicity on K562 leukemic cells. Subsequent results demonstrated that it efficiently inhibited the expansion of human CML cell line and bone marrow cells of SCL-tTA-BCL/ABL transgenic model mice via increased apoptosis. Critically, we also showed that CD34+ bone marrow leukemic cells collected from patients underwent more apoptosis after treated by the biscoumarins derivate. To extend these results into vivo, we observed a prolonged survival of bcr-abl transgenic mice treated by derivate mono-therapy or combination with imatinib compared to those of untreated or imatinib-treated CML mice. All together, these results indicated that this biscoumarins derivate may have novel potential as a therapeutic agent against CML. DisclosuresNo relevant conflicts of interest to declare.

A New AHI-1-DNM2-BCR-ABL Complex Regulates Leukemic Properties of Primitive CML Cells through Mediation of Cellular Endocytosis and ROS-Induced Autophagy

Tyrosine kinase inhibitor (TKI) therapies have been introduced into clinical practice with remarkable effects on chronic phase CML. However, early relapses, acquired drug resistance, and persistence of leukemic stem cells (LSCs) remain problematic. Improved treatment approaches to specifically target key molecular elements active in CML LSCs are needed. One candidate is the adaptor protein AHI-1 (Abelson helper integration site-1), an oncogene that is highly deregulated in LSCs. An AHI-1-mediated protein complex containing BCR-ABL and JAK2 has been shown to modulate transforming activity and TKI-response/resistance of CML LSCs. We have recently identified the large GTPase Dynamin-2 (DNM2) as another AHI-1 interacting protein using the AHI-1 SH3 domain as protein "bait" in immunoprecipitation (IP)/mass spectrometry. DNM2 plays key roles in the regulation of trafficking processes such as endocytosis, and is activated through tyrosine phosphorylation. However, its role in CML pathogenesis is unknown. We have now demonstrated that transcript levels of DNM2 are significantly increased in treatment-naive CD34+ cells from CML patients who were classified retrospectively, after Imatinib (IM) therapy, as IM-responders (n=11) and IM-nonresponders (n=15) in comparison to CD34+ normal bone marrow cells (n=7, p=0.013 and 0.037). In particular, DNM2 is more highly expressed in CML stem-enriched cells (lin-CD34+CD38-) and progenitor cells (lin-CD34+CD38+) than more mature cells (lin+CD34-, 2-fold). Interestingly, BCR-ABL+ human cells with stable knockdown of DNM2 exhibited significantly reduced cell growth and increased apoptosis as compared to control cells. They also showed enhanced sensitivity to single TKIs or MitMAB (DNM2 inhibitor) and these effects were greatly enhanced by combination treatments (2-3 fold). Mechanistically, co-IP and confocal co-localization analysis with mutant forms of AHI-1 and DNM2 (HA-AHI-1, HA-AHI-1 SH3Δ, Myc-DNM2 and Myc-DNM2 PRDΔ) in 293T cells indicated that the PRD domain of DNM2 is mainly responsible for the interaction between the SH3 domain of AHI-1 and DMN2. More importantly, we identified a new protein interaction between DNM2 and BCR-ABL in both BCR-ABL and BCR-ABL/AHI-1 co-transduced hematopoietic cells using co-IP/Western analysis; this interaction is enhanced in BCR-ABL/AHI-1 co-transduced cells. Moreover, DNM2 phosphorylation was decreased upon IM treatment in BCR-ABL-transduced cells, but remained unchanged in control and T315I mutant cells, suggesting that DNM2 is a direct target of BCR-ABL. Interestingly, we further observed that AHI-1 co-localizes with EEA-1 (early endosome marker) and LAMP-1 (late endosome marker) in cells co-transfected with full-length AHI-1 and DNM2, but not in AHI-1 and DNM2 mutant cells. Using transferrin uptake assays, increased transferrin signals were observed in BCR-ABL/AHI-1 co-transduced cells compared to BCR-ABL- and BCR-ABL/AHI-1 SH3Δ-transduced cells, while transferrin signals were much lower in DNM2-knockdown cells than control cells. These results suggest that the AHI-1-DNM2-BCR-ABL complex indeed improves the kinetics and increases efficiency of endocytosis, which may contribute to response/resistance of primitive CML cells to TKIs. This is further supported by observation of increased surface expression levels of CXCR4 in DNM2 knockdown cells, a key endocytotic target and known mediator of TKI response in CML. Moreover, reactive oxygen species (ROS) signals were found to be higher in BCR-ABL/AHI-1 co-transduced cells than BCR-ABL- and BCR-ABL/AHI-1 SH3Δ-transduced cells, while ROS accumulation was significantly decreased in DNM2-knockdown cells compared to control cells. More interestingly, ROS-induced autophagy was further observed in BCR-ABL/AHI-1 co-transduced cells, but reduced in DNM-2-knockdown cells, with protein expression changes in key autophagy proteins, including ULK-1, Beclin-1, LC3 and p62. To the best of our knowledge, this is the first study to implicate a new AHI-1-DNM2-BCR-ABL complex in the deregulation of endocytosis signaling and ROS production/autophagy in CML. This may play a unique and important role in the regulation of cellular properties of primitive CML cells, including their response/resistance to TKI. DisclosuresNo relevant conflicts of interest to declare.

Kinome RNAi Screens Reveal Synergistic Targeting of MTOR and FGFR1 Pathways for Treatment of Lung Cancer and HNSCC.

The FGFR1 is a therapeutic target under investigation in multiple solid tumors and clinical trials of selective tyrosine kinase inhibitors (TKI) are underway. Treatment with a single TKI represents a logical step toward personalized cancer therapy, but intrinsic and acquired resistance mechanisms limit their long-term benefit. In this study, we deployed RNAi-based functional genomic screens to identify protein kinases controlling the intrinsic sensitivity of FGFR1-dependent lung cancer and head and neck squamous cell cancer (HNSCC) cells to ponatinib, a multikinase FGFR-active inhibitor. We identified and validated a synthetic lethal interaction between MTOR and ponatinib in non-small cell lung carcinoma cells. In addition, treatment with MTOR-targeting shRNAs and pharmacologic inhibitors revealed that MTOR is an essential protein kinase in other FGFR1-expressing cancer cells. The combination of FGFR inhibitors and MTOR or AKT inhibitors resulted in synergistic growth suppression in vitro. Notably, tumor xenografts generated from FGFR1-dependent lung cancer cells exhibited only modest sensitivity to monotherapy with the FGFR-specific TKI, AZD4547, but when combined with the MTOR inhibitor, AZD2014, significantly attenuated tumor growth and prolonged survival. Our findings support the existence of a signaling network wherein FGFR1-driven ERK and activated MTOR/AKT represent distinct arms required to induce full transformation. Furthermore, they suggest that clinical efficacy of treatments for FGFR1-driven lung cancers and HNSCC may be achieved by combining MTOR inhibitors and FGFR-specific TKIs.

Incidence of Vascular Thrombotic Events in 183 Consecutive Patients Treated with Nilotinib: A Single Centre Experience

▪IntroductionThe spectrum of adverse events occurring in nilotinib is broadly similar to that of other tyrosine kinase inhibitors but recent reports suggest an increase in the incidence of vascular thrombotic events (VTE) compared to imatinib. Many patients treated with ponatinib, where the association of VTE with treatment is now widely accepted, have previously received nilotinib and it remains unclear as to whether the adverse events are a result of the cumulative use of the two drugs. It is important to clearly delineate the risk of VTE with nilotinib in order to estimate risk and provide better information for patients.MethodsWe conducted a chart review to identify adverse events in 183 consecutive patients who received nilotinib in our institution from February 2006 until June 2014. Patients were to be considered at risk of side effects in they had received at least 24 hours of treatment. Data were collected from out-patient consultations in which side effects and their severity were self-reported and recorded in the medical case notes. The cohort contained 93 women and 90 men and had a median age of 56 years (range 21-93). 8% of patients received nilotinib as first line therapy: 46% and 39% respectively were treated after failure of imatinib only or imatinib and dasatinib . The remainder were treated for relapse post allogeneic transplant. Of those who were treated after one or two prior tyrosine kinase inhibitors (TKI), 57% and 43% were intolerant or resistant respectively.ResultsThe median duration of treatment with nilotinib was 714 days (range 10 -2816 days). Information was available for pre-existing cardiovascular risk factors in 93% of patients and were present in 59%. We recorded 20 occurrences of VTE in 10% of patients with 9 (5%), 7 (4%) and 4 (2%) episodes of myocardial ischaemia, peripheral arterial occlusive disease and cerebrovascular disease respectively. Only one patient without pre-existing cardiovascular risk factors experienced a VTE, The median age of patients with VTE was 67 years (range 35-79) compared to 55 years (range 21-93) in those without VTE. In contrast to previously reported results VTE were more common (18%) in patients who had received two prior TKI compared to 8% in those who had been treated with a single TKI and 7% who received nilotinib upfront. 75% of VTE occurred in patients who have been treated with nilotinib for more than 2 years but this may in part be because of continuation of treatment at a time of lack of awareness of the association of nilotinib with VTE. The remaining adverse events reported on nilotinib were in accordance with published data. Side effects occurring in >10% of patients are given in the table.ConclusionsThe incidence of VTE in patients treated with nilotinib in our institution was 10%. VTE was more frequent in older patients, in those with pre-existing cardiovascular risk factors and in those who received prolonged therapy with nilotinib. Without a suitable control group matched for age and cardiovascular risk factors it is difficult to provide an accurate estimate of any potential increased risk of treatment with nilotinib. Nevertheless caution must be exercised in older patients with pre-existing risks for VTE and appropriate counselling and monitoring provided. Table 1Adverse eventIncidence (%)Rash and/or pruritus43Fatigue31Elevated transaminases21Myalgia18Abdominal pain17Headaches17Arthralgia16Nausea14Thrombocytopenia12Neutropenia12Anaemia7 DisclosuresGerrard:Novartis: Research Funding. Foroni:Novartis: Research Funding. Apperley:Ariad Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Speakers Bureau; Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Targeting Treatment-Naïve CML Stem/Progenitor Cells From Imatinib-Nonresponders with JAK2 (BMS-911543)/ABL Inhibitor Combination Therapy

AbstractAbstract 1339Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder characterized by a BCR-ABL fusion gene with constitutive tyrosine kinase activity that drives the pathogenesis of the disease. Deregulated tyrosine kinase activity of BCR-ABL perturbs several intracellular signaling pathways, including the JAK2/STAT5 pathway. In particular, JAK2 physically interacts with the C-terminal region of BCR-ABL and is one of the most prominent targets of BCR-ABL. Recent introduction of imatinib mesylate (IM) and other tyrosine kinase inhibitor (TKI) therapies have had a major impact on treatment of chronic phase CML. However, early relapse, acquired drug resistance and ineffectiveness in eliminating leukemic stem cells remain impediments to successful treatment for many patients. Improved treatment approaches to prevent development of resistant subclones, by combined targeting of other key molecular elements active in CML stem cells, are thus clearly needed. One candidate is a complex between the oncoproteins encoded by Abelson helper integration site-1 (AHI-1), BCR-ABL and JAK2 that we recently identified. This complex contributes to the transforming activity of BCR-ABL and also plays a key role in the IM response/resistance of primary CML stem/progenitor cells. We have therefore hypothesized that combined suppression of BCR-ABL and JAK2 activities might be more effective in eliminating treatment-naïve CML stem/progenitor cells in patients destined to develop resistance to TKI monotherapy. Several JAK2 inhibitors are currently in various stages of clinical trials, but their nonspecific, off-target effects on normal primitive hematopoietic cells remain a concern. We have now examined the biological effects of a new, highly specific JAK2 inhibitor (BMS-911543) in combination with a number of TKIs, including IM, dasatinib and nilotinib. First, Western blot analysis showed that combination treatment was more effective at reducing pSTAT5 levels in K562 cells and IM-resistant K562 cells than single agents. Also, in colony-forming cell (CFC) assays, combination treatment resulted in a greater reduction in colonies produced from these cells compared to single agents, especially in medium-sized colonies (50–500 cells per colony, 2–3 fold, p<0.05). To determine whether the results obtained following the simultaneous targeting of BCR-ABL and JAK2 in CML cell lines would extend to primary primitive CML cells and whether this combined BCR-ABL-JAK2 targeting approach could also be therapeutically effective for CML patients who do not respond adequately to treatment with a single TKI, we investigated primitive CML cells obtained at diagnosis from seven CML patients who were classified retrospectively as IM-nonresponders, after initiation of IM therapy. Similar to our cell line studies, intracellular staining analysis showed that combined exposure of CD34+CML cells (n=3) to BMS-911543 and a TKI produced a deeper and more prolonged suppression of pSTAT5 activity than a single agent (40–46% suppression for the combinations vs. 15–20% suppression for the single agents at 72 hrs, p<0.05). Combination treatments also caused greater inhibition in colony growth of these cells compared to single treatments (74–86% vs. 40–50%, p<0.05). Interestingly, the combination of BMS-911543 and a TKI almost completely inhibits BFU-E colony formation as compared to treatment with TKI alone (92–100% vs.63–66%, p<0.01). CFU-GM colonies were also more significantly reduced as a result of combination treatment, compared to single agents (49–71% vs. 30–39%, p<0.01). Furthermore, long-term culture-initiating cell (LTC-IC) assays showed that the more primitive cells were also more significantly eliminated by combination treatments than by single agents (2–5 fold, p<0.05), indicating the merit of combination therapy in targeting very primitive cells. Most importantly, our CFC data indicate that BMS-911543 has far less toxicity on normal bone marrow CD34+ cells (n=4) than CML samples (2–3 fold, p<0.05). This study suggests a rational strategy for improving therapeutic outcomes in CML IM-nonresponders by simultaneously targeting both BCR-ABL and JAK2 activities in primary CML stem/progenitor cells. Disclosures:Lorenzi:Bristol-Myers Squibb: Employment, Equity Ownership. Woolfson:Bristol-Myers Squibb: Employment, Equity Ownership.

Alternating Nilotinib 400 mg twice daily and Imatinib 400 mg once daily as Frontline Treatment of Ph+ Chronic Myeloid Leukemia. A Phase 2 Multicentric Study of the GIMEMA CML Working Party

Abstract 453 Background:Imatinib (IM) 400 mg daily is the standard treatment for Ph+ Chronic Myeloid Leukemia (CML) in early Chronic Phase (ECP). Nilotinib (NIL) is a 2nd generation tyrosine kinase inhibitor (TKI) with superior efficacy to IM (phase 3 ENESTnd trial). NIL has been approved for the frontline treatment of CML in many countries. The treatment with more than one TKI, according to the principles of cancer polychemotherapy, may improve the response rates and may decrease the frequency of drug-resistance. The combination of different TKIs is potentially toxic, difficult to be explored in the ECP setting. The sequential administration of IM and NIL is worth to be investigated because a significant proportion of CML patients treated with a single TKI as monotherapy develops primary or secondary resistance. Aims:To evaluate the response (either cytogenetic and molecular) and the outcome of ECP Ph+ CML patients treated with the sequential administration of NIL and IM. Methods:A phase 2 study was conducted by the GIMEMA CML WP (ClinicalTrials.gov. NCT00769327). NIL was administered first because it has a more rapid therapeutic effect. Schedule: NIL 400 mg twice daily for 3 months; IM 400 mg daily for the next 3 months; then, NIL and IM turning every 3 months, for a total duration of 24 months (study core). The 3-month rotation schedule was respected, irrespectively of temporary discontinuations. The primary end-point was the Complete Cytogenetic Response (CCgR) rate at 12 months. If the conventional cytogenetic analysis resulted not evaluable, a FISH analysis was performed. If one of the 2 drugs was permanently discontinued for adverse event (AE), the patient remained in study, continuing the treatment with the other drug (except for cardiac AEs). Definitions: Major Molecular Response (MMR): BCR-ABL/ABL ratio < 0,1%IS; failure: according to 2009 ELN recommendations; event: failure, permanent discontinuation of both drug for any reason, patient refusal. All the analysis were performed according to the ITT principle. Results:123 patients have been enrolled in 38 italian hematologic Centers; median age 56 years (range 18–84); 33% low, 45% intermediate and 22% high Sokal score; median follow-up 21 months (at least 12 months observation, 24 months by November 2011). The cumulative CCgR rate by 12 months was 87%; CCgR at each milestone: 68% at 3 months, 73% at 6 months, 67% at 12 months (primary efficacy variable). The cumulative MMR rate by 12 months was 82%, while the rates of MMR at 3, 6 and 12 months were 59%, 62% and 60%, respectively. The discrepancies between cumulative response rates and response at each timepoint were mainly due to the number of patients not evaluable at each timepoint (10% of cytogenetic analysis not evaluable at 12 months) and to protocol discontinuation in stable CCgR and/or MMR. The incidence of hematologic AEs was low. Non-hematologic AEs or lab abnormalities grade > 2 observed in >5 % of patients were as follows: NIL - skin rash, pruritus, bilirubin increase, transaminase increase, lipase increase; IM - fluid retention, periorbital edema. AEs were manageable with appropriate dose adaptations. Four patients (3%) showed a prolongation of the QTcF above 450 msec (none above 500 msec). At the end of the first 12 months, 95 patients (77%) remained on study: 6 and 3 on NIL and IM monotherapy, respectively, 86 on sequential treatment with both drugs; 15% of patients permanently reduced the NIL dose to 400 mg daily and 9% of patients permanently reduced the IM dose to 300 mg daily. During the first year, 28 patients (23%) experienced an event: 10 treatment failure (8%); 2 death in CP (2%); 16 refusal, protocol violation or AE (13%). Six out of the 10 patients who failed the treatment progressed to advanced phase (3 patients: detection of a T315I mutation). Conclusions:The cumulative response rates achieved with a sequential administration of NIL and IM seem to be superior to the historical data of IM alone. The response rates at each timepoint, lower than expected, were probably due to the high number of not evaluable patients and to the number of patients not continuing the study despite a stable CCgR/MMR. However, if compared to the excellent results of 2nd generation TKI as monotherapy in ECP CML (single and randomized trials), the present analysis do not support an alternating schedule of NIL and IM as frontline treatment of ECP CML. Acknowledgments:European LeukemiaNet, COFIN, Bologna University, BolognAIL Disclosures:Castagnetti:Novartis Pharma: Honoraria; Bristol-Myers Squibb: Honoraria. Rosti:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Soverini:Novartis: Consultancy; ARIAD: Consultancy; Bristol-Myers Squibb: Consultancy. Martinelli:Bristol-Myers Squibb: Consultancy; Novartis: Consultancy, Research Funding.

Abstract 619: Dual targeted therapies to overcome tyrosin kinase inhibitors resistant in epidermal growth factor receptor (EGFR) expression of lung cancer

Abstract Background: Non-small cell lung cancer (NSCLS) comprises about 80% of all lung cancers. The regular treatments for NSCLC are surgery, chemotherapy, radiotherapy and EGFR-targeted therapy. EGFR is frequently mutated and amplified in lung adenocarcinom as sensitive to EGFR inhibitors. The EGFR-targeted therapy, Gefitinib and Erlotinib, significantly improves disease free survival. However, it was not possible to predict super-responder of gefitinib degree of EGFR expression as a single agent. There is an imposing need for new rationally designed regimens to overcome Gefitinib resistance. The other tyrosine kinase inhibitor, Lapatinib, is a small-molecule inhibitor targeting both the HER2 and the epidermal growth factor receptor (EGFR) in solid tumors. We hypothesized that this aberrant activation of EGFR may contribute to Gefitinib resistance and dual blocking EGFR activation with EGFR and ERBB2 inhibitor will re-sensitize HER family in lung cancers. Experimental Design: In the in vitro study, cancer cells with EGFR wild type or mutation type were treated with Tyrosine kinase inhibitor, Gefitinib, Lapatinib, or combined treatments. The responses of cells to Gefitinib or/and Lapatinib treatments were evaluated by MTS proliferation, apoptosis assay, soft agar colony formation assay and western blot. In the in vivo study, pulmonary xenograft tumors were established in ICR/SCID mouse and treated with vehicle control, Gefitinib, Lapatinib, or Gefitinib plus Lapatinib for 4 weeks. Tumor volume were monitored and further studied by immunohistochemistry on collected tumors. Results: We selected multiple pairs of EGFR wild type and mutation type. The EGFR mutation cells have more effect under single tyrosine kinase inhibitor (Gefitinib) treatment compared with EGFR wild type cells. Dual inhibition of EGFR activation sensitized EGFR expression pulmonary cancer cells to Gefitinib treatment. Compared with single treatment, combined treatment led to a significantly more effective inhibition of cell proliferation and induced apoptosis. In a mouse pulmonary xenograft model, combination therapy with Gefitinib and Lapatinib dramatically inhibited EGFR wild type tumor growth. Conclusions: Since the EGFR family inhibitor, Lapatinib is currently under Phase III clinical trial, combining Gefitinib with this Lapatinib, dual EGFR inhibitors is a clinically applicable strategy and a promising combinatory targeted anti-cancer therapy to overcome EGFR wild type mediated Gefitinib resistance. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 619.

Blockade of EGFR and ErbB2 by the Novel Dual EGFR and ErbB2 Tyrosine Kinase Inhibitor GW572016 Sensitizes Human Colon Carcinoma GEO Cells to Apoptosis

Coexpression of the epidermal growth factor receptor (EGFR) family receptors is found in a subset of colon cancers, which may cooperatively promote cancer cell growth and survival, as heterodimerization is known to provide for diversification of signal transduction. Recently, efforts have been made to develop novel 4-anilinoquinazoline and pyridopyrimidine derivatives to inhibit EGFR and ErbB2 kinases simultaneously. In this study, we tested the efficacy of a novel reversible dual inhibitor GW572016 compared with the selective EGFR and ErbB2 tyrosine kinase inhibitors (TKI) AG1478 and AG879 and their combination, using the human colon adenocarcinoma GEO mode. GEO cells depend on multiple ErbB receptors for aberrant growth. A synergistic effect on inhibition of cell proliferation associated with induction of apoptosis was observed from the combination of AG1478 and AG879. Compared with AG1478 or AG879, the single TKI compound GW572016 was a more potent inhibitor of GEO cell proliferation and was able to induce apoptosis at lower concentrations. Western blot analysis revealed that AG1478 and AG879 were unable to suppress both EGFR and ErbB2 activation as well as the downstream mitogen-activated protein kinase (MAPK) and AKT pathways as single agents. In contrast, GW572016 suppressed the activation of EGFR, ErbB2, MAPK, and AKT in a concentration-dependent manner. Finally, in vivo studies showed that GW572016 treatment efficiently blocked GEO xenograft growth at a dose range of 30 to 200 mg/kg with a twice-daily schedule. In summary, our study indicates that targeting both EGFR and ErbB2 simultaneously could enhance therapy over that of single agents directed at EGFR or ErbB2 in cancers that can be identified as being primarily heterodimer-dependent.